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Aptamers are single-stranded nucleic acids, typically 20-80 nucleobases (nb) in length, which can bind different compounds with high affinity and selectively. Their ligand-binding properties can be attenuated by adding short complementary strands. These interactions open new opportunities for aptamer-based assays. Strong dependence between the length and electrophoretic mobility of short nucleic acids makes polyacrylamide gel electrophoresis a powerful tool for studying their complexes. The interactions between the 36 nb DNA G-quadruplex aptamer (5’-GAT-CGG-GTG-TGG-GTG-GCG-TAA-AGG-GAG-CAT-CGG-ACA-3’) specific to ochratoxin A and 9 complementary single-stranded DNA (ssDNA) were studied. The length of ssDNA varied from 5 to 9 nb. To maintain ligand-binding conformation of the aptamer, a high ionic strength buffer was used. The best resolution between the aptamer and its complex was provided for the gel with 15% monomer and a monomer/cross-linker ratio of 15:1. Bands of free aptamer and ssDNA were observed for all studied variants. If the ssDNA length was less than 9 nb, the position of the aptamer’s band remained unchanged, independent of the aptamer/ssDNA ratios, and additional bands did not appear. The longest ssDNA (5’-CGC-CAC-CCA-3’) did not lead to the appearance of a new band, but it slowed the aptamer’s migration depending on the ssDNA concentration. Under a 27-fold excess of the given ssDNA, the relative mobility of the aptamer band changed from 0.566 to 0.468. Thus, electrophoresis visualizes aptamer-ssDNA interactions and can be used in the development of aptamer-based analytical systems.

A new technique for immunochromatographic serodiagnosis of tuberculosis that is based on the interaction of specific anti-mycobacterial immunoglobulins from blood serum with label—antigen conjugate and subsequent binding of the formed complex with the Staphylococcus aureus protein A immobilized on a test-strip has been suggested. Unlike the conventional technique in which all the immunoglobulins in a sample react with the label—immunoglobulin-binding protein conjugate, and then the labeled specific antibodies interact with an immobilized antigen, this technique prevents binding of the label to immunoglobulins that are not specific to Mycobacterium tuberculosis. The suggested assay was implemented using the recombinant 38 kDa (Rv0934) protein of M. tuberculosis as an antigen and colloidal gold as a label. It was experimentally shown that the proposed technique allows to increase the percentage of detection for seropositive serum with low concentrations of the specific antibodies to the M. tuberculosis cause.

The development of sector of "fast-testing" i.e. test-systems permitting carrying out analysis during home visit of doctor or at primary examination of patient without any additional devices and reagents is predominant tendency in international practice. The immunochromatography is an effective technical solution in out-laboratory diagnostic, which nowadays is actively applied in controlling hundreds of diagnostically significant markers of infectious diseases, metabolic and functional disorders. However, common immunochromatography is focused on qualitative visual evaluation of results of study i.e. conclusion on presence or absence of coloration of particular zones of test-band. Therefore, the technical solutions retaining such merits of immunochromatography as expressness and technical simplicity and at the same time providing objectivity of diagnostic and increasing its informativeness are extremely in demand. The review considers main methodical solutions and tendencies of their practical implementation targeted at device documentation, processing and interpretation of results of immunochromatography analysis. The optical systems of registration in visible area of spectrum dominating in assortment of modern detectors are presented. The new solutions oriented on working with fluorescent, magnetic and electroconductive markers are presented too. The perspectives of further development of this direction are characterized including application as detectors of domestic communication devices and formation of cloud data bases for storage and processing of information concerning results of examinations.

