单个B细胞制备CRP单克隆抗体及其ELISA检测体系的初步建立
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摘要
目的:用单个B细胞抗体制备技术结合Exip293真核表达系统制备hs-CRP单克隆抗体。方法:用重组CRP蛋白免疫小鼠,荧光标记探针和流式分选技术分离出表达CRP单克隆抗体的单个B细胞,通过PCR方法扩增出编码CRP单克隆抗体蛋白的轻重链核酸序列并构建重组质粒,质粒转染到Expi 293细胞中制备出单克隆抗体蛋白。结果:用间接ELISA法筛选出两个具有良好特异性的单克隆抗体20#,22#,将这两个抗体配对并建立ELISA双抗体夹心检测方法,CRP浓度在15～250 ng/ml范围内有较好的检测线性(y=0. 003x-0. 035 8,R2=0. 997 4),精密度分析的批内变异系数CV为6. 7%～9. 37%,批间变异系数为8. 52%～9. 10%,平均回收率为98%,与临床血清检测值具有良好的相关性,达到CRP检测要求。结论:本研究所用的单个B细胞分离分选方法结合Expi 293真核表达系统在制备单克隆抗体方面具有省时、高效、特异性好、灵敏度高的特点,具有很好的应用价值。
Objective: Use single B cell antibody preparation technique and Expi 293 eukaryotic expression system to produce high-sensitivity CRP monoclonal antibody. Methods: Mice were immunized with recombinant protein of CRP and single B cells of expressing CRP monoclonal antibodies were isolated using fluorescent-labeled probes and flow sorting techniques. The heavy chain and light chain genes encoding CRP monoclonal antibody proteins were amplified by PCR and constructed recombinant plasmid,transfered the recombinant plasmid into Expi 293 cells to prepare monoclonal antibody protein. Results: The monoclonal antibody 20 # and 22 #which had good specificity were screened by indirect ELISA,pairing these two antibodies and establishing an double antibody-sandwich assay. The CRP concentration had a good detection linearity in the range of 15-250 ng/ml( y = 0. 003 x-0. 0358,R2= 0. 9974). The intraassay coefficient of variation( CV) of the precision analysis was 6. 7%-9. 37%,the inter-assay coefficient of variation was 8. 52%-9. 10%,and the average recovery was 98%. It had a good correlation with the clinical serum test values and met the requirements for CRP detection. Conclusion: The single B cell isolation and sorting method combined with the Expi 293 eukaryotic expression system in this study had the advantages of time-saving,high efficiency,good stability and high specificity in the preparation of monoclonal antibodies.
Objective: Use single B cell antibody preparation technique and Expi 293 eukaryotic expression system to produce high-sensitivity CRP monoclonal antibody. Methods: Mice were immunized with recombinant protein of CRP and single B cells of expressing CRP monoclonal antibodies were isolated using fluorescent-labeled probes and flow sorting techniques. The heavy chain and light chain genes encoding CRP monoclonal antibody proteins were amplified by PCR and constructed recombinant plasmid,transfered the recombinant plasmid into Expi 293 cells to prepare monoclonal antibody protein. Results: The monoclonal antibody 20 # and 22 #which had good specificity were screened by indirect ELISA,pairing these two antibodies and establishing an double antibody-sandwich assay. The CRP concentration had a good detection linearity in the range of 15-250 ng/ml( y = 0. 003 x-0. 0358,R2= 0. 9974). The intraassay coefficient of variation( CV) of the precision analysis was 6. 7%-9. 37%,the inter-assay coefficient of variation was 8. 52%-9. 10%,and the average recovery was 98%. It had a good correlation with the clinical serum test values and met the requirements for CRP detection. Conclusion: The single B cell isolation and sorting method combined with the Expi 293 eukaryotic expression system in this study had the advantages of time-saving,high efficiency,good stability and high specificity in the preparation of monoclonal antibodies.
引文
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[2]迟象阳,于长明,陈薇.单个B细胞抗体制备技术及应用[J].生物工程学报,2012,28(6):651-660.Chi XY,Yu CM,Chen W.Single B cell monoclonal antibody technologies and applications[J].Chin J Biotechnol,2012,28(6):651-660.
[3]Chew KS.What's new in Emergencies Trauma and Shock?C-reactive protein as a potential clinical biomarker for influenza infection:More questions than answers[J].J Emerg Trauma Shock,2012,5(2):115-117.
[4]白书玲,李建军.C反应蛋白与动脉粥样硬化[J].中华心血管病杂志,2004,32(8):355-357.Bai SJ,Li JJ.C-reactive protein and atherosclerosis[J].Chin JCardiol,2004,32(8):355-357.
[5]Boekholdt SM,Kastelein JJ.C-reactive protein and cardiovascular risk:more fuel to the fire[J].Lancet,2010,375(9709):95-96.
[6]Jiang S,Bao Y,Hou X,et al.Serum C-reactive protein and risk of cardiovascular events in middle-aged and older chinese population[J].Am J Cardiol,2009,103(12):1727-1731.
[7]刘平果,李国强,陈毅歆,等.一种定量检测人血清高敏C反应蛋白的化学发光免疫方法[J].生物工程学报,2010,26(8):1150-1156.Liu PG,Li GQ,Chen YX,et al.Chemiluminescent immunoassay for high-sensitivity C-reactive protein[J].Chin J Biotechnol,2010,26(8):1150-1156.
[8]胡春艳,刘全忠.单克隆抗体制备技术及应用的研究进展[J].安徽预防医学杂志,2008,(2):116-118.Hu CY,Liu QZ.Recent advances in the generation and application of the monoclonal antibody[J].Anhui J Preventive Med,2008,(2):116-118.
[9]Wrammert J,Smith K,Miller J,et al.Rapid cloning of high-affinity human monoclonal antibodies against influenza virus[J].Nature,2008,453(7195):667-671.
[10]Doria-Rose NA,Connors M.Antibody-secreting B cells in HIV infection[J].Curr Opinion Hiv Aids,2009,4(5):426-430.
[11]Tajiri K,Ozawa T,Jin A,et al.Analysis of the epitope and neutralizing capacity of human monoclonal antibodies induced by hepatitis B vaccine[J].Antiviral Res,2010,87(1):40-49.
[12]De KJ,Kramer A,Visser T,et al.Human immunoglobulin repertoires against tetanus toxoid contain a large and diverse fraction of high-affinity promiscuous V(H)genes[J].J Mol Biol,2009,387(3):548-558.