An alternative method for the isolation of NS-1 hybridomas using cholesterol auxotrophy of NS-1 mouse myeloma cells

Summary We have used the cholesterol auxotrophy of NS-1 mouse myeloma cells as the basis for selecting NS-1 hybridomas. The outgrowth of nascent NS-1 hybridomas in cholesterol-free serum-free medium was 3- to 9-fold more efficient than that in HAT medium and resulted in 3- to 13-times as many antigen-reactive hybridoma wells. This method of hybridoma selection can be applied with any sterol-dependent parent cell line. Hybridomas established under serum-free culture conditions were growth inhibited by fetal calf serum. This work was supported by the Japanese Ministry of Education, Science and Culture and in part by grants from the National Institutes of Health. Editor's Statement This article reports a creative technical application of the author's previous work on lipid metabolism in lymphoid cells allowing an efficient, alternative selection procedure for isolation of hybridomas.     

Enhanced formation of mouse hybridomas without hat treatment in a serum-free medium

Summary A newly developed, serum-free medium (NYSF-404) selects for antibody-producing hybridomas after fusion of antigen-sensitized mouse spleen cells with myeloma cell lines P3-X63-Ag8-U1 (P3-U1), P3-X63-Ag8-6.5.3 (Ag8.653), or P3-NSI/1-Ag4-1 (NS-1). Without the need for hypoxanthine-aminopterinthymidine (HAT) selection of hybrid cells, frequency of hybridoma formation in medium NYSF-404 is higher (twice) than that in serum- and HAT-containing medium. Colonies developed upon limiting dilution in the presence of the mortal parent myeloma cells in medium NYSF-404 and pure culture of antibody-secreting cells could be subsequently established. The results suggest that fusions can be done in serum-free medium and that the clonal growth of hybridomas is dependent on factors produced by parent myeloma cells under serum-free culture conditions. Such factors seem deficient in serum- and HAT-containing medium or are masked by serum.     

Hybridoma cell lines secreting monoclonal antibodies against foot-and-mouth disease virus. 1. Cell culturing requirements

The production of hybridoma cell lines secreting antibody against foot-and-mouth disease virus (FMDV) was more difficult than the production of similar cell lines secreting antibody against vesicular stomatitis virus or measles virus. A rapid and efficient protocol for the selection and culturing of 'anti-FMDV' hybridoma cultures was therefore developed and is described. This required the determination of the optimal culture medium (commercially available), source of serum supplement, line of myeloma cells, type of culture and routine for the subculturing of the hybridoma cells. The protocol consisted of fusion between immune splenocytes and NS-1 mouse myeloma cells, seeding into the wells of 24-well (24W) plates, culturing in RMPI 1640 medium supplemented with either foetal or donor calf serum, and passaging through 24W plates, 6W plates and 100 ml flasks (20 ml medium), respectively. The time at which aminopterin was added to kill unfused myeloma cells was also critical, with the optimum time being 24 h after fusion. In contrast, the B lymphocyte growth stimulant (2-mercaptoethanol) had no beneficial effects on the growth of the hybridomas.     

Sterol depletion reduces receptor-mediated low-density lipoprotein binding in NS-1 mouse myeloma cells

NS-1 mouse myeloma cells, a cholesterol auxotrophic cell line with a lesion in the cholesterol biosynthetic pathway at the demethylation of lanosterol to C-29 sterol, were depleted of cholesterol by incubation in cholesterol-free medium for 24 to 48 h. The low-density lipoprotein receptor activities in untreated and in cholesterol-depleted cells were then compared. The cholesterol-depleted NS-1 cells consistently exhibited a 75 to 90% reduction in receptor-mediated low-density lipoprotein binding compared to untreated cells. The decline of the low-density lipoprotein binding of cholesterol-free medium-incubated NS-1 cells was prevented by addition of free cholesterol or its biosynthetic intermediate, demosterol, to the medium. The addition of lanosterol, an intermediate upstream to the lesion site in the cholesterol biosynthetic pathway, was completely ineffective. The results indicate that proper membrane cholesterol content is necessary for the maintenance of normal low-density lipoprotein receptor function in NS-1 cells.     

The murine plasma cell antigen PC-1: purification and partial amino acid sequence

The murine plasma cell alloantigen PC-1 is selectively expressed on B lymphocytes in their terminal phase of differentiation into antibody-secreting cells. Previous work on an analytical scale has shown that PC-1 consists of two apparently identical disulfide-bonded polypeptides, each of Mr 115,000. In this paper, we describe the generation of a monoclonal antibody to PC-1 and its use in the preparative isolation of PC-1 by affinity chromatography. Final purification to apparent homogeneity was achieved by preparative polyacrylamide gel electrophoresis. It was estimated that NS-1 myeloma cells possess 1 to 4 X 10(5) PC-1 monomers per cell on their surface. The yield of PC-1 after purification was approximately 10(5) monomers per cell. Purified PC-1 was digested with trypsin, and the resulting peptides were separated by reversed-phase high-performance liquid chromatography. Purified peptides were sequenced with a gas-phase sequencer.     

