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目的:研制抗乙肝病毒G145R变异表面抗原的单克隆抗体(mAb)。方法:将重组乙肝病毒G145R变异表面抗原以Al(OH)3佐剂化,常规免疫BALB/c小鼠,采用细胞融合技术制备抗乙肝病毒G145R变异表面抗原的mAb。采用ELISA对制备物的效价与特异性进行鉴定,将其用于部分临床标本的检测,并与德国抗HBs mAb GL2.001进行比较。结果:获得1株稳定分泌抗G145R HBs mAb的杂交瘤细胞,命名为4-E12。该细胞株染色体数目为90条,所分泌mAb属IgG1亚类。其培养上清与腹水抗G145R HBs ELISA效价为(0.5~1)×103及(1~10)×106。经持续3月培养,低血清适应以及反复冻存与复苏后,其细胞增殖能力与上清抗G145R HBs效价与克隆化结束时大致相似。将其与德国抗G145R HBs mAb GL2.001相比,二者对重组乙肝表面抗原G145R变异S蛋白的反应性大致相当,灵敏度前者稍低于后者(分别为2.0μg/L与1.0μg/L)。对野生株表面抗原的检测前者在实验浓度范围内未显示有意义的反应信号,后者在与高浓度(1mg/L及以上)抗原反应时S/N值达到或高于3.9。将二者用于对一组特定患者的临床检测,其阳性率前者(17/200,8.5%)较后者(11.5%)略低,其差别经统计学比较无统计学意义(P>0.05)。结论:成功地制备了1株抗乙肝表面抗原G145R变异体的mAb,为乙肝G145R变异的基础、临床与流行病学研究提供了进一步的物质基础。
Objective: To develop a monoclonal antibody (mAb) against hepatitis B virus G145R variant surface antigen. Methods: The recombinant hepatitis B virus G145R mutant surface antigen was adjuvated with Al (OH) 3, and BALB / c mice were immunized routinely. The mAbs against hepatitis B virus G145R variant surface antigen were prepared by cell fusion technique. The titer and specificity of the preparations were identified by ELISA, which were used for the detection of some clinical specimens and compared with the German anti-HBs mAb GL2.001. Results: One hybridoma cell stably secreting anti-G145R HBs mAb was obtained and named as 4-E12. The number of chromosomes in this cell line is 90, and the secreted mAb belongs to IgG1 subclass. The titers of anti-G145R HBs in culture supernatants and ascites were (0.5 ~ 1) × 103 and (1 ~ 10) × 106. After continued cultivation in March, low serum adaptation and repeated cryopreservation and resuscitation, the cell proliferation ability and the supernatant anti-G145R HBs titers were similar to those at the end of cloning. Compared with the German anti-G145R HBs mAb GL2.001, the reactivity against the recombinant hepatitis B surface antigen G145R mutant S protein was approximately equivalent, with the former slightly lower than the latter (2.0 μg / L and 1.0 μg / L respectively ). Detection of wild-type surface antigen The former did not show significant response signals within the experimental concentration range, with S / N values at or above 3.9 when reacted with high concentrations of antigen (1 mg / L and above). The two were used for clinical testing of a specific group of patients, the positive rate (17/200%, 8.5%) was slightly lower than that of the latter (11.5%). The difference was statistically insignificant (P> 0.05 ). CONCLUSION: A mAb of G145R variant of hepatitis B surface antigen was successfully prepared, which provided the basis for further study of G145R mutation in hepatitis B virus. The clinical and epidemiological studies provided a further material basis.