NS-1 Cells Research Articles

Production and characterization of monoclonal antibodies against major allergens of American cockroach

From several fusion experiments between spleen cells obtained from BALB/c mice immunized with partially purified Cr-PI of American cockroach and NS-1 cells, growth was observed in many wells. Seven stable subclones secreting monoclonal antibodies (mAbs) against Cr-PI, as determined by enzyme-linked immunosorbent assay (ELISA) with high absorbance values and immunoblot analysis, were obtained. All seven mAbs were characterized as IgG1 subclass by immunodiffusion, and reacted strongly with 72 kilodaltons (kD) of Cr-PI which have been identified as a major allergen of American cockroach. Six mAbs were found to have similar epitope specificities against Cr-PI by ELISA. The remaining mAb was found to have different epitope specificities with others. Interestingly, all mAbs did not react with any components of crude extracts of Oriental and German cockroaches as determined by immunoblot analysis and ELISA. A mAb-based double-antibody sandwich ELISA was developed, and the ELISA was dose-dependent and capable of detecting as little as 140 ng of Cr-PI allergen.

N-linked oligosaccharides of the murine transferrin receptor from a plasmacytoma cell line. Comparison with total cellular N-glycans

The N-linked oligosaccharides synthesised by the murine plasmacytoma cell line NS-1 have been analysed by lectin affinity chromatography on columns of immobilised concanavalin A (Con A), Lens culinaris (lentil), Ricinus communis agglutinin (RCA) and leuko-phytohemagglutinin (L-PHA). The majority of complex N-glycans in this transformed cell line were branced structures with only a low level of biantennary complex chains detected. The analysis showed the major complex N-glycan fraction consisted of a minimum sialylated triantennary structure. [ 3H]Mannose-labelled transferrin receptor was isolated from NS-1 cells by immunoprecipitation followed by electroelution from SDS polyacrylamide gels. The isolated receptor was digested with Pronase and the 3H-labelled glycopeptides analysed by lectin affinity chromatography. Analysis by Con A-Sepharose indicated that approx. 50% of the labelled glycopeptides were branced complex N-glycans (unbound fraction) while the remainder were oligomannose structures (strongly bound). The presence of tri and/or tetraantennary structures in the Con A unbound fraction was further suggested by the interaction of 61% of the fraction with L-PHA. The lectin profiles obtained for the complex N-glycans of the transferrin receptor glycopeptides were similar to those for the total cellular glycopeptides of NS-1 cells. Reverse-phase HPLC analysis of tryptic glycopeptides of the isolated [ 3H]mannose-labelled transferrin receptor gave three 3H-labelled peaks, indicating that all three potential N-glycosylation sites on the receptor are utilised. The Con A-Sepharose profiles of the three fractions indicated the presence of branched complex N-glycans and high mannose chains at each site. The profiles of two of the tryptic glycopeptide fractions were very similar, while the third had a higher content of oligomannose oligosaccharides.

Characterization of Monoclonal Antibodies to Young-Adult Worms of Angiostrongylus cantonensis

Three IgG1 and one IgG2a monoclonal antibodies were obtained by fusion of NS-1 cells with spleen cells of BALB/C mice immunized with young-adult worm antigen of A. cantonensis. These 4 monoclonal antibodies were specific to A. cantonensis without cross-reactivity to any antigens of Toxocara canis, Ascaris suum, Clonorchis sinensis, Dirofilaria immitis, Paragonimus westermani, Gnathostoma spinigerum, Strongyloides stercoralis and Anisakis spp. detected by ELISA. The affinity constants of monoclonal antibodies specific to young-adult worms antigen of A. cantonensis were in the range of 10(7) M-1 to 10(9) M-1. The immunoblots showed that the 4 monoclonal antibodies recognized epitope on molecular weight of 204 kiloDaltons. They will be utilized to purify their corresponding antigen by an immunoaffinity column for the specific immunodiagnosis.

