September 2009

Environmental Technology 

Verification Report

ABRAXIS ECOLOGENIA® 17β-ESTRADIOL 

(E2) MICROPLATE ENZYME-LINKED 

IMMUNOSORBENT ASSAY (ELISA) TEST KITS

Prepared by

Battelle

Under a cooperative agreement with

 U.S. Environmental Protection Agency

September 2009

Environmental Technology Verification

Report

ETV Advanced Monitoring Systems Center

ABRAXIS ECOLOGENIA® 17β-ESTRADIOL (E2)

MICROPLATE ENZYME-LINKED IMMUNOSORBENT 

ASSAY (ELISA) TEST KITS

By

Stephanie Buehler, Zachary Willenberg, Amy Dindal, Battelle

Eric Kleiner, Michelle Henderson, and John McKernan, U.S. EPA
ii

Notice

The U.S. Environmental Protection Agency, through its Office of Research and Development, 

funded and managed, or partially funded and collaborated in, the research described herein. It 

has been subjected to the Agency’s peer and administrative review and has been approved for 

publication. Any opinions expressed in this report are those of the author (s) and do not 

necessarily reflect the views of the Agency, therefore, no official endorsement should be inferred. 

Any mention of trade names or commercial products does not constitute endorsement or 

recommendation for use.
iii

Foreword

The 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 environmental technology 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 EPA 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.
iv

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 

involvement and support of all staff from the participating laboratories who conducted testing as 

part of this verification test. In particular, we would like to thank the following staff for their 

contribution in conducting this verification at their respective laboratories: Mark Mills and Scott 

Jacobs, U.S. EPA NRMRL, Cincinnati, OH; Jennifer Gundersen, Dave Russell, Ronald Landy, 

Annie Hilliard, John Curry, and Martin Lazarus, U.S. EPA Region 3 Fort Meade, MD; Dennis 

Wesolowski, Larry Zintek, and Charles Steiner, U.S. EPA Region 5 Chicago, IL; Mike Meyer, 

Keith Loftin, Larry Barber, and James Gray, USGS, Kansas; Jim Lazorchak, Tirumuru Reddy, 

and Dan Bender, U.S. EPA NERL, Cincinnati, OH; and Jeanette Van Emon, U.S. EPA NERL 

Las Vegas, NV. Finally, we would like to thank Lisa Olsen, USGS; Paul Pennington, NOAA, 

and Marion Kelly, U.S. EPA, for their review of this verification report.
v

Contents

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

Page

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

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

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

Chapter 1 Background ................................................................................................................... 1

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

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

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

3.2 Test Facilities........................................................................................................................5

3.3 Test Procedures.....................................................................................................................6

3.3.1 Test Sample Collection and Preparation............................................................. 7

3.3.2 Test Sample Analysis Procedure ........................................................................ 8

Chapter 4 Quality Assurance/Quality Control............................................................................. 10

4.1 Quality Control Samples...............................................................................................10

4.1.1 GC-MS Blank and Surrogate Spike Results........................................................... 10

4.1.2 Method Blanks........................................................................................................ 11

4.2 Audits..................................................................................................................................11

4.2.1 Performance Evaluation Audit................................................................................ 11

4.2.2 Technical Systems Audit ........................................................................................ 12

4.2.3 Data Quality Audit.................................................................................................. 13

4.3 QA/QC Reporting ...............................................................................................................13

4.4 Data Review........................................................................................................................13

Chapter 5 Statistical Methods...................................................................................................... 15

5.1 Precision..............................................................................................................................15

5.2 Percent Bias ........................................................................................................................16

5.3 Matrix Effects .....................................................................................................................16

5.4 Operational Factors.............................................................................................................17

Chapter 6 Test Results................................................................................................................. 18

6.1 Precision..............................................................................................................................18

6.2 Percent Bias ........................................................................................................................19

6.3 Matrix Effects .....................................................................................................................20

6.5 Operational Factors.............................................................................................................22

Chapter 7 Performance Summary................................................................................................ 24

Chapter 8 References................................................................................................................... 26
vi

Figures

Figure 2-1. Abraxis 17β-Estradiol (E2) microplate ELISA Test Kit..............................................2

Tables

Table 3-1. Target Analytes ............................................................................................................ 5

Table 3-2. ELISA Test Kit Evaluation Responsibilities for Each Participating Laboratory......... 5

Table 4-1. PE Audit Sample Results............................................................................................ 12

Table 4-2. Summary of Data Recording Process......................................................................... 14

Table 6-1. ELISA Test Kit Average Concentration and Relative Standard Deviation (RSD) 

Results........................................................................................................................................... 19

Table 6-2. ELISA Test Kit Percent Bias vs. GC-MS .................................................................. 19

Table 6-3. GC-MS Average Concentration, RSD, and Percent Bias Results.............................. 20

Table 6-4. ELISA Test Kit Percent Bias vs. Expected Spike Concentration .............................. 21

Table 6-5. Percent Recovery........................................................................................................ 21

Table 6-6. Concentrations of Cross-Reactive Compounds.......................................................... 22
vii

List of Abbreviations

AMS Advanced Monitoring Systems

°C degrees Celsius

COC chain of custody

DI deionized

E1 estrone

E2 17-β-estradiol

E3 estriol

EDC endocrine-disrupting compound

EE2 17-α-ethynylestradiol

ELISA enzyme-linked immunosorbent assay

EPA U.S. Environmental Protection Agency

ETV Environmental Technology Verification

GC-MS gas chromatography–mass spectrometry

GFF glass fiber filter

HPLC high performance liquid chromatography

L liter

LC-MS liquid chromatography–mass spectrometry

MB method blank

MDL method detection limit

μL microliter

μm micron

NERL EPA ORD National Exposure Research Laboratory

ng nanogram

nm nanometer

NP nonylphenol

NRMRL EPA ORD National Risk Management Research Laboratory

ORD EPA Office of Research and Development

ppb

PE

parts per billion

performance evaluation

QA quality assurance

QC quality control

QMP quality management plan

rpm revolutions per minute

RSD relative standard deviation

S standard deviation
viii

SOP standard operating procedure

SPE solid phase extraction

TSA technical systems audit

USGS United States Geological Survey

v/v volume/volume

WWTP wastewater treatment plant
1

Chapter 1

Background

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

Verification Program (ETV) to facilitate the deployment of innovative environmental 

technologies through performance verification and dissemination of information. The goal of 

ETV 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 high-quality, peerreviewed 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 

peer-reviewed 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 Risk Management Research Laboratory (NRMRL) and its verification 

organization partner, Battelle, operate the Advanced Monitoring Systems (AMS) Center under 

ETV. The AMS Center recently evaluated the performance of the Abraxis 17β-estradiol (E2) 

microplate enzyme-linked immunosorbent assay (ELISA) test kit for determining endocrinedisrupting compounds (EDCs) in water.
2

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 report provides results for 

the verification testing of the Abraxis 17β-estradiol (E2) microplate ELISA test kit. The 

following is a description of the test kit, based on information provided by the vendor. 

