Fetuin-A ELISA Assay Kit

$745.00

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

Documents

Product Documents


Publications

Citations


  • Stenvinkel P, Wang K, Qureshi AR, Axelsson J, Pecoits-Filho R, Gao P, Barany P, Lindholm B, Jogestrand T, Heimbürger O, Holmes C, Schalling M, Nordfors L. Low fetuin-A levels are associated with cardiovascular death: Impact of variations in the gene encoding fetuin. Kidney Int. 2005 Jun;67(6):2383-92.
  • Wang AY, Woo J, Lam CW, Wang M, Chan IH, Gao P, Lui SF, Li PK, Sanderson JE. Associations of serum fetuin-A with malnutrition, inflammation, atherosclerosis and valvular calcification syndrome and outcome in peritoneal dialysis patients. Nephrol Dial Transplant. 2005 Aug;20(8):1676-85.
  • Sato H, Kazama JJ, Wada Y, Kuroda T, Narita I, Gejyo F, Gao P, Yamashita H. Decreased levels of circulating alpha2-Heremans-Schmid glycoprotein/Fetuin-A (AHSG) in patients with rheumatoid arthritis. Intern Med. 2007;46(20):1685-91. Epub 2007 Oct 15.
  • Lehtinen AB, Burdon KP, Lewis JP, Langefeld CD, Ziegler JT, Rich SS, Register TC, Carr JJ, Freedman BI, Bowden DW. Association of alpha2-Heremans-Schmid glycoprotein polymorphisms with subclinical atherosclerosis. J Clin Endocrinol Metab. 2007 Jan;92(1):345-52. Epub 2006 Oct 24
  • Lim P, Collet JP, Moutereau S, Guigui N, Mitchell-Heggs L, Loric S, Bernard M, Benhamed S, Montalescot G, Randé JL, Guéret P. Fetuin-A is an independent predictor of death after ST-elevation myocardial infarction. Clin Chem. 2007 Oct;53(10):1835-40. Epub 2007 Aug 16
  • Ix JH, Wassel CL, Kanaya AM, Vittinghoff E, Johnson KC, Koster A, Cauley JA, Harris TB, Cummings SR, Shlipak MG; Health ABC Study. Fetuin-A and incident diabetes mellitus in older persons. JAMA. 2008 Jul 9;300(2):182-8.
  • Ix JH, Wassel CL, Bauer DC, Toroian D, Tylavsky FA, Cauley JA, Harris TB, Price PA, Cummings SR, Shlipak MG; for the Health ABC Study. Fetuin-A and Bone Mineral Density in Older Persons: The Health Aging and Body Composition (Health ABC) Study. J Bone Miner Res. 2008 Nov 18.
  • Giulia Bivona, Chiara Bellia, Antonietta Caruso, Daniela Butera, Bruna Lo Sasso, Patrizia Altavilla, Rosa C. Carollo, Gaia Chiarello andMarcello Ciaccio. Low Serum Fetuin A Levels and Cardiovascular Events in End-Stage Renal Disease (ESRD) Patients. Research Journal of Medical Sciences 2 (4): 200-202, 2008
  • Mario Cozzolinoa, Andrea Galassia, Maria Luisa Biondib, Olivia Turrib, Sergio Papagnic, Nicola Mongellic, Luigi Civitac, Maurizio Gallienia, Diego Brancaccioa Serum Fetuin-A Levels Link Inflammation and Cardiovascular Calcification in Hemodialysis Patients. Am J Nephrol 2006;26:423-429
  • G. Metry, P. Stenvinkel, A. R. Qureshi, J. J. Carrero, M. I. Yilmaz, P. Bárány, S. Snaedal, O. Heimbürger, B. Lindholm and M. E. Suliman Low serum fetuin-A concentration predicts poor outcome only in the presence of inflammation in prevalent haemodialysis patients. European Journal of Clinical Investigation 2008;38(11):804 – 811
  • Doris Hendig, Veronika Schulz, Marius Arndt, Christiane Szliska, Knut Kleesiek, and Christian Go¨ tting1. Role of Serum Fetuin-A, a Major Inhibitor of Systemic Calcification, in Pseudoxanthoma Elasticum. Clin Chem 2006;52:227-234
  • G. Marhaug, V. Shah, R. Shroff, H. Varsani, L. R. Wedderburn, C. A. Pilkington and P. A. Brogan. Age-dependent inhibition of ectopic calcification: a possible role for fetuin-A and osteopontin in patients with juvenile dermatomyositis with calcinosis. Rheumatology 2008 47(7):1031-1037
  • Marcello Ciaccio, et al.  Changes in serum fetuin-A and inflammatory markers levels in end-stage renal disease (ESRD): effect of a single session haemodialysis. Clin Chem Lab Med 2008;46:212–214