Chapter 10. IFN-γ ELISPOT Assay Validation

  1. Uma Prabhakar Ph.D. and
  2. Marian Kelley Director of Compliance
  1. Manjula Reddy,
  2. Jackson Wong,
  3. Charles Pendley Ph.D. and
  4. Uma Prabhakar Ph.D.

Published Online: 8 APR 2008

DOI: 10.1002/9780470987810.ch10

Validation of Cell-Based Assays in the GLP Setting: A Practical Guide

Validation of Cell-Based Assays in the GLP Setting: A Practical Guide

How to Cite

Reddy, M., Wong, J., Pendley, C. and Prabhakar, U. (2008) IFN-γ ELISPOT Assay Validation, in Validation of Cell-Based Assays in the GLP Setting: A Practical Guide (eds U. Prabhakar and M. Kelley), John Wiley & Sons, Ltd, Chichester, UK. doi: 10.1002/9780470987810.ch10

Editor Information

  1. Department of Clinical Pharmacology & Experimental Medicine, Centocor Research and Development, Inc., 145 King of Prussia Road, Radnor, PA 19087, USA

Author Information

  1. Department of Clinical Pharmacology & Experimental Medicine, Centocor Research and Development, Inc., 145 King of Prussia Road, Radnor, PA 19087, USA

  1. To whom correspondence should be addressed.

Publication History

  1. Published Online: 8 APR 2008
  2. Published Print: 21 MAR 2008

ISBN Information

Print ISBN: 9780470028766

Online ISBN: 9780470987810

SEARCH

Keywords:

  • ELISPOT;
  • IFN-γ;
  • peripheral blood mononuclear cells;
  • phytohemagglutinin;
  • validation

Summary

The utility of the IFN-γ ELISPOT assay for detecting the frequency of antigen-specific T lymphocytes in autoimmune disease models and for immune responses in patients with infectious, autoimmune neoplastic diseases undergoing immunotherapeutic treatments has been reported in several studies. Development, optimization, validation of an IFN-γ ELISPOT assay for use in the analysis of clinical study samples will be described in this chapter. Several assay conditions such as cell number, length of culture, positive and negative controls, and antigen concentration will be addressed. Procedures validating the stability of responses in shipped samples, stability assessment of long term cryopreserved samples, assay precision including intra-assay (variability in replicates), inter-assay (plate-to-plate variability and within day variability), inter-day and inter-operator variability's will also be described. In addition, data analysis and interpretation including lower and upper limits of quantification will be discussed.