September 2004 

Environmental Technology 

Verification Report 

TETRACORE, INC.
 ANTHRAX, BOTULINUM TOXIN, AND RICIN


ENZYME-LINKED IMMUNOSORBENT ASSAY


(ELISA)


Prepared by


Battelle


Under a cooperative agreement with

 U.S. Environmental Protection Agency 
September 2004 

Environmental Technology Verification


Report


ETV Advanced Monitoring Systems Center 

Tetracore, Inc.


Anthrax, Botulinum Toxin, and Ricin 


Enzyme-Linked Immunosorbent Assay (ELISA)


by


Ryan James


Amy Dindal


Zachary Willenberg


Karen Riggs


Battelle


Columbus, Ohio 43201

Notice


The U.S. Environmental Protection Agency (EPA), through its Office of Research and 

Development, has financially supported and collaborated in the extramural program described 

here. This document has been peer reviewed by the Agency. Mention of trade names or 

commercial products does not constitute endorsement or recommendation by the EPA for use. 

ii 
Foreword


The U.S. Environmental Protection Agency (EPA) is charged by Congress with protecting the 

nation’s air, water, and land resources. Under a mandate of national environmental laws, the 

Agency strives to formulate and implement actions leading to a compatible balance between 

human activities and the ability of natural systems to support and nurture life. To meet this 

mandate, the EPA’s Office of Research and Development provides data and science support that 

can be used to solve environmental problems and to build the scientific knowledge base needed 

to manage our ecological resources wisely, to understand how pollutants affect our health, and to 

prevent or reduce environmental risks. 

The Environmental Technology Verification (ETV) Program has been established by the EPA to 

verify the performance characteristics of innovative environmental technology across all media 

and to report this objective information to permitters, buyers, and users of the technology, thus 

substantially accelerating the entrance of new environmental technologies into the marketplace. 

Verification organizations oversee and report verification activities based on testing and quality 

assurance protocols developed with input from major stakeholders and customer groups 

associated with the technology area. ETV consists of six verification centers. Information about 

each of these centers can be found on the Internet at http://www.epa.gov/etv/. 

Effective verifications of monitoring technologies are needed to assess environmental quality 

and to supply cost and performance data to select the most appropriate technology for that 

assessment. Under a cooperative agreement, Battelle has received funding to plan, coordinate, 

and conduct such verification tests for “Advanced Monitoring Systems for Air, Water, and Soil” 

and report the results to the community at large. Information concerning this specific 

environmental technology area can be found on the Internet at 

http://www.epa.gov/etv/centers/center1.html. 

iii 
Acknowledgments


The authors wish to acknowledge the support of all those who helped plan and conduct the 

verification test, analyze the data, and prepare this report. We sincerely appreciate the 

contribution of drinking water samples from the New York City Department of Environmental 

Protection (Paul Bennett), the City of Orlando (Terri Slifko), and the Metropolitan Water 

District of Southern California (Paul Rochelle). Also, thank you to the Metropolitan Water 

District of Southern California for concentrating each drinking water sample. We would also 

like to thank Karen Bradham, U.S. EPA National Exposure Research Laboratory (NERL); Steve 

Allgeier, U.S. EPA Office of Water; Ricardo DeLeon, Metropolitan Water District of Southern 

California; and Stanley States, Pittsburgh Water and Sewer Authority, for their careful review of 

the test/QA plan and this verification report. Thanks go to Linda Sheldon, U.S. EPA NERL, for 

her review of the verification reports and statements. 

iv 
Contents


Page 

Notice ..................................................................... ii 


Foreword ................................................................. iii 


Acknowledgments .......................................................... iv 


List of Abbreviations ........................................................ vii 


1 Background .............................................................. 1 


2 Technology Description ..................................................... 2 


3 Test Design and Procedures .................................................. 4 


3.1 Introduction ....................................................... 4 


3.2 Test Samples ...................................................... 5 


3.2.1 Performance Test Samples ...................................... 6 


3.2.2 Drinking Water Samples ....................................... 8 


3.2.3 Quality Control Samples ....................................... 8 


3.3 Test Procedure ..................................................... 8 


3.3.1 Laboratory Testing ............................................ 8 


3.3.2 Non-Laboratory Testing ........................................ 9 


3.3.3 Drinking Water Characterization ................................. 9 


4 Quality Assurance/Quality Control ............................................ 11 


4.1 Sample Chain-of-Custody Procedures .................................. 11 


4.2 Equipment/Calibration ............................................. 11 


4.3 Characterization of Contaminant Stock Solutions ......................... 11 


4.3.1 Characterization of Botulinum Toxin and Ricin .................... 11 


4.3.2 Characterization of Anthrax Spores .............................. 12 


4.3.3 Anthrax Enumeration Data .................................... 13 


4.4 Technical Systems Audit ............................................ 16 


4.5 Audit of Data Quality .............................................. 16 


4.6 QA/QC Reporting ................................................. 17 


4.7 Data Review ..................................................... 17 


5 Statistical Methods and Reported Parameters .................................... 18 


5.1 Qualitative Contaminant Presence/Absence .............................. 18 


5.2 False Positive/Negative Responses .................................... 18 


5.3 Consistency ...................................................... 18 


5.4 Lowest Detectable Concentration ..................................... 19 


5.5 Other Performance Factors .......................................... 19 


v 
6 Test Results .............................................................. 20 


6.1 Qualitative Contaminant Presence/Absence ............................. 20 


6.1.1 Anthrax ................................................... 20 


6.1.2 Botulinum Toxin ............................................ 22 


6.1.3 Ricin ..................................................... 23 


6.2 False Positive/Negative Responses .................................... 23 


6.2.1 Interferent PT Samples ........................................ 24 


6.2.2 DW Samples ............................................... 25 


6.2.3 Cross-Reactivity PT Samples ................................... 26 


6.3 Consistency ...................................................... 26 


6.4 Lowest Detectable Concentration ..................................... 27 


6.5 Other Performance Factors .......................................... 27 


7 Performance Summary ..................................................... 28 


8 References .............................................................. 32 


Figures 

Figure 2-1. Tetracore ELISA ................................................... 2 


Tables 

Table 3-1. Lethal Dose and Source of Contaminants ............................... 5 


Table 3-2. Performance Test Samples ........................................... 6 


Table 3-3. Drinking Water Samples ............................................ 7 


Table 3-4. ATEL Water Quality Characterization of Drinking Water Samples ........... 10 


Table 4-1. Characterization Information for Battelle Preparation of Anthrax Spores ...... 13 


Table 4-2. Anthrax Enumeration Data for PT Samples ............................. 14 


Table 4-3. Anthrax Enumeration Results for Fortified Interferent and 


Drinking Water Samples ........................................... 15 


Table 4-4. Summary of Data Recording Process .................................. 17 


Table 6-1a. Anthrax Contaminant-Only PT Sample Results .......................... 21 


Table 6-1b. Botulinum Toxin Contaminant-Only PT Sample Results ................... 23 


Table 6-1c. Ricin Contaminant-Only PT Sample Results ............................ 23 


Table 6-2. Interferent PT Sample Results ....................................... 24 


Table 6-3. DW Sample Results ............................................... 25 


Table 6-4. Potentially Cross-Reactive PT Sample Results .......................... 26 


Table 7-1. Anthrax Summary Table ........................................... 28 


Table 7-2. Botulinum Toxin Summary Table .................................... 30 


Table 7-3. Ricin Summary Table .............................................. 31 


vi 
List of Abbreviations


AMS Advanced Monitoring Systems 

ATEL Aqua Tech Environmental Laboratories, Inc. 

Ca calcium 

CDC Centers for Disease Control and Prevention 

cfu colony-forming units 

COA certificate of analysis 

DI deionized 

DW drinking water 

ELISA enzyme-linked immunosorbent assay 

EPA U.S. Environmental Protection Agency 

ETV Environmental Technology Verification 

L liter 

LOD limit of detection 

MB method blank 

Mg magnesium 

mg/L milligram per liter 

μL microliter 

mL milliliter 

PT performance test 

QA quality assurance 

QC quality control 

QMP quality management plan 

RPD relative percent difference 

SOP standard operating procedure 

TSA technical systems audit 

vii 
Chapter 1 


Background


The U.S. Environmental Protection Agency (EPA) supports the Environmental Technology 

Verification (ETV) Program to facilitate the deployment of innovative environmental technologies through performance verification and dissemination of information. The goal of the 

ETV Program is to further environmental protection by accelerating the acceptance and use of 

improved and cost-effective technologies. ETV seeks to achieve this goal by providing highquality, peer-reviewed data on technology performance to those involved in the design, 

distribution, financing, permitting, purchase, and use of environmental technologies. 

ETV works in partnership with recognized testing organizations; with stakeholder groups 

consisting of buyers, vendor organizations, and permitters; and with the full participation of 

individual technology developers. The program evaluates the performance of innovative technologies by developing test plans that are responsive to the needs of stakeholders, conducting 

field or laboratory tests (as appropriate), collecting and analyzing data, and preparing peerreviewed reports. All evaluations are conducted in accordance with rigorous quality assurance 

(QA) protocols to ensure that data of known and adequate quality are generated and that the 

results are defensible. 

