A phage-displayed single domain antibody fused to alkaline phosphatase for detection of porcine circovirus type 2
| Autor: | Youjun Shang, Shunli Yang, Di Wang, Cai Jianping, Gong Zhenli, Muyldermans Serge, Shuanghui Yin, Xiangtao Liu |
|---|---|
| Přispěvatelé: | Department of Bio-engineering Sciences, Cellular and Molecular Immunology |
| Rok vydání: | 2014 |
| Předmět: |
Circovirus
Recombinant Fusion Proteins Immunocytochemistry Blotting Western Enzyme-Linked Immunosorbent Assay Biology Sensitivity and Specificity Western blot Virology medicine Animals Surface plasmon resonance medicine.diagnostic_test Single-Domain Antibodies Surface Plasmon Resonance biology.organism_classification Alkaline Phosphatase Molecular biology Immunohistochemistry Porcine circovirus Single-domain antibody biology.protein Alkaline phosphatase Antibody Protein Binding |
| Zdroj: | Journal of virological methods. 213 |
| ISSN: | 1879-0984 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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