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.

Modern ELISA for determination of functional activity of component C2 and factors B and D, proteinases of a complement system, and component C3, substrate C3-convertases, key complex enzymes of the complement, have been developed. Essential feature of C3-convertases classical (C4bC2a) and alternative (C3bBb) pathways of the complement activation is that their substrate C3 after proteolytic cleavage is converted into C3b, carrying on the surface thioester covalent bond linking C3b with nucleophilic acceptors that results in immobilization of this proteolytic product near the activating enzyme. Cascade character of an activation of complement system allows to create artificial deficit of separate components in the experimental system and to determine (by ELISA) covalently immobilized component C3 during activation, and also to determine functional activity of any of pre-exhausted components. Use of such approach resulted in the development of the ELISA systems suitable for determination of functional activity of component C2 of classical pathway and factors B and D of the alternative pathway by testing quantity of the immobilized C3b at excess C3. The developed methods allow to investigate mechanisms of functioning of complement, inhibition of the cascade activation by endogenic and exogenous inhibitors, and also to find functional deficiency of components in serum and other biological fluids.

For research of effect of medical product at a system of a complement the methods of analysis of inhibition of a system of a complement in vitro and in vivo are designed. The first of them is ELISA for determination of inhibition of the first stage of complement activation: binding of a subcomponent C1q with immunoglobulin. Second - supression of a lethal action of a mink complement on the mouse. As medical product - inhibitors of a complement the operating known by the inhibiting effect of heparin (known anticoagulant) and suramin (drug for treatment trypanosomiasis) is studied. By the first method was retrieved, that the constant of inhibition of binding suramin with C1q is 411 ± 29 mg/ml (or 0,287 ± 0,020 mmole/l), and the constant for heparin is 36,4 ± 1,7 mg/ml (or 2,28 ± 0,10 mmole/l), i.e. the binding of heparin with C1q in 10 times is better, than those for suramin in weight relation or in 100 times it is better - in molar. The second method is based on capacity of mink serum to kill of mice at the intravenous introducing due to action of a mink complement. Was shown, that the introducing 3 mg of suramin or 0,3 mg of heparin on the mouse cancels killing of mice called by the intravenous introducing 0,08 ml of mink serum, i.e. the efficiency of action of these inhibitors of a complement in experience in vivo also differs in 10 times, and their concentrations in a blood animal approximately correspond to constants of inhibition of C1q binding, obtained method in vitro.

It is known, that the lysozyme has alongside with hydrolytic activity an immunomodulation properties. In this connection the ability of an egg lysozyme was explored to act on a complement system. The interaction of a lysozyme with a subcomponent C1q of the first component of a human complement was explored earlier (Ki 0.29±0.09 mM). In operation are studied: (1) “anticomplementary” activity of a lysozyme (Ki 0.30±0.09 mM), (2) activity inhibiting it at the stage of formation C3 convertase of a classic path of a complement (Ki 0.25±0.05 mM), and (3) inhibition at the stage of formation C5 convertase (Ki 0.26±0.01 mM). The detected direct action on a complement of a lysozyme irrelevant with its enzymatic activity, can be one of explanations of an exotic immunomodulation properties of this protein.

The difference in the functional activity of the isotypes A and B of component C4 of human complement was used to determine their ratio to detect the inherited deficiency of the isotypes. The frequency of deficiency in healthy persons blood donors was equal for C4A and C4B (0.14 for each isotype), i.e. 14% of total number (22) donors, or 28% totally. These results agree with the literary data, on which the frequency of deficiency of C4A is 0.14, and of C4B is 0.11-0.16. The inherent deficiencies of C4A and C4B for persons infected by Chlamydia were studied. For this purpose the patients (35 persons) with in blood antibodies (IgG or IgM) to Chlamydia (C. trachomatis, C. psittaci and C. pneumoniae) were investigated. The frequencies of deficiency of C4A and C4B were 0.29 and 0.46 respectively. Thus, the number of the undeficiency patients was only 25%, while among healthy persons 70-75% of individuals not having deficiencies of isotypes C4 were observed. The deficiencies of isotypes of C4 at this pathology is detected for the first time. The obtained data suggest the existence of the predisposition to the development of diseases stipulated by Chlamydia in persons with inherent deficiency of C4 component of complement.

The difference in the functional activity of the isotypes A and B of component C4 of human complement was used to determine their ratio to detect the inherited deficiency of the isotypes. The frequency of deficiency in healthy persons blood donors was equal for C4A and C4B (0.14 for each isotype), i.e. 14 % of total number (22) donors, or 28 % totally. These results agree with the literary data, on which the frequency of deficiency of C4A is 0,14, and of C4B is 0.11-0.16. The inherent deficiencies of C4A and C4B for persons infected by Chlamydia were studied. For this purpose the patients (35 persons) with in blood antibodies (IgG or IgM) to Chlamydia (C. trachomatis, C. psittaci and C. pneumoniae) were investidated. The frequencies of dificiency of C4A and C4B were 0.29 and 0.46 respectively. Thus, the number of the undeficiencyt patients was only 25 %, while among healthy persons 70-75 % of individuals not having deficiencies of isotypes C4 were observed. The deficiencies of isotypes of C4 at this pathology is detected for the first time. The obtained data suggest the existenc of the predisposition to the development of diseases stipulated by Chlamydia in persons with inherent deficiency of C4 component of complement.

