A modified immunochemical assay for the fast detection of DNA damage in human white blood cells.
An immunochemical assay to detect damage in DNA has been modified to a so-called sandwich ELISA. With this assay DNA damages can be detected that give rise to a certain level of single-strandedness in DNA of white blood cells during partial unwinding of cellular DNA under alkaline conditions. The modified method includes the following steps: incubation of alkali-treated whole blood in the wells of microtiter plates precoated with antibody directed against single-stranded DNA (ssDNA), which results in selective binding of ssDNA, and the subsequent detection of bound ssDNA by incubation with anti-ssDNA antibody alkaline phosphatase conjugate. With this method the amount of damage induced by ionizing radiation in DNA in cells of human blood can be detected within 1 h, after doses as low as 0.2 Gy. The precoating of microtiter plates with anti-ssDNA antibody enables the detection of ssDNA fragments directly in alkali-treated blood samples, isolation of the nucleated cells from the blood is not necessary. Because the DNA is released somewhat faster from lymphocytes than from granulocytes upon alkali treatment, it even appeared possible to discriminate between the effect of the radiation on these cell types in the same blood sample. The method is also applicable to other cell types that can be obtained in suspension.
Timmerman
A J
AJ
T.N.O. Nutrition and Food Research Institute, Department of Genetic Toxicology, Rijswijk, The Netherlands.
Mars-Groenendijk
R H
RH
van der Schans
G P
GP
Baan
R A
RA
D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D004247:DNA / Q000528:radiation effects; D004249:DNA Damage; D004260:DNA Repair; D004277:DNA, Single-Stranded / Q000032:analysis* / Q000276:immunology; D004307:Dose-Response Relationship, Radiation; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005720:Gamma Rays; D006801:Humans; D007962:Leukocytes / Q000737:chemistry / Q000276:immunology / Q000473:pathology* / Q000528:radiation effects
D000911:Antibodies, Monoclonal; D004277:DNA, Single-Stranded; D004247:DNA