The study on conditions of immunofluoresence-cytochemistry test of autoantibodies to human melanocytes

LIU Chenggang; WANG Dongxue; ZHU Meicai

Abstract：ObjectiveTo establish a simple and accurate immunofluoresence-cytochemistry method of autoantibodiesto melanocytes.MethodsThe cell mixtures obtained from the foreskin tissues which were digested with dispaseⅡand typesin separately were cultured in M2 melanocyte medium supplied with human melanocyte growth supplement, and then were subcultured by using Accutase. The third passage melanocyte were fixed with methanol stored at-20℃ and reacted with the sera of vitiligo patients and normal controls at 1:10 dilution, and then incubation with sheep anti-human IgG-FITC. All the sera were reconfirmed by performing Western-Blot.ResultsThe melanocytes were selectively propagated in M2 melanocyte medium and the second subcultured cells obtained about 100% purity without contamination of fibroblasts and keratinocytes. The fixed cells maintained a good shape and without phenomina of detaching from the surface during testing. The fluorescence brightness was very different according to the level of antibodies to melanocytes, and the special antigens were located in the membrane and plasm of melanocytes or in membrane only. The similar results obtained after the assay described above were repeated two times with the same samples. The results were accurately confirmed by Western-Blot.ConclusionThe method of immunofluoresence-cytochemistry established in this passage was very simple and accurate, it was suitable to detect the autoantibodies to melanocytes and the study of its related autoantigens.