[Psychological assessment of visual hemispatial neglect: standardization and approbation of the modified digit cancellation test]. / Diagnostika odnostoronnego zritel'no-prostranstvennogo nevnimaniia: standartizatsiia i aprobatsiia modifitsirovannoi tsifrovoi korrekturnoi proby.

AIM: To study the phenomena of visual-hemispatial neglect in healthy people and patients with brain diseases of different genesis. MATERIAL AND METHODS: Eighty-eight patients with schizophrenia spectrum disorders, 68 patients with exogenous organic brain diseases and 240 healthy adults of different age were included in the study. The digit cancellation test modified by the authors was used. RESULTS AND CONCLUSION: The validity of the modified digit cancellation test was approved and its age standards were obtained. In healthy right-handed people, there was the bias of attention focus to the left, the decrease of asymmetry intensity of visual-spatial inattention during physiological aging and the presence of some clinical peculiarities of neglect in schizophrenia spectrum disorders and lateralized organic damages of the brain. This variant of the test can be recommended for practical use as the sensitive psychometric tool.

Characterization of antigenic properties of horseradish peroxidase with monoclonal antibodies.

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.

[Effect of degree of aerosol OT reverse micelle hydration on the catalytic activity of peroxidase, its conjugate with cortisol and their immune complexes]. / Vliianie stepeni gidratatsii obrashchennykh mitsell aérozolia OT na kataliticheskuiu aktivnost' peroksidazy, ee kon''iugata s kortizolom i ikh immunnykh kompleksov.

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.

[Solid-phase methods of immunoenzyme analysis of the herbicides simazine and atrazine]. / Tverdofaznye metody immunofermentnogo analiza gerbitsidov simazina i atrazina.

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.

[The effect of monoclonal and polyclonal antibodies to peroxidase on combined peroxidase oxidation of 4-iodophenol with luminol and 4-aminoantipyrine]. / Vliianie monoklonal'nykh i poliklonal'nykh antitel k peroksidaze na sopriazhennoe pereoksidaznoe okislenie 4-iodfenola s liuminolom i 4-aminoantipirinom.

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.

[Interaction of monoclonal antibodies with horseradish peroxidase]. / Vzaimodeistvie monoklonal'nykh antitel s peroksidazoi khrena.

Properties of four types of monoclonal antibodies to horse-radish peroxidase were investigated. The dissociation constants and molecular-weight composition of the immune complexes were determined. The antibodies are shown to be directed to different epitopes on the polypeptide chain. Results of the theoretical prediction of the epitope localisation are presented. The interaction between the antibodies and peroxidase isozymes were studied.

[Changes in the adiabatic compressibility of mono- and polyclonal antibodies during interaction with antigens]. / Izmenenie adiabaticheskoi szhimaemosti mono- i poliklonal'nykh antitel pri vzaimodeistvii s antigenami.

The values of apparent adiabatic compressibility of free and antigen-bound antibodies were determined by means of precise density and ultrasound velocity measurements. It was shown that during the formation of soluble immune complexes (insulin--monoclonal antibodies to insulin and alpha-amylase--monovalent Fab-fragments of antibodies to alpha-amylase), the apparent compressibility of antibodies decreased by (0.3 divided by 0.9).10(-6) cm3/g.bar. During the formation of large insoluble aggregates (alpha-amylase--polyclonal antibodies to alpha-amylase), the apparent compressibility decreased by (5.5 +/- 0.7).10(-6) cm3/g.bar. It is suggested that the decrease in the magnitude of thermal fluctuations of the molecular volume of antibodies during antigen binding, manifesting itself by the decrease in their compressibility and strengthened several-fold by precipitate formation, may favour the activation of the effectory functions of antibodies.

Mapping of the immunodominant regions of the NAD-dependent formate dehydrogenase.

A panel of 4 monoclonal antibodies and 7 polyclonal antisera against NAD-dependent formate dehydrogenase from methylotrophic bacterium Pseudomonas sp. 101 has been obtained. The reactivity of the 37 overlapping proteolytic peptides with the monoclonal antibodies and polyclonal antisera has been studied with ELISA test. The data obtained were interpreted residing on the structural model of the formate dehydrogenase at 3 A resolution. The immunodominant regions in the formate dehydrogenase molecule and the epitopes for the monoclonal antibodies were elucidated.

