PHA Stimulation Assay Workflow

PHA Stimulation Assay Workflow2018-05-30T01:25:50-07:00

About Phytohaemagglutinin (PHA)

Phytohaemagglutinin (PHA) is a lectin found in red kidney bean that consists of two closely related proteins called PHA-L and PHA-E.  The letters L and E indicate these proteins agglutinate leukocytes or erythrocytes.  For immunoassays, PHA-L is well suited for functional analysis of T cells because PHA-L binds and crosslinks components of the T cell receptor to induce T cell activation.  For PHA-L, approximately 1-5 µg/ml is a good working range to stimulate human peripheral blood lymphocytes.  By 96 hours post-stimulation, this condition typically results in considerable proliferation due to activation of greater than 90% of the T-cells, which can be measured by CFSE diluation.  Similarly, cytokines, such as IL-2, IL-6, TNF-α, and IFN-γ, are produced and can be measured by cytometric bead arrays using these conditions.

How will my PHA Stimulation Assay be performed?

Our immunologists at iQ Biosciences will work together with your team on the experimental design of the PHA stimulation assay and workflow.  We primarily use PBMCs from multiple healthy donors or disease samples for this assay at optimal cell concentrations.  Additionally, stimulation assays using mouse splenocytes can be used for animal bridging studies.

We will stimulate the cells with PHA in the presence or absence of your molecule at multiple concentrations.  More importantly, we will work with you and your research team to determine the best method to present (soluble or plate coated) your test molecule.  Both the sample format and presentation format will take into account the biological nature of the target and your project end goals.

Once the sample format and presentation of the test molecule is confirmed, the samples are stimulated under the agreed upon conditions.  At specified timepoints, we will harvest the supernatant or plasma to determine the levels of multiple cytokines using cytokine bead arrays or AlphaLISA.  We generally recommend measuring IL-2, IL-6, TNF-α, and IFN-γ; however, we can measure other cytokines that may be of interest as well.  If the PBMCs were pre-labeled with CFSE, we can measure proliferation of the lymphocytes in response to PHA and your test article.  We can also perform surface receptor analysis by flow cytometry with the remaining cells from the assay.  Feasibility of the antibody panel due to the aggregating nature of the PHA-L stimulated cells for these stainings will be carefully considered.

Finally, our research team will put together a report of the findings based on your needs.  Together, we will work with you at every step of the process to ensure you are comfortable and involved.  With this open communication policy, you can feel confident that your molecule is being tested properly and the results are reliable.

We are a small biotech company that critically depends on outside reagents and reliable contract research to move therapeutic candidates forward. We have turned to IQ Biosciences for access to their quality cellular reagents (including primary lymphocytes), ability to perform a wide range of in vitro assays to support our validation path, and their experience in designing and executing preclinical in vivo studies. I have been extremely pleased with their ability to execute work plans on time, communicate effectively, and apply a high level of scientific rigor that is unparalleled in my experiences with other contract organizations. Importantly, they care about the progress of our research and development.
Kyle L. Senior Scientist, AvidBiotics

Example PHA Stimulation Assay Results

Figure 1. Example: PHA induction of T cell Proliferation. PHA induces proliferation of CFSE-labeled PBMCs.

Figure 2. Example: TIGIT expression is induced by non-antigen specific PHA stimulation on T cells. PHA stimulation results in the up-regulation of TIGIT expression on T cells.