[Determination of antibiotics using luminescent Escherichia coli and serum].

The methodical bases for detecting antibiotics using a bioluminescent assay and blood serum are briefed. Antibiotics inhibit the luminescence of a genetically engineered Escherichia coli strain. The degree of inhibition depended on the type of antibiotic, its concentration, and the time of cell incubation with antibiotic. The highest cell sensitivity was recorded towards the aminoglycoside antibiotics, which amounted to 85 +/- 10 ng/ml for gentamicin and streptomycin. The sensitivity of this system to a number of antibiotics essentially increased when the cells were previously activated with blood serum. The sensitivity of this method for gentamicin and streptomycin in the presence of blood serum amounted to 2.5 +/- 0.5 ng/ml; for tetracycline, 45 +/- 8 ng/ml. Use of the sera containing specific antibodies to the antibiotic detected provided a high sensitivity of the biosensor tested. Comparison of the luminescences of E. coli cells activated with normal and specific antisera upon incubation with an antibiotic allows the type of antibiotic and its quantitative content in the sample to be determined. Characteristic of the analysis of antibiotics with the help of recombinant E. coli are a high accuracy, sensitivity, specificity, simplicity, and a short time needed for measurement.

Evaluation of dissociation constants of antigen-antibody complexes by ELISA.

In this communication some of the advantages and constraints in the use of ELISA (enzyme-linked immunosorbent assay) procedures to evaluate antigen-antibody dissociation constants (Kd) are discussed and experimental conditions under which the effective Kd is close to the true value are proposed. Interactions between horseradish peroxidase (POD), human myoglobin and insulin with mono- and polyclonal antibodies (McAb and PcAb) were used to demonstrate that ELISA can be used to determine the average Kd, characterizing the interaction between antigens and PcAb. The Kd values obtained by ELISA were similar to those determined by luminescent immuno-cofactor analysis (LICA).

Involvement of opioid receptors in Met-enkephalin modulation of blast-transformation of mouse splenocytes.

The influence of Met-enkephalin on mitogenic stimulation of mouse splenocytes was investigated. Met-enkephalin (ME) was shown to suppress proliferation induced by Concanavalin A and activate proliferation induced by Staphylococcus enterotoxin A. Both effects were revealed at low (down to 10(-14) M) concentration of pentapeptide. Naloxone reversed ME influence on cell activation. The number of receptors for naloxone was shown to increase up to 2.5-fold during mitogenic activation. The difference in expression of various types of opioid receptors at mitogenic stimulation was demonstrated by ligand displacement experiments.

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.

Preparation of conjugates of progesterone with bovine serum albumin in the reversed micellar medium.

Progesterone-BSA (bovine serum albumin) conjugates which contain up to 47 steroid molecules linked to a BSA molecule have been prepared by the activated ester method, the conjugation step being carried out in reversed micellar solutions of sodium di(2-ethylhexyl) sulphosuccinate (AOT) in octane. The number of incorporated steroid molecules increases on passing to increased water/AOT ratios at the given activated steroid/BSA ratio. The results show that the reversed micellar medium would be useful for preparation of conjugates of hydrophobic steroids with proteins in respect to simplicity and ease in obtaining conjugates with high steroid/protein ratios.

[The effect of condition of oxidation of the carbohydrate component of peroxidase on the composition and properties of insulin-peroxidase conjugate]. / Vliianie uslovii okisleniia uglevodnogo komponenta peroksidazy na sostav i svoistva kon''iugata insulin-peroksidaza.

The influence of sodium metaperiodate concentration on kinetics and conversion degree of peroxidase carbohydrate moiety as well as the effect of the oxidation degree of the carbohydrate moiety on the composition, structure and properties of insulin-peroxidase conjugates were studied. The initial rate of peroxidase's oxidation is directly proportional to the periodate concentration; the oxidation rate constant of peroxidase carbohydrate moiety is 1.23 x 10(-3) M-1 min-1. At the molar ratio of metaperiodate to peroxidase 150:1 or higher, the maximal quantity of aldehyde groups (62 +/- 2) in the peroxidase molecule is formed and the oxidation of each carbohydrate chain leads to the formation of eight aldehyde groups. The molecular mass composition of the insulin-peroxidase conjugates was studied by HPLC. The conjugates proved to be multicomponent mixtures of oligomers (53, 83, 128, 174, 268, 440 kD and higher). The insulin-peroxidase molar ratio in the fractions of the conjugates with molecular masses higher than 83 kD is 8:1. It was shown that the affinity of insulin-peroxidase conjugates to antibodies depends on the oxidation degree of peroxidase used for production of conjugates.

[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.

[Production of rabbit precipitating antisera to subclasses of human IgG]. / Poluchenie krolich'ikh pretsipitiruiushchikh antisyvorotok k subklassam IgG cheloveka.

