Preparation of monoclonal antibodies to murine platelet glycoprotein IIb/IIIa (αIIbβ3) and other proteins from hamster-mouse interspecies hybridomas

Stephan Lengweiler; Susan S. Smyth; Marketa Jirouskova; Lesley E. Scudder; Helen Park; Thomas Moran; Barry S. Coller

doi:10.1006/bbrc.1999.1172

Preparation of monoclonal antibodies to murine platelet glycoprotein IIb/IIIa (αIIbβ3) and other proteins from hamster-mouse interspecies hybridomas

Stephan Lengweiler, Susan S. Smyth, Marketa Jirouskova, Lesley E. Scudder, Helen Park, Thomas Moran, Barry S. Coller

Internal Medicine

Research output: Contribution to journal › Article › peer-review

36 Scopus citations

Abstract

To obtain mouse-specific monoclonal antibodies (mAbs) against platelet proteins, an Armenian hamster was immunized with washed mouse platelets. Immune splenocytes were then fused with a nonsecreting murine myeloma cell line, and the resulting heterohybridomas were screened for antibody production utilizing an ELISA in which the target antigen was mouse platelets adsorbed onto microtiter plates in the presence of thrombin. Secondary screening assays included ELISA tests using murine fibrinogen or platelets from β3-integrin knockout mice, flow cytometry, immunoblotting, immunoprecipitation, and a functional assay to identify antibodies that inhibit platelet-fibrinogen interactions. Hybridoma cells producing hamster mAbs against murine glycoprotein (GP) IIb/IIIa, fibrinogen, CD9, and other platelet integrins were identified. Two hybridomas (1B5 and 9C2) producing antibodies that react with the GPIIb/IIIa complex in immunoprecipitation analysis were subcloned twice. Functional analyses by means of aggregation and adhesion assays revealed that 1B5 completely inhibits platelet-fibrinogen interactions, whereas 9C2 does not affect platelet aggregation or platelet adhesion.

Original language	English
Pages (from-to)	167-173
Number of pages	7
Journal	Biochemical and Biophysical Research Communications
Volume	262
Issue number	1
DOIs	https://doi.org/10.1006/bbrc.1999.1172
State	Published - Aug 19 1999

Bibliographical note

Funding Information:
This work was supported by Grant 19278 from the National Heart, Lung, and Blood Institute to B. S. Coller. S. Lengweiler was supported in part by a Swiss National Science Foundation Grant for Future Researchers.

Funding

This work was supported by Grant 19278 from the National Heart, Lung, and Blood Institute to B. S. Coller. S. Lengweiler was supported in part by a Swiss National Science Foundation Grant for Future Researchers.

Funders	Funder number
National Heart, Lung, and Blood Institute (NHLBI)	R37HL019278
Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung

ASJC Scopus subject areas

Biophysics
Biochemistry
Molecular Biology
Cell Biology

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10.1006/bbrc.1999.1172

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title = "Preparation of monoclonal antibodies to murine platelet glycoprotein IIb/IIIa (αIIbβ3) and other proteins from hamster-mouse interspecies hybridomas",

abstract = "To obtain mouse-specific monoclonal antibodies (mAbs) against platelet proteins, an Armenian hamster was immunized with washed mouse platelets. Immune splenocytes were then fused with a nonsecreting murine myeloma cell line, and the resulting heterohybridomas were screened for antibody production utilizing an ELISA in which the target antigen was mouse platelets adsorbed onto microtiter plates in the presence of thrombin. Secondary screening assays included ELISA tests using murine fibrinogen or platelets from β3-integrin knockout mice, flow cytometry, immunoblotting, immunoprecipitation, and a functional assay to identify antibodies that inhibit platelet-fibrinogen interactions. Hybridoma cells producing hamster mAbs against murine glycoprotein (GP) IIb/IIIa, fibrinogen, CD9, and other platelet integrins were identified. Two hybridomas (1B5 and 9C2) producing antibodies that react with the GPIIb/IIIa complex in immunoprecipitation analysis were subcloned twice. Functional analyses by means of aggregation and adhesion assays revealed that 1B5 completely inhibits platelet-fibrinogen interactions, whereas 9C2 does not affect platelet aggregation or platelet adhesion.",

author = "Stephan Lengweiler and Smyth, {Susan S.} and Marketa Jirouskova and Scudder, {Lesley E.} and Helen Park and Thomas Moran and Coller, {Barry S.}",

note = "Funding Information: This work was supported by Grant 19278 from the National Heart, Lung, and Blood Institute to B. S. Coller. S. Lengweiler was supported in part by a Swiss National Science Foundation Grant for Future Researchers.",

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month = aug,

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doi = "10.1006/bbrc.1999.1172",

language = "English",

volume = "262",

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Preparation of monoclonal antibodies to murine platelet glycoprotein IIb/IIIa (αIIbβ3) and other proteins from hamster-mouse interspecies hybridomas. / Lengweiler, Stephan; Smyth, Susan S.; Jirouskova, Marketa et al.
In: Biochemical and Biophysical Research Communications, Vol. 262, No. 1, 19.08.1999, p. 167-173.

Research output: Contribution to journal › Article › peer-review

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T1 - Preparation of monoclonal antibodies to murine platelet glycoprotein IIb/IIIa (αIIbβ3) and other proteins from hamster-mouse interspecies hybridomas

AU - Lengweiler, Stephan

AU - Smyth, Susan S.

AU - Jirouskova, Marketa

AU - Scudder, Lesley E.

AU - Park, Helen

AU - Moran, Thomas

AU - Coller, Barry S.

N1 - Funding Information: This work was supported by Grant 19278 from the National Heart, Lung, and Blood Institute to B. S. Coller. S. Lengweiler was supported in part by a Swiss National Science Foundation Grant for Future Researchers.

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N2 - To obtain mouse-specific monoclonal antibodies (mAbs) against platelet proteins, an Armenian hamster was immunized with washed mouse platelets. Immune splenocytes were then fused with a nonsecreting murine myeloma cell line, and the resulting heterohybridomas were screened for antibody production utilizing an ELISA in which the target antigen was mouse platelets adsorbed onto microtiter plates in the presence of thrombin. Secondary screening assays included ELISA tests using murine fibrinogen or platelets from β3-integrin knockout mice, flow cytometry, immunoblotting, immunoprecipitation, and a functional assay to identify antibodies that inhibit platelet-fibrinogen interactions. Hybridoma cells producing hamster mAbs against murine glycoprotein (GP) IIb/IIIa, fibrinogen, CD9, and other platelet integrins were identified. Two hybridomas (1B5 and 9C2) producing antibodies that react with the GPIIb/IIIa complex in immunoprecipitation analysis were subcloned twice. Functional analyses by means of aggregation and adhesion assays revealed that 1B5 completely inhibits platelet-fibrinogen interactions, whereas 9C2 does not affect platelet aggregation or platelet adhesion.

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Preparation of monoclonal antibodies to murine platelet glycoprotein IIb/IIIa (αIIbβ3) and other proteins from hamster-mouse interspecies hybridomas

Abstract

Bibliographical note

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