Validação de anticorpo monoclonal para detecção de vesículas extracelulares de plaquetas humanas
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Data
2021-05-28
Autores
Oliveira, Fernando Aparecido de
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Universidade Estadual Paulista (Unesp)
Resumo
A terapia transfusional, com o uso de sangue e componentes, é uma tecnologia de grande relevância na atualidade, colaborando para a melhoria e salvando vida de pacientes. No entanto, assim como outras intervenções terapêuticas, pode acarretar em complicações aguda ou tardias ao receptor. Dentre as indicações médicas de transfusão de hemocomponentes, destaca-se o Concentrado de Plaquetas (CP), obtido através de separação, por centrifugação de uma unidade de sangue total - (ST) ou por aférese. Esses hemocomponentes tem volume e quantidade de plaquetas diferentes dependendo da forma de preparo. Quanto ao armazenamento, devem ser mantidos em ambiente com temperatura de 20ºC a 24ºC, sob agitação constante e tem validade de 5 dias. A transfusão de concentrado de plaquetas ocorre diante da recorrência de plaquetopenia seja por falência medular e alto gasto de plaquetas ou por consumo ou destruição periférica. Este hemocomponentes precisam de rigoroso controle de qualidade. Este tipo de CQ tem alto valor agregado. Este trabalho validou anticorpo produzido com a finalidade de contribuir com tecnologia de ponta, baixo custo, no controle de qualidades destes hemocomponentes aumentando a segurança transfusional. Objetivo: Estabelecer novo método de controle de qualidade de plaquetas com finalidade transfusional compreendendo: 1) Caracterização e validação de anticorpos monoclonais dirigidos contra antígenos plaquetários humanos. 2) Estabelecer padrões de conformidade e não conformidade para a liberação de CP utilizando a tecnologia de monitoramento de VEs por Citometria de Fluxo e 3) Propor depósito de patente para o anticorpo monoclonal caracterizado. Resultados: Foram obtidos dos anticorpos monoclonais murinos (AcMm), após a fusão cellular, 3.125 híbridos, após a clonagem celular pela técnica de diluição limitante foram selecionados 2 clones. Um destes clones, o PAT1-10 A1 foi o selecionado para os testes de caracterização e eletroforese em gel de poliacrilamida e Western Blotting foram realizados. Para a corrida eletroforética foram usadas as proteínas extraídas da membrana plaquetária de 5 diferentes doadores. Da eletroforese 1D, em Gel de Poliacrilamida a 6% coradas com Azul de Coomassie, foi possível identificar o reconhecimento de proteína de alto peso molecular e subsequente as análises de proteômica, seguidas por citometria de fluxo com anticorpos monoclonais de especificidade anti-plaquetária, marcados com fluorocromo: anti-CD41-PE; ANTI-CD61-FITC e anti-CD62P – APC. Conclusão: Estabeleceu-se novo método de controle de qualidade de plaquetas com finalidade transfusional, baseado em citometria de fluxo com anticorpo monoclonal com a especificidade anti-fibrinogênio produzido home made e um imunossensor deverá ser construído e testado. Foi caracterizado o anticorpo monoclonal PAT1-10 A5, com especificidade anti fibrinogênio humano, molécula total.
