Handbook of ELISPOT: Methods and Protocols (Methods in Molecular Biology, 2768)

Preface
Contents
Contributors
Chapter 1: Important Considerations for ELISpot Validation
	1 Introduction
	2 Materials and Reagents
	3 Method
		3.1 Accuracy
		3.2 Precision
		3.3 Specificity
		3.4 Limit of Detection (LOD)
		3.5 Robustness
		3.6 Linearity
		3.7 Range
		3.8 Upper Limit of Quantitation (ULOQ)
		3.9 Lower Limit of Quantitation (LLOQ)
		3.10 Limit of Blank (LOB)
	4 Notes
	References
Chapter 2: Current Trends in Validating Antibody Specificities for ELISpot by Western Blotting
	1 Introduction
	2 Membrane Supports for Protein Transfer
		2.1 Nitrocellulose Membranes
			2.1.1 Mechanism of Immobilization
			2.1.2 Disadvantages of Nitrocellulose Membrane
		2.2 Polyvinylidene Difluoride
			2.2.1 Mechanism of Binding to Membranes
			2.2.2 Advantages of PVDF
		2.3 Activated Paper
		2.4 Nylon Membranes
			2.4.1 Shortcomings of Nylon
	3 Antibody Evaluation
	4 Antibody Considerations
		4.1 Polyclonal Antibodies Versus Monoclonal Antibodies
	5 Methods to Transfer Proteins from Gel to Membrane
		5.1 Simple Diffusion
		5.2 Vacuum Blotting
		5.3 Electroblotting
			5.3.1 Wet Transfer
			5.3.2 ``Semi-Dry´´ Transfer
	6 Conclusion
	References
Chapter 3: An Overview of Peptides and Peptide Pools for Antigen-Specific Stimulation in T-Cell Assays
	1 Introduction
	2 Peptide Synthesis, Purification, Analysis, and QC/QA
		2.1 Peptide Synthesis
		2.2 Peptide Analysis and Purification
		2.3 Peptide Impurities, quality assurance (QA), and quality control (QC)
	3 Peptide Length and Applications
	4 Peptide Libraries and Pools
		4.1 Pool Size and Limitations to the Number of Peptides PerPool
		4.2 Protein-Spanning Peptide Pools
		4.3 Peptide Pools Covering Sequence Variants
		4.4 Pools Covering Multiple T-CellStimulating Peptides of an Organism
		4.5 Matrix Pools for Epitope Mapping
	5 Control Pools
		5.1 Positive Control Pools
		5.2 Negative Control Pools
	6 Peptide Solutions, Storage, and Depreciation
	7 Summary and Outlook
	References
Chapter 4: The Applications of ELISpot in the Identification and Treatment of Various Forms of Tuberculosis and in the Cancer ...
	1 Introduction
		1.1 Applications of ELISpot in Tuberculosis Research
		1.2 Applications of ELISPOT in Cancer Research
	2 Materials
	3 Methods
		3.1 General Protocol for ELISpot Release Assay
	4 Notes
	References
Chapter 5: Artificial Intelligence-Based Counting Algorithm Enables Accurate and Detailed Analysis of the Broad Spectrum of Sp...
	1 Introduction
		1.1 More Diverse Spot Morphologies Are Observed in Direct ImmunoSpot Assays Than in Pan-Ig Assays
		1.2 Size Distribution of Antigen-Specific ASC Does Not Follow Log Normal Distribution and Cannot Be Analyzed Using Statistics-...
		1.3 Artificial Intelligence-Based IntelliCount Provides Accurate User- and Assay-Independent Counts of Antigen-Specific ASC-De...
