RESUMO
A homogeneous noncompetitive immunoassay based on photoaffinity labeling techniques is described. Using this method, a fluorophore (reporter) can be specifically attached to an antibody in the vicinity of its antigen-combining sites. Upon antigen binding, changes in the fluorescence spectrum of the reporter molecule are often observed. Two fluorophores, pyrene and dansyl, were evaluated for this purpose. Also, this technology is ideal for fluorescence energy-transfer immunoassays that require labeling of the antibody with either a donor or acceptor fluorophore. In such cases, a fluorescent dye can be specifically attached near the antigen-combining site, where it can participate in high-efficiency energy transfer with its complementary fluorophore attached to the antigen.
Assuntos
Marcadores de Afinidade , Anticorpos/imunologia , Biotina/imunologia , Compostos de Dansil , Fluorimunoensaio/métodos , Pirenos , Compostos de Dansil/química , Corantes Fluorescentes/química , Estrutura Molecular , Pirenos/química , Rodaminas/químicaRESUMO
Immobilization of biomolecules to solid phase materials has been widely used in many areas (e.g., purification, analytical chemistry, and catalysis). The interfacial properties of immobilized antibodies on pretreated silica and hydrogel surfaces were explored by comparing native and modified antibodies with respect to their surface activity. The antibody was modified by exposing it to a low pH solution prior to immobilization. Both physical adsorption and covalent immobilization methods were studied. It was found that the surface activity of the modified antibody is higher than that of the native antibody on two silica surfaces. The results of this study demonstrate that the adsorption properties of the antibodies play an important role in their covalent immobilization on certain types of solid supports.
