Characterization of a polyclonal antibody raised against the human melanocortin-1 receptor

We have generated a polyclonal antibody raised against a synthetic peptide corresponding to the amino acids 2-18 of the extracellular, N-terminal domain of the human melanocortin-1 receptor (MC-1R). Specificity of the affinity-purified anti-MC-1R antibody was confirmed by dot blot analysis with the antigenic peptide. The antibody detected MC-1R antigenicity on the surface of normal human melanocytes and WM35 melanoma cells, as shown by FACS and immunofluorescence analysis. The antibody was suitable for immunoperoxidase staining of deparaffinized skin sections, revealing prominent MC-1R staining of a cutaneous melanoma as opposed to undiseased skin in which normal melanocytes were only occasionally immunoreactive. Distinct adnexal structures in normal skin also displayed MC-1R immunostaining. Specificity of the MC-1R immunoreactivity in each technique was confirmed by preabsorption with the immunogenic peptide, omission, or substitution of the primary antibody with preimmune serum. Our results provide a baseline for future studies on MC-1R expression in diseased human skin.