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现有报道用斑点免疫结合试验(DIA)筛选单克隆抗体(McAb),其优点是能直接利用未纯化抗原。但在操作上即使用多孔道微量进样器,要筛选几百份上清也极费力,改良DIA 可简化和加快试验进程。简要程序如下:将业经PBS 浸润的硝酸纤维放入加有1.2mg/ml 抗原的塑料袋中,置室温10小时,洗涤后用含5%脱脂牛奶的PBS 封闭1小时,晾干。将该膜覆盖在96孔微板上,用不带头子的多孔道微量进样器垂直对准一排孔压下,使形成硝酸纤维膜圆片落入孔中,如此每孔均装一圆片(对照孔用无特异抗原的硝酸纤维膜)。检测时每二个复孔各加待筛的细胞培养上清50μl,置室温20小时后,用含5%脱脂牛奶的PBS 洗三
It has been reported in the past that monoclonal antibody (McAb) screening by Dot Immunoassay (DIA) has the advantage of directly utilizing unpurified antigen. However, even using a multichannel microinjector in operation, it is also laborious to screen hundreds of supernatants. Improving the DIA simplifies and speeds up the test. The procedure is as follows: The nitrocellulose infiltrated with PBS was placed in a plastic bag with 1.2 mg / ml antigens and kept for 10 hours at room temperature. After washing, the cells were blocked with 5% skim milk in PBS for 1 hour and allowed to dry. The membrane was covered on a 96-well microplate and vertically aligned with a row of multichannel injectors without a head to depress a row of nitrocellulose membrane discs so that each well contained a circle (Control wells with no specific antigen nitrocellulose membrane). Each duplicate test hole when the cell culture supernatant to be screened 50μl, set 20 hours at room temperature, washed with 5% skim milk PBS