Preparation and characterization of polyclonal and monoclonal antibodies against @entity1 aromatase XXXX ( @entity4819 ), and their use in its purification.

@entity1 @entity12325 @entity42634 @entity3208 @entity4819 @entity19 @entity1219 @entity2228 @entity95 @entity1110 @entity4364 @entity2975 @entity8851 @entity2671
Aromatase @entity12325 ( @entity4819 ) was partially purified from @entity1 placental microsomes by hydrophobic affinity chromatography using @entity4364 and ion-exchange chromatography on @entity8851 -cellulose. The resulting preparation had a specific activity of 2 nmol/mg protein with respect to cytochrome @entity4819 content and displayed a type I difference spectrum upon addition of the substrate @entity2671 . When the @entity12325 -enriched fractions were subjected to @entity3208 - @entity2975 gel electrophoresis and stained with Coomassie blue, there was an enrichment of two proteins having apparent molecular weights of 50,000 and 55,000. The bands containing these proteins were removed from unstained @entity2975 gels and injected separately or together into three @entity95 . An aliquot of the serum or an immunoglobulin (IgG) fraction prepared from the serum of the @entity95 injected with the @entity42634 band or with both the 50- and @entity42634 bands inhibited aromatase activity of @entity1 placental microsomes by 80%; this IgG had no effect on 17 alpha-hydroxylase or 21-hydroxylase activities of @entity1 fetal adrenal microsomes. In contrast, the serum of the @entity95 injected with the 50-kDa band had little capacity to inhibit placental aromatase activity. By immunoblot analysis, it was found that the IgG from the serum of the @entity95 immunized with the @entity42634 protein bound specifically to a protein of 55 kDa in @entity1 placental microsomes. Monoclonal antibodies were prepared from a hybridoma cell line derived from the spleen cells of @entity19 immunized against the @entity42634 protein. The monoclonal IgG was covalently linked to a Sepharose 4B column and was used for immunoaffinity chromatography of @entity4819 . The finding that @entity12325 and the @entity42634 protein were selectively retained by the affinity column and eluted with @entity1219 (2 M) and @entity2228 (0.2 M, pH 3.0) and that this fraction contained aromatase activity upon reconstitution with purified @entity1110 - @entity12325 reductase and phospholipid, is indicative that the @entity42634 protein is indeed cytochrome @entity4819 . These findings are also indicative that both the monoclonal and polyclonal IgGs are specific for @entity1 @entity4819 .
