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1-17 of 17 Resource Library

  • Application Note

    Screening for Inhibitors of PD-1 and PD-L1 Binding with AlphaLISA Technology

    Many tumor cells, which under normal, healthy conditions would be recognized by the body’s T cells and thereby targeted for destruction, have developed ways to evade the host immune system by taking advantage of immune checkpoint pathways. Among the most promising approaches to activating therapeutic antitumor immunity is through the blockade of immune checkpoints. The programmed cell death-1 (PD-1) immune checkpoint pathway is a negative regulator of T-cell immune function. When PD-1 is bound to programmed cell death-ligand 1 (PD-L1), T cell response is suppressed. Inhibitors that block PD-1/PD-L1 complex formation lead to increased activation of T-cells and immune system functions, allowing the body’s immune system to identify and attack tumor cells. So far, several anti-PD-1 or PD-L1 monoclonal antibodies have been developed to treat a variety of cancers, including non-small cell lung carcinoma (NSCLC), metastatic melanoma and renal cancer. The promise of therapeutically exploiting this pathway has created a need for more robust, straight-forward assays to identify and qualify potential inhibitors which interrupt PD-1/PD-L1 binding. Find out how AlphaLISA® technology provides a simple, homogenous, straightforward method for detecting PD-1/PD-L1 binding.

  • Application Note

    A Comparative Study of Two Immunoassay Platforms to Determine Lentivirus Titer for CAR-T Development

    The main vectors of gene therapy in research are viruses. The most popular tool for gene delivery is a genetically modified lentivirus. Modified lentivirus (HIV-1) vectors retain their ability to infect undivided cells, thereby increasing their ability to transduce a wide variety of cells, including those that are difficult to transduce. This advantage enables the stable long-term expression of a transgene.

    In immunotherapy, CAR-T cells are manufactured by transducing the CAR gene with an HIV-1 vector in T cells to express a specific chimeric p24 protein on their surface. This allows them to recognize cancer cells and destroy them. These CAR-T cells must be generated individually to treat each patient.

    This application note demonstrates a comparative quantification of the p24 titer in a lentiviral GFP control sample using Alliance HIV-1 p24 Antigen ELISA and p24 AlphaLISA immunoassay platforms.

    Check out the different sections of this application note:

    • A lentiviral vector that encodes the CAR construct
    • Determination of the efficiency of transient co-transfection by measuring viral titer
    • Detection of the presence of a p24 HIV-1 specific antibody in the sample
    • Quantification of targets present in the sample

  • Application Note

    Evaluating the Specificity of PD-1 and PD-L1 Blocking Antibodies Using AlphaLISA Human and Mouse PD-1/PD-L1 Binding Kits

    Mouse pharmacological models continue to play a large role in the study of human disease, and mouse tool reagents have shown high utility in immunology and cancer research for decades. It can often be quicker to learn about immunology and the regulation of immune responses using a syngeneic mouse model. However, working in mouse systems can often require the development of separate mouse reagents, if the therapeutic agent of interest does not cross-react with mouse. Find out how the AlphaLISA® human PD-1/PD-L1 and AlphaLISA mouse PD-1/PD-L1 binding assays provide a fast, powerful, homogeneous platform for obtain binding potencies from potential novel drug candidates.

  • Application Note

    Application Spotlight: Cell and Gene Therapy

    The collection of app notes focuses on how we can quickly analyze cellular images through machine-assisted learning and characterize profile, and test for the efficacy of the delivery vehicle and transgene using our latest technologies.

  • Article

    The 4 Pillars of Critical Quality Attributes

    As the number of cell and gene therapy applications grows, so does the requirement for the monitoring of Critical Quality Attributes (CQAs) during viral vector characterization. Analytical tools are essential for this to be done accurately, leading to the development of high-quality and safe products.

    We’ve identified four key pillars to achieve this and have solutions to support you through the whole process.

  • Application Note

    Detection and Quantification of Autophagy Using the AlphaLISA p62 Assay

    Lysosomal dysregulation is the hallmark of many diseases including neurodegenerative diseases, lysosomal storages diseases, and aging. The lysosome is responsible for eliminating cellular waste in a multistep pathway called autophagy. This process is well regulated and can be affected by a number of stimulants including nutrient starvation, physiological stress, and chemical induction. Sequestosome-1, also known as p62, is incorporated into autophagosomes then subsequently degraded in the final steps of autophagy. Assessing this protein can help decipher a block or increased flux in autophagy.

    Quantification of p62 levels within cellular lysates is often performed with labor intensive wash-based ELISA assays. In this study, chloroquine is used as an inhibitor of autophagy to show how the homogeneous no-wash AlphaLISA® p62 assay can detect changing levels of this lysosomal protein.

