# Presentation: 867615

## Laboratory of Immunobiochemistry

- Allergenic Products Advisory Committee, April 8, 2003

## Allergenic Products Advisory Committee, April 8, 2003

- Lab overview
- Staffing
- Lot release
- Reference replacement
- Operational issues
- Sheep serum replacement issues
- ISO certification report
- Research/regulatory update
- Endotoxin studies
- Cockroach antigens and antibodies

## Lab overview

- Staffing
- Lot release
- Reference replacement

- Principal Investigators
- Jay E. Slater, MD, Lab Chief – Supervisory Medical Officer  (4)
- Ronald Rabin, MD - Senior Staff Fellow (2)
- Post Doctoral Fellows
- Jonny Finlay, PhD - IRTA (2)
- Bo Chi, MD - Visiting Associate (<1)

- Research Technicians
- Albert Gam – Biologist (2)
- Mona Febus – Microbiologist (3)
- Marc Alston – Biologist  (2)
- Cherry Valerio – Biologist (2)
- Katia Dobrovolskaia – Visiting associate (2)

## LIB staffing 1998-2003

## “Routine” regulatory activities

- Lot release
- Reference distribution
- Reference maintenance
- semiannual checks
- replacement

- 357 protocols submitted and reviewed
- 1 withdrawn
- Reference distribution
- 2002: 1978 vials in 107 shipments sent to  manufacturers

## Lot release protocols submitted

## Reference distribution

## Laboratory of Immunobiochemistry

- Operational issues

## Operational issues

- Replacement of cat and ragweed antisera
- Transition to ISO compliance

## Ragweed and cat antisera need to be replaced

- S2a cat
- Released in 1998
- S6 ragweed
- Released in 2000
- Replacement programs for both initiated spring 2002

## Immunization protocol

## Immunization protocol

- plasmapheresis

- Immunization doses

## Slide 16

## Slide 17

## Transmissible spongiform encephalopathies

- Animal
- scrapie (sheep and goats)
- chronic wasting disease (mule deer, elk)
- transmissible mink encephalopathy
- bovine spongiform encephalopathy
- feline spongiform encephalopathy

**Notes:**
Scrapie is fatal progressive neurological disorder of sheep. Scrapie has been reported in many countries and was first recorded in 1732. Scrapie affects sheep and goats naturally and can be transmitted experimentally to several animal species.
CWD is a rare disease of elk and deer, predominantly in the US. Although most of the cases have occurred in captive populations, many of the animals were originally caught in the wild and are reported not to have been fed ruminant protein. Cases have been identified in wild animals too, so it must be presumed to be a natural disease rather than one introduced through feed. The name of the disease is appropriate in that the animals show progressive wasting rather than the neurological signs seen with BSE. The wasting may however be partly due to interference with brain centers that control rumination, which are also affected in BSE.
TME takes the form of a rapidly evolving epidemic, usually involving single mink farms. The disease is fatal, with neurological symptoms including locomotor incoordination, somnolence and debilitation. It appears to be associated with the feeding of contaminated food. 
BSE was recognised as a potential member of the scrapie family of diseases as soon as it was discovered in November 1986. It showed all of the pathological characteristics of a TSE.
FSE was first identified in Britain in 1990. Since then there have been 87 cases in Great Britain, one in Northern Ireland, one in Norway and one in Liechtenstein. Epidemiologically - geographically and temporally - the disease appears to be derived from BSE.

## Transmissible spongiform encephalopathies

- Human
- kuru
- Creutzfeldt-Jakob disease
- classic sporadic
- familial
- iatrogenic
**new variant (a/w bovine TSE)**
- Gerstmann-Sträussler-Scheincker
- fatal familial insomnia
- sporadic fatal insomnia

**Notes:**
Kuru: First identified in the 1950’s in isolated tribes in New Guinea. Spread by  ingestion of the brains of deceased relatives. Prevalence decreased after the practice was identified and discouraged.
CJD:
Sporadic: 1/million/year
iatrogenic: a/w corneal transplantation and dura mater grafts, as well as occupational inoculation during neorosurgery or autopsy
GSS: 2% the rate of CJD, more prominent ataxia/motor problems, less prominent dementia. Familial and sporadic.

