Research

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Open AccessArticle

Normal Distribution of CD8+ T-Cell-Derived ELISPOT Counts within Replicates Justifies the Reliance on Parametric Statistics for Identifying Positive Responses

by Alexey Y. Karulin, Richard Caspell, Marcus Dittrich and Paul V. Lehmann

Cells 2015, 4(1), 96-111; https://doi.org/10.3390/cells4010096 - 02 Mar 2015

Cited by 22 | Viewed by 7345

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Special Issue ELISPOT Research)

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Open AccessArticle

Comparative Multi-Donor Study of IFNγ Secretion and Expression by Human PBMCs Using ELISPOT Side-by-Side with ELISA and Flow Cytometry Assays

by Jodi Hagen, Ryan Zimmerman, Christine Goetz, Jody Bonnevier, Jeffrey P. Houchins, Kevin Reagan and Alexander E. Kalyuzhny

Cells 2015, 4(1), 84-95; https://doi.org/10.3390/cells4010084 - 11 Feb 2015

Cited by 16 | Viewed by 7393

Abstract

ELISPOT, ELISA and flow cytometry techniques are often used to study the function of immune system cells. It is tempting to speculate that these assays can be used interchangeably, providing similar information about the cytokine secreting activity of cells: the higher the number [...] Read more.

ELISPOT, ELISA and flow cytometry techniques are often used to study the function of immune system cells. It is tempting to speculate that these assays can be used interchangeably, providing similar information about the cytokine secreting activity of cells: the higher the number of cytokine-positive cells measured by flow cytometry, the higher the number of cytokine-secreting cells expected to be detected by ELISPOT and the larger the amount of secreted cytokine expected to be measured by ELISA. We have analyzed the expression level and secretion capacity of IFNγ from peripheral blood mononuclear cells isolated from five healthy donors and stimulated by calcium ionomycin mixed with phorbol 12-myristate 13-acetate in a non-specific manner in side-by-side testing using ELISPOT, ELISA and flow cytometry assays. In our study, we observed a general correlation in donors’ ranking between ELISPOT and flow cytometry; ELISA values did not correlate with either ELISPOT or flow cytometry. However, a detailed donor-to-donor comparison between ELISPOT and flow cytometry revealed significant discrepancies: donors who have similar numbers of IFNγ-positive cells measured by flow cytometry show 2–3-fold differences in the number of spot-forming cells (SFCs) measured by ELISPOT; and donors who have the same number of SFCs measured by ELISPOT show 30% differences in the number of IFNγ-positive cells measured by flow cytometry. Significant discrepancies between donors were also found when comparing ELISA and ELISPOT techniques: donors who secreted the same amount of IFNγ measured by ELISA show six-fold differences in the number of SFCs measured by ELISPOT; and donors who have 5–7-times less secreted IFNγ measured by ELISA show a two-fold increase in the number of SFCs measured by ELISPOT compared to donors who show a more profound secretion of IFNγ measured by ELISA. The results of our study suggest that there can be a lack of correlation between IFNγ values measured by ELISPOT, ELISA and flow cytometry. The higher number of cytokine-positive cells determined by flow cytometry is not necessarily indicative of a higher number of cytokine-secreting cells when they are analyzed by either ELISPOT or ELISA. Our ELISPOT vs. ELISA comparison demonstrates that the higher number of SFCs observed in ELISPOT does not guarantee that these cells secrete larger amounts of cytokines compared to donors with lower SFC numbers. In addition, our data indicate that ELISPOT, ELISA and flow cytometry should be performed as complementary, rather than stand-alone assays: running these assays in parallel on samples from the same donors may help to better understand the mechanisms underlying the physiology of cytokine-secreting cells. Full article

(This article belongs to the Special Issue ELISPOT Research)

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Open AccessArticle

ELISPOT Assays in 384-Well Format: Up to 30 Data Points with One Million Cells

by Jodi Hanson, Srividya Sundararaman, Richard Caspell, Edith Karacsony, Alexey Y. Karulin and Paul V. Lehmann

Cells 2015, 4(1), 71-83; https://doi.org/10.3390/cells4010071 - 29 Jan 2015

Cited by 14 | Viewed by 8324

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Special Issue ELISPOT Research)

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Open AccessArticle

ELISPOTs Produced by CD8 and CD4 Cells Follow Log Normal Size Distribution Permitting Objective Counting

by Alexey Y. Karulin, Kinga Karacsony, Wenji Zhang, Oleg S. Targoni, Ioana Moldovan, Marcus Dittrich, Srividya Sundararaman and Paul V. Lehmann

Cells 2015, 4(1), 56-70; https://doi.org/10.3390/cells4010056 - 20 Jan 2015

Cited by 12 | Viewed by 8470

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Special Issue ELISPOT Research)

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Open AccessArticle

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

by Marie Wunsch, Richard Caspell, Stefanie Kuerten, Paul V. Lehmann and Srividya Sundararaman

Cells 2015, 4(1), 40-55; https://doi.org/10.3390/cells4010040 - 09 Jan 2015

Cited by 7 | Viewed by 7764

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Special Issue ELISPOT Research)

