14:30 〜 14:45
▼ [15p-313-3] Plastic antibodies for vascular endothelial growth factors synthesized by protein-ligand interaction-based molecular imprinting
キーワード：Plastic antibody, Protein-ligand interaction, Molecular imprinting
Protein recognition polymeric materials have been recognized as plastic antibodies, which could become to be substitutes for natural antibodies. In order to achieve high binding activity comparable to natural antibody, it is important to design and synthesize well-defined binding cavities with nonspecific adsorption-free for off-targeted proteins.
Vascular endothelial growth factor-A (VEGF-A), an angiogenesis promotor in growth and metastasis of diverse cancers, can be mainly classified into three families including VEGF165 and VEGF189 that possess a common heparin binding domain(s) (HBD), and no HBD-comprised VEGF121. Since different VEGFs are produced depending on cancer cells and tissues, selective detection of these VEGF families is significantly important in early diagnosis of cancers. In this study, we have designed and synthesized plastic antibodies for VEGF165 by using protein-ligand interaction-based molecular imprinting, where the obtained plastic antibodies possess a suitable size for VEGF165 due to the imprinting effect.
VEGF165 was conjugated with the immobilized heparin on a gold-coated glass substrate bearing initiators for atom transfer radical polymerization (ATRP), and then methacrylic acid (MAA) was added as a functional monomer which can interact with VEGF165. After the addition of a hydrophilic comonomer and a crosslinker, surface-initiated ATRP (SI-ATRP) was carried out. The removal of VEGF165 from the resultant polymer resulted in molecularly imprinted polymer thin layer for VEGF165 (VEGF165-MIP), i.e. plastic antibody for VEGF165. As a reference, MAA-free VEGF165-MIP (VEGF165-MIP w/o MAA) was also prepared without adding MAA.
Binding behaviors of VEGF families towards the obtained polymer layers were investigated by using a surface plasmon resonance sensor. VEGF165-MIP (Ka=2.0×108 M-1) showed 10 times higher affinity for VEGF165 than VEGF165-MIP w/o MAA(Ka=2.5×107 M-1). Binding selectivity of VEGF families were examined using selectivity factor (SF) that is defined as a ratio of binding amounts of VEGF189 or 121 to that of VEGF165. The binding selectivity for VEGF165 (SF for VEGF189: 0.55 and for VEGF121: 0.05) were higher than VEGF165-MIP w/o MAA (for VEGF189: 0.85 and for VEGF121: 0.07). These high binding selectivity for VEGF165 is attributed to the anchored heparins and the MAA residues within the imprinted cavity bearing complementary binding sites to VEGF165. These orthogonal two different interactions enabled the plastic antibody to enhance the selectivity, and the present strategy would provide a novel way to fabricate highly sensitive and selective plastic antibodies for a diverse range of biologically important protein families with high sequence homology.
Vascular endothelial growth factor-A (VEGF-A), an angiogenesis promotor in growth and metastasis of diverse cancers, can be mainly classified into three families including VEGF165 and VEGF189 that possess a common heparin binding domain(s) (HBD), and no HBD-comprised VEGF121. Since different VEGFs are produced depending on cancer cells and tissues, selective detection of these VEGF families is significantly important in early diagnosis of cancers. In this study, we have designed and synthesized plastic antibodies for VEGF165 by using protein-ligand interaction-based molecular imprinting, where the obtained plastic antibodies possess a suitable size for VEGF165 due to the imprinting effect.
VEGF165 was conjugated with the immobilized heparin on a gold-coated glass substrate bearing initiators for atom transfer radical polymerization (ATRP), and then methacrylic acid (MAA) was added as a functional monomer which can interact with VEGF165. After the addition of a hydrophilic comonomer and a crosslinker, surface-initiated ATRP (SI-ATRP) was carried out. The removal of VEGF165 from the resultant polymer resulted in molecularly imprinted polymer thin layer for VEGF165 (VEGF165-MIP), i.e. plastic antibody for VEGF165. As a reference, MAA-free VEGF165-MIP (VEGF165-MIP w/o MAA) was also prepared without adding MAA.
Binding behaviors of VEGF families towards the obtained polymer layers were investigated by using a surface plasmon resonance sensor. VEGF165-MIP (Ka=2.0×108 M-1) showed 10 times higher affinity for VEGF165 than VEGF165-MIP w/o MAA(Ka=2.5×107 M-1). Binding selectivity of VEGF families were examined using selectivity factor (SF) that is defined as a ratio of binding amounts of VEGF189 or 121 to that of VEGF165. The binding selectivity for VEGF165 (SF for VEGF189: 0.55 and for VEGF121: 0.05) were higher than VEGF165-MIP w/o MAA (for VEGF189: 0.85 and for VEGF121: 0.07). These high binding selectivity for VEGF165 is attributed to the anchored heparins and the MAA residues within the imprinted cavity bearing complementary binding sites to VEGF165. These orthogonal two different interactions enabled the plastic antibody to enhance the selectivity, and the present strategy would provide a novel way to fabricate highly sensitive and selective plastic antibodies for a diverse range of biologically important protein families with high sequence homology.