Development of a polyclonal antibody with defined specificity against synthetic peptides from the N-myc oncoprotein using multiple antigen peptide and hemocyanin conjugation methods

Background/Purpose: The importance of determining N-myc oncoprotein rather than genomic N-myc amplification has been emphasized in neuroblastoma, espe cially in an international project to register biological risk factors in a II neuroblastomas. A method to raise a specific polyclonal antibody agains t the N-myc oncoprotein in large quantities was sought using the synthetic antigen peptide and the multiple antigen peptide (MAP) method. Methods: Two sets of peptides, HGRGPPTAGSTAQSPG and GVAPPRPGGRQTSGGDH, cons erved in the N-myc oncoprotein were synthesized. The hemocyanin-conjugated peptides and the lysine core-conjugated (multiple antigen peptide method) p eptides were injected into rabbits with adjuvant. IgG fractions precipitate d from the sera were purified on an affinity column coupled with these pept ides, and the potency and specificity of the pu rifled IgGs were examined b y immunoblotting and immunohistochemistry in small cell lung cancer cell ti n es with known positivity-negativity of amplification and expression of N- myc, c-myc, and L-myc. Results: Peptides conjugated to the lysine core raised more potent antibodi es than those conjugated to hemocyanin. Purified IgG against GVAPPRPGGRQTSG GDH reacted positively with an N-myc-amplified lung cancer cell line, but n ot with N-myc-unamplified and c-myc/L-myc-amplified cell lines on either im munoblotting or immunostaining. This IgG strongly stained the nuclei of cel ls in a series of surgical specimens and cell lines of neuroblastoma with N -myc amplification. Conclusion: A polyclonal antibody specific for a synthetic peptide from the N-myc oncoprotein was thus obtained and will find wide international use. Copyright (C) 1999 by W.B. Saunders Company.