Development and evaluation of a highly sensitive immunochromatographic strip test using gold nanoparticle for direct detection of Vibrio cholerae O139 in seafood samples

摘要

A strip test for the detection of Vibrio cholerae O139 was developed using two monoclonal antibodies (MAbs), namely VC-273 and VC-812, which specifically bind to the lipopolysaccharide and capsular polysaccharide of V. cholerae O139. The MAb VC-273 gold nanoparticle conjugate was sprayed onto a glass fiber pad that was placed adjacent to a sample chamber. MAb VC-812 and the goat anti-mouse immunoglobulin G (GAM) antibody were sprayed onto a nitrocellulose membrane in strips at positions designated as T and C, respectively. The test strips were assessed for their ability to directly detect V. cholerae O139 using samples dispersed in application buffer, and a 100μL aliquot of sample was applied to the sample chamber. The results were observable within 20min after application of the sample. In samples containing V. cholerae O139, the antigen was bound to the colloidal gold-conjugated MAb to form an antibody-antigen complex. This complex was captured by the MAbs at the T test line, resulting in the appearance of a reddish-purple band at the T position. The sensitivity of the test was determined to be 10~4cfumL~(-1). Direct detection of V. cholerae O139 in various fresh seafood samples could be accomplished with similar sensitivities. The detection limit was substantially improved to 1cfumL~(-1) of the original bacterial content after pre-incubation of the sample in alkaline peptone water for 12h. The V. cholerae strip test provides several advantages over other methods, including the speed and simplicity of use because there is no requirement for sophisticated equipment.