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

J Immunol Methods. 1994 Mar 29;170(1):125-35. doi: 10.1016/0022-1759(94)90252-6.

Abstract

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.

MeSH terms

  • Animals
  • Antibodies, Monoclonal
  • Cells, Cultured
  • Enzyme-Linked Immunosorbent Assay / methods*
  • Humans
  • Interleukin 1 Receptor Antagonist Protein
  • Interleukin-1 / analysis*
  • Luminescent Measurements*
  • Mice
  • Mice, Inbred BALB C
  • Monocytes / immunology*
  • Plasmids
  • Rabbits
  • Receptors, Interleukin-1 / antagonists & inhibitors*
  • Recombinant Proteins / analysis
  • Rheumatic Diseases / immunology
  • Sialoglycoproteins / analysis*
  • Synovial Fluid / immunology*

Substances

  • Antibodies, Monoclonal
  • IL1RN protein, human
  • Il1rn protein, mouse
  • Interleukin 1 Receptor Antagonist Protein
  • Interleukin-1
  • Receptors, Interleukin-1
  • Recombinant Proteins
  • Sialoglycoproteins