Preparation and validation of radio iodinated recombinant human IL-10 for the measurement of natural human antibodies against IL-10.
Radio iodinated recombinant human IL-10 was prepared and validated for the measurement of natural human anti-IL-10 antibodies. Iodination of IL-10 was accomplished by means of the chloramine-T method. The crude tracer was purified by size chromatography as homo-dimeric IL-10 with a specific activity of 75 cpm/pg. Validation of the tracer confirmed preserved antibody epitopes and receptor binding ability. A robust Radio Immuno Assay (RIA) was developed and validated to detect natural human anti-IL-10 antibodies based on the formation of (125)I-labeled IL-10-IgG complexes in solution and separation of the complexes by chromatography on mini-columns. The RIA was applied to 3360 plasma samples derived from normal Danish blood donors. Generally, IL-10 did not bind to plasma factors other than natural anti-IL-10 IgG antibodies. The prevalence of donors high positive for antibodies against IL-10 was 0.5%. These levels were apparently associated with IL-10 deficiency, since the antibodies blocked IL-10-induced STAT3 phosphorylation in normal blood leukocytes. This methodology will be used to explore the clinical significance of natural anti-IL-10 antibodies with respect to inflammatory disorders.
de Lemos Rieper
Carina
C
Department of Clinical Immunology, Rigshospitalet, National University Hospital, Copenhagen, Denmark. carina.de.lemos.rieper@rh.regionh.dk
Galle
Pia
P
Svenson
Morten
M
Pedersen
Bente Klarlund
BK
Hansen
Morten Bagge
MB
D000818:Animals; D001323:Autoantibodies / Q000097:blood*; D001782:Blood Donors; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D046148:Donor Selection / Q000379:methods*; D006801:Humans; D007249:Inflammation / Q000097:blood; D016753:Interleukin-10 / Q000737:chemistry* / Q000235:genetics; D007456:Iodine Isotopes; D011863:Radioimmunoassay / Q000379:methods; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics
D001323:Autoantibodies; C508609:IL10 protein, human; D007456:Iodine Isotopes; D011994:Recombinant Proteins; D016753:Interleukin-10