Mouse Myeloma Line Research Articles

Monoclonal anti-B as a new blood-typing reagent.

Stable cloned lines of anti-B-secreting cells have been derived by fusing spleen cells of a mouse immunized by group B antigen with a mouse myeloma line. The tissue culture supernatant of one of them (NB1/19.112.28) containing secreted monoclonal anti-B antibody is an especially good blood-grouping reagent, well suited for use with manual and automated methods. The unlimited availability of a potent reagent of uniform properties, cheaply produced by a single source, makes monoclonal anti-B a serious competitor to human typing serum.

Confirmation of the assignment of genes of human immunoglobulin heavy chains to chromosome 14 by analysis of Ig synthesis by man-mouse hybridomas.

Hybridomas were produced by fusing the NS1 mouse myeloma line, which does not produce mouse heavy chain Ig, with human peripheral B lymphocytes from a normal individual. Two vigorously growing colonies from this fusion were found to secrete human Ig heavy chains and were recloned. Two secondary clones, which secreted human chains, were again recloned. Among the tertiary clones, two were identified which produced intracellular human Ig chains, but did not secrete immunoglobulin. These tertiary clones were recloned, generating 6 quaternary clones which failed to produce human Ig heavy chains, and 15 quaternary clones which produced intracellular Ig chains. Hybrid clones from each successive subcloning were examined for their human chromosomal content and only those clones which were found to be individually chromosomally distinct, a total of 56 clones in all, were used to analyze the segregation of human chromosomes and human Ig heavy chain synthesis. Results of this study indicate concordant segregation of human Ig heavy chain synthesis and chromosome 14. These studies therefore confirm the previous assignment by C. M. Croce et al. (Proc. Natl. Acad. Sci. USA 1979. 76: 3416) of the genes for human Ig heavy chains to chromosome 14.

Production of specific antibody toCryptococcus neoformansby hybridomas in vitro

Cells of spleen homogenates from sensitized BALB/c or C57BL/6J mice were fused with cells of a mouse myeloma line (P3X63Ag8 or SP2/0 Ag 14) using polyethylene glycol. Nine of 40 hybridomas from a BALB/c, P3X63Ag8 cross produced anti-cryptococcal antibody. Antibodies of at least four different specificities were identified.

Monoclonal antibodies to guinea pig Ia antigens. I. Production, serologic, and immunochemical characterization.

Xenogeneic antibodies to guinea pig Ia antigens were produced by application of the hybridoma technique. BALB/c mice were immunized with the Ia-positive B cell leukemia line EN-L2C of strain 2 guinea pigs, and the spleen cells were fused to the mouse myeloma line NS-1. Culture supernatants of hybrids growing in the selective HAT medium were screened in a binding assay, and hybrids selectively reactive with EN-L2C but not with its Ia-negative variant BZ-L2C were cloned. Ascitic fluid was produced and used as a source of antibody. Four independently derived antibodies proved to be specific for Ia antigens. Two of the antibodies (25E3 and 25E11) seemed to recognize alloantigenic determinants on strain 2 Ia antigens in that they bound only to the cells of strain 2 but not to the cells of strain 13 animals. Two other antibodies (22C4 and 27E7) were found to react with both strain 2 and strain 13 cells, and immunoprecipitation experiments revealed that these antibodies were directed to common determinants shared between certain strain 2 and strain 13 Ia antigens. All antibodies were cytotoxic in the presence of complement for EN-L2C (but not for BZ-L2C) leukemia cells and for lymph node, spleen, peritoneal exudate cells, thymocytes, and purified T cells of strain 2 guinea pigs. In addition, 22C4 was cytolytic for cells of both strains. These results indicate that antibodies can be obtained through the hybridoma technique that are able to distinguish different determinants on Ia antigen molecules of te guinea pig. These antibodies should provide a useful tool for further study of the role of Ia antigens in cellular interaction and Ia-Ir gene function.

Monoclonal antibodies to beta-adrenergic receptors: use in purification and molecular characterization of beta receptors.

