Authors
Sandeep Kumar Vashist
Summary
This method describes a highly-sensitive and rapid procedure for surface plasmon resonance (SPR) immunoassays, which is based on the covalent-orientated immobilization of capture antibodies on 3-aminopropyltriethoxysilane (APTES)-functionalized gold (Au)-coated SPR chip. It involves sequentially the cleaning of Au surface; APTES-functionalization; covalent binding of protein A (PrA) by heterobifunctional crosslinking using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride) (EDC) and sulfo-N-hydroxysuccinimide (sulfoNHS); blocking with 1% (w/v) BSA; orientated immobilization of anti-human fetuin A (HFA) by PrA; and, the detection of HFA. The developed procedure is highly sensitive, rapid and more cost-effective than the conventional procedure on commercial carboxymethyldextran-coated SPR chips. The anti-HFA antibody-bound SPR chips are prepared in 1.5 h and can be stored for one month at 4° C without any loss of functional activity. They detect HFA in the range of 0.6-20 ng mL-1 in only 10 min. This method is generic and can be employed for the detection of other analytes.Further details
The protocol was published on Protocol Exchange on 6 October 2011. To see the entire protocol, click on the source link.Advertisement
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