Microplates for Assays

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

Electrophoretic Mobility Shift Assay (EMSA) is a standard technique used to study protein-DNA interactions. It is a radioactive assay, suitable only for low-throughput applications due to the need for a gel-based separation step. AlphaLISA ™ is a bead-based chemiluminescent technology, as a faster, simpler, and non-radioactive alternative to EMSA. AlphaLISA ™ is highly sensitive, requiring less nuclear extract compared to EMSA, highly reproducible, and suitable for higher throughput applications. Revvity developed an AlphaLISA immunoassay to monitor the presence of specific DNA-binding proteins in nuclear extracts. In this application note, we use HepG2 nuclear extracts to demonstrate the binding of Sp1 and HNF1 transcription factors to tagged oligonucleotides containing required cognate response elements. Read this note to see the results and access detailed data compared to EMSA.

Evaluating pharmaceutical compound efficacy and safety in regulating cell behavior can involve the study of multiple signal transduction pathways and measuring more than one cellular target can provide greater confidence of positive compound hits on the cellular target of interest. Protein phosphorylation has been identified as a useful readout of cellular activation or inhibition, and these pathways are commonly targeted for therapeutic modulation of disease. Numerous assay technologies are available to look at protein phosphorylation for drug development, including the AlphaLISA ™ SureFire ® Ultra which provides a highly sensitive, fully homogenous option for the analysis of signaling pathways. This application note details the implementation of multiplexing with the AlphaLISA SureFire Ultra Multiplex assay, which offers a homogenous and high throughput approach to multiplexing.

Pyrogens are substances that cause fever in humans and animals. They can be endogenous or exogenous. One of the characteristics of endogenous pyrogens is their ability to induce cytokine production (IL-1ß, IL-6, IL-8, and TNFa) by the body's immune system. The detection of these pyrogens in pharmaceutical products is an essential regulatory requirement to ensure product quality and patient safety. In recent years, the Monocyte Activation Test (MAT) has become the most attractive and relevant method for humans. This method enables the detection and quantification of substances with a pyrogenic effect, i.e. those causing a febrile response, and does not require the use of animals. This application note demonstrates the usefulness of the AlphaLISA IL-6 kit for the quantification of pyrogens and the ease of use of a homogeneous, wash-free assay with low sample volumes.

Leptospirosis is an infectious disease caused by Leptospira bacteria, which can spread across different species of mammals. Transmission occurs through contact or consumption of food from infected domestic animals. In cattle, Leptospira infection leads to reproductive failure, affecting productivity and profitability in the dairy and beef industries. Vaccinations are widely used to prevent infection in animals and reduce transmission to humans. This application note: demonstrates the performance of AlphaLISA bovine cytokine kits by measuring the cytokine release from antigen stimulated PBMCs isolated from cow blood shows how cytokine measurements are critical for understanding cell mediated immune responses during vaccine development.

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! Add sample Add Acceptor bead mix; incubate 1 hr Add Donor beads; incubate 30 mins 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.

AlphaLISA™ technology is a highly sensitive, easy-to-use, and reproducible method for detecting and quantifying molecules in various biological matrices. It works by using streptavidin-coated Donor beads and biotinylated anti-analyte antibodies. When these come into close proximity, the excitation of the Donor beads at 680 nM triggers an energy transfer cascade in the Acceptor beads, generating a sharp emission peak at 615 nM. However, some cell culture media contain high levels of biotin, which can interfere with AlphaLISA and other assay technologies that rely on a streptavidin-biotin binding event for detection. High levels of free biotin in the sample matrix can result in a decrease in total counts, lower signal to background ratios, and reduced AlphaLISA assay detection limits. To mitigate this, AlphaLISA biotin-free kits have been developed. This application note demonstrates the value of using AlphaLISA biotin-free kits to reduce the effects of biotin interference in sample and standard preparations.

We know you are working hard to produce the next big breakthrough and the last thing you need to worry about is sub-par quality in something so commonly used, yet often overlooked, as a microplate.

