Journal: Bioactive Materials

Article Title: Cascade-targeted delivery platform enhances antigen cross-presentation and STING activation for durable cellular immunity

doi: 10.1016/j.bioactmat.2025.08.033

Figure Lengend Snippet: 16a-HSA NEs Efficiently Induce Antigen-Specific Immune Responses In Vivo . a , Evaluation schematic illustration of antigen-specific immune responses in mice following immunization with PBS, FO, 0O, 16O, FOA, 0OA, and 16OA formulations. b - d , Antigen-specific antibody levels induced by each immunization group in mice (n = 6 per group): total antibody IgG ( b ), antibody subtype IgG1 ( c ), antibody subtype IgG2a ( d ). e , f , Antigen-specific CTL lysis induced by each immunization group in mice ( e ) (n = 6 per group) and representative flow cytometry analysis plots ( f ). g - j , Activation of B and T cells within the lymph nodes specifically induced by each immunization group (n = 6 per group): memory B cells ( g ), germinal center ( h ), activated CD4 + T cells ( i ), activated CD8 + T cells ( j ). k , Splenic lymphocytes from immunized mice were isolated and subjected to intracellular cytokine staining (ICS) and ELISPOT assays as illustrated. Created with BioRender.com . l , ICS results in splenic T lymphocytes from immunized mice: the level of CD4 + or CD8 + T cells expressing IFN-γ, TNF-α (n = 6 per group); m-o , ELISPOT analysis of IFN-γ ( m ) and IL-4 ( n ) secretion by splenic lymphocytes (n = 4 per group), along with representative images ( o ); p , spleen lymphocyte secretion levels of IL-1β, IL-4, IL-6, IFN-γ, TNF-α (pg/mL).

Article Snippet: ELISpot Plus: Mouse IFN-γ (ALP) kit and ELISpot Plus: Mouse IL-4 (ALP) kit were purchased from Mabtech.

Techniques: In Vivo, Lysis, Flow Cytometry, Activation Assay, Isolation, Staining, Enzyme-linked Immunospot, Expressing

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: Commercially ready to use human IFNγ ELISpot kit (Mabtech AB, Sweden).

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. 2 Steps of an Elispot experiment. The first step pertains to the preparation of the sample (guidance available in [6, 25]), which may include cell isolation (e.g., PBMC from whole blood), freezing, thawing, resting, counting and viability assessment, and perhaps isolation of subpopulations or expansion of cells. The second step, the assay (guidance available in [25, 31]), typically includes the coating and blocking of the Elispot plate, the plating of the sample and stimu- lants, incubation, and removal of cells and spot development. The third step, labeled by a star above the box, includes the enumeration of spots (guidance available in [24, 32]), and the analysis of raw spot numbers for obtaining mea- sures for response definition (guidance available in [33, 34]). This article focuses on the first part of step 3, the spot enumeration

Article Snippet: Commercially ready to use human IFNγ ELISpot kit (Mabtech AB, Sweden).

Techniques: Enzyme-linked Immunospot, Cell Isolation, Isolation, Blocking Assay, Incubation, Labeling

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

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

Figure Lengend Snippet: Fig. 3 True and artificially altered well images. Images on the left hand side were taken with a Zeiss reader, with the RGB alignment and exposure settings optimally set as demonstrated in Fig. 4. The real specimen (Elispot plate) exposed high bluish-grey background staining, caused by typical overstaining with BCIP/NBT, and faint spots which were barely recognizable by eye. The upper panel image was taken from a well contain- ing cells stimulated with an antigen. The negative control (cells plus medium only) is shown in the lower panel. The right side images were obtained with a different reader system that performed automated color and intensity adjustments to artificially change the image to highly intense blue staining with nonrealistically strong spots, a process that was not controlled appropriately. Due to the falsely increased presentation of signals (and high sensitivity settings), counted spot numbers in the antigen-stimulated well and the negative control are inaccurately high. While perhaps looking more pleasing to the eye; the images on the right hand side are deceitful and do not reflect the true conditions of that Elispot plate. A part of the well periphery was also cut out and is not represented in the image

Article Snippet: Commercially ready to use human IFNγ ELISpot kit (Mabtech AB, Sweden).

Techniques: Enzyme-linked Immunospot, Staining, Negative Control

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

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

Figure Lengend Snippet: Fig. 4 Optimal camera adaption of exposure and RGB alignment. The intensity of each of the three light chan- nels (RGB) is converted onto a grey scale that per definition has the lowest value set as zero (black, see arrow at left side of diagram) and the highest value at 255 (white, see arrow at right side of diagram). The camera should be adjusted so that all three signals have the same intensity (arrow pointing to the tops of the RGB curves). If this is not the case, the image may appear more red, blue, or green, depending on which signal has the highest intensity. Elispot reader systems have typically a command that allows the automated adjustment of the signal strength and the alignment of the RGB channels (also called Auto-White Balance = AWB). Further, the exposure determines how dark or bright an image will appear. It is important to adjust the exposure set- tings in a way that none of the signals from the RGB channels are lost in overexposure (“too bright,” arrow pointing to the right end bottom of RGB signal curves). Underexposure leads to too dark images

Article Snippet: Commercially ready to use human IFNγ ELISpot kit (Mabtech AB, Sweden).

