PI: Title: Antigen Detection assay for the Diagnosis of Melioidosis

Received: 12/05/2013 FOA: PA10-124 Council: 05/2014

Competition ID: ADOBE-FORMS-B1 FOA Title: NIAID ADVANCED TECHNOLOGY STTR (NIAID-AT-STTR [R41/R42])

2 R42 AI102482-03 Dual: Accession Number: 3650491

IPF: 3966401 Organization: INBIOS INTERNATIONAL, INC.

Former Number: Department:

IRG/SRG: ZRG1 IDM-V (12)B AIDS: N Expedited: N

Subtotal Direct Costs Animals: N New Investigator: N

(excludes consortium F&A) Humans: Y Early Stage Investigator: N

Year 3: Clinical Trial: N

Year 4: Current HS Code: E4

Year 5: HESC: N

Senior/Key Personnel: Organization: Role Category:

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APPLICATION FOR FEDERAL ASSISTANCE

SF 424 (R&R) 

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Pre-application Application Changed/Corrected Application

12/04/2013

InBios International Inc

562 Ist Avenue S., Suite 600

Seattle King

98104-3829

Antigen Detection assay for the Diagnosis of Melioidosis

Yes No

USA: UNITED STATES

WA: Washington

R: Small Business

National Institutes of Health

New Resubmission

Renewal Continuation Revision

8. * TYPE OF APPLICATION:

OMB Number: 4040-0001 

Expiration Date: 06/30/2011 

b. Agency Routing Identifier

12. PROPOSED PROJECT:

* Start Date * Ending Date

07/01/2014 06/30/2017

* 13. CONGRESSIONAL DISTRICT OF APPLICANT

WA 007

14. PROJECT DIRECTOR/PRINCIPAL INVESTIGATOR CONTACT INFORMATION

* First Name: Middle Name:

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* Street1:

Street2:

* City:

* Country: * ZIP / Postal Code:

* Phone Number: Fax Number:

* Email:

* State:

County / Parish:

Province:

Prefix:

98104-3829

Seattle King

562 Ist Avenue S., Suite 600

InBios International Inc

WA: Washington

USA: UNITED STATES
SF 424 (R&R) APPLICATION FOR FEDERAL ASSISTANCE Page 2

15. ESTIMATED PROJECT FUNDING 16. * IS APPLICATION SUBJECT TO REVIEW BY STATE EXECUTIVE 

ORDER 12372 PROCESS?

a. YES a. Total Federal Funds Requested

17. By signing this application, I certify (1) to the statements contained in the list of certifications* and (2) that the statements herein are

true, complete and accurate to the best of my knowledge. I also provide the required assurances * and agree to comply with any resulting 

terms if I accept an award. I am aware that any false, fictitious. or fraudulent statements or claims may subject me to criminal, civil, or 

administrative penalities. (U.S. Code, Title 18, Section 1001)

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InBios International Inc

562 Ist Avenue S., Suite 600

Seattle King

WA: Washington

USA: UNITED STATES 98104-3829

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12/05/2013

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424 R&R and PHS-398 Specific

Table Of Contents Page Numbers

SF 424 R&R Face Page------------------------------------------------------------------------------------------ 1

Table of Contents--------------------------------------------------------------------------------------------- 3

Performance Sites--------------------------------------------------------------------------------------------- 4

Research & Related Other Project Information------------------------------------------------------------------ 7

Project Summary/Abstract (Description)---------------------------------------- 8

Public Health Relevance Statement (Narrative attachment)---------------------------------------- 9

Facilities & Other Resources---------------------------------------- 10

Equipment---------------------------------------- 13

Research & Related Senior/Key Person-------------------------------------------------------------------------- 14

Biographical Sketches for each listed Senior/Key Person---------------------------------------- 17

Research & Related Budget - Year 1---------------------------------------------------------------------------- 35

Research & Related Budget - Year 2---------------------------------------------------------------------------- 38

Research & Related Budget - Year 3---------------------------------------------------------------------------- 41

Budget Justification---------------------------------------- 44

Research & Related Budget - Cumulative Budget----------------------------------------------------------------- 46

Research & Related Budget - Consortium Budget (Subaward 1)---------------------------------------------------- 47

SBIR/STTR Information----------------------------------------------------------------------------------------- 59

Research & Development Outside the U.S.---------------------------------------- 61

Commercialization Plan---------------------------------------- 62

PHS 398 Specific Cover Page Supplement------------------------------------------------------------------------ 74

PHS 398 Specific Research Plan-------------------------------------------------------------------------------- 76

Specific Aims---------------------------------------- 77

Research Strategy---------------------------------------- 78

Human Subjects Sections---------------------------------------- 90

Protection of Human Subjects---------------------------------------- 90

Women &Minorities---------------------------------------- 92

Children---------------------------------------- 93

Select Agent Research---------------------------------------- 94

Multiple PI Leadership Plan---------------------------------------- 98

Bibliography & References Cited---------------------------------------- 99

Consortium/Contractual---------------------------------------- 102

Letters of Support---------------------------------------- 104

Resource Sharing Plan---------------------------------------- 115

PHS 398 Checklist--------------------------------------------------------------------------------------------- 116

Table of Contents Page 3 
Project/Performance Site Location(s)

Project/Performance Site Primary Location

Organization Name:

* Street1:

Street2:

* City: County:

* State:

Province:

* Country:

* ZIP / Postal Code:

InBios International

562 1st Avenue S., Suite 600

Seattle King

WA: Washington

USA: UNITED STATES

98104-3829

OMB Number: 4040-0010 

Expiration Date: 08/31/2011

I am submitting an application as an individual, and not on behalf of a company, state, 

local or tribal government, academia, or other type of organization. 

DUNS Number:

* Project/ Performance Site Congressional District: WA 007

Project/Performance Site Location 1

University of Nevada, Reno

1664 North Virginia Street

Reno Washoe

NV: Nevada

USA: UNITED STATES

* ZIP / Postal Code: 89557-0240 NV 002

* Country:

Province:

* State:

* City:

Street2:

* Street1:

Organization Name:

County:

DUNS Number:

* Project/ Performance Site Congressional District: 

I am submitting an application as an individual, and not on behalf of a company, state, 

local or tribal government, academia, or other type of organization. 

Project/Performance Site Location 2

Menzies School of Health Research-Royal Darwin Hospital

PO Box 41096

Casuarina

Northern Territory

AUS: AUSTRALIA

* ZIP / Postal Code: 0811

* Country:

Province:

* State:

* City:

Street2:

* Street1:

Organization Name:

County:

DUNS Number:

* Project/ Performance Site Congressional District:

I am submitting an application as an individual, and not on behalf of a company, state, 

local or tribal government, academia, or other type of organization. 

Performance Sites 
Project/Performance Site Location(s)

Project/Performance Site Location 3

Mahidol University

420/6 Ratchawithi Road

Bangkok

THA: THAILAND

* ZIP / Postal Code: 10400

* Country:

Province:

* State:

* City:

Street2:

* Street1:

Organization Name:

County:

DUNS Number:

* Project/ Performance Site Congressional District:

I am submitting an application as an individual, and not on behalf of a company, state, 

local or tribal government, academia, or other type of organization. 

Project/Performance Site Location 4

Health Protection Agency

Research Department

Porton Down

Salisbury Wiltshire

GBR: UNITED KINGDOM

* ZIP / Postal Code: SP4 0JG

* Country:

Province:

* State:

* City:

Street2:

* Street1:

Organization Name:

County:

DUNS Number:

* Project/ Performance Site Congressional District:

I am submitting an application as an individual, and not on behalf of a company, state, 

local or tribal government, academia, or other type of organization. 

Project/Performance Site Location 5

Menzies School of Health Research-Royal Darwin Hospital

PO Box 41096

Casuarina

Northern Territories

AUS: AUSTRALIA

* ZIP / Postal Code: 0811

* Country:

Province:

* State:

* City:

Street2:

* Street1:

Organization Name:

County:

DUNS Number:

* Project/ Performance Site Congressional District:

I am submitting an application as an individual, and not on behalf of a company, state, 

local or tribal government, academia, or other type of organization. 

Performance Sites Page 5 
Project/Performance Site Location(s)

Additional Location(s) Add Attachment Delete Attachment View Attachment

Performance Sites 
RESEARCH & RELATED Other Project Information

1. Are Human Subjects Involved?

If no, is the IRB review Pending?

IRB Approval Date:

Human Subject Assurance Number:

2. Are Vertebrate Animals Used?

2.a. If YES to Vertebrate Animals

Is the IACUC review Pending?

IACUC Approval Date:

Animal Welfare Assurance Number:

3. Is proprietary/privileged information included in the application?

4.a. Does this Project Have an Actual or Potential Impact - positive or negative - on the environment? 

4.b. If yes, please explain:

4.c. If this project has an actual or potential impact on the environment, has an exemption been authorized or an environmental assessment (EA) or 

 environmental impact statement (EIS) been performed?

4.d. If yes, please explain:

6. Does this project involve activities outside of the United States or partnerships with international collaborators?

6.a. If yes, identify countries:

6.b. Optional Explanation:

7. Project Summary/Abstract

8. Project Narrative

9. Bibliography & References Cited

10. Facilities & Other Resources

11. Equipment

12. Other Attachments

Non STTR funds will be used for foreign sites

Yes No

1.a. If YES to Human Subjects

Yes No

Yes No

If yes, check appropriate exemption number.

Yes No

Yes No

Yes No

Yes No

Yes No

Yes No

Australia, Thailand, UK

1241-Phase II Abstract.pdf Add Attachment Delete Attachment View Attachment

1242-Phase II Project Narrative.pdf Add Attachment Delete Attachment View Attachment

1243-Bp Phase II REferences.pdf Add Attachment Delete Attachment View Attachment

1244-Burk_FACILITIES_Nov_2013.pdf Add Attachment Delete Attachment View Attachment

1245-Burk_Equipment_UNR_Nov_2013.pdf Add Attachment Delete Attachment View Attachment

Add Attachments Delete Attachments View Attachments

Is the Project Exempt from Federal regulations?

5. Is the research performance site designated, or eligible to be designated, as a historic place? Yes No

5.a. If yes, please explain:

OMB Number: 4040-0001 

Expiration Date: 6/30/2016

1 2 3 4 5 6

Other Information Page 7 
Abstract 

Burkholderia pseudomallei is a Gram-negative bacterium that is the causative agent of melioidosis. The 

bacterium causes significant morbidity and mortality in tropical regions and endemic areas are 

expanding. Melioidosis is difficult to treat and diagnose due to the fact that B. pseudomallei is resistant 

to common antibiotics and symptoms are non-specific. Mortality rates reach 45% and 20% in Thailand 

and Australia respectively, even with antibiotic treatment. Currently, there is no validated diagnostic 

product for the diagnosis of melioidosis. Culturing of patient samples is the “gold standard” for diagnosis, 

however this can take 3-7 days. Our goal is to develop a rapid point-of-care lateral flow immunoassay 

(LFI) for the detection of the B. pseudomallei capsular antigen (CPS) directly from patient samples. 

Phase I STTR Specific Aims focused on producing a library of anti-CPS monoclonal antibodies (mAbs), 

optimizing the LFI components and sample preparation, and performing a feasibility study on archived 

melioidosis patient samples. The objectives of this Phase II proposal is advanced development and 

validation of the prototype AMD LFI that incorporates a higher affinity CPS mAb. By completing these 

goals we hope to produce an optimized LFI that possesses sensitivity equal to or greater than culturing 

of patient samples. To achieve these goals we have established an experienced team. A strong 

partnership exists between Dr. AuCoin’s laboratory and InBios International that has lead to the 

development of an encouraging prototype LFI. In addition, both groups will work closely with experts in 

endemic areas (Thailand and Australia) who will be conducting a large preclinical evaluation of the LFI 

with different patient sample types. 

Project Description Page 8 
Project Narrative 

Melioidosis is a rapidly progressing and frequently fatal disease if left untreated. Burkholderia

pseudomallei is the causative agent of melioidosis; this soil dwelling bacterium has been listed as a Tier I 

Select Agent by the CDC. In the endemic areas of Thailand and northern Australia melioidosis is a major 

public health concern. Rapid diagnosis of melioidosis is needed in order to properly administer effective 

treatment with specific antibiotics. At present, the diagnostic “gold standard” is culturing of patient 

samples, which takes 3-7 days and significantly delays administration of proper treatment. In this 

proposal, we offer to develop a point-of-care, cost-effective, and rapid diagnostic for melioidosis that can 

deliver results within minutes. Such a rapid diagnostic test will have the capability to detect acute B.

pseudomallei infections in endemic areas as well as following a bioterrorist attack. 

Public Health Relevance Statement Page 9 
FACILITIES 

InBios International

InBios International Inc. is a ten-year old biotechnology company focusing on developing novel diagnostic 

products and reagents based on proprietary antigens and technologies. The company’s objective is to become 

a leader in the development and manufacturing of novel diagnostic tests for infectious diseases. The company 

specializes in development of diagnostic products for diagnosis of less frequent infectious diseases that may 

be overlooked due to a perceived small market size. The company’s founders and employees have extensive 

experience in developing, manufacturing and marketing diagnostic tests worldwide. In-house expertise 

includes various aspects of chemistry and immunology including immunochemistry, protein 

expression/purification, and their manipulation. InBios is also specialized in particle and conjugation chemistry 

that includes our confidential gold-based technology. The company manufacturing, quality control and 

regulatory staff are experienced in producing large-scale lots of rapid and ELISA based assay reproducibly. 

The company is FDA registered and GMP compliant. 

Laboratory: InBios occupies approximately 9,000 sq ft of laboratory space on three floors of a seven story 

secure building in downtown Seattle, WA. The core facility is designed for molecular, immunological, and 

biochemical research in support of developing and manufacturing ELISA and lateral flow based immunoassay 

diagnostics. A minimum of 7 separate benches exists for technician and scientist work. There exists a standard 

laboratory fully equipped with a BSL-2 biosafety hood for working with human biological fluids, as well as all 

equipment necessary for assay development. A dedicated dry room that is humidity (<15% RH) and 

temperature controlled, and everything needed for analyte conjugation is housed at InBios. A separate 

molecular biology laboratory and cold room exist for all protein expression/purification work. InBios also has a 

tissue culture room for culturing monoclonal antibody cell lines and a dedicated Quality Control room/staff. 

Computer: All scientific staff have dedicated computers. All computers are on a company server providing 

regular backups and access to various peripheral devices. They are supported by DNA sequence analysis and 

other scientific, data and word processing software and also provide internet and email access. 

Office: In addition to lab space, approximately 3,000 sq. ft. of office spaces exists at InBios. The facility 

contains office space for all scientists, administrative staff (including project manager, administrative assistant, 

accountant, and document control specialist) as well as general use areas including conference rooms; library 

space and staff lounge. All office supplies are kept on-hand, in addition to copiers, projector, scanner, fax, and 

telephone capabilities. 

Other: There is full-service media prep; glass wash and autoclave area located within InBios laboratories. 

InBios also has access to photography, microscopy, machine, electronic shops, and other facilities and 

services at local research companies in the area on an as needed and fee-for use basis. 

University of Nevada, Reno 

Studies at UNR will be done in the Laboratory for Diagnostics Discovery and Development (LDDD) located in 

the newly constructed (October, 2010) Center for Molecular Medicine. The LDDD houses the research 

programs of Dr. David AuCoin and Dr. Thomas Kozel. LDDD is dedicated to identification of targets for 

diagnosis of infectious diseases and the production of reagents for immunoassay construction. LDDD consists 

of 2,850 sq. ft. of bench laboratory space that includes 1,300 sq. ft. of general laboratory space and 1,550 sq. 

ft. of specialty laboratory space.

Specialty laboratories within the LDDD include i) a hybridoma production laboratory, ii) a tissue culture 

laboratory dedicated to large-scale production of engineered antibodies, iii) a serology laboratory for analysis 

of human and non-human primate samples under BSL-2 conditions, iv) a BSL-2 laboratory dedicated to growth 

of bacteria and fungi, and v) a separate laboratory that is capable of functioning under GLP. 

Capabilities of the LDDD in routine use at this time include i) immunization of mice, fusions and screening for 

production of mAbs; ii) subclass switching of mAbs to optimize antibody performance; iii) large-scale (gram 

Facilities Page 10 
amounts) production of mAbs; iv) antibody purification, fragmentation and labeling (enzyme, colloidal gold and 

fluorophore); v) molecular characterization (sequencing of variable region genes) and engineering of mAbs; vi) 

affinity enhancement via phage display; vii) production of engineered antibodies in CHO cells; viii) antibody 

characterization, including evaluation with Biacore; ix) immunoassay construction and evaluation, including 

ELISA and lateral flow immunochromatography; x) production and purification of microbial antigens; and ix) 

identification of microbial protein antigens by 2D gel immunoblotting and sequencing of peptides via mass 

spectrometry at the UNR Proteomics Core Laboratory. 

The Center for Molecular Medicine includes a state-of-art vivariam that is suitable for immunization of mice for 

mAb production. Also included in the CMM is a BSL-3 suite that meets the security and safety requirements of 

the Patriot Act. Designed through consultation with Ted Tram (World BioHazTec Corp.), commissioning and 

CDC certification of the facility are scheduled for January 2012. 

Mahidol-Oxford Tropical Medicine Research Unit, Mahidol University, Bangkok Thailand

Laboratory: Dr. Limmathurotsakul and collogues have full access to a new 5,400 sq. ft. microbiology laboratory 

which includes a 1216 sq. ft. BSL3 laboratory. The laboratory is located on the second floor of the building. 

The BSL3 laboratory can accommodate up to eight people at the same time, and is equipped with eight -80°C 

freezers, one -20°C freezer and six Class II biosafety cabinets. Two biosafety cabinets are dedicated to B. 

pseudomallei work. One -80 freezer is dedicated to select agents including B. pseudomallei. This freezer is 

locked and access is given to authorized personnel only. Freezerworks, the sample tracking system complying 

with international conference on harmonization-good clinical practice (ICH-GCP), is used for storage and 

tracking all specimens. Mahidol-Oxford Tropical Medicine Research Unit is enrolled in a Select Agent Program 

developed by CDC/NIH. 

Office: Dr Direk Limmathurotsakul has a 150 sq. ft. office on the third floor of the building for his use, and there 

is another shared office next to the microbiology laboratory for the use of lab technicians. Mahidol-Oxford 

Tropical Medicine Research Unit has a variety of support personnel, including purchasing and account agents. 

Computer: The laboratory is equipped with Windows and Mac desktops and laptop computers, local-area 

network and servers, which are sufficient for the basic support for this project. Internet access is secured by a 

well-established firewall. The server is well secured and maintained to collect data for clinical studies. 

Scientific Environment: The laboratory is within the Faculty of Tropical Medicine at Mahidol University. There 

are large communities of microbiologists, molecular microbiologists, epidemiologists, statisticians and health 

economists in the unit. Dr Direk Limmathurotsakul has been leading a study team researching bacterial 

infectious diseases, particularly melioidosis. Dr Ben Cooper is assisting with the studies involving modeling of 

infectious disease and development of preventive guidelines for melioidosis. Prof Nicholas Day is assisting 

with the design and analysis of the studies. 

Sappasithiprasong Hospital, Ubon Ratchathani, northeast of Thailand

Laboratory: Dr. Limmathurotsakul and collogues have an office within Sappasithiprasong Hospital, Ubon 

Ratchathani. The hospital laboratory is very familiar with B. pseudomallei as it is one of the most common 

bacteria isolated from clinical specimens, and safety measures of this laboratory are at the highest standard 

required by the Ministry of Public Health, Thailand. B. pseudomallei, which is isolated from clinical specimens 

from patients, will be transferred to the BSL3 laboratory of Mahidol-Oxford Tropical Medicine Research Unit 

under appropriate containment using a high level biosafety standards. Freezerworks, the sample tracking 

system, has been set up at Sappasithiprasong Hospital and will be used to track the records of specimen 

transportation. 

Office: Dr. Limmathurotsakul has an 800 sq. ft. office for use by his study team. The office at 

Sappasithiprasong Hospital has secretarial support for the projects and accounting. 

Facilities Page 11 
Computer: The office is equipped with Windows and Mac desktops and laptop computers, local-area network 

and servers. Internet access is secured by a well-established firewall. The server is well secured and 

maintained to collect data from clinical studies. 

Clinical: Dr. Limmathurotsakul and his study team have a strong collaboration with physicians from all 

departments, including pediatricians and director of the Sappasithiprasong hospital. 

Scientific Environment: The laboratory and the office are within Sappasithiprasong Hospital. The collaboration 

between Mahidol-Oxford Tropical Medicine Research Unit and Sappasithiprasong Hospital has been 

established for more than 30 years. Dr. Limmathurotsakul has been working with Sappasithiprasong Hospital 

for more than 5 years, performing clinical, laboratory and environmental studies. 

Menzies School of Health Research - Royal Darwin Hospital (Menzies)

Menzies School of Health Research is an independent research organization that has research programs 

focused on Indigenous, tropical, and remote health, and is the focal point for medical research in northern 

Australia. The mission of Menzies is to improve the health of people living in northern and central Australia, 

and regions to the near north, through multidisciplinary research and education. 

Menzies has a Melioidosis Research program within its Tropical and Emerging Infectious Diseases Division. 

Menzies is based in Darwin, on the grounds of the Royal Darwin Hospital. The Royal Darwin Hospital is the 

primary acute care facility for the Australian Northern Territory, providing a wide range of medical services. 

Royal Darwin Hospital has been accredited under the Australian Council of Health Care Standards since 1997. 

These team members are responsible for the Darwin Prospective Melioidosis Study. This study has a clinicalresearch arm, which over the last 22 years has established state-of-the-art therapy for melioidosis, and a 

laboratory-research arm, which maintains the most extensive collection of patient clinical sample DNA in the 

world, as well as a meticulously curated collection of over 3000 B. pseudomallei isolates from humans, 

animals, and the environment. 

Dr. Bart Currie is the Head of the Tropical and Emerging Infectious Diseases Division at Menzies and is an 

internationally recognized expert in melioidosis and B. pseudomallei. Menzies, in association with Royal 

Darwin Hospital, where Dr. Currie is also Head of Infectious Diseases, has generated diverse B. pseudomallei

isolates and clinical specimens for assay validation. These isolates are available to the AuCoin laboratory and 

InBios for evaluation of assay specificity. Menzies has a CDC-certified PC3 laboratory for handling of B. 

pseudomallei cultures and also designated space at PC2 level for handling patient samples. Full IT support is 

also available. 

