Análise proteômica na caracterização de anticorpos monoclonais dirigidos contra antígenos eritrocitários e leucocitários humanos
Data
2018-07-16
Autores
Santis, Laís Priscila de
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
As membranas de hemácias e leucócitos são compostas por centenas de antígenos que desempenham diversas funções relacionadas a homeostase, metabolismo celular e podem estar envolvidos em processos de rejeição de transplantes, doenças hemolíticas e reações transfusionais. Para a detecção desses antígenos são utilizados anticorpos monoclonais e a obtenção destes anticorpos envolve diversas etapas que culminam na caracterização dos produtos obtidos. Esta etapa é crítica e envolve diferentes técnicas, incluindo a Proteômica na descrição da proteína-alvo de cada anticorpo monoclonal. O objetivo deste estudo foi caracterizar anticorpos monoclonais de especificidade anti-eritrocitária e anti-leucocitária produzidos pelo Laboratório de Engenharia Celular (LEC) do Hemocentro de Botucatu. Foram selecionados um clone e um hibridoma produtores de anticorpos anti-eritrocitários, e um clone produtor de anticorpos anti-leucocitários, pertencentes ao banco de células do LEC. As células foram expandidas em cultura, foi realizado Western Blotting (WB) e cada banda proteica reconhecida pelos anticorpos (antígenos) foi analisada por Espectrometria de Massas, segundo técnicas proteômicas. Outros testes adicionais foram realizados, como técnicas imuno-hematológicas, citometria de fluxo e imuno-histoquímica. Após expansão, retestagem e verificação de reatividade contra hemácias humanas, e a seleção dos dados de outros estudos até então não explorados, na técnica de WB os anticorpos reconheceram diversas bandas. Após a análise por espectrometria de massas, identificou-se, com bom índice de confiabilidade, que a proteína reconhecida por LAMB 10 (anti-eritrocitário) pode ser ALAD, G3PD ou GPC, presentes na membrana de hemácias humanas. LAMB 11 (anti-eritrocitário) é um possível anti-CD36, Espectrina b1 eritrocítica ou ADD2, não descartando anti-ALAD e G3PD. LAMB 12 (anti-leucocitário) é um possível anti-Mieloperoxidase. Os anticorpos caracterizados têm ampla aplicabilidade, uma vez que o CD-36 pode ser um marcador para trombose venosa, diferenciando da trombose arterial. Da mesma forma, por ter um importante papel no diagnóstico, o anticorpo anti-mieloperoxidase tem grande aplicação nas rotinas imunofenotípicas e diagnósticas. A proteômica é uma ferramenta de caracterização obrigatória e importante no processo de validação de anticorpos. No entanto, técnicas complementares para validação ainda são valiosas e indispensáveis na confirmação da especificidade dos anticorpos estudados, partindo das possibilidades apresentadas pela proteômica. Os anticorpos aqui caracterizados pelos resultados do conjunto das técnicas, além das aplicabilidades em WB e Citometria de Fluxo, têm a possibilidade de contribuir dentro escopo da biotecnologia, para auxílio diagnóstico e controle de qualidade, de forma prática, rápida e precisa.