FFI: severe selective atrophy of the thalamus

sporadic fatal insomnia: NEJM 1999

KEY POINT IN PATHOGENESIS: BOTH INFECTIOUS, SPORADIC AND FAMILIAL DISEASES

ALL  FORMS - WHETHER ENDOGENOUS OR EXOGENOUS IN ORIGIN - GENERATE INFECTIOUS AGENTS

## Scrapie

- In Europe for >250 years
- In US since 1947
- >1000 flocks
- Vertical transmission
- Horizontal transmission, presumably by contamination with placenta and blood during lambing season
- Long incubation period
- No human transmission

- Prion polymorphisms associate with different susceptibility, especially at codon 171
- QQ (glutamine/glutamine) susceptible
- RR (arginine/arginine) resistant
- QR (glutamine/arginine) intermediate

## Scrapie eradication program

**Preclinical testing and surveillance**
- Live animal test (third eyelid biopsy)
- May take months to years to turn positive
**Tracking of infected and exposed animals**
**Cleanup strategies:** identify and genotype exposed animals
- Destroy QQ exposed
- QR or RR exposed are tracked but may be slaughtered for human consumption

- Not believed to pose any risk to humans
- No recognized human transmission in three centuries of exposure in Europe
- This serum is safe to use because
- No documented transmission to humans
- Contact with affected sheep was limited
- Not in adjoining pens; >30 feet distant
- Normal BSL 2 precautions in place for all work with animal sera
- However, in order to maintain a serum reagent that is as safe as possible...

## Current approach...

- Begin immunizing two new sheep
- Process plasma from ewes 6747 and 7777 now
- Conserve current stocks

## Current approach...

- If new sera are available before we run out, sera from 6747 and 7777 will be saved frozen for possible future use
- If we have a shortfall, sera from 6747 and/or 7777 will be used until the new sera are available

## Current approach...

- Advantages
- Highest degree of safety
- Large supplies for future
- Disadvantages
- Possibility of two serum switches in short period
- Time
- Expense

## Slide 27

## CBER Laboratory Quality Management Initiative

- Seek ISO-17025 compliance for official product testing
- ISO – International Organization for Standardization, Geneva, Switzerland
- 17025: “General requirements for the competence of testing and calibration laboratories” (1999)
- Set of guidelines for labs that do testing and calibration to show operation of a quality system to assure technical competence and production of valid results

## CBER Laboratory Quality Management Initiative

- Establish policy with Center level Quality Manual
- Audit for compliance with ISO-17025
- Obtain Test/Lab Accreditation where appropriate
- “Accreditation – successful 3rd party audit

## Why is CBER becoming ISO compliant?

- Establish recognized competence, assure the trust and value of our data and processes
- We require the manufacturers’ labs to be in GMP compliance
- International efforts at harmonization are attempting to equate GMP and ISO requirements
- ISO is an internationally recognized standard

## Why is CBER becoming ISO compliant? (continued)

- Implement Laboratory Quality Management policies and practices for official testing activities
- To document a high level of training, competence, and proficiency
- To establish a consistent product testing process

## Elements of ISO compliance

- Management of
- People
- Equipment
- Documents
- Processes

## Elements of ISO compliance

- Management of People
- Defined roles
- Appropriate training
- Demonstrated proficiency
- Initial and ongoing
- Authorization process

## Elements of ISO compliance

- Management of Equipment
- Program for equipment calibration
- Maintenance
- Process to assure that only calibrated and maintained equipment is used
- Assurance of measurement traceability to accepted standards

## Elements of ISO compliance

- Management of Documents
- Includes policies, procedures, specifications, equipment manuals, certifications
- Must be reviewed, issued and controlled
- Approved and issued prior to use

## Elements of ISO compliance

- Management of Processes
- Approval of testing materials
- Environmental specifications and monitoring
- Handling of samples
- Validation and suitability
- Handling of data (including non-conforming data)
- Corrective action, Preventive action systems
- Internal Audits
- Management review
- Recording and handling complaints