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. 5 Concave well membrane. A well image from an old format MAHA well plate with many spots is shown. The image was taken with a KS Elispot reader system (Carl Zeiss, Inc., Thornwood, NY, USA). While spots in the well center are in focus, the well is shaped highly concave with the well periphery being completely out of focus and spots cannot be accurately discriminated and counted

Article Snippet: Commercially ready to use human IFNγ ELISpot kit (Mabtech AB, Sweden).

Techniques: Membrane, 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. 6 Elispot well with artifact ring. PBMC were tested for IFNɣ release. A ring of small artifacts is recognizable in the well periphery (arrow), indicative of cell death. A few true spots are also detectable, among a faint cover of artifacts across the membrane. It is advisable to minimize the AOI to exclude the artifact ring in the well periphery. The image was taken with a KS Elispot reader system (Carl Zeiss, Inc., Thornwood, NY, USA)

Article Snippet: Commercially ready to use human IFNγ ELISpot kit (Mabtech AB, Sweden).

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. 8 Example of a B-cell Elispot well testing human B cells for IgG release. The well was coated with the antigen (whole protein) of interest. Spots are very large, requiring algorithm settings focusing on detecting large spots, combing small staining differences within a spot to one spot. The image was taken with an AID iSpot Reader system (Strassberg, Germany)

Article Snippet: Commercially ready to use human IFNγ ELISpot kit (Mabtech AB, Sweden).

Techniques: Enzyme-linked Immunospot, Staining

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

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

Figure Lengend Snippet: Fig. 12 DNA precipitates. The well image contains a large netlike artifact that was caused by a DNA precipitate. Two blue spots can also be seen above the artifact. The image was taken with a KS Elispot reader system (Carl Zeiss, Inc., Thornwood, NY)

Article Snippet: Commercially ready to use human IFNγ ELISpot kit (Mabtech AB, Sweden).

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. 13 Granulocyte contamination. Two well images are presented (left image developed with TMB, right image developed with AEC) from PBMC testing. High levels of white ghost spots can be recognized in both wells, and spot disintegration is evident. It is very challenging to adjust parameters to obtain accurate spot counts. It is recommended to set the algorithm with focus on large spots, in order to allow the software to “repair” some of the spot disintegration and to avoid multiple counts for the same spot. The images were taken by a KS Elispot reader system (Carl Zeiss, Inc., Thornwood, NY)

Article Snippet: Commercially ready to use human IFNγ ELISpot kit (Mabtech AB, Sweden).

Techniques: Software, 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. 15 Changing amount of artifacts within a sample. Four well images present different stimulation condi- tions for the same PBMC sample in an IFNɣ Elispot. There are high amount of artifacts present in the negative control as well as in two different stimulations with peptide antigen. When cells are stimulated with SEB, the amount of artifacts decreases dramatically, and many well-defined spots are visible. The images were taken with a KS Elispot reader system (Carl Zeiss, Inc., Thornwood, NY)

Article Snippet: Commercially ready to use human IFNγ ELISpot kit (Mabtech AB, Sweden).

Techniques: Enzyme-linked Immunospot, 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 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: Commercially ready to use human IFNγ ELISpot kit (Mabtech AB, Sweden).

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. 3 Example of nonspecific spots generated by cells sticking to PVDF membrane in the ELISpot plate. Cells sticking to the PVDF membrane in the ELISpot plate can be the source of a signal that resembles spots formed by cell-secreting proteins. This image illustrates nonspecific spots using HRP-AEC chromogenic detection (red color)

Article Snippet: Commercially ready to use human IFNγ ELISpot kit (Mabtech AB, Sweden).

Techniques: Generated, Membrane, 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. 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: Commercially ready to use human IFNγ ELISpot kit (Mabtech AB, Sweden).

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: Commercially ready to use human IFNγ ELISpot kit (Mabtech AB, Sweden).

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. 2 Spots color subdivision for ELISPOT images. Left panel—the image with large dark blue spots and small red spots. Five blue and eight red spots were selected. Right panel—image with small brown and gray spots. Eight gray and 40 brown spots were selected

Article Snippet: Commercially ready to use human IFNγ ELISpot kit (Mabtech AB, Sweden).

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 CD8 cell activation and differentiation states can be identified via their expression pattern of IFN-γ, TNF- α, IL-2, and GzB. (A): Naïve CD8 cell, (B): Terminal effector CD8 cell, (C): Central CD8 memory cell, (D): Silent CD8 killer cell, (E): Polyfunctional CD8 cell, (F): Stem-cell like CD8 memory cell, TSCM, (G): Dysfunctional CD8 cell, (H): Senescent CD8 cell. The phenotypes shown here correspond to the consensus nomenclature for CD8 T cell phenotypes established in 2015 [14]. Here we focus on the analyte secretion patterns of these subsets. The corresponding cell surface markers that can be established by flow cytometry are summarized in that publication

Article Snippet: Commercially ready to use human IFNγ ELISpot kit (Mabtech AB, Sweden).

