. The ELISA had a minimum detectable concentration (MDC) of 0.06 g/ml, which, when dilution adjusted, yielded a whole-serum MDC of 3.0 g of anti-PA IgG per ml. The reliable detection limit (RDL) was 0.09 g/ml, while the dynamic range was 0.06 to 1.7 g/ml. The diagnostic sensitivity of the assay was 97.6% and the diagnostic specificity was 94.2% for clinically verified cases of anthrax. A competitive inhibition anti-PA IgG ELISA was also developed to enhance the diagnostic specificity to 100%. We report a newly developed fluorescence covalent microbead immunosorbent assay (FCMIA) for B. anthracis PA which was Luminex xMap technology. The FCMIA MDC was 0.006 g of anti-PA IgG per ml, the RDL was 0.016 g/ml, and the whole-serum equivalent MDC was 1.5 g/ml. The dynamic range was 0.006 to 6.8 g/ml. Using this system, we analyzed 20 serum samples for anti-PA IgG and compared our results to those measured by ELISA in a double-masked analysis. The two methods had a high positive correlation (r 2 ؍ 0.852; P < 0.001). The FCMIA appears to have benefits over the ELISA for the measurement of anti-PA IgG, including greater sensitivity and speed, enhanced dynamic range and reagent stability, the use of smaller sample volumes, and the ability to be multiplexed (measurement of more than one analyte simultaneously), as evidenced by the multiplexed measurement in the present report of anti-PA and anti-lethal factor IgG in serum from a confirmed clinical anthrax infection.In response to the anthrax terrorist attacks of 2001, the Centers for Disease Control and Prevention (CDC) undertook accelerated development for a quantitative enzyme-linked immunosorbent assay (ELISA) for detection of anti-protective antigen (PA)-specific immunoglobulin G (IgG) in human serum and the development of a competitive inhibition assay to enhance diagnostic specificity (15). This assay was shown to have a diagnostic sensitivity of 97.6% and a diagnostic specificity of 94.2%. Preadsorption of sera with PA enhanced the diagnostic specificity to 100%. A potential limitation of ELISA is that it is a monoplex technology. Only one analyte can be measured per assay; measurement of numerous analytes necessitates either simultaneous or sequential assays. When the number of analytes becomes large, resource and manpower limitations can occur. An alternative to the ELISA is an assay that can multiplex analytes, i.e., measure numerous analytes simultaneously. Fluorescent covalent microsphere immunoassay (FCMIA) is a technology that can accomplish this by using uniquely dually stained microspheres for the measurement of up to 100 analytes simultaneously (18). In the present report we describe a newly developed FCMIA and compare it to a specific, sensitive, and quantitative ELISA for anti-PA IgG and also present multiplexed data for measuring anti-PA and antilethal factor (LF) IgG in serum from a confirmed case of human clinical anthrax.
MATERIALS AND METHODSSerum samples. Twenty-two serum samples (3 quality control standards, 1 negative control standard, and 16 unk...
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