IgG antibody levels to meningococcal porins in patient sera: comparison of immunoblotting and ELISA measurements

Abstract

IgG antibody levels to the meningococcal PorA and PorB proteins in 56 acute and convalescent phase sera from 25 patients with meningococcal disease were compared by immunoblotting and ELISA. Heat-treated outer membrane vesicles from strain 44/76 (B:15:P1.7,16) served as antigens for immunoblotting, whereas purified P1.7,16 PorA and P15 PorB from the same strain were used as antigens in the ELISA. In the blotting method, IgG binding to the porins was determined by digital scanning of the immunoreactive bands and calculated relative to the PorA binding of a reference serum on each blot. The coefficient of variation for the reference serum was 21.6% (a total of 144 strips) with smaller variations for each day’s experiments. Blotting of all 56 sera at the standard 1/200 dilution measured anti-PorA and anti-PorB levels that correlated with those obtained by ELISA (Spearman rank-order correlation coefficient r s=0.48; P<0.001). At this dilution, the anti-PorA ( r s=0.52; P<0.004) and anti-PorB ( r s=0.60; P<0.001) levels of the convalescent phase sera ( n=29) corresponded with the ELISA measurements, whereas no correlation was found with the results for the acute phase sera, which mostly had low ELISA antibody levels (<2 μg/ml IgG). A corresponding blot analysis of convalescent sera from the seven patients, who had received the 44/76 outer membrane vesicle vaccine, demonstrated a high correlation coefficient for the anti-PorA levels ( r s=0.95; P<0.001) vs. the ELISA results. No such correlation was observed for the PorB response in these sera, being nine-fold higher than the PorA response, because of a prozone effect on the blots at the standard dilution. However, blotting at a higher serum dilution (1/2000) resulted in anti-PorB levels that also correlated strongly ( r s=0.93 P<0.001) with the ELISA measurements.