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Non-Radioactive Natural Killer Assay

Traditionally, Natural Killer (NK) cell-mediated lysis of K562 tumor cells has required laborious, time consuming, radioactive Chromium Release Assays (CRA). No longer! Introducing TVA™ (Target cell Visualization Assay), a fast, simple, non-radioactive, direct-imagingbased Natural Killer Assay from Cellular Technology Limited (CTL).

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  • The Correlation between the Virus- and Brain Antigen-Specific B Cell Response in the Blood of Patients with Multiple Sclerosis
    Marie Wunsch 1,*, Christopher Hohmann 2, Bianca Milles 2, Christina Rostermund 1, Paul V. Lehmann 3, Michael Schroeter 4, Antonios Bayas 5, Jochen Ulzheimer 6, Mathias Mäurer 6, Süleyman Ergün 1 and Stefanie Kuerten 1
    Viruses 2016, 8, 105; doi:10.3390/v8040105

    There is a largely divergent body of literature regarding the relationship between Epstein-Barr virus (EBV) infection and brain inflammation in multiple sclerosis (MS). Here, we tested MS patients during relapse (n = 11) and in remission (n = 19) in addition to n = 22 healthy controls to study the correlation between the EBV- and brain-specific B cell response in the blood by enzyme-linked immunospot (ELISPOT) and enzyme-linked immunosorbent assay (ELISA). Cytomegalovirus (CMV) was used as a control antigen tested in n = 16 MS patients during relapse and in n = 35 patients in remission. Over the course of the study, n = 16 patients were untreated, while n = 33 patients received immunomodulatory therapy. The data show that there was a moderate correlation between the frequencies of EBV- and brain-reactive B cells in MS patients in remission. In addition we could detect a correlation between the B cell response to EBV and disease activity. There was no evidence of an EBV reactivation. Interestingly, there was also a correlation between the frequencies of CMV- and brain-specific B cells in MS patients experiencing an acute relapse and an elevated B cell response to CMV was associated with higher disease activity. The trend remained when excluding seronegative subjects but was non-significant. These data underline that viral infections might impact the immunopathology of MS, but the exact link between the two entities remains subject of controversy.

  • How frequently are predicted peptides actually recognized by CD8 cells?
    Ioana Moldovan1 · Oleg Targoni1 · Wenji Zhang1 · Srividya Sundararaman1 · Paul V. Lehmann1 2016, 4

    Detection of antigen-specific CD8 cells frequently relies on the use of peptides that are predicted to bind to HLA Class I molecules or have been shown to induce immune responses. There is extensive knowledge on individual HLA alleles’ peptide-binding requirements, and immunogenic peptides for many antigens have been defined. The 32 individual peptides that comprise the CEF peptide pool represent such well-defined peptide determinants for Cytomegalo-, Epstein–barr-, and Influenza virus. We tested the accuracy of these peptide recognition predictions on 42 healthy human donors that have been high-resolution HLA-typed. According to the predictions, 241 recall responses should have been detected in these donors. Actual testing showed that 36 (15 %) of the predicted CD8 cell responses occurred in the high frequency range, 41 (17 %) in mid-frequencies, and 45 (19 %) were at the detection limit. In 119 instances (49 %), the predicted peptides were not targeted by CD8 cells detectably. The individual CEF peptides were recognized in an unpredicted fashion in 57 test cases. Moreover, the frequency of CD8 cells responding to a single peptide did not reflect on the number of CD8 cells targeting other determinants on the same antigen. Thus, reliance on one or a few predicted peptides provides a rather inaccurate assessment of antigenspecific CD8 cell immunity, strongly arguing for the use of peptide pools for immune monitoring.

  • Normal Distribution of CD8+ T-Cell-Derived ELISPOT Counts within Replicates Justifies the Reliance on Parametric Statistics for Identifying Positive Responses
    Alexey Y. Karulin 1, Richard Caspell 1, Marcus Dittrich 2 and Paul V. Lehmann 1
    Cells 2015, 4, 96-111; doi:10.3390/cells4010096

