Detection of anthrax protective antigen (PA) using europium labeled anti-PA monoclonal antibody and time-resolved fluorescence.

Inhalation anthrax is a rare but acute infectious disease following adsorption of Bacillus anthracis spores through the lungs. The disease has a high fatality rate if untreated, but early and correct diagnosis has a significant impact on case patient recovery. The early symptoms of inhalation anthrax are, however, non-specific and current anthrax diagnostics are primarily dependent upon culture and confirmatory real-time PCR. Consequently, there may be a significant delay in diagnosis and targeted treatment. Rapid, culture-independent diagnostic tests are therefore needed, particularly in the context of a large scale emergency response. The aim of this study was to evaluate the ability of monoclonal antibodies to detect anthrax toxin proteins that are secreted early in the course of B. anthracis infection using a time-resolved fluorescence (TRF) immunoassay. We selected monoclonal antibodies that could detect protective antigen (PA), as PA83 and also PA63 and LF in the lethal toxin complex. The assay reliable detection limit (RDL) was 6.63×10(-6)µM (0.551ng/ml) for PA83 and 2.51×10(-5)µM (1.58ng/ml) for PA63. Despite variable precision and accuracy of the assay, PA was detected in 9 out of 10 sera samples from anthrax confirmed case patients with cutaneous (n=7), inhalation (n=2), and gastrointestinal (n=1) disease. Anthrax Immune Globulin (AIG), which has been used in treatment of clinical anthrax, interfered with detection of PA. This study demonstrates a culture-independent method of diagnosing anthrax through the use of monoclonal antibodies to detect PA and LF in the lethal toxin complex.

Sensitivity and specificity of serologic assays for detection of human infection with 2009 pandemic H1N1 virus in U.S. populations.

Swine origin 2009 H1N1 influenza virus has spread globally to cause the first influenza pandemic of the 21st century. Serological studies can improve our understanding of the extent of human infection and risk factors associated with the transmission of this pandemic virus. The "gold standard" for serodiagnosis of human influenza virus infection is the detection of seroconversion between acute- and convalescent-stage samples. However, the timing of seroepidemiological investigations often precludes the collection of truly acute-phase sera, requiring development of serological criteria for evaluating convalescent-phase sera that optimize detection of true positives and true negatives. To guide seroepidemiological investigations into the spread of the novel 2009 pandemic H1N1 virus, we characterized serum antibody responses to 2009 H1N1 virus in 87 individuals with confirmed viral infection and 227 nonexposed U.S. individuals using microneutralization (MN) and hemagglutination inhibition (HI) assays. Sensitivity and specificity were determined for each assay alone and in combination for detection of 2009 H1N1 virus-specific antibodies in convalescent-phase sera. Although the HI assay was more specific for detecting antibody to 2009 H1N1, the MN assay was more sensitive, particularly for detecting low-titer seroconversions. A combination of titers (MN ≥ 40 and HI ≥ 20) provided the highest sensitivity (90%) and specificity (96%) for individuals aged <60 years and 92% specificity for adults aged ≥ 60 years for detection of serologically confirmed 2009 H1N1 infections in U.S. populations during the first pandemic waves. These studies provide an approach to optimize timely serological investigations for future pandemics or outbreaks of novel influenza viruses among humans.

A two-stage, multilevel quality control system for serological assays in anthrax vaccine clinical trials.

A two-stage, multilevel assay quality control (QC) system was designed and implemented for two high stringency QC anthrax serological assays; a quantitative anti-PA IgG enzyme-linked immunosorbent assay (ELISA) and an anthrax lethal toxin neutralization activity (TNA) assay. The QC system and the assays were applied for the congressionally mandated Centers for Disease Control and Prevention (CDC) Phase 4 human clinical trial of anthrax vaccine adsorbed (AVA, BioThrax). A total of 57,284 human serum samples were evaluated by anti-PA enzyme-linked immunosorbent assay (ELISA) and 11,685 samples by anthrax lethal toxin neutralization activity (TNA) assay. The QC system demonstrated overall sample acceptance rates of 86% for ELISA and 90% for the TNA assays respectively. Monitoring of multiple assay and test sample variables showed no significant long term trends or degradation in any of the critical assay reagents or reportable values for both assays. Assay quality control data establish the functionality of the quality control system and demonstrates the reliability of the serological data generated using these assays.

