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 pre-coated with a rabbit polyclonal antibody specific for human FGF-21. Standards and samples are pipetted into the wells and any human FGF-21 present is bound by the immobilized antibody. After washing away any unbound substances, a biotin labelled polyclonal antibody specific for human 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 human 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 human 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. 

Human FGF-21 (pg/mL)	Absorbance (450 nm)	Blanked Absorbance
0	0.094	0
30	0.127	0.033
60	0.158	0.064
120	0.224	0.13
240	0.362	0.268
480	0.626	0.532
960	1.141	1.047
1920	2.14	2.046

 

B. Sensitivity

The lowest level of human FGF-21 that can be detected by this assay is 30 pg/mL.

 

C. Specificity

Cross Reactivity of recombinant proteins

Analyte	Cross Reactivity
Mouse FGF-21	No
Human FABP4	No
Human LCN2	No
Human Adiponectin	No
Human ANGPL4	No

 

D. Precision

Intra-assay Precision (Precision within an assay)

Two samples of known concentration were tested 12 times on one plate.

Sample	Mean (pg/mL)	SD (pg/mL)	CV (%)
1	93.3	3.72	4.0
2	547	27.6	5.0

Inter-assay Precision (Precision between assays)

Two samples of known concentration were tested in 10 separate assays.

Sample	Mean (pg/mL)	SD (pg/mL)	CV (%)
1	273.5	27.9	10.2
2	335	11.8	3.5

 

E. Linearity

To assess the linearity of the assay, samples containing and/or spiked with high concentrations of human FGF-21 were serially diluted with the 1×Assay buffer to produce samples with values within the dynamic range of the assay.

Sample 1

Dilution	Mean (pg/mL)	Expected (pg/mL)	Recovery (%)
Neat (non-diluted)	448	448	100
1:2	245	224	109
1:4	123	112	109
1:8	63.6	56	113

 

Sample 2

Dilution	Mean (pg/mL)	Expected (pg/mL)	Recovery (%)
Neat (non-diluted)	318	318	100
1:2	170	159	106
1:4	84.3	79.5	106
1:8	43.1	39.75	108

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