Biological inactivation and impaired detection of IL-10 by suramin.
Enzyme-linked immunosorbent assay (ELISA) measures the amount of cytokines secreted by cells but "equivalent" ELISAs from different manufacturers may show different results, dependent on the epitopes used to generate the antibodies employed in the ELISAs. This fact was dramatically illustrated in a series of experiments we conducted to analyze the effect of suramin on the lipopolysaccharide-induced interleukin-10 (LPS-induced IL-10) production in human CD14+ monocytes. Use of the human IL-10 DuoSet ELISA from R&D Systems showed no influence of suramin on IL-10 levels, whereas the human IL-10 ELISA from Biosource detected only low amounts of IL-10 in the presence of suramin. The results with the Biosource ELISA reflected not only interference of the recognition of IL-10 in the presence of suramin, but also a block in the biological activity of IL-10. Thus, in the presence of suramin the addition of recombinant biological IL-10 protein could not inhibit the LPS-induced tumor necrosis factor alpha (TNF-alpha) or interleukin-12p40 (IL-12p40) production. Furthermore, suramin very efficiently neutralizes endogenous IL-10, resulting in a pronounced increase in IL-12 and TNF-alpha synthesis. Impaired recognition of biologically inactive conformations of IL-10 was not unique to suramin, in that heat-treated or chemically reduced recombinant IL-10 was also no longer recognized by the Biosource ELISA. This suggests that at least one of the antibodies employed in the Biosource ELISA is conformation sensitive, while the antibodies employed in the R&D Systems ELISA are insensitive to a number of conformation changes. All in all, our results show that suramin neutralizes the biological activity of IL-10 produced by monocytes. Furthermore, the amount of IL-10 detected by the Biosource ELISA correlates with the biological activity of IL-10, whereas the amount detected by the R&D Systems ELISA includes active as well as several inactive forms of IL-10.
Thom
Felicitas
F
Grünenthal GmbH, Aachen 52099, Germany. Felicitas.Thom@grunenthal.de
Stockhausen
Hannelore
H
Germann
Tieno
T
Frosch
Stefanie
S
Wade
Erik J
EJ
D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D016753:Interleukin-10 / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000494:pharmacology; D018950:Lipopolysaccharide Receptors / Q000276:immunology; D009000:Monocytes / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis / Q000037:antagonists & inhibitors; D013498:Suramin / Q000494:pharmacology*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D018950:Lipopolysaccharide Receptors; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D016753:Interleukin-10; D013498:Suramin