			1.3.1 IntelliCount Automatically Establishes Accurate Spot Boundaries for High-Content Analysis
			1.3.2 IntelliCount Counting Mode Provides Extended Linear Ranges for Accurate Calculation of ASC Frequencies
		1.4 Further Advantages of AI-Based SFU Analysis
	2 Materials
		2.1 Single-Color ELISPOT Assay
		2.2 Single-, Three-, or Four-Color FluoroSpot Assays
		2.3 Four-Color Antigen-Specific Direct FluoroSpot Assay (Affinity Capture Coating)
		2.4 Single-Color, Antigen-Specific Inverted ImmunoSpot Assay
	3 Methods
		3.1 Pan-Ig ImmunoSpot Assay (total ASC, Irrespective of Specificity)
		3.2 Antigen-Specific, Direct B-Cell ImmunoSpot
		3.3 Antigen-Specific, Single-Color Human IgG Inverted ImmunoSpot Assay (see Notes 31-33)
		3.4 Automatic Scanning and Counting of ImmunoSpot Plates
	4 Notes
	References
Chapter 6: Comparing Flow Cytometry and ELISpot for Detection of IL-10, IL-6, and TNF Alpha on Human PBMCs
	1 Introduction
	2 Materials
		2.1 Isolation of Human Peripheral Blood Mononuclear Cells (PBMCs)
		2.2 Stimulation and Restimulation of PBMCs for Flow Cytometry
		2.3 Flow Cytometry Assays
		2.4 Stimulation of PBMCs for ELISpot
		2.5 ELISpot Assays
	3 Methods
		3.1 Isolation of PBMCs
		3.2 Stimulation and Restimulation of Naive PBMCs for Flow Cytometry
		3.3 Flow Cytometric Staining Protocol
		3.4 Flow Cytometric Analysis of IL-6, TNF-Alpha, and IL-10 on the Agilent Quanteon
		3.5 ELISpot Assay
		3.6 Flow Cytometry Results
		3.7 ELISpot Results
		3.8 Benefits of Flow Cytometry and ELISpot for Visualization of Secreted Cytokines
			3.8.1 Benefits of Flow Cytometry
			3.8.2 Benefits of ELISpot
	4 Notes
	References
Chapter 7: Reagent Tracker Platform Verifies and Provides Audit Trails for the Error-Free Implementation of T-Cell ImmunoSpot ...
	1 Introduction
	2 Materials
	3 Methods
		3.1 Plan Experiment and Assign Reagent Tracker Dyes to Color-Code Each Assay Condition for Verification of Pipetting Accuracy
		3.2 Preparation of Reagent Tracker Calibration Plate for Calibration of the Software
		3.3 Perform Reagent Tracker Calibration Scan to Generate Reference  Data
		3.4 Thawing of Cryopreserved PBMC Samples (Sterile Conditions)
		3.5 Preparation of Antigens and First Verification of Pipetting Accuracy Using Reagent Tracker Dyes and Software (Sterile Cond...
		3.6 Plating of PBMC into ELISPOT Assay and Second Verification of Pipetting Accuracy Using Reagent Tracker Dyes and Software (...
		3.7 Development of the ELISPOT Assay (Nonsterile Conditions)
	4 Notes
	References
Chapter 8: Detection of SARS-CoV-2-Specific Cells Utilizing Whole Proteins and/or Peptides in Human PBMCs Using IFN- ELISPOT A...
	1 Introduction
	2 Materials
		2.1 Preparation of Antigen Stocks
		2.2 Collection and Isolation of PBMCs
		2.3 Coating of ELISPOT Plates
		2.4 Thawing of Cryopreserved PBMCs
		2.5 Preparation of Antigen Dilutions (Working Solutions)
		2.6 Human IFN-γ ELISPOT Assay Setup: Plating and Culture
		2.7 Assay Development
	3 Methods
		3.1 Preparation of Antigen Stocks
		3.2 Collection and Isolation of Human PBMCs
		3.3 Coating of ELISPOT Plates
		3.4 Thawing of Cryopreserved PBMCs
		3.5 Preparation of Antigen Dilutions (Working Solutions)
		3.6 Human IFN-γ ELISPOT Assay Setup: Plating and Culture
		3.7 Assay Development
		3.8 Determination of IFN-γ Secreting Cell Frequencies (in SFUs per 2 x 105 cells) (See Notes 12-14)
	4 Notes
	References
Chapter 9: Interferon-γ/IL-2 ELISpot and mRNA Responses to the SARS-CoV2, Feline Coronavirus Serotypes 1 (FCoV1), and FCoV2 Re...