The EPA’s National Exposure Research Laboratory and its verification organization partner, 

Battelle, operate the Advanced Monitoring Systems (AMS) Center under ETV. The AMS Center 

recently evaluated the performance of the Tetracore, Inc., anthrax, botulinum toxin, and ricin 

enzyme-linked immunosorbent assays (ELISA). Immunoassay detection technologies were 

identified as a priority technology category for verification through the AMS Center stakeholder 

process. 

1

Chapter 2 


Technology Description


The objective of the ETV AMS Center is to verify the performance characteristics of environmental monitoring technologies for air, water, and soil. This verification report provides results 

for the verification testing of the Tetracore ELISA (Figure 2-1), including kits for detecting 

anthrax, botulinum toxin, and ricin. The following is a description of the Tetracore ELISA based 

on information provided by the vendor. The information provided below was not subjected to 

verification in this test. 

The antigen-capture Tetracore ELISA detects antigens in samples by capturing them between a 

sandwich of antibodies. The immunosorbent assay uses immunological reagents to identify 

antibodies. The Tetracore ELISA can be read qualitatively (visually) and recorded by hand or 

quantitatively (using a photometer that measures and prints out the optical density of fluid 

samples in the microplate). Readings were made qualitatively during this verification test. 

To perform a test, positive and negative capture antibody reagents are applied to alternating 

wells of a 96-well plate, where they are passively adsorbed. If the target antigen is present in the 

sample, it will bind to the reagent. A detector antibody forms the top of the sandwich and binds 

to any antigen in the sample after it is captured. The conjugate, to which the enzyme is 

covalently bound, is the third reagent added; and it binds to the detector antibody. The substrate, 

added after the conjugate, contains 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate), which, in 

the presence of horseradish peroxidase, changes to a bright green. The amount of color change is 

directly proportional to the amount of 

horseradish peroxidase present, which 

correlates to the amount of antigen (target 

contaminant) bound in the sandwich. The 

color change confirms the “capture” of 

antigen by the antibody reagents. For 48 

samples, the process takes approximately 5 

hours. 

The Tetracore ELISA kit includes two 96­

well plates in which the odd-numbered 

rows had been pre-plated with the 

antibodies specific for each target 

contaminant and the even-numbered rows 

Figure 2-1. Tetracore ELISA had been pre-plated with antibodies not 

2

specific to the target contaminants. The inclusion of the non-target antibodies provides 

assurance that a color change is due to an antigen-antibody interaction and is not due to a 

reaction with one of the other reagents. Also provided are dilution buffer, wash buffer, and the 

appropriate reagents needed for the analysis. The 96-well microplate is 12.5 centimeters (cm) by 

8 cm. One Tetracore ELISA kit (positive and negative coated wells) costs $400. 

3

Chapter 3 


Test Design and Procedures


3.1 Introduction 

The objective of this verification test of immunoassay test kits was to evaluate their ability to 

detect specific biological toxins and agents in water samples and to determine their 

susceptibility to specific interferents added to pure water and to interferents inherently present in 

several drinking water (DW) samples. The single-use Tetracore ELISA plates detect only one 

contaminant at a time. For Tetracore ELISA analyses, the presence of contaminants is indicated 

by a change in color of a solution within a sample well. When a water sample is added, this 

color change occurs during the last step of a procedure that takes approximately 5 hours. With 

Tetracore ELISA, usually a large number of samples (i.e., 48 samples) can be analyzed 

simultaneously. Tetracore ELISA kits can be used in conjunction with an electronic reader to 

determine the presence or absence of a contaminant or can be read visually. In this verification 

test, the Tetracore ELISA 96-well plates for anthrax, botulinum toxin, and ricin were read 

visually, without the aid of a plate reader. 

During this verification test, the Tetracore ELISA was subjected to various concentrations of 

anthrax spores, botulinum toxin, and ricin in American Society for Testing and Materials Type II 

deionized (DI) water. Table 3-1 shows the contaminants and information about their detection, 

including the vendor-stated limit of detection (LOD), the lethal dose concentrations, and the 

source of the contaminant. The Tetracore ELISA also was used to analyze contaminant-fortified 

DW samples that were collected from four water utilities that use a variety of treatment methods. 

The effect of interferents was evaluated by analyzing individual solutions of organic acids 

(humic and fulvic), magnesium (Mg) and calcium (Ca) in DI water both with and without the 

addition of the contaminants with the Tetracore ELISA. In addition, specificity was evaluated by 

exposing the Tetracore ELISA to a potentially cross-reactive compound or spore for each target 

contaminant. 

4

Table 3-1. Lethal Dose and Source of Contaminants 

Contaminant

 Vendor-Stated 

LOD 

Lethal Dose 

Concentration(a) Source of Contaminant 

Bacillus anthracis 

Ames Strain (anthrax) 

Botulinum toxin 

Types A and B 

Ricinus communis 

Agglutinin II (ricin) 

2 × 104 

spores/mL 

0.004 mg/L 

0.0015 mg/L 

200 spores/mL(1) 

0.3 mg/L(2) 

15 mg/L(3) 

Battelle and U.S. Army 

Dugway Proving Ground 

Metabiologics, Inc. 

(Madison, Wisconsin) 

Vector Laboratories, Inc. 

(Burlingame, California) 

(a) The lethal dose of each contaminant was determined by calculating the concentration at which 250 mL of water 

would probably cause the death of a 154-pound person based on human mortality data. 

mL = milliliter 

mg/L = milligrams per liter 

The verification test for the Tetracore ELISA was conducted January 14 through April 23, 2004, 

according to procedures specified in the Test/QA Plan for Verification of Immunoassay Test 

Kits.

(4) This test was conducted at Battelle laboratories in Columbus and West Jefferson, Ohio. 

Aqua Tech Environmental Laboratories, Inc. (ATEL) of Marion, Ohio, performed physicochemical characterization for each DW sample to determine the following parameters: turbidity; 

concentration of dissolved and total organic carbon; specific conductivity; alkalinity; concentration of Mg and Ca; pH; hardness; and concentration of total organic halides, trihalomethanes, 

and haloacetic acids. Battelle confirmed the presence of anthrax spores using plate enumeration. 

The Tetracore ELISA were evaluated for the following parameters: 

# Qualitative contaminant presence/absence 

# False positive/false negative response 

- Interferents 

- DW matrix effects 

- Cross-reactivity 

# Consistency 

# Lowest detectable concentration 

# Other performance factors 

- Field portability 

- Ease of use 

- Sample throughput. 

3.2 Test Samples 

Tables 3-2 and 3-3 summarize the samples analyzed for each contaminant. The ability of the 

Tetracore ELISA to individually detect various concentrations of anthrax spores, botulinum 

toxin, and ricin was evaluated by analyzing performance test (PT) and DW samples. PT samples 

included DI water fortified with either the target contaminant, an interferent, both, or only a 

5

cross-reactive species. DW samples were analyzed using the Tetracore ELISA with the addition 

of each target contaminant. All the samples listed in the test/QA plan were initially analyzed. As 

discussed below, additional concentration levels were analyzed to more thoroughly evaluate the 

performance of the Tetracore ELISA. 

Table 3-2. Performance Test Samples 

Type of PT 

Sample Sample Characteristics Approximate Concentrations 

Anthrax spores 200 to 1010 spores/mL(a) 

Contaminant-only Botulinum toxin Types A and B 0.004 to 0.3 mg/L 

Ricin 0.0015 to 15 mg/L 

Contaminants in 46 mg/L Ca 

and 18 mg/L Mg 

Anthrax - 106

 spores/mL 

Botulinum toxin (Type B only) - 0.04 mg/L 

Ricin - 0.015 mg/L 

Interferent 

Contaminants in 230 mg/L Ca 

and 90 mg/L Mg 

Anthrax - 106

 and 108 spores/mL 

Botulinum toxin (Types A and B) - 0.04 mg/L 

Ricin - 0.015 mg/L 

Contaminants in 0.5 mg/L humic 

acid and 0.5 mg/L fulvic acid 

Anthrax - 106

 spores/mL 

Botulinum toxin (Type B only) - 0.04 mg/L 

Ricin - 0.015 mg/L 

Contaminants in 2.5 mg/L humic 

acid and 2.5 mg/L fulvic acids 

Anthrax - 106

 and 108 spores/mL 

Botulinum toxin (Types A and B) - 0.04 mg/L 

Ricin - 0.015 mg/L 

Potentially 

Cross-reactive 

Bacillus thuringiensis (anthrax 

analogue) 

Lipopolysaccharide 

(botulinum toxin analogue) 

Lectin from soybean 

(ricin analogue) 

104

 spores/mL 

0.04 mg/L 

0.015 mg/L 

(a)	 This concentration range includes all samples analyzed, including spores preserved with and without phenol, 

spores prepared at Battelle and at Dugway Proving Ground, and vegetative anthrax cells. 

3.2.1 Performance Test Samples 

The contaminant-only PT samples were prepared in DI water using certified standards of ricin 

and botulinum toxin. Reference methods were not available for quantitative confirmation of the 

botulinum toxin and ricin test solutions so certificates of analysis (COA) and QA oversight of 

solution preparation were used to confirm their concentrations. Anthrax PT samples also were 

prepared in DI water using anthrax spores prepared and characterized by Battelle using standard 

methods. All test samples were prepared from the standards or stock solutions on the day of 

analysis. Spores also were obtained from Dugway Proving Ground and enumerated by Battelle 

during this verification test. 

6

Table 3-3. Drinking Water Samples 

Drinking Water Sample Description 

Approximate 

Contaminant Concentrations 

Water Utility 

Water 

Treatment 

Source 

Type 

Conc. / 

Unconc. 

