T7 RNA polymerase as a self-replicating label for antigen quantification.
Enzymes are used widely as labels in binding assays for protein analytes, because they provide signal amplification. Efforts at improving the assay sensitivity have been focused mainly on the synthesis of novel substrates, e.g. fluorogenic and chemiluminogenic ones. We report the investigation of T7 RNA polymerase (T7RP) as a label with unique characteristics for antigen quantification. In an in vitro, coupled (one-step) transcription/translation reaction, T7RP catalyzes the expression of an enzyme-coding DNA template to produce free enzyme (luciferase) in solution. We demonstrate that the generated luciferase is linearly related to the input T7RP in a range covering over four orders of magnitude. It is also shown that T7RP exhibits a significant level of self-replication (100-fold) in vitro by acting on a DNA template comprising the T7RP cDNA downstream of a T7 promoter. By combining the self-replication reaction with the expression of luciferase DNA, as low as 1400 T7RP molecules are detectable. Furthermore, the T7RP is biotinylated, complexed with streptavidin and used for antigen quantification in a microtiter well-based assay with high sensitivity and reproducibility.
Tannous
Bakhos A
BA
Department of Chemistry and Biochemistry, University of Windsor, Ontario N9B 3P4, Canada.
Laios
Eleftheria
E
Christopoulos
Theodore K
TK
D019921:Biotinylation; D004261:DNA Replication; D004279:DNA, Viral / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007118:Immunoassay / Q000379:methods; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D017430:Prostate-Specific Antigen / Q000276:immunology / Q000378:metabolism*; D015203:Reproducibility of Results; D019809:Streptavidin / Q000737:chemistry; D014764:Viral Proteins
D004279:DNA, Viral; D014764:Viral Proteins; D019809:Streptavidin; D008156:Luciferases; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D017430:Prostate-Specific Antigen