Fetuin-A ELISA Assay Kit

Additional Information

Assay Background

Fetuin-A, also known as alpha-2-HS glycoprotein, is a 59 kDa glycoprotein that consists of two amino-terminal cystatin domains and a smaller carboxyl-terminal domain. Fetuin-A is synthesized by the liver and secreted into blood stream, where its concentration in adult mammals ranges from 0.5 – 1.0 g/L. Fetuin-A occurs in high serum concentration during fetal life and involves in protease inhibitory activities and development-associated regulation of calcium metabolism and osteogenesis. It accumulates in bones and teeth as a major fraction of noncollagenous bone proteins.

Biologically, studies have demonstrated that Fetuin-A is the major calcification inhibitor found in circulation, where it interferes with calcium salt precipitation. Recent study has indicated that Fetuin-A level drops in uremic patients on hemodialysis in comparison to normal healthy controls. The low Fetuin-A level in patients with chronic kidney failure strongly associates with a higher cardiovascular mortality. On the other hand, it is demonstrated that a higher than normal serum Fetuin-A  in older population associates incident diabetes, which is independent from other markers of insulin resistance. Further, a higher Fetuin-A level may be an independent risk marker of patients with for cardiovascular diseases.

Assay Procedure

1. Place a sufficient number of antibody coated microwell strips in a holder to run human Fetuin-A standards, controls and unknown samples in duplicate.
2. Add 25 µL of standards, controls and 1:10,000 diluted patient samples into the designated microwell. Note: if urine sample is used, 1:100 diluted urine sample should be used.
3. Add 100 µL of assay buffer to each well.
4. Mix gently and cover the plate with one plate sealer and also with aluminum foil to avoid exposure to light.
5. Incubate the plate at room temperature for 2 hours.
6. Prepare Tracer Antibody Working Solution by 1:21 fold dilution of the Fetuin-A Tracer Antibody with the Tracer Antibody Diluent. For each strip, it is required to mix 1 mL of Tracer Antibody Diluent with 50 µL of Fetuin-A Tracer Antibody in a clean test tube.
7. Remove the aluminum foil and the 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 above diluted tracer antibody working solution to each of the wells.
9. Cover the plate with one plate sealer and also with aluminum foil to avoid exposure to light.
10. Incubate the plate at room temperature for 30 minutes.
11. Remove the aluminum foil and the 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.
12. Add 100 µL of ELISA HRP Substrate into each of the wells.
13. Cover the plate with aluminum foil to avoid exposure to light.
14. Incubate the plate at room temperature for 20 minutes
15. Remove the aluminum foil.  Add 100 µL of ELISA Stop Solution into each of the wells. Mix gently.
16. Read the absorbance at 450 nm within 10 minutes in a microplate reader

Typical Standard Curve