title: Immunochemical assay for deoxyribonuclease activity in body fluids.
abstract: 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.
authors.affiliation: Institute of Chemical Biology and Fundamental Medicine, Siberian Division of the Russian Academy of Sciences, 8, Lavrentiev Ave., Novosibirsk, 630090, Russia.
authors.forename: Anna
authors.initials: A
authors.lastname: Cherepanova
authors.forename: Svetlana
authors.initials: S
authors.lastname: Tamkovich
authors.forename: Dmitrii
authors.initials: D
authors.lastname: Pyshnyi
authors.forename: Maria
authors.initials: M
authors.lastname: Kharkova
authors.forename: Valentin
authors.initials: V
authors.lastname: Vlassov
authors.forename: Pavel
authors.initials: P
authors.lastname: Laktionov
mesh_terms: D000199:Actins / Q000494:pharmacology; D001360:Avidin / Q000737:chemistry; D019921:Biotinylation; D001826:Body Fluids / Q000201:enzymology*; D002138:Calibration; D002384:Catalysis / Q000187:drug effects; D045744:Cell Line, Tumor; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000097:blood / Q000378:metabolism / Q000652:urine; D003851:Deoxyribonucleases / Q000097:blood* / Q000378:metabolism / Q000652:urine*; D004492:Edetic Acid / Q000494:pharmacology; D004706:Endodeoxyribonucleases / Q000097:blood / Q000378:metabolism / Q000652:urine; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D019793:Fluorescein / Q000737:chemistry; D006735:Horseradish Peroxidase / Q000737:chemistry; D006801:Humans; D007118:Immunoassay / Q000379:methods; D007120:Immunochemistry; D008297:Male; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012337:RNA, Ribosomal, 18S / Q000235:genetics
chemical_list: D000199:Actins; D009838:Oligodeoxyribonucleotides; D012337:RNA, Ribosomal, 18S; D001360:Avidin; D004247:DNA; D004492:Edetic Acid; D006735:Horseradish Peroxidase; D003851:Deoxyribonucleases; D004706:Endodeoxyribonucleases; D003850:Deoxyribonuclease I; C019329:deoxyribonuclease II; D019793:Fluorescein