We have developed four hybridomas that produce monoclonal antibodies to the turkey erythrocyte beta 1-adrenergic receptor and one hybridoma that produces a monoclonal antibody to the calf lung beta 2 receptor. Splenic lymphocytes from BALB/c mice immunized with partially purified turkey erythrocyte beta 1 receptors or calf lung beta 2 receptors were used with the mouse myeloma line SP2/O-Ag14 to yield hybridoma cultures producing beta receptor monoclonal antibodies of the IgG class. The anti-turkey erythrocyte beta receptor antibodies precipitated partially purified beta receptors and inhibited adrenergic ligand binding. In contrast to autoantibodies to beta 2-adrenergic receptors [Venter, J. C., Fraser, C. M. & Harrison, L. C. (1980) Science 207, 1361-1363] which do not crossreact with cardiac beta 1 receptors, monoclonal antibody FV-104 directed against the adrenergic ligand binding site of turkey erythrocyte beta receptors crossreacted equally with calf liver and lung beta 2 receptors as well as calf heart beta 1 receptors. These data suggest that some molecular homology exists between beta-adrenergic receptors of substantially diverse pharmacological classes. We utilized the monoclonal antibodies in the final stage of turkey erythrocyte beta 1 receptor purification. Turkey erythrocyte beta receptors eluted from FV-104 monoclonal antibody affinity columns with Na-DodSO4 appeared as three components of Mr 70,000, 31,000, and 22,000 on NaDodSO4/polyacrylamide gels. Iodination of material eluted from immunoaffinity columns with propranolol demonstrated the existence of only a single component (Mr, 70,000), indicating that the turkey erythrocyte beta 1 receptor can be purified to homogeneity.

Establishment and characterization of permanent murine hybridomas secreting monoclonal anti-thy-1 antibodies.

hybridomas secreting anti-Thy-1 antibodies were produced by fusing cells of the mouse myeloma line P3-NSI/1-Ag4-1 (NS-1) with spleen cells from AKR/J mice immunized with C3H/Di thymus cells and by subsequent growth in tissue culture and selection of the hybrid cells. Two permanent hybridomas, 1B5 and 1aG4/C5, secreting antibodies of IgG3 subclass were isolated by repeated cloning of cells by dilution and in soft agar. Growth of the hybrid cell colonies depended on the presence of feeder cells; spleen cells at 1-2 x 10(6)/ml were most effective, then thymus cells at 1-4 x 10(6)/ml and peritoneal cells at a concentration 1-2 orders of magnitude lower. The two hybridomas were grown in vitro or vivo and their products were further analysed. In tissue culture in serum-free medium under the optimum conditions the supernatant from hybridoma 1B5 contained 0.07 mg/ml of antibodies and that from hybridoma IaG4/C5 had 0.26 mg/ml of antibodies, whereas ascites 1B5 contained 3.6 mg/ml and ascites 1aG4/C5 4.4 mg/ml of antibodies. A very low electrophoretic mobility of both antibodies facilitated their isolation. The specificity of the antibodies was tested in the cytotoxicity assay in the presence of complement and by the binding of isotopically labelled antibodies to thymus cells from A/Ph mice and other Thy-1.2+ strains and A.Thy-1.1 and AKR/J mie. Antibodies of clone 1aG4/C5 were specific for Thy-1.2+ cells, whereas antibodies of clone 1B5 at higher concentrations also reacted with Thy-1.1+ cells from the thymus and lymph nodes. Both antibodies killed more than 95% thymus cells and 60-70% lymph node cells in the cytotoxicity assay. The specificity of antibodies for T lymphocytes was confirmed in the functional test in which the antibodies eliminated the response of spleen cells to Concanavalin A but did not affect the response to lipopolysaccharide in the presence of complement.

Monoclonal antibodies to human erythrocyte glucose 6-phosphate dehydrogenase

glucose 6-phosphate dehydrogenase (G6PD, type B) have been raised in rabbits and used for the characterization of genetic variants of this enzyme protein [l-4]. Use of the antiG6PD antisera for estimating the specific activity of GGPD variants has recently been criticized and shown to be inadequate to this purpose [5]. More- over, both the specificity and strength of conventional antisera are not satisfactory enough to allow a precise characterization of G6PD variants associated with severe deficiency of activity [6]. In an attempt to overcome these limitations, we used cell fusion techniques to obtain anti-G6PD anti- body-secreting hybridomas [7]. By this approach, a number of antibodies directed to human G6PD were isolated after fusion of an 8-azaguanine-resistant mouse myeloma line with splenocytes from a mouse hyperimmunized with G6PD. 2. Materials and methods

Monoclonal antibodies to human estrogen receptor.