The 17β-estradiol (E2) microplate ELISA kit applies the principle of ELISA to determine 17βestradiol in water samples. The E2 microplate ELISA kit uses a colorimetric procedure to detect 

17β-estradiol. 

The standards and samples, and an enzyme labeled estradiol conjugate are added to a disposable 

microtiter plate (uncoated), and mixed. 100 microliter (μL) aliquots of the mixture are then 

added to antibody (monoclonal anti-estradiol) coated wells in a 96-well microplate. At this point 

a competitive reaction occurs between the estradiol which may be in the sample and the enzyme 

labeled estradiol for a finite number of antibody binding sites. The reaction is allowed to 

continue for sixty (60) minutes. At the end of the incubation period, the plate contents are 

decanted to remove the unbound reagents from the estradiol and labeled estradiol that remain

bound to the antibodies on the plate, in proportion to their original concentration. After 

decanting, the plate is washed with Washing Solution. A substrate is then added and 

enzymatically converted from a colorless to a blue solution. After an incubation period, the 

reaction is stopped by the addition of diluted acid. The estradiol concentration is determined by 

measuring the absorbance of the sample solution with a photometer (450 nm) and comparing it 

to the absorbance of standards.

The E2 microplate ELISA Kit (Figure 2-1) 

contains a 96-well microplate coated with 

estradiol antibody (mouse anti-estradiol), two 

vials of lyophilized horseradish peroxidaselabeled estradiol analog, two 7 mL vials of 

buffer solution, five 1.5 mL vials of estradiol 

standard concentrations of 0, 0.05, 0.15, 0.4, 1.0 

parts per billion (ppb) with preservatives and 

stabilizers, a 15 mL bottle of hydrogen peroxide 

and 3,3′,5,5′-tetramethylbenzidine solution in an 

organic base, a 15 mL bottle of diluted acid, a 

50 mL bottle of 6X concentrated Washing 

Buffer, one uncoated microtiter plate, and one 

adhesive plate cover.

Figure 2- 1. Abraxis 17β-Estradiol (E2) microplate 

ELISA Test Kit
3

The E2 microplate ELISA Kit measures 7 by 5 by 4 1⁄2 inches. Final results and calibration 

curves are printed from the photometric analyzer or sent directly to a laboratory computer. List 

price is $699 for a 96-test kit. Other materials that are required but are not provided with the E2 

microplate ELISA Kit are pipettes, and a plate photometer capable of reading at 450 nanometer 

(nm). These materials can be purchased separately or rented.
4

Chapter 3

Test Design and Procedures

3.1 Introduction

This verification test was conducted according to procedures specified in the Test/QA Plan for 

Verification of Enzyme-Linked Immunosorbent Assay (ELISA) Test Kits for the Quantitative 

Determination of Endocrine Disrupting Compounds (EDCs) in Aqueous Phase Samples.

1

Deviations to the test/QA plan were made due to unanticipated circumstances. As such, the test 

procedures described in this chapter are a complete description of the actual test conditions.

Because of their potential to interfere with human, domestic animal, and wildlife reproduction, 

EDCs are of increasing concern throughout the country. Several EPA Regions have undertaken 

activities to monitor for these compounds, and several states are considering including 

monitoring for EDCs in their regulatory programs. Presently, gas chromatography–mass 

spectrometry (GC-MS), high performance liquid chromatography (HPLC), and liquid 

chromatography–mass spectrometry (LC-MS) are being used for detecting these compounds. 

However, immunoassay techniques, particularly ELISA, are becoming increasingly popular in 

the field of environmental analysis due to their high sensitivity, ease of use, short analysis time, 

and cost-effectiveness. 

Immunoassay analytical detection is based on the capability of antibodies to specifically 

recognize and form stable complexes with antigens. Immunoassays employ antibodies as 

analytical reagents. In ELISA test kits, an enzyme conjugate competes with the chemical in the 

sample for a limited number of binding sites on the antibody coated plate or particle. The extent 

of color development is inversely proportional to the amount of chemical in the sample or 

standard. The higher the concentration of a specific steroid or other EDC in the sample, the less 

color reaction produced and recorded using a plate reader or tube photometer.

Testing was conducted with multiple collaborating laboratories, specifically the EPA Office of 

Research and Development (ORD) National Risk Management Research Laboratory (NRMRL), 

EPA Region 3, and the United States Geological Survey (USGS) Organic Geochemistry 

Research Laboratory in Kansas. The laboratory participation was coordinated by EPA NRMRL, 

in collaboration with Battelle. Laboratory names are removed, and simply stated as “Laboratory 

(or Lab) 1, 2, and 3” in the test results section, since inter-laboratory comparison was not an 

objective of this report.
5

This verification test evaluated the ability of the Abraxis E2 microplate ELISA test kit to 

quantitate 17-β-estradiol (E2) in four different water matrices, per the manufacturer protocols. 

More detailed information on the EDC tested is provided in Table 3-1. EPA and USGS 

laboratories used the Abraxis E2 microplate ELISA kit (according to Table 3-2) to quantitate 

triplicate spiked samples for hormones (E2), which were prepared and shipped by EPA NRMRL.

The test was conducted in four phases from June to September 2008, with each phase being a 

different aqueous matrix. As the more established method for detecting these compounds, GCMS served as the reference method2 for this test.

Table 3-1. Target Analytes

Analyte Synonyms CAS# Use

(17β)-Estra-1,3,5(10)-

triene-3,17-diol

17-β-Estradiol (E2) 50-28-2 Naturally occurring 

hormone

Table 3-2. ELISA Test Kit Evaluation Responsibilities for Each Participating Laboratory

Responsibility NRMRL Region 3 USGS-KS

Sample Collection, Processing and Distribution √

Test Kit Evaluation – E2 96 well ELISA √ √ √

Reference Measurement - E2 GC-MS √

The Abraxis E2 microplate ELISA test kit was verified by evaluating the following parameters:

• Precision

• Percent bias

• Matrix effects

• Operational factors.

Verification of the system was conducted from June to September 2008. Precision was 

determined by measuring the relative standard deviation of average concentration values as 

reported by the test kit. Percent bias was determined as positive or negative, with positive values 

indicating that ELISA concentration was higher than the reference method and negative values

indicating that it was lower. Matrix effects were determined by comparing the percent bias 

measurements for Phase I deionized (DI) water samples to the percent bias measurements for the 

Phase II through IV matrix-water samples.