Facilities Page 12 
EQUIPMENT 

InBios International 

InBios is fully equipped for the development and production of both ELISA and dipstick diagnostic tests. 

All equipment is housed in BSL-2 level facilities and secured. Equipment includes: a BSL-2 Biosafety hood, 

refrigerated centrifuges (low and high speed), microfuges, water baths, both analytical and conventional 

balances, mixers/stirrers, UV/vis spectrophotometer, pH meter, -20°C freezer, -70°C freezer, LN2 tank, 

Baxter Cryofridge, environmental chamber with recorder, microplate ELISA readers and plate washers. 

Accessories required for a full range of protein purification are available which include peristaltic pump, UV 

monitor and fraction collector. Fully equipped strip production facility with Biodot Quanti 3000, laminator, 

strip cutter, heat sealer and automatic cassette closure are also available. A PCR thermal cycler machine 

and equipment for agarose and polyacrylamide gel electrophoresis. The company also has a Perkin Elmer 

microarray reader and devices for making hand made arrays as well as a Biorad Odyssey calligrapher for 

machine made arrays. Also available is a 3D printer for making cassette designs prior to sending to 

manufacturer for scale up. 

University of Nevada, Reno 

Work for this proposal at UNR will be done in the Laboratory for Diagnostics Discovery and Development 

(LDDD) suite. Please see the Facilities section for a description of the LDDD. Relevant equipment in the 

LDDD includes standard laboratory and tissue culture equipment such as biosafety cabinets (4), HERAcell 

150i CO2 incubators (8), Isco low pressure chromatography systems (three complete systems), 

refrigerated low speed benchtop centrifuges (3), a large capacity liquid nitrogen system for preservation of 

hybridoma cell lines, -80°C freezers (2), a Sorvall RC6 high speed centrifuge, a New Brunswick Excella 

E25 incubator shaker, a Wave 2/10 EH bioreactor system for large scale growth of hybridoma and CHO 

cells, multiple electrophoresis rigs, a BioTek EL808 plate reader, and a BioTek ELx405 plate washer. 

Large equipment within the LDDD includes a Biacore X100 for assessment of mAb binding. The Biacore 

X100 has recently been upgraded to a Biacore X100 Plus with software for extended control of reaction 

conditions and determination of analyte concentrations. Additional large equipment includes a complete 

system for fabrication of lateral flow strips that includes a Biodot XYZ 3050 dispensing system, a Biodot 

guillotine strip cutter and a Biodot laminator; a Zeiss LSM 700 laser scanning microscope; a BioRad 

ChemiDoc XRS+ imaging system; and a complete BioRad high pressure liquid chromatography system. 

Finally, the LDDD has licensed the OptiCHO Antibody Express system from Invitrogen. This system is 

used for cloning and expression of recombinant antibodies in dihydrofolate reductase (DHFR)-deficient, 

Chinese hamster ovary (CHO)-derived DG44 cells in suspension culture. The system includes the 

pOptiVEC-TOPO TA cloning system, the pcDNA 3.3-TOPO TA cloning system and DG44 cells. This 

system is currently being used for production of chimeric mouse/human antibodies and for production of 

antibodies that have been affinity-enhanced via phage display. Optimization of cell lines for large scale 

antibody production with the Wave bioreactor system is currently underway.

Equipment Page 13 
Province:

PROFILE - Project Director/Principal Investigator

Prefix: * First Name: Middle Name:

* Last Name: Suffix:

Organization Name: Division:

Position/Title: Department:

* Street1:

Street2:

* Phone Number: Fax Number:

* E-Mail:

Credential, e.g., agency login:

* Project Role: Other Project Role Category:

* Country: * Zip / Postal Code:

* State:

* City: County/ Parish:

RESEARCH & RELATED Senior/Key Person Profile (Expanded)

*Attach Biographical Sketch

Attach Current & Pending Support

InBios International Inc

562 Ist Avenue S., Suite 600

Seattle King

WA: Washington

USA: UNITED STATES 98104-3829

PD/PI

Ph.D.

1973

1235- Biosketch Nov 2 Add Attachment Delete Attachment View Attachment

Add Attachment Delete Attachment View Attachment

OMB Number: 4040-0001 

Expiration Date: 06/30/2011

Degree Type:

Degree Year:

Province:

PROFILE - Senior/Key Person

Prefix: * First Name: Middle Name:

* Last Name: Suffix:

Organization Name: Division:

Position/Title: Department:

* Street1:

Street2:

* Phone Number: Fax Number:

* E-Mail:

Credential, e.g., agency login:

* Country: * Zip / Postal Code:

* State:

* City: County/ Parish:

University of Nevada, Reno School of Medicine

Washoe

1664 North Virginia Street

USA: UNITED STATES

Department of Microbiology and Immunology/320

Reno

NV: Nevada

89557-0000

* Project Role: PD/PI Other Project Role Category:

1

Degree Type: Ph.D.

Degree Year: 2002

*Attach Biographical Sketch 1236-aucoin biosketch.pdf Add Attachment Delete Attachment View Attachment

Attach Current & Pending Support Add Attachment Delete Attachment View Attachment

Key Personnel 
RESEARCH & RELATED Senior/Key Person Profile (Expanded)

Province:

PROFILE - Senior/Key Person

Prefix: * First Name: Middle Name:

* Last Name: Suffix:

Organization Name: Division:

Position/Title: Department:

* Street1:

Street2:

* Phone Number: Fax Number:

* E-Mail:

Credential, e.g., agency login:

* Country: * Zip / Postal Code:

* State:

* City: County/ Parish:

InBios International Inc

King

Suite 600

Research

USA: UNITED STATES

562 1st Avenue South

Seattle

WA: Washington

98104-3829

* Project Role: Co-Investigator Other Project Role Category:

2

Degree Type: Ph.D.

Degree Year: 1981

*Attach Biographical Sketch 1237-BIOGRAPHICAL SKETCH SRay Add Attachment Delete Attachment View Attachment

Attach Current & Pending Support 1238-Other support - July 201 Add Attachment Delete Attachment View Attachment

Province:

PROFILE - Senior/Key Person

Prefix: * First Name: Middle Name:

* Last Name: Suffix:

Organization Name: Division:

Position/Title: Department:

* Street1:

Street2:

* Phone Number: Fax Number:

* E-Mail:

Credential, e.g., agency login:

* Country: * Zip / Postal Code:

* State:

* City: County/ Parish:

Mahidol University

24 Medicine Research Unit Rajvithee 25 Rd

THA: THAILAND

420/6 Ratchawithi Road

Bangkok

10400

* Project Role: Other Professional Other Project Role Category: Collaborator

3

Degree Type: MD

Degree Year: 2000

*Attach Biographical Sketch 1239-Limmathurotsakul_BIO.pdf Add Attachment Delete Attachment View Attachment

Attach Current & Pending Support Add Attachment Delete Attachment View Attachment

Key Personnel 
RESEARCH & RELATED Senior/Key Person Profile (Expanded)

Province:

PROFILE - Senior/Key Person

Prefix: * First Name: Middle Name:

* Last Name: Suffix:

Organization Name: Division:

Position/Title: Department:

* Street1:

Street2:

* Phone Number: Fax Number:

* E-Mail:

Credential, e.g., agency login:

* Country: * Zip / Postal Code:

* State:

* City: County/ Parish:

Menzies School of Health Royal Darwin Hospital

Northern Territories

AUS: AUSTRALIA

PO Box 41096

Casuarina

* Project Role: Other Professional Other Project Role Category: Collaborator

4

Degree Type: MD

Degree Year: 1978

*Attach Biographical Sketch 1240-Currie_NIH_Biosketch_Pha Add Attachment Delete Attachment View Attachment

Attach Current & Pending Support Add Attachment Delete Attachment View Attachment

Key Personnel Page 16 
 

Travel

Domestic travel: $ /year for trips to UNR and scientific meetings on Melioidosis and 

Biodefense. 

Materials and Supplies 

All supply costs are estimates, based on current usage for similar projects. 

Annual costs include: 

 General laboratory reagents

 Disposable Plasticware

 Tissue culture media (Mab development)

 Strip materials

 Membranes, pads and backing materials

 Cassettes (as needed)

 Colloidal gold and conjugate development

 Gel electrophoresis materials for evaluating purity of purified antibodies

 Reagents for internal control e.g. goat anti mouse IgG or chicken IgY/anti IgY

 ELISA plates

 HRP antibody conjugation materials

Baseline costs: $ /year for years 1 through 3 ($ /month). 

Subawards 

As part of this STTR there is a sub contract between InBios and the University of Nevada and 

the budget for this is attached. Total is $ for a total of three years 

Shipping costs 

$2000/year for three years will cover the estimated costs of shipping all analytes and prototype 

tests. 

Fee at 7% (Including Subaward): A total of $ is requested. 

Fringe Benefit and overhead rates: 

Fringe benefit rate: 33%, overhead rate: 38% 

Determined by Andrew Sandberg, Procurement Analyst with the Office of Acquisitions, 

Management, Contracts & Grants (AMCG), HHS, at meeting at InBios on 3/29/11. 

Budget Justification Page 45 
 

Budget Justification UNR

Fringe Benefit Rate Justification: The fringe benefits percentages used are average rates by employee 

type that are approved by the University for use in budgeting fringe benefits in proposals. However, 

awards are charged the actual fringe benefit cost for each employee as they are incurred. Fringe benefits 

cover Worker's Compensation, Medicare taxes, retirement and health insurance. 

Materials and Supplies Budget Period 1:

Disposable plastics (microtiter plates, tissue culture supplies, pipettes, pipette tips, etc.) $

Immunochemicals, reagents, ELISA supplies $

Tissue culture media $

Integra bioreactors ($ /bioreactor x 10) $

Columns, gels, reagents for mAb and CPS purification and characterization $

BSL-3 PPE (tyvek suites, shrouds, gloves, scrubs, etc.) $

Biacore NTA sensor chips (3 pack) $

Total $

Funds for 2 trips per year to InBios and a yearly melioidosis meeting (AuCoin) $

Publication costs $

Biacore service contract (50% of total contract ($ ) $

Materials and Supplies Budget Period 2: 

Disposable plastics (microtiter plates, tissue culture supplies, pipettes, pipette tips, etc) $

Immunochemicals, reagents, ELISA supplies $

Tissue culture media $

Integra tissue culture flasks for large scale mAb production $

Columns, gels, reagents for mAb and CPS purification and characterization $

BSL-3 PPE (tyvek suites, shrouds, gloves, scrubs, etc.) $

Subaward 1 Budget Justification Attachment Page 57 
 

Total $

Funds for 2 trips per year to InBios and a yearly melioidosis meeting (AuCoin) $

Publication costs $

Biacore service contract (50% of total contract ($ ) $

Materials and Supplies Budget Period 3: 

Disposable plastics (microtiter plates, tissue culture supplies, pipettes, pipette tips, etc) $

Immunochemicals, reagents, ELISA supplies $

Tissue culture media $

Integra bioreactors ($ /bioreactor x 5) $

Integra tissue culture flasks for large scale mAb production $

Columns, gels, reagents for mAb purification and characterization $

BSL-3 PPE (tyvek suites, shrouds, gloves, scrubs, etc.) $

Biacore NTA sensor chips (3 pack) $

Total $

Funds for 2 trips per year to InBios and a yearly melioidosis meeting (AuCoin) $

Publication costs $

Biacore service contract (50% of total contract ($ ) $

UNR Indirect Cost Rate: 

Indirect costs are included at the University’s federally negotiated rate of 43.5%.

This rate is based on modified total direct costs for on–campus research.

Subaward 1 Budget Justification Attachment Page 58 
SBIR/STTR Information OMB Number: 4040-0001 

Expiration Date: 6/30/2016

* Program Type (select only one)

* SBIR/STTR Type (select only one)

* 1a. Do you certify that at the time of award your organization will meet the eligibility criteria for a small business as defined in the funding

opportunity announcement?

* 2. Does this application include subcontracts with Federal laboratories or any other Federal Government agencies?

* If yes, insert the names of the Federal laboratories/agencies:

* 3. Are you located in a HUBZone? To find out if your business is in a HUBZone, use the mapping utility provided by the Small Business 

* 4. Will all research and development on the project be performed in its entirety in the United States?

* Explanation:

* 5. Has the applicant and/or Program Director/Principal Investigator submitted proposals for essentially equivalent work under other 

Federal program solicitations or received other Federal awards for essentially equivalent work? 

* If yes, insert the names of the other Federal agencies:

* 6. Disclosure Permission Statement: If this application does not result in an award, is the Government permitted to disclose the title of 

your proposed project, and the name, address, telephone number and e-mail address of the official signing for the applicant organization, 

to organizations that may be interested in contacting you for further information (e.g., possible collaborations, investment)?

* 7. Commercialization Plan: If you are submitting a Phase II or Phase I/Phase II Fast-Track Application, include a 

Commercialization Plan in accordance with the agency announcement and/or agency-specific instructions.

* Attach File:

Questions 1-7 must be completed by all SBIR and STTR Applicants:

If no, provide an explanation in an attached file.

Administration at its web site: http://www.sba.gov 

SBIR STTR

Both (See agency-specific instructions to determine whether a particular agency allows a single submission for both SBIR and STTR)

Phase I Phase II

Fast-Track (See agency-specific instructions to determine whether a particular agency participates in Fast-Track)

Yes

No

30

Yes

No

Yes

No

Yes

No

Yes

No

Yes

No

1257-SBIR__STTR_4_Information M Add Attachment Delete Attachment View Attachment

1258-MELIOIDOSIS commercializat Add Attachment Delete Attachment View Attachment

* 1b. Anticipated Number of personnel to be employed at your organization at the time of award.

SBIR/STTR Information 
* 8. Have you received SBIR Phase II awards from the Federal Government? If yes, provide a company commercialization history in 

accordance with agency-specific instructions using this attachment. 

* Attach File:

* 9. Will the Project Director/Principal Investigator have his/her primary employment with the small business at the time of award?

* 10. Please indicate whether the answer to BOTH of the following questions is TRUE:

(1) Does the Project Director/Principal Investigator have a formal appointment or commitment either with the small business directly 

(as an employee or a contractor) OR as an employee of the Research Institution, which in turn has made a commitment to the 

small business through the STTR application process; AND 

(2) Will the Project Director/Principal Investigator devote at least 10% effort to the proposed project?

* 11. In the joint research and development proposed in this project, does the small business perform at least 40% of the work and the research 

institution named in the application perform at least 30% of the work?

SBIR-Specific Questions:

STTR-Specific Questions:

SBIR/STTR Information

Questions 8 and 9 apply only to SBIR applications. If you are submitting ONLY an STTR application, leave questions 8 and 9 blank and proceed to 

question 10.

Questions 10 and 11 apply only to STTR applications. If you are submitting ONLY an SBIR application, leave questions 10 and 11 blank.

Add Attachment Delete Attachment View Attachment

Yes

No

Yes

No

Yes

No

Yes

No

SBIR/STTR Information 
SBIR/STTR INFORMATION 

Melioidosis is caused by the Gram-negative bacterium Burkholderia pseudomallei transmitted through 

contaminated water and soil, and prevalent in tropical regions of the world. At present, only non-indigenous 

cases of melioidosis have been reported in the United States. The worldwide distribution is as yet unknown 

due to lack of rapid field tests. In the studies proposed we plan to further evaluate a rapid test with the view to 

eventual FDA clearance. 

In order to conduct an appropriate, statistically valid evaluation of our tests, it is necessary to perform the field 

studies with a significant number of samples (good sample size with enough power for significance 

calculation). Considering that this disease is non-endemic to the US; is difficult to treat; and the causative 

agent of this disease is listed as a bioterrorism agent we propose to collaborate by providing tests to experts in 

the endemic regions where this disease has a public health concern. 

Such centers will include Mahidol University, THAILAND and Menzies School of Health Research-Royal 

Darwin Hospital, AUSTRALIA. These sites have appropriate facilities to conduct the testing and sufficient 

number of archived melioidosis positive patient samples for the evaluation. We have also recently been asked 

to participate in the Detection of Melioidosis in Africa Working Group (DMAWG). 

In addition, we plan to test the LFI and quantitate via ELISA the CPS from B. pseudomallei and B. mallei in 

archived blood and urine samples from non-human primates. Collecting time course samples after exposure 

will provide supportive data on kinetics of CPS production and the ability of the LFI to detect B. mallei

(Glanders). Only non-STTR funds will be used to compensate these foreign sites. 

We also plan to evaluate a Deki reader with data capture, software and cloud computing capabilities to 

facilitate data analysis in the field. 

Research & Development Outside the U.S. Page 61 
 

 Active Melioidosis Detect (AMD) Commercialization Plan 

Value of the STTR Project, Expected Outcomes and Impact. 

Intended use of the product: The Active Melioidosis DetectTM (AMD) is a point 

of care diagnostic test for active melioidosis. The AMD is an in vitro 

immunochromatographic device for visual and rapid qualitative detection of 

the Burkholderia pseudomallei capsular polysaccharide (CPS) directly from 

patient samples or cultured samples (Figure 1). The disease can present as 

local to very generalized and disseminated infections. The assay will accept 

multiple sample matrices that are known to accumulate CPS during 

melioidosis. These matrices include blood, urine, pus, and respiratory 

secretions. The assay is also applicable for cultured clinical samples from 

enrichment broth. Particularly hemoculture broth, and bacterial colonies from 

cultured samples on solid medium. 

The final test is intended for use in diagnosis of B. pseudomallei infection in 

subjects with early non-specific signs and symptoms and/or individuals with 

confirmed, presumed or likely exposure to B. pseudomallei. Tests are for use 

by healthcare professionals and medical personnel, including emergency 

personnel who have been trained. AMD positive results may be confirmed by 

blood culture (the current gold standard). The test could also have the 

potential for detecting presence of B. pseudomallei in (a) soil samples where 

the organism resides (b) contaminated water as well as (c) monitoring for 

infection in laboratory animals such as non-human primates. 

Currently, there is no FDA cleared (point-of-care) POC test for the diagnosis of melioidosis. A rapid diagnosis 

of melioidosis is essential to the survival of patients infected with B. pseudomallei. The bacterium is resistant 

to commonly prescribed antibiotics; therefore, timely diagnosis is needed for administration of effective 

antibiotics. Diagnosis in endemic and non-endemic regions is extremely challenging due to a lack of 

experience of identifying B. pseudomallei and a lack of effective diagnostic tools.. Isolation of B. pseudomallei

from cultures of patient samples remains the “gold standard” for the diagnosis of melioidosis. However, 

accurate diagnosis from culturing of patient samples can take 3-7 days for bacterial isolation and another 1-2 

days for bacterial identification. Use of the Active Melioidosis Detect Assay will reduce the time to diagnosis to 

10-15 minutes from clinical specimens, in addition the assay can also be used to rapidly identify B.

pseudomallei bacterial colonies with high accuracy. 

Serious problems exist with current rapid diagnostics such as the indirect hemagglutination assay (high 

background levels of seropositive individuals) and PCR (reduced sensitivity), immunofluorescence assay 

(requires fluorescent microscope and skilled personnel) and latex agglutination (not applicable to clinical 

samples.). As a consequence there is not an FDA cleared diagnostic available for the diagnosis of melioidosis. 

Therefore, there is a critical need for a rapid, effective and low cost point-of-care (POC) diagnostic test for 

melioidosis and an improved algorithm for early diagnosis. A field test is also needed to fully assess the 

incidence of melioidosis worldwide. 

The global incidence of melioidosis. To date there have been no good estimates of the global incidence of 

melioidosis. However our collaborator in Thailand (Dr. Direk Limmathurotsakul) has put significant time and 

effort into generating a risk map for melioidosis based on epidemiological data and geospatial statistical 

modeling (Fig. 2). Importantly, it can be used to predict areas at risk of acquiring human melioidosis 

worldwide, including areas where melioidosis might be endemic but under diagnosed or undiagnosed. 

 Also, Table 2 is an estimate of the mortality of the countries that are 

within known and predicted melioidosis endemic regions. 

Established methods and models were used to generate a risk map [1]. Briefly, data from recent reviews of 

melioidosis and environmental B. pseudomallei studies [2,3] along with a systematic literature review were 

used to obtain updated evidence of clinical melioidosis worldwide. This included peer reviewed journals 

(PubMed, Google Scholar, ISI Web of Science), disease surveillance networks (ProMED archives, 

Eurosurveillance archives), Genbank database and health websites of low and middle-income countries. 

Figure 1. Active Melioidosis Detect kit

Commercialization Plan Page 62 
 

The above studies and data analysis would indicate the incidence of 

melioidosis is under reported. By providing prototype tests to potential 

collaborators we expect to better understand the prevalence of melioidosis. 

This will then lead into larger market size for finalized tests. To this end Dr. 

AuCoin and his collaborator in Thailand (Dr. Limmathurotsakul) created an

initiative to “Explore the Presence of Melioidosis in Africa”. Please see initiative 

below that has been posted to “groups.google.com/group/melioidosis”.

Exploring the Presence of Melioidosis in Africa 

Goal: To determine if Burkholderia pseudomallei, the causative agent of melioidosis, is an underreported 

cause of infection in Africa. Hospital/microbiological facilities in Africa will be offered the use of the prototype 

Active Melioidosis DetectTM Lateral Flow Immunoassay (AMD LFI) developed by the University of Nevada and

InBios International to rapidly identify B. pseudomallei isolate. 

Organizers: Detection of Melioidosis in Africa Working Group (DMAWG) 

Significance: Burkholderia pseudomallei is an environmental Gram-negative bacillus and the cause of 

melioidosis. Melioidosis is highly endemic to northeast Thailand and northern Australia, but more recent 

evidence indicates a much wider distribution that includes the Indian subcontinent and southern China, Hong 

Table 1. Predicted global incidence of melioidosis as compared to other 

infectious diseases

Disease Predicted 

incidence

Predicted 

mortality

Predicted case 

fatality rate

Source of data

Tuberculosis 8,600,000 1,300,000 15% WHO website

Malaria 219,000,000 660,000 0.3% WHO website

2009 H1N1 N/A 284,000 N/A LID 2012,

12(9)687-695

Severe 

Dengue

500,000 125,000 2.5% WHO website

Leptospirosis 1,000,000 60,000 6% ILS 2013

Table 2. Predicted 

incidence of melioidosis 

by country

Country Predicted

mortality

Figure 2. Risk map for populations in predicted and known melioidosis endemic regions. Colored areas indicate regions predicted areas of

incidence of melioidosis. Dark red areas indicate areas at highest risk.

