Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-61779-325-7

Figure Lengend Snippet: Fig. 2. Measuring T cell functions by flow cytometry vs. ELISPOT. Left panel: For the detection of secretory products by ICS, the cells need to be “poisoned” first with Golgi inhibitors to prevent secretion, then permeabilized and fixed/“mummified.” The subsequent standard flow cytometric analysis does not make the distinction whether the analyte is indeed bound for secretion and thus is biologically active, or is retained in/on the cell. Right panel: In contrast, ELISPOT measures the actual secretory activity of pharmacologically untreated, living cells. The cells survive ELISPOT assays unharmed, and can be retested, phenotyped, expanded, cloned, or cryopreserved. Graphic artist: Gabor Pesthy.

Article Snippet: Human IFN ELISPOT Kit (MABTECH AB, Nacka Strand, Sweden) for Fig. 3a and Human IFN ELISPOT Development Module (R&D Systems Inc.) for Fig. 4.

Techniques: Cytometry, Enzyme-linked Immunospot, Activity Assay, Clone Assay

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-61779-325-7

Figure Lengend Snippet: Fig. 4. The different functional avidities of antigen-reactive T cells. PBMC of an HLA-A2 positive subject were plated with different concentrations of individual A-2 restricted CEF peptides, as specified by the different symbols. A standard 24 h IFN-G ELISPOT assay was performed. Note how far apart the maximum stimulatory concentrations of the different peptides are.

Article Snippet: Human IFN ELISPOT Kit (MABTECH AB, Nacka Strand, Sweden) for Fig. 3a and Human IFN ELISPOT Development Module (R&D Systems Inc.) for Fig. 4.

Techniques: Functional Assay, Enzyme-linked Immunospot

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-61779-325-7

Figure Lengend Snippet: Fig. 1. Accumulation of ELISPOT measurement values around the panel median. The measurement values of all panelists from the 4th ELISPOT proficiency panel of the CIC/CRI for one donor against the CEF peptide pool are shown. Each lab employed their own SOP, but plated 200,000 cells per well and 1 Mg/ml peptide pool. The graph illustrates individual results as box plots with maximum, minimum, mean (triangle within box ), and median (line within box ) values of six replicate measurements. The panel median (64 spots) is presented as a line marked by an arrow. Most measurements accumulate around the panel median while some measurements are clearly out of range.

Article Snippet: Human IFN ELISPOT Kit (MABTECH AB, Nacka Strand, Sweden) for Fig. 3a and Human IFN ELISPOT Development Module (R&D Systems Inc.) for Fig. 4.

Techniques: Enzyme-linked Immunospot

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-61779-325-7

Figure Lengend Snippet: Fig. 3. Improvement of ELISPOT performance during the harmonization process. The percentage of panelists missing to detect the weak responder in dependence of the stage of the ELISPOT proficiency panel program of the CIC/CRI is depicted as the black pie part to the right. This number decreased with increasing harmonization from 47 to 14 to 7% of participants.

Article Snippet: Human IFN ELISPOT Kit (MABTECH AB, Nacka Strand, Sweden) for Fig. 3a and Human IFN ELISPOT Development Module (R&D Systems Inc.) for Fig. 4.

Techniques: Enzyme-linked Immunospot

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-61779-325-7

Figure Lengend Snippet: Fig. 1. Typical ELISPOT images of equine IFNG and IL-4. The number of spot-forming cells (SFCs) produced by the same number of plated cells from the same animal can vary depending on the cell-stimulating reagents.

Article Snippet: Human IFN ELISPOT Kit (MABTECH AB, Nacka Strand, Sweden) for Fig. 3a and Human IFN ELISPOT Development Module (R&D Systems Inc.) for Fig. 4.

