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目的建立轮状病毒(rotavirus,RV)抗原双抗体夹心ELISA定量检测方法,用于RV灭活疫苗生产过程中抗原含量的检测。方法采用RV全病毒免疫豚鼠,制备抗RV血清,纯化后作为检测抗体。以购进的羊抗A群轮状病毒多克隆抗体作为包被抗体,经辣根过氧化物酶(HRP)标记的羊抗豚鼠血清抗体作为酶标抗体,建立双抗体夹心ELISA法,检测样本中RV抗原含量,并对该方法进行验证及应用。结果所建立的方法,包被抗体的使用浓度为5μg/ml,检测抗体的使用浓度为3.2U/ml,酶标抗体使用浓度为10ng/ml。测定的线性范围为3~94ng/ml,R2在0.993以上,线性关系良好,定量限度为3ng/ml;对不同浓度的RV抗原参考品进行检测,回收率在91.5%~109.3%;变异系数均小于5.2%;与冻干乙型脑炎灭活疫苗、灭活乙型脑炎病毒液、小牛血清、人白蛋白、MA104细胞培养上清液、M199培养基均无交叉反应;通过RV灭活疫苗原液纯化过程中间品抗原含量的检测,能有效反映抗原纯化趋势。结论已建立了RV抗原双抗体夹心ELISA定量检测方法,该方法线性好,特异性强,灵敏度高,准确性好,可用于RV疫苗生产过程中间品及终产品的抗原定量检测。
OBJECTIVE To establish a sandwich enzyme-linked immunosorbent assay (ELISA) method for the detection of rotavirus (RV) antigen in the production of RV inactivated vaccine. Methods Guinea pigs were immunized with RV full virus to prepare anti-RV serum and purified as detection antibody. Using the purchased anti-A group of rotavirus polyclonal antibody as coating antibody and horseradish peroxidase (HRP) labeled goat anti-guinea pig serum antibody as enzyme-labeled antibody, the sandwich ELISA method was established to detect the sample In the RV antigen content, and verify the method and application. Results The established method, the concentration of coating antibody was 5μg / ml, the detection antibody was used at 3.2U / ml and the concentration of enzyme-labeled antibody was 10ng / ml. The linear range was 3 ~ 94ng / ml, the R2 was above 0.993, the linearity was good, and the limit of quantitation was 3ng / ml. The recovery rates of reference samples with different concentrations of RV antigen were 91.5% -109.3% Less than 5.2%; no cross-reaction with lyophilized Japanese encephalitis inactivated vaccine, inactivated encephalitis virus solution, calf serum, human albumin, MA104 cell culture supernatant, M199 medium; The detection of the content of antigen in the process of purification of live vaccine stock solution can effectively reflect the trend of antigen purification. Conclusion The quantitative sandwich ELISA method for detection of RV antigen was established. The proposed method has the advantages of good linearity, high specificity, high sensitivity and good accuracy. It can be used for the quantitative detection of antigen in intermediate and final products of RV vaccine production.