Abstract
Antigen Used for Immunization
The N-terminal half of human Chibby protein (aa 1-63) with a His tag at its N-terminal end was produced in and purified from Escherichia coli and used as an immunizing antigen.
Method of Immunization
Female BALB/c mice (8 weeks old) were injected subcutaneously with the immunogen emulsified in Freund's complete adjuvant. The titer of mouse serum was determined by ELISA.
Parental Cell Line Used for Fusion
Sp2/0-Ag14 myeloma cells.
Selection and Cloning Procedure
Culture supernatants from hybridoma clones were initially screened by ELISA. Subsequently, the ELISA-positive hybridoma supernatants were assessed by immunoblotting of total cell lysates from HEK293T cells transfected with full-length human Chibby. Selected antibody-producing hybridomas were subcloned by the limiting dilution method to ensure their monoclonality.
Heavy and Light Chains of Immunoglobulin
8-2 and 27-11 belong to IgG2a and IgG1 with light chains, respectively.
Specificity
The specificity was evaluated by Western blot analysis, immunoprecipitation, and immunohistochemistry.
Specific Antigen Identified
8-2 reacts with both human and mouse Chibby proteins, whereas 27-11 is specific to human Chibby.