Ukr.Biochem.J. 2020; Volume 92, Issue 3, May-Jun, pp. 58-70

doi: https://doi.org/10.15407/ubj92.03.058

Novel monoclonal antibody to fibrin(ogen) αC-region for detection of the earliest forms of soluble fibrin

04.08.2020   Uncategorized   No comments

N. E. Lugovska1, I. M. Kolesnikova1, Ye. M. Stohnii1, V. O. Chernyshenko1*,
A. V. Rebriev1, O. P. Kostiuchenko1, G .K. Gogolinska1, N. A. Dziubliuk2,
L. D. Varbanets2, T. M. Platonova1, S. V. Komisarenko1

1Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
2Zabolotny Institute of Microbiology and Virology,National Academy of Sciences of Ukraine, Kyiv;
*e-mail: bio.cherv@gmail.com

Received: 08 May 2020; Accepted: 30 June 2020

Obtaining new monoclonal antibodies (mAbs) towards fibrin(ogen) and its fragments is an important task for studying mechanisms of blood clot formation, searching for novel antithrombotic agents and developing immunodiagnostics. The aim of the present work was to create and characterize a new mAb towards the fibrin(ogen) αС-region. We surmise that having a specific mAb towards this flexible part of the molecule will allow us to study the role of the αС-region in fibrin polymerization and also to develop an approach for detecting the earliest forms of soluble fibrin by sandwich ELISA. Using hybridoma technology we оbtained mAb 1-5A to the αC-region of fibrinogen.. It was characterized using several variations of ELISA and Western blot. Application of specific proteases together with MALDI-TOF analysis allowed us to localize its epitope that is located in fragment 537-595 of the Aα-chain of fibrin(ogen). МAb 1-5A can be used as a detecting tag-antibody in sandwich ELISA for the quantification of the earliest forms of soluble fibrin which are uncleaved by plasmin and preserved C-terminal portions of αC-regions. These earliest forms of soluble fibrin are direct evidence of blood coagulation system activation, thrombin generation and the danger of intravascular thrombus formation. Their determination will provide additional, more accurate information about the state of the blood coagulation system and the risk of blood clotting, which is very important for the timely and correct selection of adequate antithrombotic therapy. MAb 1-5A effectively binds the αC-containing molecules of fibrinogen and fibrin in blood plasma. It also can be used for studying protein-protein and protein-cellular interactions of the αC-regions of fibrin(ogen).

Keywords: , , , , ,

References:

