Background Isotypes C4A and C4B of human complement component C4 are innate immunity factors. We found that gel electrophoresis of patient sera in the new diagnostic area of C4 complexes/aggregates (pI 4-5) followed by immunoblotting allowed evaluation of patient C4 isotype deficiency grouping in accordance to our developed functional C4 isotype analyses in microplates [1]. Solid phase peroxidase kinetic chemiluminescence (CL) in the presence of CL substrate is sensitive process to detect serum glycoproteins distribution. Objectives The objective was further study of patient C4B and C4A subisotypes within C4-isotype-(non-deficient or deficient) sera groups of patients. Methods C4 subisotype patterns were obtained using separation of desialylated sera by isoelectric focusing in plate of polyacrylamide in gradient pH 3-5 followed by gel blotting on Immobillon P (Millipore). Blots were treated with goat anti-C4 polyclonal antibodies conjugated to horse radish peroxidase (Ab-HRP) or Ab-HRP against human IgG, IgM or IgA. Protein-A–HRP was also used. Substrate (BioWest, UVP; with increased sensitivity) was added and kinetics of the blot HRP CL was stepwise registered in live imagination regime using BioChemi System (UVP). Subisotypes were revealed by blot treatment with acetate buffer pH 4.5. Analysis was especially effective in optimized by us conditions (desialylation control, blot washings, substrate using, Cl registration regimes). Results Patient C4B or C4A subisotypes were presented as combinations of mainly up to 5 bands of different contribution. Contribution of subisotype(s) in C4A region was confirmed by the presence of increased levels of IgG in less acidic band(s) and/or IgM as additional early CL of mostly acidic 5th band in cases of SLE with or without antiphospholipid syndrome (group of C4B-deficiency). In cases of non-C4-deficiency groups (examples of rheumatoid arthritis etc.) preferential localization of non-early IgM, IgA and binding Protein A within C4B region indicated infectious nature of contribution to subisotype(s) bands. Ig Fc-fragments were masked in complexes (C1q binding was not observed). Relative resistance of subisotypes patterns upon sera storing indicated partial masking glycans in C4 subisotypes complexes possessing increased hydrophobicity. Algorithm of revealing very low levels of serum subisotypes (invisible in comparison to other sera) is proposed. Conclusions Results support both biomarker diagnostic and prognostic potential of patient sera C4B and C4A subisotypes and prospects of its further development of fine resolution CL patterns of C4 associates together with increased levels of classes and subclasses of Ig and lipoproteins, glyco- and lipid-containing antigens (also of microbial origin), others sera components using extended set of Ab. Few diagnostic kinetic CL pattern states (early and later) are possible to characterize disease(s) type. References Lakhtin M et al. Lakhtin M et al. Chemiluminescent visualization and quantification of the complement glycoprotein C4 containing systems in patient whole sera separated with isoelectric focusing in polyacrylamide gel and electroblotting: sialidase- and peroxidase-based new approaches. Glycoconjugate J 2011; 28: 296. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.3511

Microbial capsular polysaccharides for many years provided a highly practical public health vaccines for preventing meningococcal, pneumococcal and Haemophilus influenza infection, and typhoid fever. Their application in the form of conjugates with protein carriers eliminate the gap in protection against these infections in children under one year. Extremely promising turned out offered us a new generation of vaccines, which have synthetic peptides conjugated to a meningococcal polysaccharide. Thus, new approaches to the solution of the problem of meningococcal disease vaccination serogroup B were open. In recent years, Russian researchers first suggested to use IgA1 protease (one of the major virulence factors of microbes and almost identical for mentioned below infections) for prevention of such diseases as meningococcal of all serogroups, pneumococcus and hemophilia infections. Patented processes for producing of the vaccine define domestic priority of its production and use.

Microbial capsular polysaccharides for many years provided a highly practical public health vaccines for preventing meningococcal, pneumococcal and Haemophilus influenza infection, and typhoid fever. Their application in the form of conjugates with protein carriers eliminate the gap in protection against these infections in children under one year. Extremely promising turned out offered us a new generation of vaccines, which have synthetic peptides conjugated to a meningococcal polysaccharide. Thus, new approaches to the solution of the problem of meningococcal disease vaccination serogroup B were open. In recent years, Russian researchers first suggested to use IgA1 protease (one of the major virulence factors of microbes and almost identical for mentioned below infections) for prevention of such diseases as meningococcal of all serogroups, pneumococcus and hemophilia infections. Patented processes for producing of the vaccine define domestic priority of its production and use.