Extranuclear estrogen receptor protein (estrophilin) of MCF-7 human breast cancer cells was purified by passage of the cytosol fraction of a cell homogenate through an affinity column of estradiol linked to Sepharose by a substituted di-n-propyl sulfide bridge in the 17 alpha position. Elution with 50 micro M [3H]estradiol in 10% (vol/vol) dimethyl formamide/0.5 M sodium thiocyanate gave 40% recovery of [3H]estradiol-estrophilin showing 14% of the specific radioactivity expected for the pure complex. Serum from a Lewis rat immunized with this partially purified estradiol-receptor complex contained antiestrophilin antibodies that reacted not only with nuclear and extranuclear estradiol-receptor complexes from MCF-7 cells but also with estrophilin from rat, calf, and monkey uterus, hen oviduct, and human breast cancers. Splenic lymphocytes from the immunized rat were fused with cells of two different mouse myeloma lines (P3-X63-Ag8 and Sp2/0-Ag14) to yield hybridoma cultures, 2% of which produced antibodies to estrophilin. After cloning by limiting dilution, three hybridoma lines secreting antiestrophilin were expanded in suspension culture and as ascites tumors in athymic mice to provide substantial quantities of monoclonal antibodies that recognize mammalian but not avian estrophilin and that show different degrees of reactivity with receptor from nonprimate sources. By growing the clone from Sp2/0 in the presence of [35S]methionine, radiolabeled monoclonal IgG has been prepared. These monoclonal antibodies should prove useful in the study of estrogen receptors of human reproductive tissues, in particular for the radioimmunochemical assay and immunocytochemical localization of receptors in breast cancers.

A new genetic marker of human T lymphoid cells detected by a xenogenic monoclonal antibody.

SummaryA hybrid cell line R3/41‐10 was produced by fusing a mouse myeloma line with spleen cells from CBA/H mice which had previously been immunized with human peripheral blood lymphocytes (PBL) obtained from a single donor. Hybrid secreting antibodies to lymphocyte antigens were detected by assaying the culture supernatant for antibody binding to the donor PBL. Those lymphocytes binding more than ten times the background were cloned in soft agar and transferred to micro‐culture plates (Limbro). Antibodies from some wells lost their binding activity. Antibodies from other clones, although retaining their binding capacity, were multispecific in cytotoxicity experiments, killing all the lympoid cells from a panel of normal donors; yet a third kind gave specific limited reactions. Supernatants from clones R3/41‐10 and R3/41‐13 gave concordant cytotoxic reactions killing 20‐40% of the PBL. There was no cytotoxicity of Ig+ cells (B cells). Lymphocytes from seven out of fourteen normal donors reacted with the antibody. The specificity of the antibody produced by clone R3/41‐10 was confirmed by absorption studies.The monoclonal antibodies (Mc+b) described in this communication are shown to react with a subpopulation of T lymphocytes of some individuals and not others, suggesting that they are detecting a polymorphic system of alloantigens like the Ly system in the mouse, provisionally designated HT‐Ly. l.

Monoclonal antibodies to estrophilin: probes for the study of estrogen receptors.

Splenic lymphocytes from a Lewis rat, immunized with purified estradiol-receptor complex of calf uterine nuclei, were fused with cells of three different mouse myeloma lines (P3-X63-Ag8, P3-NSI/I-Ag4-1, and Sp2/0-Ag14) to yield hybridoma cultures, 9% of which produced antibodies to the receptor protein (estrophilin). When cloned by limiting dilution, approximately 70% of the viable cultures secreted antiestrophilin antibody. When expanded in suspension culture, three clones derived from Sp2/0-Ag14 were found to secrete rat IgG (gamma 2a class), whereas seven other clones (from all three myeloma lines) secreted IgM. Monoclonal IgG shows comparable affinity for nuclear and extranuclear receptors, whereas IgM reacts preferentially with the nuclear form. Both classes of antibody react with unoccupied as well as with occupied receptor and do not interfere with its ability to bind to estradiol. By growing IgG-secreting clones in the presence of [35S]methionine, radiolabeled monoclonal antiestrophilin has been prepared. Unlike antiestrophilin antibody previously generated in the rabbit or the goat, which crossreacts with estrogen receptors from every animal species tested, antibodies produced by the Lewis rat and by hybridomas derived from its spleen cells react specifically with estrophilin from calf tissues. These monoclonal antibodies provide reagents for the application of immunochemical techniques to study estrogen receptors in calf target tissues.