An immunochromatographic test system was developed for the detection of T-2 toxin (T2T), which is one of priority contaminants of cereals. The detection is based on the competition between T2T in the sample and the T2T-protein conjugate immobilized on the test strip for the binding to the complexes of anti-T2T antibodies with gold nanoparticles serving as the marker. The results of the competition are recorded as the coloration in the test zone of the test strip produced by the marker. The optimum dilution of the sample for the reliable high-sensitivity analysis corresponds to the final methanol concentration equal to 20%. The deceleration of the movement of reactants along the test strip due to the use of additional membranes impregnated with 10% BSA resulted in the decrease in the detection limit of T2T. The test system was examined for the detection of T2T in water-methanol extracts of maize grains. The disappearance of the color in the test zone, which attests to the presence of mycotoxin, was observed for grain samples containing T2T at a concentration of 53 μg/kg or more (the final T2T concentration in the immunochromatorgaphic assay is 3 ng/mL). The video-digital detection limit of T2T is 16 μg/kg (0.9 ng/mL). The duration of the assay is 15 min. The results of the present study suggest that the developed test system is suitable for the control of the maximum allowable T2T content.

A test system based on immunochromatography in the sandwich format and intended for express detection of Helicobacter pylori antigens has been developed. Contact of a sample with a test strip coated with immunochemical reagents triggers the movement of the liquid along the membrane components of the test strip, immunochemical interactions, and the formation of detection zones stained by gold nanoparticles. The concentration and kinetic dependences of the immunochemical interactions have been characterized. The reagent and membrane composition of the test system has been selected to provide a minimal detection limit. The detection of H. pylori cell wall antigens at concentrations as low as 0.3 μg/mL in aqueous solution and a suspension of a clinical sample of feces has been demonstrated; the assay duration was 10 minutes. Staining enhancement by the addition of silver salts allowed for a further reduction of the detection limit to 0.03 μg/mL. The developed test system can be used for field diagnostics.

A rapid method for detection of the surface lipopolysaccharide antigen and the cells of the causative agent of bovine brucellosis was developed. The method represents a sandwich format immunochromatographic assay. The contact between the sample and the test strip with immobilized immunoreagents initiates the fluid movement along the membrane components of the test strip, immunochemical reactions, and the formation of colored bands. The novel method requires 10 minutes to determine the lipopolysaccharide antigen of the cell wall of the brucellosis causative agent at concentrations down to 10 ng/mL and the Brucella abortus cells at concentrations down to 10(6) cells/mL (5 x 10(4) cells in the sample). The specificity of the immunodetection was confirmed. The designed test system can be used for the rapid field diagnosis of brucellosis in cattle.

An immunochromatographic method for determination of beta-lactam antibiotic ampicillin has been developed. The method is based on the competitive interaction between antibiotic molecules contained in the sample and protein conjugate of penicillin immobilized on a membrane for binding with specific antibodies labeled with colloidal gold, which occurs during movement of the sample to be tested and reagents along the membrane. The completion of the test system ensures control of exceeding the maximum permissible content of the antibiotic in milk and dairy products (10 ng/ml). The possibility of testing milk, raw milk, and dairy products for 10 minutes at room temperature without sample preparation has been demonstrated.

The review is devoted to comparative characterization of immunochemical methods of detection of mycotoxin, which belongs to one of the priority groups of the food contaminants. It has been shown that the high specificity and the possibility of mycotoxin detection in low concentrations combined with existent diverse equipment allow for considering the immunochemical methods of analysis to be the most promising for wide practical application. The analytical characteristics of the existent developments are presented; the merits and demerits of the different kinds of immunoanalytical systems are compared.

An enzyme immunoassay technique for the detection of sulfamethoxypyridazine in honey, developed using rabbit polyclonal antibodies raised against N-sulfonyl-4-aminobutyric acid, which contains a structural group characteristic of sulfonamides, is proposed. Under the optimized conditions, the sulfamethoxypyridazine detection limit was 0.05 ng/ml, with the entire analysis procedure taking 2 h. In total, 24 honey samples were tested using the protocol based on tenfold dilutions of samples without their preliminary treatment.

The approaches for high sensitive and specific determination of nonylphenol (NP) with the help of immune enzymatic (ELISA) method have been developed. The process of preparation of conjugates of NP with proteins, antiserum obtaining, purification of immunoglobulin (Ig) fractions and study of specificity of the obtained antibodies were described in detail. It was shown that the antiserum and total Ig fraction do not differ in respect of specificity and binding abilities. The developed algorithm of ELISA method fulfillment is able to provide NP revealing with the sensitivity in the range of 20-50 ng/ml and working controlled concentrations--from 20-50 to 1000 ng/ml.