Colcemid treatment of myeloma prior to cell fusion increases the yield of hybridomas between myeloma and splenocyte

Effect of Colcemid treatment of myeloma (X63-Ag8-6.5.3.) prior to fusion with mouse spleen cell was studied in terms of hybridoma formation. Spleen cells from BALB/c mice immunized with various soluble antigens were fused with the myeloma cells by using polyethylene glycol solution. Colcemid treatment of myeloma cells prior to fusion increased the average number of hybridoma colonies per well by 26-570%. The yield of hybridomas producing antigen-specific antibodies was also higher with the Colcemid treatment. The results suggest that most of the proliferative hybridomas are formed by fusion of cells in the M-phase of the cell cycle.     

Monoclonal antibodies NORM-1 and NORM-2 induce more normal behavior of tumor cells in vitro and reduce tumor growth in vivo

In this study, large numbers of hybridomas (produced by syngeneic immunization with B16 mouse melanoma and fusion with NS-1 myeloma cells) were screened for the production of antibodies that affected morphology and growth of animal and human tumor cells in vitro. Two such antibodies, NORM-1 and NORM-2 (both IgG2a), inhibited the growth of B16 melanoma cells in soft agar and increased the serum requirements of tumor cells in tissue culture. Antibody NORM-2 also inhibited the growth of SV40-transformed 3T3 cells in agar and caused them to deposit more fibronectin into extracellular matrix. These antibodies thus seem to induce a more normal behavior of tumor cells in vitro. In vivo both antibodies reduced the number of growing lung tumors of B16 melanoma in C57BL/6 mice by 70%-90% when injected 3 days after the tumor cells. By immunoprecipitation of 35S-methionine-labeled cell extracts, NORM-2 antibody recognized a 59 kd protein in B16 mouse and in A375 human melanoma cells but not in 3T3 fibroblasts.     

Monoclonal Antibodies to and Immunoaffinity Purification of Choline Acetyltransferase from Bovine Brain

Three hybridomas producing monoclonal antibodies to bovine brain choline acetyltransferase (ChAT) have been established by fusion of the spleen cells from a mouse immunized with purified enzyme with myeloma NS-1 cells. All three clones produced IgGl antibodies that reacted with enzyme protein denatured with sodium dodecyl sulfate. By using one of the monoclonal antibodies, a rapid and efficient immunoaffinity purification procedure of bovine ChAT has been established. Immunoblot analysis and immunoaffinity purification indicated that bovine ChAT is a single 68-kilodalton protein. The monoclonal antibodies will offer us a good tool to isolate the cDNA clones encoding ChAT.     

Production of monoclonal antibodies to human chorionic gonadotropin

Production of monoclonal antibodies against human chorionic gonadotropin (hCG) has been studied using hCG as an immunogen. Spleen cells of BALB/c mice immunized with hCG were fused with NS-1 mouse myeloma cells. This study reports the successful isolation of a hybrid clone secreting a monoclonal antibody specific for hCG. By using PEG 4,000 as a fusion agent, the fusion rates were between 42.0 and 50.2%. In total 842 hybridomas were produced. Among them, 403 hybridomas had hCG antibody production. After cloning twice by limiting dilution and alternately screening by enzyme immunoassay and by radioimmunoassay, there were 39 cell lines having specific antibody production. Among them, the No. 57-42-2 had the highest reactivity. By Ouchterlony test, the monoclonal antibody was shown to be IgG1. The affinity constant of the antibody to hCG was 0.6 x 10(9) 1/mole. In radioimmunoassay, the cross reactivity of the antibody to human luteinizing hormone (LH) and human follicle-stimulating hormone (FSH) was 1.5% and 0.7%, respectively. There was no cross reaction with human thyrotropin-stimulating hormone (TSH).     

