Specific, Sensitive, and Quantitative Enzyme-Linked Immunosorbent Assay for Human Immunoglobulin G Antibodies to Anthrax Toxin Protective Antigen

Methods

Recombinant anthrax toxin protective antigen (rPA) with an amino acid sequence concurring with that from the Bacillus anthracis V770-NP1-R anthrax vaccine strain was obtained from the National Institute of Craniofacial and Dental Research, National Institutes of Health, Bethesda, MD. Antigen was stored frozen at -80°C in small aliquots (10-100 µL, 4.75 mg/mL) in 5 mM Hepes, pH 7.3. Antigen was expressed from the attenuated asporogenous host B. anthracis BH445 and purified to homogeneity as described.^[4]

To determine the background level of anti-PA ELISA reactivity in a cross-section of the U.S. population, a panel of 238 control sera from healthy adult persons was assembled from the CDC Occupation Health Service and the National Health and Nutrition Examination Survey (NHANES, CDC) serum collections. Donors were selected on the basis of having no known exposure to B. anthracis or anthrax and no known history of anthrax vaccination. In addition, a panel of 277 sera was assembled from persons with clinically confirmed non-anthrax-related illnesses (acute hepatitis A, acute hepatitis B, influenza A and B, brucellosis, staphylococcal toxic-shock syndrome, group A streptococcal infections, legionellosis, Chlamydia pneumoniae infection, and Mycoplasma pneumoniae infection) and from children and adults who had received non-anthrax-related vaccines (trivalent influenza, hepatitis B, tetanus toxoid, and botulinum toxoid). To determine assay sensitivity, an additional panel of 68 sera from persons who had received anthrax vaccine adsorbed (AVA) and 19 control sera from nonvaccinees was obtained. All sera were tested in duplicate without heat inactivation.

The anti-AVA standard human reference serum, AVR414, was prepared by plasmapheresis of healthy adult CDC volunteers who had received at least four subcutaneous injections of Anthrax Vaccine Adsorbed (AVA, BioPort Corp., Lansing, MI) with the licensed regimen (0, 2, and 4 weeks; 6, 12, and 18 months; and yearly boosters). Plasmapheresis and serum conversion were done at the Emory Transfusion Medicine Program, Emory University School of Medicine (Atlanta, GA) and the Scientific Resource Program at CDC, respectively. Plasmapheresis was done by the TPE DUAL- NEEDLE procedure with the COBE Spectra Apheresis System (Gambro BCT, Inc., Blood Component Technology, Lakewood, CO) and following the manufacturer's procedure manual (Manual #701900-000 1999/1). Each plasma unit was clotted with sterile glass microbeads (B. Braun Instruments, Burlingame, CA) and suspended in 1.5 M CaCl₂-2.0 M -amino-caproic acid. All units were allowed to clot overnight at room temperature and were then centrifuged at 2,200 x g at 4°C for 15 min. The serum from each unit was stored in a 500-mL sterile plastic container. The level of residual anticoagulants was not measured. The total IgG concentration of the serum pool was determined by radial immunodiffusion and nephelometry, with the U.S. National Reference Preparation for Specific Human Serum Proteins (CDC) as a standard.^[5] Anti-PA specific IgG mass value assignment to the standard serum was done by differential adsorption, homologous enzyme-linked immunoassay (EIA), and heterologous ELISA (Semenova VA, et al., manuscript in preparation), with U.S. Food and Drug Administration (FDA) 1983 Haemophilus influenzae type b (Hib) reference serum.^[6]

Polyoxyethylene sorbitol monolaurate (Tween 20) was purchased from BioRad Laboratories (Hercules, CA). Skim milk powder was obtained from Difco/Becton Dickinson (Atlanta, GA). Horseradish peroxidase (HRPO)-conjugated mouse anti-human IgG (affinity purified, -chain specific monoclonal clone HP6043) was obtained from Hybridoma Reagent Laboratories (Baldwin, MD). Peroxidase substrate 2,2'-azino-di(3-ethyl-benzthiazoline-6-sulfonate) (ABTS), hydrogen peroxide (H₂O₂), and peroxidase stop solution were obtained from Kirkegaard & Perry Laboratories (KPL, Gaithersburg, MD). All other laboratory reagents were obtained from Sigma Chemical Co. (St. Louis, MO) unless otherwise specified. Sterile, Type I endotoxin-free water was used for all ELISA procedures.

