Expression Immunoassay. Antigen quantitation using antibodies labeled with enzyme-coding DNA fragment.

UNCG Author/Contributor (non-UNCG co-authors, if there are any, appear on document)
Norman H. Chiu, Assistant Professor (Creator)
Institution
The University of North Carolina at Greensboro (UNCG )
Web Site: http://library.uncg.edu/

Abstract: A novel immunoassay is reported which uses an enzymecoding DNA fragment as label (expression immunoassay). The DNA label is determined with high sensitivity by measuring the enzymatic activity produced after expression. A DNA fragment encoding the firefly luciferase is biotinylated and complexed with streptavidin. Biotinylated, specific antibodies are used for quantitation of antigen immobilized on microtiter wells. After completion of the immunoreaction, streptavidin-DNA is bound to the immunocomplex. Subsequent expression of the solid phase-bound DNA, by an one-step (coupled) cell-free transcription/translation, produces luciferase. The enzyme catalyzes the luminescent reaction of luciferin with 02 and ATP. As few as 3000 molecules of DNA label can be detected. Also, 50000 antigen molecules can be detected, and the luminescence is a linear function of the number of antigen molecules in a range extending over 3 orders of magnitude. The high sensitivity achieved is a result of the combined amplification due to transcription/ translation and the substrate turnover.

Additional Information

Publication
Language: English
Date: 1995
Keywords
expression immunoassay, DNA, biochemistry, chemistry, genetics, enzyme-coding, antibodies