破伤风抗毒素间接ELISA检测方法的建立及应用
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摘要
目的建立检测破伤风抗毒素(TAT)效价的间接ELISA法,并进行初步应用。方法用方阵滴定法确定包被抗原和待检血清的最适工作浓度,并对各种反应条件进行优化,建立间接ELISA法。用建立的间接ELISA法检测3批TAT效价,并与小鼠中和试验法检测结果进行比较。结果建立的间接ELISA法的最适反应条件:抗原最适包被质量浓度为1.2μg/m L,血清最佳稀释浓度为400 m IU/m L;抗原与待检血清作用时间为45 min,加入酶标二抗后作用时间为60 min;抗原最适包被缓冲液为0.05 mol/L p H 9.6碳酸盐(CBS)缓冲液,4℃包被12 h;封闭液为30%BSA(5%蔗糖和5%乳糖);酶标二抗最适工作浓度为1∶8 000。3批TAT间接ELISA法效价检测结果与小鼠中和试验法效价检测结果相比,差异无统计学意义(P均>0.05)。结论建立的间接ELISA法具有较高灵敏度,可用于TAT效价的检测。
Objective To develop an indirect ELISA method for determination of titer of tetanus antitoxin(TAT).Methods The optimal working concentrations of coating antigen and serum samples were determined by chessboard titration,and the reaction condition was optimized,based on which an indirect ELISA method was developed and used for determination of titers of three batches of TAT,and the determination result was compared with that by neutralization test in mice. Results The optimal antigen concentration for coating and dilution of serum sample were 1. 2 μg/m L and400 m IU/m L,while the optimal reaction times of coating antigen with serum and enzyme-labeled second antibody were45 and 60 min,respectively. The optimal buffer for antibody coating was 0. 05 mol/L carbonate(CBS) at pH 9. 6,while the optimal temperature and time for coating were 4 ℃ and 12 h respectively. The BSA at a concentration of 30%(5% sucrose and 5% lactose)was served as blocking agent. The optimal working concentration of enzyme-labeled second antibody was 1 ∶ 8 000. The determination results of three batches of TAT by indirect ELISA showed no siginificant difference with those by neutralization test in mice(each P > 0. 05). Conclusion The developed indirect ELISA method showed high sensitivity,which might be used for determination of titer of TAT.
Objective To develop an indirect ELISA method for determination of titer of tetanus antitoxin(TAT).Methods The optimal working concentrations of coating antigen and serum samples were determined by chessboard titration,and the reaction condition was optimized,based on which an indirect ELISA method was developed and used for determination of titers of three batches of TAT,and the determination result was compared with that by neutralization test in mice. Results The optimal antigen concentration for coating and dilution of serum sample were 1. 2 μg/m L and400 m IU/m L,while the optimal reaction times of coating antigen with serum and enzyme-labeled second antibody were45 and 60 min,respectively. The optimal buffer for antibody coating was 0. 05 mol/L carbonate(CBS) at pH 9. 6,while the optimal temperature and time for coating were 4 ℃ and 12 h respectively. The BSA at a concentration of 30%(5% sucrose and 5% lactose)was served as blocking agent. The optimal working concentration of enzyme-labeled second antibody was 1 ∶ 8 000. The determination results of three batches of TAT by indirect ELISA showed no siginificant difference with those by neutralization test in mice(each P > 0. 05). Conclusion The developed indirect ELISA method showed high sensitivity,which might be used for determination of titer of TAT.
引文
[1]Chinese Pharmacopoeia Commission.Pharmacopoeia of People's Repubic of China(VolⅢ)[S].Beijing:China Med Sci Press,2015:212-213.(in Chinese)国家药典委员会.中华人民共和国药典(三部)[S].北京:中国医药科技出版社,2015:212-213.
[2]Chinese Pharmacopoeia Commission.Pharmacopoeia of People's Repubic of China(VolⅢ)[S].Beijing:China Med Sci Press,2015:General Rule123.(in Chinese)国家药典委员会.中华人民共和国药典(三部)[S].北京:中国医药科技出版社,2015:通则123.
