Laboratory of Immunochemistry

Scientific studies of Laboratory of Immunochemistry are devoted to different scientific branches. One scientific block is investigation of the activation and regulation processes of natural immunity. Next block is studying of role of Staphylococcal exotoxins in diseases caused by Staphylococcus aureus, and development of multiparametric diagnostic systems for pathogen detection based on immunochemical methods.

Pathogenic microorganisms and their bacterial toxins

One of the most common pathogens of humans and animals is the Staphylococcus aureus. Staphylococcus rapidly evolves, adapting to changes in the environment. Strains have resistance to diffrent antibiotics in humans and animals. Staphylococcus are able to produce a set of protein and peptide toxins, some of which are stable during heat treatment,  resistant to the action of proteolytic enzymes and at extreme pH values. First of all, these toxins cause human poisoning, and their repeated exposure causes a number of serious complications and leads to the development of autoimmune diseases and allergies. Immunochemical methods of analysis are used for detection toxins in human secrets, food products and the environment. Our Laboratory has the representative panel of mouse monoclonal antibodies to staphylococcal toxins. Based on these antibodies, toxin detection systems have been developed, both using instrumental methods and without special instruments in the field conditions.

In addition, the laboratory interest is directed toward other bacterial toxins produced by the C. botulinum (causative agent of botulism) and B. anthrax (causative agent of anthrax). Monoclonal antibodies were obtained to the most dangerous bacterial toxins of botulinum and anthrax, it contribute to develop some selective and sensitive methods of detection based on the "sandwich" ELISA method.

Staphylococcus-associated mastitis

Mastitis is the most important cause of losses in the production of milk, even in prosperous countries like Holland or Switzerland. In Russia, about 30% of the mastitis causes are associated with staphylococcus. Staphylococcal toxins may be present in milk and products derived from it. The Laboratory studies are devoted to toxicity of staphylococcus in mastitis in the Russian Federation and the production of toxins by toxicogenic strains isolated from patients with mastitis of animals, the immune response of animals to staphylococcal toxins is studied.

Role of GMDP-RN peptide in natural immunity reactions

Investigation of the natural immunity activation and the role of glucosaminylmuramyl dipeptide (GMDP), the bacteria cell wall fragment, play an important place in the Laboratory research. Using antibodies to the GMDP obtained at the Laboratory, as well as the technology of the peptide phage display, the researchers opened a 15-dimensional peptide – GMDP mimetic, termed the terminal amino acid residues RN peptide. The study of the amino acids role in the recognition of peptide antibodies and the ability to influence the immune system showed the following effects: at the body level - adjuvant at doses 1-3 ng and 1-1.5 mcg; at the cell level – an increase in the expression of NFκB2, regulation of NFκB2 expression as a function of the concentration of YB-1 in the culture medium; at the molecular level – binding to YB-1 with Kd = 4 ∙ 10-9, competition with GMDP for the binding site of YB-1. Based on the investigation data was proposed a model for the activation of natural immunity of the GMDP – RN peptide with the participation of NOD2 and the cold-shock protein Yb-1.

Preparation of Recombinant Antibodies of Animals and Human

Next of the research Laboratory area is costructione of recombinant antibodies, preferably are single-stranded miniantibodies, developing methods for stabilization – refolding and directed evolution of miniantibodies. The Laboratory has a representative library of human miniantibodies in phage display format. Miniantibodies to enterotoxins of staphylococcus were obtained and characterized. The role of the antibody format for specificity in a number of enterotoxins was investigated.

Toxicity analysis of enterotoxins and toxin-neutralizing potential of antibodies

The Laboratory in cooperation with the Laboratory of Biological Testing develops methods for valuation the toxicity of staphylococcal toxins and the toxin-neutralizing potential of antibodies to enterotoxins. As model animals they use mice grown in Nursery for Laboratory Animals. The approaches used are described in this paper.

According to the research of pathogenic microorganisms and their bacterial toxins, the Laboratory cooperates with the Institute of Molecular Biology RAS (Moscow), the Prokhorov General Physics Institute RAS (Moscow), the Institute of Theoretical and Experimental Biophysics RAS (Pushchino), the Gamaleya Federal Research Centre for Epidemiology and Microbiology, Ministry of Health (Moscow), the Institute of Applied Microbiology Federal Service for the Oversight of Consumer Protection and Welfare (Obolensk). Studies of staphylococcus-associated mastitis are conducted jointly with the Ernst All-Russian Research Institute for Animal Husbandry (Dubrovitsy, Moscow region). Work on the RN peptide study is conducted with the Laboratory of Peptide Chemistry of the IBCh RAS and the Laboratory of Regulation of Protein Biosynthesis at the Institute of Protein RAS. Recombinant antibodies of animals and humans are obtained together with the Gamaleya Research Institute of Epidemiology and Microbiology RAMS, the Institute of Applied Microbiology and the Laboratory of Protein Physics.

