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

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.

Immunoassay-Compatible Inactivation of SARS-CoV-2 in Plasma Samples for Enhanced Handling Safety.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) inactivation is an important step toward enhanced biosafety in testing facilities and affords a reduction in the biocontainment level necessary for handling virus-positive biological specimens. Virus inactivation methods commonly employ heat, detergents, or combinations thereof. In this work, we address the dearth of information on the efficacy of SARS-CoV-2 inactivation procedures in plasma and their downstream impact on immunoassays. We evaluated the effects of heat (56 °C for 30 min), detergent (1-5% Triton X-100), and solvent-detergent (SD) combinations [0.3-1% tri-n-butyl phosphate (TNBP) and 1-2% Triton X-100] on 19 immunoassays across different assay formats. Treatments are deemed immunoassay-compatible when the average and range of percentage recovery (treated concentration relative to untreated concentration) lie between 90-110 and 80-120%, respectively. We show that SD treatment (0.3% TNBP/1% Triton-X100) is compatible with more than half of the downstream immunoassays tested and is effective in reducing SARS-CoV-2 infectivity in plasma to below detectable levels in plaque assays. This facile method offers enhanced safety for laboratory workers handling biological specimens in clinical and research settings.