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Time-resolved fluoroimmunoassay of 5-methyl-2′-deoxycytidine

Authors
Journal
Analytical Biochemistry
0003-2697
Publisher
Elsevier
Publication Date
Volume
154
Issue
2
Identifiers
DOI: 10.1016/0003-2697(86)90011-4
Keywords
  • Dna Methylation
  • 5-Methyl-2′-Deoxycytidine
  • Time-Resolved Fluoroimmunoassay
  • Modified Nucleosides

Abstract

Abstract We describe time-resolved fluoroimmunoassay of 5-methyl-2′-deoxycytidine (5MedCyd). The assay is based on the use of a highly specific antiserum raised in rabbits against BSA-conjugated 5-methylcytidine (5MeCyd). The tracer in the solid-phase time-resolved fluoroimmunoassay (TR-FIA) was antigen-selected anti-5MedCyd labeled with Europium. Thyroglobulin-linked 5MeCyd served as the solid-phase antigen. The measuring range for the fluoroimmunoassay was from less than 1 to 5000 pmol per assay of 5MedCyd. A good correlation between the results obtained with the TR-FIA and HPLC was demonstrated when the methods were applied to the measurement of methylation in human leukemic cells and other DNA samples. TR-FIA has several advantages over the more laborious techniques available so far: (i) high sensitivity, (ii) large assay ranges, (iii) rapidity and large number of simultaneous assays, (iv) simplicity, and (v) low cost provided that the laboratory has equipment for time-resolved fluorometry.

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