## CBER’s commitment to implementation of a Lab Quality System

- Establishment of CBER Quality Board
- Development of Quality Assurance structure within CBER
- Appointment of Center Level Quality Manager
- Hiring Office quality Managers
- Appointment and hiring of Division Quality Coordinators
- Preparation of CBER Quality Manual
- Purchase of Integrated Quality Management computer software

## What will this mean to LIB?

- No substantive protocol revisions
- Documentation
- Formatting
- Substance
- Formal separation of research and regulatory equipment
- Internal audits
- External audits

## Timetable (tentative)

- Implementation in stages over the next 3 years... Seek accreditation in 2005
- Software operational during 2003
- Policies and Quality Manual issued 2003
- Initiation of compliance audits (ongoing)
- Training and process development (ongoing)

## Laboratory of Immunobiochemistry

- Research/regulatory update

## Active research projects

- PI Slater
**Cockroach allergen standardization**
- Determination of optimal surrogate test
**Depletion analysis of CR extracts**
- Cockroach IgE combinatorial library
**Endotoxin in allergen vaccines**
- PI Rabin
- MDR proteins in T cell activation
- RSV responses in human tonsil

## Publications

- Patterson ML, Slater JE. Characterization and comparison of commercially available German and American cockroach allergen vaccines. *Clin Exp Allergy* 2002;32:721-727
- Sutherland MF, Drew A, Rolland JM**, **Slater JE, Suphioglu C, O'Hehir RE.  Specific monoclonal antibodies and human IgE show Hev b 5 is an abundant allergen in high protein powdered latex gloves. *Clin Exp Allergy* 2002;32:583-589
-  Trivedi B, Valerio C,** **Slater JE. Endotoxin content of standardized allergen vaccines. *J Allergy Clin Immunol *2003 (in press).

## Publications (reviews)

- Lockey RF, Slater JE, Esch R.  Preparation and standardization of allergen vaccines. In Middleton’s Allergy: Principles and Practice, 6th ed. St. Louis: Mosby (in press).

## Abstracts

- Rabin RL, Alston MA, Huang H, Slater JE. Cytokine secretion by activated T cells is dependent on multidrug resistance protein-1 (MRP-1). *J Allergy Clin Immunol *2003; 111:S153.
- Valerio C, Slater JE.  The effects of lipopolysaccharide (LPS) on immune responses in C57Bl/6 mice. *J Allergy Clin Immunol *2003; 111:S166.
- Slater JE, Valerio C, Trivedi B. Endotoxin in standardized allergen vaccines. *J Allergy Clin Immunol *2003; 111:S243.
- Finlay WJJ, Rabin RL, Slater JE.  Analysis of IgE heavy chain V-gene usage in human tonsil.  *J Allergy Clin Immunol *2003; 111:S313.

## Endotoxin content of allergen vaccines

- Allergenic extracts are not required to undergo evaluation for the presence of pyrogens (21CFR 610.13(b))
- Prior studies confirmed variable endotoxin content (Siraganian, et al. J Allergy Clin Immunol 1979; 64:526-533)

## Products exempted in 21 CFR 610.13(b)

- Blood products
- Horse serum
- Bacterial, rickettsial and viral vaccines
- Toxoids
- Toxins
- Allergenic extracts

## LAL gel-clot method

## LAL gel-clot method

## ENDOTOXIN

- Act. factor C

- Factor B

- Proclotting enzyme

- Clotting enzyme

- Factor G

- Act. factor B

- Act. factor G

- Coagulogen

- Coagulin

*ENDOTOXIN*

- –D- glucan

## Endotoxin content of allergen vaccines – possible interference

- Non-endotoxin (1,3)--D-glucans may induce clotting by an alternative pathway in the standard LAL assays
- Proteases (especially in cat and mite extracts) may also induce clotting

## Endotoxin content of allergen vaccines - approach

- Determine endotoxin content using gel-clot method
- Assess the contribution of non-endotoxin components ((1,3)--D-glucans, enzymes)

