Evans D L, Harris D T, Leary J, Jaso-Friedmann L
Department of Medical Microbiology, College of Veterinary Medicine, University of Georgia, Athens 30602.
Cell Immunol. 1992 May;141(2):293-305. doi: 10.1016/0008-8749(92)90149-j.
Monoclonal antibodies were generated against idiotopes on an NK target antigen-specific IgM monoclonal antibody (mab). This mab (18C2) was originally produced against (NC-37) human EBV-transformed B cells. The 18C2 mab inhibits natural killer cell lysis of NC-37 and other target cells by preventing conjugate formation. Anti-18C2(id) mabs were tested for binding to effector cells and screened by ELISA, flow cytometry, and by inhibition of NK cytotoxicity. Two of the anti-18C2(id) (anti-id) mabs (12H1.C5 and 6D9.B11) were chosen for further study. The idiotypic specificity of these anti-id mabs was confirmed by testing their binding to 18C2 hybridoma cells in the presence of homologous and heterologous "cold" inhibitor mabs. Experiments were also conducted to determine the functional properties of these mabs. Anti-18C2(id) mab 12H1.C5 inhibited the cytotoxic activity of rat splenic NK (nylon wool nonadherent cells, NWNA) and rat ALAK cells. Flow cytometric (FCM) analysis of the binding of the anti-18C2(id) mabs demonstrated that mab 12H1.C5 bound 75.43% rat NWNA spleen cells, 43.74% rat ALAK cells, and 74.33% rat CRC- cells. Anti-id mab 6D9.B11 bound 45.20% NWNA cells, 70.45% rat ALAK cells, and 55.86% CRC- cells. Two-color FCM analysis demonstrated that the anti-id mabs not only bound to the same molecule on NK cells, but also these mabs bound to the same molecule as 5C6, an anti-NK cell mab. Biochemical analysis of the antigen recognized by mab 12H1.C5 was determined by Western blotting. The determinant on NWNA cells recognized by mab 12H1.C5 had an M(r) of 40 kDa and appeared to be identical to that recognized by mab 5C6. The same experiment using a transformed rat RNK-16 (CRC-) cell extract and Western blot analysis, demonstrated an M(r) of 42 and 48 kDa in the presence of mabs 5C6 and 12H1.C5. Monoclonal antibody 5C6 was previously shown to recognize a vimentin-like function-associated molecule on NK cell membranes. The anti-id mabs were also shown to have cross-reactivity with the intermediate filament vimentin as determined by Western blot analysis.
针对一种NK靶抗原特异性IgM单克隆抗体（mab）上的独特型表位产生了单克隆抗体。这种mab（18C2）最初是针对（NC - 37）人EB病毒转化的B细胞产生的。18C2 mab通过阻止共轭形成来抑制NK细胞对NC - 37和其他靶细胞的裂解。检测了抗18C2（独特型）mab与效应细胞的结合，并通过酶联免疫吸附测定（ELISA）、流式细胞术以及NK细胞毒性抑制试验进行筛选。选择了两种抗18C2（独特型）（抗独特型）mab（12H1.C5和6D9.B11）进行进一步研究。通过在同源和异源“冷”抑制mab存在的情况下检测它们与18C2杂交瘤细胞的结合，证实了这些抗独特型mab的独特型特异性。还进行了实验以确定这些mab的功能特性。抗18C2（独特型）mab 12H1.C5抑制大鼠脾脏NK（尼龙毛非黏附细胞，NWNA）和大鼠ALAK细胞的细胞毒性活性。抗18C2（独特型）mab结合的流式细胞术（FCM）分析表明，mab 12H1.C5结合75.43%的大鼠NWNA脾细胞、43.74%的大鼠ALAK细胞和74.33%的大鼠CRC - 细胞。抗独特型mab 6D9.B11结合45.20%的NWNA细胞、70.45%的大鼠ALAK细胞和55.86%的CRC - 细胞。双色FCM分析表明，抗独特型mab不仅与NK细胞上的同一分子结合，而且这些mab与抗NK细胞mab 5C6结合同一分子。通过蛋白质印迹法对mab 12H1.C5识别的抗原进行生化分析。mab 12H1.C5在NWNA细胞上识别的决定簇的相对分子质量（M(r)）为40 kDa，似乎与mab 5C6识别的相同。使用转化的大鼠RNK - 16（CRC -）细胞提取物和蛋白质印迹分析进行的相同实验表明，在mab 5C6和12H1.C5存在的情况下，相对分子质量为42和48 kDa。先前已表明单克隆抗体5C6识别NK细胞膜上一种波形蛋白样功能相关分子。通过蛋白质印迹分析还表明，抗独特型mab与中间丝波形蛋白具有交叉反应性。