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Open AccessArticle

High Reproducibility of ELISPOT Counts from Nine Different Laboratories

by 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 2015, 4(1), 21-39; https://doi.org/10.3390/cells4010021 - 09 Jan 2015

Cited by 15 | Viewed by 12382

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Special Issue ELISPOT Research)

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Open AccessArticle

Improvement of IFNg ELISPOT Performance Following Overnight Resting of Frozen PBMC Samples Confirmed Through Rigorous Statistical Analysis

by Radleigh Santos, Alcinette Buying, Nazila Sabri, John Yu, Anthony Gringeri, James Bender, Sylvia Janetzki, Clemencia Pinilla and Valeria A. Judkowski

Cells 2015, 4(1), 1-18; https://doi.org/10.3390/cells4010001 - 24 Dec 2014

Cited by 22 | Viewed by 10657 | Correction

Abstract

Immune monitoring of functional responses is a fundamental parameter to establish correlates of protection in clinical trials evaluating vaccines and therapies to boost antigen-specific responses. The IFNg ELISPOT assay is a well-standardized and validated method for the determination of functional IFNg-producing T-cells in [...] Read more.

Immune monitoring of functional responses is a fundamental parameter to establish correlates of protection in clinical trials evaluating vaccines and therapies to boost antigen-specific responses. The IFNg ELISPOT assay is a well-standardized and validated method for the determination of functional IFNg-producing T-cells in peripheral blood mononuclear cells (PBMC); however, its performance greatly depends on the quality and integrity of the cryopreserved PBMC. Here, we investigate the effect of overnight (ON) resting of the PBMC on the detection of CD8-restricted peptide-specific responses by IFNg ELISPOT. The study used PBMC from healthy donors to evaluate the CD8 T-cell response to five pooled or individual HLA-A2 viral peptides. The results were analyzed using a modification of the existing distribution free resampling (DFR) recommended for the analysis of ELISPOT data to ensure the most rigorous possible standard of significance. The results of the study demonstrate that ON resting of PBMC samples prior to IFNg ELISPOT increases both the magnitude and the statistical significance of the responses. In addition, a comparison of the results with a 13-day preculture of PBMC with the peptides before testing demonstrates that ON resting is sufficient for the efficient evaluation of immune functioning. Full article

(This article belongs to the Special Issue ELISPOT Research)

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Open AccessArticle

Triple Cytokine FluoroSpot Analysis of Human Antigen-Specific IFN-γ, IL-17A and IL-22 Responses

by Tomas Dillenbeck, Eva Gelius, Jenny Fohlstedt and Niklas Ahlborg

Cells 2014, 3(4), 1116-1130; https://doi.org/10.3390/cells3041116 - 27 Nov 2014

Cited by 13 | Viewed by 7429

Abstract

The involvement of T-helper (Th)1, Th17 and Th22 cell subsets, in immunity, as well as in pathological inflammatory reactions, makes it important to determine their relative proportion. A triple FluoroSpot detecting the hallmark cytokines of Th1 (IFN-γ), Th17 (IL-17A) and Th22 (IL-22) was [...] Read more.

The involvement of T-helper (Th)1, Th17 and Th22 cell subsets, in immunity, as well as in pathological inflammatory reactions, makes it important to determine their relative proportion. A triple FluoroSpot detecting the hallmark cytokines of Th1 (IFN-γ), Th17 (IL-17A) and Th22 (IL-22) was developed and evaluated using human peripheral blood mononuclear cells from healthy donors incubated with tetanus toxoid, Candida albicans extract, mycobacterial purified protein derivative or medium only. Antigen stimulation yielded mainly cells secreting IFN-γ, IL-17A or IL-22 alone but lower proportions of double-secreting cells were also found; triple-secreting cells were rare. The response to C. albicans contrasted in that higher proportions of IL-17A single secreting as well as co-secreting cells, in particular IL-17A/IL-22, were found. The FluoroSpot analysis correlated well with single cytokine ELISpot assays ran in parallel and the methods displayed a comparable sensitivity. The results demonstrate the functionality of the FluoroSpot assay for simultaneous analysis of distinct Th1, Th17, Th22 as well as intermediate cell populations. The method provides a mean for a simple and rapid analysis of the involvement of these cells in immunity and disease. Full article

(This article belongs to the Special Issue ELISPOT Research)

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Open AccessArticle

Stepping up ELISpot: Multi-Level Analysis in FluoroSpot Assays

by Sylvia Janetzki, Markus Rueger and Tomas Dillenbeck

Cells 2014, 3(4), 1102-1115; https://doi.org/10.3390/cells3041102 - 27 Nov 2014

Cited by 32 | Viewed by 11385

Abstract

ELISpot is one of the most commonly used immune monitoring assays, which allows the functional assessment of the immune system at the single cell level. With its outstanding sensitivity and ease of performance, the assay has recently advanced from the mere single function [...] Read more.

