Monoclonal antibody based enzyme-linked and chemiluminescent assays for the human interleukin-1 receptor antagonist. Application to measure hIL-1ra levels in monocyte cultures and synovial fluids.
Interleukin-1 receptor antagonist (IL-1ra) has the potential to counteract at least part of the biological effects of interleukin-1. The outcome of an inflammatory reaction may therefore be determined by the balance between IL-1 and IL-1ra, rather than by IL-1 alone. We have developed an immunoassay to address this issue as well as to assess the effects of anti-inflammatory agents on the expression of IL-1 and IL-1ra in vitro or in body fluids. Recombinant human IL-1ra was expressed in an E. coli system, purified to homogeneity, and used to derive monoclonal antibodies in mice as well as polyclonal antibodies in rabbits. A sandwich ELISA was constructed with F(ab')2 fragments of a high affinity monoclonal antibody and the rabbit serum as a source of secondary antibody. The assay required no sample treatment to avoid interference by rheumatoid factor. The measuring range was 0.020-2 ng/ml. By labelling a second monoclonal antibody with an acridinium ester, a chemiluminescence assay with a wider measuring range (0.050-15 ng/ml) was generated. In accord with published data, we found that IL-1ra was secreted by human monocytes stimulated with LPS, Zymosan, IL-1 alpha, or human IgG. After an induction phase of ca. 4 hours and depending on the stimulus, IL-1ra accumulated linearly for periods up to 96 h. IL-1ra levels in synovial fluids of 19 patients suffering from various inflammatory joint diseases were compared with the cytokine levels of IL-1 beta, IL-6, IL-8, and TNF-alpha. Highest positive correlations were found with IL-8 and IL-1 beta. In normal blood donors IL-1ra serum levels were 150-800 pg/ml (Median: 387 pg/ml). Owing to its sensitivity and large measuring range the newly developed assays appear to be suitable for measuring IL-1ra in cell cultures as well as in biological fluids.
Towbin
H
H
CIBA-Geigy Ltd, Basel, Switzerland.
Schmitz
A
A
van Oostrum
J
J
Seitz
M
M
Dewald
B
B
Zingel
O
O
Motz
J
J
Vosbeck
K
K
Rordorf
C
C
D000818:Animals; D000911:Antibodies, Monoclonal; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D053590:Interleukin 1 Receptor Antagonist Protein; D007375:Interleukin-1 / Q000032:analysis*; D008163:Luminescent Measurements; D051379:Mice; D008807:Mice, Inbred BALB C; D009000:Monocytes / Q000276:immunology*; D010957:Plasmids; D011817:Rabbits; D017472:Receptors, Interleukin-1 / Q000037:antagonists & inhibitors*; D011994:Recombinant Proteins / Q000032:analysis; D012216:Rheumatic Diseases / Q000276:immunology; D012795:Sialoglycoproteins / Q000032:analysis*; D013582:Synovial Fluid / Q000276:immunology*
D000911:Antibodies, Monoclonal; C507763:IL1RN protein, human; C507764:Il1rn protein, mouse; D053590:Interleukin 1 Receptor Antagonist Protein; D007375:Interleukin-1; D017472:Receptors, Interleukin-1; D011994:Recombinant Proteins; D012795:Sialoglycoproteins