Temperature dependent rise in activity of horseradish peroxidase caused by non-ionic detergents and its use in enzyme-immunoassay

Porstmann, B; Porstmann, T; Gaede, Doerte; Nugel, E; Egger, E

doi:10.1016/0009-8981(81)90332-6

Cited by 33 publications

(9 citation statements)

References 6 publications

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“…The cells were scraped from the dish and incubated in the lysis buffer for 10 min at 0 mC before centrifugation at 10 000 g for 10 min at 4 mC. The supernatant was collected and the HRP concentration determined spectrophotometrically with o-dianisidine at 460 nm or with equivalent results at 405 nm in a microplate reader after quenching of the colour reaction with 100 mM HCl [35]. Assays were routinely performed in triplicate for each condition.…”

Section: Fluid-phase Pinocytosismentioning

confidence: 99%

Insulin and secretagogues differentially regulate fluid-phase pinocytosis in insulin-secreting β-cells

Howland

Rothenberg

1996

Biochemical Journal

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The physiological role of the beta-cell insulin receptor is unknown. To evaluate a candidate function, the insulin regulation of fluid-phase pinocytosis was investigated in a clonal insulinoma cell line (beta TC6-F7) and, for comparison, also in Chinese hamster ovary cells transfected with the human insulin receptor (CHO-T cells). In CHO-T cells, the net rate of fluid-phase pinocytosis was rapidly increased 3-4-fold over the basal rate by 100 nM insulin, with half-maximal stimulation at 2 nM insulin, as assayed by cellular uptake of horseradish peroxidase from the medium. Wortmannin, an inhibitor of phosphatidylinositol (PI)-3-kinase, blocked insulin-stimulated pinocytosis with an IC50 of 7.5 nM without affecting the basal rate of pinocytosis. In insulin-secreting beta TC6-F7 cells, the secretagogues glucose and carbachol (at maximally effective concentrations of 15 mM and 0.5 mM respectively) augmented fluid-phase pinocytosis 1.65-fold over the basal rate. Wortmannin also inhibited secretagogue-stimulated pinocytosis in these beta-cells with an IC50 of 7 nM but did not affect the basal rate of pinocytosis measured in the absence of secretagogues. Wortmannin did not influence either basal or secretagogue-induced insulin secretion. Although these beta TC6-F7 cells have cell-surface insulin receptors, adding exogenous insulin or insulin-like growth factor 1 did not affect their rate of fluid-phase pinocytosis, either in the absence or presence of secretagogues. From these observations, we conclude that: (1) in both insulin-secreting beta-cells and in conventional, insulin-responsive CHO-T cells, a common, wortmannin-sensitive reaction, which probably involves PI-3-kinase, regulates fluid-phase pinocytosis; (2) the insulin-receptor signal transduction pathway is dissociated from the regulation of fluid-phase pinocytosis in the insulin-secreting beta-cell line we studied; and (3) the enhancement of fluid-phase pinocytosis associated with secretagogue-induced insulin release in beta TC6-F7 cells is not attributable to autocrine activation of beta-cell surface insulin receptors.

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Section: Fluid-phase Pinocytosismentioning

confidence: 99%

Insulin and secretagogues differentially regulate fluid-phase pinocytosis in insulin-secreting β-cells

Howland

Rothenberg

1996

Biochemical Journal

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“…As with other enzymes, POase activity depends on the temperature. The presence of non-ionic detergents, Tween 20 and Triton X-100, delays inactivation of the enzyme (Portsmann et al 1981), and necessitates an increase in temperature (from 15 to 20°C) for optimum timing of the assay. In the present procedure, an incubation time of between 15-20min was found to be sufficient for colour development.…”

Section: Optimization Of Elisa Assaymentioning

confidence: 99%

Immunochemical quantification of cytochrome f in leaves of a non-yellowing senescence mutant of Festuca pratensis

1990

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A non-competitive enzyme-linked immunosorbent assay (ELISA) which enables detection of as little as 0.1 ng cytochrome f in leaf extracts has been developed. No evidence for specific or non-specific interference by proteins other than cytochrome f was found. The assay was applied to a comparative study of age-related changes in the cytochrome f content of leaves of Festuca pratensis Huds. cv. Rossa, and a non-yellowing mutant genotype (Bf993) having a lesion in the mechanism responsible for thylakoid membrane disassembly. Cytochrome f in senescent leaves of the latter genotype was found to be present at significantly higher levels than in the wild-type, implying an inability on the part of the mutant to degrade this protein. The results obtained by ELISA were confirmed by antibody probing of Western blots.

