Novel moNocloNal aNtibody to fibriN(ogeN) αc-regioN for detectioN of the earliest forms of soluble fibriN

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).

Fibrinogen is a crucial protein of the blood coagulation system that is converted to fibrin under the action of thrombin, followed by spontaneous polyme rization of fibrin to form the core of a thrombus [7,9]. These are the reasons why the study of the structure and functions of fibrinogen and fibrin as well as their fragments is of interest for investigation of blood clot formation, development of new antithrombotic drugs and immunodiagnostics. The mAbs that were previously produced against fibrinogen, fibrin or its fragments have allowed identification of new fibrin polymerization sites [7,[10][11][12][13][14][15] and creation of numerous immunodiagnostic techniques [7,[16][17][18][19][20][21][22][23][24]. Development of antithrombotic agents doi: https://doi.org/10.15407/ubj92.03.058 based on immunoglobulins has also been widely discussed [25].
One of most promising targets for production of mAbs towards the fibrinogen molecule are the αСregions of fibrinogen formed by the Аα221-610 fragment. The functional role of these regions is still not fully understood [26]. Structurally organized residues Аα392-610 of the αС-regions are the most interesting objects for study [27] and for development of specific mAbs targeted to them [28]. Such anti-αС-region antibodies can also be used as the tags for the detection of fibrinogen or soluble fibrin, in particular the earliest forms that preserve αС-regions uncleaved by plasmin that are extremely important for the determination of dangerous intravascular thrombus formation [29][30][31].
Taking into account the value of broadening the available panel of mAbs towards different fragments of fibrinogen αС-regions, in this study we aimed to create and characterize such an antibody for its further use in fibrin polymerization studies and for developing immunodiagnostic approaches.
Blood plasma samples. Patients (36 women, 52 men) with stage V chronic kidney disease who were treated by program hemodialysis were recruited from the Research Institute of Rehabilitation of Vin-nytsia National Pirogov Memorial Medical University (Vinnytsia, Ukraine). Blood plasma was collected into sterile plastic 10 ml tubes where it was mixed immediately with 38 g/l sodium citrate (9 parts blood to 1 part sodium citrate). Blood was spun down at 160 g for 30 min at 25 °C. Platelet poor plasma was obtained by centrifugation at 300 g for 15 min. Studies were conducted according to the Ethical Committee Approval N 14 form 01.03.2018 (Vinnytsia National Pirogov Memorial Medical University).
Fibrinogen-Sepharose was synthesized using commercially available BrCN-activated Sepharose CL-4B (Sigma-Aldrich) according to the method of Heene et al. [32]. For this synthesis the matrix was washed for 15 min on glass filter using 0.001 M HCl. The buffer was then changed to 0.1 M K 2 HPO 4 , рН 7.8 and the matrix was mixed with 20 ml of 0.5-0.75% solution of fibrinogen in the same buffer . The matrix was incubated for 3 hours at room temperature with agitation. Then the matrix was placed on the glass filter and washed with 0.1 М Tris-Н 3 РО 4 , рН 8.6 with 1 М NaCl and 0.025 М of ɛ-aminocapronic acid. Then the buffer was serially changed to 0.1 М K 2 HPO 4 , рН 7.8; 0.1 М Tris-Н 3 РО 4 , рН 4.5 with 1 М NaCl and 0.025 М ɛ-aminocapronic acid; 0.1 М K 2 HPO 4 , рН 7.8 and finally to 0.1 М K 2 HPO 4 , рН 7.8, with 0.2 М glycine and left at agitation for 40 min. The ready for use preparation of Fibrinogen-Sepharose was washed on the glass filter with 0.05 М Tris-Н 3 РО 4 , рН 7.6 and stored at 4 °С.
Proteins and their fragments. Fibrinogen was isolated from human blood plasma by a Na 2 SO 4 salting-out method as previously described [33]. The content of protein that was able to form a clot under the action of thrombin was 96-98%. The homogeneity of fibrinogen chains was characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in the presence of 0.2% β-mercaptoethanol.
Fibrin desA and desAB were obtained from fibrinogen using thrombin that cleaved fibrinopeptides A and B, and the thrombin-like enzyme ancistron that cleaved fibrinopeptides A exceptionally. The produced fibrin was dissolved in 0.125% acetic acid [34].
