| Avain infectious bronchitis (IB) is a highly contagious, acute infectious disease caused by avain infectious bronchitis virus (IBV). This disease is widespread around the word. It causes respiratory in broilers, such as cough, sneeze and tracheal sound, and reduces egg production, quality variation in layers and breeders. The humoral immunity plays a very important role in the prevention and treatment of IB. The test of IBV antibody level is a crucial evidence to judge the quality of vaccine antibody level, however the indirect ELIS A is still the best for mass tests. This experiment discussed about the indirect ELISA for IBV for IBV antibody test through homemade HRP labelled antibody and laid the foundation of clinical application of the approach.In this experiment, chicken IgG Whole serum was purified by ammonium sulfate precipitation method, DEAE-52Cellulose ion exchange chromatography, and Sephadex G200 gel filtration chromatography. The 65KD and 25KD protein of the IgG were showed in the SDS-PGE electrophoretogram which proved the purified IgG reached a high purity. The the concentration of purified and concentrated chicken IgG was 3.8mg/mL by the ultraviolet detector assay. The rabbit anti-children IgG reached 1:32 titer. The rabbit anti-children hyper-immune serum was purifed in the same way. HRP labelled rabbit anti-chicken IgG was prepared by sodium periodate oxidation method, the working concentration was 0.25μg/mL. Conditions were optimized for using these preparations for an IBV indirect ELISA. The virion coating concentration was 18.5μg/mL. The best coating condition was 37℃ 2h or 4℃ stay overnight. The best block liquid was 5% skim milk powder and best sealing time was 2h at the temperature of 37℃. The best serum diluent was 0.1% BSA. The best the dilution of serum sample was 1:400 and the best reaction condition with coating antigen was 37℃ 1h. The best working concentration of IgG-HRP was 0.25μg/mL and the best reaction condition with serum sample was 37℃ 1h.The substrate for ELIS A was incubated at 37℃ for 15 min before reading the According to the ELIS A method this research used, the the threshold value of negative and positive serum was 0.133. The known IBV CK/CH/SCEM/09I, CK/CH/SCMS/10I,4/91, 28/86, W93, CK/CH/SCMY/10I, CK/CH/SCYA/10I, Ma5, H120, M41 strain’s antiserum, which were tested by this method were all positive. There was no cross reaction with known SPF chicken IgG, AIV (H5) antiserum and AIV (H9) antiserum, which proved the indirect ELIS A has good specificity. The result of the re-productivity test showed that the coefficient CV was between 4.2%-7.0% and among batch CV was between 6.6%-7.4%. Both of them were under 10%, which meant the repeatability of this method was good. The minimum detection amount of anti-Sczy3 IgG was 1.359μg/mL.22 IBV positive antiserum and 8 IBV negative antiserum were done the parallel tested by this method and IDEXX kit. The coincidence rate was 93.3%. In the environment of 4℃, the coating reaction plate, HRP labelled antibody, positive serum, negative serum and other reagent could last 12 month.This experiment established a method to use sulfate precipitation DEAE-52Cellulose ion exchange chromatography-Sephadex G200 gel chromatography to purify chicken and rabbit IgG serum and laid a foundation for massive purification of chicken and rabbit IgG serum. The use of homemade HRP labelled antibody to establish the indirect ELISA for the test of IBV antibody had clinical application prospect and enriched the methods to test IBV antibody. |
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