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Aim: Construction of an immunologic test-system for detection of causative agents of enteropathogenic Yersinia (Yersinia pseudotuberculosis and Y. enterocolitica) by dot-immunoassay., Materials and Methods: Nenoparticles of colloid silver sized 5-9 nm were used as a marker of specific antibodies. IgG fraction was isolated from commercial antisera to Y. pseudotuberculosis (Ο:1) and Y. enterocolitica (Ο:3 and Ο:9). Testing of the obtained test-system was carried out on 20 strains of Y.pseudotuberculosis and Y. enterocolitica (10 of each species)., Results: Dot-analysis had a specific character and detected enteropathogenic Yersinia at a level of 5-10⁵ - 8.10⁶ CFU/ml (100 - 1000 CFU in sample). Wherein cross-reaction with heterologic studied microorganisms - Escherichia coli, Salmonella typhimurium, Shigella flexneri, Yersina pestis EV - as not observed. A possibility of simultaneous detection and serotyping of Y. enterocolitica is shown, that is necessary for confirmation of their epidemic significance., Conclusion: The developed test-systems allow to study micro volumes of the samples under study (1 μ1), are express (1.5 - 2h), highly sensitive and specific, technically simple and do not require the use of high-cost equipment, special training of the staff, may be successfully used in practical healthcar in laboratories with varying equiptment levels.

The comparative evaluation was carried out concerning systems of detection using conjugates on the basis of peroxidase, alkaline phosphatase, colloid gold and system of amplification “Super-CARD" for multiplex dot-immune assay of antibodies. It is established that sensitivity of system of detection with colloid gold is approximately 8 times higher than with amplification “SuperCARD"; 30 times higher than with conjugate of alkaline phosphatase and 250 times higher than with peroxidase conjugate. The immunesols of gold maintain limit of detection of human IgG 10 pg with dynamic range of signal alteration from 5 ng to10 pg overlapping the range of concentrations of specific IgG in case of typical natural immune response. The most specific results provides conjugate of colloid gold with monoclonal antibodies to human IgG.

Affinity haemoadsorbents based on WY, WTY, WNY ligands and polysaccharide matrix were developed for the human immunoglobulin G binding. The characteristics of new sorbents such as the binding of total IgG and binding of IgG subclasses were compared. It was found that all new sorbents well extract the IgG from the blood plasma. It was evidenced that WNY-based sorbent is more effective for binding of IgG subclass 3. The determination of physic-chemical characteristics of IgG binding revealed that desorption constants for IgG are 10 ± 3, 28 ± 4 and 13 ± 3 µM for WY, WTY, WNY based sorbents respectively. Maximum sorption capacities for IgG are 43 ± 2, 45 ± 3 and 46 ± 3 mg IgG per ml of sorbent for WY, WTY, WNY based sorbents respectively. Also it was shown that the new sorbents are compatible with blood and are suitable for the medical purposes.

The use of proteomic analysis to find potential diagnostic biomarkers is limited by the presence of serum albumin (HSA) and immunoglobulin (IgG) at high concentrations in patients' blood; these substances impede the detection of serum proteins with similar molecular weights. Recombinant HSA- and IgG-binding polypeptides are used as ligands in creating sorbents for complete removal of the proteins by affinity chromatography. The binding specificity of the sorbents for HAS and IgG is higher than that of the conventionally used antibodies. A composite sorbent enabling the depletion of HSA and IgG from serum by single-step affinity chromatography is obtained. The. developed sorbents were used to prepare serum for proteomic analysis.

Immunoelectrophoresis and immunodiffusion analysis with antibodies to whole intact cells of the type strain of nitrogen-fixing soil bacteria Azospirillum brasilense Sp7 revealed at least three conservative surface immunogenic proteins of azospirilla. Cross-reactions with these proteins made it possible to use the above antibodies for detection of azospirilla as a genus-specific probe conjugated with horseradish peroxidase as an enzymatic label. Direct immune-enzyme analysis of soil suspensions (typical chernozem, Saratov oblast) confirmed applicability of the conjugates based on genus-specific antibodies to the surface proteins of azospirilla for direct detection of this bacterial genus in environmental samples. These results provide a basis for broad application of this method for analysis of Azospirillum occurrence in soil.

