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Current Trends in Validating Antibody Specificities for ELISpot by Western Blotting

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Handbook of ELISPOT

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2768))

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Abstract

The enzyme-linked immunospot (ELISpot) assay is a highly useful and sensitive method to detect total immunoglobulin and antigen-specific antibody-secreting cells. In addition, this method can measure biological activity and immunological secretions from immune cells. In general, membrane-bound antigen allows binding of antibody secreted by B cells, or a membrane-bound analyte-specific antibody binds to the specific analyte (e.g., cytokines) elicited from cells added to the well containing the bound antibody. The response from added cells is then detected by using an anti-Ig antibody and a colorimetric substrate, while in the case of non-B cells, the elicited antigen is detected with appropriate antibodies and enzyme-conjugated antibodies. Specificity of antibodies binding the protein of interest is necessary to achieve correct results. Western blotting can be used for this with/without siRNA knockdown of proteins of interest or with the use of peptide inhibitors to inhibit the binding of specific antibodies to the target protein. Despite its general simplicity, western blotting is a powerful technique for immunodetection of proteins (notably low abundance proteins) as it provides simultaneous resolution of multiple immunogenic antigens within a sample for detection by specific antibodies. Now, we have plethora of immunoblotting methods to validate antibodies for ELISpot.

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Author information

Authors and Affiliations

  1. Arthritis and Clinical Immunology Program, Oklahoma Medical Research Foundation, Oklahoma, OK, USA

    Biji T. Kurien & R. Hal Scofield

  2. Department of Veterans Affairs Medical Center, Oklahoma City, Oklahoma, OK, USA

    Biji T. Kurien & R. Hal Scofield

  3. Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma, OK, USA

    R. Hal Scofield

Authors
  1. Biji T. Kurien
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  2. R. Hal Scofield
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Corresponding author

Correspondence to Biji T. Kurien .

Editor information

Editors and Affiliations

  1. Biotechne, Minneapolis, MN, USA

    Alexander E. Kalyuzhny

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© 2024 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Kurien, B.T., Scofield, R.H. (2024). Current Trends in Validating Antibody Specificities for ELISpot by Western Blotting. In: Kalyuzhny, A.E. (eds) Handbook of ELISPOT . Methods in Molecular Biology, vol 2768. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3690-9_2

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  • DOI: https://doi.org/10.1007/978-1-0716-3690-9_2

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3689-3

  • Online ISBN: 978-1-0716-3690-9

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