Application

Key components:

1. Capturing antibody
2. Detecting antibody
3. Human Fc protein standard
4. ELISA Protocol

The following reagents are not included in the human Fc ELISA reagent kits and must be provided by the end users:
1. 96-well or 384-well ELISA plates
2. Assay Diluent (PBS + 1% BSA)
3. Wash buffer (PBS + 0.01% Tween20)
4. HRP substrate (TMB)
5. Stop solution (0.5N HCl)
6. Absorbance microplate reader (we recommend BMG LABTECH microplate readers)

Background

Fc is the tail region of an immunoglobulin G (IgG) that interacts with cell surface receptors called Fc receptors and some proteins of the complement system. The ~230 amino acid fragment generally exists as a dimer, although under reduced condition, it exists as a monomer. It is the basis of prolonged pharmacokinetics of antibodies and is commonly used as a fusion to extend half-life of fusion proteins.

This assay employs the quantitative sandwich enzyme immunoassay technique. An affinity purified polyclonal antibody specific for human or mouse Fc has been coated onto a 96-well microplate. Standards, control, and samples are pipetted into the wells and any human or mouse Fc present is bound by the immobilized antibody. After washing away any unbound substances, an enzyme-linked polyclonal antibody specific for human or mouse Fc is added to the wells. Following an incubation and a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells. The enzyme reaction yields a blue product that turns yellow when the stop solution is added. The intensity of the color measured is in proportion to the amount of human or mouse Fc bound in the initial step. The sample values are then read off the standard curve.