Mouse Myeloma Line Research Articles

Effect of the presence or absence of J chain on expression of recombinant anti‐Kell immunoglobulin M

The aim of this study was to determine the effect of expressing a recombinant anti-Kell immunoglobulin (Ig) M from two cell lines, CH0 and NS0, on its ability to function as a diagnostic antibody. As a polymeric immunoglobulin, IgM is able to directly agglutinate red blood cells (RBCs), making it a useful blood grouping reagent. To simplify expression, recombinant human IgM (rIgM) from NS0 (a mouse myeloma line) and CHO (Chinese hamster ovary line) cells was expressed in the absence of human J chain. Whereas NS0 expresses mouse J chain, rIgM expressed from CH0 cells lack J chain. Although the ability to polymerize resides within the tailpiece of IgM heavy chain, J chain can influence the polymeric state. This in turn could affect the ability of rIgM to bind its antigen. The variable region of the heavy chain of an anti-Kell IgG was grafted onto the constant region of human IgM and co-expressed with light chain derived from the same antibody. rIgM was purified from each cell line and the strength of direct agglutination assessed. Both cell lines produced polymeric rIgM that was able to specifically bind the target antigen and to directly agglutinate RBCs to the same degree. The presence or absence of J chain did not affect the ability of the rIgM to bind the Kell antigen or the strength of agglutination. The presence of J chain is not required for the production of a functional rIgM for use as a diagnostic reagent. CHO and NS0 lines are both suitable for production of such a reagent.

César Milstein (1927–2002)

César Milstein (1927–2002)

Induction of specific cytotoxic T-cell activity against xenogeneic target cells in carp (Cyprinus carpio).

To investigate the induction of cytotoxic T cells in carp (Cyprinus carpio) after inoculation of fish with 2 xenogeneic line cells and to examine specificity of the cytotoxic activity. 22 carp. Fish were inoculated with mouse myeloma line cells P3.NS-1/1Ag4.1 (NS-1) or chicken Marek's disease tumor-derived lymphoma line cells (MDCC MSB-1). Cytotoxic activity of immune lymphocytes was evaluated by incubating effector cells with homologous and heterologous target cells. Populations of effector cells were identified by blocking T-lymphocytes from effector cells, using anti-carp T-cell monoclonal antibody and complement. Lymphocytes in blood, spleen, and head kidney of carp inoculated with NS-1 cells or MDCC MSB-1 cells had dose-dependent cytotoxic effects against homologous target cells but not against heterologous target cells. Lymphocytes from noninoculated carp did not have cytotoxic effects. Depletion of T-lymphocytes in spleen cells from NS-1-inoculated carp resulted in a decrease of cytotoxic activity against NS-1 cells. Cytotoxic activity of spleen lymphocytes from NS-1-inoculated or noninoculated carp was not evident when cytotoxic tests were performed after addition of anti-NS-1 carp serum. Inoculation with xenogeneic target cells induces a specific cytotoxic T-cell response in carp. Thus, cell-mediated immunity plays a role in defense against infection of parasitic organisms such as protozoa and helminths.

Monoclonal Antibodies Against Hepatitis B S Antigen: Production, Characterization, and Use for Diagnosis

Three different hybridoma clones secreting anti-HBsAg antibody were constructed by fusing cells of mouse myeloma line Ag8-X63 with splenocytes from BALB/c mice immunized with recombinant HBsAg and natural HBV. The monoclonal antibodies obtained were characterized immunologically, and two were used to develop UMELISA for detection of HBsAg. This monoclonal assay enabled the detection of 0.1 UPE/mL with reference to the standard of the Paul Ehrlich Institute (Frankfurt, Germany). The assay compared well with a commercially available kit (UMELISA HBsAg) and was used for detection of HBsAg in blood donors.

A panel of CAb antibodies recognize endogenous and ectopically expressed ING1 protein.

Nine monoclonal antibodies (MAbs) against human and rodent ING1 protein have been generated using an IL6-secreting mouse myeloma line. These antibodies are all effective in recognizing ING1 protein in ELISAs, Western blot assays, and by indirect immunofluorescence. Combining different CAb monoclonal antibodies in a Western blot assay also allows detection of the very low levels of endogenous ING1 found in fibroblast cells in culture and the identification of at least two isoforms of ING1 in normal human diploid fibroblasts and established brain cancer cell lines.

