Guesdon J L, Bouges Bocquet B, Débarbouillé M, Hofnung M
J Immunol Methods. 1985 Nov 28;84(1-2):53-63. doi: 10.1016/0022-1759(85)90414-4.
We describe an immunological method which allows the in situ colorimetric detection of translated DNA fragments in bacteria. In the absence of lysis only cell surface proteins are detected. For cytoplasmic proteins, lysis is required. The procedure comprises the following steps: bacteria are lysed, the proteins are transferred onto a disc of nitrocellulose sheet, the remaining protein sites are blocked, the disc is successively soaked in a solution of antibodies specific for the protein to be detected and in a solution of peroxidase-labelled anti-IgG antibody solution. Finally, the immune complexes are made visible by enzyme substrate incubation. We describe the application of this method to the detection of the LamB protein, the LacZ protein, and a LamB-polio VP1 chimera translated from cloned DNA fragment in E. coli.
我们描述了一种免疫学方法，该方法可对细菌中翻译的DNA片段进行原位比色检测。在不进行裂解的情况下，仅能检测到细胞表面蛋白。对于细胞质蛋白，则需要进行裂解。该程序包括以下步骤：裂解细菌，将蛋白质转移到硝酸纤维素薄片圆盘上，封闭剩余的蛋白质位点，将圆盘依次浸泡在针对待检测蛋白质的抗体溶液和过氧化物酶标记的抗IgG抗体溶液中。最后，通过酶底物孵育使免疫复合物可见。我们描述了该方法在检测大肠杆菌中从克隆DNA片段翻译而来的LamB蛋白、LacZ蛋白以及LamB-脊髓灰质炎病毒VP1嵌合体中的应用。
