Nonspecific staining in ELISpot assay is a major obstacle in accurate quantification of experimental data. The appearance of nonspecific spots may be caused by different factors including cell- and immunoassay-related issues. In our study, we have shown that nonspecific spots can result from either cells or their debris sticking to the membranes in ELISpot plates, as well as by impurities in wash buffers and precipitation of aggregated detection antibodies. Although there is a growing interest in using Fluorospot assays allowing for simultaneous detection of multiple cell-secreted proteins, it appears that these fluorescence assays are more susceptible to developing nonspecific profiles resembling specific spots. In this chapter, we outline necessary ELISpot controls that need to be employed to tell the difference between bona fide spots vs. stained artifacts.
Download full-text PDF |
Source |
|---|---|
| http://dx.doi.org/10.1007/978-1-4939-8567-8_3 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
Theoretical and practical considerations for validating antigen-specific B cell ImmunoSpot assays.
J Immunol Methods
January 2025
Research & Development Department, Cellular Technology Limited, Shaker Heights, OH 44122, USA. Electronic address:
Owing to their ability to reliably detect even very rare antigen-specific B cells in cellular isolates such as peripheral blood mononuclear cells (PBMC), and doing so robustly in a high throughput-compatible manner, B cell ELISPOT/FluoroSpot (collectively "B cell ImmunoSpot") tests have become increasingly attractive for immune monitoring in regulated settings. Presently, there are no guidelines for the qualification and validation of B cell ImmunoSpot assay results. Here, we propose such guidelines, building on the experience acquired from T cell ImmunoSpot testing in an environment adhering to the requirements of regulatory bodies yet taking the unique features of B cell assays into account.
View Article and Find Full Text PDFImmunogenicity of HIV-1 mRNA and VLP mRNA Vaccines in Mice.
Vaccines (Basel)
January 2025
National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 100052, China.
Background: The development of a protective vaccine is critical for conclusively ending the human immunodeficiency virus (HIV) epidemic.
Methods: We constructed nucleotide-modified mRNA vaccines expressing HIV-1 Env and Gag proteins. Env-gag virus-like particles (VLPs) were generated through co-transfection with env and gag mRNA vaccines.
Optimizing Microneutralization and IFN-γ ELISPOT Assays to Evaluate Mpox Immunity.
Vaccines (Basel)
December 2024
Division of Infectious Diseases, Allergy and Immunology, Saint Louis University, St. Louis, MO 63104, USA.
Background: Available assays to measure pox virus neutralizing antibody titers are laborious and take up to 5 days. In addition, assays to measure T cell responses require the use of specific antigens, which may not be the same for all pox viruses. This study reports the development of robust assays for the measurement of mpox-specific neutralizing antibodies and IFN-γ-producing T-cell responses.
View Article and Find Full Text PDFVACCIMEL, an allogeneic melanoma vaccine, efficiently triggers T cell immune responses against neoantigens and alloantigens, as well as against tumor-associated antigens.
Front Immunol
January 2025
Centro de Investigaciones Oncológicas (FUCA), Fundación Cáncer, Ciudad Autónoma de Buenos Aires, Argentina.
VACCIMEL is a therapeutic cancer vaccine composed of four irradiated allogeneic human melanoma cell lines rationally selected to cover a wide range of melanoma tumor-associated antigens (TAA). We previously demonstrated that vaccination in the adjuvant setting prolonged the distant-metastasis-free survival of cutaneous melanoma patients and that T cells reactive to TAA and the patient's private neoantigens increased during treatment. However, immune responses directed to vaccine antigens that may arise from VACCIMEL's somatic mutations and human polymorphisms remain unexplored.
View Article and Find Full Text PDFAge-associated alterations in immune and inflammatory responses in captive olive baboons ().
Front Aging
January 2025
The University of Texas MD Anderson Cancer Center Bastrop, Department of Comparative Medicine TX, Bastrop, TX, United States.
Introduction: Advanced age is a primary risk factor for many chronic diseases and conditions; however, age-related immune dysregulation is not well understood. Animal models, particularly those that resemble human age-related physiological changes, are needed to better understand immunosenescence and to improve health outcomes. Here, we explore the utility of the olive baboon (Papio anubis) in studying age-related changes to the immune system and understanding mechanisms of immunosenescence.
View Article and Find Full Text PDFWant AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!