A novel technique for preparation affinity sorbent based on tyramine and tryptamine was proposed. It was shown that tryptamine-Sepharose and tyramine-Sepharose effectively bind IgG, IgA, lipoprotein (a) (Lp(a)) and low density lipoproteins (LDL) from blood plasma. The sorption capacity is 4-9 mg of IgG, 2-4 mg IgA, 3-5:mg of Lp(a) and 5-7 mg of LDL per mL of gel. It was found that new sorbents can bind Lp(a) and IgG as themselves or in a complex of Lp(a) with IgG. The existence of this complex may indicate the presence of anti-Lp(a) autoantibodies in the blood of some patients. The advantages of new sorbents are easiness of its synthesis and stability during use and storage. In practice they can be applied for medical and biotechnological purposes where it is necessary to bind Lp(a), LDL, IgG, IgA.

Impact of living organism on nonorganic materials, which are applied for the implants manufacturing, was studied. There was established, that organism of a recipient patient actively effects on exogenic material, destroying its superficial layer and forming the connective tissue capsule around the implant. The implant surface may start to dissolve selectively as a result of aseptic inflammation, keeping around, in such a situation it become necessary to remove the prostheses, which have lost their function. Impact of organism on surface of different implants of nonorganic origin and the ways of it prophylaxis were investigated. Instrumental method of the preoperative compatibility testing between a recipient organism and the implant material, using the atomic-power microscope, was proposed.

The thermodynamic parameters of the stabilization of the native structure C(H)2 domains of human myeloma immunoglobulin LOM and SIN second subclass and their firstly obtained hFc fragments were determined using the methods of scanning microcalorimenry. A decrease in the termal stability and energy stability of the native of these domains in the intact proteins correlated in the "phisiological" range of temperatures (20-37 degrees C), mainly, with entropy factor.

Human myeloma immunoglobulin second subclass LOM and SIN, their Fc fragment and firstly obtained hFc fragment in which there is not only low portion of the hinge region, but also its core portion (Cys-Cys-Val-Glu-Cys-Pro-Pro-Cys), have been studied by number of physical methods (scanning calorimetry, fluorescence spectroscopy, analytical centrifugation). Joint analysis of calorimetric and optical melting curves revealed that only first (low-temperature) heat absorption peak at all the melting curves corresponds to the melting of the two CH2 domains. It was shown that CH2 domains of intact IgG2 are destabilized relative to those domains in hFc and Fc fragments.

The antigenic properties of virus isolates of tobacco mosaic, cucumber mosaic, bean common and yellow mosaic, soybean mosaic, Y-, X-, and A-potato viruses, Vicia unijuga mosaic, affecting agricultural crops and wild plants in the Far East of Russia were studied. Universal immunodiagnosticums fit for testing the broad range of virus isolates have been obtained on the basis of the most antigen-active strains. They allow to struggle effectively against viruses rendering economic damage to agriculture.

New chromatographic material based on tryptophil-threonil-tirosine was prepared. This sorbent effectively binds human, sheep, goat and cow immunoglobulins G. New sorbent shows high selectivity for removing immunoglobulins from blood plasma. Effective sorption capacity is 15-25 mg of immunoglobulin G per ml of matrix. Optimal method of covalent attachment ligand to polysaccharide matrix allows achieving high stability of the sorbents in terms of use and storage. This sorbent can be used in medicine and biotechnology.

pFh fragments from the hinge region of human IgG3 Kuc and Sur can fold into compact form, resulting the formation of proteins with secondary (super-secondary) structure, which is represented almost exclusively double poly-L-proline helix. It was demonstrated by several methods that the thermal denaturation of compact form pFh fragment (hinge region) IgG3 Kuc and Sur occurs in two stages. The "two-state" model described the disintegration of the compact structure with preservation of the secondary structure (double poly-L-proline helix). In the second stage melts itself helix consisting of four cooperative units, which are formed by the sections with a high content of proline residues. Poliproline conformation of secondary structure and large number of disulfide bonds is responsible for high specific enthalpy of denaturation and high thermal stability.

It has been shown using scanning microcalorimetry and densitometry that partial specific heat and specific partial volume of two pFh fragments of IgG3 increase during the decay of its tertiary structure, the secondary structure, the double poly-L-proline helix, being unchanged. This effect may be explained by a high degree of hydration, which increases on globule decompactization due to increased accessibility of peptide groups of the helix to solvent.