Anthrax 

(spores/mL) 

Botulinum 

Toxin (mg/L) Ricin (mg/L) 

Metropolitan 

Water District of 

California (CA) 

filtered 

chloraminated 

surface conc. unspiked 

106 

108 

unspiked 

0.04 (Type B) 

0.04 (Type A) 

unspiked 

0.015 

New York City, 

New York (NY) 

unfiltered 

chlorinated 

surface conc. unspiked 

106 

108 

unspiked 

0.04 (Type B) 

0.04 (Type A) 

unspiked 

0.015 

Metropolitan 

Water District of 

California (CA) 

filtered 

chloraminated 

surface unconc. unspiked 

106 

unspiked 

0.04 (Type B) 

unspiked 

0.015 

New York City, 

New York (NY) 

unfiltered 

chlorinated 

surface unconc. unspiked 

106 

unspiked 

0.04 (Type B) 

unspiked 

0.015 

Columbus, Ohio 

(OH) 

filtered 

chlorinated 

surface both unspiked 

106 

unspiked 

0.04 (Type B) 

unspiked 

0.015 

Orlando, Florida 

(FL) 

filtered 

chlorinated 

ground both unspiked 

106 

unspiked 

0.04 (Type B) 

unspiked 

0.015 

Initially, the test/QA plan called for the analysis of PT samples with concentrations including the 

lethal dose; the vendor-stated LOD; and approximately 5, 10, and 50 times the LOD. These 

samples were analyzed using the Tetracore ELISA. Preliminary results indicated that anthrax was 

not detectable; therefore, the original test/QA plan was amended to include the analysis of higher 

concentration levels of anthrax, as well as a second source of anthrax spores that were never 

preserved in phenol and vegetative anthrax cells. This testing and the subsequent results are fully 

described in Section 6.1. 

The interferent PT samples consisted of samples of humic and fulvic acids isolated from the 

Elliott River (obtained from the International Humic Substances Society) and Ca and Mg 

(prepared from their chlorides), each spiked into DI water at two concentration levels. These 

solutions were analyzed both with the addition of each target contaminant at one concentration 

level and without the addition of any target contaminant. To be able to evaluate the susceptibility 

of the ELISA test kit to false negative results due to interferents, the test/QA plan was amended to 

include the fortification of detectable concentrations of anthrax spores into interferent solutions. 

The last type of PT sample was a cross-reactivity check sample to determine whether the plates 

produce false positive results in response to similar analytes. Bacillus thuringiensis (for anthrax), 

lectin from soybean (for ricin), and lipopolysaccharide (for botulinum toxin) are chemically or 

biologically similar to the specified targets. Solutions of these were prepared in DI water at 

7

concentrations similar to the vendor-stated LOD of the test kits for the specified targets and 

analyzed using the Tetracore ELISA. 

Three replicates of each PT sample were analyzed. A total of 162 PT samples was analyzed by 

the Tetracore ELISA for this test. The results provided information about how well the Tetracore 

ELISA detected the presence of each contaminant at several concentration levels, the consistency 

of the responses, and the susceptibility of the Tetracore ELISA to some selected interferents and 

possibly cross-reactive species. 

3.2.2 Drinking Water Samples 

Table 3-3 lists the DW samples collected from four geographically distributed municipal sources 

to evaluate the performance of the Tetracore ELISA with various sample matrices. These samples 

were unique in terms of their source and treatment and disinfection process. All collected samples 

were finished DW either ready for the distribution system or from within the distribution system. 

Approximately 120 L of each of the DW samples were collected in pre-cleaned high-density 

polyethylene containers. All but 20 L of the DW samples were shipped to the Metropolitan Water 

District of Southern California, dechlorinated with sodium thiosulfate, and then concentrated 

through ultra-filtration techniques to a final volume of 250 mL. This concentration factor was 

selected because it is the goal of an EPA onsite ultra-filtration method which is currently being 

developed. The remaining 20 L of each DW sample was shipped to ATEL for water quality 

analysis. Each DW sample (non-concentrated and concentrated) was analyzed without adding any 

contaminant, as well as after fortification with individual contaminants at a single concentration 

level. A total of 156 DW samples was analyzed by the Tetracore ELISA for this test. 

3.2.3 Quality Control Samples 

In addition to the 318 PT and DW samples analyzed, 33 method blank (MB) samples consisting 

of DI water also were analyzed to confirm negative responses in the absence of any contaminant 

and to ensure that no sources of contamination were introduced during the analysis procedures. 

With each set of samples, 12 concentration levels (one replicate each) of the target contaminant 

were analyzed to develop a calibration curve for use if a reader was used. In addition to serving as 

a calibration curve, those samples served as positive control samples to confirm the function of 

the Tetracore ELISA plate. Because of this control procedure, other positive control samples were 

not analyzed. 

3.3 Test Procedure 

3.3.1 Laboratory Testing 

The scope of this verification test required that most of the test samples be analyzed within 

Battelle laboratories staffed with technicians trained to safely handle anthrax, botulinum toxin, 

and ricin. Each day, fresh samples were prepared from standards or stock solutions in either DI 

water, an interferent matrix, or a DW matrix. Each sample was prepared in its own container and 

8

labeled only with a sample identification number that also was recorded in a laboratory record 

book along with details of the sample preparation. Prior to the analysis of each sample, the 

verification staff recorded the sample identification number on a sample data sheet; then, after the 

analysis was complete, the result was recorded on the sample data sheet. Three replicates of each 

test sample were analyzed. The testing procedure included the following steps for analyzing liquid 

samples for the presence of anthrax spores, botulinum toxin, or ricin: (1) 50 :L of dilution buffer 

were added to all wells that would contain a sample; (2) 50 :L of test sample were added to the 

dilution buffer, and the plates were incubated for one hour at room temperature; (3) plates were 

washed three times with 200 :L of Tetracore ELISA wash buffer per well; (4) 100 :L of detector 

antibody were added to each sample well, and plates were incubated for one hour at room 

temperature; (5) plates were washed three times with 200 :L of Tetracore ELISA wash buffer per 

well; (6) 100 :L of conjugate were added to each sample well, and the plates were incubated for 

one hour at room temperature; (7) the plates were washed three times with 200 :L of Tetracore 

ELISA wash buffer per well; (8) 100 :L of substrate solution were added to each sample well, and 

the plates were incubated for 30 minutes at room temperature; and (9) the results were read 

visually. Wells that changed to a color different than the negative control were recorded as having 

given positive results. The procedure took approximately 5 hours. 

3.3.2 Non-Laboratory Testing 

The test/QA plan called for testing the Tetracore ELISA in a non-laboratory setting with both a 

trained operator and an operator that had not been trained to operate the Tetracore ELISA and had 

no laboratory experience. This approach was designed to test the performance of the Tetracore 

ELISA in a non-laboratory setting, not to thoroughly evaluate the effect of changing conditions 

such as temperature and humidity on the Tetracore ELISA. However, two characteristics of the 

Tetracore ELISA prompted amending the test/QA plan to omit field portable verification 

parameters for both the technically trained and untrained operators. First, the Tetracore ELISA 

requires the use of a multichannel micropipettor to precisely manipulate solutions. Using a 

multichannel pipettor is a skill that requires training and practice. The objective of this portion of 

the testing was to evaluate the effectiveness of the Tetracore ELISA as a detection tool for 

untrained operators. Therefore, the test/QA plan was amended to omit this testing parameter for 

technologies such as the Tetracore ELISA that cannot be operated without some acquired 

technical skill. Second, the testing procedure does not change at all when performing tests outside 

the laboratory. The Tetracore ELISA plates and reagents could be packaged into a shoebox-sized 

container and transported to a non-laboratory location. In the manner that the Tetracore ELISA 

was tested, there was no requirement for electrical power. The only items required were a waste 

container and a flat surface. Because observing the analyses in the laboratory was adequate for 

determining the feasibility of using the Tetracore ELISA outside the laboratory, the nonlaboratory testing including the trained operator was omitted from the test/QA plan. 

3.3.3 Drinking Water Characterization 

An aliquot of each DW sample, collected as described in Section 3.2.2, was sent to ATEL prior to 

concentration to determine the following water quality parameters: turbidity; concentration of 

dissolved and total organic carbon; conductivity; alkalinity; pH; concentration of Ca and Mg; 

hardness; and concentration of total organic halides, trihalomethanes, and haloacetic acids. 

9

Table 3-4 lists the methods used to characterize the DW samples, as well as the characterization 

data from the four water samples collected as part of this verification test. Water samples were 

collected and water quality parameters were measured by ATEL in January. Samples were then 

transported and test strips were analyzed from January through March. Because of this, some of 

the water quality parameters may have changed from the time of analysis by ATEL until testing 

with the Tetracore ELISA test strips. 

