مقايسه واكنش پروتيين هاي طبيعي و اسرشته پيكره بروسلا آبورتوس ...

Reaction of Native and Denatured Brucella abortus (S19) Proteins with Antibody Using Affinity Chromatography and Immunoblotting

Western blotting or immunoblotting commonly use for study of reaction between antigens and antibodies. Denaturation of many proteins in immunoblotting can affect greatly the reactivity of antibodies and outcome of the procedure. In this study proteins of Brucella abortus (S19) was extracted by a mild method and reaction of the extracted proteins with serum of infected human and goat and immunized rabbit compared by affinity chromatography and immunoblotting. Gamma globulin (mostly IgG) fraction of the sera was precipitated by half saturation of ammonium sulfate and Iinked to activated sepharose 4B. The extracted proteins were loaded on the affinity column. Attached proteins was eluted by low pH and analyzed by SDS-PAGE Reaction of the total extract and eluted fractions with IgG fraction of sera was evaluated by Western blotting. Upon the results of affinity chromatography and immunoblotting, Brucella proteins can be classified in four groups: 1- The proteins that adsorbed to the affinity column and react with IgG in westemblotting. 2- Proteins that react with IgG in native state but no in denatured state. 3- Proteins that do not react with IgG in native state but react in denatured state. 4- Proteins that do not react with IgG in native and denatured state.