Sara Tamir

The presence of non-isotype-specific antibodies in polyclonal anti-IgE reagents: Demonstration of their binding to specifically selected Epstein-Barr virus-transformed cell lines

Polyclonal anti-human IgE reagents were earlier shown to contain variable amounts of nonisotype-specific antibodies depending on the strategy used for their preparation. The presence of these antibodies in two commercial anti-IgE reagents was demonstrated in this work by (a) their binding to human Ig-surface-positive lymphoblastoid cells specifically selected by one of the polyclonal anti-human IgE reagents and (b) their binding to the non-IgE immunoglobulins secreted by those lymphoblastoid cells. Peripheral blood B lymphocytes from two normal and two atopic patients were immortalized with Epstein-Barr virus (EBV) and then selected for cells that rosette with anti-IgE-coated erythrocytes. Selection was repeated four times and cells were then cloned. The cloned cells formed rosettes and their supernatants agglutinated erythrocytes coated with rabbit anti-IgE. The immunoglobulins of these clones were positive in an ELISA for IgE, using two different polyclonal anti-human IgE reagents. They were shown, however, to be 19 S IgMs. This discrepancy was due apparently to substantial contamination of anti-non-IgE-isotype-specific antibodies in the polyclonal anti-IgE reagents used both in the selection of cells and in the ELISA. The human monoclonal B-cell lines which were applied here as targets amplified the non-IgE-isotype specific antibody contamination present in the polyclonal anti-human IgE reagents. Because of the normally very low frequency of IgE-positive cells, the use of polyclonal anti-IgE reagents to detect these cells has to be carefully evaluated.

In vitro produced monoclonal rheumatoid factor: Purification, radiolabel, and possible applications

Abstract A human monoclonal rheumatoid factor (IgM, λ) produced in vitro by Epstein-Barr virus (EBV) immortalized cell line was purified from cell culture medium. Purification, based on the binding of this antibody to protein A, was carried out by adsorption on protein A-Sepharose and elution with low pH buffer. The eluted preparation (×400 purified) was radiolabeled. The specific binding of the purified rheumatoid factor (RF) to immune complexes was kept intact. This unique human monoclonal antibody is applied as a universal reagent to detect low titers of antibodies directed toward cellular antigens.

Human monoclonal antibodies produced by Epstein-Barr virus transformed cell lines bind protein A

Epstein-Barr virus (EBV) is a polyclonal T-independent activator of viral receptor positive human B lymphocytes. Lymphocytes infected in vitro with the virus are transformed into immortalized cell lines [Nilsson, K, and Klein, G. (1982) Adv. Cancer Res. 37, 319]. In this way human cell lines that secrete specific IgM, IgG and IgA monoclonal antibodies are established. Protein A is also a polyclonal T-independent B cell activator [Langone, J. J. (1982) Adv. Immunol. 32, 157], the targets of which are surface immunoglobulin and C3d receptor positive cells, as are the targets of EBV. We found that almost all (16 out of 17) of the specific monoclonal antibodies (IgM, IgG and IgA) produced in vitro by EBV cell lines bind protein A. Unlike these in vitro produced antibodies, a substantial fraction of the immunoglobulins in human serum does not bind protein A. Thus, those plasma cells which in vivo secrete protein A nonbinding immunoglobulins originate from precursors of B cell that were EBV noninfective. Alternatively, during in vivo B differentiation some immunoglobulins undergo a change from protein A binding to protein A nonbinding molecules.

Human Monoclonal Anti-Idiotypic Antibodies against a Murine Monoclonal Antibody (Mab17-1A) as an Anti-Tumor Vaccine

Specifically selected anti-idiotypic antibodies can be used for immunization in place of the nominal antigen. Aiming toward this, human monoclonal anti-idiotypic antibodies Mab2s) specific for a mouse monoclonal anticolon carcinoma antibody (Mab17-1A)(Ab1) were produced by Epstein-Barr virus (EBV)-immortalized cells (Steinitz et al., 1988b). Ten of these human IgGs, which inhibited the binding of Mab17-1A to colon carcinoma cells, bound to the same or to a close epitope in the combining site of Ab1. The Mab2s did not inhibit the binding of another mouse monoclonal antibody (M74) directed to the same molecule to which Mab17-1A was directed.

The coating of erythrocytes with detergent-solubilized molecules: a general method for improved coupling of antigens and antibodies

A method is described by which different antigens and antibodies in solutions containing deoxycholate (DOC) may be coated to glutaraldehyde (GA)-fixed erythrocytes. This method, based on the use of CrCl3, yields erythrocytes which preserve the antigenicity and antibody activity of the coated molecules and can thus be applied to various assays in which indicator red cells are required. The sensitivity of the assays increases when these red cells are used. The cells form rosettes with the appropriate receptor positive cells. They sediment in Ficoll-Isopaque as do native erythrocytes and do not aggregate spontaneously even when coated at very high or low concentrations of CrCl3 or antigen. This coupling method, which is performed in the presence of DOC, and which requires only a small amount of antigen, is proposed for the coupling of cell membrane dissolved antigens.

Separation of rare cell subpopulations with the aid of biotin-labelled ligands

A universal method for selection of surface marker-positive cells is described. The cells, admixed with an excess of surface marker-negative cells, are In-st labelled with a specific biotinylated ligand and then isolated with the aid of monoclonal, anti-biotin coated beads. The method enables selection and isolation of cells with a frequency as low as 10-4. The ligand can be an antigen (for selection of infrequent antibody-producing cells), an antibody (for selection of surface antigen-positive cells) or other molecules (for selection of specific receptor-positive cells).

A sensitive method for detecting cells coated with antibodies with human monoclonal rheumatoid factor produced in vitro

A human monoclonal rheumatoid factor (RF) produced in vitro by an Epstein-Barr virus immortalized cell line has been used to detect and quantitate antibodies specifically bound to cells. The RF is purified from cell culture supernatant by a simple procedure and then radiolabeled. In this assay, the binding of 125I-labeled RF to cells coated with specific antibodies is determined. This RF method detects very low titers of antibodies directed against specific cellular antigens and also minorities of antibody coated tumor cells in mixtures with identical non-coated cells. The advantages of this unique human monoclonal antibody are discussed.