Commercialization Plan Page 63 
Kong, and Taiwan [4]. There are also an increasing number of reports of sporadic and possibly endemic 

disease in the Caribbean, Central and South America and East and West Africa [4-8]. The extent of 

melioidosis in Africa remains uncertain. Melioidosis may pass unrecognized because diagnostic confirmation 

relies on microbiological culture, which is often unavailable in resource-restricted regions of the Africa. Even 

with such facilities, B. pseudomallei may be dismissed as a culture contaminant, or be misidentified as 

Pseudomonas spp. or other organisms by standard identification methods including API 20NE and automated 

bacterial identification systems. 

Assay: The AMD LFI is a point of care diagnostic test, based on the detection of capsular polysaccharide 

(CPS) produced by Burkholderia pseudomallei (Fig. 1). The assay is an immunochromatographic strip that is 

similar in design to the rapid Strep test or home pregnancy test. The assay is designed to accept all routinely 

collected clinical samples from patients with suspected melioidosis (blood, urine, pus, respiratory secretions, 

etc.) and to identify the organism isolated from culture. Preliminary sensitivity and specificity of AMD LFI to 

identify a B. pseudomallei isolate is 98.7% and 97.2%, respectively. For the current initiative, the AMD LFI is

to be used for testing all Gram-negative, oxidase positive bacilli that are isolated from blood culture 

and that cannot be simply identified as Pseudomonas aeruginosa. 

Participants: Hospital/microbiological facilities in geographic areas of Africa 

This is an open call for hospital/microbiological facilities in Africa to receive free AMD LFI kits. 

Work plan: 

1) This initiative is for hospital/microbiology facilities that perform blood culture for either routine practice or

research, and have capacities to perform Gram strain and oxidase tests in Africa.

2) The University of Nevada and InBios International will provide a set of LFI tests (e.g. 20-50 strips) to be

used with all Gram-negative oxidase-positive bacilli that are isolated from blood culture.

3) Isolates that are positive by the AMD LFI test should be suspected to be B. pseudomallei and should be

further evaluated by the participating sites using SOPs for identification that will be provided by the

DMAWG.

4) Alternatively, the Centers for Disease Control and Prevention has agreed to offer confirmation for any

positive isolates and genotyping them as well (See CDC contacts below).

5) There is usually no need for Ethical Clearance for work of this nature. However, if the participating site

considers that it is required, it is the responsibility of the participating site to obtain EC clearance prior to

the participation.

6) The overall result that the DMAWG would like to receive back is, i) how many tested were used?, ii) how

many were positive? and iii) what is the final identification of those positive isolates? The DMAWG will

release the results only as an overall result.

7) The participating site can write up the case report with full clinical details. If needed, collaboration for

writing case report can be sought from the DMAWG.

8) Clinical and public health advice from experts in the field will be made available through the network to any

site where melioidosis is discovered.

9) If you are interested to participate to this initiative, please email Dr David P Aucoin

Figure 1. Prototype Active Melioidosis Detect (AMD) LFI.

(A) Schematic of AMD LFI components. (B) B. pseudomallei

colony grown on an agar plate was picked and suspended in 2 

drops of lysis buffer. The lysate was added to the sample pad 

followed by three drops of LFI chase buffer (top LFI). The LFI 

was imaged following a 15 min run time. The same test 

condition were used with a colony of E. coli (bottom LFI).

Commercialization Plan Page 64 
 

Assay Contact:

Clinical contact:

WHO contact:

CDC contact:

Isolate analysis by CDC:

Current members of DMAWG 

The DMAWG was formed following the 7th World Melioidosis Congress 2013. This was in response to 

increased reports of melioidosis in Africa. Following release of this initiative we have requests for the 4C4 

AMD LFI from around the world. The Africa initiative will be modified to include any region predicted to be 

endemic for melioidosis. In just one month, we have received requests from 13 laboratories from around the 

globe. Some examples are Papua New Guinea, Singapore, Brazil, Nairobi, Nepal, and Seychelles. 

Importantly we have formed a collaboration with 

We anticipate by 

spearheading these initiatives and collaborations we will expand the market significantly for the AMD 

LFI.

In Phase I we identified a “lead candidate” antigen (B. pseudomallei CPS) and a “lead candidate” monoclonal

antibody (mAb 4C4) for the diagnosis of melioidosis. A prototype Active Melioidosis Detect Assay kit was 

initially developed by InBios and UNR based on mAb 3C5 was tested for feasibility in Thailand and Australia 

(Figure 1) with various sample types. With the isolation of IgG1 mAb 4C4 the assay achieves improved 

analytical sensitivity This latest generation of test is available and is being scaled up for more extensive testing. 

Therefore, the goal of the project is assay optimization (improved sensitivity and specificity), improved process 

development, and validation of the diagnostic assay in a variety of patient samples. A prototype antigencapture ELISA and a prototype lateral flow immunoassay for the detection of B. pseudomallei CPS based on 

4C4 are two intermediate products that have been developed and will be improved upon during the Phase II 

award period. Both of these diagnostic products possess encouraging limit of detection values for CPS, 

however improvements can surely be made. Both tests will also be made available for testing in archived 

samples from non-human primates. Tests will also be used to understand prevalence levels of melioidosis 

outside of Thailand and Australia. 

Following the completion of the product development plan the goal is submission of a 510(k) application (by 

the end of Project Year 3) and FDA clearance. The assay would be approved for use with a variety of patient 

samples that are relevant to a melioidosis infection (blood, urine, pus, respiratory secretions, and pleural fluid). 

Data that support the selection of the candidate product for further development, including and assessment of 

the present capacity of the diagnostic to meet the performance specifications: 

• InMAD (In vivo Microbial Antigen Discovery) has identified CPS as a secreted antigen that is shed into

samples from animal models of melioidosis and humans infected with B. pseudomallei.

• The qCPS ELISA and AMD LFI identified shed CPS in filtered urine and serum samples from

melioidosis patients.

Commercialization Plan Page 65 
• AMD LFI is able to identify CPS in a variety of melioidosis patient samples (blood, urine, pus, sputum,

and pleural fluid)

• Preliminary studies have determined that the CPS is highly conserved.

• CPS is a well-established virulence factor and its expression is necessary for disease in animal models

of melioidosis.

• The LFI is ideal for the point-of-care and resource poor setting (rapid, sensitive, specific, easy to use,

adaptable, allows for random access and cost effective).

• Clinicians in Thailand (Dr. Direk Limmathurotsakul) and Australia (Dr. Bart Currie) are quite encouraged

by the performance of the prototype CPS LFI (see letters of support).

• Both the Department of Homeland Security and the Defense Threat Reduction Agency are very

interested in the AMD LFI and are potential end users (see letters of support).

 Clinicians in Laos, India, Singapore and Africa as well as WHO requested evaluation of the AMD LFI

(see letters of support)

Discussions with the FDA, which are relevant to development for the candidate product: 

A major goal of the proposal is to submit a 510(k) to the FDA. InBios International has a well-established 

history of product development with five approved medical diagnostics (Table 4). InBios and UNR will take the 

lead in drafting the pre-submission to FDA (Project Year 2) to seek advice and requirements for prospective 

studies and review of in house performance studies. Estela Raychaudhuri at InBios who is experienced in 

FDA submissions will provide regulatory assistance. It is anticipated that Dr Steven Hawes, a consultant to 

InBios, will perform Biostatistics. 

Regulatory items to be covered by the InBios team: 

• Development of pre-market submission strategies

• Premarket submission preparation (510(k)s, PMAs, IDEs, reclassification petitions, etc.) and reviews

• FDA correspondence

• Procedure and user manual development

• Assistance with FDA compliance matters, GMP/QSR reviews, development and auditing (including

SOP development and review)

• Good clinical practice auditing

• Clinical protocol development

• Statistical support services

• FDA staff meeting preparation and participation

• FDA Advisory Panel preparation and participation

• Technology and regulatory due diligence assessments

Compliance with GLP defined by 21 CFR (58) and cGMP defined by 21 CRF (211): 

Prototype LFIs will be produced under GLP (Project Years 1-2) and the optimized LFI will be produced under 

GMP (Project Years 2-3). A Research Associate, who has recently completed and FDA GLP training course 

for pre-clinical testing, will oversee all GLP requirements in the AuCoin laboratory. InBios is a FDA registered 

and an ISO (International Organization for Standardization) certified company who has established a set of 

general manufacturing principles in compliance with Good Manufacturing Practice (GMP) ensuring that 

products are consistently produced and controlled according to quality standards. InBios manufacturing 

processes are clearly defined, controlled and documented. All critical processes are validated to ensure 

consistency and compliance with design specifications. All operators are trained to carry out and document 

procedures. Deviations are investigated and documented. Records of manufactured goods that enable the 

complete history of a batch to be traced are retained in a comprehensible and accessible form. 

The entire staff at InBios manufacturing is trained for GMP practices. James Needham who is also responsible 

for final development and transition efforts to manufacturing leads the InBios manufacturing team. The 

manufacturing team includes an engineer, wet chemistry application specialists and a team of assemblers. 

A design plan will be written to establish the overall design goals, specifications, input and output objectives, 

project schedule, studies to be performed, and when formal design reviews shall be held. As required by CFR 

820.30, The Contractor shall maintain a design history file that contains or references the design of this assay. 

It will include the design plan, design specifications, protocols for the studies to be conducted, study results 

and associated data, a record of project meetings minutes and design review minutes, and copies of all 

communications with collaborators and FDA. At the beginning of this project, InBios shall establish a design 

Commercialization Plan Page 66 
review body consisting of scientists, clinicians, and experts in regulatory affairs, quality control and 

manufacturing. The team will meet on a regular basis and assess the developmental work and shall 

recommend future efforts. All input discussion shall be documented and design output will be followed for the 

product’s continued development. In Table 3 we have described our preliminary product specification for 

Active Melioidosis DetectTM assay. This will be considered as input of the AMD assay system and will be constantly

monitored and modified depending on the feedback and results.

Table 3. PRODUCT SPECIFICATION 

 Product Identification: Active Melioidosis DetectTM, device not yet classified by FDA

 Device: Active Melioidosis DetectTM (AMD; Lateral flow immunoassay (LFI))

 Intended Use: In vitro diagnosis of active melioidosis for visual and rapid qualitative detection of the 

Burkholderia pseudomallei capsular polysaccharide (CPS) antigen 

 Analyte: Whole blood (venous and/or capillary blood), blood, urine, pus, and respiratory 

secretions 

 Antibodies to be used: Proprietary antibodies to the B. pseudomallei CPS 

 Detection agent: Selected antibody(s) to CPS conjugated with gold 

 Buffer system: Chase buffer that should not require cold storage 

 Instrument size/weight: No additional instrumentation required. 

Performance input: 

 Clinical Sensitivity Pus > 98% 

Sputum > 98% 

Urine > 95% 

Blood > 90% 

 Clinical Specificity: ≥95% 

 Analytical sensitivity: Limit of detection ≤ 1 ng/ml CPS 

 Analytical Specificity: Reactivity with B. pseudomallei and B. mallei strains but not other 

Burkholderia spp. 

 Cross Reactivity: No reactivity with other pathogenic bacteria e.g. Pseudomonas

aeruginosa, Staphylococcus aureus, Streptococcus pneumonia etc. 

 Comparative Methods PCR, Immunofluorescence, Culture 

 Sample preparation time: None (depending on the matrix to be used)

 Reading time (time to result): 5 to 20 minutes (TBD)

 Sample throughput rate: Unlimited. Many samples can be run in parallel on separate strips.

 General assay characteristics: Clear test and control lines

 Shelf life: 24 months at room temperature (≤30C)

Company Background: InBios is a 12-year old FDA-registered ISO-certified biotechnology company 

focusing on developing novel immunologic diagnostic products and reagents based on proprietary 

antigens/antibodies and technologies. The company’s objective is to become a leader in the development and 

manufacturing of novel diagnostic tests for infectious diseases. The company’s primary focus has been on: 1) 

infectious diseases that plague developing countries and have a great potential to impact the United States 

due to immigration; and on 2) emerging infectious diseases such as Chagas (T. cruzi), West Nile Virus, 

Dengue fever, and Japanese Encephalitis 3) biodefense e.g. anthrax and melioidosis. The company believes 

that major diagnostic firms are not focused on emerging infectious disease diagnostic markets or third world 

diseases because of their perceived small market size and/or profit margin. InBios believes that this niche 

market needs to be filled and is committed to developing high quality, cost-effective diagnostics tests. To that 

end InBios has developed the following diagnostic assay services: 

 Twenty plus years of experience in assay development, both lateral flow and ELISA based

 Expertise in production of conjugates, including various gold sized particles

 Feasibility studies

 Prototype and beta test development

 Full FDA compliance-design plan and design history files prepared

 Laboratory studies- accelerated stability, cross reactivity, interference and precision

Commercialization Plan Page 67 
 Analytical sensitivity and specificity studies

 Clinical trial management

 FDA product submissions (510(k) & PMA)

 Small and large scale manufacturing of rapid and ELISA based immunoassays

The company’s founders and employees have extensive experience in developing, manufacturing, and

marketing diagnostic tests worldwide. In-house expertise includes various aspects of chemistry and 

immunology including immunochemistry, protein expression and purification, and protein manipulation. InBios 

also specializes in particle and conjugation chemistry that includes our proprietary gold-based technology for 

assay read-out. Regulatory approval and commercialization of a new diagnostic test is confronted with many 

significant scientific and regulatory hurdles. InBios has an excellent track record of obtaining funds to develop 

diagnostic tests/products. Over 75% of InBios’ externally-funded projects have successfully obtained 

competitive renewal, indicating that InBios performs excellent science and achieves significant progress. 

Recently, InBios released an FDA-cleared Dengue virus IgM detection diagnostic test which is the first and 

only FDA-cleared dengue test available in the market. Here is the NIAID webpage link to the dengue 

diagnostic test: http://www.niaid.nih.gov/topics/DengueFever/Research/Diagnosis/Pages/default.aspx

Please note we outcompeted a number of big players in our race to gain FDA clearance of our dengue IgM 

ELISA product. The competition included companies like Alere (Inverness), Panbio, Standard Diagnostics, etc. 

to name a few. We have a number of advanced levels of products that are progressing towards the FDA 

submission process. Our typical FDA submission process ranges between 3-5 years. A brief overview of 

Inbios selected product portfolio is shown in Table 1 below. 

The company has successfully applied for and received SBIR funding for the development of its TB test 

(Phase I) and for its T. cruzi, Dengue and Leishmania assays (Phase I and II). Its test for visceral leishmaniasis 

received FDA 510(k) clearance May 2003; commercialization of this product is well underway and sales are 

being realized. InBios has also received competitive funding from the Department of Defense for development 

of multi-pathogen dipstick rapid tests for enteric diseases, febrile illnesses and sand fly fever viruses as well as 

cutaneous leishmaniasis. Development of diagnostics to meet a growing need in the military and abroad 

presents unique commercialization opportunities for a variety of the company’s product portfolio.

The company has significant regulatory experience. It is registered with the Food and Drug Administration and 

several of its products have gained FDA clearance (see Table 4 below). Establishment licensure with the 

USDA has been received and several tests with veterinary applications will be marketed in the coming year. 

The company adheres to the Quality Systems regulations of the FDA from product inception by following 

Design Controls mandated by CFR 820 to the validation of its equipment and processes. FDA inspection of its 

facility has not yielded any complaints (483s) regarding its quality system. InBios became ISO 13485:2003 

certified in 2011 which has increased its ability to market its products internationally. Several products are also 

CE marked and can be sold in Europe. 

The vision of InBios is simple: develop and market high quality diagnostic assays with superb performance at 

an affordable price. The founders’ of InBios have maintained that vision since the company’s inception. To 

accomplish that goal, the company has invested the majority of the private investment dollars it has received 

as well as product sales revenue into research and development. Overhead at InBios is kept low and the “burn

rate” is below average for a company of its size. The company believes that its new products, such as the 

West Nile Virus and Dengue ELISA will result in significant sales and revenue in years to come. The company 

understands that as it grows, changes in management will take place. The company already plans to add 

positions in management for sales and marketing. At present, the company relies on consultants and focuses 

its employees on its core competencies – research and development as well as in-house manufacturing and 

regulatory affairs. 

Market, Customer, and Competition: We anticipate that Active Melioidosis Detect TM (AMD) marketing will 

be analogous to our FDA cleared Kalazar Detect TM test. Kala-azar or Visceral leishmaniasis (VL) is a severe 

disease of children and women. There is an effective therapy if an accurate diagnosis is made. Many of the 

symptoms are similar to malaria, dengue and other tropical diseases. We developed a rapid test for early 

diagnosis of VL that changed VL disease management completely. We started with an export use only 

product, obtained FDA approval and created this market when the only diagnosis was splenic and/or bone 

marrow aspirate, microscopy and culture. At this time we are the leader in this field and our customers include 

Commercialization Plan Page 68 
US and UK Army who are/were engaged in Gulf and Afghanistan conflict. We are selling hundreds of 

thousands of this test every year. However, the price ranges between 1$ US and 10$ US, depending on the 

markets. Similar to the VL target the meliodosis target is proprietary and IP protected. 

Melioidosis will follow a very similar development plan. B. pseudomallei is found all over the world, but 

Melioidosis is most frequently reported in Southeast Asia and Northern Australia (Fig. 2). It also occurs in 

South Pacific, Africa, India, and the Middle East. People acquire the disease by inhaling dust contaminated by 

the bacteria and when the contaminated soil comes in contact with an open wound of the skin. Infection most 

commonly occurs during the rainy season. Melioidosis symptoms most commonly stem from lung disease 

where the infection can form a cavity of pus (abscess). The effects can range from mild bronchitis to severe 

pneumonia. As a result, patients also may experience fever, headache, loss of appetite, cough, chest pain, 

septicemia and general muscle soreness. The symptoms are very much like many other diseases like dengue, 

leptospirosis, and rickettsial diseases. The septicemia caused by B. pseudomallei infection is clinically similar 

to that caused by other major pathogens. Subacute and chronic disease may mimic tuberculosis clinically. 

Considering the rate of mortality of this disease, acute and early diagnosis is critical. 

If untreated, melioidosis is fatal. When treated with antibiotics, severe forms of the illness have a 50% chance 

of recovery, but overall the mortality rate is 40% in resource poor settings. Early detection is critical. In the 

United States, confirmed cases range from 0 to 5 each year and occur among travelers and immigrants, 

according to the U.S. Centers for Disease Control and Prevention (CDC). However, in the US, the melioidosis 

is further complicated because it has the potential to be developed as a biological weapon. It is classified by 

the US Centers for Disease Control (CDC) Tier 1 select agent. The Department of Defense and Homeland 

Security is very interested in the diagnosis and control of melioidosis (see support letters). 

Current diagnosis is limited to specialized laboratories and the number of competitors is also limited. We 

believe that number of cases is much higher than reported. Many cases are not reported because of a lack of 

diagnosis. Recently, more and more cases are reported because of awareness of the disease from the level of 

Government agencies (CDC, Army, local universities, etc.). We also believe that the testing will be performed 

on a greater number of people with unrelated febrile symptoms, just to rule out the possibility of melioidosis. 

The diagnostic assay InBios is developing for melioidosis is very robust, field deployable, and simple to use 

and does not require any special equipment or any trained personnel. 

Intellectual Property (IP) Protection:

Patent Title: Method of Diagnosing and Treating Melioidosis 

PCT Patent Application No. PCT/US2011/032155, filed on April 12, 2011 

This application claims the benefit of U.S. Provisional Application: 

U.S. Provisional Patent Application No. 61/323,236, filed on April 12, 2010 

U.S. Provisional Patent Application No. 61/452,388, filed on March 14, 2011 

Inventors: David P. AuCoin; Thomas R. Kozel (University of Nevada School of Medicine) 

Table 4. InBios selected product portfolio

Product Test 

type

510(k) No. Status

Generic Blood Glucose Dipstick K001092 Cleared; de-listed

Kalazar Detect (a test for Visceral

Leishmaniasis; Class I)- NIH funded

Dipstick K023483 Cleared

West Nile Virus (Class III)- CDC 

collaboration

ELISA K041817 Cleared

Dengue IgM (Class II)- NIH funded ELISA K100534 Cleared

Cutaneous Leishmaniasis (class I) 

– DOD funded

Dipstick - FDA submission 1st Qtr 2014

Dengue NS1 – NIH funded ELISA, 

dipstick

- FDA submission of ELISA product 

is imminent

Chagas (T. cruzi) – NIH funded Dipstick - Clinical studies have begun

Scrub typhus – DOD funded Dipstick - Pre-IDE communication stage

Japanese Encephalitis (class III) –

CDC collaboration

ELISA - In clinical

AMD Rapid In development

AAD Rapid In development

Commercialization Plan Page 69 
Abstract: Non-invasive methods are provided herein for the diagnosis of melioidosis with specific antibodies 

capable of detecting molecules associated with melioidosis in a biological fluid, such as urine or serum. These 

molecules can be identified using proteomic methods, including but not limited to antibody based methods, 

such as an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), or a lateral flow 

immunoassay. Methods of inducing an immune response to melioidosis are also disclosed. The methods 

include the use of the immunogenic melioidosis polypeptides, nucleic acids encoding these polypeptides, 

and/or viral vectors encoding an immunogenic melioidosis polypeptide, alone or in conjunction with other 

agents, such as traditional melioidosis therapies. Also disclosed are methods for treating a subject having 

melioidosis. These methods include inducing an immune response to melioidosis and/or using an inhibitory 

nucleic acid, such as a siRNA or antisense molecule, to decrease a melioidosis associated molecule 

expression in order to treat melioidosis. InBios’ innovation in Melioidosis diagnostics is protected by intellectual 

properties through the University and Inbios know how and trade secrets. 

Finance Plan: The maturity of AMD candidate is to a point where achieving FDA clearance within 3-4 years is 

likely. While challenging, FDA clearance is critical to provide dependable products manufactured under GMP. 

