Multicenter comparison of Neisseria meningitidis serogroup C anti-capsular polysaccharide antibody levels measured by a standardized enzyme-linked immunosorbent assay

Abstract

A standardized enzyme-linked immunosorbent assay (ELISA) was used by 11 laboratories to measure levels of total serum antibody to Neisseria meningitidis serogroup C capsular polysaccharide in 16 unpaired pre-and postvaccination serum samples. Twelve serum samples were from adults, and four were from children aged 2, 3, 5, and 9. The between-laboratory coefficient of variation for pre-and postvaccination sera ranged from 16 to 59% and 11 to 21%, respectively. The average percent difference (absolute value) from the betweenlaboratory means for all prevaccination sera measured by each laboratory was 24%, whereas the average percent difference was 13% for all postvaccination sera. A postvaccination quality control serum was diluted three times to give optical densities on the high, middle, and low portions of the standard reference curve. The three dilutions were assayed by the 11 laboratories a total of 241 times and yielded an overall coefficient of variation of 20%. Antibody-binding inhibition curves showed that the standardized ELISA was specific for N. meningitidis serogroup C capsular polysaccharide antibody. Fifty percent inhibition of seven serum samples was obtained after reaction with an average concentration of 0.9 ,ug of meningococcal serogroup C polysaccharide per ml; an average of 93% inhibition was obtained with 50 ,ug of polysaccharide per ml. The acceptance and use of this standardized ELISA will reduce between-laboratory assay variability and ensure a more accurate and reproducible assessment of immunogenicity for vaccines under development.

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

“…The importance of using standardised assay parameters can be seen when comparing studies for example the age dependent response to serogroup C polysaccharide as described by Maslanka et al (1998) [16]. This study utilised standardised assays for serogroup C-speci¢c IgG ELISA [12] (with Oac antigen) and standardised serogroup C SBA [11] with baby rabbit serum, on specimens from young North American children from Montana [16]. Results of serogroup C-speci¢c IgG levels of 2.8 Wg ml 31 (95% CI 1.9^4.3) for 6 2-year-olds (age range 4^23 months) in the present study and 3.63 Wg ml 31 (95% CI 2.96^4.49) (age range 12^23 months) for the Montana study were obtained.…”

“…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].…”

Development of antibodies against tetravalent meningococcal polysaccharides in revaccinated complement-deficient patients

“…The concentration of IgG against the polysaccharides C, Y and W135 in the reference serum was arbitrarily considered to be 1000 U/ml. For polysaccharide A the IgG levels were de®ned as 4000 U/ml, because they appeared to be four times higher than the antibody levels against polysaccharide C [17].…”

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.…”