Development and evaluation of various enzyme-linked immunosorbent assays for the detection of Clostridium perfringens beta anti-toxins.
The aim of our work was to develop an enzyme-linked immunosorbent assay for the detection of antibodies against the Clostridium perfringens beta toxin. For this purpose, five different ways of performing an enzyme-linked immunosorbent assay were investigated. Positive and negative sera of different animals and partially purified beta toxin were used. In all enzyme-linked immunosorbent assay tests, microplates were first coated with monoclonal antibodies against the C. perfringens beta toxin. Actually, the first three ways of performing enzyme-linked immunosorbent assay proved to be an inhibition or a blocking enzyme-linked immunosorbent assay. In the first of these modifications, the examined serum was added on a microplate after the toxin. In the second two tests, they were added simultaneously after they were incubated together (60 min at room temperature or overnight at 4 degrees C, respectively). An anti-toxin conjugate was used for the detection. It was also used in a competitive enzyme-linked immunosorbent assay, where it was added together with the examined serum on the microplate, to which the toxin was already bound. The fifth way of performing an enzyme-linked immunosorbent assay differed from others by the use of conjugated anti-species immunoglobulin for the detection. The biggest differences in absorbances between positive and negative sera were found in the blocking enzyme-linked immunosorbent assay, where the mixture of the toxin and the examined serum were previously incubated overnight at 4 degrees C. The smallest differences in absorbance were found when anti-species conjugates were used.
Krt
B
B
Institute for Microbiology and Parasitology, Veterinary faculty, University of Ljubljana, Slovenia. krtbr@vf.uni-lj.si
D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood / Q000276:immunology*; D000942:Antigens, Bacterial / Q000276:immunology*; D001427:Bacterial Toxins / Q000276:immunology*; D002417:Cattle; D002418:Cattle Diseases / Q000097:blood / Q000276:immunology*; D003015:Clostridium Infections / Q000097:blood / Q000276:immunology / Q000662:veterinary*; D003016:Clostridium perfringens / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005069:Evaluation Studies as Topic; D006734:Horse Diseases / Q000097:blood / Q000276:immunology*; D006736:Horses; D011817:Rabbits; D012756:Sheep; D012757:Sheep Diseases / Q000097:blood / Q000276:immunology*; D013552:Swine; D013553:Swine Diseases / Q000097:blood / Q000276:immunology*
D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D001427:Bacterial Toxins; C031884:CPB protein, Clostridium perfringens