Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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 method using p-benzoquinone for coupling antigens and antibodies to enzymes and erythrocytes is described. The method involves the treatment of proteins (or polysaccharides) at pH 6 or 7 with an excess of p-benzoquinone. After removal of the unreacted reagent by gel filtration, the "activated" proteins were coupled at pH 8-9 with enzymes or erythrocytes. Biological activities of the proteins were not substantially modified by this treatment since 80-100% of the antigen binding capacity was found to be preserved in p-benzoquinone treated antibodies or Fab fragments. Anti-Ig antibodies (or Fab) were coupled by this procedure to peroxidase, alkaline phosphatase, lactoperoxidase, glucose oxidase and beta-galactosidase, and the conjugates obtained were found to be highly effective in detecting intracellular Ig by immunohistochemical techniques. Erythrocytes coated with sheep anti-mouse Ig antibody or Fab were used to titrate by passive hemagglutination serum Ig. The same erythrocytes were employed to detect by plaque assay mouse Ig secreting cells. Erythrocytes coated with peroxidase, alkaline phosphatase, bovine serum albumin, ribonuclease, Salmonella polysaccharide (B 27 +) and pneumoccocal polysaccharide SIII were employed to titrate serum antibody by passive hemagglutination and hemolysis and to detect mouse antibody secreting cells by plaque assay. All the antigens and antibodies coated erythrocytes prepared gave highly satisfactory and reproducible results.
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PMID:A new method using p-benzoquinone for coupling antigens and antibodies to marker substances. 0 79

At least three gluconic acid forming enzymes were identified in cell-free extracts of Aspergillus niger: glucose oxidase (EC 1.1.3.4), a glucose dehydrogenase (EC 1.1.99.10), and an enzyme or a mixture of enzymes catalyzing the cleavage of 6-phosphogluconate into gluconate and inorganic phosphate. 2,6-dichlorphenolindophenol was one of the hydrogen acceptors in vitro of the glucose dehydrogenase. Some properties of this enzyme (Km values, pH-dependence, substrate and hydrogen acceptor specificity), as determined in cell-free extracts, were found to be in good agreement with properties described in literature for a glucose dehydrogenase which has been purified from Aspergillus oryzae. The formation of Pi from 6-phosphogluconate and other phosphate esters was found to have an optimum between pH 7 and 8 , and another below pH 4. This suggests that it is catalyzed by an alkaline and an acid phosphomonoesterase (EC 3.1.3.1, 3.1.3.2), both enzymes exhibiting only low substrate specificity. The influence of extraction and assay buffers on the activity of gluconate forming enzymes was investigated. Loss of activity during preparation of cell-free extracts, as calculated from loss of activity storage of cell-free extracts at 4 degrees C, was found to be lower than 4%. Purified glucose oxidase added before homogenization was found in the extract almost quantitatively.
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PMID:[Gluconic acid forming enzymes in Aspergillus niger (author's transl)]. 1 16

Peroxidase (PO), alkaline phosphatase, and glucose oxidase, as well as Fab anti-PO, were coupled with varying molar ratios of trinitrophenyl (TNP) hapten. These reagents were evaluated for their ability to detect anti-TNP antibodies in the lymph node cells of Balb/c mice immunized with heavily-substituted TNP45 alkaline phosphate, which gave rise only to anti-hapten antibody-forming cells (AFC). The best results were obtained with lightly-substituted TNP-Fab anti-PO plus PO, TNP-alkaline phosphatase, and TNP-glucose oxidase. These reagents gave strong, specific staining of AFC, and negative background staining. Anti-hapten and anti-carrier AFC could be stained in contrasting colors on the same slide, when immunization was performed with lightly-substituted TNP5.9 alkaline phosphatase. Anti-hapten AFC were detected with TNP-Fab anti-PO or TNP-glucose oxidase, and unsubstituted alkaline phosphatase was used to reveal anti-carrier AFC. The number of AFC detected with these reagents was compared with the number of direct and indirect anti-TNP plaque-forming cells (PFC). At three and five weeks after primary immunization, 40 and 70% more AFC than PFC were detected. These methods can be employed alone, to enumerate anti-TNP AFC and, if desired, anti-carrier AFC; they can also be used in parallel with anti-TNP PFC assays, to determine the fractions of AFC that are not actively involved in antibody secretion.
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PMID:Detection of anti-TNP antibody-forming cells (AFC) with TNP-enzyme and TNP-Fab anti-enzyme conjugates. 77 69

