Development and Validation of an Antibody for Detection of Lysine Homocysteinylation in Cobalamin Disorders

Requirements:

The proposed development of a novel post-translational modification (PTM)-specific antibody directly supports the U.S. Government's mission to advance biomedical research on rare genetic diseases, improve public health outcomes, and accelerate the development of precision medicine approaches

Currently there is no commercially available antibody against N-homocysteine-lysine (N-homocysteinylation) so we will need to have one generated. By generating it, we are able to provide specifications for the peptide sequence based on preliminary findings in our scientific models of disease insuring it will work effectively. Our objectives are as follows:  

1. Synthesize and purify peptide antigens with N-homocysteinylated lysine residues and matched control peptides with unmodified lysines.  Conjugate peptides to carrier proteins (e.g., KLH) for immunization 
2. Immunize appropriate host species (rabbit for polyclonal antibody generation) 
3. Generate polyclonal sera prior to immunization and 2 to 3 times after following antigen boost. Test sera on lab samples of N-homocysteinylated proteins from mouse models of MMACHC delicacy in our lab. Also perform ELISA screening of antibody on modified and unmodified peptide.  
4. Terminal bleed on immunized animal and purification of working antibody from serum 
5. With working antibody we will characterize the proteins affected by this aberrant posttranslational modification to better understand disease development and in future design targeted therapies for it.