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首页> 外文期刊>Talanta: The International Journal of Pure and Applied Analytical Chemistry >A combinatorial strategy of a new monoclonal ELISA and immunoaffinity chromatography using sodium deoxycholate to increase the recovery of multimeric proteins like r-HBsAg
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A combinatorial strategy of a new monoclonal ELISA and immunoaffinity chromatography using sodium deoxycholate to increase the recovery of multimeric proteins like r-HBsAg

机译：一种新的单克隆ELISA和免疫亲和色谱法的组合策略，使用脱氧胆酸钠来提高r-HBsAg等多聚体蛋白的回收率

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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.
机译：在这项工作中，用于定量从酵母细胞中获得的HBsAg的基于三明治单克隆的ELISA进行了标准化和验证。此测定法中使用的单克隆抗体与r-HBsAg的不同分子亚型均匀反应。免疫测定允许r-HBsAg的定量分析范围为11.9-191.7 ng / mL。批内和批内精密度变异系数分别在0.77-3.43％和1.95-8.89％之间，回收率在98.2-100.8％之间。这证实了它的可靠性。 r-HBsAg是碳水化合物，蛋白质和脂质的复合物，组装成球形颗粒，平均直径为24 nm。在纯化过程中，许多宿主污染物伴随着该蛋白质，这会干扰免疫亲和基质对抗原的识别。为了解决这个问题，研究了几种去污剂在r-HBsAg定量和纯化中的作用。在此ELISA中添加0.1％的表面活性剂脱氧胆酸钠（NaDoc），使r-HBsAg的识别和定量提高了未经处理样品的2.4倍。在免疫亲和层析中观察到相似的结果，其中回收率显示增加了1.5倍。 NaDoc的应用可以减少对抗原抗体识别的抑制作用，从而提高定量和免疫亲和层析的效率。这种分析组合可以应用于多聚体蛋白，例如HB疫苗的r-HBsAg。

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