Standardization and a multilaboratory comparison of Neisseria meningitidis serogroup A and C serum bactericidal assays. The Multilaboratory Study Group

Abstract

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.

Meningococcal serogroup C-specific IgG antibody responses and serum bactericidal titres in children following vaccination with a meningococcal A/C polysaccharide vaccine

“…Although the early SBA advocated the use of human complement [17], in more recent years, with the need for standardisation and also the unavailability of large volumes of suitable human complement, baby rabbit serum was suggested as an alternative complement source [16,28,29]. Recently, studies for both serogroups A and C, including the present one, have highlighted that meningococci are more sensitive to baby rabbit complement than human complement resulting in higher titres.…”

“…The calibration factor of the standard CDC 1992 serum for serogroup C-speci¢c IgG used in both Oac 3 and Oac assays was 24.1 Wg ml 31 for the standard assay and 19.6 EU ml 31 for the assay to detect primarily high avidity antibody. SBAs were performed as described by Maslanka et al [16] with either baby rabbit sera (Pel-Freeze Incorporated, Rodgerson, AZ, USA) or human sera as an exogenous complement source. The target strain used throughout was C11 (C :16:P1.1,7 a ).…”

“…The functional assay of choice, the serogroup C serum bactericidal assay (SBA) [16] can be performed with either baby rabbit sera or human sera as an exogenous complement source. Uncertainty exists over the exact SBA titre which correlates with protection for serogroup C meningococci but is thought to be v 4 when using human complement [17].…”

Neisseria meningitidis serogroup B: laboratory correlates of protection

“…Probably, other mechanisms besides the bactericidal response can also provide protective immunity against serogroup B meningococci. For serogroups A and C a standardized assay was designed using rabbit serum as a complement source, since this is readily available [25]. However, the use of infant rabbit complement results in much higher SBA titers than the use of human complement and a recent paper showed that this assay lacks sensitivity in predicting a protective titer as assessed by Goldschneider et al [1,2] using human complement [26].…”

Bordetella pertussis fimbriae are effective carrier proteins in Neisseria meningitidis serogroup C conjugate vaccines

“…Serum bactericidal antibodies (SBA) were determined according to standardised methodology [28] using a complement source of 25% baby rabbit complement (Pelfreeze) and N. meningitidis serogroup C strain C11 as the target strain. Titres are expressed as the reciprocal of the ¢nal dilution producing v 50% bactericidal killing after 60 min [29].…”