Development of IgY-Based Sandwich ELISA as a Robust Tool for Rapid Detection and Discrimination of ToxigenicVibrio cholerae

Abstract

Background. The conventional methods for diagnosis ofVibrio choleraeare time consuming, complicated, and expensive. Development of rapid detection tests is critical for prevention and management of cholera. This study aimed to introduce two sensitive sandwich ELISAs based on avian antibodies (IgY) targeting outer membrane protein W (OmpW) and cytotoxin B (CtxB) antigens ofV. cholerae.Methods. The sequences ofompWandctxBgenes were cloned into pET28a vector.Escherichia coliBL21 (DE3) was transformed with the recombinant vectors, and gene expression was induced by IPTG. The expressed proteins were purified by affinity chromatography using Ni-NTA resins. Two groups of white Leghorn chickens were immunized by recombinant proteins, and the generated antibodies were purified from egg yolks of chickens by PEG precipitation. The antibodies were used for the development ofα-OmpW andα-CtxB ELISAs.Results. The expression and purification yielded 59 and 38 mg of recombinant OmpW and CtxB, respectively, per one liter of bacterial culture. PEG precipitation and purification of egg yolk antibodies yielded on average (±SD) 66.5 ± 1.80 and 50.9 ± 2.23 mg of purifiedα-OmpW andα-CtxB per egg, respectively. The analytical sensitivity ofα-OmpW ELISA was 103 cfu/mL ofV. choleraeand that ofα-CtxB ELISA was 33 pg/mL of recombinant cytotoxin B. The two developed ELISAs did not show any cross-reactivity to any tested bacteria grown in common conditions.Discussion. The current study is the first report on using IgY for detection ofV. cholerae. The developed ELISAs were shown to have considerable analytical sensitivity and specificity. Therefore, the assays can be one of the convenient methods for sensitive and specific detection of toxigenicV. choleraestrains in clinical and environmental samples.