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

For many decades,feline infectious disease has been among themost 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. Earlydiagnosis is essential to increase the chance of successful treatment.Generally, measurement of the IgG level is considered to be indicativeof an individual's immune status for a particular pathogen.The antibodies specific to feline IgG are crucial components for thedevelopment of a detection kit. In this study, feline IgG-bound scFvwas selected using phage display technology. Three rounds of biopanningwere conducted against purified feline IgG. Through an indirect enzyme-linkedimmunosorbent assay (ELISA), two scFv clones demonstrating the bestbinding ability to feline IgG were chosen for biochemical characterization.In addition, the selected scFv (N14) was expressed and purified ina bacterial system. Sodium dodecyl sulfate-polyacrylamide gel electrophoresisrevealed that the size of the purified N14 was 29 kDa. A sandwichELISA was used to evaluate the binding capacity of the purified scFvto feline IgG. As expected, the purified N14 had the capacity to bindfeline IgG. Furthermore, N14 was modified to create a scFv-alkalinephosphatase (scFv-AP) fusion platform. The surface plasmon resonance(SPR) results revealed that N14-AP bound to feline IgG with an affinitybinding value of 0.3 & PLUSMN; 0.496 & mu;M. Additionally, the directELISA demonstrated the binding capacity of N14-AP to feline IgG inboth cell lysate and purified protein. Moreover, N14-AP could be appliedto detect feline IgG based on electrosensing with a detection limitof 10.42 nM. Overall, this study successfully selected a feline IgG-boundscFv and developed a scFv-AP platform that could be further engineeredand applied in a feline infectious disease detection kit.