Selected publications

  1. Fursova KK, Shchannikova MP, Loskutova IV, Shepelyakovskaya AO, Laman AG, Boutanaev AM, Sokolov SL, Artemeva OA, Nikanova DA, Zinovieva NA, Brovko FA (2018). Exotoxin diversity of Staphylococcus aureus isolated from milk of cows with subclinical mastitis in Central Russia. J Dairy Sci 101 (5), 4325–4331
  2. Rudenko NV, Karatovskaya AP, Noskov AN, Shepelyakovskaya AO, Shchannikova MP, Loskutova IV, Artyemieva OA, Nikanova DA, Gladyr EA, Brovko FA (2018). Immunochemical assay with monoclonal antibodies for detection of staphylococcal enterotoxin H. J Food Drug Anal 26 (2), 741–750
  3. Artykov AA, Fursova KK, Ryazantsev DY, Shchannikova MP, Loskutova IV, Shepelyakovskaya AO, Laman AG, Zavriev SK, Brovko FA (2017). Detection of staphylococcal enterotoxin a by phage display mediated immuno-PCR method. Russ. J. Bioorganic Chem. 43 (5), 540–543
  4. Rudenko NV, Karatovskaya AP, Tsfasman IM, Brovko FA, Vasilyeva NV (2017). Molecular forms of AlpA and AlpB lytic endopeptidases from Lysobacter sp. Xl1: immunochemical determination of their intra- and extracellular localization. Russ. J. Bioorganic Chem. 43 (5), 526–530
  5. Артыков ФФ, Фурсова КК, Рязанцев ДЮ, Щанникова МП, Лоскутова ИВ, Шепеляковская АО, Ламан АГ, Завриев СК, Бровко ФА (2017). Детекция стафилоккокового энтеротоксина А методом фаговой иммуно-ПЦР. 43 (0), 518–522
  6. Karatovskaya AP, Rudenko NV, Tsfasman IM, Guseva KA, Laman AG, Boziev KM, Brovko FA, Vasilyeva NV (2016). A method for the quantitation of homologous endopeptidases AlpA and AlpB from Lysobacter sp. XL1. Process Biochem 51 (10), 1521–1526
  7. Fursova KK, Shchannikova MP, Shepelyakovskaya AO, Pavlik LL, Brovko FA (2016). Staphylococcal enterotoxin a detection with phage displayed antibodies. Russ. J. Bioorganic Chem. 42 (3), 294–299
  8. Laman AG, Lathe R, Savinov GV, Shepelyakovskaya AO, Boziev KM, Baidakova LK, Chulin AN, Brovko FA, Svirshchevskaya EV, Kotelevtsev Y, Eliseeva IA, Guryanov SG, Lyabin DN, Ovchinnikov LP, Ivanov VT (2015). Innate immunity: Bacterial cell-wall muramyl peptide targets the conserved transcription factor YB-1. FEBS Lett 589 (15), 1819–1824
  9. Rukavtsova EB, Rudenko NV, Puchko EN, Zakharchenko NS, Buryanov YI (2015). Study of the immunogenicity of hepatitis B surface antigen synthesized in transgenic potato plants with increased biosafety. J Biotechnol 203 (0), 84–88
  10. Tarlachkov SV, Dyachenko OV, Cherevatenko AM, Rudenko NV, Shevchuk TV (2014). Cloning, purification and characterization of translationally fused protein DNA methyltransferase M•HhaI-EGFP. Process Biochem 49 (12), 2170–2173
  11. Savinov GV, Shepelyakovskaya AO, Boziev KM, Brovko FA, Laman AG (2014). Search for ligand of N-acetylglucosaminyl-N-acetylmuramyl dipeptide using its peptide mimetic. Biochemistry (Mosc) 79 (2), 131–138
  12. Rudenko NV, Tsfasman IM, Latypov OR, Ledova LA, Krasovskaya LA, Karatovskaya AP, Brovko FA, Vasileva NV, Stepnaya OA (2014). Detection of propeptides of AlpA and AlpB lytic endopeptidases from Lysobacter sp. XL1 by the sandwich enzyme immunoassay based on monoclonal antibodies. Russ. J. Bioorganic Chem. 40 (3), 272–278
  13. Lyubavina IA, Brovko FA, Valyakina TI, Vertiev YV, Grishin EV (2014). Methods of express analysis of staphylococcal enterotoxin a in food products. Russ. J. Bioorganic Chem. 40 (2), 170–178