Precipitating antisera to human subclasses IgG were obtained by immunization of rabbits by whole molecules IgG2, IgG3, IgG4 and gamma 1-chains derived from IgG1H (Pr). Analysis of the antisera obtained demonstrated that rabbits produced specific antibodies to the antigenic subclass determinants IgG3 well, to IgG2, IgG4--much worse, and failed to produce specific antibodies to subclass IgG1 (in immunization with whole molecules of this protein). Antisera contained antibodies to the antigenic determinants common of IgG, and antibodies to light chains which were removed by immunosorption, for which purpose a sorbent on the basis of BrCN sepharose conjugated with IgG of the three other subclasses and Fab-fragment was used.

[Determination of the binding constants of immune serum antibodies to human IgG]. / Opredelenie konstant sviazyvaniia antitel immunnykh syvorotok k IgG cheloveka.

The possibility of using two variants of the enzyme-linked fluorescent cofactor immunoassay for the determination of antibody binding constants has been demonstrated. The determination of binding constants for antibodies isolated by affinity chromatography techniques has been carried out. These techniques permit the isolation of fractions, differing in their affinity by 5-10 times, from the whole population of antibodies.

[Isotachophoretic isolation of IgD and IgE and production of monospecific antisera against them]. / Vydelenie metodom izotakhoforeza IgD i IgE i poluchenie k nim monospetsificheskikh antisyvorotok.

The method of preparative isotachophoresis in acrylamide gel ensuring a high yield of IgD and IgE with insignificant admixtures of IgG, etc. was used for the isolation of IgD and IgE from the blood sera of myeloma patients. As a result of immunization with these antigens, monospecific IgD and IgE antisera were obtained. These antisera, alongside with specific antibodies, contained antibodies to admixtures; the latter were eliminated by the method of immune absorbtion carried out with the use of a sorbent based on Sepharose activated with bromo-cyanogen and conjugated with normal human blood serum. Ig D antisera were also shown to contain antibodies to idiotypical IgD determinants located in the Fab fragment of this immunoglobulin.

[Use of immunoenzyme analysis for controlling the activity of antisera to insulin]. / Ispol'zovanie immunofermentnogo analiza dlia kontrolia aktivnosti antisyvorotok k insulinu.

Antisera to insulin were obtained from guinea pigs hyperimmunized with the use of Freund's adjuvant. To control the specific activity of antisera, the highly specific modern method of ELISA was used. Insulin was allowed to adsorb on the surface of the wells of polystyrene microplates, then consecutive dilutions of the tested antisera to insulin were placed into the wells. The insulin-antibody complexes thus obtained were detected by means of immunoperoxidase conjugate. Antibody titers in antisera obtained from different animals varied from 5 X 10(-3) to 10(-5). A good correlation between the results obtained by ELISA and by radioimmunoassay was observed.

[Properties of the human anti-IgG-IgG system in various modifications of immunoenzyme analysis]. / Svoistva sistemy anti-IgG-IgG cheloveka v razlichnykh modifikatsiiakh immunofermentnogo analiza.

The physico-chemical characteristics of EIA, and in particular sensitivity, accuracy, reproducibility, can undergo essential changes with variations occurring in the course of the experiment, which makes it necessary to optimize conditions at each stage of the assay. For this purpose the human anti-IgG-IgG system has been studied in two modifications of EIA: the sandwich and competitive methods.

[Immunoenzyme method of sequential saturation for determining antigens using the model of insulin]. / Immunofermentnyi metod posledovatel'nogo nasyshcheniia dlia opredeleniia antigenov na modeli insulina.

The method for the determination of insulin by means of the enzyme immunoassay, based on the use of insulin-peroxidase conjugates, has been developed. In this assay the scheme of the successive saturation of the active sites of antibodies is used. The antigenic properties of two conjugates differing in the method of their preparation are compared. The conjugates were obtained by the covalent binding of peroxidase, oxidized in its carbohydrate component, with insulin (conjugate 1) or hexamethylene-diamine-modified insulin (conjugate 2). The conjugates represented a mixture of oligomers differing in their molecular weight. Conjugate 1 possessed higher affinity to antibodies and higher enzymatic activity than conjugate 2. The method for evaluating the quality of antisera to insulin used in the assay has been proposed. The time of the insulin assay is 5-16 hours, the limit of insulin detection is 5 microU/ml, the variation factor is 3-12%.

[Determination of the subclasses of monoclonal human immunoglobulins G and of the light chain type with the aid of specific antisera]. / Opredelenie subklassov monoklonal'nykh IgG cheloveka i tipa legkikh tsepeí s pomoshch'iu spetsificheskikh antisyvorotok.

Sera of 86 patients suffering from G-myeloma were studied for the purpose of determination of subclasses of monoclone IgG. Investigations were carried out by means of antisera to subclasses IgG by the double diffusion method in gel after Ouchterlony. The following distribution of myeloma Ig was revealed: G1--70%, G2--17%, G3--11%,and G4--2%. In typing of the light igG chains by the method of immunoelectrophoresis, using antisera to the light chains of immunoglobulins of the chi and lambda type it was found that IgG1 chi was encountered more frequently than IgG1 lambda (3:1 ratio). The amount of the sera with the IgG2, IgG3, and IgG4 was insufficient for the reliable conclusion of their distribution by the type of light chains.

