| Eperythrozoonosis is a zoonosis caused by E. ovis.In order to diagnose the disease quickly and accurately, and provide a technical support for controlling this disease, an accurate and efficient E.ovis specific antigen preparation technique was explored in this study, so as to establish a specific, sensitive double-antibody sandwich ELISA.Anticoagulant blood samples whose erythrocyte positive infection rates being larger than 90% were collected, the water bath method and in vitro drug anthelmintic method were used to dissociate E.ovis. Eperythrozoon infection rate (%), erythrocyte infection intensity (number), Eperythrozoon number (1×104/mm3), amount of impurity, and Eperythrozoon movement were observed both before and after dissociation. Eperythrozoon antigen was extracted, Eperythrozoon antigen somatic protein suspensions was prepared. Then determined the protein content, compared the dissociation effect. SDS-PAGE test on Eperythrozoon antigen somatic protein was used to observe the consistent in band between the two preparations made from different method.The results showed that adding 1mL "Shuang Xiang Hong Lian Mie" per 200mL infection blood, reacting 36h in 4℃, could achieve a better separation effect. Comparing with water bath method, after dissociation erythrocyte infection rate and intensity were decreased significantly, the protein content increased (high yield) and got good purity. The SDS-PAGE test results showed that the antigen protein bands of Eperythrozoon antigen protein suspensions from in vitro anthelmintic preparation were same as those from water bath method.The E.ovis antigen protein was prepared with drug anthelmintic in vitro, and was purified with gel purification, horizontal electrophoresis and dialysis, then the immunogenic specific antigen protein composition was screened out with SDS-PAGE and immunoblotting technique.The specific antigen protein composition was collected again, and was used to immunize rabbits to obtain IgG, to estabilish double-antibody sandwich ELISA test.The results showed that there were 4 main antigen protein bands in E.ovis antigen proteins, their molecular weight were 115.35kDa,74.13 kDa,68.87 kDa,32.96 kDa through the analysis. Western blotting test showed that the band whose molecular weight was 115.35kDa and 68.87 kDa respectedly was positive, and was determined as the E.ovis specific antigen protein. The rest bands was only part of composition reacted with antibody or was negative. Double-antibody sandwich ELISA test showed that, the specific immune antigen protein concentration was 1.8mg/mL,35 days after immunizing rabbits, rabbit serum antibody titer reached height of 1:5120. The sensitive test showed that antigen minimum detectable amount was 3.55μg/mL,the sensitivity had been improved comparing with the ELISA method established by crude antigen preparation antibodies.The specificity test results showed that the purification antibodies reacted only to E. ovis, having no reaction to Mycoplasma pneumoniae, Escherichia coli and Staphylococcus aureus. The cross test results showed that purified antibody only reacted to E. ovis, Whereas had no reaction to Eperythrozoon from other animal. It indicated that the test specificity was good. The 100 blood samples from some sheep farms of different counties in Zhangjiakou City were collected and were detected using the established double-antibody sandwich ELISA,the result showed that the positive rate was 79% which was more sensitive than microscopic method (positive rate was 62%), the difference was significant(P<0.05). |
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