[Selection of optimal conditions for synthesis of protein A-laccase conjugates and their use in different variants of immunoenzyme analysis].

A study to determine optimal molar ratios of Protein A to laccase for the synthesis of their conjugate by periodate method is presented. No loss of enzymatic or immunological activity of the conjugate developed was observed during 6 month. The conjugate could be effectively used in various techniques of enzyme immunoassay (competitive or sandwich techniques, dot immunoblotting) for immunoglobulin G.

[Quantitative determination of cattle angiogenin].

Polyclonal antibodies to a milk antigen were obtained by a standard immunization procedure. The possibility was demonstrated to use the peroxidase-antibody conjugate in a competitive immunoenzymatic test. The minimal amount of the antigen determined by this method is 10 ng/ml.

[Immunoenzyme analysis based on laccase conjugates with fluorometric detection of the enzymatic reaction product].

The possibility of using homovanillic acid as a substrate of laccase (produced by the basidiomycete Coryolus hirsutus) has been demonstrated for the first time. The reaction was shown to result in the formation of a fluorescent product. Several kinetic parameters and optimal conditions were determined for this enzymatic reaction.

[Study of physiological functions of human ceruloplasmin. The effect of ceruloplasmin on immunocytes in a normal state and in pathology].

The immunomodulating effect of ceruloplasmin (CP) on the major components of the immunocompetent system of the organism--the natural resistance system and the specific immune response--has been established. CP can exert various influences on the level of expression of specific markers of T- and B-lymphocytes (as determined by various modifications of the rosette-forming test), on the phagocytic activity of neutrophils and monocytes as well as on the activity of "respiratory burst" enzymes. CP modulation was found to depend predominantly on the initial level of the immunological parameters to be determined, i.

Modulatory effects of ceruloplasmin on lymphocytes, neutrophils and monocytes of patients with altered immune status.

We investigated the effect of plasma ceruloplasmin (Cp) on the different types of lymphocyte rosetting, and phagocytosis of polystyrene particles and culture Candida albicans by peripheral blood neutrophils and monocytes. Lymphocytes, neutrophils, and monocytes were isolated from the blood of patients with elevated immuno-status (n = 9), healthy donors (n = 21), and patients with reduced immuno-status (n = 21). The ability of Cp to decrease the number of lymphocytes forming E- and EAC-rosettes and rosettes with auto-erythrocytes was shown for both patients and healthy donors.

[The effect of conditions of synthesis of immunolaccase conjugates on characteristics and composition of the compounds obtained].

Optimal conditions for preparing laccase conjugates by the periodate method have been selected. The effect of the initial molar ratio of IgG to laccase and pH of the medium on the composition of laccase conjugates was studied by the HPLC method. The maximum yield of the conjugates was observed, when laccase was oxidized with 0.

[Laccase from Coriolus hirsutus--a new marker enzyme for immunoenzyme analysis].

A new immunochemical reagent is proposed which contains laccase, isolated from the culture liquid of the basidial fungus Coriolus hirsutus, as a marker enzyme. The feasibility of immunolaccase conjugates for different variants of immunoassay, i.e.

A new approach to the construction of potentiometric immunosensors.

An electrochemical immunosensor based on a new detection principle was developed. Laccase, which is able to catalyse the electroreduction of oxygen via the direct (mediatorless) mechanism was used as an enzyme label. The new detection method does not require the presence of an electrochemically active mediator, and the reaction substrates are atmospheric oxygen and electrons, the latter being taken by the active site of the enzyme label directly from the electrode.

[Interconnection between the structure and protective action of normal and pathological ceruloplasmin preparations in copper-induced erythrocyte lysis].

It was found that the differences in the protective effects of ceruloplasmin (CP) isolated from the blood of healthy donors and of the ceruloplasmin-like protein (pat-CP) isolated from the blood of patients with hepatovertebral dystrophy (HCD) during Ca(2+)-induced lysis of erythrocytes (RBC) result from significant changes in the carbohydrate fragment of pat-CP, the bulk of which (65%) is devoid of mannose and acetylglucosamine residues. According to the data from lentil-lectin Sepharose chromatography, only 4% of pat-CP molecules contain the [formula; see text] fragment necessary for the binding to ER receptors. The curves reflecting the Cu2+ accumulation in healthy donor ER and in pat-CP during the Cu(2+)-induced lysis were found to differ significantly.