Operational factors were determined based on documented observations of the testing staff and 

the Verification Test Coordinator. Operational factors were described qualitatively, not 

quantitatively; therefore, no statistical approaches were applied to the operational factors.

3.2 Test Facilities

Laboratory analyses of the Abraxis E2 microplate ELISA test kit were conducted in three

different collaborating laboratories by the laboratory staff. These laboratories were: EPA ORD 

NRMRL laboratory in Cincinnati, OH; EPA Region 3; and USGS - Kansas. 
6

3.3 Test Procedures

This verification test was conducted in four phases. Phase I consisted of a clean water sample 

(DI water) spiked with a single concentration of E2, split into single samples, and submitted to 

the ELISA kit users in each collaborating laboratory to measure the concentration, in triplicate. 

The split sample, as well as the un-spiked, matrix background sample, were also simultaneously 

sent for reference GC-MS analysis of E2 and for various compounds which are known to cause 

cross-reactivity with some ELISA kits. Phase II consisted of environmental surface water 

samples subjected to the same spiking and splitting process as Phase I. Phase III consisted of a 

complex matrix of wastewater treatment plant (WWTP) effluent samples subjected to the same 

spiking and splitting process as Phase I and II. Phase IV consisted of a complex matrix of 

WWTP influent samples, spiked and split as in previous phases. Details on the sample matrices, 

spiking levels, and spiking procedures for each Phase are provided in Section 3.3.1. All E2 spike 

concentrations used in each phase of this verification test were based on real-world 

concentrations found in environmental samples, per the procedure described in the test/QA plan.1

Background concentrations of E2 were taken for each matrix for each phase. These GC-MS

measurements were made to determine if any measureable amounts of E2 might exist in the 

sample matrix prior to the addition of any sample spikes. If a detectable concentration was 

found, this concentration was then added to the spiked amount of E2 to calculate the total 

concentration for all spiked samples of a particular phase. Specific concentrations of E2, as 

presented in Section 3.3.1, were spiked into the sample matrix for each phase, regardless of any 

background concentrations of these compounds that may have been present in the collected 

water. For Phase III and Phase IV, 4.03 ng/L and 4.00 ng/L of E2, respectively, were found in 

the background matrix samples. The nominal concentration of each sample was then calculated 

using the measured background concentration and the expected spiked concentrations for each 

phase. The ELISA kit results from the various laboratories for the Abraxis test kit were

compared to each other and compared to GC-MS results. 

 

The E2 microplate ELISA test kit was tested only under laboratory controlled conditions, as 

opposed to field conditions which would have been more variable. The analyses were performed 

according to the vendor’s recommended procedures as described in the user’s instructions or 

manual. Simple cleanup procedures, as directed by the manufacturer of the test kit, were used 

for the four different matrices. Each sample was analyzed after solid phase extraction (SPE)

cleanup using the procedure detailed in the kit instructions and provided in Section 3.3.2. Each 

sample for ELISA analysis was filtered through a 1 micron (μm) glass fiber filter prior to SPE. 

Calibration and maintenance of the technology reader (i.e., microplate reader) was performed as 

specified by the vendor. 

A US EPA NRMRL GC-MS standard operating procedure (SOP) was followed for reference 

measurements.2

 The GC-MS method for estrone (E1), E2, estriol (E3), EE2, testosterone, 

dihydrotestosterone, androstenedione, and progesterone operated within a concentration range of 

2-50 ng/L. Samples for the GC-MS methods went through an extraction step to concentrate (or 

dilute, depending upon the sample) to ensure the samples were within the method’s analytical 

range.2

 The procedures for preparing, storing, and analyzing the test samples are provided 

below.
7

3.3.1 Test Sample Collection and Preparation

All sample bottles and glassware associated with hormone samples, including the glass carboy, 

were cleaned and silanized using a procedure included in the test/QA plan1. All samples were 

thoroughly mixed and were thus assumed to contain the same concentration. Samples were 

spiked with E2 as one large stock solution and then split into smaller sub-samples in bottles. All 

sample bottles were amber glass to prevent photodegradation of the analytes. All samples were 

prepared and shipped by NRMRL, immediately after being made, in coolers on ice or freezer 

packs to maintain a 4 degrees Celsius (oC) temperature. When samples were received by each 

laboratory, the condition of the samples, i.e., temperature, broken bottles etc., was noted by the 

receiving laboratory operator and the samples were then immediately placed in a refrigerator at 

4oC until analyzed. Holding times of hormone samples are currently unknown; therefore, all 

samples were either analyzedor solvent exchanged within 24 hours of receipt to reduce error 

associated with analyte degradation during sample holding. All laboratories performing 

quantitative analysis, ELISA or GC-MS, received split samples from the same bulk sample. 

Each laboratory that participated in the ELISA analysis received one 2.5 L spiked sample plus 

one 500 mL DI water method blank. The laboratory that performed the reference analysis 

received one 4 L spiked sample and one 1 L DI water method blank to be processed by the GCMS method. 

3.3.1.1 Phase I Samples

A sample of DI water was collected in a cleaned, 20 L, glass carboy from the USEPA laboratory 

in Cincinnati, Ohio. The water was spiked with E2 to a concentration of 10 ng/L. This

concentration was selected because they are on the higher end of the range of concentrations 

expected to be encountered in a real-world situation and is representative of the anticipated midrange of the test kit. The carboy was thoroughly mixed, by inserting a stir bar and stirring on a 

stir plate at 300 revolutions per minute (rpm) for 2 hours, to ensure homogeneous concentrations 

of the analyte throughout the carboy. One 2.5 L spiked sample was collected for each 

participating laboratory as well as one 4L sample for each reference laboratory. DI water blanks 

were also prepared and shipped in separate 500 mL bottles. The blank samples were analyzed 

after SPE but only in two wells on the kits as opposed to three wells for all other samples. 

Before spiking, the DI water was sampled and analyzed by GC-MS to confirm the background 

levels of E2. Samples of the spiked mixtures were taken and the concentrations of these samples 

and blank were determined using the E2 microplate ELISA test kit and GC-MS.

3.3.1.2 Phase II Samples

Grab samples of stream water were collected in three, clean, five gallon buckets from the South 

Hasha Tributary to Eastfork Lake in Clermont County, Ohio. The tributary was accessed from 

where it crosses Williamsburg-Bantam Road. Before the stream water was spiked, a single 

sample of the collected stream water was taken, split into triplicate aliquots, and analyzed by 

GC-MS to confirm the background levels of E2. Background levels of E2 measured in the 

samples were added to the spiked concentration of E2 once results were obtained. Next, a 

cleaned, 20 L, glass carboy was used to collect 20 L of the stream water, which was then spiked 

to contain a 10 ng/L concentration of E2. The carboy was thoroughly mixed by inserting a stir 

bar and stirring on a stir plate at 300 rpm for 2 hours, to ensure homogeneous concentration of 

the analyte throughout the carboy. Split samples were taken, as noted in Phase I. DI water 
8

method blanks were filled with DI water at the same time as the stream water in the 20 L 

carboys. 