Techniques: Enzyme-linked Immunospot, Produced

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-61779-325-7

Figure Lengend Snippet: Fig. 2. FIV p24-specific IL-2 and IFNJ responses of PBMC from prototype IWV-vaccinated cats. The IL-2 (a) and IFNJ (b) responses of the PBMC were from four cats vaccinated with pro- totype IWV vaccine for 7× to 9× over the course of 4 years. The number below the peptide pool, FIV p24, FIV IWV, or ConA is the total number of responders (t50 SFU threshold) with a responder frequency of t75%. The numbers in dark color represent those peptide-pool responses common between the two cytokine groups while the numbers in light color are observed predominantly by the corresponding cytokine group than the other. Many cats had both IL-2 and IFNJ responses to Fp6, Fp10, and Fp12. The peptide pools Fp14 and Fp16 induced pre- dominantly IL-2 responses in majority of cats while Fp3, Fp5, and Fp17 induced predominantly IFNJ responses. Overall, FIV p24-specific IFNJ responses of the PBMC from IWV-vaccinated cats correlate with IL-2 responses for certain peptide pools. When the IFNJ results of these cats (BDA, BDM, QVD, QVF) after 4 years of vaccination (post 8–9 vaccinations) (b) are compared to their responses from the first year (post 2–4 vaccinations) in Fig. 1b, IFNJ responses to Fp3 and Fp10 are clearly retained over 4 years while those to Fp1 and Fp13 are lost with only one cat each retaining a response to a peptide pool long term. An interesting observation was that responses to Fp7 and Fp8 are lost by QVD and QVF (QVs are siblings), but not by BDA and BDM (BDs are from same parents but different litters), indicating a genetic influence. Thus, ELISPOT results provide information on the immune modulation over a period of time.

Article Snippet: Human IFN ELISPOT Kit (MABTECH AB, Nacka Strand, Sweden) for Fig. 3a and Human IFN ELISPOT Development Module (R&D Systems Inc.) for Fig. 4.

Techniques: Enzyme-linked Immunospot

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-61779-325-7

Figure Lengend Snippet: Fig. 4. IFNJ responses to FIV and HIV-1 p24 and reverse transcriptase (RT) peptide pools by HIV-1-infected subjects, IWV-vaccinated cat, and HIV-1 p24-immunized cat. Top five rows are actual IFNJ ELISPOT results from PBMC or T-cell populations of HIV-infected subjects with no stimulation (column 3 ) or after stimulation with the FIV-p24 peptide pool Fp14 (column 1 ), counterpart HIV-p24 peptide pool Hp15 (column 2 ; sequence counterpart of Fp14), FIV-RT peptide pool FRT3 (column 6 ), counterpart HIV-1 peptide pool HRT3 (column 5, counterpart of FRT3), or T-cell mitogen (positive control concanavalin A, ConA) (column 4). The description of the subject or the cat is shown on the left along with the multiplication

Article Snippet: Human IFN ELISPOT Kit (MABTECH AB, Nacka Strand, Sweden) for Fig. 3a and Human IFN ELISPOT Development Module (R&D Systems Inc.) for Fig. 4.

Techniques: Reverse Transcription, Infection, Enzyme-linked Immunospot, Sequencing, Positive Control

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-61779-325-7

Figure Lengend Snippet: Fig. 1. Typical ELISPOT images of secreted cytokines from human PBMCs. Note the prominent inhibitory effect of oxidative stress on secretion of all cytokines, except IL-4 and IL5.

Article Snippet: Human IFN ELISPOT Kit (MABTECH AB, Nacka Strand, Sweden) for Fig. 3a and Human IFN ELISPOT Development Module (R&D Systems Inc.) for Fig. 4.

Techniques: Enzyme-linked Immunospot

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-61779-325-7

Figure Lengend Snippet: Fig. 1. Typical ELISPOT image of TNFA secreted by microglial BV2 cells. BV2 cells were plated at 1,000 cells per well (a) and 120 cells per well (b). Note that, unlike uniform round spots formed by TNFA secreted by immune system cells, many spots formed by TNFA secreted from BV2 microglial cells have “zigzag tails” as a result of active movement of BV2 cells during the incubation.

Article Snippet: Human IFN ELISPOT Kit (MABTECH AB, Nacka Strand, Sweden) for Fig. 3a and Human IFN ELISPOT Development Module (R&D Systems Inc.) for Fig. 4.

Techniques: Enzyme-linked Immunospot, Incubation

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-61779-325-7

Figure Lengend Snippet: Fig. 1. Macrokinetic model of ELISPOT. Analyte-secreting cell (G) is located on the surface of the membrane. Secreted analyte can be either bound by capture antibodies on the membrane surface or diffuse away from the cell into media above the membrane.

Article Snippet: Human IFN ELISPOT Kit (MABTECH AB, Nacka Strand, Sweden) for Fig. 3a and Human IFN ELISPOT Development Module (R&D Systems Inc.) for Fig. 4.