Immunopotentiometric electrodes based on bioelectrocatalysis in the absence of mediators.

A new method of immunoelectrochemical analysis employing laccase as the enzyme label is described. The ability of the enzyme to catalyze electroreduction of oxygen via a direct mechanism allows the detection of the biospecific interaction of a laccase-labeled receptor, or antibody, with a ligand-modified electrode. Formation of a complex between the laccase-labeled antibody and the antigen on the electrode surface resulted in a considerable (greater than 300 mV) change in the electrode potential.

Interrelation between structure and protective action of normal and pathological ceruloplasmins during copper-induced lysis of red blood cells.

The origin of the difference between the protective action of ceruloplasmin (CP) from healthy donors blood and of ceruloplasmin-like protein (p-CP) from blood of patients with Wilson disease which they exert during copper-induced lysis of red blood cells (RBC) was elucidated. The difference is due to a significant change in the carbohydrate moiety of p-CP the major proportion of which (65%) does not contain mannose and acetylglucosamine residues. The data of chromatography on lentil lectin reveal that only 4% of p-CP molecules contain the fragment [table: see text] required for binding to RBC receptors.

[Protective effect of ceruloplasmin during human erythrocyte lysis induced by Fe2+ ions].

In order to elucidate the nature of linkage between the oxidase activity and protective effect of ceruloplasmin during the Fe2(+)-induced lysis of erythrocytes, the both factors were identified in ceruloplasmin samples prepared from blood sera of healthy donors and patients with hepatocerebral dystrophy (HCD). It was found that the oxidase activity of healthy donor ceruloplasmin markedly exceeds that of HCD patients, whereas the protective effect of the HCD protein, contrariwise, markedly exceeds that of normal ceruloplasmin. The data obtained suggest that the protective effect of ceruloplasmin during Fe2(+)-induced erythrocyte lysis is not correlated with its oxidase (ferroxidase, in particular) activity.

The protective effect of normal and pathological (Wilson disease) ceruloplasmins on human erythrocytes.

The binding of the ceruloplasmin (CP) from the healthy donor blood and of ceruloplasmin-like protein (p-CP) isolated from the Wilson patients' blood with erythrocytes (RBC) of healthy donors and with RBC of Wilson's patients (p-RBC) was investigated. It was shown, that the number of CP binding sites both on the RBC and p-RBC was significantly lower than that for p-CP, but Kd value for p-CP binding with both types of erythrocytes was approximately ten times higher than Kd value for CP. The protective action of CP on copper stimulated hemolysis is significantly higher than that of p-CP.

Protective action of blood ceruloplasmin obtained from normal individuals on red blood cells compared with that from patients with Wilson's disease.

A ceruloplasmin-like protein (pathological CP, p-CP) was isolated from the serum of patients with Wilson's disease whose antigenic determinant was identical to that of ceruloplasmin (CP) obtained from the blood of normal individuals. Because the protective action of CP involves its interaction with receptors on red blood cells (RBC), the binding of CP and p-CP to the RBC from normal individuals and to those from patients with Wilson's disease (p-RBC) was investigated. The number of binding sites for CP on both types of RBC was significantly lower than that for p-CP.

[Protective effect rendered by human ceruloplasmin on erythrocytes in hepatocerebral dystrophy].

In order to elucidate the protective effect of human ceruloplasmin (CP) on erythrocytes in patients with hepatocerebral dystrophy (HCD), the parameters reflecting the interaction of CP from the blood of healthy donors (n-CP) and of HCD patients (h-CP) with erythrocytes from healthy donors (n-ER) and from HCD patients (h-ER) were estimated. The protective effects of n-CP and h-CP on n-ER and h-ER during the Cu2+-induced lysis were compared. It was shown that the ability of h-CP to prevent the human ER breakdown upon Cu2+-induced lysis is much lower (approximately 3-fold) than that of n-CP.