Analysis of O6-methylguanine-DNA methyltransferase in individual human cells by quantitative immunofluorescence microscopy.
A quantitative assay of immunofluorescence is described that can be performed on individual cells from standard pathologic specimens using fluorescence microscopy. The technique has been applied to measurement of O6-methylguanine-DNA methyltransferase, a DNA repair protein that is a molecular marker for resistance to chloroethylnitrosources used in cancer chemotherapy. The immunofluorescence assay makes use of monoclonal antibodies with specificity for human transferase, fluorescence microscopy with digital imaging, fluorescent bead internal standards, and computerized image analysis. This method is specific for the transferase, produces results correlated with activity measurements, and yields new data about tissue heterogeneity and subcellular localization previously unavailable with standard assay methods.
Belanich
M
M
Applied Genetics Inc., Freeport NY 11520.
Ayi
T C
TC
Li
B F
BF
Kibitel
J T
JT
Grob
D W
DW
Randall
T
T
White
A B
AB
Citron
M L
ML
Yarosh
D B
DB
D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D016650:Fluorescein-5-isothiocyanate; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes; D006801:Humans; D007211:Indoles; D008099:Liver / Q000166:cytology / Q000201:enzymology*; D008780:Methyltransferases / Q000032:analysis* / Q000276:immunology; D008856:Microscopy, Fluorescence / Q000379:methods; D019853:O(6)-Methylguanine-DNA Methyltransferase; D011994:Recombinant Proteins / Q000276:immunology; D012015:Reference Standards; D012867:Skin / Q000166:cytology / Q000201:enzymology*; D014407:Tumor Cells, Cultured / Q000201:enzymology
D000911:Antibodies, Monoclonal; D005456:Fluorescent Dyes; D007211:Indoles; D011994:Recombinant Proteins; C007293:DAPI; D008780:Methyltransferases; D019853:O(6)-Methylguanine-DNA Methyltransferase; D016650:Fluorescein-5-isothiocyanate