Caracterização e validação de anticorpo monoclonal murino anti-Linfócitos B humanos para uso em citometria de fluxo e imunoquímica
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Data
2010-02-24
Autores
Guilherme, Gabrielle Reinoldes Bizarria [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
O sistema imunológico é dividido em imunidade natural e adquirido (humoral e celular). Os linfócitos B são os principais efetores da resposta humoral. Junto aos linfócitos T, mediam diversas reações imunológicas. Todos os leucócitos possuem antígenos de superfície (clusters of differentiation – CD) determinados que possuem as mais diferentes funções. As expressões destes CDs podem variar na maturação e na presença de patologias, sendo as de maior prevalência e gravidade as leucemias e linfomas, tornando-se marcadores importantes que podem ser avaliados por citometria de fluxo ou imunoquímica através do uso de anticorpos monoclonais murinos (AcMm). Após produção dos AcMm é necessário caracterizar e validá-los. Utilizou-se 11 clones que apresentaram especificidade somente contra linfócitos B. Pela técnica de Western Blotting, 5 anticorpos (3 do tipo IgM e 2 do tipo IgG) foram escolhidos de acordo com sua possível especificidade e importância clínica. A validação dos anticorpos tipo IgM foi realizada por citometria de fluxo utilizando anticorpo comercial para comparação de quantidade de células marcadas, sendo testados em 20 amostras de indivíduos normais e 20 de indivíduos portadores de neoplasias hematológicas diversas. O LINB B, que foi comparado com o anti-CD171 e anti-CD45RA, apresentando diferença estatística somente em relação ao anti-CD45RA, e identidade com o anti-CD171. O LINB C, que foi comparado com o anti-CD20 e anti-CD19, não apresentou diferença estatística significante quando frente a ambos anticorpos comerciais. No teste de regressão linear, houve maior correlação dos resultados com o anti-CD19. O LINB E foi comparado somente contra o anti-CD107b, havendo grande identidade entre os dois. Dos resultados apresentados, conclui-se que o LINBs B e E apresentam grandes chances de serem específicos contra...
The immunological system is divided into: natural immunity and acquired immunity (celular and humoral responses). The B lymphocytes are the main effectors of the humoral response. Together with the T lymphocytes, they make several immunological reactions. All leucocytes have antigens on the surface (clusters of differentiation – CD) that possess lot of functions. The expressions of these CDs may be altered during maturation and pathologies, like leukemia and lymphomas, becoming important markers that can be evaluated by flow cytometry or immunochemistry thought murine monoclonals antibodies (Mab). After production of Mabs it´s necessary characterize and validated them. We used 11 clones that presented Mab against B lymphocytes only. By Western Blotting method 5 Mab (3 IgM and 2 IgG) were chosen according you possible especifity and clinical importance. The validation of IgM Mabs were made by flow cytometry using commercial antibody to compare the quantity of marked cells, being used 20 samples from normal people and 20 samples from person with hematological cancer. The LINB B, compared to anti-CD171 and anti-CD45RA, presented statistical difference from anti-CD45RA and identity to anti-CD171. The LINB C, compared to anti-CD19 and anti-CD20, didn´t presented any statistical difference from both commercial antibodies, although it correlates better with anti-CD19. The LINB E was compared to anti-CD107b, where it appears great identity between then. By the present results, we conclude that LINB B and E need multicentre studies to expand validation, and LINB C, needs to increase the samples to have a statistical validation. The two IgGs LINB, possible anti-CD138, weren´t test yet.
The immunological system is divided into: natural immunity and acquired immunity (celular and humoral responses). The B lymphocytes are the main effectors of the humoral response. Together with the T lymphocytes, they make several immunological reactions. All leucocytes have antigens on the surface (clusters of differentiation – CD) that possess lot of functions. The expressions of these CDs may be altered during maturation and pathologies, like leukemia and lymphomas, becoming important markers that can be evaluated by flow cytometry or immunochemistry thought murine monoclonals antibodies (Mab). After production of Mabs it´s necessary characterize and validated them. We used 11 clones that presented Mab against B lymphocytes only. By Western Blotting method 5 Mab (3 IgM and 2 IgG) were chosen according you possible especifity and clinical importance. The validation of IgM Mabs were made by flow cytometry using commercial antibody to compare the quantity of marked cells, being used 20 samples from normal people and 20 samples from person with hematological cancer. The LINB B, compared to anti-CD171 and anti-CD45RA, presented statistical difference from anti-CD45RA and identity to anti-CD171. The LINB C, compared to anti-CD19 and anti-CD20, didn´t presented any statistical difference from both commercial antibodies, although it correlates better with anti-CD19. The LINB E was compared to anti-CD107b, where it appears great identity between then. By the present results, we conclude that LINB B and E need multicentre studies to expand validation, and LINB C, needs to increase the samples to have a statistical validation. The two IgGs LINB, possible anti-CD138, weren´t test yet.
Descrição
Palavras-chave
Imunologia, Imuno-histoquímica, Monoclonal antibodies, Cancer
Como citar
GUILHERME, Gabrielle Reinoldes Bizarria. Caracterização e validação de anticorpo monoclonal murino anti-Linfócitos B humanos para uso em citometria de fluxo e imunoquímica. 2010. 100 f. Dissertação (mestrado) - Universidade Estadual Paulista, Faculdade de Medicina de Botucatu, 2010.