ELISA assays and alcohol: increasing carbon chain length can interfere with detection of cytokines

Abstract

Enzyme-linked immunosorbent assays (ELISA) are frequently used in studies on cytokine production in response to treatment of cell cultures or laboratory animals. When an ELISA assay is performed on cell culture supernatants, samples often contain the treatment agents. The purpose of the present study was to determine if some of the agents evaluated might inhibit cytokine detection by interfering with the ELISA, leaving the question of whether cytokine production was inhibited unanswered. Mouse and human cytokine ELISA kits from BD Biosciences were used according to the manufacturer's instructions. Cytokine proteins were subjected to one to five carbon alcohols at 86.8 mM (methanol, ethanol, 1-propanol, 2-propanol, n-butanol, and npentanol). After treating cell cultures with alcohols of different carbon chain lengths, we found that some of the alcohols interfered with measurement of some cytokines by ELISA, thus making their effects on cytokine production by cells in culture unclear. Increasing carbon chain length of straight chain alcohols positively correlated with their ability to inhibit detection of TNF-α and IL-10, but not with the detection of IL-6, IL-8, and IL-12. To avoid misinterpretation of treatment effects, ELISA assays should be tested with the reference protein and the treatment agent first, before testing biological samples. These results along with other recent results we obtained using circular dichroism indicate that alcohols with 2 or more carbons can directly alter protein conformation enough to disrupt binding in an ELISA (shown in the present study) or to inhibit ligand induced conformational changes (results not shown). Such direct effects have not been given enough consideration as a mechanism of ethanol action in the immune system.

Bioanalytical chemistry of cytokines – A review

“…Other sample preparation problems have included cytokine removal during albumin depletion steps [82] and protein/antibody interference of mM alcohol concentrations [83]. …”

The Antiviral Effect of High-Molecular Weight Poly-Gamma-Glutamate against Newcastle Disease Virus on Murine Macrophage Cells

“…The proinflammatory cytokine inducing effect of HM- γ -PGA on RAW 264.7 cells was examined using commercial ELISA kits for murine interleukin-6 (IL-6), interleukin-12 (IL-12), tumor necrosis factor-alpha (TNF- α ) (BD Bioscience, USA), and murine interferon- β (IFN- β ) (PBL Interferon Source, USA) [ 13 , 22 , 23 ]. Briefly, a suitable number (2 × 10 6 /well) of RAW 264.7 cells were seeded to each well of a 6-well TC plate (Nunc, Denmark) and cultured.…”