Abstract
High quality, well-validated antibodies are needed to mitigate irreproducibility and clarify conflicting data in science. We describe an epitope-directed monoclonal antibody (mAb) production method that addresses issues of antibody quality, validation and utility. The workflow is illustrated by generating mAbs against multiple in silico-predicted epitopes on human ankyrin repeat domain 1 (hANKRD1) in a single hybridoma production cycle. Antigenic peptides (13–24 residues long) presented as three-copy inserts on the surface exposed loop of a thioredoxin carrier produced high affinity mAbs that are reactive to native and denatured hANKRD1. ELISA assay miniaturization afforded by novel DEXT microplates allowed rapid hybridoma screening with concomitant epitope identification. Antibodies against spatially distant sites on hANKRD1 facilitated validation schemes applicable to two-site ELISA, western blotting and immunocytochemistry. The use of short antigenic peptides of known sequence facilitated direct epitope mapping crucial for antibody characterization. This robust method motivates its ready adoption for other protein targets.
Highlights
High quality, well-validated antibodies are needed to mitigate irreproducibility and clarify conflicting data in science
Synthetic peptides may be chemically conjugated to large carrier proteins such as bovine serum albumin (BSA), ovalbumin or keyhole limpet hemocyanin (KLH), or prepared as branched peptides known as multiple antigen peptides (MAP)
A cocktail of three Trx-tripeptide antigens corresponding to short sequences in the N, C- and internal regions of human ankyrin repeat domain 1 (hANKRD1) yielded 24 stable hybridoma clones and 12 monoclonal antibody (mAb) were selected for detailed evaluation and validation
Summary
Well-validated antibodies are needed to mitigate irreproducibility and clarify conflicting data in science. The workflow is illustrated by generating mAbs against multiple in silico-predicted epitopes on human ankyrin repeat domain 1 (hANKRD1) in a single hybridoma production cycle. The use of short antigenic peptides of known sequence facilitated direct epitope mapping crucial for antibody characterization. This robust method motivates its ready adoption for other protein targets. Spatially distant, B-cell epitope-predicted sequences are independently cloned into the surface exposed loop of a highly soluble His-tagged thioredoxin (Trx) carrier This facilitates high-yield production and easy purification of bacterially expressed fusion peptides, which are combined into a mixed immunogen cocktail for animal immunization. The availability of well-characterized mAbs directed towards functionally relevant protein domains would facilitate mechanistic studies of hANKRD1 processing, cellular localization, binding profile with interacting partners and pathophysiological dysregulation within the cell and in circulation
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