Si-Wu Fu1, Ya-Li Zhang, Dian-Yuan Zhou. 1. PLA Institute for Digestive Medicine, Nanfang Hospital, First Military Medical University, Guangzhou 510515, Guangdong Province, China. fusiwu2001@sina.com
Abstract
AIM: 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. CONCLUSION: 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.
AIM: 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. CONCLUSION: 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.
Authors: C S Merz; C Kramer; M Forman; L Gluck; K Mills; K Senft; I Steiman; N Wallace; P Charache Journal: J Clin Microbiol Date: 1994-05 Impact factor: 5.948