Variable estimates of cytokine levels produced by commercial ELISA kits: results using international cytokine standards.
Commercially available ELISA kits now make it possible to measure cytokines in biological samples and cell culture supernatants. We have compared the levels of IL-1 beta, IL-6, IL-8 and TNF-alpha in various pathological plasma and synovial fluids, and in supernatants of human monocytes activated by lipopolysaccharide (LPS). Measurements were performed using ELISA kits from different companies. A wide variation in values was obtained when measurements were deduced from the standard curves formed with the standard provided by the manufacturers. We also performed calibration curves for all ELISA kits, using the international standards provided by the NIBSC (UK). The coefficients of variation were then significantly improved for IL-6 and IL-8 measurements but not for IL-1 beta and TNF alpha assays. However, despite this attempt to obtain uniform measurements, none of the kits gave similar values for individual samples. These results suggest that the nature of the different pairs of monoclonal antibodies employed in each ELISA does not permit comparable recognition of cytokines in samples. Further work with the various kits is required to establish whether (i) denaturation of the recognized epitope within the natural cytokine, (ii) fragmentation of the cytokine following enzymatic cleavage, (iii) depolymerization, (iv) binding of cytokines to undefined ligands, (v) variable glycosylation of the natural cytokines (vi) recognition of precursor forms, interferes with the measurements.
Ledur
A
A
INSERM U430, Hôpital Broussais, Paris, France.
Fitting
C
C
David
B
B
Hamberger
C
C
Cavaillon
J M
JM
D000911:Antibodies, Monoclonal / Q000276:immunology; D001168:Arthritis / Q000378:metabolism; D002478:Cells, Cultured; D017077:Culture Media, Conditioned / Q000032:analysis; D004797:Enzyme-Linked Immunosorbent Assay / Q000592:standards*; D000939:Epitopes / Q000276:immunology; D005188:False Negative Reactions; D006801:Humans; D007375:Interleukin-1 / Q000032:analysis; D015850:Interleukin-6 / Q000032:analysis; D016209:Interleukin-8 / Q000032:analysis; D007378:Interleukins / Q000032:analysis*; D007390:International Agencies; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D011489:Protein Denaturation; D011933:Reagent Kits, Diagnostic / Q000592:standards*; D012015:Reference Standards; D015203:Reproducibility of Results; D018805:Sepsis / Q000097:blood; D013582:Synovial Fluid / Q000737:chemistry; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis*
D000911:Antibodies, Monoclonal; D017077:Culture Media, Conditioned; D000939:Epitopes; D007375:Interleukin-1; D015850:Interleukin-6; D016209:Interleukin-8; D007378:Interleukins; D008070:Lipopolysaccharides; D011933:Reagent Kits, Diagnostic; D014409:Tumor Necrosis Factor-alpha