Deficient melanosome formation in some coat-color mutant mice revealed by a monoclonal antibody against melanosome.

Abstract

Some coat-color loci in mice are considered to control melanosome formation. In order to investigate genetic control of melanosome-associated proteins, we prepared monoclonal antibodies against mouse melanosomes. Melanosomes were isolated from B16 mouse melanoma through differential fractionation. BALB/c mice were immunized with an SDS-solubilized melanosome fraction. The spleen cells were subsequently fused with mouse myeloma cells, the resulting hybridomas cloned. Their secreted IgG was screened for reactivity to the SDS-solubilized melanosome fraction. One monoclonal antibody, M10, was shown to react to melanosomes by immunoelectronmicroscopy. It recognized a single protein band of 61,000 dalton on immunoblots of gel-fractionated melanosomes. The reactivities of M10 to skin homogenates from various coat-color mutants were examined by the ELISA method. Five congenic genotypes, non-agouti (a/a), brown (b/b), albino (c/c), dilute (d/d), and pink-eyed dilution (p/p) were examined. Among these, b/b and p/p showed significantly lower reactivities than a/a. Our results seem to suggest that the pigment abnormalities in these mutants result from abnormalities of the melanosomal proteins. In the case of albino mice, the reactivity of M10 to skin homogenate was almost the same as the wild-type mouse. It seems that the albino mice are capable of producing the melanosomal protein.