Detection of BCR-ABL transcripts from the Philadelphia translocation by hybridization in microtiter wells and time-resolved immunofluorometry.

[摘要] 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%.

[学科分类] 过敏症与临床免疫学