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Issue 21, 2011
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Effect of antibody immobilization strategies on the analytical performance of a surface plasmon resonance-based immunoassay

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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.

Graphical abstract: Effect of antibody immobilization strategies on the analytical performance of a surface plasmon resonance-based immunoassay

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Publication details

The article was received on 18 Apr 2011, accepted on 26 Jul 2011 and first published on 08 Sep 2011


Article type: Paper
DOI: 10.1039/C1AN15325K
Citation: Analyst, 2011,136, 4431-4436
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    Effect of antibody immobilization strategies on the analytical performance of a surface plasmon resonance-based immunoassay

    S. K. Vashist, C. K. Dixit, B. D. MacCraith and R. O'Kennedy, Analyst, 2011, 136, 4431
    DOI: 10.1039/C1AN15325K

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