含前S1、前S2和S表位的乙型肝炎病毒表面抗原不同组分的SDS-PAGE定量分析

目的 通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)对含前S1、前S2和S三种抗原成分的乙型肝炎病毒表面抗原SS1S2(由SS1和SS2两种抗原组成)不同组分进行定量分析,建立SS1S2原液中SS1和SS2抗原的SDS-PAGE定量测定方法.方法对SDS-PAGE条件进行优化,确立SS1S2中SS1和SS2抗原的分离条件.用Gel-Pro Analyzer图形分析软件分析电泳图谱上不同蛋白带的相对含量.取3批制品对该含量测定方法进行重复性验证.运用该方法测定不同批次SS1S2原液中SS1和SS2抗原的相对含量并进行统计分析,确定SS1和SS2抗原的取值范围.结果通过优化SDS-PAGE电泳条件,SS1S2中的SS1和SS2组分得以有效分离,进而通过软件分析得到其含量百分比.利用3批制品对该方法进行重复性验证,SS1和SS2抗原含量的变异系数分别为5.38%～6.21%和6.27%～6.97%.通过对12批SS1S2原液的SS1和SS2抗原含量进行测定,以均数±3×标准差(99%可信区间)作为取值标准,确定SS1S2原液中SS1和SS2抗原的取值范围分别为55.10%±16.20%和44.90%±16.20%.结论建立了SS1S2原液中SS1和SS2抗原的SDS-PAGE定量测定方法并初步确定了该疫苗原液不同组分的限量标准.

Quantitative SDS-PAGE analysis for different components in recombinant HBsAg vaccine bulks containing preS1, preS2 and S epitopes

Objective To develop a quantitative SDS-PAGE method to determine the percentages of SS1 and SS2 polypeptides in recombinant HBsAg vaccine bulks containing preS1,preS2 and S epitopes. Methods Optimization of SDS-PAGE procedure was performed to ensure the separation of SS1 and SS2 subunits and their relative percentages were determined according to the band intensities in the gel with Gel-Pro Analyzer software. The repeatability of the test was validated based on multi-measurements of 3 separate samples,and then the quality standards of SS1 and SS2 contents in bulks of the vaccine were infered by statistic analysis of the testing results from different batches of vaccine bulks. Results SS1 and SS2 bands were effectively separated after optimization of SDS-PAGE procedure and their relative contents were obtained. The repeatability validation results showed that the coefficients of variation of SS1 and SS2 contents were 5.38%-6.21% and 6.27%-6.97%, respectively. The quality control limits for the SS1 and SS2 contents were determined as 55.10%±16.20% and 44.90%±16.20%, respectively, based on the criterion of mean±3×SD. Conclusion The quantitative SDS-PAGE method for the meas-urement of SS1 and SS2 contents has been established and validated. The quality standards of SS1 and SS2 contents in bulks of the vaccine have also been established based on the test method.