Express immunochromatographic test-strip assays were developed for detection of five plant viruses varying in shape and size of virions: spherical carnation mottle virus, bean mild mosaic virus, rod-shaped tobacco mosaic virus, and filamentous potato viruses X and Y. Multimembrane composites (test strips) with immobilized polyclonal antibodies against viruses and colloidal gold-conjugated antibodies were used for the analysis. The immunochromatographic test strips were shown to enable the detection of viruses both in purified preparations and in leaf extracts of infected plants with a sensitivity from 0.08 to 0.5 microg/ml for 10 min. The test strips may be used for express diagnostics of plant virus diseases in field conditions.

An enzyme immunoassay with colorimetric detection of sulfamethoxipyridazine (SMP), the most widely used sulfamide, was developed with the soybean anionic peroxidase as an enzyme marker. The range of SMP detection is 1.3-63.0 ng/ml with a detection limit of 0.4 ng/ml. The root square deviation of detection results did not exceed 6%. It was demonstrated that 0.15% casein added to the working buffer prevented the effect of the milk matrix on the detection. The results obtained demonstrate that the assay developed is promising, displaying a sensitivity that exceeds the maximum permissible concentration of sulfamides in milk (100 microg/l) by several orders of magnitude.

A method of the competitive immunochromatographic assay of the pesticides 2,4-D (2,4-dichlorophenoxyacetic acid) and simazine (2-chloro-4,6-bis(N-ethylamino)-1,2,5-triazine) in aqueous samples was developed. Monoclonal antibodies to these pesticides labeled with colloidal gold were used to visualize the results. The sensitivity of the 2,4-D and simazine assay is 12 ng/ml, and the time of analysis is 3-7 min. The method does not differ in sensitivity from the competitive EIA using conjugates of monoclonal antibodies to the pesticides with horseradish peroxidase; however, the time of the EIA is 1.5 h. The immunochromatographic method of the pesticide detection is available and simple and may be recommended for the development of assays of any other low-molecular compounds. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 2; see also http://www.maik.ru.

Mathematical models of three levels of complexity are proposed, describing the interactions between antibodies and polyvalent liposomal antigens. The models take into account the contribution of high-affinity bivalent complexes and clusters of several antigenic groups to the immunochemical reaction. The models were analyzed numerically at different values of thermodynamic and kinetic parameters of the antigen-antibody reaction. Conditions, under which models of different levels of complexity provide a satisfactory description of the antigen-antibody interaction, were determined. The adequacy of the results of modeling was confirmed experimentally in liposomal preparations differing from each other by the surface density of monovalent hapten (atrazine) conjugated with a lipid.

A portable optoelectronic reflectometric photocolorimeter was designed. The photocolorimeter implements two operating modes (transmission and reflection), and its performance was tested in two systems of testosterone immunoenzyme assay: microplate ELISA and membrane dot-ELISA. The detection threshold for microplate ELISA and membrane dot-ELISA was 0.1 and 0.6 ng/ml, respectively. The coefficient of correlation between the photocolorimeter readings and conventional photometric methods is r = 0.999. Relative standard deviation of the results of photocolorimetric measurements (n = 4) within the optical density range from 0.03 to 1.00 ranged from 3.4 to 0.7%. Simple and versatile design of the photocolorimeter make it appropriate for testing various substances both in laboratory settings and in the field.

Detecting labels based on water dispersions of colloidal textile dyes were developed that are useful in various analytical and diagnostic test systems for a simple visual assessment of the assay. Colored water-insoluble particles of dyes were used for the sorptional immobilization of streptavidin on their surface. The resulting streptavidin-dye (STR-DYE) complexes possessed a high visualizing capacity and were used for the combined detection of pesticides (simazine and 2,4-dichlorophenoxyacetic acid) by noninstrumental immunoassay (DYE-comb-assay, competitive dot-immunoassay in the comb format). The detection limits and the duration of our DYE-comb-assay (4 ng/ml, 20-25 min), HRP-comb-assay (competitive dot-immunoassay in the comb format using the enzymic conjugate of STR with horseradish peroxidase) (16 ng/ml), and the traditional competitive ELISA (12-16 ng/ml, 1.5 h) were compared. This DYE-comb-assay is simple enough and can be used under field conditions.

