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Development of a novel competitive ELISA method for Typhoid Vi Polysaccharide Quantitation
Ashwani Kumar Mandyal1, Rajasekaran Angannan2, Balananda Dhurjati Kumar Putcha2
1Department of Biotechnology, Vignan's Foundation for Science, Technology and Research, Vadlamudi, Guntur District, Andhra Pradesh, 522213, India; R&D Department, Auro Vaccines Private Limited, Indrakaran, Sangareddy District, Hyderabad, Telangana, 502329, India.
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.
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.
