Clinical Chemistry
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Clinical Chemistry 45: 1954-1959, 1999;
This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (10)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chiu, N. H.L.
Right arrow Articles by Christopoulos, T. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chiu, N. H.L.
Right arrow Articles by Christopoulos, T. K.
Related Collections
Right arrow Proteomics and Protein Markers
Right arrow Automation and Analytical Techniques
(Clinical Chemistry. 1999;45:1954-1959.)
© 1999 American Association for Clinical Chemistry, Inc.

Articles

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

Norman H.L. Chiu and Theodore K. Christopoulosa

Department of Chemistry and Biochemistry, University of Windsor, 401 Sunset Ave., Windsor, Ontario, N9B 3P4 Canada.
a Author for correspondence. Fax 519-973-7098; e-mail tkc{at}uwindsor.ca

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-µL sample) with a signal-to-background ratio of 2.3, and the linear range extended to 3 µg/L. The results obtained from the proposed assay agreed well to those determined by IMx immunoassay (y = 0.98x + 0.74 µg/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.




The following articles in journals at HighWire Press have cited this article:


Home page
 Clin. Chem.Home page
Y. Huang, T.-H. Wang, J.-H. Jiang, G.-L. Shen, and R.-Q. Yu
Prostate Specific Antigen Detection Using Microgapped Electrode Array Immunosensor with Enzymatic Silver Deposition
Clin. Chem., May 1, 2009; 55(5): 964 - 971.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
B. A. Tannous, E. Laios, and T. K. Christopoulos
T7 RNA polymerase as a self-replicating label for antigen quantification
Nucleic Acids Res., December 15, 2002; 30(24): e140 - e140.
[Abstract] [Full Text] [PDF]


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Copyright © 1999 by the American Association for Clinical Chemistry.