Antibody Production: Activity-Dependent Neuroprotective Protein (ADNP) as an Example

Activity-dependent neuroprotective protein (ADNP, human calculated molecular mass 123,562.8 Da) is a newly discovered glial protein that it is essential for embryonic development and brain formation. ADNP includes an active neuroprotective site, an 8 amino acid peptide NAPSVIPQ (NAP). The current study was set out to prepare antibodies to ADNP that will recognize different sites on the molecule. Four peptides of 8–20 amino acids that span the ADNP molecule, including NAPVSIPQ, were prepared. Peptides (containing a Cys residue attached to the N-terminal amino acid) were conjugated to keyhole limpet hemocyanin (KLH) and injected to respective rabbits in the presence of Freund’s complete adjuvant. Following five booster injections in incomplete Freund’s adjuvant, the respective antisera were collected and assayed by enzyme-linked immunosorbent assay (ELISA) and purified by affinity chromatography on peptides conjugated to SulfoLink Coupling Gel (Pierce). Mouse brain proteins were prepared (4 months old) and separated into cytoplasmic and nuclear fractions. Proteins were further separated by SDS-PAGE (SDS-PolyAcrylamide Gel Electrophoresis) and transferred to nitrocellulose membranes. Membranes were then probed with the antibodies followed by a secondary antibody horseradish peroxidase conjugated anti-rabbit IgG prepared in goat and developed with ECL. All antibodies recognized intact ADNP in both cytoplasmic and nuclear fractions. This work developed antibodies against ADNP and NAP that will be utilized for further experimentations to elucidate the distribution and mechanisms of ADNP and NAP neuroprotection.