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 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.

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.

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.

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.

scholarly journals Monoclonal antibody to murine gamma interferon inhibits lymphokine-induced antiviral and macrophage tumoricidal activities.

1984 ◽  
Vol 159 (5) ◽  
pp. 1560-1565 ◽  
Author(s):  
G L Spitalny ◽  
E A Havell
Keyword(s):  
Monoclonal Antibody ◽  
Antiviral Activity ◽  
Recombinant Dna ◽  
Gamma Interferon ◽  
Spleen Cells ◽  
Tumoricidal Activity ◽  
Antiviral Activities ◽  
Immune Spleen Cells ◽  
Mouse Myeloma

Fusion of rat immune spleen cells with mouse myeloma cells resulted in the formation of a stable hybridoma that secretes monoclonal antibody (MAb) directed against murine gamma interferon ( MuIFN -gamma). This MAb specifically neutralized the antiviral activity of a variety of MuIFN -gamma preparations, including a sample produced by recombinant DNA technologies. In contrast, the antiviral activities of a mixture of MuIFN -alpha plus MuIFN -beta, as well as those of rat or human IFN-gamma, were not neutralized by this antibody. The ability of the MAb to inhibit lymphokine-induced macrophage activation was also tested. It was found that in relation to the quantity of antibody needed to completely neutralize antiviral activity, much higher concentrations of MAb were required to abolish the capacity of lymphokine preparations to induce macrophage tumoricidal activity in vitro. The MAb was also coupled to cyanogen bromide-activated Sepharose beads and used as an immunoadsorbent. By reacting lymphokines with MAb coupled to an insoluble matrix, it was possible to show that this immobilized antibody completely and specifically removed from the lymphokine preparations the ability both to invoke macrophage tumoricidal activity and to mediate antiviral activity.

scholarly journals Characterization of a monoclonal antibody specific to a flavonol 2′-O-glucosyltransferase

1989 ◽  
Vol 67 (4-5) ◽  
pp. 210-213 ◽  
Author(s):  
Lilian Latchinian ◽  
Ragai K. Ibrahim
Keyword(s):  
Monoclonal Antibody ◽  
Supernatant Fraction ◽  
Enzyme Linked Immunosorbent Assay ◽  
Spleen Cells ◽  
Native Form ◽  
In Vitro Immunization ◽  
Culture Supernatants ◽  
Mouse Myeloma

A monoclonal antibody to a partially purified preparation of 2′-O-glucosyltransferase was produced by in vitro immunization of spleen cells from BALB/c mice, followed by fusion with mouse myeloma cells. Hybridoma culture supernatants were screened by enzyme-linked immunosorbent assay for (i) their ability to produce immunoglobulins and (ii) their immunoreactivity with a partially purified enzyme preparation. The majority of the immunoglobulin-producing hybridomas were IgM secretors. Two highly immunoreactive IgM-secreting clones were chosen for further characterization. The supernatant fraction from a culture of one of these clones displayed 50% inhibition of the 2′-O-glucosyltransferase activity. The native form of the 2′-O-glucosyltransferase was essential for recognition, suggesting that the epitope recognized by the antibody is a conformational discontiguous one.Key words: monoclonal antibody, in vitro immunization, flavonoid, O-glucosyltransferase.

scholarly journals IMMUNE RESPONSES IN VITRO

1971 ◽  
Vol 134 (2) ◽  
pp. 395-416 ◽  
Author(s):  
Carl W. Pierce ◽  
Barbara M. Johnson ◽  
Harriet E. Gershon ◽  
Richard Asofsky
Keyword(s):  
Culture Conditions ◽  
Antibody Concentration ◽  
Spleen Cells ◽  
Immunoglobulin Class ◽  
Cell Densities ◽  
Erythrocyte Antigen ◽  
First Time ◽  
Kinetics Of

