Leading ELISPOT

Reliable detection of rare antigen-specific T and/or B cells has been a major challenge for immune monitoring. For over two decades, CTL has been leading this field with its ImmunoSpot® platform. View a lecture in which Prof. Dr. Paul V. Lehmann (see CV), CTL's Founder and CEO, explains how.

Prof. Lehmann and scientists working with him were the FIRST to:

• Build an ELISPOT reader (US Patent 6,410,252 B1; filed 12/95) – marking the beginning of the CTL ImmunoSpot® Analyzer line which introduced objectivity, reproducibility, and audit trails in ELISPOT/FluoroSpot analysis. Today, thousands of our readers serve the “who is who” of the academic and regulated market world-wide.
• Introduce PVDF plates for ELISPOT assays (Science, 1996, 271:1728 and US Patent 6,140,252; filed 12/95), which transformed the ELISPOT assay that previously had the reputation of “cold fusion” into the robust platform it is today. The PVDF membrane is now universally used for ELISPOT/FluoroSpot. Read more about this discovery (pages 227-29 in Critical Reviews in Immunology, 2020, 40: 225).
• Define which cytokines and spot sizes are T cell-derived (and thus relevant for T cell diagnostics) and which are “bystander” cytokine spots (Journal of Neuroimmunology, 2005, 170:105and Journal of Immunology, 2002,168:545), introducing the basics for using ELISPOT as a scientifically-validated T cell diagnostic tool.
• Use ELISPOT to understand different qualities of immunity induced by vaccination, delineating different T cell effector classes (Science, 1996, 271:1728).
• First to establish that CpG PAMS induce Th1 immunity (Journal of Experimental Medicine, 1997, 186:1623).
• Establish the scientific principles of ELISPOT analysis, such as how to interpret different spot sizes (Journal of Immunology, 2001, 167:1353), introducing the basics of automated gating strategies that ImmunoSpot® applies.
• Use ELISPOT for measuring T cell avidity for antigen (Journal of Experimental Medicine, 1998, 187:2055), introducing an important additional parameter of T cell immunity that ELISPOT assays can provide.
• Apply ELISPOT to comprehensive determinant mapping (Journal of Immunology, 2000, 165:1641), also introducing the high-throughput capabilities of the assay.
• Show that, when following the CTL protocols, PBMC can be cryopreserved without loss of function in ELISPOT assays (Journal of Immunological Methods 2003, 278:79), enabling the testing of clinical samples in specialized central laboratories and to generate cryopreserved PBMC libraries for reference samples and streamlined human research.
• Perform assays with a minute amount of cell material, such as isolates of mouse brain or blood (Journal of Immunology, 2001,166:4757 and Journal of Immunology, 2005, 174:4598), showing ELISPOT’s unique efficacy in cell utilization.
• Introduce double-color ELISPOT analysis with the ability to detect cytokine co-expressing / “polyfunctional” T cells with the same sensitivity as ICS (Journal of Immunology, 2000, 164:1862).
• Introduce granzyme B (Journal of Immunological Methods, 2000, 240:143) and perforin ELISPOT assays (AIDS Research and Human Retroviruses 2007, 24: 62) for detecting effector CD8 cells, and distinguishing them from memory cells that produce IFN-γ only, and TRAIL assays to detect helpless CD8 cells (AIDS Research and Human Retroviruses, 2008, 24:1175).
• Use ELISPOT to study transplant immunity (Transplantation 1997, 64:292, and US Patent 5,939,281; filed 9/96).
• Pioneer autoimmunity using ELISPOT (Journal of Experimental Medicine, 1996, 183:1561).
• Study adjuvant-guided T cell responses (Journal of Experimental Medicine, 1997, 186:1623).
• Use ELISPOT to study T cell immunity to HIV (Journal of Immunology, 2000, 164:3723-32).
• Use ELISPOT to study anti-tumor immunology (Journal of Immunology, 2003, 171:3941-6).
• First to establish that ImmunoSpot® is a robust T cell monitoring platform that generates highly reproducible results in different laboratories (Journal of Immunotoxicology, 2009, 6:227).
• First to introduce reference standards for regulated ELISPOT testing
• Introduce ELISPOT to diagnosing Multiple Sclerosis (Clinical Immunology, 2014, 152:20).
• First to harmonize ELISPOT between laboratories (Journal of Immunotoxicology, 2009, 6:227, and Cells, 2015, 4:21).
• To characterize Th1, Th2, Th17 immunity and polyfunctional T cells in HCMV infection (Viruses, 2015, 7:4417).
• Introduce and validate 384 well ELISPOT assays (Cells, 2015, 4:71).
• Study the impact of apoptotic cells within PBMC on ELISPOT results (Cells, 2015, 4:40-55).
• Systematically study the (lack of) benefit of PBMC resting (Cells 2012, 1:313).
