Abstract：
Rabbit anti-thymocyte globulin (rATG) has been widely used to prevent graft-versus-host disease (GvHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). The therapeutic window of rATG is narrow, and it may increase the risk of relapse, viral reactivation, delayed immune reconstitution and GvHD when overexposed or underexposed. Therefore, a reliable method for detecting the rATG concentration in human serum by flow cytometry was established and fully validated for therapeutic drug monitoring. In this method, Jurkat T cells were used to capture active rATG in human serum, and PE-labeled donkey anti-rabbit IgG was used as a secondary antibody. The method showed good specificity, selectivity and excellent linearity at concentration of 0.00300-20.0 AU/mL. The intra- and interday precision values were all within 20% at four concentration levels for the analyte. The stock solutions of rATG showed no significant degradation after storage at ambient temperature for 8 h and at - 80 °C for 481 days. No significant degradation of rATG in serum was observed at ambient temperature for 6 h, during six freezethaw cycles and at - 80 °C for at least 373 days. This method was fully validated and successfully applied to monitor active rATG concentration in serum of patients with haploid-identical hematopoietic stem cell transplantation.
摘要：
兔抗胸腺细胞球蛋白（rATG）已被广泛用于预防异基因造血干细胞移植（allo-HSCT）后的移植物抗宿主病（GvHD）。rATG的治疗窗口狭窄，它可能会增加复发的风险，病毒再激活，过度曝光或曝光不足时延迟免疫重建和GvHD。因此,建立了通过流式细胞术检测人血清中rATG浓度的可靠方法,并对治疗药物监测进行了充分验证.在这种方法中，JurkatT细胞用于捕获人血清中的活性rATG，使用PE标记的驴抗兔IgG作为第二抗体。该方法具有良好的特异性,在0.00300-20.0AU/mL浓度下的选择性和优异的线性。在分析物的四个浓度水平下，日内和日间精度值均在20％以内。rATG的储备溶液在环境温度下储存8h和在-80°C下储存481天后未显示显著降解。在环境温度下6小时内未观察到血清中rATG的显着降解，在六个冷冻循环期间，在-80°C下持续至少373天。该方法得到充分验证，并成功应用于单倍体相合造血干细胞移植患者血清中活性rATG浓度的监测。
