Ultrasensitive ELISA for measurement of human cytokine responses in primary culture.
ELISAs offer excellent specificity and, once fully optimized, sensitivity that rivals that of bioassays. The major variables that need to be experimentally determined when developing an ELISA are the optimal number of fresh cells required per well, the optimal antigen concentrations for stimulation, period of culture, and the anticipated intensity of the response. In this chapter, we review the major factors to be considered in the development and application of ultrasensitive ELISAs to the analysis of human immune responses. We specify the conditions we have found to be optimal for quantifying a number of cytokines of demonstrated relevance to human immune regulation and discuss the major pitfalls inherent in this approach.
Stefura
William P
WP
Department of Immunology, University of Manitoba, Winnipeg, Canada.
Campbell
J Darren
JD
Douville
Renée
R
Stinson
Monique J
MJ
Simons
F Estelle
FE
Becker
Allan B
AB
HayGlass
Kent T
KT
D002478:Cells, Cultured; D016207:Cytokines / Q000032:analysis* / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000191:economics / Q000379:methods* / Q000592:standards; D006801:Humans; D008163:Luminescent Measurements; D012015:Reference Standards; D012680:Sensitivity and Specificity
D016207:Cytokines