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J Immunol Methods ; 266(1-2): 185-96, 2002 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-12133636


To overcome poor immunogenicity of prothymosin alpha, a small and highly acidic nuclear protein involved in cell proliferation, production of anti-prothymosin alpha antibodies in mice immunized with free human prothymosin alpha, with prothymosin alpha coupled to different carriers and with prothymosin alpha fused to green fluorescent protein was assessed. Fusing prothymosin alpha to green fluorescent protein turned out to be the superior approach resulting in production of high titer anti-prothymosin alpha antibodies. From these studies, two highly specific anti-prothymosin alpha monoclonal antibodies recognizing epitopes within the amino terminal (2F11) and middle (4F4) portions of the human prothymosin alpha molecule were obtained and characterized. As expected, the 2F11 antibody displayed broad species specificity, whereas the 4F4 antibody appeared to be species-specific permitting discrimination of human versus rat protein. Furthermore, a combination of point mutations in prothymosin alpha that alter the properties of the protein precluded recognition by the 4F4 antibody. Intramolecular masking of the 4F4 epitope in prothymosin alpha fused to the Tat transduction peptide of human immunodeficiency virus type 1 was observed. The anti-prothymosin alpha antibodies obtained were suitable for precipitation of human prothymosin alpha from HeLa cell lysates and for immunolocalization of the endogenous prothymosin alpha within the cells. Fusion with green fluorescent protein may thus be helpful in raising antibodies against 'problematic' proteins.

Anticorpos Monoclonais/imunologia , Precursores de Proteínas/imunologia , Timosina/análogos & derivados , Timosina/imunologia , Animais , Especificidade de Anticorpos , Mapeamento de Epitopos , Produtos do Gene tat/genética , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Proteínas Luminescentes/genética , Camundongos , Camundongos Endogâmicos BALB C , Mutação Puntual , Conformação Proteica , Precursores de Proteínas/química , Precursores de Proteínas/genética , Proteínas Recombinantes de Fusão/imunologia , Especificidade da Espécie , Timosina/química , Timosina/genética