Giovanni Galfre

Production of monoclonal antibodies to group A erythrocytes, HLA and other human cell surface antigens-new tools for genetic analysis

Antibody-secreting hybrid cells have been derived from a fusion between mouse myeloma cells and spleen cells from a mouse immunized with membrane from human tonsil lymphocyte preparations. Hybrids secreting antibodies to cell surface antigens were detected by assaying culture supernatants for antibody binding to human tonsil cells. Six different antibodies (called W6/1, /28, /32, /34, /45 and /46 were analyzed. These were either against antigens of wide tissue distribution (W6/32, /34, and /46) or mainly on erythrocytes (W6/1 and W6/28). One of the anti-erythrocyte antibodies (W6/1) detected a polymorphic antigen, since blood group A1 and A2 erythrocytes were labeled while B and O were not. Antibodies W6/34, /45 and /46 were all against antigens which were mapped to the short arm of chromosome 11 by segregation analysis of mouse-human hybrids. Immunoprecipitation studies suggest that W6/45 antigen may be a protein of 16,000 dalton, apparent molecular weight, while W6/34 and /46 antigens could not be detected by this technique. Antibody W6/32 is against a determinant common to most, if not all, of the 43,000 dalton molecular weight chains of HLA-A, B and C antigens. This was established by somatic cell genetic techniques and by immunoprecipitation analysis. Tonsil leucocytes bound 370,000 W6/32 antibody molecules per cell at saturation. The hybrid myelomas W6/32 and W6/34 have been cloned, and both secrete an IgG2 antibody. W6/32 cells were grown in mice, and the serum of the tumor-bearing animals contained greater than 10 mg/ml of monoclonal antibody. The experiments established the usefulness of the bybrid myeloma technique in preparing monospecific antibodies against human cell surface antigens. In particular, this study highlights the possibilities not only of obtaining reagents for somatic cell genetics, but also of obtaining mouse antibodies detecting human antigenic polymorphisms.

Analysis of cell surfaces by xenogeneic myeloma-hybrid antibodies: differentiation antigens of rat lymphocytes.

Abstract A new approach to study differentiation antigens by means of monoclonal xenogeneic antibodies produced by myeloma-hybrid lines in culture is described. Spleen cells from mice immunized with rat thymocyte membranes were fused with a mouse myeloma. Hybrids were selected and screened for the production of antibodies to rat thymocytes by a binding assay. A number of such antibodies was detected, and five specificities (W34, /6, /13, /15 and /25) were further investigated. Two of the hybrid myelomas were cloned, and the monoclonal antibodies were shown to contain heavy chains of molecular weight 50,000 daltons (W313) and about 70,000 daltons (W34), respectively. All antibodies were shown to detect different, previously undefined antigens on different subpopulations of lymphoid cells. The different labeling patterns on thymocytes, thoracic duct lymphocytes and bone marrow cells were investigated by the fluorescence-activated cell sorter. Antibodies W36, /13, /15 and /25 labeled most thymocytes, while W34 labeled very few. Among thoracic duct lymphocytes, W313 labeled most, if not all, of the T cells, and W325 labeled a large fraction of them; W34, on the other hand, labeled mainly B cells. In bone marrow, W313 and W315 labeled sub-populations which were largely independent of each other. Absorption studies showed that brain, but not kidney or liver, absorbed W313, while all these tissues were markedly less effective than lymphoid cells for absorption of W325. The number of antibody molecules per cell binding at saturation to thymocytes was: 5000 (W36); 17,000 (W315 and W325); 36,000 (W313). None appears, therefore, to detect major proteins in the membrane. The method is therefore extremely sensitive and allows identification down to minor membrane molecules and also of antigens on small subpopulations of a heterogeneous mixture of cells.

