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Information × Registration Number 0218U000724, 0113U002496 , R & D reports Title Surface plasmon resonance imunosensor express-analyzer for the quantitative determination of markers of hemostasis system for medicine and veterinary medicine: metrological ensuring and trial operation. popup.stage_title Head Komisarenko Serhiy, Доктор біологічних наук Registration Date 12-02-2018 Organization A.V.Palladin Institute of Biochemistry of National Academy of Sciences popup.description2 In the course of the experimental exploitation of the active prototype of the SPR express-analyzer of hemostasis markers, the two-channel immunosensor chamber was improved, the method of depositing gold on the chip was optimized, the elemental composition and gold film structure were characterized, the methods for forming a self-assembling monolayer (SAM) from short chain thio-compounds - mercaptoethanol and mercaptoethylamine and the method of functionalizing the surface of the gold plate of the chip with the use of carboxymethyldextran were optimized; worked out the technology of preparation of fibrin (ogen) - imunosensor chips based on mAb developed at the A.V. Palladin Institute of Biochemistry of NAS of Ukraine, to determine fibrinogen, fibrin and D - dimer in plasma and model systems from purified proteins; a technology for determining markers of the hemostasis system has been developed, which includes the preparation of blood plasma samples for analysis by dilution in a buffer containing inhibitors of fibrin polymerization, a cocktail of inhibitors of proteolytic enzymes, and regeneration of the operating surface of the immunosensor chip; calibration curves were obtained to determine the concentration of fibrinogen and fibrin in human blood plasma, the concentration of fibrinogen and fibrin in the blood plasma and in the model system from the purified proteins was determined. Methods for preparing protein-calibrators for the construction of calibration curves have been developed. It has been found that the use of sodium fluoride instead of sodium chloride doubles the response of the sensor to the binding of fibrin to monoclonal antibodies. To increase the sensitivity of the immunobiosensor, selective gold nanoparticles conjugated to GPRP peptide (NPG-GPRP), which is a fibrin polymerization site, were designed and characterized. It was found that NPG-GPRP increased by an order of magnitude the responses of fibrin(ogen)- specific immunobiosensor. Experimental operation of the SPR express-analyzer as a new method for studying in real time the process of thrombin-dependent transformation of fibrinogen into fibrin and the mechanism formation of a fibrin clot - the basis of a thrombus, allowed to establish that the rate of transformation of fibrinogen into fibrin after cleavage of fibrinopeptides A by thrombin is regulated by Cl ions; binding of peptides to mAb I-3c unambiguously points to the fibrin B?-chain 126-135 as to the site on the fibrin molecule involved in the lateral association of protofibrils; the site where conformational changes are observed covers 123-135 amino acid residues of the B?-chain of the molecule; peptides that model amino acid sequences in the hinge region of fibrin and block the lateral association of fibrin protofibrils, bind to mAb I-3c, the epitope of which is also localized in the hinge region of fibrin. A method has been developed for determining the kinetic parameters of the antigen-antibody interaction reactions (ka, kd, and Kd) for the analysis of individual sensorograms obtained using the Plasmont-6 SPR spectropolarimeter. The analysis of sensorograms of fibrinogen binding to mAb II-4d and fibrin desAB with mAb I-3c in a system of purified proteins and in blood plasma, as well as X fragments of desAB fibrin with mAb I-3c in a system of purified proteins, and kinetic parameters of the association reactions were found. In cooperation with the V.E. Lashkaryov IPS of NAS of Ukraine developed the program "Itera" for the automated quantitative determination of the kinetic parameters of binding of markers of the hemostasis system in blood plasma samples on the basis of analysis of the sensorogram of the device. Product Description popup.authors Березницький Г.К. Гоголінська Г.К. Колеснікова І.М. Костюченко О.П. Луговський Е. В. Макогоненко Є. М. Назаренко В.І. Урвант Л. П. Ушенін Ю. В. Чернишенко В.О. popup.nrat_date 2020-04-02 Close