Monoclonal antibodies: methods and clinical laboratory applications

Kohler and Milstein have shown that individual clones of normal antibody-secreting lymphocytes could be immortalized by fusion with myeloma cells. These investigators initiated a new era of technology with the successful in vitro production of monoclonal antibodies via somatic cell hybridization. With the use of monoclonal antibodies, many major problems arising from the limited specificity and reproducibility of conventional antisera can be solved. Some of the commonly employed methods for the production of monoclonal antibody are: (1) fusion of sensitized lymphocytes and myelomas from different sources to produce continuous antibody-producing cell lines; (2) in vitro viral transformation of sensitized lymphocytes to form continuous antibody-producing cells; (3) hybrid fusion of sensitized lymphocytes and continuous B lymphocyte cell lines. During the past few years, monoclonal antibody methodology has been used in almost every area of biological research. Monoclonal antibodies have been used as structural probes for proteins and hormones, and as highly specific agents for histocompatibility testing, tumor localization, immunotherapy, purification of molecules, identification of new surface antigens on lymphocytes and tumor cells, and detection of drug levels and microbial and parasitic diseases. In addition, several investigators have developed alternative methods for the production of human as well as mouse and rat monoclonal antibodies. The new technology of in vitro production of animal and human monoclonal antibodies will have many future applications in diagnosis and therapy in laboratory and clinical medicine.     

The use of tolerization in the production of monoclonal antibodies against minor antigenic determinants

An initial attempt to prepare monoclonal antibodies specific for the Dictyostelium discoideum lysosomal enzyme beta-glucosidase was unsuccessful. All of the antibodies resulting from this fusion recognized an extremely immunogenic epitope that is present on all of the lysosomal enzymes of Dictyostelium. In two succeeding fusions, changes in the immunization schedule intended to increase the immune response to enzyme-specific epitopes were not entirely successful. Although nine hybridomas producing antibodies specific for beta-glucosidase resulted from these two fusions, most (70%) of the cell lines isolated secrete antibodies that recognize the shared, immunodominant epitope. Moreover, the nine beta-glucosidase-specific antibodies proved to be of limited utility since none recognize the native enzyme. Therefore, we attempted to tolerize a BALB/c mouse to the common epitope by injecting the lysosomal enzyme, N-acetylglucosaminidase, within 40 h after birth. As an adult, this animal was immunized with beta-glucosidase. Fusion of the spleen cells from this mouse with myeloma cells resulted in the isolation of nine hybridoma lines that produce antibodies specific for beta-glucosidase. No antibodies reactive with the common epitope were detected. These results suggest that tolerization may provide a means whereby an undesired class of antibody-producing cell lines can be selectively eliminated from the products of a fusion.     

Myeloma with multiple rearranged immunoglobulin kappa genes: only one kappa gene codes for kappa chains

In many myelomas more than one kappa gene is rearranged (2-5). We are reporting here the results of studies undertaken to determine whether all the rearranged genes are expressed. It was found that in the myeloma NS-1 three different rearranged kappa genes exist. In a subline of NS-1 and several hybridomas produced by fusion of mouse spleen cells with NS-1 it was found that production of NS-1 kappa chains was correlated with the presence of one of the three kappa genes. Loss of this "expressed" gene eliminated the synthesis of the NS-1 kappa chains, loss of one of the other two rearranged kappa genes did not. It is hypothesized, that allelic exclusion (20) of kappa genes generally operates by the functional rearrangement of one kappa gene; other rearrangements are relatively frequent, at least in myelomas, but mostly they are nonfunctional and thus scrambled antibody molecules do not arise.     

Mouse macrophage hybridomas secreting a cytotoxic factor and interleukin 1

Stable mouse macrophage hybridomas were produced by somatic cell fusion between proteose peptone-elicited peritoneal macrophages and NS-1 myeloma cells. Three cloned hybrid cell lines, designated as N/P-5-3, -6-2, and -7-1, exhibited typical macrophage-like morphology. Moreover, their macrophage characteristics were confirmed by the manifestation of intracellular nonspecific esterase, the detection of Mac-1 antigens and Fc-receptors on the cell surface, and the demonstration of phagocytic and antigen-presenting activities. Furthermore, these cell lines, stimulated with LPS, secreted considerable amounts of a cytotoxic factor and interleukin 1. Cultured cells of various tumor cell lines were sensitive to the cytotoxic factor, but normal thymocytes, spleen cells, and liver cells were not killed by the factor.     

Biochemical characterization of Junonia coenia densovirus nonstructural protein NS-1.

