RESUMO
A methodology using biosensor technology for combined kinetic and thermodynamic analysis of biomolecular interactions is described. Rate and affinity constants are determined with BIAcore. Thermodynamics parameters, changes in free energy, enthalpy and entropy, are evaluated from equilibrium data and by using rate constants and transition state theory. The methodology using van't Hoff theory gives complementary information to microcalorimetry, since only the direct binding is measured with BIAcore whereas microcalorimetry measures all components, including e.g. hydration effects. Furthermore, BIAcore gives possibilities to gain new information by thermodynamic analysis of the rate constants.
Assuntos
Técnicas Biossensoriais/métodos , Proteínas/química , Animais , Anticorpos Monoclonais/química , Reações Antígeno-Anticorpo , Calorimetria , Proteínas de Transporte/química , Proteínas de Transporte/imunologia , Proteínas de Ligação a Ácido Graxo , Anticorpos Anti-HIV/química , Proteína do Núcleo p24 do HIV/química , Humanos , Técnicas In Vitro , Cinética , Muramidase/química , Muramidase/imunologia , Ligação Proteica , TermodinâmicaRESUMO
In biological systems, weak-affinity interactions (association constant, Ka, of less than approximately 10(4) M-1) between biomolecules are common and essential to the integrity of such units. However, studies of weak biological interactions are difficult due to the scarcity of analytical methods available for the bioscientist. In this communication, we report on the use of biosensors based on surface plasmon resonance to detect and characterize weak affinity antibody-antigen interactions. Monoclonal antibodies towards carbohydrate antigens were immobilized on sensor surfaces and were used to detect weak binding of the carbohydrate tetraglucose of dissociation constant, Kd, in the millimolar range. Sensorgrams were received in the form of square pulses where the kinetic rate constants were difficult to assess due to the rapid association and dissociation of the antigen to/from the immobilized antibody.
Assuntos
Reações Antígeno-Anticorpo , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Técnicas Biossensoriais , Sequência de Carboidratos , Dados de Sequência Molecular , Oligossacarídeos/imunologia , Oligossacarídeos/metabolismo , Ligação Proteica , RefratometriaRESUMO
Antibody binding to surfaces with differing amounts of immobilised antigen was measured in a biosensor system using surface plasmon resonance detection. Binding rates obtained during the initial binding phase on high density antigen surfaces were proportional to antibody concentration and independent of antigen-antibody affinity. One antibody calibration curve covering the range from 0.5 to 160 nM (0.08-25 micrograms/ml) antibody was valid for IgG antibodies with different antigen specificities. To illustrate the use of this methodology active antibody concentrations were analysed in culture media and in rabbit serum.