Christopoulos T K, Chiu N H
Department of Chemistry and Biochemistry, University of Windsor, Ontario, Canada.
Anal Chem. 1995 Dec 1;67(23):4290-4. doi: 10.1021/ac00119a014.
A novel immunoassay is reported which uses an enzyme-coding DNA fragment as label (expression immunoassay). The DNA label is determined with high sensitivity by measuring the enzymatic activity produced after expression. A DNA fragment encoding the firefly luciferase is biotinylated and complexed with streptavidin. Biotinylated, specific antibodies are used for quantitation of antigen immobilized on microtiter wells. After completion of the immunoreaction, streptavidin-DNA is bound to the immunocomplex. Subsequent expression of the solid phase-bound DNA, by an one-step (coupled) cell-free transcription/translation, produces luciferase. The enzyme catalyzes the luminescent reaction of luciferin with O2 and ATP. As few as 3000 molecules of DNA label can be detected. Also, 50,000 antigen molecules can be detected, and the luminescence is a linear function of the number of antigen molecules in a range extending over 3 orders of magnitude. The high sensitivity achieved is a result of the combined amplification due to transcription/translation and the substrate turnover.
报道了一种新型免疫测定法，该方法使用编码酶的DNA片段作为标记（表达免疫测定法）。通过测量表达后产生的酶活性，可高灵敏度地测定DNA标记。编码萤火虫荧光素酶的DNA片段经生物素化后与链霉亲和素复合。生物素化的特异性抗体用于定量固定在微量滴定板孔上的抗原。免疫反应完成后，链霉亲和素-DNA与免疫复合物结合。随后，通过一步（偶联）无细胞转录/翻译对固相结合的DNA进行表达，产生荧光素酶。该酶催化荧光素与氧气和ATP的发光反应。可检测到低至3000个DNA标记分子。此外，可检测到50000个抗原分子，并且在超过3个数量级的范围内，发光是抗原分子数量的线性函数。所实现的高灵敏度是转录/翻译和底物周转导致的联合扩增的结果。
