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Clinical and Diagnostic Laboratory Immunology, January 2004, p. 50-55, Vol. 11, No. 1
1071-412X/04/$08.00+0     DOI: 10.1128/CDLI.11.1.50-55.2004

Comparison of a Multiplexed Fluorescent Covalent Microsphere Immunoassay and an Enzyme-Linked Immunosorbent Assay for Measurement of Human Immunoglobulin G Antibodies to Anthrax Toxins

Raymond E. Biagini,1* Deborah L. Sammons,1 Jerome P. Smith,1 Barbara A. MacKenzie,1 Cynthia A. F. Striley,1 Vera Semenova,2 Evelen Steward-Clark,2 Karen Stamey,2 Alison E. Freeman,2 Conrad P. Quinn,2 and John E. Snawder1

Biological Monitoring Laboratory Section, Biomonitoring and Health Assessment Branch, National Institute for Occupational Safety and Health, Cincinnati, Ohio,1 Microbial Pathogenesis and Immune Response Laboratory, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia2

Received 4 August 2003/ Returned for modification 12 September 2003/ Accepted 2 October 2003

Recently, the Centers for Disease Control and Prevention reported an accurate, sensitive, specific, reproducible, and quantitative enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G (IgG) antibodies to Bacillus anthracis protective antigen (PA) in human serum (C. P. Quinn, V. A. Semenova, C. M. Elie et al., Emerg. Infect. Dis. 8:1103-1110, 2002). The ELISA had a minimum detectable concentration (MDC) of 0.06 µg/ml, which, when dilution adjusted, yielded a whole-serum MDC of 3.0 µg of anti-PA IgG per ml. The reliable detection limit (RDL) was 0.09 µg/ml, while the dynamic range was 0.06 to 1.7 µg/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 µg of anti-PA IgG per ml, the RDL was 0.016 µg/ml, and the whole-serum equivalent MDC was 1.5 µg/ml. The dynamic range was 0.006 to 6.8 µg/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 (r2 = 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.

* Corresponding author. Mailing address: Division of Applied Research and Technology, Biomonitoring and Health Assessment Branch, Biological Monitoring Laboratory Section, CDC/NIOSH MS C-26, Robert A. Taft Laboratories, 4676 Columbia Parkway, Cincinnati, OH 45226. Phone: (513) 533-8196. Fax: (513) 533-8494. E-mail: rbiagini{at}cdc.gov.

Clinical and Diagnostic Laboratory Immunology, January 2004, p. 50-55, Vol. 11, No. 1
1071-412X/04/$08.00+0     DOI: 10.1128/CDLI.11.1.50-55.2004




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