| 丘立文,汤汉文,王亚娣,廖金娥,郝卫,温坤,何秀敏,车小燕.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[J].Chinese journal of Epidemiology,2005,26(4):277-281 |
| 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 |
| Received:November 04, 2004 |
| DOI： |
| KeyWord: 严重急性呼吸综合征冠状病毒 核衣壳蛋白 单克隆抗体 |
| English Key Word: Severe acute respiratory syndrome-CoV Nucleocapsid protein Monoclonal antibodies |
| FundProject:广东省防治非典型肺炎科技攻关项目资助(粤科社字:200380) |
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| Abstract: |
| 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。 |
| English Abstract: |
| 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 ELISA was established for detecting N antigen of SARS-CoV,which might apply to early diagnosis, source tracing and epidemiological studies of SARS. |
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