Detection of BCR-ABL transcripts from the Philadelphia translocation by hybridization in microtiter wells and time-resolved immunofluorometry.
Two hybridization assays have been developed to detect BCR-ABL mRNA transcripts arising from the Philadelphia translocation. Both assays use time-resolved immunofluorometric detection of polymerase chain reaction-amplified BCR-ABL mRNA sequences hybridized to specific probes. In configuration I, biotinylated amplified target is immobilized onto streptavidin-coated wells and hybridized to a probe labeled with the hapten digoxigenin. Hybrids are detected via an alkaline phosphatase-labeled antibody and fluorosalicylylphosphate as substrate. The fluorosalicylate produced forms highly fluorescent complexes with Tb(3+)-EDTA. In configuration II, biotinylated probe is immobilized onto streptavidin-coated wells. PCR, performed in the presence of hapten-labeled deoxyribonucleotide, generates labeled product, which is hybridized to immobilized probe and quantified as above. BCR-ABL transcripts from one leukemic cell amidst mRNA from 500,000 normal granulocytes are detectable with signal/background ratios as high as 36.4 and 24.6 for configurations I and II, respectively. The respective CVs for the assays were 6.6-9.0% and 5.1-12.5%.
Bortolin
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Department of Chemistry and Biochemistry, University of Windsor, Ontario, Canada.
Christopoulos
T K
TK
D001483:Base Sequence; D015200:Fluoroimmunoassay; D016044:Fusion Proteins, bcr-abl / Q000235:genetics*; D006801:Humans; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000235:genetics; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D010677:Philadelphia Chromosome; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis*; D014407:Tumor Cells, Cultured
D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D016044:Fusion Proteins, bcr-abl