Enzyme-linked immunosorbent assay for detection of Salmonella typhi protein antigen

Abstract

A double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) was designed for the detection of Salmonella typhi protein antigen. The optimal concentration of antibody for coating the plate was found to be 5 μg/ml. The optimum conditions for antibody coating and antigen and conjugate incubation were 37°C for 3 h, 37°C for 2 h, and 4° C overnight, respectively. The enzyme-substrate reaction was allowed to take place at 30°C for 1 h. The established ELISA was found to be reproducible, with an inter-run coefficient of variation of less than 12% for the detection of an S. typhi protein antigen concentration of 0.5 to 50 μg/ml. The minimal detectable level of the antigen was 0.5 μg/ml. Cross-reactions were observed with the high level (50 μg/ml) of protein antigens obtained from Salmonella typhimurium, Escherichia coli, Salmonella paratyphi A, and Salmonella enteritidis. The ELISA established was used for the detection of S. typhi protein antigen in serum from 62 patients with typhoid, 30 patients with clinically diagnosed typhoid fever, 21 patients with parathyroid, 17 patients with pyrexia caused by other bacteria, and 160 normal, healthy individuals. It was found that the sensitivity, specificity, accuracy, positive predictive value, and negative predictive value of this assay was 83.87, 89.04, 87.93, 67.53, and 95.31%, respectively.