scholarly journals A monoclonal antibody to VIII:C produced by a mouse hybridoma

Blood ◽  
1981 ◽  
Vol 58 (5) ◽  
pp. 1000-1006
Author(s):  
HP Muller ◽  
NH van Tilburg ◽  
J Derks ◽  
E Klein-Breteler ◽  
RM Bertina
Keyword(s):  
Monoclonal Antibody ◽  
Myeloma Cell ◽  
Hybridoma Cells ◽  
Spleen Cells ◽  
Myeloma Cell Line ◽  
Myeloma Cells ◽  
Heavy Chains ◽  
Normal Plasma ◽  
Cofactor Activity ◽  
Mouse Myeloma

Spleen cells of a BALB/c mouse immunized with factor VIII procoagulant activity (VIII:C) (isolated by affinity chromatography) were fused with mouse myeloma cells (P3 x 63 Ag8). After the fusion 12/32 wells produced an inhibitor to VIII:C. Cells from one well (1B3) were subcloned four times in order to isolate the hybridoma that produces the anti-VIII:C antibody. Injection of hybridoma cells in pristane pretreated BALB/c mice results in anti-VIII:C titers of 5000–10,000 Bethesda U/ml. Analysis of the produced immunoglobulin demonstrated heavy chains of IgG1 (produced by the myeloma cell line) and IgG2b subclass. The 1B3 antibody neutralizes VIII:C in LMW FVIII, crysosupernatant, cryoprecipitate, and normal plasma. It was found that binding of the IgG to FVIII results in a delay in its activation and not in an inhibition of its cofactor activity. The antibody removes VIII:C from pooled normal plasma when coupled to Sepharose; when coupled to plastic tubes, it binds VIIICAG from isolated VIII:C, purified FVIII, and pooled normal plasma; it does not bind VIIIR:AG, fibrogen, or serum VIIICAG. The 1B3 antibody can be used successfully in an IRMA for VIIICAG.

scholarly journals A monoclonal antibody to VIII:C produced by a mouse hybridoma

Blood ◽  
1981 ◽  
Vol 58 (5) ◽  
pp. 1000-1006 ◽  
Author(s):  
HP Muller ◽  
NH van Tilburg ◽  
J Derks ◽  
E Klein-Breteler ◽  
RM Bertina
Keyword(s):  
Monoclonal Antibody ◽  
Myeloma Cell ◽  
Hybridoma Cells ◽  
Spleen Cells ◽  
Myeloma Cell Line ◽  
Myeloma Cells ◽  
Heavy Chains ◽  
Normal Plasma ◽  
Cofactor Activity ◽  
Mouse Myeloma

Abstract Spleen cells of a BALB/c mouse immunized with factor VIII procoagulant activity (VIII:C) (isolated by affinity chromatography) were fused with mouse myeloma cells (P3 x 63 Ag8). After the fusion 12/32 wells produced an inhibitor to VIII:C. Cells from one well (1B3) were subcloned four times in order to isolate the hybridoma that produces the anti-VIII:C antibody. Injection of hybridoma cells in pristane pretreated BALB/c mice results in anti-VIII:C titers of 5000–10,000 Bethesda U/ml. Analysis of the produced immunoglobulin demonstrated heavy chains of IgG1 (produced by the myeloma cell line) and IgG2b subclass. The 1B3 antibody neutralizes VIII:C in LMW FVIII, crysosupernatant, cryoprecipitate, and normal plasma. It was found that binding of the IgG to FVIII results in a delay in its activation and not in an inhibition of its cofactor activity. The antibody removes VIII:C from pooled normal plasma when coupled to Sepharose; when coupled to plastic tubes, it binds VIIICAG from isolated VIII:C, purified FVIII, and pooled normal plasma; it does not bind VIIIR:AG, fibrogen, or serum VIIICAG. The 1B3 antibody can be used successfully in an IRMA for VIIICAG.

Development of the ELISAs for detection of hormone-disrupting chemicals

2000 ◽  
Vol 42 (7-8) ◽  
pp. 81-88 ◽  
Author(s):  
Y. Goda ◽  
A. Kobayashi ◽  
K. Fukuda ◽  
S. Fujimoto ◽  
M. Ike ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Phthalate Esters ◽  
Hybridoma Cells ◽  
Enzyme Linked Immunosorbent Assay ◽  
Spleen Cells ◽  
Myeloma Cells ◽  
Structural Resemblance ◽  
Reaction Test ◽  
Mouse Myeloma

