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目的 :建立一种新的检测人类HLA Ⅰ类抗体方法———酶联免疫斑点法 (ELISpot)。方法 :以鼠B细胞系HB95为模型 ,建立一系列特异性ELISpot检测HLA Ⅰ类抗体的参数 ,包括HLA抗原的来源、包被抗体最适宜浓度的选择、B细胞最适宜孵化时间 ,同时以经EBV转化的敏感NTP患者B细胞系为干扰因素 ,检验该方法的敏感性和特异性。结果 :以B细胞溶胞体作为HLA抗原来源 ,当抗原浓度为 0 2 5mg/ml时 ,相同细胞密度下所获得的斑点频数均高于其它三种抗原浓度 (0 12 5、0 5和 1 0mg/ml,P <0 0 5 ) ;当包被抗体浓度为 1 8mg/ml、稀释度为 1∶12 5时 ,相同孵育时间下所获得的斑点频数高于其它稀释浓度 (1∶2 5 0、1∶5 0 0和 1∶10 0 0 ,P <0 0 5 ) ;且在抗体稀释度相同情况下 ,B细胞孵育时间为 2 4小时所获得的斑点最清晰、背景着色最浅、最容易被分析仪器识别。此外 ,当同时加入经EBV转化的人B细胞时 ,所获得的斑点频数与单纯加鼠B细胞相似 ,无明显差异 (P >0 0 5 )。结论 :当B细胞溶胞体作为HLA抗原且浓度为 0 2 5mg/ml、包被抗体浓度为 1 8mg/ml且稀释度为 1∶12 5、B细胞孵育时间为 2 4小时时 ,该ELISpot方法能特异地、敏感地、可信地检测人HLA Ⅰ类抗体。
OBJECTIVE: To establish a new method for detecting human HLA class Ⅰ antibody by ELISpot. Methods: Using the murine B cell line HB95 as a model, a series of specific ELISpot detection parameters of HLA class I antibodies were established, including the origin of HLA antigens, the optimal concentration of coating antibody, the optimum incubation time of B cells, The B cell lines of EBV-transformed NTP patients were interfering factors to test the sensitivity and specificity of the method. Results: Using B cell lysates as the source of HLA antigens, the frequency of spots obtained at the same cell density was higher than that of the other three antigens when the antigen concentration was 0 25 mg / ml (0 12 5, 05 and 10 mg / ml, P <0 05). When the coating antibody concentration was 18mg / ml and the dilution was 1:12 5, the frequency of spots obtained at the same incubation time was higher than that of other dilutions (1: 250 , 1:5 0 0 and 1:10 0 0, P <0 05). And when the antibody dilution was the same, the incubation time of B cells was 24 hours and the spots obtained were the clearest and the background was shaded the most Easily recognized by analytical instruments. In addition, when the EBV-transformed human B cells were added at the same time, the frequency of spots obtained was similar to that of the mouse B cells alone (P> 0.05). CONCLUSIONS: When the B cell lysate is used as an HLA antigen and has a concentration of 025 mg / ml, a coating antibody concentration of 18 mg / ml and a dilution of 1: 125, and a B cell incubation time of 24 hours, the ELISpot method Human HLA class I antibodies can be detected specifically, sensitively and reliably.