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 tr anscripts arising from the Philadelphia translocation. Both assays use time-resolved immunofluorometric detection of polymerase chain reacti on-amplified BCR-ABL mRNA sequences hybridized to specific probes. In configuration I, biotinylated amplified target is immobilized onto str eptavidin-coated wells and hybridized to a probe labeled with the hapt en digoxigenin. Hybrids are detected via an alkaline phosphatase-label ed antibody and fluorosalicylylphosphate as substrate. The fluorosalic ylate produced forms highly fluorescent complexes with Tb3+-EDTA. In c onfiguration II, biotinylated probe is immobilized onto streptavidin-c oated wells. PCR, performed in the presence of hapten-labeled deoxyrib onucleotide, generates labeled product, which is hybridized to immobil ized probe and quantified as above. BCR-ABL transcripts from one leuke mic cell amidst mRNA from 500000 normal granulocytes are detectable wi th signal/background ratios as high as 36.4 and 24.6 for configuration s I and II, respectively. The respective CVs for the assays were 6.6-9 .0% and 5.1-12.5%.