Abstract
Antigen Used for Immunization
Peptide fragment (KCSFNQKPRAPGDEEAQVEN) of FXYD6 was analyzed by epitope prediction analysis software (Gold Key) and obtained by solid-phase synthesis.
Method of Immunization
BALB/c mice were immunized four times with FXYD6 synthetic peptide fragment (200 μg/rat) at days 0, 14, 28, and 31. Complete and incomplete Freund's adjuvant (1:1) was used for the first and other immunizations respectively; intraperitoneal and subcutaneous injection was used for the last and other immunizations respectively.
Parental Cell Line Used for Fusion
SP 2/0 myeloma cell line.
Selection and Cloning Procedure
The hybridoma cells were cultured in RPMI 1640 medium supplemented with HAT medium and 15% fetal bovine serum. After 2 weeks, the HAT medium was replaced by HT medium for 2 weeks. Positive clones were detected with indirect ELISA. The positive cells were dealt by limiting dilution cloning three to five times until their antibody secretion was 100%. The strain of hybridoma cells that established stable growth with the strongest positive expression was selected.
Heavy and Light Chains of Immunoglobulin
Monoclonal antibody against human FXYD6 is an immunoglobulin of IgG1 subclass. Its light chain is κ chain.
Specificity
Specificity of FXYD6 monoclonal antibody was assessed by indirect ELISA.
Specific Antigen Identified
FXYD6 synthetic peptide.