C-Terminal FGF-21 ELISA Assay Kit

$550.00

The Eagle Biosciences C-Terminal FGF-21 ELISA Assay Kit is intended for use in the quantitative determination of C-terminal FGF-21 levels in human serum and EDTA plasma. It measures both the intact FGF-21 and the C-terminal FGF-21 fragments that must not be C-terminally truncated while the N-terminal end of the FGF-21 may be truncated. The Eagle Biosciences C-Terminal FGF-21 ELISA Assay Kit is intended for research use only.

C-Terminal FGF-21 ELISA Assay Kit

For Research Use Only

Size: 1×96 wells
Sensitivity: 3.7 pg/mL
Dynamic Range: 54.0 – 2100 pg/ml
Incubation Time: 2.5 hours
Sample Type: Serum, Plasma
Sample Size: 100 µL

Controls Included

Product Developed and Manufactured in the USA


Additional FGF-21 Assays Available

Additional Information

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 FGF-21 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. The N-terminal non-truncated FGF-21 is a potent cell membrane FGF receptor binder. Whereas, 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 are increased in patients with nonalcoholic fatty liver disease (NAFLD), type 2 diabetes, gestational diabetes and obesity. An increase of circulating FGF-21 is also found in patients with Cushing’s syndrome, patients with lipodystrophy induced by HIV-1 and patients with chronic renal disease or end-stage renal disease (ESRD).

Assay Principle


The Eagle Biosciences C-Terminal FGF-21 ELISA Assay Kit is designed, developed and produced for the quantitative measurement of human C-terminal 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 FGF-21. One of the antibodies specifically binds to the C-terminal human FGF-21 (175-181) and the other is to the multi-epitopes of mid-regional and N-terminal human FGF-21.

Assay calibrators, controls and patient samples are added directly to wells of a microplate that is coated with an anti-human FGF-21 (175-181) specific antibody. After the first incubation period, a horseradish peroxidase-conjugated anti-human FGF-21 polyclonal antibody is added to each well. After the second incubation period, the antibody on the wall of microtiter well captures human C-terminal FGF-21 in the sample and further forms “sandwich” with the tracer antibody. Unbound proteins in each microtiter well are washed away. An immunocomplex of “anti-FGF-21 antibody — human C-terminal 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 C-terminal FGF-21 on the wall of the microtiter well is directly proportional to the amount of C-terminal FGF-21 in the sample. A calibrator curve is generated by plotting the absorbance versus the respective human intact FGF-21 concentration for each calibrator on point-to-point or 4 parameter curve fit. The concentration of human C-terminal FGF-21 in test samples is determined directly from this calibrator curve.

Assay Procedure


  1. Place a sufficient number of antibody coated microwell strips in a holder to run human cFGF-21 calibrators, controls and unknown samples in duplicate.
  2. Add 100 µL of calibrators, controls and patient plasma/serum samples into the designated microwell.
  3. Cover the plate with one plate sealer and incubate plate with orbital shaking 170 rpm (big radius) or 400 rpm (smaller radius) at room temperature for 1 hour.
  4. 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 completely aspirating the contents. Alternatively, an automated microplate washer can be used.
  5. Add 100 µL of 1:21 diluted tracer antibody to each well.
  6. Cover the plate with one plate sealer and incubate plate with orbital shaking 170 rpm (big radius) or 400 rpm (smaller radius) at room temperature for 1 hour.
  7. 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 completely aspirating the contents. Alternatively, an automated microplate washer can be used.
  8. Add 100 µL of ELISA HRP Substrate into each of the wells.
  9. 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.
  10. Remove the aluminum foil and plate sealer. Add 100 µL of ELISA Stop Solution into each of the wells. Mix gently.
  11. Read the absorbance at 450/650 nm within 10 minutes in a microplate reader using point-to-point curve fitting

Typical Standard Curve


Manual

Product Manual


Publications

Citations


Parmar, Biraj, et al. “Eccentric Exercise Increases Circulating Fibroblast Activation Protein α but Not Bioactive Fibroblast Growth Factor 21 in Healthy Humans.” Experimental Physiology, vol. 103, no. 6, 1 June 2018, pp. 876–883., doi:10.1113/ep086669.

Soeberg S, et al. (2018). FGF21, a Liver Horomone That Inhibits Alcohol Intake in Mice, Increases in Human Circulation After Acute Alcohol Injestion and Sustained Binge Drinking at Oktober Fest. Molecular Metabolism. 

Keipert, S;Kutschke, M;Ost, M;Schwarzmayr, T;van Schothorst, EM;Lamp, D;Brachthäuser, L;Hamp, I;Mazibuko, SE;Hartwig, S;Lehr, S;Graf, E;Plettenburg, O;Neff, F;Tschöp, MH;Jastroch, M;, (2017). Long-Term Cold Adaptation Does Not Require FGF21 or UCP1. Cell Metab., 26(1), 437-446.e5-

Shenoy, VK;Beaver, KM;Fisher, FM;Singhal, G;Dushay, JR;Maratos-Flier, E;Flier, SN;, (2016). Elevated Serum Fibroblast Growth Factor 21 in Humans with Acute Pancreatitis. PLoS ONE, 11(11), e0164351-

Singhal, G;Douris, N;Fish, AJ;Zhang, X;Adams, AC;Flier, JS;Pissios, P;Maratos-Flier, E;Fibroblast growth factor 21 has no direct role in regulating fertility in female mice; Molecular Metabolism.2016: 5(8):690-698.

Coppage, AL;Heard, KR;DiMare, MT;Liu, Y;Wu, W;Lai, JH;Bachovchin, WW; Human FGF-21 Is a Substrate of Fibroblast Activation Protein, PLoS ONE; 11:3:e0151269, doi: 10.1371/journal.pone.0151269

Keipert, S. et al. “Genetic disruption of uncoupling protein 1 in mice renders brown adipose tissue a significant source of FGF21 secretion.”Molecular Metabolism. 2015: 4(7):537-542.