A new method, which allows evaluation of the avidity of bivalent antibodies when they react with bivalent antigen, is suggested. The theory is based on the idea that was proposed by us earlier. That theory considered the relation between affinity constants for bi-molecular and mono-molecular reactions of the reagents, which either are connected or not connected by a flexible linker. Using the suggested theory it makes possible to calculate antibody avidity if the affinity and molecular structure of the antibody are known. The obtained by us theoretical results completely coincide with the experimental values of antibody avidity that are known from literature.

This study was devoted to the creation of biomagnetic sorbents, capable of removing the virus particles from the human blood serum. With this purpose specific globulins were isolated from reconvalescents sera after acute hepatitis B virus (HBV) and hepatitis C virus (HCV), which were later used as molecules-vectors (ligands). Ligands were covalently bound to magnetite encapsulated by the matrix, carrying aminopropyl groups on its surface. The dynamics of immunoglobulin sorption with magnet controllable bearer was studied. Sera which were positive by DNA HBV, by RNA HCVin polymerase-chain reaction (PCR) and by surface antigen HBsAg in immunosorbent assay (ELISA) were approbated with the purpose of virus separation by immunomagnetic sorbents. Completed virus decontamination of the serum was achieved according to results of PCR on DNA HBV, at the same time the results of ELISA testify to the presence of HBsAg in the tested samples of the serum, it can be associated with the presence of many defect virus particles in population or with the lack of biomagnetic sorbent for complete elimination. Results ofquality PCR on RNA HCV of sera after decontamination testify to the absence of complete HCV elimination.

The quantitative dependences of the immune complement-dependent lysis of a monodisperse suspension of 200-nm liposomes whose membrane was sensitized by monovalent hapten (2,4-DNP-epsilon-caproyl-DPPE) or a polyvalent antigen (LPS from F. tularensis) on the initial concentration of specific antibodies (IgG) to hapten and the antigen have been investigated. The quantity of antibodies binding sites on the surface of liposome was evaluated. The difference between the complement-dependent immune lysis of poly- and monodisperse suspensions of liposomes was shown. The experimental results are well described by the direct binding model.

The effect of precipitation of complexes of streptavidin with biotinylated proteins under conditions of simple (according to Mancini) and double (according to Ouchterlony) radial diffusion in agar gel was studied. The position and form of precipitation lines depended primarily on the initial concentration of components and the degree of protein biotinylation. Free biotin, 1% SDS, and 6 M urea contained in the gel, as well as thermal denaturation of streptavidin inhibited the precipitate formation. Mannose, glucose, fucose, galactose, sucrose, and NaCl at high concentrations had no effect on biospecific precipitation. A model of interaction of streptavidin with biotinylated macromolecules is suggested, which accounts for the observed effect, and the prospects of practical application of the precipitation effect are discussed.

Complex formation between immunoglobulins and ligands immobilized on mica was studied by atomic force microscopy in two different systems. In the first system, 60-kDa ligands possessing only one site for antibody recognition were used. In the other system, a more complex interaction of human immunoglobulin with immobilized polyclonal antibodies was studied. In both systems, specific complexes with proper ligand appeared, and unspecific interaction was not detected. The method of revealing immunocomplexes by image atomic force microscopy can be used in the development of modern diagnostic systems.

The effect of immunocortin, an ACTH-like decapeptide VKKPGSSVKV corresponding to the 11-20 sequence of the variable part of the human IgG1 heavy chain on the content of 11-hydroxycorticosteroids (CS) in rat adrenal glands and blood serum in vivo was studied. An intramuscular injection of immunocortin at a dose of 10 microg/kg was found in an hour to induce a twofold decrease in CS content in the adrenal glands and a 1.8-fold increase in the blood serum CS content. At the same time, an immunocortin dose of 100 microg/kg exerted practically no effect on the CS content and its dose of 1000 microg/kg increased the CS content both in adrenal glands and in blood serum by 1.6 and 2.2 times, respectively. Four hours after the injection of any of the three doses of immunocortin, the CS content in adrenal glands did not differ from the control value, and after 24 h the content decreased threefold. Immunocortin was shown to be bound by the ACTH receptors in the membranes of the rat adrenal cortex with a high affinity and specificity (inhibiting the specific binding of 125I-labeled ACTH-(11-24) peptide with Ki of 1.2 nM).

