Development and Characterization of a Polyvalent Polyclonal Antibody as a Common Capture Antibody for the Detection of Enterotoxigenic Escherichia coli in a Sandwich ELISA.

Abstract

Due to its low cost and simplicity, the sandwich enzyme-linked immunosorbent assay (sELISA) is a traditional technique for identifying foodborne pathogens. However, most sELISAs are designed for single foodborne pathogen detection using two specific antibodies, which capture and detect the target bacteria. This study aimed to produce and characterize a common capture polyclonal antibody for Enterotoxigenic Escherichia coli (ETEC), Salmonella Typhimurium, and Shigella flexneri (S. flexneri) by a sELISA. Rabbit polyclonal antibodies (pAbs) were generated against recombinant proteins of CsgA, FhuA, and OmpA, which we called anti-mix. The recombinant proteins generated are conserved in Escherichia coli (E. coli), Salmonella enterica serovar Typhimurium, and S. flexneri species, but not in Listeria monocytogenes (L. monocytogenes) and Enterococcus faecalis (E. faecalis). The anti-mix serum gave a title higher than 1:32,000 by an indirect ELISA using purified recombinant proteins and whole bacteria cultures of the bacteria expressing the antigens but failed to recognize L. monocytogenes and E. faecalis. In addition, a recombinant protein A was purified and used to orient the capture antibodies (anti-mix) in the sELISA. However, no statistically significant difference was found in the assay sensitivity for ETEC detection in spiked milk samples with or without protein A. The assay linearity of sELISA for ETEC detection in Phosphate-buffered saline (PBS) was from 1 × 10⁸ to 1 × 10⁴ cells/mL, and for spiked milk samples was 1 × 10⁸ to 1 × 10⁵ cells/mL. In spiked milk samples, the detection limit of ETEC was lower than PBS, which suggests a negative effect from the matrix analyzed (milk) compared to PBS.