Indrapichate K, Meehan D, Lane T A, Chu S Y, Rao C V, Johnson D, Chen T T, Wimalasena J
Department of Physiology, University of Nebraska Medical Center, Omaha 68198-4575.
Biol Reprod. 1992 Feb;46(2):265-78. doi: 10.1095/biolreprod46.2.265.
Several recent studies have elucidated the structure of the mammalian LH/hCG receptor; as reported in the present work, we have developed a series of monoclonal antibodies (mAbs) against the rat ovarian LH/hCG receptor using highly purified receptor as immunogen and by screening hybridomas with purified LH/hCG receptors. The mAbs were able to specifically immunoprecipitate LH/hCG receptors from solubilized preparations of rat ovarian membranes as well as from partially purified preparations. Western blotting with mAb P1B4 detected a probable receptor dimer and a receptor fragment in rat and porcine ovarian tissue but not in other tissues. This mAb also partially inhibited hCG binding to rat and porcine ovarian tissues. The receptor mAbs were able to inhibit hCG-induced progesterone synthesis in cultured human and porcine granulosa cells without affecting cAMP- and FSH-induced progesterone synthesis. The mAb P1B4 was used to demonstrate that the majority of ovarian receptors are internalized after hCG treatment and that in pseudopregnant rats receptors are present in the rough endoplasmic reticulum and in microvesicles. Bovine corpus luteal cells also contained P1B4 binding sites, as detected by immunohistochemical technique. Taken together, these results suggest that the mAbs are specific for the LH/hCG receptor, mAb P1B4 recognizes an epitope that is highly conserved among mammals, and this epitope is probably in the extracellular domain.
最近的几项研究已经阐明了哺乳动物促黄体生成素/人绒毛膜促性腺激素（LH/hCG）受体的结构；正如本研究报道的那样，我们以高度纯化的受体作为免疫原，并通过用纯化的LH/hCG受体筛选杂交瘤，制备了一系列针对大鼠卵巢LH/hCG受体的单克隆抗体（mAb）。这些单克隆抗体能够从大鼠卵巢膜的可溶制剂以及部分纯化制剂中特异性免疫沉淀LH/hCG受体。用单克隆抗体P1B4进行蛋白质免疫印迹分析，在大鼠和猪的卵巢组织中检测到一种可能的受体二聚体和一个受体片段，但在其他组织中未检测到。该单克隆抗体也部分抑制了hCG与大鼠和猪卵巢组织的结合。这些受体单克隆抗体能够抑制hCG诱导的培养的人及猪颗粒细胞中孕酮的合成，而不影响cAMP和FSH诱导的孕酮合成。单克隆抗体P1B4被用于证明大多数卵巢受体在hCG处理后会被内化，并且在假孕大鼠中，受体存在于粗面内质网和微泡中。通过免疫组织化学技术检测发现，牛黄体细胞中也含有P1B4结合位点。综上所述，这些结果表明这些单克隆抗体对LH/hCG受体具有特异性，单克隆抗体P1B4识别的表位在哺乳动物中高度保守，并且该表位可能位于细胞外结构域。