[Isolation and characterization of monoclonal antibodies to horseradish peroxidase]. / Poluchenie i issledovanie monoklonal'nykh antitel k peroksidaze iz kornei khrena.

Monoclonal antibodies to horseradish peroxidase were obtained. The interaction of two antibody clones with the enzyme was studied. Antibodies of one clone were found to inhibit the enzyme activity during the oxidation of 2.2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate) diammonium salt and the cooxidation of luminol and luciferin. The latter was concomitant with a complete inhibition of the peroxidase activity. The values of binding constants as determined by the solid phase immunoenzymatic and homogeneous methods are equal to (1.2 +/- 0.5).10(8) M-1 and (1.8 +/- 0.2).10(11) M-1, respectively.

[Cooperative effects during interaction of monoclonal antibodies with insulin dimers]. / Issledovanie kooperativnykh éffektov pri vzaimodeistvii monoklonal'nykh antitel s dimerom insulina.

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.

[Preparation of an insulin conjugate with beta-galactosidase from E. coli for immunoenzyme assay]. / Poluchenie kon''iugata insulina s beta-galaktozidazoi iz E. coli dlia tselei immunofermentnogo analiza.

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.

Study of the monoclonal antibody-insulin interaction.

We generated four stable hybridoma lines producing monoclonal antibodies. All antibodies were as reactive with insulin from other species as with swine insulin. The apparent affinity of the monoclonal antibodies for insulin varied in the range from 2 X 10(8) M-1 to 2 X 10(10) M-1. According to their affinity constants some antibodies could be used for the preparation of immunosorbent, and others for the development of immunoassay methods. Bioluminescent cofactor immunoassay is a method of the choice for the detection of insulin concentration in physiological fluids due to its extreme sensitivity and reproducibility. High affinity antibodies of clones I and II may serve as immunospecific constituents for immunoassay method. Epitope specificity of all monoclonal antibodies has not been studied yet in detail and will be the subject of further investigation.

[Effect of specific antibodies on association of glyceraldehyde 3-phosphate dehydrogenase subunits]. / Vliianie spetsificheskikh antitel na assotsistsiiu sub"edinits glitseral'degid-3-fosfatdegidrogenazy.

The rabbit antibodies against glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12) from baker's yeast or rat muscle are strictly specific to the corresponding antigens and are not involved in the cross reaction. The interaction of specific antibodies with Fab-fragments does not affect the activity of the yeast enzyme which is in agreement with the previous data on the rat muscle enzyme. The antibodies against yeast dehydrogenase immobilized on BrCN-activated Sepharose were used for the preparation of the enzyme linked to the matrix by the antigen--antibody complex. The tetrameric enzyme molecule thus immobilized completely retains its activity and the ability to dissociate into dimers in the cold in the presence of ATP. The dimer which remains bound within the antigen--antibody complex retains its activity in the presence of agents causing inactivation and release of the second dimer into solution. The tetramer of yeast dehydrogenase covalently bound to the BrCN-activated Sepharose by one subunit is completely stable under conditions causing dissociation, when it exists in the complex with three molecules of specific antibodies. The binding of four molecules of Fab-fragments of specific antibodies per molecule of tetramer protects the rat muscle apoenzyme against anion-dependent cold inactivation and thermal inactivation in solution. The binding of two antibody molecules specific to rat muscle glyceraldehyde 3-phosphate dehydrogenase to the hybrid tetramer made up of yeast dehydrogenase dimer covalently linked to Sepharose and of rat muscle dehydrogenase dimer, prevents the dissociation of the tetramer (i.e. release of the "muscle" type dimer into solution). Gel filtration through Sepharose 6B of the apo-glyceraldehyde phosphate dehydrogenase complex with the Fab-fragments of specific antibodies demonstrated that the binding of the Fab-fragments shifts the tetramer in equilibrium or formed from dimers equilibrium in the apoenzyme solution towards the tetramer. It is concluded that the specific antibodies increase the intersubunit interactions in the oligomer by stabilizing the native tertiary structure of individual subunits.

[Use of immobilization for investigation of glyceraldehyde 3-phosphate dehydrogenase. Immobilized tetramers]. / Ispol'zovanie immobilizatsii dlia izucheniia glitseral'degid-3-fosfatdegidrogenazy. Immobilizovannye tetramery.