Techniques: Activation Assay, Expressing, Flow Cytometry

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

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

Figure Lengend Snippet: Fig. 2 Four color ImmunoSpot® results measuring IFN-γ, TNF-α, IL-2, and GzB. The four color assay was per- formed as described here testing CEF-peptide-reactive CD8 cells. For each analyte, a separate channel was established permitting detection of that analyte without cross-bleeding of color. Panel (a) shows the IFN-γ channel in green (b) the IL-2 channel in red, (c) the TNF-α channel in yellow, and (d) the GzB channel in blue. The overlay of the four color planes is shown in (e). Polyfunctional CD8 cells positive for all four analytes have been counted in (f). The fact that these cells are indeed quadruple positive, as opposed to resulting from ran- dom overlays was established by serial dilution of the cells

Article Snippet: Commercially ready to use human IFNγ ELISpot kit (Mabtech AB, Sweden).

Techniques: Serial Dilution

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

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

Figure Lengend Snippet: Fig. 1 Assay principle of the B cell FluoroSpot used for the analysis of cross-reactive B cells. Immobilized anti- mouse IgG antibodies were used to capture IgG secreted by splenocytes from a mouse immunized with cat interferon gamma (IFN-γ). IgG secreted by B cells was allowed to bind two structurally similar antigens, tagged with biotin (cat IFN-γ; blue antigen) or a peptide tag (dog IFN-γ; red antigen), respectively. By using fluores- cently labeled detection systems, i.e., streptavidin (SA) with red fluorophore and anti-tag antibody with green fluorophore, respectively, cross-reactivity of IgG antibodies from single B cells was analyzed. Antibodies having bound only one of the antigens were detected by only one detection system while cross-reactive antibodies were identified by their binding of both tagged antigens

Article Snippet: Commercially ready to use human IFNγ ELISpot kit (Mabtech AB, Sweden).

Techniques: Labeling, Binding Assay

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

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

Figure Lengend Snippet: Fig. 2 FluoroSpot analysis of anti-cat interferon gamma (IFN-γ)-reactive B cells, potentially cross-reactive with dog IFN-γ. (a) Separate images from wells demonstrating splenocyte reactivity with cat (left) and dog IFN-γ (right) using tagged antigens and fluorescently labeled detection systems. (b) Computerized overlay of the two separate well images enables the identification of co-localized spots, i.e., cross-reactive splenocytes. The mag- nified image displays a single-positive spot (red) and two double-stained co-localized spots (yellow). (c) Number of spots in the separate images. All green spots represent cross-reactive B cells (co-localized spots)

Article Snippet: Commercially ready to use human IFNγ ELISpot kit (Mabtech AB, Sweden).

Techniques: Labeling, Staining

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

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

Figure Lengend Snippet: Fig. 2 Counting of individual color spots in the triple-color human IFN-γ (green), TNF-α (blue), and IL-2 (red) T-cell FLUOROSPOT assay. Magnified regions of the well scanned and counted using individual color detection is shown on top panels a, b, and c. Single-color spots count overlays are also shown on the merged triple-color images (bottom panels d, e, and f). Partially overlapping spots were separated by spot separation function of ImmunoSpot® Software

Article Snippet: Commercially ready to use human IFNγ ELISpot kit (Mabtech AB, Sweden).

Techniques: Flurospot, Software

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

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

Figure Lengend Snippet: Fig. 7 Counting of dual- and triple-color spots in the human IFN-γ (green), TNF-α (blue), and IL-2 (red) T-cell FLUOROSPOT assay (magnified region of the well is shown). All combinations of two color spot overlays (panels a, b, and c) and triple-color spot overlays (panel d) are shown superimposed over the merged tri- ple-color image

Article Snippet: Commercially ready to use human IFNγ ELISpot kit (Mabtech AB, Sweden).

Techniques: Flurospot

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

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

Figure Lengend Snippet: Fig. 8 Multi-color counting results: single-color spot outlines are shown for the individual fluorescence chan- nels: GzB—450 nm blue (a), IFN-γ—520 nm green (b), TNF-α—580 nm yellow (c), and IL-2—690 nm red (d). Triple-color and four-color outlines are shown for GzB/IFN-γ/TNF-α triple-positive (e) and GzB/IFN-γ/TNF-α/ IL-2 qudrouple-positive spots on the four-color merged image (f)

Article Snippet: Commercially ready to use human IFNγ ELISpot kit (Mabtech AB, Sweden).

Techniques: Fluorescence

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: Commercially ready to use human IFNγ ELISpot kit (Mabtech AB, Sweden).

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. 2 Representative ELISpot images of tetanus toxin-specific IgG ASCs detected from PBMCs. PBMCs were isolated from a healthy donor 2 weeks after receiving a booster of tetanus toxin. ELISpot plate was pre-coated with 50 μL/well of tetanus toxin (10 μg/mL in PBS). Aliquots of 5 × 105, 2.5 × 105, and 1.25 × 105 PBMCs (#1, #2 and #3) were seeded into wells of the ELISpot plate, respectively. Tetanus toxin-specific IgG ASCs were detected and illustrated

Article Snippet: Commercially ready to use human IFNγ ELISpot kit (Mabtech AB, Sweden).

Techniques: Enzyme-linked Immunospot, Isolation

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: Commercially ready to use human IFNγ ELISpot kit (Mabtech AB, Sweden).