An SL2 lectin was isolated from human serum and characterized previously; cellular localization of the lectin was studied using polyclonal rabbit antibodies. According to cytofluorimetry, anti-SL2 antibodies bound only to lymphocytes and monocytes but not to other blood cells. Antibodies bound to Jurkat T cell lymphoma but did not interact with IM-9 cells of B cell origin. Moreover, the Jurkat cells bound oligosaccharides having the highest affinity to SL2 (GalNAcalpha_and Fucalpha1-2Gal), and this interaction was inhibited by anti-SL2 antibodies. Lysis of the Jurkat cells with subsequent electrophoresis and Western blotting indicates that anti-SL2 antibodies recognized a 14-kD protein.

The difference in the functional activity of the isotypes A and B of component C4 of human complement was used to determine their ratio and to detect the inherited deficiency of the isotypes. ELISA methods were developed for the quantitative assay of component C4 (conventional sandwich method) and its functional activity. When determining the functional activity, the classic pathway of the complement and therefore of component C4 was activated by activators sorbed on ELISA microplates (immunoglobulin IgG3 or Liposaccharide of the Shigella sonnei cell walls, which activates the complement by binding component C1). The nascent fragment C4b is covalently bound to the target activator; C4Ab binds better to the target protein (immunoglobulin), and C4Bb to the target carbohydrate (liposaccharide). Therefore, when immunoglobulin is a target activator, isotype C4A is bound and determined; and when the complement is activated by liposaccharide, isotype C4B is determined. The ratio of the activities determined by the two methods indicates a deficiency in the individual isotypes of component C4 or its absence. The rabbit polyclonal monospecific antibodies against the human component C4 and the conjugates of these antibodies with horseradish peroxidase were used in the methods described.

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 theNeisseria 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 by 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 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 use of the Langmuir equation for processing ELISA data (the sandwich variant) helped to ascribe a physical sense to the parameters of the optimization of the antigen-antibody titration curve: the maximum response that characterizes complete binding corresponds to the saturation of all epitopes of the antigen, and the concentration at which half of the maximum response is attained corresponds to the dissociation constant of the immune complex, i.e., to the average affinity of the antibodies. The algorithm was tested for systems in which antibodies against IgE and IgD were sorbed on a support, and the antigen bound was determined by the antibodies conjugated with peroxidase. A good fit of the experimental and theoretical curves and reasonable values for the affinity constants were found. In another system, the binding of specific IgG, IgM, and IgA antibodies with the polysaccharide from Neisseria meningitidis serotype A was studied during vaccine testing. The structural simplicity of the antigen molecule made it possible to suggest the presence of two main epitopes and to reveal the dynamics of formation of the antibodies to them.

In this work the results of the study of specific antibodies (Ab), isotypes IgM, IgG, IgA, types kappa and lambda, in 235 serum samples from 27 adults immunized with group A meningococcal polysaccharide vaccine (AMPV) in a single injection of 50 microg and from 20 control subjects are presented. The study was made by the method of sandwich EIA. The study revealed that in a month after the injection of the vaccine the intensive synthesis of IgA, IgG and IgA Ab and their subsequent circulation for 2 years were observed; 3 years after immunization (the term of observation) the prevalence of IgG and IgA antibodies was registered. Prior to immunization the ratio of kappa and lambda Ab was 1.7. In a month after immunization the maximum ratio of 3.2 was achieved and in all subsequent terms of examination this ratio remained higher than prior to immunization (3.1 -- 2.3). As revealed in this study, the injection of AMPV induced the intensive synthesis of antibodies of types kappa and lambda during the first year after immunization, then the production of type lambda Ab decreased by the second year and in 2 and 3 years after immunization the circulation of type kappa Ab prevailed.

Blood serum IgG1, IgG2, IgA, and IgM were assayed by radial immunodiffusion after exposure of guinea pig skin to oil refinery products, mineral oil distillate D-11 (MOD) and furfurol (F), applied both separately and together. Application of MOD, F, and MOD + F subthreshold concentrations was found to be associated with a tendency to a reduction of IgG1 level; isolated applications of the agents in threshold concentrations involved statistically significant lowering of IgG1 (in exposure to MOD) and imbalanced levels of IgG1, IgG2, IgA, and IgM (in exposure to F). Combined application of both agents induced immunity shifts of other type as against isolated exposure.

The results of the production and analysis of monospecific rabbit antisera to guinea pig IgG1, IgG2, IgA and IgM are presented. Isolated immunoglobulins of different isotypes, as well as immune precipitates obtained by immunoelectrophoresis, were used for immunization. After adsorption antisera of each type there formed one precipitation line with guinea pig serum in immunoelectrophoresis, thus indicating that they contained antibodies to immunoglobulins of the definite isotype.

Antigenic features of monoclonal IgM kappa components of three mixed IgM-IgG cryoglobulins were studied by gel-diffusion precipitin analysis. An unusual kappa-chain determinant was revealed in the IgM components but not in other monoclonal immunoglobulins (IgM kappa, BJ kappa) or normal IgG. Antibodies to this determinant were found not only in anti-kappa, but also in anti-lambda sera, and in antisera to hidden determinants of L-chains.

The present report summarizes the main clinical and immunochemical features of 17 patients with IgD myeloma and compares than with the evidence reported in the literature. The difficulties inherent in the immunodiagnosis of this disease, particularly in detection of the M-component, typing of IgD and demonstrating its monoclonal nature, are discussed on the basis of personal observations and those of other investigations. Special emphasis is placed on clinical and immunochemical characteristics of IgD kappa myeloma. Immunoquantitation of serum IgD is considered to be the most reliable method of immunodiagnosis.