We have demonstrated for the first time that mouse spleen cells stimulated in vitro with heterologous erythrocytes developed immunoglobulin class-specific γM, γ1, γ2a+2b, and γA plaque-forming cell (PFC) responses. A modification of the hemolytic plaque technique, the addition of goat anti-mouse µ-chain antibody to the assay preparation, specifically prevented development of all γM PFC and enabled accurate and reproducible enumeration of immunoglobulin class-specific PFC after treatment with appropriate monospecific anti-globulins and complement. Culture conditions, with regard to medium, atmosphere, agitation, and spleen cell densities, were similar to those previously shown to support only γM PFC responses. Evaluation of the kinetics of appearance of PFC showed that γM PFC reached maximum numbers on days 4–5; the magnitude of this response was 3–10 times greater than γ1 γ2a+2b, or γA PFC which reached maximum numbers on days 5–6. Optimal erythrocyte antigen dose for γM PFC responses was 107/culture, whereas a dose of 106 erythrocytes/culture consistently stimulated optimal γ1 γ2a+2b, or γA PFC responses. Investigations of the effects of anti-erythrocyte antibody on γM and γG PFC responses indicated that antibody suppressed these responses by neutralizing the effective antigenic stimulus at the macrophage-dependent phase of the response. At the same antibody concentration, γG PFC responses were more effectively suppressed than γM PFC responses. Further, γG responses could be almost completely suppressed by antibody as long as 48 hr after initiation of cultures, whereas γM PFC responses could only be completely suppressed during the first 24 hr. These results were discusssed in terms of the role of antigen in the stimulation γM and γG antibody.

Preparation Of Factor IX-Deficient Human Plasma Using RFF-IX/1 Monoclonal Antibody

1981 ◽  
Author(s):  
F Rotblat ◽  
A H Goodall ◽  
G Janossy ◽  
G Kemble ◽  
D P O’Brien ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Cell Line ◽  
Factor Ix ◽  
Hybrid Cells ◽  
Spleen Cells ◽  
High Affinity ◽  
A Cell ◽  
A Site ◽  
Monoclonal Cell Line ◽  
Mouse Myeloma

A cell line that secretes a monoclonal antibody to factor IX has been produced by fusing spleen cells from a mouse that had been hyper immunised to purified factor IX with mouse myeloma cells (line P3-NSI/I-Ag4-1). Hybrid cells were selected and a monoclonal cell line has been established in culture. This cell line secretes an IgGl(k) antibody (RFF-IX/1) with high affinity for a site related to the coagulant function of factor IX.Monoclonal antibody was partially purified from ascitic fluid from mice implanted with the RFF-IX/1 secreting cells by precipitation at 50% saturation with ammonium sulphate. This fraction has typically 630 NIH units/ml anti IX activity and 13.5 mg/ml protein. It was coupled to cyanogen bromide activated Sepharose 2B in the ratio of 9 mg. protein/1 ml gel. A column containing 10 ml of this gel removed all the assayable factor IX from the first 280 ml of normal ci.trated plasma that was passed over it. After that volume small amounts of factor IX could be detected in the effluent. Subsequently 10-20% of the factor IX activity adsorbed could be recovered by eluting the column with 3 M potassium iodide.Immuno-affinity depleted plasma could be used as substrate in a one-stage factor IX assay under routine laboratory conditions and was undistinguishable for that purpose from severe Christmas disease plasma.

scholarly journals Differential regulation of spleen cell-mediated eosinophil and neutrophil-macrophage production

Blood ◽  
1980 ◽  
Vol 55 (3) ◽  
pp. 489-493 ◽  
Author(s):  
SH Bartelmez ◽  
WH Dodge ◽  
DA Bass
Keyword(s):  
Growth Factor ◽  
Contrast Media ◽  
Spleen Cell ◽  
Trichinella Spiralis ◽  
Cell Types ◽  
Differential Regulation ◽  
Spleen Cells ◽  
Secretory Products ◽  
Kinetics Of

Abstract Nonadherent spleen cells of mice infected with Trichinella spiralis released growth stimulatory factors (GSFs) in vitro when challenged with excretory/secretory products of muscle stage larvae. The assay of GSF was based on proliferation of normal, nonadherent syngeneic marrow cells in liquid tube cultures. Media conditioned for 1 day by challenged spleen cells stimulated eosinophil production but failed to stimulate production of other cell types. In contrast, media conditioned for 5 days supported eosinophil, neutrophil, and macrophage production. The kinetics of cell production were also different. Eosinophil production started within 1 day, reached a peak at day 2, and was down to control levels by day 4. In contrast, neutrophil/macrophage production began between 2 and 4 days and reached a peak at 6--8 days. The short duration of eosinophil production was evidently due to depletion of growth-factor-responsive cells.

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