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. 2 Examples of TNTC, positive, and negative ELISPOT wells. After development and drying, ELISPOT wells were imaged using an ELISPOT plate reader. The first well is an example of a well that is too numerous to count, or TNTC. The second well is a positive well with distinct and easily separated spots. The third well is a negative well containing only cell debris and no spots

Article Snippet: Commercially ready to use human IFNγ ELISpot kit (Mabtech AB, Sweden).

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: Commercially ready to use human IFNγ ELISpot kit (Mabtech AB, Sweden).

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: Commercially ready to use human IFNγ ELISpot kit (Mabtech AB, Sweden).

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 Representative wells for IFN-γ and IL-4 ELISpots carried out for dengue patients and controls. When PBMCs of both patients and controls (healthy individuals) were stimulated with α-GalCer, IFN-γ responses were clearly observed but the IL-4 responses were extremely low

Article Snippet: Commercially ready to use human IFNγ ELISpot kit (Mabtech AB, Sweden).

Techniques:

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: Commercially ready to use human IFNγ ELISpot kit (Mabtech AB, Sweden).

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. 2 IFN-γ responses measured by the dendritic cell-based ELISpot correlated with the immune status of the subjects suggesting the assay is antigen specific. SPUs to dengue serotype-specific E proteins in seven immune subjects (a) and seven naïve subjects (b)

Article Snippet: Commercially ready to use human IFNγ ELISpot kit (Mabtech AB, Sweden).

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. 3 IFN-γ memory response was detected 9 years after the exposure to dengue infection. Three dengue- immune subjects (a, b, and c) received live-attenuated dengue vaccine in year 2001 and IFN-γ response was measured in samples obtained in 2007, 2008, and 2010

Article Snippet: Commercially ready to use human IFNγ ELISpot kit (Mabtech AB, Sweden).

Techniques: Infection

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: Commercially ready to use human IFNγ ELISpot kit (Mabtech AB, Sweden).

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. 2 IL2, IFNγ, and IL10 responses of MAP-vaccinated cats at 6 weeks post-second vaccination. The IL2 (a), IFNγ (b), and IL10 (c) responses were after the in vitro stimulation of the PBMC from the vaccinated cats with FIV MAPs and peptides. One set of seven cats was subcutaneously vaccinated with the MAP vaccine (grey bars), and another set of seven was intradermally vaccinated with MAP vaccine (blue bar). Each bar represents the value after subtraction of the average value of the results from the nine control cats, with the exception of the mitogens (ConA and PMA/PHA). The results for the mitogens (a–c) are without any subtraction. The p-value (t-test) in red above the bars of each FIV stimulant indicates that the results from the combined vaccine group are significantly elevated in response to the corresponding FIV stimulant when compared to the results from the control cat group. Note that FIV stimulant IWV, which is inactivated whole viruses, do not stimulate any cytokine responses since no cats were immunized in this study with IWV. Furthermore, T cells from MAP- vaccinated cats can recognize the FIV MAPs and/or the FIV peptides present in the vaccine

Article Snippet: Commercially ready to use human IFNγ ELISpot kit (Mabtech AB, Sweden).

Techniques: In Vitro, Control

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

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

Figure Lengend Snippet: Fig. 3 Constitutive IL2, IFNγ, and IL10 production of the PBMC of vaccinated and non-vaccinated cats. The constitutive production or the spontaneous release of IL2 (a), IFNγ (b), and IL10 (c) are shown for the PBMC of the seven subcutaneous vaccinated cats (Vaccine Group 1, grey bars), seven intradermally vaccinated cats (Vaccine Group 2, blue bars), and nine non-vaccinated control cats (Control Group, black bars) at 6 weeks post-second vaccination. The average (ave) value of each group is shown above the bars of each group. Note that the magnitude of the IL10 spontaneous release is about 1000-fold higher (see y-axis) than those of IL2 and IFNγ

Article Snippet: Commercially ready to use human IFNγ ELISpot kit (Mabtech AB, Sweden).

Techniques: Control

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

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

Figure Lengend Snippet: Fig. 6 IL2, IFNγ, and IL10 responses of MAP-vaccinated cats at 6 weeks post-third vaccination. The IL2 (a), IFNγ (b), and IL10 (c) responses were after the in vitro stimulation of the PBMC from vaccinated cats with FIV MAPs and peptides. One set of seven cats was subcutaneously vaccinated with the MAP vaccine (grey bars), and another set of seven was intradermally vaccinated with MAP vaccine (blue bars). Each bar for the IL2 (a) and the IFNγ (b) responses represents the value after the subtraction of the average value of the results from the nine control cats, with the exception of the mitogens (ConA and PMA/PHA). Each bar for the IL10 responses (c) represents the value without any subtractions. The results for the mitogens (a–c) are shown without any subtraction. The p-value (t-test) in red above the bar(s) for the FIV stimulant indicates that the results from the combined vaccine group were significantly elevated in response to the corresponding FIV stimulant when compared to the results from the control cat group. The p-values on the bottom of the bars represent the statistical comparison between combined vaccine group and their media controls. The p-values in red repre- sent significant increase or elevation, whereas those in blue represent significant decrease or suppression

Article Snippet: Commercially ready to use human IFNγ ELISpot kit (Mabtech AB, Sweden).