Techniques: Enzyme-linked Immunospot, Membrane

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-61779-325-7

Figure Lengend Snippet: Fig. 1. Effects of illumination conditions on the results of the automated search process. (a) Field of view in bottom of the ELISPOT well is evenly illuminated which results in almost a uniform background. (b) Illumination is uneven and as a result sharp round spots can be easily detected by the software, whereas blurry spots that have irregular form may remain undetected.

Article Snippet: Human IFN ELISPOT Kit (MABTECH AB, Nacka Strand, Sweden) for Fig. 3a and Human IFN ELISPOT Development Module (R&D Systems Inc.) for Fig. 4.

Techniques: Enzyme-linked Immunospot, Software

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-61779-325-7

Figure Lengend Snippet: Fig. 2. Analysis of a dual-analyte ELISPOT assay. QuantiHub 4.1 is capable of recognizing and detecting spots that have different colors as well as different shades of the same color within a large dynamic range.

Article Snippet: Human IFN ELISPOT Kit (MABTECH AB, Nacka Strand, Sweden) for Fig. 3a and Human IFN ELISPOT Development Module (R&D Systems Inc.) for Fig. 4.

Techniques: Enzyme-linked Immunospot

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-61779-325-7

Figure Lengend Snippet: Fig. 3. Typical dual-color ELISPOT detection results using the “Fast” counting mode in QuantiHub 4.1 software.

Article Snippet: Human IFN ELISPOT Kit (MABTECH AB, Nacka Strand, Sweden) for Fig. 3a and Human IFN ELISPOT Development Module (R&D Systems Inc.) for Fig. 4.

Techniques: Enzyme-linked Immunospot, Software

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-61779-325-7

Figure Lengend Snippet: Fig. 4. Typical dual-color ELISPOT detection results using the “Enhanced” counting mode in QuantiHub 4.1 software.

Article Snippet: Human IFN ELISPOT Kit (MABTECH AB, Nacka Strand, Sweden) for Fig. 3a and Human IFN ELISPOT Development Module (R&D Systems Inc.) for Fig. 4.

Techniques: Enzyme-linked Immunospot, Software

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-61779-325-7

Figure Lengend Snippet: Fig. 1. Statistics-based automated ELISPOT counting. Raw images of a medium control well (“Media”) and the correspond- ing antigen-stimulated well (“Antigen”) are shown in the top two panels (a). Underneath (b) are the spot counts obtained without gating, with each spot highlighted. (The ImmunoSpot® software establishes the spot recognition parameters auto- matically by learning spot morphologies, instead of requiring users to subjectively set a multitude of parameters inevitably leading to variability in counts.) The spots automatically recognized are highlighted by the software, creating complete transparency of the (ungated, intermediate) counting result. While all spots in this medium well are “real” (being produced either by the cells of the innate immune system or by antigen-stimulated T cells), and recognized precisely as spots by the software, for establishing the T-cell count this ungated result is completely wrong. The next step is gating. The ImmunoSpot® software automatically establishes size distributions for the media and the antigen-triggered wells and sets the gates automatically, based on the statistical analysis of the distributions as we established in J. Immunol. 2000 164:1862–72 and J. Immunol. 2001, 167:1353–61, among several other publications. After re-counting with these gates, the counts are now correct and objective (c). Thus, there is no human judgment involved in the analysis – anyone in the world analyzing these images with the ImmunoSpot® software would come up with exactly the same scientifically validated count.

Article Snippet: Human IFN ELISPOT Kit (MABTECH AB, Nacka Strand, Sweden) for Fig. 3a and Human IFN ELISPOT Development Module (R&D Systems Inc.) for Fig. 4.