Fc Gamma Receptors (FcγRs) are critical for the function of therapeutic antibodies and mediate many effector functions. Therapeutic antibody engineering programs may aim to increase the affinity for certain FcγRs while decreasing the affinity for others. Robust, transferable assays are needed to determine the binding affinity for a particular therapeutic antibody to all of the FcγRs, to generate a ‘characterization binding profile’ that can help determine a therapeutic antibody’s MOA and potential off-target effects. In this application note we demonstrate the ease with which AlphaLISA toolbox reagents can be used to develop any FcγR binding assay/ across the various stages of biologics research and development, including therapeutic screening and GMP Lot Release.

G protein-coupled receptors (GPCRs) are a large family of transmembrane receptors that are significant drug targets. They function as enzymes that activate G-proteins. GPCRs couple to three main families of Gα subunits: Gαi/o, Gαs, and Gαq. The coupling specificity depends on the GPCR and its cellular environment. Gαs and Gαi subunits operate through the cAMP pathway, either activating or inhibiting adenylate cyclase, which converts ATP to cAMP. In this application note, the AlphaScreen™-based cAMP detection technology has been used to study Gαi-coupled receptors, specifically the 5-hydroxytryptamine-1A (5-HT1A) serotonergic receptor. The note demonstrates the utility of the AlphaScreen-based cAMP Assay kit for the pharmacological characterization and potential high-throughput screening of Gαi-coupled receptors.

G protein-coupled receptors (GPCRs) play a crucial role in various cellular and physiological processes, including cell proliferation, differentiation, neurotransmission, development, and apoptosis. GPCR activity is commonly assessed by measuring levels of intracellular cAMP upon stimulation by agonists. Abnormal GPCR activity is associated with many diseases, including cancer, making GPCRs an important class of pharmaceutical drug targets. Multiple assay formats have been developed to measure the level of cAMP generated, which can determine the pharmacological potency of different agonists and antagonists. The ideal assay is homogeneous, non-radioactive, and allows for sensitive and reproducible detection. AlphaScreen ™ technology provides a quantitative detection of molecules in a homogeneous, no-wash format and can be applied to GPCR research. Download this application note to learn more on the cAMP AlphaScreen assay: Detailed protocol, Data illustrated by graph and tables, Dynamic range description and validation.

The measurement of protein phosphorylation is a useful tool for measuring the modulation of receptor activation by both antibodies and small molecules. CCR7 and CXCR2 receptors, which are expressed in immune cells and are therapeutic targets for disorders like lupus erythematosus, adult leukemia, lymphomas, chronic obstructive pulmonary disease (COPD), and sepsis. AlphaLISA ™ SureFire ® Ultra ™ and Alpha SureFire ® Ultra ™ Multiplex assays are automation-friendly, applicable to both small and large-scale screens, and can assess phosphorylation status in complex matrices. The LANCE Ultra cAMP assay is measures cyclic AMP (cAMP) produced upon modulation of adenylyl cyclase activity by G-protein coupled receptors (GPCRs). This application note demonstrates how the SureFire Ultra and LANCE Ultra cAMP assays can be used for measuring inhibitors to CCR7 and CXCR2 cell surface receptors using a cellular model system where these receptors are overexpressed in CHO cells. The assays were optimized to measure receptor blockage and assayed receptor activity modulation by detecting ERK and AKT phosphorylation status and cAMP modulation. For more details, download the application note!

AlphaLISA and LANCE (TR-FRET) biomarker assays can be used to measure ECM-associated protein modulation caused by human transforming growth factor-beta (TGF-β) induction of EMT in a 3D Spheroid model of human prostate carcinoma.

Over time, Adeno-associated virus (AAV) vectors have become the leading platform for gene delivery in the potential treatment of a variety of human diseases. Researchers and bioanalytical scientists within the gene therapy community have focused on characterizing various AAV serotypes using conventional and next-generation technologies to deliver critical quality attributes along their workflow. In this application note, discover how AlphaLISA ™ and LabChip ® GXII Touch ™ microfluidics-based CE technology can be used as orthogonal methodologies to detect and characterize AAV8 serotype capsid proteins.

