PERT-ELISA法用于细胞逆转录病毒检测的再研究

Re-evaluation on PERT-ELISA for Detection of Retrovirus in Cells

目的进一步分析PERT-ELISA法在细胞逆转录病毒检测应用中的意义。方法分析不同细胞培养液及其添加成分对PERT-ELISA法检测结果的影响及检测方法的精密性和检测用试剂的稳定性,将该方法用于不同来源细胞的逆转录酶活性的检测,并通过共培养法对我国金黄地鼠肾细胞中逆转录酶活性是否具有感染性进行初步分析。结果不同细胞培养液及其添加成分逆转录酶活性检测均为阴性,不会影响样本的检测结果。PERT-ELISA法具有良好的试验间精密性及重现性,检测用试剂在保存条件下,至少在1年内可保持稳定。近250株细胞的检测结果显示,含有逆转录病毒颗粒的细胞,无论是否具有感染性,均为逆转录酶活性阳性;除部分人淋巴瘤来源的细胞外,大部分人源细胞为逆转录酶活性阴性;8种91株猴肾来源的细胞包括74株Vero细胞均为逆转录酶活性阴性;而鼠源细胞则大部分为阳性,其中20株CHO来源的细胞均为阳性;3株猪源细胞及1株猫源细胞为阳性,而2株水貂细胞、2株牛源细胞及1株犬源细胞均为阴性。被检测的15株组织工程产品用人源细胞逆转录酶活性均为阴性,而我国不同地区来源的金黄地鼠肾细胞上清中,逆转录酶活性均为阳性。金黄地鼠肾细胞与人2BS细胞共培养后连续传代7次,细胞上清中逆转录酶活性仍为阳性,但随着传代次数的增加呈下降趋势。结论PERT-ELISA法可用于细胞逆转录病毒的检测,但对于阳性细胞,还需要进一步分析其与逆转录病毒或逆转录病毒样颗粒的相关性及是否具有感染性。

Objective To further analyze the significance of PERT-ELISA in detection of retrovirus in cells. Methods The effects of various cell culture media and their additives on detection result by PERT-ELISA as well as the precision and stability of the detection kit were analyzed. The reverse-transcriptase（RTase） activities in cells of various origins were detected by PERT-ELISA, and the infectivity of RTase activity in golden hamster kidney cells was preliminarily analyzed by co-culture with human diploid 2BS cells. Results All the detection resuhs of RTase activities in various cell culture media and their additives were negative, indicating no effect on the detection results of test samples. PERT-ELISA showed good intra-precision and reproducibility. All the prepared PERT- ELISA kits showed good stability after storage at the corresponding conditions for more than 12 month. The detection results of nearly 250 cell strains showed that the cells containing retrovirus particles, whether infectious or not, were positive for RTase activity. Except a part of human leucoma-derivcd cells, most of cells from human origin were negative for RTase activity. A total of 91 strains of monkey kidney-derived cells of 8 species, including 74 Veto cells strains, were negative for RTase activity. However, most of cells from mouse origin were positive for RTase activity, of which all the 20 CHO-derived cell strains were positive. Three porcine-derived and 1 feline-derived cell strains were positive, while 2 mink-derived, 2 bovine-derived and 1 canine-derived cell strains were negative for RTase activity. All the 15 cell strains from human origin, used for production of tissue engineering products, were negative, while all the supernatant of golden hamster kidney cells from various regions in China were positive for RTase activity. After the golden hamster kidney cells co-cultured with human 2BS cells were subcuhured for 7 passages, the RTase activity was still detected in cuhure supernatant, though showed a decreasing tendency with increased passages. Conclusion PERT-ELISA may be used for the detection of retrovirus in cells. However, the positive cells shall be further analyzed for relationship to retrovirus or retrovirus-like particles and for infectivity.