田来明;杨滨僮;李智鹏;张桐嘉;宫鹏涛;李建华;王全楷;杨举;李赫;张西臣;
TIAN Lai-ming;YANG Bin-tong;LI Zhi-peng;ZHANG Tong-jia;GONG Peng-tao;LI Jian-hua;WANG Quan-kai;YANG Ju;LI He;ZHANG Xi-chen;College of Veterinary Medicine, Jilin University;

• 1:吉林大学动物医学学院
• 2:长春市农业科学院
• 3:吉林农业大学动物科学技术学院

摘要(Abstract)：

目的建立检测鹿新孢子虫感染的双抗夹心Dot-ELISA方法,并进行初步应用。方法以双抗夹心ELISA方法的A490值对应Dot-ELISA方法显色结果的方式,建立双抗夹心Dot-ELISA方法检测结果判定标准,确定HRP标记的抗犬新孢子虫MIC6单克隆抗体1H11、包被抗体(抗犬新孢子虫MIC6单克隆抗体1A1)、待测血清的最佳工作浓度,并对该方法的特异性、重复性及灵敏度进行验证。用建立的方法检测79份鹿血清,并与双抗夹心ELISA方法进行比较。结果确定该方法 HRP-H11抗体的最佳稀释度为1∶100,包被抗体最适工作浓度为0.3μg/片,待测血清稀释度为1∶8。自然感染犬新孢子虫的鹿阳性血清的敏感性稀释度为1∶64,与弓形虫阳性血清无交叉反应,敏感性、特异性较强;自然感染新孢子虫的鹿阳性血清3次检测结果重复性较好。该方法检测的76份鹿血清中,阳性12份,阳性率为15.2%,与双抗夹心ELISA方法检测结果一致。结论建立的双抗夹心Dot-ELISA方法可用于检测鹿群感染新孢子虫循环抗原,为新孢子虫病的诊断提供了新的方法。
Objective To develop and preliminarily apply a double antibody sandwich Dot-ELISA for Neospora caninum infection in deer. Methods The standard for judgment of determination result by double antibody sandwich Dot-ELISA was developed by corresponding the A490 value of double antibody sandwich ELISA to the chromogenic result of DotELISA. The working concentrations of HRP-labeled monoclonal antibody(Mc Ab)1H11 against canine N. caninum MIC6,coating antibody(Mc Ab 1A1 against canine N. caninum MIC6) and serum samples to be tested were optimized, and the developed method was verified for specificity, reproducibility and sensitivity. A total of 79 deer serum samples were deter-mined by the developed method, and the result was compared with that by double antibody sandwich ELISA. Results The optimal dilutions of HRP-H11 antibody and serum sample to be tested were 1 ∶ 100 and 1 ∶ 8 respectively, while the optimal working concentration of coating antibody was 0. 3 μg / tablet. No cross reaction with Toxoplasma gondii positive serum was observed, indicating high sensitivity and specificity of the method. Three determination results of deer sera nat-urally infected with N. caninum indicated good reproducibility of the method. Of the 76 deer serum samples determined by the developed method, 12 were positive, indicating a positive rate of 15. 2%, which was consistent with that by double an-tibody sandwich ELISA. Conclusion The developed method might be used for determination of infection with circulating antigen of N. caninum in deer, which provided a new method for diagnosis of diseases caused by N. caninum.

关键词(KeyWords)： 新孢子虫;鹿;双抗夹心Dot-ELISA方法;MIC6
Neospora caninum;Deer;Double antibody sandwich Dot-ELISA;MIC6

Abstract:

Keywords:

基金项目(Foundation): 国家重点基础研究发展计划(973计划)(2015CB150300);; 国家质检公益性行业专项(201410061);; 吉林省科技发展计划项目(20100222)

作者(Authors): 田来明;杨滨僮;李智鹏;张桐嘉;宫鹏涛;李建华;王全楷;杨举;李赫;张西臣;
TIAN Lai-ming;YANG Bin-tong;LI Zhi-peng;ZHANG Tong-jia;GONG Peng-tao;LI Jian-hua;WANG Quan-kai;YANG Ju;LI He;ZHANG Xi-chen;College of Veterinary Medicine, Jilin University;

DOI: 10.13200/j.cnki.cjb.000862

参考文献(References)：

• [1]Yin JG,Qu G,Cao L,et al.Characterization of Neospora caninum microneme protein 10(Nc MIC10)and its potential use as a diagnostic marker for neosporosis[J].Vet Parasitol,2012,187(1-2):28-35.
• [2]Cervantes-Landín AY,Martínez-Martínez I,Reyes PA,et al.Standardization of Dot-ELISA for detection of anti-Trypanosoma cruzi antibodies,compared to ELISA and Western blot[J].Enferm Infecc Microbiol Clin,2014,32(6):363-368.
• [3]He PF.Screening of Neospora caninum specific genes,establishment of detection methods and their applications[D].Changchun:Jilin University,2013.(in Chinese)贺鹏飞.犬新孢子虫特异基因筛选、检测方法建立及应用[D].吉林长春:吉林大学,2013.
• [4]Dubey JP,Hattel AL,Lindsay DS,et al.Neonatal Neospora caninum infection in dogs:isolation of the causative agent and experimental transmission[J].J Am Vet Med Assoc,1988,193(10):1259-1263.
• [5]Packham AE,Sverlow KW,Conrad PA,et al.A modified agglutination test for Neospora caninum:development,optimization,and comparison to the indirect fiuorescent-antibody test and enzyme-linked immunosorbent assay[J].Clin Diagn Lab Immunol,1998,5(4):467-473.
• [6]Hosseininejad M,Hosseini F,Mosharraf M,et al.Development of an indirect ELISA test using an affinity purified surface antigen(P38)for sero-diagnosis of canine Neospora caninum infection[J].Vet Parasitol,2010,171(3-4):337-342.
• [7]Ibrahim HM.Seroprevalence of Neospora caninum antibodies in chicken samples from Delta Egypt using a recombinant Nc SAG1protein-based ELISA[J].Egypt J Immunol,2013,20(1):29-37.
• [8]Ma L.Preparation of monoclonal antibodies against SAG3 protein of Toxoplasma gondii and its applied research[D].Changchun:Jilin University,2012.(in Chinese)马亮.弓形虫表面抗原SAG3单克隆抗体的制备及应用研究[D].吉林长春:吉林大学,2012.