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FGF-21 (Intact) ELISA Assay Kit:  
For Research Use Only
Size:  1x96 wells
Sensitivity:  1.7 pg/mL
Dynamic Range:  32.5 - 2000 pg/ml
Incubation Time:  2.5 hours
Sample Type:  Serum, Plasma   
Sample Size: 50 µL
Controls Included
Product Developed and Manufactured in the USA

Intended Use
The Eagle Biosciences Human Intact Fibroblast Growth Factor 21 (FGF-21) ELISA Assay Kit (enzyme-linked immunoassay kit) is intended for the quantitative determination of human Intact Fibroblast Growth Factor 21 (FGF-21) levels in serum. The Eagle Biosciences Human Intact Fibroblast Growth Factor 21 (FGF-21) ELISA Assay Kit is for research use only and not to be used in diagnostic procedures.

Assay Principle
The Eagle Biosciences Human Intact Fibroblast Growth Factor 21 (FGF-21) ELISA Assay Kit is designed, developed and produced for the quantitative measurement of human intact FGF-21 in serum and EDTA-plasma sample. The assay utilizes the two-site “sandwich” technique with two selected antibodies that bind to different epitopes of human intact FGF-21. One of the antibodies is specifically binds to the N-terminal human FGF-21 (1-7) and the other is specifically to the C-terminal human FGF-21 (175-181).

Assay standards, controls and patient samples are added directly to wells of microplate that is coated with an anti-human FGF-21 (1-7) specific antibody. Simultaneously, a horseradish peroxidase conjugated anti-human FGF-21 (175-181) specific antibody is added to each well. After the first incubation period, the antibody on the wall of microtiter well captures human FGF-21 in the sample and an unbound protein in each microtiter well is washed away. A “sandwich” of “anti-FGF-21 antibody --- human intact FGF-21 --- HRP conjugated tracer antibody” is formed. The unbound tracer antibody is removed in the subsequent washing step. For the detection of this immunocomplex, the well is then incubated with a substrate solution in a timed reaction and then measured in a spectrophotometric microplate reader. The enzymatic activity of the immunocomplex bound to human intact FGF-21 on the wall of the microtiter well is directly proportional to the amount of intact FGF-21 in the sample. A standard curve is generated by plotting the absorbance versus the respective human intact FGF-21 concentration for each standard on point-to-point or 4 parameter curve fit. The concentration of human intact FGF-21 in test samples is determined directly from this standard curve.

1. Place a sufficient number of murine IgG coated microwell strips/wells in a holder to run human FGF-21 standards, controls and unknown samples in duplicate.
2. Add 50 µL of standards, controls and patient plasma/serum samples into the designated microwell.
3. Add 50 µL of 1:21 diluted tracer antibody to each well
4. Cover the plate with one plate sealer and incubate plate with orbital shaking 170 rpm at room temperature for 2 hours.
5. Remove plate sealer. Aspirate the contents of each well. Wash each well 5 times by dispensing 350 µL of working wash solution into each well and then compley aspirating the contents. Alternatively, an automated microplate washer can be used.
6. Add 100 µL of ELISA HRP Substrate into each of the wells.
7. Cover the plate with one plate sealer and also with aluminum foil to avoid exposure to light. Incubate plate at room temperature for 20 minutes.
8. Remove the aluminum foil and plate sealer.  Add 100 µL of ELISA Stop Solution into each of the wells. Mix gently.
9. Read the absorbance at 450/650 nm within 10 minutes in a microplate reader

Assay Background

Fibroblast Growth Factor 21 (FGF-21) belongs to the FGF-19 subfamily, which includes FGF-19, FGF-21 and FGF-23. The FGF-19 family members are potent endocrine hormones in the regulation of a diverse physiological homeostasis. 

The intact FGF-21 is a small protein comprising 181 amino acids. Administration of recombinant FGF21 lowered plasma glucose and insulin levels, reduced hepatic and circulating triglycerides and cholesterol levels, and improved insulin sensitivity, energy expenditure, hepatic steatosis and obesity in a range of insulin resistant animal models. The physiological functions of FGF-21 are relied on the intact molecular structure and amino acid sequence in its N-terminal and C-terminal region. An N-terminal truncated FGF-21 (7-181) is a potent inhibitor that competitively inhibits the biological activity of intact FGF-21 (1-181). Therefore, it is important to measure the circulation intact FGF-21 level in the assessment of the physiological and pathophysiological condition. An assay that determines the fragment of the FGF-21 might overestimate the biological activity of the protein in test sample.

Circulation FGF-21 is a biomarker and its levels is increased in patient with nonalcoholic fatty liver disease (NAFLD), type 2 diabetes, gestational diabetes and obesity. An increase of circulating FGF-21 is also found in patient with Cushing’s syndrome, patient with lipodystrophy induced by HIV-1 and patient with chronic renal disease or end-stage renal disease (ESRD).