DELFIA® immunoassays are particularly well suited for discovery of specific, high-affinity monoclonal antibodies (mAbs), especially at the low concentrations that may be present in hybridoma supernatants. An assay used in these conditions must be sensitive, specific, reliable, reproducible and easy enough to handle 1,000 to 1,500 samples in one batch.

Evaluation of the therapeutic profile of anti-oncogene compounds in various cell lines with AlphaLISA™ and HTRF™ KRAS is a proto-oncogene known to be mutated in many cancer subtypes, inducing uncontrolled proliferation and cell metabolism changes. Like most small GTPases, KRAS will bind to GDP in its inactive form or to GTP in its active form. KRAS G12C is one of the most commonly found mutant forms in cancers, and leads to a permanently active state of KRAS. The upregulation of KRAS interaction with the exchange factor SOS1 leads to cancer phenotypes. Reducing KRAS activity and associated pathways could control the biological processes involved in cancer growth. Furthermore, it is well known that KRAS induces activation of mitogen-activated protein kinase (MAPK), thus playing a central role in human cancers. This application note provides a convincing demonstration of the reliability of the AlphaLISA and HTRF KRAS portfolios to evaluate compound in vitro therapeutic profiles in a cellular context: Determine the effects of KRAS and SOS1 inhibitors in different human cancer cell lines Discriminate the cellular action of KRAS-targeting compounds and evaluate their effectiveness in modulating KRAS downstream pathways.

Due to limitations driven by circulatory half-life and drug target bioavailability, injected biologics often require the injection of high doses, which can result in patient discomfort, unwanted side effects, a limited therapeutic window, and higher costs. To sidestep these pain points, Tenza has engineered probiotic microbes to synthesize and deliver protein therapeutics directly to the target tissue. The functional activity of the secreted protein biologic is assessed by its binding to a target protein relevant to its therapeutic indication. The pre-existing assay format for testing functional activity was a standard ELISA, which had limited dynamic range and throughput, required large sample volumes, and involved multiple tedious wash steps. Download this application note and discover how the authors switched to a custom developed AlphaLISA ® assay to overcome the limitations they had observed in the use of their ELISA assays.

Anti-inflammatory monoclonal antibody drugs that specifically target TNF alpha 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 TNF alpha.

A Bruton Tyrosine Kinase (BTK) case study PROteolysis Targeting Chimeras (PROTACs) are one of the latest trending tools used in drug discovery. This note compiles experiments carried out to characterize PROTAC compounds using Bruton Tyrosine Kinase (BTK) as a model on Ramos cells. Both AlphaLISA and HTRF technologies were used in biochemical and cell-based contexts. Featured in this note: Assay principle and workflow Consistent results (data and graphs) Informative materials and methods, including references

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.

Read this brochure to get more information about the chemagic Prime Jr intrument for automated nucleic acid purification and assay setup and its application fields.

Fc Receptors (FcRs) are cell-surface proteins found on various cell types and play a role in the adaptive immune system. The Fc-Gamma Receptors (FcγRIIIa) are part of the immunoglobulin superfamily and are crucial for the function of therapeutic antibodies. This application note demonstrates how AlphaLISA assay can be used to determine the binding activity of human IgG Fc fragment to human FcγRIIIa and to study how other antibodies bind to FcγRIIIa.

This application note shows you how to determine the relative affinity of different therapeutic antibodies to FcRn, and how it can be used to compare biosimilars. It contains experiments and data that investigate the specificity of the assays for binding the Fc portion of an antibody, and the binding performance of different subtypes. It features: Assay principle and workflow Consistent results (data and graphs) Informative materials and methods, including references

G protein-coupled receptors (GPCRs) are cell surface transmembrane receptors that activate G-proteins. GPCR activity is often assessed by measuring intracellular cAMP levels upon stimulation by agonists. Since GPCRs are important pharmaceutical drug targets, there’s a high demand for methods to detect and quantify cAMP. The ideal assay is homogenous, non-radioactive, and allows for sensitive and reproducible detection of cAMP. The AlphaScreen™ cAMP assay is one such method. It was evaluated for its ability to detect agonist or antagonist-induced cAMP responses in cells expressing either endogenous or recombinant receptors. Read this application note to access detailed data and results on GPCR studies using AlphaScreen cAMP detection kit.