  1. Gao Y, Huang X, Zhu Y, Lv Z. A brief review of monoclonal antibody technology and its representative applications in immunoassays. J Immunoassay Immunochem. 2018;39(4):351-364.    PubMed, CrossRef
  2. Okda F, Lawson S, Liu X, Singrey A, Clement T, Hain K, Nelson J, Christopher-Hennings J, Nelson EA. Development of monoclonal antibodies and serological assays including indirect ELISA and fluorescent microsphere immunoassays for diagnosis of porcine deltacoronavirus. BMC Vet Res. 2016;12:95. PubMed, PubMedCentral, CrossRef
  3. Ansar W, Ghosh S. Monoclonal antibodies: A tool in clinical research. Indian J Clin Med. 2013; 4: 9-21. CrossRef
  4. Temrikar ZH, Suryawanshi S, Meibohm B. Pharmacokinetics and clinical pharmacology of monoclonal antibodies in pediatric patients. Paediatr Drugs. 2020;22(2):199-216. PubMed, PubMedCentral, CrossRef
  5. Lu RM, Hwang YC, Liu IJ, Lee CC, Tsai HZ, Li HJ, Wu HC. Development of therapeutic antibodies for the treatment of diseases. J Biomed Sci. 2020;27(1):1. PubMed, PubMedCentral, CrossRef
  6. Siddiqui MZ. Monoclonal antibodies as diagnostics; an appraisal. Indian J Pharm Sci. 2010;72(1):12-17. PubMed, PubMedCentral, CrossRef
  7. Lugovskoy EV, Makogonenko EM, Komisarenko SV. Molecular mechanisms of formation  and destruction of fibrin. K.: Nauk. Dumka, 2013. 230 p. (In Russian).
  8. Lugovskoy EV, Komisarenko SV. Monoclonal antibodies as an instrument to study fibrin polymerization. Bioorg Khim. 2000;26(12):883-891. (In Russian). PubMed
  9. Wolberg AS, Campbell RA. Thrombin generation, fibrin clot formation and hemostasis. Transfus Apher Sci. 2008;38(1):15-23. PubMed, PubMedCentral, CrossRef
  10. Lugovskoy EV, Gritsenko PG, Kolesnikova IN, Zolotarova EN, Chernishov VI, Nieuwenhuizen W,  Komisarenko  SV. Two monoclonal antibodies to D-dimer-specific inhibitors of fibrin polymerization. Thromb Res. 2004;113(3-4):251-259. PubMed, CrossRef
  11. Lugovskoi EV, Makogonenko EM, Chudnovets VS, Derzskaya SG, Gogolinskaya GK, Kolesnikova IN, Bukhanevich AM,  Sitak  IN, Lyashko ED, Komissarenko SV. The study of fibrin polymerization with monoclonal antibodies. Biomed Sci. 1991;2(3):249-256. PubMed
  12. Lugovskoy EV, Gritsenko PG, Kapustianenko LG, Kolesnikova IN, Chernishov VI, Komisarenko SV. Functional role of Bbeta-chain N-terminal fragment in the fibrin polymerization process. FEBS J. 2007;274(17):4540-4549. PubMed, CrossRef
  13. Lugovskoy EV, Chudnovets VS, Makogonenko EM,  Derzskaia SG, Gogolinskaia GK, Kolesnikova IN, Mikhalovskaia LI, Komissarenko SV. Study of the polymerization of fibrin using monoclonal antibodies 2D-2A and their Fab-fragments. Ukr Biokhim Zhurn. 1995;67(1):64-70. (In Russian). PubMed
  14. Lugovskoy EV, Gritsenko PG, Kolesnikova IN, Lugovskaya NE, Komisarenko SV. A neoantigenic determinant in coiled coil region of human fibrin beta-chain. Thromb Res. 2009;123(5):765-770.   PubMed, CrossRef
  15. Gaffney PJ, Edgell TA, Walker JM. Fibrin polymerisation. Evidence for a secondary polymerisation site on the carboxy terminal end of the AA chain using a human fibrin specific murine monoclonal antibody. Ukr Biokhim Zhurn. 1996;68(4):43-44.  PubMed
  16. Soe G, Kohno I, Inuzuka K, Itoh Y, Matsuda M. A monoclonal antibody that recognizes a neo-antigen exposed in the E domain of fibrin monomer complexed with fibrinogen or its derivatives: its application to the measurement of soluble fibrin in plasma. Blood. 1996; 88(6): 2109-2117.
  17. Török-Nagy B, Antal J, Dénes B. Generation and characterization of D-dimer specific monoclonal antibodies for use in latex agglutination test. PLoS One. 20194;14(2):e0212104. PubMed, PubMedCentral, CrossRef
  18. Koga S. A novel molecular marker for thrombus formation and life prognosis–clinical usefulness of measurement of soluble fibrin monomer-fibrinogen complex (SF). Rinsho Byori. 2004;52(4):355-361. (In Japanese).  PubMed
