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

Hanson, Jodi; Sundararaman, Srividya; Caspell, Richard; Karacsony, Edith; Karulin, Alexey Y.; Lehmann, Paul

doi:10.3390/cells4010071

Cited by 18 publications

(16 citation statements)

References 14 publications

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“…Thus, at least two cell types are involved in T cell assays such as ELISPOT. In order to detect all antigen specific T cells, one needs to plate the PBMC dense enough so that each antigen-specific T cell can interact with an APC otherwise the T cells would go undetected [ 48 ]. We showed for peptide antigen-reactive CD8 cells producing IFN-γ, that the numbers of PBMC plated per well and the spot counts elicited by the peptide follow a linear function between 100,000 and 800,000 PBMC per well [ 40 , 48 , 49 , 50 ].…”

Section: Resultsmentioning

confidence: 99%

Characterization of the HCMV-Specific CD4 T Cell Responses that Are Associated with Protective Immunity

Wunsch

Zhang²,

Hanson³

et al. 2015

Viruses

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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 mononuclear cell (PBMC). Using the enzyme-linked Immunospot® assay (ELISPOT) that 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)-γ 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-γ 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.

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Section: Resultsmentioning

confidence: 99%

Characterization of the HCMV-Specific CD4 T Cell Responses that Are Associated with Protective Immunity

Wunsch

Zhang²,

Hanson³

et al. 2015

Viruses

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“…To become activated by an antigen, T-cells must make contact with APC. Therefore, the numbers at which PBMC are plated will affect the results: when plated at less than 5 × 10 4 PBMC per well in a 96-well plate, the cells no longer form a monolayer, and recall responses become undetectable [ 23 ]. On the other hand, when plated at more than 10 6 PBMC per well, the cells become crowded and settle in several layers, also leading to a loss of accurate ELISPOT counts.…”

Section: Resultsmentioning

confidence: 99%

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

Karulin¹,

Caspell²,

Dittrich

et al. 2015

Cells

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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.

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“…Recently 384-well ELISOT plates have been introduced that permit to test one-third of the PBMC cell number per well compared to standard 96-well plates, resulting in a precisely proportional (one-third) reduction of the spot count per well over a wide range of PBMC numbers plated per well [ 28 ]. Thus, the 384-well approach lends itself to detecting dominant and subdominant peptide responses.…”

Section: Discussionmentioning

confidence: 99%

How frequently are predicted peptides actually recognized by CD8 cells?

Moldovan¹,

Targoni²,

Zhang³

et al. 2016

Cancer Immunol Immunother

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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 antigen-specific CD8 cell immunity, strongly arguing for the use of peptide pools for immune monitoring.

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

Cited by 18 publications

References 14 publications

Characterization of the HCMV-Specific CD4 T Cell Responses that Are Associated with Protective Immunity

Characterization of the HCMV-Specific CD4 T Cell Responses that Are Associated with Protective Immunity

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

How frequently are predicted peptides actually recognized by CD8 cells?

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