Utility of lyophilized PMA and ionomycin to stimulate lymphocytes in whole blood for immunological assays

Belouski, S. S.*, Wilkinson, J.^, Thomas, J.*, Kelley, K.*, Wang, S.-W.*, Suggs, S.* and Ferbas, J.* (2010), . Cytometry, 78B: 59–64.

doi: 10.1002/cyto.b.20492

The need to implement robust biomarkers in clinical trials has never been greater, and such efforts can be easily compromised by reagent instability or simple human error during assay set-up. Many biotechnology and pharmaceutical companies are introducing efforts to conduct biomarker studies under more rigorous settings, and the use of plates or tubes pre-loaded with stimulation or staining reagents could be of value for studies that involve flow cytometry.

Author Information:

* Amgen Inc., Department of Medical Sciences

^ Beckman Coulter Inc., Custom BioPharma Group, Miami, Florida (author Julie Wilkinson current address is ImmunoSite Technologies, Fort Lauderdale, FL)

Email: John Ferbas (jferbas@amgen.com)

*One Amgen Center Drive, Mailstop 30E-3-C, Thousand Oaks 91320-1799, CA

IST quite literally helped “write the book” on cell analysis testing.  They were asked to contribute a chapter of a book titled Validation of Cell-Based Assays in the GLP Setting: A Practical Guide, by authors/editors Uma Prabhakar and Marian Kelley (Publisher John Wiley and Sons, 2008, ISBN 0470028769, 9780470028766).  Chapter 8 of this book entitled “Intracellular cytokine detection by flow cytometry” was authored by IST scientists Julie G. Wilkinson, Carlos L. Aparicio, and Wade E. Bolton.

Readers gain an understanding of the details and the high level considerations of assay qualification for difficult cell-based assays.  The level of optimization described for cell-based assays lends itself to biomarker assay automation which can be used for biomarker qualification and validation studies to the satisfaction of the FDA, EMEA, and other regulatory agencies.   Cell-based assay platforms covered are flow cytometry, intracellular cytokine ICS, immunophenotyping, elispot, IHC, cylex, neutralization bioassays, and endpoint assays.