	1 Introduction
	2 Materials
		2.1 Human Subjects
		2.2 Animal Subjects
		2.3 Peripheral Blood Mononuclear Cell Preparation
		2.4 Stimulants
		2.5 IFNγ and IL2 ELISpot for Humans and Cats
		2.6 PBMC Culture Medium
		2.7 Primers and Probes for Cytokine mRNAs
		2.8 mRNA Extraction
		2.9 Reverse Transcription and PCR of cDNA
	3 Methods
		3.1 IFNγ and IL2 Analyses of Subjects Y3
		3.2 IFNγ Analysis of Other Vaccinated Subjects
		3.3 IFNγ Analysis of FCoV1-Infected Cats
		3.4 Isolation of Human and Feline  PBMC
		3.5 Human and Feline Cytokine ELISpot Analyses
		3.6 Human Cytokine mRNA Analyses
	4 Notes
	References
Chapter 10: Frequencies of SARS-CoV-2 Spike Protein-Specific Memory B Cells in Human PBMCs, Quantified by ELISPOT Assay
	1 Introduction
	2 Materials
		2.1 SARS-CoV-2 Spike-Specific Recombinant Proteins
		2.2 Collection and Isolation of Peripheral Blood Mononuclear Cells (PBMCs)
		2.3 Thawing of Cryopreserved PBMCs
		2.4 Resting PBMCs in the Presence of IL-2 and R848
		2.5 Coating of ELISPOT Plates
		2.6 Human B-Cell ELISPOT Assay Setup: Plating and Culture
		2.7 Assay Development
		2.8 Determination of Memory B-Cell Frequencies/MBCs (in SFUs per 2 x 105 Cells)
	3 Methods
		3.1 Collection and Isolation of Peripheral Blood Mononuclear Cells (PBMCs)
		3.2 Thawing of Cryopreserved PBMCs
		3.3 Resting PBMCs in the Presence of IL-2 and R848 (Nonspecific Stimulation of B Cells)
		3.4 Coating of ELISPOT Plates
		3.5 Human B-cell ELISPOT Assay Setup: Plating and Culture
		3.6 Assay Development (See Note 12)
		3.7 Determination of MBC Frequencies (in SFUs per 2 x 105 Cells)
	4 Notes
	References
Chapter 11: Monitoring Memory B Cells by Next-Generation ImmunoSpot Provides Insights into Humoral Immunity that Measurements ...
	1 Introduction
		1.1 Differential Fate Decisions for B Cell Differentiation into the Memory vs. Plasma Cell Lineages
		1.2 Serum Antibodies Reflect the First Wall of Adaptive Humoral Defense, Bmem the Second
		1.3 Emerging Evidence for Serum Antibody Levels and Memory Cell Frequencies Providing Divergent Information on the Magnitude o...
		1.4 Emerging Evidence for Bmem Detection Being More Reliable for Revealing Past Antigen Exposure than Serum Antibody Measureme...
		1.5 Assessing Bmem Might Be Important, but how, and  why?
		1.6 Affinity Coating: Enabling B Cell ImmunoSpot Assays ``to See´´
		1.7 High-Content Information Provided by Assessing Individual ASC via ImmunoSpot vs. Serum Antibody Measurements
		1.8 Frequency of Antigen-Specific, Bmem-Derived ASC Reflect Memory Potential
		1.9 Defining the Ig Class/Subclass Usage of Antigen-Specific Bmem
		1.10 Assessing the Affinity Distribution of the Antigen-Specific Bmem Repertoire
		1.11 Measuring Cross-Reactivities of Individual Bmem-Derived ASC
		1.12 Concluding Remarks
	2 Materials
		2.1 Thawing of Cryopreserved  PBMC
		2.2 In Vitro Polyclonal Stimulation of B Cells in  PBMC
		2.3 Four-Color, Antigen-Specific FluoroSpot Assay (Affinity Capture Coating)
		2.4 Single-Color, Antigen-Specific Inverted ImmunoSpot Assay
	3 Methods
		3.1 Thawing of Cryopreserved PBMC (Sterile Conditions)
		3.2 In Vitro Polyclonal Stimulation of B Cells in PBMC (Sterile Conditions)
		3.3 Four-Color, Antigen-Specific FluoroSpot Assay (Affinity Capture Coating)
		3.4 Single-Color, Antigen-Specific Human IgG-Inverted ImmunoSpot Assay
	4 Notes
	References
Chapter 12: Tracking Circulating HLA-Specific IgG-Producing Memory B Cells with the B-Cell ImmunoSpot Assay
	Abbreviations
	1 Introduction
	2 Materials
		2.1 Isolation and Culture of Human PBMC
		2.2 ImmunoSpot Assay (See Table for Lab-Specific Details)
	3 Methods
		3.1 PBMC Stimulation
		3.2 ImmunoSpot Assay (Focusing on Methods in Publications:)
			3.2.1 Plate Coating (Day 5), See Fig. 1 for Diagrammatic Representation (See Note 3)
			3.2.2 Plate Seeding (Day 6)
			3.2.3 Detection (Day 7)
			3.2.4 Data Analysis
		3.3 Conclusion
	4 Notes
	References