Techniques: In Vitro, Control, Comparison

Journal: Methods in Molecular Biology

Article Title: Handbook of ELISPOT

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

Figure Lengend Snippet: Fig. 8 Constitutive IL2, IFNγ, and IL10 production of the PBMC of vaccinated and non-vaccinated cats at post- third vaccination. The constitutive production or the spontaneous release of IL2 (a), IFNγ (b), and IL10 (c) are shown for the PBMC of the seven subcutaneous vaccinated cats (Vaccine Group 1, grey bars), seven intrader- mally vaccinated cats (Vaccine Group 2, blue bars), and nine non-vaccinated control cats (Control Group, black bars) at 6 weeks post-third vaccination. The average (ave) value of each group is shown above the bars of each group. Note that the magnitude of the IL10 spontaneous release is 2000-fold to 20,000-fold higher (see y-axis) than those of IL2 and IFNγ, respectively

Article Snippet: Commercially ready to use human IFNγ ELISpot kit (Mabtech AB, Sweden).

Techniques: Control

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: Commercially ready to use human IFNγ ELISpot kit (Mabtech AB, Sweden).

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. 3 Example of programming: The figure represents a short impression how the automated script of the ELISpot assay can be programmed. Modules like “sub-routine” or “group” are included, as well as variables, loops, and the standard basic commands “aspirate” and “dispense”

Article Snippet: Commercially ready to use human IFNγ ELISpot kit (Mabtech AB, Sweden).

Techniques: Enzyme-linked Immunospot

Journal: Cell Reports Medicine

Article Title: Engineering lipid nanoparticle-stabilized emulsions for spatiotemporal mRNA delivery and enhanced T cell immunity

doi: 10.1016/j.xcrm.2026.102667

Figure Lengend Snippet: Coordinating mRNA expression kinetics with immune dynamics for Th1-biased cellular immunity (A) Immunization regimen and timeline for immune kinetics analysis. C57BL/6J mice ( n = 6) received intramuscular injections of LNP or LSE formulations containing 5 μg Spike mRNA on days 0 and 28. GC B cell and Tfh cell responses in the dLNs were analyzed by flow cytometry on days 7, 14, 21, 28, 29, and 35 (blue). Serum antigen-specific IgG titers were measured by ELISA on days 7, 14, 21, 28, 35, 42, 49, 56, 63, 77, 91, 188, and 268 (green). T cell dynamics in splenocytes were evaluated by intracellular cytokine staining (ICS) on days 21, 28, 29, 35, and 56 (red). (B) Recruitment of DCs ( n = 6) at the injection site via flow cytometry at the indicated time points. (C–F) GC B cell (B220 + GL-7 + FAS + , C and E) and Tfh cell (CD4 + CXCR5 + PD-1 + , D and F) responses in dLNs. Gating strategy is shown in B. (G and H) Humoral immune responses. (G) Serum Spike-specific IgG titers. (H) IgG2a/IgG1 ratios at days 56 as an indicator of Th1/Th2 polarization. (I) Frequencies of Spike-specific CD4 + T cells producing TNF-α, IL-2, or IFN-γ in splenocytes, with LSE shown as red solid lines and LNP as blue dashed lines. Sample sizes were n = 6 for days 21–35 and n = 8 for day 56. Gating strategy is shown in C. (J and K) ELISpot analysis of cytokine-producing splenocytes at day 56 ( n = 8). IFN-γ-producing (G) and IL-4-producing (H) spot-forming cells were quantified among antigen-specific peptide-restimulated splenocytes, reported as spot-forming units per 10 6 cells. Representative ELISpot images (left) and corresponding statistical analyses (right) are shown. In (J and K), data were presented as mean ± SEM. Statistical significance was determined by one-way ANOVA compared to LSE-injected mice with post hoc Dunnett’s multiple comparisons tests. ∗∗∗∗ p < 0.0001.

Article Snippet: ELISpot Plus: Mouse IFN-γ (ALP) , Mabtech , Cat# 3321-4AST-2.

Techniques: Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Staining, Injection, Enzyme-linked Immunospot

Journal: Cell Reports Medicine

Article Title: Engineering lipid nanoparticle-stabilized emulsions for spatiotemporal mRNA delivery and enhanced T cell immunity