## Study design

- Standardized allergen vaccines
- LAL gel-clot assay
- Adsorption of selected allergens with ENP-silica resin, followed by LAL assay
- Endotoxin neutralizing protein (ENP) is a ~12 kDa, cationic, amphipathic protein that binds to and neutralizes the biological activity of lipopolysaccharide.
- Pre-treat selected allergens at 95°C for 15 min, followed by LAL assay

## P

## Slide 54

## Slide 55

## Slide 56

## Slide 57

## Heat inactivation

## Slide 59

## Slide 60

## Conclusions

- The observed LAL gel-clot activity probably represents real endotoxin content, not -glucan or protease activity

## Is this amount of endotoxin physiologically significant?

- Mean endotoxin content: 1,900 EU/mL (allergen immunotherapy dose 0.5 – 2.0 mL per month)
- 40 – 50 EU/kg (2,800 to 3,500 EU) (administered IV) can elicit a rise in temperature, heart rate, and white blood cell count

- Wolff SM. J Infect Dis 1973; 128:Suppl-64.
- Michie HR, et al. N Engl J Med 1988 Jun 9 318;1481-6.

## Specific endotoxin limits

- Product-specific
- Generally based on
- Drug dose
- 5.0 EU/kg limit

## Specific endotoxin limits

- * Based on USP limits and estimated maximum therapeutic doses

**Notes:**
Doxorubicin HCl: 2.2 EU/mg * 75 mg/m(2) * 1.73 m(2)
Fentanyl: 50 EU/mg * 1.6 mg
Insulin (pork): 10 EU/mg * 0.0342 mg/U * 1 U/kg/d * 70 kg
Insulin (human): 0.8 EU/U * 1 U/kg/d * 70 kg
Prepen 5833 EU/mL * 0.1 mL

## The clinical consequences of endotoxin in allergen vaccines have not been studied

- No data that adverse events from IT are associated with endotoxin levels
- No data to support a beneficial effect of endotoxins
- Future studies of allergen IT should be controlled for endotoxin dose
- Role of endotoxin in safety and efficacy of IT should be assessed

## Conclusions

- Pollens < cat and mite
- Cat hair < cat pelt
- D. pteronyssinus << D. farinae
- Bioburden?
- Endogenous heat-stable ENP-binding LAL activator in D. farinae?
- Endogenous ENP in D. pteronyssinus?

## Plan

- Expand study of endotoxin content
- Additional standardized and non-standardized extracts
- Different methods (GC mass spec)
- Investigate differences between D. farinae and D. pteronyssinus

## Cockroach allergen standardization

- Clinical studies
- Developing the appropriate surrogate
- Correlation
- Depletion studies
- IgE combinatorial library to cockroach

## Cockroach allergen standardization

-  Clinical studies
-  Developing the appropriate surrogate
-  Correlation
-  Depletion studies
-  IgE combinatorial library to cockroach

## Cockroach allergen standardization

-  Clinical studies
-  Developing the appropriate surrogate
-  Correlation
-  Depletion studies
-  IgE combinatorial library to cockroach

## Current standardized allergens

- D. farinae
- D. pteronyssinus
- Cat hair
- Cat pelt
- Short ragweed pollen
- Hymenoptera
- Honey bee
- Wasp
- Yellow jacket
- Yellow hornet
- White-faced hornet
- Mixed vespid

- Grass pollens
- Bermuda grass
- Red top
- June (Kentucky blue)
- Perennial rye
- Orchard
- Timothy
- Meadow fescue
- Sweet vernal

## Allergen standardization

- Establish a US standard, _and_
- Establish a testing procedure
- Manufacturers may use the established procedure, or may develop equivalent procedures

## Which allergens should be standardized? Impact criteria...

- Availability of stable, preferably lyophilized material for use as long-term reference extracts.
- Consistency of currently marketed product.
- Widespread use as a diagnostic and/or therapeutic reagent in the U.S.
- Number of manufacturers producing the product.
- Potential use in immunotherapy or diagnostics.
- Public health impact of correct diagnosis and/or adequate treatment.

