Ultrasensitive ELISA for Measurement of Human Cytokine Responses in Primary Culture

Abstract

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.

Vitamin D [1,25(OH)2D3] Differentially Regulates Human Innate Cytokine Responses to Bacterial versus Viral Pattern Recognition Receptor Stimuli

“…Primary culture supernatant cytokine and chemokine levels were quantified via ELISA (32). In brief, titrations of four 2-fold dilutions of each sample were assessed with anti-cytokine/anti-chemokine capture and biotinylated Abs purchased from Biolegend (San Diego, CA), Peprotech (Rocky Hill, NJ), and R&D Systems (Minneapolis, MN).…”

Early Urinary CCL2 is Associated With the Later Development of Interstitial Fibrosis and Tubular Atrophy in Renal Allografts

“…Urinary chemokine measurements were performed with pH-adjusted urine samples following previously described protocols (42). The chemokines CXCL9, CXCL10, and CCL2 were analyzed by sandwich ELISA using commercially available antibodies (Peprotech, Rocky Hill, NJ) on a microplate reader (Vmax Kinetic Microplate Reader; Molecular Devices, Sunnyvale, CA) with good reproducibility.…”

Epigenetic regulation of established human type 1 versus type 2 cytokine responses

“…16,17 Antibodies and standards were from BD Pharmingen (San Diego, Calif), Biolegend (San Diego, Calif), PeproTech (Rocky Hill, NJ), and R&D Systems (Minneapolis, Minn), except for IFN-g standard (1 U 5 115 pg; National Institutes of Health Research Reference Reagent GX01-902-535). Sensitivities were 7.8 pg/mL (IL-5), 3.9 pg/mL (IL-13), 7.8 pg/mL (IL-10), 2 pg/mL (IL-2), 18 pg/mL (IFN-g), and 7.8 pg/mL (CXCL-10/IP-10).…”