On the base of nucleotide sequence, coding IgA1 protease from Neisseria meningitidis serogroup B, strain ????58, which was determined from data base http://www.ncbi.nlm.nih.gov/Genbank), recombinant plasmide DNA was created comprising nucleotide sequence of IgA1 protease, strain H44/76, providing IgA1 protease expression in the host-cell (pBIGAPS1). The method of expression, isolation, purification and refolding of recombinant enzyme was developed. IgA1 protease exhibits high specificity and cleaves only IgA1, but not IgG. Immunogenic and protective properties of IgA1 protease to meningococcus of serogroup ??, ?? and ?? were shown. Obtained enzyme can be considered as perspective polyvaccine candidate for prophylaxis against meningococcus infection, induced by bacteria N. meningitidis and especially against serogroup B. References Kazeeva T.N., Shevelev A.B. / Biohimija. 2007. T.72. ??? 5. S. 603-614. Kilian M., Thomsen B., Petersen T.E., Bleeg H. / Mol.Immunol. 1983. V. 20. P. 1051–1058. Monteiro R. C., J. G. J. Van de Winkel. / Annu. Rev. Immunol. 2003. V. 21. P. 177–204 Mulks M.H., Plaut A.G. / N. Engl. J. Med. 1978. V. 299. P. 973–976 Mistry D., Stockley R.A. / Int. J. Biochem. Cell. Biol. 2006. V. 38. P. 1244–1248. Tajmurazov M.G. / Poluchenie i nekotorye svojstva meningokokkovoj IgA1-proteazy. Diss. kand. nauk. Gosudarstvennyj Nauchnyj Centr prikladnoj mikrobiologii. Obolensk, 2006. Arzese, A., & Botta, G. A. / Clinical Infectious Diseases. 1995. V. 20 (Suppl. 2): 169–171 Bachovchin WW, Plaut AG, Flentke GR, Lynch M, Kettner CA. / J Biol Chem. 1990. V. 265(7). P. 3738-3743. Plaut AG, Bachovchin WW. / Methods Enzymol. 1994. V. 244. P. 137-151. Jagudaeva E. Ju, L. S. Zhigis, O. A. Razguljaeva, V. S. Zueva, Je. Je. Mel'nikov, V. P. Zubov, L. V. Kozlov, A. M. Bichucher, O. V. Kotel'nikova, A. P. Alliluev, A. Je. Avakov, L. D. Rumsh. / Zhurn. bioorgan. himii. 2010, ??? 1. C.96-105 Alliluev A.P., Anohina I.V., Rumsh L.D., Kotel'nikova O.V., Drozhzhina E.Ju. Zhigis L.S., Jagudaeva E. Ju., Razguljaeva O.A., Zueva V.S., Kozlov L.V., Bichucher A.M., Avakov A.Je. / Vestnik RUDN. ser. Medicina. 2010. ??? 1. C. 7-12 Maniatis, T., Fritsch, E.F. and Sambrook J. / Molecular Cloning: a Laboratory Manual. Cold Spring Harbor Laboratory Press. NY.1982. O.V. Kotel'nikova, O.V. Chibiskova, V.A. Nesmejanov, A.P. Alliluev, O.M. Vol'pina, D.O. Koroev, M.N. Zhmak, M.A. Titova, V.T. Ivanov / BJeBIM. 2005. ???5. C. 553-556.

