PubMed Health⌕ Search
Home/Archive/PubMed

PubMed · 8633774

Expression immunoassay. Antigen quantitation using antibodies labeled with enzyme-coding DNA fragments.

T K Christopoulos·N H Chiu
View original source ↗Export metadata ↓

Abstract

A novel immunoassay is reported which uses an enzyme-coding 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 O2 and ATP. As few as 3000 molecules of DNA label can be detected. Also, 50,000 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.

Explore related subjects

AntigensDNAImmunoassayLuciferasesProtein BiosynthesisTranscription, GeneticJournal ArticleResearch Support, Non-U.S. Gov't

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

T K Christopoulos, N H Chiu. 1995-12-01. Expression immunoassay. Antigen quantitation using antibodies labeled with enzyme-coding DNA fragments.. https://doi.org/10.1021/ac00119a014

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2007

Multianalyte immunoassay based on insulating-controllable PoPD film at arrayed electrodes integrated on a silicon chip.

A novel, simple and label-free multianalyte immunoassay system is presented here by integrating arrayed electrodes on a silicon chip via MEMS. The chip is consisted of six Au disk electrodes, an Au counter electrode and an Ag/AgCl reference electrode. Semi-insulating poly(o-phenylenediamine) (PoPD) was utilized to co-polymerize and immobilize antibodies at the arrayed Au electrodes, and wider linear detection range was obtained than those prepared with completely insulating PoPD. Electrochemical cyclic voltammogram (CV), AC impedance spectroscopy, AFM and fluorescence microscopy were employed to characterize the system. The arrayed electrodes offered exact control of deposition position via electrochemical operation, allowing selectively immobilization of different antibodies at desired positions on a single chip. Specific recognition of antibody (Ab) to corresponding antigen (An) was quantitatively monitored by cyclic voltammograms in the presence of electrochemical redox probe, ferrocene methanol. The proposed immunoassay chips showed sensitive response to three liver fibrosis markers, hyaluronic acid (HA), collagen type IV (IV-C) and lamin (LN) at ng/mL level simultaneously and specifically in a tiny amount of volume, usually 50 microL. The results obtained via chips were well consistent with those obtained by commercial radio immunoassays (RIA).

Antigens↗
PubMed2007

The fibrinogen antigenic turbidimetric assay (FIATA): the X2x test--the corrected chi-square comparison against the control-mean.

Vancomycin precipitates fibrinogen. The turbidity induced by this vancomycin-fibrinogen interaction is used to establish a simple standardized antigenic assay for plasmatic fibrinogen, the FIATA. 1 mM vancomycin or 2 mM chloramine-T inactivates 50% of fibrinogen in human plasma. In contrast to chloramine-T, vancomycin does not react in NaJ-based photometric assay for chloramines,vancomycin does not inactivate the singlet oxygen-sensible antithrombin III, and the vancomycin action against fibrinogen is not changed in spite of the presence of the 1O2 quenchers methionine or ascorbic acid. The FIATA is performed as follows: to 25 microL plasma 50 microL PBS are added and the absorbance (A) at 405 nm is read. Then 50 microL FIATA-reagent, consisting of 4.4 mM vancomycin in PBS, are added. After 2 minutes (RT) DeltaA is determined and standardized against a plasma pool of 100% of norm (2.8 g/L) fibrinogen. The FIATA is nearly linear up to a fibrinogen concentration of about 150% of norm (4.2 g/L), resulting in a DeltaA of about 600 mA. The lower detection limit is 4% of norm (0.1 g/L). The intra-assay and interessay CV values are < 4%. The normal range of FIATA is 100% +/-20% (x- +/- 1 SD). In = 321 or 344 unselected patient plasmas the FIATA (x- = 130%; SD = 52% or 43%) correlated with the functional fibrinogen assays a) modified Clauss-Method (x- = 4.1 g/L; SD =1.7 g/L) with r = 0.755 and b) FIFTA (x- = 124%; SD = 40%) with r = 0.813. The vancomycin/fibrinogen interaction (binding of about 16 molecules of vancomycin/molecule of fibrinogen) can be used to purify fibrinogen out of plasma. Vancomycin also clouds dysfunctional fibrinogen (fibrinogen in presence of EDTA or chloramine-T)or soluble fibrin. Vancomycin-reacted fibrinogen stimulates tissue type plasminogen activator (t-PA) up to about 20-fold. The experimental data are analyzed by a new significance test: the two foldYates-corrected chi-square comparison against the mean value ofthe control-collective, called the Chi2x - Test. The P < .05 significance barrier calculated with the Chi2x - Test is equivalent to that calculated with the Fisher's Exact Test. The FIATA might be considered an interesting screening test for inactive fibrinogen forms or soluble fibrin, as eg in disseminated intravascular coagulation. Fibrinogen precipitation by vancomycin within the blood vessel might explain why vancomycin has to be infused slowly (< 10 mg/min) to prevent nephrotoxicity. The FIATA is of such a simplicity that the determination of fibrinogen antigen in plasma can be performed anywhere--even outside a hospital--within seconds. Thus, the presented FIATA might contribute to extra hospital testing of patients for assessing their risk for myocardial or cerebral ischemia/infarction.

Antigens↗