SP278
VALIDATIONOF A C-TERMINAL FGF23MULTI-MATRIX SANDWICH ELISA FOR THE DETECTION OF FGF23 IN HUMAN SERUM AND PLASMA

INTRODUCTION AND AIMS: Fibroblast growth factor 23 (FGF23) is secreted by osteoblasts and osteocytes and mainly regulates phosphate homeostasis and calcitriol levels. The bioactive intact FGF23 contains 251 amino acids and is glycosylated and phosphorylated. Its activity is mediated by binding to FGFR/Klotho receptor complex at the target cell surface. Intact FGF23 is cleaved between Arg179 and Ser180 to an N-terminal and a C-terminal fragment. Several studies revealed that FGF23 concentrations are increased in chronic kidney disease, oncogenic osteomalacia and several rare hereditary disorders. Most of these measurements were performed by using immunoassays, which detect only intact (intact FGF23 ELISA) or both intact and C-terminal fragments (C-terminal FGF23 ELISA).

METHODS: Here, we show the validation and characterization of a C-terminal multi-matrix FGF23 ELISA. Epitopes of both polyclonal antibodies were analyzed by overlapping linear peptides spotted to a microarray and also determination of binding kinetics with biolayer interferometry was performed. The assay was validated according to standard quality guidelines with a special focus on matrix comparison (serum and EDTA, heparin and citrate plasma) and analyte stability.

RESULTS: Linear epitopes of both polyclonal antibodies are distributed throughout the C-terminal part of FGF23 and antibodies bind with high affinity to FGF23. The assay generates highly specific FGF23 signals (>90%), a mean accuracy and dilution linearity of 85-110% and 90-108%, respectively. Both, intra- as well as inter-assay precision are within the standard of acceptance. Compared with other C-terminal FGF23 ELISA on the market, that detect FGF23 only in plasma, this C-terminal assay can be used for the measurement of serum as well as. Matrix comparison of serum and plasma of apparently healthy and dialysis patients show a good correlation with R² > 0.85. Endogenous FGF23 is freeze-thaw stable in serum and plasma for at least 4 cycles, whereas whole blood stability is better for plasma than for serum samples.

CONCLUSIONS: This well-characterized and fully validated C-terminal FGF23 ELISA can be used for the measurement of human serum and plasma samples and may support further FGF23 research.