Two-site expression immunoassay using a firefly luciferase-coding DNA label.
BACKGROUND
We report the first two-site, "sandwich type" expression immunoassay using as a label an expressible DNA fragment encoding firefly luciferase.


METHODS
The DNA label consisted of a T7 RNA polymerase promoter, a firefly luciferase-coding sequence, and a poly(dA/dT) tail. The 3' end of the DNA label was biotinylated and complexed with streptavidin. A sandwich immunoassay for prostate-specific antigen (PSA) was developed in which the antigen was first bound to an immobilized monoclonal antibody and then reacted with a biotinylated polyclonal antibody. The streptavidin-luciferase-coding DNA complex was then bound to the immunocomplex. The DNA label was subsequently expressed in vitro by coupled transcription and translation. The generated luciferase was measured by its characteristic bioluminescent reaction.


RESULTS
The bioluminescence was linearly related to the concentration of PSA in the sample. As low as 30 ng/L PSA was measured (12.5-microL sample) with a signal-to-background ratio of 2.3, and the linear range extended to 3 microg/L. The results obtained from the proposed assay agreed well to those determined by IMx immunoassay (y = 0.98x + 0.74 microg/L; r = 0.971; n = 44).


CONCLUSIONS
The use of the newly developed DNA label in a two-site immunoassay was demonstrated for the first time. The assay was applied successfully to the measurement of serum PSA.
Chiu
N H
NH
Department of Chemistry and Biochemistry, University of Windsor, 401 Sunset Ave., Windsor, Ontario, N9B 3P4 Canada.
Christopoulos
T K
TK
D000818:Animals; D001517:Coleoptera / Q000737:chemistry*; D004247:DNA / Q000096:biosynthesis / Q000737:chemistry*; D049454:Firefly Luciferin; D006801:Humans; D007118:Immunoassay / Q000379:methods; D008156:Luciferases / Q000737:chemistry / Q000235:genetics*; D008163:Luminescent Measurements; D017430:Prostate-Specific Antigen / Q000097:blood* / Q000737:chemistry; D014176:Protein Biosynthesis; D012680:Sensitivity and Specificity; D014158:Transcription, Genetic
D049454:Firefly Luciferin; D004247:DNA; D008156:Luciferases; D017430:Prostate-Specific Antigen