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 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.

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.

Solid-phase iodothyronine-5′-deiodinase (5′-D) assays applied in production of monoclonal antibodies against 5′-D

1988 ◽  
Vol 118 (3) ◽  
pp. 439-445
Author(s):  
N. Boye ◽  
H. Frøkiaer ◽  
K. Kaltoftt ◽  
P. Laurberg
Keyword(s):  
Monoclonal Antibodies ◽  
Microsomal Fraction ◽  
Solid Phase ◽  
Rat Kidney ◽  
Enzyme Linked Immunosorbent Assay ◽  
Cortical Tissue ◽  
Spleen Cells ◽  
Hybridoma Cell Lines ◽  
Mouse Myeloma

ABSTRACT Characterization of iodothyronine-deiodinating enzymes has been difficult due to loss of enzyme activity during purification. To obtain a new tool for studying these enzymes we investigated the possibility of developing monoclonal antibodies (MAbs) against iodothyronine-5′-deiodinase (5′-D). Two specific and sensitive solid-phase microassays were developed for screening hybridoma supernatants for the presence of antibodies inhibiting rat kidney 5′-D. and antibodies binding to but not inhibiting the enzyme. BALB/c mice were immunized with a 3-((3-cholamidopropyl) -dimethylammonio) -1- propanesulphonate (CHAPS)-solubilized 5′-D-rich membrane preparation from rat kidney cortical tissue. Spleen cells were fused with NSI-Ag 4/1 mouse myeloma cells by means of polyethylene glycol. Two hybridoma cell lines (AF5 and BE8) secreting MAbs specifically binding to without inhibiting 5′-D were produced. The AF5 antibody was of the IgG2a subclass and the BE8 antibody of the IgG2b subclass. Binding of one of the antibodies to the enzyme inhibited binding of the other in both an enzyme-linked immunosorbent assay (ELISA) and a specific enzymebinding assay. CHAPS-solubilized kidney microsomal fraction was chromatographed on a Sepharose 6B column. Elution profiles of 5′-D activity and MAb-binding antigens, as measured by ELISA with both AF5 and BE8, were identical. Monoclonal antibodies should be valuable probes in the further elucidation of the nature of the iodothyronine-deiodinating activity in various tissues. J. Endocr. (1988) 118, 439–445

scholarly journals Production and characterization of a monoclonal antibody to biotin

1986 ◽  
Vol 237 (2) ◽  
pp. 477-482 ◽  
Author(s):  
K Dakshinamurti ◽  
R P Bhullar ◽  
A Scoot ◽  
E S Rector ◽  
G Delespesse ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Myeloma Cell ◽  
Enzyme Linked Immunosorbent Assay ◽  
Spleen Cells ◽  
Myeloma Cell Line ◽  
Biotin Deficiency ◽  
Keyhole Limpet Haemocyanin ◽  
Limiting Dilution

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.

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.

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’.

Histopathological Characterization of a Novel Monoclonal Antibody, MLuC1, Reacting with Lung Carcinomas

1988 ◽  
Vol 74 (4) ◽  
pp. 401-410 ◽  
Author(s):  
Roberto Agresti ◽  
Rachele Alzani ◽  
Salvatore Andreola ◽  
Vittorio Bedini ◽  
Silvia Gianì ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Monoclonal Antibody ◽  
Carcinoma Cell ◽  
Immunoperoxidase Method ◽  
Spleen Cells ◽  
Paraffin Sections ◽  
Ovarian Carcinomas ◽  
Lung Carcinomas ◽  
Mouse Myeloma

A monoclonal antibody (MoAb), MLuC1, derived from the fusion of P3 - X63 - Ag 8 - U1 mouse myeloma cells with spleen cells from an HR mouse immunized with the carcinoma cell line SW626, was studied to define its reactivity profile on normal and neoplastic human tissues and its potential clinical applications in lung cancer. Evaluation of paraffin sections using the ABC immunoperoxidase method showed a « pan-epithelial » reactivity; a large majority of epithelial components of organs in the respiratory, digestive and urogenital systems (except liver, rectum and ovary) were immunostained. As regard to neoplastic tissues MLuC1 recognized 84 % of lung carcinomas (82 % of small cell, 100 % of squamous cell, 74 % of adenocarcinomas), 86 % of breast and 62 % of ovarian carcinomas. On the contrary, MLuC1 was non-reactive with the other normal and tumoral non-epithelial tissues. Due to its spectrum of reactivity this MoAb could be useful for different diagnostic purposes such as differential diagnosis and lung cancer cytology.

scholarly journals Production of monoclonal antibodies against calmodulin by in vitro immunization of spleen cells.

1983 ◽  
Vol 96 (4) ◽  
pp. 1149-1154 ◽  
Author(s):  
R L Pardue ◽  
R C Brady ◽  
G W Perry ◽  
J R Dedman
Keyword(s):  
Monoclonal Antibodies ◽  
Mitotic Spindle ◽  
Calcium Binding ◽  
Initial Screening ◽  
Spleen Cells ◽  
Monoclonal Antibody Production ◽  
In Vitro Immunization ◽  
Antigenic Proteins ◽  
Mouse Myeloma

Monoclonal antibodies against the highly conserved ubiquitous calcium-binding protein, calmodulin (CaM), were produced by immunization of mouse primary spleen cell cultures. Dissociated spleen cells were cultured for 5 d in the presence of mixed thymocyte culture conditioned media (TCM) and purified bovine testes CaM (50 ng-1 mg). Following immunization, cells were fused with mouse myeloma cells (SP2/0, Ag 8.653) and cultured for 2-3 wk before initial screening for antibody. In five independent immunizations there was a range of 25-44% of the initial polyclonal cultures which produced antibodies reacting with purified CaM as determined by immunoassay. 80% of the cloned hybridoma produced IgM immunoglobulins while the remaining clones were IgG producers. This ratio was changed to 50% IgM and 50% IgG by subsequent extension of the in vitro immunization periods and reduced amounts of antigen and extended in vitro culturing. In vitro immunization introduces a new dimension to monoclonal antibody production where limited antigen or poorly antigenic proteins are of interest. The monoclonal antibodies produced in this study have enabled us to to selectively localize CaM in association with distinct subcellular structures, mitochondria, stress fibers, centrioles, and the mitotic spindle.

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