Alpha 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.

G-protein coupled receptors (GPCRs) are crucial transmembrane proteins involved in cellular signal transduction. This technical note outlines a method for preparing GPCR membrane model extracts from stable cell lines, specifically for use with the HTRF GTP Gi binding assay. Get this technical note and discover: Key Highlights such as the Importance of GPCRs and the advantages of using HTRF GTP Gi Binding Assay Detailed Method with Cell Culture Preparation, Cell Lysis, Membrane Preparation and Assay Optimization For research use only. Not for use in diagnostic procedures.

Reliable, Quantitative, and No-Wash Assay Platform for AAV Capsid Detection AAVs are widely used as viral vectors in human gene therapy implementing recombinant DNA technology. Measurement of AAV concentration in complex biological matrices is essential for and effective manufacturing of AAV gene therapies. Revvity has developed and manufactured a line of immunoassays that detect and quantify AAV capsid with AlphaLISA™ technology. These assays can detect AAV1, AAV2, AAV3B, AAV5, AAV6, AAV8, and AAV9 serotypes, and measure AAV particles present in cell culture media, lysis buffer, and cell lysate. Get your application note to: Learn more about AAV capsid detection kits Access data on detection limit, assay reproducibility, variability, specificity, and recovery from multiple analyte matrices through explained diagrams See details on experimental conditions

Bispecific antibodies (bsAbs) have emerged as an attractive class of therapeutic agents with the potential to revolutionize the treatment of various disorders. While the concept of bsAbs has been around for several decades, it has taken time for the technology and understanding of antibody engineering to advance to a point where its practical application has become feasible. The development of bsAbs demands intricate screening processes to identify candidates with desired binding properties, ensuring they induce specific biological activities. Additionally, achieving optimal tissue tropism and potency requires careful consideration of factors such as antibody size, affinity, and the implementation of innovative characterization strategies. In this context, this whitepaper explores novel approaches contributing to bsAb characterization, offering real-world examples that highlight their practical applications.

What Are the Challenges and Solutions? All across the globe, AAVs are getting the attention of scientists and companies working in gene therapy, as they provide the right combination of characteristics that make them one of the most promising vector today. In 2021 only, the global gene therapy market was 4.1 billion USD, and AAV vector therapies made up more than 43% of that market value! Designing and manufacturing AAV vectors is complex, and to be successful, certain challenges must be addressed. This implies monitoring and optimtizing production for a thorough quality control process, including reliable ongoing characterization of process intermediates and the final product. Key features include: Overview and data on the gene therapy market Description of AAV vectors’ genomes and transfer Challenges associated with AAV vectors’ design and manufacturing Analytical methods for AAV vector quality control with a description of innovative no-wash AlphaLISA™ assays

In the dynamic landscape of pharmaceuticals, biotherapeutics research stands out as the fastest-growing sector. Scientists worldwide are on a quest for ever more efficient tools to develop therapeutic proteins. From lead selection to bioprocessing qualification, rigorous analytical characterization is essential. That’s where Revvity steps in. Our comprehensive line of no-wash tools revolutionizes biologics screening, mechanism-of-action studies, and biomanufacturing. With robustness and convenience at the forefront, we empower researchers to accelerate discoveries and drive innovation. Welcome to a new era of precision and efficiency in biotherapeutics research. For research use only. Not for use in diagnostic procedures.

Detecting and quantifying HCPs with automatable homogeneous immunoassays During biotherapeutic manufacturing and production, the host cells - a great majority of them being Chinese Hamster Ovary (CHO) cells – produce protein impurities that are called Host Cell Proteins (HCPs). Even if more than 99% of them are removed from the final product, the residual CHO HCPs can induce immunogenicity in individuals or reduce the potency, stability, or effectiveness of a drug. Therefore, to meet regulatory organizations’ guidelines (such as FDA or EMA) on CHO HCP levels, biopharmaceutical companies spend significant amounts of money on tools and strategies for their detection. Illustrated with robust results, this Application Note explains the many ways in which HTRF™ and AlphaLISA™ kits can improve the workflow for CHO HCP detection: Wide antibody coverage Compatibility with most commonly used buffers No cross-reactivity between CHO HCP detection and drug substance Good dilutional linearity and antigen spike recovery

Alpha technolgy enables the rapid and straightforward mesaure of virtually any target. This includes enzymes, receptor-ligand interactions, low-affinity interactions, second messenger levels, DNA, RNA, proteins, protein-protein interactions, peptides, sugars and small molecules. Explore the vast selection of homogenous Alpha assays and reagents, including AlphaLISA ™ , AlphaLISA ™ SureFire ® Ultra ™ , AlphaScreen ™ , and Alpha Toolbox. For research use only. Not for use in diagnostic procedures.

Alpha has been used to study a wide variety of interactions, including protein:protein, protein:peptide, protein:DNA, protein:RNA, protein:carbohydrate, protein:small molecule, receptor:ligand, and nuclear receptor:ligand interactions. Both cell-based and biochemical interactions have been monitored, and applications such as phage display, ELISA, and EMSA (electrophoretic mobility shift assay) have been adapted to Alpha.

Discover Alpha SureFire ®   Ultra ™ assays, the no-wash cellular kinase assays leveraging Revvity's exclusive bead-based technology and sandwich immunoassays for detecting phosphorylated proteins in cells. Offering a quantitative alternative to Western Blotting, Alpha SureFire assays are automation-friendly, easily miniaturized, and proficient in detecting both endogenous and recombinant proteins. Explore our comprehensive portfolio of SureFire Assays, designed to help you elevate and expedite your drug discovery journey.