Table 3-4. ATEL Water Quality Characterization of Drinking Water Samples 

Sources of Drinking Water Samples 

Columbus, Orlando, New York City, MWD, 

Ohio Florida New York California 

Parameter Unit Method (OH DW) (FL DW) (NY DW) (CA DW) 

Turbidity NTU EPA 180.1(5) 0.2 0.5 1.3 0.1 

Dissolved mg/L SM 5310(6) 2 2 2 2 

organic carbon 

Total organic mg/L SM 5310(6) 2 2 2 2 

carbon 

Specific :S/cm2 SM 2510(6) 357 325 85 740 

conductivity 

Alkalinity mg/L SM 2320(6) 55 124 4 90 

pH EPA 150.1(7) 7.33 7.93 6.80 7.91 

Calcium mg/L EPA 200.8(8) 42 41 5.7 35 

Magnesium mg/L EPA 200.8(8) 5.9 8.4 19 1.5 

Hardness mg/L EPA 130.2(7) 125 137 28 161 

Total organic :g/L SM 5320(6) 360 370 310 370 

halides 

Trihalomethanes :g/L/ EPA 524.2(9) 26.9 80.9 38.4 79.7 

analyte 

Haloacetic acids :g/L/ EPA 552.2(10) 23.2 41.1 40.3 17.6 

analyte 

NTU = nephelometric turbidity unit 

MWD = Metropolitan Water District 

:S/cm2 = microSiemen per square centimeter 

10

Chapter 4 


Quality Assurance/Quality Control


Quality assurance/quality control (QC) procedures were performed in accordance with the quality 

management plan (QMP) for the AMS Center

(11)

 and the test/QA plan(4) for this verification test. 

4.1 Sample Chain-of-Custody Procedures 

Sample custody was documented throughout collection, shipping, and analysis of the samples. 

Sample chain-of-custody procedures were in accordance with ASAT II-007, Standard Operating 

Procedure for Chain of Custody for Dioxin/Furan Analysis. The chain-of-custody forms 

summarized the samples collected and analyses requested and were signed by the person 

relinquishing samples once that person had verified that the custody forms were accurate. The 

original sample custody forms accompanied the samples; the shipper kept a copy. Upon receipt at 

the sample destination, sample custody forms were signed by the person receiving the samples 

once that person had verified that all samples identified on the custody forms were present in the 

shipping container. 

4.2 Equipment/Calibration 

The Tetracore ELISA and all appropriate reagents and supplies specific for the detection of 

anthrax, botulinum toxin, and ricin were provided to Battelle by the vendor. Because no plate 

reader was used, the Tetracore ELISA test results were read visually; and, therefore, no calibration 

was required. For DW characterization and confirmation of the possible interferents, analytical 

equipment was calibrated by ATEL according to the procedures specified in the appropriate 

standard methods. Pipettes used during the verification test were calibrated according to Battelle 

Standard Operating Procedure (SOP) VI-025, Operation, Calibration, and Maintaining Fixed 

and Adjustable Volume Pipettes. 

4.3 Characterization of Contaminant Stock Solutions 

4.3.1 Characterization of Botulinum Toxin and Ricin 

Certificates of analysis for botulinum toxin and ricin were provided by the supplier. Because 

standard reference methods do not exist, the concentration of botulinum toxin and ricin were not 

11

independently confirmed. The COAs stated that the ricin standard (Vector Laboratories, Inc., 

Burlingame, California) had a concentration of 1,000 mg/L and the botulinum toxin standards 

(Metabiologics, Inc., Madison, Wisconsin) had concentrations of 2,000 mg/L for Type B and 

1,000 mg/L for Type A. Test samples containing these contaminants were prepared by diluting 

aliquots of these stock solutions with DI water. 

4.3.2 Characterization of Anthrax Spores 

Multiple sources of the Ames strain of Bacillus anthracis (anthrax) were evaluated during this 

verification test. The primary source was a lot of spores prepared by Battelle and stored in a 

1% stock solution of phenol in water as a means to prevent vegetative cell growth. This lot of 

spores is referred to in this report as Battelle-prepared, phenol-preserved. Prior to testing, an 

aliquot of the stock solution described above was centrifuged, the phenol/water solution was 

removed, and the spores were reconstituted with DI water. This process was repeated two times to 

ensure that the spores were suspended only in DI water. This lot of spores was characterized with 

an 11-step characterization process prior to use in the verification test. For confidentiality reasons, 

Table 4-1 gives the outcome of only five of the characterization parameters, as well as the location 

at which each step was performed. These characterization steps were performed when this lot of 

spores was prepared in September 2003. It should be noted that, once a stock solution of spores is 

characterized, less concentrated solutions of spores can be prepared from the stock solution 

without questioning the integrity of the spores. This lot of spores met all 11 acceptance criteria. 

Two parts of the characterization process—DNA sequencing and gene identification—were 

performed by Dr. Alex Hoffmaster at the Epidemiologic Investigations Laboratory, Meningitis 

and Special Pathogens Branch of the Centers for Disease Control and Prevention (CDC). The 

CDC analyses confirmed that the spores were Ames strain anthrax spores, and the guinea pig LD50 

study confirmed their virulence. The stock solution of spores was enumerated after preparation to 

determine its original concentration. In addition, a vegetative cell analysis showed that the stock 

solution was 99.94% anthrax spores. Because at least one spore is needed to spur the growth of a 

colony during an enumeration, the concentrations determined represented a minimum 

concentration of spores. Care was taken to spread the samples to avoid clumping; but, if clumping 

occurred, the spore concentrations would only be higher than shown in the data tables. 

Another lot of anthrax spores from Dugway Proving Ground was obtained and used to investigate 

the sensitivity of Tetracore ELISA to a different spore preparation (referred to as Dugwayprepared in this report). No characterization data except for enumerations were performed on this 

lot of spores. Vegetative Bacillus anthracis was also analyzed during this verification test. 

Vegetative cells from an enumeration of the Battelle-prepared, phenol-preserved spores were 

collected, placed in solution, and then enumerated to determine the concentration of vegetative 

Bacillus anthracis in the solution. No further characterization was performed on these vegetative 

anthrax cells. Solutions of these cells were used to determine the sensitivity of the Tetracore 

ELISA to vegetative cells. 

12

Table 4-1. Characterization Information for Battelle Preparation of Anthrax Spores 

Characterization Outcome Analysis Performed By 

% vegetative cells 0.06% Battelle 

Viable spore count 5.26 ×109

 Battelle 

Guinea pig 10 day LD50 10 spores Battelle 

DNA fingerprinting MLVA Genotype 62 CDC 

PA gene sequencing Protective Antigen Type I CDC 

Regardless of the source and type of anthrax stock solution used to make test samples, its 

concentration was confirmed by a plate enumeration method. This was done within 24 hours of 

any stock solution being used for test sample preparation and is described in Battelle SOP MREF 

X-054, Enumeration of BL-2 and BL-3 Bacteria Samples Via the Spread Plate Technique. In 

addition, four times during the verification test the serial dilution method was validated by 

enumerating the PT samples. For example, for a 109

 spores/mL sample to be enumerated, the 

method requires that it be diluted to at least 103 spores/mL so 100 :L of sample will provide a 

countable number of spores on a culture plate. Therefore, if 100 :L of the 103 spores/mL solution 

provided the correct number of spores to the plate, the concentration of every serial dilution made 

to obtain that concentration was confirmed. 

4.3.3 Anthrax Enumeration Data 

Table 4-2 gives the results of all plate enumerations performed throughout the verification test on 

anthrax solutions prepared in DI water. The data from enumerations to validate the serial dilution 

method are also given in Table 4-2. The expected concentration, as determined from a previous 

enumeration (if available), the actual concentration, and the relative percent difference between 

the two are given in the table. Relative percent difference (RPD) is determined using the 

following equation, where E is the expected concentration and A is the actual concentration as 

determined by the enumeration. 

For the Battelle-prepared, phenol-preserved spores, only one enumeration resulted in a concenE A− RPD = ×100% E 

tration that was more than 25% different from the expected concentration. The average 

concentration of the Battelle stock solution was 6 ×109

 spores/mL (ranging from 5.3 × 109 to 

8.2 × 109

 spores). Over the two-month period that the stocks were used and the enumeration 

performed, the relative standard deviation of the eight results was 15%. The accuracy and 

13

Table 4-2. Anthrax Enumeration Data for PT Samples 

Spore Solution Expected Actual 

Description (units) Date Concentration Concentration(a) RPD 

January 28 53 58 9 

January 28 58 53 9 

January 30 53 61 15 

Battelle-prepared, phenolpreserved stock solution 

(108 spores/mL) 

February 2 

February 10 

February 26 

61 

61 

82 

53 

82 

63 

14 

55 

23 

March 1 63 67 5 

March 23 67 57 14 

Battelle-prepared, phenol- January 28 10 7.8 22 

preserved serial dilution 

validations 

January 30 40 32 20 

(104

 spores/mL) March 2 10 7.7 24 

March 23 1,000 992 1 

Vegetative anthrax March 23 Unknown 26 NA 

(104

 cfu/mL) March 24 260 350 35 

March 22 Unknown 666 NA 

Dugway-prepared 

(106

 spores/mL) March 23 

March 24 

0.010 

10 

0.0081 

8.0 

19 

20 

(a) Each enumeration involved the development of three to five plates. The average, standard deviation, and relative 

standard deviation for each set of Battelle-prepared, phenol-preserved enumeration data were determined, and the 

average relative standard deviation of all enumerations was calculated to estimate the variability in the enumeration 

process, which was 15%. 

NA = not applicable. 

precision of these enumerations indicate that the concentration of the spore stock solution was 

consistent over several months and was usually close to the expected concentration. The serial 

dilution validation data confirm that the PT samples containing the Battelle-prepared, phenolpreserved spores were prepared accurately at various concentration levels. Also shown in Table 4­

2 are the enumerations performed to determine the concentration of the vegetative anthrax cells 

and a stock solution of spores obtained from Dugway Proving Ground. For enumerations with 

unknown expected concentrations, the concentration of that particular solution or the stock from 

which it had been prepared had not previously been determined. 