    Accurate assessment of positive ELISPOT responses for low frequencies of antigen-specific T-cells is controversial. In particular, it is still unknown whether ELISPOT counts within replicate wells follow a theoretical distribution function, and thus whether high power parametric statistics can be used to discriminate between positive and negative wells. We studied experimental distributions of spot counts for up to 120 replicate wells of IFN-γ production by CD8+ T-cell responding to EBV LMP2A (426 – 434) peptide in human PBMC. The cells were tested in serial dilutions covering a wide range of average spot counts per condition, from just a few to hundreds of spots per well. Statistical analysis of the data using diagnostic Q-Q plots and the Shapiro-Wilk normality test showed that in the entire dynamic range of ELISPOT spot counts within replicate wells followed a normal distribution. This result implies that the Student t-Test and ANOVA are suited to identify positive responses. We also show experimentally that borderline responses can be reliably detected by involving more replicate wells, plating higher numbers of PBMC, addition of IL-7, or a combination of these. Furthermore, we have experimentally verified that the number of replicates needed for detection of weak responses can be calculated using parametric statistics.

  • Characterization of the HCMV-Specific CD4 T Cell Responses that Are Associated with Protective Immunity
    Marie Wunsch, Wenji Zhang, Jodi Hanson, Richard Caspell, Alexey Y. Karulin, Mascha S. Recks, Stefanie Kuerten, Srividya Sundararaman and Paul V. Lehmann
    Viruses August 2015, 7, 4414-4437; dio:10.3390/v7082828

    Most humans become infected with human cytomegalovirus (HCMV). Typically, the immune system controls the infection, but the virus persists and can reactivate in states of immunodeficiency. While substantial information is available on the contribution of CD8 T cells and antibodies to anti-HCMV immunity, studies of the TH1, TH2, and TH17 subsets have been limited by the low frequency of HCMV-specific CD4 T cells in peripheral blood excels in low frequency measurements, we have established these in a sizable cohort of healthy HCMV controllers. Cytokine recall responses were seen in all seropositive donors. Specifically, interferon (IFN)- and/or interleukin (IL)-17 were seen in isolation or with IL-4 in all test subjects. IL-4 recall did not occur in isolation. While the ratios of TH1, TH2, and TH17 cells exhibited substantial variations between different individuals these ratios and the frequencies were relatively stable when tested in samples drawn up to five years apart. IFN- and IL-2 co-expressing polyfunctional cells were seen in most subjects. Around half of the HCMV-specific CD4 cells were in a reversible state of exhaustion. The data provided here established the TH1, TH2, and TH17 characteristic of the CD4 cells that convey immune protection for successful immune surveillance against which reactivity can be compared when the immune surveillance of HCMV fails.

  • ELISPOT Assays in 384-Well Format: Up to 30 Data Points with One Million Cells
    Jodi Hanson, Srividya Sundararaman, Richard Caspell, Edith Karacsony, Alexey Y. Karulin and Paul V. Lehmann
    Cells January 2015, 4, 71-83; doi:10.3390/cells4010071

    Comprehensive immune monitoring requires that frequencies of T cells, producing different cytokines, are measured to establish the magnitude of Th1, Th2, and Th17 components of cell-mediated immunity. Antigen titration provides additional information about the affinity of T cell response. In tumor immunity, it is also advisable to account for determinant spreading by testing multiple epitopes. Efforts for comprehensive immune monitoring would require substantial numbers of PBMC to run the above tests systematically, which in most test cases is limiting. Immune monitoring with ELISPOT assays have been performed, thus far, in a 96-well format. In this study we show that one can increase cell utilization by performing the assay in 384-well plates whose membrane surface area is one third that of 96-well plates. Systematic testing of PBMC for antigen-specific T cell response in the two formats demonstrated that the 384-well assay corresponds to a one-in-three miniaturization of the 96-well assay. The lowest number of cells that can be used in the 384-well format, while allowing for sufficient contact with APC, is 33,000 PBMC/well. Therefore, with one million PBMC typically obtained from 1 mL of blood, a 30 well T cell ELISPOT assay can be performed in a 384-well format.

