Handbook of ELISPOT

This fourth edition volume expands on the previous editions with discussions on the latest protocols that can be applied in different laboratory landscapes ranging from purely research to clinical diagnostic environments. This volume particularly focuses on ELISpot validation and evaluation of antibodies used in this assay. The chapters in this volume cover topics such as peptides in T-Cell assays and ELISpot applications for monitoring infectious diseases like tuberculosis and SARS-CoV2; FluoroSpot; the use of AI for studying ELISpot; and comparing ELISpot with flow cytometry, feline coronavirus (FCoV2), and memory b-cells. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Cutting-edge and comprehensive, Handbook of ELISPOT: Methods and Protocols, Fourth Edition is a valuable resource to aid in the advancement of science and technology, and inspire scientists and researchers to determine and pursue new discoveries in this developing field.

Chapters 5, 7, 11, 13, and 15 are available open access under Creative Commons Attribution 4.0 International License via link.springer.com.

Includes cutting-edge methods and protocols Provides step-by-step detail essential for reproducible results Contains key notes and implementation advice from the experts

Inhalt

Important Considerations for Elispot Validation.- Current Trends in Validating Antibody Specificities for Elispot by Western Blotting.- An Overview of Peptides and Peptide Pools for Antigen-Specific Stimulation in T-Cell Assays.- The Applications of ELISpot in the Identification and Treatment of Various Forms of Tuberculosis and in the Cancer Immunotherapies.- Artificial Intelligence-Based Counting Algorithm Enables Accurate and Detailed Analysis of the Broad Spectrum of Spot Morphologies Observed in Antigen-Specific B Cell ELISPOT and FluoroSpot Assays.- Comparing Flow Cytometry and ELISpot for Detection of IL-10, IL-6, and TNF Alpha on Human PBMCs.- Reagent Tracker™ Platform Verifies and Provides Audit Trails for the Error-Free Implementation of T Cell ImmunoSpot® Assays.- Detection of SARS-CoV-2-Specific Cells Utilizing Whole Proteins and/or Peptides in Human PBMCs using IFN- ELISpot Assay.- Interferon-/IL-2 ELISpot and mRNA Responses to the SARS-CoV2, Feline Coronavirus Serotypes 1 (FCoV1), and FCoV2 Receptor Binding Domains by the T Cells from COVID-19-Vaccinated Humans and FCoV1-Infected Cats.- Frequencies of SARS-CoV-2 Spike Protein-Specific Memory B Cells in Human PBMCs, Quantified by ELISPOT Assay.- Monitoring Memory B Cells by Next Generation ImmunoSpot® Provides Insights into Humoral Immunity that Measurements of Circulating Antibodies Do Not Reveal.- Tracking Circulating HLA-Specific IgG-Producing Memory B Cells with the B Cell ImmunoSpot Assay.- Assessing the Affinity Spectrum of the Antigen-Specific B Cell Repertoire via ImmunoSpot®.- Using PBMCs in a Multiplex FluoroSpot Assay for Detection of Innate Immune Response Modulating Impurities (IIRMIs).- Four-Color ImmunoSpot® Assays Requiring Only 1-3 mL of Blood Permit Precise Frequency Measurements of Antigen-Specific B Cells Secreting Immunoglobulins of All Four Classes and Subclasses.- High-Plex Elispot: FOLISPOT Based on Fluorescence Detection and DNA Complementary Pairing.- Triple-Color FluoroSpot Analysis of Polyfunctional Antigen-Specific T Cells by Quantification of Spot-Forming Units and Relative Spot Volumes.- Performance and Stability of New Class of Fetal Bovine Sera (FBS) and Its Lyophilized Form in ELISpot and FluoroSpot Assays: Applications for Monitoring the Immune Response in Vaccine, and Cell and Gene Immunotherapy in Clinical Trials.