3.3.1.3 Phase III Samples

Grab samples of final effluent wastewater were collected in three, clean, five gallon buckets from 

the Metropolitan Sewer District of Greater Cincinnati in Hamilton County, Ohio. After the 

sample was transported back to the NRMRL laboratory, the effluent was measured and then 

transferred into a clean, 20 L carboy. Before spiking, a single sample of the effluent was taken, 

split into triplicate aliquots, and analyzed by GC-MS to confirm the background levels of E2. In 

a cleaned, 20 L, glass carboy, 20 L of WWTP effluent was prepared containing 10 ng/L of E2. 

The carboy was thoroughly mixed by inserting a stir bar and stirring on a stir plate at 300 rpm for 

2 hours, to ensure homogeneous concentration of the analyte throughout the carboy. Split 

samples were collected, as noted for Phase I. 

3.3.1.4 Phase IV Samples

Grab samples of influent wastewater were collected in three, clean, five gallon bucket from the 

Metropolitan Sewer District of Greater Cincinnati in Hamilton County, Ohio. After the sample 

was transported back to the NRMRL laboratory, the influent was measured and transferred into a

20 L carboy. Before spiking, a single sample of the influent was taken, split into triplicate 

aliquots, and analyzed by GC-MS to confirm the background levels of E2. In a cleaned, 20 L, 

glass carboy, 20 L of WWTP influent was prepared containing 10 ng/L concentration of E2. The 

carboy was thoroughly mixed by inserting a stir bar and stirring on a stir plate at 300 rpm for 2 

hours, to ensure homogeneous concentration of the analyte throughout the carboy. Split samples 

were collected as noted in Phase I. 

3.3.2 Test Sample Analysis Procedure

The ELISA test kit users followed simple cleanup procedures as directed in the vendor’s 

instructions. The 2.5L sample was split into three 500 mL aliquots. Each of the three aliquots 

was analyzed by utilizing glass fiber filter (GFF) cleanup and SPE. Each aliquot sample was 

transferred in triplicate to the 96-well microplate for quantification, per the test kit protocols. 

The cleanup procedures are described below. 

Each sample for ELISA analysis was filtered through a 1 μm GFF. Three 500 mL aliquots were 

removed from the filtered sample for SPE. These three aliquots were treated as three 

independent samples. SPE directions entitled “Extractions for EE2 from Water Sample for 

ELISA”, which were based on the vendor’s protocols and summarized by EPA NRMRL, were 

followed.

1

 The SPE protocol consists of the following steps: 

1. Filter 500 mL of the sample, or the remainder of liquid in the sample bottle noting the volume 

for later calculation, through 1 μm glass fiber filter. 

2. Rinse a C18 SPE cartridge with 5mL of methanol and then 10 mL of distilled water at a flow 

rate not exceeding 20 mL/min (preconditioning). 

3. Pour the filtered sample through the C18 SPE cartridge at a flow rate, no faster than 20

mL/min. 
9

4. Wash the cartridge with 5 mL of distilled water (up to 20 mL/min). Keep suctioning for about 

a minute to dry the cartridge. 

5. Wash the cartridge with 5 mL of hexane (up to 20 mL/min). 

6. Elute the analyte with 5 mL of dichloromethane at a rate, no faster than 3 mL/min. 

7. Evaporate the solvent with nitrogen gas to dryness. 

8. Add 1 mL of 100% methanol to the residue and stir the mixture with a vortex mixer. To 

adjust the content to 10% methanol (volume/volume (v/v)) add 9 mL of distilled water for a total 

volume of 10 mL.

After the SPE column, the E2 samples were reconstituted with 10 mL of a 10% methanol 

solution. For the spiked samples, this process effectively increased the overall spike 

concentration 50 times to an expected level of 505 ng/L. All reconstituted samples were applied 

to three wells (100 μL for each well) according to the manufacturer’s instructions. Samples 

were quantified by reading their photometric responses at a wavelength of 450 nm using a plate 

reader following the manufacturer’s instructions. The general steps for operating the Abraxis E2 

microplate ELISA test kit that were followed during this verification test are provided below. 

The Abraxis ELISA E2 96 well microtiter plate kit assay procedure consists of the following 

steps: 

1. Take the kit out of the refrigerator approximately half an hour before use and let come to room 

temperature (18-25o

C). Filter the sample through a 1μm glass fiber filter and add methanol to 

obtain a final methanol concentration of 10% (v/v). 

2. Dilute the E2 standard concentrate 10 fold with 10% methanol to prepare a 10 μg/L E2 diluted 

stock solution. Then, further dilute this solution to prepare E2 standards of designated 

concentrations, from 50 ng/L to 1000 ng/L. 

3. Reconstitute the antigen-enzyme conjugate powder with 7mL out of 8mL buffer solution. 

4. Mix 100 μL of conjugate solution and 100 μL of E2 standard (or sample) in each well of 

uncoated plate. Dispense the conjugate solution first, then add standard solution of sample. 

Each standard will be added to two wells while each sample will be added to four wells. 

5. Dispense 100 μL aliquots of the standard/sample and conjugate mixture into each coated well 

of the microplate. Incubate it for 60 minutes at room temperature (18-25o

C). 

6. Dilute wash solution (6-fold concentration) in 5 times of its volume of distilled water to 

prepare a wash solution. 

7. Rinse each microplate well with approximately 300 μL of the wash solution and repeat the 

step twice more. Firmly tap out the plate on a lint-free paper towel to remove solution from the 

microplate. 

8. Mix chromogen solution and substrate solution at a ratio of 1:100 to prepare coloring reagent. 

9. Dispense 100 μL of the color solution into each well and incubate it for 30 minutes at room 

temperature (18-25oC). Then, add 100 μL of stop solution to terminate reaction. 

10. Measure the absorbance at 450 nm for each standard solution and generate a standard curve. 

The quantity of E2 in the sample is then calculated from an absorbance reading and interpolation 

from the standard curve.
10

Chapter 4

Quality Assurance/Quality Control

QA/Quality Control (QC) procedures were performed in accordance with the quality 

management plan (QMP) for the AMS Center3 and the test/QA plan for this verification test.1 

Test procedures were as stated in the test/QA plan1; however deviations to the test/QA plan were 

made due to unanticipated circumstances. As such, the test procedures described in Chapter 3 are 

a complete description of the actual test conditions. The statistical calculations intended for 

analysis of the test kit results were also changed. This deviation is further described in Chapter 

5. This change had no impact on the quality of the results. QA/QC procedures and results are 

described below.

