Date of Award:

5-1985

Document Type:

Dissertation

Degree Name:

Doctor of Philosophy (PhD)

Department:

Biology

Committee Chair(s)

Nabil N. Youssef

Committee

Nabil N. Youssef

Committee

Mark C. Healey

Committee

Raymond T. Sanders

Committee

Reed P. Warren

Committee

James LeGrand Shupe

Abstract

The objective of this study was to produce a monoclonal antibody specific for the microgametocytes of Eimeria tenella, examine the site and stage specificity of the antibody, and investigate the immunopotency of the antibody. BALB/c mice were immunized with antigen containing Eimeria tenella microgametocytes isolated from in vitro systems. After three intraperitoneal immunizations with the antigen and one booster immunization administered by tail vein injection, the mice were sacrificed and their spleen cells fused with SP2/0 mouse myeloma cells using polyethylene glycol as a fusing agent. Resultant hybridomas were screened by immunoelectrophoresis, indirect immunofluorescent antibody assay, and immunoelectron microscopy to determine the isotype, subisotype, site and stages pecificity of the antibody. Of four 96 well plates seeded with fusion products, four hybridomas were found to be producing antibody specific for the target antigen. Only the most strongly positive of these hybridomas, clone T1A3B9, was used for the study. The antibody produced by this hybridoma was found to be of sub isotype IgG2b.

T1A389 monoclonal antibody was introduced into Eimeria tenella infected cell cultures on days four, five, and six post-infection. At seven days post-infection, oocyst production was assayed by fixing, staining, and counting the resultant oocysts. Results of the in vitro experiments showed a greater than 50% reduction in oocyst production in experimental cultures over controls. Statistical significance of the data were confirmed by a Mann-Whitney U Test. These results indicate that the monoclonal antibody was exerting an inhibitory effect on the fertilization process.

T1A3B9 monoclonal antibody was incubated with Eimeria tenella infected cecal scrapings and cell culture material, immunolabeled with colloidal gold conjugates, and observed by electron microscopy. Results showed that the antibody was binding to the microgametocytes and to no other life cycle stages of the parasite, nor was it binding to host tissue. This indicates that the antibody is stage specific. Additionally, the antibody was seen to bind only to areas in close proximity to the budding flagella of developing microgametes, thus indicating distinct site specificity.

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