On the base of nucleotide sequence, coding IgA1 protease from Neisseria meningitidis serogroup B, strain МС58, which was determined from data base http://www.ncbi.nlm.nih.gov/Genbank), recombinant plasmide DNA was created comprising nucleotide sequence of IgA1 protease, strain H44/76, providing IgA1 protease expression in the host-cell (pBIGAPS1). The method of expression, isolation, purification and refolding of recombinant enzyme was developed. IgA1 protease exhibits high specificity and cleaves only IgA1, but not IgG. Immunogenic and protective properties of IgA1 protease to meningococcus of serogroup А, В and С were shown. Obtained enzyme can be considered as perspective polyvaccine candidate for prophylaxis against meningococcus infection, induced by bacteria N. meningitidis and especially against serogroup B. References Kazeeva T.N., Shevelev A.B. / Biohimija. 2007. T.72. № 5. S. 603-614. Kilian M., Thomsen B., Petersen T.E., Bleeg H. / Mol.Immunol. 1983. V. 20. P. 1051–1058. Monteiro R. C., J. G. J. Van de Winkel. / Annu. Rev. Immunol. 2003. V. 21. P. 177–204 Mulks M.H., Plaut A.G. / N. Engl. J. Med. 1978. V. 299. P. 973–976 Mistry D., Stockley R.A. / Int. J. Biochem. Cell. Biol. 2006. V. 38. P. 1244–1248. Tajmurazov M.G. / Poluchenie i nekotorye svojstva meningokokkovoj IgA1-proteazy. Diss. kand. nauk. Gosudarstvennyj Nauchnyj Centr prikladnoj mikrobiologii. Obolensk, 2006. Arzese, A., & Botta, G. A. / Clinical Infectious Diseases. 1995. V. 20 (Suppl. 2): 169–171 Bachovchin WW, Plaut AG, Flentke GR, Lynch M, Kettner CA. / J Biol Chem. 1990. V. 265(7). P. 3738-3743. Plaut AG, Bachovchin WW. / Methods Enzymol. 1994. V. 244. P. 137-151. Jagudaeva E. Ju, L. S. Zhigis, O. A. Razguljaeva, V. S. Zueva, Je. Je. Mel'nikov, V. P. Zubov, L. V. Kozlov, A. M. Bichucher, O. V. Kotel'nikova, A. P. Alliluev, A. Je. Avakov, L. D. Rumsh. / Zhurn. bioorgan. himii. 2010, № 1. C.96-105 Alliluev A.P., Anohina I.V., Rumsh L.D., Kotel'nikova O.V., Drozhzhina E.Ju. Zhigis L.S., Jagudaeva E. Ju., Razguljaeva O.A., Zueva V.S., Kozlov L.V., Bichucher A.M., Avakov A.Je. / Vestnik RUDN. ser. Medicina. 2010. № 1. C. 7-12 Maniatis, T., Fritsch, E.F. and Sambrook J. / Molecular Cloning: a Laboratory Manual. Cold Spring Harbor Laboratory Press. NY.1982. O.V. Kotel'nikova, O.V. Chibiskova, V.A. Nesmejanov, A.P. Alliluev, O.M. Vol'pina, D.O. Koroev, M.N. Zhmak, M.A. Titova, V.T. Ivanov / BJeBIM. 2005. №5. C. 553-556.

A method of the isolation and purification of IgA1 protease from a culture of Neisseria meningitidis serogroup A has been developed. Three inactivated intermediates of the production of the meningococcal vaccine, a culture liquid, as well as a supernatant and precipitate obtained by the precipitation of bacterial cells by cetavlon, served as a starting material. The purity of IgA1 protease was determined by SDS-PAGE. An immunoenzyme assay for determining the IgA1 protease activity has been developed. The yield of the enzyme with a specific activity of 0.5 to 4 million units/mg from 103 g of the cetavlon precipitate (40 l of culture liquid) was about 600 µg. It was shown that IgA1 protease isolated from serogroup A meningococcus is capable of protecting experimental animals (mice) infected with meningococcus of serogroup B.

IgA1-protease allocated from the culture N. meningitidis serogroup A. As original materials were used three different intermediate products of vaccine production: cultural fluid, cetavlon supernatant and cetavlon precipitate. IgA1-protease was used to evaluate their protectivity and immunogenity. It was shown, that isolated IgA1 protease from the meningococcus serogroup A is able to protect mice, infected by meningococcus serogroup B.

IgA1-protease allocated from the culture N. meningitidis serogroup A. As original materials were used three different intermediate products of vaccine production: cultural fluid, cetavlon supernatant and cetavlon precipitate. IgA1-protease was used to evaluate their protectivity and immunogenity. It was shown, that isolated IgA1 protease from the meningococcus serogroup A is able to protect mice, infected by meningococcus serogroup B.

