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.

Production of monoclonal antibodies against prostatic acid phosphatase by in vitro immunization of human spleen cells

1985 ◽  
Vol 84 (1-2) ◽  
pp. 105-116 ◽  
Author(s):  
Noboru Yamaura ◽  
Masanao Makino ◽  
Linda J. Walsh ◽  
Andrew W. Bruce ◽  
Byung-Kil Choe
Keyword(s):  
Monoclonal Antibodies ◽  
Acid Phosphatase ◽  
Prostatic Acid Phosphatase ◽  
Spleen Cells ◽  
Human Spleen ◽  
In Vitro Immunization

scholarly journals In Vitro Immunization of Mouse Spleen Cells and the Production of Monoclonal Antibodies.

1983 ◽  
Vol 37b ◽  
pp. 647-648 ◽  
Author(s):  
Carl A. K. Borrebaeck ◽  
Liselotte Nielsen ◽  
William H. Pirkle ◽  
Ulla Björkroth ◽  
Lu Yi-An ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Mouse Spleen ◽  
Spleen Cells ◽  
In Vitro Immunization

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.

Monoclonal antibodies against surface antigens of schistosomula of Schistosoma mansoni

Parasitology ◽  
1982 ◽  
Vol 84 (1) ◽  
pp. 65-82 ◽  
Author(s):  
D. W. Taylor ◽  
A. F. Butterworth
Keyword(s):  
Monoclonal Antibodies ◽  
Gel Electrophoresis ◽  
Primary Infection ◽  
Polyacrylamide Gel Electrophoresis ◽  
Apparent Molecular Weight ◽  
Surface Antigens ◽  
Soft Agar ◽  
Surface Proteins ◽  
Spleen Cells

SUMMARYMonoclonal antibodies have been produced after fusion of NS-1 murine myeloma cells with spleen cells from mice immunized either by chronic primary infection or with irradiated cercariae: in both cases, animals were challenged with live cercariae 7 days before fusion. The initial cultures were screened for anti-schistosomular antibodies both by a radioimmunoassay with whole schistosomulum extracts and by immunofluorescence. There was no correlation between the two techniques and subsequent screening was carried out by immunofluorescence. Cloning was carried out in soft agar and 7 cloned cell lines, from 5 initial cultures, were selected for detailed study. Products of 6 of these 7 lines were monoclonal, as judged by isoelectricfocusing of [35S]methionine-labelled supernatant fluids, and their binding to live schistosomula was specific. None of the antibodies showed detectable activity in mediating eosinophil- or complement-dependent damage to schistosomula in vitro. However, 2 antibodies were successfully used to isolate surface proteins with an apparent molecular weight of 24000 on SDS-polyacrylamide gel electrophoresis.

Secreted modular calcium-binding protein 1 binds and activates thrombin to account for platelet hyperreactivity in diabetes

Blood ◽  
2021 ◽  
Vol 137 (12) ◽  
pp. 1641-1651
Author(s):  
Fredy Delgado Lagos ◽  
Amro Elgheznawy ◽  
Anastasia Kyselova ◽  
Dagmar Meyer zu Heringdorf ◽  
Corina Ratiu ◽  
...  
Keyword(s):  
Type 2 Diabetes ◽  
Monoclonal Antibodies ◽  
Platelet Function ◽  
Calcium Binding ◽  
Transforming Growth Factor ◽  
Binding Protein ◽  
Calcium Binding Protein ◽  
Matricellular Protein

Abstract Secreted modular calcium-binding protein 1 (SMOC1) is an osteonectin/SPARC-related matricellular protein, whose expression is regulated by microRNA-223 (miR-223). Given that platelets are rich in miR-223, this study investigated the expression of SMOC1 and its contribution to platelet function. Human and murine platelets expressed SMOC1, whereas platelets from SMOC1+/− mice did not present detectable mature SMOC1 protein. Platelets from SMOC1+/− mice demonstrated attenuated responsiveness to thrombin (platelet neutrophil aggregate formation, aggregation, clot formation, Ca2+ increase, and β3 integrin phosphorylation), whereas responses to other platelet agonists were unaffected. SMOC1 has been implicated in transforming growth factor-β signaling, but no link to this pathway was detected in platelets. Rather, the SMOC1 Kazal domain directly bound thrombin to potentiate its activity in vitro, as well as its actions on isolated platelets. The latter effects were prevented by monoclonal antibodies against SMOC1. Platelets from miR-223–deficient mice expressed high levels of SMOC1 and exhibited hyperreactivity to thrombin that was also reversed by preincubation with monoclonal antibodies against SMOC1. Similarly, SMOC1 levels were markedly upregulated in platelets from individuals with type 2 diabetes, and the SMOC1 antibody abrogated platelet hyperresponsiveness to thrombin. Taken together, we have identified SMOC1 as a novel thrombin-activating protein that makes a significant contribution to the pathophysiological changes in platelet function associated with type 2 diabetes. Thus, strategies that target SMOC1 or its interaction with thrombin may be attractive therapeutic approaches to normalize platelet function in diabetes.

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.

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