DIRECT IMMOBILIZATION OF GANGLIOSIDES ONTO GOLD-CARBOXYMETHYLDEXTRAN SENSOR SURFACES BY HYDROPHOBIC INTERACTION - APPLICATIONS TO ANTIBODY CHARACTERIZATION

We describe a novel immobilization technique to investigate interactio ns between immobilized gangliosides (GD3, GM1, and GM2) and their resp ective antibodies, antibody fragments, or binding partners using an op tical biosensor. Immobilization was performed by direct injection onto a carboxymethyldextran sensor chip and did not require derivatization of the sensor surface or the ganglioside, The ganglioside appeared to bind to the sensor surface by hydrophobic interaction, leaving the ca rbohydrate epitope available for antibody or, in the case of GM1, chol era toxin binding, The carboxyl group of the dextran chains on the sen sor surface did not appear to be involved in the immobilization as evi denced by equivalent levels of immobilization following conversion of the carboxyl groups into acyl amino esters, but rather the dextran lay er provided a hydrophilic coverage of the sensor chip which was essent ial to prevent nonspecific binding, This technique gave better reactiv ity and specificity for anti-ganglioside monoclonal antibodies (anti-G D3: KM871, KM641, R24; and anti-GM2: KM966) than immobilization by hyd rophobic interaction onto a gold sensor surface or photoactivated cros s-linking onto carboxymethydextran. This rapid immobilization procedur e has facilitated detailed kinetic analysis of ganglioside/antibody in teractions, with the surface remaining viable for a large number of cy cles (> 125), Kinetic constants were determined from the biosensor dat a using linear regression, nonlinear least squares and equilibrium ana lysis. The values of k(d), k(a), and K-A obtained by nonlinear analysi s (K-A KM871 = 1.05, KM641 = 1.66, R24 = 0.14, and KM966 = 0.65 x 10(7 ) M-1) were essentially independent of concentration and showed good a greement with data obtained by other analytical methods.