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

BackgroundTo 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. MethodsWe coupled LOS from gonococcal strains 1291wt (nLc4 chain), 1291a (Lc3 chain) and 1291c (Lc2 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. ResultsConcentrations 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. ConclusionsThis accurate LOS immunoassay can be expanded to include any number of LOS specificities. Short SummaryWe developed a multiplexed fluorescent microsphere immunoassay of IgG antibodies to glycose antigens of the Neisseria Lipooligosaccharide in clients of an STI clinic who were sexually exposed to gonorrhea.