An efficient procedure for obelin conjugation with immunoglobulins was developed. The possibility was shown of using the resulting conjugates instead of a radioisotope label for the immunoassay of thyroid stimulating hormone in sera; the conjugates provide a sensitivity of 0.01 microIU/ml. The results of bioluminescent immunoassay (sera of 34 patients) satisfactorily correlate with the results of radioisotope assay (R 0.99).

Optimal conditions of protein scanning by atomic force microscopy were developed. Proteins of different molecular masses (950-11.5 kDa) and different three-dimensional organization were used to investigate the structural features of proteins. The most distinct images of proteins were obtained using a tip with the free amplitude in the range of 5-15 nm and with the set-point amplitude in the regime of repulsion from the sample. The method allowed one to clearly recognize the structural details of large molecules such as immunoglobulins IgM and IgG1 and Ricinus agglutinin. The revealing of the structural properties of proteins with molecular masses of 60 kDa and less was limited by the sharpness of probe tips used in the present study. It was shown that, by a quantitative analysis of the geometric parameters of molecules, it is possible to distinguish IgG1, Ricinus agglutinin, and ricin.

Methods of analysis of inhibition of complement system in vitro and in vivo have been developed for study of effects of medical drugs on the complement. The first one, ELISA method, for determination of inhibition of the first stage of complement activation includes binding of C1q subcomponent to immunoglobulin. The second method is based on capacity of mink serum to kill mice at the intravenous administration due to the action of mink complement. The effects of heparin, known anticoagulant, and suramin, used for treatment of trypanosomiasis, have been studied using these systems. The inhibition constants of binding suramin and heparin binding evaluated by the first method C1q were 411 +/- 29 micrograms/ml (or 0.287 +/- 0.020 mumole/l) and 36.4 +/- 1.7 micrograms/ml (or 2.28 +/- 0.10 mmole/l), respectively. This indicates that heparin binding with C1q in 10 times is higher, than that for suramin (as weight ratio) or 100 times higher in molar ratio. Administration of 3 mg of suramin or 0.3 mg of heparin to mice protected them against lethal action of intravenously injected 0.08 ml of mink serum. Blood concentrations of these compounds approximately correspond to inhibition constants for C1q binding, obtained using in vitro method.

It was found in the experiments in vivo and in vitro that the contact of perfluorocarbon emulsion stabilized with proxanol 268 with blood plasma leads to the sorption of various plasma proteins on the surface of emulsion particles. The profile of the proteins sorbed is complex and includes proteins with molecular weights ranging from 14 to 94 kDa. Among proteins sorbed on the emulsion particles circulating in blood, IgG was identified. Incubation of the emulsion stabilized with proxanol 268 with human blood plasma in vitro was shown to result in the sorption of IgG and IgA the perfluorocarbon particles. The sorbtion of serum proteins and immune complexes circulating in blood on the surface of perfluorocarbon particles stabilized with proxanol 268 was revealed to activate the complement system.

By spin labeling the monoclonal IgM and normal IgG at the carbohydrate moiety with 2,2,6,6-tetramethyl-4-aminopiperidine-1-oxyl, preparations were obtained whose ESR spectra indicate rapid exchange spin-spin interactions between two spin labels. It was shown that, in the case of spin-labeled IgM, this spectrum is determined by a glycopeptide noncovalently bound to IgM, which incorporates two spin labels.

The inhibition of covalent binding of the nascent C4b fragment of the human complement component to its natural target, immunoglobulin G, was studied. To this end, an immunoenzyme system was developed. In this ELISA method, the complement was activated on the sorbed IgG molecules and the resulting nascent C4b fragment acylated IgG or interacted with a competitive inhibitor added to the system. The inhibition constants for binding of the nascent C4b to its target were determined for immunoglobulins G1, G2, G3, G4, M, and A1, as well as for ferritin, yeast mannan, capsid polysaccharides of the Neisseria meningitidis A, B, and C serotypes, diphtheria anatoxin, epinephrine, and salicylic acid. On the basis of the experimental data, the immunoglobulin role at the activation stage of the complement regulation cascade, the relationship between the antigen immunogenicity and its ability to interact with C4b, and the direct effect of a number of therapeutic agents on the complement system were discussed. Lectins of various specificities were shown to inhibit the enzymic activation of C4 by the first complement component and the subsequent C4b sorption to its target, which allowed us to suggest that some oligosaccharide fragments of the C1s and C4 molecules are spatially close to the C1s active site and to the thioester bond of C4.

