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Programmed cell death protein 1 (PD-1), also known as cluster of differentiation 279 (CD279), belongs to immunoglobulin superfamily and is a transmembrane receptor protein. PD-1 is present on T –cells or B-cells and binds to ligands, such as PD-L1 and PD-L2, to down regulate the immune system by modulating many other cytokines and cytokine receptors. Anti PD-1 antibody drugs been shown to enhance the immune systems of several types of cancer patients.
AlphaLISA technology allows the detection of molecules of interest in a no-wash, highly sensitive, quantitative assay. In an AlphaLISA assay, a biotinylated anti-analyte antibody binds to the Streptavidin-coated Donor beads while another anti-analyte antibody is conjugated to AlphaLISA Acceptor beads. In the presence of the analyte, the beads come into close proximity. The excitation of the Donor beads causes 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.
|Assay Target Class||Protein|
|Experimental Type||In vitro|
|Product Brand Name||AlphaLISA|
|Shipping Condition||Blue Ice|
|Unit Size||500 assay points|
The introduction of enzyme-linked immunosorbent assays (ELISAs) in the early 1970’s offered researchers a non-radiometric immunoassay platform without compromising sensitivity. Over the last 50 years scientists have made huge strides in disease research and drug discovery and a demand for greater assay throughput and sensitivity has evolved. In response, more robust immunoassays have been developed to address some of the limitations of the standard, colorimetric ELISA.
Find out about the most common limitations of traditional ELISAs and how different ELISA alternative technologies address these limitations.
One approach to immunotherapy is the modulation of immune checkpoints that are critical in regulating the degree and duration of immune system responses and preventing autoimmunity.
In this application note, you will learn:
Immune checkpoints serve a critical role in the immune system to prevent autoimmunity and manage the degree and duration of an immune response. One immune checkpoint of significant interest involves the inhibitory (anti-inflammatory) transmembrane protein cytotoxic T-Lymphocyte-associated protein 4 (CTLA-4, or CD152). When CTLA-4 is expressed (after T-lymphocyte activation) and competes with CD28 for its ligands, 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.
Herein, AlphaLISA technology was demonstrated in a screen for novel CTLA-4 blocking drugs by utilizing the AlphaLISA CTLA-4/CD80 binding assay. First, to demonstrate the binding assay, a titration of CTLA-4 was performed with a fixed amount of CD80 protein. Further, unlabeled proteins CLTA-4, CD80, and a negative control were used to block CTLA-4/CD80 binding. Next, a variety of antibodies were tested to try to block CTLA-4/CD80 binding. Utilizing a successful blocking antibody, Ipilimumab, different buffers, media and DMSO were tested to show the effect on Alpha technology performance. Finally, the Z-prime (Z’) was determined using an anti-CD80 antibody to demonstrate the exceptional quality of the assay for drug screening.