Purification of soluble cytokine receptors from normal human urine by ligand-affinity and immunoaffinity chromatography.
Affinity chromatography of crude human urinary proteins on either human recombinant interleukin-6 (rIL-6) or human recombinant interferon-gamma (rIFN-gamma) or anti IFN-gamma receptor (IFN-gamma-R) monoclonal antibodies (McAb) yielded the two respective soluble receptors in significant amounts. A single sequence of 30 amino acid residues was obtained by N-terminal microsequencing of the protein peak purified in tandem by affinity chromatography on an IL-6 column and reversed-phase high-performance liquid chromatography. This sequence was identical with the predicted N-terminal sequence of IL-6-R as previously reported. The purified IL-6-R retained its biological activity. It was used for the preparation of specific anti IL-6-R monoclonal antibodies. Analysis of the eluted proteins from both IFN-gamma and anti IFN-gamma-R columns by inhibition of solid-phase radioimmunoassay, enzyme-linked immunosorbent assay, sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting proved the existence of soluble IFN-gamma-R in normal urine. This finding together with the already known presence of soluble TNF receptors and a soluble IL-2 receptor found both in plasma and in urine indicates that release of soluble cytokine receptors into body fluids is a general phenomenon which occurs under normal physiological conditions.
Novick
D
D
Department of Molecular Genetics and Virology, Weizmann Institute of Science, Rehovot, Israel.
Engelmann
H
H
Wallach
D
D
Leitner
O
O
Revel
M
M
Rubinstein
M
M
D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006825:Hybridomas / Q000032:analysis; D007120:Immunochemistry; D007371:Interferon-gamma / Q000652:urine; D015850:Interleukin-6 / Q000652:urine*; D011507:Proteinuria / Q000652:urine; D011863:Radioimmunoassay; D011971:Receptors, Immunologic / Q000652:urine*; D017471:Receptors, Interferon; D019947:Receptors, Interleukin-6; D011994:Recombinant Proteins / Q000032:analysis
D015850:Interleukin-6; D011971:Receptors, Immunologic; D017471:Receptors, Interferon; D019947:Receptors, Interleukin-6; D011994:Recombinant Proteins; D007371:Interferon-gamma