Development of a novel competitive ELISA method for Typhoid Vi Polysaccharide Quantitation.

Licensed typhoid vaccines contain Vi polysaccharide (ViPS), a capsular antigen of Salmonella. Typhi (S.typhi), either unconjugated or conjugated to carrier proteins like tetanus toxoid (TT), diphtheria toxoid (DT), and Cross-reactive material 197 (CRM-197). Quantitation of ViPS during vaccine development, is challenging, as ViPS demonstrates limited adsorption to solid phases, resulting in poor binding and reduced sensitivity in conventional ELISA formats. We developed a novel competitive enzyme-linked immunosorbent assay (c-ELISA) using biotinylated ViPS as a detector probe for ViPS quantitation. The performance of c-ELISA with a novel Biotinylation approach was evaluated with WHO recommended orthogonal colorimetry based Hestrin assay and High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD) method. Both c-ELISA and HPAEC-PAD demonstrated comparable specificity, accuracy and repeatability, however c-ELISA showed a higher sensitivity than HPAEC-PAD in the estimation of unconjugated polysaccharide (free-PS) in typhoid conjugates with a range of 90-10,000 ng/ml. Overall, the newly developed c-ELISA is a reliable and sensitive immunochemical method for the quantification of ViPS, which complements the HPAEC-PAD. Together, these tests can serve as orthogonal analytical platforms for the monitoring of the ViPS during the production of typhoid vaccines and for the quality control.