Studies on monoclonal anti-isotypic and anti-idiotypic antibodies against leukemia and myeloma: I. preparation and characteristics of monoclonal anti-isotypic antibodies to igm from B chronic lymphocytic leukemia

Five hybridomas producing McAbs against human serum IgM-isotype were obtained by fusing the myeloma NS-1 cell and the BALB/C murine spleen cell which had been immunized by serum IgM of patient with B chronic lymphocytic leukemia (B-CLL). These McAbs only reacted with human IgM, not with other immunoglobulins in ELISA and immune double diffusion test. An approximate positive rate of peripheral blood lymphocyte (PBL) was got when these McAbs and the McAbs against B cell were tested by indirect immunofluorescent assay. The positive rate was similar to that obtained by direct immunofluorescent test. Immunoblotting showed that the molecular weight of the antigen to these McAbs was 70-90 Kd, indicating that the antigen was IgM. The practical value of the McAbs against human IgM was also discussed.

The use of tissue culture for the detection of mycotoxins.

After incubation with 20 different mycotoxin standards and extracts from fungi and feed stuffs, fluorescent flow cytometry was used to measure viability of NS-1 cells and compared to microscopic assessment of cytotoxicity on stained HEp-11 monolayers. Both methods gave essentially the same results but the cytometric analysis offered a more quantitative approach and was particularly appropriate in the screening of extracts containing fats. The potential uses of a cytotoxic test in the analysis of feed stuffs and fungal extracts for the presence of mycotoxins are discussed.

Establishment and characterization of somatic hybrids between human differentiated macrophages and mouse myeloma ns1 cells

Human macrophages obtained from circulating monocytes matured in vitro by culture for seven days in hydrophobic flexible teflon bags were successfully fused with murine myeloma NS1 cells. Six of 20 clones, selected for their adherence properties, were further studied. All possessed human chromosomes (mean number ranging from 4 to 14 depending on the clones studied), exhibited non-specific esterases (but no peroxidase activity) and expressed CD14 antigen and C3 receptors (but no MAX-1 antigen). Moreover, the hybridomas retained phagocytic activity and high interferon plus lipopolysaccharide-activable cytolytic activity against tumor cells.

Effect of glycosylation inhibitors on the structure and function of the murine transferrin receptor

The murine transferrin receptor is a disulphide-linked dimer with three N-glycosylation sites. We have investigated the structural and functional properties of the transferrin receptor from murine plasmacytoma cells (NS-1 cells) treated with the glycosylation inhibitor, tunicamycin and the glycosylation-processing inhibitors, swainsonine and castanospermine. 1. Tunicamycin (1 microgram/ml) inhibited mannose incorporation in NS-1 cells by greater than 90%, but also inhibited methionine incorporation by up to 50%. Both swainsonine (1 microgram/ml) and castanospermine (50 micrograms/ml) resulted in mannose incorporation greater than 100% of untreated cells and neither drug affected methionine incorporation. 2. Incubation of NS-1 cells with tunicamycin resulted in a shift in the apparent molecular mass of the transferrin receptor from 96 kDa and 94 kDa to approximately 82 kDa. 3. Peptide N-glycosidase F digestion of the receptor from untreated cells resulted in the fully deglycosylated 82 kDa component as well as an 87 kDa component which represents partially deglycosylated receptor resistant to peptide N-glycosidase F digestion. 4. The receptor from swainsonine-treated cells was equally sensitive to peptide N-glycosidase F and endo-beta-N-acetylglucosaminidase H (endo H; resulting in both 87-kDa and 82-kDa components), whereas the receptor from castanospermine-treated cells was only partially sensitive to endo H. 5. Analysis of mannose- and fucose-labelled cellular glycopeptides by concanavalin-A--Sepharose chromatography showed that swainsonine (1 microgram/ml) treatment resulted in approximately 90% inhibition of the synthesis of complex N-glycans and an accumulation of fucosylated hybrid structures. In contrast, castanospermine (100 micrograms/ml) treatment resulted in only partial inhibition (60%) of the synthesis of complex N-glycans. 6. Analysis of the receptor from tunicamycin, swainsonine and castanospermine treated cells under nonreducing conditions showed a single component corresponding to the dimer, indicating that dimerisation of newly synthesised murine receptor is independent of carbohydrate. 7. The non-glycosylated receptor from tunicamycin-treated cells appears to bind transferrin as demonstrated by interaction with transferrin-Sepharose. 8. Surface expression of the receptor was not significantly altered in the presence of either swainsonine or castanospermine as judged by flow cytometry.