Techniques: Enzyme-linked Immunospot, Control, Software, Produced, Cell Counting

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-61779-325-7

Figure Lengend Snippet: Fig. 4. Quality control in ELISPOT analysis. The image on the left-hand side shows the counting results obtained in auto- mated analysis mode on a well that contains an artifact (in this case, a cell clump caused the spot cluster in the upper left-hand quadrant). The cluster was treated as a group of individual spots, resulting in a spot count of 63. In quality control mode, this artifact-containing region can be outlined (in green) and excluded from the analysis, as shown on the right-hand side. The software then normalizes the spot count by correcting for the size of that region. In the example shown, 33 spots were actually detected, and this count was increased by 8 to compensate for the exclude area, resulting in an adjusted spot count of 41. (The asterisk beside the spot count of 41 indicates that this is a re-calculated value, rather than a direct measurement.) In keeping with Good Laboratory Practices, the software saves and annotates all such subjective adjust- ments made to the objective automated count. This same example also contains a spot near the center of the well that exceeds the upper gate threshold. This spot was automatically outlined in dark blue during automated analysis, indicating that it was treated as a cluster. In such cases, the software automatically calculates the number of spots required to gener- ate such a cluster based on the average spot size and density distribution, and re-computes the spot count accordingly (the asterisk beside the spot count of 63 likewise indicates that this value was re-calculated, as does the automatically generated A11 annotation code).

Article Snippet: Human IFN ELISPOT Kit (MABTECH AB, Nacka Strand, Sweden) for Fig. 3a and Human IFN ELISPOT Development Module (R&D Systems Inc.) for Fig. 4.

Techniques: Control, Enzyme-linked Immunospot, Software, Generated

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-61779-325-7

Figure Lengend Snippet: Fig. 5. ELISPOT data management. The spot counts in 96-well format are linked to the plate layout. For each well, the antigen, the test subject, the cytokine, and the number of cells plated are specified. All these data are linked and processed for exporting into a database.

Article Snippet: Human IFN ELISPOT Kit (MABTECH AB, Nacka Strand, Sweden) for Fig. 3a and Human IFN ELISPOT Development Module (R&D Systems Inc.) for Fig. 4.

Techniques: Enzyme-linked Immunospot

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-61779-325-7

Figure Lengend Snippet: Fig. 1. Measured versus theoretical distribution of ELISPOT counts. IFN-G-transfected CHO cells were plated into an IFN-G ELISPOT assay at 15, 30, 60, and 120 cells per well, with 192 replicate wells per cell number, developed and counted with ImmonoSpot® Software. Distributions for spot counts are depicted for a Poisson distribution (dotted lines ) and a normal distribution (the solid line) with the means and a standard deviation corresponding to the square root of the means.

Article Snippet: Human IFN ELISPOT Kit (MABTECH AB, Nacka Strand, Sweden) for Fig. 3a and Human IFN ELISPOT Development Module (R&D Systems Inc.) for Fig. 4.

Techniques: Enzyme-linked Immunospot, Transfection, Software, Standard Deviation

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-61779-325-7

Figure Lengend Snippet: Fig. 2. Overdispersion of ELISPOT count data. Left panel: Simulated count data drawn from a Poisson distribution with mean parameter lambda for 15, 30, 60, and 120 cells per well (n = 192 each) exhibit a variance equal to the mean as expected. Right panel : Real spot count data obtained with the transfected cell lines display a classical overdispersion effect, that is, the variance increases with the mean.

Article Snippet: Human IFN ELISPOT Kit (MABTECH AB, Nacka Strand, Sweden) for Fig. 3a and Human IFN ELISPOT Development Module (R&D Systems Inc.) for Fig. 4.

Techniques: Enzyme-linked Immunospot, Transfection

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-61779-325-7

Figure Lengend Snippet: Fig. 1. Example of ex vivo ELISPOT assays used to assess vaccine-induced cellular immunity: in vitro cellular immune responses after vaccination. PBMCs were stimulated in vitro peptide pool containing the complete set of 14 overlapping peptides covering the Rv3019c sequence. (a) IFN responses measured by ELISPOT and expressed as mean SFC/106

Article Snippet: Human IFN ELISPOT Kit (MABTECH AB, Nacka Strand, Sweden) for Fig. 3a and Human IFN ELISPOT Development Module (R&D Systems Inc.) for Fig. 4.

Techniques: Ex Vivo, Enzyme-linked Immunospot, In Vitro, Sequencing

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-61779-325-7

Figure Lengend Snippet: Fig. 2. Flow chart of ex vivo and cultured ELISPOT assay.

Article Snippet: Human IFN ELISPOT Kit (MABTECH AB, Nacka Strand, Sweden) for Fig. 3a and Human IFN ELISPOT Development Module (R&D Systems Inc.) for Fig. 4.

