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Clinical and Vaccine Immunology, August 2008, p. 1188-1193, Vol. 15, No. 8
1071-412X/08/$08.00+0     doi:10.1128/CVI.00478-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Development of a Fluorescent-Bead-Based Multiplex Immunoassay To Determine Immunoglobulin G Subclass Responses to Neisseria meningitidis Serogroup A and C Polysaccharides{triangledown} ,{dagger}

Richarda M. de Voer,1,2 Fiona R. M. van der Klis,1 Carla W. A. M. Engels,2 Ger T. Rijkers,3,4 Elisabeth A. Sanders,3 and Guy A. M. Berbers1*

Laboratory for Infectious Diseases and Screening, National Institute of Public Health and the Environment, Bilthoven, The Netherlands,1 Department of Immunology, University Medical Center, Utrecht, The Netherlands,2 Department of Pediatric Immunology and Infectious Diseases, University Medical Center Utrecht, Utrecht, The Netherlands,3 Laboratory of Medical Microbiology and Immunology, St. Antonius Hospital, Nieuwegein, The Netherlands4

Received 6 December 2007/ Returned for modification 8 February 2008/ Accepted 3 June 2008

A fluorescent-particle-based multiplex flow cytometric immunoassay (MIA) for the detection of serum immunoglobulin G (IgG) and two IgG subclasses, IgG1 and IgG2, specific for Neisseria meningitidis serogroup A (MenA) and C (MenC) polysaccharides (PS) was developed. The assay comprised three separate duplex assays, one for the detection of the IgG response to MenA and MenC PS, another for the detection of the IgG1 response to MenA and MenC PS, and a third for the detection of the IgG2 response to MenA and MenC PS. Next, the three separate duplex assays were combined and analyzed as a hexaplex assay. No interference between monoplex, duplex, and hexaplex assays was observed, and the assay was found to have low intra- and interassay variation (<9.0% and <27%, respectively). Comparison of the meningococcal subclass MIA to the in-house enzyme-linked inmmunosorbent assays showed a good correlation (R ≥ 0.85) for each of the subclasses. We conclude that the hexaplex meningococcal subclass MIA is an easy and specific assay for the determination of anti-MenA and anti-MenC PS subclass IgG, requiring minimal amounts of serum to study IgG subclass responses to vaccines.

* Corresponding author. Mailing address: Laboratory for Infectious Diseases and Screening, National Institute of Public Health, Antonie van Leeuwenhoeklaan 1, PO-Box 1, Bilthoven, The Netherlands. Phone: 31 30 2742496. Fax: 31 30 2748888. E-mail: guy.berbers{at}rivm.nl

{triangledown} Published ahead of print on 11 June 2008.

{dagger} Supplemental material for this article may be found at http://cvi.asm.org/.

Clinical and Vaccine Immunology, August 2008, p. 1188-1193, Vol. 15, No. 8
1071-412X/08/$08.00+0     doi:10.1128/CVI.00478-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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