Quantitative bead–based multiplex assay for simultaneous determination of IgG concentrations of pertussis toxin, filamentous hemagglutinin, pertactin, diphtheria, tetanus, Haemophilus influenzae b, and hepatitis B in human serum samples

An equi-mix of an international reference standard for Hep B (07/164) and Hib (09/222) provided the best dynamic range for the seven-plex assay. Method validation was conducted using a panel of human serum samples that included samples from vaccinated healthy volunteers, non-vaccinated volunteers, negative controls, and international reference standards. Assay specificity using inhibition experiments demonstrated specificities of 98%, 95%, 93%, 98%, 97%, 97%, and 98% for DT, TT, FHA, PRN, PT, Hib, and Hep-B, respectively. Spike recoveries of 80%-120% were demonstrated in different matrices, including those of hemolytic and lipemic sera samples. The precision and accuracy were confirmed by evaluating a panel of human serum samples obtained from vaccinated individuals. The assay demonstrated coefficients of variation (CV) of ≤ 20% across all assays, regardless of run, day, or analyst. This method demonstrated strong agreement with conventional commercially available assays, highlighting the advantages of multiplexing over traditional enzyme-linked immunosorbent assays (ELISAs).