doi: 10.1016/j.xcrm.2026.102667

Figure Lengend Snippet: Potent and long-term VZV gE-specific T cell immune responses (A–F) LSE-induced enhanced VZV gE-specific T cell responses, compared with LNP. (A) gE-specific CD4 + T cell responses following prime-boost immunization of LSE and LNP (5 μg gE mRNA per C57BL/6 mouse, n = 6) with a 4-week interval. PBS served as a control. PBMCs collected at days 56, 70, 120, and 300 post-primary immunization were stimulated with gE-overlapping peptides and analyzed by ICS using flow cytometry. (B) Frequencies of gE-specific IFN-γ + (left) and IL-2 + (right) CD4 + T cells following immunization ( n = 6). (C) Cytokine secretion profiles (IL-2, IFN-γ, TNF-α, IL-21) of splenocytes at day 120 post-primary dosing, measured via ELISA assay and presented as a heatmap ( n = 6). (D) IFN-γ (top) and IL-2 (bottom) ELISpot assay of splenocytes following stimulation on day 120 after primary immunization ( n = 6). Representative ELISpot images (left) and corresponding spot-forming cell counts (right) are shown. (E) T CM (CD44 + CD62L + ) and T EM (CD44 + CD62L − ) in CD4 + and CD8 + T cells among the splenocytes at day 120. (F) Proliferation of CD4 + and CD8 + T cells at day 300 post-primary administration. T cell proliferation was evaluated using a CFSE dilution assay. Splenocytes were labeled with CFSE and stimulated with gE-overlapping peptides. After 72 h, CFSE dilution in CD4 + or CD8 + T cells was analyzed by flow cytometry to determine proliferation. (G–R) VZV gE-specific T cell response with enhanced clonal expansion and repertoire diversity, compared with Shingrix. (G) Schematic workflow of vaccination, T cell immunity assessment, and TCR sequencing. Female C57BL/6 mice ( n = 6) were immunized with LNP or LSE formulations containing 5 μg gE mRNA per mouse using a prime-boost regimen with a 4-week interval; PBS served as the control. Splenocytes collected on days 27 and 56 were sorted into T cell populations and subjected to antigen-stimulated and unstimulated TCR sequencing, and cellular immunity was analyzed at day 120. Image created with BioRender.com . (H and I) Frequencies of VZV gE-specific CD4 + (H) and CD8 + (I) T cells producing IFN-γ, IL-2, and TNF-α at day 120 post-immunization, assessed by ICS. (J) IFN-γ (left) and IL-2 (right) ELISpot assay of splenocytes following stimulation, at day 120 after immunization ( n = 6). (K and L) T CM and T EM subsets within CD4 + (K) and CD8 + (L) T cell populations among splenocytes on day 120 after primary immunization ( n = 6). (M) T cell proliferation on days 28, 56, and 120 post-primary dosing, assessed using a CFSE dilution assay. (N) Schematic of total and gE-reactive TCR repertoires analysis. Set A corresponds to total TCR repertoire from T cells sorted pre-stimulation, Set B includes gE-reactive TCR repertoire from proliferated T cells post-stimulation. Set C is intersection of two TCR repertoires. (O) Clonality of clonotypes at various stages of immunity. Bubble chart (left) shows TCR clonotypes for LSE, and Shingrix groups, with circle size indicating clonal fraction. The clonality index (right) compares the total TCR repertoire (Set A) across three groups at different immunity stages. (P) Cumulative fraction of gE-reactive clonotypes within the total TCR repertoire. (Q) Clonotype diversity of gE-reactive TCR repertoires quantified by Shannon’s index. (R) The proportion of high clonotypes (clone fraction ≥0.01%) within Set B, as a fraction of the total clonotype population. In (B, H, and I), for boxplots, the box extends from the minimum to the maximum values, with the line indicating the median. Statistical significance was determined by two-way ANOVA with post hoc Šídák’s multiple comparisons tests. In (D, K, and L), data were presented as mean ± SEM, statistically analyzed by two-way ANOVA with post hoc Šídák’s multiple comparisons tests. In (F and M), data were presented as mean ± SEM, statistically analyzed by two-way ANOVA compared to LSE-injected mice with post hoc Dunnett’s multiple comparisons tests. In (J), data were presented as mean ± SEM, statistically analyzed by one-way ANOVA with post hoc Tukey’s multiple comparisons tests. ns, p ≥ 0.05, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: ELISpot Plus: Mouse IFN-γ (ALP) , Mabtech , Cat# 3321-4AST-2.

Techniques: Control, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot, Dilution Assay, Labeling, Sequencing, Injection

Journal: Cell Reports Medicine

Article Title: Engineering lipid nanoparticle-stabilized emulsions for spatiotemporal mRNA delivery and enhanced T cell immunity

