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Y K Sykulev

Publications and source records attributed to Y K Sykulev.

6 recordsLinked to original sources
PubMed1992

Quantitation of reversible binding by particle counting: hapten-antibody interaction as a model system.

With a view toward developing a general method for measuring intrinsic equilibrium constants for the reversible interactions between two ligands, we used an antibody-hapten model system [2,4-dinitrophenyl (DNP) hapten and anti-DNP antibody] to explore an approach based on particle counting of uniform polystyrene spheres to which the hapten is coupled covalently. This approach was made possible by an optical pulse particle size analyzer that accurately counts individual sphere clusters and quantitates with high precision specific aggregation of spheres crosslinked by antibody. The reduction in crosslinking that results from competition for antibody binding sites between a soluble DNP ligand and immobilized DNP groups on the spheres provides the basis for measuring the intrinsic equilibrium constant for the soluble ligand-antibody interaction. The binding constants measured in this way for several DNP ligands and an anti-DNP antibody (2A1) agreed with the values obtained by conventional methods. The range of intrinsic equilibrium constants that can be determined by particle counting is likely to be exceptionally wide and a value as low as 10(3) liters/mol has been measured. And since all soluble antigens, regardless of their mass, acquire the same ability to scatter light as a result of their immobilization on the much larger uniform spheres (0.36 microns), the approach described here should be applicable to virtually any molecularly dispersed antigen and its monoclonal antibody.

Antibodies↗
PubMed1991

Galactose-containing epitopes on the surface of IgG model immune complexes are accessible for specific binding with the high molecular weight ligand, Ricinus agglutinin, in solution-light-scattering studies.

Immunoglobulin G (IgG) molecules contain covalently linked carbohydrate chains with galactose residues in their branched "antennae". The ability of galactose-containing epitopes on the surface of IgG model immune complexes (IC) to interact with a high mol. wt ligand in solution has been elucidated. Different types of IgG model IC with pre-determined molecular mass were mixed with Ricinus Agglutinin (RCI), which is known to bind specifically to galactose-containing oligosaccharides. The relative light-scattering increases (delta I) in the reaction mixture were measured as a function of time. The galactose-associated epitopes of the IgG model IC were accessible for binding with RC1. The rate of the interaction between IgG model IC and RC1 was dependent on the molecular mass of the complexes; the larger the model IC molecular mass, the faster the rate of interaction. The binding of RC1 to IgG model IC was highly specific because it was completely abolished in the presence of lactose. The galactose-containing epitopes of monomeric IgG were also able to interact with RC1 but the kinetics of the interaction was much slower. We suggest than an increase in the density of the epitopes on the surface of the model IC, by close attachment of the IgG molecules, mainly determines the ability of galactose-containing epitopes to be recognized by RC1. The data presented support the importance of IgG glycans in recognition events of IgG by biologically active molecules.

Antigen-Antibody Complex↗