ELISpot is one of the most commonly used immune monitoring assays, which allows the functional assessment of the immune system at the single cell level. With its outstanding sensitivity and ease of performance, the assay has recently advanced from the mere single function cell analysis to multifunctional analysis by implementing detection reagents that are labeled with fluorophores (FluoroSpot), allowing the detection of secretion patterns of two or more analytes in a single well. However, the automated evaluation of such assays presents various challenges for image analysis. Here we dissect the technical and methodological requirements for a reliable analysis of FluoroSpot assays, introduce important quality control measures and provide advice for proper interpretation of results obtained by automated imaging systems. Full article

(This article belongs to the Special Issue ELISPOT Research)

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Open AccessArticle

CD8+ T Lymphocyte Epitopes From The Herpes Simplex Virus Type 2 ICP27, VP22 and VP13/14 Proteins To Facilitate Vaccine Design And Characterization

by Rebecca J. Platt, Tansi Khodai, Tim J. Townend, Helen H. Bright, Paul Cockle, Luis Perez-Tosar, Rob Webster, Brian Champion, Timothy P. Hickling and Fareed Mirza

Cells 2013, 2(1), 19-42; https://doi.org/10.3390/cells2010019 - 04 Jan 2013

Cited by 5 | Viewed by 7267

Abstract

CD8+ T cells have the potential to control HSV-2 infection. However, limited information has been available on CD8+ T cell epitopes or the functionality of antigen specific T cells during infection or following immunization with experimental vaccines. Peptide panels from HSV-2 proteins ICP27, [...] Read more.

CD8+ T cells have the potential to control HSV-2 infection. However, limited information has been available on CD8+ T cell epitopes or the functionality of antigen specific T cells during infection or following immunization with experimental vaccines. Peptide panels from HSV-2 proteins ICP27, VP22 and VP13/14 were selected from in silico predictions of binding to human HLA-A*0201 and mouse H-2Kd, Ld and Dd molecules. Nine previously uncharacterized CD8+ T cell epitopes were identified from HSV-2 infected BALB/c mice. HSV-2 specific peptide sequences stabilized HLA-A*02 surface expression with intermediate or high affinity binding. Peptide specific CD8+ human T cell lines from peripheral blood lymphocytes were generated from a HLA-A*02+ donor. High frequencies of peptide specific CD8+ T cell responses were elicited in mice by DNA vaccination with ICP27, VP22 and VP13/14, as demonstrated by CD107a mobilization. Vaccine driven T cell responses displayed a more focused immune response than those induced by viral infection. Furthermore, vaccination with ICP27 reduced viral shedding and reduced the clinical impact of disease. In conclusion, this study describes novel HSV-2 epitopes eliciting strong CD8+ T cell responses that may facilitate epitope based vaccine design and aid immunomonitoring of antigen specific T cell frequencies in preclinical and clinical settings. Full article

(This article belongs to the Special Issue ELISPOT Research)

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Open AccessArticle

Neuroantigen-Specific CD4 Cells Expressing Interferon-γ (IFN-γ), Interleukin (IL)-2 and IL-3 in a Mutually Exclusive Manner Prevail in Experimental Allergic Encephalomyelitis (EAE)

by Alexey Y. Karulin, Stefan Quast, Maike D. Hesse and Paul V. Lehmann

Cells 2012, 1(3), 576-596; https://doi.org/10.3390/cells1030576 - 24 Aug 2012

Cited by 5 | Viewed by 8733

Abstract

Experimental allergic encephalomyelitis (EAE) is mediated by neuroantigen-specific pro-inflammatory T cells of the Th1 and Th17 effector class. Th-17 cells can be clearly defined by expression of IL-17, but not IFN-γ, IL-2 or IL-3. Th1 cells do not express IL-17, but it is [...] Read more.

Experimental allergic encephalomyelitis (EAE) is mediated by neuroantigen-specific pro-inflammatory T cells of the Th1 and Th17 effector class. Th-17 cells can be clearly defined by expression of IL-17, but not IFN-γ, IL-2 or IL-3. Th1 cells do not express IL-17, but it is unclear presently to what extent they co-express the cytokines canonically assigned to Th1 immunity (i.e., IFN-γ, IL-2 and IL-3) and whether CD4 cells producing these cytokines indeed belong to a single Th1 lineage. It is also unclear to what extent the Th1 response in EAE entails polyfunctional T cells that co-express IFN-γ and IL-2. Therefore, we dissected the Th1 cytokine signature of neuroantigen-specific CD4 cells studying at single cell resolution co-expression of IFN-γ, IL-2 and IL-3 using dual color cytokine ELISPOT analysis. Shortly after immunization, in the draining lymph nodes (dLN), the overall cytokine signature of the neuroantigen-specific CD4 cells was highly type 1-polarized, but IFN-γ, IL-2, and IL-3 were each secreted by different CD4 cells in a mutually exclusive manner. This single cell – single cytokine profile was stable through the course of chronic EAE–polyfunctional CD4 cells co-expressing IL-2 and IFN-γ presented less than 5% of the neuroantigen-specific T cells, even in the inflamed CNS itself. The neuroantigen-specific CD4 cells that expressed IFN-γ, IL-2 and IL-3 in a mutually exclusive manner exhibited similar functional avidities and kinetics of cytokine production, but showed different tissue distributions. These data suggest that Th1 cells do not belong to a single lineage, but different Th1 subpopulations jointly mediate Th1 immunity. Full article

(This article belongs to the Special Issue ELISPOT Research)

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Open AccessArticle

Resting of Cryopreserved PBMC Does Not Generally Benefit the Performance of Antigen-Specific T Cell ELISPOT Assays

by Stefanie Kuerten, Helena Batoulis, Mascha S. Recks, Edith Karacsony, Wenji Zhang, Ramu A. Subbramanian and Paul V. Lehmann

Cells 2012, 1(3), 409-427; https://doi.org/10.3390/cells1030409 - 30 Jul 2012

Cited by 19 | Viewed by 11629

Abstract

T cell monitoring is increasingly performed using cryopreserved PBMC. It has been suggested that resting of PBMC after thawing, that is, culturing them overnight in test medium, produces higher antigen-induced spot counts in ELISPOT assays. To evaluate the importance of overnight resting, we [...] Read more.