Proteases. Protease II was obtained from the culture media of Bacillus thuringiensis var. israelensis IMV B-7465. It was saturated from the supernatant received by precipitation with ammonium sulphate and then obtained as previously described [40].
Protease III was obtained from the culture media of Bacillus sp. that was found in periphyton of a dolphin basin in the Black Sea. Samples were provided by the Scientific Research Centre of Military Forces of Ukraine "State Oceanarium" (Odesa, Ukraine). Bacillus sp. was cultivated for 24 h in Erlenmeyer flasks during constant agitation (250 rpm) at 28 °С in culture liquid containing KH 2 PO 4 -1.6 mg/ml; MgSO 4 •7H 2 O -0.75 mg/ml; ZnSO 4 •7H 2 O -0.25 mg/ml; (NH 4 ) 2 SO 4 -0.5 mg/ml; maltose -1.0 mg/ml; gelatine -10.0 mg/ml; yeast extract -0.15 mg/ml; and рН -6.5-6.7 [41]. The fraction of protease was obtained by sedimentation using 90% (NH 4 ) 2 SO 4 . The pellet was collected by centrifugation at 5000 g for 30 min and was dissolved in 0.01 M Tris-HCl buffer (pH 7.5). The solution was then applied to the column (2.5×40 cm) with the anion exchanger TSK DEAE 650 (M) (Sigma-Aldrich), with the speed of elution set at 0.85 ml/min.
Methods. ELISa. Antigens solutions (10 µg/ml) were added to the wells of a 96-well plate (Nunc, Roskilde, Denmark). Fibrinogen was in an 0.2 М ammonium-acetate buffer, pH 8.5. Fibrin was in the same buffer but with 3 M urea added. D-dimer was in bicarbonate buffer, pH 9.5. Proteins were adsorbed during 16 h at 4 °С. Then wells were washed using an automatic washing system and poured with a 0.02 М potassium-phosphate buffer that had 0.14 М NaCl and 0.05% Tween-20 (ТPBS), and then incubated for 3 min at ambient temperature. After removing the liquid, the solution of primary mAb to human fibrinogen in TPBS was added to the wells in concentrations ranging from 10 to 0.078 µg/ ml and incubated for 1 hour at 37 °С. The wells were washed as described above. Secondary antibody (HRP-conjugated goat anti-mouse antibody (Sigma-Aldrich) was added to the wells at the titer of 1:1000 and incubated for 1 hour at 37 °С. After the washing procedure, the wells were poured with 0.1 ml of 0.05 М potassium-phosphate buffer рН 6.0 that contained 0.03-0.04% o-phenylenediamine. The reaction was terminated by the addition of 50 µl of 2 М Н 2 SО 4 . Optical density of the solutions was determined at 492 nm using the multiplate reader RT 2100C (Rayto, Shenzhen, China).
Production of mab. 0.1 mg of antigen mixed 1:1 with complete Freund adjuvant was intraperi-toneally injected into BALB/c mice. Immunization was repeated after 3 weeks using incomplete Freund adjuvant. Presence of specific antibody in the blood serum of immunized mice was determined at 10 days after each successive immunization using ELISA. At 4 weeks after the last injection, before the hybridization the mice were injected with 0.1 mg of antigen in 0.15 M NaCl. Hybridization was performed as previously described [42]. Spleen cells of immunized mice and myeloma cells X63-Ag8.6.5.3 (Flow Laboratories, Sunnyvale, California, United States) were used for the hybridization. It was performed in the presence of 50% polyethylene glycol, 1450 kDa (Sigma-Aldrich) during 1 min. Cells of the hybridoma were then re-suspended in HAT culture medium (hypoxanthine, aminopterin, thymidine; (Sigma-Aldrich) and 20% solution of fetal bovine serum Gibco, (Thermo Scientific). Then hybridomas were translocated to a 24-well plate Costar (Sigma-Aldrich) and cultivated using standard procedure. After 10 days the HAT medium was changed to HT medium (containing hypoxanthine and thymidine) and we performed screening of hybridomas to find the antigen-specific antibody by ELISA. Positive hybridomas were cultivated for multiplication. Antibody was purified from the culture media using affinity chromatography on Fibrinogen-Sepharose or (alternatively) on G-Sepharose (Pharmacia) in 0.01 M potassium-phosphate buffer pH 7.2 with 0.14 M NaCl and 0.02% of NaN 3 (PBS). Material that bound to G-Sepharose was eluted by 0.1 M glycine-HCl buffer pH 3.0. Elution from Fibrinogen-Sepharose was performed using 0.2 M glycine-HCl buffer pH 2.8. The pH of the eluted solution of antibody was immediately changed to neutral by adding PBS and concentrating it on the Centrifugal Filter Unit Amicon PM-10 (Merck, Kenilworth, New Jersey, USA).