Establishment and characterization of monoclonal antibody against androgen receptor

Hybrid cell lines were prepared by the fusion of BALB/c myeloma NS-1 cells with the lymphocytes of BALB/c mice that were immunized with partially purified androgen receptor (AR) from human prostates. Nine clones of the hybrid progeny were determined for the production of antibodies against AR by immunoprecipitation assay. One of the clones, referred to as “5F4”, was chosen for analysis of the detailed specificity. The clone “5F4” secreted IgM class antibodies against AR. Competition study demonstrated that “5F4” antibody inhibited androgen binding of AR, suggesting that the antibody identifies androgen binding site of AR. Immunoblotting analysis showed that the antibody identified the ARs as two proteins, 95kD and 41kD proteins, on a sodium dodecyl sulfate polyacrilamide gel. It is suspected that a 95kD protein should be a monomeric AR and a 41kD protein is a proteolytic fragment of AR. Immunohistochemical analysis demonstrated that androgen-dependent tissues—human prostatic hypertrophy tissues, an AR abundant prostatic cancer tissue and fibroblast cells from human genital skin—were stained intensely with “5F4” monoclonal antibody, while androgen-independent tissues—fibroblast cells from lymph nodes, an AR deficient prostatic cancer tissue and human prostatic cancer cell line, PC-3—showed no staining. These results also support the specificity of the antibody for AR.

Coxsackievirus-B3-induced myocarditis: virus receptor antibodies modulate myocarditis.

Two variants of coxsackievirus group B, type 3 (CVB3) differ in ability to induce myocarditis in Balb/cCUM mice. Infection with the highly pathogenic variant (CVB3M) stimulates autoimmunity to normal cardiocyte antigens, and tissue injury results primarily from an autoreactive cytolytic T lymphocyte (ACTL). Animals infected with the less pathogenic CVB3o variant do not develop ACTL, although CVB3o replicates to high titers in the heart and polyclonal neutralizing antisera fail to distinguish between the two variant virions. The present study uses two IgM mAb derived by fusing spleen cells from CVB3M-infected mice with NS-1 cells. These mAb investigate important differences between the virus variants that may explain why only selected infections trigger autoimmunity. mAb 8A6 is a virus-neutralizing antibody that prevents infection of HeLa cells and cultured cardiocytes by attaching to the virus. mAb 10A1 also interferes with infection but presumably reacts to the virus receptor on the susceptible cells and shows little or no binding to the virions. While 8A6 is equally effective in neutralizing both CVB3o and CVB3M, suggesting that antigenic epitopes on both variants are either identical or highly cross-reactive, 10A1 distinguishes between the variants, suggesting that the pathogenic and less pathogenic viruses use distinct cell surface receptors. Competitive binding studies using radiolabeled CVB3M and either of the unlabeled variants confirm this hypothesis. Both mAb effectively prevent CVB3M-induced cardiac damage in vivo. mAb 10A1 also inhibits autoreactive ACTL lysis of cardiocytes, indicating that the autoimmune effectors may recognize the virus receptor, and that the receptor utilized by a virus may prove important in triggering auto-sensitization.

Enhanced transcription of the 78,000-dalton glucose-regulated protein (GRP78) gene and association of GRP78 with immunoglobulin light chains in a nonsecreting B-cell myeloma line (NS-1).

The 78,000-dalton glucose-regulated protein (GRP78) is a stress-inducible protein localized in the endoplasmic reticulum. It has been identified as the immunoglobulin heavy-chain-binding protein. We report here a high level of GRP78 expression in a B-cell myeloma line, NS-1, which produces only kappa light-chain proteins but is unable to secrete them. GRP78 transcription was enhanced in NS-1 cells, resulting in higher levels of GRP78 mRNA and protein than in non-immunoglobulin-producing cells. Furthermore, the nonsecreted light chains in NS-1 cells were found in specific association with GRP78. We hypothesize that in nonsecreting lymphoid cells, the presence of free, unassembled light chains in the endoplasmic reticulum could result in increased transcription of the GRP78 gene and that GRP78 can also bind to immunoglobulin light chains.

Enhanced Transcription of the 78,000-Dalton Glucose-Regulated Protein (GRP78) Gene and Association of GRP78 with Immunoglobulin Light Chains in a Nonsecreting B-Cell Myeloma Line (NS-1)

The 78,000-dalton glucose-regulated protein (GRP78) is a stress-inducible protein localized in the endoplasmic reticulum. It has been identified as the immunoglobulin heavy-chain-binding protein. We report here a high level of GRP78 expression in a B-cell myeloma line, NS-1, which produces only kappa light-chain proteins but is unable to secrete them. GRP78 transcription was enhanced in NS-1 cells, resulting in higher levels of GRP78 mRNA and protein than in non-immunoglobulin-producing cells. Furthermore, the nonsecreted light chains in NS-1 cells were found in specific association with GRP78. We hypothesize that in nonsecreting lymphoid cells, the presence of free, unassembled light chains in the endoplasmic reticulum could result in increased transcription of the GRP78 gene and that GRP78 can also bind to immunoglobulin light chains.

