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. 2023:2578:121-141.
doi: 10.1007/978-1-0716-2732-7_9.

One-Shot Generation of Epitope-Directed Monoclonal Antibodies to Multiple Nonoverlapping Targets: Peptide Selection, Antigen Preparation, and Epitope Mapping

Oi Wah Liew  1 , Samantha Shi Min Ling  2 , Shera Lilyanna  2 , Jenny Pek Ching Chong  2 , Jessica Yan Xia Ng  2 , Arthur Mark Richards  2
Affiliations

One-Shot Generation of Epitope-Directed Monoclonal Antibodies to Multiple Nonoverlapping Targets: Peptide Selection, Antigen Preparation, and Epitope Mapping

Oi Wah Liew et al. Methods Mol Biol. 2023.

Abstract

This chapter describes an epitope-directed approach to generate antipeptide monoclonal antibodies to multiple nonoverlapping protein sites using a cocktail of fusion peptides as immunogen. It provides a step-by-step protocol on how antigenic peptides on a target protein can be identified by in silico prediction and discusses considerations for final peptide selection. Each antigenic peptide (10-20 amino acids long) is displayed as three-copy inserts on the surface exposed loop of a thioredoxin scaffold protein. The corresponding DNA coding sequence specifying the tripeptide insert flanked by Gly-Ser-Gly-Ser-Gly linkers is cloned in-frame into the Rsr II site of the thioredoxin gene in the pET-32a vector. The presence of a C-terminal polyhistidine tag (His6-tag) allows the soluble fusion proteins to be purified by one-step native immobilized metal affinity chromatography (IMAC) to greater than 95% purity. Multiple thioredoxin fusion proteins are mixed in equimolar concentrations and used as an immunogen cocktail for animal immunization. The use of short antigenic peptides of known sequence facilitates direct epitope mapping requiring only small mutagenesis scan peptide libraries in the multipin peptide format.

Keywords: Epitope prediction; Gly/Ser linkers; antigen cocktail; bacteria expression; epitope mapping; immobilized metal affinity chromatography; monoclonal antibodies; multipin peptide library; peptide selection; thioredoxin.

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