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“…Chiids (12). The activity jise of conjugated due to detergent, described in this paper, was (4,6). We assume that detergent might also delay the formation of Compound III, shifting the reaction into the'classical route, or increase the decomposition of Compound III, rendering enhanced access of chromogen to the inactive compound, this again might explain why, in some chromogens, only a minor rise in activity was obtainable from detergents.…”

Section: Discussionmentioning

confidence: 63%

Comparison of Chromogens for the Determination of Horseradish Peroxidase as a Marker in Enzyme Immunoassay

Porstmann¹,

Porstmann²,

Nugel³

1981

CCLM

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o-Phenylenediamine, 2,2'-azino-di(3-ethylbenzthiazoline sulphonic acid-6) (ABTS), odianisidine and 4-aminoantipyrine were compared as chromogens for the determination of horseradish peroxidase. Highest sensitivity in the determination of horseradish peroxidase-IgG conjugates in dissolved form was obtained with o-phenylenediamine. When these conjugates were used in a two-site binding enzyme immunoassay for hepatitis B surface antigen (HBsAg), the steepest calibration curve and the lowest detection limit were obtained when ABTS was used to determine the immune complexes bound to the solid phase. Non-ionic detergents, such as polyoxyethylene^sorbitol ester, retarded horseradish peroxidase inactivation, resulting in a chromogen-dependent activity rise of horseradish peroxidase. An optimised determination of horseradish peroxidase is reported, in which the sensitivity of the solid phase enzyme immunoassay is doubled by the use of 0-dianisidine. Vergleich von zur Aktivitätsbestimmung von Meerrettich-Peroxidase als Marker im Enzymimmunoassay verwendeter ChromogeneZusammenfassung: Die Empfindlichkeit der Aktivitätsbestimmung von Meerrettich-Peroxidase wurde mit den Chromogenen 0-Phenylendiamin, 2,2'-Azino,di(3-ethylbenzthiazolin-sulfonat-6) (ABTS), o-Dianisidin und 4-Aminoantipyrin verglichen. An IgG gekoppelte Meerrettich-Peroxidase wird in gelöster Form am empfindlichsten mit ö-Phenylendiarnin bestimmt. Nach Einsatz der Konjugate in einem zwei-Seiten Enzymimmunoassay zur Bestimmung des Hepatitis B-Oberflächenantigens (HBsAg) und quantitativer Bestimmung der an die feste Phase gebundenen Immunkomplexe werden die steilste Ständardkurve und geringste untere Nächweisgrenze mit ABTS erhalten. Nichtionische Detergenzien wie Polyoxyethylen*orbitester verzögern die Inaktivierung von Meerrettich-Peroxidase während der Reaktion und fuhren zu einer chromogenabhängigen Aktivitätssteigerung der Meerettich-Peroxidase. Es wird eine .optimierte Bestimmung von Meerrettich-Peroxidase mit <7,Dianisidin vorgestellt, wodurch die Empfindlichkeit des Festphase-OEnzymimmunoassays verdoppelt werden kann.

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Temperature dependent rise in activity of horseradish peroxidase caused by non-ionic detergents and its use in enzyme-immunoassay

Cited by 33 publications

References 6 publications

Insulin and secretagogues differentially regulate fluid-phase pinocytosis in insulin-secreting β-cells

Insulin and secretagogues differentially regulate fluid-phase pinocytosis in insulin-secreting β-cells

Immunochemical quantification of cytochrome f in leaves of a non-yellowing senescence mutant of Festuca pratensis

Comparison of Chromogens for the Determination of Horseradish Peroxidase as a Marker in Enzyme Immunoassay

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