Table 4-3 gives the enumeration data for all of the interferent PT (shaded) and DW samples that 

were spiked with anthrax spores. For possible interferent samples and samples prepared in DW, 

the addition of spores was confirmed by enumeration for at least one sample representing each 

matrix. The results of the DW samples enumerated in late January and early February indicated 

that the relative difference between the expected concentration and the actual concentration 

ranged from 17 to 96%. The larger percent differences for the DW samples as compared with the 

PT samples were not a surprise, considering that DW is presumably an interferent-prone matrix. 

These data suggest that spore health is dependent on whether the solution is in DI water or DW. 

14

Table 4-3. Anthrax Enumeration Results for Fortified Interferent and Drinking Water 

Samples 

Sample 

Description 

Date 

(2004) 

Expected 

Concentration 

(105

 spores/mL) 

Actual 

Concentration(a) 

(105 spores/mL) RPD 

Conc. CA DW January 28 10 0.38 96 

Conc. CA DW January 30 100 8.7 91 

Unconc. CA DW January 30 40 8 80 

0.5 mg/L OC February 2 15 16 9 

2.5 mg/L OC February 3 15 16 9 

230 mg/L Ca 

90 mg/L Mg February 3 15 5.6 63 

46 mg/L Ca 

18 mg/L Mg February 3 15 8.3 45 

Conc. CA DW February 3 15 6.9 54 

Unconc. CA DW February 3 15 6.5 57 

Conc. OH DW February 3 15 5.7 62 

Unconc. OH DW February 3 15 6.9 54 

Conc. NY DW February 3 15 13 17 

Unconc. NY DW February 3 15 12 21 

Conc. FL DW February 3 15 9.1 39 

Unconc. FL DW February 3 15 7.5 50 

Conc. NY DW March 3 1,000 933 7


Conc. CA DW March 3 1,000 1,100 10


2.5 mg/L OC March 3 1,000 993 1 

230 mg/L Ca 

90 mg/L Mg March 3 1,000 1,000 0 

2.5 mg/L OC March 23 1,000 962 4 

Conc. CA DW March 23 1,000 448 55 

230 mg/L Ca 

90 mg/L Mg March 24 1,000 788 21 

Conc. NY DW March 24 1,000 486 51 

OC = Organic carbon (humic and fulvic acids)


Shading on table distinguishes the interferent and cross-reactivity PT samples from the DW samples.


(a) The uncertainty of the enumeration technique is approximately 15%. 

15 
However, the effect of DW on spore health seemed to be less significant when the concentration 

of spores was higher. For example, in March, when the DW and interferent samples were spiked 

with higher concentrations of anthrax spores, the difference between the expected concentration 

and the actual concentration for the interferent samples was between 0 and 21% and for the DW 

samples between 7 and 55%. Enumerations were performed to characterize the concentration of 

spores in each sample matrix. For each test matrix, spores were enumerated within a day of 

testing. In the Chapter 6 tables, the actual concentrations of the test samples have been corrected 

for the result of the appropriate enumeration for that sample. Because not every test sample was 

enumerated and some of the test samples were the result of dilutions of enumerated samples, not 

every actual concentration will be represented directly in Table 4-2 or Table 4-3. 

The concentrations of the possible cross-reactive interferents of soybean lectin (analogue of ricin) 

and lipopolysaccharide (analogue of botulinum toxin) were not confirmed independent of the 

COA received from the supplier because of the lack of available analytical methodologies for 

these analytes. However, samples containing Bacillus thuringiensis (analogue of anthrax) were 

enumerated and were determined to be approximately an order of magnitude less than expected 

because some spores were lost during washing with water. Because the lowest detectable concentration of anthrax was much more concentrated than Tetracore, Inc., had claimed, additional PT 

samples containing higher concentration levels of anthrax were prepared and analyzed. Additional 

resources were not expended to determine the cross-reactivity of Bacillus thuringiensis at 

comparable concentration levels. 

4.4 Technical Systems Audit 

The Battelle Quality Manager conducted a technical systems audit (TSA) to ensure that the 

verification test was performed in accordance with the test/QA plan(4)

 and the AMS Center 

QMP.

(11)

 As part of the audit, the Battelle Quality Manager reviewed the standards and methods 

used, compared actual test procedures with those specified in the test/QA plan,(4)

 and reviewed 

data acquisition and handling procedures. Observations and findings from this audit were documented and submitted to the Battelle Verification Test Coordinator for response. No findings 

were documented that required any significant action. The records concerning the TSA are 

permanently stored with the Battelle Quality Manager. 

4.5 Audit of Data Quality 

At least 10% of the data acquired during the verification test was audited. Battelle’s Quality 

Manager or designee traced the data from the initial acquisition, through reduction and statistical 

analysis, to final reporting, to ensure the integrity of the reported results. All calculations 

performed on the data undergoing the audit were checked. 

16

4.6 QA/QC Reporting 

Each internal assessment and audit was documented in accordance with Sections 3.3.4 and 3.3.5 

of the QMP for the ETV AMS Center.

(11)

 Once the assessment report was prepared, the Battelle 

Verification Test Coordinator responded to each potential problem and implemented any 

necessary follow-up corrective action. The Battelle Quality Manager ensured that follow-up 

corrective action was taken. The results of the TSA were sent to the EPA. 

4.7 Data Review 

Records generated in the verification test were reviewed before they were used to calculate, 

evaluate, or report verification results. Table 4-4 summarizes the types of data recorded. The 

review was performed by a technical staff member involved in the verification test, but not the 

staff member who originally generated the record. The person performing the review added 

his/her initials and the date to a hard copy of the record being reviewed. 

Table 4-4. Summary of Data Recording Process 

Responsible How Often Disposition 

Data to Be Recorded Party Where Recorded Recorded of Data 

Dates and times of test 

events 

Battelle ETV data sheets Start/end of test, 

and at each 

change of a test 

parameter 

Used to organize/check 

test results; manually 

incorporated in data 

spreadsheets as 

necessary 

Sample collection and 

preparation information, 

including chain-ofcustody 

Battelle ETV data sheets 

and chain-ofcustody forms 

At time of sample 

collection and 

preparation 

Used to organize/check 

test results; manually 

incorporated in data 

spreadsheets as 

necessary 

Detection device 

procedures and sample 

results 

Battelle ETV data sheets Throughout test 

duration 

Manually incorporated 

in data spreadsheets 

Anthrax enumeration 

data 

Battelle Enumeration data 

forms 

With every 

enumeration 

Manually incorporated 

in data spreadsheets 

Reference method 

procedures and sample 

results 

ATEL Data acquisition 

system, as 

appropriate 

Throughout 

sample analysis 

process 

Transferred to 

spreadsheets and 

reported to Battelle 

17

Chapter 5 


Statistical Methods and Reported Parameters


The methods presented in this chapter were used to verify the performance parameters listed in 

Section 3.1. The Tetracore ELISA test kits produce qualitative results; i.e., the color change 

within a sample well on a Tetracore ELISA plate indicates only the presence or absence of a 

contaminant, not a measure of the concentration present. Therefore, the data evaluation methods 

were used in that context. 

5.1 Qualitative Contaminant Presence/Absence 

Accuracy was assessed by reporting the number of positive results out of the total number of 

samples tested for the Tetracore ELISA at each concentration level of contaminant-only PT 

sample tested for anthrax spores, botulinum toxin, and ricin. 

5.2 False Positive/Negative Responses 

A false positive response was defined as a positive response when the DI water or DW sample was 

spiked with a potential interferent, a cross-reactive compound, or not spiked at all. A false 

negative response was defined as a negative response when any sample was spiked with a 

contaminant at a concentration greater than the lowest detectable concentration of the Tetracore 

ELISA for each analyte in DI water. Interferent PT samples, cross-reactivity PT samples, and DW 

samples were included in the analysis. The number of false positive and negative results is 

reported. 

5.3 Consistency 

The reproducibility of the results was assessed by calculating the percentage of individual test 

samples that produced positive or negative results without variation within replicates. 

18

5.4 Lowest Detectable Concentration 

The lowest detectable concentration for each contaminant was determined to be the concentration 

level at which at least two out of the three replicates generated positive responses. These concentration levels are determined for each target contaminant in solutions of DI water. 

5.5 Other Performance Factors 

Aspects of the Tetracore ELISA performance such as ease of use, field portability, and sample 

throughput are discussed in Section 6. Also addressed are qualitative observations of the 

verification staff pertaining to the performance of the Tetracore ELISA. 

19

Chapter 6 


Test Results


6.1 	Qualitative Contaminant Presence/Absence 

The responses for the Tetracore ELISA using the contaminant-only PT samples containing 

anthrax, botulinum toxin, and ricin are discussed in the following sections. The Tetracore ELISA 

provides indication of only a positive or negative response based on whether the color of a 

solution in a sample well changes to a color different from that of the method blank sample. 

