title: Effect of antibody immobilization strategies on the analytical performance of a surface plasmon resonance-based immunoassay.
abstract: Antibody immobilization strategies (random, covalent, orientated and combinations of each) were examined to determine their performance in a surface plasmon resonance-based immunoassay using human fetuin A (HFA) as the model antigen system. The random antibody immobilization strategy selected was based on passive adsorption of anti-HFA antibody on 3-aminopropyltriethoxysilane (APTES)-functionalized gold (Au) chips. The covalent strategy employed covalent crosslinking of anti-HFA antibody on APTES-functionalized chips using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC) and sulfo-N-hydroxysuccinimide (SNHS). The orientation strategy used passive adsorption of protein A (PrA) on Au chips, with subsequent binding of the anti-HFA antibody in an orientated fashion via its fragment crystallisable (Fc) region. In the covalent-orientated strategy, PrA was first bound covalently, to the surface, which in turn, then binds the anti-HFA antibody in an orientated manner. Finally, in the most widely used strategy, covalent binding of anti-HFA antibody to carboxymethyldextran (CM5-dextran) was employed. This immobilization strategy gave the highest anti-HFA antibody immobilization density, whereas the highest HFA response was obtained with the covalent-orientated immobilization strategy. Therefore, the covalent-orientated strategy was the best for SPR-based HFA immunoassay and can detect 0.6-20.0 ng/mL of HFA in less than 10 min.
authors: Centre for Bioanalytical Sciences (CBAS), National Centre for Sensor Research, Dublin City University, Dublin 9, Ireland.
authors: Sandeep Kumar
authors: SK
authors: Vashist
authors: Chandra Kumar
authors: CK
authors: Dixit
authors: Brian D
authors: BD
authors: MacCraith
authors: Richard
authors: R
authors: O'Kennedy
mesh_terms: D055910:Antibodies, Immobilized / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D003911:Dextrans / Q000737:chemistry; D006046:Gold / Q000737:chemistry / Q000378:metabolism; D007118:Immunoassay / Q000379:methods*; D011437:Propylamines; D012821:Silanes / Q000737:chemistry; D013205:Staphylococcal Protein A / Q000737:chemistry; D020349:Surface Plasmon Resonance / Q000379:methods*; D060749:alpha-2-HS-Glycoprotein / Q000032:analysis* / Q000276:immunology
chemical_list: D055910:Antibodies, Immobilized; D003911:Dextrans; D011437:Propylamines; D012821:Silanes; D013205:Staphylococcal Protein A; D060749:alpha-2-HS-Glycoprotein; D006046:Gold; C014392:carboxymethyl dextran; C477625:amino-propyl-triethoxysilane