T cell monitoring is increasingly performed using cryopreserved PBMC. It has been suggested that resting of PBMC after thawing, that is, culturing them overnight in test medium, produces higher antigen-induced spot counts in ELISPOT assays. To evaluate the importance of overnight resting, we systematically tested cryopreserved PBMC from 25 healthy donors. CEF peptides (comprising CMV, EBV and flu antigens) were used to stimulate CD8 cells and mumps antigen to stimulate CD4 cells. The data show that resting significantly increased antigen-elicited T cell responses only for CEF high responder PBMC. The maximal gain observed was doubling of spot counts. For CEF low responders, and for mumps responders of either low- or high reactivity levels, resting had no statistically significant effect on the observed spot counts. Therefore, resting is not a generally applicable approach to improve ELISPOT assay performance, but can be recommended only for clinical subject cohorts and antigens for which it has a proven benefit. Because resting invariably leads to losing about half of the PBMC available for testing, and because doubling the PBMC numbers plated into the assay reliably doubles the antigen-induced spot counts, we suggest the latter approach as a simple and reliable alternative to resting for enhancing the performance of ELISPOT assays. Our data imply that resting is not required if PBMC were cryopreserved and thawed under conditions that minimize apoptosis of the cells. Therefore, this study should draw attention to the need to optimize freezing and thawing conditions for successful T cell work. Full article

(This article belongs to the Special Issue ELISPOT Research)

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Open AccessArticle

Can ELISPOT Be Applied to A Clinical Setting as A Diagnostic Utility for Neuroborreliosis?

by Marika Nordberg, Pia Forsberg, Dag Nyman, Barbro H. Skogman, Clara Nyberg, Jan Ernerudh, Ingvar Eliasson and Christina Ekerfelt

Cells 2012, 1(2), 153-167; https://doi.org/10.3390/cells1020153 - 08 Jun 2012

Cited by 19 | Viewed by 7508

Abstract

The aim of this prospective study was to investigate the diagnostic performance of Borrelia (Bb)-induced interferon (IFN)-γ secretion detected by ELISPOT modified to be feasible for clinical laboratories as a supplementary test to the laboratory diagnosis of Lyme neuroborreliosis (LNB) in an [...] Read more.

The aim of this prospective study was to investigate the diagnostic performance of Borrelia (Bb)-induced interferon (IFN)-γ secretion detected by ELISPOT modified to be feasible for clinical laboratories as a supplementary test to the laboratory diagnosis of Lyme neuroborreliosis (LNB) in an endemic setting. Between 2002 and 2004, patients with symptoms of suspected clinical LNB were included in a study conducted on the Åland islands in the Finnish archipelago, which is a hyper-endemic area for Lyme borreliosis (LB). Fourteen patients with confirmed LNB and 103 patients with non-LNB were included, and the numbers of spontaneous and Bb-induced IFN-γ-secreting cells were assayed by the ELISPOT test. The ELISPOT assay showed a weak diagnostic performance with a sensitivity of 36% and a specificity of 82%. The findings in this study show that this ELISPOT-assay modified to be feasible in clinical routine laboratories is not useful as a supplementary diagnostic tool in the laboratory diagnosis of patients with clinically suspected LNB. Full article

(This article belongs to the Special Issue ELISPOT Research)

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Open AccessArticle

Dissecting the T Cell Response: Proliferation Assays vs. Cytokine Signatures by ELISPOT

by Donald D. Anthony, Kimberly A. Milkovich, Wenji Zhang, Benigno Rodriguez, Nicole L. Yonkers, Magdalena Tary-Lehmann and Paul V. Lehmann

Cells 2012, 1(2), 127-140; https://doi.org/10.3390/cells1020127 - 10 May 2012

Cited by 14 | Viewed by 9034

Abstract

Chronic allograft rejection is in part mediated by host T cells that recognize allogeneic antigens on transplanted tissue. One factor that determines the outcome of a T cell response is clonal size, while another is the effector quality. Studies of alloimmune predictors of [...] Read more.