4.1 Quality Control Samples 

Steps taken to maintain the quality of data collected during this verification test included 

analyzing specific quality control samples for both the reference method (GC-MS) and the test 

kits. 

4.1.1 GC-MS Method Blank and Surrogate Spike Results

This verification test included a comparison of the Abraxis E2 microplate ELISA test kit results 

to those of the GC-MS reference method for E2. Samples analyzed for each phase included PE 

samples, test samples, background samples, and blank samples. The quality of the reference 

measurements was evaluated by adherence to the requirements of the GC-MS method for this 

compound, including requirements for method blanks (MBs), instrument solvent blanks, and 

surrogate spikes, as indicated in the test/QA plan.

1

 Method blank samples were analyzed to 

ensure that no sources of contamination were present. If the analysis of a method blank sample 

indicated a concentration above five times the method detection limit, contamination was 

suspected. Any contamination source(s) were corrected and samples were reanalyzed or flagged 

before proceeding with the analyses. Surrogate spikes were also included in each sample. 

Average acceptable recoveries for these samples were between 60 and 140%. Samples outside of 

the acceptable range were generally flagged and/or reanalyzed. D4-EE2 was used as a surrogate 

standard for the GC-MS analysis of E2 in the samples. No levels of E2 were detected in any of 

the reference method blank samples.

Surrogate recoveries in Phase I – IV samples varied across phases. Phase I surrogate recoveries 

ranged from 59 – 96% and averaged 85 ± 10% across 13 samples. All recoveries were 

considered in the acceptable range. Phase II recoveries ranged from 77 to 155% and averaged 
11

132 ± 25% over 11 samples. Surrogate recoveries for six of the samples were outside of the 

acceptable range. Compared to the surrogates, the peak shapes for the target analytes were good 

and the baselines were clean in the chromatogram. Phase III surrogate recoveries ranged from 

154% to 197% and averaged 176 ± 14% over 8 samples. Surrogate recoveries for all samples 

were outside of the acceptable range for Phase III. Phase IV surrogate recoveries ranged from 

61% to 93% and averaged 76 ± 10% over 11 samples. Surrogate recoveries for all Phase IV 

samples were within the acceptable range.

4.1.2 Test Kit Method Blanks

Method blank samples were run in duplicate after SPE clean-up with each set of samples for all 

four phases. Method blank samples were unspiked DI water. Because concentrations for 

samples analyzed with the test kit are calculated based on the interpolations from a curve 

constructed from the standards run with each batch of samples, it is possible to obtain 

concentration values for all samples. However, the E2 microplate test kit has a stated method 

detection limit (MDL) of 0.05 – 1 μg/L. Based on this MDL, it is assumed that sample 

concentrations lower than this level cannot be reliably determined or reported. Thus, any 

samples, including method blank samples, with concentrations lower than the manufacturer’s 

stated MDL were considered non-detects. 

The E2 microplate test kit was evaluated by three laboratories (see Table 3-2). Concentrations of 

E2 were not detected in any of the method blank samples from two of the participating 

laboratories. For the USGS results, levels of E2 above the MDL were not detected in Phase II –

IV method blanks. However, during the analysis of Phase I samples, concentrations of E2 above 

the MDL were found for one of the two replicates in both sets of duplicate method blanks for one 

test kit. Two Abraxis E2 microplate ELISA test kits were evaluated by this laboratory for each 

phase of testing. The second test kit operated during Phase I did not show any detectable levels 

of E2 in any of the method blanks analyzed. The detectable levels of E2 found in the method 

blanks were only slightly above the MDL threshold, and averaging the duplicate samples put the 

overall concentration below the MDL. All method blanks for this phase came from the same 

initial sample. 

4.2 Audits

Three types of audits were performed during the verification test: a PE audit of the reference 

method measurements (GC-MS analyses), a technical systems audit (TSA) of the verification 

test performance, and a data quality audit. Audit procedures are described further below.

4.2.1 Performance Evaluation Audit

A PE audit was conducted to assess the quality of the reference method measurements (GC-MS 

analyses) made in this verification test. The reference method PE audit was performed by 

supplying an independent second standard solution of E2 prepared from a different source other 

than that used in verification testing. The PE audit samples were analyzed in the same manner as 

all other samples and the analytical results for the PE audit samples were compared to the 

nominal concentration. The target criterion for this PE audit was agreement of the analytical 

result within 30% of the expected concentration. This audit was performed once during each 
12

phase of testing. Table 4-1 shows the percent error results for the PE samples for each phase. 

The percent error was calculated based on the difference between the actual and expected E2 

concentrations divided by the expected concentration. The E2 PE audit samples were within 

30% of the expected concentration for Phases I, II and IV, while Phase III PE audit samples were 

outside of this agreement range (94% error). The PE results were consistent with the surrogate 

results presented in Section 4.1.1, where Phases I and IV were within specifications; Phase II 

was slightly outside, and Phase III did not meet requirements. The PE audit sample results were 

also similar to the GC-MS sample results for Phase III. This could have impacted the 

comparison of the ELISA test kit results to the GC-MS data for Phase III. No adjustments were 

made to the standards nor were PE audit samples reanalyzed based on these results. It does not 

appear that the reference laboratory results for Phase II and III were overall of lower quality than 

Phases I and IV, since reference results were fairly consistent within phases for the actual 

samples (e.g., see Table 6-2). However, the test kit results were more comparable to the expected 

spiked concentrations (Table 6-3 and Table 6-4) than to the GC-MS results (Table 6-2).

Table 4-1. PE Audit Sample Results

Phase Expected Actual

Concentration (ng/L) Concentration (ng/L) % Error

I 10 9.03 -10

II 10 7.43 -26

III 10 19.4 94

IV 10 8.17 -18

4.2.2 Technical Systems Audit

The Battelle Quality Manager performed a TSA twice during this verification test. Because the 

testing was taking place in multiple laboratories across the country, Battelle’s Quality Manager 

visited only two laboratories for in-person TSAs. Battelle conducted TSAs at the Cincinnati, OH

facility on July 23-24, 2008 and at the Fort Meade, MD facility on July 31, 2008. All TSA 

findings were reported to the Verification Test Coordinator. 

