Monoclonal antibodies (mcAbs) specific to alkaline isoenzymes of horseradish peroxidase were used to characterize the antigenic properties of horseradish peroxidase. The results of a competitive binding assay indicated that monoclonal antibodies can be divided into three groups directed against distinct parts of the protein. The interaction of monoclonal antibodies with native and modified horseradish peroxidase showed also three different patterns of reactivity. Antibodies from groups I and II are directed against epitopes which are conformational and formed by tertiary structure elements. Epitopes recognized by these antibodies are sensitive to heme removal or partial denaturation of peroxidase. Antibodies from group III bind specifically with epitopes consisting of primary or secondary structure elements. The antigenic determinants recognized by antibodies from group III PO(1) and 36F(9) were shown to be linear (continuous) and formed by amino acid residues 261-267 and 271-277, respectively, as determined by the peptide scanning method (PEPSCAN). The location of revealed linear antigenic determinants in the molecular structure of peroxidase is analyzed.

Bioluminescence and Chemiluminescence, pp. 165-168 (2002) No AccessMODULATION OF THE ENHANCED CHEMILUMINESCENCE WITH ANTI-PEROXIDASE SPECIFIC ANTIBODIESMYU RUBTSOVA, O V IGNATENKO, T V CHEREDNIKOVA, and A M EGOROVMYU RUBTSOVADept Chem Enzym, MV Lomonosov Moscow St University, 119899 Moscow, Russia, O V IGNATENKODept Chem Enzym, MV Lomonosov Moscow St University, 119899 Moscow, Russia, T V CHEREDNIKOVADept Chem Enzym, MV Lomonosov Moscow St University, 119899 Moscow, Russia, and A M EGOROVDept Chem Enzym, MV Lomonosov Moscow St University, 119899 Moscow, Russiahttps://doi.org/10.1142/9789812776624_0037Cited by:0 PreviousNext AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsRecommend to Library ShareShare onFacebookTwitterLinked InRedditEmail Abstract: The effective application of horseradish peroxidase (HRP) in analytical biochemistry and biotechnology depends on its stability, especially when it is used repeatedly or in lengthy processes. HRP was shown to undergo irreversible time-dependent inactivation when catalysing different oxidation reactions. The peroxidation of some donor substrates results in the formation of highly reactive radical intermediates, which can in turn attack and modify the prosthetic group 1,2 or apo-protein 3 causing the loss of enzymatic activity… FiguresReferencesRelatedDetails Bioluminescence and ChemiluminescenceMetrics History PDF download

In Aerosol OT (AOT) reversed micelles in heptane, the oxidation kinetics of ortho-phenylenediamine (PDA) catalyzed by peroxidase (HRP), its conjugate with nine cortisol molecules and their immunocomplexes with monoclonal antibodies AT-2C specific to domain I of HRP has been studied with regard to the hydration degree of micelles W(0) = [H2O]/[A0T] and the concentration of solubilized biocatalysts. The profiles of dependencies of the initial PDA oxidation rate, v(0), on W(0) varied with an increase in the concentrations of HRP, HRP-cortisol and their immunocomplexes in micellar systems. The rise in HRP concentration from 0.5 up to 1.5 nM increases the v(0) values at a constant hydration degree, W(0) and is characterized by a limiting value of the initial rate at W(0) > 30. At HRP-cortisol concentrations of 1.2 and 1.5 nM, the dependences v(0)-W(0) have three maxima with gradually increasing values of the initial rate which may be accounted for by self-association of the hydrophobized conjugate, HRP-cortisol, in AOT micelles. The profiles of dependencies, v(0)-W(0) for HRP immunocomplexes (HRP-cortisol) with monoclonal antibodies, AT-2C, at various relationships of the components are characterized by three maxima with increasing values of v(0) which may by related to initial biocatalysts (HRP or HRP-cortisol) and their immunocomplexes with one or two molecules of AT-2C.

Horseradish peroxidase (HRP)-specific monoclonal antibodies (MABs) 2C, 9D, and 9F, interacting with domain I of the enzyme, have been shown to activate HRP in the reaction of o-phenylenediamine (OPD) oxidation at moderate H2O2 concentrations (<10 mM). In contrast, the monoclonal antibody 3E, interacting with domain II, inhibited the oxidation of OPD over a broad range of H2O2 concentrations. HRP-specific polyclonal antibodies (PABs) were inhibitory irrespective of H2O2 Concentration. Similar effects were observed when OPD was oxidized by a hydrophobic conjugate of HRP with nine molecules of cortisol. Possible mechanisms underlying the activation and inhibition of the enzyme are discussed.

