Development Of Indirect ELISA Kit For Detecting Aleutian Mink Disease Antibody And Serological Investigation

Aleutian disease of mink(MAD) is a chronic, wasting infectious disease. It is caused by Aleutian mink disease parvovirus [1](AMDV), which has autonomous replicating capability and can infringe immune cells so that the body’s immune system dysfunction. The disease is almost spread in all mink-raising countries around the world. In China, the serum positive rate of some mink groups can reach 30% ~ 80%, and minks of all ages are susceptible to Aleutian disease of mink, causing it one of the most serious infectious diseases for minks. At present, the main method for the detection of the disease is Convection immunoelectrophoresis(CIEP). However, its high testing costs and relatively small number of samples for each test make CIEP not practical for mass detection, but just suitable for laboratory test. Therefore, the independent research and development of a more effective and cheap detection kit is of great significance to the prevention of the disease as well as the purification of mink farms.In this study, the artificial antigen peptide(named sd) which had been identified well in reactogenicity and specificity, was induced expression in Escherichia coli BL21. Then after the purification of the Niz+ affinity chromatography column as well as ultrafiltration concentration, the sd recombinant protein with high purity and good reactogenicity was obtained, whose density was 12 mg/m L. In accordance with the detection method of ELISA(the antibody of MAD), which had already been established in the laboratory, the recombinant protein sd was developed into coating antigen and the indirect ELISA kit for detecting Aleutian mink disease antibody was obtained. After evaluating the preservation period, repeatability, specificity and sensitivity of the kit, we found that it can be stored at-20 ℃ for six months, and at 4℃ for one months. It can specifically detect the standard positive serum of Aleutian disease of mink, which had no cross reactions with other positive labeled serums of viruses such as canine distemper virus, mink enteritis virus, canine parvovirus and pseudorabies virus; its sensitivity is also higher than CIEP in the detection; the repeatability test showed that the batch coefficient of variation was 0.667% ~ 5.811%, and the inter batch coefficient of variation was 0.882% ~ 6.046%, both being less than 10%, which indicated that the repeatability of the kit is good among batches as well as within batches. By applying the ELISA kit and CIEP simultaneously to detect the 379 mink serums saved by the laboratory, we found that the positive detection rate of sd-ELISA kit was 82.3%, and that of CIEP was 74.6%, which proved the kit had a higher positive detection rate.With this method, we conducted an Aleutian disease serological survey with 2190 mink serums in China’s main mink-raising provinces including Shandong, Dalian, Jilin, Heilongjiang and Henan, we found that the infection rates were respectively 96.3%, 82.1%, 71.2%, 64.2%, 69.8%.The experimental results proved that the indirect sd-ELISA kit has the advantages of good repeatability, high specificity and sensitivity etc. Compared to CIEP, its lower costs and good detection performance make it more suitable for batch operations and to meet the practical need of mink farms, scattered farmers as well as the import and export inspection and quarantine.