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 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.

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 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.

scholarly journals An antifibrin monoclonal antibody useful in immunoscintigraphic detection of thrombi

Blood ◽  
1989 ◽  
Vol 74 (2) ◽  
pp. 708-714 ◽  
Author(s):  
MN Wasser ◽  
PW Koppert ◽  
JW Arndt ◽  
JJ Emeis ◽  
RI Feitsma ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Degradation Product ◽  
Spleen Cells ◽  
Fibrinogen Degradation Product ◽  
Myeloma Cells ◽  
High Concentrations ◽  
Preformed Plasma

Abstract Balb/c mice were immunized with human plasmin-generated fibrinogen degradation product Y. Spleen cells were fused with P3X63-Ag8.653 myeloma cells. A clone (Y22) was found that produces monoclonal antibodies (MoAbs) with a strong reactivity with human fibrin and only a weak reactivity with fibrinogen in an enzyme immunoassay (EIA). Y22 also reacts with fibrin of rabbits, rats, sheep, and dogs. The antibodies are of the IgG1 kappa-type and appear to be directed against a conformation-dependent epitope in the D-domain of fibrin. Experiments with 99mTc-labeled Y22 in vitro show that Y22 binds rapidly to forming clots. 99mTc-Y22 also binds to preformed plasma clots in a plasma milieu, even in the presence of high concentrations of heparin. Clot localization experiments in rabbits and rats confirm the high fibrin specificity and the potential of 99mTc-Y22 for thrombus imaging in vivo.

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 An antifibrin monoclonal antibody useful in immunoscintigraphic detection of thrombi

Blood ◽  
1989 ◽  
Vol 74 (2) ◽  
pp. 708-714
Author(s):  
MN Wasser ◽  
PW Koppert ◽  
JW Arndt ◽  
JJ Emeis ◽  
RI Feitsma ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Degradation Product ◽  
Spleen Cells ◽  
Fibrinogen Degradation Product ◽  
Myeloma Cells ◽  
High Concentrations ◽  
Preformed Plasma

Balb/c mice were immunized with human plasmin-generated fibrinogen degradation product Y. Spleen cells were fused with P3X63-Ag8.653 myeloma cells. A clone (Y22) was found that produces monoclonal antibodies (MoAbs) with a strong reactivity with human fibrin and only a weak reactivity with fibrinogen in an enzyme immunoassay (EIA). Y22 also reacts with fibrin of rabbits, rats, sheep, and dogs. The antibodies are of the IgG1 kappa-type and appear to be directed against a conformation-dependent epitope in the D-domain of fibrin. Experiments with 99mTc-labeled Y22 in vitro show that Y22 binds rapidly to forming clots. 99mTc-Y22 also binds to preformed plasma clots in a plasma milieu, even in the presence of high concentrations of heparin. Clot localization experiments in rabbits and rats confirm the high fibrin specificity and the potential of 99mTc-Y22 for thrombus imaging in vivo.

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 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 Generating and characterizing the anti-human CD45 monoclonal antibody

2020 ◽  
Vol 23 (3) ◽  
pp. 665-672
Author(s):  
Giang Huong Ta ◽  
Huy Quoc Nguyen ◽  
Quan Dang Nguyen
Keyword(s):  
Monoclonal Antibody ◽  
Western Blotting ◽  
Jurkat Cells ◽  
Spleen Cells ◽  
Myeloma Cells ◽  
E Coli ◽  
Common Marker ◽  
Sds Page ◽  
Hybridoma Technology ◽  
Mab Production

Introduction: CD45 is a common marker of leukocytes. Anti-human CD45 monoclonal antibody (MAb) has been used widely in diagnosing and monitoring hematologic diseases. The aim of this study was to generate an anti-human CD45 MAb, which can be used in research and diagnosis. Methods: Recombinant human CD45RO antigen was expressed from E. coli BL21 (DE3), purified and analyzed by SDS-PAGE and Western blotting. The purified CD45RO antigen was used to immunize Balb/c mice. Spleen cells from immunized mouse were collected and fused with P3X63Ag8.653 myeloma cells to form hybridoma. Anti-CD45 antibody-secreting capacity of hybridoma clones was evaluated by ELISA assay. Anti-CD45 MAb from the culture supernatant of the chosen hybridoma clone was purified by affinity chromatography. The MAb was characterized the biochemical characteristics and biological activity. Results: Recombinant human CD45RO antigen was expressed and purified from E.coli BL21 (DE3). Injection of purified CD45RO antigen provoked the immune response in Balb/c mice. Hybridoma clones were generated successfully by the fusion of spleen cells from the selected immunized-mouse and myeloma cells. Among these hybridoma clones, one with the highest yield of MAb production was identified. The isotype of the anti-CD45 MAb created in this work is IgG2b, while its the light chain is kappa (k) type. The affinity of this MAb with CD45RO antigen is high with Kd value at the picomolar level. The anti-CD45 MAb can interact with CD45 naturally expressed on the surface of Jurkat cells in Western blotting and fluorescent immuno-staining assay. Conclusion: We have developed successfully an anti-human CD45 MAb using hybridoma technology, which can recognize CD45 in ELISA, Western blotting, and fluorescent immuno-staining analysis. Although further investigations are necessary, obviously, our anti-human CD45 MAb is potential for research and diagnosis applications.

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.

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