Research paper Development of a multiplexed fluorescent immunoassay for the quantitation of antibody responses to four Neisseria meningitidis serogroups Thomas B. Martins a, ⁎, Troy D. Jaskowski a , Anne Tebo a,b , Harry R. Hill a,b a Associated Regional and University Pathologists (ARUP) Institute for Clinical and Experimental Pathology, Salt Lake City, UT, USA b Department of Pathology, Pediatrics and Medicine, University of Utah School of Medicine, Salt Lake City, UT, USA article info abstract Article history: Received 29 August 2008 Received in revised form 17 December 2008 Accepted 19 December 2008 Available online 19 January 2009 Neisseria meningitidis is a gram-negative bacterium causing disease world wide with a fatality rate of 5–10%. Five serogroups, A, B, C, Yand W-135 are responsible for virtually all cases of the disease in humans. We have developed a multiplexed assay for the simultaneous quantitation of IgG antibody responses to the four most immunogenic (A, C, Y, and W-135) N. meningitidis serogroups. A simple and less manipulative method was employed for conjugation of the capsular polysaccharide antigens to the microspheres. The multiplex assay compared well with traditional individual ELISAs, but demonstrated greater than 1 log increase in dynamic range and sensitivity. Specificity studies of the multiplex assay showed greater than 95% homologous inhibition and less than 5% heterologous inhibition for all four serogroups. Intra and inter-assay CVs were generally less than 10% and the limit of detection was b 600 pg/ml. The multiplexed assay proved to be reproducible as well as specific and sensitive when compared to the standardized ELISAs. Advantages included a greater dynamic range and simultaneous detection of antibody responses to the four serogroups contained in the tetravalent meningococcal polysaccharide vaccine. © 2008 Elsevier B.V. All rights reserved. Keywords: Neisseria meningitidis Multiplex assay Polysaccharide conjugation Antibody quantitation 1. Introduction Neisseria meningitidis is a gram-negative bacterium caus- ing serious disease world wide which often results in substantial morbidity and mortality, especially in children under 5 years of age. It has become the leading cause of bacterial meningitis in the United States surpassing both Streptococcus pneumoniae (Whitney et al., 2003) and Hae- mophilus influenzae type b (Schuchat et al., 1997). N. meningitidis has 13 different serogroups which are classified according to the antigenic structure of their poly- saccharide capsule. Five serogroups, A, B, C, Y and W-135 are responsible for virtually all cases of disease in humans. Serogroup A causes major epidemics of meningitis in sub- Sahara Africa with nearly 200,000 cases reported to World Health Organization (WHO) in 1996. Serogroup B is the most lethal strain, comprising 40% of cases in the United Kingdom (UK) and approximately one third of cases in the United States. It is also the leading serogroup in infections among infants less than 1 year of age causing greater than 50% of cases. The capsular polysaccharide of this strain is poorly immunogenic, and there currently is no effective vaccine for this serogroup. Serogroup C is responsible for approximately 35% of menin- gococcal disease in the United States. In the last decade, serogroup Y has become more common in the United States Journal of Immunological Methods 342 (2009) 98–105 Abbreviations: ELISA, Enzyme-linked immunosorbant assay method; mHSA, methylated human serum albumin; EDC, 1-Ethyl-3-[3-dimethylami- nopropyl]carbodiimide hydrochloride; MES, 2-[N-Morpholino]ethanesulfo- nic acid; PBS, phosphate buffered saline; PBST, 10 mM phosphate buffered saline (with 0.02% TWEEN 20, pH 7.4); MFI, median fluorescent intensity; PE, phycoerythrin. ⁎ Corresponding author. ARUP Institute for Clinical and Experimental Pathology, 500 Chipeta Way, Salt Lake City, UT 84108, USA. Tel.: +1801 583 2787; fax: +1 801 584 5109. E-mail address: martintb@aruplab.com (T.B. Martins). 0022-1759/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2008.12.003 Contents lists available at ScienceDirect Journal of Immunological Methods journal homepage: www.elsevier.com/locate/jim