Study of the isotype of the mAb was performed using ELISA with goat anti-serum to mice immunoglobulin isotypes (Clinical Credential; ICN Immunobiologicals, Lisle, IL, USA).
Determination of dissociation constant (k D ) of mAb and fibrin(ogen) was performed using indirect ELISA by the method of Friguet [43] with correction according to Stevens [44].
antibody biotinylation. For the biotinylation of the mAb it was dissolved in 0.1 M NaHCO 3 buffer рН 9.0 and concentrated on the Centrifugal Filter Unit Amicon PM-10 (Merck) to a final concentration of 1 mg/ml. The N-hydroxysuccinimide derivative of biotin (Sigma, USA) was dissolved in dimethyl sulfoxide (DMSO) to the same concentration. Resulting solutions were mixed in a volume:volume ratio of 8:1 (activated biotin vs antibody) and incubated at ambient temperature for 4 h. Then dialysis against PBS in argon atmosphere was performed using the Centrifugal Filter Unit Amicon PM-10 (Merck).
SDS-PagE. The molecular weights and purity of proteins were determined by SDS-PAGE using 10% or 12% gels accordingly to Laemli [45]. Hydroly sis of fibrinogen and fibrin by proteases was also analyzed by SDS-PAGE under reducing conditions.
Western Blotting was performed by a previously described technique [46]. The separated proteins after SDS-PAGE were transferred to a nitrocellulose membrane in order to specify the bands by immunoprobing. The membrane was blocked with 5% milk in PBS for one hour, incubated with a mouse mAb for another hour and then developed with a secondary HRP-conjugated goat anti-mouse antibody. The bands were visualized using 0.001 M 4-chloro-1-naphtol solution in 0.5 M Tris-НСl, pH 7.5 and 0.03% H 2 O 2 .
To work out the mass of amino acid sequence we used Peptide Mass Calculator (Peptide Protein Research Ltd., Hampshire, UK). It handles n-terminal modifications, oxidized cysteines and phosphorylated amino acids.
Cleavage of chromogenic substrates was studied in microtiter plates by mixing 0.05 М Tris-НСl buffer рН 7.4 containing 0.13 М NaCl with chromogenic substrates in the concentration range from 25 to 160 µМ, and protease III (0.005 mg/ml), at 25 °С. Amidase activity of protease III was con-tinuously monitored at 405 nm. The amount of hydrolyzed substrate was calculated using a molar extinction coefficient of 10.500 M −1 ×cm −1 for free pNA on reader Multiskan EX (Thermo Scientific) [48].
Sandwich ELISa. Catch-antibody was immobilized on a 96-well plate MaxiSorp (Nunc) in 0.11 ml of PBS (0.01 mg/ml) and incubated for 18 hours at 4 °C. Then the wells were washed using an automatic washing system, poured with the same volume of ТPBS and incubated for 30 minutes at ambient temperature. TPBS was removed from the wells that were then poured with the solutions of antigens in serial dilutions starting from the concentration of 0.01 mg/ml. Plates were incubated for 1 hour at 37 °C. After washing, the wells were poured with 0.1 ml of biotinylated tag-antibody (1 µg/ml) and incubated for 1 hour at 37 °C. Plates were washed as described above and secondary antibody (polymeric HRP-conjugated goat anti-mouse antibody, Sigma) was added to the wells at the titer of 1:6000 and incubated for 1 hour at 37 °С. After the washing procedure, wells were poured with 0.1 ml of 0.05 М potassium-phosphate buffer рН 6.0 that contained 0.03% of 0.04% o-phenylenediamine. The reaction was terminated by the addition of 0.05 ml of 2 М Н 2 SО 4 . Optical density of the solutions was determined at 492 nm using the multiplate reader RT 2100C (Rayto, China).
