Immunodetection of the MCHR1 antibody in vitiligo patient sera

Abstract

Human vitiligo is an acquired depigmenting skin disorder characterized by milk-white skin macules resulting from a chronic and progressive loss of melanocytes. It has been suggested that autoimmune mechanisms are involved in this disorder. We undertook this project to obtain an MCHR1-C linear peptide containing four main MCHR1 B-cell epitopes in an attempt to detect the IgG antibody against MCHR1 in vitiligo. The target gene encoding the MCHR1-C peptide was cloned into a pGEX-4T-2 expression vector, expressed in Escherichia coli BL21, and purified using a GST column. The molecular weight was analyzed by SDS-PAGE and western blotting. The IgG antibody response to MCHR1 was detected by ELISA with MCHR1-C as a coated antigen, and the absorption experiment was used to assess the binding ability of the Ab detected by MCHR1-C with a membrane protein in human epidermal melanocytes. The purified peptide MCHR1-C with a molecular weight of 33 kDa was obtained, and could bind to the MCHR1 antibody in human sera. Of the vitiligo patient sera examined, 24 of 145 (16.55%) tested positive for the MCHR1 antibody, and the frequency in the vitiligo patient group was significantly greater compared to the normal control. However, no significant difference between gender, disease duration, clinical subtype or family history between the two groups was observed. In addition, the Ab detected by MCHR1-C in sera was specifically absorbed by the membrane protein obtained from human epidermal melanocytes. Collectively, our data suggest that the MCHR1-C peptide can be used to detect the MCHR1 antibody in vitiligo patients as a coated antigen instead of MCHR1 protein. We conclude that the level of MCHR1 antibody in active vitiligo patients is significantly higher than that in healthy individuals, while gender, disease duration, clinical subtype and family history have no association with the level of MCHR1 antibody in vitiligo patients.