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ImmunoSpot® Kits

Standardized T Cell and B Cell ELISPOT Kits. Single- and Double-color Enzymatic ELISPOT. Up to Four-color T Cell & B Cell FluoroSpot Kits.
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ImmunoSpot® Analyzers

High-resolution Imaging. Intelligent Software.
Workflow Management. Data Audit Trails. Scientifically-validated Counting Technique.
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ePBMC® Cryopreserved Human PBMC

Extensive Library of Characterized and Uncharacterized HLA-typed Donors. Perfect biological controls for basic and clinical research.
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Serum-free Media

Optimized for Consistent, Standardized Performance in T Cell Assays.
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Contract Laboratory Services

GLP Compliant & CLIA Certified.
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Empowering the Immune Monitoring Community Worldwide

CTL introduces new HDR imaging platform for FluoroSpot analysis

Conventional FluoroSpot relies on the analysis of a single image (for each channel in multi-color assays) of a well, captured using a single exposure level. While more than adequate for accurate counts when the intensities of the spots to be analyzed are within a normal range, this approach may miss very faint spots, and overly bright spots will most likely be over-exposed, with details of the intensity profile lost from the image.

In HDR imaging, a series of incremental exposure times is used for the well images captured (shown in the video to the left).

Spots at both the low and high ends of the intensity range are captured, at exposure times optimal for accurate detection of each, and thus the full dynamic range of relative spot intensities is captured.

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The video [above] shows the ImmunoSpot software’s High Dynamic Range (HDR) FluoroSpot analysis using the CTL Analyzer’s RAW image files to capture the full range of fluorescence intensities of spots in an assay well.

Fluorescence-capable ImmunoSpot® analyzers now come with high-throughput live/dead/apoptotic PBMC counting capability

Serial Measurements of Apoptotic Cell Numbers Provide Better Acceptance Criterion for PBMC Quality than a Single Measurement Prior to the T Cell Assay

ImmunoSpot® analyzers now permit measuring
cell-mediated cytotoxicity via the
Target cell Visualization Assay

High-Throughput GLP-Capable Target Cell Visualization Assay for Measuring Cell-Mediated Cytotoxicity

Multi-color ImmunoSpot® can accomplish what flow cytometry has been struggling to do

The cytokine co-expression profile of individual T cells determined by multicolor ImmunoSpot® analysis permits to define T cell lineages and activation states. For our recent publications, see:

Natural T cell autoreactivity to melanoma antigens: clonally expanded melanoma-antigen specific CD8 + memory T cells can be detected in healthy humans.

Detection of Antigen-Specific T Cell Lineages and Effector Functions Based on Secretory Signature.

Four Color ImmunoSpot® Assays for Identification of Effector T Cell Lineages.

CTL scientists highlight ImmunoSpot®’s suitability for high-throughput immune monitoring

This recent publication details how comprehensive ImmunoSpot® testing against all potential determinants of an antigen is technically feasible, and calls into question the usage of single proteins or peptides for reliable immune monitoring.

Comprehensive Evaluation of the Expressed CD8+ T Cell Epitope Space Using High-Throughput Epitope Mapping

New ImmunoSpot® Release: SpotStat™. Software for the statistical analysis of ELISPOT data

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Recent additions to our multi-color ImmunoSpot® portfolio: Seven color Human B cell, and triple color mouse IFN-γ/TNF-α/IL-2 FluoroSpot

Detecting all Immunoglobulin Classes and Subclasses in Multiplex 7 Color ImmunoSpot® Assay.

Product specifications

Cellular Technology Limited ranked among top 10 image analysis solution providers for 2019

According to the Image Analysis Special edition of Pharma Tech Outlook Magazine, Cellular Technology Limited is the industry leader in the development, use, and manufacture of standardized tools for cellular immune assays.

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  • Detection of Antigen-Specific T Cell Lineages and Effector Functions Based on Secretory Signature
    Greg A. Kirchenbaum, Jodi Hanson, Diana R. Roen, Paul V. Lehmann*
    Journal of Immunological Sciences 2019

  • Clonally expanded, stem cell-like melanoma-antigen specific CD8 memory cells can be detected in healthy humans.
    PrzybyƂa A., Zhang T., Li R., Roen D.R., Mackiewicz A., and P. V. Lehmann (2018)
    Cancer Immunol. Immunotherapy 2019

  • Comprehensive evaluation of the expressed CD8+ T cell epitope space using high-throughput epitope mapping.
    Paul V. Lehmann, Maneewan Suwansaard, Ting Zhang, Diana R. Roen, Greg A. Kirchenbaum, Alexey Y. Karulin, Alexander Lehmann, and Pedro A. Reche
    Frontiers in Immunology 2019