  19. Hamano A, Tanaka S, Takeda Y, Umeda M, Sakata Y. A novel monoclonal antibody to fibrin monomer and soluble fibrin for the detection of soluble fibrin in plasma. Clin Chim Acta. 2002;318(1-2):25-32. PubMed,  CrossRef
  20. Lugovskoy EV, Kolesnikova IN, Lugovskaia NE, Litvinova LM, Gritsenko PG, Gogolinskaia GK, Liashko ED, Kostiuchenko EP, Remizovskiy GA, Pedchenko VN. Komisarenko SV. Quantification of D-dimer and soluble fibrin in blood plasma of people with ischemic heart disease and hypertension. Ukr Biokhim Zhurn. 2004; 76(6): 136-141. (In Russian).
  21. Lugovskoy EV, Kolesnikova IN, Lugovskaya NE, Gritsenko PG, Litvinova LM, Gogolinskaia GK, Liashko ED, Kostiuchenko EP, Golota VIa, Kurochka VV, Komisarenko SV. Soluble fibrin and D-dimer at normal pregnancy and pregnancy with risk of miscarriage. Ukr Biokhim Zhurn. 2006;78(4):120-129. (In Russian). PubMed
  22. Lugovskoy EV, Efimov DA, Gritsenko PG, Kolesnikova IN, Lugovskaya NE, Litvinova LM, Kostyuchenko EP, Efimov AS, Komisarenko SV. Soluble fibrin and D-dimer as molecular markers of blood vessels complications at diabetes mellitus. Rep Nat Acad Sci Ukraine. 2009;(12):190-193. (In Russian).
  23. Kramareva VN, Gritsenko PG, Kolesnikova IN, Lugovskaya NE, Litvinova AL, Kostyuchenko EP, Lugovskoi EV. Levels of thrombinemia and thrombosis markers in patients with essential hypertension and cardiovascular risk. Nauk Visnyk Med Univ Bogomolets. 2009;(4):177-181.
  24. Lugovska NE.  Inventive activity of the Departments of Protein Structure and Function, and Molecular Immunology of the Palladin Institute of Biochemistry of NAS of Ukraine. Part II. National breakthrough in the study and diagnostics of human hemostasis system. Ukr Biochem J. 2016; 88(3):106-118.   (In Ukrainian).  PubMed, CrossRef
  25. Elgundi Z, Reslan M, Cruz E, Sifniotis V, Kayser V. The state-of-play and future of antibody therapeutics. Adv Drug Deliv Rev. 2017;122:2-19. PubMed, CrossRef
  26.  Mosesson MW. Fibrinogen and fibrin structure and functions. J Thromb Haemost. 2005;3(8):1894-904.  PubMed, CrossRef
  27. Tsurupa G, Mahid A, Veklich Y, Weisel JW, Medved L. Structure, stability, and interaction of fibrin αC-domain polymers. Biochemistry. 2011;50(37):8028-8037. PubMed, PubMedCentral, CrossRef
  28. Weisel JW, Litvinov RI. Mechanisms of fibrin polymerization and clinical implications. Blood. 2013;121(10):1712-1719.  PubMed, PubMedCentral, CrossRef
  29. Dempfle CE. The use of soluble fibrin in evaluating the acute and chronic hypercoagulable state. Thromb Haemost. 1999;82(2):673-83.  PubMed, CrossRef
  30. Lugovskoi EV, Gritsenko PG, Lugovskaya NE, Kolesnikova IN, Komisarenko SV. Molecular composition of soluble fibrin and fibrin degradation products. Methods of their assay. Hematol Transfusiol. 2006;51(5):39-43.
  31. Suzuki A, Ebinuma H, Matsuo M, Miyazaki O, Yago H. The monoclonal antibody that recognizes an epitope in the C-terminal region of the fibrinogen α-chain reacts with soluble fibrin and fibrin monomer generated by thrombin but not with those formed as plasmin degradation products. Thromb Res. 2007;121(3):377-385.  CrossRef
  32. Heene DL, Matthias FR. Adsorbtion of fibrinogen derivatives on insolubilized fibrinogen and fibrin monomer. Thromb Res. 1973; 2(2): 137-154.
  33. Varetskaya TV. Microheterogeneity of fibrinogen. Cryofibrinogen. Ukr Biokhim Zhurn. 1960;32:13–24.
  34. Belitser VA, Varetskaja TV, Malneva GV. Fibrinogen-fibrin interaction. Biochim Biophys Acta. 1968;154(2):367-375. PubMed,  CrossRef
  35. Lugovskoy EV, Kolesnikova IN, Grisenko PG, Zolotareva EN, Gaffney P, Nieuwenhuizen W, Komisarenko SV. A neoantigenic determinant in the D-dimer fragment of fibrin. Thromb Res. 2002;107(3-4):151-156. PubMed, CrossRef
  36. Platonova TN, Musialkovskaia AA, Tolstykh VM, Belitser VA. Inhibition of fibrin assembly by fragment D and its dimer derived from fibrinogen and stabilized fibrin. Evidence for the two-step type of inhibition. Biokhimiia. 1980;45(10):1780-1787. (In Russian).  PubMed