Chronic allograft rejection is in part mediated by host T cells that recognize allogeneic antigens on transplanted tissue. One factor that determines the outcome of a T cell response is clonal size, while another is the effector quality. Studies of alloimmune predictors of transplant graft survival have most commonly focused on only one measure of the alloimmune response. Because differing qualities and frequencies of the allospecific T cell response may provide distinctly different information we analyzed the relationship between frequency of soluble antigen and allo-antigen specific memory IFN-g secreting CD4 and CD8 T cells, their ability to secrete IL-2, and their proliferative capacity, while accounting for cognate and bystander proliferation. The results show proliferative responses primarily reflect on IL-2 production by antigen-specific T cells, and that proliferating cells in such assays entail a considerable fraction of bystander cells. On the other hand, proliferation (and IL-2 production) did not reflect on the frequency of IFN-γ producing memory cells, a finding particularly accentuated in the CD8 T cell compartment. These data provide rationale for considering both frequency and effector function of pre-transplant T cell reactivity when analyzing immune predictors of graft rejection. Full article

(This article belongs to the Special Issue ELISPOT Research)

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Open AccessArticle

Increased Level of IFN-γ and IL-4 Spot-Forming Cells on ELISPOT Assay as Biomarkers for Acute Graft-Versus-Host Disease and Concurrent Infections

by Masahiro Hirayama, Eiichi Azuma and Yoshihiro Komada

Cells 2012, 1(2), 61-73; https://doi.org/10.3390/cells1020061 - 30 Apr 2012

Viewed by 6416

Abstract

Acute graft-versus-host disease (aGVHD) remains a significant cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation. Infections may coexist and in certain circumstances aggravate aGVHD. It was described that type 1 as well as type 2 cytokines are important [...] Read more.

Acute graft-versus-host disease (aGVHD) remains a significant cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation. Infections may coexist and in certain circumstances aggravate aGVHD. It was described that type 1 as well as type 2 cytokines are important mediators of aGVHD. We measured spot-forming cells (SFCs) for interferon (IFN)-γ, interleukin (IL)-4, IL-10, and IL-17 in unstimulated peripheral blood from 80 patients with hematological disorders who underwent allogeneic hematopoietic stem cell transplantation by using the enzyme-linked immunospot (ELISPOT) assay that reflects the ongoing in vivo immune status. A serial monitoring showed that both type 1 and type 2 cytokine SFCs were correlated with aGVHD activity. The numbers of IFN-γ and IL-4 SFCs in patients with grade II-IV aGVHD were significantly higher than those in patients with grade 0 and/or I aGVHD. Elevation of IFN-γ and IL-4 SFCs was significantly correlated with the severity of aGVHD, but not with infection itself, e.g., cytomegalovirus infection. Cytokine SFCs are clinically relevant biomarkers for the diagnostic and therapeutic evaluation of aGVHD and concurrent infection. Full article

(This article belongs to the Special Issue ELISPOT Research)

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Open AccessArticle

Characterization of Spontaneous Immune Responses against Long Peptides Derived from Bcl-X(L) in Cancer Patients Using Elispot

by Stine Kiaer Larsen, Morten Hansen, Inge Marie Svane, Per Thor Straten and Mads Hald Andersen

Cells 2012, 1(2), 51-60; https://doi.org/10.3390/cells1020051 - 26 Apr 2012

Cited by 2 | Viewed by 6721

Abstract

In recent years we and others have used the ELISPOT assay successfully to identify novel tumor antigens by the characterization of spontaneous HLA class I restricted immune responses against a number of minimal 9–10 amino acid long peptide epitopes. In the present study, [...] Read more.

In recent years we and others have used the ELISPOT assay successfully to identify novel tumor antigens by the characterization of spontaneous HLA class I restricted immune responses against a number of minimal 9–10 amino acid long peptide epitopes. In the present study, we examined the capability of using longer peptides when scrutinizing Peripheral Blood Mononuclear Cells (PMBC) from melanoma patients for spontaneous immunity by means of ELISPOT IFN-γ secretion assay. To this end, we examined PBMC for the presence of specific T-cell responses against long peptides derived from the tumor associated antigen BCL-X(L). The protein product of the larger BCL-X(L) differs from Bcl-X(S) protein by an inserted region (amino acids 126–188). Thus, we scrutinized eight long peptides covering this inserted region for spontaneous immunity. The peptides were overlapping and consisted of 20–23 amino acids. PBMC were pre-stimulated with peptide-pulsed autologous dendritic cells (DC) and subjected to the IFN-γ ELISPOT assay. Four of the BCL-X(L) derived peptides elicited very frequent responses in several patients. Additionally, in all patients responses against more than one of the peptides could be detected. In conclusion several long BCL-X(L) derived peptide epitopes exist, which may be used in anti-cancer immunity. Furthermore, the ELISPOT assay offers an attractive and sensitive method for the characterization of spontaneous immune reactivity against long peptides. Full article

(This article belongs to the Special Issue ELISPOT Research)

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Open AccessArticle

Cytomegalovirus (CMV)-Specific Perforin and Granzyme B ELISPOT Assays Detect Reactivation of CMV Infection in Inflammatory Bowel Disease

by Tobias M. Nowacki, Dominik Bettenworth, Matthias Ross, Jan Heidemann, Paul V. Lehmann and Andreas Lügering

Cells 2012, 1(2), 35-50; https://doi.org/10.3390/cells1020035 - 23 Apr 2012

Cited by 10 | Viewed by 9785

Abstract

The role of cytomegalovirus (CMV) infection in the pathogenesis and exacerbation of Inflammatory Bowel Disease (IBD) has been unresolved. Typically, the CMV genome remains dormant in infected cells, but a breakdown of immune surveillance can lead to re-activation of viral replication in the [...] Read more.

