Two enzyme linked immunosorbent assays for detecting antibodies against meningococcal capsular polysaccharides A and C.

Abstract

Aims-To evaluate two of the recent methods of coating microtitre plates in the enzyme linked immunosorbent assay (ELISA) for detecting human antibodies against meningococcal capsular polysaccharides A and C with a view to validating a specific meningococcal antibody assay for routine clinical use. Methods-Two four-layer ELISA protocols were standardised: one method utilised meningococcal polysaccharides conjugated to poly-L-lysine polypeptide for coating the microtitre plates; another used polysaccharides mixed with methylated human serum albumin (mHSA). Titration curves were plotted for the ELISAs and the squared Pearson correlation coefficient (R2) was used to determine the degree of accuracy of fit of the curves. Specificity tests were performed by inhibition and adsorption studies. Results-Both methods gave good titration curves with a high R2 of >0-98, indicating a high degree of accuracy in forming the curves. The titration end point after vaccination, obtained by the mHSA method, was 20 times higher, however, than that obtained by the poly-L-lysine method. Specificity tests showed that in the ELISA using polysaccharide/poly-L-lysine, antibody activity of a pre-vaccination serum sample was inhibited by 37%, and of post-vaccination serum by 50%/ with 1000-fold excess antigen. Antibody activity (postvaccination) was reduced by 51% and 59o/o, respectively, by adsorption with antigen-coated Sepharose beads or adsorption with suspensions of killed meningococci. In contrast, antibody activity of a pre-vaccination serum was inhibited by 60% and a post-vaccination serum by 90% in ELISA employing polysaccharides mixed with mHSA. Reproducibility was better with the use of methylated human serum albumin than with poly-L-lysine; the former showed intrabatch and interbatch coefficients of variation of 4% and 2%, respectively, compared with 43% (intrabatch) and 16% (interbatch) obtained with the poly-Llysine. Conclusion-It is concluded that the antibody assay using meningococcal polysaccharides groups A and C mixed with mHSA is much better than that using polysaccharides coupled with poly-Llysine.

Protection against meningococcal serogroup ACYW disease in complement-deficient individuals vaccinated with the tetravalent meningococcal capsular polysaccharide vaccine

“…An ELISA was used to measure specific IgG, IgM and IgA against the capsular polysaccharides. The capsular polysaccharides of serogroups A, C, Y, W135 were coated unto the ELISA plates with methylated albumin, as described previously [25][26][27]. The purified polysaccharides used as antigen in this ELISA were provided by SmithKline Beecham.…”

Memory immune response generated inCercopithecus aethiopsagainst meningococcal polysaccharide serogroup C conjugate vaccine

“…After three washes with T‐PBS the plates were incubated with anti‐monkey IgG‐HRP or with anti‐human IgG‐HRP at rt for 2 h. Finally plates were washed as indicated and the substrate H 2 O 2 (3%) and o ‐phenylenediamine (0.5 mg ml −1 ) in citrate buffer 0.1 M, pH 5, was used to develop the chromogenic reaction that was chemically stopped after 15 min. The optical density (OD) was read at 492 nm, in a plate reader [15].…”

Immunoassay of HumanNeisseria MeningitidisSerogroup a Antibody

“…Methylated human serum albumin prepared as described by Gheesling et al (6) and N. meiiiiigitidis group A polysaccharide were mixed to form a final concentration of 5 pg/rnl of each in lOmM PBS, pH 7.4 (coating buffer). 100 p1 of this mixture were added to Immulon I microtiter plates (Dynatech Laboratories, Inc., Chantilly, VA) which were sealed and incubated for 6-7 hours at room temperature.…”