## Allergens and asthma

- Indoor allergens
- dust mites*
- cat*
- cockroach
- molds
- dog
- Outdoor allergens
- molds
-                          * = already standardized

## Cockroaches

## Why is cockroach allergy important?

- Ubiquitous
- Difficult to control
- Associated with asthma

## Why is cockroach allergen standardization important?

- To the patient
- More accurate diagnosis
- Safer and more effective immunotherapy
- To the physician/scientist
- Better science (if you can’t measure it, you can’t study it...)
- Pathophysiology
- Epidemiology
- Environmental control
- To the FDA
- Safer, more effective product

## Phase I - Laboratory

- Develop/adapt methods for allergen determination
- Compare allergen content of different lots

## Goals

- Determine consistency of available US products:
- protein content
- specific allergen content
- overall allergenicity
- Determine best lot release measures

## Extracts used as reference

- E2-Cg and E2-Ca
- Previously characterized
- Limited skin test data
- Lyophilized; available in large quantities

## Cockroach extracts studied

- From all nine allergen extract manufacturers

## Relative Potency of All Cockroach Extracts (determined by competition ELISA)

## Relative Potency of All Cockroach Extracts (determined by competition ELISA)

## Relative Potency of All Cockroach Extracts (determined by competition ELISA)

## Relative Potency of All Cockroach Extracts (determined by competition ELISA)

## Bla g 1 and Bla g 2 Levels in glycerinated German cockroach

## Correlation of protein concentrations and ELISA results

- Protein concentration

- Bla g 1 levels

- Bla g 2 levels

- Relative potency

- R2 = 0.83

- R2 = 0.92

- R2 = 0.93

- R2 = 0.96

- R2 = 0.92

- R2 = 0.92

**Notes:**
Protein concentrations were correlated with the relative potency (r2=0.94) and Bla g 1 (r2=0.92) but not Bla g 2 levels (r2 = 0.03).
Relative potency values were correlated with Bla g 1 (r = 0.97) but not Bla g 2 (r = 0.01) levels.
Bla g 1 levels were not correlated with Bla g 2 levels (r = 0.03).
This data along with the greater variation in measured Bla g 2 levels (previous slide) led us to question if the Bla g 2 allergen rapidly degrades over time.  Correlation analysis of Bla g 2 levels with age did not support this hypothesis. 
False: There is no correlation of Bla g 2 levels and the age of the extract (r = 0.34).

## Conclusions

- Commercially available cockroach allergen extracts:
- vary widely in protein content, Bla g 2 content, SDS-PAGE banding pattern, and overall allergenicity.
- appear to be less potent and contain less Bla g 1 than the candidate reference extracts

## Conclusions

- Established that cockroach allergen vaccines need to be standardized

## What we need now...

- New cockroach references
- Characterize the references
- ID50EAL testing (**I**ntradermal **D**ilution for **50** mm Sum of **E**rythema Determines Bioequivalent **Al**lergy Units)
- Proceed to next phase

## Phase II - Clinical

- skin testing, histamine release, IT data
- establish biological unitage and ideal dosing ranges

## ID50EAL testing

- Proficiency
- Recruitment
- Testing
- Analysis

## ID50EAL testing

## Recruitment

- Inclusion criteria
- 18 to 65 years of age
- history of allergic disease, such as allergic rhinitis, related to exposure to the allergen of interest
- puncture sum of erythema diameter responses (ΣE) to the allergen concentrate of ≥30 mm.

## Recruitment

- Exclusion criteria
- Asthma with use of systemic steroids in the past 12 months
- Peak flow < 75% predicted at the time of testing
- Skin coloring or condition that would preclude the measurement of erythema responses
- Dermographism (> 4 mm ΣE following saline skin test)
- Immunotherapy – past or present - with the test allergen
- Current use of antihistamines, tricyclic antidepressants, MAO inhibitors, and beta-blockers

## How many study subjects?

- Rabin et al., Sample Size Considerations for Establishing Clinical Bioequivalence of Allergen Formulations. Arb.Paul Ehrlich Inst.Bundesamt Sera Impfstoffe Frankf.A.M, in press

**Notes:**
Let’s plug in some numbers and see what values of n we obtain.

In all of these examples we accept the default values of  and. In the top panel we accept the default value of  , the acceptable difference between the reference and test values, as 10%.  In the top row, we use the given  of 2 (about 20% of the measured quantity), and we find that we need at least 50 study subjects to make a statistically valid comparison.

