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

Journal Article · Mon Apr 01 00:00:00 EST 1991 · Lymphokine Cytokine Research; (United States)

OSTI ID:5266205

Mae, N; Liberato, D J; Chizzonite, R; Satoh, H ^[1]

---

1. Hoffmann-La Roche Inc., Nutley, NJ (USA)

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 {times} 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 {times} 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}.

• OSTI ID: 5266205
• Journal Information: Lymphokine Cytokine Research; (United States), Vol. 10:1-2; ISSN 1056-5477
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
LYMPHOKINES
ENZYME IMMUNOASSAY
MONOCLONAL ANTIBODIES
BIOLOGICAL FUNCTIONS
BIOCHEMICAL REACTION KINETICS
BLOOD SERUM
IODINE 125
ION EXCHANGE CHROMATOGRAPHY
LIQUID COLUMN CHROMATOGRAPHY
MAN
MOLECULAR WEIGHT
RECEPTORS
TRACER TECHNIQUES
ANIMALS
ANTIBODIES
BETA DECAY RADIOISOTOPES
BIOASSAY
CHROMATOGRAPHY
DAYS LIVING RADIOISOTOPES
ELECTRON CAPTURE RADIOISOTOPES
GROWTH FACTORS
IMMUNOASSAY
INTERMEDIATE MASS NUCLEI
INTERNAL CONVERSION RADIOISOTOPES
IODINE ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
MAMMALS
MEMBRANE PROTEINS
MITOGENS
NUCLEI
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
PRIMATES
PROTEINS
RADIOISOTOPES
REACTION KINETICS
SEPARATION PROCESSES
VERTEBRATES
550201* - Biochemistry- Tracer Techniques