Simultaneous measurement of specific serum IgG responses to five select agents.

Select Agents are defined by CDC and the USDA Animal and Plant Health Inspection Service (APHIS) as biological agents or toxins deemed a threat to public, animal, or plant health, or to animal or plant products. They are classified on the basis of their ease of dissemination, mortality/morbidity rate, and potential for social disruption. A subset of these agents includes Bacillus anthracis, Yersinia pestis, Francisella tularensis, ricin toxin (RT), and staphylococcal enterotoxin B (SEB). Infection or intoxication with these agents has been shown to elicit an antigen-specific serum IgG response. We describe a fluorescent covalent microsphere immunoassay (FCMIA) for measurement of specific IgG antibodies to seven different antigens from five different select agents; B. anthracis [protective antigen (PA) and lethal factor (LF)], Y. pestis (F1 and V antigens), F. tularensis, RT and SEB simultaneously in human B. anthracis vaccinee sera (containing anti-PA and anti-LF IgG) which had been spiked with animal specific IgG antibodies to the other select agents. Inter-assay and intra-assay coefficients of variation were 6.5 and 13.4%, respectively (N = 4). There were no significant differences (P > 0.70) between assay responses when the assays were performed individually or multiplexed. When the observed versus expected interpolated concentrations were compared, highly linear relationships were observed (r2 values from 0.981 to 0.999, P < 0.001). Minimum detectable concentrations (MDC) ranged from 0.3 ng mL(-1) (Y. pestis F1) to 300 ng mL(-1) (RT). Finally, the curves showed responses were linear for most analytes from their MDC to 125 (SEB) to 1,300 (Y. pestis F1) x their MDC. These data indicate that multiplexed FCMIA is a sensitive and accurate method for simultaneous measurement of specific IgG in serum to CDC select agents and may be of value in screening either decontamination workers or the general population for exposure to/infection with these agents.

Determination of serum IgG antibodies to Bacillus anthracis protective antigen in environmental sampling workers using a fluorescent covalent microsphere immunoassay.

AIMS: To evaluate potential exposure to Bacillis anthracis (Ba) spores in sampling/decontamination workers in the aftermath of an anthrax terror attack. METHODS: Fifty six serum samples were obtained from workers involved in environmental sampling for Ba spores at the American Media, Inc. (AMI) building in Boca Raton, FL after the anthrax attack there in October 2001. Nineteen sera were drawn from individuals both pre-entry and several weeks after entrance into the building. Nine sera each were drawn from unique individuals at the pre-entry and follow up blood draws. Thirteen donor control sera were also evaluated. Individuals were surveyed for Ba exposure by measurement of serum Ba anti-protective antigen (PA) specific IgG antibodies using a newly developed fluorescent covalent microsphere immunoassay (FCMIA). RESULTS: Four sera gave positive anti-PA IgG results (defined as anti-PA IgG concentrations > or = the mean microg/ml anti-PA IgG from donor control sera (n = 13 plus 2 SD which were also inhibited > or = 85% when the serum was pre-adsorbed with PA). The positive sera were the pre-entry and follow up samples of two workers who had received their last dose of anthrax vaccine in 2000. CONCLUSION: It appears that the sampling/decontamination workers of the present study either had insufficient exposure to Ba spores to cause the production of anti-PA IgG antibodies or they were exposed to anthrax spores without producing antibody. The FCMIA appears to be a fast, sensitive, accurate, and precise method for the measurement of anti-PA IgG antibodies.

Development of a sensitivity enhanced multiplexed fluorescence covalent microbead immunosorbent assay (FCMIA) for the measurement of glyphosate, atrazine and metolachlor mercapturate in water and urine.

Body burdens from exposures to pesticides may be estimated from urinary analyses of pesticide parent/metabolite concentrations. Pesticide applicators and others are often exposed to numerous unrelated pesticides, either sequentially or simultaneously. Classically, body burdens of pesticides are analyzed using chemical/instrumental analysis (CIM) or enzyme immunoassays (EIAs). Both of these technologies can usually be used to quantitate one analyte (or closely related groups of analytes) per analysis. Alternatively, multiple analytes can be measured simultaneously using a multiplexed fluorescence covalent microbead immunoassay (FCMIA). We developed a multiplexed FCMIA to simultaneously measure glyphosate (Gly), atrazine (Atz), and metolachlor mercapturate (MM) in water and urine. The assay had least detectable doses (LDDs) in water/diluted urine of 0.11/0.09 ng/ml (Gly, water/urine LDD), 0.10/0.07 ng/ml (Atz) and 0.09/0.03 ng/ml (MM). The sensitivity for the measurement of Gly was enhanced by derivatization. All assays gave linear responses from the LDDs for each respective pesticide to 300 ng/ml. There was no cross-reactivity between the three analytes. Using a 96-well microplate and an autosampler, as many as 288 separate analyses can be completed in approximately 120 min with precision, sensitivity, and specificity equivalent to, if not better, than that found when these same analytes are measured by CIM or EIA.