Preparation On Monoclonal Antibodies Against NcP40 And Development Of ELISA And Colloidal Gold To Detect Infection Of Neospora Caninum In Cattles

Neospora caninum is an obligate intracellular protozoan from the phylum Apicomplexa,closely related to Toxoplasma gondii.There are about 30 countries in Europe,Asia,and Africa that have reported infection with N.caninum infection,the rate is 10%-40%,as much as 82%.Dogs and canidae are the terminal hosts of Neospora caninum,while having been a wide range of intermediate hosts,including cattle,sheep,deer,horses,dogs,rabbits,and other livestock and marine mammals.Neosporosis,which leads to significant economic losses in both the dairy and beef industries around the world,is a major cause of abortions in the cattle industry,and has been associated with neonatal mortality and neurological clinical signs incongenitally infected calves.However,the detected methods are limited,especially for the detection of circulating antigens.Therefore,it is very important to establish a convenient,sensitive,specific and rapid detection method for the diagnosis and prevention of bovine neosporosis.This study took firstly in the preparation of monoclonal antibodies of P40 protein of N.caninum,and then the localization of Nc P40 protein antigen in N.caninum tachyzoide was determined.Subsequently,a double-antibody sandwich ELISA for detection of N.caninum circulating antigens was established for the first time.The indirect ELISA,Dot-ELISA and colloidal gold test strips were established using purified Nc P40 recombinant protein as the coating antigen.Summary,a quicker and easier method for the diagnosis of neosporosis in cattles was provided.Monoclonial antibody of Nc P40 being Preparated and purified.In this study,successfully expressed and purified Nc P40 protein and proved to have good immunogenicity and reactogenicity,Balb/c mice were immunized with Nc P40 recombination protein.The spleen cells of immunized mice were fused with SP2/0 myeloma cells and the cell fusions,6 strains of the positive hybridoma cells were selected and named NCLIV-004380-1,2,9,13,25,36,respectively.After identification of antibody subtype,NCLIV-004380-1,2,13,36 were Ig G1 subtype,NCLIV-004380-9 was Ig G2? subtype,NCLIV-004380-25 was Ig G2 b subtype.NCLIV-004380-1 and-25 strains were purified by affinity chromatography.The efficiency of purification was perfect.ELISA diction showed the antibody titer of NCLIV-004380-1 and-25 were 1×105 and 4×105,respectively.The localization of Nc P40 in Neospora caninum tachyzoide.Immunofluorescence technique was used to locate the Nc P40 protein and found that the protein was expressed on the surface of N.caninum.The establishment of Double-antibody sandwich ELISA detection method of Neospora caninum Infection.A double-antibody sandwich ELISA for the detection of N.caninum infection was established.And the optimal coating amount of the monoclonal antibody of NCLIV₀04380-1 was 0.125 ?g/well,the optimal dilution for serum was 1:200,and the best blocker was 5% of skimmed milk powder;optimal concentration of HRP-labeled NCLIV₀04380-25 monoclonal antibody is 1:3000;optimal substrate reaction time is 20min;sensitivity can reach 1:12800.There was no cross reactivity with the positive serum samples of Cryptosporidium parvum and Theileria annulata.The intra-assay batch test variation co-efficient was less than 6%.Using the double-antibody sandwich ELISA method,120 samples of bovine serum from Jilin Province were detected,and 4 positive samples were detected,the positive rate was 3%;compared with N.bovis test kits,same results can be getten.The detection method for Neospora caninum Infection established with indirecting ELISA.The Nc P40 recombinant protein was used as antigen to establish an indirect ELISA method for detection of N.caninum infection.The optimal conditions were as follows: optimal coated antigen amount was 0.5?g/well,optimal serum dilution was 1:100,optimum blocking agent was 1% gelatin,and optimal dilution of enzyme-labeled antibody was 1:5000.The sensitivity of the established method can reach 1:800,and there was no cross-reaction with Theileria annulata.The intra-assay batch variation coefficient is less than 10%,indicating that the established method has good repeatability.The method was used to detect 120 bovine serume samples from Jilin Province.The result showed that 26 samples were positive,the positive rate was 21.7%.Detection method for Neospora caninum Infection established with Dot-ELISA.In this study,Nc P40 recombinant protein was used to establish Dot-ELISA detection method of N.caninum infection.Through the optimization of reaction conditions,the optimum dilution ratio for the sample to be examined was 1:200,the best dilution ratio for the enzyme-labeled secondary antibody was 1:8000,and the optimal amount of antigen coating was 0.5?g/well.The sensitivity of Dot-ELISA detection method was 1:1600.There was no cross reactivity with the positive serum samples of Theileria annulata.The method was used to detect 120 bovine serume samples from Jilin Province.The result showed that 23 samples were positive,the positive rate was 19.17%.The establishment of colloidal gold strip detection method of Neospora caninum Infection.In this study,Nc P40 recombinant protein was used as antigen to establish colloidal gold strip detection method of N.caninum infection.And the optimal reaction conditions were determined by a series of tests.The optimal p H of colloidal gold solution was 3?L K2CO3?2mol/L?.The optimal labeling value of SPA is 6?g,the best T line incubation concentration was 1mg/m L,the best C line antibody incubation concentration was 0.5mg/m L.The sensitivity of strip detection method was 1:320.There was not cross reactivity with the positive serum samples of Theileria annulata.The test strips were assembled,sealed and dried,then stored at 4°C,room temperature?20°C?and 37°C respectively.The color changes of the test strips were observed at 1d,7d,30 d,and 60 d.The results showed that the test strips were at 4°C and can reach 60 d.The prepared colloidal gold test strips were used to detect 120 samples of clinical bovine serum samples from Jilin Province.The result showed that 21 samples were positive,the positive rate was 17.5%.