Techniques: Ex Vivo, Cell Culture, Enzyme-linked Immunospot

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-61779-325-7

Figure Lengend Snippet: Fig. 3. Example of cultured ELISPOT application: Correlation of memory responses with protection. Memory cell responses were determined by cultured IFN-G ELISPOT assay. Results of ELISPOT analysis are expressed as mean spot-forming cells (SFCs)/million cells. Cultured ELISPOT assays were performed before Mycobacterium bovis infection 14 weeks post-BCG vaccination and compared to outcome of infection with M. bovis at week 28 post-vaccination (animals were challenged with M. bovis at week 14 post- vaccination). Protection has been determined by bacterial load (log CFU/g tissue). Shown is the correlation of mean cultured ELISPOT responses and mean bacterial loads using data from unvaccinated cattle as well as cattle vaccinated with three different vaccines that induced various degrees of protection (from ref. 2).

Article Snippet: Human IFN ELISPOT Kit (MABTECH AB, Nacka Strand, Sweden) for Fig. 3a and Human IFN ELISPOT Development Module (R&D Systems Inc.) for Fig. 4.

Techniques: Cell Culture, Enzyme-linked Immunospot, Infection, Vaccines

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-61779-325-7

Figure Lengend Snippet: Fig. 4. 96-well filter-bottomed plate used for ELISPOT and HTS applications.

Article Snippet: Human IFN ELISPOT Kit (MABTECH AB, Nacka Strand, Sweden) for Fig. 3a and Human IFN ELISPOT Development Module (R&D Systems Inc.) for Fig. 4.

Techniques: Enzyme-linked Immunospot

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-61779-325-7

Figure Lengend Snippet: Fig. 5. 96-well filter-bottomed plate used specifically for ELISPOT.

Article Snippet: Human IFN ELISPOT Kit (MABTECH AB, Nacka Strand, Sweden) for Fig. 3a and Human IFN ELISPOT Development Module (R&D Systems Inc.) for Fig. 4.

Techniques: Enzyme-linked Immunospot

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-61779-325-7

Figure Lengend Snippet: Fig. 6. Membrane-bottomed 8-well strip plate used specifically for ELISPOT (especially diagnostic applications).

Article Snippet: Human IFN ELISPOT Kit (MABTECH AB, Nacka Strand, Sweden) for Fig. 3a and Human IFN ELISPOT Development Module (R&D Systems Inc.) for Fig. 4.

Techniques: Membrane, Stripping Membranes, Enzyme-linked Immunospot, Diagnostic Assay

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-4939-8567-8

Figure Lengend Snippet: Fig. 1 Initial Elispot Harmonization guidelines, as published in [4]

Article Snippet: Feline (Fe) IFNγ ELISPOT Development Module, FeIL-2 ELISPOT Development Module, and FeIL-10 ELISPOT Development Module (R&D Systems Inc., Minneapolis, MN) included a matching pair of antibodies used for capture and detection of individual feline cytokine and ELISPOT Blue Color Module (R&D Systems Inc.): (a) Respective feline cytokine (IFNγ, IL-2, IL-10) Capture Antibody Concentrate reconstituted in 1 mL PBS and diluted 1 in 50 parts PBS. (b) Respective feline cytokine (IFNγ, IL-2, IL-10) Detection Antibody Concentrate reconstituted in 1 mL reagent diluent and diluted in 50 parts reagent diluent Fig. 1 Structure and sequences of the four multiple antigenic peptides (MAPs).

Techniques: Enzyme-linked Immunospot

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-4939-8567-8

Figure Lengend Snippet: Fig. 1 Anatomy of a typical spot. After finishing the ELISpot assay developed with BCIP/NBT chromogen and immobilizing cells to the PVDF membrane, the mem- brane was removed from the plate and sectioned on the cryostat in a perpen- dicular direction. DAPI was used to counterstain the nuclei of immobilized cells (arrowhead). Spot boundaries indicate that cell-secreted proteins can migrate away from the cell in distances that exceed the size of the cell 3–5 times

Article Snippet: Feline (Fe) IFNγ ELISPOT Development Module, FeIL-2 ELISPOT Development Module, and FeIL-10 ELISPOT Development Module (R&D Systems Inc., Minneapolis, MN) included a matching pair of antibodies used for capture and detection of individual feline cytokine and ELISPOT Blue Color Module (R&D Systems Inc.): (a) Respective feline cytokine (IFNγ, IL-2, IL-10) Capture Antibody Concentrate reconstituted in 1 mL PBS and diluted 1 in 50 parts PBS. (b) Respective feline cytokine (IFNγ, IL-2, IL-10) Detection Antibody Concentrate reconstituted in 1 mL reagent diluent and diluted in 50 parts reagent diluent Fig. 1 Structure and sequences of the four multiple antigenic peptides (MAPs).