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[4]GAN S D,PATEL K R.Enzyme immunoassay and enzymelinked immunosorbent assay[J].J Invest Dermatol,2013,133(9):e12.doi:10.1038/jid.2013.287.
[5]WU K,FAN H L,ZHAO Z H,et al.Development of an indirect ELISA method for detection of Salmonella typhi antibody in mouse serum using typhoid Vi polysaccharide as coating antigen[J].Chin J Biologicals,2014,27(8):1070-1075.(in Chinese)吴凯,樊会兰,赵志宏,等.包被伤寒Vi多糖抗原检测鼠血清中伤寒沙门菌抗体的间接ELISA方法的建立[J].中国生物制品学杂志,2014,27(8):1070-1075.
[6]宁磊,吴祥林.实验动物福利与"3R"原则[J].微生物学免疫学进展,2006,34(3):53-56.
[7]STOKES W S,KULPA-EDDY J,MCFARLAND R.The international workshop on alternative methods to reduce,refine,and peplace the use of animals in vaccine potency and safety testing:introduction and summary[J].Procedia in Vaccinology,2011,5(1):1-15.
[8]CASEY W,SCHMITT M,MCFARLAND R,et al.Improving animal welfare and reducing animal use for human vaccine potency testing:state of the science and future directions[J].Procedia in Vaccinology,2011,5(1):33-46.
[9]COOMBES L,TIERNEY R,RIGSBY P,et al.In vitro antigen ELISA for quality control of tetanus vaccines[J].Biologicals,2012,40(6):466-472.
[10]SCICLUNA M T,AUTORINO G L,NOGAROL C,et al.Validation of an indirect ELISA employing a chimeric recombinant gag and envpeptide for the serological diagnosis of equine infectious anemia[J].J Virol Mthods,2018,251(1):111-117.
[2]Chinese Pharmacopoeia Commission.Pharmacopoeia of People's Repubic of China(VolⅢ)[S].Beijing:China Med Sci Press,2015:General Rule123.(in Chinese)国家药典委员会.中华人民共和国药典(三部)[S].北京:中国医药科技出版社,2015:通则123.
[3]LEQUIN R M.Enzyme immunoassay(EIA)/enzyme-linked immunosorbent assay(ELISA)[J].Clin Chem,2005,51(12):2415-2418.
[4]GAN S D,PATEL K R.Enzyme immunoassay and enzymelinked immunosorbent assay[J].J Invest Dermatol,2013,133(9):e12.doi:10.1038/jid.2013.287.
[5]WU K,FAN H L,ZHAO Z H,et al.Development of an indirect ELISA method for detection of Salmonella typhi antibody in mouse serum using typhoid Vi polysaccharide as coating antigen[J].Chin J Biologicals,2014,27(8):1070-1075.(in Chinese)吴凯,樊会兰,赵志宏,等.包被伤寒Vi多糖抗原检测鼠血清中伤寒沙门菌抗体的间接ELISA方法的建立[J].中国生物制品学杂志,2014,27(8):1070-1075.
[6]宁磊,吴祥林.实验动物福利与"3R"原则[J].微生物学免疫学进展,2006,34(3):53-56.
[7]STOKES W S,KULPA-EDDY J,MCFARLAND R.The international workshop on alternative methods to reduce,refine,and peplace the use of animals in vaccine potency and safety testing:introduction and summary[J].Procedia in Vaccinology,2011,5(1):1-15.
[8]CASEY W,SCHMITT M,MCFARLAND R,et al.Improving animal welfare and reducing animal use for human vaccine potency testing:state of the science and future directions[J].Procedia in Vaccinology,2011,5(1):33-46.
[9]COOMBES L,TIERNEY R,RIGSBY P,et al.In vitro antigen ELISA for quality control of tetanus vaccines[J].Biologicals,2012,40(6):466-472.
[10]SCICLUNA M T,AUTORINO G L,NOGAROL C,et al.Validation of an indirect ELISA employing a chimeric recombinant gag and envpeptide for the serological diagnosis of equine infectious anemia[J].J Virol Mthods,2018,251(1):111-117.