[Isolation and properties of human immunoglobulin-peroxidase complex].
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The complexes of human immunoglobulin G (IgG) with peroxidase produced by a two-step synthesis using glutaraldehyde retain high enzymatic and immunochemical activities. The effectiveness of the complex formation depends on the concentration of glutaraldehyde used for enzyme modification. Optimal molar ratios of the constituent components during the synthesis of the complex and localization of the enzyme on the IgG molecule are discussed. Preliminary heating of IgG or the overall complex at 50 degrees C results in an increase of the complex stability (20-fold) under storage.Keywords:
Glutaraldehyde
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A method is described for the demonstration of specific immunoglobulin in plasma cells and other lymphoid cells in sections taken from routine surgical histology specimens which have been formalin fixed and paraffin embedded. An indirect sandwich technique was employed using specific rabbit antihuman immunoglobulin antisera (anti-K, L, G, A, and M) and a swine antirabbit serum Ig G, conjugated with horseradish peroxidase. The presence of plasma cells was revealed by staining the tissue-bound peroxidase-labelled antibody, having previously stained the endogenous peroxidase a contrasting colour. It was possible to demonstrate clearly immunoglobulin in the plasma cells of tissues processed and embedded several years previously. Some of the potential uses of the method are discussed.
Horseradish peroxidase
Histology
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The complexes of human immunoglobulin G (IgG) with peroxidase produced by a two-step synthesis using glutaraldehyde retain high enzymatic and immunochemical activities. The effectiveness of the complex formation depends on the concentration of glutaraldehyde used for enzyme modification. Optimal molar ratios of the constituent components during the synthesis of the complex and localization of the enzyme on the IgG molecule are discussed. Preliminary heating of IgG or the overall complex at 50 degrees C results in an increase of the complex stability (20-fold) under storage.
Glutaraldehyde
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Inorganic pyrophosphatase isolated from Escherichia coli has been proposed as a label in heterogeneous enzyme immunoassays. The enzyme is remarkably stable and insensitive to sodium azide. Enzyme-antibody conjugates were prepared with glutaraldehyde and purified by gel filtration. Enzyme activity was measured by means of a sensitive colour reaction between phosphomolybdate and malachite green. A 5-10-fold increase is sensitivity in terms of absorbance readings was observed compared to peroxidase-based assays. The colour change (yellow/greenish blue) inherent in the use of pyrophosphatase as the labelling agent is highly suitable for visual analysis.
Malachite green
Glutaraldehyde
Sodium azide
Inorganic pyrophosphatase
Pyrophosphatases
Absorbance
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Suspension cultures of carrot (Daucus carrota L.) which had an absolute requirement for exogenously supplied auxin were grown in medium containing indoleacetic acid (IAA) as the sole auxin source. Putative cell surface proteins were extracted from the intact cells. Resupply of IAA to cultures partially depleted of auxin resulted in rapidly increased activities of three enzyme activities subsequently extracted. Two of the enzyme activities which increased, peroxidase and pectinesterase, have been implicated in the literature as important to cell wall development, structure, and growth. The other enzyme activity which was increased, IAA oxidase, may be involved in the degradation of IAA In vivo. Polypeptides in the extracts were found to increase equally as rapidly as the enzymes in response to IAA as determined with sodium dodecyl sulfate-polyacrylamide electrophoretic gels stained with silver. It is not known whether the changes in enzyme and polypeptide levels in the protein extracts were due to auxin effects on protein synthesis, transport, or extractability.
Pectinesterase
Daucus carota
Sodium dodecyl sulfate
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Glutaraldehyde
Protein A
Rabbit (cipher)
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Peroxidase-labeled antibody conjugates were prepared by a two-step conjugation procedure using glutaraldehyde. Immunoadsorbent-purified antibody and the gamma-globulin fraction of sheep anti-human IgG antiserum were employed for these preparations, Procedures for the determination of the antibody and enzymatic activity, as well as the specificity of the enzy-e-antibody reactions, were outlined. Peroxidase-anti-human-IgG conjugates were prepared with approximately a 1:1 molar ratio of peroxidase to IgG, which demonstrated a loss of 10-15% of precipitating antibody activity. Standardization procedures for use of peroxidase-labeled antibody in the indirect ANA test were established. The peroxidase-labeled antibody in the indirect ANA test were established. The peroxidase-antibody procedure was found to demonstrate a reproducible plateau endpoint which was comparable to that of the fluorescent antibody preparations.
Conjugate
Glutaraldehyde
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DyP-type peroxidases are heme-containing enzymes that have received increasing attention over recent years with regards to their potential as biocatalysts. A novel DyP-type peroxidase (CboDyP) was discovered from the alkaliphilic cellulomonad, Cellulomonas bogoriensis, which could be overexpressed in Escherichia coli. The biochemical characterization of the recombinant enzyme showed that it is a heme-containing enzyme capable to act as a peroxidase on several dyes. With the tested substrates, the enzyme is most active at acidic pH values and is quite tolerant towards solvents. The crystal structure of CboDyP was solved which revealed atomic details of the dimeric heme-containing enzyme. A peculiar feature of CboDyP is the presence of a glutamate in the active site which in most other DyPs is an aspartate, being part of the DyP-typifying sequence motif GXXDG. The E201D CboDyP mutant was prepared and analyzed which revealed that the mutant enzyme shows a significantly higher activity on several dyes when compared with the wild-type enzyme.
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ABSTRACT OG-oxidases (OGOXs) and CD-oxidase (CELLOX) are plant berberine bridge enzyme-like oligosaccharide oxidases that oxidize oligogalacturonides (OGs) and cellodextrins (CDs), cell wall fragments with nature of damage-associated molecular patterns (DAMPs). The oxidation of OGs and CDs attenuates their elicitor activity by concomitantly releasing H 2 O 2 . Here, we demonstrate that the H 2 O 2 generated downstream of the combined action between a fungal polygalacturonase and OGOX1 or an endoglucanase and CELLOX can be directed by plant peroxidases (PODs) either towards a reaction possibly involved in plant defence such as the oxidation of monolignol or a reaction possibly involved in a developmental event such as the oxidation of auxin (IAA), pointing to OGOX1 and CELLOX as enzymatic transducers between microbial glycoside hydrolases and plant PODs.
Oligosaccharide
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Horseradish peroxidase was conjugated to immunoglobulin G via glutaraldehyde by a two-step procedure using an increasing excess of peroxidase in the second step reaction. The yield of conjugated monomeric IgG and the amount of free IgG were analyzed by SDS-polyacrylamide electrophoresis and gel-filtration. The antigen binding capacity of the enzyme-antibody conjugates was evaluated by radial immunodiffusion. Conjugation of peroxidase to IgG with a 1:20 molar:molar excess of glutaraldehyde-activated peroxidase resulted in a high yield of conjugated IgG without any detectable amounts of polymers of IgG or residual free IgG. The antigen binding capacity of the conjugate varied between different antigen-antibody systems, but in general it was not significantly different from that of native IgG. The enzyme activity was reduced to 70% of the activity of native peroxidase.
Horseradish peroxidase
Glutaraldehyde
Conjugate
Polyacrylamide
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