ORIGINAL PAPER R. E. Biagini Æ D. L. Sammons Æ J. P. Smith B. A. MacKenzie Æ C. A. F. Striley Æ S. A. Robertson J. E. Snawder Æ C. P. Quinn Simultaneous measurement of specific serum IgG responses to five select agents Received: 10 January 2005 / Revised: 24 February 2005 / Accepted: 8 March 2005 / Published online: 2 June 2005 Ó Springer-Verlag 2005 Abstract 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 re- sponse. 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 anti- gens), F. tularensis, RT and SEB simultaneously in hu- man B. anthracisvaccinee 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 relation- ships were observed (r 2 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)·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. Keywords Anthrax Æ Tularemia Æ Plague Æ Staphylococcal enterotoxin B Æ Ricin Introduction The Department of Health and Human Services/Centers for Disease Control and Prevention (DHHS/CDC) has classified several agents which may be used in a bioter- rorism attack as select agents [1]. They are classified as such on the basis of ease of dissemination, mortality/ morbidity rate, and potential for social disruption. These agents include viruses, bacteria, rickettsiae, fungi, and toxins. A subset of these agents includes Bacillus anthracis, Yersinia pestis, Francisella tularensis, ricin toxin (RT), and staphylococcal enterotoxin B (SEB). Exposure to, infection with, and vaccination with B. anthracis [2, 3], Y. pestis[4–6], F. tularensis [7], RT [8] or SEB [9] have been shown to cause production of specific IgG antibodies. Comparison of pre-bioterrorism and post-bioterrorism-incident IgG antibody levels has been shown to be a potentially useful method for biological monitoring of decontamination and clean-up workers for potential exposure to bioterrorism agents [2]. The potential exists for exposure to multiple select agents in a single bioterrorism event [3]. In this paper we describe a multiplexed fluorescent covalent microsphere R. E. Biagini (&) Æ D. L. Sammons Æ J. P. Smith B. A. MacKenzie Æ C. A. F. Striley Æ S. A. Robertson J. E. Snawder Biomonitoring and Health Assessment Branch, Division of Applied Research and Technology, Centers for Disease Control and Prevention, National Institute for Occupational Safety and Health, 4676 Columbia Parkway, Cincinnati, OH 45226, USA E-mail: reb4@cdc.gov Tel.: +1-513-5338196 Fax: +1-513-5338494 C. P. Quinn Microbial Pathogenesis and Immune Response (MPIR) Laboratory, Centers for Disease Control and Prevention, National Center for Infectious Diseases, Atlanta, GA 30333, USA Anal Bioanal Chem (2005) 382: 1027–1034 DOI 10.1007/s00216-005-3204-6