Bioluminescence hybridization assays using recombinant aequorin. Application to the detection of prostate-specific antigen mRNA.
We developed microtiter well-based bioluminescence hybridization assays using the photoprotein aequorin as a reporter molecule. The target DNA was hybridized simultaneously with a capture probe and a detection probe. The capture probe was immobilized on the wells through digoxigenin/anti-digoxigenin interaction. The detection probe was biotinylated. The hybrids were determined by using aequorin covalently attached to streptavidin or complexes of biotinylated aequorin with streptavidin. The luminescence was then measured in the presence of excess Ca2+. The optimized protocols showed linearity in the range from 5 amol to 10 fmol of target DNA. In combination with reverse transcriptase polymerase chain reaction, the proposed assay was applied to the detection of the mRNA for prostate-specific antigen (PSA). PSA mRNA from a single cell, in the presence of one million cells that do not express PSA, was detected with a signal-to-background ratio of 2.5. Typical CVs obtained were 6%.
Galvan
B
B
Department of Chemistry and Biochemistry, University of Windsor, Ontario, Canada.
Christopoulos
T K
TK
D000331:Aequorin / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry; D001710:Biotin / Q000737:chemistry; D002118:Calcium / Q000737:chemistry; D004247:DNA / Q000032:analysis / Q000096:biosynthesis / Q000737:chemistry; D017931:DNA Primers / Q000737:chemistry / Q000235:genetics; D006801:Humans; D008163:Luminescent Measurements; D008297:Male; D009693:Nucleic Acid Hybridization / Q000379:methods*; D017430:Prostate-Specific Antigen / Q000032:analysis* / Q000737:chemistry; D012333:RNA, Messenger / Q000032:analysis* / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D012680:Sensitivity and Specificity; D019809:Streptavidin; D014407:Tumor Cells, Cultured / Q000276:immunology
D001426:Bacterial Proteins; D017931:DNA Primers; D012333:RNA, Messenger; D011994:Recombinant Proteins; D000331:Aequorin; D001710:Biotin; D004247:DNA; D019809:Streptavidin; D017430:Prostate-Specific Antigen; D002118:Calcium