Comparison of methods for immobilization to carboxymethyl dextran sensor surfaces by analysis of the specific activity of monoclonal antibodies.
The authors have recently described the development of a carboxymethyl dextran-based sensor surface for biospecific interaction analysis by surface plasmon resonance. Ligands are immobilized via primary amine groups after activation of the carboxymethyl groups on the sensor surface with a mixture of N-hydroxysuccinimide and N-ethyl-N'-(dimethylaminopropyl) carbodiimide. Methods have now been developed for efficient immobilization via thiol/disulfide exchange, aldehyde coupling and biotin-avidin coupling. The specific activity of monoclonal antibodies immobilized by the four different methods was investigated by altering the immobilization conditions, e.g., activation time, protein concentration, ionic strength and the degree of modification, etc. Investigations have also been made concerning possible differences in the specific activity for antibodies immobilized using optimized conditions with respect to the four different chemistries. These studies show that, with the flexible carboxymethyl dextran matrix used here, the immobilization methods give rise to only minor differences in specific activity. Thus, with this solid support, a 'site directed' immobilization strategy for monoclonal antibodies has no advantage. In general the specific activity for optimized systems was approximately 75% for the binding of beta 2 mu-globulin to an immobilized monoclonal antibody directed against beta 2 mu-globulin. Reduced specific activities of immobilized antibodies induced by variation of the coupling conditions could be attributed to the deterioration of the active site of the antibody.
Johnsson
B
B
Pharmacia Biosensor AB, Uppsala, Sweden.
Löfås
S
S
Lindquist
G
G
Edström
A
A
Müller Hillgren
R M
RM
Hansson
A
A
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D000915:Antibody Affinity; D000918:Antibody Specificity; D001426:Bacterial Proteins; D001609:Beta-Globulins / Q000276:immunology; D001710:Biotin; D002846:Chromatography, Affinity; D003432:Cross-Linking Reagents; D003911:Dextrans; D007074:Immunoglobulin G; D007202:Indicators and Reagents; D007700:Kinetics; D051379:Mice / Q000276:immunology; D019809:Streptavidin; D014168:Transferrin / Q000276:immunology / Q000302:isolation & purification*
D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D001609:Beta-Globulins; D003432:Cross-Linking Reagents; D003911:Dextrans; D007074:Immunoglobulin G; D007202:Indicators and Reagents; D014168:Transferrin; D001710:Biotin; D019809:Streptavidin; C014392:carboxymethyl dextran