The Human EGF Bioassay ELISA Kit allows for the detection and quantification of human EGF proteins in serum, plasma and cell supernatants. Detection Range: 8-1000pg/ml Assay Principle: The Human EGF Bioassay ELISA Kit contains the components necessary for quantitative determination of natural or recombinant concentrations within any experimental sample including cell supernatants, serum and plasma. This particular immunoassay utilizes the quantitative technique of a ''Sandwich Enzyme-Linked Immunosorbent Assay (ELISA) where the target protein (antigen) is bound in a ''sandwich format by the primary capture antibodies coated to each well-bottom and the secondary detection antibodies added subsequently by the investigator. The capture antibodies coated to the bottom of each well are specific for a particular epitope on while the user-added detection antibodies bind to epitopes on the captured target protein. Amid each step of the procedure, a series of wash steps must be performed to ensure the elimination of nonspecific binding between proteins to other proteins or to the solid phase. After incubation and ''sandwiching of the target antigen, a peroxidase enzyme is conjugated to the constant heavy chain of the secondary antibody (either covalently or via Avidin/Streptavidin-Biotin interactions), allowing for a colorimetric reaction to ensue upon substrate addition. When the substrate TMB (3, 3, 5, 5-Tetramethylbenzidine) is added, the reaction catalyzed by peroxidase yields a blue color that is representative of the antigen concentration. Upon sufficient color development, the reaction can be terminated through addition of Stop Solution (2N Sulfuric Acid) where the color of the solution will turn yellow. The absorbance of each well can then be read by a spectrophotometer, allowing for generation of a standard curve and subsequent determination of protein concentration. Kit Components: 472029A: Microtiter Strips, 1x96 wells *472029B: EGF Standard, 1x1vial *472029C: Antibody (Biotin), 1x1vial 472029D: Streptavidin (HRP), 400X, 1x30ul 472029E: Assay Diluent, 1x50ml 472029F: Wash Buffer, 15X, 1x60ml 472029G: TMB Substrate, 1x12ml 472029H: Stop Solution, 2N H2SO4, 1x12ml Storage and Stability: Store all components at 4C. Reconstituted *472029B and *472029C are stable for 1 month at 4C.. Other components are stable for 6 months after receipt. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.
