Chicken-derived phage display antibodies against ASARM for calcification and phosphate research.

Abstract

Human phosphorylated acidic serine- and aspartate-rich motif (pASARM), derived from matrix extracellular phosphoglycoprotein, exhibits anti-calcification activity and induces hypophosphatemia by inhibiting phosphate uptake in the intestine and kidneys. pASARM is considered a potential therapeutic candidate for hyperphosphatemia and ectopic calcification in chronic kidney disease-mineral and bone disorder owing to its inhibitory effects on phosphate uptake and mineralization. However, developing effective monoclonal antibodies is challenging because of the high amino acid sequence homology among mammalian species and the low immunogenicity of pASARM in mice and rabbits. In contrast, chickens have attracted attention as an alternative immune animal model because of their greater phylogenetic distance from humans. In this study, we aimed to generate anti-ASARM mAbs using chicken-derived phage display technology. First, a highly diverse library of single-chain variable fragment (scFv) antibodies was constructed from chickens immunized with ASARM. Second, biopanning and cloning were performed using ASARM to isolate highly reactive scFv antibodies. Third, the selected scFv antibodies were converted into chicken-human chimeric anti-ASARM IgG₄ (anti-ASARM IgG₄) for future effector-function applications. An enzyme-linked immunosorbent assay demonstrated that the two anti-ASARM IgG₄ antibodies possessed high reactivity toward ASARM. Immunohistochemical analyses showed that one antibody successfully detected ASARM localization in the femoral tissues of 8-week-old male mice and age-matched male rats. The anti-ASARM IgG₄ generated in this study exhibited low non-specificity and high reproducibility, making them potentially valuable tools for basic research involving ASARM.