Human FGF-23 ELISA Kit
Assay range: 78.125 - 5000pg/mL
Kit Size: 96 wells/kit
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 +2xSDblank) 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%