Estrophilin: Pro and anti

Antibodies to the estrogen-receptor protein (estrophilin) of calf uterus have been prepared in the rabbit, the goat and two strains of rat. Because these antibodies react with the receptor without preventing or displacing estradiol binding, antibody-antigen interaction is demonstrated and studied conveniently by its effect on the gel filtration and sedimentation properties of estrophilin, using radioactive estradiol as a marker. Antibodies to the nuclear form of calf uterine estrophilin produced in the rabbit and the goat cross react with nuclear as well as extranuclear estradiol-receptor complexes from reproductive tissues of every species tested; antiestrophilin obtained recently in the ACI rat appears similarly cross reactive. In contrast, antiestrophilin generated in the Lewis rat reacted only with receptor from calf uterus. Polyethylene glycol-mediated fusion of spleen cells from the immunized Lewis rat with three different mouse myeloma lines produced viable hybridoma cultures yielding monoclonal hybridoma cell lines secreting either rat IgG or IgM. which react specifically with calf uterine estrophilin. Production and cloning of hybridomas derived from spleen cells of the ACI rat, in an attempt to obtain cross reacting monoclonal antibody, are in progress. This assortment of either cross reacting or species-specific antibodies to estrophilin, especially those produced by monoclonal hybridomas, offers immunochemical approaches to receptor assay and purification as well as novel probes of receptor structure and function, including the question of whether estrophilin is synthesized in target cells by way of a proestrophilin incapable of binding steroid.

Monoclonal Immunoglobulin Rescue From a Patient With Chronic Lymphocytic Leukemia and Autoimmune Hemolytic Anemia

In order to test whether autoimmune hemolytic anemia in a patient with chronic lymphocytic leukemia was due to autoantibody production by the neoplastic clone, somatic cell hybrids were constructed between the patient's leukemic cells and a mouse myeloma line. Light-chain-restricted human immunoglobulin was secreted by 4 of 11 of the established hybrid lines. No binding of this immunoglobulin to red cell surface antigens could be detected. Thus, we conclude that the autoantibody in this patient represented a concomitant reactive response and was not the product of the malignant clone of B cells.

Properties and Applications of Monoclonal Antibodies Directed against Determinants of the Thy-1 Locus

Fusion of cells of the mouse myeloma line, P3/X63-Ag8 with spleen cells from AKR/J mice immunized against C3H thymocytes or from (BALB/c x BALB.K)F1 mice immunized against AKR/J thymocytes gave rise to hybrid cell lines that continuously secrete antibodies specific for the Thy-1.2 and Thy-1.1 antigens, respectively. Monoclonal antibodies from four such cell lines were analyzed in detail. All were 19S IgM, and, in the presence of complement (C), had high lytic titers on T cells of the appropriate antigenicity. Their specificity was shown by lysis of thymocytes from Thy-1 congenic mouse strains, A/J(Thy-1.2) and A. Thy 1.1. Furthermore, they lyse only 60 to 70% of lymph node cells, suggesting cytotoxicity for mature T cells and not B cells. Treatment of peripheral lymphocyte populations with monoclonal antibody plus C eliminated effector cytotoxic T lymphocytes, their precursors, and the mitogenic response to Con A, but did not affect the response to LPS. Purified, fluorescein-labeled monoclonal anti-Thy-1 antibody could be used to distinguish T and B cells. Purified antibody coupled to Sepharose 6MB was used to separate viable T and B cells. Two independently isolated anti-Thy-1.2 hybridomas are indistinguishable and bind the same determinant whereas a third is unique and may bind a separate site.

Monoclonal xenogeneic antibodies to murine cell surface antigens: identification of novel leukocyte differentiation antigens.

