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目的 建立一种新的基于PCR的乙型肝炎病毒(HBV)体外中和试验,将其用于HBV中和抗体的检测,为新型HBV疫苗的评估提供一种体外模型。方法 待检免疫血清与已知HBV作用后接种HepG2 细胞,吸附、洗涤、培养后提取细胞核酸,PCR检测HBVDNA以判断中和试验结果。结果 免疫血清与10倍最小PCR感染剂量的HBV作用后接种细胞,培养2 4h后检测表明,其中和滴度高,结果稳定。市售疫苗免疫小鼠血清、多数抗HBsELISA阳性人血清和2份含HBVS区的新型候选疫苗的免疫血清中和试验阳性,而正常小鼠血清、戊型肝炎病毒重组蛋白免疫血清和抗 HBs阴性人血清中和试验阴性。结论 基于PCR的HBV体外中和试验简单、快速、经济,具有良好的特异性和敏感性,可以用于新型HBV疫苗免疫效果的评估。
OBJECTIVE: To establish a new PCR-based in vitro neutralization test for hepatitis B virus (HBV) and to use it in the detection of neutralizing antibodies against HBV, so as to provide an in vitro model for the evaluation of novel HBV vaccines. Methods HepG2 cells were inoculated with the immune serum to be tested and the known HBV, and then adsorbed, washed and cultured to extract the nucleic acid of the cells. PCR was used to detect the HBVDNA to determine the neutralization test results. Results The immune sera were inoculated with HBV at a dose of 10 times the minimum PCR. After 24 h of culture, the cells were seeded with high titers of neutralization and the results were stable. Serum from commercially available vaccines, most anti-HBs ELISA-positive human sera and two new vaccine candidates containing HBVS were tested for immune sera neutralization, whereas normal mouse sera, hepatitis E virus recombinant protein serum and anti-HBs negative Human serum neutralization test negative. Conclusion The PCR-based in vitro neutralization assay of HBV is simple, rapid, economical, and has good specificity and sensitivity. It can be used to evaluate the immunogenicity of novel HBV vaccines.