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Enzyme-Linked ImmunoSorbent Assay (ELISA) for the Quantitative Determination of Mouse Fibroblast Growth Factor 23 Levels in Plasma, Serum* or Cell Culture Media

INTENDED USE

This kit is intended for research use only in the quantitative determination of mouse FGF-23 levels in plasma, serum* or cell culture media. This assay is also useful in the determination of rat FGF-23 levels. * See Specimen Collection Section.

INTRODUCTION

Fibroblast growth factor 23 (FGF-23), which is produced by bone cells, is a novel member of a large family of related proteins. Its gene encodes a 251 amino acid protein. The amino-terminal portion of FGF-23 (aa 1-24) is hydrophobic and is likely to serve as a signal peptide allowing its secretion into the blood circulation. Its carboxylterminal portion (aa 180-251) shares only limited amino acid homology with other members of the FGF family of proteins. Renal phosphate wasting disorders leading to hypophosphatemia are among the causes of defective mineralization of bone and growth plate development. Autosomal dominant hypophosphatemic rickets (ADHR), a rare genetic disorder, results from one of several different FGF-23 mutations that make the protein resistant to proteolytic cleavage. Furthermore, tumors that cause oncogenic osteomalacia (OOM) have been shown to overexpress FGF-23 mRNA making it likely that elevated concentrations of FGF-23 in the blood are the cause of renal phosphate wasting. Consistent with this conclusion, the administration of recombinant FGF-23 to rodents was shown to increase urinary excretion of phosphate thus leading to hypophosphatemia and osteomalacia/rickets. Recent studies with chronic kidney disease (CKD) patients have shown FGF-23 to be an early predictor of abnormal renal tubular function, bone mineralization, disease severity and over-all mortality risk. Taken together, all currently available data suggest that the measurement of FGF-23 levels may provide an important diagnostic tool for the evaluation of hypophosphatemic and hyperphosphatemic disorders.

TEST PRINCIPLE

This Mouse FGF-23 (Intact) ELISA Kit is a homologous, two-site enzymelinked immunosorbent assay (ELISA) for the measurement of intact FGF-23. Two affinity purified goat polyclonal antibodies have been selected to detect epitopes within the amino-terminal and carboxylterminal regions of mouse FGF-23. The amino-terminal antibody is biotinylated for capture and the carboxyl-terminal antibody is conjugated with the enzyme horseradish peroxidase (HRP) for detection. In a two-step reaction a sample containing mouse FGF-23 is first incubated with the biotinylated antibody in a streptavidin coated microtiter well. After washing the well to remove any unbound antibody and other components, the well is incubated with the HRP conjugated antibody. FGF-23 contained in the sample is immunologically bound by the capture antibody and the detection antibody to form a “sandwich” complex: Well/Avidin— Biotin Anti-mFGF23 — Mouse FGF23 — HRP Anti-mFGF23 (NH2-terminal) (C-terminal) Following another wash the enzyme antibody bound to the well is incubated with a substrate solution in a timed reaction and then measured in a spectrophotometric microtiter plate reader. The enzymatic activity of the antibody complex bound to the well is directly proportional to the amount of FGF-23 in the sample. A standard curve is generated by plotting the absorbance versus the respective FGF-23 concentration for each standard on linear or logarithmic scales. The concentration of mouse FGF-23 in the samples is determined directly from this curve.

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