Chapter 13: Assessing the Affinity Spectrum of the Antigen-Specific B Cell Repertoire via ImmunoSpot
	1 Introduction
		1.1 Definition of Affinity, Avidity, and Functional Affinity
		1.2 Biological Benefits of Antibodies with High Functional Affinity
		1.3 Affinity Maturation of the Antibody Response
		1.4 Measuring Antibody Functional Affinity in the Serum vs. the B Cells Themselves
		1.5 Measuring Functional Affinity by B Cell ImmunoSpot
		1.6 B Cell Hybridoma Studies Validating ImmunoSpot Affinity Measurements
		1.7 B Cell Affinity Distribution Measurements in PBMC with Inverted ImmunoSpot Tests
		1.8 B Cell Affinity Distribution Measurements in PBMC with Direct ImmunoSpot Tests
	2 Materials
		2.1 Thawing of Cryopreserved  PBMC
		2.2 In Vitro Polyclonal Stimulation of B Cells in PBMC
		2.3 Antigen-Specific, Single-Color Inverted FluoroSpot Assay
		2.4 Antigen-Specific FluoroSpot Assay (Affinity Capture Coating)
	3 Methods
		3.1 Thawing of Cryopreserved PBMC (Sterile Conditions)
		3.2 In Vitro Polyclonal Stimulation of B Cells in PBMC (Sterile Conditions)
		3.3 Antigen-Specific, Single-Color Human IgG Inverted FluoroSpot Assay (Limiting Antigen Probe) (See Notes 19, 28, and 29)
		3.4 Antigen-Specific (Affinity Capture Coating) FluoroSpot Assay (Limiting Antigen-Coating Concentrations)
	4 Notes
	References
Chapter 14: Using PBMCs in a Multiplex FluoroSpot Assay for Detection of Innate Immune Response-Modulating Impurities (IIRMIs)
	1 Introduction
	2 Materials
		2.1 Instrumentation and Software
		2.2 FluoroSpot Kits
		2.3 Buffers
		2.4 Media
		2.5 Cells
		2.6 Stimuli (Fig. 2)
	3 Methods
		3.1 Trippel-Color FluoroSpot
	4 Notes
	References
Chapter 15: Four-Color ImmunoSpot Assays Requiring Only 1-3 mL of Blood Permit Precise Frequency Measurements of Antigen-Speci...
	1 Introduction
	2 Materials
		2.1 Isolation and Cryopreservation of PBMC from Whole Blood
		2.2 Thawing of Cryopreserved  PBMC
		2.3 In Vitro Polyclonal Stimulation of B Cells in PBMC
		2.4 Single-Color ELISPOT Assay
		2.5 Single-, Three- or Four-Color FluoroSpot Assays
		2.6 Four-Color Antigen-Specific FluoroSpot Assay (Affinity Capture Coating)
	3 Methods
		3.1 Isolation and Cryopreservation of PBMC from Whole Blood (Sterile Conditions)
		3.2 Thawing of Cryopreserved PBMC (Sterile Conditions)
		3.3 Polyclonal In Vitro Stimulation of B Cells in PBMC (Sterile Conditions)
		3.4 Four-Color, Antigen-Specific FluoroSpot Assay (Affinity Capture Coating)
	4 Notes
	References
Chapter 16: High-Plex ELISPOT: FOLISPOT Based on Fluorescence Detection and DNA Complementary Pairing
	1 Introduction
	2 Materials
	3 Methods
		3.1 Antibody-DNA Conjugation and Purification
		3.2 Characterization of DNA-Conjugated Antibodies by MALDI-TOF
		3.3 Preparation of Concatemers
		3.4 Verification and Purification of Concatemers
		3.5 Detection Strands
		3.6 Cell Culture
		3.7 Signal Amplification
		3.8 Fluorophore Hybridization and Dehybridization
		3.9 Detection of Multiple Targets by FOLISPOT
		3.10 Detection of Antigens by Traditional ELISPOT
		3.11 Detection of Four Targets by FOLISPOT with a Single-Stage Amplification System
		3.12 Detection of Six Targets with FOLISPOT
		3.13 Comparison of FOLISPOT with Traditional ELISPOT
		3.14 Conclusion
	4 Notes
	References
Chapter 17: Triple-Color FluoroSpot Analysis of Polyfunctional Antigen-Specific T Cells by Quantification of Spot-Forming Unit...
	1 Introduction
	2 Material
	3 Methods
		3.1 Triple-Color FluoroSpot
		3.2 Analysis of Spots
	4 Notes
	References
Chapter 18: Performance and Stability of New Class of Fetal Bovine Sera (FBS) and Its Lyophilized Form in ELISpot and FluoroSp...
	1 Introduction
	2 Materials
	3 Methods
		3.1 For Cultured ELISpot or FluoroSpot Continue with These Procedures. If Not Performing a Cultured ELISpot or FluoroSpot Proc...
		3.2 For Direct (Ex Vivo) ELISpot/FluoroSpot Assay, Thaw Cryopreserved PBMCs and Rest Overnight
		3.3 Preparation of Precoated Plates and Seeding of Stimulated PBMC
	4 Notes
	References
Index