| An indirect enzyme-linked immunosorbent assay (ELISA) was standardized and an ELISA test kit was developed for mornitoring antibodies against infectious bursal disease virus (IBDV).Several measures of purifying immunoglobulin G (IgG) were compared, which included ammonium sulfate precipitation, caprylic acid-ammonium sulfate precipitation, hydrochloric acid-ammonium sulfate precipitation, acetic acid-ammonium sulfate precipitation and ammonium sulfate precipitation-caprylic acid. The results of sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and protein concentration analysis demonstnated that the purity and protein content of IgG prepared by the method of ammonium sulfate precipitation-caprylic acid were higher than others. Prepared anti-IBDV rabbit IgG and anti-chicken rabbit IgG were conjugated with horseradish peroxidase (HRP) by the reformative sodium periodate method and achieved preferable purpose. Their work concentrations were 1:1000 and 1:12000 respectively.Application of the MTT colorimetric method on IBDV propagated in cell culture was investigated,vvhich revealed that the method was practicable for detecting IBDV passaged in CEF for the first time.The D78 strain and Hb strain of IBDV multiplicated in three host systems (vero cells , chicken embryo fibroblast (CEF) cells and chicken embryo) were investigated. The results showed that the period of IBDV propagated in vero cells was longer than in CEF cells (generally four to five days). However, the titration and protein concentration of IBDV in vero cells was higher than that in CEF cells. SO we selected heterogenous vero cells for replicating IBDV abundantly and adopted different methods to purify them. The results suggested that filtration chroraatograpliy method could purify antigen partly, but it was trivial, wasted time and manpower. While the method with chloroform distill and polyethylene glycol (6000) precipitation was simple and had good potential for the extend value.The optimum work concentration of the indirect ELISA was established. Polystyrene42microtitration plates were coated by the purified ELISA antigen at the amount of 5.84g in 100l 0.05M sodium bicarbonate buffer (pH9.6) per well. The coated plates were blocked by 0.5% bovine serum albumin in 0.01M phosphate buffered saline solution with 0.05% Tween-20 and the working concentration of serum samples was 1:100 dilution.All the incubation steps above were done at 37"C for 60min.A linear relationship was existed between the values of positive/negative ratio of serum at 1:100 and the logarithm of ELISA titer (IgET) determined by a standard serum dilution method of 30 field sera. Regression was y=10.1029-3.0461x. The ET of a serum sample could be determined by a single serum dilution by the equation. The samples were judged as positive based on the ELISA data from 32 sera of specific-pathogen-free chickens. An ELISA kit was developed for detection of antibodies against infectious bursal disease (IBD). The reagents' character, storage, quality control were studied and the kit' specificity, sensibility, repeatability, coincidence rate, storage time and comparation with import kit were also investigated detailedly and roundly. The reagents were stable well when storing at -20癈 for six months. The coincidence rate between the kit and the imported kit or agar gel precipitin test (AGP) was 86.7% and 92.5% for the same samples respectively. The kit was simple, convenient , special, sensitive and rapid for monitoring antibodies against EBDV and diagnosing improvisely.
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