RESUMO
Polyclonal (pAb) and monoclonal (mAb) anti-human aorta elastin antibodies were reacted with a series of overlapping hexapeptides along the human tropoelastin sequence covering exons 2-7 and 23-36 from the N-terminus to the C-terminus, advancing 1 amino acid residue each time. ELISA indicated reactive epitopes. mAb A2.1 recognized sequences containing Ala-Lys, mAb G8.1, A7.1 and pAb, hydrophobic sequences. None of them reacted with the hexapeptide VGVAPG, or with desmosine or isodesmosine. pAb L85 reacted with a His-containing sequence coded in exon 26A. pAb kappaE(L), kappaE(S) and L85 reacted with the Cys-containing sequence of exon 36. A synthetic 14-residue peptide containing the three proximal tyrosines coded in exon 13 did not react with any of the antisera tested. It appears therefore that the most frequently recognized epitopes are hydrophobic sequences. One polyclonal antibody detected several isoforms of tropoelastin in the medium of cultured vascular smooth muscle cells. Monoclonal and polyclonal antibodies stained elastic fibers on tissue sections, suggesting that the epitopes recognized are available on the native fibers for reaction with the antibodies.
Assuntos
Elastina/imunologia , Mapeamento de Epitopos , Epitopos/imunologia , Tropoelastina/imunologia , Sequência de Aminoácidos , Aminoácidos/genética , Aminoácidos/imunologia , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Células Cultivadas , Desmosina/imunologia , Elastina/genética , Elastina/isolamento & purificação , Éxons , Humanos , Imuno-Histoquímica , Isodesmosina/imunologia , Isomerismo , Dados de Sequência Molecular , Músculo Liso/imunologia , Músculo Liso/metabolismo , Peptídeos/síntese química , Peptídeos/imunologia , Peptídeos/isolamento & purificação , Tropoelastina/genéticaRESUMO
A radioimmunoassay was developed for the determination of isodesmosine as the tetraacetyl derivative. Isodesmosine tetraacetate conjugated with bovine albumin was injected into rabbits which developed useful titers of antibodies after five months. The radioligand for the assay was prepared by acetylating isodesmosine with [3H] acetic anhydride. The bound ligand was separated from free ligand by coprecipitation with human gamma-globulin in 46% saturated ammonium sulfate solution. The sensitivity of the assay was 2 ng isodesmosine. The antiserum was specific for isodesmosine tetraacetate and only desmosine tetraacetate gave appreciable cross-reactivity (4%). The assay was found to be suitable for the accurate estimation of elastin in small samples (5 mg dry weight) of rat and ovine fetal lung tissue and for elastin degradation products in amniotic fluid (0.5 ml).
