Total FGF-21 ELISA Assay

Additional Information

Assay Principle

The Eagle Biosciences Total FGF-21 ELISA Assay Kit is designed, developed and produced for the quantitative determination of human N-terminal and C-terminal FGF-21 level in EDTA-plasma or serum. It measures the intact FGF-21, the N-terminal and C-terminal FGF-21 fragments that must not be N-terminally and C-terminally truncated. 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 N-terminal and 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 N-terminal and 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 N-terminal and C-terminal FGF-21 on the wall of the microtiter well is directly proportional to the amount of N-terminal and 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 the combined N-terminal and 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 ncFGF-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