Immulon II-HB flat-bottom 96-well microtiter plates (Thermo Labsystems, Franklin, MA), were coated for 16 hrs at +4°C with 100 µL/well of rPA at a concentration of 2.0 µg/mL in 0.01 M phosphate-buffered saline (PBS), pH 7.4 (Life Technologies, Gaithersburg, MD). Plates were stored at +4°C without blocking and used within 7 days of preparation. Antigen-coated plates were then washed three times (ELX405 microplate washer, BioTek Instruments Inc., Winooski, VT) with PBS containing 0.1% Tween 20 and blotted dry by inversion on clean paper towels. Control and serum antibodies were tested without a separate blocking step. Serum standards and sera for testing were prepared at the appropriate dilutions in PBS containing 5% skim milk and 0.5% Tween 20, pH 7.4. The human standard reference serum and test sera were serially diluted twofold in the plate in the same buffer solution. The minimum dilution of test serum was 1/50. Three positive control sera from three separate donors and one negative control serum were each used at single dilution factors selected to give a range of optical density (OD) values across the standard reference curve. The final volume in all wells was 100 µL.

Test and standard sera were incubated in a humidified chamber (covered tray) for 60 min at 37°C, and the plates were then washed three times with PBS containing 0.1% Tween 20. Bound anti-PA IgG was then detected by using HRPO-conjugated mouse anti-human IgG Fc PAN monoclonal HP6043 diluted in PBS containing 5% skim milk and 0.5% Tween 20 (100 µL/well), and plates were incubated in a humidified chamber (covered tray) for 60 min at 37°C. Plates were again washed three times with PBS containing 0.1% Tween 20, and bound conjugate was detected colorimetrically by using ABTS/H₂O₂ substrate (100 µL/well). Color development was over 30 min (±5 min) and was stopped by addition of 100 µL of Peroxidase Stop Solution (KPL) to all wells of the test plates. OD values were read within 30 min of addition of the stop solution with a MRX Revelation microtiter plate reader (Thermo Labsystems, Franklin, MA) at a wavelength of 410 nm with a 610-nm reference filter. Data were analyzed by using a four-parameter (4-PL) logistic-log curve fitting model with ELISA for Windows software.^[7] A calibration factor for the standard reference serum was used to determine the concentration of anti-PA IgG in micrograms per milliliter of serum (µg/mL).

To enhance specificity, a supplementary rPA competitive inhibition ELISA (CI-ELISA) was developed based on the qualified anti-PA IgG ELISA. The CI-ELISA was a direct extension of the standard ELISA procedure with the following modifications. The anti-PA antibody concentrations of the test sera were first determined by using the standard ELISA. Only sera with a minimum reactivity level of 10 µg/mL anti-PA antibody were suitable for evaluation in the CI-ELISA. The 10-µg/mL threshold was determined empirically as the minimum level for which a reduction in ELISA reactivity could be assigned with statistical significance. A concentration of 50 µg rPA/500 µL diluted sample was chosen as the absorbing concentration after a preliminary study with ranges between 0 and 200 µg/mL.^[8] Test sera were then diluted to a concentration calculated to provide an OD value of approximately 1.0, based on their reactivity in the standard anti-PA ELISA. A 1-mL volume of each diluted serum was prepared and divided into two aliquots of equal volume. To one of these aliquots, rPA was added to a final concentration of 100 µg/mL. Both tubes were capped tightly and mixed by inversion for 16-18 hrs at +4°C. After this incubation, the tubes were centrifuged at 4°C for 10 min at 8,000 x g to remove precipitated materials. Test sera were incubated in the presence and absence of an excess of rPA in solution before analysis in the standard ELISA.

The supernatants were used without further dilution in the standard ELISA described above. Based on defined sera from anthrax vaccine recipients and confirmed clinical cases, a ≥85% suppression of reactivity in the competitive ELISA was identified as the threshold to discriminate between true positives and false positives.

Accuracy describes the exactness of the assay to measure a known, true value of anti-PA IgG and to measure it repeatedly. In this study, accuracy was determined by repeated analysis of a positive control human anti-AVA antiserum for which differential absorption and heterologous ELISA had determined the anti-PA IgG concentration. Accuracy is expressed as the percent error between the assay-determined value and the assigned value for that serum. A percent error of ≤20% is an acceptable level of accuracy for an enzyme immunoassay.^[9] Precision, a measure of the degree of repeatability of an assay under normal operating conditions, is expressed as the coefficient of variation of the concentrations calculated for the standard reference curve dilutions within a single assay plate (intraassay precision) and between different assay plates (interassay precision) determined over time and controlling for different operators. Acceptable levels of intraassay and interassay precision are 10% and 20%, respectively,^[9] and these can be used to define the range of the assay and the upper and lower limits of quantification. The range of the assay is the interval between the upper and lower levels of antibody (inclusive) that have been demonstrated to be determined with these levels of precision and accuracy.

