Alonso-Whipple C, Couet M L, Doss R, Koziarz J, Ogunro E A, Crowley W F
Reproductive Endocrine Unit, Massachusetts General Hospital, Boston 02114.
Endocrinology. 1988 Oct;123(4):1854-60. doi: 10.1210/endo-123-4-1854.
To establish structure-function relationships for human (h) LH, 14 murine monoclonal antibodies (MABs) to hLH were characterized in terms of their affinity of binding (Ka), their specificity for intact glycoproteins and their subunits, their paratopic relationships, and their ability to interfere with the biological activity of hLH. The Ka values obtained ranged between 2.4 x 10(6) and 1.0 x 10(10) for intact hLH and between 2.3 x 10(6) and 7.5 x 10(8) liters/M for the free alpha- and beta-subunits, indicating that, in general, the antibodies showed higher avidity for the intact hormone. Six MAB recognized both the intact and free alpha-subunit of hLH, cross-reacted with intact hCG, hFSH, and hTSH, and thus appeared to be alpha-directed. Four MAB were beta-directed, recognizing both intact hLH and its free beta-subunit. One of these beta-directed MABs also cross-reacted with intact hCG, hFSH, and hTSH, while two others recognized both intact and free beta-subunits of hLH and hCG. The fourth beta-directed MAB was quite specific for intact hLH and its beta-subunit. The remaining four MABs recognized epitopes only on the intact hormone; three recognized intact hLH and hCG, and the fourth was specific for intact hLH. Their paratopic relationships tested in competitive binding studies resulted in either mutual competition or complementarity, sometimes with cooperativity. Biointerference, defined as the ability to inhibit hLH-induced testosterone biosynthesis in dispersed rat Leydig cells, indicated that three of the alpha- and one of the beta-directed antibodies neutralized the biological response of hLH in this bioassay in a dose-responsive manner. Their ability to inhibit hLH bioactivity largely paralleled their affinity constants. Our data have allowed us to establish a tentative topographic relationship of epitopes to the biological region of the molecule of hLH, foreshadowing (in additive binding studies) some of the possible combinations of antibodies that might allow us to design two- or multiple-site immunometric assays in which measurement of immunoactive LH reflects biological activity. In addition, these studies suggest that both the alpha- and beta-subunits participate in LH receptor binding and/or biological activity.
为了建立人促黄体生成素（hLH）的结构-功能关系，对14种抗hLH的鼠单克隆抗体（MABs）进行了表征，包括它们的结合亲和力（Ka）、对完整糖蛋白及其亚基的特异性、互补位关系以及干扰hLH生物活性的能力。完整hLH的Ka值在2.4×10⁶至1.0×10¹⁰之间，游离α和β亚基的Ka值在2.3×10⁶至7.5×10⁸升/摩尔之间，这表明一般来说，抗体对完整激素的亲和力更高。六种MAB识别hLH的完整和游离α亚基，与完整hCG、hFSH和hTSH发生交叉反应，因此似乎是α导向的。四种MAB是β导向的，识别完整hLH及其游离β亚基。其中一种β导向的MAB也与完整hCG、hFSH和hTSH发生交叉反应，而另外两种识别hLH和hCG的完整和游离β亚基。第四种β导向的MAB对完整hLH及其β亚基具有相当的特异性。其余四种MAB仅识别完整激素上的表位；三种识别完整hLH和hCG，第四种对完整hLH具有特异性。在竞争性结合研究中测试的它们的互补位关系导致相互竞争或互补，有时具有协同作用。生物干扰定义为抑制分散的大鼠睾丸间质细胞中hLH诱导的睾酮生物合成的能力，表明三种α导向和一种β导向的抗体在该生物测定中以剂量反应方式中和hLH的生物反应。它们抑制hLH生物活性的能力在很大程度上与它们的亲和常数平行。我们的数据使我们能够建立表位与hLH分子生物区域的初步拓扑关系，（在加和性结合研究中）预示了一些抗体的可能组合，这些组合可能使我们能够设计双位点或多位点免疫测定法，其中免疫活性LH的测量反映生物活性。此外，这些研究表明α和β亚基都参与LH受体结合和/或生物活性。