The purpose of this audit was to ensure that the verification test was being performed in 

accordance with the AMS Center QMP,3 the test/QA plan,

1 and the GC-MS SOP2 used during 

the verification test. In the TSA, the Battelle Quality Manager reviewed the reference methods 

used, compared actual test procedures to those specified or referenced in test/QA plan, and 

reviewed data acquisition and handling procedures. The Battelle Quality Manager also toured the 

laboratory where verification and reference testing were taking place,1 inspected sample chain of 

custody (COC) documentation, reviewed technology-specific record books, checked standard 

certifications and technology data acquisition procedures, and conferred with technical staff. A 

TSA report was prepared, including a statement of findings and the actions taken to address any 

adverse findings, and a copy of Battelle’s TSA report was sent to the EPA AMS Center QA

Manager. No adverse findings were reported. The TSA findings were communicated to 

technical staff at the time of the audit.
13

4.2.3 Data Quality Audit 

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

Manager 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. Minor transcription errors and errors due to rounding 

were identified and corrected before the results were used for the calculations described in 

Chapter 5.

4.3 QA/QC Reporting

Each audit was documented in accordance with Section 3.3.4 of the AMS Center QMP.

3 Once 

the audit reports were prepared, the Battelle Verification Test Coordinator ensured that a 

response was provided for each adverse finding or 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 submitted to the EPA.

4.4 Data Review

Records generated in the verification test received an independent internal review before these 

records were used to calculate, evaluate, or report verification results. Table 4-2 summarizes the 

types of data recorded. Data were reviewed by a Battelle technical staff member involved in the 

verification test. The person performing the review added his/her initials and the date to a hard 

copy of the record being reviewed. 
14

Table 4-2. Summary of Data Recording Process

Data Recorded Where Recorded

How Often 

Recorded By Whom

Disposition of 

Data

Dates times and 

details of test 

events

Laboratory record 

books or data 

recording forms, or 

electronically

Start/end of test 

procedure, and at 

each change of a 

test parameter or 

change of 

technology status

Participating 

laboratories

Used to organize 

and check test 

results; manually 

incorporated into 

data spreadsheets 

as necessary

Technology 

calibration 

information

Laboratory record 

books, data 

recording forms, or 

electronically

At technology 

reader calibration 

or recalibration, as 

applicable

Participating 

laboratories

Incorporated into 

verification report 

as necessary

Technology 

readings

Recorded 

electronically or 

manually by the 

operator or 

electronically by 

the technology 

reader, as 

appropriate

Each sample and 

QC analysis

Participating 

laboratories

Converted to or 

manually entered 

into spreadsheets 

for statistical 

analysis or 

comparisons

Sample collection 

and reference 

method analysis 

procedures, 

calibrations, etc. 

Laboratory record 

books, chain-ofcustody, 

electronically, or 

other data 

recording forms

Throughout 

sampling and 

analysis processes

Participating 

laboratories

Retained as 

documentation of 

sample collection 

or reference 

method 

performance

Reference method 

results

Electronically from

reference 

measurement 

technology

Every sample or 

QC analysis

Participating 

laboratories

Transferred to 

spreadsheets for 

calculation of 

results and 

statistical analysis 

or comparisons
15

Chapter 5

Statistical Methods

The statistical methods used to evaluate the quantitative performance factors listed in Section 3.1

are presented in this chapter. Qualitative observations were also used to evaluate verification test 

data. 

Per the test/QA plan,

1 repeatability and reproducibility were intended to be calculated as 

performance parameters for this verification test. However, after further discussion with EPA, 

and in agreement with EPA, it was determined that higher level summary statistics provided a 

better synopsis of the test kit results. Thus, the mean and relative standard deviations (precision) 

were calculated for the test kit results.

5.1 Precision

The standard deviation (S) of the results for the replicate analyses of the same sample was 

calculated as follows:

∑=

− − =

n

k

M M

n

S

1

2 1 / 2

2 ( ) ] 1

1 [

where n is the number of replicate samples, M

k 

is the ELISA test kit measurement for the k

th 

sample, and M is the average ELISA test kit measurement of the replicate samples. The precision 

for each sample is reported in terms of the relative standard deviation (RSD), which was 

calculated as follows:

(%) = ×100

M

S RSD

The RSD was calculated for each laboratory that participated in the verification test and for each 

test kit that was tested. The RSD was also calculated across all laboratories and test kits for each 

phase of testing.

(1)

(2)
16

5.2 Percent Bias

Percent bias was calculated as a percentage for each measurement in each phase for each kit 

using Equation 3:

% Bias = [ 

n

ji

y

x – 1 ] ×100 (3)

where j = 1, 2, 3 denotes the laboratory, i = 1, 2 denotes the ELISA test kit within laboratory, n = 

1, 2 denotes the reference method, xji is the ELISA concentration for the j

th laboratory and the ith

test kit, yn is the concentration of the reference method GC-MS or the concentration of the spike.

Ideally percent bias results will be within ±25%. 

5.3 Matrix Effects

Matrix effects were examined by comparing the percent bias measurements for the Phase I DI 

water samples to the percent bias measurements for the Phase II – IV samples. Percent bias was

determined as described in Section 5.2. 

General observations of potential matrix effects, such as false negatives, if observed, were 

documented but were not used in statistical calculations. False negatives were defined as a 

negative (zero) response in a sample that is spiked with contaminant at a detectable 

concentration. 

General observations on potential cross-reactivity were documented. Blank samples of each 

matrix were evaluated by GC-MS to determine background levels of the compounds with which 

the kits have cross-reactivity, as stated by the vendor. 

Percent recovery results were calculated on a per-sample and per-phase basis and were based on 

the expected spiked concentration of the analyte in each sample matrix. Percent recovery was 

calculated using the Equation 4:

 % Recovery = ×100

E

A (4)

Where A is the actual ELISA test kit measurement and E is the expected concentration. The 

expected concentration includes the known spike concentration as well as any detected 

background levels of E2 in the matrix water (see Section 3.3). Percent recovery results are 

presented to provide another measure of test kit performance to the end user. Ideal percent 

recovery values are near 100%.
17

5.4 Operational Factors

Operational factors were determined based on documented observations of the testing staff. 

Operational factors are described qualitatively, not quantitatively; therefore, no statistical 

approaches were applied to the operational factors.
18

Chapter 6

Test Results

The results of the verification tests of the Abraxis E2 microplate ELISA test kit are presented 

below for each of the performance parameters.

6.1 Precision

The relative standard deviation (RSD) is used as a means of evaluating the precision of the 

ELISA test kit. Three laboratories operated the E2 microplate ELISA test kit. Two laboratories 

(Lab 1 and Lab 2) ran identical samples on two separate test kits (kit “a” and kit “b”). Lab 3 ran a 

single kit. Table 6-1 presents the resulting RSD for each participating laboratory and test kit 

along with the overall average concentrations per phase of E2 found using the E2 microplate

ELISA test kit for all analyses. RSD values are also presented across all results for each phase. 

