Direct immobilization of gangliosides onto gold-carboxymethyldextran sensor surfaces by hydrophobic interaction: applications to antibody characterization.
We describe a novel immobilization technique to investigate interactions between immobilized gangliosides (GD3, GM1, and GM2) and their respective antibodies, antibody fragments, or binding partners using an optical 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 carbohydrate epitope available for antibody or, in the case of GM1, cholera toxin binding. The carboxyl group of the dextran chains on the sensor surface did not appear to be involved in the immobilization as evidenced by equivalent levels of immobilization following conversion of the carboxyl groups into acyl amino esters, but rather the dextran layer provided a hydrophilic coverage of the sensor chip which was essential to prevent nonspecific binding. This technique gave better reactivity and specificity for anti-ganglioside monoclonal antibodies (anti-GD3: KM871, KM641, R24; and anti-GM2: KM966) than immobilization by hydrophobic interaction onto a gold sensor surface or photoactivated cross-linking onto carboxymethydextran. This rapid immobilization procedure has facilitated detailed kinetic analysis of ganglioside/antibody interactions, with the surface remaining viable for a large number of cycles (>125). Kinetic constants were determined from the biosensor data using linear regression, nonlinear least squares and equilibrium analysis. The values of kd, ka, and KAobtained by nonlinear analysis (KAKM871 = 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 agreement with data obtained by other analytical methods.
Ludwig Institute for Cancer Research, Melbourne, Australia, Tumour Targeting Program, Ludwig Institute for Cancer Research and Austin Hospital, Heidelberg, Victoria, Australia.
B
B
Catimel
A M
AM
Scott
F T
FT
Lee
N
N
Hanai
G
G
Ritter
S
S
Welt
L J
LJ
Old
A W
AW
Burgess
E C
EC
Nice
D000911:Antibodies, Monoclonal; D000915:Antibody Affinity; D000918:Antibody Specificity; D015374:Biosensing Techniques; D002772:Cholera Toxin / Q000378:metabolism; D005677:G(M1) Ganglioside / Q000737:chemistry / Q000276:immunology; D005678:G(M2) Ganglioside / Q000737:chemistry / Q000276:immunology; D005732:Gangliosides / Q000737:chemistry / Q000276:immunology*; D016018:Least-Squares Analysis; D055095:Optics and Photonics; D013499:Surface Properties
D000911:Antibodies, Monoclonal; D005732:Gangliosides; D005678:G(M2) Ganglioside; D005677:G(M1) Ganglioside; C026226:ganglioside, GD3; D002772:Cholera Toxin