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An Overview C. difficile Toxin A+B Antigen Detection Microwell ELISA
Summary Clostridium difficile may be part of the normal bacterial flora of the human intestinal tract, but can become an opportunistic pathogen when the intestinal tract has been compromised or altered, as with patients undergoing antibiotic therapy. Hall and O’Toole isolated the bacteria and described its toxigenic characteristics in 1935. Toxin-producing strains of C. difficile produce two toxins: toxin A, an enterotoxin, and toxin B, a cytotoxin. C. difficile was not considered an opportunistic pathogen until the late 1970’s when a correlation between the bacteria and pseudomembranous colitis (PMC) was established. PMC is an antibiotic-associated disease that progresses from diarrhea and mucosal inflammation to the formation of colonic pseudomembranes composed of fibrin, mucous, necrotic epithelial cells and leukocytes.
Though up to 50% of infants are colonized by toxigenic C. difficile and exhibit high levels of toxin A and B, few develop PMC, instead remaining asymptomatic. Hypotheses for this phenomenon include colostrum’s ability to neutralize toxin A and B, a diminished sensitivity of toxin A by fetal intestinal cells, and the possible lack of toxin receptors. A less studied population exhibiting reduced susceptibility to PMC is cystic fibrosis patients. Rapid methods of isolation and identification of C. difficile or its toxin(s) are readily available. The most common clinical diagnostic procedures for C. difficile antibiotic-associated colitis are cell culture cytotoxicity and latex agglutination assays. The cell culture cytotoxicity assay (CTA) detects the presence of toxin B by the observation of cytopathic effect on cell culture. The CTA assay is very sensitive (50 pg/ml toxin B) but requires a minimum of two days to complete. Latex agglutination is a common stool screening method for detection of proteins associated with C. difficile, though cross-reactivity and detection of nontoxigenic C. difficile has been reported. .
ASSAY PRINCIPLE During the first incubation, antibodies attached to the wells capture C. difficile toxin A+B present in the stool supernatant. The second incubation adds an additional anti-toxin A+B antibody that “sandwiches” the antigen. The third incubation attaches a anti-second antibody conjugated to horseradish peroxidase to the sandwich. After washings to remove unbound enzyme, a chromogen is added which develops a color in the presence of the enzyme complex and peroxide. The stop solution ends the reaction. The reaction may then be read visually or with an ELISA reader. The IVD Research C. difficile assay utilizes a separate positive control for Toxin A and Toxin B to insure that the assay is performing to specifications. The use of a Toxin A and Toxin B combined control does not adequately insure that the assay is detecting both Toxin A and Toxin B at the lower levels set in the manufacturer’s specifications.
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