Even if the standard deviation of the test is smaller - say, equal to  - we still need at least 13 subjects in the study. It is only when becomes quite small, just a little over half of , that four study subjects would be adequate.

In the lower panel, we look at the consequences of liberalizing . If we double it - that is we say that a 20% difference is acceptable - then, in this example,  and again we need to study 13 subjects. It is only when we increase to 3.5 - that is, 35% of the measured quantity - that four study subjects would be adequate.

## for D50 should be 10%

**BAU/mL= 3****-(14 - mean D50)**** * 100,000**

##  for D50 is about 10-20%

- Smith et al. Annals Allergy Asthma Immunol 1995; 75:317-323

##  for D50 is about 10-20%

## How many study subjects?

- Estimate n = 40 to establish D50 for each extract (based on /= 1.5): 80
- Geographic diversity: 80 x 3 = 240
- Overlap between American CR and German CR allergic subjects may permit reduction in n (150-200)

## Conclusions

- Established that cockroach allergen vaccines need to be standardized
- Need to establish the potency of candidate US reference materials by bioassay (ID50EAL)

## NIAID Inner City Asthma Consortium

- “...established in FY 2002 to explore and evaluate promising new strategies for the treatment of asthma among minority children residing in the inner city. This consortium of basic scientists and clinical investigators will conduct clinical studies to elucidate the immunopathogenesis and natural history of asthma in this population.”

- From the FY 2003 Budget Justification Narrative, NIAID,
- http://www.niaid.nih.gov/director/congress/2002/cj/narrative.htm

## NIAID Inner City Asthma Consortium steering committee

- _[Busse](http://www.icacweb.org/committee/single_mail_form.asp?recipient=wwb@medicine.wisc.edu&cid=2)__[, William W., MD](http://www.icacweb.org/committee/single_mail_form.asp?recipient=wwb@medicine.wisc.edu&cid=2)_ Chair
- _[Adams, Kenneth, PhD](http://www.icacweb.org/committee/single_mail_form.asp?recipient=kadams@niaid.nih.gov&cid=2)_
- _[Eggleston, Peyton A., MD](http://www.icacweb.org/committee/single_mail_form.asp?recipient=pegglest@jhmi.edu&cid=2)_
- _[Gruchalla](http://www.icacweb.org/committee/single_mail_form.asp?recipient=Rebecca.Gruchalla@utsouthwestern.edu&cid=2)__[, Rebecca S., MD, PhD](http://www.icacweb.org/committee/single_mail_form.asp?recipient=Rebecca.Gruchalla@utsouthwestern.edu&cid=2)_
- _[Kattan](http://www.icacweb.org/committee/single_mail_form.asp?recipient=meyer.kattan@mssm.edu&cid=2)__[, Meyer, MD](http://www.icacweb.org/committee/single_mail_form.asp?recipient=meyer.kattan@mssm.edu&cid=2)_
- _[Kercsmar](http://www.icacweb.org/committee/single_mail_form.asp?recipient=Carolyn.kercsmar@uhhs.com&cid=2)__[, Carolyn M., MD](http://www.icacweb.org/committee/single_mail_form.asp?recipient=Carolyn.kercsmar@uhhs.com&cid=2)_
- _[Liu, Andrew H., MD](http://www.icacweb.org/committee/single_mail_form.asp?recipient=liua@njc.org&cid=2)_
- _[Malveaux](http://www.icacweb.org/committee/single_mail_form.asp?recipient=fmalveaux@howard.edu&cid=2)__[, Floyd J., MD, PhD](http://www.icacweb.org/committee/single_mail_form.asp?recipient=fmalveaux@howard.edu&cid=2)_
- _[Mitchell, Herman, PhD](http://www.icacweb.org/committee/single_mail_form.asp?recipient=hmitchell@rhoworld.com&cid=2)_