Six kinds of enzyme-linked immunosorbent assay (ELISA) systems were developed for the quantitative analysis of hormone-disrupting chemicals (HDCs), such as estrogen (ES: the total amount of estrone (E1), 17 β-estra (E2) and estriol (E3)), E2, bisphenol A (BPA), alkylphenol (AP), phthalate esters (PE) and chlorophenols (CP). To generate specific monoclonal antibodies against BPA, AP, PE, CP, hybridoma cells were produced by the fusion of mouse myeloma cells and spleen cells from mice immunized with carboxylated derivatives, while anti E2 monoclonal antibody was selected from those available on the market, and anti ES monoclonal antibody was purchased from Teikoku Hormone Mfg Co. Ltd. The detection limits of ES, E2, BPA, AP, PE and CP ELISAs were 0.1, 0.1, 5, 10, 200, 10 μg/L, when E2, E2, BPA, Nonylphenol (NP), Dibutylphthalate (DBP), 2,4-CP were used as standard, respectively, and the specificity of each ELISA was confirmed with the cross-reaction test using several compounds which have structural resemblance to the compounds of interest.

A Monoclonal Antibody To VIII:C Produced By A Mouse Hybridoma

1981 ◽  
Author(s):  
H P Muller ◽  
N H van Tilburg ◽  
R M Bertina ◽  
J Derks ◽  
E Klein-Breteler
Keyword(s):  
Monoclonal Antibody ◽  
Hybridoma Cells ◽  
Immunoradiometric Assay ◽  
Spleen Cells ◽  
Isoelectric Focussing ◽  
Heavy Chains ◽  
In Vitro Cell Cultures ◽  
Kinetics Of ◽  
Mouse Myeloma

Spleen cells of a Balb-c mouse immunized with VIII:C (isolated by affinity chromatography) were fused with mouse myeloma cells (MOPC-21 derivative). After the fusion 12/32 wells produced an inhibitor to VIII:C. After subclonation (3 x) a stable hybridoma line was obtained. The antibody in the supernatant was detected with a modified VIII: Cinhibitor technique. The supernatant of in vitro cell cultures of the hybridoma cells contained anti-VIII:C titers (Bethesda) of about 0.3-1.0 units/ml. Injection of the hybridoma cells in pristane pretreated Balb-c mice results in anti-VIII:C titers of 5,000-10,000 units/ml ascites.Analysis of the produced immunoglobulin demonstrated the presence of one band after isoelectric focussing, which contained heavy chains both of IgG1 and IgG2B subclass. Because of the unusual kinetics of the monoclonal antibody with VIII:C extensive characterisation of the nature of its VIII: C neutralising properties was necessary.The monoclonal antibody does not bind 125I-fibrinogen or isolated VIIIR:AG, it reacts with isolated VIII:C and can be used in a two-site immunoradiometric assay for VIIICAG. The epitope against which the antibody is directed is not present on ‘serum-VIIICAG’.

scholarly journals Mouse myeloma cells that make short immunoglobulin heavy chains: pleiotropic effects on glycosylation and chain assembly.

1984 ◽  
Vol 98 (6) ◽  
pp. 2215-2221 ◽  
Author(s):  
A L Kenter ◽  
T Warren ◽  
D Shields ◽  
B K Birshtein
Keyword(s):  
Conformational Changes ◽  
Myeloma Cell ◽  
In Vitro Translation ◽  
Myeloma Cell Line ◽  
Present Evidence ◽  
Heavy Chains ◽  
Mouse Myeloma Cell ◽  
Mouse Myeloma Cell Line ◽  
Mouse Myeloma

Two variants in immunoglobulin heavy chain production, derived from the MPC 11 mouse myeloma cell line, make short heavy (H) chains with identical precise deletions of the CH3 domain. The CH3 domain is expressed in the H chain mRNA from both variants. Although in vitro translation of this mRNA produces one H chain species, deleted heavy chains are secreted as heavy-light (HL) and H2L2 moieties in contrast to MPC 11, which secretes only H2L2 . The heavy chains of HL apparently contain more carbohydrate (CHO+) than do the H chains of H2L2 , and inhibition of N-linked glycosylation results in the secretion of relatively more H2L2 . Here we present evidence suggesting that (a) the absence of the CH3 domain has led to conformational changes in these molecules, (b) these changes permit posttranslational glycosylation, and (c) unrestrained glycosylation can frequently yield unusual CHO+ structures that make complete assembly unlikely.

scholarly journals Production and Characterization of Monoclonal Antibodies to Psittacine Beak and Feather Disease Virus

1992 ◽  
Vol 4 (1) ◽  
pp. 13-18 ◽  
Author(s):  
Branson W. Ritchie ◽  
Frank D. Niagro ◽  
Kenneth S. Latimer ◽  
W. L. Steffens ◽  
Denise Pesti ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Myeloma Cell ◽  
Enzyme Linked Immunosorbent Assay ◽  
Spleen Cells ◽  
Myeloma Cell Line ◽  
Elisa System ◽  
Mouse Myeloma Cell Line ◽  
Feather Disease Virus ◽  
Mouse Myeloma

