Development And Preliminary Application Of ELISA And Colloidal Gold Immunochromatographic Strips For The Detection Of Toxoplasma Infection In Cat

Toxoplasma gondii(T.gondii)is an important zoonotic parasite,the current global estimated incidence of congenital toxoplasmosis is 190,100 cases a year in humans.The disease has a wide range of host,all warm-blooded animals,and some cold blood animals can be infected by it.There are many recessive infections in healthy people with no symptom,but patients with immunodeficiency disease,such as AIDS,always have obvious symptoms and may even die.The cats are both final host and intermediate host.Human toxoplasmosis is mainly caused by the ingestion of cysts from contaminated food or undercooked meat with cysts.The infected pregnant animal may show abortion,stillbirth,feeble body,mummy body and other symptoms.At present,the diagnostic methods of T.gondii include pathogenic diagnosis,molecular biology diagnosis and immunological diagnosis.This study is based on immunological diagnostic techniques for the establishment of the method.SAG3 is a kind of surface antigen of T.gondii with a molecular weight of 43 k Da that take part in all the invasion steps.Similar with SAG1,SAG3 is a tachyzoite-specific protein as well as a highly immunogenic antigen which regards as a good choice for vaccine.SAG3 also has the similar structure and function as SAG1.There are many reports about SAG1,but fewer about SAG3.The aim of the present study was to use SAG3 for the establishment of toxoplasma detection in cat.Establishment of indirect ELISA for detection of toxoplasmosis in cat.In this study,the ELISA method was established with the purified SAG3 protein as the antigen,and the conditions were optimized.The most appropriate amount of antigen was 0.75μg /well,the best serum dilution ratio was 1:800,and the optimal dilution of peroxidase-labeled Anti-cat was 1:5000.The sensitivity of this method was 1:12800.The feline calicivirus positive sera and cat transmissible gastroenteritis virus positive serum cross-reaction test results were negative.By select the kit in different storage period,shows the variation coefficient between ot within the batch are less than 6%.Detection of 96 cat sera samples were compared with commercialized indirect hemagglutination kits.The results showed that the positive rate of indirect ELISA was 61.46%(59/96),and the positive rate of indirect hemagglutination kit was 56.25%(54/96).Establishment of indirect Dot-ELISA for detection of toxoplasmosis in cat.In this study,coated with purified SAG3 protein of T.gondii,while selecting nitrocellulose membrane(NC)as the solid carrier.The best dilutions of serum sample and HRP-Ig G cat were 1:1600 and 1:5000 respectively,the most optimal working concentration of coating antibody was 0.125μg/pice,the optimal working concentration of sensitivity was 1:12800.The result indicated that the method was specific and there was no cross-reaction with others.Detection of 96 cat sera were compared with commercialized indirect hemagglutination kits.The results showed that the positive rate of Dot-ELISA was 62.5%(52/96),and the positive rate of indirect hemagglutination kit was 56.25%(54/96).Development of Gold immunogold assay for detection of toxoplasmosis in cat.In this study,a colloidal gold solution was prepared by reduction of chloroauric acid with trisodium citrate,SPA was used to establish a method for the development of colloidal gold test strip of cat’s toxoplasmosis.The optimum p H of the solution is reached when 3μL K2CO3(0.2M/L)be added,the best labeling amount of SPA was 6μg,and the best line(T)and the line(C)were antibody incubation concentration 0.5mg/m L and 1 mg/m L.In the performance test,the sensitivity was up to 1: 640,and there was no cross-reaction in the positive serum of feline calicivirus and feline gastroenteritis virus,and the test of different batches was repeated.The results of the storage test of different storage conditions(room temperature,4 ℃,37 ℃)and different storage period(1d,7d,30 d,60d,90 d,120d)showed that with 4℃ storage,the gold immunogold assay could be saved for 120 days.Room temperature preservation with drying agent of the test strip is relatively good,storage period was up to 90d(still visible slight band).37 ℃ stored test strip effect is relatively poor,the shelf life of 90 d has been invalid.Detection of clinical samples of 96 samples and compared with commercialized indirect hemagglutination kits.The positive rate of immunogold detection method was 57.29%(52/96),and the positive rate of indirect hemagglutination kit was 56.25%(54/96).