A protease activity may be determined by means of immunoglobulins. Since proteolytic products apparently do not retain antigenic determinants of the initial substrate, the monitoring of enzymatic process may employ ELISA methods. The ELISA determination of functional activity of specific IgA1 protease has been used not only for detection of this enzyme, but also for measurement of its inhibition constants. IgG adsorbed onto a microplate was used for evaluation of total proteolytic activity. Varying pH values of the reaction medium it is possible to measure activity of neutral, alkaline and acid proteases. This approach was used for estimation total proteolytic activity of neutral proteases in blood serum. Due to high sensitivity of this method it was possible to dilute serum up to the level when serum inhibitors had not blocked enzyme activity. Assay of serum enzyme activity at acidic pH results in activation of pepsinogens and determination of pepsin activity. Measurement of a total level of serum pepsinogen activity may have diagnostic importance in gastroenterology, due to decisive contribution of pepsinogen I to the detectable activity.

Microbial spectrum and non-specific as well as specific IgA1 protease activity of isolated microorganisms were investigated in gingival liquid of patients with periodontitis. Microorganisms from the gingival liqud of these patients belonged to conditional-pathogenic obligate and facultatively anaerobic bacteria. 24 strains of microorganisms have been identified. Nonspecific proteolytic activity was found in the following microorganisms: Actinomyces israelii, Actinomyces naeslundii, Aerococcus viridans, Bifidobacterium longum, Neisseria subflave, Streptococcus parvulus, Eubacterium alactolyticum, Lactobaccilus catenoforme, Bacillus spp. Specific IgA1-protease activity and lack of proteolytic activity towards IgG was found in Streptococcus acidominimus, Streptococcus hansenii, Streptococcus salivarius, Leptotrychia buccalis, Staphylococcus haemolyticus and Neisseria sicca. No proteolytic activity was found in cultivation medium of Eubacterium alactolyticum (1 strain), Prevotella buccalis, Aerococcus viridans and Streptococcus sanguis.

Since undesirable activation of the complement system through the classical pathway is associated with tissue damage and other pathologic proinflammatory consequences at ischemia/reperfusion injury, autoimmune diseases, and rejection of allo- and xenografts, creation of selective inhibitors of the classical pathway leaving the alternative pathway intact is of great importance. Classical pathway is triggered by binding of its recognizing unit, protein C1q, to a number of targets like antibodies, pentraxins, apoptotic cells, and others. In order to obtain inhibitors blocking the first step of the classical cascade, synthesis of sulfates of steroids (Δ5-3β-hydroxycholenic, Δ5-3β-hydroxyetiocholenic, deoxycholic, and cholic acids) and triterpenoids (betulin, 20,29-dihydro-20,29-dichloromethylenbetulin, betulinic, ursolic, and oleanolic acids) has been performed. Testing of the compounds in classical pathway inhibition assay has displayed derivatives of triterpenoid betulin (betulin disulfate and betulinic acid sulfate) to be the most potent inhibitors. Further studies of the two compounds established that their activity to inhibit the classical pathway had been due to their capability to block the interaction of C1q with antibodies. Betulin disulfate and betulinic acid sulfate have shown weak inhibition of the alternative route of activation, what makes them promising inhibitors for the selective suppression of the classical complement pathway at the earliest possible level as well as perspective agents for blocking the interaction of C1q with its other targets.

An approach is proposed to detect deficiencies in isotypes A and B of the C4 component of human complement, based on the calculation of the ratio of their IEA activities and the ratio of their quantities determined by isoelectrofocusing of their desialated forms with chemiluminescent detection in an immunoblot. The ratios of the quantities and activities of C4A/C4B practically coincided when determined in blood serum of 20 patients, many of which had inherited deficiencies in the C4 component isotypes.

A great number of natural substances affect the complement system in addition to its natural regulators. Among the complement effectors, the most important are inhibitors of the activation cascade. The necessity of searching for preparations capable of a purposeful effect on complement by inhibition of single stages of the activation cascade and without influence on its other functions is connected with the current importance of use in medicine of novel therapeutic regulators of the complement system. Important directions are the search for complement inhibitors that (a) interfere with the rejection of transplants; (b) can replace C1 inhibitor in hereditary angioedema; and (c) have a high anti-inflammatory activity in the therapy of rheumatic diseases, diabetes, and other autoimmune disorders. It is expedient to use the available techniques for the directed detection of the action of medicinal substances on complement, which allow the determination of their action on the complement system at various stages of the cascade of its activation.

