A Multiplexed Fluorescent Microsphere Immunoassay of IgG Antibodies to the Individual Glycose Antigens of the Neisseria Lipooligosaccharide α Chain in Clients of an STI Clinic

Background: To evaluate the immunogenicity of lipooligosaccharide (LOS) based gonococcal vaccines, we developed a multiplexed fluorescent microsphere immunoassay that distinguishes and quantifies antibodies that bind the various LOS antigens. Methods: We coupled LOS from gonococcal strains 1291wt (nLc4 α chain), 1291a (Lc3 α chain) and 1291c (Lc2 α chain), to different fluorescent microsphere regions (BioRad) after disaggregation in 1% deoxycholate. The microspheres were incubated with sera from 37 participants in a study of gonococcal risk factors. Laser excited fluorescence from each region was converted to ng/mL of IgG with use of affinity purified human IgG specific for each LOS. IgG specific to the nLc4 terminal galactose was quantified by subtraction of Lc3 IgG from 1291wt IgG; that specific to the Lc3 terminal glucosamine by subtraction of Lc2 IgG from Lc3 IgG. Results: Concentrations of nLc4, Lc3 and Lc2 IgG summed to the concentration of 1291wt IgG. Visual inspection of the data revealed a non-normally distributed, bimodal distribution of nLc4 IgG concentrations; 31/37 had a mean serum nLc4 IgG concentration of 6.22 ng/mL, and the other six had a mean nLc4 IgG concentration of 11.2 ng/ml. Upon statistical comparison of these groups, conducted prior to knowledge of infection status, those participants whose nLc4 IgG concentration centered around a higher mean, were more likely to be asymptomatic (p = 0.03) and were less likely to be infected (p = 0.05) than those with lower IgG concentrations. Conclusions: This accurate LOS immunoassay can be expanded to include any number of LOS specificities.