EXPRESSION IMMUNOASSAY - ANTIGEN QUANTITATION USING ANTIBODIES LABELED WITH ENZYME-CODING DNA FRAGMENTS

A novel immunoassay is reported which uses an enzyme-coding DNA fragme nt as label(expression immunoassay). The DNA label is determined with high sensitivity by measuring the enzymatic activity produced after ex pression, A DNA fragment encoding the firefly luciferase is biotinylat ed 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 t he 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 luciferi n with O-2 and ATP, As few as 3000 molecules of DNA label can be detec ted. Also, 50000 antigen molecules can be detected, and the luminescen ce 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/translati on and the substrate turnover.