For melioidosis where there is no huge market at this time (besides the military use), the FDA clearance is 

hindered by the limited availability of well-defined samples in the US. The cost of organizing clinical studies in 

the endemic areas (primarily international sites) is also cost prohibitive unless strong financial support is in 

place. We anticipate seeking help from our existing collaborators with pre-clinical and clinical studies. Some 

of the current funding resources will support in house performance studies and enhance our documentation 

capability, GMP process development, GMP manufacturing and overall QC manpower and capability of 

performing in house studies. All these are needed for FDA submission and continued GMP manufacturing of 

the AMD product. As needed we will seek additional funds through Phase III as well funds from sales of AMD 

research use only (RUO) and analyte specific reagent (ASR).

Production and Marketing Plan: InBios has the facility and manufacturing experience to produce LFI and 

ELISA tests on a large scale. It has automatic, quantitative reagent dispensing equipment, automated strip 

cutter machine, high speed labeling and package sealer as well validated manufacturing processes to produce 

all rapid tests. The company is currently producing several lots a week of its Visceral Leishmania test and are 

making small lots of prototype melioidosis tests. The manufacturing process for a rapid melioidosis test will be 

similar to the process currently used for its Visceral Leishmaniasis test and other rapid tests in development. 

However, we will gear towards more automation in the near future. Therefore, in-house manufacturing of this 

product will follow under a routine process. The work that will be required to manufacture this AMD assay will 

be finalization of documentation of the manufacturing and quality control as well as validation of the design and 

process. The company is confident its manufacturing processes are under quality control as it has been 

audited recently by the FDA and did not receive any complaints during the inspection. 

InBios, in collaboration with UNR, has already created awareness regarding availability of the rapid melioidosis 

diagnostic market by introducing the first rapid test which is being evaluated by a number of clinicians and has 

received considerable interest from government agencies. InBios also has distributors in India, Pakistan, 

Vietnam, Singapore, Thailand, South America, and the U.S. Once developed, we will approach all the 

distribution channels and reference laboratories. In the U.S., we are connected with all major State Health and 

reference laboratories such as ARUP and the Mayo clinic. However, for marketing in the US we will need FDA 

approval. We shall market the test in the US as a research use only (RUO). We shall definitely market outside 

US as an export use only product. We have many years of experience with a number of products for “export 

use only” category. In the context of RUO we have already established a contract with the Navy (DTRA) to 

provide a cassette version of rapid test that has combined capability of detecting Dengue NS1 as well as CPS 

(See letter of support). This test is designed only for using large volumes of whole blood or serum and is not 

adapted or intended for the multi sample type described in this application.

We strongly believe that having a FDA cleared AMD test will be needed for creating and penetrating markets 

not familiar with this novel test. Our experience confirms that end users are looking for quality and affordability. 

FDA clearance will provide a stamp of approval, consistency and quality. In addition, solid publications from 

the opinion makers will definitely help to bring in public awareness about this disease and expand markets for 

AMD. FDA clearance will also be important for marketing to government agencies e.g. DoD, DTRA and 

Department of Homeland Security. 

To maintain our competitive advantage and thwart competition we shall employ several strategies: (a) pursue 

FDA clearance and CE marking for our products to demonstrate product quality; (b) consider manufacturing 

Commercialization Plan Page 70 
 

cost-reduction strategies so that we could maintain a competitive and affordable price for the AMD; (c) reenforce alliance with existing distributors and bring in additional well-known international distributors, if 

warranted, who can further provide a reputable presentation for our products and saturate the competitive 

market; (d) use customer feedback to continuously improve the product quality and our services. 

Revenue Stream: InBios is a privately-held, for-profit company with operation resources from contract 

manufacturing, product sales, and federal-funded Small Business programs. Commercialization costs for these 

products will be funded by the company’s revenue and potential future Government funding. InBios forecasts 

its service and sales revenue for fiscal year 2013 to be close to $2MM, not including grant funding. Our 2012 

sales have reached close to $1.5MM, not including grant revenues. Table 5 shows the selective revenues 

generated and forecast by major product/service type. Our dengue IgM and NS1 ELISA business is growing 

faster and therefore will generate increased revenues essential for additional hiring and new product 

development. Please note we do not have a dedicated in house marketing and sales team. The revenues are 

all generated through our web site and via references. We anticipate recruiting sales and marketing once 

sales reach $5MM. The sales of AMD will move slowly (similar to Kalazar Detect). Once the incidence of 

melioidosis worldwide is better understood we expect sales to increase significantly. We are looking for a 

steady growth year after year once the technology is proven and markets are aware of the test. We are hoping 

to sell close to 10 to 20K tests in the first year once a final prototype and selection of matrix or matrices are 

established. Overall pricing structure, government requirement and insurance reimbursement, if allowed will 

dictate the final pricing. 

InBios has also received USDA approval to begin selling its Equine West Nile test, which will generate 

additional revenue. 

Future Developments: Product development to be completed following the award period: Following FDA 

clearance of the Melioidosis Antigen Detect Assay “multiplexing” will be evaluated. Dr. Thomas Kozel 

(University of Nevada, Reno) is currently developing an LFI for the detection of the capsular polypeptide of 

Bacillus anthracis, the causative agent of anthrax. This project is in collaboration with InBios and a pre-IDE for 

the diagnostic has recently been submitted. The goal will be to combine these two FDA-approved tests to 

produce an LFI that has the ability to diagnosis both a melioidosis or anthrax infection. The AuCoin laboratory 

is also teaming with InBios to develop a diagnostic product for tularemia and plague. Therefore, the final 

product could potentially be a rapid LFI that can identify all of the Tier 1 bacterial pathogens as outlined by the 

Federal Experts Security Advisory Panel – Recommendations Concerning the Select Agent Program (June 13, 

2011; Department of Health and Human Services).

As part of our contract with DTRA we are in the process of evaluating the portable Deki Reader (Fio Corp). 

This enables image analysis of the rapid tests in cassettes, determines positivity and negativity and stores 

clinical data in the cloud. Information and benefits on the Deki reader is provided below. Fio Corp has provided 

InBios two Deki Readers to evaluate the AMD LFI strips in a cassette supplied by Fio. (See attached letter of 

support). 

Table 5.: Selected Product/Service Revenue of 2012 and Forecast 2013

Product description Revenue earned 2012, Forecast for revenue for 2013

Kalazar Detect TM

(FDA-cleared)

$ $

West Nile Detect 

(FDA-cleared)

$ (burst of 

sales compared to last 

year due to outbreak)

$ (not anticipating any 

outbreak this year)

Dengue ELISA 

(FDA-cleared)

$ $

Dengue NS1 Detect (ELISA 

and RAPID) 

(FDA studies have begun)

$ $

T cruzi Human 

(FDA studies underway)

$ $

Commercialization Plan Page 71 
Fio Technology Overview - Fionet System: The Fionet system represents a significant advance in point-ofcare (POC) rapid testing and patient case management. Fio’s technologies provide healthcare providers faster, 

more accurate, and more cost effective rapid test results. Fionet makes it possible to collect and monitor 

diagnostic and clinical data in real time, to support fast, targeted and economic health interventions. 

The Fionet system integrates mobile diagnostic devices with cloud information services. Fionet is comprised of 

Deki, a companion system for health workers, airFio, a secure cloud data system, and Spiri, a web-based 

gateway to information services for health managers. Deki mobile software applications and companion 

devices for health workers guide clinical workflow and facilitate digital data capture at the point of care. Deki is 

designed for use with standard Android phones (“Deki Phones”) and tablets (“Deki Tablets”), and the Deki 

Reader. Deki captures and transfers critical patient information to airFio, giving managers high-quality data. 

airFio aggregates and stores data transmitted by Deki units (readers, tablets and phones) over wireless mobile 

phone or WiFi networks. Spiri controls access to airFio and provides two-way communication between Deki 

units and Spiri. airFio and Spiri provide real-time access to aggregated data from all deployed Deki Readers 

and generate automated reports for health program monitoring, evaluation and surveillance. In addition, Spiri 

enables managers to monitor and communicate with health workers, and to disseminate workflow protocols, 

custom survey forms and software updates. 

The Deki Reader is a portable, rugged, rechargeable battery-powered rapid diagnostic test (RDT) reader with 

diagnostic software developed on Google’s Android operating system. The user interface consists of a liquid 

crystal display (LCD) display with a touch screen, and a drawer that holds the RDT cassettes (or dipsticks) in 

place during image analysis. The Deki Reader is programmed to function with a predefined menu of 

commercially available RDTs that have been validated for use with the reader. Hence, the Deki Reader is a 

universal RDT reader that can accommodate the majority of RDT cassette formats. 

References 

1. Bhatt S, Gething PW, Brady OJ, Messina JP, Farlow AW, et al. (2013) The global distribution and burden of

dengue. Nature 496: 504-507.

2. Limmathurotsakul D, Dance DA, Wuthiekanun V, Kaestli M, Mayo M, et al. (2013) Systematic review and

consensus guidelines for environmental sampling of Burkholderia pseudomallei. PLoS Negl Trop Dis 7: 

e2105.

3. Limmathurotsakul D, Peacock SJ (2011) Melioidosis: a clinical overview. Br Med Bull 99: 125-139.

Commercialization Plan Page 72 
4. Currie BJ, Dance DA, Cheng AC (2008) The global distribution of Burkholderia pseudomallei and

melioidosis: an update. Trans R Soc Trop Med Hyg 102 Suppl 1: S1-4.

5. Wiersinga WJ, Currie BJ, Peacock SJ (2012) Melioidosis. N Engl J Med 367: 1035-1044.

6. Cuadros J, Gil H, Miguel JD, Marabe G, Gomez-Herruz TA, et al. (2011) Case report: melioidosis imported

from West Africa to Europe. Am J Trop Med Hyg 85: 282-284.

7. Salam AP, Khan N, Malnick H, Kenna DT, Dance DA, et al. (2011) Melioidosis acquired by traveler to

Nigeria. Emerg Infect Dis 17: 1296-1298. 

8. Morosini MI, Quereda C, Gil H, Anda P, Nunez-Murga M, et al. (2013) Melioidosis in traveler from Africa to

Spain. Emerg Infect Dis 19: 1656-1659.

 

Principal Investigator/Program Director (Last, first, middle): 

Commercialization Plan Page 73

PHS 398 Cover Page Supplement

1. Project Director / Principal Investigator (PD/PI)

* Title:

* Street1:

* City:

* Country:

Street2:

County/Parish:

* State:

* Zip / Postal Code:

Prefix: * First Name:

Middle Name:

* Last Name:

Suffix:

2. Human Subjects

Clinical Trial?

* Agency-Defined Phase III Clinical Trial?

* First Name:

Middle Name:

* Last Name:

Suffix:

3. Applicant Organization Contact

Person to be contacted on matters involving this application

* Phone Number: Fax Number:

Email:

OMB Number: 0925-0001 

Province:

Prefix:

No Yes

No Yes

562 1st Avenue S., Suite 600

Seattle

USA: UNITED STATES

WA: Washington

98104-3829

Clinical Trial & HESC 
4. Human Embryonic Stem Cells

* Does the proposed project involve human embryonic stem cells?

If the proposed project involves human embryonic stem cells, list below the registration number of the 

specific cell line(s) from the following list: http://stemcells.nih.gov/research/registry/. Or, if a specific 

stem cell line cannot be referenced at this time, please check the box indicating that one from the 

registry will be used: 

Specific stem cell line cannot be referenced at this time. One from the registry will be used.

PHS 398 Cover Page Supplement

No Yes

Cell Line(s):

Clinical Trial & HESC 
PHS 398 Research Plan

1. Application Type:

From SF 424 (R&R) Cover Page. The response provided on that page, regarding the type of application being submitted, is repeated for your

reference, as you attach the appropriate sections of the Research Plan.

*Type of Application:

OMB Number: 0925-0001 

2. Specific Aims

3. *Research Strategy

6. Protection of Human Subjects

7. Inclusion of Women and Minorities

8. Targeted/Planned Enrollment Table

9. Inclusion of Children

10. Vertebrate Animals

13. Consortium/Contractual Arrangements

14. Letters of Support

15. Resource Sharing Plan(s)

16. Appendix

1. Introduction to Application

(for RESUBMISSION or REVISION only)

Human Subjects Sections

2. Research Plan Attachments:

Please attach applicable sections of the research plan, below.

Other Research Plan Sections

5. Progress Report Publication List

11. Select Agent Research

12. Multiple PD/PI Leadership Plan

4. Inclusion Enrollment Report

New Resubmission Renewal Continuation Revision

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1247-Bp Phase II SA.pdf Add Attachment Delete Attachment View Attachment

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1252-Select_Agents_ Research Add Attachment Delete Attachment View Attachment

1253-Multiple PI Melioidosis Add Attachment Delete Attachment View Attachment

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1255-Letters of support fina Add Attachment Delete Attachment View Attachment

1256-Resource Sharing Plan M Add Attachment Delete Attachment View Attachment

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List of Research Plan Attachments 
SPECIFIC AIMS 

Melioidosis is a devastating tropical disease caused by the Gram-negative bacterium Burkholderia pseudomallei. 

The clinical manifestations of melioidosis are broad and include disseminated disease with organ abscesses, 

severe sepsis, and mild infection of the skin and soft tissue. The highest number of reported cases occurs in 

endemic regions of Thailand and Australia. Of major concern is that the global distribution of melioidosis is 

expanding as laboratories become more familiar with identifying B. pseudomallei. 

Isolation of B. pseudomallei by culturing clinical samples remains the diagnostic gold standard, unfortunately this 

takes 3-7 days. Culture is routinely performed on multiple sample types (blood, urine, pus, sputum etc.) and 

isolation of B. pseudomallei from any one of these samples is diagnostic for melioidosis. A major roadblock is 

that many diagnostic laboratories misidentify B. pseudomallei through lack of experience or validated diagnostic 

reagents. Any delay in diagnosis is critical as B. pseudomallei is resistant to many antibiotics. A rapid diagnosis 

is essential so patients can be treated with effective antibiotics.

For the past two years InBios 

and the AuCoin laboratory have 

been developing the Active 

Melioidosis DetectTM lateral flow 

immunoassay (AMD LFI) under 

an NIAID Phase I STTR award 

(Figure). The AMD LFI is an 

immunochromatographic strip 

assay that detects the capsular 

polysaccharide (CPS) of B. pseudomallei within a variety of melioidosis patient samples. Phase I Specific Aims 

focused on producing a library of anti-CPS monoclonal antibodies (mAbs), optimizing the LFI components and 

sample preparation, and performing a feasibility study on archived melioidosis patient samples. The objectives 

of this Phase II proposal is advanced development and validation of the prototype AMD LFI. The Phase II Specific 

Aims are as follows: 

1. Scale up production of purified CPS and lead candidate monoclonal antibody 4C4 (Year 1)

2. Optimize the 4C4 AMD LFI for detection of CPS in all relevant clinical matrices

a. Spike all available sample matrices with CPS and determine analytical sensitivity (limit of detection) in

each sample (Year 1)

b. Fine-tune and standardize LFI prototype for compatibility with all matrices by finalizing selection of buffer,

pads, membranes, blockers etc. (Year 1-2)

c. Provide prototype tests to collaborators for further testing of all matrices and cultures using archived

specimens. Develop diagnostic algorithm (Years 1 - 2)

d. Seek pre-submission guidance from FDA for studies required for 510(k) submission (Year 2)

3. Produce multiple lots of the 4C4 AMD LFI under document and design control and GMP for large

preclinical evaluation of performance characteristics with archived patient samples

a. Complete in house validation following pre- submission guidance from FDA; Studies include: analytical

sensitivity, reactivity, and specificity,cross reactivity, reproducibility/precision studies at 3 sites,

interference and stability studies (Years 2 & 3)

b. Validate AMD LFI in field studies at two geographical sites using archived sera and other matrices to

confirm diagnostic algorithm and compare with current gold standard diagnostic (Years 2 & 3)

c. Prevalence studies in melioidosis high and low endemic countries (Years 2 & 3)

4. Determine the in vitro and in vivo expression and secretion profile of CPS

a. Determine variation of CPS expression across genetic diversity of B. pseudomallei (Years 1 & 2)

b. Evaluate 4C4 AMD LFI with mouse and NHP aerosol model of melioidosis (Years 2 & 3)

c. Quantify CPS in human sample with the qCPS ELISA (Years 2 & 3)

This is a mature project that has developed from i) identification of a melioidosis biomarker, ii) construction of 

an promising prototype immunoassay, iii) successful feasibility studies in endemic sites and iv) assembly of an 

optimized lateral flow immunoassay ready for a larger preclinical analysis. 

C. 

Control'

Test'

Test'line' Control'line'

Prototype Active Melioidosis Detect lateral flow immunoassay.

Specific Aims Page 77 
 

RESEARCH STRATEGY 

Significance 

B. pseudomallei is a major cause of community-acquired sepsis in endemic areas. The clinical 

manifestations of melioidosis include mild infection of the skin, disseminated disease with organ abscesses and 

severe sepsis [1]. The highest numbers of cases occur in endemic regions of Thailand and Australia. Rising 

incidence rates have been recorded in Thailand between 1997-2006 during which the average mortality rate was 

42.6% [2]. In 2006, melioidosis and tuberculosis mortality rates in northeast Thailand were equivalent and 

second only to HIV/AIDS for infectious disease deaths [2]. In regions of northern Australia, where intensive care 

treatment is more readily available, the mortality rate is still alarmingly high at 20% [3,4]. 

The global distribution of melioidosis is expanding. Melioidosis is highly endemic to northeast Thailand and 

northern Australia, but more recent evidence indicates a much wider distribution that includes the India, southern 

China, Hong Kong, and Taiwan [6]. There are also an increasing number of reports of possible endemic disease 

in the Caribbean, Central and South America and East and West Africa [1,6-10]. In these regions melioidosis be 

overlooked because diagnostic confirmation relies on microbiological culture, which is often unavailable in 

resource-restricted regions. Even with such facilities, B. pseudomallei is often dismissed as a culture 

contaminant, or misidentified as a Pseudomonas spp. or other organisms by identification methods including API 

20NE and automated bacterial identification systems.

B. pseudomallei has been classified as a Tier 1 select agent. Health and Human Services has compiled a 

list of roughly forty-five biological select agents and toxins (BSATs). The Federal Security Advisory Panel [11] 

was recently tasked with classifying the most dangerous BSATs as Tier I. Ten BSATs were classified as Tier I; 

B. pseudomallei is included on this list. Tier I classification is based on the ability to produce a mass casualty 

event or devastating effects to the economy, communicability, low infectious dose, and a history of or current 

interest in weaponization based on threat reporting [11]. 

Melioidosis presents with many diagnostic challenges. Isolation of B.

pseudomallei from clinical samples remains the “gold standard” against which 

other melioidosis diagnostics are compared [13]. Culture is routinely performed 

on multiple sample types such as blood, urine, pus, sputum etc. [12,14]. A critical 

barrier to accurate diagnosis is the highly variable efficiency with which the 

bacterium is cultured from different clinical samples. Table 1 summarizes 

quantitative culture results from a variety of melioidosis patient samples. What 

is clear is blood contains the lowest levels of B. pseudomallei (~ 1 CFU/ml). In 

endemic areas blood cultures are routinely performed and nearly half are 

positive. 

An additional barrier is culturing of clinical samples takes 3-7 days [15]. A problem that is compounded by the 

fact that many diagnostic laboratories misidentify B. pseudomallei [16]. B. pseudomallei requires therapy with 

ceftazidime or a carbapenem drug, which are not agents of choice for empirical therapeutic regimes. Taken 

together, these factors point to a clear need for a simple and rapid diagnostic test for accurate identification of 

B. pseudomallei directly on clinical samples or cultures. 

The current rapid technologies for diagnosing melioidosis are unacceptable. Many laboratories in 

endemic areas have developed “home-brew” assays in an attempt diagnose melioidosis prior to culture results 

becoming available. PCR has been developed but is not in routine practice because it is limited by low sensitivity, 

stemming from the low concentration of B. pseudomallei in blood and the co-purification of PCR inhibitors with 

target DNA [17-19]. The indirect hemagglutination assay (IHA) is a rapid and inexpensive method used to detect 

B. pseudomallei antibodies. However, a large percentage of healthy individuals in endemic areas are 

seropositive [20,21]. This point is underscored by the fact that nearly 70% of children in northeast Thailand are 

seropositive for B. pseudomallei antigens [22,23]. Consequently, the IHA (or any serological test for melioidosis) 

has limited clinical utility in the endemic setting [1,24]. 

Antigen detection by immunofluorescence assay (IFA) or latex agglutination is commonly used in endemic areas. 

IFA is used in northeast Thailand for rapid diagnosis directly from patient samples containing high levels of B.

Table 1. B. pseudomallei culture 

results from 414 melioidosis patientsa

Sample

(# collected)

Median

CFU/mL

% Culture 

positive

Blood (414) 1.1 49%

Urine (268) 1.5 x 104 21%

Sputum (120) 1.1 x 105 78%

Pus (28) 1.1 x 107 82%

a modified from [12]

Research Strategy Page 78 
pseudomallei (sputum, pus, urine and respiratory secretions) [25,26] and from blood cultures [27]. The main 

drawback of IFA is the requirement for a fluorescent microscope and the requisite expertise, which is not feasible 

in most endemic settings. In addition, although specificity of the IFA is high, the sensitivity ranges from 45-48% 

when used directly on clinical samples [25]. Latex agglutination is an inexpensive technique that is effective at 

identifying B. pseudomallei from cultures of patient samples grown on agar plates or within liquid broth [28-31]. 

The agglutination assay is able to detect B. pseudomallei at concentrations of 1-2 x 106 CFU/ml; this limits its 

utility to cultured patient samples or colonies isolated on solid agar [30,31]. 

Development of this test provides an opportunity to test for Burkholderia pseudomallei in a broad spectrum of 

clinical samples. This has the potential to lead to an improved diagnostic algorithm for testing for melioidosis. 

Availability of the test will also enable clinicians worldwide to assess the true prevalence rate of the disease. Our 

approach to commercializing the product is outlined in the commercialization plan. In addition in the plan we 

discuss the opportunity to couple the simple field test with a portable Deki reader which allows for scanning of 

tests, data analysis of line intensity and storage of clinical information and test data in the cloud. This would 

greatly improve the interpretation and record keeping at remote sites. 

