Preparation And Application Of Latex Agglutination Test For Detection Of Antibody Against Aleutian Mink Disease

Aleutian disease of mink(AD)is a chronic,progressive infectious disease caused by Aleutian disease virus(ADV).The disease can not only lead to a decline in the breeding capacity of the mink population,but also can cause adult mink weight loss,reduced skin quality,bring huge economic losses to the Mink industry,and also seriously hinder the healthy development of China's fur animal husbandry industry.The currently accepted method is to eliminate the disease by testing,so as to achieve the role of decontamination.Detection of ADV-specific antibodies is the best means of diagnose the disease.Convection immunoelectrophoresis(CIEP)is currently recognized as the most commonly used method for detection of ADV serum antibodies.This method is relatively complicated and costly to detect.Limited in clinical applications.Therefore,it is urgent to establish a rapid,accurate,and efficient detection method for the effective diagnosis of the disease.In this study,the antigenic peptide-SD microspheres of mink Aleutian virus was covalently coupled with carboxylated polystyrene microspheres designed and expressed in a series.A microsphere agglutination test was established for the detection of specific antibodies in AD serum.The results showed that the optimal conditions for the preparation of the peptide-SD microsphere complex were as follows: carboxylated polystyrene microspheres with EDC as the activator,activated for 15 min,p H 7.5 MES buffer reaction system,coupling for 30 min.The protein concentration is 0.4 mg/ml.The antigenic peptide-SD microsphere complex was ready to detect the antigen.The method of antibody detection for Aleutian disease agglutination test of mink was established as follows: the tested serum and standard negative,positive serum were diluted 20 ?l was added to the slide respectively.The 20 ?l antigenic peptide-SD microspheres were added to the serum and the standard negative,and the positive serum was mixed to observe the results of 30s~60s.The results showed that the peptide-SD microsphere complex could react strongly with ADV positive serum,but not with pseudorabies virus,canine distemper virus and mink enteritis virus.The weak agglutination reaction between the positive serum diluted at 1: 32 and the antigenic peptide-SD complex showed that the method had high sensitivity,and there was no agglutination reaction between the complex and ADV negative serum,distilled water,MES buffer,etc.and there is no self-coagulation occurs.According to the method outlined in the present experiment,a microsphere agglutination kit for on-site detection of ADV antibodies was initially developed,and 404 serum samples of mink were stored together with CIEP.The coincidence rate is 89.6%.In October 2017,the kit was used to test 373 serum from three mink farms(A,B and C)of Wafangdian and Dalian City during the period of skin-peening.The results showed that the total positive infection rate in the three fields was 67.85%,66.93% and 66.42% respectively.Subsequently,an intermediate trial test was performed on the kit.The results showed that the kit performed well in terms of specificity,sensitivity and reproducibility.In conclusion,the agglutination method of antigenic peptide-SD microspheres and the kit assembled in this study has a short detection time,good sensitivity and specificity,and high coincidence rate with CIEP,and the results do not need complex treatment.It provides a rapid diagnostic method for mink population,which is worth popularizing and can also provide technical guarantee for better prevention and control of AD.