Clostridium difficile Toxin A+B Ag
The Clostridium difficile Toxin A+B Ag ELISA is an in vitro diagnostic device for direct detection of the toxins A and B of Clostridium difficile in stool specimens and culture suspensions.
Clostridium difficile is a bacterium causing nosocomial diarrhea in adults during or after the treatment with antibiotics such as 3rd generation cephalosporines (1). Although 2-3% of healthy adults and 20-50% of healthy children are colonized with Clostridium difficile, the infection is usually of exogenous origin and results from the contact either to hospital staff or to Clostridium difficile spores which may contaminate toilets, bed clothes etc. Both exotoxins A and B of this spore-forming bacteria cause the depolymerisation of actin filaments due to the intracellular enzymatic modification of rho-proteins. Consequently, the permeability of cell membrane is raised and neutrophiles may invade leading to expression of the clinical picture of the so-called Clostridium difficile-associated diarrhea and colitis or finally the pseudomembraneous colitis (PMC) (1). As the production of toxins and the outbreak of disease is correlated, diagnosis of Clostridium difficile infection is based mainly on a direct detection of the toxins in stool specimens. To date the cytotoxicity test has been considered as the gold standard for detection of Clostridium difficile toxins. Recently it has been replaced to a large extent by immunological tests such as enzyme immunoassay (2).
The Clostridium difficile Toxin A + B ELISA is an indirect two-site-immunoassay for the qualitative determination of both Clostridium difficile toxins A and B based on polyclonal and monoclonal antibodies.
Clostridium difficile toxins of stool specimens or culture suspensions and the positive control react with monoclonal antitoxin A and polyclonal anti-toxin B antibodies coated on the solid phase of the microplate. After incubation non-bound material is removed by a washing step. Subsequently bound toxins specifically react with biotinylated polyclonal anti-toxin A and monoclonal anti-toxin B antibodies during a second incubation period. Non-bound material is separated from the solid-phase immune complexes by a subsequent washing step.
During the next incubation period horseradish peroxidase (HRP) conjugated streptavidin reacts with the bound biotinylated antibodies. Unbound conjugate is removed by a washing step.
HRP converts the subsequently added colourless substrate solution of 3,3’,5,5’-tetra¬methylbenzidine (TMB) into a blue product. The enzyme reaction is terminated by sulphuric acid dispensed into the wells turning the solution from blue to yellow.
The optical density (OD) of the solution read at 450/620 nm is directly proportional to the specifically bound amount of Clostridium difficile toxin A and B. After consideration of the cut-off value, results are interpreted as positive or negative.
- Rambaud J-C., LaMont J-T. (Hrsg.): “Ökosystem Darm Special- Updates on Clostridium difficile” Springer Verlag 1995
- Wilkins T.D. and Lyerly D.M. (2003): „Clostridium difficile Testing: after 20 Years, Still Challenging“ Journal Of Clinical Microbiology, Vol. 41, No. 2, p. 531-534