Oriented Immobilization of Antibodies with GST-Fused Multiple Fc-Specific B-Domains on a Gold Surface

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• 作者：Tai Hwan Ha ; Sun Ok Jung ; Jeong Min Lee ; Kun Yeong Lee ; Yan Lee ; Jong Sang Park ; Bong Hyun Chung
• 刊名：Analytical Chemistry
• 出版年：2007
• 出版时间：January 15, 2007
• 年：2007
• 卷：79
• 期：2
• 页码：546 - 556
• 全文大小：564K
• 年卷期：v.79,no.2(January 15, 2007)
• ISSN：1520-6882

文摘

We have constructed a novel platform for the orientedbuildup of immunoglobulins on a gold surface for asurface plasmon resonance imaging microarray. To thisend, genetically engineered glutathione S-transferaseproteins bearing one, two, and three F_c-specific B-domains in protein G from Streptococci (GST-GB₁, -GB₂,and -GB₃, respectively) were produced. In order to tetherthese GST-GB_x proteins specifically, a novel glutathione-derivatized ligand (LA-GSH) was also synthesized from abiaminated tri(ethylene glycol) backbone. Each end of thebackbone was further functionalized with a maleimidegroup for a glutathione modification and a lipoic acid forsurface immobilization. The glutathione ligand demonstrated a negligible nonspecific protein adsorption towardother spectator proteins while showing a strong specificassociation toward GST-GB_x proteins. This F_c-specificsurface exhibited at least a 2-fold enhancement in theimmunoglobulin density (from human and mouse) withits antigen capture capability totally conserved comparedto a covalently tethered GB_x proteins. A single antibodytethered on the GST-GB₃ is estimated to capture twoantigens (enhanced green fluorescent protein), and thisantigen capture ratio seems to be the most efficient valueever observed.