Competitive methods of enzyme-linked immunoassay (EIA) for detecting simazine and atrazine were developed, and conditions providing the optimal performance were found. The sensitivity of EIA increased by an order of magnitude if the samples were preincubated with the antibody, chloride ions were omitted from, the medium, and the herbicide-peroxidase conjugate was treated with urea. The sensitivity thresholds of EIA using labelled antibodies toward simazine and atrazine were 0.05 and 0.1 ng/ml, respectively. The total duration of ELA was 1 to 2 h. These methods can be used for water quality tests.

Competitive methods of enzyme immuno assay (EIA) for detecting simazine and atrazine were developed, and conditions providing optimal performance were found. EIA sensitivity was shown to increase by an order of magnitude if samples were preincubated with antibodies; chloride ions were omitted; a herbicide-peroxidase conjugate was treated with urea. In EIA using labelled antibodies sensitivities thresholds towards simazine and atrazine were 0.05 and 0.1 ng/ml, respectively. EIA took 1-2 h to be done. The methods developed might be applied for quality control of water.

Peroxidase-dependent cooxidation of 4-iodophenol and 4-aminoantipyrine (AAP), and 4-iodophenol and luminol has been studied The influence of polyclonal and monoclonal antibodies (polyAb and monoAb) to horseradish peroxidase on the kinetics of the reaction was investigated over a wide range of H2O2 concentrations (0.01-10.0 Mm). Three types of MonoAb (2C, 3E, and 9D) in the concentration range 0.05-500 Nm inhibited the cooxidation of AAP and 4-iodophenol at H2O2 concentrations above 1.0 Mm but activated the cooxidation of luminol and 4-iodophenol. Binary and ternary mixtures of monoAb activated the cooxidation of AAP and 4-iodophenol at the same H2O2 concentrations but inhibited or did not affect the cooxidation of luminol and 4-iodophenol. PolyAb activated the cooxidation of AAP and 4-iodophenol more effectively and only slightly activated (or inhibited) the cooxidation of luminol and 4-iodophenol. PolyAb diminished rate constants for the interaction of peroxidase active intermediates, E1 and E2, with 4-iodophenol, AAP, and luminol. Possible mechanisms for the influence of the antibodies on the cooxidation of the substrate pairs are discussed

The kinetics of peroxidase-dependent cooxidation for two substrate pairs [p-iodophenol + 4-aminoantipyrine (AAP) and p-iodophenol + luminol was studied both in the absence and presence of polyclonal antibodies (polyAB), three types of peroxidase-specific monoclonal antibodies (monoAB) and their double or triple mixtures in a wide range of H2O2 concentrations (0.01-10.0 mM). MonoAB 2C, 3E and 9D at concentrations of 0.05-500 nM inhibited the cooxidation of p-iodophenol + AAP at H2O2 concentration above 1.0 mM but activated the cooxidation of p-iodophenol + luminol. The double and triple mixtures of monoAB activated the cooxidation of p-iodophenol + AAP at the same H2O2 concentrations without any effect on the p-iodophenol + luminol cooxidation. PolyAB activated the cooxidation of p-iodophenol + AAP more effectively and only slightly activated (or inhibited) that of p-iodophenol + luminol. PolyAB diminished the values of rate constants for the interaction of the peroxidase active intermediates, E1 and E2, with p-iodophenol, AAP or luminol. Possible modes of monoAB and polyAB effects on the two substrate pair cooxidation are discussed.

The interaction of monoclonal antibodies of three types with the ATP-labeled insulin dimer was studied by the luminescent immunocofactor method. It was shown that the effective equilibrium binding constant increases at equimolar antigen/antibody concentrations. This can be due to the formation of multimolecular complexes between the antigens and antibodies. The feasibility of the binding constants increase during the formation of cyclic tetramolecular complexes is considered. A theoretical model for the description of interaction between the bivalent antigen and antibodies based on the increase of the binding constant during the formation of cyclic complexes is proposed. The coefficients of binding constant increase for antigens belonging to three different clones were calculated.

A modified procedure has been worked out for preparing a conjugate of porcine insulin with E. coli beta-galactosidase employing a heterobifunctional reagent, N-hydroxysuccinimidyl m-maleimidobenzoate. Optimal conditions for insulin acylation and subsequent coupling with beta-galactosidase were selected that afforded the conjugate in a high yield. The ability of the modified antigen to react with antibody was evaluated in the reaction of conjugate binding with immobilized monoclonal antibody to insulin. The conjugate almost completely retained the enzymatic activity and reacted with high specificity with the antibody to insulin. The conjugate can be used in competitive ELISA of insulin.