  • Application Note

    Simultaneous Detection of Total and Phosphorylated Protein in Cellular Kinase Assay Using Alpha SureFire Ultra Multiplex Technology

    Cellular kinase signal transduction pathways are involved in the regulation of many important cellular processes such as cell survival, differentiation, and apoptosis. Kinase signaling networks are typically characterized by multiple kinases arranged in cascades containing nodes with feedback loops and crosstalk between pathways. Numerous assay technologies have been developed for studying kinase signaling pathways, and for screening compound libraries in search of agents to modify receptors or kinase activities. The quality of these assays can be impacted by data variability due to cell seeding inhomogeneity or from compound toxicity. A common method for controlling for variability is to normalize the assay signal to a cellular protein whose level does not change as a function of the treatment.

    Alpha SureFire® Ultra Multiplex Phospho and Total assays utilize two types of Alpha Acceptor beads to simultaneously measure two signals from each assay well to easily normalize the assay. Here, we demonstrate the benefit and utility of normalizing assay signal of phosphorylated protein levels to total protein levels in two different cellular models: ERK 1/2 phosphorylation in human melanoma A375 cells and AKT 1/2/3 phosphorylation in mouse myoblast C2C12 cells.

  • Case Study

    NIH NCATS Case Study: Using Accelerated Screening Research for SARS-CoV-2 Drug Repurposing Candidates

    Drug Repurposing can be an effective way to identify treatments for diseases, especially when time is of the essence.

    Research scientists at National Center for Advancing Translational Sciences (NCATS), a part of the NIH, embarked on a drug repurposing strategy as the quickest route to generating data that would help the pharma industry drive towards an effective treatment for the COVID-19 virus.

    Find out how in a matter of months the NCATS team was able to screen 3,384 molecular entities and narrow them down to a field of 25 quality "hits" capable of disrupting SARS-Cov-2 S1 protein:ACE2 receptor binding in this case study.

  • Application Note

    Applicability of AlphaLISA Technology to a Wide Spectrum of Complex Biological Samples

    Alpha (Amplified Luminescent Proximity Homogeneous Assay) technology is a bead-based, no-wash alternative to traditional ELISAs. Instead of detection with an HRP substrate, the Alpha assay signal is generated by the excitation of an Eu+-coated bead that has been conjugated to the detection antibody.

    Alpha technology offers a simple, straight forward workflow. No wash needed!

    1. Add sample
    2. Add Acceptor bead mix; incubate 1 hr
    3. Add Donor beads; incubate 30 mins
    4. Read on Alpha-enable microplate reader

    Download this application note to see how no-wash AlphaLISA® technology provides a more-convenient alternative to ELISA for quantitation of biomarkers in complex sample types, including tissue, serum, and plasma.

  • Technical Note

    Assessing AST Released in a Cell Culture Model of Liver Toxicity Using AlphaLISA

    Aspartate transaminase (AST) or aspartate aminotransferase, also known as AspAT/ASAT/AAT or serum glutamic oxaloacetic transaminase (SGOT), is an important enzyme in amino acid metabolism. AST catalyzes the reversible transfer of an a-amino group between aspartate and glutamate. The enzyme is found in metabolically active organs and tissues, such as the liver, heart, skeletal muscle, kidneys, brain, and red blood cells. Inflamed or injured liver cells leak higher than normal amounts of AST, and other enzymes, resulting in elevated levels found in the bloodstream. A blood test for AST is commonly used as one measure in the detection of liver damage.

    In this technical note we demonstrate here the utility and benefits of using AlphaLISA assays for identifying and quantifying levels of AST protein present in cellular lysate and released into the supernatant from a human hepatoma cell line. The data illustrate the benefits of using AlphaLISA assays as a fast, powerful, homogeneous platform for screening modulators of AST expression and release in cultured human cells. The technology has a number of distinct advantages including high signal to background, wide dynamic range, and an extremely simple, straight-forward, no-wash protocol. Download our technical note to learn more.

  • Application Note

    Measuring PD-L1 Expression in Breast Cancer Cell Lines with AlphaLISA

    Too many candidates, too little time. The lack of robust, rapid, high-throughput assays to identify and qualify potential therapeutic targets in areas such as cancer research continues to cost valuable time. What if you could increase assay throughput without compromising sensitivity, obtain more data points from each sample and eliminate tedious wash steps? Find out how AlphaLISA® assay technology, combined with the EnVision® multimode plate reader, provides a fast, powerful, homogeneous platform for screening potential inhibitors of PD-L1 (a protein associated with breast cancer tumor cells) expression in human cells.

  • Application Note

    Determination of Cytokines Present in a CAR-T Co-Culture Environment by AlphaLISA and HTRF Technologies

    While fundamental knowledge about tumor immunology has exploded recently, a new therapeutic approach to cancer is taking off: immunotherapy. Instead of directly attacking tumor cells, the idea is to help the immune system recognize and destroy them.