The p-nitrophenyl ester method was assessed as an enzyme labeling technique. The active ester of a carboxylated testosterone derivative was treated with alkaline phosphatase and glucose oxidase to give labeled antigens, using various molar ratios of steroid to enzyme. Satisfactory immunoreactivities with an anti-testosterone antibody in an enzyme immunoassay system were obtained with the labeled antigens prepared at pH 8.5 by the use of molar ratios higher than 30 and 10, respectively, in the alkaline phosphatase and glucose oxidase labelings.
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PMID:Enzyme labeling in steroid enzyme immunoassays. The p-nitrophenyl ester method for alkaline phosphatase and glucose oxidase labelings. 139 54

The sensitivities of monoclonal antibody-based enzyme immunoassays for 11-deoxycortisol using alkaline phosphatase (AP), horseradish peroxidase (HRP), beta-galactosidase (beta-GAL) and glucose oxidase (GOD) as labels were compared. The anti-11-deoxycortisol antibody used was that produced in ascites by inoculating antibody-secreting hybridoma cells into mice. Enzyme labeling of 11-deoxycortisol was carried out by the N-succinimidyl ester method. The activated ester of 4-(2-carboxyethylthio)-11-deoxycortisol was treated with each enzyme to give a homologous enzyme-labeled antigen. In the competitive immunoassay, the bound and free enzyme-labeled antigens were separated by a double antibody method and the enzymic activity of the immune precipitate was determined by colorimetric and fluorimetric methods. The AP activity was measured in three ways, using p-nitrophenyl phosphate, nicotinamide adenine dinucleotide phosphate (NADP), and 4-methylumbelliferyl phosphate as substrates. o-Nitrophenyl beta-D-galactopyranoside and 4-methylumbelliferyl beta-D-galactopyranoside were used for beta-GAL, and 3,3',5,5'-tetramethylbenzidine (TMB) and 3-(p-hydroxyphenyl)propionic acid (HPPA) for HRP. In the case of GOD, TMB and HPPA were used in combination with HRP. A dose-response curve with a high sensitivity was obtained in each 11-deoxycortisol assay system by the use of a minimum amount of the enzyme-labeled antigen at an appropriate dilution of monoclonal anti-11-deoxycortisol antibody (Ka = 2 x 10(10) M-1). The amounts of 11-deoxycortisol needed to displace 50% of the bound label ranged from 5 to 15 pg in the colorimetric methods, and 4-9 pg in the fluorimetric methods.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Sensitivity of steroid enzyme immunoassays. Comparison of four label enzymes in an assay system using a monoclonal anti-steroid antibody. 268 Jan 24

Batch cultures of Aspergillus niger grown from conidia on a medium with high C/N ratio accumulated gluconate from glucose with a yield of 57%. During almost the whole time of accumulation there was no net synthesis of total protein in the mycelium but the activity per flask and the specific activity of glucose oxidase (EC 1.1.3.4) in mycelial extracts increased whereas both values decreased for glucose dehydrogenase (EC 1.1.99.10) 'gluconate 6-phosphatase' (cf. EC 3.1.3.1, 3.1.3.2), gluconokinase (EC 2.7.1.12), glucose 6-phosphate and phosphogluconate dehydrogenases (EC 1.1.1.49, EC 1.1.1.44), phosphoglucomutase (EC 2.7.5.1), and most enzymes of the Embden-Meyerhof pathway and the tricarboxylic acid cycle. Gluconate dehydratase (EC 4.2.1.39), gluconate dehydrogenase (EC 1.1.99.3) and enzymes of the Entner-Doudoroff pathway could not be detected. By cycloheximide the increase of glucose oxidase activity was inhibited. It is concluded that the high yield of gluconate was due mainly to the net (de novo) synthesis of glucose oxidase which occurred during protein turnover after the exhaustion of the nitrogen source, and which was not accompanied by a net synthesis of the other enzymes investigated. Some gluconate may also have been formed by hydrolytic cleavage of gluconate 6-phosphate.
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PMID:Gluconate accumulation and enzyme activities with extremely nitrogen-limited surface cultures of Aspergillus niger. 301 15

