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 (2013) Production and Characterisation of Specific Antibodies to Bovine Interleukin 5 and 6 and Their Use in the Development of Sensitive Elisa for their Measurement. PhD thesis, Kenyatta University.

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Abstract

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 Immunosorbent 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 antibodies (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 objective of the study was to produce and characterize antibodies (Abs) to boIL-5 and 6 and to use these Abs in developing sensitive ELISA to measure the respective cytokines. Four murine anti boIL-5, four murine anti boIL-6 mAbs, two rabbit anti boIL-5 and two rabbit anti boIL-6 pAbs were produced and their binding specificities determined by indirect ELISA, Western blotting, Ouchterlony‘s double immunodiffusion technique and flow cytometry. Sandwich ELISA for boIL-5 was developed using mAb 25.1 as the capture antibody and the pAb R1 as the detector antibody. Sandwich ELISA for boIL-6 was also developed using mAb 56.2 as the capture antibody and pAb R6 as the detector antibody. The developed ELISA was used to detect both recombinant and natural boIL-5 and boIL- 6 present in supernatant of peripheral 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 these assays to a number of in vitro culture systems. Some of the antibodies also showed promising results in intra cellular cytokine staining and flow cytometry. It is therefore recommended that these immunoassays be further optimized with a view to making them available for commercial use.

Item Type:	Thesis (PhD)
Subjects:	Q Science > QR Microbiology
Divisions:	Africana
Depositing User:	Tim Khabala
Date Deposited:	28 Mar 2018 13:28
Last Modified:	28 Mar 2018 13:28
URI:	http://thesisbank.jhia.ac.ke/id/eprint/3628

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