    The use of CAR-T cells (Chimeric Antigen Receptor-T Cells), a new avenue of immunotherapy, consists in genetically modifying the patient's immune cells to arm them against a tumor. Concretely, T lymphocytes are taken from the patient's blood and modified in vitro. This leads to their expression of specific surface receptors, which recognize a tumor antigen. Once modified, these CAR-T cells are multiplied and re-injected into the patient's body in large quantities. There they go on to destroy cancer cells after binding to the tumor antigen, releasing a mixture of cytokines and pro-inflammatory chemokines.

    This application note focuses on detecting cytokine and chemokine secretion using two orthogonal no-wash immunoassays, AlphaLISA® and HTRF®, in an in vitro co-culture model with CAR-T cells and CD19 positive Raji cells targeting tumors.

  • Application Note

    Utilizing AlphaLISA Technology to Screen for Inhibitors of the CTLA-4 Immune Checkpoint

    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.

  • Application Note

    Development of Pharmacokinetic (PK) Assays for Detecting Biosimilars Targeting TNF alpha using AlphaLISA

    Anti-inflammatory monoclonal antibody drugs that specifically target TNFα, such as Humira®, have been highly successful in the market. As patents expire on these top-selling drugs, effort has been placed on developing biosimilars. Biosimilars differ from small molecule generic drugs in that their chemical structure does not have to be exactly the same as the patented drug. Therefore, the FDA has stringent requirements for proving that the biosimilars have the same efficacy and safety profile as the patented drug. Companies that develop biosimilars are tasked with proving that the biosimilar shows equivalent pharmacokinetics as the patented drug.

    Proving “biosimilarity” involves comparing parameters such as overall exposure, absorption, half-life, and clearance time using patient samples. Sensitive, robust, and fast assays are needed to measure these parameters. Traditional methods for detecting and quantifying these drugs in patient samples include time-consuming, wash-based ELISA and MSD methods. In contrast, AlphaLISA allows for fast, no-wash, high-throughput detection and quantification of the drug of interest in a variety of sample matrices. Here, we demonstrate the application of AlphaLISA for detecting biosimilars targeting TNFα.

  • Application Note

    Soluble PD-L1 Detection in Cellular Supernatants Using AlphaLISA

    When PD-1, which is expressed on the T cell, binds to PD-L1 expressed on the tumor cell, the T cell response is suppressed. Utilization of this pathway leads to tumor immune escape and promotes tumor cell growth. In fact, PD-L1 expression increases with tumor severity in many types of cancer. Release of a soluble form of PD-L1 (sPD-L1) into circulation is one mechanism that tumors may use to evade the immune response; however, it is unclear whether sPD-L1 can bind PD-1 and deliver an inhibitory signal. Previous studies have shown that soluble forms of PD-L1 have been detected in supernatants of cancer cell lines.

    Traditional methods for assessing soluble and membrane-associated PD-L1 are wash-based ELISA assays, which typically require 5-6 hours of assay time. AlphaLISA® technology provides a rapid, no-wash bead-based alternative to traditional ELISAs. In this Application Note, we demonstrate how AlphaLISA is used to detect the presence of PD-L1.

  • Application Note

    Applicability of DELFIA Fluorescence-Based Immunoassay Technology to Detect and Quantitate Biomarker

    DELFIA® (Dissociated-Enhanced Lanthanide Fluorescent Immunoassay) TRF is a proven, robust immunodetection platform with over 20 years of history. DELFIA has a similar assay principle and workflow to that of a traditional ELISA, but with the added benefits of a stable, time-resolved fluorescent signal, and improved assay dynamic range.

    Download this application note to see how DELFIA time-resolved fluorescence (TRF) technology serves as an alternative to traditional ELISA with a wider dynamic range and superior signal stability for quantitation of biomarkers in complex sample types, including serum, plasma, and blood.

  • Technical Note

    Quantifying TNFR1 in Both Soluble and Membrane Bound Form Using AlphaLISA Technology

    Tumor Necrosis Factors (TNFs) are cytokines that are the primary modifiers of inflammatory and immune response. Researchers have shown that a soluble form of TNFR1 (sTNFR1) is a truncated version of the receptor produced by the disintegration and extracellular release of membranous protein on the cell surface (ectodomain shedding). sTNFR1 is found in healthy and diseased patients alike, however increased sTNFR1 levels are an indicator for disease states such as inflammation, infection, and asthma.

    Here we present a way to measure TNFR1 using homogeneous bead-based AlphaLISA assay. The human TNFR1 AlphaLISA® detection kit was designed for the quantitative determination of soluble TNFR1 in serum and cell culture media. This technical note further demonstrates the functionality of the kit by detecting sTNFR1 in cell supernatant as well as TNFR1 on the cell membrane.