Fast, Antigen-Saving Multiplex Immunoassay To Determine Levels and Avidity of Mouse Serum Antibodies to Pertussis, Diphtheria, and Tetanus Antigens

Abstract

To enhance preclinical evaluation of serological immune responses to the individual diphtheria, tetanus, and pertussis (DTP) components of DTP combination vaccines, a fast hexavalent bead-based method was developed. This multiplex immunoassay (MIA) can simultaneously determine levels of specific mouse serum IgG antibodies to P antigens P.69 pertactin (P.69 Prn), filamentous hemagglutinin (FHA), pertussis toxin (Ptx), and combined fimbria type 2 and 3 antigens (Fim2/3) and to diphtheria toxin (Dtx) and tetanus toxin (TT) in a single well. The mouse DTP MIA was shown to be specific and sensitive and to correlate with the six single in-house enzyme-linked immunosorbent assays (ELISAs) for all antigens. Moreover, the MIA was expanded to include avidity measurements of DTP antigens in a multivalent manner. The sensitivities of the mouse DTP avidity MIA per antigen were comparable to those of the six individual in-house avidity ELISAs, and good correlations between IgG concentrations obtained by both methods for all antigens tested were shown. The regular and avidity mouse DTP MIAs were reproducible, with good intra-and interassay coefficients of variability (CV) for all antigens. Finally, the usefulness of the assay was demonstrated in a longitudinal study of the development and avidity maturation of specific IgG antibodies in mice having received different DTP vaccines. We conclude that the hexaplex mouse DTP MIA is a specific, sensitive, and high-throughput alternative for ELISA to investigate the quantity and quality of serological responses to DTP antigens in preclinical vaccine studies.

The Performance of Multiplex Immunoassays for Antibody Determination to Diphtheria, Tetanus and Pertussis: A Need for Standardisation

“…In our recent study [1], the capture antigen concentration for the three pertussis antigens (pertussis toxin PT, filamentous hemagglutinin FHA and pertactin Prn) used for coupling to magnetic beads was identical to that in previously described studies [7,8,10] but for diphtheria toxoid DT and tetanus toxoid TT, the best results were obtained with less antigen than described by Van Gageldonk et al [8] and Stenger et al [10] (Table 1).…”

“…Various buffers have been used especially for the coupling step of antigens to the beads. In our study and the one of Prince et al, beads were activated in a monobasic sodium phosphate (NaH 2 PO 4 pH 6.2) buffer and coupling buffer contained 2-(N-morpholino) ethane-sulfonic acid (MES pH 5), in contrast with the other studies, which used PBS buffer for both steps [3,6,10,12].…”

“…Among the six reported publications (Table1), three [6,8,10] tested the specificity by homologous and heterologous inhibition. In our study, we first performed five separate monoplex assays and compared the data with the pentaplex data.…”

“…Using a smaller assay volume [8,10] could reduce assay sensitivity and shorter incubation times [3,6,7] could miss antibodies with low avidity [11]. Various buffers have been used especially for the coupling step of antigens to the beads.…”

“…Pickering et al [7] described the measurement of antibodies to diphtheria and tetanus toxin and Haemophilus influenza type b capsular polysaccharide using the two step carbodiimide coupling procedure. In the studies of Van Gageldonk et al [8] and Stenger et al [10], the performance of the MIA was evaluated by comparing it with an in-house ELISA using the same capture antigens in MIA and in ELISA. If comparisons are made between MIA and ELISA which use identical capture antigens, as well as similar buffers, variability will be minimized, correlations will be good, and similar quantitative values will be achieved.…”