The Preparation Of Specific E. Wenyoni Antigen And Establishment Of Antibody-Sandwich ELISA For Detection Of E. Wenyoni

To improve specificity and sensitivity of serum diagnostic method to bovine Eperythrozoonosis, this study explored an accurate, efficient and specific antigen preparation technology, and established double-antibody sandwich ELISA diagnostic method to detect bovine Eperythrozoonosis. Further more, its clinical feasibility was investigated.The positive anticoagulant blood (erythrocyte infection rate> 90%) for Eperythrozoon wenyoni was collected, and Eperythrozoon suspension was prepared using conventional methods. Eperythrozoon suspension was broke and cracked using the repeated freezing and thawing-ultrasonic lysis method to prepare crude antigen solution. The crude antigen solution was used to immunize rabbits to prepare rabbit anti-bovine Eperythrozoon positive serum.The prepared antigen protein was adopted for SDS-PAGE experiment. The concentration of separating gel is 11%,The concentration of stacking gel is 5%. Electrophoresis for 1.5h, staining for 1h, decoloration for 1h, then, the whole Eperythrozoon wenyoni antigen protein component was observed. Four deeper staining antigen proteins were selected to cut out from gel and purify. After crude antigen by SDS-PAGE electrophoresis, protein bands in the 0.4 mol/LNaAc solution (pH 13) were dyed to brown. Four protein bands were cut out from gel, put into distilled water, decolourized in swing bed, and installed in dialysis bags for horizontal electrophoresis respectively, and then dialysis in PBS solution 4℃overnight. With polyethylene glycol (PEG-6000) condensation, the supernate was got after precipitation. Then, SDS-PAGE was applied to examine purified results. Moreover, the Western-blotting technology was applied. Through the transfer electrophoresis and immune staining, the color of imaging protein band in nitrocellulose membrane can confirm whether these antigen proteins have the immunogenicity.The immunogenic antigen proteins were collected using gel purification technology. Rabbits were immunized by conventional methods to obtain immune serum. Extraction and purification IgG with the caprylic acid-ammonium sulfate. The IgG antibody was labeled using improved sodium periodate method. The double-antibody sandwich ELISA test was adopted using of crude antigen solution, purified IgG and horseradish peroxidase labeled IgG.The best antibody coated amount, the work concentration of enzyme labeled antibody and minimum detectable amount of antigen were explored. The specific test, cross-test, repeated test and expand test were made to establish specific and sensitive double antibody sandwich ELISA.SDS-PAGE results showed that the four deeper color protein bands of the Eperythrozoon wenyoni entire antigen protein were selected. Molecular weight of four separated protein bands (band 1,band 2, band 3 and band 4) using cutting gel technology was 114.9kDa,70.1 kDa,60.6 kDa,49.4 kDa respectively. Western blot test showed that band 1,2 and 3 were positive, and part ingredient in band 2 responsed with antibody. The band 4 was negative.The results of double antibody sandwich ELISA showed that, the optimal coated amount of antibody was 78.1μg/ml, the optimal working concentration of enzyme labeled antibody was 1:400, and the minimum detectable amount of antigen was 4.71μg/mL. The sensitivity was very high. Specific blocking test results show rabbit anti-bovine Eperythrozoon positive serum could block antigen-positive color reaction, but negative serum could not block. The cross test results found that only Eperythrozoon produce positive results, but Toxoplasma Gondii, Blood Cryptosporidium, E. coli are negative. These proved that the method had good specificity. Adopting this method to detect 100 dairy cattle blood sample collected from three different dairy farms, the positive detection rate was 52.0%. The detectable ratio improved obviously compared with microscopic examination(41.0%).The test results show that the preparation technology of Eperythrozoon wenyoni specific antigen provided by this test is entirely feasible. The diagnostic method of double antibody sandwich ELISA is a specific, sensitive and rapid diagnostic method. This technology has great significance to clinical diagnosis.