FGF21 is a metabolic hormone mainly produced in liver. It regulates glucose and lipid metabolism through pleiotropic actions in multiple tissues, and protects against metabolic damages under various stresses.
 In humans, high circulating FGF21 levels are found in obesity and multiple cardiometabolic disorders,  including metabolic syndrome, type 2 diabetes, non-alcoholic fatty liver disease and coronary artery disease. Serum FGF21 is a potential biomarker for the early detection and risk prediction of these diseases.

This assay is a quantitative sandwich ELISA. The immunoplate is precoated with a rabbit polyclonal antibody specific for mouse FGF-21. Standards and samples are pipetted into the wells and any mouse FGF-21 present is bound by the immobilized antibody. After washing away any unbound substances, a biotin labelled polyclonal antibody specific for mouse FGF-21 is added to the wells. After wash step to remove any unbound reagents, 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 mouse FGF-21 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 mouse FGF-21, the unknown sample concentration can be interpolated from a reference curve included in each assay.

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. 

FGF-21 (pg/mL)	Absorbance (450 nm)	Blanked Absorbance
0	0.109	0
31.2	0.132	0.023
62.5	0.158	0.049
125	0.209	0.1
250	0.309	0.2
500	0.571	0.462
1000	1.031	0.992
2000	1.9	1.791

 

B. Sensitivity

The lowest level of mouse FGF-21 that can be detected by this assay is 31.2 pg/mL.

 

C. Specificity

Cross reactivity of recombinant proteins

Analyte	Cross Reactivity
Human FGF-21	No
Mouse Mup-1	No
Mouse LCN2	No
Mouse Adiponectin	No
Mouse ANGPL4	No

 

D. Precision

Intra-assay Precision (Precision within an assay) C.V. <8%.

Inter-assay Precision (Precision between assays) C.V. <10%.

 

E. Recovery

The recovery of the assay was determined by adding various amounts FGF21 to a sample. The measured concentration of the spiked sample in the assay was compared to the expected concentration. The average recovery was 92%.

 

F. Linearity

To assess the linearity of the assay, a sample with high level of FGF-21 was serially diluted with the 1×Assay buffer to produce samples with values within the dynamic range of the assay.

Dilution	Measured (pg/mL)	Expected (pg/mL)	Recovery (%)
1/2	1360	1360	100
1/4	680	680	101
1/8	380	340	111
1/16	200	170	115

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