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AlphaLISA technology allows detecting the binding of target proteins in a highly sensitive, quantitative, reproducible and user-friendly mode. In this AlphaLISA assay, a biotinylated LAG-3 binds to the Streptavidin-coated Alpha Donor beads, while His-tagged HLA DRA is captured by Anti-His AlphaLISA Acceptor beads. When LAG-3 binding to HLA DRA happens, Donor beads and Acceptor beads come into close proximity. The excitation of the Donor beads provokes the release of singlet oxygen molecules that triggers a cascade of energy transfer in the Acceptor beads, resulting in a sharp peak of light emission at 615 nm.
Human Leukocyte Antigen (HLA) is an MHC (major histocompatibility complex) class II cell surface receptor. HLA DRA is one of Antigen D related HLA class II (HLA DR) alpha chain paralogues. This class II molecule is a heterodimer consisting of an alpha and a beta chain, both anchored in the membrane. It plays a central role in the immune system by presenting peptides derived from extracellular proteins. Lymphocyte activation gene-3 (LAG-3), also known as CD223, is a member of the immunoglobulin superfamily. LAG-3 binds to MHC class II with higher affinity, providing negative regulation of T cell receptor signaling. Binding of a homodimerized LAG-3/Ig fusion protein to MHC class II molecules induces maturation of immature dendritic cells and secretion of cytokines. Deletion of LAG-3 facilitates anti-cancer immune response, also blocks self-tolerance and increases susceptibility to autoimmune diseases. Because of its profound inhibitory role, blocking HLA DR and LAG-3 binding has been considered as promising therapeutic target for human autoimmune disease and cancers.
|Assay Target||HLA-DRA, LAG3|
|Assay Target Class||Protein|
|Product Brand Name||AlphaLISA|
|Unit Size||500 Assay Points|
Immune checkpoints serve a critical role in the immune system to prevent autoimmunity and manage the degree and duration of an immune response. Cytotoxic T-Lymphocyte-associated protein 4 (CTLA-4 or CD152) is an inhibitory transmembrane protein involved in an immune checkpoint of significant interest for therapeutic development. When CTLA-4 is expressed and competes with CD28, the immune system response is downregulated. As a result of this immune system response balance, immune checkpoints provide an opportunity for therapeutic intervention to modulate immune system activity.
There is a high demand for new drugs to block CTLA-4 and modulate immune system activity. In this application note, we demonstrate how to screen for novel CTLA-4 blocking drugs by utilizing the AlphaLISA CTLA-4/CD80 binding assay.