Innovation 

A new addition to the diagnostic algorithm for melioidosis. As mentioned above, diagnosis of melioidosis 

is challenging. There are advantages and disadvantages to all of the rapid melioidosis assays mentioned, 

however the AMD LFI was designed to offer a significant improvement over the current rapid techniques. We

strongly feel that the AMD LFI can replace all unapproved rapid tests thus, simplifying the melioidosis 

diagnostic algorithm. Lateral flow devices are inexpensive, rapid, sensitive, and stable at room temperature. 

In addition, LFIs do not require expensive equipment and they can accept multiple sample matrices, two 

characteristics that are essential for the diagnosis of melioidosis in resource poor settings. 

InMAD is a novel methodology for the identification of microbial biomarkers. The innovation of this project 

began with determining which B. pseudomallei antigens are detectable within patient samples that contain an 

overwhelming amount of host proteins. In an attempt to identify circulating bacterial antigens that possess 

diagnostic potential, we developed a platform technology termed “In vivo Microbial Antigen Discovery” or “InMAD”

[32,33]. The process involves immunizing a mouse with filtered serum collected from an animal model of 

melioidosis. The immunized mouse develops an antibody response to the shed/secreted B. pseudomallei

antigens present in the filtered serum. The serum from the immunized mouse is used to probe a B. pseudomallei

lysate by Western blot. Reactive antigens are identified as candidate diagnostic biomarkers. We identified a 

number of biomarkers, however the CPS was the most encouraging target [32]. Research and development 

performed in Phase I confirmed that the CPS was indeed shed into melioidosis patient samples, thus validating 

the InMAD procedure. Since then we are using the InMAD procedure to identify diagnostic biomarkers for 

invasive aspergillosis (IA) under the auspices of NIAID R21/R33 award: “Innovative Approaches to Target 

Identification and Assay Development for Fungal Diagnosis”. 

InBios and UNR have refined the selection of antibodies 

that should be used for diagnostic assays. Two practical 

characteristics of antibodies have been discovered during 

the Phase I period. First, we have determined that IgG3 

antibodies may not be an ideal choice of mAb for LFI 

applications due to their ability to self-associate [34-36]. Our 

first prototype AMD LFI incorporated IgG3 mAb 3C5 as both 

the gold-labeled and test line mAb. The self-association 

properties of IgG3 mAb 3C5 made it difficult to label with gold 

particles for LFI conjugate production. In addition, when 

increased volumes of melioidosis negative patient samples 

were tested on the LFI self-association of the gold-labeled 

mAb and test line mAb occurred. This produced a small 

percentage of false positive results. We were able to 

measure this interaction via surface plasmon resonance 

(SPR). IgG3 mAb 3C5 was linked to a SPR sensor chip and 

the same mAb was allowed to flow over the chip. The data 

indicated that the antibodies were binding, however, other 

IgG subclasses did not bind to the IgG3 mAb that was linked to the chip. We also determined that IgG3 mAbs 

Figure 1. IgG3-IgG3 interaction. IgG3 mAb 3C5 was linked to 

a Biacore sensor chip. IgG3 mAb F26G3 (specific to the capsule 

of B. anthracis) was then flowed over the chip at the concentration 

listed. The increase in response units indicates interaction of 

mAbs. 

Research Strategy Page 79 
that bind to different epitopes also associated. Fig. 1 illustrates that IgG3 mAb 3C5 interacts with IgG3 mAb 

F26G3 (specific to the capsule of Bacillus anthracis). 

To avoid the self-association roadblock we isolated mAb 3C5 subclass switch antibodies (IgG1, IgG2b, and 

IgG2a) from the IgG3 hybridoma culture. There is a low frequency of subclass switching of hybridoma cells in 

culture. We developed a technique to isolate subclass variants that was initially used for B. anthracis mAbs 

[37]. As shown in Table 2, these subclass switch antibodies had considerably lower affinity than the parent IgG3 

mAb and did not improve assay performance. This drastic shift in affinity occurs even though the subclass switch 

mAbs have identical variable regions. Our working hypothesis is that the IgG3 mAbs “selfassociate” and produce higher affinity through avidity effects. This may benefit a mouse trying to survive a 

bacterial infection but it does not improve the performance of lateral flow immunoassays. 

Approach 

Research and development efforts during the Phase I period 

began with a prototype AMD LFI in hand. The prototype was 

constructed with mAb 3C5 on the test line and as the gold-labeled 

conjugate antibody. Our approach was to optimize the 3C5 AMD 

LFI in the laboratory and at endemic sites, while simultaneously 

trying to isolate a library of high affinity CPS mAbs. Our Phase I 

STTR Specific Aims and Results are listed below; we are 

confident all or our goals were met. 

1. Produce and characterize a library of high-affinity CPS

mAbs. mAb 3C5 performed well when incorporated into the 1st

generation AMD LFI. However, we believed we could improve 

performance by incorporating higher affinity CPS specific mAbs 

into the LFI. As described above an additional reason for 

producing a library of CPS mAbs relates to the characteristics of 

mAb 3C5, which is a murine IgG3 antibody. IgG3 antibodies are 

difficult to label with gold and tend to self associate. We 

anticipated that incorporating high affinity, non-IgG3 mAbs into 

the LFI would simplify process development and produce a more 

accurate and sensitive assay. 

Listed in Table 2 are the anti-CPS mAbs that were isolated during Phase 

I. mAb 3C5 IgG3 was our first and only CPS mAb isolated by 

immunization of BALB/c mice with heat killed B. pseudomallei. The 

IgG1, IgG2b, and IgG2a 3C5 mAbs were isolated from the parent 3C5 

IgG3 hybridoma cell line. Interestingly, the affinities of these switch 

mAbs were 10-50 fold lower than the IgG3 3C5 mAb. The only subclass 

that was comparable to the IgG3 subclass via antigen-capture ELISA 

was the IgG2a. 

The additional mAbs listed in Table 2 were isolated by immunization with 

a BSA-CPS glycoconjugate that was provided to the AuCoin laboratory 

by Dr. Paul Brett (University of South Alabama). As expected the 

glycoconjugate induced a robust IgG response in the immunized mice. 

We were able to isolate two IgM mAbs and five IgG1 mAbs. The IgM 

mAbs did not perform well, however two of the IgG1 mAbs (1A2 & 2A5) 

were comparable to mAb 3C5 as determined by ELISA. Additionally, 

one mAb (4C4) performed better via ELISA (data not shown). These 

mAbs were then incorporated into prototype LFIs. Each mAb was 

tested as the gold conjugate and test line mAb. Different combinations 

of mAbs were used to determine the best performing prototype. Fig. 2 

is an example of one of the mAb combination experiments. InBios 

determined that mAb 4C4 was superior as the gold conjugate and the 

test line mAb. Therefore, a prototype 4C4 AMD LFI was developed and 

Table 2. Characteristics of CPS specific mAbs

mAb Subclass KD (nM) Improvement

over 3C5 IgG3*

3C5 IgG3 73 -

3C5 IgG1 1460 No

3C5 IgG2b 788 No

3C5 IgG2a 377 Same

4C4 IgG1 12 Improvement

2A5 IgG1 43 Same

1A2 IgG1 36 Same

3B4 IgG1 139 No

1D3 IgG1 Low No

2B3 IgM Low No

1D4 IgM Low No

*As determined by ELISA and/or LFI

Figure 2. Optimization of mAb combinations. 

Example of testing different prototypes with goldlabeled mAb (indicated with *) along with a test 

line mAb. 

Research Strategy Page 80 
tested. As shown in Fig. 3 the 4C4 AMD LFI produces a limit of 

detection (LOD) of 0.04 ng/ml when CPS is diluted in 150 l of 

chase buffer (the chase buffer is a component of the LFI kit). This 

is lower than the 3C5 AMD LFI, which achieved a LOD of 0.2 

ng/ml. In addition, the 4C4 AMD LFI test line appears more 

intense at all CPS concentrations. 

While producing a library of CPS mAbs we concurrently tested

the analytical reactivity and specificity of the 3C5 AMD LFI 

against a panel of B. pseudomallei, B. mallei, and near neighbor 

species. This provided valuable data on the extent of 

conservation of CPS among Burkholderia species. Strain panels 

tested included isolates selected by the Stakeholder Panel on 

Agent Detection Assay (SPADA) Working Group. A large panel of 

B. mallei was also tested as it has recently been shown to produce 

the identical manno-heptose capsule as B. pseudomallei [38]. We have previously shown mAb 3C5 reactivity to 

B. mallei CPS by Western blot [32]. 

The LFI testing was performed at the Centers for Disease Control and Prevention (CDC). The CDC requested 

to test the assay for possible inclusion in the Laboratory Response Network which is the network of State Health 

Laboratories in the U.S. Three colonies from each isolate were tested separately on the LFI. Of the B.

pseudomallei isolates tested 76/77 (98.7%) were positive and 30/33 (90.9%) of the B. mallei isolates were also 

positive. In addition, 35/36 (97.2%) of near neighbor species were negative by LFI. Eight B. thailandensis

isolates were tested, seven were negative. Other near neighbor species where also tested, including B.

humptydooensis sp. nov., B. oklahomensis and Burkholderia cepacia complex species, all were negative. 

Both the B. pseudomallei negative and B. thailandensis positive LFI results can be explained through sequencing 

analysis. The negative isolate originated from a patient with a persistent asymptomatic B. pseudomallei infection 

in Australia. A frameshift mutation was identified within the wcbR gene of this isolate [39]. The B. thailandensis

LFI positive isolate was recently shown to encode the CPS operon [40,41]. 

The 4C4 AMD LFI was recently tested for reactivity to B. pseudomallei and to other near neighbor species. A 

panel of 40 B. pseudomallei isolates was acquired from the laboratory of Dr. Paul Keim (Northern Arizona 

University). The 4C4 AMD LFI was positive for 39/40 of these isolates. mAb 3C5 was reactive with 38/40 of the 

isolates via antigen-capture ELISA. In addition, 6 Gram-negative bacteria and 9 Gram-positive bacteria were 

tested for reactivity against the 4C4 AMD LFI, and all were negative. In addition to the testing mentioned, the 

4C4 AMD LFI will be tested at UNR against the panel of bacterial isolates identical to the panel used by the CDC. 

The Department of Homeland Security (DHS) will provide this panel (see letter of support). 

2. Optimize LFI components for enhanced detection of CPS in serum and urine samples. Phase I began

with fine-tuning the 3C5 AMD LFI to achieve the lowest analytical sensitivity (limit of detection) without causing 

background reactions (false positive tests). When the LFI was tested with 20 different serum and urine samples 

a small percentage of false positive reactions were occurring. This occurred only when increased volumes of 

sample (50 - 100 l) were analyzed and did not occur when 25 l of sample was used. Ideally, we wanted the 

option of testing higher volumes (100 l) on the LFI; this would be ideal for blood and urine samples that 

contained low levels of CPS. This is a fairly common problem and often relates to the pH of individual samples. 

However, we believed two possible factors could be exacerbating the false positive reactions. First, as described 

the gold-labeled IgG3 mAb 3C5 could be binding to IgG3 mAb 3C5 on the test line. Second, human anti-mouse 

antibody (HAMA) reactions could be occurring. This can occur in blood or serum samples that contain human 

antibodies that bind to mouse antibodies. Thus, the human antibody binds to the gold-labeled mouse mAb in 

addition to the mouse mAb on the test line. 

We took two approaches to eliminate the false positive reactions. First, non-IgG3 CPS specific mAbs were 

tested (Fig. 2). In addition, commercially available HAMA blockers were added as a component of the sample 

pad. Initial work focused on IgG1 mAb 2A5 since mAb 4C4 had not been isolated. An LFI composed of mAb 

2A5 deposited on the test line and a gold-labeled mAb 3C5 was able to accept a higher volume of sample, 

however the LOD was not acceptable (Fig. 2). When mAb 4C4 was incorporated into the optimized LFI as the 

gold-labeled and test line mAb 75 l of sample could analyzed with virtually no false positive reactions. The 

sample pad was then exchanged to accept whole blood and additional sample volume. The current 4C4 AMD 

Figure 3. LOD comparison between the 3C5 and 4C4 

AMD LFIs. A five-fold serial dilution of CPS was spiked 

into chase buffer followed by application to the sample pad.

Research Strategy Page 81 
LFI is able to accept 100 l of blood or urine. It also produces the highest signal, improved LOD and clean 

background when tested with multiple normal sera and whole blood samples (heparin, EDTA and citrate). 

3. Evaluate optimized LFIs and quantify CPS in a variety of melioidosis patient samples. During the Phase

I period we used our quantitative CPS (qCPS) ELISA [32] to analyze filtered serum and urine samples collected 

from patients with culture-confirmed melioidosis in Thailand (Table 3). Quantitative cultures were performed on 

urine samples prior to testing and are reported as colony forming units/ml (CFU/ml). Blood cultures were also 

tested however the CFU/ml was not determined (too low). Each serum (isolated from blood) and urine sample 

was filtered to remove intact bacterial cells prior to shipment. CPS was detected in 

6/10 filtered urine samples at concentrations ranging from 0.78 – 448 ng/ml. The 3C5 AMD LFI was positive for 

all of the urine samples were positive by the qCPS ELISA (below Table 

3). As expected, the concentration of CPS was higher in samples that 

contained a higher CFU/ml. CPS was detected in 5/10 filtered serum 

samples at concentrations ranging from 0.85 to 6.7 ng/ml. This is 

encouraging since the mean concentration of B. pseudomallei in 

patient blood is ~1 CFU/ml [12,14]. We anticipate that CPS may be 

shed from internal abscesses into blood; so theoretically, even if the 

CFU/ml is low the concentration of CPS may be within the detectable 

range of the LFI. Also, if the samples were not filtered we believe more 

would have been positive. Patient serum samples were not tested on 

the LFI due to insufficient volumes, but half contained concentrations 

of CPS that could be detected by the AMD LFI. 

Menzies School of Health Research (MSHR) in Darwin, Australia 

performed a pre-clinical evaluation of the prototype 3C5 AMD LFI over 

the past 18 months. In total, 81 archived samples from 38 melioidosis 

and two non-melioidosis patients were tested. Many of these samples 

were tested on the LFI multiple times in order to optimize sample 

preparation. This was not a trivial task. An optimized sample 

preparation protocol listed in Table 4. This protocol will be used to test 

the 4C4 AMD LFI. 

In addition to sample preparation MSHR compared the performance 

of the 3C5 AMD LFI to both the current gold standard (culture) and 

real-time PCR for detection of B. pseudomallei specific Type III 

secretion system (TTS1) [42]. While all samples have been tested 

with LFI not all clinical specimens have been assayed with all three detection methods. Forty-eight of the samples 

have culture results allowing direct comparison of the sensitivity of AMD LFIs to the gold standard, with a subset 

of samples verified as negative or positive using the TTS1 assay. 

Urine specimens (17 samples): A total of 17 melioidosis patient urine samples were evaluated. Our results show 

good correlation with culture results. We obtained the best correlation by first centrifuging the cellular 

components in the urine followed by use of the supplied lysis buffer. Initially, results indicated a decreased 

sensitivity of the LFIs in comparison to culture with two culture positive samples negative with the LFI tests. 

However, following optimization of our methods we saw an increase in LFI sensitivity and interestingly in two 

cases (both melioidosis patients) we saw an LFI positive while the culture results were negative. 

Sputum (12 samples): Sputum performs the quite well with the LFI with strong correlation between culture results. 

13 matched with culture results (11 positive, 2 negative) with one false negative. The main difficulty we 

encountered with sputum was the ability for the specimen to migrate along the LFI strip, necessitating variations 

in dilution based on sputum viscosity. The ability to diagnose melioidosis from sputum by LFI would be 

particularly useful in a bioterrorism setting since aerosolized B. pseudomallei would cause a severe pulmonary 

infection. 

Serum (8 samples): Eight out of eight LFI tests correlated with the blood culture results (seven positive and one 

negative) showing promising sensitivity. Blood is a notoriously difficult clinical sample type for use in diagnosis. 

In this pre-clinical experimentation we saw increased sensitivity with the LFIs in comparison to the TTS1 PCR 

assay. This is a important result since over half of melioidosis patient blood cultures are positive. 

Table 3. Quantitation of CPS in filtered melioidosis

patient samples

Urinea Serum

Sample CFU/mL

[CPS]

ng/ml

Sample Culture 

resultb

[CPS]

ng/ml

UID1 2.3 x 104 2.7 MSID1 + 5.4

UID2 >1 x 105 448 MSID2 + 0

UID3 7.5 x 104 20 MSID3 + 6.7

UID4 1.2 x 104 0.78 MSID4 + 3.3

UID5 >1 x 105 66 MSID5 + 0

UID6 3.5 x 103 0 MSID6 + 0.85

UID7 >1 x 105 187 MSID7 + 0

UID9 <1 x 103 0 MSID8 + 0

UID10 1 x 103 0 MSID9 + 1.6

UID12 1 x 103 0 MSID10 + 0

a Serum and urine were collected from different patients 

b Blood cultures (serum) were all positive (+)

Research Strategy Page 82 
Other Sample types: Pus, CSF and lung swabs were also tested. Four pus samples were analyzed, all showed 

direct correlation with culture. The one culture positive CSF sample tested failed to give a positive LFI result. 

The false negative result is probably indicative of the low bacterial load present in the CSF. All three lung fluid 

samples showed positive culture and positive LFI results. 

Phase II - Specific Aim 1: Scale up production of purified CPS and lead candidate monoclonal 

antibody 4C4 

Rationale: Our first prototype AMD LFI incorporated mAb 3C5 as the conjugate and test line mAb. During 

Phase I studies a significant amount of time was spent characterizing this mAb and the LFI. The mAb and the 

LFI performed well, however, based on our preliminary 

data, mAb 4C4 is now the current lead antibody. It has 

a higher affinity than mAb 3C5, it is a non-IgG3 antibody, 

and it performs better that mAb 3C5 when incorporated 

into the AMD LFI (Fig. 3). For these reasons we will 

scale up and validate large batches of mAb 4C4. In 

addition, we will purify CPS, an indispensible reagent for 

AMD LFI production. 

Monoclonal antibody purification. We have begun 

scaling up production of mAb 4C4 in order to produce a 

large lot of 4C4 AMD LFIs for pre-clinical testing. We 

are currently growing mAb 4C4 hybridoma cells in 

CELLine bioreactors (Integra Biosciences); production 

from each bioreactor is 5-10 mg/week. mAb is purified 

via affinity chromatography over protein-A Sepharose 

followed by concentration. A number of quality control 

steps will be followed to verify batch purity and mAb 

performance. First, a non-denaturing Coomassie gel will 

be used to confirm the mAb has not fragmented during 

the purification process. Next, the batch is evaluated for 

binding to purified CPS via ELISA. The binding affinity 

(KD) of each batch will also be evaluated by surface 

plasmon resonance (SPR). To perform the affinity 

analysis CPS must first be conjugated to biotin, which 

allows for binding to a streptavidin coated SPR sensorchip. The binding affinity (KD) between mAb and CPS can 

then be calculated as a function of the kinetic constants (on- and off-rates). This analysis was performed for 

each of the mAbs listed in Table 2 and is commonly performed in the AuCoin laboratory. Each lot of mAb will 

be compared to previous lots to ensure there is no variation. 

Purification of CPS. Purified CPS is an essential quality control (QC) reagent that is required to validate AMD 

LFI performance. The AuCoin laboratory has been optimizing the purification of CPS during the Phase I period. 

Purification of CPS is being performed as previously described [32,43]. The B. pseudomallei LPS O-antigen and 

CPS co-purify by the method described, therefore, an O-antigen-deficient mutant isolate of B. pseudomallei

(DD503) is being used. The CPS is isolated from overnight cultures of DD503 by a modified hot aqueous-phenol 

procedure. The structure and purity of CPS will be verified by the Complex Carbohydrate Center (University of 

Georgia). Both mAb and CPS will be produced under GLP. A senior technician, who has completed FDA GLP 

training for pre-clinical testing, will oversee GLP requirements in the AuCoin laboratory. 

Limitations and alternative strategies. This aim is fairly straightforward. Purification of mAb 4C4 and CPS 

has been initiated during the Phase I period. In addition we will produce 100 mg of each of the alternative mAbs. 

The alternative mAbs are 3C5, 1A2, and 2A5. These mAbs performed well via ELISA and LFI and will be 

available if needed. These mAbs may be incorporated into the LFI if by rare chance mAb 4C4 does not meet 

design specifications described in Specific Aims 2 & 3. For example, we strongly feel that the 3C5 AMD LFI 

would be an acceptable alternative to the 4C4 AMD LFI. 

Timeline and benchmarks for success. We will produce 100 mg of purified CPS and 1 gram of mAb 4C4 by 

the end of Project Year 2. Success will be measured by purifying enough mAb and CPS needed to support the 

large preclinical analysis that will occur in Project Year 3. 

Table 4: Optimized sample preparation protocol

Sample type Sample preparation

Whole blood, 

serum, and plasma

Combine 35 l with 3 drops of chase buffer

Urine & Culture 

fluid (concentrated)

Centrifuge 1-10 ml, 3000 x g, 10 min

Resuspend cell pellet in 2 drops of lysis buffer

Combine 20l with 3 drops of chase buffer

Urine (straight) Combine 50 l with 3 drops of chase buffer

Pus Combine 20 l with 2 drops of lysis buffer, vortex

Add 20 l of lysate to sample pad followed by 3

drops of chase buffer

Sputum (viscous) Combine 20 l with 3 drops of lysis buffer; vortex

Add 20 l of lysate to sample pad followed by 3

drops of chase buffer

Sputum (nonviscous)

Combine 50 l with 2 drops of lysis buffer; vortex

Add 20 l of lysate to sample pad followed by 3

drops of chase buffer

Bacterial colonies Resuspend a single bacterial colony in 2 drops

of lysis buffer

Apply lysate to sample pad followed by 3 drops

of chase buffer

Research Strategy Page 83 
Specific Aim 2: Optimize the 4C4 AMD LFI for detection of CPS in all relevant clinical matrices 

Rationale. An LFI must be constructed that can accept a variety of melioidosis patient samples. LFI components 

and buffers have been optimized for the 3C5 AMD LFI. This Aim will determine whether these components will 

be compatible with the 4C4 AMD LFI. We feel if we can optimize the 4C4 AMD LFI to perform well in blood 

products (that contain low CFU/ml) this test should work well in other matrices that contain higher CFU/ml (Table 

1). 

