Compositions and methods for linear and conformational site-specific antibodies and methods of making the same

An in vivo immunization method comprising:

• a. immunizing an animal at least once with a modified peptide having an amino acid sequence of about 10 to about 20 amino acids identical to an amino acid sequence of a protein of interest except wherein one internal native amino acid has been substituted with a non-native amino acid (nnAA), wherein the nnAA is a phosphorylated amino acid, an acetylated amino acid, an isocyanated amino acid, a sulfated amino acid, or a nitrated amino acid;
• b. boosting the animal at least once with a first unmodified peptide comprising a core amino acid sequence that is identical to the modified peptide of step (a), except wherein the nnAA has been substituted with the native amino acid, a first N-terminal amino acid sequence that is not native to the protein of interest, and a first C-terminal amino acid sequence that is not native to the protein of interest;
• c. cloning B-cells obtained from the animal;
• and
• d. identifying a B-cell clone of step (c) that produce an antibody that:
• i. binds to the first unmodified peptide and a second unmodified peptide comprising
• the core amino acid sequence of the first unmodified peptide,
• a second N-terminal amino acid sequence that is not native to the protein of interest, and
• a second C-terminal amino acid sequence that is not native to the protein of interest,
• wherein the first N-terminal amino acid sequence, the first C-terminal amino acid sequence, the second N-terminal amino acid sequence, and the second C-terminal amino acid sequences are different amino acid sequences.