[The Homogeneous immunocofactor method of determining insulin]. / Gomogennyi immunokofaktornyi metod opredeleniia insulina.

An immunocofactor quantitative method of measuring insulin in solution was developed. The method uses antibody competitive binding of free and NAD labeled insulin. The NAD-insulin conjugate was obtained by covalent binding of the C(6)-aminogroup modified cofactor with insulin by means of soluble carbodiimide. To determine the NAD-insulin conjugate, which was not bound with antibodies, in the presence of antibodies and free insulin, an enzymic system of the cofactor regeneration with conjugated substrates of horse liver alcohol dehydrogenase, cyclohexanol and p-nitroso-N,N-dimethyl aniline, was employed. The sensitivity of insulin assay was about 5.10(-7) M.

[Comparison of luciferase activity of homogenates from fireflies of different species]. / Sravnitel'noe izuchenie liutsiferaznoi aktivnosti gomogenatov raznykh vidov svetliakov.

The stability and catalytic properties of enzymes from fireflies of three species - Lucida mingrelica, Lucida mongolica and Lampris sp.-were investigated. The enzyme of the homogenate from Lampris sp. was found most stable. The enzyme specific activities of the three species differed greatly. The specific activity of the homogenate from Luciola mingrelica was 7 times higher than that from Lampris sp. The Km constants with respect to Mo ATP and luciferine were identified for each enzyme.

[Quantitative determination of immunoglobulin A in human serum by an immunoenzyme technic]. / Kolichestvennoe opredelenie immunoglobulina V v syvorotke krovi cheloveka metodom immunofermentnogo analiza.

A complex of immunoglobulin A (IgA) with peroxidase was obtained as a result of interaction of IgA with the enzyme, modified with sodium periodate. The molar ratio of IgA/peroxidase was equal to 1 in the complex, as shown by gelfiltration and sedimentation analysis. Peroxidase maintained 75% of the initial activity in the complex. The antigenic and enzymatic properties of the complex were retained completely within 5 months, if the preparation was kept at 4 degrees in phosphate buffer, pH 7.4. The complex isolated was used for quantitative estimation of IgA in human blood serum. The method is based on the reaction of competitive binding between free and labelled with peroxidase IgA molecules and antibodies, immobilized on inorganic porous carrier--sylochome. The method enables to estimate quantitatively IgA concentration within the limits of 102=105 ng/ml.

[Various causes of decreased aldehyde dehydrogenase activity in the rat liver and blood in chronic alcoholic intoxication]. / Nekotorye prichiny umen'sheniia aktivnosti al'degiddegidrogenazy pecheni i krovi krys pri khronicheskoi alkogol'noi intoksikatsii.

Dynamics of accumulation of alcohol- and aldehyde dehydrogenases inhibitors in liver tissue of rats with chronic alcohol intoxication showed that content of the bioinhibitors of protein nature was increased during the animals alcoholization, whereas inhibition of aldehyde dehydrogenase was more distinct as compared with alcohol dehydrogenase. When the nature of aldehyde dehydrogenase inhibitor was studied using the immunoenzyme analysis, the inhibitor proved to be an autoantibody, produced in chronic alcoholization of rats as a result apparently of the enzyme modification. Titre of specific antibodies to "autoantigen" (aldehyde dehydrogenase from liver tissue of alcohol consuming rats) was 12-16-fold higher in liver tissue and blood serum of rats with alcoholism as compared with the corresponding preparations of control animals. An immuno-enzymological mechanism, responsible for an increase of acetaldehyde content in blood developed after ethanol consumption in alcoholism, is discussed.

[Synthesis of conjugates of L-lysine alpha-oxidase with antibodies]. / Poluchenie kon''iugatov L-lizin-al'fa-oksidazy s antitelami.

The conjugation of the drugs with vector molecules enables to obtain therapeutic preparation, which may be transported to the selected target organ. In the present work the methods of conjugation of antineoplastic enzyme L-lysine alpha-oxidase with antibodies were elaborated. Conjugates were worked out through the attachment of amino groups on the antibody surface either with the aldehyde groups which were created in L-lysine alpha-oxidase molecule (0.2% of initial enzymatic activity) or with the aldehyde groups of cross-linking molecules. Maximal (78%) L-lysine alpha-oxidase activity in conjugates was observed when oxidized peroxidase which contained the aldehyde groups was used as crosslinking agent. The glutaraldehyde method yielded 70% of initial enzyme activity.

[Enzyme-linked immunosorbent assay for determination of cyclosporin A in whole blood].

A test-system based on enzyme-linked immunosorbent assay (ELISA) for quantitative determination of cyclosporin A (CSA) in human whole blood has been developed. The detection limit of the method was 25 ng/ml, the linearity of the method in the concentration range 60-1400 ng/ml varied from 94 to 105%, the variation coefficient did not exceed 8%. The novel method exhibited good correlation with radio-immunoassay and polarization fluoroimmunoassay methods; the linear regression coefficients were 0.965 and 0.983, respectively. The developed test system is stable for at least 9 months when stored at 4 degrees C and can be used in clinical practice.