Cherepanova Anna, Tamkovich Svetlana, Pyshnyi Dmitrii, Kharkova Maria, Vlassov Valentin, Laktionov Pavel
Institute of Chemical Biology and Fundamental Medicine, Siberian Division of the Russian Academy of Sciences, 8, Lavrentiev Ave., Novosibirsk, 630090, Russia.
J Immunol Methods. 2007 Aug 31;325(1-2):96-103. doi: 10.1016/j.jim.2007.06.004. Epub 2007 Jun 27.
We have developed two microtiter plate assays to quantify the deoxyribonuclease activity in biological fluids. Both assays are based on hydrolysis of biotinylated and fluorescein-labeled DNA substrates, with subsequent immunochemical detection of non-digested DNA. The assay based on hydrolysis of 974 bp PCR product labeled with biotinylated forward and fluorescein-labeled reverse primers is more sensitive (0.05 U/ml) and convenient for quantifying the DNase activity in biological fluids than the assay based on hydrolysis of double-labeled 20 bp oligonucleotide. The DNase activity in urine and blood plasma of healthy donors was measured using the PCR product-based assay. Urine samples revealed greater activity, 1.49+/-1.41 U/ml; blood plasma DNase I-like activity was 0.36+/-0.20 U/ml. DNase II-like activity was not detected in the plasma samples. The data obtained confirm that DNase I-like enzymes are responsible for the majority of deoxyribonuclease activity in blood plasma.
我们开发了两种微量滴定板测定法来定量生物体液中的脱氧核糖核酸酶活性。两种测定法均基于生物素化和荧光素标记的DNA底物的水解，随后对未消化的DNA进行免疫化学检测。与基于双标记20bp寡核苷酸水解的测定法相比，基于用生物素化正向引物和荧光素标记反向引物标记的974bp PCR产物水解的测定法更灵敏（0.05 U/ml），且便于定量生物体液中的DNase活性。使用基于PCR产物的测定法测量了健康供体尿液和血浆中的DNase活性。尿液样本显示出更高的活性，为1.49±1.41 U/ml；血浆DNase I样活性为0.36±0.20 U/ml。在血浆样本中未检测到DNase II样活性。获得的数据证实，DNase I样酶是血浆中大部分脱氧核糖核酸酶活性的原因。
