Development of small fragment antibodies against feline immunoglobulin G by phage display technology for detection kit

Name: Natchaya Rasri

LCSH: Kasetsart University -- Theses. M.S. (Biochemistry) 2022

Classification :.LCCS: QR186.8.G2

LCSH: Kasetsart University. -- Department of Biochemistry -- Theses

LCSH: Immunoglobulin G

LCSH: Feline immunodeficiency virus

LCSH: Feline leukemia virus

LCSH: Monoclonal antibodies

Abstract: Feline infectious disease is one of the most common health problems and leading cause of death for over decades such as toxoplasmosis, feline leukemia virus (FeLV) disease, especially feline immunodeficiency virus (FIV) disease. The early diagnosis is essential to increase the chance for successful treatment. Measurement of IgG are generally considered an individual’s immune status for particular pathogen. In addition, the antibodies specific to feline IgG is an essential component for development the detection kit. However, conventional monoclonal antibodies have been concerned in term of time-and cost-consuming production, animal requirement, and unstable under harsh conditions. Currently, recombinant antibody fragment technology becomes an effective strategy to rapidly produce binder molecules such as single-chain variable fragment (scFv) that are time-and cost-effective, batch consistency, and pilot scale production. Herein, this study aimed to select feline IgG-bound scFv by phage display technology. Three rounds of biopanning was done against purified feline IgG. The soluble 8 scFv clones were subjected to determine the binding ability against the target by indirect enzymelinked immunosorbent assay (ELISA). N8 and N14 clones elicited the highest binding capacity against the purified feline IgG. The results from PCR and western blot analysis revealed the expected molecular size in term of DNA (~1000 bp) and protein (~29 kDa). The selected scFv (N14) was further expressed and purified in a bacterial system. From the optimization, 0.1 mM of isopropyl β-D-1- thiogalactopyranoside (IPTG) were used for scFv expression. Protein purification was done by hybrid condition through Ni2+ -column chromatography, and the band of protein was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis. The result showed that the size of purified scFv was 29 kDa. The purified scFv were used to determine the binding capacity to purified feline IgG by sandwich ELISA. As a result, purified N14 scFv demonstrated the binding capacity to feline IgG. In addition, N14 scFv was modified to create scFvalkaline phosphatase (scFv-AP) fusion and used as reagents to utilize in the detection kit. The result from direct ELISA demonstrated the binding capacity of scFv-AP to feline IgG in both cell lysate and purified protein. Taken together, this study successfully selected the feline IgG-bound scFv and developed scFv-AP platform that could be further engineered and utilized for feline infectious disease detection kit.

Kasetsart University. Office of the University Library

Address: Bangkok

Email: tdckulib@ku.ac.th

Name: Kiattawee Choowongkomon

Role: Thesis Advisor

Name: Chomdao Sinthuvanich

Role: Thesis CO-Advisor

Created: 2022

Modified: 2025-07-24

Issued: 2025-07-24

วิทยานิพนธ์/Thesis

application/pdf

URL: https://www.lib.ku.ac.th/KUthesis/2565/natchaya-ras-all.pdf

CallNumber: QR186.8.G2 .N38

eng

DegreeName: Master of Science

Level: Master's Degree

Descipline: Biochemistry

Grantor: Kasetsart University

©copyrights Kasetsart University

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