Rat × rat hybrid myelomas and a monoclonal anti-Fd portion of mouse IgG

MONOCLONAL antibodies can be produced by cultures of permanent cell lines derived by fusion of suitable myelomas with spleen cells from immunised animals1. So far, mainly two mouse myelomas, X63-Ag8 and NSI/1-Ag4.1, have been used for this purpose1,2. When cells from immunised mice are used in the fusion, the mouse × mouse hybrid myelomas can be grown as tumours in appropriate mouse strains. We describe here the preparation and use of a new clone of a rat myeloma, which is suitable for the derivation of rat × rat hybrid myelomas producing specific rat antibodies. The spent medium of hybrid myeloma cultures usually contains 1–20 μg ml−1 antibody. The serum and ascites of the tumour-bearing mice, with few exceptions, yield 1–20 mg ml−1 of antibody, which is 1,000 times more concentrated and very convenient for larger preparations.

Monoclonal antibodies as probes for differentiation and tumor-associated antigens: a Forssman specificity on teratocarcinoma stem cells

A set of monoclonal antibodies derived by fusing P3-NS1/1-Ag4-1 myeloma cells with spleen cells from a rat immunized with mouse spleen were screened for activity against a tumor cell panel. One of these antibodies was found to react only with mouse embryonal carcinoma cells and no other tumor cell type tested, including differentiated derivatives of teratocarcinomas. In the adult mouse, this antigen is expressed by subpopulations of cells in the spleen, bone marrow, lymph node, brain, kidney and testes, although not in liver and thymus. This antigen has a species and tissue distribution consistent with that of Forssman antigen. The molecules which carry this specificity on the embryonal carcinoma cells appear to be glycolipids.

Monoclonal Antibodies as Tools to Analyze the Serological and Genetic Complexities of Major Transplantation Antigens

The rat MHC resembles that of other species in displaying extensive polymorphism for a variety of MHC-characteristic functions: antigens detected by serological assays, antigens detected by cellular assays such as the MLR, GVH and CML, and immune response genes for a variety of cellular and non-cellular antigens (Gunther & Stark 1977, Gasser 1977). Nevertheless very little is known about the genetic structure of the region because of the shortage of laboratory recombinants. The present study grew out of the realisation that the high resolving power of monoclonal antibodies against complex polymorphic antigens could compensate for lack of resolution at the genetic level. Granted suitable monoclonal alloantibodies, the number and antigenic structure of the polymorphic molecules specified by the MHC can in principle be examined with greater precision than is possible by analysis of recombinants using planned immunizations and absorptions of conventional sera. This review describes the preparation of monoclonal antibodies by fusion of spleen cells from alloimmunized rats with mouse plasmacytoma cells and some results of an analysis of the properties of these antibodies. Some preliminary data have been published elsewhere (Galfre et al. 1977, Howard et al. 1978).

Monoclonal antibodies and cell surface antigens

Antibody chains are encoded in three gene clusters containing genes for the variable and constant regions. V and C genes are separated in germ line and during differentiation a rearrangement takes place. But even after this rearrangement the V and C coding sequences are not contiguous. A final splicing must take place in committed cells between the transcription of a discontinuous V-and C-region DNA and the expression of a continuous mRNA coding for an antibody chain. Analysis by cell fusion indicates that the splicing is cis. When two antibody-producing cell lines are fused, the resulting hybrids express the two antibodies that characterize the parental lines. Permanent cell lines producing antibody of predefined specificity have now been derived in this way. Spleen cells from hyperimmunized donors are fused with myeloma cells and a proportion of the hybrids that are established synthesize and secrete antibodies directed against the immunogen. The heterogeneous cell population can be cloned and propagated. This is a potent way of producing monospecific antibodies to complex antigens such as cell membranes and transplantation antigens. Monoclonal xenogeneic antibodies to rat cell-surface membranes have proved very valuable for characterizing and separating rat lymphocyte subpopulations. In more recent experiments, monoclonal xenogeneic antibodies to mouse and human cell-surface antigens have also been produced which permit the characterization of the hitherto undescribed differentiation antigens.