Negative regulation of rat natural killer cell activity by major histocompatibility complex class I recognition.

The cytolytic activity of human and mouse natural killer (NK) cells is negatively regulated by self major histocompatibility complex (MHC) class I molecules on potential target cells. In the rat, protection by RT1 class I gene products has so far not been formally shown although the complex effects of foreign and self RT1 genes on polyclonal NK cell activity suggest that MHC recognition can have both stimulatory and inhibitory effects. Here we report that the expression of self-MHC class I molecules on target cells strongly inhibits lysis by a long term NK cell line derived from LEW (RT1l) rats and by LEW NK cells activated by short-term culture in the presence of interleukin-2. This was demonstrated with mouse-rat hybridoma target cells expressing different rat MHC alleles and with mouse tumor target cells transfected with classical (RT1.Al) and nonclassical (RT1.Cl) rat MHC class I genes. With hybridoma target cells, the strongest reduction in lysis as compared to the parental mouse myeloma line was observed when "self" (LEW) MHC was expressed, while hybridomas expressing other MHC alleles showed less and variable reduction. Transfection of RT1.Al protected both L-929 fibroblasts and P815 mastocytoma cells from lysis by the NK cell line, while RT1.Cl only protected P815 cells, indicating that additional target cell properties regulate rat NK cell activity.

Characterization of a monoclonal antibody to the capsule of Haemophilus influenzae type b, generated by in vitro immunization

Monoclonal antibodies to polyribosylribitolphosphare (PRP), the capsular polysaccharide of Haemophilus influenza type b ( Hib), are useful tools in the investigation of the molecular and cellular mechanisms causing Hib meningitis. A better understanding of these mechanisms may lead to improved therapeutic strategies. A number of different in vivo immunization techniques in BALB/c mice were used, which did not however reveal detectable serum levels of antibodies to PRP. Therefore a modified in vitro immunization technique, originally established for in vitro immunization of human B lymphocytes, was used for this weak immunogen in mice. After 5 days of in vitro stimulation with purified PRP the splenic lymphocytes of BALB/c mice were fused with the mouse myeloma line P3-X63-Ag8.653. One hybridoma produced an IgM antibody (12E7) which recognized the capsular polysaccharide in ELISA and specifically labelled all tested Hib strains in immune fluorescent microscopy. The blotted polysaccharide PRP was immunostained with monoclomal antibody 12E7. Preincubation of Hib with this antibody enhanced the oxygen radical metabolism of polymorphnuclear leucocytes in a chemiluminescence assay. There was no cross-reactivity with the supernatants of other Haemophilus influenzae serotypes and other bacterial species, as shown by counterimmunoelectrophoresis.

Production and Characterisation of a Human Monoclonal Thyroid Peroxidase Autoantibody

A human-mouse hybridoma has been produced by fusion of Hashimoto thyroid lymphocytes with the mouse myeloma line X63-Ag8.653. The cloned hybridoma secreted 2.5 micrograms per 10(6) cells per day of an IgG kappa thyroid peroxidase (TPO) autoantibody (2G4) with high affinity (2.5 x 10(9) molar-1) and specificity for human TPO. 2G4 did not react with lactoperoxidase, horseradish peroxidase or human myeloperoxidase or with porcine TPO or with human thyroglobulin. Plastic tubes coated with 2G4 bound about 50% of 125I-labelled human TPO added and the binding was inhibited by IgGs prepared from 18/18 TPO autoantibody-positive sera. This indicated that all 18 sera contained autoantibodies which recognised the same (or closely related) epitope as 2G4. Plastic tubes coated with IgGs from different TPO autoantibody-positive patient sera also bound 125I-labelled TPO but inhibition by 2G4 in this system was not complete. This suggested that the sera contained at least 2 types of TPO autoantibodies, with only one type of autoantibody reactive with the same epitope as 2G4.

Analysis of mature guinea pig T cells with a monoclonal antibody directed against a framework determinant of the T-cell receptor for antigen.