Blood transfusion therapy, which uses blood and components, is a great relevant technology nowadays and helps to improve and save the life of the patient. However, like other therapic interventions, this therapy may lead to acute or late complications for the blood receptor. Among medical recommendations for blood transfusion, the Platelet Concentration (PC) underlines, it can be obtained through the separation by centrifugation from a whole blood unit (WBU) or by apheresis. These blood components have different volume and amount of platelets depending on the form of their preparation. Regarding to storage, blood components must be stored in an environment of a temperature between 20 to 24ºC, under constant agitation, and valid for up to 5 days. The transfusion of PC may be required when thrombocytopenia occurs, whether due to marrow failure and high platelet use or due to platelet consumption or peripheral destruction. It is necessary to have a strict quality control (QC) with a high value when it comes to blood components. This study validated the antibody produced from the blood components in order to contribute not only with with cutting-edge technology and low cost but also with the QC of them, consequently, increase the safety of blood transfusions. Objetive: This study established a new method of QC for platelets under transfusion purposes: 1) Characterization and validation of monoclonal antibodies against human platelet antigens. 2) Compliance and non-compliance standards for CP release using the EVs monitoring tehcnology by Flow Cytometry. 3) Patent purpose for the characterized monoclonal antibody. Results: After cell fusion and cell cloning by limiting dilution technique, 3.125 hydrids were obtained from murine monoclonal antibodies (mAbs) and 2 clones were chosen, respectively. PAT1-10 A1 was selected for the characterization tests, polyacrylamide gel electrophoresis and Western Blotting were also performed. Proteins from platelet membrane of 5 different donors were extracted for the electrophoresis performance. Through 1D eletrophoresis method, 6% polyacrylamide gel stained with Coomassie Blue, was possible to identify the recognition of high molecular weight proteins and subsequently proteomics analysis, followed by Flow Cytometry with labeled anti-platelet specific monoclonal antibodies with fluorochrome: anti-CD41PE, anti-CD61-FITC, and anti-CD62P-APC. Conclusion: Our results indicate that a new method of QC of platelets for transfusion purposes was established, based on Flow Cytometry with monoclonal antibody with in house specificity of anti-fibrinogen, however, an immunosensor should be constructed and tested. Furthermore, the monoclonal antibody PATI-10 A5 was characterized with specificity agains human fibrinogen, total molecule.
Blood transfusion therapy, which uses blood and components, is a great relevant technology nowadays and helps to improve and save the life of the patient. However, like other therapic interventions, this therapy may lead to acute or late complications for the blood receptor. Among medical recommendations for blood transfusion, the Platelet Concentration (PC) underlines, it can be obtained through the separation by centrifugation from a whole blood unit (WBU) or by apheresis. These blood components have different volume and amount of platelets depending on the form of their preparation. Regarding to storage, blood components must be stored in an environment of a temperature between 20 to 24ºC, under constant agitation, and valid for up to 5 days. The transfusion of PC may be required when thrombocytopenia occurs, whether due to marrow failure and high platelet use or due to platelet consumption or peripheral destruction. It is necessary to have a strict quality control (QC) with a high value when it comes to blood components. This study validated the antibody produced from the blood components in order to contribute not only with with cutting-edge technology and low cost but also with the QC of them, consequently, increase the safety of blood transfusions. Objetive: This study established a new method of QC for platelets under transfusion purposes: 1) Characterization and validation of monoclonal antibodies against human platelet antigens. 2) Compliance and non-compliance standards for CP release using the EVs monitoring tehcnology by Flow Cytometry. 3) Patent purpose for the characterized monoclonal antibody. Results: After cell fusion and cell cloning by limiting dilution technique, 3.125 hydrids were obtained from murine monoclonal antibodies (mAbs) and 2 clones were chosen, respectively. PAT1-10 A1 was selected for the characterization tests, polyacrylamide gel electrophoresis and Western Blotting were also performed. Proteins from platelet membrane of 5 different donors were extracted for the electrophoresis performance. Through 1D eletrophoresis method, 6% polyacrylamide gel stained with Coomassie Blue, was possible to identify the recognition of high molecular weight proteins and subsequently proteomics analysis, followed by Flow Cytometry with labeled anti-platelet specific monoclonal antibodies with fluorochrome: anti-CD41PE, anti-CD61-FITC, and anti-CD62P-APC. Conclusion: Our results indicate that a new method of QC of platelets for transfusion purposes was established, based on Flow Cytometry with monoclonal antibody with in house specificity of anti-fibrinogen, however, an immunosensor should be constructed and tested. Furthermore, the monoclonal antibody PATI-10 A5 was characterized with specificity agains human fibrinogen, total molecule.
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Palavras-chave
Plaquetas, Vesículas extracelulares, Anticorpo monoclonal, Platelets, Extra-cellular vesicles, Monoclonal antibody