Equilibrium and kinetic constants were determined for interactions between alpha-amylase from Bacillus subtilis and polyclonal antibodies (with immobilization of either reagent). The effects of desorption of immobilized molecules and intermolecular complexes on the immunochemical reaction were studied. Models of sandwich enzyme immunoassay, whereby complexes of immobilized antibody-determined antigen-labeled antibody were formed, were developed from these results. The results obtained from the model and experiments were compared. The desorption was shown to cause the hook-effect, that is, a decrease in the label binding at high concentrations of the antigen.

The cytokinin family includes biologically active polypeptide molecules secreted by haemopoietic and immunocompetent cells which control cell proliferation and differentiation. Cytokinin interactions with specific receptors of the cell surface results in oligomerization of these receptors, i.e. in association of two or more membrane molecules. It is becoming obvious that oligomerization of receptors is an indispensable stage in the manifestation by cytokinins of their biological activity. In this context, studies of regularities of cytokinin receptor interactions resulting in receptor oligomerization is important for both elucidation of molecular mechanisms underlying kinin action and construction of compounds having the properties of agonists (or antagonists) of cytokinin-induced oligomerization of membrane receptors. A conclusion is draw about the important role of polyvalent cytokinin interactions with cell receptors in the initiation of oligomerization and subsequent formation of functionally active receptor complex.

Competitive solid-phase enzyme immunoassays for the detection of 2,4-dichlorophenoxyacetic (2,4-D) and 2,4,5-trichlorophenoxyacetic (2,4,5-T) acids have been developed and optimized. The sensitivity of the assay is 3 ng/ml for 2,4-D and 5 ng/ml for 2,4,5-T. The time of the assay is 1.5 h. The sensitivity of the assays increases after immobilization of antibodies on staphylococcus protein A, in the case of using monovalent derivatives of antibodies, and as a result of chemical modification of hapten. The detection limit for the pesticides is 0.1 ng/ml in this case, which is close to the sensitivity of the assay for the haptens conjugated with proteins.

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.

A simple and convenient technique has been developed for detection of beta-galactosidase from E. coli on nitrocellulose sheets using a mixture of 5-bromoindol-3-yl-beta-D-galactopyranoside and nitro blue tetrazolium, which enables rapid detection of fmole (10(-15) mole) quantities of the enzyme at pH 9.5. The technique has the following advantages: the substrates are stable for a long period; reaction products give non-fading intense blue colour, resolution is extremely good with essentially no diffusion.

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.

As revealed by double immunodiffusion and the competitive immunoenzyme assay, the immunization of animals with ribosomes isolated from group A streptococci leads to the formation of antibodies to M-protein and does not lead to the formation of antibodies to A-polysaccharide and lipoteichoic acid, which is seemingly caused by the presence of M-protein in ribosomes. Antiribosomal serum has permitted the detection of more antigenic determinants in M-protein than commercial antiserum to homologous M-protein. The immunoenzyme assay for the quantitative determination of ribosomes has been developed.

The behaviour of an immunochemical analytical system based on the sequential binding of determined and labeled antigens to the specific antibodies (saturation analysis) was investigated by means of computer modelling. Using the mathematical model, optimal ratios of reagent concentrations and duration of immunochemical reactions were determined. To get satisfactory calibration curves the incubation time of the reaction with labelled antigen should be approximately equal to 0.1 k-1, where k-1 is a kinetic constant of dissociation of the antigen-antibody complex. Changes in the shape of the calibration curves caused by the presence of two populations of antibodies with different affinities for antigen and by positive cooperativity in the antigen binding to two binding sites of a single antibody were analysed. A satisfactory correlation between experimental and calculated results was illustrated by the ELISA of insulin.

