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Method Article
Sandeep Kumar Vashist
NUS Nanoscience & Nanotechnolocy Initiative-NanoCore, National University of Singapore, T-Lab Level 11, 5A Engineering Drive1, Singapore-117580, Singapore
Corresponding Author
License:
This work is licensed under a CC BY-NC 3.0 License. Read Full License
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.
Keywords
surface plasmon resonance, immunoassay, covalent-orientated, antibody immobilization, 3-aminopropyltriethoxysilane, EDC, sulfoNHS, protein A, human fetuin A.
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The authors declare no competing financial interests.
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Subject Areas
Biological techniques
Associated Publications
Multisubstrate-compatible ELISA procedures for rapid and high-sensitivity immunoassays
by Chandra Kumar Dixit, Sandeep Kumar Vashist, Brian D MacCraith, +1
Nature Protocols (06 October, 2011)
Effect of antibody immobilization strategies on the analytical performance of a surface plasmon resonance-based immunoassay
by Sandeep Kumar Vashist, Chandra Kumar Dixit, Brian D. MacCraith, +1
The Analyst (05 October, 2011)
An open repository of community-contributed protocols sponsored by Nature Research.
Learn more about Protocol Exchange
Method Article
Sandeep Kumar Vashist
NUS Nanoscience & Nanotechnolocy Initiative-NanoCore, National University of Singapore, T-Lab Level 11, 5A Engineering Drive1, Singapore-117580, Singapore
Corresponding Author
Version History
CURRENT STATUS: Posted
Version 1
Posted 06 Oct, 2011
No community comments so far
Metrics
Comments: 0
HTML Views: ...
Subject Areas
Biological techniques
License:
This work is licensed under a CC BY-NC 3.0 License. Read Full License
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.
Figure 1
Figure 2
Figure 3
Figure 4
The authors declare no competing financial interests.
Loading...
Associated Publications
Multisubstrate-compatible ELISA procedures for rapid and high-sensitivity immunoassays
by Chandra Kumar Dixit, Sandeep Kumar Vashist, Brian D MacCraith, +1
Nature Protocols (06 October, 2011)
Effect of antibody immobilization strategies on the analytical performance of a surface plasmon resonance-based immunoassay
by Sandeep Kumar Vashist, Chandra Kumar Dixit, Brian D. MacCraith, +1
The Analyst (05 October, 2011)