Statistical analysis. Statistical analysis of the data was performed by Student's t-test. Statistical calculations were made using MS Excel software (Microsoft Corporation, Redmond, WA, USA). The experimental data were processed with the variation statistics method using OriginPro 8 software (OriginLab, Northampton, MA, USA).

results and discussion
Producing the mAb against the αС-region of human fibrinogen. To produce the mAb against the αС-region of the fibrin(ogen) molecule by the method of hybridoma technology, we used the mixture of isolated chains of fibrinogen for immunization. As a result, hybridoma 1-5A was obtained. It was able to produce antibody towards fibrinogen and fibrin that was purified using Fibrinogen-Sepharose.
Specificity of the mAb was determined by indirect ELISA using the following antigens: fibrinogen, fibrin desAB, D-dimer and NDSK (Fig. 1). It was shown that mAb 1-5A selectively bound fibrinogen and fibrin desAB but did not interact with D-dimer or NDSK fragments. D-dimer and the NDSK frag-ments together comprise almost the entire core of the molecule. Thus, we assumed that the epitope of the newly obtained antibody 1-5A was located in the αC-regions.
Goat anti-serum produced to immunoglobulin isotypes of mice allowed us to determine that mAb 1-5A belonged to the IgG1 family of immunoglobulins. Antibodies of this isotype are effectively bound to G-Sepharose which can be advantageous for the scaling of their preparation. Direct ELISA by the methods of Friguet [43] and Stevens [44] allowed us to determine the dissociation constant (k d ) of mAb 1-5A in the reaction with fibrinogen (Fig. 2). It was shown that fibrinogen at the concentration of 2.8 µg/ ml decreased by two times the binding of mAb 1-5A adsorbed in the well of the 96-well plate. The calculated k d was 3.41×10 -9 М and indicated the high affinity of mAb 1-5A to fibrinogen.
Localization of the epitope of mAb І-5А. For the preliminary localization of the epitope in the fibrinogen molecule we identified the polypeptide chain that interacted with mAb 1-5A in the fibrinogen molecule. For this aim, we performed SDS-PAGE of fibrinogen under reducing conditions (0.2% of β-mercaptoethanol) in which three chains of fibrinogen moved in the gel separately. mAb 1-5A was used for the detection of the protein in Western blotting. Thus, we showed that the target of mAb 1-5A was specifically the Аα-chain of the fibrinogen molecule (Fig. 3).  For further localization of the epitope we used two proteases with different specificities, that possessed fibrinogenase activity and cleaved the fibrinogen Aα-chain. Both enzymes called protease II and protease III were purified from the different Bacillus strains. We studied their action on fibrinogen using Western blotting (Fig. 4).
Previously it was reported that protease II cleaved the peptide bond Аα504-505 [49]. The resulting fragment Аα505-610 was clearly detected using mAb 1-5A (Fig. 4, a). However no hydrolytic products of protease III were observed in the same Western blot which may indicate that the epitope of mAb 1-5A is was destroyed by proteolysis, therefore it overlapped the residues cleaved by protease III. To prove this concept we compared the hydrolysis of fibrinogen by the studied proteases in Western blots using mAb 1-5A and previously described antibody ІІ-5С with the epitope located in the Aα20-78 fragment of fibrinogen [50] (Fig. 5).
Western blotting using mAb ІІ-5С demonstrated that protease ІІІ cleaved off the С-terminal portion of the Аα-chain of fibrinogen with an apparent molecular weight of 6 kDa (Fig. 5, central panel). Western blotting using the novel mAb 1-5A showed the disappearance of the Аα-chain of fibrinogen and no accumulation of hydrolytic products that can be detected using this antibody (Fig. 5, bottom panel). Thus we concluded that the epitope of mAb 1-5A is located in fibrinogen fragment Аα505-610 and is destroyed under the action of protease III from Bacillus thuringiensis var. israelensis IМV В-7465. So by determining the site of proteolytic cleavage we can detect the epitope of mAb 1-5А.
