White S R, Chiu N H, Christopoulos T K
Department of Chemistry and Biochemistry, University of Windsor, Ontario.
Analyst. 1998 Jun;123(6):1309-14. doi: 10.1039/a706408j.
An immunoassay is reported which uses, as a label, an expressible DNA fragment encoding the alpha-peptide of beta-galactosidase. This inactive peptide consists of 97 amino acid residues containing an amino-terminal portion of the enzyme. Antigen (an anti-thyrotropin immunoglobulin) immobilized in microtiter wells is allowed to react with specific antibodies which are then linked to the DNA label via biotin-streptavidin interaction. After completion of the immunoreaction, the solid phase bound DNA is subjected to a cell-free, one-step transcription/translation reaction to produce the alpha-peptide. The alpha-peptide is allowed to react (complementation reaction) with the remaining part of the beta-galactosidase (M15 protein, also inactive) to give fully active enzyme molecules. 4-Methylumbelliferyl galactoside is used as a substrate. The fluorescence is linearly related to the amount of antigen in the well. As little as 3 fmol of antigen can be detected. The RSDs (within-run) obtained for 8 and 20 fmol of antigen were 10.7 and 9.3%, respectively (n = 4). The present work illustrates the utility of expressing a non-detectable peptide capable of triggering a signal generating system.
报道了一种免疫测定法，该方法使用编码β-半乳糖苷酶α-肽的可表达DNA片段作为标记。这种无活性肽由97个氨基酸残基组成，包含该酶的氨基末端部分。固定在微量滴定孔中的抗原（抗促甲状腺素免疫球蛋白）与特异性抗体反应，然后通过生物素-链霉亲和素相互作用与DNA标记物相连。免疫反应完成后，对固相结合的DNA进行无细胞一步转录/翻译反应以产生α-肽。使α-肽与β-半乳糖苷酶的其余部分（M15蛋白，也无活性）反应（互补反应），以产生完全有活性的酶分子。使用4-甲基伞形基半乳糖苷作为底物。荧光与孔中抗原的量呈线性关系。可检测到低至3 fmol的抗原。8 fmol和20 fmol抗原的批内相对标准偏差（RSD）分别为10.7%和9.3%（n = 4）。本研究说明了表达一种能够触发信号产生系统的不可检测肽的实用性。
