Preparation and validation of radio iodinated recombinant human IL-10 for the measurement of natural human antibodies against IL-10
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Preparation and validation of radio iodinated recombinant human IL-10 for the measurement of natural human antibodies against IL-10. / de Lemos Rieper, Carina; Galle, Pia; Svenson, Morten; Pedersen, Bente Klarlund; Hansen, Morten Bagge.
I: Journal of Immunological Methods, Bind 350, Nr. 1-2, 2009, s. 46-53.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
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TY - JOUR
T1 - Preparation and validation of radio iodinated recombinant human IL-10 for the measurement of natural human antibodies against IL-10
AU - de Lemos Rieper, Carina
AU - Galle, Pia
AU - Svenson, Morten
AU - Pedersen, Bente Klarlund
AU - Hansen, Morten Bagge
N1 - Keywords: Animals; Autoantibodies; Blood Donors; CHO Cells; Cricetinae; Cricetulus; Donor Selection; Humans; Inflammation; Interleukin-10; Iodine Isotopes; Radioimmunoassay; Recombinant Proteins
PY - 2009
Y1 - 2009
N2 - 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.
AB - 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.
U2 - 10.1016/j.jim.2009.07.005
DO - 10.1016/j.jim.2009.07.005
M3 - Journal article
C2 - 19632236
VL - 350
SP - 46
EP - 53
JO - Journal of Immunological Methods
JF - Journal of Immunological Methods
SN - 0022-1759
IS - 1-2
ER -
ID: 20010526