6.1.1 Anthrax 

The results obtained for the performance test samples containing anthrax spores are given in 

Table 6-1a. The first five concentration levels listed were initially analyzed, and the results 

indicated that none of those samples (up to 50 times the vendor-stated LOD) produced detectable 

results. The Battelle-prepared, phenol-preserved serial dilution validation enumeration on 

February 2 confirmed the concentration of the stock solution used to prepare the solutions 

analyzed by the Tetracore ELISA. In addition, the serial dilution validation enumeration on 

January 30 (1 × 105

 spores/mL expected) confirmed that anthrax solutions can be accurately 

diluted using standard techniques. After discussions with Tetracore, Inc., the following 

speculative explanations for these results were considered: 

1.	 The target proteins on the spore’s surface may have been stripped off or chemically altered by 

phenol in the storage solution. (The absence or alteration of these proteins would probably 

decrease the sensitivity of the Tetracore ELISA to the affected spores.) 

2.	 The sensitivity of the Tetracore ELISA to anthrax spores is dependent on the method used to 

prepare the spores; therefore, the spores prepared at Battelle may result in decreased 

responsiveness compared with spores prepared elsewhere. 

20

Table 6-1a. Anthrax Contaminant-Only PT Sample Results 

Purpose of 

Analysis 

Actual Fortified 

Concentration(a) 

Anthrax 

Description 

Prep 

Location 

PhenolPreserved 

Positive 

Results Out 

of Total 

Replicates 

200 spores/mL(b) Spores Battelle Yes 0/3 

Original test/QA 

plan PT samples 

2 × 104 spores/mL(c) 

8 × 104 spores/mL 

2 × 105

 spores/mL 

Spores 

Spores 

Spores 

Battelle 

Battelle 

Battelle 

Yes 

Yes 

Yes 

0/3 

0/3 

0/3 

8 × 105 spores/mL Spores Battelle Yes 0/3 

Expanded 

sensitivity 

determination 

8 × 108 spores/mL 

8 × 107 spores/mL 

8 × 106 spores/mL 

Spores 

Spores 

Spores 

Battelle 

Battelle 

Battelle 

Yes 

Yes 

Yes 

3/3 

3/3 

3/3 

8 × 106

 spores/mL Spores Dugway No 0/3 

Alternate spore 

preparation 

8 × 105

 spores/mL 

8 × 104

 spores/mL 

Spores 

Spores 

Dugway 

Dugway 

No 

No 

0/3 

0/3 

8 × 103

 spores/mL Spores Dugway No 0/3 

3 × 105 cfu/mL Vegetative Battelle NA 3/3 

Vegetative cell 

sensitivity 

3 × 104 cfu/mL

3 × 103

 cfu/mL 

 Vegetative 

Vegetative 

Battelle 

Battelle 

NA 

NA 

3/3 

0/3 

3 × 102

 cfu/mL Vegetative Battelle NA 0/3 

(a)	 Actual concentrations were corrected for the enumeration of the stock solution from which each sample was 

prepared. The uncertainty of the enumeration technique is approximately 15%. (b)	 Lethal dose concentration. 

(c) Vendor-stated LOD. 

NA = not applicable. Vegetative cells were not prepared from any stock solution; they were grown and placed in 

solution. 

Additional testing beyond that described in the test/QA plan was performed to explore these 

possible explanations and to gain more information about the performance of the Tetracore 

ELISA. It included evaluating whether Battelle’s storage of the stock solution of anthrax spores in 

a 1% solution of phenol had any impact on the performance of the Tetracore ELISA, increasing 

the concentration of spores beyond what was required by the test/QA plan, and subjecting the 

plates to Ames strain anthrax spores prepared by Dugway Proving Ground using a preparation 

method that is different from the one Battelle uses. 

21

To address the possibility that storing spores in phenol affected the sensitivity of the Tetracore 

ELISA, as well as the possibility that the Tetracore ELISA could not detect the spores prepared by 

Battelle as readily as the spores prepared elsewhere, a series of samples was prepared using one 

anthrax spore stock solution that had been stored in a phenol solution and one that had not. Using 

the Battelle preparation, samples containing approximately 109

, 108, and 107 spores/mL were 

prepared and analyzed. All three concentration levels were detectable, whereas 106

 spores/mL had 

been shown to be not detectable during prior testing. Therefore, the lowest detectable concentration for Battelle-prepared spores was approximately 107

 spores/mL. Spores that were prepared at 

Dugway Proving Ground and received at Battelle in 2001 were then analyzed. Since 2001, the 

Dugway stock solution had been refrigerated as a solution of spores in spent media. The solution 

was washed in DI water as described for the phenol storage solution above and diluted by tenfold 

factors several times to prepare solutions with various concentration levels (107

, 106, 105, and 

104

 spores/mL). Both the stock solution concentration and the dilution methodology were 

confirmed by plate enumeration as shown in Table 4-2. These samples were analyzed to determine 

the approximate sensitivity to these spores. None of these concentration levels were detectable. 

Tetracore informed Battelle that its ELISA is more sensitive to vegetative anthrax than spores. To 

evaluate this premise, a solution of vegetative cells was prepared by collecting a single vegetative 

anthrax colony from an enumeration plate, placing it into DI water, and mixing it well. This 

solution was diluted by a factor of 10 four times, and then the stock solution and two diluted 

samples were enumerated to determine the concentration of vegetative cells in each sample. These 

samples were analyzed to determine the approximate sensitivity for these vegetative cells. The 

lowest detectable concentration of vegetative cells was 3 × 104

 colony-forming units (cfu)/mL, 

approximately the concentration of the vendor-stated LOD for anthrax spores. 

6.1.2 Botulinum Toxin 

The results obtained for the PT samples containing botulinum toxin Types A and B are given in 

Table 6-1b. The results showed that the Tetracore ELISA was reproducibly sensitive to botulinum 

toxin Type A at concentrations as low as 0.02 mg/L. However, for botulinum toxin Type B, the 

results were very inconsistent. The lowest concentrated sample (0.004 mg/L) generated 2 out of 3 

positive results, and the next two higher concentration levels (0.02 and 0.04 mg/L) resulted in 

only 1 positive result out of 6 total replicates. The 0.2-mg/L samples generated 3 out of 3 

detectable results, and the 0.3-mg/L sample generated 1 out of 3 detectable results. These results 

are especially puzzling because the Tetracore ELISA was able to reproducibly detect 0.04 mg/L of 

botulinum toxin Type B when it was spiked into possible interfering matrices, as well as DW. 

There is no certain explanation for the inconsistent botulinum toxin Type B PT sample results. 

22

Table 6-1b. Botulinum Toxin Contaminant-Only PT Sample Results 

Purpose Concentration Positive Results Out of Positive Results Out of Total 

of Analysis (mg/L) Total Replicates (Type A) Replicates (Type B) 

0.004(a) 0/3 2/3 

Botulinum toxin 0.02 3/3 0/3 

PT samples 0.04 3/3 1/3 

0.2 3/3 3/3 

0.3(b) NA(c) 1/3 

(a)

 Vendor-stated LOD. 

(b)

 Lethal dose concentration. 

(c)

 This concentration level was not analyzed using Type A botulinum toxin. 

6.1.3 Ricin 

The results obtained for the PT samples containing ricin are given in Table 6-1c. With the 

exception of the 0.0015 mg/L sample (which generated all negative results), all replicate samples 

analyzed generated positive results. 

Table 6-1c. Ricin Contaminant-Only PT Sample Results 

Purpose Concentration Positive Results Out of 

of Analysis (mg/L) Total Replicates 

0.0015(a) 0/3 

0.0075 3/3 

Ricin PT samples 0.015 3/3 

0.075 3/3 

15(b) 3/3 

(a)

 Vendor-stated LOD. 

(b)

 Lethal dose concentration. 

6.2 False Positive/Negative Responses 

Three types of samples were analyzed to evaluate the susceptibility of Tetracore ELISA to false 

positive and negative results. These included interferent PT samples, made up of DI water 

fortified with Ca and Mg and samples fortified with humic and fulvic acids with and without the 

addition of target contaminants; cross-reactivity PT samples, made up of DI water fortified with a 

contaminant similar biologically or chemically with each specific target contaminant; and DW 

samples both concentrated and unconcentrated and both with and without the addition of target 

contaminants. A false positive result was defined as a positive result in the absence of the target 

contaminant, and a false negative result was defined as a negative result from a sample containing 

detectable levels of each target contaminant. 

23

6.2.1 Interferent PT Samples 

The results from the interferent PT samples are given in Table 6-2. For Tetracore ELISA plates 

coated with antibodies specific to each contaminant, the number of positive results out of the 

number of replicates is given for PT samples containing only the possible interferents and those 

possible interferents in the presence of the listed concentration of target contaminant. For anthrax, 

expanded testing included additional interferent PT samples with a higher concentration of 

anthrax. Results for botulinum toxin Types A and B and ricin are presented. 

For anthrax, no false positive results were generated from the interferent samples that did not 

contain anthrax spores (blanks). The two spiked Ca and Mg interferent samples contained 6 and 

8 × 105 spores/mL and were falsely negative with respect to the vendor-stated LOD. However, 

because these concentrations were very similar to the concentration of the PT samples that were 

also determined to be not detectable, it is not clear if the negative results were due to the 

interferents. For both spiked humic and fulvic acid interferent samples containing 2 × 106 

spores/mL, a concentration that had previously been shown not to be detectable in the PT 

samples, the results were positive. There is not a clear explanation for these positive results. 

Expanded testing was performed to evaluate the performance of the Tetracore ELISA when 

analyzing higher concentrations of anthrax spores. During that testing, 1 × 108 spores/mL, a 

detectable concentration in DI water, were spiked into two interferent solutions. In Ca and Mg, as 

well as in humic and fulvic acids, none of the three replicates had false negative results. 