Effects of a reduced dose schedule and intramuscular administration of anthrax vaccine adsorbed on immunogenicity and safety at 7 months: a randomized trial.

CONTEXT: In 1999, the US Congress directed the Centers for Disease Control and Prevention to conduct a pivotal safety and efficacy study of anthrax vaccine adsorbed (AVA). OBJECTIVE: To determine the effects on serological responses and injection site adverse events (AEs) resulting from changing the route of administration of AVA from subcutaneous (s.q.) to intramuscular (i.m.) and omitting the week 2 dose from the licensed schedule. DESIGN, SETTING, AND PARTICIPANTS: Assessment of the first 1005 enrollees in a multisite, randomized, double-blind, noninferiority, phase 4 human clinical trial (ongoing from May 2002). INTERVENTION: Healthy adults received AVA by the s.q. (reference group) or i.m. route at 0, 2, and 4 weeks and 6 months (4-SQ or 4-IM; n = 165-170 per group) or at a reduced 3-dose schedule (3-IM; n = 501). A control group (n = 169) received saline injections at the same time intervals. MAIN OUTCOME MEASURES: Noninferiority at week 8 and month 7 of anti-protective antigen IgG geometric mean concentration (GMC), geometric mean titer (GMT), and proportion of responders with a 4-fold rise in titer (%4 x R). Reactogenicity outcomes were proportions of injection site and systemic AEs. RESULTS: At week 8, the 4-IM group (GMC, 90.8 microg/mL; GMT, 1114.8; %4 x R, 97.7) was noninferior to the 4-SQ group (GMC, 105.1 microg/mL; GMT, 1315.4; %4 x R, 98.8) for all 3 primary end points. The 3-IM group was noninferior for only the %4 x R (GMC, 52.2 microg/mL; GMT, 650.6; %4 x R, 94.4). At month 7, all groups were noninferior to the licensed regimen for all end points. Solicited injection site AEs assessed during examinations occurred at lower proportions in the 4-IM group compared with 4-SQ. The odds ratio for ordinal end point pain reported immediately after injection was reduced by 50% for the 4-IM vs 4-SQ groups (P < .001). Route of administration did not significantly influence the occurrence of systemic AEs. CONCLUSIONS: The 4-IM and 3-IM regimens of AVA provided noninferior immunological priming by month 7 when compared with the 4-SQ licensed regimen. Intramuscular administration significantly reduced the occurrence of injection site AEs. Trial Registration clinicaltrials.gov Identifier: NCT00119067.

Standardized, mathematical model-based and validated in vitro analysis of anthrax lethal toxin neutralization.

Quantification of anthrax lethal toxin (LTx) neutralization activity (TNA) is pivotal in assessing protective antibody responses to anthrax vaccines and for evaluation of immunotherapies for anthrax. We have adapted and redesigned the TNA assay to establish a unifying, standardized, quantitative and validated technology platform for LTx neutralization in the J774A.1 murine cell line. Critical design features of this platform are 1) the application of a free-form or constrained 4 parameter logistic (4-PL) function to model neutralization responses within and between boundary limits of 100% cell survival and 95% cell lysis and 2) to exploit innovative assay curve recognition algorithms for interpretive endpoints. The assay was validated using human serum ED50 (dilution of serum effecting 50% neutralization) as the primary reportable value (RV). Intra-operator and intermediate precision, expressed as the coefficient of variation (%CV), were high at 10.5-15.5%CV and 13.5-14.5%CV respectively. TNA assay dilutional linearity was demonstrated for human sera using linear regression analysis of log(10) transformed data with slope=0.99, intercept=-0.03 and r(2)=0.985. Assay accuracy, inferred from the precision and linearity data and using a spike-recovery approach, was high with a percent error (%E) range of only 3.4-20.5%E. The lower limit of detection (LLOD) was ED50=12 and the lower limit of quantification (LLOQ) was ED50=36. The cell-based assay was robust, tolerating incubation temperatures from 35 to 39 degrees C, CO(2) concentrations from 3% to 7% and reporter substrate (MTT) concentrations of 2.5-7.5 mg/ml. Strict assay quality control parameters were met for up to 25 cell culture passages. The long term (50 month) assay stability, determined using human reference standards AVR414 and AVR801, indicated high precision, consistent accuracy and no detectable assay drift. A customized software program provided two additional assay metrics, Quantification Titer (QT) and Threshold Titer (TT), both of which demonstrate acceptable accuracy, precision and dilutional linearity. The TT was also used to establish the assay reactivity threshold (RT). The application of the assay to sera from humans, Rhesus macaques and rabbits was demonstrated separately and by aggregate dilutional linearity analysis of the ED50 (slope=0.98, intercept=0.003, r(2)=0.989). We propose this TNA assay format with a qualified standard reference serum and customized interpretive software as a unifying platform technology for determination of functional serologic responses to anthrax vaccines and for evaluation of anthrax immunotherapeutics.

