体外诱导和检测人类B细胞产生抗HLA抗体的研究

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

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5d，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ELISA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HIA抗体一致。结论PWM和SAC体外刺激PBMC，结合HLA特异的ELISPOT方法，能诱导和检测到人类B细胞产生的HLA抗体。

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 volunteers were isolated by density gradient centrifugation, and cultured with the stimulation of PWM alone or PWM ＋ SAC for 5 d. The Ig levels in culture supernatants and the number of Ig producing B cells were determined 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-culture 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 IgM levels were increased （ P = 0.03 ） in the presence of the combination 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-culture supernatants supported the specificity. Conclusion The activation of PBMC with the combination of PWM and SAC, and the application of HLA-specific ELISPOT assay can succeed in the induction and detection of human B cells producing HLA antibodies.