HMB-45 antibody demonstrates melanosome specificity by immunoelectron microscopy

The intracellular localization of antigenic sites recognized by the monoclonal antibody HMB-45 was investigated in melanomas of the choroid and skin by postembedding immunoelectron microscopy. Antigenic sites were detected by a three-step procedure, consisting of incubating sections with the monoclonal HMB-45 antibody (protein G affinity-purified ascites from Enzo Diagnostics Inc or tissue culture supernatant from Dako Corp), followed by incubation with an affinity-purified rabbit anti-mouse IgG and finally with protein A-gold complex. Gold particles, indicative of HMB-45 immunoreactivity, were restricted to melanosomes in the malignant melanocytes. Early stages in melanosome formation (stages I through III) were most intensely stained, while late-stage melanosomes (stage IV) were only sparsely labeled or not stained at all. Melanophages adjacent to a cutaneous melanoma showed intense immunoreactivity in the cytoplasm and especially over electron-dense portions of lysosomes with the HMB-45 antibody from Enzo. In marked contrast, only very sparse labeling was detected over melanophages using a similar concentration of the HMB-45 antibody from Dako. Subsequently, when the Enzo antibody was diluted 40 times above the recommended working dilution, most of the melanophage staining disappeared, while melanocyte-specific staining was maintained. Immunolabeling of melanosomes with HMB-45 was drastically reduced or absent following section pretreatment with neuraminidase, confirming an earlier report that the HMB-45 antigen is partially composed of sialic acid. Our immunoelectron microscopic results show that HMB-45 antibody specifically stains melanosomes, rather than diffuse cytoplasmic antigen, as described by light microscopic immunohistochemical analysis, thus explaining its specificity for melanocytes. In addition, the elimination of HMB-45 immunoreactivity by neuraminidase pretreatment supports the idea that sialylation of antigen is crucial to HMB-45 binding, and suggests that the absence of staining in normal adult melanocytes, dermal nevi, and other melanocytic lesions may be a result of differential sialylation.