Laios E, Ioannou P C, Christopoulos T K
Department of Chemistry and Biochemistry, University of Windsor, Ontario, Canada.
Anal Chem. 2001 Feb 1;73(3):689-92. doi: 10.1021/ac0004815.
The sensitivity of aequorin-based bioluminometric hybridization assays was enhanced by introducing, enzymically, multiple aequorin labels per DNA hybrid. The target DNA was hybridized in microtiter wells with an immobilized capture probe and a digoxigenin-labeled detection probe. The hybrids were reacted with an anti-digoxigenin antibody conjugated to horseradish peroxidase. Peroxidase catalyzed the oxidation of digoxigenin-tyramine by hydrogen peroxide, resulting in the attachment of multiple digoxigenin moieties to the solid phase. Aequorin-labeled anti-digoxigenin antibody was then allowed to bind to the immobilized digoxigenins. The bound aequorin was determined by its characteristic Ca2+-triggered bioluminescence. As low as 20 fmol/L (1 amol/ well) target DNA was detected with a signal-to-background ratio of 2.7. A hybridization assay that used only aequorin-labeled anti-digoxigenin antibody without the peroxidase amplification step gave a signal-to-background ratio of 2 for 160 fmol/L target DNA. The signal enhancement of the amplified assay was in the range of 14-38 times. The analytical range of the amplified assay extended up to 2600 fmol/L. The CVs were in the range of 5.5-7.3%.
通过酶促反应在每个DNA杂交体上引入多个水母发光蛋白标签，增强了基于水母发光蛋白的生物发光杂交检测的灵敏度。将靶DNA在微量滴定孔中与固定化的捕获探针和地高辛配基标记的检测探针进行杂交。杂交体与与辣根过氧化物酶偶联的抗地高辛配基抗体反应。过氧化物酶催化过氧化氢氧化地高辛-酪胺，导致多个地高辛部分附着到固相上。然后使水母发光蛋白标记的抗地高辛配基抗体与固定化的地高辛配基结合。通过其特征性的Ca2+触发生物发光来测定结合的水母发光蛋白。检测到低至20 fmol/L（1 amol/孔）的靶DNA，信噪比为2.7。仅使用水母发光蛋白标记的抗地高辛配基抗体而没有过氧化物酶扩增步骤的杂交检测，对于160 fmol/L的靶DNA，信噪比为2。扩增检测的信号增强在14-38倍范围内。扩增检测的分析范围扩展到2600 fmol/L。变异系数在5.5-7.3%范围内。