Fine-tune and standardize LFI prototype for 

compatibility with all matrices by finalizing selection 

of buffer, pads, membranes, blockers etc. The 

components of the 3C5 AMD LFI were optimized and 

tested in endemic sites during the Phase I period. We 

expect that many of the same components will be 

used for the finalized 4C4 AMD LFI. As summarized 

in Table 5, these components can be modified to 

improve the performance of the LFI. Importantly, the 

3C5 AMD LFI was originally designed with a sample 

pad that did not accept whole blood. The 4C4 AMD 

LFI contains a sample pad that accepts whole blood 

and a careful evaluation of the pad will be performed. 

Spike all available sample matrices with CPS and 

optimize detection. 4C4 AMD LFI components and 

buffers will be selected based on the ability to detect 

the lowest concentration of CPS in all sample 

matrices. This will be performed by spiking blood, 

serum, urine, sputum, pus with a concentration range of purified CPS. This work has been initiated at InBios 

with the 4C4 AMD LFI prototype. Shown in Fig. 5 CPS was spiked into either 35 l of control urine, whole blood 

and serum followed by addition of chase buffer. In each of these sample matrices CPS is detectable at 0.25 

ng/ml. 

Provide prototype tests to collaborators for further testing of all matrices and cultures using archived specimens.

Our collaborators in Thailand and Australia have access to a melioidosis patient population that is not available 

in the US. Therefore, Drs. Currie and Limmathurotsakul will evaluate the prototype 4C4 AMD LFI on de-identified 

melioidosis patient samples (see support letters). As summarized in Table 4, sample preparation guidelines for 

the 3C5 AMD LFI have been developed by the Currie laboratory. The goal is to determine whether these 

protocols are valid for the 4C4 AMD LFI and make adjustments to the procedures if necessary. Batches of 50 

LFIs will be shipped to each endemic location for evaluation. 

The same archived samples that were used to evaluate the 3C5 AMD LFI will be used to evaluate the 4C4 AMD 

LFI (as volume permit). This should provide valuable performance data of the 4C4 LFI compared to the 3C5 LFI 

(same samples and sample preparation). If performance of the 4C4 LFI is not optimal, adjustments to sample 

preparation or to the components of the LFI (Table 4) can be made at this stage. Following feedback from 

endemic sites additional batches of 50 LFIs will be shipped to each site. We anticipate that 250 LFIs per site will 

be needed to validate 4C4 LFI performance and sample preparation. 

Routine practice within endemic areas is to culture samples from suspected melioidosis patients in broth for 

seven days and plate samples from this broth on solid agar on day 2 and day 7. The agar plate is then incubated 

for another 24-72 hours at which time a latex agglutination test or immunofluorescence assay (IFA) can be 

performed from a bacterial colony. Our collaborators stationed Laos, Drs. Dance and Newton (Lao-Oxford 

University-Wellcome Trust-Research Unit), strongly believe the AMD LFI will be extremely effective at identifying 

B. pseudomallei at the very early stages of the culturing process. Therefore, they will evaluate the 4C4 LFI on 

culture broth (see letter of support). Experiments will include spiking blood culture bottles with a range of locally 

relevant pathogens and testing the 4C4 AMD LFI for reactivity over specific time points during the incubation 

period. Following validation of this method they will perform a similar study with archived samples collected from 

patients diagnosed with febrile illness of unknown origin. This group performed an identical study with a 

diagnostic test specific for Salmonella enterica serovar Typhi [45] and determined this was a rapid, inexpensive 

and accurate technique. Interestingly, the study also found that of 196 clinically significant Gram-negative rods 

that were identified following culture 34 (17.3%) were eventually identified as B. pseudomallei. 

Figure 5. Optimization of the 4C4 AMD LFI in a variety of sample 

matrices. Purified CPS was spiked into control urine, whole blood and 

serum at the indicated concentrations. The same CPS concentrations 

were used to spike each sample type. 

Research Strategy Page 84 
Seek pre-submission guidance from FDA for studies required for 510(k) submission. A major goal of the proposal 

is to submit a 510(k) by the end of Project Year 3. The FDA approval process can be quite daunting. Fortunately, 

InBios International has a well-established history of product development with four approved medical 

diagnostics. InBios will take the lead establishing the initial interaction with the FDA. The goal is to understand 

FDA requirements, regulations, and guidance documents and define the needs for a 510(k) submission. Please 

see “Commercialization Plan” for more details regarding interaction with the FDA. 

Limitations and alternative strategies. Ideally, the 4C4 AMD LFI performs optimally with the LFI components 

and the sample preparation protocols used for the 3C5 AMD LFI. However, we cannot be sure of this. To date 

the 4C4 AMD LFI is performing well when CPS is spiked into blood, serum and urine in the laboratory setting 

(Fig 5). The real test will be to evaluate how the LFI performs in the endemic setting with multiple sample 

matrices. During the Phase I period we were receiving real time feedback form the endemic locations on sample 

preparation and LFI performance. This will also occur during the Phase II period and we feel this is essential for 

timely optimization of the 4C4 AMD LFI. 

Timeline and benchmarks for success. We will finalize the LFI components and buffers by the middle of 

Project Year 2. Evaluation of the 4C4 AMD LFI at endemic sites will be completed by the end of Project Year 2. 

Success will be measured by i) finalizing optimized LFI components, ii) developing a validated set of sample 

preparation guidelines and iii) producing a 4C4 AMD LFI with improved performance over the 3C5 AMD LFI. 

Also, from the pre-submission we will have a clear understanding of the data needed to support a 510(k) 

submission by the end of Project Year 3 based on archival samples or the need for future prospective studies. 

Specific Aim 3: Produce multiple lots of the 4C4 AMD LFI under document and design control and GMP 

for large preclinical evaluation of performance characteristics with archived patient samples 

Rationale: In house validation of multiple GMP lots of LFIs will be performed. Studies will include analytical 

reactivity/specificity, reproducibility/precision, interference and stability analysis. These studies will be followed 

by evaluation of GMP LFIs at endemic sites. The list of collaborators requesting the AMD LFI is expanding (see 

Commercialization Plan). Multiple lots of optimized 4C4 AMD LFIs need to be produced and validated prior to 

shipment to collaborators in endemic sites. We anticipate requiring 5000 4C4 AMD LFIs produced in multiple 

lots to complete the large pre-clinical analysis during Project Year 3. These potential collaborators can 

substantially increase the knowledge of the presence of Melioidosis worldwide. 

Production of 4C4 AMD LFIs under GMP. InBios is a FDA registered and an ISO (International Organization for 

Standardization) certified company who has established a set of general manufacturing principles in compliance 

with Good Manufacturing Practice (GMP) ensuring that products are consistently produced and controlled 

Table 5. Optimization of components of a lateral flow immunoassay.

Sample pad The sample pad is used for buffering samples for optimal reaction with gold-labeled mAbs (conjugate) and the test antibody 

immobilized on membranes. The LFI for detection of CPS in a drop of blood will require a sample pad with filtering properties to 

remove erythrocytes. The pad will also incorporate an optimized buffer that will need to neutralize a variety of sample types.

Sample 

volume

Optimal samples volume to be determined; - typically 10-100 l sample volumes are used. A number of factors to be considered:

 pH and buffer capacity of samples will be carefully considered (e.g. if respiratory secretions and urine samples are found

to be viable matrices

 Depending on the concentration of analytes, a concentration step will be devised in place

Chase 

buffer

The chase buffer must i) buffer all samples; especially urine, which can cause false positive reactions due to pH variations and (ii) 

enhance product shelf life.

Conjugate 

pad

The conjugate pad must stabilize the gold-labeled mAb over its entire shelf life and must consistently release the conjugate from the 

pad to avoid variation in assay performance. 

mAb 

labeling

Colloidal gold is the label of choice. A number of other labels may be selected including latex particles. Labeling efficiency can vary 

depending on the diameter of the label and the method of labeling. Europium based detection systems will also be explored to 

achieve greater sensitivity, especially in situations where very low concentrations of CPS is present and may enable detection without 

concentration.

Membrane A variety of nitrocellulose membranes from a number of sources will be evaluated. Membranes will be screened based on their pore 

size, flow rate, and consistency. Also, nitrocellulose must be converted from a hydrophobic to hydrophilic membrane through 

treatment with surfactant formulations.

Test line 

mAb

Repeating epitopes on CPS allows use of the same mAb for the conjugate and the immobilized test line mAb. Using different mAbs 

for the conjugate and test line that bind to different epitopes may improve sensitivity.

Absorbent 

pad

The only function of the absorbent pad is to serve as a sink for the liquid that is processed through the LFI. The size and 

composition of the absorbent pad will be adjusted following a determination of the sample size needed to achieve sensitivity targets.

Research Strategy Page 85 
according to quality standards. InBios manufacturing processes are clearly defined, controlled and 

documented. All critical processes are validated to ensure consistency and compliance with design 

specifications. All operators are trained to complete and document procedures. Deviations are investigated and 

documented. Records of manufactured goods are retained in an accessible form, which enables the complete 

history of a batch to be traced. The entire InBios manufacturing staff is trained for GMP practices. The 

manufacturing team includes an engineer, wet chemistry application specialists and a team of assemblers. 

Analytical sensitivity – Limit of Detection (LOD): LOD is the concentration of CPS that yields a positive result 

50% of the time and a negative result 50% of the time. We will follow the LOD protocol that has been established 

by the Clinical and Laboratory Standards Institute (CLSI) entitled, User Protocol for Evaluation for Qualitative

Test Performance [44]. LOD will be determined on GMP produced LFIs. We will run a number of replicates to 

accurately determine the LOD for each sample type. LOD determination for CPS spiked into blood, serum and 

urine will be determined at InBios and UNR. We will rely on our collaborators in Australia and Thailand to spike 

patient samples, including pus, sputum, and other biological samples that are not archived at InBios and UNR. 

Deki Readers (Fio corporation) will also be implemented at this stage (see letter of support). The Deki Reader 

is a CE marked medical device that can read LFI test strips. The unit digitally captures LFI test and control line 

data and mobile Fionet software stores test results in a secure cloud network. Deki Readers will be used at 

InBios, UNR, Menzies and Mahidol to facilitate interpretation of the LFI data. Please see the commercialization 

plan for more information regarding the Deki Reader. 

Analytical reactivity and specificity testing. Preliminary testing on the 4C4 AMD LFI produced results identical to 

mAb 3C5. A panel of 40 B. pseudomallei isolates were acquired from the laboratory of Dr. Paul Keim (Northern 

Arizona University). The 4C4 AMD LFI was positive for 39/40 of these isolates. mAb 3C5 was reactive with 

38/40 of the isolates via antigen-capture ELISA. In addition, 15 clinically relevant bacteria (6 Gram-negative 

and 9 Gram-positive) bacteria were not reactive with the 4C4 AMD LFI. 

As described, the 3C5 AMD LFI was tested against a large panel of B. pseudomallei, B. mallei, and Burkholderia

near neighbor species at the CDC. The same panel of isolates will be acquired from DHS (see letter of support) 

in order to establish the analytical reactivity and specificity for the 4C4 AMD LFI and the alternative anti-CPS 

mAbs. The panel consists of 77 B. pseudomallei, 33 B. mallei, and 36 near neighbor strains. Additional isolates 

will include a panel of common community and hospital-acquired bacteria in addition to 7 bacterial biothreats. 

Our collaborators at Menzies, Mahidol and Laos will test the LFI against B. pseudomallei and other bacterial 

pathogens previously isolated from patients with severe sepsis. It is expected that there will be more than 400 

strains of B. pseudomallei and 300 strains of other bacteria isolated yearly. Therefore, they will test a large panel 

of these isolates during the process of evaluation and optimization of the 4C4 AMD LFI. 

Analytical reproducibility (test replicates at different agent concentrations, at different sites, etc.): Three sites will 

be used to test the reproducibility of each optimized LFI lot, Deki Readers will be used to quantify results. 

Reproducibility studies shall be performed according to the CLSI document, EP15-A2 (Clinical and Laboratory 

Standards Institute, 2005, User Verification of Performance for Precision and Trueness; Approved Guideline—

Second Edition (EP15-A2)). A panel of samples shall be prepared in relevant analyte-negative matrices 

containing CPS antigen at different concentrations. The CPS concentration selected shall ensure that the 

analyte concentration in the specimens represents assay specification. The test panel shall include high negative, 

low positive and moderate positive samples. Positive and negative control in a stable buffer system shall be 

provided along with the reproducibility panel package. Before LFIs will be used in pre-clinical evaluations 

concordant results will need to be obtained at each site. 

Data analysis. The Reproducibility (Precision) study uses a QC panel that is tested at various sites by multiple 

users. The precision is analyzed by following NCCLS guideline EP5-T2, “Evaluation of Precision Performance

of Clinical Chemistry Devices”. This will establish within-run, day-to-day, site-to-site and total standard deviations 

and coefficients of variation. 

Ruggedness: Ruggedness testing shall examine the behavior that results from small changes in environmental 

and/or operating conditions, such as temperature of samples or of reagents, accuracy of obtaining sample 

quantity, time to test once collected, sample throughput rate and time to result, concentration of reagents, age 

of reagents, mixing time (if required), temperature of analysis, flow rates, variations in measuring apparatus. We 

shall have a detailed protocol describing how different parameters shall be studied. 

Data analysis. Ruggedness testing will evaluate a QC panel and directly compare samples/tests that have 

undergone the ruggedness protocols and compare these to a control group. Paired student’s t-tests will be 

used to evaluate for statistical significance with the control group. 

Research Strategy Page 86 
Stability and shelf life: The typical shelf life for rapid tests prepared with the proposed LFI format is 24 months 

at room temperature. However, that shall depend on active components used in the assay. Therefore, the final 

assay design shall be evaluated using three different lots of test strips, according to the same SOP. Reagent 

shelf life is routinely measured by a combination of real-time and accelerated stability testing. 

Data analysis. The stability study shall evaluate a QC panel and directly compare tests that have undergone 

both accelerated and real-time aging to a control group and to expected values. Results shall be analyzed with 

a paired student’s t-test to evaluate for significance with the control group. 

Validate AMD LFI in field studies at two geographical sites using archived sera and other matrices to confirm 

diagnostic algorithm and compare with appropriate gold standard for melioidosis confirmation 

Clinical sensitivity and specificity. By the end of Project Year 2 an optimized 4C4 AMD LFI and validated sample 

preparation protocols will be available. A large lot of LFIs will be produced and distributed to endemic sites. 

However, a large number of archived samples will be available given the melioidosis rates that occur in Thailand 

and Australia every year. Our collaborators estimate that a minimum of 300 culture-confirmed melioidosis cases 

will occur in these regions per year. An average of three patient sample types are collected from each patient. 

Therefore, the total number of samples to be evaluated will be 900. 

Collaborators in Thailand (Mahidol) and Australia (MSHR) will compare all LFI results with culture results. In 

addition, LFI results will be compared with common diagnostic procedures that are used in each endemic region. 

The LFI will be used to test de-identified samples from patients with culture confirmed melioidosis and also 

patients presenting with sepsis. At Mahidol alone there are at least 1000 suspected cases of melioidosis per 

year of which 100 are confirmed by culture. Over the last 2.5 years MSHR has confirmed 254 melioidosis cases 

by culture. Therefore, a variety of sample types will be available from melioidosis confirmed and non-melioidosis 

patients presenting with sepsis to optimize the LFI. 

In addition to culture, the LFI will be compared to two rapid diagnostics used for melioidosis. MSHR will test the 

TTS1 real-time PCR assay [46]. Mahidol will compare LFI results with an immunofluorescence assay (IFA) that 

is routinely performed directly on clinical samples from suspected melioidosis patients. Taken together the LFI 

will be methodically compared to the current gold standard and two commonly used rapid techniques in endemic 

regions. 

Further studies will test ≥100 human whole blood samples (capillary, venous blood and plasma) obtained through 

commercial suppliers. InBios and the AuCoin laboratory maintain a large bank of such samples. These samples 

include more than 100 random blood donors, as well as samples from patients with unrelated Third World 

diseases and various febrile diseases such as tuberculosis, T. cruzi, leishmania, West Nile virus, Dengue virus, 

and Japanese encephalitis virus infections. Preliminary studies indicate that the 4C4 AMD LFI does not produce 

false positive reactions when tested against a panel twenty sera and urine samples. However, in case some 

samples do react in the AMD, further studies will be undertaken to characterize the response and optimize the 

assay to prevent it. All results shall be expressed in a specificity table summarizing analysis of normal samples 

and cross-reactivity; appropriate statistical methods will be used for data analysis. 

The % sensitivity and % specificity will be calculated based on the number of AMD-positive or negative samples 

that correlated with culture positive or negative data. Samples that are AMD-positive and culture-negative may 

occur in patients who are diagnosed with “clinical melioidosis”, have internal abscesses on the liver and spleen, 

and/or respond to antibiotics effective against B. pseudomallei. The diagnosis of “clinical melioidosis” is

described in detail in a published article by our collaborator Dr. Limmathurotsakul [47]. 

With collaborators from other regions where melioidosis may be prevalent it is anticipated that prevalence rates 

may be assessed and compared to known rates in Thailand and Northern Australia enabling a prediction of 

expected cases in these regions. 

Table 6. PRODUCT SPECIFICATION 

 Product Identification: Active Melioidosis DetectTM, device not yet classified by FDA

 Device: Active Melioidosis DetectTM (AMD; Lateral flow immunoassay (LFI) 

 Intended Use: In vitro diagnosis of active melioidosis for visual and rapid qualitative detection of the 

Burkholderia pseudomallei capsular polysaccharide (CPS) antigen 

 Analyte: Whole blood (venous and/or capillary blood), blood, urine, pus, and respiratory secretions 

 Antibodies to be used: Proprietary antibodies to capsular polysaccharide antigen CPS Burkholderia pseudomallei

 Detection agent: Selected antibody(s) to CPS conjugated with gold 

 Buffer system: Chase buffer that should not require cold storage 

 Instrument size/weight: No additional instrumentation required. 

Research Strategy Page 87 
Performance input: 

 Sensitivity Pus > 98% 

Sputum > 98% 

Urine > 95% 

Blood > 90% 

 Specificity: ≥95% 

 Analytical sensitivity: Limit of detection ≤ 1 ng/ml 

 Comparative Methods PCR, Immunofluorescence, Culture 

 Sample preparation time: None (depending on the matrix to be used)

 Reading time (time to result): 5 to 20 minutes (TBD)

 Sample throughput rate: Unlimited. Many samples can be run in parallel on separate strips.

 General assay characteristics: Clear test and control lines

Shelf life: 24 months at room temperature (≤30C)

Matrix comparison: Matrix comparison for serum and plasma, whole blood (venous and/or capillary blood), urine, pus, and respiratory 

secretions matrices will be compared. The purpose of this study is to determine which of the matrix or matrices will be most suitable for 

the current LFI format. For this purpose, we will spike purified CPS and/or CPS containing clinical samples into the above matrices at 

concentrations close to LOD of the AMD system and test. The matrix study will be performed in-house or by our collaborators before 

clinical studies can begin. The results will be reported in the FDA application. 

Limitations and alternative strategies. The GMP produced LFIs need to perform consistently as outlined in 

above. If major problems arise (high false positive rates, poor sensitivity, etc.) then InBios will make the proper 

modifications to the LFI and produce additional GMP lots for testing. We do not anticipate changes will need to 

be made to sample preparation guidelines, however, this can also occur during this larger LFI analysis. 

Timeline and benchmarks for success. Analytical reactivity and specificity testing is currently being performed 

and will be a major focus throughout the grant period. Large lots of 4C4 AMD LFIs will be ready for testing at 

the start of Project Year 3. The large preclinical analysis will occur throughout Project Year 3. Product 

specifications and performance targets are listed in Table 6. There is currently no approved rapid diagnostic for 

melioidosis. However, there are a number of non-validated rapid diagnostics that have been evaluated (latex 

agglutination, immunofluorescence, PCR, etc.). The 4C4 AMD LFI will need to be superior to any current rapid 

diagnostic for melioidosis while performing as well as culture.

Specific Aim 4: Determine the in vitro and in vivo expression and secretion profile of CPS 

Rationale. Development of a CPS detection immunoassay for the diagnosis of melioidosis will require a detailed 

understanding of the expression and secretion profile of CPS. To this end, the qCPS ELISA will be used to 

establish the concentration of CPS within (i) in vitro grown cultures, (ii) a variety of de-identified melioidosis 

patient samples, and (iii) within samples from animals challenged via the aerosol route with B. pseudomallei. 

The advantage of the animal studies is that the exact time of infection is known and the concentration of CPS 

within a variety of samples can be determined at defined time points post-exposure. 

Determine variation of CPS expression across the genetic diversity of B. pseudomallei. Burkholderia encode 

very complex and variable genomes that have developed from its ability to acquire sequences from other bacteria 

[1]. However, we have shown that expression of CPS is highly conserved among B. pseudomallei isolates. We 

will determine if there is variation in the expression and secretion profile of CPS over a diverse panel of isolates 

that includes representatives from each the major clades of B. pseudomallei. The isolates will be selected from 

the panel of B. pseudomallei supplied by DHS. Isolates will be grown in culture broth and a time-course of 

samples will be collected. Quantitative cultures will be performed on each sample and the production of 

CPS/bacterial cell and CPS/ml of supernatant fluid will be determined with the qCPS ELISA. Purified CPS will 

be used to create a standard curve in order to determine the concentration of CPS within the samples. 

Evaluate 4C4 AMD LFI with mouse and NHP aerosol model of melioidosis. The AuCoin laboratory has 

established a collaboration with Dr. Simon Funnell (see support letter) who is the Scientific Leader/Program 

Coordinator of Microbiology Services for Public Health England (PHE, Porton Down, United Kingdom). PHE has 

secured funding from NIAID for two projects in which BALB/c mice and rhesus macaques will be used to study 

the effects of aerosol challenge with B. pseudomallei. The PHE team is interested in determining if B.

pseudomallei CPS is detectable in samples taken during the BALB/c and macaque studies of infection. A pilot 

study was recently performed with the 3C5 AMD LFI. Initially, LFI performance was confirmed with macaque 

urine (naïve and spiked with CPS), followed by testing urine collected from a macaque on day 4 post B.

pseudomallei infection (Fig. 5). The macaque sample was positive by LFI indicating accumulation of CPS and/or 

Research Strategy Page 88 
B. pseudomallei in urine. Based on the pilot study PHE has requested the use of the 4C4 AMD LFI and the 

qCPS ELISA. 