Techniques: Enzyme-linked Immunospot, Membrane

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-4939-8567-8

Figure Lengend Snippet: Fig. 5 Controls recommended for running side by side with experimental groups. Several controls are recom- mended to make sure that experimental conditions and reagents are not causing the formation of nonspecific spots: (1) Background control—complete ELISpot development with no cells added to wells; (2) No-detection antibody control—cells are added to the wells but ELISpot development omits adding detection antibodies; (3) Non-stimulated cells control—complete ELISpot development using non-stimulated cells (see Note 12); and (4) Immunoassay control—complete ELISpot development adding a corresponding recombinant protein rather than stimulated cells

Article Snippet: Feline (Fe) IFNγ ELISPOT Development Module, FeIL-2 ELISPOT Development Module, and FeIL-10 ELISPOT Development Module (R&D Systems Inc., Minneapolis, MN) included a matching pair of antibodies used for capture and detection of individual feline cytokine and ELISPOT Blue Color Module (R&D Systems Inc.): (a) Respective feline cytokine (IFNγ, IL-2, IL-10) Capture Antibody Concentrate reconstituted in 1 mL PBS and diluted 1 in 50 parts PBS. (b) Respective feline cytokine (IFNγ, IL-2, IL-10) Detection Antibody Concentrate reconstituted in 1 mL reagent diluent and diluted in 50 parts reagent diluent Fig. 1 Structure and sequences of the four multiple antigenic peptides (MAPs).

Techniques: Control, Enzyme-linked Immunospot, Recombinant

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-4939-8567-8

Figure Lengend Snippet: Fig. 1 Effects of illumination conditions on the results of the automated search process. (a) Field of view in bottom of the ELISPOT well is evenly illuminated which results in almost a uniform background. (b) Illumination is uneven and as a result, the software easily detects sharp round spots, whereas blurry spots that have irregu- lar form may remain undetected

Article Snippet: Feline (Fe) IFNγ ELISPOT Development Module, FeIL-2 ELISPOT Development Module, and FeIL-10 ELISPOT Development Module (R&D Systems Inc., Minneapolis, MN) included a matching pair of antibodies used for capture and detection of individual feline cytokine and ELISPOT Blue Color Module (R&D Systems Inc.): (a) Respective feline cytokine (IFNγ, IL-2, IL-10) Capture Antibody Concentrate reconstituted in 1 mL PBS and diluted 1 in 50 parts PBS. (b) Respective feline cytokine (IFNγ, IL-2, IL-10) Detection Antibody Concentrate reconstituted in 1 mL reagent diluent and diluted in 50 parts reagent diluent Fig. 1 Structure and sequences of the four multiple antigenic peptides (MAPs).

Techniques: Enzyme-linked Immunospot, Software

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-4939-8567-8

Figure Lengend Snippet: Fig. 1 A schematic flowchart of ELISpot assay to detect Ag-specific ASCs. (A) Pre-coat the wells of the ELISpot plate with an Ag, e.g., tetanus toxin. (B) Seed serial diluted PBMCs into wells of the plate, respectively. Culture overnight (minimum: 8 h). (C) Wash off cells with PBS-T. (D) Add AP-conjugated detection Abs specific to IgM, IgG, or IgA. (E) Wash off unbound Abs. (F) Develop the spots with BCIP/NBT substrate solution

Article Snippet: Feline (Fe) IFNγ ELISPOT Development Module, FeIL-2 ELISPOT Development Module, and FeIL-10 ELISPOT Development Module (R&D Systems Inc., Minneapolis, MN) included a matching pair of antibodies used for capture and detection of individual feline cytokine and ELISPOT Blue Color Module (R&D Systems Inc.): (a) Respective feline cytokine (IFNγ, IL-2, IL-10) Capture Antibody Concentrate reconstituted in 1 mL PBS and diluted 1 in 50 parts PBS. (b) Respective feline cytokine (IFNγ, IL-2, IL-10) Detection Antibody Concentrate reconstituted in 1 mL reagent diluent and diluted in 50 parts reagent diluent Fig. 1 Structure and sequences of the four multiple antigenic peptides (MAPs).

