Tzeng J J, Barth R F, Johnson C W, Adams D M
Department of Pathology, Ohio State University, Columbus 43210.
Proc Soc Exp Biol Med. 1990 Apr;193(4):285-92. doi: 10.3181/00379727-193-43037.
Two rat anti-B16 melanoma monoclonal antibodies (MoAb), designated IB16-6 and IB16-8, recognize an epitope expressed with high density on the surface of B16 parental cells and B16-F1, F10, F10FLR, and BL6 sublines. The purpose of this study was to define by means of cytolytic and clonogenic assays whether these MoAbs reacted with the same or distinct determinants as those recognized on B16 targets by lymphokine-activated killer (LAK) cells. Using 125I-labeled antibody and Scatchard analysis, the affinity constant (KA) of IB16-6 was determined to range from 5.6 to 9.4 x 10(8) liter/M and the number of receptor sites per B16 cell was 4.8 x 10(4) to 2.5 x 10(5). The effects of anti-B16 MoAb on LAK activity were determined by either preincubating 51Cr-labeled B16 target cells with varying concentrations of MoAb, followed by the cytolytic assay, or exposing unlabeled B16 cells to MoAb, and then carrying out a 10-day clonogenic assay. Over a wide range of antibody concentrations, IB16-6 and IB16-8 had minimal effects on LAK activity, and even at MoAb concentrations up to 1 mg there were no changes in target cell sensitivity or colony-forming ability. Enzymatic treatment of B16 melanoma cells with either trypsin or pronase completely removed the epitope recognized by MoAb IB16-6 but did not alter B16 sensitivity to LAK cells. These observations indicate that the LAK recognition unit was distinct from the epitope reactive with MoAb IB16-6 and that the B16 determinant(s) recognized by LAK cells is resistant to proteolytic enzymes. The molecular structure of each of these remains to be determined.
两种大鼠抗B16黑色素瘤单克隆抗体（MoAb），命名为IB16 - 6和IB16 - 8，识别在B16亲本细胞以及B16 - F1、F10、F10FLR和BL6亚系细胞表面高密度表达的一个表位。本研究的目的是通过细胞溶解和克隆形成试验来确定这些单克隆抗体是否与淋巴因子激活的杀伤（LAK）细胞在B16靶细胞上识别的是相同还是不同的决定簇。使用125I标记的抗体和Scatchard分析，确定IB16 - 6的亲和常数（KA）范围为5.6至9.4×10(8)升/摩尔，每个B16细胞的受体位点数量为4.8×10(4)至2.5×10(5)。抗B16单克隆抗体对LAK活性的影响通过以下两种方法确定：一是将51Cr标记的B16靶细胞与不同浓度的单克隆抗体预孵育，然后进行细胞溶解试验；二是将未标记的B16细胞暴露于单克隆抗体，然后进行为期10天的克隆形成试验。在很宽的抗体浓度范围内，IB16 - 6和IB16 - 8对LAK活性的影响极小，即使在单克隆抗体浓度高达1毫克时，靶细胞敏感性或集落形成能力也没有变化。用胰蛋白酶或链霉蛋白酶对B16黑色素瘤细胞进行酶处理可完全去除单克隆抗体IB16 - 6识别的表位，但不会改变B16对LAK细胞的敏感性。这些观察结果表明，LAK识别单位与与单克隆抗体IB16 - 6反应的表位不同，并且LAK细胞识别的B16决定簇对蛋白水解酶具有抗性。它们各自的分子结构仍有待确定。