Junonia coenia densovirus (JcDNV) is an autonomous parvovirus that infects the larvae of the common buckeye butterfly, Junonia coenia. Unlike vertebrate parvoviruses, the genes encoding the structural protein and nonstructural (NS) proteins of JcDNV are in opposite orientations; thus, each strand contains a sense and antisense open reading frame (ORF). The promoter at map position 93 controls expression of NS ORFs 2, 3, and 4, which encode three NS proteins, NS-1, NS-2, and NS-3. These proteins are likely to be involved in viral DNA replication, among other functions. In contrast to the nonstructural proteins of the vertebrate parvoviruses, the NS proteins of the Densovirinae have not been characterized. Here, we describe biochemical properties of the NS-1 protein of JcDNV. The NS-1 ORF was cloned in frame with the Escherichia coli malE gene, which encodes the bacterial maltose binding protein (MBP). Using electrophoretic mobility shift and DNase I protection assays, we identified the region of the JcDNV terminal sequence that is recognized specifically by the MBP-NS-1 fusion protein. The site consists of (GAC)4 and is located on the A-A' region of the terminal palindrome. In addition, the MBP-NS-1 fusion protein catalyzes the cleavage of single-stranded DNA (ssDNA) substrates derived from the JcDNV putative origin of replication, primarily at two sites in the motif 5'-G*TAT*TG-3'. One cleavage site is between the thymidine dinucleotide at positions 92 and 93 and the other site corresponds to thymidine at nucleotide 95; both sites are on the complementary strand of the sequence assigned GenBank accession number A12984. Cleavage of ssDNA is dependent on the presence of a divalent metal cofactor but does not require nucleoside triphosphate hydrolysis. Parvovirus NS proteins contain the phylogenically conserved Walker A- and B-site ATPase motifs. These sites in JcDNV NS-1 diverge from the consensus, yet despite these atypical motifs our analyses support that MBP-NS-1 has ATP-dependent helicase activity. These results indicate that JcDNV NS-1 possesses activities common to the superfamily of rolling-circle replication initiator proteins in general and the parvovirus replication proteins in particular, and they provide a basis for comparative analyses of the structure and function relationships among the parvovirus NS-1 equivalents.     

Growth of NS0 cells in protein-free, chemically defined medium

Many hybridoma and recombinant myeloma cell lines have been successfully adapted to growth in protein-free media. Compared with serum-supplemented media, use of protein-free media promotes superior cell growth and protein expression and facilitates downstream purification of the expressed product. Owing to its sterol auxotrophy, the NS0 myeloma is normally grown in either a serum-supplemented medium or a serum-free medium supplemented with an animal-derived lipoprotein. CD Hybridoma Medium (a protein-free, chemically defined formulation) grows many cell lines that do not exhibit lipid dependence, but this medium does not support growth of NS0 cells without further lipid supplementation. We tested several commercially available lipid supplements in CD Hybridoma Medium, including bovine EX-CYTE VLE. None of the tested supplements supported long-term growth of NS0 cells in CD Hybridoma Medium. Sustained long-term growth of NS0 cells was achieved in CD Hybridoma Medium supplemented with various animal- or plant-derived lipids complexed with cyclodextrin. NS0 cells adapted to CD Hybridoma Medium supplemented with cyclodextrin-lipid complex reached peak cell densities that were more than double those observed in serum-supplemented medium and were cultured for more than 15 passages. These cultures were also successfully cryopreserved and recovered in this defined medium. Through the use of cyclodextrin-based additives to CD Hybridoma Medium, it is possible to solubilize significant quantities of sterols and other lipids and to maintain a protein-free, chemically defined cultivation environment for NS0 cells. The culture system can be kept entirely free of animal-derived components if the supplement is made with plant-derived or synthetic lipids.     

The culture of rat myeloma and rat hybridoma cells in a protein-free medium

Y0 is a rat x rat hybridoma cell line, which does not secrete immunoglobulin, produced using a fusion partner derived from the Y3 (Y3,Ag.1.2.3) rat myoloma cell line. Y0 and Y3 have both been widely used as fusion partners in the production of rat x rat hybridomas. Y0 has also been used in recombinant gene technology. Y0 cells grown in shake flask culture, using RPMI 1640 medium with 4mM l-glutamine and 5% foetal bovine serum, reached a maximal cell density of 1.5×10⁶ cells ml^–1 with 86% viability. Y0 cells which has been adapted to grow in ABC protein-free medium reached a maximal density, in shake flask culture, of 8.75×10⁵ cells ml^–1 with 79% viability. An improved protein-free medium, designated W38 medium, was developed. In shake flask culture, W38 medium supported Y0 cell growth to a density of 2.02×10⁶ cells ml^–1 with 96% viability. Two Y3 hybridomas, YID 13.9.4 cells and SAM 618 cells were adapted to growth in W38 medium. For both hybridomas, cell growth and product yield in shake flask culture using W38 medium was superior to that obtained with serum-containing RPMI 1640 medium.Abbreviations F12 Hams F12 medium - DMEM Dulbeccos medium - RPMI RPMI 1640 medium - FBS foetal bovine serum     

