Production and Characterisation of specific Antibodies to Bovine Interleukin 5 and 6 and their use in the Development of Sensitive Elisa for their Measurement
Arodi, Washingtone Ouma
MetadataShow full item record
Lack of food especially animal proteins is a major problem in the developing world. Livestock provide the bulk of these proteins hence the need to increase the supply through improved livestock health . Enzyme Linked Immunos orbent Assays (ELISA) for bovine interleukin 5 and interleukin 6 (boIL - 5 and boIL - 6) could be applied for analyses of immune responses and advance understanding of a variety of diseases of cattle. Biological and biochemical characteristics of monoclonal an tibodies (mAbs) and polyclonal antibodies (pAbs) against rboIL - 5 and rboIL - 6 are described as well as their use in developing specific and sensitive ELISA for the detection and measurement of these cytokines. The ob j ective of the study was to produce and characterize an tibodies (Abs) to boIL - 5 and 6 and to use the se Abs in developing sensitive ELISA to measure the respective cytokine s . Four murine anti boIL - 5, four murine anti boIL - 6 mAbs, two rabbit anti boIL - 5 and two rabbit anti boIL - 6 pAbs were prod uced and their binding specificities determined by in direct ELISA, Western blotting , Ouchterlony‘s double immunod iffusion technique and flow cytometry. Sandwich ELISA for boIL - 5 was developed using mAb 25 .1 as the capture antibody and the pAb R 1 as the det ector antibody. Sandwich ELISA for boIL - 6 was also developed using mAb 56.2 as the capture antibody and pAb R 6 as the detector antibody. The developed ELISA was used to detect both recombinant and natural boIL - 5 and boIL - 6 present in supernatant of periph eral blood mononuclear cells ( PBMC ) stimulated with Concanavalin A ( Con A ) and other mitogens. The ELISA developed was able to detect as little as 48pg/ml of boIL - 5 and 24pg/ml of boIL - 6 . Using this method it was possible to detect native boIL - 5 and boIL - 6 in supernatants of PBMC from cattle as well as in cell lines infected with Theileria parva, indicating the applicability of the se assay s to a number of in vitro culture systems. Some of the antibodies also showed promising results in intra cellular cytok ine staining and flow cytometry. It is therefore recommended that these immunoassays be further optimized with a view to making them available for commercial use.