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ProMap® naïve CFSE T Cell Proliferation Assays
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ProSpot™ T Cell ELISpot Assays
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ProScern® CFSE DC-T Cell Assays
COVID-19 related Assays & Services
ProImmune’s ProMap® T cell proliferation assay can be used to identify epitope sequences that elicit helper T cell proliferation and therefore potentially cause a helper T cell immune response.
Unlike traditional assays which are based on radioactive thymidine incorporation, this assay utilizes powerful flow cytometry methods, enabling accurate determination of the percentage of proliferating cells and detailed phenotyping of T cell responses, all with significantly improved overall sensitivity.
Why choose ProMap® CFSE proliferation assays?
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CFSE dye dilution is a tried and tested method referenced in thousands of publications
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Much more sensitive than radioactive assays
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Flow cytometric readout resolves the correct cell populations to be analysed, such as live CD4+ T cells
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Further sub-set phenotyping is easy to add and proliferation of sub-sets can be read out simultaneously
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Integral assay measures all proliferation over proliferation timeframe and not over a time window at the end of the incubation time
ProMap® T cell Proliferation Assay Principle.
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PBMC for 20-50 HLA typed donors are selected from the ProImmune Biobank
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A peptide library is synthesized according to project requirements
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Donor PBMC are labelled with CFSE dye.
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Each peptide is co-cultured with the PBMC from each donor.
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Proliferation of CD4+ T cells in response to peptide causes dilution of the CFSE label.
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Flow cytometry analysis of CFSE in conjunction with staining for CD4, is used to quantitate cell division, and provide a measure of antigenicity.
Key publication:
Validating antibody humanization with ProMap® T cell assays published by [dstl], part of the UK Ministry of Defence (MoD)
Figure 1 comparing overlapping peptide T cell responses in murine and humanized V-regions of an antibody.
Figure 2 Example staining data from naïve T cell assay,
(1) CFSE-labeled T cells cultured in media alone (unstimulated), (2) CFSE-labeled T cells cultured with peptide derived from antigen of interest, (3) CFSE-labeled T cells cultured with control protein (Tuberculin PPD).
ProImmune provides a seamless, high-throughput, core facility service for measuring cell mediated immunity in qualified T cell ELISpot assays for cyokines, such as IFN-gamma, IL-2, IL-4, IL-10, IL-13, IL-17, Granzyme B, and many others.
Save yourself the cost and effort of setting up these time-consuming ELISpot assays in your lab. Let our experienced ELISpot CRO team do the work for you, with high reproducibility and rapid turnaround. We have the capability to process tens to hundreds of samples in a project in a high throughput format, freeing up your time for core work and project planning.
By relying on our team to carry out your ELISpot assays using optimized and validated protocols, you can avoid the pitfalls of variability in cellular assays and benefit from the efficiency and accuracy of a dedicated facility.
Step-by-step Process
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Customer cryopreserves cells in accordance with optimized protocol recommended by ProImmune or optimized customer protocol. Alternatively ProImmune receives fresh blood in blood collection tubes and carries out cryopreservation.
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ProImmune collects cells from customer site using our trusted shipper (customer may also use their own shipper), or, where ProImmune has processed cells, retrieves cells from ProImmune cryostores.
- ProImmune carries out HLA tissye typing as an option if required.
- ProImmune carries out custom peptide synthesis as an option if required.
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ProImmune thaws cells with optimized protocol and carries out ELISpot assays in accordance with one or more of the following assay formats:
a. ELISpot assay for detecting CD8+ and CD4+ T cell responses on frozen unmodified PBMC.
b. ELISpot assay for detecting only CD4+ T cell responses on frozen PBMC that are depleted of CD8+ T cells after thawing.
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Customer receives assay report and data; in addition to full analysis, the assay report includes raw data and images from the automated ELISpot reader.
On request these processes can be adapted to any additional or alternative requirements.
Validation Data for IFN-g T cell ELISpot Assay
Figure 1 Low inter-assay variability in ProImmune ELISpot Assays. Normalized data from two users across six IFN gamma ELISpot assays performed over three days. The data show the consistency of the response for positive control and two test peptides.