The "goodness of fit" of the assay is, for comparative purposes, an indication of how closely the data points of the reference serum standard curve fit the 4-PL model. Goodness of fit is expressed as the regression coefficient (R²) of the standard curve. An R² value that approaches unity is indicative of a good fit for the data to the curve.^[9]

The 4-PL function was used to model the characteristic curve for the standards data. These data exhibit a sigmoidal shape when plotted on an OD-log₁₀ dilution scale. The 4-PL function fits these data with a high degree of accuracy and extends the range of the assay, thus providing a more precise measurement of antibody concentration for patient sera.^[10] The lowest concentration of analyte (anti-PA IgG) that can be detected with a specific degree of probability in a diluted serum sample is defined as the minimum detectable concentration (MDC). The lowest concentration of analyte that has a high probability of producing a response significantly greater than the response at zero concentration of analyte is defined as the reliable detection limit (RDL). The MDC and RDL of the anti-PA IgG ELISA were derived from a 4-PL fit applied to the AVR414 standard reference serum.^[9] The MDC is the concentration of anti-PA antibody corresponding to the interpolated intersection of the lower asymptote of the upper 95% confidence interval (95% CI) with the 4-PL fit of the standards data. The RDL is the concentration of anti-PA antibody corresponding to the interpolated intersection of the upper 95% CI asymptote with the lower -95% CI of the standards data. The MDC and RDL are thus both derived from the 95% CIs of the standard curve. They are distinct and statistically robust measurements of the lower limits of detection of the assay; the RDL is the more conservative of the two. An illustration of the relationship of MDC and RDL to the standard curve is shown (Figure).

The reactivity threshold (Figure) is used to categorize a serum as reactive or nonreactive and to determine the diagnostic sensitivity (DSN) and diagnostic specificity (DSP) of the assay. The reactivity threshold of this assay was determined from the frequency distribution^[11] of log₁₀-transformed OD values from a panel of sera from humans with non-anthrax-related clinical infections (554 observations) and a panel of control human sera (476 observations). The reactivity threshold was determined as the upper 95% CI of the frequency distribution from log₁₀-transformed OD values of control human sera tested at 1/50 dilution. This OD value was converted to an anti-PA IgG concentration by using the standard curve calibration factor. Where this calculated value is below the MDC of the assay, the MDC becomes the default reactivity threshold. Ideally, the MDC, RDL, and reactivity threshold will all fall within the limits of quantification as defined above.

The DSP and DSN of the anti-PA IgG ELISA were determined. The quantitative test results were categorized into reactive or nonreactive by application of the reactivity threshold. The DSP of the assay was calculated as [TN/(TN+FP)], where TN = true negatives and FP = false positives. The DSN of the assay was calculated as [TP/(TP+FN)], where TP = true positives and FN = false negatives. Initially, serum specimens from clinical anthrax cases were insufficient to be useful in determining the DSN of the anti-PA IgG ELISA. Thus, the DSN was calculated by using sera from a cohort of anthrax vaccine recipients who had received a minimum of four subcutaneous injections of AVA. The DSN of the assay was reevaluated at the end of the anthrax emergency, when a greater number of specimens from clinical cases had accumulated.

B. anthracis Spores

The qualified anti-PA ELISA was applied to sera from persons with confirmed or suspected anthrax cases and from persons exposed to B. anthracis spores. Blood was collected in serum separation tubes and allowed to clot; the serum was then separated from clotted cells by low-speed centrifugation. Serum was shipped to CDC with a unique identification number. All clinical serum samples were blinded to the laboratory team and tested in duplicate. All ELISA-reactive sera were tested a minimum of twice. The CI-ELISA was applied to single serum specimens with a reactivity of ≥10 µg/mL and when persons' paired sera indicated reactivity in the absence of changing anti-PA antibody concentrations over time. A ≥4-fold rise over the calculated value for the acute serum or the assay reactivity threshold was used to define seroconversion.

Anti-PA IgG concentrations in test sera were calculated by interpolation to the standard reference calibration curve by using the ELISA for Windows Software Version 1.0;^[7] anti-PA IgG concentrations were expressed in micrograms per milliliter of the original serum sample. For results to be reportable, the assay was required to meet a set of quality control acceptance criteria. For an acceptable level of precision, the mean anti-PA IgG concentrations for three separate quality control sera were required to calculate within 3 standard deviations (SDs) of their assigned mean concentrations; at least two of the mean anti-PA IgG concentrations for these sera were required to be within 2 SDs of their respective assigned mean values. Assay plates were also evaluated for parallelism between the standard curve and the test samples.^[12]

Emerging Infectious Diseases. 2002;8(10) © 2002 Centers for Disease Control and Prevention (CDC)

Cite this: Specific, Sensitive, and Quantitative Enzyme-Linked Immunosorbent Assay for Human Immunoglobulin G Antibodies to Anthrax Toxin Protective Antigen - Medscape - Oct 01, 2002.