For the identification of hydrolytic products of proteinase III we used MALDI-TOF analysis. Sev- eral polypeptides were found among the hydrolytic products and were identified as fragments of Aαchains of fibrinogen using the computer server Peptide Mass Calculator (Fig. 6). Core products of fibrinogen hydrolysis by protease III were polypeptides with the molecular weights of 3928 Da and 3484 Da (Fig. 6). Using Peptide Mass Calculator we identified them as fragments Aα550-583 and 553-583. As shown in Fig. 6, both sites of cleavage of fibrinogen by protease III are located in the hydrophobic clusters: IAEF, YKMA. To prove the conclusion that protease III preferentially cleaves peptide bonds surrounded by hydrophobic amino acids, we used substrate analysis and compared the ability of protease III for cleavage of chromogenic peptide substrates that contained different amino-acid residues in S1 and S2 centers. Chromogenic peptide substrates of elastase, thrombin and plasmin (Suc-Ala-Ala-Ala-pNa, H-D-Phe-Pip-Arg-pNa and H-D-Val-Leu-Lys-pNa, respectively) were studied. Protease III effectively cleaved the Suc-Ala-Ala-Ala-pNa substrate. Thus, it was specific for the peptide bond formed by the COOH-terminal group of alanine in the hydrophobic surrounding.

Fig. 4. Western blots of fibrinogen hydrolytic products obtained from the action of (A) protease II and (B) protease III. Time of hydrolysis -45 min. Monoclonal antibody (mAb) 1-5A was used for the band detection. Lanes 1 -sample of fibrinogen before hydrolysis. Lanes 2 -sample of fibrinogen after hydrolysis. Upper outlined zone -native Aα-chain of fibrinogen. Lower outlined zone -505-610 fragment of Aα-chain of fibrinogen. No fragments were detected in the case of proteolysis by protease III
Taking into account the data on protease III specificity towards Aα549-550, 552-553 and 583-584  , lanes 1-11) and corresponding Western blot (central, bottom panels) using monoclonal antibody (mAb) ІІ-5С and mAb І-5А. Samples were taken after 2. 5, 5, 7.5, 10, 15, 20, 25, 30, 40, 50,  Aα Aα peptide bonds we can conclude that the epitope of mAb 1-5A must be destroyed by one of these three cleavage events during the hydrolysis. It is known that epitopes range from 4 to 12 amino acids in size [51]. That is the reason why in determining the epitope of mAb 1-5A we may count a maximum of 12 amino acid residues to the C-terminal point from the 550th amino acid and a maximum of 12 amino acid residues to the N-terminal point from the 583rd amino acid of the Aα-chain. Thus, the epitope can be located in the 537-595 fragment of the Aα-chain. Approbation of mAb 1-5A as tag-antibody for immunodiagnostics. The high affinity of the created mAb 1-5A and its specificity towards the Aα537-595 residue of the αC-region of fibrin(ogen) allowed us to conclude it has potential use in immunodiagnostics.

and 60 min of incubation with protease. Top panel -SDS-PAGE. Aα-chain of fibrinogen is disappearing during incubation with protease III. Central panel -Western blot using mAb ІІ-5С that interacts with N-terminal portions of the Aα-chain of fibrinogen. Accumulation of hydrolytic product desAα that contains uncleaved N-terminus identified by mAb ІІ-5С indicates that protease III preferentially cleaves the C-terminal peptide of the Aα-chain. Bottom panel -Western blot using mAb І-5A that interacts with C-terminal portions of the Aα-chain of fibrinogen. The Aα-chain of fibrinogen is disappearing . No C-terminal peptide containing fragments are found
We previously developed a highly sensitive and effective test-system for the determination of soluble fibrin in human blood plasma [52]. As the catchantibody in this test-system we used fibrin-specific mAb FnI-3C. As the tag-antibody we used another mAb (II-4d) that has an epitope in the NH2-terminal fragment of the γ-chain of the D-region of the fibrin(ogen) molecule [53]. This test-system based on sandwich ELISA allows to perform quantitative determination in human blood plasma of soluble fibrin that is composed of monomers, dimers and oligomers of fibrin, possibly with fibrinogen molecules at the sticky ends and also initial products of plasmin hydrolysis of fibrin lacking αС-regions [53].
As far as the epitope of mAb 1-5A is located in the Aα537-595 fragment of fibrin(ogen), this antibody can be used as a tag-antibody in sandwich ELISA and exclusively detect the earliest forms of soluble fibrin that were not cleaved by plasmin and preserve αC-regions in their structure. It should be emphasized that these uncleaved forms of soluble fibrin are extremely important as early markers of blood coagulation activation and the danger of thrombus formation [55].