Rats were immunized with guinea pig T lymphocytes and the spleen cells were fused with cells of a mouse myeloma line. The resulting hybrids were screened for the production of antibodies selectively reacting with guinea pig T cells. The monoclonal antibody (MoAb) H159 was analysed in detail because it bound to T lymphocytes, but not to B lymphocytes or macrophages. Cellular ELISA, cytofluorometry and immunohistology revealed that the antigen detected by H159 is selectively expressed on the majority of peripheral mature T lymphocytes (about 95%). In contrast, it stained only a minor population of thymocytes in FACS analysis. H159 precipitated from NP40 lysate of T cells a protein with a molecular weight of about 90 kDa when separated under non-reducing conditions in SDS-PAGE. Under reducing conditions bands with molecular weights of about 50 kDa were found. After binding to anti-rat Ig coated beads, the MoAb H159 had a mitogenic effect for guinea pig T lymphocytes whereas soluble MoAb H159 in the presence or absence of macrophages was not mitogenic. The cellular expression and molecular characteristics of the H159 antigen together with the mitogenic activity of the antibody for T cells indicate that the MoAb H159 recognizes the guinea pig T-cell receptor for antigen via a constant region determinant.

Preparation and characterization of a chimeric CD 19 monoclonal antibody

FMC63 is an IgG2a mouse monoclonal antibody belonging to the CD19 cluster. CD19 antibodies react with a 95kDa protein expressed by cells of the B lymphocyte lineage, from pre-B cells to mature B lymphocytes. CD19 antibodies have been suggested as candidates for immunological attack on leukaemic and lymphoma cells of the B lineage because the antigen is restricted to the B lineage. With the potential use of FMC63 in immunotherapy in mind, we have produced a mouse-human chimera in which the genes coding for the VDJ region of the heavy chain and the VJ region of the light chain derive from the FMC63 mouse hybridoma, while the C region genes code for human IgG1. The genes have been transfected back into a mouse myeloma line, which secretes low levels of immunoglobulin. (Ig). This Ig was purified and biotinylated in order to determine the specificity of the antibody. The chimeric antibody has a reaction profile concordant with the original FMC63 antibody, but has the properties of a human IgG1, including the ability to fix human complement. However, the antibody is not cytotoxic in vitro in the presence of complement or cells capable of mediating antibody-dependent cellular cytotoxicity. Possible reasons for this and ways of using the antibody are discussed.

Characterization of a panel of neurofilament antibodies recognizing N‐terminal epitopes

Peptides corresponding to sequences from the amino-terminal "head" regions of the low, middle, and high molecular weight neurofilament proteins (NF-L, NF-M, and NF-H) were synthesized by a modification of the Merrifield solid-phase method, and a panel of polyclonal antibodies to these epitopes were prepared in rabbits by the injection of synthetic peptides conjugated to the carrier protein keyhole limpet hemocyanin (KLH). An additional, monoclonal antibody recognizing both glial fibrillary acidic protein (GFAP) and vimentin was also produced, by fusion of cells of the mouse myeloma line NS-1 with spleen cells from a mouse immunized with cytoskeletal extracts. Antibody specificities were confirmed by a combination of Western blotting against cytoskeletal extracts and immunofluorescence using both rat brain sections and fibroblasts transfected with fully encoding cDNAs for each neurofilament protein, driven by viral promoters.

Production and characterisation of monoclonal antibodies to parathyroid hormone (1–34)

Monoclonal antibodies to the biologically active N terminal region of parathyroid hormone (PTH) suitable for use in the measurement of circulating PTH concentrations have proved difficult to produce. In this study, no serum PTH antibody titres could be detected in mice using synthetic human PTH (1–34) (free or coupled to albumin) or PTH (1–10) (coupled to keyhole limpet haemocyanin) as immunogen. A consistent response to PTH (1–34) peptide was obtained in DA rats. We have produced five monoclonal antibodies to PTH (1–34) derived from the fusion of DA rat spleen cells and the mouse myeloma line X63 Ag.8.653. Bulk production of the antibodies was achieved using congenitally athymic mice for ascites production. Antibody assessment studies revealed the antibodies to be sensitive to the oxidation state of the methionine residues in PTH (1–34). Two of the antibodies, 3B3 and 6E3, were shown to be of potential use in measuring circulating PTH (1–84) when used in combination with available antibodies to C terminal PTH. A third antibody, 4G3, which failed to recognise PTH (1–84) when used in combination with 3B3, formed the basis of a specific assay for PTH (1–34).

