Comparison of antibody functionality using different immobilization methods.
This study investigates the influence of antibody immobilization methods on antigen capture. Adsorption and two surface chemistries, an aminosilane chemistry and a common heterobifunctional crosslinker (N-gamma-maleimidobutyryloxy-succinimide ester, GMBS), were compared and evaluated for their ability to immobilize antibodies and capture antigen. The role of protein A as an orienting protein scaffold component in each of these techniques was also evaluated. Through experimentation it was determined that the GMBS technique immobilized the highest amount of antibody and minimized nonspecific binding. For all techniques, the most functional antibodies were found to be those immobilized with protein A. Interestingly, the aminosilane technique demonstrated the highest antigen capture with antibody alone but also exhibited the highest level of nonspecific binding.
Danczyk
R
R
Purdue University, 500 Central Drive, West Lafayette, Indiana 47907-2022, USA.
Krieder
B
B
North
A
A
Webster
T
T
HogenEsch
H
H
Rundell
A
A
D000465:Algorithms; D000704:Analysis of Variance; D000818:Animals; D000937:Antigen-Antibody Reactions / Q000276:immunology; D000941:Antigens / Q000276:immunology; D015374:Biosensing Techniques / Q000379:methods*; D003432:Cross-Linking Reagents / Q000737:chemistry; D004140:Dinitrophenols / Q000276:immunology; D005898:Glass / Q000737:chemistry; D006241:Haptens / Q000737:chemistry; D007118:Immunoassay / Q000379:methods; D007120:Immunochemistry / Q000379:methods; D007074:Immunoglobulin G / Q000737:chemistry* / Q000276:immunology; D051379:Mice; D008863:Microspheres; D018448:Models, Immunological; D011437:Propylamines / Q000737:chemistry; D011485:Protein Binding / Q000276:immunology; D012821:Silanes / Q000737:chemistry; D013205:Staphylococcal Protein A / Q000737:chemistry / Q000276:immunology; D013388:Succinimides / Q000737:chemistry; D013499:Surface Properties
D000941:Antigens; D003432:Cross-Linking Reagents; D004140:Dinitrophenols; D006241:Haptens; D007074:Immunoglobulin G; D011437:Propylamines; D012821:Silanes; D013205:Staphylococcal Protein A; D013388:Succinimides; C088294:3-aminopropyltrimethoxysilane; C032138:N-(gamma-maleimidobutyryloxy)succinimide