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Immuno-slot-blot: A highly sensitive immunoassay for the quantitation of carcinogen-modified nucleosides in DNA

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Summary

We have established a highly sensitive immuno-slot-blot (ISB) procedure that can be routinely applied for detection and quantitation of any heat- or alkali-stable structural DNA modification (caused by carcinogens or mutagens, for example) for which a specific (monoclonal) antibody (MAB) is available. The essential step in this assay is the immobilization on nitrocellulose filters of the structurally modified DNA in its single-stranded form. The immobilized DNA is first reacted with an MAB specifically directed against a particular modified DNA component (e.g., an alkyldeoxynucleoside), and thereafter with a second antibody directed against the first one. The second antibody can be either labeled with 125I or linked to an enzyme complex capable of eliciting a color reaction with a suitable substrate. The sensitivity of the ISB is demonstrated for two different alkyldeoxynucleosides, O6-ethyldeoxyguanosine (O6-EtdGuo) and O4-ethyldeoxythymidine (O4-EtdThd), both of which are produced in cellular DNA exposed to the alkylating N-nitroso carcinogen N-ethyl-N-nitrosourea and both of which represent DNA lesions miscoding during DNA replication and transcription. Using anti-(O6-EtdGuo) and anti-(O4-EtdThd) MABs, respectively, O6-EtdGuo and O4-EtdThd are detected at levels as low as ≧0.3×10-15mol O6-EtdGuo/3 μg DNA (O6-EtdGuo/deoxyguanosine molar ratio in DNA, ≧2×10-7) and ≧0.1×10-15 mol of O4-EtdThd/3 μg DNA (O4-EtdThd/deoxythymidine molar ratio in DNA, ≧4×10-8).

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Abbreviations

dGuo :

2′-deoxyguanosine

dThd :

2′-deoxythymidine

EDTA :

ethylenediamine tetraacetic acid

EtNU :

N-ethyl-N-nitrosourea

fmol :

femtomol (10-15 mol)

Ig :

immunoglobulin

IgG :

immunoglobulin class G

ISB :

immuno-slot-blot

K A :

affinity constant

MAB :

monoclonal antibody

NC :

nitrocellulose

O 6-EtdGuo:

O6-ethyl-2′-deoxyguanosine

O 4-EtdThd:

O4-ethyl-2′-deoxythymidine

PBS :

phosphate-buffered saline according to Dulbecco and Vogt (J Exp Med 99:167; 1954)

PBS-NaCl :

PBS containing additional 0.16 M NaCl

pmol :

picomol (10-12 mol)

RIA :

radioimmuno-assay

SSC :

standard saline citrate (0.15 M NaCl, 0.015 M tri-sodium citrate)

ssDNA :

single-stranded DNA

TE-buffer :

10 mM Tris-HCl, 1 mM EDTA; pH 7.2

Tris-NaCl-buffer :

0.1 M Tris-HCl, pH 7.5, 1 M NaCl, 2 mM MgCl2, 0.05% Triton X-100

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Authors and Affiliations

  1. Institut für Zellbiologie (Tumorforschung), Universität Essen (GH), Hufelandstrasse 55, D-4300, Essen 1, Federal Republic of Germany

    P. Nehls, J. Adamkiewicz & M. F. Rajewsky

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  1. P. Nehls
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  2. J. Adamkiewicz
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  3. M. F. Rajewsky
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Dedicated to Professor Hermann Druckrey on the occasion of his 80th birthday

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Nehls, P., Adamkiewicz, J. & Rajewsky, M.F. Immuno-slot-blot: A highly sensitive immunoassay for the quantitation of carcinogen-modified nucleosides in DNA. J Cancer Res Clin Oncol 108, 23–29 (1984). https://doi.org/10.1007/BF00390969

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  • DOI: https://doi.org/10.1007/BF00390969

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