Various visualization methods were compared for quantitation of proteins by the dot-immunobinding assay. Comparisons were carried out using a multi-subunit protein, eukaryotic initiation factor 2, and monospecific antibodies directed against two of the factor's subunits. The protein was spotted onto nitrocellulose and the membranes were incubated with primary antibody. The antigen-antibody complex was visualized by one of six methods using either alkaline phosphatase-, horseradish peroxidase-, or glucose oxidase-conjugated IgG, or colloidal gold-labelled IgG, colloidal gold-labelled IgG with silver enhancement, or 125I-labelled protein A. The amount of secondary antibody bound was quantitated by densitometric scanning of the nitrocellulose membrane after staining or autoradiography. The sensitivity of each of the methods was similar; each of the visualization methods could detect less than 1 ng of protein by the dot-immunobinding assay. Curves of protein concentration vs. densitometric absorbance were found to fit a parabolic relationship (r2 = 0.99) over a wide range of concentrations.
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PMID:Quantitation of proteins by dot-immunobinding assay. A comparison of visualization methods using eukaryotic initiation factor 2 and a monospecific antibody. 327 93

A murine monoclonal antibody specific for aspergillus niger glucose oxidase has been prepared and used in an unlabeled antibody bridge technique for the detection of monoclonal antibodies. This procedure--the monoclonal glucose oxidase anti-glucose oxidase (GAG) immunosandwich assay--provides excellent immunocytochemical labeling of routine hematological films in combination with optimal preservation of cellular details. In contrast to conventional immunofluorescence procedures, routine hematological films can be used, and these can be stored before and after the immunolabeling. Compared with other immunoenzyme techniques such as those using alkaline phosphatase or peroxidase, the GAG assay is as sensitive and has the advantage that no problems with endogenous enzyme activity are encountered. The availability of alcohol-resistant disclosing reagents allows for routine hematological counterstaining which provides a very clear visualization of both the immunoreaction and the individual morphology of the blood cells.
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PMID:Monoclonal glucose-oxidase-anti-glucose-oxidase (GAG) immunosandwich assay for the detection of monoclonal antibodies on routine hematological smears. 328 28

The production of monoclonal antibodies against peroxidase, alkaline phosphatase and glucose oxidase and the use of the respective enzyme monoclonal anti-enzyme complexes in immunoassays are described. A micromethod using nitrocellulose membranes as solid phase was developed. This microassay has the advantage of being a rapid and simple test procedure, using only 0.2 microliter of antigen solution and 25 microliter of test reagents. A high sensitivity was achieved by repeated incubation of monoclonal enzyme anti-enzyme complexes bridged by anti-mouse immunoglobulin. As little as 200 pg human IgG and 400 pg human IgM could be detected. The glucose oxidase assay together with the alkaline phosphatase assay displays the highest sensitivity and has significant advantages including easy evaluation due to high color contrast of the enzymatic reaction, availability of non-toxic substrate reactions, and no interference with endogenous enzyme activity in mammalian antigen preparations.
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PMID:Production of monoclonal antibodies against glucose oxidase, alkaline phosphatase and peroxidase. Their application in a highly sensitive antigen spot microassay. 330 48

Highly sensitive direct visual detection of potyviruses was achieved using a dot blot immunobinding assay (DBIA). The small sample volumes required permit the detection of as little as 0.5 pg virus in purified preparations. The binding of rabbit antibodies could be visualized using goat anti-rabbit IgG (GAR) conjugated to alkaline phosphatase, beta-D-galactosidase, glucose oxidase, or horseradish peroxidase and histochemical substrates. The avidin-biotin system was also useful, but somewhat less sensitive than GAR-enzyme conjugates. Detection of potyviruses in an aphid vector was also attempted, but without success due to endogenous aphid enzymes.
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PMID:Detection of picogram quantities of potyviruses using a dot blot immunobinding assay. 393 55


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