  • ELISPOTs Produced by CD8 and CD4 Cells Follow Log Normal Size Distribution Permitting Objective Counting
    Alexey Y. Karulin, Kinga Karacsony, Wenji Zhang, Oleg S. Targoni, Ioana Moldovan, Marcus Dittrich, Srividya Sundararaman and Paul V. Lehmann
    Cells January 2015, 56-70; doi:10.3390/cells4010056

    Each positive well in ELISPOT assays contains spots of variable sizes that can range from tens of micrometers up to a millimeter in diameter. Therefore, when it comes to counting these spots the decision on setting the lower and the upper spot size thresholds to discriminate between non-specific background noise, spots produced by individual T cells, and spots formed by T cell clusters is critical. If the spot sizes follow a known statistical distribution, precise predictions on minimal and maximal spot sizes, belonging to a given T cell population, can be made. We studied the size distributional properties of IFN-γ, IL-2, IL-4, IL-5 and IL-17 spots elicited in ELISPOT assays with PBMC from 172 healthy donors, upon stimulation with 32 individual viral peptides representing defined HLA Class I-restricted epitopes for CD8 cells, and with protein antigens of CMV and EBV activating CD4 cells. A total of 334 CD8 and 80 CD4 positive T cell responses were analyzed. In 99.7% of the test cases, spot size distributions followed Log Normal function. These data formally demonstrate that it is possible to establish objective, statistically validated parameters for counting T cell ELISPOTs.

  • High Reproducibility of ELISPOT Counts from Nine Different Laboratories
    Srividya Sundararaman, Alexey Y. Karulin, Tameem Ansari, Nadine BenHamouda, Judith Gottwein, Sreenivas Laxmanan, Steven M. Levine, John T. Loffredo, Stephanie McArdle, Christine Neudoerfl, Diana Roen, Karina Silina, Mackenzie Welch, and Paul V. Lehmann
    Cells January 2015, 21-39; doi:10.3390/cells4010021

    The primary goal of immune monitoring with ELISPOT is to measure the number of T cells, specific for any antigen, accurately and reproducibly between different laboratories. In ELISPOT assays, antigen-specific T cells secrete cytokines, forming spots of different sizes on a membrane with variable background intensities. Due to the subjective nature of judging maximal and minimal spot sizes, different investigators come up with different numbers. This study aims to determine whether statistics-based, automated size-gating can harmonize the number of spot counts calculated between different laboratories. We plated PBMC at four different concentrations, 24 replicates each, in an IFN-γ ELISPOT assay with HCMV pp65 antigen. The ELISPOT plate, and an image file of the plate was counted in nine different laboratories using ImmunoSpot® Analyzers by (A) Basic Count™ relying on subjective counting parameters set by the respective investigators and (B) SmartCount™, an automated counting protocol by the ImmunoSpot® Software that uses statistics-based spot size auto-gating with spot intensity auto-thresholding. The average coefficient of variation (CV) for the mean values between independent laboratories was 26.7% when counting with Basic Count™, and 6.7% when counting with SmartCount™. Our data indicates that SmartCount™ allows harmonization of counting ELISPOT results between different laboratories and investigators.

  • Serial Measurements of Apoptotic Cell Numbers Provide Better Acceptance Criterion for PBMC Quality than a Single Measurement Prior to the T Cell Assay
    Marie Wunsch, Richard Caspell, Stefanie Kuerten, Paul V. Lehmann and Srividya Sundararaman
    Cells January 2015, 40-55; doi:10.3390/cells4010040

    As soon as Peripheral Blood Mononuclear Cells (PBMC) are isolated from whole blood, some cells begin dying. The rate of apoptotic cell death is increased when PBMC are shipped, cryopreserved, or stored under suboptimal conditions. Apoptotic cells secrete cytokines that suppress inflammation while promoting phagocytosis. Increased numbers of apoptotic cells in PBMC may modulate T cell functions in antigen-triggered T cell assays. We assessed the effect of apoptotic bystander cells on a T cell ELISPOT assay by selectively inducing B cell apoptosis using α-CD20 mAbs. The presence of large numbers of apoptotic B cells did not affect T cell functionality. In contrast, when PBMC were stored under unfavorable conditions, leading to damage and apoptosis in the T cells as well as bystander cells, T cell functionality was greatly impaired. We observed that measuring the number of apoptotic cells before plating the PBMC into an ELISPOT assay did not reflect the extent of PBMC injury, but measuring apoptotic cell frequencies at the end of the assay did. Our data suggest that measuring the numbers of apoptotic cells prior to and post T cell assays may provide more stringent PBMC quality acceptance criteria than measurements done only prior to the start of the assay.


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
July 17-21, 2017

Multiplexing ELISPOT 2-day Workshop
Venue to be announced
Dates to be announced


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