抗哇巴因抗体IgY的制备及其应用

Preparation and application of anti-ouabain IgY antibody

目的制备高度特异性的抗哇巴因多克隆抗体,并将其应用于组织中内源性哇巴因的免疫学检测。方法采用哇因-牛血清蛋白耦联物作为免疫原免疫母鸡。免疫后1周收集鸡蛋。鸡蛋黄采用PEG-6000方法进行纯化分离,并采12%SDS-PAGE分析其纯度。酶联免疫吸附法检测IgY抗体效价。然后应用IgY的竞争性酶联免疫吸附法检测哇巴水平,并将IgY应用于大鼠肾上腺组织中内源性哇巴因的检测。结果IgY抗体效价在第二次免疫后迅速升高,最高效达到1:10240,并持续至少4周左右。竞争性酶联免疫吸附法检测哇巴因的批内差异及批间差异分别为2.03%、2.34%免疫组化结果显示内源性哇巴因主要分布于大鼠肾上腺皮质的网状带,髓质未见明显阳性颗粒。结论免疫母鸡简单快速,并且高效价抗体持续时间较长。同时本研究结果提示IgY抗体可用于常规免疫学检测。

Objective To prepare highly specific anti-ouabain polyclonal antibody for detecting endogenous ouabain in tissues. Methods Ouabain-BSA compound was used to immunize hens, and the eggs were collected one week after the first immunization. The IgY antibodies in the egg yolk were separated and purified by PEG-6000 Method, and analyzed by 12% SDS-PAGE and enzyme-linked immunosorbent assay （ELISA） for titration. The IgY antibodies obtained were applied subsequently in ELISA and immunohistochemistry. Results The IgY titer increased rapidly after the second immunization, with the highest titer of 1：10240 that lasted for at least 4 weeks. Competitive ELISA for IgY detection showed an average intraassay coefficient of variation （CV） of 2.03% and an inter-assay CV of 2.34%. Immunohistochemistry visualized the location of the endogenous ouabain mainly in the cytoplasm of the zona reticularis of rat adrenal cortex. Condusion Immunization of hens allows efficient preparation of IgY antibody which can be used in routine immunoassays.