Development Of ELISA And Colloidal Gold Immunochromatographic Strips For The Detection Of Cryptosporidium Parvum In Bovine

Cryptosporidiosis is a globally distributed zoonosis.There are many types of Cryptosporidium that can infect bovine,including Cryptosporidium andersoni,Cryptosporidium parvum,Cryptosporidium bovis and Cryptosporidium ryanae,among which Cryptosporidium parvum has a wide range of host and is most harmful to human health.In recent years,with the rapid development of the cattle industry,cattle have become the main source of human cryptosporidiosis.As there is no specific vaccine or drug against Cryptosporidiosis,a rapid and accurate method for the diagnosis of Cryptosporidium is of great significance to the human health.It has been found that a virus exists in the oocysts of Cryptosporidium parvum,that is Cryptosporidium parvum virus?CSpV1?,which is stable in the oocysts and has a large number,CSpV1 can be used as an important diagnostic indicator of Cryptosporidium parvum infection in bovine.In this study,the CSpV1 monoclonal antibody was selected as the capture antibody and the polyclonal antibody was used as the detection antibody,We established three diagnostic immunoassay methods.It provides reference of dynamic monitoring of Cryptosporidium parvum in the future.Development of double-antibody sandwich ELISA:Firstly,weexpressed the Cryptosporidium parvum virus capsid protein and purified the polyclonal antibody of Cryptosporidium parvum virus capsid protein.In this study,the monoclonal antibody was used as the capture antibody,and the polyclonal antibody labeled by HRP as the detection antibody.Conditions were optimized and the ELISA method was established.The most appropriate amount of monoclonal antibody is0.25?g/holes,the best sample dilution ratio is 1:4,the optimal dilution of detection antibody is 1:200;the sensitivity is 1:160,The cattle C.andersoni and Giardia positive fecal samples test results were negative,The variation coefficient of variation within the batch is less than 6%,and the coefficient of variation is less than9%.Detection of 120 cattle fecal samples in Changchun area and compared with commercialized ELISA kits.The results showed that the positive rate of double-antibody sandwich ELISA was 5%?6/120?,and the positive rate of commercialized ELISA kits was 3%?4/120?.Development of Dot-ELISA for detection of Cryptosporidium parvum in bovine:In this study,we used CSpV1 5#monoclonal antibody and HRP-labeled CSpV1 polyclonal antibody and established the Dot-ELISA method for detection of Cryptosporidium parvum infection in bovine.The optimal dilution ratio for the determination of fecal samples was 1:16;the optimal dilution ratio for HRP-labeled CSpV-S antibody was 1:500,the optimal working concentration of coating antibody was 0.5?g/tablet,the sensitivity was 1:40.The result indicated that the method was specific and there was no cross reaction.Detection of 120 cattle samples in Changchun area and the results showed that the positive rate of Dot-ELISA was 5%?6/120??Development of Gold immunogold assay:Colloidal gold solution was prepared by reduction of chloroauric acid with trisodium citrate,establish a method of colloidal gold test strip of Cryptosporidium parvum in bovine.The optimum pH is when added 4?L K₂CO₃?0.2M/L?,the best labeling amount of antibody was 6?g,and the optimum dilution concentration of line?T?and the line?C?was 0.5mg/m L and 0.25mg/m L.the sensitivity was 1:64,and there was no cross reaction in the positive fecal samples of C.andersoni and Giardia.The results of the different storage conditions?room temperature,4?,37??and different storage period?7d,30d,60d,90d,?showed that the preservation effect of 4?,could be saved for 90days.Room temperature and 37?preservation of the test strip could be saved for 60days.Detection of 120 clinical samples in Changchun area and the positive rate of immunogold detection method was 5%?6/120?.