RSDs among the four phases ranged from 2 to 44%. The overall RSDs for the average 

concentration of Phases II and IV were similar (18% and 16%, respectively). The Phase III 

average concentration had the lowest RSD at 11%, close to the RSDs for Phases II and IV. 

Phase I had the highest RSD among all analyses at 30%, which was approximately twice as high 

as the RSD found among the remaining phases. The average concentration in Phase III was 

higher than that of Phase IV, though both were expected to have the same concentration. 

Similarly, the average concentration of Phase I was higher than that of Phase II. 
19

Table 6-1. ELISA Test Kit Average Concentration and Relative Standard Deviation (RSD) 

Resultsa

Overall Expected

Average Average Phase

Conc (ng/L) RSD Conc (ng/L) RSD Conc (ng/L)

Phase I Lab 1 kit a 597 19%

Lab 1 kit b 595 25%

Lab 2 kit a 443 33% 540 30% 505

Lab 2 kit b 408 44%

Lab 3 655 9%

Phase II Lab 1 kit a 458 12%

Lab 1 kit b 463 5%

Lab 2 kit a 543 8% 489 18% 505

Lab 2 kit b 387 19%

Lab 3 596 7%

Phase III Lab 1 kit a 857 10%

Lab 1 kit b 940 6%

Lab 2 kit a 949 13% 925 11% 706

Lab 2 kit b 916 13%

Lab 3 Out of rangeb

Phase IV Lab 1 kit a 890 3%

Lab 1 kit b 880 11%

Lab 2 kit a 883 5% 874 16% 705

Lab 2 kit b 889 4%

Lab 3 931 2%

a The average concentration and RSD are based on all replicates within the detectable range of the test kit. 

b Test kit results were above the upper end of the test kit’s range.

6.2 Percent Bias

Bias is a systematic error that causes measurements to err in one direction, either high or low. 

For this section, percent bias was calculated relative to the GC-MS reference method results. A 

positive percent bias indicates that the ELISA test kit concentration is higher than the reference 

method, while a negative percent bias indicates that the ELISA test kit concentrations are lower 

than the reference method. Table 6-2 presents the percent bias results. 

Table 6-2. ELISA Test Kit Percent Bias vs. GC-MS

Phase I Phase II Phase III Phase IV

Lab 1 kit a 25 -28 -7 93

Lab 1 kit b 25 -27 2 91

Lab 2 kit a -7 -15 3 91

Lab 2 kit b -14 -39 -1 93

Lab 3 37 -7 14 102
20

Phase I percent bias results were mixed, with some showing positive bias and some showing 

negative. Results from both test kits for Lab 1 and Lab 3 results showed positive bias while 

those for Lab 2 showed negative. Phase III samples were also mixed, with percent bias split 

across test kits evaluated within a laboratory although the results indicated that the test kit and 

GC-MS results for Phase III were similar since the percent bias values were small in magnitude. 

All results for Phase II samples showed a negative bias while all Phase IV samples showed a 

positive bias. Phase IV samples showed the highest bias consistently across all laboratories. All 

biases in Phase IV were positive, covering a small range of positive bias from 91 – 102%. Lab 3 

had the highest bias (either positive or negative) in most phases. Phase III had the least bias 

across all laboratories, with three of the five tests having <5% (positive or negative) bias. 

For comparison, average concentrations, RSD, and percent bias for the GC-MS measurements 

with regard to the expected concentration are presented in Table 6-3 for each phase. RSD values 

were less than 30% for all phases, and percent bias results were within ±35% of the expected 

concentration. These results demonstrate that the GC-MS results were biased high (in Phases II 

and III) and low (in Phases I and IV) as compared to the expected concentration. 

 

Table 6-3. GC-MS Average Concentration, RSD, and Percent Bias Results

Phase

Average

Conc (ng/L) RSD

% Bias 

(vs. Expected Conc)

I 9.53 10% -6

II 12.76 3% 27

III 18.50 1% 31

IV 9.26 7% -34

6.3 Matrix Effects

To understand how the matrix of each phase of testing might have affected the results, percent 

bias and percent recovery were calculated for the test kit results in comparison to the expected 

spiked concentration of E2. A positive percent bias indicates that the ELISA test kit 

concentration is higher than the expected spike concentration, while a negative percent bias 

indicates that the ELISA test kit concentrations are lower than the expected spike concentration. 

Table 6-4 presents the percent bias results. No false negatives were observed during this 

verification test.

Phase III and Phase IV percent bias results were all positive. Though these phases generally 

have the highest bias of all of the phases, all but one of the results for these phases was below 

35% bias. Phase I and II have a mix of positive and negative bias, indicating that the test kit 

results erred both high and low as compared to the expected E2 spike concentrations. Lab 2 kit b 

bias results were negative for both phases while kit a results for Lab 2 were negative in Phase I 

and positive in Phase II. Regardless of whether the bias was positive or negative, bias results for 

all samples tested were similar within phases. Lab 3 percent bias results were greater than 

results from any other laboratory for Phases I, III, and IV. 
21

Table 6-4. ELISA Test Kit Percent Bias vs. Expected Spike Concentration

Phase I Phase II Phase III Phase IV

Lab 1 kit a 18 -9 21 26

Lab 1 kit b 18 -8 33 25

Lab 2 kit a -12 8 34 25

Lab 2 kit b -19 -23 30 26

Lab 3 30 18 >110 32

Phase III and Phase IV percent bias results were all positive. Though these phases generally 

have the highest bias of all of the phases, all but one of the results for these phases was below 

35% bias. Phase I and II have a mix of positive and negative bias, indicating that the test kit 

results erred both high and low as compared to the expected E2 spike concentrations. Lab 2 kit b 

bias results were negative for both phases while kit a results for Lab 2 were negative in Phase I 

and positive in Phase II. Regardless of whether the bias was positive or negative, bias results for 

all samples tested were similar within phases. Lab 3 percent bias results were greater than 

results from any other laboratory for Phases I, III, and IV. 

In terms of positive or negative bias, the comparison of the test kit results to the expected spike 

concentrations (Table 6-4) was similar to the comparison to the GC-MS results (Table 6-2). The 

comparison to the expected concentrations usually showed a smaller amount of bias with the 

exception of Phase III where the magnitudes were higher for the comparison to the expected 

concentrations.

As another measure of accuracy, percent recovery results, comparing the test kit results against 

the expected spiked concentration, were also calculated on a per sample and per phase average 

basis. Table 6-5 presents these results. 

