轮状病毒抗原ELISA定量检测方法的建立  

Development of a quantitative ELISA for detection of rotavirus antigen

作　　者：李云富[1] 付臻鹏[1] 李刚[1] 罗翀[1] 黄海彬[1] 黄勇[1] 刘春庭[1] 林上炎[1] 周淑芳[1] LI Yun-fu FU Zhen-peng LI Gang LUO Chong HUANG Hai-bin HUANG Yong LIU Chun-ting LIN Shang-yan ZHOU Shu-fang(Lizhu Vaccine Engineering Corporation, Zhuhai , Guangdong 519090, China)

机构地区：[1]丽珠集团疫苗工程股份有限公司,广东珠海519090

出　　处：《中国病毒病杂志》2016年第4期288-293,共6页Chinese Journal of Viral Diseases

基　　金：广东省重大科技专项基金项目(2013A022100018)

摘　　要：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 develop a double antibody sandwich ELISA for quantitative detection of rotavirus antigen during the production of inactivated rotavirus vaccine. Methods Polyclonal antibodies against rotavirus antigen were prepared by immunizing rabbits with complete rotavirus.A double antibody sandwich ELISA was developed using polyclonal antibody for coating and HRP-labeled polyclonal antibody to detect rotavirus antigen. Results The linear range and R2 value of the developed ELISA were 3-94ng/ml and greater than0.993,respectively.The quantitation limit of the method was 3ng/ml.The recovery rates of internal reference of rotavirus antigen at various concentrations ranges from 91.5% to 109.3%,with a variation coefficient of less than 5.2%.No cross reactions were observed with frozen-dried inactivated Japanese encephalitis vaccine,inactivated Japanese encephalitis virus liquid,calf serum,human albumin,MA104 cells culture supernatant,M199. Conclusions A double antibody sandwich ELISA for quantitative detection of rotavirus antigen was developed with good linearity and high specificity and sensitivity.

关 键 词：轮状病毒抗原 双抗体夹心ELISA 线性范围 

分 类 号：R392-33[医药卫生—免疫学]