导　　师： 崔言顺;张秀美

学科专业： I0602

授予学位： 硕士

作　　者： ;

机构地区： 山东农业大学

摘　　要： 该课题对ibd免疫监测的间接elisa方法进行了标准化研究,并制备了相应的诊断试剂盒.在酶标抗体的制备方面,该研究对几种提纯igg的方法（硫酸铵法、盐酸-硫酸铵法、醋酸-硫酸铵法、辛酸-硫酸铵法和硫酸铵-辛酸法）作了比较.sds-page电泳和蛋白含量测定分析表明,辛酸-硫酸铵法和硫酸铵-辛酸法提取的igg纯度均较高,且硫酸铵-辛酸法提取的蛋白含量高于辛酸-硫酸铵法.采用改良过碘酸钠法将提纯的兔抗ibdv igg和兔抗鸡igg进行辣根过氧化物酶（hrp）标记,效果比较理想,二者工作浓度分别为1：1000,1：1200.该研究探讨了mtt比色法用于检测病科中ibdv在细胞培养物中增殖情况的应用.经反复试验,该研究确立了间接elisa检测ibd抗体的最佳反应条件.根据对32份spf鸡阴性血清检测结果的平均值制定了间接elisa判定标准.在此基础上研制出ibd间接elisa快速诊断试剂盒,并对盒内各组分的性状、保存、质量控制以及诊断试剂盒的特异性、敏感性、可重复性、符合率、与国外同类产品的比较、保存期等进行了全面、详细的研究.结果表明研制的试剂盒在-20℃保存至6个月时各项性能都很好.该试剂盒与进口试剂盒对同样血清样品检测,符合率为86.7﹪.间接elisa试剂盒与琼扩试验的符合率为92.5﹪.该试剂盒可对大量血清样品进行检测,操作简单方便、结果特异性强、敏感性高、快速,更适合于大型鸡场ibd免疫抗体水平的监测及现地疫病诊断等需要. 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. Polystyrene 42 microtitration 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.

关 键 词： 酶联免疫吸附试验 诊断试剂盒 鸡 传染性法氏囊病 监测方法 免疫监测

分 类 号： [S858.31]

领　　域： [农业科学] [农业科学] [农业科学]