Method establishment and preliminary application of sandwich ELISA for detecting Cryptosporidium parvum antigen
ZHANG Lu1,2, LIU Zhen-zhen1,2, CHEN Kai-li1,2, WANG Rong-jun1,2, ZHANG Long-xian1,2, NING Chang-shen1,2, JIAN Fu-chun1,2
1.College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002, China; 2.International Joint Research Laboratory of Zoonosis of Henan Province,Zhengzhou 450002,China
Abstract:We established the method of Sandwich ELISA to detect Cryptosporidium parvum antigen. Purified anti-Cryptosporidium IgG and IgY were used as a capture antibody and detection antibody respectively to develop sandwich ELISA. A checkerboard titration study was carried out to determine the optimal conditions of ELISA. The PCR based on 18SrRNA was used to evaluate the pre-treatment effect of three methods (saturated sucrose solution floating, saturated salt water floating and PBST detergent washing). The optimum concentration of coated antibody, antigen, detection antibody and enzyme-labelled antibody were 1∶800, 2.5 μg/mL, 1∶100 and 1∶5 000 respectively. The coating condition, antigen antibody reaction, optimum reaction time of enzyme-labelled antibody were 4 ℃ through the night after 37 ℃, incubated at 37 ℃ for 30 min and 45 min respectively; the optimal termination condition was 2 mol/L H2SO4,50 μL/well; TMB developed 10 minutes at room temperature. The developed sandwich ELISA has no cross reaction with the eggs/oocyts of Nematode, Coccidium and Ascarid; coefficient of variation of intra-assay and inter-assay were all less than 10%. The results showed that the total coincidence rate of the three pre-treatment methods with nested PCR were 95.83%, 91.67% and 83.33%, respectively, and the Saturated Sucrose Floatation method was the best one among the three methods, the sensitivity of the method was lower than the Cryptosporidium detection kit of IDEXX (6×103/mL), and whole test process was longer than the kit. While, its specificity and reproducibility were consistent with that of IDEXX kit, and the developed method was more economical. The method is simple, rapid, sensitive, and can be used for clinical epidemiological investigation of Cryptosporidiosis or pathogen detection.
[1] Leitch GJ, He Q.Cryptosporidiosis-an overview[J]. J Biomedical Res, 2012, 25(1): 1-16. DOI:10.10 16/S1674-8301(11)60001-8 [2] Baldursson S, Karanis P.Waterborne transmission of protozoan parasites: review of worldwide outbreaks-an update 2004-2010[J]. Water Res, 2011, 45(20): 6603-6614. DOI:10.1016/j.watres.2011.10.013 [3] Xiao L, Fayer R.Molecular characterization of species and genetypes of Cryptosporidium and Giardia and assessment of zoonotic transmission[J]. Int J Parasitol, 2008, 3(6): 1239-1255. DOI: 10.1016 /j.ijpara. 2008. 03.006 [4] Xiao L.Molecular epidemiology of cryptosporidiosis: an update[J]. Exper Parasitol, 2010, 124(1): 80-89. DOI: 10.1016/j.exppara.2009.03.018 [5] Baldursson S, Karanis P.Waterborne transmission of protozoan parasites: review of worldwide outbreaks-an update 2004-2010[J]. Water Res, 2011, 45(20): 6603-6614. DOI: 10.1016/j.watres.2011.10.013 [6] Qi M, Zhang XS, Ning CS, et al. Advances in immunological diagnosis of giardiasis[J]. Chin J Zoonoses, 2008, 24(11): 1066-1069. ( in Chinese) 齐萌, 张小三, 宁长申, 等. 贾第虫病免疫学诊断方法研究进展[J]. 中国人兽共患病学报, 2008, 24(11): 1066-1069. [7] Deng MJ, Xiao XZ, Sun T, et al. Progress on detection methods for Giardia lamblia[J]. Progr Vet Med, 2008, 24(11): 1066-1069. ( in Chinese) 邓明俊, 肖西志, 孙涛, 等. 