In this webinar sponsored by GenScript, Dorothea Höpfner, Chemistry Doctoral Candidate at the Technical University of Munich, talked about her work developing and characterizing monoclonal antibodies to detect the post-translational modification AMPylation.
AMPylation refers to the post-translational modification of protein residues by the addition of adenosine monophosphate (AMP). AMPylation occurs by the covalent modification of serine, threonine, and tyrosine residues at side-chain hydroxyl groups. In humans, the enzyme adenylyltransferase, FICD, is involved in catalyzing AMP transfer from ATP to target proteins. AMPylation plays a role in bacterial infections and protein homeostasis.
Because specific and reliable antibodies for detecting AMPylated proteins are not available,
Dorothea’s
project focused on developing a strategy to generate monoclonal antibodies that could specifically detect
this
post-translational modification. Ideally, antibody leads would recognize the small AMP modification,
discriminate between AMPylation and other closely related modifications, and detect AMPylation irrespective
of
its protein context. To achieve this goal, Dorothea used a short and simple synthetic AMPylated peptide,
with
a non-immunogenic backbone, for animal immunizations.
Development of antibodies for specific and sensitive detection of AMPylated proteins relied on GenScript’s Custom Monoclonal Antibody Generation Service. For this project, a total of 10 mice were immunized with the designed AMPylated synthetic peptide, and antibody-producing B cells were isolated from spleens for fusion with myeloma cells for the development of hybridomas.
For selecting antibody candidates, Dorothea used a two-step process whereby antibodies were evaluated first through ELISA and secondly by Western blot analysis for interactions with AMPylated protein, AMPylated peptide, and their respective unmodified controls. By following this approach, Dorothea obtained a large number of hybridoma clones, from which she selected the top ten best ELISA performing clones for further Western blot evaluation.
Ultimately, a total of three monoclonal antibody leads were identified and further characterized for their sensitivity, ability to detect AMP-modifications irrespective of protein context, and cross-reactivity with structurally related post-translational modifications. Validated monoclonal antibody leads could then be sequenced and expressed for recombinant antibody production.
Overall, Dorothea successfully identified monoclonal antibodies with high sensitivity and specificity for the detection of AMP-modified proteins, which will significantly facilitate understanding the roles of AMPylation in health and disease.
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