猪瘟病毒抗体间接ELISA检测方法的建立及优化

doi:10.16431/j.cnki.1671-7236.2016.03.007

Abstract

Abstract: In this study,through a series of screening and optimization of reactions condition,an indirect ELISA method was developed for detections the antibodies against classical swine fever virus (CSFV) with the purified recombinant CSFV E2 protein.The results showed that the optimal concentration for coating antigen was 1.0 μg/mL and incubated at 37 ℃ for 1 h.The proper serum sample was diluted by 1:200 and the sealing time was 37 ℃ for 1 h.Enzyme labeled second antibody was diluted by 1:10 000 and the reaction time was 45 min incubating at 37 ℃.The D450 nm<0.30 of sample defined as negative of CSFV antibody in the serum sample.The intra-batch and inter-batch variation coefficients were 4.8% and 6.9%,respectively.It indicated that the method in this study had a high stability.Specific test showed that the indirect ELISA had no crossing reactions with the antibodies against PRV,PRRSV,PCV and PPV.80 serum samples were detected in this study,the positive coincidence rate of indirect ELISA was 83.58%,the negative coincidence rate was 76.92%,and the total coincidence rate was 80.25% compared to the results of import blocking ELISA kit.The results suggested that the established indirect ELISA method in this study had an excellent clinical application.

Key words: classical swine fever virus (CSFV); E2 protein; indirect ELISA; antibody detection

CLC Number:

S852.65⁺

FENG Chun-hua, ZHU Yan-ping, GUO Dong-guang, YUE Feng. Development and Optimization of Indirect ELISA Detection Method of Classical Swine Fever Virus Antibody[J]. , 2016, 43(3): 608-614.

References

[1] Risatti G R,Borca M V,Kutish G F,et al.The E2 glycoprotein of classical swine fever virus is a virulence determinant in swine[J].Journal of Virology,2005,79(6):3787-3796.
[2] Büttner M,Ahl R.Classical swine fever[J].Tierarztl Prax Ausg G Grosstiere Nutztiere,1998,26(5):278-286.
[3] 董浩,李菲,王鑫,等.猪瘟病毒野毒株与疫苗株酶切检测方法的建立、优化及应用[J].中国畜牧兽医,2011,38(9):187-189.
[4] 马超英.4种猪瘟病毒抗体检测方法的比较[J].动物医学进展,2012,33(10):128-131.
[5] 庞然,张小敏,周斌,等.4种商品化试剂盒检测猪瘟病毒抗体的比较分析[J].中国兽医杂志,2011,47(9):40-42.
[6] 李娇,沈志强,祖立闯,等.间接 ELISA在猪瘟抗体检测上的应用[J].动物医学进展,2012,33(6):126-129.
[7] van Rijn P A,van Gennip H G,de Meijer E J,et al.Epitope mapping of envelope glycoprotein E1 of hog cholera virus strain Brescia[J].J Gen Viral,1993,74:2053-2060.
[8] Peng W P,Hou Q,Xia Z H,et al.Identification of a conserved linear B-cell epitope at the N-terminus of the E2 glycoprotein of Classical swine fever virus by phage-displayed random peptide library[J].Virus Research,2008,135(2):267-272.
[9] Liu S G,Yua X L,Wang C L,et al.Quadruple antigenic epitope peptide producing immune[J].Vaccine,2006,6(42):7175-7180.
[10] Xu X G,Liu H J.Baculo virus surface display of E2 envelope glycoprotein of classical swine fever virus and immunogenicity of the displayed proteins in a mouse model[J].Vaccine,2008,26(43):5455-5460.
[11] He D M,Qian K X,Shen G F,et al.Recombination and expression of classical swine fever virus (CSFV) structural protein E2 gene in Chlamydomonas Reinhardtii chloroplasts[J].Colloids Surf Biointerfaces,2007,55(1):26-30.
[12] 郭东光,朱艳平,银梅,等.猪瘟病毒E2蛋白抗原表位集中区的原核表达及鉴定[J].动物医学进展,2013,34(3):15-20.
[13] 温国元,李坤,邵华斌,等.猪瘟病毒 E2蛋白的原核表达及抗体间接ELISA检测方法的建立[J].湖北农业科学,2009,48(11):2632-2635.
[14] 李文良,毛立,江杰元,等.猪瘟病毒E2蛋白主要抗原区的原核表达及其间接ELISA检测方法的建立与应用[J].动物医学进展,2012,33(5):8-13.

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Development and Optimization of Indirect ELISA Detection Method of Classical Swine Fever Virus Antibody

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