AlphaPlate-384, Light gray, untreated, Case of 50

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

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.

A variety of chemotherapeutic drugs with different modes of action have been developed and tested as potential therapies for colorectal cancer. Characterizing the effects of potential drugs with different modes of action is a key part of the process.

In this application note you will learn:

• How to rapidly measure multiple biomarkers in both cell culture supernatant and lysates from the same wells of a microplate to examine complex protein expression profiles from a colorectal cancer cellular model
• Benefits of using AlphaLISA^® technology for characterizing the effects of potential drugs with different modes of action on a cell culture model of human colorectal cancer
• How to measure the effects of drug treatments, such as 5FU and Sorafenib, on cellular proliferation by automated well-imaging and cell counting using the EnSight^® multimode plate reader
• Examples of the data and analysis you can generate for such biomarker and proliferation assays

Technical advancements in antibody engineering has brought about greater interest in more novel antibody therapeutic design and the emergence of new classes of antibody therapeutics called bispecific antibodies (bsAbs). The principle behind bispecific antibody design is to create an antibody / antibody fragment to two or more binding sites to help with the treatment of complex diseases.

As more bsAbs are produced as therapeutics, fast and accurate methods for functionally evaluating and characterizing the stability of these antibodies are necessary during both discovery and development stages, as well as during formulation and quality analysis.

In this application note, we demonstrate how AlphaLISA^® assay technology with the EnVision^® multimode plate reader can be used for bispecific antibody detection, through an example application to characterize the binding and specificity of a mouse bispecific antibody targeting mouse TIGIT and mouse PD-L1.

You will find out:

• What bispecific antibodies are and how they differ from classical antibody formats
• How to design quick and easy experiments to measure binding of the bispecific antibody to the target proteins
• How a no-wash multiplex assay can show direct binding of each site to its respective target simultaneously in one well
• How these assays could be used to evaluate the stability and functional reproducibility between batches of the bsAb preparations