ENINGOCOCCAL DISEASE caused predominantly by Neisseria meningitidis serogroups A, B, and C occurs predominantly in young children and remains a substantial cause of morbidity and mortality worldwide. 1,2 In addition to causing endemic disease globally, meningococci, unlike other encapsulated bacteria, cause epidemics. Serogroup B epidemics, problematic in Norway and throughout much of Latin America in the 1980s and 1990s, 1 have recently emerged in New Zealand 3 and the United States. [4][5][6] Response to serogroup B epidemics, unlike serogroup A and C epidemics, is difficult because existing serogroup B vaccines have not been shown to be efficacious on an international scale. [7][8][9][10] Quadrivalent meningococcal polysaccharide vaccine is efficacious against meningococcal disease caused by the A, C, W-135, and Y serogroups. [11][12][13] Serogroup B polysaccharide antigen, however, is poorly immunogenic in humans, 14,15 and the elicitation of antibodies to serogroup B polysaccharide antigen is of concern because this antigen is present in human neonatal neural tissue. 16,17 Therefore, alternative Author Affiliations are listed at the end of this article.

A standardized serum bactericidal assay (SBA) is required to evaluate the functional activity of antibody produced in response to Neisseria meningitidis serogroup A and C vaccines. We evaluated assay parameters (assay buffer, target strains, growth of target cells, target cell number, complement source and concentration, and methods for growth of surviving bacteria) which may affect the reproducibility of SBA titers. The various assay parameters and specificity of anticapsular antibody to five serogroup A strains (A1, ATCC 13077, F8238, F9205, and F7485) and four serogroup C strains (C11, G7880, G8050, and 1002-90) were evaluated with Centers for Disease Control and Prevention meningococcal quality control sera. The critical assay parameters for the reproducible measurement of SBA titers were found to include the target strain, assay incubation time, and complement. The resulting standardized SBA was used by 10 laboratories to measure functional anticapsular antibody against serogroup A strain F8238 and serogroup C strain C11. In the multilaboratory study, SBA titers were measured in duplicate for 14 pairs of sera (seven adults and seven children) before and after immunization with a quadrivalent polysaccharide (A, C, Y, and W-135) vaccine. The standardized SBA was reliable in all laboratories regardless of experience in performing SBAs. For most sera, intralaboratory reproducibility was ؎1 dilution; interlaboratory reproducibility was ؎2 dilutions. The correlation between median titers (interlaboratory) and enzyme-linked immunosorbent assay total antibody concentrations was high for both serogroup A (r ‫؍‬ 0.86; P < 0.001; slope ‫؍‬ 0.5) and serogroup C (n ‫؍‬ 0.86; P < 0.001; slope ‫؍‬ 0.7). The specified assay, which includes the critical parameters of target strain, incubation time, and complement source, will facilitate interlaboratory comparisons of the functional antibody produced in response to current or developing serogroup A and C meningococcal vaccines.

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Salmonella isolates have traditionally been classified by serotyping, the serologic identification of two surface antigens, O-polysaccharide and flagellin protein. Serotyping has been of great value in understanding the epidemiology of Salmonella and investigating disease outbreaks; however, production and quality control of the hundreds of antisera required for serotyping is difficult and time-consuming. To circumvent the problems associated with antiserum production, we began the development of a system for determination of serotype in Salmonella based on DNA markers. To identify flagellar antigen-specific sequences, we sequenced 280 alleles of the three genes that are known to encode flagellin in Salmonella, fliC, fljB, and flpA, representing 67 flagellar antigen types. Analysis of the data indicated that the sequences from fliC, fljB, and flpA clustered by the antigen(s) they encode not by locus. The sequences grouped into four clusters based on their conserved regions. Three of the four clusters included multiple flagellar antigen types and were designated the G complex, the Z4 complex, and the ␣ cluster. The fourth cluster contained a single antigen type, H:z 29 . The amino acid sequences of the conserved regions within each cluster have greater than 95% amino acid identity, whereas the conserved regions differ substantially between clusters (75 to 85% identity). Substantial sequence heterogeneity existed between alleles encoding different flagellar antigens while alleles encoding the same flagellar antigen were homologous, suggesting that flagellin genes may be useful targets for the molecular determination of flagellar antigen type.

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