The BALB/c study involves challenge with the K92643 strain 

of B. pseudomallei. The rhesus macaques will be challenged 

with a different laboratory strain of B. pseudomallei (NCTC 

13392). For each of these studies, urine, blood and oral 

swabs (macaques) will be collected prior to and following 

infection at defined time points. Although we would expect 

CPS concentrations to be correlated to CFU/ml of B.

pseudomallei in the animal samples, it may be possible to detect CPS in the absence of bacteremia. The AuCoin 

laboratory will provide the reagents to run the qCPS ELISA to enable PHE to determine the concentration of 

CPS in the collected samples. In addition, the 4C4 AMD LFI will also be tested on as many samples as possible 

(as volumes permit). 

Quantify CPS in human samples with the qCPS ELISA. Filtered urine and serum samples from Thailand were 

recently shipped to the AuCoin laboratory and analyzed with the qCPS ELISA (Table 3). The data was quite 

encouraging with six out of ten urine samples containing readily detectable levels of CPS. Serum as expected, 

contained less CPS however; in five out of 10 samples CPS was detectable. The AuCoin laboratory will continue 

to perform a qCPS ELISA on samples tested by LFI in endemic areas. Small aliquots of de-identified patient 

samples will be shipped to the AuCoin laboratory and the qCPS ELISA will be performed in a BSL-3 laboratory. 

The samples will not be filtered, unlike the samples used to generate the data in Table 3. Culture (CFU/ml) will 

have been previously performed; this data will be compared with the concentration of CPS in each sample. We 

anticipate detection of higher concentrations of CPS in patient samples when whole bacterial cells are not 

removed by filtration. Also, since we have shown that CPS is a shed/secreted antigen, there is a good chance 

that although a blood culture is negative it still may contain CPS that is shed from a distant site, e.g. an abscesses, 

which is a hallmark characteristic of melioidosis. Therefore, blood and urine samples that are culture negative 

from melioidosis positive patients will be evaluated for the presence of CPS.

Limitations and alternative strategies. The qCPS ELISA will be performed on as many de-identified patient 

samples as possible. It may be the case that not every patient sample tested by LFI will be tested with the qCPS 

ELISA due to volume limitations. However, we are confident that enough samples will be tested by ELISA to 

understand the accumulation of CPS in a variety of patient samples. Another potential problem is that the 

concentration of CPS in blood samples may be too low to routinely detect with the prototype qCPS ELISA. This 

potential problem may be addressed with use of mAb 4C4 instead of mAb 3C5 as part of the qCPS ELISA. 

Additionally, our laboratory has recently estimated the concentration of antigens in patient samples by SPR. This 

will involve linking mAb 4C4 to a SPR sensor chip followed by flowing patient samples over the chip. The 

advantages of this method are that low volumes (~10 l) of samples are analyzed and detection limits are far 

below those seen with ELISA. Another potential problem is whether the qCPS ELISA will perform consistently 

performed in Funnell laboratory in the UK. We will also ship the appropriate control CPS samples to ensure that 

results are consistent prior to testing the animal samples. We have previously shipped the qCPS ELISA to the 

laboratory of Dr. Gregory Bancroft (London School of Hygiene and Tropical Medicine, UK) where the assay was 

used to successfully identify CPS in tissue from mice infected with B. pseudomallei. 

Timeline and benchmarks for success. We will perform the in vitro CPS expression and secretion studies 

and the animal model studies during Project Year 1 & 2. Quantification of CPS in patient samples will occur 

during the Project Years 1-3. Success will be measured by having a clear understanding of the expression and 

secretion profile of B. pseudomallei CPS across its genetic diversity and during infection. 

Figure 5. Detection of CPS in macaque urine. Urine sample 

was collected at day 4 post B. pseudomallei infection. Sample 

was diluted 1:1 in chase buffer and tested on the 3C5 AMD LFI.

Research Strategy Page 89 
Protection of Human Subjects 

a. Human Subjects Involvement and Characteristics: The diagnostic assay developed in the course

of this project will be applicable to both genders, all age groups, and all ethnicities and races. The studies 

described in this proposal will utilize only archived and commercial human samples without personal 

identifiers from patients with symptoms of particular disease. Subjects will not be selected based on age, 

gender, or ethnicity. The targeted racial and ethnic mix composition will be reflective of demographic 

regions of sample origin. 

Fetuses, neonates, pregnant women, prisoners or institutionalized individuals will not be used at 

any time in this project. 

The proposed research is in compliance with DHHS and NIH regulations on protection of human subjects 

(HHS regulations at 45 CFR 46.102(f), and NIH regulations on inclusion of women, minorities, and children. 

This research project will use small quantities (less than 1 ml) of human plasma/serum, urine or respiratory 

secretions obtained through collaborations and via commercial sources. These will be archived samples 

with no personal identifiers collected as part of routine clinical procedures and not specifically for these 

studies. Since these are archival samples we are requesting Exemption 4. Testing of the LFI will be done 

with a variety of human samples, however i) no specimens will be collected specifically for this study and 

ii) there are no subject identifiers.

b. Source of Materials:

University of Nevada. Serum and urine samples from controls are maintained within our laboratory at 

UNR. The consenting process included informing subjects that the samples will be used for a wide variety 

of diagnostic studies. The samples have been de-identified. Specifically, i) studies will be limited to existing 

archived samples, and ii) the samples will be de-identified in such a manner that subjects cannot be 

identified, directly or through identifiers linked to the subjects. These samples will be used as negative 

controls for the antigen-capture ELISA for detection of CPS. 

InBios. InBios maintains a bank of specimens from normal subjects and patients with a variety of infectious 

diseases. Consent for use of these samples for a variety of diagnostic studies has been obtained. These 

samples contain no subject identifiers. These samples will be used as negative controls and will also be 

spiked with purified CPS to determine the LOD of the LFI. No melioidosis samples will be tested at the 

InBios facility. 

Menzies School of Health Research. All samples are coded and tested delinked to patient name. Written 

informed consent is obtained for sample collection from the patients and the collection has been approved 

by the Human Research Ethics Committee (IRB) of the NT Dept of Health and the Menzies School of 

Health Research (HREC 04/09). The samples are not obtained for a specific study and are used for a 

variety of diagnostic studies. The samples will be used to test optimized LFIs and determine proper sample 

preparation. 

Mahidol University. The melioidosis laboratory at Sappasithiprasong Hospital received a number of 

specimens from patients with suspected melioidosis for rapid diagnostic tests, including 

immunofluorescent assays, and confirmatory tests, such as latex agglutination tests. Therefore, they are 

not collected for a specific study. All samples used in this proposal will be previously coded and tested 

delinked to patient names and identifiers. 

c. Potential Risks: No potential risks to patients are expected as we will use only archived and

commercial human samples. Normal universal precautions for handling samples will be adhered to. 

d. Potential benefits of the proposed research to the subjects and others and importance

of the knowledge to be gained: 

Protection of Human Subjects Page 90 
The archived and commercial samples obtained for this project will be used to assist the design 

of a novel diagnostic directed to expedite the early diagnosis of Melioidosis. These samples will 

be used to evaluate performance of assay(s), which will help to select successful assay 

prototypes for further studies directed to assembly of the full diagnostic panel. The 

development of proposed diagnostic assay(s) can assist in developing an improved diagnostic 

algorithm for active melioidosis. 

InBios is already approved as a site of medical research using human subjects by the NIH and 

has both IRB and FWA registration (FWA00001139). 

Protection of Human Subjects Page 91 
INCLUSION OF WOMEN and MINORITIES 

INCLUSION of WOMEN 

1. A description of the subject selection criteria and rationale for selection in terms of the

scientific objectives and proposed study design. In this study we will use archived samples 

only. Melioidosis affects both men and women; therefore, the study samples will not be gender or 

age specific. Samples included in this study will be from male and female individuals having 

symptoms of melioidosis, as well as subjects with other febrile illnesses, and normal individuals. 

2. A compelling rationale for proposed exclusion of any sex/gender or racial/ethnic group.

There will be no selection/exclusion based upon racial/ethnic group. 

The proposed dates of enrollment (beginning and end). Archived samples will be used 

shortly after the grant is funded and continue throughout the grant period. 

3. A description of proposed outreach programs for recruiting women and minorities in

clinical research as subjects. An outreach program specifically for recruiting women and 

minorities will not be done as this study will not conduct recruitment. Samples will be archived 

and include women and minorities. Most probably the positive samples will represent 

racial/sexual mix representative of the total population in the specified endemic areas. 

INCLUSION OF MINORITIES 

1. A description of the subject selection criteria and rationale for selection in terms of the

scientific objectives and proposed study design. Melioidosis is not a disease that affects a 

particular racial or ethnic background. Therefore, subjects used in this study will include 

individuals from all racial groups. Clinics providing samples typically serve an ethnically diverse 

population and will encompass samples from individuals with African-American, Asian, Hispanic 

and whites that present at that particular geographic area. 

2. A compelling rationale for proposed exclusion of any racial/ethnic group. There will be

no selection/exclusion based on racial/ethnic group. 

3. Proposed dates of enrollment (beginning and end). The acquisition of archived samples

will begin shortly after the grant is approved for funding and will continue throughout the grant 

period. 

4. A description of outreach programs for recruiting minorities in clinical research as

subjects. An outreach program specifically for recruiting minorities will not be done as this study 

will not conduct recruitment. 

Women &Minorities Page 92 
INCLUSION OF CHILDREN 

If children will be included, please describe your plans for including children. We will use archived 

samples only. Archived samples will include children. Special recruitment of children, however, will not 

take place. 

If children will be excluded from the Research, please provide an acceptable justification for the 

exclusion. Children are not excluded from this study. 

Children Page 93 
Select Agents Research 

InBios International 

No work with Select Agents will be done at InBios. 

University of Nevada, Reno 

Select agent to be used. The select agent to be used is Burkholderia pseudomallei and Burkholderia

mallei. The Department of Homeland Security (DHS) will ship large B. pseudomallei and B. mallei strain 

panel to the BSL3 facility at UNR. Also, if needed the Currie laboratory will supply additional B.

pseudomallei strains in order to test the analytical reactivity and specificity of the CPS specific mAbs. 

Provide the registration status of all entities where Select Agent will be used. Select Agent 

Registration at the University of Nevada, Reno is done under the auspices of the Department of 

Environmental Health and Safety at the University of Nevada, Reno (UNR). The Select Agent Registration 

Number for the UNR campus is C20110303-1196, effective until March 3, 2014. 

Provide a description of all facilities where the Select Agent will be used. Work for the proposed 

study will be done in a newly constructed and CDC certified BSL-3 laboratory located within the Center for 

Molecular Medicine (CMM), a new $78 M biomedical research facility on the medical school campus. The 

BSL-3 laboratory is located adjacent to the AuCoin laboratory in the CMM. The BSL-3 laboratory has been 

designed and constructed from its inception for use of Select Agents. The BSL-3 was designed through 

consultation with Ted Traum of World BioHazTec. Mr. Traum is a world authority on design and 

commissioning of BSL-3 laboratories and is one of the co-authors of the NIH document for Biosafety Level

3 Laboratory Certification Requirements. Under Mr. Traum’s supervision, a team from World BioHazTec 

has conducted a certification of the BSL-3 laboratory and confirmed it meets NIH and BMBL facility 

performance requirements. 

Procedures that will be used to monitor possession, use and transfer of Select Agent. Records of 

all B. pseudomallei and all Select Agent possession will be maintained in the form of an inventory log. A 

Select Agent suite (314G, see attached BSL-3 map) has been designated within the BSL-3 and contains 

a locked Select Agent freezer. Bacterial strains will be stored upon arrival in the Select Agent freezer 

within suite 314G. Access to suite 314G and the freezer within is allowed only to select agent approved 

laboratory employees. Information to be noted on the Select Agent freezer inventory log includes: user 

name, agent, date and time removed from freezer, date and time returned to freezer, purpose of use, and 

date and method of disposal. The inventory list is kept locked in the BSL-3 laboratory, accessible only to 

approved microbiologists and administrative personnel. A monthly inventory is made of all Select Agents 

in storage to determine if documentation of amounts in storage are correct. All materials to be discarded 

will be autoclaved at the end of every work session and the sterilization verified. Other reusable equipment 

will be decontaminated at the end of every work session. Destruction/disposal of the Select Agents is 

documented on a log sheet.

Plans for appropriate biosafety, biocontainment, and security of the Select Agent. 

Biosafety and biocontainment considerations. All work with viable B. pseudomallei will take place in the 

CMM BSL-3. Any open cultures will handled within a biosafety cabinet within the BSL-3 suite 314G. 

Biosafety Class II cabinets are used, with HEPA filtration of single pass hood air to the outside, in addition 

negative pressure ventilation of the BSL-3 area is functional. Every reasonable precaution is taken to 

ensure that these organisms are handled safely in an environment designed to minimize the risk to 

laboratory personnel. All personnel working with open cultures of B. pseudomallei will be required to where 

appropriate PPE including a powered air-purifying respirator (PAPR). 

Security measures. Eight video surveillance cameras are mounted and in use to monitor the BSL-3 area. 

Select Agent Research Page 94 
These are monitored at a central campus location, and can be accessed for investigation if tampering with, 

or removal of agents is suspected or detected. There is no outside facing windows and the BSL3 is located 

on the third floor of the CMM. Card-key access along with a pass-code is needed to enter the BSL-3, all 

entries and exits are logged in real-time. There are four suites located within the BSL-3, suite 314G has 

been designated a Select Agent suite. All of the Select Agents are stored in a -80°C freezer within suite 

314G. Card key access to suite 314G is given to Select Agent approved employees only (as defined in 

the Public Health Security and Bioterrorism Preparedness and Response Act of 2002 [42 CFR]). The select 

agent freezer within 314G is secured with a combination lock, thus limiting access to only those persons 

previously designated as approved persons. No visitors are permitted into the laboratory unless 

accompanied by a Select Agent approved laboratory employee. All routine maintenance and janitorial 

services are carried out during customary working hours of the laboratory. No casual traffic is allowed in 

the BSL-3 laboratory. By laboratory policy, only approved employees are allowed entrance into that area; 

no employee may use his/her card-key to give access to an unapproved employee, family member, or any 

other non-approved persons. “Piggy-backing,” the practice of two or more employees (even if approved

for access to the BSL3 area) entering upon use of a single card-key, is not permitted. Each person must 

individually use his or her card to enter the area. Video surveillance of the area is in place to detect any 

violations of these policies. 

Menzies School of Health Research - Royal Darwin Hospital (Menzies) 

The select agent to be used is Burkholderia pseudomallei. Menzies Burkholderia repository has over 3000 

isolates for use in various projects. 

Registration at the Menzies School of Health Research, Darwin, Australia is through the Biosafety 

Committee and Laboratory Safety Committee (AQIS registration/Australian Standard AS2243, Safety in 

Laboratories). BSL3 laboratory registration is by The Office of the Gene Technology Regulator (OGTR) a 

part of the Australian Commonwealth Department of Health and Ageing. Menzies has select agent 

approval in the United States of America (USA) from the National Institute of Allergy and Infectious 

Diseases/NIH to work in collaboration with the select agent. 

Menzies School of Health Research is an independent research organization located on the grounds of 

the Royal Darwin Hospital. Menzies operates three BSL3/PC3 suites located within a BSL2/PC2 

laboratory. Select agent work is subject to approval from the Biosafety Committee and Laboratory Safety 

Committee (AQIS registration/Australian Standard AS2243, Safety in Laboratories) Menzies conforms with 

IATA Dangerous Goods Regulations and they are subject to rulings of the Office of the Gene Technology 

Regulator.

B. pseudomallei and all Select Agent possession will be monitored in the form of inventory log sheet 

documentation. The Select Agent will only be worked on within the BSL3/PC3 suite rooms. Bacterial 

strains will be stored in the locked Select Agent freezer. Access to the BSL3 laboratory and freezer is 

allowed only to select agent approved laboratory employees. Information to be recorded on the Select 

Agent freezer inventory log includes: user name, agent, date and time removed from freezer, date and 

time returned to freezer, purpose of use, and date and method of disposal. The inventory list is kept locked 

in the BSL-3 laboratory, accessible only to approved microbiologists. A quarterly inventory is made of all 

Select Agents in storage to determine if documentation of amounts in storage are correct. All materials to 

be discarded will be autoclaved at the end of every work session and the sterilization verified. Other 

reusable equipment will be decontaminated at the end of every work session. Destruction/disposal of the 

Select Agents is documented on a log sheet.

Work with viable B. pseudomallei will take place in the BSL-3/PC3 suites. Any open cultures will be handled 

within a biosafety cabinet within the BSL3/PC3 suites. Biosafety Class II cabinets are used, with HEPA 

filtration of single pass hood air to the outside; in addition the BSL3 is under negative pressure. Every 

reasonable precaution is taken to ensure that these organisms are handled safely in an environment 

designed to minimize the risk to laboratory personnel. All personnel working with open cultures of B.

Select Agent Research Page 95 
pseudomallei will be required to where appropriate PPE including a powered air-purifying respirator 

(PAPR). 

Mahidol-Oxford Tropical Medicine Research Unit, Mahidol University, Bangkok Thailand 

Dr. Limmathurotsakul and collogues have full access to a new 5,400 sq. ft. microbiology laboratory which 

includes a 1216 sq. ft. BSL3 laboratory. The laboratory is located on the second floor of the building. The 

BSL3 laboratory can accommodate up to eight people at the same time, and is equipped with eight -80C 

freezers, one -20C freezer and six Class II biosafety cabinets. Two biosafety cabinets are dedicated to B.

pseudomallei work. One -80 freezer is dedicated to select agents including B. pseudomallei. This freezer 

is locked and access is given to authorized personnel only. Freezerworks, the sample tracking system 

complying with international conference on harmonization-good clinical practice (ICH-GCP), is used for 

storage and tracking all specimens. We will perform all bacterial culture within the BSL3 laboratory, under 

an established standard of procedures, and with the approvals from the Ministry of Public Health, Thailand. 

Mahidol-Oxford Tropical Medicine Research Unit is also enrolled in a Select Agent Program developed by 

CDC/NIH. 

Sappasithiprasong Hospital, Ubon Ratchathani, northeast of Thailand 

Dr. Limmathurotsakul and collogues have an office within Sappasithiprasong Hospital, Ubon Ratchathani. 

The hospital laboratory is very familiar with B. pseudomallei as it is one of the most common bacteria 

isolated from clinical specimens, and safety measures of this laboratory are at the highest standard 

required by the Ministry of Public Health, Thailand. B. pseudomallei, which is isolated from clinical 

specimens from patients, will be transferred to the BSL3 laboratory of Mahidol-Oxford Tropical Medicine 

Research Unit under appropriate containment using a high level biosafety standards. Freezerworks, the 

sample tracking system, has been set up at Sappasithiprasong Hospital and will be used to track the 

records of specimen transportation. 

Select Agent Research Page 96 
 

Multiple PD/PI Leadership Plan: 

The rationale for choosing the multiple PD/PI approach relates to the unique abilities of the CoPIs. is well-established investigator who has extensive experience immunoassay 

development. is the UNR Hybridoma Laboratory Director who has produced many 

mAbs specific to protein and polysaccharide antigens. This collaboration works well since InBios 

does not have a hybridoma laboratory and UNR lacks the skills needed to bring an immunoassay 

to market. Both investigators will contribute to all aspects of the project; however, each 

investigator has a distinct role in making the final decisions on scientific direction. The 

organizational structure of the leadership team is as follows: 

 is the Contact PD/PI for the project and is responsible for communication with NIH, 

assembling application materials, and coordinating progress reports. He will direct the studies of 

LFI optimization and production and evaluation studies for FDA clearance. 

 is a PD/PI for the project and will oversee production of the 4C4 mAb (or others as 

needed). He will also direct the evaluation of and Quality control of monoclonal antibodies by 

Biacore and studies involving quantification of CPS in patient or NHP samples by antigen-capture 

immunoassay. He will also be responsible for scale up and purification of CPS. In addition, he will 

guide experiments performed in the BSL3 laboratory relating to testing prototype LFI against a 

large panel of B. pseudomallei and B. mallei isolates. Finally, Dr. AuCoin will assist Dr. 

in assembling manuscripts and progress reports and in data analysis. Both PIs will interact with 

Fio Corp with regards evaluating LFIs with the Deki reader. 

To enhance communication among the team the InBios and the AuCoin laboratory will arrange a 

conference call every two weeks to discuss progress and future directions. Also, monthly 

conference calls or other forms of communication (Skype) with collaborators will take place to 

discuss LFI evaluation. Also, will travel to InBios twice per year for one-day meetings 

to review data and plan future experiments. The meetings will also allow time to resolve any 

conflicts that have arisen between researchers. Conflicts that are particularly difficult to resolve 

will be decided by consensus of the PD/PI’s and collaborators.

The PD/PI’s will both have access to project funds through separate accounts. A subaward will 

be issued from InBios to the AuCoin laboratory. will make the budget decisions for 

InBios while will make the budget decisions for UNR. 

Multiple PI Leadership Plan Page 98 
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18. Limmathurotsakul D, Peacock SJ (2011) Melioidosis: a clinical overview. British medical bulletin 99:

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19. Richardson LJ, Kaestli M, Mayo M, Bowers JR, Tuanyok A, et al. (2012) Towards a rapid molecular

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26. Wuthiekanun V, Desakorn V, Wongsuvan G, Amornchai P, Cheng AC, et al. (2005) Rapid

immunofluorescence microscopy for diagnosis of melioidosis. Clin Diagn Lab Immunol 12: 555-

556.

27. Chantratita N, Tandhavanant S, Wongsuvan G, Wuthiekanun V, Teerawatanasuk N, et al. (2013)

Rapid Detection of Burkholderia pseudomallei in Blood Cultures Using a Monoclonal AntibodyBased Immunofluorescent Assay. Am J Trop Med Hyg. 