Glyceraldehyde-3-phosphate dehydrogenase from yeast and rat skeletal muscle was covalently linked to CNBr-activated Sepharose 4B. When the activation was as high as 4-5 mg of CNBr per ml of Sepharose, the enzymes had their maximal activity and were linked to the carrier only by one of the four subunits. The specific activity of immobilized dehydrogenases makes up to 50-60% of that of soluble preparations, since the rate of the substrate diffusion into Sepharose granules is too low. The Km values for NAD and substrate and the pH dependence of the immobilized enzymes were determined. It was found that the enzymes used as adsorbents for isolation of specific antibodies reveal their maximal activity when CNBr concentration reaches 150-200 mg per ml of gel. No inhibition of activity of the immobilized dehydrogenase from yeast or stabilizing effect of antibodies on the enzyme structure were observed.

Study of subunit interactions in immobilized D-glyceraldehyde-3-phosphate dehydrogenase.

Under conditions which cause dissociation of soluble tetrameric glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) into inactive dimers, immobilized apoenzymes from yeast and rat skeletal muscle coupled to CnBr-activated Sepharose via one subunit retain 50% of matrix-bound protein with unaltered specific activity. The solubilized dissociated species are inactive. Two molecules of NAD+ (NADH) firmly bound to the immobilized rat muscle tetramer can prevent the dissociation. Immobilized dimer was demonstrated to bind one molecule of coenzyme with high affinity. Using various combinations of immobilized and soluble rat muscle and yeast dimers, we succeeded in reconstituting tetramers, containing one molecule of NAD+ bound either to a matrix-linked or to a non-covalently bound dimer. In the latter case, the dissociation of the tetramer was completely prevented. This suggests that the binding of a single coenzyme molecule is sufficient to stabilize the interdimeric contacts provided the neighbouring dimer is stabilized independently. Such stabilization is produced by the covalent binding of one of the subunits comprising the dimer to the matrix. The structure of the dimer as a whole becomes resistant to the action of the dissociating agent. The effect appears to be cooperative and similar to that of NAD+ or NADH. The dissociation of the immobilized tetramer is, most likely, the result of conformational changes, affecting the structure of the non-covalently bound dimer. Any factor, capable of preventing these changes, would stabilize the interdimeric contacts. The latter conclusion is substantiated by the effect of specific antibodies, which prevent the dissociation of the immobilized tetramer by forming a complex with the dimer, non-covalently bound to the matrix. The evidence obtained in the present investigation supports the conclusion that the isolated dimer of glyceraldehyde-3-phosphate dehydrogenase represents a relatively independent structural and functional 'unit' of the enzyme. It can be stabilized in a catalytically active form by interactions other than those involved in inter-dimeric contacts in the tetramer. The kinetics of the association of immobilized and soluble dimers have been studied. Association rate constants were determined for homologous (yeast-yeast, rat-rat) and heterologous (yeast-rat, yeast-rabbit) dimer combinations. The binding of one molecule of specific antibody to the immobilized dimer was shown to increase the rate constant of association.

[Immobilization of glyceraldehyde-3-phosphate dehydrogenase by non-covalent binding to specific antibodies and Fab-fragments coupled to Sepharose]. / Nekovalentnaia immobilizatsiia glitseral'degid-3-fosfatdegidrogenazy na antitelakh i Fab-fragentakh antitel, sviazannykh s sefarozoi.

Rabbit antibodies to rat skeletal muscle glyceraldehyde-3-phosphate dehydrogenase, as well as monovalent Fab fragments of these antibodies were coupled to CNBr-activated Sepharose 4B. Rat skeletal muscle glyceraldehyde-3-phosphate dehydrogenase was then immobilized on a matrix by non-covalent binding to specific antibodies. Immobilized enzyme retains approximately 90% catalytic activity of the soluble dehydrogenase; pH optimum of activity and the Km value observed are changed as compared to the enzyme in solution. Glyceraldehyde-3-phosphate dehydrogenase immobilized on specific antibodies is shown to undergo adenine nucleotide-induced dissociation into dimers. The immobilized dimeric form of the enzyme thus obtained is catalytically active and capable of reassociating with the dimers of apoglyceraldehyde-3-phosphate dehydrogenase added in solution to the suspension of Sepharose.