Cardiovascular and Oxidative Stress Assay Kit

Fetuin-A ELISA Assay Kit

$835.00

The Fetuin A ELISA Assay Kit is intended for the quantitative determination of human Fetuin-A, also known as alpha-2-HS glycoprotein (AHSG), in serum, plasma, cell culture supernatant, tissue extraction and urine. The Eagle Biosciences Fetuin A ELISA Assay kit is for research use only and not to be used in diagnostic procedures.

SKU: FET39-K01 Categories: , ,

Fetuin-A ELISA Assay Kit

Fetuin-A ELISA Assay Kit Developed and Manufactured in the USA

Sensitivity: 5 ng/ml
Dynamic Range: 12.5 – 370 ng/ml
Incubation Time: 3 hours
Sample Type: Serum, Plasma, Urine, Tissue, Biological Fluids
Sample Size: 10 µL
Alternative Names: AHSG, alpha-2-HS glycoprotein
For Research Use Only

Controls Included

Assay Principle

This Fetuin A ELISA Assay Kit is designed, developed and produced for the quantitative measurement of human Fetuin-A in serum samples. The assay utilizes the two-site “sandwich” technique with two selected goat anti-human Fetuin-A polyclonal antibodies that bind to different epitopes of human Fetuin-A.

Assay standards, controls and prediluted patient serum samples containing human Fetuin-A is added to microtiter wells of microplate that was coated with a high affinity polyclonal goat anti-human Fetuin-A antibody. After the first incubation period, the antibody on the wall of microtiter well captures human Fetuin-A in the sample and unbound proteins in each microtiter well is washed away. Then a horseradish peroxidase (HRP) conjugated polyclonal anti-human Fetuin-A antibody is added to each microtiter well and a “sandwich” of “capture antibody – human Fetuin-A – HRP conjugated tracer antibody” is formed. The unbound tracer antibody is removed in the subsequent washing step. HRP conjugated tracer antibody bound to 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 tracer antibody bound to the Fetuin-A on the wall of the microtiter well is directly proportional to the amount of Fetuin-A in the sample. A standard curve is generated by plotting the absorbance versus the respective human Fetuin-A concentration for each standard on point-to-point or cubical scales. The concentration of human Fetuin-A in test samples is determined directly from this standard curve.

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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

Fetuin-A ELISA Assay Kit

Package Inserts

Product Documents

 

Please note: All documents above are for reference use only and should not be used in place of the documents included with this physical product. If digital copies are needed, please contact us.