  • Direct detection of T and B memory lymphocytes reveals HCMV exposure that serum antibodies fail to identify.
    Fredrik Terlutter, Tobias M. Nowacki, Richard Caspell, Ruliang Li, Krisztian Horvath, Charlotte von Gall, Stefanie Kurten, Srividya Sundararaman, and Paul V. Lehmann (2018)
    Cells 2018, 9, 75

    It is essential to identify donors who have not been infected with human cytomegalovirus (HCMV) in order to avoid transmission of HCMV to recipients of blood transfusions or organ transplants. In the present study, we tested the reliability of seronegativity as an indicator for the lack of HCMV exposure in healthy human blood donors. Eighty-two HCMV seronegative individuals were identified, and their peripheral blood mononuclear cells (PBMC) were tested in ImmunoSpot® assays for the presence of HCMV-specific T- and B-memory lymphocytes. Eighty-two percent (67 of 82) of these HCMV seronegative individuals featured at least one memory cell that was lineage specific for HCMV, with the majority of these subjects possessing CD4+ and CD8+ T cells, as well as B cells, providing three independent lines of evidence for having developed immunity to HCMV. Only 15 of these 82 donors (18%) showed neither T- nor B-cell memory to HCMV, consistent with immunological naïveté to the virus. The data suggest that measurements of serum antibodies frequently fail to reveal HCMV exposure in humans, which may be better identified by direct detection of HCMV-specific memory lymphocytes.

  • B cells and B cell blasts withstand cryopreservation while retaining their functionality for producing antibody.
    Fecher P., Caspell R., Naeem, V., Karulin A.Y., Kuerten S.and P.V. Lehmann.
    Cells 2018, 7, 50

    In individuals who have once developed humoral immunity to an infectious/foreign antigen, the antibodies present in their body can mediate instant protection when the antigen re-enters. Such antigen-specific antibodies can be readily detected in the serum. Long term humoral immunity is, however, also critically dependent on the ability of memory B cells to engage in a secondary antibody response upon re-exposure to the antigen. Antibody molecules in the body are short lived, having a half-life of weeks, while memory B cells have a life span of decades. Therefore, the presence of serum antibodies is not always a reliable indicator of B cell memory and comprehensive monitoring of humoral immunity requires that both serum antibodies and memory B cells be assessed. The prevailing view is that resting memory B cells and B cell blasts in peripheral blood mononuclear cells (PBMC) cannot be cryopreserved without losing their antibody secreting function, and regulated high throughput immune monitoring of B cell immunity is therefore confined to—and largely limited by—the need to test freshly isolated PBMC. Using optimized protocols for freezing and thawing of PBMC, and four color ImmunoSpot® analysis for the simultaneous detection of all immunoglobulin classes/subclasses we show here that both resting memory B cells and B cell blasts retain their ability to secrete antibody after thawing, and thus demonstrate the feasibility of B cell immune monitoring using cryopreserved PBMC.

  • High-throughput GLP-capable target cell visualization assay for measuring cell-mediated cytotoxicity.
    Welter A., Sundararaman S., Li R., Zhang T., Karulin A.Y., Lehmann A., Naeem V., Roen D.R., Kuerten S, and P. V. Lehmann (2018)
    Cells 2018, 7, 35

    One of the primary effector functions of immune cells is the killing of virus-infected or malignant cells in the body. Natural killer (NK) and CD8 effector T cells are specialized for this function. The gold standard for measuring such cell-mediated cytolysis has been the chromium release assay, in which the leakage of the radioactive isotope from damaged target cells is being detected. Flow cytometry-based single cell analysis of target cells has recently been established as a non-radioactive alternative. Here we introduce a target cell visualization assay (TVA) that applies similar target cell staining approaches as used in flow cytometry but based on single cell computer image analysis. Two versions of TVA are described here. In one, the decrease in numbers of calcein-stained, i.e., viable, target cells is assessed. In the other, the CFSE/PI TVA, the increase in numbers of dead target cells is established in addition. TVA assays are shown to operate with the same sensitivity as standard chromium release assays, and, leaving data audit trails in form of scanned (raw), analyzed, and quality-controlled images, thus meeting requirements for measuring cell-mediated cytolysis in a regulated environment.

  • Reagent Tracker Dyes permit quality control for verifying plating accuracy in ELISPOT tests.
    Lehmann, A., Megyesi Z., Przybyla A., and P.V. Lehmann
    Cells 2018, 7, 3

    ELISPOT assays enable the detection of the frequency of antigen-specific T cells in the blood by measuring the secretion of cytokines, or combinations of cytokines, in response to antigenic challenges of a defined population of PBMC. As such, these assays are suited to establish the magnitude and quality of T cell immunity in infectious, allergic, autoimmune and transplant settings, as well as for measurements of anti-tumor immunity. The simplicity, robustness, cost-effectiveness and scalability of ELISPOT renders it suitable for regulated immune monitoring. In response to the regulatory requirements of clinical and pre-clinical immune monitoring trials, tamper-proof audit trails have been introduced to all steps of ELISPOT analysis: from capturing the raw images of assay wells and counting of spots, to all subsequent quality control steps involved in count verification. A major shortcoming of ELISPOT and other related cellular assays is presently the lack of audit trails for the wet laboratory part of the assay, in particular, the assurance that no pipetting errors have occurred during the plating of antigens and cells. Here, we introduce a dye-based reagent tracking platform that fills this gap, thereby increasing the transparency and documentation of ELISPOT test results.