The fractional composition of commercial immunoglobulin was studied by the method of gel filtration on ultragel AcA-34 and the possibility of its use for the calibration of a chromatographic column was shown. The fractionation of a specimen of IgG revealed the presence of 4 fractions. Their molecular weights corresponded to dimers, monomers, Fab fragments and low-molecular peptides. The qualitative fractional composition of the preparation was shown to be stable during 3 years of storage, as well as after keeping the preparation at 37 degrees C for 1 month. The attested characteristic of each IgG fraction, the distribution coefficient (Kav), was established. The use of a specimen of immunoglobulin obtained with the use of the modified Cohn's method--as calibrant will make it possible to calibrate the columns for a shorter period and to control the correctness of the analysis.

Exposed lysine residues of human IgG were modified by a spin-label, 2,2,5,5-tetramethyl-3-male-imidopyrrolidine-1-oxyl at pH 9.2. Under these conditions, the degree of modification was about 10 lysine residues per protein molecule. The ESR spectrum of the spin-labeled immunoglobulin was much more mobile than that of spin-labeled immunoglobulin with the modification degree of about 1 residue that was obtained at pH 7.0. Thus, the sharp increase in the modification degree due to the increase in pH by two units leads to a marked loosening of the tertiary structure of the protein in solution, which is just indicated by the mobile ESR spectrum. Lithium chloride added to the solution of spin-labeled immunoglobulin induces a similar "immobilization" of its ESR spectra as sucrose.

Heat denaturation of glycosylated and aglycosylated human immunoglobulin G1 Fc fragments was investigated by differential scanning microcalorimetry. The enthalpy of the interaction between aglycosylated CH2 and CH3 domains is significantly reduced at 37 degrees C (but not at 0 degree C) as compared to the glycosylated form. The temperature dependence is consistent with the data on restricted proteolysis by trypsin.

The authors examined the possibility of replacing immunoperoxidase conjugates with silver sol (mean diameter of particles 9 nm) adsorption-bound to antispecies IgG in solid-phase enzyme immunoassay in microtitration plates. Experiments with commercial test systems Antigen and Recombitest-antiHIV-1,2 manufactured by the Vektor State Research Center of Virology and Biotechnology, Ministry of Health and Medical Industry of the Russian Federation, showed that the sensitivity of detecting anti-HIV with silver immunosols and boosting of the signal with physical development was no less sensitive than standard EIA with immunoperoxidase conjugate. Preparation of silver immunosols is simple, easily reproducible, and is based on sparing and economic use of immunoreagents.

Human blood plasma fibronectin immobilised on agarose in physiological interacts with soluble myeloperoxidase (Kd = 2.43 mM). Interaction of myeloperoxidase with fibronectin adsorbed on immobilised gelatin a, natural fibronectin ligand, resulted in formation more stable complex (Kd = 0.94 mM). The presence of thermoaggregated (but not native) IgG in the liquid phase increased a stability of the complex (0.06 mM). It is suggested that myeloperoxidase could represent a component of complex super molecular structure, real immune complex.

