Graduate Thesis Or Dissertation

Identification of an inguinal adipocyte-specific protein using monoclonal antibody selected by cyclophosphamide

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  • Evidence suggests that adipose tissue is not a homogeneous entity, but rather that it differs functionally and morphologically according to its anatomical location. Production of a site-specific monoclonal antibody (MAb) is therefore of interest, not only for the study of adipocyte development and differentiation, but also for the elimination of adipocyte tissue at a specific site in vivo. Three adipocyte plasma membrane (APM) preparations were isolated from inguinal, epididymal, and perirenal fat depots of Sprague-Dawley rats. Two immunization approaches were applied in this study. In the original approach, BALB/c mice were immunized with inguinal APM proteins. An enzyme-linked immunosorbent assay (ELISA) method was developed to screen the hybridoma producing MAb specific to inguinal APM. The results of the screening procedure showed that all positive clones appeared to react with both epididymal and perirenal APM as strongly as with inguinal APM. In the alternative approach, BALB/c mice were immunized with a mixture of epididymal and perirenal preparations. The immunization was followed by the injection of cyclophosphamide to eliminate the antigen-stimulated lymphocytes selectively. Following a three-week rest, the mice were immunized with the inguinal APM preparation. The same ELISA method as in the first approach was used to screen the hybridoma clones. Positive clones producing inguinal-specific antibody were selected. A unique protein with molecular weight estimated at 25 KDa was identified only in the inguinal plasma membrane preparation by using sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and western blot techniques. Results from indirect-immunofluorescence demonstrated that this antigen is located on the plasma membrane of inguinal fat cells.
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