To visualize the possibility of using mAb 1-5A as a detecting tag-antibody in sandwich ELISA for the detection of the earliest forms of soluble fibrin we created a calibration curve with different concentrations of fibrin desAB using the fibrin-specific catch-antibody FnI-3C. We also compared this curve with that obtained using the standard tag-antibody ІІ-4d. Both tag-antibodies were biotinylated (Fig. 7). The result shown in Fig. 7 confirmed that newly created mAb 1-5А can be used in sandwich ELISA as a tag-antibody. Being applied in pair with catchantibody FnI-3C it would allow detection of the earliest forms of soluble fibrin. The next task was to prove the possibility of application of this method directly in blood plasma. To achieve this goal we collected samples of blood plasma from patients with stage V chronic kidney disease who were treated by program hemodialysis and determined the earliest forms of soluble fibrin using 1-5А as a tag-antibody. We also determined the total content of soluble fibrin by the same algorithm using ІІ-4d as a tag-antibody (Fig. 8).
The group of patients was selected as a heterogeneous population that is known to manifest thrombophilia, however we could expect patients would have low or high risk of intravascular thrombus formation as well as patients with bleeding [54]. Selection of these patients allowed us to not only test the newly obtained mAb in ELISA, but to also try it with blood plasma samples that have a potentially wide range of soluble fibrin concentrations -from normal to increased concentrations. Preliminary analysis allowed us to conclude that the earliest forms of soluble fibrin constitute a small part of total soluble fibrin content. Using ІІ-4d as the tag-antibody allowed us to determine an elevated content of soluble fibrin in blood plasma from 50% of the patients. Only several patients also had the earliest forms of soluble fibrin at a concentration of more than 3 µg/ml according to the results of the test performed using 1-5А as the tag-antibody.
We obtained novel mAb 1-5A that had high affinity towards fibrinogen and fibrin but did not react with D-dimer or the NDSK fragment of fibrin. Being specific to the fragment 537-595 of the Aα-chain of the fibrin(ogen) molecule this mAb can be used both for laboratory diagnostics as well as for probing the fibrinogen molecule structure and functions.
1-5A can be used as a tag-antibody in sandwich ELISA together with the fibrin-specific FnI-3C catch-antibody and allow the determination of the earliest forms of soluble fibrin that are direct evidence of pathological thrombin generation and the danger of intravascular thrombus formation. Being the direct evidence of activation of blood coagulation and generation of thrombin, the concentration of the earliest forms of soluble fibrin can provide additional information of the state of the blood coagulation system in patients and indicate the activation of blood coagulation that is occurring. An additional diagnostic approach developed on the basis of the newly obtained antibody can be applied simultaneously with the existing method of determination of the total content of soluble fibrin indicating the danger of intravascular thrombus formation, allowing estimation of the efficiency of antithrombotic therapy or predicting disease progression. Selection of the conditions for using the newly obtained mAb for application in the sandwich ELISA technique will allow us to approbate the proposed method and to prove its diagnostic ability in different pathologies connected with the risk of intravascular thrombus formation.
Having a unique specificity towards the structurally labile and functionally active part of the fibrinogen molecule, mAb 1-5A can also be used for studies of the protein-protein and protein-cell interactions of the αC-regions of the fibrin(ogen) molecule. In particular, residue 537-595 of the Aαchain of the molecule of fibrin(ogen) that was determined to be an epitope of mAb 1-5A can play important roles in platelet receptor recognition, polymerization of fibrin and interactions of endothelial cells during wound healing. Further studies with the use of mAb 1-5A as the specific inhibitor will allow us to evaluate these roles more precisely.
conclusion An anti-Aα537-595 mAb was obtained and characterized. Its application will contribute to the study of structure and functions of fibrinogen αCregions and can also be useful for the detection of the earliest forms of soluble fibrin which can be evidence of the risk of intravascular thrombus formation.
This study was funded in the frame of the Ukrainian Government order scientific program according to Agreement № DZ/59-2018 "Development of test-system for simultaneous determination of three molecular markers of intravascular blood coagulation".

Conflict of interest.
Authors have completed the Unified Conflicts of Interest form at http://ukrbiochemjournal.org/wp-content/uploads/2018/12/ coi_disclosure.pdf and declare no conflict of interest.