Influence of antibody immobilization strategy on molecular recognition force microscopy measurements.
A systematic evaluation of the effects of antibody immobilization strategy on the binding efficiency and selectivity (e.g., ability to distinguish between specific and nonspecific interactions) of immunosurfaces prepared with F(ab') antibody fragments of rabbit Immunoglobulin G (IgG) is described. F(ab') was attached to gold surfaces either (1) directly via the formation of a gold-thiolate bond or (2) indirectly through a series of a bifunctional linkers containing an alkane chain or ethylene glycol spacer. Immobilization of F(ab') via the sulfhydryl reactive group located opposite the antigen binding site ensured optimum orientation of the antigen binding site. X-ray photoelectron spectroscopy (XPS) and surface plasmon resonance (SPR) were used to confirm surface modification with the bifunctional linkers and antibody immobilization, respectively. Binding efficiency assays performed with SPR indicated that increasing the length of the linker increased the antigen binding efficiency. Atomic force microscopy (AFM) adhesion force measurements indicated that AFM probes functionalized with directly immobilized F(ab') more effectively discriminated between specific and nonspecific surface-bound proteins than probes modified indirectly via linker-immobilized F(ab'). In addition, a greater number of antibody-antigen binding events were observed with directly immobilized F(ab')-functionalized probes.
Brogan
Kathryn L
KL
Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3290, USA.
Schoenfisch
Mark H
MH
D000268:Adhesiveness; D000818:Animals; D000906:Antibodies / Q000737:chemistry* / Q000648:ultrastructure*; D000941:Antigens / Q000737:chemistry / Q000276:immunology; D003432:Cross-Linking Reagents / Q000737:chemistry; D018625:Microscopy, Atomic Force; D015394:Molecular Structure; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D011817:Rabbits; D013057:Spectrum Analysis; D020349:Surface Plasmon Resonance; D013499:Surface Properties; D014867:Water / Q000737:chemistry
D000906:Antibodies; D000941:Antigens; D003432:Cross-Linking Reagents; D010446:Peptide Fragments; D014867:Water