Pose Alaín González, Rodríguez Elsa Rodríguez, Piñeiro Marisdania Joglar, Montesino Raquel, Sánchez Oliberto, Toledo Jorge Roberto
Biotechnology and Biopharmaceutical Laboratory, Pathophysiology Department, School of Biological Sciences, Universidad de Concepción, Victor Lamas 1290, P.O. Box 160-C, Concepción, Chile; Animal Biotechnology Department, Center for Genetic Engineering and Biotechnology (CIGB), P. O. Box 6162, Havana 10600, Cuba.
Animal Biotechnology Department, Center for Genetic Engineering and Biotechnology (CIGB), P. O. Box 6162, Havana 10600, Cuba.
J Immunol Methods. 2018 Aug;459:70-75. doi: 10.1016/j.jim.2018.05.008. Epub 2018 May 24.
Analytical techniques are essential in the process of standardizing and validating vaccines. In this study we described a methodology to establish an ELISA sandwich for the quantification of a new vaccine against avian influenza virus H5N1 based on the main antigenic determinant of the virus, the extracellular domain of the glycoprotein hemagglutinin (HA), fused to the extracellular domain of the chicken CD154 glycoprotein (HACD). The chimerical proteins HA and HACD were produced in SiHa cells and the experiments were performed by using three monoclonal antibodies (MAb-HA1, MAb-HA2 and MAb-HA3), alone or conjugated to horseradish peroxidase (HRP-HA1, HRP-HA2 and HRP-HA3). The hemagglutination inhibition assay was carried out with a negative and a positive H5N2 reference serum, together with the antigen H5N1 A/Mallard/Italy/3401/05, all purchased from the "Istituto Zooprofilattico delle Venezie", Italy. After demonstrating the similar recognition pattern between the HA and the HACD proteins, the MAb-HA2 at a concentration of 2,5 μg/mL was selected as the capture antibody and the HRP-HA3 at a dilution of 1/20000 was selected as the detection antibody due to their optimal values of optical density at these conditions. The best dynamic range of the standard curve using the protein HACD was achieved at concentrations from 100 to 1,56 ng/mL. There were no significant differences when five batches of HACD were quantified by the ELISA sandwich and the bicinchoninic acid method linked to densitometry. In conclusion, the final parameters for the quantification of the chimeric protein HACD using an ELISA sandwich were described, which could contribute to develop a large-scale process for the final vaccine production.
分析技术在疫苗标准化和验证过程中至关重要。在本研究中，我们描述了一种建立酶联免疫吸附测定（ELISA）夹心方法的流程，用于基于禽流感病毒H5N1的主要抗原决定簇（糖蛋白血凝素（HA）的胞外结构域，与鸡CD154糖蛋白的胞外结构域融合（HACD））对一种新型H5N1禽流感疫苗进行定量分析。嵌合蛋白HA和HACD在SiHa细胞中产生，实验使用三种单克隆抗体（MAb-HA1、MAb-HA2和MAb-HA3）单独或与辣根过氧化物酶偶联（HRP-HA1、HRP-HA2和HRP-HA3）进行。血凝抑制试验使用阴性和阳性H5N2参考血清以及抗原H5N1 A/野鸭/意大利/3401/05进行，所有这些均购自意大利“威尼托动物卫生研究所”。在证明HA和HACD蛋白具有相似的识别模式后，选择浓度为2.5μg/mL的MAb-HA2作为捕获抗体，选择稀释度为1/20000的HRP-HA3作为检测抗体，因为它们在这些条件下具有最佳光密度值。使用蛋白HACD时，标准曲线的最佳动态范围在100至1.56ng/mL的浓度范围内实现。当用ELISA夹心法和与密度测定法相关的二辛可宁酸法对五批HACD进行定量时，没有显著差异。总之，描述了使用ELISA夹心法定量嵌合蛋白HACD的最终参数，这可能有助于开发最终疫苗生产的大规模流程。
