Process Control Department, Center for Genetic Engineering and Biotechnology, 31st Ave /158 and 190, P.O. Box 6162, Havana 10600, Cuba.
Talanta. 2010 Apr 15;81(1-2):314-9. doi: 10.1016/j.talanta.2009.12.002. Epub 2009 Dec 16.
In this work, a sandwich monoclonal-based ELISA for quantifying the HBsAg obtained from yeast cells was standardized and validated. The monoclonal antibody employed in this assay reacts uniformly with different molecular isoforms of r-HBsAg. Immunoassay allowed the r-HBsAg quantification in an analytical range 11.9-191.7 ng/mL. Inter- and intra-assay precision variation coefficients were between 0.77-3.43% and 1.95-8.89%, respectively, and the recovery ranged 98.2-100.8%; which confirms its reliability. r-HBsAg is a complex of carbohydrates, proteins and lipids assembled into spherical particles with an average diameter of 24 nm. Many host contaminants accompany this protein during purification process, which can interfere the antigen recognition by the immunoaffinity matrix. To solve this problem, the effect of several detergents in the quantification and purification of r-HBsAg were studied. The addition of the surfactant sodium deoxycholate (NaDoc) at 0.1% in this ELISA improved the recognition and quantification of r-HBsAg by 2.4-fold higher than untreated samples. Similar results were observed in the immunoaffinity chromatography where a 1.5-fold increasing recovery values was shown. The application of NaDoc allows to reduce the inhibitory effect upon the antigen-antibody recognition, increasing the quantification and immunoaffinity chromatography efficiency. This analytical combination could be applied to multimeric proteins like r-HBsAg of HB vaccine.
本工作建立了一种基于单克隆抗体的 ELISA 法定量检测酵母表达的 HBsAg，对该方法进行了标准化和验证。该检测方法中使用的单克隆抗体能均匀地与不同的 r-HBsAg 分子异构体反应。免疫分析允许在 11.9-191.7ng/mL 的分析范围内定量 r-HBsAg。批内和批间精密度变异系数分别为 0.77-3.43%和 1.95-8.89%，回收率范围为 98.2-100.8%；这证明了其可靠性。r-HBsAg 是一种由碳水化合物、蛋白质和脂质组成的复合物，组装成平均直径为 24nm 的球形颗粒。在纯化过程中，许多宿主污染物会伴随这种蛋白质，这可能会干扰免疫亲和基质对抗原的识别。为了解决这个问题，研究了几种去污剂对 r-HBsAg 定量和纯化的影响。在 ELISA 中添加 0.1%的去氧胆酸钠（NaDoc）可提高 r-HBsAg 的识别和定量，比未处理的样品高 2.4 倍。在免疫亲和层析中也观察到了类似的结果，回收率值增加了 1.5 倍。NaDoc 的应用可以减少对抗原-抗体识别的抑制作用，提高定量和免疫亲和层析的效率。这种分析组合可应用于 HB 疫苗等多聚体蛋白的 r-HBsAg。
