Description

The Diagnostic Automation, Inc. C. difficle ELISA kit is intended for the qualitative determination of C. difficile toxins A+B in feces. It is a double antibody (sandwich) ELISA using an anti-toxin A+B antibodies to capture the antigen from the stool supernatant. A second set of anti-toxin A+B antibodies are added which sandwiches the captured antigen. This reaction is visualized by the addition of an anti-second antibody conjugated to peroxidase and the chromogen tetramethylbenzidine (TMB). The resulting blue color development indicates the presence of C. difficile toxins A+B being bound by the antibodies.

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 1935.1 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.2,3 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.4,5 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.5 A less studied population exhibiting reduced susceptibility to PMC is cystic fibrosis patients.5 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.5 The cell culture cytotoxicity assay (CTA) detects the presence of toxin B by the observation of cytopathic effect on cell culture. The assay is very sensitive (50 pg/ml toxin B)5 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 crossreactivity and detection of nontoxigenic C. difficile has been reported.

C. difficile EIA methods have been researched by a number of investigators, with a reported sensitivity to either toxin A or toxin B of 1-10 ng/mL.

During the first incubation, C. difficile toxins A+B present in the stool supernatant are captured by
antibodies attached to the wells. The second incubation adds additional anti-toxin A+B antibodies that \\\"sandwiches\\\" the antigen. The next incubation adds an anti-second antibody conjugated to peroxidase.
After washings to remove unbound enzyme, a chromogen is added which develops a blue color in the presence of the enzyme complex and peroxide. The stop solution ends the reaction and turns the blue color to yellow.