Distribution and organelle specificity of melanoma-associated antigens identified by Mo 465.12 in human malignant melanoma.

Mo 465.12 (Mo 465) is a unique monoclonal antibody that detects cytoplasmic antigens highly restricted to the cells of human malignant melanoma (MM). As yet, it is not known which cytoplasmic components are reactive with Mo 465 within MM cells. This study, using the enzyme-linked immunosorbent assay (ELISA) technique, examines the distribution and specificity of Mo 465-reactive proteins in MM tissues that were isolated by cell fractionation and solubilized by a nonionic detergent (Brij 35). Light microscopically, Mo 465 bound strongly to the cytoplasm of MM cells, being localized as patchy or microgranular, but not to the normal epidermal melanocytes. When the cultured MM cells were fractionated, Mo 465 reacted with both soluble and membrane fractions and with the spent culture medium. To further identify the organelle specificity of Mo 465-reactive proteins, metastatic human MM and normal human liver tissues were separated into the fractions of large granules, small granules, Golgi apparatus, mitochondria, rough endoplasmic reticulum (ER), smooth ER, microsomes, and melanosomes. Mo 465-reactive proteins were detected in the fractions of crude homogenate, microsomes, and rough ER of MM tissue, and their reactivities were in parallel to the concentration of solubilized protein. the findings indicate that Mo 465-reactive proteins are localized in rough ER, that they are released into the cytoplasm, and that in the in vitro condition they are shed into the culture medium.