抗巨细胞病毒早晚期抗原单克隆抗体制备及在病毒培养物鉴定中的初步应用

目的 制备抗CMV早晚期抗原单克隆抗体并在病毒培养物鉴定中初步应用.方法 CMV感染MRC-5细胞24h、48h和72h后甲醛灭活的可溶性抗原免疫BALB/c小鼠,小鼠脾细胞与骨髓瘤细胞NS-1进行融合.酶免法筛查阳性杂交瘤并有限性稀释法进行克隆.免疫荧光及印迹对筛选后的克隆进行鉴定,最终获得的杂交瘤制备腹水并使用Protein G进行纯化.纯化后单抗应用于CAP室间质评标本以及临床标本CMV培养物的鉴定.结果 3只小鼠脾细胞与骨髓瘤细胞融合后初步筛查出约110株阳性克隆.剔除与其它抗原有交叉反应、抗体效价低、非全覆盖早晚期抗原的克隆最终获得抗CMV单抗杂交瘤1株,即23B5-1.免疫荧光显示23B5-1株单抗与CMV感染后3h-120h的MRC-5细胞均反应,即覆盖即刻、早期和晚期抗原.免疫印迹试验显示23B5-1株单抗与CMV抗原25KD-50KD之间的5个蛋白条带结合.23B5-1株单抗免疫球蛋白亚型为IgG1.Protein G纯化腹水后的单抗效价≥1∶12800.纯化单抗染色鉴定6份室间质评及10份临床标本CMV培养物全部符合.结论 初步应用显示制备的抗CMV早晚期抗原单克隆抗体性能良好.

Monoclonal antibody against immediate-early,early and late antigens of cytomegalovirus and its preliminary applications in testing of viral cultures

Objective To prepare monoclonal antibody against early and late antigens of cytomegalovirus (CMV) and to use the antibody for the identification of isolates from proficiency testing and clinical samples.Methods BALB/C mice were immunized with soluble and formaldehyde to inactivate CMV antigens from the CMV-infected MRC-5 cells at 24,48 and 72 hours after inoculation.Mouse myeloma NS-1 cells were fused with spleen cells of the immunized mice.Positive hybridomas were screened with EIA and were cloned with limited dilution method.The immunofluorescent and immunoblotting tests were applied to confirm the positive clones after screening.The ascites was prepared with the identified hybridoma and was purified by Protein G.The purified monoclonal antibody was used to identify CMV isolates from CAP proficiency testing samples and clinical samples.Results A total of 110 positive hybridomas clones were preliminary identified from fused cells of three immunized mice.One hybridoma clone,23B5-1 was finally obtained while other clones were eliminated due to cross-reactive with other antigens,low antibody titers or without reactions with both CMV early and late antigens.It was shown that 23B5-1 reacted with CMV infected MRC-5 cells from 3 hours to 120 hours (5 days) after inoculation.All of immediate-early,early and the late CMV antigens were recognized by the monoclonal antibodies simultaneously.It was also shown that 23B5-1 reacted with five CMV protein bands between 25KD to 50 KD in western blot.The immunoglobulin subclass of antibody was IgG1 and the titer of the purified antibody was equal or more than 1:12 800.The results of staining with the antibody were consistence with predicted results for all six CMV isolates from proficiency testing and ten isolates from clinical samples.Conclusions The performance of our self-prepared monoclonal antibody was good according to the preliminary application.