The role of cytomegalovirus (CMV) infection in the pathogenesis and exacerbation of Inflammatory Bowel Disease (IBD) has been unresolved. Typically, the CMV genome remains dormant in infected cells, but a breakdown of immune surveillance can lead to re-activation of viral replication in the gut mucosa, which is not necessarily associated with viremia or changes in antibody titers. We hypothesized that the detection of CMV-specific CD8 effector T cells should permit the distinction between dormant and active CMV infection. As CD8 effector T cells, unlike memory CD8 T cells, have perforin (PFN) and granzyme B (GzB) preformed in their cytoplasmic granules, we employed single cell resolution ELISPOT assays to measure the CMV antigen-triggered release of these molecules by CD8 T cells isolated from subjects with IBD, and age-matched healthy controls. The frequencies of CMV-specific (GzB) and PFN-producing CD8 T cells were increased in IBD patients compared to healthy controls. Furthermore, the increased CMV reactivity was associated with active IBD disease and with longer disease duration. Notably, PCR on serum frequently failed to detect CMV DNA during flares. The data show that during active IBD there is a flare of CD8 T cell activity against CMV in a substantial proportion of IBD patients, suggesting CMV reactivation that serum PCR does not detect. While it remains open whether CMV reactivation is a cause or consequence of IBD, our data suggest that monitoring CMV antigen-specific effector CD8 T cells with GzB and PFN ELISPOT analysis can provide novel insights into the role of CMV infection in IBD. Additionally, our data have implications for the fields of transplantation, HIV, cancer, and autoimmune diseases, in all of which patient care critically depends on sensitive and reliable detection of a reactivation of CMV infection. Full article

(This article belongs to the Special Issue ELISPOT Research)

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Open AccessArticle

Comparison of ELISpot and FluoroSpot in the Analysis of Swine Flu-Specific IgG and IgA Secretion by in Vivo Activated Human B Cells

by Gun Kesa, Per H. Larsson, Niklas Ahlborg and Bernt Axelsson

Cells 2012, 1(2), 27-34; https://doi.org/10.3390/cells1020027 - 20 Apr 2012

Cited by 9 | Viewed by 8344

Abstract

We have evaluated a novel B-cell FluoroSpot assay for the analysis of antibody responses in healthy individuals vaccinated intramuscularly with Influenza A (H1N1) antigen (Pandemrix^®, GlaxoSmithKline). Using the FluoroSpot assay and an ELISpot assay run in parallel for comparison, we measured [...] Read more.

We have evaluated a novel B-cell FluoroSpot assay for the analysis of antibody responses in healthy individuals vaccinated intramuscularly with Influenza A (H1N1) antigen (Pandemrix^®, GlaxoSmithKline). Using the FluoroSpot assay and an ELISpot assay run in parallel for comparison, we measured the frequency of cells secreting antigen-specific as well as total IgG or IgA antibodies seven days post vaccination. The assays were based on high affinity monoclonal antibodies for capture and detection of human IgG and IgA. Whereas conventional ELISpot analyzes IgG- and IgA-secreting B cells separately, fluorescent detection enabled simultaneous enumeration of B cells secreting IgG or IgA in the same well. The FluoroSpot protocol was also simpler as the assay could be performed without the need for an amplifying detection step. While having all the advantages of a conventional ELISpot assay, including high sensitivity, robustness and ease of performance, the FluoroSpot assay adds further value in reducing costs, time and material. Full article

(This article belongs to the Special Issue ELISPOT Research)

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Open AccessArticle

Simultaneous Detection of Antigen-Specific IgG- and IgM-Secreting Cells with a B Cell Fluorospot Assay

by Mattia Bonsignori and M. Anthony Moody

Cells 2012, 1(1), 15-26; https://doi.org/10.3390/cells1010015 - 21 Mar 2012

Cited by 1 | Viewed by 7443

Abstract

The traditional enzyme-linked immunospot (ELISpot) assay is the gold standard for the enumeration of antigen-specific B cells. Since B cell availability from biological samples is often limited, either because of sample size/volume or the need of performing multiple analyses on the same sample, [...] Read more.

The traditional enzyme-linked immunospot (ELISpot) assay is the gold standard for the enumeration of antigen-specific B cells. Since B cell availability from biological samples is often limited, either because of sample size/volume or the need of performing multiple analyses on the same sample, the implementation of ELISpot assay formats that allow the simultaneous detection of multiple antibody types is desirable. While dual-color ELISpot assays have been described, technical complexities have so far prevented their wide utilization as well as further expansion of their multicolor capability. An attractive solution is to replace the chromogenic reaction of the traditional ELISpot assay with a fluorescent detection system (fluorospot assay). Fluorospot assays using fluorophore-conjugated secondary antibodies in conjunction with fluorescence enhancers, FITC/anti-FITC and biotin/avidin amplification systems and dedicated equipment for spot detection have been developed to enumerate T-cells secreting two or three specific cytokines and, more recently, IgG and IgA antibody-secreting cells (ASCs). We hereby report a method for a multiplex B cell fluorospot assay that utilizes quantum-dot nanocrystals as reporters without further amplification systems or need of dedicated equipment. With this method we simultaneously enumerated HIV-1 gp41 envelope glycoprotein-specific IgG and IgM antibody-secreting cells with sensitivity comparable to that of the traditional ELISpot assay. Full article

