酶联免疫吸附试验间接抗体夹心检测SARS-CoV抗原方法的建立和应用  

Development and application of triple antibodies-based sandwich ELISA for detecting nucleocapsid protein of SARS-associated coronavirus

作　　者：丘立文[1] 汤汉文 王亚娣[1] 廖金娥 郝卫[1] 温坤[1] 何秀敏 车小燕[1] 

机构地区：[1]南方医科大学珠江医院中心实验室,广州510282 [2]珠海经济特区海泰生物制药有限公司

出　　处：《中华流行病学杂志》2005年第4期277-281,共5页Chinese Journal of Epidemiology

基　　金：广东省防治非典型肺炎科技攻关项目资助(粤科社字:200380)

摘　　要：目的制备和鉴定严重急性呼吸综合征冠状病毒(SARSCoV)核衣壳(N)蛋白单克隆抗体(mAb)和多克隆抗体建立SARSCoVN抗原捕获抗体夹心酶联免疫吸附试验(ELISA)方法用于SARSCoV感染的早期诊断。方法用基因重组SARSCoVN蛋白免疫BALB/c小鼠和新西兰大白兔制备mAb和多克隆抗体,采用ELISA间接法、免疫荧光和免疫印迹进行筛选和鉴定,用单克隆抗体与兔多克隆抗体进行配对试验,建立抗原捕获抗体夹心ELISA法测定SARSCoVN抗原。结果获得9株特异性针对SARSCoVN蛋白的mAb和高效价的兔多克隆抗体,通过高亲和力的mAb与兔多抗的配对试验,筛选出3株单抗N1E8、N8E1和N10E4混合作为捕获抗体,与兔多克隆抗体和辣根过氧化物酶标记羊抗兔IgG组合作为测定抗体,建立了抗体夹心ELISA法,测定重组SARSCoVN蛋白最高灵敏度为50pg/ml,特异性达99.86%,测定420份血清学确诊的SARS患者血清,其中发病1-10天阳性检出率为90.1%,11-20天检出率为23%,21天以上均为阴性,与其他呼吸道病毒和冠状病毒无交叉反应。结论获得特异性好、亲和力高的单克隆抗体和高效价的兔多克隆抗体,经过抗体的配对和优化,建立了一种灵敏度高、特异性强的SARSCoV抗原的ELISA捕捉法,可应用于SARS早期诊断、溯源及流行病学研究。Objective To prepare and characterize monoclonal antibodies (mAb) and polyclonal antibodies against nucleocapsid (N) protein of severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) and to establish antibodies-based sandwich ELISA for detecting N protein of SARS-CoV, which might apply to early diagnosis of patients with SARS-CoV infection. Methods BALB/c mice were immunized with purified recombinant N protein of SARS-CoV for producing mAbs,and New Zealand white rabbits were immunized for producing polyclonal antibodies.The identification of antibodies was performed using indirect enzyme-linked immunosorbent assay (ELISA),indirect fluorescent-antibody assay (IFA),and Western immunoblotting.Capturing and detecting antibodies were selected by pairing the mAbs and polyclonal antibodies one by one and an antibodies-based sandwich antigen capture ELISA was used for detecting N antigen of SARS-CoV. Results Nine mAbs and hyperimmune rabbit polyclonal antibodies,specifically against SARS-CoV nucleocapsid protein were obtained.Using paired ELISA assay,three mAbs N1E8,N8E1 and N10E4 were selected as capturing antibody and rabbit polyclonal antibodies as detecting antibody then triple antibodies-based sandwich ELISA was established following horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G.The recombinant N protein was used as a standard to establish a detection sensitivity of approximated 50 pg/ml with this assay.When tested with 420 serum specimens from serologically confirmed SARS patients,the positive rates of serum N protein were 90.1 %,23% and 0%,in which sera collected from 1 to 10 days,11 to 20 days and beyond 21 days respectively after the onset of symptoms.The specificity of the assay was 99.86 % in 715 control serum specimens.There was no cross-reaction with other respiratory viruses and coronaviruses. Conclusion Specific and high affinity mAbs and rabbit polyclonal antibodies were obtained.By paired and optimized sandwich ELISA,a sensitive and specific antigen capture

关 键 词：酶联免疫吸附试验(ELISA) 冠状病毒(SARS-CoV) 夹心ELISA法 SARS-COV感染 严重急性呼吸综合征 多克隆抗体 BALB/c小鼠 ELISA间接法 ELISA捕捉法 单克隆抗体 原方 检测 捕获抗体 新西兰大白兔 辣根过氧化物 SARS患者 流行病学研究 

分 类 号：R446.6[医药卫生—诊断学]