  • A positive control for detection of functional CD4 T cells in PBMC: The CPI pool.
    Annemarie Schiller, Ting Zhang, Ruliang Li, Andrea Duechting, Srividya Sundararaman, Anna Przybyla, Stefanie Kuerten and Paul V. Lehmann
    Cells 2017, 6, 47

    Testing of peripheral blood mononuclear cells (PBMC) for immune monitoring purposes requires verification of their functionality. This is of particular concern when the PBMC have been shipped or stored for prolonged periods of time. While the CEF (Cytomegalo-, Epstein-Barr and Flu-virus) peptide pool has become the gold standard for testing CD8 cell functionality, a positive control for CD4 cells is so far lacking. The latter ideally consists of proteins so as to control for the functionality of the antigen processing and presentation compartments, as well. Aiming to generate a positive control for CD4 cells, we first selected 12 protein antigens from infectious/environmental organisms that are ubiquitous: Varicella, Influenza, Parainfluenza, Mumps, Cytomegalovirus, Streptococcus, Mycoplasma, Lactobacillus, Neisseria, Candida, Rubella, and Measles. Of these antigens, three were found to elicited interferon (IFN)-γ-producing CD4 cells in the majority of human test subjects: inactivated cytomegalo-, parainfluenza-, and influenza virions (CPI). While individually none of these three antigens triggered a recall response in all donors, the pool of the three (the ‘CPI pool’), did. One hundred percent of 245 human donors tested were found to be CPI positive, including Caucasians, Asians, and African-Americans. Therefore, the CPI pool appears to be suitable to serve as universal positive control for verifying the functionality of CD4 and of antigen presenting cells.

  • Delayed activation kinetics of Th2 and Th17 vs. Th1 Cells.
    Andrea Duechting, A., Przybyla, A., Kuerten, S. and Lehmann, P.V
    Cells 6, 47

    During immune responses, different classes of T cells arise: Th1, Th2, and Th17. Mobilizing the right class plays a critical role in successful host defense and therefore defining the ratios of Th1/Th2/Th17 cells within the antigen-specific T cell repertoire is critical for immune monitoring purposes. Antigen-specific Th1, Th2, and Th17 cells can be detected by challenging peripheral blood mononuclear cells (PBMC) with antigen, and establishing the numbers of T cells producing the respective lead cytokine, IFN-γ and IL-2 for Th1 cells, IL-4 and IL-5 for Th2, and IL-17 for Th-17 cells, respectively. Traditionally, these cytokines are measured within 6 h in flow cytometry. We show here that 6 h of stimulation is sufficient to detect peptide-induced production of IFN-γ, but 24 h are required to reveal the full frequency of protein antigen-specific Th1 cells. Also the detection of IL-2 producing Th1 cells requires 24 h stimulation cultures. Measurements of IL-4 producing Th2 cells requires 48-h cultures and 96 h are required for frequency measurements of IL-5 and IL-17 secreting T cells. Therefore, accounting for the differential secretion kinetics of these cytokines is critical for the accurate determination of the frequencies and ratios of antigen-specific Th1, Th2, and Th17 cells.


Apply Adhesive Plate Sealer
To maximize the use of plates, an adhesive plate-sealing sheet is including in each kit. Using fingers and thumb, adhere firmly to plate. Use a razor to cut sheet and expose wells to be used in assay.

Balance & Spin Vials for PBMC Isolation
Carefully balance the weight of the tubes in centrifuge buckets and spin at 800g for 30 minutes.

Collect Mononuclear Cells
Collect mononuclear cells after density gradient centrifugation.

Create Humidified Chamber After coating the plate with Capture Solution, incubate plate at 4°C overnight in humidified chamber.

CTL BioSpot Yeast Viability Software Demo
Quick overview of CTL BioSpot Yeast Viability Software for counting live and dead cells.

CTL Cell Counting Software Demo
High-throughput image analysis of individual cells using fluorescence detection. Determines viability of mammalian and yeast cells by the individual analysis of live, dead, and apoptotic cells.

CTL TVA™ Software Demo
A demo of the CTL Cell Counting Software in the NK-TVA module.

Diluting Blood Before Isolating PBMC
Diluting the blood before density gradient centrifugation to isolate PBMC for use in an ELISPOT assay.


Immune Monitoring Hands-on 5-day ELISPOT Workshop
CTL Headquarters
Cleveland, OH

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