Immunochemical assay for deoxyribonuclease activity in body fluids.
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.
Cherepanova
Anna
A
Institute of Chemical Biology and Fundamental Medicine, Siberian Division of the Russian Academy of Sciences, 8, Lavrentiev Ave., Novosibirsk, 630090, Russia.
Tamkovich
Svetlana
S
Pyshnyi
Dmitrii
D
Kharkova
Maria
M
Vlassov
Valentin
V
Laktionov
Pavel
P
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
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