Sullivan W P, Beito T G, Proper J, Krco C J, Toft D O
Endocrinology. 1986 Oct;119(4):1549-57. doi: 10.1210/endo-119-4-1549.
In an effort to obtain additional probes for analysis of the avian progesterone receptor, this receptor was isolated and used to prepare several monoclonal antibodies. Progesterone receptor purified from oviduct cytosol by chromatography on deoxycorticosterone-Sepharose and heparin-agarose was used as the immunizing antigen. Twenty-nine hybridoma cultures which tested positive in an enzyme-linked immunosorbent assay against the receptor preparation were subcloned resulting in establishment of 12 stable cell lines. Of these, 5 produced antibodies capable of complexing receptor-bound progesterone from cytosol as measured by adsorption of receptor-antibody complexes onto antimouse immunoglobulin G-agarose. Each was used to generate ascites and the purified antibodies were designated alpha PR 6, 11, 13, 16, and 22. In addition to precipitating receptor-bound progesterone from cytosol, the antibodies were also effective in increasing the sedimentation velocity of progesterone receptor centrifuged on glycerol gradients, and in recognizing receptor proteins that were resolved by denaturing gel electrophoresis and transferred to nitrocellulose (Western blots). Immunoisolation of receptor was also demonstrated using receptor labeled covalently with the synthetic progestin, R5020. The antibodies were specific for progesterone receptor and did not cross-react with estrogen receptor from the oviduct or glucocorticoid receptor from chick liver. Two antibodies, alpha PR 6 and alpha PR 22, also recognized some mammalian forms of the progesterone receptor. Both antibodies reacted with progesterone receptor from the rabbit uterus and alpha PR 6 recognized human progesterone receptor. Four of the antibodies recognized both A and B forms of the avian receptor while alpha PR6 was specific for the B form.
为了获得更多用于分析禽类孕酮受体的探针，对该受体进行了分离，并用于制备几种单克隆抗体。从输卵管胞质溶胶中通过脱氧皮质酮 - 琼脂糖凝胶和肝素 - 琼脂糖柱层析纯化的孕酮受体用作免疫抗原。对受体制剂进行酶联免疫吸附测定呈阳性的29个杂交瘤培养物进行了亚克隆，从而建立了12个稳定的细胞系。其中，有5个产生的抗体能够通过将受体 - 抗体复合物吸附到抗小鼠免疫球蛋白G - 琼脂糖上来结合胞质溶胶中与受体结合的孕酮。每个抗体都用于产生腹水，纯化后的抗体分别命名为αPR 6、11、13、16和22。除了从胞质溶胶中沉淀与受体结合的孕酮外，这些抗体还能有效提高在甘油梯度上离心的孕酮受体的沉降速度，并能识别经变性凝胶电泳分离并转移到硝酸纤维素膜上（Western印迹）的受体蛋白。还使用与合成孕激素R5020共价标记的受体证明了受体的免疫分离。这些抗体对孕酮受体具有特异性，不会与输卵管中的雌激素受体或鸡肝中的糖皮质激素受体发生交叉反应。两种抗体，αPR 6和αPR 22，还能识别某些哺乳动物形式的孕酮受体。这两种抗体都能与兔子宫中的孕酮受体发生反应，αPR 6能识别人类孕酮受体。其中四种抗体能识别禽类受体的A和B两种形式，而αPR6对B形式具有特异性。
