Development and optimization of in-house made indirect ELISA kit for the detection of antibodies against Pasteurella multocida in chicken.

Fowl cholera is caused by P. multocida. The currently available commercial kit is relatively expensive for Ethiopian laboratories. To address this issue indirect ELISA kit to detect antibodies against P. multocida in chicken was developed. In-house made indirect ELISA kit was developed and optimized. Re-constituted local avian P. multocida biotype A was used for the preparation of coating antigen. The purity of the P. multocida biotype A inoculum was checked by gram staining and centrifugation at 4500 rpm for 30 min. The assay was developed and evaluated using 128 samples of chicken serum. Optimizations of various components were carried out against both anti-P. multocida hyper immune and pre immune serum. To develop an indirect ELISA test, washing buffer concentration of 500 µl of T-20, blocking buffer concentration of 200 µl, antigen concentration of 0.1 µg/ml, serum samples with a final dilution of 1:500 and optimal dilution of Rabbit anti-sheep IgG HRP-conjugate of 1: 1000 µg/ml are determined by checker board titration (CBT) method. The cut-off value was determined to be 0.23. Sensitivity, specificity and accuracy of in-house made indirect ELISA were 96%, 90% and 93%, respectively. This test result indicated good correlation with that of the commercial kit with a Cohen's κ agreement of 0.86 with a 95% confidence interval of 0.77 to 0.95. We found that in-house made indirect ELISA was more sensitive and convenient to perform. Consequently, in-house made indirect ELISA was as good as the commercial indirect ELISA in screening chicken serum samples for detection of antibodies against P. multocida.