Isolation, native purification of RGD motif binding integrins from LFBK cells and evaluation of its potential to capture foot and mouth disease virus in the ELISA to titrate serum antibodies against capsid proteins.

Solid phase competitive ELISA (SPCE), a WOAH-accepted serological assay for foot-and-mouth disease (FMD), commonly employs rabbit hyperimmune serum for capturing foot-and-mouth disease virus (FMDV). In accordance with the principles of the 3Rs for animal experimentation, this study evaluated native integrin, the cellular receptor for FMDV, as an alternative coating reagent for SPCE.BHK-21, MDBK, and LFBK cell lines were screened for relative abundance of integrin subunits (αV, α5, β1, β3, β5, β6, and β8) by qRT-PCR and for FMDV reactivity by sandwich ELISA. LFBK cells exhibited the highest integrin expression (β8 > αV > β5 > β6) and strongest ELISA reactivity; therefore, the LFBK was selected for integrin isolation. Optimal cell lysis was achieved using 1mM Triton X-100 for 30min. Affinity chromatography was performed using full-length fibronectin, GRGDSP peptide, and recombinant fibronectin type III9-10 (FN-III9-10) domain as ligands. Western blot confirmed that all three ligands captured αV integrin; however, FN-III9-10 was selected due to its scalability. Integrin was eluted using NaCl concentrations of 250, 500, and 1,000mM, with maximum yield obtained at 250mM NaCl. The eluted integrin retained reactivity with anti-αV antibodies and demonstrated the ability to capture FMDV in ELISA. Coating concentration of 50ng/well was optimized by ELISA titration. Although integrin-coated wells produced lower absorbance than polyclonal serum, specific binding and dilution-dependent inhibition were observed. Antibody titers were comparable to conventional SPCE (Wilcoxon signed-rank test, p = 0.324). These findings provide proof-of-concept that native integrin can be a potential alternate to polyclonal serum in SPCE.