T细胞受体工程化T细胞的流式检测方法的建立

Establishment of flow cytometry assay for detection of T cell receptor-engineered T cells

[目的]建立特异性识别乙肝病毒表面抗原的T细胞受体工程化T细胞(T cell receptor-engineered T cells, TCR-T)的流式检测方法。[方法]将3个检测抗体分别进行梯度稀释(死细胞指示剂FVS780:0.020~10.000μg/mL、鼠抗人的CD3抗体:0.039~20.000μg/mL和结合TCR的多肽-MHC复合物蛋白Dextramer-PE:1∶5至1∶2 560倍稀释)后,分别与同一TCR-T细胞共孵育,得出最佳使用浓度。两个不同分析人员采用此方法分别同时对3批TCR-T细胞样本进行3次检测,计算TCR阳性T细胞比例的CV值考察方法的中间精密度。同一分析人员分别检测2批TCR-T细胞的6个平行样本,计算TCR阳性T细胞比例的CV值考察方法的重复性。经组合染色后的TCR-T细胞采用荧光减一对照细胞样本进行梯度稀释得到12个不同TCR阳性T细胞比例的样本,检测后分析结果得到检测限和定量限。对同一工艺生产的3批TCR-T细胞的检测结果进行统计学分析。[结果]确定最佳抗体使用浓度分别为:FVS780:0.313μg/mL,CD3抗体:2.500μg/mL,PE-Dextramer:1∶20稀释。中间精密度考察3批TCR-T细胞检测结果的CV分别为3.2%、7.4%和2.2%。重复性考察2批TCR-T细胞检测结果的CV分别为2.1%、2.2%。3批产品批内差异统计的P值分别为0.706、0.706和0.09,均>0.05;批间差异统计的P值为0.171,>0.05。[结论]建立了特异性、精密度(CV<10%)和灵敏度(定量限为0.85%,检测限为0.45%)均良好的识别乙肝病毒表面抗原的T细胞受体工程化T细胞的流式检测方法。

[Objective]A flow cytometry method for detection of T cell receptor-engineered T cells(TCR-T)that recognize hepatitis B virus surface antigen were established.[Method]The three detection antibodies were diluted in gradient(Dead cell indicator FVS780:0.020-10.000μg/mL,mouse anti human CD3 antibody:0.039-20.000μg/mL and hepatitis B surface antigen peptide-MHC complex protein Dextramer-PE:1:5-1:2560 dilution)and incubated with the same TCR-T cell for detection respectively,and the best experimental concentration was determined according to the result.In order to investigate the intermediate precision of the method,three batches of TCR-T cell samples were tested by two different analysts at the same time using the above optimized method for three times respectively,and the CV value of the proportion of TCR positive T cells was calculated.In order to investigate the repeatability of the method,six parallel samples of two batches of TCR-T cells were tested by the same analyst,and the CV value of TCR positive T cells was calculated.Twelve samples with different TCR positive T cell proportions were prepared by mixing the fluorescent minus one control cell sample with the TCR-T cell sample after combined staining,and were tested respectively.The detection limit and quantitative limit were obtained through the result analysis.The test results of three batches of TCR-T cells produced by the same process were statistically analyzed.[Result]The optimal antibody concentration were determined(FVS780:0.313μg/mL,CD3 antibody:2.500μg/mL,Dextramer-PE:1:20 dilution).Intermediate precision:CV of three batches of TCR-T cells was 3.2%,7.4%and 2.2%respectively.Repeatability:CV of two batches of TCR-T cells was 2.1%and 2.2%respectively.The P values of intra batch difference statistics of the three batches of products were 0.706,0.706 and 0.09 respectively,which were greater than 0.05;The P value of inter batch difference statistics is 0.171,greater than 0.05.[Conclusion]JA flow cytometry method for the detection of engineered T cells targeting hepatitis B virus surface antigen was established,which has good specificity,precision(CV<10%)and sensitivity(LOQ:0.85%,LOD:0.45%).