doi: 10.1016/j.xcrm.2026.102667

Figure Lengend Snippet: Enhanced anti-tumor effects in mice (A) Schematic illustration of the vaccination regimen. Female C57BL/6 ( n = 5) mice were intramuscularly injected with LNP or LSE formulations containing 5 μg OVA mRNA per mouse. (B) Flow cytometry analysis of cell frequencies of SIINFEKL-MHCI (left), IFN-γ (middle), and granzyme B (right) T cells (CD3 + CD8 + ) in spleen ( n = 5) before and after booster immunization. (C–E) Therapeutic EG7-OVA tumor model. (C) Schematic illustration of the vaccination and tumor inoculation. C57BL/6 mice ( n = 5) were inoculated subcutaneously with EG7-OVA cells and 7 days later, received a single intramuscular injection of LNP or LSE formulations containing 5 μg OVA mRNA per mouse. (D and E) Average tumor volumes (D) and survival curves (E) are shown. (F–I) Prophylactic B16-OVA tumor model. (F) Schematic illustration of the vaccination and tumor inoculation. C57BL/6 mice ( n = 5) received two intramuscular injections of LNP or LSE formulations containing 5 μg OVA mRNA per mouse at a 4-week interval, followed by subcutaneous inoculation of OVA-expressing melanoma (B16-OVA) cells 4 weeks after the booster dose. (G–I) Survival curves (G), average tumor volumes (H), and represented tumor volumes (I) are shown. Mice were euthanized when the tumor volume exceeded 2,000 mm 3 . (J–M) Therapeutic B16-OVA tumor model and tumor microenvironment (TME) analysis. (J) C57BL/6 mice ( n = 6) were inoculated subcutaneously with B16-OVA cells and 7 days later, received two intramuscular injections of LNP or LSE formulations containing 5 μg OVA mRNA per mouse at a 1-week interval. At 6 days post-booster immunization, tumor was extracted and T cell dynamics and cytokine secretion profiles were analyzed. (K) Average tumor volumes. (L) Expression of CD3 + T cell exhaustion markers (PD-1 and Lag-3) in the TME. (M) Cytokine levels (IFN-γ, IL-2, and TNF-α) in tumor lysates measured by ELISA and normalized by total protein content. (N and O) Metastatic B16-OVA melanoma model. (N) Schematic illustration of the vaccination and tumor inoculation. C57BL/6 mice ( n = 5) were inoculated intravenously with B16-OVA cells and 7 days later, received two intramuscular injections of LNP or LSE formulations containing 5 μg OVA mRNA per mouse at a 1-week interval. (O) Representative lung photographs (left) and quantification of tumor nodules in the lungs (right) collected on day 28. (P–R) Tumor rechallenge model using B16-OVA melanoma. (P) Schematic illustration of the vaccination and tumor inoculation regimen. C57BL/6 mice ( n = 6) were inoculated subcutaneously with B16-OVA cells to establish the primary tumor. When the size reached 100 mm 3 , primary tumors were surgically removed; 7 days later, mice received two intramuscular injections of LNP or LSE formulations containing 5 μg OVA mRNA per mouse at a 1-week interval. (Q and R) Average tumor volumes (Q) and survival curves (R). (S–Z) Prophylactic, therapeutic, and rechallenge tumor models using NY-ESO-1-highly expressing LLC. (S and T) Prophylactic LLC model. (S) Schematic illustration of the vaccination and tumor inoculation. C57BL/6 mice ( n = 6) received two intramuscular injections of LNP or LSE formulations containing 5 μg NYESO-1 mRNA per mouse at a 2-week interval, followed by subcutaneous inoculation of NY-ESO-1-highly expressing LLC cells 7 weeks after the booster dose. (T) Average tumor volumes are shown. (U–W) Therapeutic LLC model. (U) Schematic illustration of the vaccination and tumor inoculation. C57BL/6 mice ( n = 6) were inoculated subcutaneously with NY-ESO-1-highly expressing LLC cells and 7 days later, received two intramuscular injections of LNP or LSE formulations containing 5 μg OVA mRNA per mouse at a 1-week interval. (V and W) Average tumor volumes (V) and survival curves (W) are shown. (X–Z) Rechallenge LLC model. (X) Schematic illustration of the vaccination and tumor inoculation. C57BL/6 mice ( n = 5) were inoculated subcutaneously with NY-ESO-1-highly expressing LLC cells to establish the primary tumor. When the size reached 100 mm 3 , primary tumors were surgically removed; 7 days later, mice received two intramuscular injections of LNP or LSE formulations containing 5 μg NYESO-1 mRNA per mouse at a 1-week interval. (Y and Z) Average tumor volumes (Y) and survival curves (Z) are shown. In (B, K, Q, V, and Y), data were presented as mean ± SEM, statistically analyzed by two-way ANOVA comparing LNP-injected mice to LSE-injected mice with post hoc Šídák’s multiple comparisons tests. In (D), data were presented as mean ± SEM, statistically analyzed by two-way ANOVA with post hoc Šídák’s multiple comparisons tests. In (H), data were shown as mean ± SEM, statistically analyzed by two-way ANOVA compared to LSE-injected mice with post hoc Dunnett’s multiple comparisons tests. In (L and M), data were shown as mean ± SEM, statistically analyzed by one-way ANOVA with post hoc Tukey’s multiple comparisons test. ns, p ≥ 0.05, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: ELISpot Plus: Mouse IFN-γ (ALP) , Mabtech , Cat# 3321-4AST-2.

Techniques: Injection, Flow Cytometry, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Poultry Science

Article Title: A novel self-amplified RNA vaccine co-expressing NA and HA1 delivered by Salmonella confers potent protection against H9N2 influenza in chickens

doi: 10.1016/j.psj.2026.107072

Figure Lengend Snippet: Intracellular cytokine production. Chicken splenic lymphocytes were isolated for analysis. Cell proliferation was assessed using CCK-8 analysis with ConA (A), mixed HA1 peptides (B), and NA protein (C). Additionally, the production of IFN-γ by splenic T lymphocytes was measured via an ELISpot assay, utilizing NA and HA1 proteins as stimulators for 36 h (D).

Article Snippet: IFN-γ production was assessed using a commercial Chicken IFN-γ ELISpot kit (Mabtech, Sweden).

Techniques: Isolation, CCK-8 Assay, Enzyme-linked Immunospot

Journal: Poultry Science

Article Title: A novel self-amplified RNA vaccine co-expressing NA and HA1 delivered by Salmonella confers potent protection against H9N2 influenza in chickens

doi: 10.1016/j.psj.2026.107072

Figure Lengend Snippet: Intracellular cytokine production. The intracellular mRNA expression levels of IL-4 (B, D) and IFN-γ (A, C)—as well as the relative concentrations of these cytokines in cell culture supernatants stimulated by the NA peptide (E, F) or HA1 protein (G, H) for 48 h—were determined using qRT-PCR and ELISA, respectively. Data are expressed as the mean ± SEM and analyzed using one-way ANOVA (* P < 0.05, ** P < 0.01, and *** P < 0.001; n = 4).