Table 6-2. Interferent PT Sample Results 

Interferent 

Sample 

Positive Results Out of Total Replicates 

Anthrax (spores/mL) 

Blank 2×106(a) 1×108 

Botulinum Toxin (mg/L) 

Blank 

Type B 

0.04 

Type A 

0.04 

Ricin (mg/L) 

Blank 0.015 

46 mg/L Ca 

18 mg/L Mg 

230 mg/L Ca 

90 mg/L Mg 

0.5 mg/L 

humic and 

fulvic acid 

2.5 mg/L 

humic and 

fulvic acid 

0/3 0/3 

8 × 105(b) 

NA 

0/3 0/3 

6 × 105(b) 

3/3 

8 × 107(b) 

0/3 3/3 

2 × 106(b) 

NA 

0/3 3/3

 2 × 106(b) 

3/3 

1 × 108(b) 

0/3 3/3 NA 

0/3 3/3 3/3 

0/3 0/3 NA 

0/3 3/3 1/3 

0/3 3/3 

0/3 3/3 

0/3 3/3 

0/3 3/3 

NA = not applicable. Sample not analyzed during expanded testing of anthrax or testing of botulinum toxin Type A. (a)

 Expected concentration. 

(b)

 Actual concentration. 

24 
For botulinum toxin, no false positive results were generated from the interferent samples that did 

not contain botulinum toxin. When botulinum toxin Type B was added to the interferent samples 

at 0.04 mg/L, all the results were positive except for the 0.5 mg/L humic and fulvic acid sample. 

This apparent interferent was unexpected because the higher concentration of humic and fulvic 

acid did not cause false negative results. Two interferent samples were fortified with botulinum 

toxin Type A. The Ca and Mg sample generated three out of three positive results (no false 

negatives), but the humic and fulvic acid sample generated only one out of three positive results 

(two false negatives). For ricin, there were no false positive or false negative results with respect 

to the interferent samples. 

6.2.2 DW Samples 

The results from the DW samples are given in Table 6-3. For Tetracore ELISA plates coated with 

antibodies specific to each contaminant, the number of positive results out of the number of 

replicates is given for the DW samples containing no target contaminants and also the DW 

samples in the presence of the listed concentration of each target contaminant. For anthrax, 

expanded testing included additional DW samples containing a higher concentration of anthrax. 

Results for botulinum toxin Types A and B and ricin are also given. 

Table 6-3. DW Sample Results 

DW Sample 

Positive Results Out of Total Replicates 

Anthrax (spores/mL) 

Blank 2×106(a) 1×108 

Botulinum Toxin (mg/L) 

Blank 

Type B 

0.04 

Type A

 0.04 

Ricin (mg/L) 

Blank 0.015 

Unconcentrated 

CA DW 

Concentrated 

CA DW 

Unconcentrated 

FL DW 

Concentrated 

FL DW 

Unconcentrated 

NY DW 

Concentrated 

NY DW 

Unconcentrated 

OH DW 

Concentrated 

OH DW 

0/3 0/3 

7 × 105(b) 

NA 

0/3 0/3 

7 × 105(b) 

3/3 

5 × 107(b) 

0/3 0/3 

8 × 105(b) 

NA 

0/3 0/3 

9 × 105(b) 

NA 

0/3 0/3 

1 × 106(b) 

NA 

0/3 0/3 

1 × 106(b) 

3/3 

5 × 107(b) 

0/3 0/3 

7 × 105(b) 

NA 

0/3 0/3 

6 × 105(b) 

NA 

0/3 3/3 NA 

0/3 3/3 3/3 

0/3 3/3 NA 

0/3 3/3 NA 

0/3 3/3 NA 

0/3 3/3 3/3 

0/3 3/3 NA 

0/3 3/3 NA 

0/3 3/3 

0/3 3/3 

0/3 3/3 

0/3 3/3 

0/3 3/3 

0/3 3/3 

0/3 3/3 

0/3 3/3 

NA = not applicable. Sample not analyzed during expanded testing. 

(a)

 Expected concentration. 

(b)

 Actual concentration. 

25 
For anthrax, there were no false positive results for the unspiked DW samples. Also, the DW 

samples initially spiked with an expected concentration of 2 × 106

 spores, but with actual 

concentrations of between 7 × 105 and 2 × 106

 spores/mL, generated false negative results with 

respect to the vendor-stated LOD. As had been observed for the humic and fulvic acid interferent 

samples, the negative results cannot necessarily be attributed to the DW matrix. The DW samples 

spiked with approximately 1 × 108

 spores/mL generated no false negative results. 

For botulinum toxin, there were no false positive results for the unspiked DW samples or false 

negative results for either the Type B or Type A botulinum toxin. In 24 out of 24 Type B replicate 

samples spiked with 0.04 mg/L botulinum toxin Type B, all the results were positive, again 

contrasting with the PT sample results, where 0.04 mg/L was only detected in 1 out of 3 

replicates. Similar results were generated for ricin. No false positives were observed for the 

unspiked DW samples, and no false negative results were observed for the 0.015 mg/L spiked DW 

samples. 

6.2.3 Cross-Reactivity PT Samples 

The results from the cross-reactivity PT samples are given in Table 6-4. For Tetracore ELISA 

plates coated with antibodies specific to each target contaminant, a PT sample fortified with a 

spore or chemical similar to each target contaminant was analyzed in the absence of any of the 

target contaminant. The number of positive results out of the number of replicates is given for 

each sample. All of the results were correctly reported as negative. 

Table 6-4. Potentially Cross-Reactive PT Sample Results 

Positive Results Out of Total Replicates 

Botulinum 

Anthrax Toxin Ricin 

Bacillus thuringiensis (1 × 104

 spores/mL)

(a) 0/3 

Lipopolysaccharide (0.04 mg/L) 

Lectin from soybean (0.015 mg/L) 0/3 

0/3 

(a)

 Concentration was determined after the fact to be below the lowest detectable concentration. Therefore, the nondetectable results may not indicate a lack of cross-reactivity. 

6.3 Consistency 

For the Tetracore ELISA analysis of anthrax spores and ricin, the results were 100% consistent. 

The Tetracore ELISA plates coated with antibodies specific for ricin generated results for 30 

sample sets of three replicates, and all of them produced results that were either all positive or all 

negative. Similarly, for anthrax, all 47 sets were entirely positive or negative. The botulinum toxin 

results were not as consistent as the anthrax and ricin results. The PT samples containing 

botulinum toxin Type B generated 3 out of 5 sample sets in which the results were not consistent. 

26

All four botulinum toxin Type A PT sample sets, however, generated consistent results. The 

botulinum toxin Type B interferent and DW samples were 100% consistent over 16 sample sets. 

Overall, 98% of the Tetracore ELISA samples analyzed generated results in sets of three in which 

all the individual replicates had the same result, whether positive or negative. 

6.4 Lowest Detectable Concentration 

The lowest detectable concentration of each target contaminant was defined as the lowest 

concentration of contaminant-only PT sample to have at least two out of three positive results. For 

anthrax spores, that concentration was 8 × 106

 spores/mL (for Battelle-prepared spores) and 3 × 

104

 cfu/mL for the vegetative anthrax cells. The spores from the Dugway preparation were not 

detectable at levels up to 8 × 106 spores/mL. For botulinum toxin Type A, the lowest detectable 

concentration was 0.02 mg/L; but, for Type B, it was less clear because of the inconsistent results 

for the PT samples. The PT sample results indicated that the lowest detectable concentration was 

0.004 mg/L, but some concentrations above that were not detectable. However, 0.04 mg/L was 

consistently detectable in the interferent and DW samples, so the LOD is at least 0.04 mg/L. For 

ricin, the lowest detectable concentration was 0.0075 mg/L. 

6.5 Other Performance Factors 

Battelle technicians, who had been trained by Tetracore to perform testing using the Tetracore 

ELISA, performed all of the required laboratory testing. The technicians had no problem 

performing the tests as they were trained. In addition to the antibody-coated Tetracore ELISA 

plates and other reagents, a multichannel pipette, disposable tips, and plastic troughs for filling 

the multichannel pipette with solution were required for testing. Because the Tetracore ELISA 

plates were made from transparent plastic, a color change in a sample well could be observed 

visually against a white background, such as a piece of paper. At times it was difficult to 

determine whether a sample was different from the negative control sample. A plate reader would 

eliminate that judgment call by the operator. 

The performance of the Tetracore ELISA was not demonstrated outside the laboratory because the 

functioning of the tools/equipment required for the Tetracore ELISA is independent of location. 

Also, the procedure for laboratory analysis would not change if testing was moved from the 

laboratory. No carrying case is provided, but a small box could easily be used for transporting the 

supplies needed. The main requirement for a non-laboratory setting would be a well-lighted, flat 

work space. Depending on the conditions, it may be beneficial to cover the ELISA plates during 

incubation to avoid contamination. 

Similarly, testing using an untrained operator was not performed because of the background 

knowledge necessary to understand the detailed procedures for performing the testing and to 

correctly use a multichannel pipettor. The operators with a technical background could analyze 48 

samples on a single plate in approximately five hours. 