By changing pH within the pH range 2-7, the processes of intramolecular rearrangement in the multidomain structure of rabbit IgG were induced which resulted in the formation of four IgG conformers - N, NA, I and NI, differing in the amount of structure in the CH2 domain and as well as in its interaction with the neighboring domains. The stability of the IgG conformers was studied by differential scanning calorimetry and expressed in terms of thermodynamic parameters, delta H, Tm, and delta G. The pH-dependent changes in thermal stability of IgG and the range of stability for the conformers were described by conformational (phase) diagrams. The native N-conformer exists at pH 7.0-5.5. At pH < 5.5 the formation of the NA conformer showing significant decooperation (lower extent of interaction) of the domains occurs. Decooperation permits the CH2 domain in the NA conformer to undergo conformational transitions independently of the rest of the molecule-the property which cannot be observed for the more cooperative N-conformer of IgG. The formation of the intermediate I-state occurs at pH < 3 as a consequence of unfolding of the tertiary structure of the CH2 domain, while its secondary structure and compact tertiary structure of the remainder of the IgG domains remain unchanged. Such partially structured and stable states of immunoglobulins have not yet been described. Reversibility of pH-induced transitions in IgG was studied using calorimetry and ligand-binding assays involving the C1q component of the complement, protein A, antigen and monospecific anti-CH2 domain antibodies as conformational probes reporting local conformational changes in the CH2 domain and its interdomain interfaces. Refolding of IgG from the I-state by increasing pH results in the formation of a functionally active N1 conformer differing from native IgG by a changed balance of interdomain interactions. In addition to previously reported data suggesting a greater extent of the CH2 and CH1 domain interactions in the NI conformer, this conformer was shown to possess a lower extent of interaction of the CH2 and CH3 domains. The changed stability and a balance of domain interactions in the NI conformer results in changes in some of the effector functions including enhanced two-site binding to protein A and antigen-dependent binding to C1q with a concomitant decrease in the affinity of protein A binding site and reduced antigen binding capacity of the high affinity IgG. Low affinity IgG does not change the antigen-binding parameters after refolding to the N1 conformer.

We apply the thermal perturbation difference spectroscopy to demonstrate that immunoglobulins G (IgG) isolated from blood serum of a patient with stomach cancer are more stable to the influence of 12% urea than IgG of a healthy donor. IgG at the same pathology have greater tendency to aggregation than IgG at norm according to the laser correlation spectroscopy data.

Horseradish peroxidase (HRP) and its conjugates with IgG (HRP-IgG) and cortisol (HRP-COR) interactions with polyvalent antibodies against peroxidase (ANTI-HRP) have been studied by homogeneous enzyme immunoassay. The total catalytic activity in o-phenylenediamine oxidation for free peroxidase or its conjugates and their immunocomplexes with antibodies against the enzyme (ANTI-HRP) has been measured. Linearization of time dependencies of the initial rate for peroxidation in the ln (v-v infinity)-time coordinates, where v and v infinity are the reaction rates at a definite moment of time and after the onset of the equilibrium, as well as in the [ANTI-HRP]/(v-v infinity)-[ANTI-HRP]0 and I/[ANTI-HRP]0k1 + k-1)-[ANTI-HRP]0 made it possible to determine the dissociation constant, Kd, and rate constant for the formation and dissociation, for the immunocomplexes k1 and k-1. The Kd values were in good agreement with those determined by the classical Scatchard method.

Some dynamic and conformational changes of Lys residue in molecules of human IgG have been studied by the spin label method using N-(1-oxyl-2,2,5,5-tetramethyl-3- pyrrolidinyl)maleimide, known to selectively modify the Lys residue in human IgG. The value of tau 20 C of the spin labelled IgG was found to be 26 +/- 2 ns, what is known to be characteristic for the labelling of the peptide residue in IgG. It has also been found that the spin-labelled Lys residues exist in two forms with different mobility and rigidity with the ratio of mobile to rigid forms of 3:1. The hypothesis that the reason for immobility of Lys residues in IgG lies in their drawing in the H-bonds formation is being proved. The study by the spin label method has shown that the often observed mobilization of the EPR spectra of the spin labelled IgG is caused by the process of autolysis in the stored samples. The use of the phenylmethylsulfonylfluoride can completely eliminate this phenomena.

