Preparation Of The Aleutian Mink Disease Detect Antigen And Development Of Indirect ELISA

Aleutian disease (AD) is an autoimmune and infectious disease in mink industry,which is caused by the Aleutian disease virus (ADV). In vivo anti-ADV antibodiescannot effectively remove the virus. In contrast, ADV infection can be enhanced bythe Fc receptor-mediated ADE, which can make immune system disorder in mink.This disease not only can cause a sharp decline of the reproduction in minkpopulations, but also can lead to death in some situation. So the disease bring a hugeeconomic loss in the mink industry and seriously hinder the development of animalfur farming in our country. However, there is no effective vaccine for preventing thisdisease yet. In order to eradicate the Aleutian disease, a variety of antibody detectiontechniques such as CIEP, ELISA were used to determine the infected minks forelimination.Our study aimed at developing the method of indirect ELISA for detecting theAleutian disease. In this study, based on the structural protein VP2of the AmericanADV-G strain, we acquired the SD sequence with higly conserved B cell antigenepitopes by B-cell epitope prediction and screening conserved sequences of VP2.With the E. coli expression system, we obtained the soluble protein of SD which had ahigh expression and molecular weight is20KDa. After the Nickel column purificationand ultrafiltration concentrated, the high purity and high concentration of SD proteinwas obtained. Western Blot indicated that the SD protein had good immuneoreactivity.When SD protein was used as the detect antigen in CIEP, its test result was similar tothe result determined by the commercial ones.To develop the indirect ELISA assay,the SD protein and the APTES were coated.According to the phalanx titration method to optimize the reaction conditions, thecoated concentration of SD protein and APTES were determined as10ug/mL and1%,respectively, the dilution of primary antibody was1:100, the dilution of secondaryantibody was1:3000, and the coated time and the blocked time were shortened to30minutes which are shorter than those of traditional indirect ELISA. The specific testsindicated that the antigen protein could only be recognized by the anti-ADV antibody.The sensitivity tests showed that the sensitivity of the established method was higherthan that of CIEP. The repeatability test showed that the coefficient of variation of intra-assay and inter-assay were0.612%~5.956%and0.135%~7.614%,respectively, less than10%. The coincidence rate between CIEP and our method was93.2%in detection of the clinical samples.In summary, the artificial antigen protein of SD acquired in this study had a goodimmuneoreactivity and can be used for establishing detection method. Based on theSD protein, indirect ELISA method had the characteristics of good specificity, highsensitivity, good repeatability and it was convenient to operate. Besides, it also havehigher coincidence rate compare with CIEP. In this study we not only provided a moresuitable genetically engineered antigen protein for the common detection method ofCIEP, but also provided the basis for developing the Aleutian Mink Disease ELISAkits and automated ELISA assay.