蓝氏贾第鞭毛虫检测技术研究进展[J]. 动物医学进展, 2013, 33(12): 168-173. [8] Rossle NF, Latif B.Cryptosporidiosis as threatening health problem: a review[J]. Asian Pacif J Trop Biomed, 2013, 3(11): 916-924. DOI:10.1016/S2221-1691(13)60179-3 [9] Liu SJ, Zhu MY, Gu SB,et al. Comparison of three methods of purifying IgG from swine serum[J]. Chin Agr Sci Bull, 2007, 23(11): 38-43. ( in Chinese) 刘生杰, 朱茂英, 顾士彬,等. 免疫球蛋白G(IgG)三种提取方法比较[J]. 中国农学通报, 2007, 23(11): 38-43. [10] Hutchings GH, Ferris NP.Indirect sandwich ELISA for antigen detection of African swine fever virus: comparison of polyclonal and monoclonal antibodies[J]. J Virological Methods, 2006, 131(2): 213-217. DOI: 10.1016/j.jviromet.2005.08.009 [11] Johnson AJ, Langevin S, Wolff KL, et al.Detection of anti-West Nile virus immunoglobulin M in chicken serum by an enzyme-linked immunosorbent assay[J]. J Clin Microbiol, 2003, 41(5): 2002-2007. DOI: 10.1128/JCM.41.5.2002-2007.2003 [12] Wang CR, Qi M, Li JQ, et al. Species and subtype identification of Cryptosporidium spp. from dairy cattle in an imported farm and biosafety evaluation[J]. Chin J Zoonoses, 2015,31(11): 1005-1009. ( in Chinese) 王臣荣, 齐萌, 李俊强, 等. 规模化引种场进口奶牛隐孢子虫种类, 基因亚型鉴定及其生物安全评价[J]. 中国人兽共患病学报, 2015,31(11): 1005-1009. [13] Santín M.Clinical and subclinical infections with Cryptosporidium in animals[J]. New Zealand Vet J, 2013, 61(1): 1-10. DOI: 10.1080/00480169.2012.731681 [14] Wang R, Zhang L, Axén C, et al.Cryptosporidium parvum IId family: clonal population and dispersal from Western Asia to other geographical regions[J]. Scientific Reports, 2014, 4: 4208. DOI:10.1038/srep04208 [15] Amer S, Zidan S, Adamu H, et al.Prevalence and characterization of Cryptosporidium spp. in dairy cattle in Nile River delta provinces, Egypt[J]. Exper Parasitol, 2013, 135(3): 518-523. DOI: 10.10 16/j.ex ppara. 20 -13.09.002 [16] Widmer G, Sullivan S.Genomics and population biology of Cryptosporidium species[J]. Parasite Immunol, 2012, 34(2/3): 61-71. DOI:10.1111/j.1365-3024.2011.01301.x [17] Zhu HL, Zhang LX, Ning CS,et al. Genotypes and genotypes of Cryptosporidium zoonoconidia[J]. Acta Parasitologica et Medica Entomologica Sinica, 2007,14(1):48-55. ( in Chinese) 朱惠丽,张龙现,宁长申,等. 人兽共患隐孢子虫种类及基因型[J]. 寄生虫与医学昆虫学报,2007,14(1):48-55. [18] Wang XH, Yang LR, Zhao LL.et al. Immunofluorescence technique and its application in detection of parasite[J]. Chin Ani Vet, 2012, 39(3): 81-84. ( in Chinese) 王小环, 杨莲如, 赵林立, 等. 免疫荧光检测技术及其在寄生虫检测中的应用进展[J]. 中国畜牧兽医, 2012, 39(3): 81-84. [19] ten Haaf A, Kohl J, Pscherer S, et al. Development of a monoclonal sandwich ELISA for direct detection of bluetongue virus 8 in infected animals[J]. J Virological Methods, 2017, 243: 172-176. DOI: 10.1016/j.jv- iromet.2017.02.003 [20] Saita T, Yamamoto Y, Hosoya K, et al.An ultra-specific and sensitive sandwich ELISA for imatinib using two anti-imatinib antibodies[J]. Analytica Chimica Acta, 2017, 969: 72-78. DOI:10.1016/j.aca.2017.03.034 [21] Yang HY.Establishment and application of monoclonal antibody mediated IFA and sandwich ELISA methods for detecting Cryptosporidium Oocysts[D]. Zhengzhou:Henan Agricultural University, 2009. (in Chinese) 杨红玉. 检测隐孢子虫卵囊的单抗介导IFA和夹心ELISA方法的建立及初步应用[D]. 郑州:河南农业大学, 2009. [22] Thompson RC, Olson ME, Zhu G, et al.Cryptosporidium and cryptosporidiosis[J]. Adv Parasitol, 2005: 77-158. DOI: 10.1016/S0065-308X(05)59002-X