Techniques: Enzyme-linked Immunospot

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-4939-8567-8

Figure Lengend Snippet: Fig. 1 Flowchart detailing the ELISPOT protocol at a glance

Article Snippet: Feline (Fe) IFNγ ELISPOT Development Module, FeIL-2 ELISPOT Development Module, and FeIL-10 ELISPOT Development Module (R&D Systems Inc., Minneapolis, MN) included a matching pair of antibodies used for capture and detection of individual feline cytokine and ELISPOT Blue Color Module (R&D Systems Inc.): (a) Respective feline cytokine (IFNγ, IL-2, IL-10) Capture Antibody Concentrate reconstituted in 1 mL PBS and diluted 1 in 50 parts PBS. (b) Respective feline cytokine (IFNγ, IL-2, IL-10) Detection Antibody Concentrate reconstituted in 1 mL reagent diluent and diluted in 50 parts reagent diluent Fig. 1 Structure and sequences of the four multiple antigenic peptides (MAPs).

Techniques: Enzyme-linked Immunospot

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-4939-8567-8

Figure Lengend Snippet: Fig. 1 A typical cultured ELISpot response to the panel of DENV serotype specific peptides. (a) The layout of the ELISpot for the 17 DENV serotype specific peptides is shown, with each peptide response done in duplicate. (b) A response to these peptides from one dengue seropositive donor is shown with responses to DENV2 pep- tide 11 and 18, DENV4 peptide 5 and 19 and PHA. Therefore, as this individual responds to SS peptides of DENV2 and DENV4, it is likely that he/she has been infected with these two DENV serotypes in the past

Article Snippet: Feline (Fe) IFNγ ELISPOT Development Module, FeIL-2 ELISPOT Development Module, and FeIL-10 ELISPOT Development Module (R&D Systems Inc., Minneapolis, MN) included a matching pair of antibodies used for capture and detection of individual feline cytokine and ELISPOT Blue Color Module (R&D Systems Inc.): (a) Respective feline cytokine (IFNγ, IL-2, IL-10) Capture Antibody Concentrate reconstituted in 1 mL PBS and diluted 1 in 50 parts PBS. (b) Respective feline cytokine (IFNγ, IL-2, IL-10) Detection Antibody Concentrate reconstituted in 1 mL reagent diluent and diluted in 50 parts reagent diluent Fig. 1 Structure and sequences of the four multiple antigenic peptides (MAPs).

Techniques: Cell Culture, Enzyme-linked Immunospot, Infection

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-4939-8567-8

Figure Lengend Snippet: Fig. 1 A typical ex vivo ELISpot assay with PHA as the positive control, DENV NS3 overlapping peptides and media as the negative control

Article Snippet: Feline (Fe) IFNγ ELISPOT Development Module, FeIL-2 ELISPOT Development Module, and FeIL-10 ELISPOT Development Module (R&D Systems Inc., Minneapolis, MN) included a matching pair of antibodies used for capture and detection of individual feline cytokine and ELISPOT Blue Color Module (R&D Systems Inc.): (a) Respective feline cytokine (IFNγ, IL-2, IL-10) Capture Antibody Concentrate reconstituted in 1 mL PBS and diluted 1 in 50 parts PBS. (b) Respective feline cytokine (IFNγ, IL-2, IL-10) Detection Antibody Concentrate reconstituted in 1 mL reagent diluent and diluted in 50 parts reagent diluent Fig. 1 Structure and sequences of the four multiple antigenic peptides (MAPs).

