Principle of Assay
This Human sCD40 Ligand ELISA Kit allows for the quantitative determination of natural or recombinant sCD40 Ligand within any experimental sample including cell lysates, serum, and plasma. This Human sCD40 Ligand 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 sCD40 Ligand. Standards or samples are then added to the appropriate microtiter plate wells and incubated. If present, sCD40 Ligand will bind and become immobilized by the antibody pre-coated on the wells. sCD40 Ligand then becomes “sandwiched” by the primary capture antibodies and the secondary detection antibodies subsequently added. After incubation and “sandwiching” of sCD40 Ligand, 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.
