| Toxoplasma gondii is an obligate intracellular parasitic protozoa,which can infect a wide variety of animals,including mammals,birds,and some cold-blooded animals.Toxoplasmosis was popular in worldwide,there were about 10 million people infected in the whole world,and infection rate of the population in our country was 7.88%.The positive rate of toxoplasma in livestock and poultry was very high,and the positive rate of pigs is 30.16%.Toxoplasmosis had seriously affected the health of human and the development of animal husbandry.Toxoplasma gondii was an opportunistic protozoan,and people infected most no obvious symptoms,but when pregnant women,newborn infants or immunosuppressed patients with acute infection would have serious consequences,therefore,early diagnosis and early treatment of toxoplasmosis had important significance.Toxoplasma circulating antigen(CAg)was the earliest specific marker in the infection process of Toxoplasma gondii,and we could detect the early phase of acute infection or active phase of toxoplasmosis by detection of circulating antigen in serum.In this study,a double antibody sandwich ABC-ELISA and colloidal gold immuno chromatography test paper strip were established to detect toxoplasma circulating antigen,in order to realize the early diagnosis of toxoplasmosis,which is an effective supplement to the current methods by detection of antibodies to Toxoplasma gondii.Test ? Anti-CAg monoclonal antibody and preparation of anti ESA polyclonal antibody preparationToxoplasma circulating antigen(CAg)was an important index in the early diagnosis of toxoplasmosis.It was very important to prepare high specific antibody against CAg for the early diagnosis of Toxoplasma gondii infection.In this study,animals were immunized with Toxoplasma gondii excretory-secretory antigens(ESA),anti-CAg monoclonal antibodies were raised after hybridoma cell supernatant screened by three kinds of antigens,and the obtained properties of monoclonal antibodies were identified.The results showed that the 3A5,3E5 and 10F5-3 three strains of monoclonal antibody were obtained,whose titer were 1:64000,1:32000 and 1:32000 respectively and recognized different epitopes.Immunofluorescence localization showed that the three antibodies were reacted with Toxoplasma gondii.Indicating that the three monoclonal antibodies were highly specific to the CAg of Toxoplasma gondii,which could be used in the diagnosis of acute infection or active phase with Toxoplasma gondii.Test ? Establishment of double antibody sandwich ABC-ELISA methodA double antibody sandwich ELISA method based on ABC(avidin biotin-peroxidase complex)amplification system was established to detect toxoplasma circulating antigen in order to realize the early diagnosis of acute infection of Toxoplasma gondii.In this study,the optimal concentration of coating antibody and optimal dilution of detection antibody were determined by square titration method.Experimental toxoplasma infection in canines and toxoplasma positive serum samples were detected by this method to determine the detection time and the accuracy of the method,and this method was applied to detection of CAg in clinical samples.Results showed that double antibody sandwich ELISA reaction condition:the optimum coating concentration of rabbit polyclonal antibody was 3.7 ?g/mL,the working concentration of biotin-labeled monoclonal antibody is 0.1 ?g/mL,0.13 ?g/mL and 0.12 ?g/mL for 3A5,3E5 and 10F5-3 respectively,biotinylated peroxidase should be mixed with streptavidin at 2:3 in volume.The lowest detection limit of ESA was 11.9 ng/ml by this method.The ELISA technique had no cross-reaction with cryptosporidium,schistosoma japonicum,eimeria tenella,canine distemper and canine parvovirus.CAg was detected in canine serum 2 days after infection with tachyzoites by double antibody sandwich ELISA method.The method can clearly distinguish positive from negative sera of Toxoplasma gondii infection.The detection result of ELISA for 2 positive serum which mixed in 68 serum samples were same as that of Nest PCR.These results indicated that the double antibody sandwich ABC-ELISA was a highly sensitive and specific method for diagnosis of T.gondii infection,which was feasible to detect CAg in acute infection or active phase with Toxoplasma gondii.Test ? Development of colloidal gold immunochromatographic test strip A simple and rapid colloidal gold immuno chromatography test(ICT)was established to detect toxoplasma circulating antigen(CAg)in serum.In this study,colloidal golds were prepared by trisodium citrate reduction method,which were used to the labeling of anti-CAg monoclonal antibody,anti-ESA rabbit polyclonal antibody was coated on NC membrane,so we established the immune chromatography detection method based on the double antibody sandwich method.The toxoplasma circulating antigen in serum and the gold labeled monoclonal antibody were combined and the conjugation moved along the NC membrane,then,the conjugation combined with anti-ESA rabbit polyclonal antibody on the membrane to form a red line visible to the naked eye.By this method,the positive sera of dogs with different time after infection were detected,toxoplasma positive serum and negative samples were detected by this method to determine the accuracy of the method,and to compare with the double antibody sandwich ABC-ELISA.Results showed that CAg was detected in canine serum in 2 to 7 days after infection with tachyzoites by this method,which had no cross-reaction with cryptosporidium,schistosoma japonicum,eimeria tenella,canine distemper and canine parvovirus.The coincidence rate of detection results with Toxoplasma gondii ELISA was 100%.It indicated that the immune chromatography test paper had high specificity and sensitivity to the toxoplasma circulating antigen in the serum,and the method is simple and rapid,and has wide application value. |
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