Kirkley J, Fabre J W
Hum Immunol. 1980 Dec;1(4):369-81. doi: 10.1016/0198-8859(80)90113-5.
The binding assay as described in this paper is a very versatile system, and in this study it has been evaluated specifically for the detection of allo- or autoantibodies to platelets in man. The basic assay involves the incubation of a standard number of platelets with dilutions of test sera and the detection of platelet bound immunoglobulin by a second incubation with 125I labeled, immunoadsorbent purified rabbit F(ab') anti-human F(ab')2 (RAH). Of most importance, by varying the number of target platelets in the titrations and looking for binding plateaus, one can readily define conditions of optimum sensitivity for particular serum/platelet combinations. In addition, the assay can be used in conjunction with quantitative absorptions to subdivide complex sera into subspecificities and to give an estimate of the relative amounts of particular antigens on different platelets or other tissue or cell suspensions. One can also use saturating concentrations of RAH in the second incubation, in which case the amount of platelet bound radioactivity is directly related to the amount of first antibody bound to the platelets, and this can be manipulated to give information about serum antibody concentrations and amounts of antigen on the target tissue. The problem of ABO antibodies in this system, optimal conditions for platelet storage for the assay, and techniques for reducing assay backgrounds resulting from immunoglobulin adsorbed to the platelet surface are all evaluated.
本文所述的结合试验是一个非常通用的系统，在本研究中，它已被专门评估用于检测人类血小板的同种或自身抗体。基本试验包括将标准数量的血小板与测试血清稀释液孵育，并通过与125I标记的、免疫吸附纯化的兔F(ab')抗人F(ab')2（RAH）进行第二次孵育来检测血小板结合的免疫球蛋白。最重要的是，通过在滴定中改变靶血小板的数量并寻找结合平台，可以很容易地确定特定血清/血小板组合的最佳灵敏度条件。此外，该试验可与定量吸收结合使用，将复杂血清细分为亚特异性，并估计不同血小板或其他组织或细胞悬液上特定抗原的相对含量。在第二次孵育中也可以使用饱和浓度的RAH，在这种情况下，血小板结合的放射性量与结合到血小板上的第一抗体量直接相关，并且可以对其进行操作以提供有关血清抗体浓度和靶组织上抗原量的信息。对该系统中ABO抗体的问题、用于试验的血小板储存的最佳条件以及减少因吸附在血小板表面的免疫球蛋白导致的试验背景的技术都进行了评估。
