Fetuin-A ELISA

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 - 1.5 g/L. Fetuin-A occurs in high serum concentration during fetal life. It accumulates in bones and teeth as a major fraction of noncollagenous bone proteins. Studies have demonstrated that Fetuin-A is the major calcification inhibitor found in serum, where it interferes with calcium salt precipitation.

This ELISA 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 serum samples containing human Fetuin-A are added to microtiter wells of a microplate 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 are 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.