体外诱导和检测人类B细胞产生抗HLA抗体的研究

目的 体外诱导和采用酶联免疫斑点法(ELISPOT)检测人类B细胞产生的抗HLA抗体.方法 对7名健康志愿者采用密度梯度离心法分离外周血单个核细胞(PBMC)并体外培养5 d,以B细胞多克隆刺激原美洲商陆丝裂原(PWM)、葡萄球菌A蛋白菌体(SAC)体外刺激PBMC,采用EHSA和ELISPOT方法分别测定培养上清中Ig浓度和抗体分泌B细胞数,探索体外诱导PBMC产生Ig的适宜方法.采用该刺激方法和HLA特异的ELISPOT法,诱导和检测9例拟行肾移植预致敏对象PBMC产生的抗HLA抗体.结果 与PWM单刺激相比,PBMC在PWM和SAC联合刺激后,有更多细胞存活的趋势(P=0.052),且培养上清中IgM的水平明显增加(P=0.03),而IgG的水平无明显变化(P>0.05).6例预致敏对象诱导和检测到抗HLA抗体.其特异性与各自培养上清中检测到的抗HLA抗体一致.结论 PWM和SAC体外刺激PBMC,结合HLA特异的ELISPOT方法,能诱导和榆测到人类B细胞产生的HLA抗体.

In vitro induction and detection of human B cells producing HLA antibodies

Objective To induce and detect human B cells producing HLA antibodies in vitro and by an ELISPOT assay. Methods Human peripheral blood mononuclear cells (PBMC) from 7 healthy vol-unteers were isolated by density gradient centrifugation, and cultured with the stimulation of PWM alone or PWM + SAC for 5 d. The lg levels in culture supernatants and the number of Ig producing B cells were de-termined by ELISA and ELISPOT assay, respectively. The optimal stimulation protocol for PBMC to produce optimal Ig in vitro was anticipated to be obtained. Using the optimal pre-cuhure and ELISPOT conditions, PBMC from 9 alloimmunized subjects were analyzed for the presence of B cells secreting HLA antibodies. Results There was a tendency towards more viable cells following the activation with PWM and SAC vs PWM alone in a 5-day culture (P =0. 052). The IgG levels in the supernatants were found to be comparable for the two culture conditions whereas the lgM levels were increased ( P = 0.03 ) in the presence of the com-bination of PWM and SAC. In 6 subjects a specific signal was obtained with our ELISPOT assay. The presence of HLA antibodies in the respective pre-cuhure supernatants supported the specificity. Conclusion The activation of PBMC with the combination of PWM and SAC, and the application of HLA-specifie ELIS-POT assay can succeed in the induction and detection of human B cells producing HLA antibodies.