Sensitive, colorimetric enzyme amplification cascade for determination of alkaline phosphatase and application of the method to an immunoassay of thyrotropin

Abstract

A highly sensitive flavin adenine dinucleotide-3'-phosphate (FADP)-based enzyme amplification cascade has been developed for determining alkaline phosphatase (ALP; EC 3.1.3.1). The cascade detects ALP via the dephosphorylation of the novel substrate FADP to produce the cofactor FAD, which binds stoichiometrically to inactive apo D-amino acid oxidase (D-AAO). The resulting active holo D-AAO oxidizes D-proline to produce hydrogen peroxide, which is quantified by the horseradish peroxidase-mediated conversion of 3,5-dichloro-2-hydroxybenzenesulfonic acid and 4-aminoantipyrine to a colored product. The FADP-based enzyme amplification cascade has been used in a novel releasable linker immunoassay (RELIA) to quantify thyrotropin (TSH). In the assay, TSH is first captured onto antibody-coated chromium dioxide particles. After formation of an antibody-TSH sandwich with a dethiobiotinylated second antibody, the complex is reacted with a streptavidin-ALP conjugate. Biotin is then used to release the conjugate into solution, and ALP is quantified in an automated version of the FADP-based amplification cascade on the aca discrete clinical analyzer (Du Pont). The sensitivity of the colorimetric RELIA assay for TSH (less than 0.1 milli-int. unit/L) is comparable with that of fluorometric assays. This technology provides a way to adapt to the aca high-sensitivity immunoassays for a wide range of analytes via colorimetric detection.

Enhanced Colorimetric Immunoassay Accompanying with Enzyme Cascade Amplification Strategy for Ultrasensitive Detection of Low-Abundance Protein

“…The first classical approach is to utilize the molecular biology techniques, e.g. by using polymerized enzymes, enzyme-anti-enzyme complexes, liposome-entrapped enzymes, and the biotin-avidin conjugate chemistry 20 21 . This approach usually needs multiple labels or multi-enzyme labels, thus relatively complicated and time-consuming.…”

A fast, sensitive and easy colorimetric assay for chitinase and cellulase activity detection

“…Upon oxidation of these substrates, a stoichiometric amount of H 2 O 2 is produced by reduction of molecular oxygen. The hydrogen peroxide is used by HRP to convert 4-aminoantipyrine (AAP) and 3,5-dichloro-2-hydroxybenzenesulfonic acid (DCHBS) into a pink and stable compound [ 12 ]. As a result, the intensity of the pink color is proportional to the concentration of the available ChitO substrates.…”

A sensitive, accurate, and high-throughput gluco-oligosaccharide oxidase-based HRP colorimetric method for assaying lytic polysaccharide monooxygenase activity

“…The properties of the purified recombinant SsGOOX were studied with cellobiose as the substrate. The HRP colorimetric assay was used to monitor the production of H 2 O 2 , with 4-amino-antipyrine (4-AAP) and 3, 5-dichloro-2-hydroxybenzenesulfonic acid (DCHBS) as chromogenic substrates [23]. Upon the oxidation of cellobiose by SsGOOX, a stoichiometric amount of H 2 O 2 was produced, which was used by HRP to convert 4-AAP and DCHBS into a pink substance with Scheme 1 Schematic diagram of the GOOX-based HRP colorimetric method for assaying AA9 LPMO activity.…”