Table 6-5. Percent Recovery

Phase I Phase II Phase III Phase IV

SPE SPE SPE SPE

% Recovery

Lab 1 kit a 118 91 121 126

Lab 1 kit b 118 92 133 125

Lab 2 kit a 88 108 134 125

Lab 2 kit b 81 77 130 126

Lab 3 130 118 >149 132

Average 107 97 130 127

Percent recoveries were close to 100% for Phases I and II and close to 130% for Phases III and 

IV. Phase IV percent recoveries showed little variation between all testing laboratories. Percent 

recoveries were similarly close in Phase III for Labs 1 and 2. Of the 20 samples analyzed for all 

phases, 15 were over-recovered. The percent recoveries for all phases were within the range of 

acceptable recoveries for the GC-MS reference method. Overall, the test kit results were more 

comparable to the expected spiked concentrations (Table 6-4 and Table 6-5) than to the GC-MS 

results (Table 6-2).
22

Some ELISA kits will react with compounds similar to the target compound, known as crossreactivity. The Abraxis E2 microplate ELISA E2 96 well plate test kit will react with known 

percent reactivities to multiple hormones. During each phase of the study, some of the 

compounds with which the test kit has cross-reactivity were measured alongside background 

levels of the kit’s target compound in that matrix by GC-MS. For some of the compounds for 

which there is known cross-reactivity with the E2 microplate ELISA test kit, there are no 

established analytical methods available by GC-MS at these concentrations and in these 

matrices. Therefore some error will have to be accepted from influence of cross-reactive 

compounds that cannot be identified via GC-MS. According to the test kit instructions, there is 

expected to be minimal error from these compounds compared to the primary target compound.

Table 6-6 lists concentrations found in each matrix blank sample from each phase along with the 

known percent reactivities for the cross-reactive compounds to the E2 microplate ELISA test kit

that were measured during this verification. Unfortunately, matrix blank samples used for 

background analysis were not analyzed on the E2 microplate ELISA test kit. Because of this, the

potential for cross-reactive compounds present in the matrix for each phase to interfere with the 

test kit results cannot be truly evaluated. 

Table 6-6. Concentrations of Cross-Reactive Compounds

Steroid Hormones

Concentration (ng/L)

Phase I Phase II Phase III Phase IV Cross Reactivity (%)

 Dihydrotestosterone ND ND ND 43.7 0.38

 Estrone (E1) ND 1.4 44.9 16.8 1.3

 Testosterone (TEST) ND ND 1.7 23.2 <0.03

 Estriol (E3) ND ND 1.4 11.6 0.6

ND = not detected

6.5 Operational Factors

In general, training is needed to effectively and properly operate ELISA test kits. The vendor 

trained staff on the operation of the test kit, but these trained staff were, in some cases, not 

available for the verification test because of testing delays and staff turn over. Therefore, staff 

that operated the test kits during the verification test may not have been trained by the vendor.

Operational concerns or issues were not reported from any of the three participating laboratories. 

The test kit instructions were readily followed by each of the operators. Operation of the test kit 

from the introduction of the sample until the reaction was stopped and the results were read, took 

approximately 2-2.5 hours. Preparation time was required prior to the introduction of the sample 

to allow all reagents time to come to room temperature before using them. Calibrated pipettes, 

test tubes, and a plate reader capable of reading at 450 nm are required for the operation of the 

test kit, but are not supplied with the test kit. GFF or SPE equipment used with the samples were 
23

not supplied with the test kit. For at least one laboratory, the concentration step after SPE was 

time consuming, requiring 1-5 hours. 

Each purchased test kit is capable of conducting 96 tests and costs $699. For comparison, GCMS analyses of these samples are estimated to cost between $500 and $900 per sample. 1

 
24

Chapter 7

Performance Summary

The ability of the Abraxis E2 microplate ELISA test kit to detect E2 in water was evaluated 

using four different water matrices. The test kit was operated by three different laboratories with 

the use of SPE cleanup. The test kit results were evaluated against the expected spike 

concentrations and the reference measurements of the same samples made using GC-MS.

Relative standard deviations (RSDs) among the four phases ranged from 2 to 44%. The overall 

RSDs for the average concentration of Phases II and IV were similar (18% and 16%, 

respectively). The Phase III average concentration had the lowest RSD at 11%, close to the 

RSDs for Phases II and IV. Phase I had the highest RSD among all analyses at 30%, which was 

approximately twice as high as the RSD found among the remaining phases. The average 

concentration in Phase III was higher than that of Phase IV, though both were expected to have 

the same concentration. Similarly, the average concentration of Phase I was higher than that of 

Phase II. 

Percent bias, as compared to the GC-MS reference analysis results, was mixed (positive and 

negative) for Phases I and III. All results for Phase II samples showed a negative bias while all 

Phase IV samples showed a positive bias. Phase III had the lowest percent bias across all 

laboratories. Percent bias, as compared to the expected spiked E2 concentration, was positive for 

Phase III and Phase IV. Phase I and II have a mix of positive and negative bias, indicating that 

the test kit results erred both high and low as compared to the expected E2 spike concentrations. 

Regardless of whether the bias was positive or negative, bias results for all samples tested were 

similar within phases. 

No false negatives were observed during this verification test. Average percent recoveries were 

close to 100% for Phases I and II and close to 130% for Phases III and IV. Of the 20 samples 

analyzed for all phases, 15 were over-recovered. The percent recoveries for all phases were 

within the range of acceptable recoveries for the GC-MS reference method. 

Operational concerns or issues were not reported from any of the three participating laboratories. 

The test kit instructions were readily followed by each of the operators. Operation of the test kit 

from the introduction of the sample until the reaction was stopped and the results were read took 

approximately 2-2.5 hours. Preparation time was required prior to the introduction of the sample 

to allow all reagents time to come to room temperature before using them. Calibrated pipettes, 

test tubes, and a plate reader capable of reading at 450 nm are required for the operation of the 

test kit but are not supplied with the test kit. GFF and SPE equipment used with the samples was 
25

not supplied with the test kit. For at least one laboratory, the concentration step after SPE was 

time consuming, requiring 1-5 hours. Each purchased test kit is capable of conducting 96 tests 

and costs $699. 
26

Chapter 8 

References

1. Test/QA Plan for Verification of Enzyme-Linked Immunosorbent Assay (ELISA) Test Kits 

for the Quantitative Determination of Endocrine Disrupting Compounds (EDCs) in Aqueous 

Phase Samples, Battelle, Columbus, Ohio, July 1, 2008.

2. U.S. EPA NRMRL Standard Operating Procedure (SOP) for the Analysis of Steroid 

Hormones in Aqueous Samples, QA ID 503-P3-0, 09/29/05.

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

Version 6.0, U.S. EPA Environmental Technology Verification Program, Battelle, 

Columbus, Ohio, November 2005.