Direct immobilization of gangliosides onto gold-carboxymethyldextran sensor surfaces by hydrophobic interaction: applications to antibody characterization

We describe a novel immobilization technique to investigate interactionsbetween immobilized gangliosides (GD3, GM1, and GM2) and their respectiveantibodies, antibody fragments, or binding partners using an opticalbiosensor. Immobilization was performed by direct injection onto acarboxymethyldextran sensor chip and did not require derivatization of thesensor surface or the ganglioside. The ganglioside appeared to bind to thesensor surface by hydrophobic interaction, leaving the carbohydrate epitopeavailable for antibody or, in the case of GM1, cholera toxin binding. Thecarboxyl group of the dextran chains on the sensor surface did not appearto be involved in the immobilization as evidenced by equivalent levels ofimmobilization following conversion of the carboxyl groups into acyl aminoesters, but rather the dextran layer provided a hydrophilic coverage of thesensor chip which was essential to prevent nonspecific binding. Thistechnique gave better reactivity and specificity for anti- gangliosidemonoclonal antibodies (anti-GD3: KM871, KM641, R24; and anti-GM2: KM966)than immobilization by hydrophobic interaction onto a gold sensor surfaceor photoactivated cross-linking onto carboxymethydextran. This rapidimmobilization procedure has facilitated detailed kinetic analysis ofganglioside/antibody interactions, with the surface remaining viable for alarge number of cycles (>125). Kinetic constants were determined fromthe biosensor data using linear regression, nonlinear least squares andequilibrium analysis. The values of kd, ka, and KAobtained by nonlinearanalysis (KAKM871 = 1.05, KM641 = 1.66, R24 = 0.14, and KM966 = 0.65 x10(7) M- 1) were essentially independent of concentration and showed goodagreement with data obtained by other analytical methods.