Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.3.1 (alkaline phosphatase)
 47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A multianalyte immunoassay concept based on the geometric separation of different analyte-specific antibodies has been demonstrated. The assay and amperometric detection are done in a cell with two working electrodes controlled at the same potential, and the amperometric signal at each electrode is monitored. The distance between any two adjacent electrodes in this prototype is 2.5 mm, and during the course of amperometric measurement, the product formed at one electrode does not reach the other working electrode within 20 min after the addition of enzyme substrate. Thus, the method relies on the spatial resolution between the different antibodies being such that measurements are taken before cross-interference due to diffusion can occur. Identical enzyme labels (alkaline phosphatase, ALP) and substrates (p-aminophenyl phosphate, PAPP) are used for all analytes. Alkaline phosphatase-conjugated rat anti-mouse IgG was immobilized by passive adsorption. Our studies showed that this concept is feasible and can be applied to the simultaneous measurement of multiple analytes.
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PMID:Feasibility studies of simultaneous multianalyte amperometric immunoassay based on spatial resolution. 1069 77

We report on the use of flow-through electrodes fabricated from a composite of superporous agarose (SPA) and reticulated vitreous carbon (RVC) for carrying out sandwich bioassays via a model sandwich assay scheme. The flow-through design of the SPA-RVC electrodes allows for ease in solution handling with the use of micropipettors while allowing sandwich assays to be performed on the SPA matrix inside the RVC. A sandwich bioassay was devised for detecting biotinylated bovine serum albumin (b-BSA) as a proof-of-concept scheme to demonstrate applicability of SPA-RVC electrodes to carry out sandwich assays. In this bioassay scheme, SPA-RVC electrodes with avidin molecules immobilized on the SPA matrix were incubated with low quantities of b-BSA followed by incubation with avidinylated alkaline phosphatase (av-ALP). This construct creates a sandwich bioassay whereby b-BSA is sandwiched between the two avidin complexes. Av-ALP labels captured on the bound b-BSA catalytically hydrolyze conversion of 4-aminophenylphosphate (PAPP) to electrochemically active 4-aminophenol (PAP) which is then voltammetrically detected inside the RVC. The lower concentration detection limit for b-BSA was 0.32+/-0.1 ng mL(-1) and the absolute detection limit was 32+/-10 pg. Non-specific binding of av-ALP enzyme labels onto the avidin-activated SPA-RVC electrodes was low. Catalytic generation of PAP by non-specifically bound av-ALP occurs at a rate less than 2% of that for PAP generation by av-ALP in [(SPA-av)-(b-BSA-b)-(av-ALP)] sandwich constructs.
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PMID:Superporous agarose--reticulated vitreous carbon electrodes for electrochemical sandwich bioassays. 1892 7

Electrochemical assay using HeLa cell lines transfected with various plasmid vectors encoding SEAP (secreted alkaline phosphatase) as the reporter has been performed by using SECM (scanning electrochemical microscopy). The plasmid vector contains different responsive elements that include GRE (glucocorticoid response elements), CRE (cAMP responsive elements), or kappaB (binding site for NFkappaB (nuclear factor kappa B)) upstream of the SEAP sequence. The transfected HeLa cells were patterned on a culture dish in a 4x4 array of circles of diameter 300 microm by using the PDMS (poly(dimethylsiloxane)) stencil technique. The cellular array was first exposed to 100 ng mL(-1) dexamethasone, 10 ng mL(-1) forskolin, or 100 ng mL(-1) TNF-alpha (tumor necrosis factor alpha) after which it was further cultured in an RPMI culture medium for 6 h. After incubation, the cellular array was soaked in a measuring solution containing 4.7 mM PAPP (p-aminophenylphosphate) at pH 9.5, following which electrochemical measurements were performed immediately within 40 min. The SECM method allows parallel evaluation of different cell lines transfected with pGRE-SEAP, pCRE-SEAP, and pNFkappaB-SEAP patterned on the same solid support for detection of the oxidation current of PAP (p-aminophenol) flux produced from only 300 HeLa cells in each stencil pattern. The results of the SECM method were highly sensitive as compared to those obtained from the conventional CL (chemiluminescence) protocol with at least 5x10(4) cells per well.
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PMID:Development of electrochemical reporter assay using HeLa cells transfected with vector plasmids encoding various responsive elements. 1936 25