Malignant CD5 B Cells-Biased Immunoglobulin Variable Gene Usage and Autoantibody Production

Malignant CD5 B cells obtained from patients with chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL) were analyzed for immunoglobulin variable gene usage, CD5 gene expression and autoantibody production. A statistically significant biased usage of the VH5, VH6 and VKIII immunoglobulin variable gene families was observed. It is important to point out that both VH5 and VH6 are extremely small families which are located at the 3' extremity of the immunoglobulin variable gene locus. We determined that the transcription of the CD5 gene in T cell malignancies, CLL, SLL and a selected group of EBV transformed lines was identical. Autoantibody production was studied in a panel of heterohybridomas obtained by the fusion of CLL cells with mouse myeloma line SP2/0. A large fraction of these heterohybridomas secrete autoantibodies; some were monospecific, some bispecific and some polyspecific.

Human monoclonals against erythrocyte antigens

Mice are not able to respond in making specific antibodies against a variety of human antigens. One of these antigens is the erythrocyte D-antigen of the Rh-system. Until now, it has been possible only to make murine monoclonals against structures of the Rh-proteins. These antibodies were not suitable for routine use in blood grouping. As an alternative, the human immune system is able to produce alloantibodies against Rh-D with high specificity. As a result, with the development of reproducible methods for the generation of human monoclonals, we have started to establish protocols for the production of antibodies against the Rh-D antigen. In addition, we selected a heteromyeloma line, MD33, which is a non-secretor line with high fusion rate giving rise to hybridomas which are high producers. Here, we compare different methods for their efficiency in producing human monoclonals against the Rh-D antigen. We tested the direct fusion of human PBL with either mouse myeloma or human lymphoblastoid cell line, immortalization by EBV-transformation, EBV-transformation followed by fusion with mouse myeloma line, and EBV-transformation followed by fusion with heteromyeloma line. We could demonstrate that most success can be achieved by fusions of EBV-transformed PBL with mouse myeloma line P3X63-Ag8.653 and heteromyeloma line MD33 which resulted in several stable and antigen-specific lines per fusion.

Monoclonal antibodies against hepatitis B e antigen: Production, characterization, and use for diagnosis

Five different hybridoma clones secreting anti-HBeAg antibody were constructed by fusing cells of mouse myeloma line SP2/0 with splenocytes from BALB/c mice immunized with recombinant HBeAg. The monoclonal antibodies obtained were characterized immunologically and one was used to develop ELISA for detection of HBeAg and anti-HBeAg antibody. These monoclonal assays enabled the detection of 3 U HBeAg/ml and 1 U anti-HBeAg/ml with reference to standards of the Paul Ehrlich Institute, Frankfurt, F.R.G. Both assays compared well with a commercially available kit (Abbott Laboratory) and were used for detection of HBeAg and anti-HBeAg antibody in clinical serum samples.

Production of monoclonal antibodies against recombinant HBcAg.

Three monoclonal antibodies against recombinant HBcAg were obtained from hybridomas fused between mouse myeloma line NS1 and splenocytes of immunized Balb/C mice. They specifically bound to recombinant HBcAg. Subtypes of these monoclonal antibodies were IgM and IgA.

Generation of a sheep × mouse heterohybridoma cell line (1C6.3a6T.1D7) and evaluation of its use in the production of ovine monoclonal antibodies

A stable aminopterin-sensitive sheep × mouse heterohybridoma cell line (1C6.3a6T.1D7) for use in the generation of sheep monoclonal antibodies is described. The line was first constructed by fusing the mouse myeloma line, NSO, to normal sheep lymphocytes obtained from the efferent lymphatic vessel of a cannulated popliteal lymph node. The line was rendered sensitive to aminopterin through a combination of irradiation and treatment with the anti-metabolite drug 6-thioguanine. Characterisation of the cloned cell line showed that it did not secrete sheep immunoglobulin (Ig) molecules, express Ig on the surface membrane, or express normal sheep B or T cell surface markers. The 1C6.3a6T.1D7 line has remained stable in tissue culture for over 2 years, showing no signs of reversion to aminopterin resistance. The 1C6.3a6T.1D7 cells have been used as fusion partners with lymphocytes from antigen primed sheep to generate sheep monoclonal antibodies to human chorionic gonadotropin (hCG) or a synthetic peptide analogue of the VP1 capsid protein of foot and mouth disease virus (FMDV). To optimise the efficiency of heterohybridoma generation, comparisons were made of peripheral blood, efferent lymph or excised lymph nodes as sources of antigen-stimulated lymphocytes for fusion. The results showed that lymphocytes prepared from either efferent lymph or lymph node on the fourth day following antigenic stimulation gave similar high fusion efficiencies, and both were vastly superior to peripheral blood lymphocytes. Results were also obtained which showed that the blast cells present in lymphoid tissues due to antigenic stimulation were the major cell types involved in the generation of viable antibody-secreting sheep × sheep × mouse heterohybridomas.