  14. Lomakina GY, Istrate A, Rudenko NV, Ugarova NN (2014). Synthesis and application of firefly luciferase antibody conjugates in a bioluminescent immunoassay of Salmonella cells. Moscow University Chemistry Bulletin 69 (2), 49–55
  15. Krasovskaya LA, Rudenko NV, Shuvalova OP, Sukharicheva NA, Abbasova SG, Skiba NP, Stepnaya OA (2013). Optimization of in vivo crosslinking technique for the study of AlpB-protein interactions in Lysobacter sp. XL1 cells. Process Biochem 48 (8), 1203–1207
  16. Orlov AV, Khodakova JA, Nikitin MP, Shepelyakovskaya AO, Brovko FA, Laman AG, Grishin EV, Nikitin PI (2013). Magnetic immunoassay for detection of staphylococcal toxins in complex media. Anal Chem 85 (2), 1154–1163
  17. Shlyapnikov YM, Shlyapnikova EA, Simonova MA, Shepelyakovskaya AO, Brovko FA, Komaleva RL, Grishin EV, Morozov VN (2012). Rapid simultaneous ultrasensitive immunodetection of five bacterial toxins. Anal Chem 84 (13), 5596–5603
  18. Tikunova N, Dubrovskaya V, Morozova V, Yun T, Khlusevich Y, Bormotov N, Laman A, Brovko F, Shvalov A, Belanov E (2012). The neutralizing human recombinant antibodies to pathogenic Orthopoxviruses derived from a phage display immune library. Virus Res 163 (1), 141–150
  19. Krasovskaya LA, Rudenko NV, Abbasova SG, Shuvalova OP, Vidyagina EO, Sukharicheva NA, Ledova LA, Stepnaya OA, Kulaev IS (2011). Monoclonal antibodies against the propeptide of endopeptidase AlpB of Lysobacter sp. XL1 for studying AlpB-protein interactions in bacterial cells. Dokl Biochem Biophys 441 (1), 298–301
  20. Shepelyakovskaya AO, Laman AG, Lomonosova AV, Fursova KK, Savinov GV, Vertiev YV, Brovko FA, Grishin EV (2011). Effect of the format of antibodies on their specificity. Mol Immunol 49 (3), 433–440
  21. Lomonosova AV, Laman AG, Fursova KK, Shepelyakovskaya AO, Vertiev YV, Brovko FA, Grishin EV (2011). Generation of scFv phages specific to Staphylococcus enterotoxin C1 by panning on related antigens. MAbs 3 (6), 513–6
  22. Laman AG, Shepelyakovskaya AO, Boziev KM, Savinov GV, Baidakova LK, Chulin AN, Chulina IA, Korpela T, Nesmeyanov VA, Brovko FA (2011). Structural modification effects on bioactivities of the novel 15-mer peptide adjuvant. Vaccine 29 (44), 7779–7784
  23. Abbasova SG, Rudenko NV, Gorokhovatskii AY, Kapralova MV, Vinogradova ID, Vertiev YV, Nesmeyanov VA, Grishin EV (2011). Monoclonal antibodies to botulinum neurotoxins of types A, B, E, and F. Russ. J. Bioorganic Chem. 37 (3), 307–315
  24. Rudenko NV, Abbasova SG, Grishin EV (2011). Production and characterization of the monoclonal antibodies to Bacillus anthracis protective antigen. Russ. J. Bioorganic Chem. 37 (3), 316–321
  25. Rubina AY, Filippova MA, Feizkhanova GU, Shepeliakovskaya AO, Sidina EI, Boziev KM, Laman AG, Brovko FA, Vertiev YV, Zasedatelev AS, Grishin EV (2010). Simultaneous detection of seven staphylococcal enterotoxins: Development of hydrogel biochips for analytical and practical application. Anal Chem 82 (21), 8881–8889
  26. Brovko FA, VasilEva VS, Lushnikova AL, Selivankina SY, Karavaiko NN, Boziev KM, Shepelyakovskaya AO, Moshkov DA, Pavlik LL, Kusnetsov VV, Kulaeva ON (2010). Cytokinin-binding protein (70 kDa) from etioplasts and amyloplasts of etiolated maize seedlings and chloroplasts of green plants and its putative function. J Exp Bot 61 (12), 3461–3474
