Cat No.: 31295 Other Names: Other Names: UNQ3027/PRO9828
Introduction
FGF-23 is a 32-kDa protein that is secreted mainly by osteocytes in bone. It has been identified that has a physiological role in
regulating mineral homeostasis. FGF-23 exerts its biological functions by binding to its cognate fibroblastic growth factor receptor
(FGFR) in the presence of its coreceptor Klotho. FGF-23 is produced and secreted in response to hyperphosphatemia and increased 1,25 dihydroxyvitamin D3 levels. FGF-23 concentrations increase progressively as glomerular filtration rate (GFR) declines and the
level can be 1000-fold higher in patients with end-stage renal disease compared with healthy individual. This increase is considered as one of the earliest biochemical abnormality in chronic kidney disease. Therefore, FGF-23 concentration is a kind of standard to
determine the existence of kidney failure.
Principle of the Assay
This assay is a rapid quantitative sandwich ELISA. The immunoplate is pre-coated with a polyclonal antibody specific for human
FGF-23. Standards or samples and a biotin labelled polyclonal antibody specific for human FGF-23 are pipetted into the wells and any human FGF-23 present is bound by the immobilized antibody. After washing away any unbound substances, streptavidin-HRP
conjugate (STP-HRP) is added. After the last wash step, an HRP substrate solution is added and colour develops in proportion to the amount of human FGF-23 bound initially. The assay is stopped and the optical density of the wells determined using a microplate
reader. Since the increases in absorbance are directly proportional to the amount of captured human FGF-23, the unknown sample
concentration can be interpolated from a reference curve included in each assay.
Assay Performance
A. Typical representation of standard curve
The following standard curve is provided for demonstration only. A standard curve should be generated for each set of sample assay.
Human FGF-23 (pg/mL) | Absorbance (450 nm) | Blanked Absorbance |
0 | 0.076 | 0 |
78.125 | 0.123 | 0.047 |
156.25 | 0.173 | 0.097 |
312.5 | 0.256 | 0.180 |
625 | 0.425 | 0.349 |
1250 | 0.707 | 0.631 |
2500 | 1.376 | 1.300 |
5000 | 2.225 | 2.149 |
B. Sensitivity
Limit of Detection (LOD) (defined as concentration of analyte giving
absorbance higher than mean absorbance of blank plus three standard deviations of the absorbance of blank: Ablank +2 x SDblank)
is calculated from the real FGF-23 values in wells and is 30.74 pg/ml.
C. Precision
Intra-assay Precision (Precision within an assay)
Two samples of known concentration were tested 20 times on one plate.
Sample | n | Mean (pg/ml) | SD (pg/ml) | CV (%) |
1 | 20 | 1100.7 | 35.1 | 3 |
2 | 20 | 388.2 | 23.1 | 6 |
D. Linearity
To assess the linearity of the assay, samples containing and/or spiked with high concentrations of human FGF-23 were serially
diluted with the 1×Sample diluent to produce samples with values within the dynamic range of the assay.
Low Range
Dilution | Measured | Expected | Recovery |
1 | 729.934 | 729.934 | 100% |
0.9 | 665.125 | 656.9406 | 101% |
0.8 | 603.839 | 583.9472 | 103% |
0.7 | 497.406 | 510.9538 | 97% |
0.6 | 389.917 | 437.9604 | 89% |
0.5 | 323.502 | 364.967 | 89% |
0.4 | 255.595 | 291.9736 | 88% |
0.3 | 175.904 | 218.9802 | 80% |
0.2 | 125.898 | 145.9868 | 86% |
0.1 | 65.1536 | 72.9934 | 89% |
High Range
Dilution | Measured | Expected | Recovery |
1 | 4870.822 | 4870.822 | 100% |
0.9 | 4598.373 | 4383.74 | 105% |
0.8 | 4143.354 | 3896.658 | 106% |
0.7 | 3615.286 | 3409.575 | 106% |
0.6 | 3251.289 | 2922.493 | 111% |
0.5 | 2730.108 | 2435.411 | 112% |
0.4 | 2241.409 | 1948.329 | 115% |
0.3 | 1512.905 | 1461.247 | 104% |
0.2 | 1165.669 | 974.1644 | 120% |
0.1 | 560.093 | 487.0822 | 115% |
Linearity of Human FGF-23 ELISA range from 72.9934 to 4870.822 pg/ml.
The recovery rate ranges from 80% to 120%