title: ELISPOTs Produced by CD8 and CD4 Cells Follow Log Normal Size Distribution Permitting Objective Counting.
abstract: Each positive well in ELISPOT assays contains spots of variable sizes that can range from tens of micrometers up to a millimeter in diameter. Therefore, when it comes to counting these spots the decision on setting the lower and the upper spot size thresholds to discriminate between non-specific background noise, spots produced by individual T cells, and spots formed by T cell clusters is critical. If the spot sizes follow a known statistical distribution, precise predictions on minimal and maximal spot sizes, belonging to a given T cell population, can be made. We studied the size distributional properties of IFN-γ, IL-2, IL-4, IL-5 and IL-17 spots elicited in ELISPOT assays with PBMC from 172 healthy donors, upon stimulation with 32 individual viral peptides representing defined HLA Class I-restricted epitopes for CD8 cells, and with protein antigens of CMV and EBV activating CD4 cells. A total of 334 CD8 and 80 CD4 positive T cell responses were analyzed. In 99.7% of the test cases, spot size distributions followed Log Normal function. These data formally demonstrate that it is possible to establish objective, statistically validated parameters for counting T cell ELISPOTs.
authors.affiliation: Cellular Technology Ltd., 20521 Chagrin Blvd. Shaker Heights, OH 44122, USA. ayk@immunospot.com.
authors.forename: Alexey Y
authors.initials: AY
authors.lastname: Karulin
authors.affiliation: Cellular Technology Ltd., 20521 Chagrin Blvd. Shaker Heights, OH 44122, USA. kinga.karacson@immunospot.com.
authors.forename: Kinga
authors.initials: K
authors.lastname: Karacsony
authors.affiliation: Cellular Technology Ltd., 20521 Chagrin Blvd. Shaker Heights, OH 44122, USA. wenji.zhang@immunospot.com.
authors.forename: Wenji
authors.initials: W
authors.lastname: Zhang
authors.affiliation: Cellular Technology Ltd., 20521 Chagrin Blvd. Shaker Heights, OH 44122, USA. oleg.targoni@immunospot.com.
authors.forename: Oleg S
authors.initials: OS
authors.lastname: Targoni
authors.affiliation: Cellular Technology Ltd., 20521 Chagrin Blvd. Shaker Heights, OH 44122, USA. ioana.moldovan@immunospot.com.
authors.forename: Ioana
authors.initials: I
authors.lastname: Moldovan
authors.affiliation: Ph.D., Biocenter, University of Wuerzburg, Am Hubland, 97074 Würzburg, Germany. marcus.dittrich@biozentrum.uni-wuerzburg.de.
authors.forename: Marcus
authors.initials: M
authors.lastname: Dittrich
authors.affiliation: Cellular Technology Ltd., 20521 Chagrin Blvd. Shaker Heights, OH 44122, USA. srividya.sundararaman@immunospot.com.
authors.forename: Srividya
authors.initials: S
authors.lastname: Sundararaman
authors.affiliation: Cellular Technology Ltd., 20521 Chagrin Blvd. Shaker Heights, OH 44122, USA. pvl@immunospot.com.
authors.forename: Paul V
authors.initials: PV
authors.lastname: Lehmann