Principle of Assay
This Human MIP-1 alpha ELISA Kit allows for the quantitative determination of natural or recombinant MIP-1 alpha within any experimental sample including cell lysates, serum, and plasma. This Human MIP-1 alpha ELISA Kit applies a technique called quantitative sandwich immunoassay. The microtiter plate provided in this kit has been pre-coated with a monoclonal antibody specific for MIP-1 alpha. Standards or samples are then added to the appropriate microtiter plate wells and incubated. If present, MIP-1 alpha will bind and become immobilized by the antibody pre-coated on the wells. MIP-1 alpha then becomes “sandwiched” by the primary capture antibodies and the secondary detection antibodies subsequently added. After incubation and “sandwiching” of MIP-1 alpha, 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 (2 N 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.
