title: A phage-displayed single domain antibody fused to alkaline phosphatase for detection of porcine circovirus type 2.
abstract: The fusion of single domain antibodies (sdAbs) and alkaline phosphatase (AP) has been demonstrated to be useful as an immunodiagnostic reagent. In this work, a porcine circovirus type-2 (PCV2) specific sdAb (psdAb) was expressed as fusion with an AP. The binding activity of psdAb-AP fusion was examined by Western blot, enzyme linked immunosorbent assay (ELISA), and surface plasmon resonance (SPR). To assess the practicality of psdAb-AP fusion as a diagnostic reagent, the fusion was used in a Western blot, a direct ELISA, and an immunocytochemistry assay (ICC) for PCV2 detection. The results indicated that the binding activity and specificity of psdAb-AP fusion was similar with psdAb, but the functional affinity of psdAb-AP fusion was about 5 times greater than psdAb as determined by SPR. As a tracer, psdAb-AP fusion could detect PCV2 cap protein down to 0.01 μg/lane and 0.05 μg/ml in Western blot and direct ELISA respectively. When compared with a control indirect fluorescence assay (IFA), the ICC psdAb-AP fusion was more efficient, needed less operation steps and ended in a shorter time. The results demonstrate that the fusion of psdAb to AP provides a valuable route to the development of psdAb-based immuno-reagents, which offers a simple, convenient, and sensitive method for PCV2 detection.
authors.affiliation: State Key Laboratory of Veterinary Etiological Biology, National Foot and Mouth Disease Reference Laboratory, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (CAAS), Lanzhou 730046, China.
authors.forename: Shunli
authors.initials: S
authors.lastname: Yang
authors.affiliation: State Key Laboratory of Veterinary Etiological Biology, National Foot and Mouth Disease Reference Laboratory, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (CAAS), Lanzhou 730046, China.
authors.forename: Youjun
authors.initials: Y
authors.lastname: Shang
authors.affiliation: State Key Laboratory of Veterinary Etiological Biology, National Foot and Mouth Disease Reference Laboratory, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (CAAS), Lanzhou 730046, China. Electronic address: yinshuanghui@caas.cn.
authors.forename: Shuanghui
authors.initials: S
authors.lastname: Yin
authors.affiliation: State Key Laboratory of Veterinary Etiological Biology, National Foot and Mouth Disease Reference Laboratory, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (CAAS), Lanzhou 730046, China.
authors.forename: Di
authors.initials: D
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Veterinary Etiological Biology, National Foot and Mouth Disease Reference Laboratory, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (CAAS), Lanzhou 730046, China.
authors.forename: Jianping
authors.initials: J
authors.lastname: Cai
authors.affiliation: State Key Laboratory of Veterinary Etiological Biology, National Foot and Mouth Disease Reference Laboratory, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (CAAS), Lanzhou 730046, China.
authors.forename: Zhenli
authors.initials: Z
authors.lastname: Gong
authors.affiliation: Cellular & Molecular Immunology, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium; Structural Biology Research Center, Vlaams Inter Universiteit Instituut voor Biotechnologie, Brussels, Belgium.
authors.forename: Muyldermans
authors.initials: M
authors.lastname: Serge
authors.affiliation: State Key Laboratory of Veterinary Etiological Biology, National Foot and Mouth Disease Reference Laboratory, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (CAAS), Lanzhou 730046, China. Electronic address: liuxiangtao@caas.cn.
authors.forename: Xiangtao
authors.initials: X
authors.lastname: Liu
mesh_terms: D000469:Alkaline Phosphatase / Q000235:genetics; D000818:Animals; D015153:Blotting, Western / Q000379:methods*; D017925:Circovirus / Q000302:isolation & purification*; D004797:Enzyme-Linked Immunosorbent Assay; D007150:Immunohistochemistry / Q000379:methods*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012680:Sensitivity and Specificity; D061905:Single-Domain Antibodies / Q000235:genetics; D020349:Surface Plasmon Resonance
chemical_list: D011993:Recombinant Fusion Proteins; D061905:Single-Domain Antibodies; D000469:Alkaline Phosphatase
keywords: Alkaline phosphatase; Porcine circovirus type 2; Single domain antibody