Hybrid myeloma cell lines secreting monoclonal antibodies to mouse cell surface antigens have been prepared. Spleen cells from a DA rat immunized with B10 mouse spleen cells that had been enriched for T cells were fused to cells from a nonsecreting mouse myeloma line (NSI). The presence in the culture supernatants of antibodies binding to mouse spleen cells was tested by a binding assay with 125I-labeled anti-rat IgG. From a large number of positive cultures, ten independent hybrid clones were purified, each secreting a different antibody. Each antigenic target was analyzed by (a) gel electrophoresis of immunoprecipitated 125 I-labeled cell surface molecules, (b) heat stability, (c) strain and species distribution and (d) cross-inhibition of binding of different monoclonal antibodies. It was concluded that the ten monoclonal antibodies regognized four types of antigen. One was the heterophile, heat-stable, Forssman antigen. The second (mol.wt. 210 000) appears to be a major 125I-labeled lymphoid cell surface protein. The third, a minor component of spleen cells, was precipitated as two polypeptides of mol.wt. 190 000 and 105 000. Five IgG-secreting clones identify the fourth antigen, a heat-stable, possibly glycolipid component expressed on mouse red blood cells and also on thymocytes. Cross-inhibition studies suggest that these last monoclonal antibodies bind to overlapping, but not identical, determinants. The class and chain composition of the monoclonal antibodies were studied by gel electrophoresis, isoelectric focusing and ability to lyse red blood cells and thymocytes.

Monoclonal antibodies against human lymphocyte antigens

MONOSPECIFIC antibodies produced by clones derived from appropriate hybridomas represent a new immunological tool of great analytical potential1,2. In this report several antibodies against different surface antigens of human lymphocytes are described. They were obtained and isolated as individual monoclonal products after fusion of splenocytes from an immunised mouse with a mouse myeloma line.

A myeloma hybrid producing antibody specific for an allotypic determinant on "IgD-like" molecules of the mouse.

A hybrid cell line was produced by fusing a mouse myeloma line with spleen cells from BALB/c mice immunized with B10 cells. The hybrid line grew in tissue culture and in syngeneic mice and produced IgM antibody specific for "IgD-like" molecules of mice with the Igb haplotype. The concentration of monoclonal antibody in the serum of tumor-bearing animals reached about 2 mg/ml and gave cytotoxic titers of up to 1:800 000. The derivation of the line, some properties of the antibody secreted and the nature of its antigenic target are described.

Continuous cultures of fused cells secreting antibody of predefined specificity.

THE manufacture of predefined specific antibodies by means of permanent tissue culture cell lines is of general interest. There are at present a considerable number of permanent cultures of myeloma cells1,2 and screening procedures have been used to reveal antibody activity in some of them. This, however, is not a satisfactory source of monoclonal antibodies of predefined specificity. We describe here the derivation of a number of tissue culture cell lines which secrete anti-sheep red blood cell (SRBC) antibodies. The cell lines are made by fusion of a mouse myeloma and mouse spleen cells from an immunised donor. To understand the expression and interactions of the Ig chains from the parental lines, fusion experiments between two known mouse myeloma lines were carried out.

Automatic monitoring of biochemical parameters in tissue culture. Studies on synchronously growing mouse myeloma cells.

An instrument is described that will maintain a population of mammalian cells at constant cell density while automatically monitoring the growth rate of the culture and the extent of precursor incorporation into a variety of cell products. The apparatus was used in an investigation of cyclic changes in the incorporation of labelled precursors into the DNA, RNA, total protein and myeloma protein synthesized in synchronous cultures of a mouse myeloma line. The incorporation of [(3)H]uridine into trichloroacetic acid-insoluble material reveals a slight periodicity, with maxima and minima corresponding to late S phase and the mitotic phases respectively. The incorporation of [(3)H]lysine into total intracellular protein also shows a slight oscillation, with maxima and minima occurring during the respective G2 and mitotic phases. Cyclical changes in the synthesis of serologically precipitable myeloma protein were found to vary somewhat according to the conditions used to synchronize the cells. In experiments conducted with 4.0mm-thymidine, maximal incorporation of label took place during S phase or early G2 phase. Experiments with 1.0mm-thymidine revealed a significantly less marked periodicity of myeloma protein synthesis.