Submitted to: Journal of Biochemical and Biophysical Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/25/2000
Publication Date: N/A
Citation: Interpretive Summary: Leptin is a protein hormone encoded by the obesity (ob) gene that functions in the regulation of feeding behavior, energy balance and reproduction in humans and animals. In mammals, the amount of leptin in blood increases in direct proportion to body fat mass. Circulating leptin signals the brain about the status of body energy (fat) stores, and the brain, in turn, activates specific neural pathways that modulate food intake and energy expenditure to help maintain energy stores at a set level. Thus, a negative feedback loop exists between adipose tissue and the central nervous system with leptin serving as an afferent signal to the brain. Recessive mutations in the genes encoding leptin and its receptor have been identified and linked with the pathological conditions of morbid obesity, diabetes and infertility. Therefore, it is important to be able to accurately monitor leptin protein levels in blood. In addition, we are interested in studying the role of leptin in regulating food intake in domestic animals. Our objective in this study was to develop an antiserum that could be used to detect and quantify leptin protein produced by any animal species. Such an immunological reagent would allow us to develop 'universal' assay techniques for leptin that could be utilized in a wide range of experimental studies. Moreover, this antiserum has been able to detect chicken leptin (both recombinant and native proteins) which was not possible previously. Thus, the development and application of immunoassay techniques for different species will add to our understanding of the function of leptin in the regulation of appetite in domestic animals.
Technical Abstract: A polyclonal antiserum was produced in rabbits immunized against a synthetic 15 amino acid peptide (QRVTGLDFIPGLHPV) derived from the reported sequence for porcine leptin. This peptide contained a core 8 amino acid sequence (GLDFIPGL) that is totally conserved for all reported leptins. Recombinant human, mouse, pig, and bovine leptins were subjected to SDS- PAGE and electroblotted onto nitrocellulose. Western immunoblots were developed with an alkaline-phosphatase-conjugated second antibody chromogenic system. The antiserum was highly specific for leptin which demonstrated a molecular weight of approximately 16 kDa. Sensitivity of the Western assay did not permit the direct detection of leptin in serum, however, we were able to employ this assay to detect native leptin in a extract of chicken plasma prepared using column chromatography. Immuno- slot-blotting demonstrated a potential use for the antiserum in semi- quantitative analyses of leptin. This antiserum was also utilized to localize leptin protein in sections of subcutaneous adipose (back fat) tissue from pigs by immunohistochemical staining. This study constitutes the first report of an anti-leptin antiserum demonstrating universal recognition characteristics.