The immunoenzyme analysis and the method for the determination of IgG-containing immune complexes, carrying C1q component of the complement, were developed. In human blood sera the functional activity of components C3, complex C1r2s2, the content of C1 inhibitor and complement-activating immune complexes were determined. The comparative analysis of the activity of components C3 and C1r2s2, as well as between the content of C1 inhibitor and the activity of complex C1r2s2 for seropositive and seronegative sera, was made. Pronounced correlation for seropositive sera was observed. In addition, for seropositive sera correlation between an increase in IgG immune complexes and a drop in the functional activity of complex C1r2s2, as well as a drop in the functional activity of complex C1r2s2 and a growth in the titers of IgG antibodies to Chlamydia trachomatis, were established. The decreased functional activity of key complement components, simultaneously with the presence of complement-activating immune complexes and high titers of specific antibodies could be the diagnostic criteria of carrier state.

In order to obtain strong inhibitors of classical pathway of complement activation the low weight negative charged compounds have been investigated. On the basis of bisphenol A anionic derivatives with one or two carboxylic, sulphate and phosphate groups the critical role of negative charged groups for complement-inhibiting activity has been established. It was determined that two sulphate or phosphate groups in the molecule provide the most inhibiting effect. At the next stage a set of bisphenol disulphates of varying structures has been synthesized and investigated. Bulky hydrophobic groups (cyclohexyliden, fluorenyliden, anthronyliden) at the central part of the bisphenol molecule it was found to increase complement-inhibiting activity markedly. The replacement of the ortho-positions to the charged group by halogens or alkyl groups (allyl, propyl) increases the inhibiting effect. It was showed by ELISA that several compounds studied interact with C1q, C1r /C1s components of complement. For the set of bisphenol disulphates the QSAR equation with hydrophobic coefficient and electronic parameters has been formulated. Both hydrophobic and electrostatic interactions it was established to have a great significance for the inhibition of classical pathway of complement activation.

Aetiology of angioedema (and therefore the scheme of its treatment) can be different. Angioedema may be subdivided into four categories: hereditary and acquired angioedemas, allergies and vasculitis. To establish the reason of the hereditary and acquired form of angioedema analyses of functional activity of complement components, quantities and activity of C1 inhibitor, presence (or absence) autoantibodies to C1 inhibitor allow. Sorption of the puried enzymes of activated subcomponent C1s or plasmin on micropanels allows to connect specifically in cells of plate C1 inhibitor from serum and with the help conjugate of antibodies against C1 inhibitor with a horse-radish peroxidase to determine quantity of connected functionally active C1 inhibitor. Addition of this test-system ELISA system for determination of quantitative contents of C1 inhibitor in serum, and also systems for definition IgG, IgA and IgM autoantibodies against C1 inhibitor finishes creation of a necessary set of methods of differential diagnostics.

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.

Polyethyleneimine (PEI, 50 kDa) and polymethacrylic acid (PMA, 200 kDa) were shown to inhibit the lysis of sheep erythrocytes induced by the guinea pig complement. They twofold suppress the hemolysis at the concentrations of 0.47 and 0.89 microgram/ml, respectively. The inhibitory effect on the binding of the C1q subunit of human complement to the sensitized sheep erythrocytes (EA) was found to depend on the component of the reaction with which the inhibitors were preliminarily incubated. When an inhibitor, C1q, and EA were simultaneously incubated, the inhibition constants for PEI and PMA were 17 +/- 6 and 8.1 +/- 0.1 micrograms/ml, respectively. The preincubation of EA with PEI and the subsequent washing out of the inhibitor resulted in the inhibition constant of 22 +/- 3 micrograms/ml. No inhibitory effect was observed after a similar preincubation of EA with PMA. No inhibition was also detected when the inhibitors were added after the formation of the C1q complex with antibodies. These observations suggest that the binding of antibodies to cationic PEI prevents the C1q-antibody complex formation, while the binding of anionic PMA to the active site of C1q impedes the interaction of this subunit with immunoglobulins. Moreover, within the range of concentrations studied, the studied inhibitors did not affect the subsequent C1q binding to the C1r and C1s enzymes.