DeBell K E, Taplits M S, Hoffman T, Bonvini E
Division of Blood and Blood Products, U.S. Food and Drug Administration, Bethesda, Maryland 20892.
Cell Immunol. 1990 Apr 15;127(1):159-71. doi: 10.1016/0008-8749(90)90122-8.
An assay has been developed to quantitate the binding of beads coated with anti-T cell receptor (TCR) monoclonal antibodies (MoAb) to T lymphocytes. The Ab used were a hamster MoAb, 145.2C11 (2C11), directed against the epsilon chain of the CD3 complex of the murine TCR, and a murine MoAb, F23.1, directed against the V beta 8-encoded determinant of the alpha/beta heterodimer of the TCR. Ab were adsorbed onto polystyrene beads and the beads labeled with [125I]bovine serum albumin [( 125I]BSA). The labeled, Ab-coated beads were mixed at 4 degrees C with murine, cloned T-helper (Th) cells and contact between beads and cells was promoted by centrifugation. The mixtures were incubated at 37 degrees C for 10-20 min, and unbound beads were separated from cell-bound beads by Percoll gradient centrifugation. Beads coated with anti-TCR Ab formed stable conjugates with Th cells; an average of 6-10 2C11 Ab-coated beads/cell, or 10-15 F23.1 Ab-coated beads/cell was measured under optimal conditions. Beads coated with control Ab (hamster or mouse IgG) did not appreciably bind to the cells. Conjugation with 2C11 Ab-coated beads could be prevented by coating the cells with soluble 2C11 Ab, but not with soluble F23.1 Ab. Blocking the CD3 epsilon chain with soluble 2C11 Ab also reduced conjugate formation with F23.1 Ab-coated beads, suggesting a steric hindrance phenomenon. The extent of conjugation depended on the density of immobilized Ab. Maximum conjugation was observed when 100 micrograms of 2C11 Ab was used to coat 10(6) beads; higher Ab amounts did not further increase binding. Increasing the bead to cell ratio in the mixture increased binding, reaching optimal binding at 300:1, irrespectively of the amount of Ab adsorbed onto the beads. Stable binding of anti-TCR Ab-coated beads to T cells was temperature and energy dependent. It was prevented when glucose was removed from the medium and the glycolysis inhibitor, 2-deoxy-D-glucose was added, or when cells were treated with sodium azide. Conjugate formation was prevented by pretreatment of the cells with cytochalasins, indicating that microfilament assembly was essential. Microtubules were not involved, as vinca alkaloids were without effect. This novel assay system provides a simple means of studying aspects of TCR function including its physical and metabolic regulation.
已开发出一种检测方法，用于定量包被抗T细胞受体（TCR）单克隆抗体（MoAb）的珠子与T淋巴细胞的结合。所使用的抗体是一种仓鼠MoAb，即145.2C11（2C11），它针对小鼠TCR的CD3复合物的ε链，以及一种小鼠MoAb，F23.1，它针对TCR的α/β异二聚体的Vβ8编码决定簇。将抗体吸附到聚苯乙烯珠子上，并用[125I]牛血清白蛋白[（125I]BSA）标记珠子。将标记的、包被抗体的珠子在4℃下与小鼠克隆的辅助性T（Th）细胞混合，并通过离心促进珠子与细胞之间的接触。混合物在37℃下孵育10 - 20分钟，然后通过Percoll梯度离心将未结合的珠子与细胞结合的珠子分离。包被抗TCR抗体的珠子与Th细胞形成稳定的结合物；在最佳条件下，平均每个细胞可检测到6 - 10个包被2C11抗体的珠子，或10 - 15个包被F23.1抗体的珠子。包被对照抗体（仓鼠或小鼠IgG）的珠子与细胞没有明显结合。用可溶性2C11抗体包被细胞可阻止与包被2C11抗体的珠子结合，但用可溶性F23.1抗体则不能。用可溶性2C11抗体封闭CD3ε链也会减少与包被F23.1抗体的珠子的结合，提示存在空间位阻现象。结合程度取决于固定化抗体的密度。当用100微克2C11抗体包被10(6)个珠子时观察到最大结合；更高的抗体量不会进一步增加结合。增加混合物中珠子与细胞的比例会增加结合，在300:1时达到最佳结合，与吸附在珠子上的抗体量无关。包被抗TCR抗体的珠子与T细胞的稳定结合依赖于温度和能量。当从培养基中去除葡萄糖并添加糖酵解抑制剂2 - 脱氧 - D - 葡萄糖时，或者当细胞用叠氮化钠处理时，结合被阻止。用细胞松弛素预处理细胞可阻止结合物形成，表明微丝组装是必不可少的。微管不参与其中，因为长春花生物碱没有作用。这种新的检测系统为研究TCR功能的各个方面提供了一种简单的方法，包括其物理和代谢调节。