(This article belongs to the Special Issue ELISPOT Research)

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Open AccessArticle

ELISPOT Refinement Using Spot Morphology for Assessing Host Responses to Tuberculosis

by Laura S. Sibley, Andrew D. White, Alice Marriott, Michael J. Dennis, Ann Williams, Philip D. Marsh and Sally A. Sharpe

Cells 2012, 1(1), 5-14; https://doi.org/10.3390/cells1010005 - 13 Mar 2012

Cited by 10 | Viewed by 7846

Abstract

Tuberculosis is a global health problem. The Mycobacterium bovis Bacille Calmette Guerin (BCG) vaccine has variable efficacy (0–80%) so there is a drive to develop novel vaccines. The cytokine, interferon gamma (IFNγ), is an essential component of the protective response to M. tuberculosis [...] Read more.

Tuberculosis is a global health problem. The Mycobacterium bovis Bacille Calmette Guerin (BCG) vaccine has variable efficacy (0–80%) so there is a drive to develop novel vaccines. The cytokine, interferon gamma (IFNγ), is an essential component of the protective response to M. tuberculosis (M. tb) infection and is also produced in response to BCG vaccination. Induction of an IFNγ response is used as a biomarker of successful vaccination in the assessment of new tuberculosis (TB) vaccines. The IFNγ ELISPOT assay provides an important tool for TB research. It is used for both the diagnosis of infection (T.Spot assay), and for the evaluation of the immunogenicity of new TB vaccine candidates in human clinical trials, in the non-human primate (NHP) model of TB infection studies. The ELISPOT assay captures IFNγ produced by peripheral blood mononuclear cells (PBMCs) following specific stimulation, onto a membrane so individual cells can be enumerated and the frequency of responding cells determined. Hence spot forming units (SFU) per 10⁶ cells provide the traditional measure for ELISPOT assays. The discriminatory power of SFU is limited. In some situations, the number of SFU in BCG vaccinated, and unvaccinated, subjects was found to be similar, although the spots were observed to be larger in vaccinated subjects. Spot size potentially provides a measure of the quantity of cytokine produced by individual cells. The AID ELISPOT plate reader software used to determine frequency of spots also has the capability to determine the size of each spot. Consideration of spot size in combination with spot forming units was investigated in our studies of BCG immunogenicity. This additional readout was found to enhance the discriminatory power of the ELISPOT assay, and provide more information on the immune response to BCG vaccination and infection with M.tb. Full article

(This article belongs to the Special Issue ELISPOT Research)

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Review

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Enzyme Linked Immuno-Spot; a Useful Tool in the Search for Elusive Immune Markers in Common Pediatric Immunological Diseases

by Maria Faresjö

Cells 2012, 1(2), 141-152; https://doi.org/10.3390/cells1020141 - 29 May 2012

Cited by 4 | Viewed by 6593

Abstract

In order to provide better therapy we strive to increase our knowledge of how the immune system behaves and communicates in common pediatric immunological diseases, such as type 1 diabetes, allergic and celiac diseases. However, when dealing with pediatric diseases, where study subjects [...] Read more.

In order to provide better therapy we strive to increase our knowledge of how the immune system behaves and communicates in common pediatric immunological diseases, such as type 1 diabetes, allergic and celiac diseases. However, when dealing with pediatric diseases, where study subjects are almost exclusively children, blood volumes available for immunological studies are limited and as such must be carefully handled and used to their full extent. Single immune markers can easily be detected by a traditional Enzyme Linked Immunosorbent Assay (ELISA), whereas multiple markers can be detected by a fluorochrome (Luminex) or electrochemiluminescence (MSD) technique. These techniques however are sometimes not sensitive enough to detect low levels of secreted immune markers in limited sample sizes. To detect immune markers at the single-cell level, an Enzyme Linked Immuno-spot (ELISPOT) can be used to pin-point elusive immune markers in common pediatric immunological diseases. Full article

(This article belongs to the Special Issue ELISPOT Research)

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Open AccessReview

ELISPOT Assay for Monitoring Cytotoxic T Lymphocytes (CTL) Activity in Cancer Vaccine Clinical Trials

by Anatoli M. Malyguine, Susan Strobl, Kimberly Dunham, Michael R. Shurin and Thomas J. Sayers

Cells 2012, 1(2), 111-126; https://doi.org/10.3390/cells1020111 - 10 May 2012

Cited by 51 | Viewed by 11467

Abstract

The profiling and monitoring of immune responses are key elements in the evaluation of the efficacy and development of new biotherapies, and a number of assays have been introduced for analyzing various immune parameters before, during, and after immunotherapy. The choice of immune [...] Read more.

