Monoclonal antibodies for carcinoembryonic antigen and related antigens as a model system: determination of affinities and specificities of monoclonal antibodies by using biotin-labeled antibodies and avidin as precipitating agent in a solution phase immunoassay.
Monoclonal antibodies for carcinoembryonic antigen and related antigens as a model system: determination of affinities and specificities of monoclonal antibodies by using biotin-labeled antibodies and avidin as precipitating agent in a solution phase immunoassay.
复制标题
癌胚抗原和相关抗原的单克隆抗体作为模型系统：在溶液相免疫测定中使用生物素标记的抗体和亲和素作为沉淀剂测定单克隆抗体的亲和力和特异性。
作者：
C. Wagener;B. Clark;K. J. Rickard;J. Shively
描述了测定单克隆抗体的亲和常数和抗原交叉反应性的通用方法。该方法在同相、竞争性放射免疫测定中采用生物素标记的抗体、放射性标记的抗原和抗生物素蛋白作为沉淀剂。该方法消除了在使用单克隆抗体的免疫测定中经常遇到的抗原-抗体复合物的不完全或可变沉淀。使用该检测系统，我们能够快速确定多种单克隆抗体对癌胚抗原 (CEA) 的亲和力常数。上一篇论文表明，五种单克隆抗体可识别 CEA 上的不同表位。在使用这五种单克隆抗体的抗原结合实验中，抗体过量结合的放射性标记 CEA 的百分比范围为 30% 至 92%。 CEA 交叉反应抗原、正常交叉反应抗原 (NCA) 和肿瘤提取的 CEA 相关抗原 (TEX) 与五种抗体中的一种显着结合，而与两种抗体结合程度较低。两种抗体未结合大量的 NCA 或 TEX。在抑制研究中，导致 125I 标记 CEA 结合抑制 50% 的未标记 CEA 量在每管 3.7 至 760 ng 范围内。显示相同抑制程度的 TEX 量比两种抗体的 CEA 量高 23 倍，比第三种抗体的 CEA 量高 351 倍。 CEA 的亲和常数在 1.0 x 10(8) 至 5.1 x 10(10) M-1 范围内。针对其中一种抗体测定的 NCA 和 TEX 亲和常数比 CEA 低三个数量级。由单克隆抗体之一结合的低分数所表明的放射性标记 CEA 的异质性很可能是由放射性碘标记损伤引起的伪影。本报告中描述的方法的应用应该消除在确定单克隆抗体的亲和常数或在竞争性同相免疫测定中使用单克隆抗体时最常遇到的问题。
A general method is described for the determination of affinity constants and antigen cross-reactivities of monoclonal antibodies. The method employs biotin-labeled antibody, radiolabeled antigen, and avidin as a precipitating agent in a homogeneous phase, competitive radioimmunoassay. This method eliminates incomplete or variable precipitation of antigen-antibody complexes often encountered in immunoassays in which monoclonal antibodies are employed. Using this assay system, we were able to rapidly determine the affinity constants for a number of monoclonal antibodies elicited to carcinoembryonic antigen (CEA). In the preceding paper it was shown that five of the monoclonal antibodies recognized distinct epitopes on CEA. In antigen-binding experiments with these five monoclonal antibodies, the percent of radiolabeled CEA bound in antibody excess ranged from 30 to 92%. The CEA cross-reacting antigens, normal cross-reacting antigen (NCA), and tumor-extracted, CEA-related antigen (TEX) were significantly bound by one, and to a lesser degree, by two of the five antibodies. Two antibodies did not bind significant amounts of NCA or TEX. In inhibition studies, the amount of unlabeled CEA leading to 50% inhibition of 125I-labeled CEA-binding was in the range of 3.7 to 760 ng per tube. The amount of TEX showing the same degree of inhibition was 23-fold greater than the amount of CEA for two antibodies and 351-fold greater than the amount of CEA for a third antibody. The affinity constants for CEA were in the range of 1.0 x 10(8) to 5.1 x 10(10) M-1. The affinity constants for NCA and TEX, determined for one of the antibodies, were three orders of magnitude lower in comparison to CEA. The heterogeneity of radiolabeled CEA as indicated by the low fraction bound by one of the monoclonal antibodies is shown to be most probably an artifact resulting from radioiodination damage. The application of the approach described in this report should eliminate the problems most commonly encountered in the determination of affinity constants for monoclonal antibodies or the use of monoclonal antibodies in competitive, homogeneous-phase immunoassays.