Design and application of a polyclonal peptide antiserum for the universaldetection of leptin protein

An epitope-specific polyclonal antiserum was produced in rabbits immunized against a synthetic 15 amino acid peptide (QRVTGLDFIPGLHPV) derived from th e coding sequence reported for the porcine leptin gene (GenBank Accession N o. U59894). This peptide contains a core sequence comprised of eight amino acids (GLDFIPGL) that is totally conserved in all leptin proteins studied t o date. Purified recombinant human, mouse, rat, pig, and chicken leptin pro teins were separated by polyacrylamide gel electrophoresis (SDS-PAGE) and e lectro-blotted onto PVDF membranes. Western blots were developed employing the leptin-specific peptide antiserum with an alkaline-phosphatase-conjugat ed anti-rabbit IgG second antibody chromogenic system. The peptide antiseru m was found to be highly specific for leptin which exhibited an estimated m olecular weight of about 16 kDa for all species analyzed. The sensitivity o f the Western blot assay was not sufficient to permit the direct detection of leptin in chicken serum or plasma. However, with this assay we were able to detect native leptin protein in an enriched fraction prepared from chic ken plasma using a combination of gel filtration and ion exchange column ch romatography. Slot blots indicated a potential application of the immunosta ining technique for quantitative analysis of leptin protein. Finally, the p eptide antiserum was successfully employed to localize leptin protein by im munohistochemical staining of thin sections prepared from adipose (chicken and pig) and liver (chicken) tissue samples. This study is the first to rep ort a polyclonal peptide antiserum that apparently recognizes intact leptin protein, both native and recombinant, regardless of the species of origin. (C) 2000 Elsevier Science B.V. All rights reserved.