Serum-free growth medium for the cultivation of a wide spectrum of mammalian cells in stirred bioreactors

A serum free medium was developed, that could be used for the large scale propagation of various cell lines in bioreactors. The medium is based on a 1:1 mixture of Iscove's Modified Dulbecco's Medium and Ham's Medium F12, supplemented with transferrin, insulin and a BSA/oleic acid complex. Several myelomas, hybridomas derived from different myelomas and spleen cells, and other lymphoid and non-lymphoid cell lines were cultivated at growth rates comparable to those observed using serum-supplemented media. There was furthermore no reduction in the formation of products such as monoclonal antibodies or recombinant human interleukin-2.Abbreviations Ag8 Mouse myeloma cell line P3-X63-Ag8.653 - BME Basal Medium Eagle - BSA Bovine Serum Albumin - DMEM Dulbecco's Modified Eagle's Medium - EDTA Ethylenediaminete-traacetic Acid - e-PC Phosphatidyl choline from egg yolk - FCS Fetal Calf Serum - FGF Fibroblast Growth Factor - GHL Glycyl-histidyl-lysine - HDL High Density Lipoprotein - HPL Human Plasma Lipid - IF 1:1 mixture of IMDM and Ham's F12 - IMDM Iscove's Modified Dulbecco's medium - LDL Low Density Lipoprotein - NS1 Mouse myeloma cell line NSI-1-Ag4-1 - PBS Phosphate Buffered Saline - s-PC Phosphatidylcholine from soy beans - s-PE Phosphatidylethanolamine from soy beans - s-lecithin lecithin from soy beans     

Homologous chromosome recombination generating immunoglobulin allotype and isotype switch variants.

We investigated whether spontaneous isotype switching in monoclonal antibody-producing hybridomas always occurs with genes on the same chromosome. Spleen cells of (BAB/ 25 X AKR/J) F1 mice, immunized with dansyl-keyhole limpet hemocyanin (DNS-KLH), were hybridized with NS-1 to generate hybridomas producing monoclonal anti-DNS antibodies of either the b or d haplotype of the BAB/25 or AKR/J parent, respectively. We selected isotype switch variants of such hybridomas using the fluorescence-activated cell sorter (FACS). Although in most cases the allotypic haplotype expressed by the parent and switch-variant hybridomas are the same, in one family of variants we noted a switch in haplotype along with the switch in isotype. This was noted in the selection of IgG2a switch variants from an IgG1 switch variant originally derived from an IgG3-producing parent. Biochemical and molecular studies confirm that the allotype switch variant expresses the same heavy-chain variable region gene complex as its parent hybridomas. As such, the allotype switch represents an example of spontaneous mitotic recombination between immunoglobulin heavy-chain genes, generating a single actively transcribed gene from loci previously positioned on different chromosomes.     

NS-417, a Novel Compound with Neurotrophic-Like Effects

NS-417 (5-(4-Chlorophenyl)-8-methyl-6-7-8-9-tetrahydro-1-H-pyrrolo[3.2-h]isoquinoline-2,3-dione-3-oxim hydrochloric acid salt) belongs to a new chemical series of compounds. NS-417 rescued differentiated PC12 cells from death induced by withdrawal of serum and nerve growth factor. Furthermore, NS-417 stimulated neurotrophic factor-induced neurite outgrowth in undifferentiated PC12 cells. In accordance with this observation, NS-417 potentiated NGF-induced signaling, such as activation of the extracellular signal-regulated kinases ERK1 and ERK2 and the Akt kinase. NS-417 also enhanced ERK activation induced by 10 minutes stimulation with NGF, bFGF or EGF in PC12 cells. In addition to the effect in PC12 cells, NS-417 increased the number of tyrosine hydroxylase (TH) positive cells in cultures established from dissociated E14 rat ventral mesencephali.     

Production and characterization of a monoclonal antibody to biotin.

Monoclonal antibodies to biotin have been prepared by using biotin linked to keyhole limpet haemocyanin (KLH) as the antigen. Spleen cells obtained from mice immunized with biotin-KLH were fused with the myeloma cell line NS-1. The resulting hybridomas were screened for the production of antibodies to biotin using an enzyme-linked immunosorbent assay. Clones producing antibodies to biotin were isolated by limiting dilution methods. Four cell lines, each derived originally from a different fusion, were chosen for the production of monoclonal antibodies. The monoclonal antibodies obtained have been characterized with respect to their ability to interact with biotin, biotin-bovine serum albumin, biotin-KLH and biocytin as well as to inhibit biotin-dependent enzymes. They have been used to produce cellular biotin deficiency in vitro for studies of biotin function.