Vi PS Capture ELISA for Typhoid Vaccine Identification

A Vi PS capture ELISA was performed at NIBSC to determine the identity of Vi PS in typhoid vaccines, according to Hitri et al. [15 (link)] with some modifications. Following the coating of plates (Nunc Maxisorp) with horse anti-mouse IgG and a blocking step with 1% w/v BSA, TCV and Vi PS vaccine samples were diluted from 1:100 to 1:102,400 in assay buffer (PBS with 0.1% v/v Brij-35 (Thermo Scientific, Waltham, MA, USA 20150) and 1% w/v BSA) in 2-fold dilutions across the plate, with a final volume of 100 µL. Plates were incubated at room temperature for 1 h, washed, and 100 µL of rabbit anti-Vi serum (NIBSC 04/152) diluted 1:5000 in assay buffer was added to the wells and incubated at room temperature for 2 h. Plates were washed, and bound IgG was detected by incubation with 100 µL goat anti-rabbit IgG-HRP (Sigma, Kawasaki City, Japan) diluted 1:10,000 in assay buffer per well at room temperature for 1 h. Plates were developed, and ODs were read at 450 nm. CombiStats software was used to evaluate the binding curves. The sample identity is positive if the Vi PS content is calculated to be NLT 40 µg/mL based on the dose–response curve of the reference Vi PS preparation (NIBSC 16/126), and the curve should be comparable with NIBSC 16/126 with no significant deviations from parallelism or linearity.