ELISA kit of monoclonal antibody to Clostridium difficile toxin A / 国际检验医学杂志

ABSTRACT

Objective To establish the ELISA kit of monoclonal antibodies to Clostridium difficile toxin A.Methods A sandwich ELISA was used.Flat-bottomed 96 well polystyrene microtitre plates were coated with 100 ?l of purified rabbit monospecific antitoxin(8 ?g/ml, capturing antibody) in carbonate buffer(pH9.6) and incubated overnight at 4℃, the plates were washed once in PBS containing 0.05% Tween-20, pH7.4 (PBST). After 200 ?l of 10% BSA in PBS-T was added to the wells and incubated at 37℃ for 2h, washed 5 times in PBS-T with 3 min incubation at room temperature between each wash, 100 ?l of C. difficile toxin A or test samples in PBS-T were added to each well and incubated for 1h at 37℃, washed 5 times. Then 100 ?l of 11000 diluted monoclonal antibodies IgG-Horseradish peroxidase conjugate(detecting antibody) was added for 1h at 37℃, wells were washed five times with PBS-T, and 0.1ml of tetramethylbenzidines substrate was added to each well. After 15 min at 37℃ in dark, the reactions were stopped by the addition of 1 drop of 2M sulfuric acid and the A450 was measured.Results The tested specimens included culture filtrates of 2 strains of toxigenic C. difficile, 2 non-toxigenic strains of C. difficile, 26 strains of E. coli, 2 strains of S. dysenteriae, 1 strains of Bifidobacterium, 5 strains of V. cholera, 2 strain of S. typhi, 7 strains of C. botulinum, 1 strain of toxigenic C. sordllii, and 1 strain of C. butyricum. The ELISA demonstrated high specificity and good sensitivity, it detected amounts of toxin A as low as 0.1ng/ml.Conclusion An ELISA kits with high specificity and good sensitivity for the rapid detection of C. difficile toxin A was presented. It will be a beneficial tool to clinical detection of Clostridium difficile toxin A.