Abstract
Antigen Used for Immunization
An EGFRvIII-specific synthetic peptide (14-amino acid peptide LEEKKGNYVVTDHC corresponding to the amino acid sequence at the fusion junction of the EGFRvIII, conjugated to the bovine serum albumin) was used for immunization.
Method of Immunization
BALB/c mouse was immunized with 40 μg of the immunogen LEEKKGNYVVTDHC-BSA in complete Freund's adjuvant intraperitoneally, and booster injections were carried out with incomplete Freund's adjuvant biweekly with 20 μg of immunogen for 3 months.
Parental Cell Line Used for Fusion
Sp2/0-Ag14 mouse myeloma cells.
Selection and Cloning Procedure
Hybridoma cells were cultivated in HAT medium and subcloned thrice by limiting dilution.
Heavy and Light Chains of Immunoglobulin
The heavy chain is IgG2b; the light chain is kappa.
Specificity
The specificity was determined using immunoblotting and ELISA. Immunoblotting was performed by lysates of A431 (EGFRwt-overexpressing cells), NIH 3T3, and HC2 20d2/c (EGFRvIII transfected 3T3) cell lines. Immunoreactivity of the monoclonal antibody with Ck19, CD147, and NuMA, recombinant proteins, or Ck19 and CD147 related peptides, was investigated using ELISA. Selected clone toward EGFRvIII was specific for this variant form of EGFR but unreactive with the wild-type EGFR and also was not reactive with some recombinant proteins or their related synthetic peptides.
Specific Antigen Identified
Epidermal growth factor receptor variant III.