28. Ekpo P, Rungpanich U, Pongsunk S, Naigowit P, Petkanchanapong V (2007) Use of protein-specific

monoclonal antibody-based latex agglutination for rapid diagnosis of Burkholderia pseudomallei 

infection in patients with community-acquired septicemia. Clin Vaccine Immunol 14: 811-812. 

29. Wuthiekanun V, Anuntagool N, White NJ, Sirisinha S (2002) Short report: a rapid method for the

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66: 759-761. 

30. Samosornsuk N, Lulitanond A, Saenla N, Anuntagool N, Wongratanacheewin S, et al. (1999) Short

report: evaluation of a monoclonal antibody-based latex agglutination test for rapid diagnosis of 

septicemic melioidosis. Am J Trop Med Hyg 61: 735-737. 

31. Anuntagool N, Naigowit P, Petkanchanapong V, Aramsri P, Panichakul T, et al. (2000) Monoclonal

antibody-based rapid identification of Burkholderia pseudomallei in blood culture fluid from 

patients with community-acquired septicaemia. J Med Microbiol 49: 1075-1078. 

32. Nuti DE, Crump RB, Dwi Handayani F, Chantratita N, Peacock SJ, et al. (2011) Identification of

circulating bacterial antigens by in vivo microbial antigen discovery. mBio 2. 

33. AuCoin DP (2012) In vivo microbial antigen discovery: finding the 'needle in the haystack'. Expert

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34. Cooper LJ, Schimenti JC, Glass DD, Greenspan NS (1991) H chain C domains influence the

strength of binding of IgG for streptococcal group A carbohydrate. J Immunol 146: 2659-2663. 

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36. Cooper LJ, Shikhman AR, Glass DD, Kangisser D, Cunningham MW, et al. (1993) Role of heavy

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37. Hovenden M, Hubbard MA, Aucoin DP, Thorkildson P, Reed DE, et al. (2013) IgG subclass and

heavy chain domains contribute to binding and protection by mAbs to the poly gamma-Dglutamic acid capsular antigen of Bacillus anthracis. PLoS Pathog 9: e1003306. 

38. Heiss C, Burtnick MN, Wang Z, Azadi P, Brett PJ (2012) Structural analysis of capsular

polysaccharides expressed by Burkholderia mallei and Burkholderia pseudomallei. 

Carbohydrate research 349: 90-94.

39. Price EP, Sarovich DS, Mayo M, Tuanyok A, Drees KP, et al. (2013) Within-host evolution of

Burkholderia pseudomallei over a twelve-year chronic carriage infection. MBio 4. 

40. Sim BM, Chantratita N, Ooi WF, Nandi T, Tewhey R, et al. (2010) Genomic acquisition of a capsular

polysaccharide virulence cluster by non-pathogenic Burkholderia isolates. Genome Biol 11: R89. 

41. Glass MB, Gee JE, Steigerwalt AG, Cavuoti D, Barton T, et al. (2006) Pneumonia and septicemia

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42. Novak RT, Glass MB, Gee JE, Gal D, Mayo MJ, et al. (2006) Development and evaluation of a realtime PCR assay targeting the type III secretion system of Burkholderia pseudomallei. J Clin 

Microbiol 44: 85-90. 

43. Brett PJ, Burtnick MN, Heiss C, Azadi P, DeShazer D, et al. (2011) Burkholderia thailandensis oacA

mutants facilitate the expression of Burkholderia mallei-like O polysaccharides. Infection and 

immunity 79: 961-969. 

44. CLSI (2008) User protocol for evaluation of qualitative test performance; approved guideline-second

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45. Castonguay-Vanier J, Davong V, Bouthasavong L, Sengdetka D, Simmalavong M, et al. (2013)

Evaluation of a simple blood culture amplification and antigen detection method for diagnosis of 

Salmonella enterica serovar typhi bacteremia. J Clin Microbiol 51: 142-148.

46. Kaestli M, Richardson LJ, Colman RE, Tuanyok A, Price EP, et al. (2012) Comparison of TaqMan

PCR Assays for Detection of the Melioidosis Agent Burkholderia pseudomallei in Clinical 

Specimens. Journal of clinical microbiology. 

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Consortium/Contractual Page 102 

Re: STIR Certification 

Project Title: Antigen Dectection Assay for the Diagnosis of Melioidosis 

UNR PI(s): Dr. David AuCoin 

UNR SP No.: 1400380 

Dear Ms. Fisher, 

The University of Nevada, Reno (UNR) will be pleased to participate in the proposed STIR project entitled 

"Antigen Dectection Assay for the Diagnosis of Melioidosis" that InBios International (Company), a small 

business concern, and University of Nevada, Reno (UNR), a research institution, have jointly prepared. 

The small business concern and the research institution certify jointly that: (1) the proposed STIR project will be 

conducted jointly by the small business concern and the research institution in which not less than 40 percent of 

the work will be performed by the small business concern and not less than 30 percent of the work will be 

performed by the research institution ("cooperative research and development"); (2) the proposed STIR project is 

a cooperative research or research and development effort to be conducted jointly by the small business concern 

and the research institution in which not less than 40 percent of the work will be performed by the small business 

concern and not less than 30 percent of the work will be performed by the research institution ("performance of 

research and analytical work"); and (3) regardless of the proportion of the proposed project to be performed by 

each party, the small business concern will be the primary party that will exercise management direction and 

control of the performance of the project 

Furthermore, UNR hereby provides its standard intellectual property agreement terms in the event your company 

receives an award under the Prime Sponsor's SB IR/STIR program: 

Patents and Inventions. The Company shall own all right, title and interest in all inventions and improvements 

conceived or reduced to practice by the Company in the performance of the research which is the subject of this 

proposal. The University shall own all right, title and interest in all inventions and improvements conceived or 

reduced to practice by University or University personnel in the performance of the Research (hereinafter 

collectively "Invention") and may, at its election, file all patent applications relating thereto. In consideration of 

Company's support of University in performance of the Research, University grants to Company an option for an 

exclusive license. The rights to said option shall expire three months after University has provided written notice 

to Company of any such invention, improvement, application or patent ("Option Period"). Upon execution of the 

option in writing, the parties will meet within thirty (30) days to begin negotiating the terms of the license. The 

parties agree to negotiate in good faith and the terms of the license will be reasonable in relation to licenses in the 

field and industry. In the event a license is not executed within three (3) months from the exercise of the option, 

Sponsored Projects 

Ross Hall, Room 204 

University of Nevada, Reno/325 

Reno, Nevada 89557-0325 

www.unr.edu/ospa 
Consortium/Contractual Page 103 

Board of Regents, Nevada System of Higher Education, on behalf of the University of Nevada, Reno 

Sponsored Projects/Mailstop 325 

University of Nevada, Reno 

Reno, NV 89557-0240 

For questions of a technical or program nature, please contact the project director. For contractual or business 

questions, please contact this office and refer to the above SP number. 

We look forward to a successful collaboration with your company on this project. 

Sincerely, 

Manager, Pre-Award, Sponsored Projects 
November 4, 2013 

Dear David, 

I am drafting this letter in full support for your STTR Phase II proposal “Antigen Detection Assay for the 

Diagnosis of Melioidosis”. A rapid, inexpensive, point-of-care immunoassay for the detection of B.

pseudomallei antigen is greatly needed in endemic areas in order to properly treat melioidosis. 

Your collaboration with InBios International has produced a prototype AMD LFI for the detection of CPS. 

My research group has been testing small batches of the prototype LFIs on de-identified patients 

samples. We are GREATLY encouraged by the results to date. As you know we have done an 

extensive analysis on patient sample preparation. We feel we have developed a validated set of 

protocols for the 3C5 AMD LFI. We would like to now test the sample preparation protocols on the 4C4 

AMD LFI. 

The LFI is adaptable to test many patient samples (blood, urine, pus, respiratory secretions) directly and 

also has the ability to test cultured patient samples. This is essential for the diagnosis of melioidosis. 

There is no doubt this simple test should be included in the melioidosis diagnostic algorithm. Therefore, I 

look forward evaluating optimized LFIs and to continue our established collaboration. 

Good luck with the proposal, 

PDF processed with CutePDF evaluation edition www.CutePDF.com

Letters of Support Page 104 
 

. November 4, 2013 

Dear David, 

This letter is to confirm my role in the development of the Active Melioidosis Detect lateral 

flow immunoassay (LFI) for the rapid diagnosis of melioidosis. We fully support the 

submission of the STTR Phase II grant “Antigen Detection Assay for the Diagnosis of 

Melioidosis” that will be submitted to NIAID. The Phase II you are drafting along with InBios 

will hopefully lead to continued support for this encouraging project. As you know we have 

tested a small number of prototype LFIs for the detection of B. pseudomallei CPS. We are 

excited with the results we have seen so far and look forward to testing optimized LFIs in 

the future. 

As we discussed we are preparing for a large pre-clinical analysis of melioidosis patient 

samples and we would like to request as many as 2000 4C4 AMD LFIs. 

My laboratory will continue to test de-identified samples and optimize sample preparation. 

We test a number of samples in order to diagnose melioidosis (blood, urine, pus, and 

respiratory secretions). Most often we need to rely on the “gold standard” of culturing for 

diagnosis, unfortunately this can take 3-7 days and the sensitivity of the culture is only about 

60%. Therefore, we anticipate that the melioidosis LFI will fill a much-needed gap.

In addition, I a personally excited about the use of the lateral flow immunoassay to explore 

the prevalence of melioidosis across the globe. I have no doubt this assay will be useful all 

over Southeast Asia, India, and Africa. 

Kind Regards, 

Principal Investigator/Program Director (Last, first, middle): 

Letters of Support Page 105 
 

Lao-Oxford University-Mahosot Hospital-Wellcome Trust-Research Unit, 

Microbiology, Mahosot Hospital,Vientiane, Lao PDR. Tel/Fax:

12!November!2013

To!whom!it!may!concern

We!would!like!to!express!our!support!for!the!Phase!II!study!being!planned!by!Dr!

David!AuCoin!and!InBios!to!develop!and!evaluate!their!lateral!flow!tests!for!the!

detection!of!Burkholderia,pseudomallei.!!We!are!a!research!group!based!in

Microbiology!Laboratory!of!Mahosot!Hospital!in!the!Lao!People’s!Democratic

Republic,!where!melioidosis!is!emerging!as!an!important!public!health!issue.!!In

the!past!10!years!we!have!seen!more!than!670!patients!with!cultureOpositive

melioidosis!in!our!hospital!in!Vientiane,!but!it!is!likely!that!this!is!just!the!tip!of!

the!iceberg!as!there!are!very!few!other!laboratories!across!the!country!that!are!

capable!of!making!the!diagnosis.!!This!is!important,!as!melioidosis!does!not

respond!to!the!antibiotics!used!routinely!for!the!empirical!treatment!of!sepsis

and!pneumonia!in!Laos!and!so,!by!analogy!with!northeast!Thailand,!many

hundreds!of!patients!are!likely!to!be!dying!without!a!diagnosis!or!appropriate

specific!treatment.!!New!diagnostic!tools!are!urgently!needed.

One!of!the!research!interests!of!our!group!is!the!evaluation!of!new!diagnostic

approaches!appropriate!to!a!developing!country!such!as!Laos,!where!there!are!

currently!few!clinical!microbiology!laboratories.!!We!are!especially!interested!in

simple!techniques!that!do!not!need!highly!trained!and!skilled!technical!staff,!such

as!lateral!flow!tests.!!We!have!had!success!using!commercially!available!rapid

diagnostic!tests!for!typhoid!on!blood!culture!fluids (CastonguayOVanier!et,al.

Evaluation!of!a!simple!blood!culture!amplification!and!antigen

detection!method!for!diagnosis!of!Salmonella,enterica!serovar!Typhi

bacteremia.!J!Clin!Microbiol!2013;!51:!142O8)!and!are!keen!to!evaluate!similar

approaches!for!other!bacteria!of!public!health!importance!in!this!region,!such!as

B.,pseudomallei.!!We!have!agreed!to!collaborate!with!Dr!AuCoin!and!his

colleagues!in!an evaluation!of!the!use!of!their!lateral!flow!test!in!a!realOlife!

diagnostic!setting.!!Initially!we!plan!to!validate!of!the!method!with!simulated

blood!culture!bottles!and!a!range!of!locally!relevant!pathogens,!as!we!did!for!the

S.!Typhi!RDTs.!!We!would!then!follow!this!up!with!a!prospective!study!on!real

samples!during!the rainy!season,!when!B.,pseudomallei!is!one!of!the!commonest

clinically!significant!isolates!we!see!in!blood!cultures.!!Since!urine!is!a!readily

accessible!and!nonOinvasive!sample,!and!urinary!antigen!detection!has

revolutionized!the!diagnosis!of!some!infectious!diseases!such!as!legionellosis,!we

Letters of Support Page 106 
 

would!also!like!to!investigate!the!use!of!the!B.,pseudomallei lateral!flow!test on

urine!samples!from!patients!with!febrile!illness!in!our!hospital.

Yours!sincerely

Letters of Support Page 107 
Letters of Support Page 108 

• Microbial Services/Research T +44 (0)1980 612 569 

Public Health Manor Farm Road F +44 (0)1980 619 894 

Porton Down 

England Salisbury SP4 OJG UK www.gov.uk/phe 

27 November 2013 

Dear David 

Re: Letter of Intent 

I am writing to confirm our continued enthusiastic support for your Phase 11 STTR proposal 

entitled "Antigen Detection Assay for the Diagnosis of Melioidosis". Based on your data and 

our preliminary findings it appears that the Burkholderia pseudomallei capsular 

polysaccharide (CPS) maybe a valuable antigen that can be targeted for a rapid diagnosis of 

acute melioidosis. 

As you know, we have secured funding for aerosol challenge studies in which primates and 

Balb/c mice will be infected via the pulmonary route with Burkholderia pseudomallei. These 

studies will provide a unique opportunity to collect and store diagnostic samples pre-infection 

and at defined time-points following infection. As part of our study we would like to 

determine if there are significant amounts of B. pseudomallei antigens shed into samples 

from infected animals. Therefore, we would like to perform the following experiments at our 

research facility: 

• Continue to evaluate your prototype CPS LFI with animal blood, urine, and possibly 

respiratory secretions 

• Use or to adapt if necessary, your quantitative ELISA to determine the concentration 

of CPS within animal samples 

• Determine if CPS can be detected in the absence of viable Burkho/deria 

pseudomallei in animal samples 

This collaboration is very likely to enhance our current study and your proposed study. We 

look forward to participating in the continued success of your research. Good luck with the 

proposal. 

Yours sincerely 

Simon 

Simon Funnell 

Scientific Leader/ 

Pro ramme Co-ordinator 

Victoria 

Victoria Graham 

Senior Immunologist 

Project T earn leader 

Miles 

Miles Carroll 

Deputy Director 

Head of MS Research 
Letters of Support Page 109 

DEPARTMENT OF MICROBIOLOGY 

THE KASTURBA MEDIC L COLLEGE 

MANIPAL- 576104, KARNATAKA, INDIA 

Ref No Datg 

Dear David, 

This letter of support is to confirm our collaboration to explore the presence of melioidosis in India 

through the use of your Active Melioidosis DetectTM lateral flow immunoassay (AMO LFI). My 

colleagues strongly agree that melioidosis is under-diagnosed in India, therefore I am also in full 

support of your Phase 11 STIR application entitled "Antigen Detection Assay for the Diagnosis of 

Melioidosis" 

Ideally, with your assistance we will adopt the same initiative used for Africa - Detection of Melioidosis 

in Africa Working Group (DMAWG). The disease was long considered under-recognized in India but 

has now gained the status of emerging infectious disease. Increasing isolation of Burkholderia 

pseudomaflei, the causative agent of melioidosis has been encountered in recent years multiple 

geographic locations within India_ What is desperately needed is a rapid and accurate diagnostic test 

to identify 8 . pseudomallei directly from patient samples and from cultures of patient samples. 

My collaborators and I look forward to establishing a more accurate picture of the true incidence of 

melioidosis in India. We feel the currently recognized cases are merely the "tip of the iceberg". 
Letters of Support Page 110 

t~\ World Health QI Organization 

20, AVENUEAPPIA- CH-1211 GENEVA 27 - SWlTZERWIO - TEL c:eN1AAL --11 22 791 2111- FAX CENTRAL -+41 22 791 3111 - WWW.V>tfO.INT 

Tel. direct: 

Fax direct: 

E-mail : 

In reply please 

refer to: EZD/EB 

Y QUI' n:ference: 

25 November2013 

Dear David, 

This letter of support is to confirm the Wor]d Health Organization's interest in the Active 

Melioidosis DetectTM Lateral Flow Immunoassay (AMD LFI) and your Phase II STIR 

application entitled, "Antigen Detection Assay for the Diagnosis ofMelioidosis". 

1 would personally like to thank you for your contribution to the Detection ofMelioidosis 

in Africa Working Group (DMA WG). The goal of the DMA WG is to determine if Burkho/deria 

pseudomallei, the causative agent of melioidosis, is an underreported cause of infection in Africa. 

Within the framework of an exploratory multicenter study, hospital/microbiological facilities in 

Africa will be offered by OMA WG for the use of the prototype AMD LFI developed by your 

laboratory at the University of Nevada and InBios International to rapidly identify B. 

pseudomal/ei isolates. 

As you know, melioidosis is highly endemic to northeast Thailand and northern Australia, 

but more recent evidence indicates a much wider distribution that includes the Indian 

subcontinent and southern China, Hong Kong, and Taiwan. There are also an increasing number 

of reports of possibly endemic disease in the Caribbean, Central and South America and East and 

West Africa. The extent of melioidosis in Africa remains uncertain. 

I look forward to collaborating with your team. 
Page 111 


Letters of Support 
Letters of Support Page 112 

November 25, 2013 

Dear David, 

I wanted to extend my support and interest in your Phase II STTR application, "Antigen Detection 

Assay for the Diagnosis of Melioidosis". As you know I have been tasked with leading the 24 Month 

Diagnostics Challenge for the U.S. Defense Threat Reduction Agency (DTRA). The diagnostic assay 

development includes pathogen targets for antigen detection of dengue, malaria, melioidosis and 

plague. We are extremely interested in the progress you have made through your Phase I STTR to 

develop the Active Melioidosis Detect lateral flow immunoassay with lnBios International because 

there are limited reagents for the detection of Burkholderia pseudomallei. 

I am also following the progress of your contract to supply DTRA with a melioidosis/dengue multiplex 

lateral flow immunoassay. We provided funds for your group through the Naval Research Laboratory 

to optimize the melioidosis assay to work in combination with a dengue test developed by lnBios 

International. The test needs to simultaneously detect both pathogens from within blood only. This is 

quite different from your singleplex melioidosis assay that can be used with multiple patient samples. 

The contract was based on a purchase price of roughly $12 per melioidosis/dengue test. We have 

requested 1000 combined tests for delivery in November and 5-10,000 tests for delivery starting 

January of 2014. I understand that these tests were purchased as "Research Use Only" and will be 

evaluated by one of our Navy laboratories. 

If your assay performs well there is the possibility for Next Generation Diagnostics System Increment 

2, a program of record for the Medical Countermeasure Systems Joint Program Executive Office. If 

this technology was selected, the Department of Defense would be a potential acquisition partner in 

future years. 

We look forward to working with your team in the future, 
 

U.S. Department of Homeland 

Security 

Washington, DC 20528 

December 4, 2013

Dear David, 

We at the Department of Homeland Security (DHS) wanted to express our full support for your 

Phase II STTR application, “Antigen Detection Assay for the Diagnosis of Melioidosis”. As you 

know DHS is interested in the Active Melioidosis Detect lateral flow immunoassay. The project 

aligns with the mission of the Department of Homeland Security Science and Technology 

Directorate to deter, detect and mitigate a biological attack on the nation. 

DHS can supply a panel of Burkholderia pseudomallei and near-neighbor strains. This panel is 

identical to the one used to evaluate the Active Melioidosis Detect LFI that incorporated mAb 3C5 

at the Centers for Disease Control and Prevention. These bacterial isolates will be essential to 

develop an inclusivity and exclusivity panel for the new anti-capsular mAbs you have isolated 

during the STTR Phase I. Also, the panel will be useful in your DHS project to produce antibodies 

to Burkholderia mallei specific diagnostic targets. 

DHS has experience in navigating diagnostics products through the FDA approval process. We 

are currently taking the lead on an FDA approval process for a lateral flow immunoassay for the 

detection of Bacillus anthracis in blood samples. This is a project that InBios International and 

your laboratory in collaboration with Dr. Thomas Kozel are currently involved in. Therefore, 

when the time is appropriate we would like to offer assistance in the FDA approval process for 

your Melioidosis assay. 

Good luck with your proposal. 

Sincerely, 

Letters of Support Page 113 
Letters of Support Page 114 

November 27. 2013 

Dear David, 

DSD NATIONAL LABORATORIES 

DMERl@DSO 

27 Medical Drive 

My research team from Singapore heard your presentation at the World Melioidosis Congress in 

Bangkok just recently and we are very interested in your Active Melioidosis Detect lateral flow 

immunoassay (AMO LFI). We also wish you good luck for your Phase II STIR application entitled, 

"Antigen Detection Assay for the Diagnosis of Melioidosis". 

I would like to personally thank you for agreeing to send a batch of your AMD LFI to assist in our soil· 

sampling project described below: 

Our project is an environmental sampling study focused on isolating 8. pseudomallei from soil. The 

project covers 10 locations in Singapore, including military training grounds, nature reserves and 

parks. At each location, we take 100 samples as per protocol recommended by the Detection of 

Environmental Burkflo/deria pseudomallei Working Party (DEBWorP). Currently we have completed 4 

locations (400 samples). Water samples are collected at various sites. largely correlating to 

fioodwaters. We have yet to commence on our air sampling yet. This project is will continue into 2016 

Kind Regards, 
Resource Sharing Plan 

Research results will be presented at national and international meetings and published in a timely fashion. 

MAbs produced as a result of this grant will be made available to academic investigators or licensed to 

commercial sources should a product arise. LFIs will be provided to academic investigators initially as 

Research Use Only or For Export Only. 

We do not anticipate the development of any model organisms and are not generating genome-wide 

association studies.

Resource Sharing Plan Page 115