Many laboratories developing and producing antibodies still rely on traditional enzyme-linked immunosorbent assay (ELISA) to perform clonal selection and characterization. Whilst well established, ELISAs can lack sensitivity and reproducibility, and are difficult to automate. In this white paper, you will learn how Alpha Technologies can provide a faster and simpler workflow for antibody detection and characterization. It will take you through key steps such as antibody clonal selection, measuring antibody affinity and characterizing antibodies, providing examples and demonstrating the advantages Alpha technology can provide as an alternative to ELISA. Download the white paper to discover how to advance your antibody detection and characterization.

Orthogonal systems to cell-based assays are a key requirement in EMA/FDA guidelines for potency estimations and require cross-validation with complementary approaches to prove and strengthen the reliability of results. In this application note published in collaboration with IBR Inc., you will learn: Why Alpha technology represents an ideal cell-free orthogonal system for potency assays How AlphaLISA assays can be used to determine Bevacizumab/VEGF165 potency An example of how to run an AlphaLISA potency assay and the type of data that can be generated Why it is suitable for assessing lot-to-lot consistency and equivalence of Bevacizumab and biosimilars

Quantifying H3K27ac Levels with AlphaLISA Assay In this technical note, you will discover how AlphaLISA immunodetection assay easily detects alterations in acetylated histone H3 lysine 27 (H3K27ac) levels within cellular extracts.

Quantifying H3K9ac in Cellular Extracts with AlphaLISA This technical note gives you details about how AlphaLISA immunodetection assay monitors changes in the levels of acetylated histone H3 lysine 9 (H3K9ac) in cellular extracts.

The AlphaLISA technology allows the detection of molecules of interest in serum, plasma, cell culture supernatants or cell lysates in a very sensitive, quantitative, reproducible, and user-friendly assay. Almost, any sandwich assay can be developed to detect an analyte of interest. Discover how to simply your immunoassay workflow with this guide! For research use only. Not for use in diagnostic procedures.

Quantifying Immune Checkpoints: AlphaLISA Biomarker Detection Kits This technical note focuses on the role of checkpoint molecules in cancer immunotherapies. Checkpoint molecules are molecular markers that can regulate an immune system attack on cancer cells. They have gained prominence as promising candidates for immunotherapies. AlphaLISA detection kits are designed to detect and quantify the levels of these molecules in cell culture media, serum, and cell lysates.

Decoding Histone Modifications: A Targeted Assay for H3K4me2 In this study, you will find details about an optimized assay designed to quantify changes in H3K4me2 levels following treatment with sodium butyrate and Trichostatin A (TSA), both of which are non-selective histone deacetylase (HDAC) inhibitors. Using a standardized histone extraction procedure, we detect the target mark by employing a biotinylated anti-Histone H3 (C-terminus) antibody and AlphaLISA Acceptor beads conjugated to an antibody specific to the mark.

Quantification of H3K27me3 Levels Using AlphaLISA Technology In this technical note discover how AlphaLISA ™ immunodetection assay tracks variations in cellular extract levels of di-methylated histone H3 lysine 27 (H3K27me2-1).

Unlocking the secrets of epigenetics, our technical not dives into optimizing a DOT1L enzymatic assay This technical document outlines the optimization of a DOT1L enzymatic assay using oligonucleosomes as the substrate. Discover how the assay easily detects the dimethylated product of histone H3 lysine 79 by leveraging a biotinylated anti-H3K79me2 antibody, which binds to the epigenetic mark of interest.

Lysine 27 Dimethylation Detection with AlphaLISA Technology In this technical note discover how AlphaLISA immunodetection assay detects dimethylation of a biotinylated Histone H3 (21-44) peptide at lysine 27. It uses anti-di/mono-methyl-Histone H3 Lysine 27 (H3K27me2-1) as the detection antibody.

AlphaLISA ™ Detects β2-Microglobulin in Urine with High Sensitivity When kidney tubular function is impaired, elevated levels of β2-microglobulin in urine indicate renal filtration or reabsorption disorders. Measuring urinary β2-microglobulin can help assess tubular function, but variability in urine composition may affect results. In this study, AlphaLISA ™ technology is demonstrated for the first time in the complex matrix of urine, and is shown to detect the renal tubular injury marker Beta2-microglobulin (β2-microglobulin), in urine at a sensitivity of 77 pg/mL.

Quantification of Histone H3 Lysine 9 Di-Methylation Using AlphaLISA Assay In this technical note discover how The AlphaLISA assay detects di-methylation of a biotinylated Histone H3 (1-21) peptide at lysine 9. In this assay, a biotinylated histone H3-derived peptide serves as the substrate, and the modified substrate is detected using Streptavidin (SA) Alpha Donor beads and AlphaLISA Acceptor beads conjugated to an antibody specific to the epigenetic mark of interest.

AlphaLISA Assay Insights: Tracking Deacetylation Dynamics on Histone H3 Peptides In this technical note discover how this AlphaLISA assay is designed to detect the removal of acetyl groups from a biotin-tagged peptide derived from Histone H3, specifically the segment from amino acids 21 to 44, which has been acetylated at the lysine 27 position. For research use only. Not for use in diagnostic procedures.

This quick guide presents a fast proof-of concept experiment to easily convert a immunoassay to a no-wasy AlphaLISA assay.