鸡柔嫩艾美耳球虫抗体ELISA检测方法的建立  

Development of an ELISA for Detecting the Specific Antibody against Eimeria tenella

作　　者：田崇瑜 段小超 陈彦华 霍乃蕊[1] 韩克光[1] 古少鹏[1] 郑明学[1] TIAN Chongyu;DUAN Xiaochao;CHEN Yanhua;HUO Nairui;HAN Keguang;GU Shaopeng;ZHENG Mingxue(College of Veterinary Medicine,Shanxi Agricultural University,Taigu 030801,China;Shanxi Anhong Testing Technology Co.,Ltd.,Taiyuan 030008,China)

机构地区：[1]山西农业大学动物医学学院,山西太谷030801 [2]山西安弘检测技术有限公司,山西太原030008

出　　处：《山西农业科学》2021年第3期361-365,共5页Journal of Shanxi Agricultural Sciences

基　　金：山西省研究生教育创新项目(2019SY211);山西省重点研发计划项目(201803D221023-4)。

摘　　要：建立鸡柔嫩艾美耳球虫特异性抗体的ELISA检测方法,为柔嫩艾美耳球虫的免疫机制研究和口服卵黄抗体制剂研发奠定基础。以柔嫩艾美耳球虫的孢子化卵囊免疫雏鸡制备阳性血清和可溶性抗原,优化ELISA检测条件,以阴性血清确定检测临界值,以阳性抗体绘制标准曲线。结果表明,经过优化,确定包被缓冲液为0.05 mol/L碳酸盐缓冲液(pH值9.6),可溶性抗原的包被质量浓度为21.46μg/mL,4℃包被16 h;3%BSA 37℃封闭2 h;待检血清和兔抗鸡IgG-HRP的最佳稀释倍数分别为20倍和500倍,阴性和阳性结果判定的临界OD_(450)值分别为0.1275和0.1210,建立的标准曲线线性关系良好(R^(2)=0.9921)。该方法的建立可为相应试剂盒的研发提供技术基础,并可促进鸡球虫抗体免疫应答研究和有效防控。The study was designed to develop an ELISA method for detecting the specific antibody against chicken Eimeria tenella,facilitating the investigation of the immune mechanism against Eimeria tenella and the development of any orally-taken yolk antibody agents.Sporulated oocysts were used to inoculate chicken to prepare the positive serum and the soluble antigen.The negative serum were used to determine the critical value for detecting,and the standard cure was made.The results suggested that the optimized conditions were as follows:0.05 mol/L carbonate buffer(pH 9.6)was selected as the coating buffer,the coating concentration of the soluble antigen was 21.46μg/mL,the ELISA plate was placed at 4℃for 16 h and blocked with 3%BSA at 37℃for 2 h.The working concentration of the tested serum and the rabbit anti-chicken IgG-HRP were 20 times and 500 times,respectively.The critical value of OD_(450)to judge the negative and positive result were 0.1275 and 0.1210,respectively.The standard cure showed a good linear relationship(R^(2)=0.9921).The method established in this study will provide the technical basis for the development of the corresponding detecting kit and facilitate the study of the immune response and the effective prevention and control for the disease of avian coccidiosis.

关 键 词：鸡球虫病 柔嫩艾美耳球虫 ELISA 孢子化卵囊 抗体检测 

分 类 号：S858.31[农业科学—临床兽医学] S852.723[农业科学—兽医学]