  37. Chudnovets VS, Lugovskoі EV, Gogolinskaia GK, Derzskaia SG, Nazimov IV, The isolation of the NH2-terminal disulfide nodes of human fibrinogen and fibrin and of their constituent polypeptide chain fragments. Dokl Akad Nauk SSSR. 1991;317(6):1496-1499. (In Russian). PubMed
  38. Gårdlund B, Hessel B, Marguerie G, Murano G, Blombäck B. Primary structure of human fibrinogen. Characterization of disulfide-containing cyanogen-bromide fragments. Eur J Biochem. 1977;77(3):595-610. PubMed, CrossRef
  39. Blombäck B, Blombäck M, Henschen A, Hessel B, Iwanaga S, Woods KR. N-terminal disulphide knot of human fibrinogen. Nature. 1968;218:130-134. CrossRef
  40. Matselyukh ОV, Nidialkova NA, Varbanets LD. Purification and physicochemical properties of Bacillus thuringiensis IMB B-7324 peptidase with elastolytic and fibrinolytic activity. Ukr Biokhim Zhurn. 2012;84(6):25-36. (In Ukrainian). PubMed
  41. Koltukova NV, Vaskivniuk VT. Selection of methods for the isolation of the proteolytic complex from Bacillus mesentericus 316m at deep cultivation. Microbiol Z. 1980; 42(2): 245-248. (In Russian).
  42. Köhler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 1975;256(5517):495-497. PubMed, CrossRef
  43. Friguet B, Chaffotte AF, Djavadi-Ohaniance L, Goldberg ME. Measurements of the true affinity constant in solution of antigen-antibody complexes by enzyme-linked immunosorbent assay. J Immunol Methods. 1985;77(2):305-319. PubMed, CrossRef
  44. Stevens FJ. Modification of an ELISA-based procedure for affinity determination: correction necessary for use with bivalent antibody. Mol Immunol. 1987;24(10):1055-1060. PubMed, CrossRef
  45. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227(5259):680-685.  PubMed, CrossRef
  46. Tsang VCW, Peralta JM, Simons AR. Enzyme-linked immunoelectrotransfer blot techniques (EITB) for studying the specificities of antigens and antibodies separated by gel electrophoresis. Methods Enzymol. 1983;92:377-391. PubMed, CrossRef
  47. Chapman JR. Mass Spectrometry of Proteins and Peptides. Humana Press, 2000, 538 p.  CrossRef
  48. Gershkovich AA, Kibirev VK. Chromogenic and fluorogenic peptide substrates of proteolytic enzymes. Bioorg Khim. 1988;14(11):1461-1488. (In Russian). PubMed
  49. Stohniy EM, Chernyshenko VO, Nidialkova NA, Rebriev AV, Varbanets LD, Hadzhynova VE, Chernyshenko TM, Kolesnikova IM, Lugovskoy EV.  Mapping of residues of fibrinogen cleaved by Protease II of Bacillus thuringiensis var. israelensis IMV B-7465. Ukr Biochem J. 2016; 88(Special Issue): 79-86.  CrossRef
  50. Urvant LP, Makogonenko ЕМ, Pozniak ТА, Pydiura NА, Kolesnikova IN, Tsap PY, Bereznitzkiy GК, Lugovskoy EV, Komisarenko SV. Binding of mAb II-5c to Aα20–78 fragment of fibrinogen inhibits aneoantigenic determinant exposure within Bβ126–135 site of a molecule.  Dopov Nac Akad  Nauk Ukr. 2014;5:149-156. (In Ukrainian). CrossRef
  51. Buus S, Rockberg J, Forsström B, Nilsson P, Uhlen M, Schafer-Nielsen C. High-resolution mapping of linear antibody epitopes using ultrahigh-density peptide microarrays. Mol Cell Proteomics. 2012;11(12):1790-1800. PubMed, PubMedCentral, CrossRef
  52. Lugovskoy EV, Kolesnikova IN, Komisarenko SV. Usage of monoclonal antibodies for determination of localization of antigenic determinants and fibrin polymerization sites within fibrinogen and fibrin molecules and their application in test-systems for diagnostics and the threat of thrombus formation. Biotechnologia Acta. 2013; 6(4): 33-42.  CrossRef
  53. Lugovskoi EV, Gritsenko PG, Komisarenko SV. Molecular mechanisms of the polymerization of fibrin and the formation of its three-dimensional network. Bioorg Khim. 2009;35(4):437-456. (In Russian). PubMed, CrossRef
  54. Grupp C, Troche-Polzien I, Stock J, Bramlage C, Müller GA, Koziolek M. Thrombophilic risk factors in hemodialysis: Association with early vascular access occlusion and patient survival in long-term follow-up. PLoS One. 2019;14(9):e0222102. PubMed, PubMedCentral, CrossRef
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