The Preparation And Preliminary Application Of Hemagglutination Inhibition Antigen For Infectious Bronchitis Virus By Ultrafiltration System

Hemagglutination Inhibition(HI)test of infectious bronchitis virus(IBV)is simple and fast,and it is suitable for rapid diagnosis of infectious bronchitis(IB).At present,there is no reliable commercial IBV HI antigens in China,only expensive imported antigens can be purchased from abroad.The studies have shown that IBV can be condensed and treated with PLCI to obtain hemagglutination antigen.And differential centrifugation is the main method of concentration,but supercentrifuge is expensive,which is not conducive to mass production.This study aims to prepare HI antigen by IBV ultrafiltration system and differential centrifugation,and screen suitable concentration of PLCI or Clostridium welchii to treat the antigen,in order to obtain a high-quality and low-cost preparation method,so that it can be prepared in general laboratory,and lay a foundation for wider clinical application.Firstly,by comparing the concentrated antigens of 100 kd and 300 kd pore size ultrafiltration tubes,using PLCI or Clostridium welchii for hemagglutination treatment,and comparing with the IBV HI antigens prepared by differential centrifugation and Dutch GD antigens,the optimum IBV HI treatment method was selected and preserved by freeze-drying.Then,the pilot-scale IBV HI antigens were prepared by ultrafiltration system and compared with Dutch GD antigens,their specificity,sensitivity and coincidence rate were analyzed.Finally,the IBV HI antigen prepared by ultrafiltration system and IDEXX-IBV kit were used to detect the same batch of clinical serum samples,and the coincidence rate was compared.The results showed that all the IBV HA titer were 1:512 after IBV antigens of 100 kd,300 kd and differential concentration were treated with 1 mL/mL PLCI + Clostridium welchii mixed solution and 3 U/mL PLCI,respectively.After 1‰ formaldehyde inactivation and freeze-drying,the storage period was 8 months at 4? and 12 months at-20?.Three batches of pilot-scale IBV were concentrated by 100 kd membrane and one batch by 300 kd hollow fiber column,with 100 times of hemagglutination.The sensitivity and specificity of three batches of IBV antigen,which were concentrated by 100 kd membrane and 100 times by 300 kd hollow fiber column,and treated by hemagglutination,were not different from those of the commercial antigens produced by GD Company of the Netherlands,and the coincidence rate was 96%.Compared to IDEXX-IBV kit,the coincidence rate of the same batch of clinical serum samples was 95.3%.In a word,the method of HIV antigen prepared by concentration of ultrafiltration system is simple,sensitive and specific.It has high coincidence with the results of Dutch GD antigen and IDEXX-IBV kit.It is suitable for poultry farm monitoring and IB vaccine immune effect detection.