Monoclonal antibodies specific for the virus that causes psittacine beak and feather disease (PBFD) were produced by fusing spleen cells from mice immunized with purified concentrated PBFD virus with mouse myeloma cell line Sp2/0. The resulting hybridomas were tested for reactivity against whole purified virus by an enzyme-linked immunosorbent assay (ELISA) system. Four clones, designated 15H8, 8E3, 11G12, and 2C3, were subcloned by limiting dilution. Isotyping indicated that clone 15H8 was secreting IgG, whereas the remaining clones secreted IgM. The secreted immunoglobulins were characterized by reactivity against purified PBFD virus using immunoblotting procedures, by immunohistochemical staining of virus-induced lesions in infected tissues, and by inhibition of PBFD virus agglutination of cockatoo erythrocytes. Antibodies secreted by clones 15H8 and 8E3 had the strongest activity against purified whole virus. Only immunoglobulin secreted by the clone 15H8 could be used to detect viral antigen in infected tissues. None of the monoclonal antibodies had hemagglutination-inhibition activity.

A Monoclonal Anti Human Factor Ix Produced By A Mouse Hybridoma

1981 ◽  
Author(s):  
R M Bertina ◽  
I K van der Linden ◽  
H P Muller ◽  
J Derks ◽  
E Klein-Breteler
Keyword(s):  
Protein A ◽  
Factor Ix ◽  
Hybridoma Cells ◽  
Spleen Cells ◽  
Main Band ◽  
Heavy Chains ◽  
Human Factor Ix ◽  
Fusion Products ◽  
Mouse Myeloma

Spleen cells of a Balb-c mouse immunized with purified human FIX were fused with mouse myeloma cells (MOPC-21 derivative]. Among the fusion products one hybridoma was found producing an inhibitor of factor IX procoagulant activity. After sub- clonation (5 x) a stable hybridoma was obtained. In vitro cell culture of the hybridoma cells gives anti-FIX titers (Bethesda units] of about 0.8 units/ml. Injection of the hybridoma cells in pristane pretreated mice results in antiFIX titers of 600-1000 units/ml ascites. Analysis of the produced immunoglobulin demonstrated the presence of one main band after iso-electric focussing, which contained heavy chains both of IgG1 and IgG2B subclass.Monoclonal anti-factor IX was isolated from ascites liquid using affinity chromatography on protein-A-Sepharose. Using the purified antibody a radioimmunoassay was developed for the epitope on factor IX, against which this antibody is directed. The epitope is present both on FIX and activated FIXa; however, the affinity of the antibody for binding to FIXa is at least 10 times less. The antibody has no significant affinity for binding to the isolated heavy and light chain of FIXa.About 27 different genetic variants of factor IX [from haemophilia B patients were tested for the presence of this epitope. All FIX-variants possessed the epitope. At least 2 variants demonstrated a reduced affinity for binding to the antibody.

scholarly journals Application of a unique monoclonal antibody as a marker for nonproliferating subpopulations of cells of some tissue.

1985 ◽  
Vol 33 (6) ◽  
pp. 587-594 ◽  
Author(s):  
E Wang ◽  
J G Krueger
Keyword(s):  
Monoclonal Antibody ◽  
Basal Layer ◽  
Positive Staining ◽  
Staining Reaction ◽  
Spleen Cells ◽  
Cultured Fibroblasts ◽  
Myeloma Cells ◽  
Culturing Conditions ◽  
Mouse Myeloma

A monoclonal antibody (clone S-30), directed to a protein of 57,000 daltons, was developed from the fusion of mouse myeloma cells and the spleen cells of mice injected with cytoskeletal extracts of fibroblasts that have been aged in in vitro culturing conditions according to a schedule of serial passaging (Cristofalo VJ, Charpentier R: J Tissue Culture Meth 6:117, 1981; Wang E: J Cell Biol, submitted). The staining activity of S-30 antibody was observed exclusively in the nuclei of nonproliferating senescent fibroblasts, but not in their young counterparts. Immunolocalization of S-30 antibody in frozen tissues from various sites reveals the positive staining reaction in the nuclear envelope region in those cells that are at the final stage of differentiation and are no longer replicating. These tissue sites include epithelial cells of the suprabasal layer of epidermis, hair sheath, and tongue, a subpopulation of fibroblasts in the dermis, chondrocytes, hepatocytes, and cells of cardiac muscle. The absence of S-30 staining activity was noted in tissues such as simple epithelium located in the gastrointestinal tract and kidney, and keratinocytes in the basal layer. These results suggest that the S-30 antibody can be used as a marker for nonproliferating cells both in cultured fibroblasts and in some tissues. It seems that the mechanism that controls the cessation of cell proliferation is related, in part, to the postmitotic expression of the 57,000 dalton protein.

