RESUMO
We have analyzed by different immunological methods the proteolytic degradation of cardiac troponin I (cTnI) in human necrotic tissue and in serum. cTnI is susceptible to proteolysis, and its degradation leads to the appearance of a wide diversity of proteolytic peptides with different stabilities. N- and C-terminal regions were rapidly cleaved by proteases, whereas the fragment located between residues 30 and 110 demonstrated substantially higher stability, possibly because of its protection by TnC. We conclude that antibodies selected for cTnI sandwich immunoassays should preferentially recognize epitopes located in the region resistant to proteolysis. Such an approach can be helpful for a much needed standardization of cTnI immunoassays and can improve the sensitivity and reproducibility of cTnI assays.
Assuntos
Miocárdio/metabolismo , Fragmentos de Peptídeos/análise , Troponina T/metabolismo , Sequência de Aminoácidos , Anticorpos Monoclonais , Western Blotting , Endopeptidases/metabolismo , Epitopos , Fluorimunoensaio , Humanos , Hidrólise , Dados de Sequência Molecular , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Miocárdio/enzimologia , Miocárdio/patologia , Fragmentos de Peptídeos/imunologia , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Fatores de Tempo , Troponina T/análise , Troponina T/imunologiaRESUMO
Two groups of monoclonal antibodies (MAbs) specific to human cardiac troponin I (cTnI) were generated by immunization of mice by isolated cTnI (group I, 16 MAbs) or by the whole troponin complex (group II, 15 MAbs). Two sets of overlapping decapeptides covering the complete sequence of cTnI were prepared and used for epitope mapping by SPOT technique. Majority of MAbs (28 out of 31) interacts with synthetic peptides thus indicating that they recognize liner epitopes. MAbs raised against isolated cTnI preferentially recognize epitopes located at the N- or C-terminal ends of cTnI. Nine out of fifteen MAbs raised against whole troponin complex interact with epitopes located in the N-terminal part of cTnI. Generation of MAbs recognizing both isolated cTnI and cTnI inside of troponin complex and mapping their epitopes provides reliable detection of TnI in serum of patients with acute myocardial infarction.
Assuntos
Anticorpos Monoclonais/imunologia , Mapeamento de Epitopos , Troponina I/imunologia , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos , Humanos , Epitopos Imunodominantes/imunologia , Camundongos , Dados de Sequência Molecular , Fragmentos de Peptídeos/imunologiaRESUMO
Tyrosine phosphorylation in human blood lymphocytes was studied as a function of stimulation with concanavalin A (ConA) and treatment of the cells with interferon alpha 2 (IFN alpha 2) and/or an IFN-derived C-terminal synthetic peptide 2438 (amino acid residues 124-138). Both IFN alpha 2 and the peptide 2438 decreased the level of protein tyrosine phosphorylation in the ConA-stimulated cells. In unstimulated cells, IFN alpha 2 increased, and the peptide 2438 decreased the level of the tyrosine phosphorylation. A possible correlation of these effects with stimulation of cell proliferation is discussed.
Assuntos
Interferon Tipo I/metabolismo , Leucócitos/metabolismo , Tirosina/metabolismo , Western Blotting , Divisão Celular , Células Cultivadas , Humanos , Fragmentos de Peptídeos/metabolismo , Peptídeos/metabolismo , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Proteínas RecombinantesRESUMO
A biologically active synthetic peptide, 2438, representing the 124-138 amino acid sequence of the human interferon alpha-2 (IFN alpha-2) molecule, which is known to possess IFN-like antiproliferative activity, specifically binds to human blood leukocytes. Scatchard plots reveal two different Kd values, for the 'low' and 'high' affinity binding. The interaction of the 125I-labelled peptide 2438 with the cells is not impaired by human IFN alpha-2 or cholera toxin.