• Systematically study interference of peptides within peptide pools (Viruses 2012, 4:2636).
• First to establish a CLIA certified, GLP-compliant (registered 6/09) laboratory.
• First to introduce rules for objective ELISPOT counting based on the statistical analysis of spot size distributions (Cells, 2015, 4:56).
• First to establish the rules for identifying positive ELISPOT test results (Cells, 2015, 4:96).
• First to introduce Reagent Tracker Dyes permitting to quality control for verifying plating accuracy in ELISPOT tests. (Cells, 2018, 7:3).
• First to establish that the detection of Th2 and Th17 cells by ELISPOT needs to account for their prolonged activation kinetics (Cells, 2017, 6:47).
• First to point out that experimentally verified CD8+ T cell determinants are not predictably immune dominant (Cancer Immunology, Immunotherapy, 2016, 65:847).
• First to experimentally work out the rules for multi-color FluoroSpot analysis establishing that simple center of mass algorithms are insufficient for identifying cytokine co-expressing T cells (Methods in Molecular Biology, 2018, 1808:95).
• First to perform 4 color T cell FluoroSpot for T cell lineage definition (Methods in Molecular Biology, 2018, 1808:51).
• First to perform 7 color B cell FluoroSpot to simultaneously detect all antibody classes and subclasses (Methods in Molecular Biology, 2018, 1808:85).
• To introduce imaging-based cytotoxicity assays that can be read on ImmunoSpot® Analyzers (Cells, 2018, 7:35).
• To draw attention to the unreliability of serum antibodies to detect immune memory to HCMV (Cells, 2018, 7:75).
• To demonstrate the feasibility of brute force epitope mapping involving the testing of 552 peptides on individual human donors with only 70 ml blood (Frontiers in Immunology, 2019, 10:655).
• To introduce a universal positive control for CD8+ T cell monitoring, CERI (Cells, 2021, 10:248).
• To introduce a positive control for CD4+ T cell monitoring, CPI (Cells, 2017, 6:47).
• To successfully use mega peptide pools for cancer immune monitoring (Cancers, 2021, 13:223).
• To draw attention to the discordance between the predicted vs. the actually recognized CD8+ T cell epitopes (Frontiers in Immunology, 2021, 11: 618428).
• To draw attention to highly variable (aleatory) nature of CD8+ T cell epitope dominance even in HLA-allele matched individuals (Frontiers in Immunology, 2021, 11: 618428, Crt. Rev. Immunol, 2020, 40: 225).
• To draw attention that there is no consistently immune dominant antigen within HCMV and EBV proteins (and possibly in other complex antigen systems) (Frontiers in Immunology, 2019, 10:655).
• To introduce negative control mega peptide pools. Frontiers in Immunology. 2021, 12: 982 Frontiers in Immunology. 2021, 12: 982
• To deconvolute specificity versus chance- and cognate cross-reactivity in the T cell response to SARS-CoV-2. Frontiers in Immunology. 2021, 12: 982
• Polyclonal B cell stimulator mimicking T cell help for monitoring antigen-specific memory B cells by ELISPOT/FluoroSpot. Cells, 2020,9:433
• Affinity tag coating to enable reliable detection of antigen-specific B cells in ImmunoSpot® assays. Cells, 2021,10:1843
• Quality control for successful and even pre-coating of ImmunoSpot® plates. Learn more.
• Introducing full exome characterized ePBMC. Learn more.
• Introducing accurate frequency measurements to antigen -specific B memory cell detection by ELISPOT/FluoroSpot. Learn more.
• To show that virus-specific memory B cells by ImmunoSpot® more reliably reveal past infections than serum antibody measurements do. Learn more.
• Enable the development of B cell ImmunoSpot® assays for any antigen of interest Learn more.
• Introduce protocols that enable to measure the accurate frequency of antigen-specific B cells secreting all four immunoglobulin classes (or IgG subclasses) with only 0.4 ml of blood? Learn more.
• Introduce protocols that enable the use of B cell ImmunoSpot® assays for assessing the affinity distribution of the antigen-specific memory B cell repertoire? Learn more.
• Introduce artificial intelligence for high content ImmunoSpot® analysis Learn more.
• Draw attention why standard serum antibody measurements cannot substitute for the direct assessment of memory B cells by ImmunoSpot® Learn more.
• To introduce a theoretic framework and provide recommendations for the validation of B cell ImmunoSpot® assays Learn more.

More of CTL/Prof. Lehmann’s publications on ELISPOT are sorted here by theme, providing more information on the basics of ELISPOT and how it can be put to innovative use, possibly offering useful references for you.

If you have any questions about immune monitoring by ELISPOT/FluoroSpot, contact us, as we take pride in sharing our expertise with you.