Use of Different Hapten-Protein Conjugates Immobilized on Nitrocellulose to Screen Monoclonal Antibodies to Abscisic Acid

The dot-immunobinding method for screening antibodies to proteins on sheets of nitrocellulose has been modified to allow monoclonal antibodies (McAb) to the hapten abscisic acid (ABA) to be screened. Several methods for conjugating ABA to proteins using new bifunctional coupling reagents, specific for hapten keto groups, are described. Hybridomas secreting McAb with a defined specificity for the hapten can be identified by screening supernatants against the carrier protein and other hapten-protein conjugates with different conjugation bridges or modified hapten structure. Inhibition of binding to conjugates by free hapten is used to determine the relative avidity of the McAb for free and bound hapten. All of these tests could be done with no more than about 50 microliter of antibody solution. Dot immunobinding is a useful alternative to radioimmunoassay for screening McAb to haptens.

Monoclonal antibody screening: Two methods using antigens immobilized on nitrocellulose

The dot-immunobinding assay of Hawkes et al. [R. Hawkes, E. Niday, and J. Gordon (1982) Anal. Biochem. 119, 142-147] has been modified such that many antibody solutions are screened simultaneously on dots of antigen applied to a single sheet of nitrocellulose using very small amounts of both antigen and antibody solutions. This method is also used in the assay of antibody binding on dots of components of the antigen after enzymatic digestion and chromatographic fractionation and to dots of compounds which may be chemically related to the antigen. An aluminum template for screening Western blots with a number of different antibody solutions on a single sheet of nitrocellulose is also described. Possible applications of the dot-immunobinding assay in screening for virus are discussed.

Monoclonal Anti-Rat MHC (H-1) Alloantibodies

We wish to report the isolation of 6 clones of hybrid myelomas secreting rat antibodies with specificity for alloantigens of the rat major histocompatibility complex H-1 (AgB). An account of the isolation of the first such clone including details of the immunization and fusion techniques has been published (1). Briefly two AO (H-1w, AgB2) rats were hyperimmunized against DA (H-1a, AgB4) lymphoid cells and finally boosted with an intravenous injection of DA cells 2.5 days or 4 days before fusion. Spleen cells were fused with P3-X63-Ag8 BALB/c mouse myeloma cells in the presence of Polyethylene Glycol (PEG) 1500. We used the intravenous route to boost before fusing to favour localization of the antibody response in the spleen (2). After fusion, aliquots initially containing 2 x 106 spleen cells were grown in the wells of 6 x 4 Linbro trays in HAT medium for two weeks. Active growth of presumptive hybrids was seen in all wells. Supernatants obtained at this stage were assayed for specific antibody by 51Cr release from AO and DA RBC in a complement dependent lytic assay. 106/113 wells contained significant lytic activity against DA RBC while no autoantibody activity against AO RBC was detected.

Monoclonal Xenogeneic Antibodies to Mouse Leukocyte Antigens: Identification of Macrophage-Specific and Other Differentiation Antigens

The identification and study of cell surface molecules which have specific immune functions and are markers of differentiated white blood cell subpopulations is of great interest in immunology. Antibodies are versatile probes with which to study these molecules. However, it is difficult to obtain highly specific antibodies recognizing individual cell surface molecules, because cell surfaces are complex mosaics of many different immunogenic glycoproteins and glycolipids. A general approach to this problem stems from the experiments of Kohler and Milstein (1). They fused myeloma cells and spleen cells from mice immunized with sheep red blood cells to derive continuous hybrid cell lines secreting antibodies to sheep red blood cells. Such lines can be manipulated in culture so that the multispecific response to a complex immunogen can be resolved into a set of monospecific responses by cloning. Hybrids have been obtained which secrete antibodies to rat major histocompatibility antigens(2), rat cell surface xenoantigens (3), mouse IgD allotypes (4), and mouse H-2K antigens (5).