The profiling and monitoring of immune responses are key elements in the evaluation of the efficacy and development of new biotherapies, and a number of assays have been introduced for analyzing various immune parameters before, during, and after immunotherapy. The choice of immune assays for a given clinical trial depends on the known or suggested immunomodulating mechanisms associated with the tested therapeutic modality. Cell-mediated cytotoxicity represents a key mechanism in the immune response to various pathogens and tumors. Therefore, the selection of monitoring methods for the appropriate assessment of cell-mediated cytotoxicity is thought to be crucial. Assays that can detect both cytotoxic T lymphocytes (CTL) frequency and function, such as the IFN-γ enzyme-linked immunospot assay (ELISPOT) have gained increasing popularity for monitoring clinical trials and in basic research. Results from various clinical trials, including peptide and whole tumor cell vaccination and cytokine treatment, have shown the suitability of the IFN-γ ELISPOT assay for monitoring T cell responses. However, the Granzyme B ELISPOT assay and Perforin ELISPOT assay may represent a more direct analysis of cell-mediated cytotoxicity as compared to the IFN-γ ELISPOT, since Granzyme B and perforin are the key mediators of target cell death via the granule-mediated pathway. In this review we analyze our own data and the data reported by others with regard to the application of various modifications of ELISPOT assays for monitoring CTL activity in clinical vaccine trials. Full article

(This article belongs to the Special Issue ELISPOT Research)

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Open AccessReview

Role of ELISPOT Assays in Risk Assessment Pre- and Post-Kidney Transplantation

by Jennifer R. Zitzner and Anat R. Tambur

Cells 2012, 1(2), 100-110; https://doi.org/10.3390/cells1020100 - 10 May 2012

Cited by 7 | Viewed by 5603

Abstract

Immunologic risk in kidney transplantation is typically minimized by avoiding, or at least limiting, the potential of donor specific humoral responses by testing for the presence of donor-specific antibodies (DSA). Additionally, selecting donor and recipient pairs with the least number of human leukocyte [...] Read more.

Immunologic risk in kidney transplantation is typically minimized by avoiding, or at least limiting, the potential of donor specific humoral responses by testing for the presence of donor-specific antibodies (DSA). Additionally, selecting donor and recipient pairs with the least number of human leukocyte antigen (HLA) mismatches has been shown to play a role in transplant outcome. However, numerous other factors may play a role in the success of transplant outcome and patient health. Specifically, the use of T-cell allospecific ELISPOT assays have helped elucidate the role of pre-formed cellular responses as additional factors in post-transplant outcome. In this review, we will evaluate numerous uses of ELISPOT assays to assess the pre- and post-transplant immunologic risk of rejection episodes, graft survival and even viral susceptibility as well as the utility of ELISPOT assays in monitoring tolerance and withdrawal of immunosuppressive medications following kidney transplantation. Full article

(This article belongs to the Special Issue ELISPOT Research)

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Open AccessReview

Distinguishing Latent from Active Mycobacterium tuberculosis Infection Using Elispot Assays: Looking Beyond Interferon-gamma

by Camilla Tincati, Amedeo J. Cappione III and Jennifer E. Snyder-Cappione

Cells 2012, 1(2), 89-99; https://doi.org/10.3390/cells1020089 - 07 May 2012

Cited by 12 | Viewed by 6707

Abstract

Mycobacterium tuberculosis (MTB) is a global heath epidemic, its threat amplified by HIV infection and the emergence of multidrug-resistant tuberculosis (MDR-TB). Interferon (IFN)-gamma release assays (IGRAs) have improved the accuracy of detection of MTB exposure in some subject groups as compared to the [...] Read more.

Mycobacterium tuberculosis (MTB) is a global heath epidemic, its threat amplified by HIV infection and the emergence of multidrug-resistant tuberculosis (MDR-TB). Interferon (IFN)-gamma release assays (IGRAs) have improved the accuracy of detection of MTB exposure in some subject groups as compared to the Tuberculin Skin Test (TST). However, as IFN-gamma is produced by both fully rested and more recently activated populations of memory T cells, it is not surprising that the measurement of this cytokine alone cannot accurately distinguish Latent TB Infected (LTBI) subjects from those with active (infectious) disease. Accurate and rapid diagnosis of infectious individuals would allow medication to be properly allocated and other actions taken to more effectively curtail MTB spread. Analysis of multi-cytokine profiles ex vivo after stimulation of PBMCs from LTBI and active MTB subjects indicate the real possibility of successfully discerning these two disease states within 24 hours of a subject’s blood draw. Due to the unparalleled sensitivity, low cost, and ease of use of Elispot assays, we propose that via a multiplex Elispot platform the accurate distinction of LTBI from active MTB-infected individuals is within reach. Full article

(This article belongs to the Special Issue ELISPOT Research)

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633 KiB

Open AccessCorrection

Correction: Santos, R.S., et al. Improvement of IFNγ ELISPOT Performance Following Overnight Resting of Frozen PBMC Samples Confirmed Through Rigorous Statistical Analysis. Cells 2015, 4, 1-18

by Radleigh Santos, Alcinette Buying, Nazila Sabri, John Yu, Anthony Gringeri, James Bender, Sylvia Janetzki, Clemencia Pinilla and Valeria A. Judkowski

Cells 2015, 4(2), 133-134; https://doi.org/10.3390/cells4020133 - 21 Apr 2015

Cited by 2 | Viewed by 4701

Abstract

The authors wish to make the following corrections to this paper [1]: [...] Full article

(This article belongs to the Special Issue ELISPOT Research)

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