Thermal stability and functional activity of rabbit IgG in its native conformation and after incubation at pH 2.0 were studied using differential scanning calorimetry and binding of conformational probes, i.e., the C1q component of the complement and two monospecific anti-IgG antibodies. The latter reacted selectively with the "hinge" region joining the Fab and Fc fragments of IgG or with the CH2 domain in the Fc fragment. At pH 2.0 complete unfolding of rabbit IgG did not occur: the protein demonstrated the presence of secondary and compact tertiary structures but differed from the native conformation by decreased overall enthalpy and TM of thermal denaturation as well as by changed secondary structure parameters as could be evidenced from CD spectroscopy and scanning calorimetry data. Incubation at pH 2.0 followed by renaturation at neutral pH led to irreversible conformational changes in IgG. The most significant differences between the two IgG conformers were demonstrated by calorimetry at pH 3.5 which revealed that the acid-treated conformer differs from the native one by enhanced thermal stability of the CH2 domain. Using the combination of thermodynamic and functional studies, it was shown that the origin of stabilization was the increase in the extent of interaction between the CH2 domain in the Fc fragment and the CHI domain in the Fab fragment. This resulted in the increase of the functional link between the antigen-binding domain and the C1q binding site in the CH2 domain of the acid-induced IgG conformer. In parallel with the increase in stability of the CH2 domain, conformational changes in the "hinge" region were found, together with the absence of intrinsic conformational changes in the CH2 domain proper as could be judged from C1q and monospecific anti-IgG binding assays. The results obtained demonstrate one of possible mechanisms whereby functionally significant rearrangements in the IgG molecule can be induced by changes in the interactions between invariably folded domains rather than by intrinsic changes in the domain conformation.

Theoretical conformational analysis was carried out for the 285-292 fragment of human immunoglobulin G (His-Asn-Ala-Lys-Thr-Lys-Pro-Arg) and its analogues containing Arg, Glu, Gly, Lys, or Trp residue instead of the His residue in position 1. Spectropolarimetic investigation of these peptides showed the analogues to have different activities in the C1q-mediated erythrocytes hemolysis assay. Comparison of the low-energy structures sets of the compounds tested allowed to suggest a model of the "biological active" conformation for the peptide molecule in the course of the C1q complement component binding.

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 0.12 M sodium periodate the pH of the medium was 8.5, and the initial molar ratio of IgG to laccase was 2:1. The conjugates can be stored for one year without any loss in immunological activity.

Modification of rabbit IgG by coproporphyrin I activated by Woodward's reagent K as well as antigen-binding and fluorescent properties of coproporphyrin-IgG have been studied. It was shown that coproporphyrin I modified IgG retains its capacity to bind the antigen. The formation of the immune antigen-antibody complex increases the intensity of coproporphyrin-IgG fluorescence.

Since immunoglobulins are used in a vast variety of immunoassays, the problem of obtaining antibodies with enhanced antigen-binding activity is of great importance. In order to discriminate between putative approaches to activating antibody modification, some functional characteristics of rabbit IgG modified at the hinge disulfide by three reagents: iodoacetamide, N-ethylmaleimide, and 2.2'-dipyridyl disulfide, have been studied. As can be judged from gel-permeation chromatography data, the molecular sizes of modified rabbit IgG were slightly increased in comparison with the native protein. Using enzyme immunoassay, it was shown that modification by each of the above reagents results in the same degree of activation of the antibody binding to the protein polyvalent antigen-human ferritin, due to the increase in segmental flexibility, i.e., Fab motion around the Fo fragment of IgG. The type of concentration dependencies of antigen binding suggest that another determinant stimulating the antigen binding in addition to the increase in segmental flexibility, can be attributed to intra- or interdomain flexibility of domains constituting the Fab fragments. Using protein A and anti-IgG as conformational probes for the antibody Fo fragment, the conformation and conformational dynamics of both CD2 domain epitopes and the switch region between the CD2 and CH3 domains have been shown to be essentially unaffected by modification.

A proximate method for conformational changes detection in immunoglobulin G preparations used in research and medical practice is introduced. This method is based on the measurement of IgG-connected 1,8-anilinenaphtalenesulphonate fluorescent probe (ANS) time of damping. When the protein structure is broken fluorescence damping time of IgG-connected ANS rises up.

To elucidate the effect of the antigen binding fluorescent thiol reagent, N-dansylaziridine (DAZ) which is sensitive to microenvironmental changes, was used for modification of the rabbit IgG hinge region cystine residue. DAZ binds to the hinge region Cys 226 as could be evidenced from the structural analysis data. Labelling of IgG with DAZ does not alter either its conformation and hydrodynamic behaviour or its antigen binding properties. Upon antigen binding the fluorescence intensity of modified IgG increases up to about 80%. This finding suggests that the interaction of antibodies with the antigen is accompanied by conformational changes in the IgG hinge region.