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Generation of a monoclonal antibody recognizing the extracellular domain of cell‐surface protein tyrosine phosphatase alpha
Author(s) -
Mullersman Jerald
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.6.lb67-c
Subject(s) - monoclonal antibody , microbiology and biotechnology , biology , polyclonal antibodies , extracellular , protein tyrosine phosphatase , immunoprecipitation , antibody , kinase , immunology
Receptor‐type protein tyrosine phosphatase alpha (RPTPalpha) is an integral membrane protein containing a small, highly glycosylated extracellular domain and an intracellular domain with two tyrosine phosphatase domains. While its function is still poorly understood, it has been found to activate Src‐family kinases and to be overexpressed in malignancies like colon carcinoma. We have explored whether monoclonal antibodies potentially useful in flow cytometric studies of malignancies of hematopoietic origin can be generated from immunization with the extracellular domain PTPa‐2F8. Candidate hybridomas (96) obtained by immunizing Armenian hamsters with a recombinant protein that included the N‐terminal 122 amino acids of mouse RPTPalpha were identified by initial ELISA screening. Of these 96, only one (PTPa‐2F8) bound fully glycosylated RPTPalpha on a cell surface. In flow cytometric studies PTPa‐2F8 detected RPTPalpha on most mouse cells tested (including 70Z/3 cells, NIH 3T3 cells, bone marrow cells, splenocytes, and thymocytes) but gave negative results with controls. PTPa‐2F8 specifically immunoprecipitated RPTPalpha species in the range of 90–130 kDa from transiently transfected HeLa cells as did a polyclonal rabbit antibody directed against the cytoplasmic domain. Immunoprecipitation of metabolically labeled proteins from 70Z/3 cells using PTPa‐2F8 in a pulse‐chase protocol with and without tunicamycin gave expected results. In conclusion, we have found that, despite the highly glycosylated state of mature RPTPalpha protein, it is possible to generate a very useful monoclonal antibody directed to the peptide backbone of the extracellular domain of RPTPalpha.