Some regularities of the enzyme immunoassay (EIA) of whole bacterial cells have been studied on one of the bacillary species of contaminant microflora. Early detection of this microorganism is highly important for the microbiological production of alpha-amylase and alkaline protease (produced by Bacillus subtilis). The effective kinetic and equilibrant parameters of the interaction of peroxidase-labeled antibodies with the cells of the contaminant microflora in the solution and on the surface of the polystyrene plates have been defined. Two methods for the separation of cells after their interaction with peroxidase-labeled antibodies have been optimized: filtration involving the use of special filter plates and centrifugation in plates. The method for the immobilization of cells in the wells of standard assay plates by centrifugation has been proposed. Four EIA methods for measurement of contaminant microflora have been developed and optimized. These methods permit the determination of the microflora at concentration of 5 X 10(5)-5 X 10(4) cells/ml, depending on the scheme of the assay, within 1-3.5 minutes.

The interaction between immobilized antibodies against human immunoglobulin G (IgG) and the immunoenzyme complex IgG-peroxidase (IgG-P) was studied. The complex was obtained by covalent binding of IgG to peroxidase modified by sodium periodate. Study of the IgG-P binding kinetics and dissociation of the antibody-(IgG-P) complex showed that the antibodies immobilized on Sepharose reversibly interacted with IgG-P, similar to the antigen-antibody reaction in solution. The efficient values of the binding constants for the antibodies binding to Sepharose covalently and through the antigen-antibody bond are (2,2+/-0,5) 10(8) M-1 and (4,2+/-0,2) 10(8) M-1, respectively. The nature of a carrier and the immobilization method used do not significantly affect the rate of the complex binding to the antibodies. The activation energy of the reaction of IgG-P binding to the antibodies immobilized on Sepharose covalently and through the antigen-antibody bond is 7,3 and 4,1 kcal/mole, respectively. A procedure of titration of immobilized antibodies active sites with the antigen-enzyme complex is discussed.

A technique for quantitative determining horseradish peroxidase was developed using an amperometric sensor that makes it possible to register up to 50 pM of the enzyme in the reaction of the peroxidase oxidation of iodide ions by hydrogen peroxide. The sensor, composed of combined carbon and Ag/AgCl electrodes, is intended for enzyme immunoassay and may be used for registering the results in the very wells of the polystyrene plates widely used in clinics and laboratories. The dependences of the initial rate of the enzymatic reaction on the substrate concentration and pH of the medium were determined, on the basis of which the conditions for the optimal operation of the sensor were found. The amperometric and spectrophotometric methods of registration on a model system for determining antibodies against X-virus of potato were compared in order to show a possibility of the sensor applying to enzyme immunoassay.

To control the effectiveness of vaccination against influenza, the optimum conditions for making the enzyme-linked immunosorbent assay (ELISA) with a view to determine the level of anti-influenza antibodies in human blood sera have been established. The kinetics of influenza virus adsorption in the wells of ELISA polystyrene plates and the kinetics of the interaction between the immobilized antigen and species-specific peroxidase-labeled antibodies have been studied. The method has been shown to be more sensitive than the hemagglutination inhibition test in the determination of seroconversion in persons immunized with influenza vaccine.

Studies aimed at the development of a variant of ELISA for the determination of hepatitis B virus surface antigen (HBsAg) showed the assay to be most effective when a modified periodate method of conjugation of highly purified horseradish peroxidase with the IgG-fraction of antiserum to HBsAg was used. With the resulting conjugates, the "sandwich"-test on polystyrene solid phase could detect HBsAg in concentrations up to 5 ng/ml which is several thousand-fold higher in sensitivity than counter immunoelectrophoresis, one order of magnitude more sensitive than the passive hemagglutination test, and comparable with the radioimmunoassay (RIA). The specificity of the method was confirmed by positive results of the neutralization test. By this method, HBsAg in the sera of hepatitis B patients was detected as frequently as by the RIA.