Modification of rapid human immunodeficiency virus (HIV) antibody assay protocols for detecting recent HIV seroconversion.

Assay protocols of three rapid human immunodeficiency virus (HIV) assays, OraQuick-1/2, SeroStrip-1/2, and Determine-1/2, were modified to detect recent HIV seroconversion using a higher dilution of serum specimens. Optimal predilution of specimens resulted in negative test results during early periods of seroconversion (about 6 months), when antibody levels were low. A total of 269 seropositive specimens from routine HIV type 1 testing and from commercial sources (low-titer and seroconversion panels) were tested, and results were recorded as negative (score=0) or positive using intensity scores from 0.5 (weak positive) to 4 (strongly positive). The same specimens were previously tested by a less sensitive (LS) enzyme immunoassay (EIA), Abbott 3A 11-LS, and were classified as recent or long-term infections based on the standardized optical density (SOD) cutoff of 0.75. Overall concordance of >94% was observed between 3A 11-LS and modified rapid tests (RT-LSs) for detecting and distinguishing recent HIV seroconversion from long-term HIV infection (kappa statistics=0.894 to 0.901). Moreover, intensity scores on RT-LSs correlated well with median 3A 11-LS SOD values (R(2)>0.98). Our results indicate that rapid HIV tests can be modified to detect recent seroconversion with results comparable to those from less sensitive EIA.

The use of simple, rapid tests to detect antibodies to human immunodeficiency virus types 1 and 2 in pooled serum specimens.

BACKGROUND: The use of pooled specimens has been proposed as a means of expanding testing for human immunodeficiency virus (HIV) antibodies in population studies and in blood screening, while reducing laboratory costs. OBJECTIVES: To develop a strategic specimen pooling method to be used with rapid HIV antibody assays to detect positive specimens and to evaluate its performance in comparison with testing with commercial EIA and WB. STUDY DESIGN: Two lateral flow rapid HIV antibody assays, Seroz*Strip HIV-1/2(1) and Determine HIV-1/2, were evaluated for their ability to detect HIV-1 antibodies in serum and/or plasma specimens pooled in sizes ranging from two to 20 following the respective manufacturers' protocols. One thousand prospectively collected specimens and 55 seroconversion specimens were prepared in pools of five for evaluation by the two rapid HIV assays. RESULTS: Optimal detection and discrimination of HIV-1 antibody-positive and HIV-1 antibody-negative specimens was observed in pool sizes of five to ten for both assays. The ability of the two rapid assays to detect HIV-1 antibody-positive samples from commercial HIV-1 seroconversion panels contained in the pools was equivalent to that of commercial enzyme immunoassays (EIAs) and Western blot (WB) to detect HIV-1 antibody in the non-pooled samples. Application of the pooling method in prospectively collected specimens yielded excellent concordance with EIA/WB results in both sensitivity (98.88% for Seroz*Strip HIV-1/2, 100% for Determine HIV-1/2) and specificity (99.56% for Seroz*Strip HIV-1/2, 99.45% for Determine HIV-1/2). CONCLUSION: Use of a pooling strategy with either assay reduced the number of tests required by almost 50% and could provide substantial cost reductions for HIV screening in settings where HIV-1 prevalence is less than 10%.