Development of an ELISA kit using monoclonal antibody to Clostridium difficile toxin A.
OBJECTIVE
To establish an ELISA kit using monoclonal antibodies against Clostridium difficile (C. difficile) toxin A.


METHODS
An indirect sandwich ELISA was described using the purified rabbit monospecific antiserum as capturing antibody. After the polystyrene microtitre plates with 96 flat-bottomed wells were coated with rabbit antiserum, the wells were blocked with 100 g/L BSA in PBS-T. C. difficile toxin A or culture filtrates were added to each well and then monoclonal antibodies IgG-horseradish peroxidase conjugate was added as detecting antibody, tetramethylbenzidine was used as substrate and A450 of the stopped reacting product was recorded in an automated plate reader.


RESULTS
The tested specimens included culture filtrates of 2 strains of toxigenic C. difficile, 2 strains of non-toxigenic C. difficile, 26 strains of E. coli, 2 strains of S. dysenteriae, 1 strain of Bif. infantis, 5 strains of V. cholera, 2 strains of S. typhi, 7 strains of C. botulinum, 1 strain of toxigenic C. sordllii, and 1 strain of C. butyricum. A total of 47 strains of culture filtrates were all negative except for 2 strains of toxigenic C. difficile. The detective limitation of toxin A was 0.1 ng/mL.


CONCLUSIONS
An ELISA kit with high specificity and excellent sensitivity for the rapid detection of C. difficile toxin A was established. It will be a useful tool for diagnostic test of C. difficile toxin A.
PLA Institute for Digestive Medicine, Nanfang Hospital, First Military Medical University, Guangzhou 510515, Guangdong Province, China. fusiwu2001@sina.com
Si-Wu
SW
Fu
Ya-Li
YL
Zhang
Dian-Yuan
DY
Zhou
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D001427:Bacterial Toxins / Q000276:immunology*; D016360:Clostridioides difficile / Q000276:immunology / Q000302:isolation & purification*; D004761:Enterocolitis, Pseudomembranous / Q000175:diagnosis* / Q000276:immunology; D004768:Enterotoxins / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006825:Hybridomas; D051379:Mice; D008807:Mice, Inbred BALB C; D012680:Sensitivity and Specificity
D000911:Antibodies, Monoclonal; D001427:Bacterial Toxins; D004768:Enterotoxins; C041348:tcdA protein, Clostridium difficile