Red blood cell and leukocyte membranes are composed of hundreds of antigens that perform various functions related to homeostasis, cell metabolism and may be involved in transplant rejection, hemolytic disease and transfusion reactions. Monoclonal antibodies are used to detect these antigens and the obtaining of these antibodies involves several steps that culminate in the characterization of the obtained products. This step is critical and involves different techniques, including Proteomics to descript the target protein of each monoclonal antibody. The aim of this study was to characterize anti-erythrocyte and anti-leukocyte monoclonal antibodies produced by the Laboratory of Cellular Engineering (LEC) of the Blood Center of Botucatu. Anti-erythrocytes clone and hybridoma antibody producers and a clone that produces anti-leukocyte antibodies, belonging to the LEC cell bank were selected. Cells were expanded in culture, it was realyzed Western Blotting (WB) technique and each protein band recognized by the antibodies (antigens) was analyzed by Mass Spectrometry according to proteomic techniques. Others tests were realized, such as immunohematology techniques, flow cytometry and immunohistochemistry. After expansion, retesting and verification of reactivity against human red blood cells, and selection of data from other studies not exploited, in the WB technique, the antibodies recognized several spots. After analysis by Mass Spectrometry, it was identified, with good reliability, that the protein recognized by LAMB 10 (anti-erytrocytic) may be ALAD or G3PD, present in the membrane protein structure of human red blood cells. LAMB 11 (anti-erytrocytic) is a possible anti-CD36, spectrin β1 erythrocytic or ADD2, not disregarding anti-ALAD and G3PD. LAMB 12 (anti-leukocytic) is a possible anti-Myeloperoxidase. Characterized antibodies have wide applicability. CD-36, for example, may be a marker for venous thrombosis, differentiating from arterial thrombosis. Similarly, due to its important role in diagnosis, the anti-myeloperoxidase antibody has great application in the immunophenotypic and diagnostic routines. Proteomics is a mandatory and important characterization tool in the antibody validation process. However, complementary techniques for validation are still valuable and indispensable in confirming the specificity of the antibodies, starting from the possibilities presented by the proteomics. The antibodies here characterized by the results of all the techniques, besides the applicability in WB and Flow Cytometry, have the possibility of contributing in biotechnology scope, for diagnostic assistance and quality control, in a practical, fast and precise way.
Red blood cell and leukocyte membranes are composed of hundreds of antigens that perform various functions related to homeostasis, cell metabolism and may be involved in transplant rejection, hemolytic disease and transfusion reactions. Monoclonal antibodies are used to detect these antigens and the obtaining of these antibodies involves several steps that culminate in the characterization of the obtained products. This step is critical and involves different techniques, including Proteomics to descript the target protein of each monoclonal antibody. The aim of this study was to characterize anti-erythrocyte and anti-leukocyte monoclonal antibodies produced by the Laboratory of Cellular Engineering (LEC) of the Blood Center of Botucatu. Anti-erythrocytes clone and hybridoma antibody producers and a clone that produces anti-leukocyte antibodies, belonging to the LEC cell bank were selected. Cells were expanded in culture, it was realyzed Western Blotting (WB) technique and each protein band recognized by the antibodies (antigens) was analyzed by Mass Spectrometry according to proteomic techniques. Others tests were realized, such as immunohematology techniques, flow cytometry and immunohistochemistry. After expansion, retesting and verification of reactivity against human red blood cells, and selection of data from other studies not exploited, in the WB technique, the antibodies recognized several spots. After analysis by Mass Spectrometry, it was identified, with good reliability, that the protein recognized by LAMB 10 (anti-erytrocytic) may be ALAD or G3PD, present in the membrane protein structure of human red blood cells. LAMB 11 (anti-erytrocytic) is a possible anti-CD36, spectrin β1 erythrocytic or ADD2, not disregarding anti-ALAD and G3PD. LAMB 12 (anti-leukocytic) is a possible anti-Myeloperoxidase. Characterized antibodies have wide applicability. CD-36, for example, may be a marker for venous thrombosis, differentiating from arterial thrombosis. Similarly, due to its important role in diagnosis, the anti-myeloperoxidase antibody has great application in the immunophenotypic and diagnostic routines. Proteomics is a mandatory and important characterization tool in the antibody validation process. However, complementary techniques for validation are still valuable and indispensable in confirming the specificity of the antibodies, starting from the possibilities presented by the proteomics. The antibodies here characterized by the results of all the techniques, besides the applicability in WB and Flow Cytometry, have the possibility of contributing in biotechnology scope, for diagnostic assistance and quality control, in a practical, fast and precise way.
Descrição
Palavras-chave
Anticorpos monoclonais, Imunologia, Antígenos de grupo sanguíneo, Leucócitos, Monoclonal antibodies, Immunology, Blood group antigens, Leukocytes