Generation of a Single-Chain Variable Fragment Antibody against Feline Immunoglobulin G for Biosensor Applications

Natchaya Rasri; Lueacha Tabtimmai; Charoenkwan Kraiya; Montarop Yamabhai; Chomdao Sinthuvanich; Jatuporn Rattanasrisomporn; Kiattawee Choowongkomon

doi:10.1021/acsomega.3c03581

Generation of a Single-Chain Variable Fragment Antibody against Feline Immunoglobulin G for Biosensor Applications

ACS Omega. 2023 Jul 19;8(30):27688-27696. doi: 10.1021/acsomega.3c03581. eCollection 2023 Aug 1.

Authors

Natchaya Rasri¹, Lueacha Tabtimmai², Charoenkwan Kraiya³, Montarop Yamabhai⁴, Chomdao Sinthuvanich¹, Jatuporn Rattanasrisomporn⁵, Kiattawee Choowongkomon¹

Affiliations

¹ Department of Biochemistry, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand.
² Department of Biotechnology, Faculty of Applied Science, King Mongkut's University of Technology North Bangkok, Bangkok 10800, Thailand.
³ Electrochemistry and Optical Spectroscopy Center of Excellence, Department of Chemistry, Faculty of Science, Chulalongkorn University, Patumwan, Bangkok 10330, Thailand.
⁴ Molecular Biotechnology Laboratory, School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand.
⁵ Department of Companion Animal Clinical Sciences, Faculty of Veterinary Medicine, Kasetsart University, Bangkok 10900, Thailand.

Abstract

For many decades, feline infectious disease has been among the most common health problems and a leading cause of death in cats. These diseases include toxoplasmosis, feline leukemia virus (FeLV), and particularly feline immunodeficiency virus (FIV) disease. Early diagnosis is essential to increase the chance of successful treatment. Generally, measurement of the IgG level is considered to be indicative of an individual's immune status for a particular pathogen. The antibodies specific to feline IgG are crucial components for the development of a detection kit. In this study, feline IgG-bound scFv was selected using phage display technology. Three rounds of biopanning were conducted against purified feline IgG. Through an indirect enzyme-linked immunosorbent assay (ELISA), two scFv clones demonstrating the best binding ability to feline IgG were chosen for biochemical characterization. In addition, the selected scFv (N14) was expressed and purified in a bacterial system. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the size of the purified N14 was 29 kDa. A sandwich ELISA was used to evaluate the binding capacity of the purified scFv to feline IgG. As expected, the purified N14 had the capacity to bind feline IgG. Furthermore, N14 was modified to create a scFv-alkaline phosphatase (scFv-AP) fusion platform. The surface plasmon resonance (SPR) results revealed that N14-AP bound to feline IgG with an affinity binding value of 0.3 ± 0.496 μM. Additionally, the direct ELISA demonstrated the binding capacity of N14-AP to feline IgG in both cell lysate and purified protein. Moreover, N14-AP could be applied to detect feline IgG based on electrosensing with a detection limit of 10.42 nM. Overall, this study successfully selected a feline IgG-bound scFv and developed a scFv-AP platform that could be further engineered and applied in a feline infectious disease detection kit.