Production and characterization of a monoclonal antibody to partially purified estrogen receptor from human breast tumor.

A hybridoma cell line that secretes monoclonal antibody, MAb-ER-Br-1-15-4-18 is established. The MAb is highly specific for estrogen receptor (ER) from human breast tumor cells. In order to raise the antibody, the ER was first isolated from human breast tumor. Mice were immunized with the partially purified ER and the fusion of the spleen cells from the mouse, showing the highest serum titer, with the cells of the NS-1 mouse myeloma line, produced hybrid cells which continuously secreted antibodies specific for ER. Three of the hybridoma cultures which tested strongly positive were cloned using limiting dilution method and one of the cell lines was selected for further study. The recovery of the MAb from the cell culture was done by ammonium sulfate precipitation followed by dialysis and then hydroxylapatite liquid chromatography using linear gradients. The purity of the antibody was checked by polyacrylamide gel electrophoresis. The MAb was isotyped and found to be IgG1. When checked against other antigens the MAb showed a minimal cross-reactivity to ER from rabbit uterus and none to ovalbumin or rat liver ferritin. Further experiments showed that the MAb recognized the ER bound to the hormone and ER in the nucleus of breast tumor cells.

A Human-Mouse Hybridoma which Secretes Monoclonal Thyroglobulin Autoantibody with Properties Similar to those of the Donor Patient's Serum Autoantibody

Human monoclonal antibodies produced by Epstein Barr (EB) virus transformation and/or cell fusion are frequently IgM antibodies which tend to cross react with a range of antigens and often bear little relationship to the highly specific IgG antibodies associated with human autoimmune disease. By fusing EB virus transformed B lymphocytes from a Hashimoto patient with a mouse myeloma line and selecting for synthesis of IgG class thyroglobulin (Tg) antibody, we have developed a hybridoma (VB/5) secreting Tg antibody of IgG2 subclass and lambda light chain type which has the characteristics of a monoclonal antibody on isoelectric focussing. The antibody has a high affinity for human Tg and recognises Tg from other primates but not non-primate Tg. However, it does not react with human thyroid peroxidase or a panel of other autoantigens. In terms of affinity constant, functional affinity and affinity heterogeneity, the antibody closely resembles the IgG2 lambda Tg antibody present in the serum of the Hashimoto patient whose B lymphocytes were used to develop the hybridoma. In addition to providing a useful reference standard for Tg antibody IgG subclass assays, VB/5 antibody and the hybridoma line provide a valuable starting point for detailed studies of Tg autoantibodies and the genes coding for the variable regions of their heavy and light chains.

Mink-mouse hybridomas that secrete mink immunoglobulin G

Optimum conditions were established to obtain mink-mouse interspecific hybridomas secreting mink IgG in fusions of mouse myelomas with mink immune spleen cells. Minks were immunized with allogeneic IgG, and the spleen cells were fused with three mouse myeloma lines P3-X63-Ag8.653, NSO and Sp2/0-Ag14. Of these, P3-X63-Ag8.653 and NSO were found to be the best fusion partners giving the highest yield of hybrid clones and number of IgG secreting clones. Cloning of mink-mouse hybridomas was efficient when BALB/c nu/nu peritoneal and spleen cells were used as feeders. The ten clonal lines produced secreted intact mink IgG molecules as shown by SDS-PAGE and subsequent immunoblotting. The secretion level of IgG ranged from 5 to 200 ng/ml in the clonal lines.