Two-Site Expression Immunoassay Using a Firefly Luciferase-coding DNA Label

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

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Bibliographic Details
Published in:Clinical chemistry (Baltimore, Md.) Md.), 1999-11, Vol.45 (11), p.1954-1959
Main Authors: Chiu, Norman H.L, Christopoulos, Theodore K
Format: Article
Language:English
Subjects:
Animals
Biological and medical sciences
Coleoptera - chemistry
DNA - biosynthesis
DNA - chemistry
Firefly Luciferin
Humans
Immunoassay - methods
Investigative techniques, diagnostic techniques (general aspects)
Luciferases - chemistry
Luciferases - genetics
Luminescent Measurements
Medical sciences
Pathology. Cytology. Biochemistry. Spectrometry. Miscellaneous investigative techniques
Prostate-Specific Antigen - blood
Prostate-Specific Antigen - chemistry
Protein Biosynthesis
Sensitivity and Specificity
Transcription, Genetic
Urinary system
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Summary:We report the first two-site, "sandwich type" expression immunoassay using as a label an expressible DNA fragment encoding firefly luciferase. 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. 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). 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.
ISSN:0009-9147
1530-8561
DOI:10.1093/clinchem/45.11.1954