Epitope And Functional Study Of Anti-platelet GPIbα Monoclonal Antibody

[Objective]To investigate the function of monoclonal antibody 9C9 against human platelet GPIba in vivo and in vitro experiments.And the antibody epitopes were studied.[Methods]Monoclonal antibody(mAb)9C9 against human platelet GPIbα was obtained by immunizing Balb/C mice with hybrioma cells to prepare mouse ascites.The ascites was collected and purified to obtain mAb 9C9.The mAb 9C9 was incubated with platelets,and the recognition specificity of mAb 9C9 against GPIba was detected by western blot and flow cytometry(FCM).The Fab was obtained by enzymatic hydrolysis of 9C9 antibody by papain,and the Fab fragment was verified by routine electrophoresis and coomassie bright blue staining.The function of mAb 9C9 was verified in vitro and vivo.In vitro experiments:1)Platelet aggregation was performed to detect platelet aggregation induced by mAb 9C9 after intervention with Ristocetin,Thrombin,U46619,ADP,Collagen and Collagen respectively.2)Human platelets were incubated with mAb 9C9,and the apoptosis and activation of platelets was detected by FCM.In vivo experiments:1)9C9 and 9C9-Fab were transfused into 7-8 weeks of hGPIba mice through orbit.Platelet count apparatus was used to detect changes in platelet count at different times after antibody intervention.2)Platelets were washed by Calcein labeled hGPIbα mice.Then fluorescent-labeled platelets were injected into orbit of hGPIbα mice of younger.The mesenteric arterioles were damaged by FeCl3,and the formation of arterial thrombosis was observed and recorded under microscope.3)Orbital infusion of mAb 9C9 and 9C9-Fab was used to intervene in hGPIbα mice to establish tail hemorrhage model and record the tail bleeding time.Next,we identified the epitope of mAb 9C9.The mAb 9C9 was fully bound to GPIbα protein,and the non-binding site was digested by trypsin and eluted.The binding site was sequenced by mass spectrometry(MS).The peptide collected by MS was compared with the database,and the epitope of mAb 9C9 was obtained by Mascot and MQ analysis.The epitopes were synthesized and coated with plates,and the binding of the short peptide at the recognition site to mAb 9C9 was verified by indirect ELISA.In order to explore the function of mAb 9C9 in a more comprehensive way,we transformed mAb 9C9 to form chimeric antibody.The chimeric antibody was incubated with platelets,and the binding between antibody and platelet GPIba was detected by western blot and FCM.Platelet aggregation was detected by ristocetin and thrombin after intervention with chimeric antibody.[Results]Monoclonal antibody 9C9 was produced by immunizing mice with hybridoma cells,and 9C9-Fab fragment was extracted.9C9 and 9C9-Fab induced the auto-aggregation of human platelets less than 10%,and both could dose-dependently inhibit ristocetin-induced aggregation,and 9C9-Fab inhibited ristocetin-induced aggregation more significantly.9C9 inhibited platelet aggregation induced by low concentration of thrombin,collagen and U46619.However,there was no inhibitory effect on ADP-induced aggregation.9C9 induces apoptosis and activation of platelets in vitro.In vivo tests showed that 9C9 antibodies reduced platelet count in hGPIba mice,while 9C9-Fab had little effect.9C9-Fab inhibits the formation of mesenteric artery thrombosis and tail bleeding time.The binding site of mAb 9C9 against human platelet GPIba was 154TLPPGLLTPTPK166 by MS.Western blot and FCM verified the binding of chimeric antibody to platelets,and the chimeric antibody inhibited platelet aggregation induced by ristocetin and thrombin.[Conclusion]Anti-platelet GPIba mAb 9C9 inhibited inducer-induced platelet aggregation in vitro and inhibited the formation of thrombus and tail bleeding time in vivo.The identification of mAb 9C9 epitopes and the transformation of chimeric antibodies provide certain reference value for the discovery of antithrombotic drugs and the further research and clinical diagnosis of platelet-related diseases.