Sandeep Kumar Vashist - Effect of antibody immobilization strategies on the analytical performance of a surface plasmon resonance immunoassay

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        ANALYST,   (in Press),   2011


      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 carboxymethyl dextran   (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 0.6-20.0ng/mL of HFA in less than 10 min.


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