  27. Laman AG, Shepelyakovskaya AO, Boziev KM, Savinov GV, Brovko FA, Nesmeyanov VA (2010). A method for the preparation of adjuvant peptide mimetics of GMDP with the use of monoclonal antibodies and combinatorial libraries of peptides in the format of phage display. Russ. J. Bioorganic Chem. 36 (2), 157–163
  28. Samarkina ON, Popova AG, Gvozdik EY, Chkalina AV, Zvyagin IV, Rylova YV, Rudenko NV, Lusta KA, Kelmanson IV, Gorokhovatsky AY, Vinokurov LM (2009). Universal and rapid method for purification of GFP-like proteins by the ethanol extraction. Protein Expr Purif 65 (1), 108–113
  29. Vasileva VS, Lushnikova AL, Pavlik LL, Moshkov DA, Brovko FA (2008). Elaboration of the method for immunocytochemical studying cytokinin-binding protein CBP70 localization in maize seedling tissues. Russ J Plant Physiol 55 (4), 552–559
  30. Brovko FA, Vasileva VS, Shepelyakovskaya AO, Selivankina SY, Kudoyarova GR, Nosov AV, Moshkov DA, Laman AG, Boziev KM, Kusnetsov VV, Kulaeva ON (2007). Cytokinin-binding protein (70 kDa): Localization in tissues and cells of etiolated maize seedlings and its putative function. J Exp Bot 58 (10), 2479–2490
  31. Rudenko NV, Sinegina LL, Arzhanov MA, Ksenzenko VN, Ivashina TV, Morenkov OS, Shaloiko LA, Vinokurov LM (2007). Barnase-barstar high affinity interaction phenomenon as the base for the heterogenous bioluminescence pseudorabies virus' immunoassay. J Biochem Biophys Methods 70 (4), 605–611
  32. Laman AG, Shepelyakovskaya AO, Berezin IA, Boziev KM, Rodionov IL, Chulina IA, Malakhova GV, Brovko FA, Murashev AN, Korpela TK, Nesmeyanov VA (2007). Identification of pentadecapeptide mimicking muramyl peptide. Vaccine 25 (15), 2900–2906
  33. Dubrovskaya VV, Ulitin AB, Laman AG, Gileva IP, Bormotov NI, Ilyichev AA, Brovko FA, Shchelkunov SN, Belanov EF, Tikunova NV (2007). Construction of a combinatorial immune library of human single-chain antibodies to orthopoxviruses and selection of antibodies to recombinant prA30L of the variola virus. Mol Biol 41 (1), 157–167
  34. Ulitin AB, Kapralova MV, Laman AG, Shepelyakovskaya AO, Bulgakova EV, Fursova KK, Abbasova SG, Volkov SK, Brovko FA, Nesmeyanov VA (2005). The library of human miniantibodies in the phage display format: Designing and testing. Dokl Biochem Biophys 405 (16), 437–440
  35. Kostesha NV, Laman AG, Shepelyakovskaya AO, Zaitseva IS, Orlov VP, Dykman LA, Brovko FA, Sokolov OI (2005). Selection and characterization of phage miniantibodies to actins of different origin. Biochemistry (Mosc) 70 (8), 884–889
  36. Zagranichny VE, Rudenko NV, Gorokhovatsky AY, Zakharov MV, Balashova TA, Arseniev AS (2004). Traditional GFP-type cyclization and unexpected fragmentation site in a purple chromoprotein from Anemonia sulcata, asFP595. Biochemistry 43 (42), 13598–13603
  37. Gorokhovatsky AY, Marchenkov VV, Rudenko NV, Ivashina TV, Ksenzenko VN, Burkhardt N, Semisotnov GV, Vinokurov LM, Alakhov YB (2004). Fusion of Aequorea victoria GFP and aequorin provides their Ca2+-induced interaction that results in red shift of GFP absorption and efficient bioluminescence energy transfer. Biochem Biophys Res Commun 320 (3), 703–711
  38. Zagranichny VE, Rudenko NV, Gorokhovatsky AY, Zakharov MV, Shenkarev ZO, Balashova TA, Arseniev AS (2004). zFP538, a Yellow Fluorescent Protein from Coral, Belongs to the DsRed Subfamily of GFP-Like Proteins but Possesses the Unexpected Site of Fragmentation. Biochemistry 43 (16), 4764–4772
