Use of thiol-terminal silanes and heterobifunctional crosslinkers for immobilization of antibodies on silica surfaces.
A procedure for covalent immobilization of functional proteins on silica substrates was developed using thiol-terminal silanes and heterobifunctional cross-linkers. Using this procedure, a high density of functional antibodies was bound to glass cover slips and silica fibers. The amount of anti-IgG antibody immobilized was determined to be in the range of 0.66 to 0.96 ng/mm2 using radiolabeled antibody. The relative amount of IgG antigen bound by the immobilized antibody (0.37 to 0.55 mol antigen/mol antibody) was three to five times greater than other investigators have reported. In addition, the amount of protein nonspecifically adsorbed to the antibody-coated surface was further reduced by the addition of blocking agents so that nonspecific adsorption of protein antigens represented only 2-6% of the total antigen binding. With this low background, IgG antigen binding could be measured at levels as low as 150 fmol when an antigen concentration of 3 pmol/ml was applied. The process for antibody immobilization is straightforward, easy to perform, and adaptable for modifying mass quantities of biosensor components.
Bhatia
S K
SK
Geo-Centers, Inc., Newton Centre, Massachusetts 02159.
Shriver-Lake
L C
LC
Prior
K J
KJ
Georger
J H
JH
Calvert
J M
JM
Bredehorst
R
R
Ligler
F S
FS
D000906:Antibodies; D000937:Antigen-Antibody Reactions; D003432:Cross-Linking Reagents; D012822:Silicon Dioxide; D013438:Sulfhydryl Compounds
D000906:Antibodies; D003432:Cross-Linking Reagents; D013438:Sulfhydryl Compounds; D012822:Silicon Dioxide