用ELISpot检测人类HLA2Ⅰ抗体的方法

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酶联免疫斑点法（ELISpot）

廖爱华　VERA Rebmann ①　HANS Grosse2Wilde ①　(华中科技大学同济医学院生殖医学中心,武汉430030)

[摘　要] 　目的:建立一种新的检测人类HLA2 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HLA2 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0125 mg/ 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(01125、 015 和110 mg/ ml , P < 0105) ;当包被抗体浓度为118 mg/ ml 、稀释度为1∶125 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∶250、1∶500 和1∶1 000 , P < 0105) ;且在抗体稀释度相同情况下,B 细胞孵育时间为24 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 > 0105) 。结论:当B 细胞溶胞体作为HLA 抗原且浓度为0125 mg/ ml、包被抗体浓度为118 mg/ ml 且稀释度为1∶ 125、B 细胞孵育时间为24 小时时,该ELISpot 方法能特异地、敏感地、可信地检测人HLA2 Ⅰ类抗体。

[关键词] 　酶联免疫 斑点法;HLA 抗体;异体移植

ELISPOT :a new tool for detecting human HLA2 Ⅰantibody LIAO Ai2Hua , VERA Rebmann 3 , HANS Grosse2Wilde 3 . Center of Reproductive Medicine , Tongji Medical College , Huazhong University of Science and Technology , Wuhan 430030 , China ; 3 Institute of Immunology , University Hospital of Essen , Essen 45147 , Germany Abstract 　Objective :To establish a new ELISpot method for detecting human HLA2 Ⅰantibodies.Methods :By applying mouse B cell line (HB95) as a model ,a series of parameters for establishing a specific HLA2 Ⅰantibody2detecting ELISpot method were under construction , including the source of HLA antigen ,the optimal concentration of coated antibody and the optimal incubation time of B cells. At the mean time , the sensitivity and specificity of this method were checked by using EBV2transforming human B cell line to interfer with mouse B cell line. Re2 sults :B cell lysate was applied as source of HLA antigen.When antigen concentration was 0125 mg/ ml ,the spot’s frequnency was the highest among the other three concentrations (01125 ,015 and 110 mg/ ml ,respectively , P < 0105) under the same B cell density. Additionally ,when coated antibody’s concentration was 118 mg/ ml and dilution was 1∶125 ,the spot’s frequency was the highest among the other three dilution (1 ∶250 ,1∶500 and 1∶1 000 ,respectively , P < 0105) at the same incubation time. Besides ,when B cell incubation time was 24 h ,the detected spots were the clearest and the background was the lightest in contrast to the other two incubation time as 48 h and 72 h.Lastly ,comparing sup2 plement of combination of EBV2transforming human B cell line and mouse B cell line with that of mouse B cell line alone ,the detected spots were almost the same and had no significant statistic differences ( P > 0105) . Conclusion :When B cell lysate was applied as source of HLA antigen and concentration was 0125 mg/ ml ,coated antibody’s concentration was 118 mg/ ml and dilution was 1∶125 ,and B cell incubation time was 24 h ,the established ELISpot assays represent a sensitive ,specific and reliable tool for detecting human HLA classⅠantibodies.

Key words 　ELISPOT ;HLA antibody ;Allograft