- _[Morgan, Wayne, MD, CM](http://www.icacweb.org/committee/single_mail_form.asp?recipient=wmorgan@resp-sci.arizona.edu&cid=2)_
- _[O'Connor, George T., MD, MS](http://www.icacweb.org/committee/single_mail_form.asp?recipient=goconnor@lung.bumc.bu.edu&cid=2)_
- _[Pongracic](http://www.icacweb.org/committee/single_mail_form.asp?recipient=j-pongracic@northwestern.edu&cid=2)__[, Jacqueline A., MD](http://www.icacweb.org/committee/single_mail_form.asp?recipient=j-pongracic@northwestern.edu&cid=2)_
- _[Sampson, Hugh A., MD](http://www.icacweb.org/committee/single_mail_form.asp?recipient=hugh.sampson@mssm.edu&cid=2)_
- _[Smartt](http://www.icacweb.org/committee/single_mail_form.asp?recipient=esmartt@niaid.nih.gov&cid=2)__[, Ernestine, RN](http://www.icacweb.org/committee/single_mail_form.asp?recipient=esmartt@niaid.nih.gov&cid=2)_
- _[Strunk](http://www.icacweb.org/committee/single_mail_form.asp?recipient=strunk@kids.wustl.edu&cid=2)__[, Robert C., MD](http://www.icacweb.org/committee/single_mail_form.asp?recipient=strunk@kids.wustl.edu&cid=2)_
- _[Szefler](http://www.icacweb.org/committee/single_mail_form.asp?recipient=szeflers@njc.org&cid=2)__[, Stanley J., MD](http://www.icacweb.org/committee/single_mail_form.asp?recipient=szeflers@njc.org&cid=2)_

## Timetable

- Steering committee approval - done
- Study centers identified
- Order extracts
- IRB approvals
- IND approval
- Distribute materials
- Proficiency testing
- Proceed with study

## Endotoxin content of cockroach vaccines

## Conclusions

- Established that cockroach allergen vaccines need to be standardized
- Need to establish the potency of candidate US reference materials by bioassay (ID50EAL)
- Endotoxin issue to be studied in depth

## Will overall allergenicity measurements be sensitive to changes in specific allergen levels?

- In mite stability study (1998-1999), RP was stable at -20°C and 4°C for up to 12 months
- Degradation of specific allergens (group 1 and 2; specific bands) was observed at 4°C

- Soldatova LN, Paupore EJ, Burk SH, Pastor RW, Slater JE. *J Allergy Clin Immunol *2000; 105:482-8.

## RID with monospecific antiserum

- Examples: cat, ragweed
- Advantages
- quantitative
- monospecific
- Disadvantages
- need to identify relevant allergen(s)

## Competition ELISA with pooled allergic human sera

- Examples: mites, grasses
- Advantages
- quantitative
- reflects spectrum of allergen recognition
- does not require identification of relevant allergens
- Disadvantages
- use of pooled sera
- effects of fluctuations in individual allergens difficult to measure

## Specific loss of a single allergen

## Will overall allergenicity measurements be sensitive to changes in specific allergen levels?

- Depletion analysis
- Raise specific antibodies to Bla g 1, 2, 4 and 5
- Selectively adsorb
- Test for specific allergen levels
- Test for overall allergenicity

## SDS-Page of Bla g 1 Absorbed Cockroach Antigen

- Cr       Sham Abs

## Selective adsorption of Bla g 1

## Selective adsorption of Bla g 1 does not reduce the RP as measured by competition ELISA

## Conclusions

- Established that cockroach allergen vaccines need to be standardized
- Need to establish the potency of candidate US reference materials by bioassay (ID50EAL)
- Endotoxin issue to be studied in depth
- Surrogate test may not be the competition ELISA

## Conclusions

- Standardized German and American cockroach allergen vaccines will
- facilitate definitive studies on the role of cockroach allergens in inner city asthma, and on the best methods for eradication and treatment
- make for safer and more effective products