Abstract
Antigen Used for Immunization
The SNAP peptide was synthesized by the company GeneCust (Dudelange, Luxembourg) after identification of a sequence with high diversity compared with CLIP. The synthesized peptide was coupled to KLH to increase the immune response in mouse.
Method of Immunization
KLH-SNAP peptide was mixed with GERBU MM adjuvant (GERBU Biotechnik GmbH), 60 μg priming followed by 30 μg boosting, according to the manufacturer's instructions.
Parental Cell Line Used for Fusion
Cell line Sp2/0-Ag14 (ATCC: CRL-1581).
Selection and Cloning Procedure
Monoclonal antibody was selected by limiting dilutions.
Heavy and Light Chains of Immunoglobulin
Heavy chain: VQLQQSGTILARPGASVKMSCKASGYSFTGYWMNWIKQRPGQGLEWIGSLYPENSDTSYSQKFKGKAKLTAVTSASTAYMELSSLTTEDSAVYYCRFGNLGYWGQGTTLTVSS
Light chain not available.
Specificity
The specificity was determined in ELISA, Western Blot, flow cytometry, and immunohistochemistry. In ELISA and Western Blot, SNAP- and CLIP-tag proteins were used to identify specific binding. In flow cytometry, an scFv-SNAP fusion protein was used to identify the binding of the mAb to EGFR-positive cells through the protein (EGFR-positive cells recognize the scFv). The specificity in the IHC was identified using HER2-positive, RD-derived tumor tissues, an scFv-SNAP fusion protein (scFv against HER2-positive), and the mAb.
Specific Antigen Identified
SNAP-tag, SNAPf-tag.
Availability
Footnotes
Author Disclosure Statement
No competing financial interests exist.