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目的:建立快速测定铝胶吸附的疫苗中抗原含量的OPA荧光检测法。方法:利用邻苯二甲醛(OPA)在2-巯基乙醇存在下与氨基酸的N端或L-谷氨酸侧链反应,在460nm处生成荧光衍生物的原理,建立了无需进行抗原蛋白提取的检测方法,并对该方法的特异性、线性、精密度、回收率、重复性进行考察。同时,配合钠十二烷基的硫酸盐-聚丙烯酰胺凝胶电泳法(SDS-PAGE),研究在配制过程中抗原蛋白与佐剂是否分离。结果:OPA荧光法,在抗原蛋白含量为0.02～0.16mg/ml时,线性良好,准确度达90%～115%,批内和批间相对标准偏差≤15%。电泳结果显示,CpG的加入可能会导致少量抗原蛋白从铝胶中解离。结论:该方法快速、准确、灵敏度较高,重复性好,可以应用于含铝胶疫苗制剂的实验室甚至生产质控过程。
OBJECTIVE: To establish an OPA fluorometric assay for the rapid determination of the antigen content of vaccines adsorbed on aluminum. Methods: The principle of using OPA to react with N-terminal or L-glutamic acid side chain of amino acid in the presence of 2-mercaptoethanol to generate fluorescent derivative at 460nm, Detection methods, and the specificity of the method, linearity, precision, recovery, repeatability were investigated. At the same time, with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), in the preparation process of antigen protein and adjuvant is separated. Results: The OPA fluorescence method showed a good linearity with the accuracy of 90% ~ 115% when the antigen protein content was 0.02 ~ 0.16mg / ml. The relative standard deviation (RSD) within and between batches was ≤15%. Electrophoresis results show that the addition of CpG may lead to the dissociation of a small amount of antigen protein from the aluminum gel. Conclusion: The method is rapid, accurate, sensitive and reproducible. It can be applied to the quality control of laboratories and even production of aluminum-containing vaccine preparations.