A Modified Enzyme-Linked Immunosorbent Assay for Measurement of Antibody Responses to Meningococcal C Polysaccharide That Correlate with Bactericidal Responses

Abstract

The standardized enzyme-linked immunosorbent assay (ELISA) for measurement of serum immunoglobulin G (IgG) antibody responses to meningococcal C polysaccharide has been modified to employ assay conditions that ensure specificity and favor detection primarily of high-avidity antibodies. The modified and standard assays were used to measure IgG antibody concentrations in sera of toddlers vaccinated with meningococcal polysaccharide vaccine or a meningococcal C conjugate vaccine. The results were compared to the respective complement-mediated bactericidal antibody titers. In sera obtained after one or two doses of vaccine, the correlation coefficients, r, for the results of the standard assay and bactericidal antibody titers were 0.45 and 0.29, compared to 0.85 and 0.87, respectively, for the modified assay. With the standard assay, there were no significant differences between the geometric mean antibody responses of the two vaccine groups. In contrast, with the modified assay, 5- to 20-fold higher postvaccination antibody concentrations were measured in the conjugate than in the polysaccharide group. Importantly, the results of the modified assay, but not the standard ELISA, paralleled the respective geometric mean bactericidal antibody titers. Thus, by employing conditions that favor detection of higher-avidity IgG antibody, the modified ELISA provides results that correlate closely with measurements of antibody functional activity that are thought to be important in protection against meningococcal disease.

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

“…The addition of 75 mM thiocyanate to serum diluent to detect predominantly high avidity antibody is important. Currently licensed MACP vaccines induce a higher proportion of lower avidity antibody than the MCC vaccines [13]. For this study, signi¢cantly lower titres were found when using the high avidity assay than the standard ELI-SA.…”

“…There was no correlation for this age group however with either the high avidity or standard ELISA with SBA when using the Oac 3 antigen. A possible reason for lack of evident improvement in correlation when the high avidity assay was used in preference to the standard ELISA may be that the high avidity assay was optimised using derivatised Oac antigen [13] and conditions may not be optimal for Oac 3 antigen. Further studies should be performed to determine the best ELISA for correlation with functional antibody.…”

“…Each serum was assayed using 5 Wg ml 31 of either (i) Oac 3 (Centre of Applied Microbiology and Research, Porton Down, Salisbury, UK) or (ii) Oac (Pasteur Merieux, Lyon, France) serogroup C polysaccharide with 5 Wg ml 31 of methylated human serum albumin. The standardised serogroup C ELISA as previously described [12] measured total serogroup C-speci¢c IgG antibody whilst the assay to detect primarily high avidity antibody was a modi¢cation of the above assay incorporating 75 mM thiocyanate in the serum diluent as described by Grano¡ et al [13]. Apart from the addition of thiocyanate to the serum diluent the only other deviation from the standardised ELISA methodology was the use of 0.05% Tween 20 (Sigma-Aldrich, Poole, Dorset, UK) in place of 0.1% Brij 35 detergent.…”

“…As e¤cacy trials will not be undertaken before introduction of MCC vaccines, much attention has focussed on determining which immunological assays provide the best correlate of protection. Two forms of ELISA may be used, a standard ELISA measuring all serogroup C-speci¢c IgG antibody [12] or one which detects primarily high avidity antibody [13]. Additionally there is a choice of antigens, namely de-O-acetylated (Oac 3 ) or O-acetylated (Oac ) serogroup C polysaccharide used by di¡erent manufacturers in their respective vaccines [14,15].…”

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

“…Interplate variation was corrected for by using a pool of day 14 sera, and sera showing a titre less than the detection limit were assigned an arbitrary titre of 50 for calculation of geometric means. An avidity ELISA using thiocyanate as the chaotropic agent [26,27] for anti-menCPs antibodies was carried out on day 35 sera.…”