Effects of proximate cholesterol precursors and steroid hormones on mouse myeloma growth in serum-free medium.

The proximate cholesterol precursors lathosterol, 7-dehydrocholesterol and desmosterol supported the growth of NS-1 and X63 mouse myeloma cells. These cells and X63.653 cells are cholesterol auxotrophs, yet each was able to convert [3H]lathosterol to [3H]cholesterol. These results are consistent with the conclusion that cholesterol auxotrophy in these myeloma cells is due to a deficiency in 3-ketosteroid reductase activity. The steroid hormones testosterone, progesterone and hydrocortisone could not replace cholesterol as a medium supplement. These results provide a greater understanding of the cholesterol auxotrophy characteristic of cell lines clonally-derived from the MOPC 21 myeloma tumor, and they provide a rational basis for the use of sterols in defined culture medium for mouse myeloma cells.

Polyunsaturated fatty acids stimulate superoxide formation in tumor cells: A mechanism for specific cytotoxicity and a model for tumor necrosis factor?

Some neoplastic cell lines are readily killed when incubated in the presence of polyunsaturated fatty acids (PUFA). In an attempt to elucidate this phenomenon, we studied PUFA-driven superoxide (O2-) production by cultured NS-1 cells (murine lymphoid tumor cells). We find: (1) Even in the absence of added PUFA, NS-1 cells generate O2- (i.e., reduce nitroblue tetrazolium). (2) addition of PUFA increases O2- by greater than 50%. (3) Artificial loading of NS-1 cells with liposome encapsulated superoxide dismutase prevents the majority of spontaneous and PUFA-driven NBT reduction. We conclude that PUFA drives O2- generation by tumor cells, that this generation is largely intracellular, and that this phenomenon may help explain toxicity of PUFA for tumor cells.

Purification of a nuclear trans-acting factor involved in the regulated transcription of a human immunoglobulin heavy chain gene

The expression of the rearranged human immunoglobulin γ1 heavy chain gene (HIG1) was shown to be induced through its enhancer by the positive regulatory trans-acting factor(s) that was contained only in cells of B lineage. The trans-acting factors were purified from mouse myeloma NS1 cells, and HIG1-inducing activity was found mainly in fractions of molecular weight 53–127 kd and in a fraction eluted from a heparin-Sepharose column with 0.5 M KCI. This semipurified fraction contained proteins binding to the conserved octamer sequence, ATGCAAAT, in the promoter region, as well as to sequences in the enhancer region. The 0.5 M KCI eluates from a heparin-Sepharose column were applied to a DNA affinity column of synthetic oligonucleotides of the octamer sequence and the sequence TATTTTAGGAAGCAAA in the Hpall-Bglll region of the HIG1 gene enhancer. The protein eluted from the enhancer sequence-specific DNA affinity column showed a strong inducing activity for the HIG1 gene, and the molecular weight of a predominant protein was 96 kd.

Characteristics of Five Monoclonal Antibodies to Major Allergens of the Short Ragweed Pollen

Monoclonal antibodies against major allergens of the short ragweed pollen were produced by fusion of NS-1 cells with splenic cells from BaLB/C mice that had been immunized separately with the major allergens, AgE-B + E-C and AgK, of the short ragweed pollen. These monoclonal antibodies were detected by enzyme-linked immunosorbent assay and further characterized by immunoblot analysis using the crude extract and highly purified allergens of the ragweed pollen. Three monoclonal antibodies obtained by immunization with AgE-B + E-C, designated as 36-6, 27-2 and 48-5, reacted mainly with the beta (36-6) and alpha (27-2) subunit of AgE and both AgE and AgK (48-5), respectively. Two monoclonal antibodies obtained by immunization with AgK, 4-7 and 8-5, had a similar reactivity with AgE-B + E-C, AgK, AgK-A and AgK-B. In addition, however, antibody 4-7 also reacted with AgE-B2 as well as the 36- and the 24- to 22-kilodalton antigens of the crude extract. All 5 monoclonal antibodies were characterized as IgGl subclass. Besides, monoclonal antibody 48-5 also showed cross-reactivity to components of sage pollen. Beyond academic interests, the monoclonal antibodies described here may also be useful in clinical allergy.

