Development and validation of bead-based assay quantifying Tetanus, Diphtheria, Pertussis Toxin, Filamentous haemagglutinin and Pertactin specific IgG in human serum

Abstract

Conventional ELISA platforms have been used for vaccine immunogenicity testing. However, due to limitations in sourcing and accessibility to human serum samples, we report the development and validation of Luminex-based multiplex immunoassay (MIA), using monovalent beads, which would reduce the analysis time, cost, and sample volume while simultaneously measuring the concentration of serum immunoglobulin G (IgG) antibodies specific for tetanus (TT), diphtheria (DT), pertussis toxin (PT), filamentous hemagglutinin (FHA) and pertactin (PRN), using the NIBSC reference standards. Additionally, we also report the development of a multiplex reference standard (MRS) focused on the simultaneous evaluation of antibodies against T, D, PT, PRN, and FHA in healthy human sera samples. As an assay evaluation parameter, the precision, accuracy, dilutional linearity, minimum and maximum detectable limit, robustness, stability, etc were assessed. The assay exhibited a wide dynamic range for all the five antigens and could quantify the IgG concentrations down to minimum concentrations, demonstrating antigen specificity with no cross-talks among the beads. The results obtained with MIA were consistent with commercially available assays. Thus, to conclude, the study provided a pentaplex assay with increased sensitivity, reproducibility and high throughput capabilities which would allow design of large and robust clinical studies for evaluating natural and vaccine-induced immunity.