  39. Zubova NN, Rudenko NV, Savitsky AP (2003). Aggregation strongly influences the pH-profile of fluorescence of the yellow fluorescent protein zFP538. Proc SPIE Int Soc Opt Eng 4967 (0), 88–99
  40. Vinokurov LM, Gorokhovatsky AY, Rudenko NV, Marchenkov VV, Skosyrev VS, Arzhanov MA, Zakharov MV, Burkhardt N, Semisotnov GV, Alakhov YB (2003). Detection of protein-protein interactions using Aequorea victoria bioluminescence resonance energy transfer. Proc SPIE Int Soc Opt Eng 4967 (0), 46–54
  41. Gorokhovatsky AY, Rudenko NV, Marchenkov VV, Skosyrev VS, Arzhanov MA, Burkhardt N, Zakharov MV, Semisotnov GV, Vinokurov LM, Alakhov YB (2003). Homogeneous assay for biotin based on Aequorea victoria bioluminescence resonance energy transfer system. Anal Biochem 313 (1), 68–75
  42. Skosyrev VS, Rudenko NV, Yakhnin AV, Zagranichny VE, Popova LI, Zakharov MV, Gorokhovatsky AY, Vinokurov LM (2003). EGFP as a fusion partner for the expression and organic extraction of small polypeptides. Protein Expr Purif 27 (1), 55–62
  43. Laman AG, Shepelyakovskaya AO, Ulitin AB, Markova EV, Mareeva TY, Bystrov NS, Brovko FA, Nesmeyanov VA (2002). The production of miniantibodies against human granulocyte colony-stimulating factor using the murine scFv combinatory library. Russ. J. Bioorganic Chem. 28 (2), 108–115
  44. Shepelyakovskaya AO, Teplova IR, Veselov DS, Burkhanova EA, Bosiev KM, Laman AG, Vasileva VS, Kudoyarova GR, Hall MA, Brovko FA, Kulaeva ON (2002). A cytokinin-binding 70-kD protein is localized predominantly in the root meristem. Russ J Plant Physiol 49 (1), 99–106
  45. Skosyrev VS, Gorokhovatsky AY, Vinokurov LM, Rudenko NV, Ivashina TV, Ksenzenko VN, Alakhov YB (2001). The Dependence of Stability of the Green Fluorescent Protein-Obelin Hybrids on the Nature of Their Constituent Modules and the Structure of the Amino Acid Linker. Russ. J. Bioorganic Chem. 27 (5), 323–329
  46. Brovko FA, Shepelyakovskaya AO, Burchanova EA, Boziev KM, Laman AG, Vasilyeva VS, Hall MA, Kulaeva ON (2001). Predominant localization of a cytokinin-binding 70-kD protein in the root apical meristem. Dokl Biochem Biophys 381 (0), 403–404
  47. Laman AG, Shepelyakovskaya AO, Bulgakova EV, Shavkunov AS, Kurdyukov SG, Brovko FA, Lipkin VM, Kulaeva ON (2000). Isolation of cDNA encoding cytokinin-binding proteins in maize. Russ J Plant Physiol 47 (1), 76–83
  48. Ivanova EG, Doronina NV, Shepelyakovskaya AO, Laman AG, Brovko FA, Trotsenko YA (2000). Facultative and obligate aerobic methylobacteria synthesize cytokinins. Mikrobiologiia 69 (6), 646–651
  49. Pashkov VN, Tsyuryupa GP, Grico NB, Bulgakov OV, Rudenko NV, Yakhnina EB, Grishin EV (1996). Anti-Idiotypic Monoclonal Antibodies against Latrotoxin Interact with Rat Brain Synaptosomes and Modify Latrotoxin-Induced Influx of Calcium into Synaptosomes. Bioorg Khim 22 (6), 406–407
  50. Pashkov VN, Tsyuryupa GP, Griko NB, Bulgakov OV, Rudenko NV, Yakhnina EB, Grishin EV (1996). Anti-idiotypical monoclonal antibodies against latrotoxin interact with rat brain synaptosomes and modify latrotoxin-induced influx of calcium into synaptosomes. Russ. J. Bioorganic Chem. 22 (6), 349–352

Fedor Brovko

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