Techniques: Ex Vivo, Enzyme-linked Immunospot, Positive Control, Negative Control

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-4939-8567-8

Figure Lengend Snippet: Fig. 1 Typical IFN-γ ELISpot images showing antigen-specific responses to serotypes of DENV E protein from a representative DENV-immune and a DENV-naïve subject

Article Snippet: Feline (Fe) IFNγ ELISPOT Development Module, FeIL-2 ELISPOT Development Module, and FeIL-10 ELISPOT Development Module (R&D Systems Inc., Minneapolis, MN) included a matching pair of antibodies used for capture and detection of individual feline cytokine and ELISPOT Blue Color Module (R&D Systems Inc.): (a) Respective feline cytokine (IFNγ, IL-2, IL-10) Capture Antibody Concentrate reconstituted in 1 mL PBS and diluted 1 in 50 parts PBS. (b) Respective feline cytokine (IFNγ, IL-2, IL-10) Detection Antibody Concentrate reconstituted in 1 mL reagent diluent and diluted in 50 parts reagent diluent Fig. 1 Structure and sequences of the four multiple antigenic peptides (MAPs).

Techniques: Enzyme-linked Immunospot

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-4939-8567-8

Figure Lengend Snippet: Fig. 1 Structure and sequences of the four multiple antigenic peptides (MAPs). The two MAPs on the left (top and bottom) consist of FIV p24 peptides (Fp9-3 and Fp14-3/4), and those on the right consist of FIV reverse transcriptase (RT) peptides (FRT3-3/4 and FRT7-1/2). The sequences of the long peptide and its smaller over- lapping peptides are shown for Fp14-3/4 (Fp14-3, Fp14-4), FRT3-3/4 (FRT3-3, FRT3-4), and FRT7-1/2 (FRT7- 1, FRT7-2). Four identical FIV peptides are on the amino-end of the MAP attached to a branched lysine backbone with palmitic acid (Pam) on the carboxyl-end. The final MAP vaccine consisted of all four MAPs at equal amounts (100 μg/dose each; total 400 μg/dose). Each MAP and its long and short peptides were used as FIV peptide stimulants in the IL2, IFNγ, and IL10 ELISpot assays and the T-cell proliferation analysis

Article Snippet: Feline (Fe) IFNγ ELISPOT Development Module, FeIL-2 ELISPOT Development Module, and FeIL-10 ELISPOT Development Module (R&D Systems Inc., Minneapolis, MN) included a matching pair of antibodies used for capture and detection of individual feline cytokine and ELISPOT Blue Color Module (R&D Systems Inc.): (a) Respective feline cytokine (IFNγ, IL-2, IL-10) Capture Antibody Concentrate reconstituted in 1 mL PBS and diluted 1 in 50 parts PBS. (b) Respective feline cytokine (IFNγ, IL-2, IL-10) Detection Antibody Concentrate reconstituted in 1 mL reagent diluent and diluted in 50 parts reagent diluent Fig. 1 Structure and sequences of the four multiple antigenic peptides (MAPs).

Techniques: Reverse Transcription, Enzyme-linked Immunospot

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

doi: 10.1007/978-1-4939-8567-8

Figure Lengend Snippet: Fig. 1 Influenza A/H1N1-specific memory B cell response pre- and post-influenza vaccination. Figure 1 demonstrates the dynamics of influenza-specific memory B cell ELISPOT response after influenza vaccination (in a cohort of 106 healthy older adults, 50–74 years old) and has been previously published in Viral Immunology [7] and Plos One [8]. The top (bottom) of the box indicates the 75th (25th) percentiles, respectively, while the bold line within the box indicates the median. The “whiskers” extend up to 1.5 times the interquartile range above or below the 75th or 25th percentiles, respectively. Beyond that point, individual points are plotted. B cell ELISPOT counts representing the influenza A/H1N1- specific IgG-producing memory-like B cell response plotted for each timepoint as spot forming units (SFUs) per 2 × 105 PBMCs

Article Snippet: Feline (Fe) IFNγ ELISPOT Development Module, FeIL-2 ELISPOT Development Module, and FeIL-10 ELISPOT Development Module (R&D Systems Inc., Minneapolis, MN) included a matching pair of antibodies used for capture and detection of individual feline cytokine and ELISPOT Blue Color Module (R&D Systems Inc.): (a) Respective feline cytokine (IFNγ, IL-2, IL-10) Capture Antibody Concentrate reconstituted in 1 mL PBS and diluted 1 in 50 parts PBS. (b) Respective feline cytokine (IFNγ, IL-2, IL-10) Detection Antibody Concentrate reconstituted in 1 mL reagent diluent and diluted in 50 parts reagent diluent Fig. 1 Structure and sequences of the four multiple antigenic peptides (MAPs).

Techniques: Enzyme-linked Immunospot