Bortolin S, Christopoulos T K
Department of Chemistry and Biochemistry, University of Windsor, Ontario, Canada.
Clin Chem. 1995 May;41(5):693-9.
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%.
已开发出两种杂交检测方法来检测由费城染色体易位产生的BCR-ABL mRNA转录本。两种检测方法均使用时间分辨免疫荧光法检测与特异性探针杂交的聚合酶链反应扩增的BCR-ABL mRNA序列。在配置I中，生物素化的扩增靶标固定在链霉亲和素包被的孔中，并与用半抗原地高辛标记的探针杂交。通过碱性磷酸酶标记的抗体和氟水杨酸磷酸酯作为底物检测杂交体。产生的氟水杨酸与Tb(3+)-EDTA形成高荧光复合物。在配置II中，生物素化的探针固定在链霉亲和素包被的孔中。在半抗原标记的脱氧核糖核苷酸存在下进行的PCR产生标记产物，该产物与固定的探针杂交并如上所述进行定量。在500,000个正常粒细胞的mRNA中，一个白血病细胞的BCR-ABL转录本在配置I和II中分别以高达36.4和24.6的信号/背景比可检测到。两种检测方法各自的变异系数分别为6.6-9.0%和5.1-12.5%。
