As infection with Pasteurella multocida is common in cattle and buffalo, a monoclonal antibody based sandwich ELISA kit was developed for its rapid and easy detection. The test was optimized and standardized so that maximum concordance could be maintained with the standard procedures of hemorrhagic septicemia diagnosis recommended by the WHO expert committee. HS-1, a Pasteurella multocida type B specific monoclonal antibody developed in mice was used as tracing antibody to capture P. multocida serotype B:2 in a sandwich ELISA. The test was standardized for whole killed bacterial cell, sonicated and the LPS antigens of P. multocida type B:2. An anti-pasteurella hyper immune serum raised in rabbit acted as the coating antibody was selected since it was previously shown to be a major immunogen during P. multocida infection in rabbits and contain antibodies against several conserved epitopes. The sensitivity of the sandwich ELISA determined with ELISA well module (8x2) for whole killed bacterial cells, and with two fold serial dilutions of an antigen (12 steps and in triplicate) for sonicated and LPS antigen, were cfu/ml, 385 ng/ml and mg/ml respectively. Specificity, evaluated against the cultured P. multocida type A antigen of bovine strain, was 100%. The coefficient of variation for sonicated and the LPS antigens calculated from intra and inter plate for same-day and inter-day tests were found within 20% indicating good reproducibility with few exceptions when CV varied more than 20%. | Development of a specific monoclonal antibody based Sandwich ELISA for rapid detection of haemorrhagic septicemia in bovine blood
