Development and evaluation of various enzyme‐linked immunosorbent assays for the detection of Clostridium perfringens β anti‐toxins

The aim of our work was to develop an enzyme‐linked immunosorbent assay for the detection of antibodies against the Clostridium perfringens β 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 β toxin were used. In all enzyme‐linked immunosorbent assay tests, microplates were first coated with monoclonal antibodies against the C. perfringens β 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°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°C. The smallest differences in absorbance were found when anti‐species conjugates were used.