27

Chapter 7 

Performance Summary 

Table 7-1. Anthrax Summary Table 

Parameter Sample Information 

Actual Fortified 

Anthrax 

Concentration(a) 

Positive Results Out of 

Total Replicates 

Battelle-prepared, phenolpreserved spores 

8 × 108

 spores/mL 3/3 

8 × 107

 spores/mL 3/3 

8 × 106 spores/mL 3/3 

8 × 105

 spores/mL 0/3 

Contaminantonly PT 

samples 

Vegetative cells 

3 × 105

 cfu/mL 3/3 

3 × 104 cfu/mL 3/3 

3 × 103

 cfu/mL 0/3 

3 × 103

 cfu/mL 0/3 

Qualitative 

contaminant 

results 

Dugway-prepared spores 

8 × 106 spores/mL 0/3 

8 × 105 spores/mL 0/3 

8 × 104 spores/mL 0/3 

8 × 103 spores/mL 0/3 

Interferent 

PT samples 

230 mg/L Ca 

90 mg/L Mg 

8 × 107

 spores/mL(b) 3/3 

2.5 mg/L humic acid 

2.5 mg/L fulvic acid 1 × 108

 spores/mL(b) 3/3 

Humic acid and fulvic acid 2 × 106

 spores/mL(b) 6/6 

Ca and Mg 2 × 106

 spores/mL 0/6 

DW samples 

Concentrated CA 5 × 107

 spores/mL(b) 3/3 

Concentrated NY 5 × 107

 spores/mL(b) 3/3 

Unconcentrated DW 2×106 spores/mL 0/24 

Crossreactivity 1 × 104 spores/mL 

Bacillus thuringiensis unspiked 0/3 

False positives 

No false positives resulted from the analysis of the interferent, DW, or crossreactivity samples. However, two humic and fulvic acid samples, spiked at 

concentrations below what was detectable in DI water, generated positive results. 

Bacillus thuringiensis was prepared at concentrations much lower than the lowest 

detectable concentration of Bacillus anthracis. Therefore, negative results with 

these samples do not necessarily indicate a lack of cross-reactivity. 

28

Parameter Sample Information 

False negatives 

No false negative results were generated for the analysis of interferent or DW 

samples spiked with detectable levels of anthrax. Tetracore ELISA was not able to 

detect anthrax at the vendor-stated LOD, but was able to at much higher 

concentrations. All of the unconcentrated DW and six Ca and Mg samples were 

spiked at concentrations less than detectable by the test strips and, therefore, were, 

as expected, negative. 

Consistency 

Lowest detectable 

concentration 

Other performance factors 

100% (47 out of 47) of the results were obtained in replicate sets in which all the 

individual replicates had the same result, whether positive or negative. 

8 × 106

 spores/mL - Battelle prep (vendor-stated LOD: 2 × 104 spores/mL); 3 × 

104

 cfu/mL - vegetative anthrax (no vendor-stated LOD); the Dugway preparation 

of spores was not detectable at concentrations up to 8 × 106

 spores/mL 

A technically trained operator easily performed the Tetracore ELISA analysis. 

Untrained, non-technical, first-time users would not likely be able to perform the 

testing because of the need to use a multichannel pipettor, prepare solutions, and 

read a technical operating procedure. The Tetracore ELISA could be used outside 

the laboratory without a problem. At times it was difficult to determine whether 

the color of the sample had changed; no reader was used. Sample throughput was 

48 samples in 5 hours. 

(a) The uncertainty of the enumeration technique was approximately 15%. (b) Battelle-prepared, phenol-preserved spores. 

29

Table 7-2. Botulinum Toxin Summary Table 

Parameter Sample Information 

Botulinum Toxin 

Concentration (mg/L) 

Positive Results Out 

of Total Replicates 

ContaminantType A 

0.004 0/3 

0.02 3/3 

0.04 3/3 

0.2 3/3 

Qualitative 

contaminant 

positive 

results 

only PT 

samples 

Type B 

0.004 2/3 

0.02 0/3 

0.04 1/3 

0.2 3/3 

0.3 1/3 

Interferent 

PT samples 

Ca and Mg 0.04 3/3 Type A 

6/6 Type B 

Humic acid and fulvic acid 0.04 1/3 Type A 

3/6 Type B 

DW samples 

Concentrated DW 0.04 6/6 Type A 

12/12 Type B 

Unconcentrated DW 0.04 6/6 Type A 

12/12 Type B 

Crossreactivity 0.04 mg/L 

Lipopolysaccharide unspiked 0/3 

False positives There were no false positive results for the interferent, DW, or cross-reactivity 

samples. 

False negatives 

Two out of three results were false negative when 0.04 mg/L botulinum toxin 

Type A was spiked into 2.5 mg/L humic and fulvic acids, and three out of three 

were false negatives when botulinum toxin Type B was spiked into 0.5 mg/L humic 

and fulvic acids. There were no false negatives for the spiked DW samples. 

Consistency 

With the exception of 2.5 mg/L humic and fulvic acids spiked with 0.04 mg/L 

botulinum toxin Type A (1 out of 3 positive), results generated for botulinum toxin 

Type A were 100% consistent. The DW and interferent samples spiked with 

botulinum toxin Type B were equally consistent, but the contaminant PT samples 

containing botulinum toxin Type B generated consistent results in just 2 out of 5 

sample sets. Overall, 98% of the results were from sample sets that were either all 

positive or all negative. 

Lowest detectable concentration 

0.02 mg/L (Type A); not clear for Type B because of sporadic results. (vendorstated LOD for botulinum toxin [non-specific]: 0.004 mg/L) 

Other performance factors 

A technically trained operator easily performed the Tetracore ELISA analysis. 

Untrained, non-technical, first-time users would not likely be able to perform the 

testing because of the need to use a multichannel pipettor, prepare solutions, and 

read a technical operating procedure. The Tetracore ELISA could be used outside 

the laboratory without a problem. At times it was difficult to determine whether the 

color of the sample had changed; no reader was used. Sample throughput was 48 

samples in 5 hours. 

30

Table 7-3. Ricin Summary Table 

Parameter Sample Information 

Ricin 

Concentration 

(mg/L) 

Positive Results Out of Total 

Replicates 

Qualitative 

contaminant 

positive 

results 

Contaminantonly PT 

samples 

Ricin PT samples 

0.0015 0/3 

0.0075 3/3 

0.015 3/3 

0.075 3/3 

15 3/3 

Interferent PT 

samples 

Ca and Mg 0.015 6/6 

Humic acid and fulvic 

acid 0.015 6/6 

DW samples 

Concentrated CW 0.015 12/12 

Unconcentrated DW 0.015 12/12 

Crossreactivity 0.015 mg/L 

Lectin from soybean unspiked 0/3 

False positives No false positive results were generated for ricin in DW or interferent samples. 

False negatives There were no false negative results for interferent or DW samples spiked with 

detectable concentrations of ricin. 

Consistency 100% of the results for ricin were obtained in replicate sets in which all the 

individual replicates had the same result, whether positive or negative. 

Lowest detectable concentration 0.0075 mg/L (vendor-stated LOD: 0.0015 mg/L) 

Other performance factors 

A technically trained operator easily performed the Tetracore ELISA analysis. 

Untrained, non-technical, first-time users would not likely be able to perform 

the testing because of the need to use a multichannel pipettor, prepare solutions, 

and read a technical operating procedure. The Tetracore ELISA could be used 

outside the laboratory without a problem. At times it was difficult to determine 

whether the color of the sample had changed; no reader was used. Sample 

throughput was 48 samples in 5 hours. 

31

Chapter 8 


References


1.	 Personal communication with Dick Burrows, U.S. Army Center for Health Promotion and 

Preventive Medicine. 

2.	 U.S. EPA threat prioritization study provided by Steve Allgeier, U.S. EPA Office of Water. 

3.	 Center for Defense Information Fact Sheet: Ricin, www.cdi.org/terrorism;ricin-pr.cfm. 

4.	 Test/QA Plan for Verification of Immunoassay Test Kits, Battelle, Columbus, Ohio, 

January 2004. 

5.	 U.S. EPA Method 180.1, “Turbidity (Nephelometric),” Methods for the Determination of 

Inorganic Substances in Environmental Samples, EPA/600/R-93/100, August 1993. 

6.	 American Public Health Association, et al. Standard Methods for the Examination of Water 

and Wastewater. 19th Edition, Washington, D.C., 1997. 

7.	 U.S. EPA, Methods for Chemical Analysis of Water and Wastes, EPA/600/4-79/020, 

March 1983. 

8.	 U.S. EPA Method 200.8, “Determination of Trace Elements in Waters and Wastes by 

Inductively-Coupled Plasma Mass Spectrometry,” in Methods for the Determination of 

Organic Compounds in Drinking Water, Supplement I, EPA/600/R-94/111, October 1994. 

9.	 U.S. EPA Method 524.2, “Permeable Organic Compounds by Capillary Column GC/Mass 

Spectrometry,” Methods for the Determination of Organic Compounds in Drinking 

Water—Supplement III, EPA/600/R-95/131, August 1995. 

10. U.S. EPA Method 552.2, “Haloacetic Acids and Dalapon by Liquid-Liquid Extraction, 

Derivatization and GC with Electron Capture Detector,” Methods for the Determination of 

Organic Compounds in Drinking Water—Supplement III, EPA/600/R-95/131, August 1995. 

11. Quality Management Plan (QMP) for the ETV Advanced Monitoring Systems Center, 

Version 5.0, U.S. EPA Environmental Technology Verification Program, Battelle, Columbus, 

Ohio, March 2004. 

32