Radioimmunoassay of 0/sup 6/-methyldeoxyguanosine in DNA of cells alkylated in vitro and in vivo

Journal Article · Thu Dec 01 04:00:00 EST 1983 · Carcinogenesis (N.Y.); (United States)

DOI:https://doi.org/10.1093/carcin/4.12.1605· OSTI ID:6834152

Wild, C P; Smart, G; Saffhill, R; Boyle, J M ^[1]

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1. Christie Hospital and Holt Radium Inst., Manchester (UK). Paterson Labs.

Mouse monoclonal and rabbit polyclonal antibodies have been prepared against O/sup 6/-methylguanosine complexed with bovine serum albumin. In radioimmunoassay 50% inhibition of binding of (/sup 3/H)O/sup 6/-methyldeoxyguanosine (O/sup 6/-MedG) was obtained with 0.3 pmol or 0.16 pmol unlabelled O/sup 6/-MedG using monoclonal or polyclonal antibodies having affinity constants of 3.0 x 10/sup 9/ and 5.9 x 10/sup 9/ I mol/sup -1/ respectively. Cross-reactivity with normal nucleosides and other adducts was determined. Sensitivity and reproducibility were improved by chromatographic separation using an Aminex A6 column eluted with 10 mM NH/sub 4/HCO/sub 3/ buffer which allowed quantitation of one molecule O/sup 6/-MedG per 10/sup 7/ molecules dG in 2 mg calf thymus DNA. Values for O/sup 6/-MedG from 14 samples of rat liver and kidney DNA analysed by the method described and by radiochromatography on Sephadex G10 were almost identical (correlation coefficient, 0.98). A modified procedure for the purification of 5-25 ..mu..g DNA from 1-5 x 10/sup 6/ fibroblasts applied to polycarbonate filters was used in providing further validation of the RIA system by measuring the persistence of O/sup 6/-MedG in cell lines of known repair capacity.

• OSTI ID: 6834152
• Journal Information: Carcinogenesis (N.Y.); (United States), Journal Name: Carcinogenesis (N.Y.); (United States) Vol. 4:12; ISSN CRNGD
• Country of Publication: United States
• Language: English

Related Subjects

550601* -- Medicine-- Unsealed Radionuclides in Diagnostics
62 RADIOLOGY AND NUCLEAR MEDICINE
ANIMAL CELLS
ANIMALS
ANTIBODIES
ANTIGEN-ANTIBODY REACTIONS
BODY
CATTLE
CELL CULTURES
CHEMICAL REACTIONS
CONNECTIVE TISSUE CELLS
DIGESTIVE SYSTEM
DNA
DOMESTIC ANIMALS
FIBROBLASTS
GLANDS
HAMSTERS
IN VITRO
ISOTOPE APPLICATIONS
KIDNEYS
LABELLED COMPOUNDS
LIVER
LYMPHATIC SYSTEM
MAMMALS
MAN
METHYLATION
MICE
MONOCLONAL ANTIBODIES
NUCLEIC ACIDS
NUCLEOTIDES
ORGANIC COMPOUNDS
ORGANS
PRIMATES
RADIOASSAY
RADIOIMMUNOASSAY
RATS
RODENTS
RUMINANTS
SOMATIC CELLS
SPECIFICITY
THYMUS
TRACER TECHNIQUES
TRITIUM COMPOUNDS
TUMOR CELLS
VERTEBRATES