Establishment of Monoclonal Antibody to Human Androgen Receptor and Its Clinical Application for Prostatic Cancers

Hybrid cell lines were prepared by the fusion of BALB/c myeloma NS-1 cells with the lymphocytes of BALB/c mice that were immunized with partially purified androgen receptor (AR) from human prostates. One of the clones, 5F4, was chosen for detailed specificity analysis. The avidin-biotin-peroxidase complex (ABC) procedure was used for immunohistochemical staining of the 5F4 monoclonal antibody. In human benign prostatic hypertrophy (BPH) tissues, cytoplasm and nuclei were stained. Of 16 prostatic cancer tissues, 2 were composed of AR-positive cells exclusively, 7 were composed of AR-negative cells, and 7 contained both AR-positive cells and AR-negative cells (mixed). Of nine cases that were AR-positive or mixed, seven cases responded to the hormone therapy, and two were not determined for responsiveness because the patients died early of other diseases. Of seven AR-negative cases, all but one inestimable case had no response to the hormone therapy. Immunohistochemical analysis of AR by using the monoclonal antibody 5F4, was a useful tool for determining androgen dependency of prostatic cancers.

Monoclonal antibodies against human oral squamous cell carcinoma reacting with keratin proteins

Two mouse monoclonal antibodies (MoAbs), B10 and 1H5, were generated by fusing mouse myeloma NS-1 cells with spleen cells from a BALB/c mouse immunized with Ueda-1 cells derived from human squamous cell carcinoma (SCC) of the floor of the mouth. Immunohistochemical analysis revealed that these MoAbs recognize the filamentous components of cytoplasm which were protein in nature. While the pattern of antigen distribution in various cell lines was not cell-type specific, reactivity of these antibodies with tissue sections was informative. MoAb 1H5 was preferentially reactive with well-differentiated squamous cell carcinoma, however, reaction with adenocarcinoma was observed infrequently. This antibody also preferentially reacted with the spinous layer of normal stratified squamous epithelium. MoAb B10, however, was reactive with nonepithelial tissues as well as with epithelial ones, and its level of binding bore no relationship to the grade of histologic malignancy. SDS-PAGE and Western blotting analysis, using cytokeratin extracts of Ueda-1 cells and human epidermis, demonstrated that MoAb B10 reacted with a wide range of keratin proteins of 65-67K, 58K, 56.5K, 56K, 52K, 50K, 48K, 45K, 40K, 38K, 36K, and 34K molecular weight (MW), while MoAb 1H5 reacted with keratin proteins of 65-67K, 58K, 56.5K, 56K, 52K, 48K, and 34K MW. These results suggest that MoAb 1H5 may recognize keratin subfamilies related to squamous differentiation, whereas MoAb B10 recognizes a wide range of keratin proteins, and may even react with other kinds of intermediate filament proteins (IFPs).

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.

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.

Monoclonal antibodies to hydroxyindole O-methyltransferase from bovine pineal gland.

Hybridomas that produce monoclonal antibodies to bovine hydroxyindole O-methyltransferase have been established by immunizing a mouse with hydroxyindole O-methyltransferase partially purified from bovine pineal glands followed by fusion of the spleen cells with myeloma NS-1 cells. Twenty-three clones have been isolated by screening the medium by two different methods. These clones produced various antibodies with different binding properties. Most of the antibodies belonged to IgG1 subtype, while 7 clones produced IgG2 alpha or IgG2 beta antibodies. Three antibodies inhibited enzymatic activity, whereas others did not. Antibodies of 7 clones recognized enzyme protein denatured with sodium dodecyl sulfate, while other antibodies reacted only with native enzyme protein. Thus a variety of monoclonal antibodies will offer us a good tool for immunohistochemical localization of the enzyme, immunoaffinity purification of hydroxyindole O-methyltransferase, and isolation of the cDNA clones encoding it. We also report here on the immunohistochemical demonstration of hydroxyindole O-methyltransferase in bovine pineal gland and a new immunoaffinity procedure to purify the enzyme to homogeneity by use of monoclonal antibodies.