邻苯二甲醛(OPA)法检测铝吸附疫苗中抗原含量

Determination of Protein Content of Alhydrogel-absorbed Vaccine Using O-phthalaldehyde(OPA) Fluorometric Method

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 develop a generic method for the determination of protein content in alhydrogel （aluminum） absorbed vaccine formulation using o-phthalaldehyde （OPA）. Method： based on the theory that OPA can react with amino acids under alkaline conditions and give strong fluorescence, in the presence of 2- mercaptoethanol, a fluorescent method without antigen extraction was developed and the specificity, linearity range, accuracy and precision were evaluated. The integrity of the vaccine formulation was also investigated by SDS-PAGE. Results： a good linearity was obtained from 0. 02mg/ml to 0. 16mg/ml of antigen content, the accuracy of 90% - 115% and CV% less than 15% were achieved. Little protein disassociation was also observed after CpG was added into the formulation. Conclusion： the OPA fluorescent method was simple and can be further used not only in the laboratory but also in routine quality control of the vaccine formulation with alhydrogel.