The Statistics of ELISpot
You may think of Shot Noise as a concept from quantum physics. In fact it is a limiting factor in cellular assays. Where such assays measure responding cells they can only do so in integer increments. For example if you are looking for a one in a million cells responding and an assay that measures e.g. 250,000 cells per condition will only have a one in four occurrence of a single response. These limitations dictate the limit of assay variance that can actually be achieved, based on the assay setup. So the question is: How close can we get to this limit?
The CV achieved by ProImmune is close to the statistical CV driven by shop noise alone. Graph showing theoretical 'shop noise' based CV compared to the CV values achieved in ProImmune's IFN gamma ELISpot inter assay comparison.
SFC = Spot Forming Cells.
The highly sensitive and versatile ProsScern® DC-T cell assay measures if candidate proteins induce helper CD4+ T cell proliferation that may lead to anti-drug antibody responses or other unwanted immunogenicity.
CD4+ T cell proliferation is measured by flow cytometry using CFSE dye dilution, a sensitive measure for cell proliferation.
Fully-formulated proteins are used in the assay, which gives an excellent in vitrocomparison of the relative potential antigenicities of proteins.
ProScern® DC-T Cell Assays for Whole Protein Immunigenicity Screening
The ProScern® Dendritic Cell (DC)-T cell assay for screening whole proteins has wide-ranging applications. It allows for an overall comparison of the T cell driven antigenicity of any number of drug candidates at a pre-clinical stage. Crucially it can also be used for assessing the impact on antigenicity of factors other than protein sequence. Such differences may include a comparison of biosimilars, protein modifications, degradation products, chemical entities given in combination therapies, and other parameters related to manufacturing processes, excipients, drug formulation and stability.
Additionally, in some cases it may not be possible to use the antigen to stimulate PBMC directly, particularly if the antigen involved modifies the function of responding T cells. To avoid such assay interference, antigens can be presented using dendritic cells, allowing the relative antigenicity of different leads to be compared directly.
ProScern® DC-T Cell AssayMethod Overview
The assay uses samples from ProImmune’s bank of healthy high-resolution MHC Class II tissue-typed donor samples. A donor cohort can be selected to provide representative coverage of a particular population of interest, or to mimic the allele frequencies found in the global population. Typically a cohort of 40 to 50 donors is used, depending on the desired scale of the study.
Donor PBMC are used as a source of monocytes that are cultured in defined media to generate immature dendritic cells. Dendritic cells are loaded with test antigen (whole protein), and are then induced into a more mature phenotype by further culture in defined media. PBMC from the same donor sample are labeled with CFSE then cultured with the antigen-primed DCs for 7 days. Each DC-T cell culture includes a set of untreated control wells. The assay also incorporates reference antigen controls, comprising two potent whole protein antigens.
Figure 1 Example staining data from DC-T cell assay, (1) CFSE-labeled T cells incubated with DCs that were not co-cultured with antigen, (2) CFSE-labeled T cells incubated with DCs that had been previously co-cultured with whole protein antigen (Drug 1), (3) CFSE-labeled T cells incubated with DCs that had been previously co-cultured with control antigen (Tuberculin PPD).
Response Index for Evaluating Immunogenicity
When evaluating immunogenicity, it is appropriate to take account of the frequency of donor cell responses across the study cohort. A positive response (percentage stimulation above background > 0.5%) in 2 or more independent donor samples is considered indicative of a potential in vivo T cell response. It is also important to consider the strength of positive donor cell responses. This is determined by taking an average of the percentage stimulation above background obtained across accepted donors for each drug. A Response Index is calculated by multiplying the value of the strength of response by the frequency of the donors responding. This index is more representative of the level of immunogenicity than methods of analysis that rely on the frequency of response alone.
If a positive response is observed in a donor sample in either of the two T cell assays, then a proliferative immune response has been mounted through at least one of the six HLA class II alleles presented by that donor.
Figure 2 Results of a DC-T cell assay performed using 40 donors and 4 test proteins (in this case, therapeutic antibodies) and 2 controls.
As can be clearly demonstrated in figure 1, the fully human antibody drug 4 had the lowest response index, indicating the lowest in vitro immunogenicity in the ProScern® DC-T Cell Assay. As expected, the humanized antibodies (drug 2 and drug 3) showed the next lowest immunogenicity and the chimeric antibody (drug 1) showed the highest immunogenicity. Different therapeutic proteins will yield specific levels of immunogenicity and this can be directly quantified in the ProImmune DC-T cell assay.