Jerome A. Langer

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.

Procedures for studying binding of interferon to human cells in suspension cultures.

Publisher Summary This chapter describes a simple and rapid assay for the binding of interferon to cells in suspension culture. Although the method is applicable to the cells of any species, human cells are used in the examples provided. It is desirable to have a simple method for measuring the binding of interferon to cells which grow in suspension. Such cells include various naturally occurring circulating cells, both normal and malignant, including many which function in immune responses and for which interferon is a biological effector. There are also a large number of established human cell lines that grow in suspension. Many of these lines are derived from leukemic cells (e.g., Daudi, HL60, and KG-I) and may prove valuable in studying the various roles of interferon. Indeed, the Daudi lymphoblastoid cell line is probably the best characterized human cell line in terms of its interaction with interferon and has been useful in correlating the growth inhibition by interferon with its binding. Many suspension lines are relatively easy to grow in large quantities, making such cells attractive for work on the interferon receptor. The method described is rapid and convenient. Because the cells are sedimented through a sucrose cushion into an elongated tip, no additional washing of the cells after pelleting is required.

Monoclonal antibodies can discriminate between some active and inactive forms of leukocyte interferon

Antiviral activity of recombinant human leukocyte A interferon was inactivated by heating at 65 degrees C or by reduction of disulfide bonds. The specific immunoreactivity, as measured by radioimmunoassays measuring binding to monoclonal antibodies, decreased concomitantly with the antiviral activity. Although the monoclonal antibodies did bind to inactivated interferon, their binding affinity to inactivated interferon was in general very much lower than their binding affinity to active interferon. Therefore, this immunoassay could replace the antiviral assay for detection of biologically active interferon. In addition, most of these antibodies should be especially useful for purification of the interferons since they discriminate between the native active and inactive denatured species. Screening for such antibodies is convenient and simple. The general use of antibodies that preferentially interact with native molecules provides a powerful new principle for choosing monoclonal antibodies with extraordinary potential in assay and purification.

[34] Procedure for reduction and reoxidation of human leukocyte interferon

Publisher Summary This chapter discusses the procedure for the reduction and reoxidation of human leukocyte interferon. The reduction and reoxidation of interferon (IFN) is of interest both in understanding the structure–function relationship of the disulfides in IFN and toward the practical goal of recovering or regenerating active interferon after reductive procedures. Three procedures are described in the chapter: (1) the reduction and (presumably) direct reoxidation of IFN-αA, (2) the reduction and reversible covalent modification of the sulfhydryls by sulfitolysis, with subsequent reoxidation, and (3) the irreversible modification of sulfhydryls by reaction with iodoacetate or iodoacettamide. The importance of the disulfides for maximal interferon activity has been clear from the early experiments. The procedures presented in the chapter give several methods for their manipulation, including the controlled recovery of the native-like active form from the completely reduced insoluble form. Future studies on the role of disulfides will be enhanced by the synthesis of the proteins of modified sequence generated by oligonucleotide-directed sitespecific mutagenesis of the corresponding DNA coding sequences.