Characterization of antihuman IFNAR-1 monoclonal antibodies: epitope localization and functional analysis.
The type I interferon receptor (IFNAR) is composed of two subunits, IFNAR-1 and IFNAR-2, encoding transmembrane polypeptides. IFNAR-2 has a dominant role in ligand binding, but IFNAR-1 contributes to binding affinity and to differential ligand recognition. A panel of five monoclonal antibodies (mAb) to human IFNAR-1 (HuIFNAR-1) was produced and characterized. The reactivity of each mAb toward HuIFNAR-1 on native and transfected cells and in Western blot and ELISA formats was determined. In functional assays, one mAb, EA12, blocked IFN-a2 binding to human cells and interfered with Stat activation and antiviral activity. Epitopes for the mAb were localized to subdomains of the HuIFNAR-1 extracellular domain by differential reactivity of the mAb to a series of human/bovine IFNAR-1 chimeras. The antibody EA12 seems to require native HuIFNAR-1 for reactivity and does not map to a single subdomain, perhaps recognizing an epitope containing noncontiguous sequences in at least two subdomains. In contrast, the epitopes of the non-neutralizing mAb FB2, AA3, and GB8 mapped, respectively, to the first, second, and third subdomains of HuIFNAR-1. The mAb DB2 primarily maps to the fourth subdomain, although its reactivity may be affected by other determinants.
Department of Molecular Genetics and Microbiology, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA.
L A
LA
Goldman
M
M
Zafari
E C
EC
Cutrone
A
A
Dang
M
M
Brickelmeier
L
L
Runkel
C D
CD
Benjamin
L E
LE
Ling
J A
JA
Langer
D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis*; D000998:Antiviral Agents / Q000378:metabolism; D019556:COS Cells; D018604:Epitope Mapping; D006801:Humans; D016898:Interferon-alpha / Q000378:metabolism; D017434:Protein Structure, Tertiary; D015398:Signal Transduction / Q000502:physiology
D000911:Antibodies, Monoclonal; D000998:Antiviral Agents; D016898:Interferon-alpha