Increased Copy Number of Interleukin-6 Receptor Gene Is Associated with Adverse Survival In Multiple Myeloma Patients Treated with Autologous Stem Cell Transplantation

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1895-1895
Author(s):  
Seon Young Kim ◽  
Hyun Jung Min ◽  
Hyun Kyung Park ◽  
Bora Oh ◽  
Tae Young Kim ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Monoclonal Antibody ◽  
Stem Cell ◽  
Cell Line ◽  
Copy Number ◽  
Myeloma Cell ◽  
Newly Diagnosed ◽  
Myeloma Cell Line ◽  
Myeloma Cells

Abstract Abstract 1895 Background: Interleukin-6 (IL-6) is a potent pleiotropic cytokine that regulates plasma cell (PC) growth via IL-6 receptor (IL-6R). We hypothesized that upregulation of IL-6R in myeloma cells might confer the growth privilege to myeloma cells over bone marrow (BM) hematopoietic cells. We investigated the frequency and prognostic implication of increased copy number of IL-6R gene by fluorescence in situ hybridization (FISH) in patients with newly diagnosed multiple myeloma (MM). To confirm the role of IL-6R, the change of proliferative potentials after blocking of IL-6R by monoclonal antibody was observed in a myeloma cell line. Methods: 102 patients with newly diagnosed MM were enrolled. FISH study for IL-6R was performed using home-made probe for the locus. The BAC (bacterial artificial chromosome) clone RP11 350G8 (BACPAC Resources, Oakland, CA) was used as the IL-6R gene-positive clone and the FISH probe was labeled by a nick translation reaction. FISH signals were counted among BM plasma cells sorted by immunoglobulin light chain staining (cIg FISH). U266 myeloma cell line was treated with IL-6R monoclonal antibody (MRA, Chugai, Japan) at concentration of 0.06 to 1.25 μM and then cell viability test were performed. Results: The amplification of IL-6R was detected in 53/102 patients (52.0%). The 5-year overall survival (OS) rate of patients with IL-6R gene amplification was 41.3% versus 44.8% for those with a normal IL-6R (P = 0.425). In 44 patients treated with high-dose chemotherapy and autologous stem cell transplantation (ASCT), patients with 3.1 or more mean IL-6R gene copy number per PC showed worse 5-year OS compared to those who had less than 2.1 mean copies of IL-6R gene (44.4% versus 78.0%, P = 0.024). In multivariate analysis, the increase of IL-6R copy numbers (mean copy/PC ≥ 3.1) could be considered as an independent prognostic factor for MM patients underwent ASCT (hazard ratio, 30.9; 95% CI, 1.74–548.6; P = 0.020). Treatment of IL-6R monoclonal antibody on the U266 cell line induced marked reduction of cell viability compared with control, ranged from 7.1% to 98.7% according to the increase in treated antibody level. Conclusions: The gain of IL-6R gene is frequent in myeloma, showing an association with adverse prognosis in MM patients treated with ASCT. In vitro suppression of cell proliferation by IL-6R blocker suggests the potential role of IL-6R in myeloma cell growth and therapeutic implication of IL-6R blocker in the future. Disclosures: No relevant conflicts of interest to declare.

Use of monoclonal antibodies for radioimmunoassay of water buffalo milk progesterone

1987 ◽  
Vol 54 (4) ◽  
pp. 471-477 ◽  
Author(s):  
Rosanna Capparelli ◽  
Domenico Iannelli ◽  
Aldo Bordi
Keyword(s):  
Monoclonal Antibodies ◽  
Water Buffalo ◽  
Myeloma Cell ◽  
Buffalo Milk ◽  
Spleen Cells ◽  
Myeloma Cell Line ◽  
Mouse Myeloma Cell ◽  
Mouse Myeloma Cell Line ◽  
The Mean ◽  
Mouse Myeloma

SummaryIn order to standardize a radioimmunoassay of milk progesterone as a routine method for confirmation of oestrus and diagnosis of pregnancy in water buffalo, monoclonal antibodies against progesterone were produced. Hybridomas were prepared by fusing spleen cells from a Balb/c mouse immunized with progesterone 11α-hemisuccinate–bovine serum albumin conjugate with the mouse myeloma cell line NS-1. Thirty wells out of 94 secreted anti-progesterone antibodies. Of the ten independent hybridomas derived, one (AF65) was suitable for the quantification of milk progesterone by radioimmunoassay. The tracer used in the assay was progesterone-11α-hemisuccinate ([2-125I]iodohistamine). The sensitivity of the assay was 50 pg/tube. The mean progesterone concentration at oestrus was 0·8±0·2 ng/ml increasing to 8·5±0·8 ng/ml 24 d later in pregnant animals.

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