Identification of high-affinity anti-IL-1 alpha autoantibodies in normal human serum as an interfering substance in a sensitive enzyme-linked immunosorbent assay for IL-1 alpha.
A highly reproducible, sensitive, and specific sandwich enzyme-linked immunosorbent assay (ELISA) for recombinant human IL-1 alpha (rhIL-1 alpha) has been developed. Results from this ELISA have demonstrated that the concentration of rhIL-1 alpha added to normal human serum (NHS) decreased by 16.3% after 3 h and 24.9% after 6 h at room temperature. Molecular exclusion column chromatography with Sephacryl S-300 HR revealed that 125I-labeled IL-1 alpha added to normal human serum rapidly formed higher molecular weight complexes without indication of proteolytic degradation. The observed reduction in immunoreactivity was correlated with this protein complex formation and accounted for the apparent instability of rhIL-1 alpha in NHS. Immunoblot analysis indicated that the molecular weight of the binding protein was 150-160K, and the IL-1 alpha binding activity was removed and recovered from NHS by Protein-G affinity chromatography; indicating that the binding protein was IL-1 alpha-specific IgG. The binding of 125I-labeled IL-1 alpha to the serum binding proteins could be inhibited by unlabeled IL-1 alpha (IC50 = 7.4 x 10(-11) M) but not by unlabeled IL-1 beta. Kinetic analysis with 125I-labeled IL-1 alpha revealed that the average binding affinity of these IL-1 alpha-specific IgGs was 4.7 x 10(10) M-1. These results suggest that these autoantibodies may interfere with the detection of IL-1 alpha in human serum by various assay systems and also could be a regulator of circulating IL-1 alpha.
Mae
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N
Department of Drug Metabolism, Hoffmann-La Roche Inc., Nutley, NJ 07110.
Liberato
D J
DJ
Chizzonite
R
R
Satoh
H
H
D001323:Autoantibodies / Q000097:blood*; D002850:Chromatography, Gel; D004355:Drug Stability; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D015151:Immunoblotting; D007375:Interleukin-1 / Q000097:blood / Q000276:immunology*; D007457:Iodine Radioisotopes; D007700:Kinetics; D011971:Receptors, Immunologic / Q000097:blood; D017472:Receptors, Interleukin-1; D011994:Recombinant Proteins / Q000276:immunology; D012016:Reference Values; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D001323:Autoantibodies; D007375:Interleukin-1; D007457:Iodine Radioisotopes; D011971:Receptors, Immunologic; D017472:Receptors, Interleukin-1; D011994:Recombinant Proteins