Calibration and Preliminary Application of Linear Quantitation Standard for Anti-HEV IgG Antibody
- 生命科学－已发表论文 
目的建立抗戊型肝炎病毒(Anti-HEV)IgG抗体的定量线性标准品,并进行初步应用。方法利用抗-HEV IgG和抗-HEV IgM ELISA检测试剂筛选出1份抗-HEV IgG阳性血清L9,经基因1型和4型的HEV ORF2C-端抗原及239抗原进行Western blot确认后,用WHO定量标准品,由3个实验室协作标定,利用量反应平行线法计算其抗-HEV IgG的含量。考察已标定的L9血清的稳定性,并用所标定的1.5倍系列稀释的血清对国内外6家抗-HEV IgG试剂的灵敏度进行检测。选择一灵敏度较高的试剂,在其线性范围内取L9的5个稀释度作为抗-HEV IgG抗体定量线性标准,对高、中、低浓度的3份临床血清重复检测5次,考察其重复性;对实验感染猴的系列血清中抗-HEV IgG含量进行定量检测,考核该定量线性标准品的应用效果;并对每次定量试验中的线性方程进行分析,确定相关系数r值和斜率k值的范围。结果经国内外试剂检测筛选出的阳性血清L9与基因1型和4型的HEV ORF2 C-端抗原及239抗原均有阳性反应。经协作标定,L9血清抗-HEV IgG含量为16.9U/ml。L9血清在-20℃下保存6、12、18个月,2～8℃保存24、48、96h后,定量结果均在95%置信区间内,且抗-HEV IgG含量均未明显下降。6家抗-HEVIgG检测试剂灵敏度差异较大,范围为0.03～5.00U/ml。确定L9血清从0.42U/ml开始的5个1.5倍系列稀释度,作为某一试剂抗-HEVIgG抗体定量线性标准品。利用该线性定量标准检测高、中、低浓度的3份临床血清,定量结果重复性较好;对实验感染猴系列血清进行定量检测,结果可有效地反映抗体水平变化趋势;94%的定量检测试验,r≥0.98,1.15≥k≥0.95。结论已建立了抗-HEVIgG抗体定量线性标准品,可用于疫苗免疫原性评价和流行病学调查。Objective To develop a linear quantitation standard for anti-hepatitis E virus (HEV) IgG antibody. Methods An anti-HEV IgG positive serum sample L9 was screened by using Anti-HEV IgM ELISA kit and Anti-HEV IgG ELISA kit and confirmed by Western blotting with HEV ORF2 C-terminal antigen of genotypes 1 and 4 and 239 antigen, then calibrated by 3 laboratories using WHO quantitation standard. Calculate the anti-HEV IgG content by dose-response parallel line assay, and evaluate the stability of calibrated serum sample. The sensitivities of 6 domestic and imported anti-HEV IgG ELISA kits were evaluated with the calibrated serum sample diluted 1. 5-fold serially. A linear quantitation standard for anti-HEV IgG antibody consisted of 5 dilutions of L9 within the linear determination range of a highly sensitive kit and evaluated for reproducibility by repeat test for 3 clinical serum samples, at high, moderate and low anti-HEV IgG contents respectively, for 5 times. The anti-HEV IgG contents in serum samples of HEV-infected monkeys were determined by the standard, and the determination curves were analyzed to define correlation coefficient r and slope k. Results Serum sample L9 showed positive reaction with HEV ORF2 C-terminal antigen of genotypes 1 and 4 and 239 antigen, and its anti-HEV IgG content was calibrated as 16. 9 U / ml. After storage at -20℃ for 6, 12 and 18 months or at 2 ～ 8℃ for 24, 48 and 96 h, all the quantitative determination results were within the 95% CI, and anti-HEV IgG content showed no significant decrease. The sensitivities of 6 kits evaluated with the L9 ranged from 0. 03 to 5. 00 U / ml. The linear quantitation standard for anti-HEV IgG antibody consisted of 5 dilutions of L9, starting from a concentration of 0. 42 U / ml. The determination results of 3 clinical serum samples showed good reproducibility of the standard. The determination results of sera of HEV-infected monkeys reflected the change of antibody level effectively. The r values of 94% of quantitative determination curves were not less than 0. 98, and the k values ranged from 1. 15 to 0. 95. Conclusion A linear quantitation standard for anti-HEV IgG antibody was established, which was suitable for the evaluation of immunogenicity and epidemical investigation of vaccine.