γ-干扰素时间分辨免疫荧光分析方法的建立

Two site Time resolved Immunofluorometric Assay of Human Interferon gamma.

采用生物素 Ｅｕ３ ＋ 标记链亲和素双抗体夹心时间分辨免疫荧光分析技术 （ Ｔ Ｒ Ｉ Ｆ Ｍ Ａ） ， 建立新的γ干扰素检测技术， 提高γ干扰素检测方法的灵敏度． 用固相包被的兔多抗捕获样品中 Ｉ Ｆ Ｎγ， 以具有中和 Ｉ Ｆ Ｎγ抗病毒活性的生物素化单抗作为二抗体， 再加 Ｅｕ３ ＋ 标记链亲和素并荧光检测． 已知不同浓度标准 Ｉ Ｆ Ｎγ Ｃ Ｐ Ｓ 值的标准曲线， 判断待检样品中 Ｉ Ｆ Ｎγ量． 本方法最低检测值为００２ μｇ／ Ｌ， 检测范围为００２ ～４００ μｇ／ Ｌ， 而 Ｔ Ｎ Ｆα， Ｉ Ｌ２ 和 Ｉ Ｆ Ｎα等细胞因子无交叉反应． 对基因工程 Ｉ Ｆ Ｎγ的生产， 纯化过程中定性， 定量监控以及对培养细胞上清中 Ｉ Ｆ

To establish a highly sensitive time resolved immunofluorometric assay of human interferon gamma . A two site “Sandwich” type time resolved immunoflourometric assay for human interferon gamma is described. It is based on usage of specific polyclonal and monoclonal antibodies. The polyclonal antibody to bind the interferon gamma in samples are immoblailized on the surface of microtiter plate strip wells.Following capture of IFN γ present in sample. Then monoclonal antibody, which is biotinylated ,is added to wells.The second biotin labeled antibody will allow the subsequent specific binding of Eu 3+ labeled streptavidin. After the immunoreactions completed, the bound fraction of Eu 3+ label is quantified by dissociating it in a fluorescence enhancement solution and measuring its fluorescence with Wallac 1234 fluorometer. The sensitivity of the assay is 0 02 μg/L. The standard curve is linear from 0 02 μg/L to 400 μg/L. The time resolved immunofluorometric IFN γ assay is quick, sensitive and suitable for testing large numbers of samples, and may be useful in both industrial producing and clinical studies.