Article Snippet: IFN-γ production was assessed using a commercial Chicken IFN-γ ELISpot kit (Mabtech, Sweden).

Techniques: Expressing, Cell Culture, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Translational Cancer Research

Article Title: Micropeptide colorectal neoplasia differentially expressed (CRNDE) 84aa encoded by CRNDE elicits a T-cell immune response to breast cancer

doi: 10.21037/tcr-2025-1-2809

Figure Lengend Snippet: CRNDE 84aa is immunogenic. (A) The binding predictions for CRNDE 84aa to HLA-A2 via the IEDB (version 2.5) analysis resource NetMHCpan (ver. 4.1) algorithm. (B) CD8 + T cells were primed by autologous DCs loaded with CRNDE antigens. The CTLs were restimulated with peptides overnight. The peptide-specific T-cell response was evaluated via an IFN-γ ELISpot. Representative ELISpot images are shown. (C) The primed T cells were restimulated with CRNDE antigens overnight and evaluated by flow cytometry; representative flow cytometry plots of intracellular cytokine staining are shown. The percentages indicate the proportions of CD8 + T cells that produced different cytokines (mean ± SD, n=3). P values were determined by two-tailed one-way ANOVA with Dunnett’s multiple-comparisons test. ANOVA, analysis of variance; CRNDE, colorectal neoplasia differentially expressed; CRNDE 84aa, CRNDE encodes an 84-amino acid peptide; DC, dendritic cell; CTL, cytotoxic T lymphocyte; ELISpot, enzyme-linked immunospot; poly(I:C), polyinosinic:polycytidylic acid; IEDB, Immune Epitope Database; IFN-γ, interferon-γ; IL-2, interleukin-2; PBS, phosphate-buffered saline; PHA, phytohemagglutinin; SD, standard deviation; TNF-α, tumor necrosis factor-α.

Article Snippet: To quantitatively assess T cell responsiveness, we performed an interferon-γ (IFN-γ) ELISpot assay using commercially available kits [Catalog No. 3420-4HST-2 (human); Mabtech, Stockholm, Sweden].

Techniques: Binding Assay, Enzyme-linked Immunospot, Flow Cytometry, Staining, Produced, Two Tailed Test, Saline, Standard Deviation

Journal: Translational Cancer Research

Article Title: Micropeptide colorectal neoplasia differentially expressed (CRNDE) 84aa encoded by CRNDE elicits a T-cell immune response to breast cancer

doi: 10.21037/tcr-2025-1-2809

Figure Lengend Snippet: CRNDE 84aa generates an immunostimulatory epitope recognized by human autologous T cells. (A,B) T cells were primed by autologous DCs loaded with CRNDE 84aa antigens. The production of IFN-γ and TNF-α by T cells in response to CRNDE 84aa was assessed via ELISA. (C,D) The primed T cells were restimulated with CRNDE 84aa antigens overnight and evaluated by qRT-PCR. ***, P<0.001, compared with untreated T cells (−). P values were determined by two-tailed one-way ANOVA with Dunnett’s multiple-comparisons test. ANOVA, analysis of variance; CRNDE, colorectal neoplasia differentially expressed; CRNDE 84aa, CRNDE encodes an 84-amino acid peptide; DC, dendritic cell; ELISA, enzyme-linked immunosorbent assay; IFN-γ, interferon-γ; mRNA, messenger RNA; qRT-PCR, quantitative reverse transcription-polymerase chain reaction; TNF-α, tumor necrosis factor-α.

Article Snippet: To quantitatively assess T cell responsiveness, we performed an interferon-γ (IFN-γ) ELISpot assay using commercially available kits [Catalog No. 3420-4HST-2 (human); Mabtech, Stockholm, Sweden].

Techniques: Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Two Tailed Test, Reverse Transcription, Polymerase Chain Reaction

Journal: Translational Cancer Research

Article Title: Micropeptide colorectal neoplasia differentially expressed (CRNDE) 84aa encoded by CRNDE elicits a T-cell immune response to breast cancer

doi: 10.21037/tcr-2025-1-2809

Figure Lengend Snippet: The CRNDE 84aa peptide, encoded by the highly expressed CRNDE in tumor cells, can activate anti-tumor immunity. CRNDE 84aa may serve as a vaccine therapeutic to activate T cells via DCs. When primed T cells recognize the CRNDE 84aa epitope presented by tumor cells, an anti-tumor immune response is elicited. CRNDE, colorectal neoplasia differentially expressed; CRNDE 84aa, CRNDE encodes an 84-amino acid peptide; DC, dendritic cell; GZMB, granzyme B; HLA-I, human leukocyte antigen class I; IFN-γ, interferon-γ; TCR, T cell receptor.

Article Snippet: To quantitatively assess T cell responsiveness, we performed an interferon-γ (IFN-γ) ELISpot assay using commercially available kits [Catalog No. 3420-4HST-2 (human); Mabtech, Stockholm, Sweden].

Techniques: