title: A monoclonal antibody against leptin.
abstract: Leptin is an important protein that regulates energy storage and homeostasis in humans and animals. Leptin deficiency results in various abnormalities such as diabetes, obesity, and infertility. Producing a high affinity monoclonal antibody against human leptin provides an important tool to monitor and trace leptin function in different biological fluids. In this study, recombinant human leptin was conjugated to KLH and injected into mice. After immunization, mouse myeloma SP2/0 cells were fused with murine splenocytes followed by selection of antibody-producing hybridoma cells. After screening of different hybridoma colonies by ELISA, a high affinity antibody was selected and purified by affinity chromatography. The affinity constant of the antibody was measured by ELISA. Western blot, immunocytochemistry, and flow cytometry experiments were used to characterize the antibody. The anti-leptin antibody had a high affinity (around 1.13 × 10(-9) M) for its antigen. The saturation of the antibody with leptin (20 moles leptin per 1 mole antibody) in Western blot analysis proved that the antibody had specific binding to its antigen. Immunocytochemistry and flow cytometry on JEG-3 (human placental choriocarcinoma cell) cells revealed that the anti-leptin antibody recognized intracellular leptin. In conclusion, we report here the production and characterization of a murine anti-leptin antibody with high affinity for human leptin.
authors.affiliation: Monoclonal Antibody Research Center, Avicenna Research Institute, Academic Center for Education, Culture, and Research, Tehran, Iran.
authors.forename: Jafar
authors.initials: J
authors.lastname: Mahmoudian
authors.forename: Mahmood
authors.initials: M
authors.lastname: Jeddi-Tehrani
authors.forename: Ali Ahmad
authors.initials: AA
authors.lastname: Bayat
authors.forename: Ahmad Reza
authors.initials: AR
authors.lastname: Mahmoudi
authors.forename: Yasaman
authors.initials: Y
authors.lastname: Vojgani
authors.forename: Banafsheh
authors.initials: B
authors.lastname: Tavangar
authors.forename: Reza
authors.initials: R
authors.lastname: Hadavi
authors.forename: Saeed
authors.initials: S
authors.lastname: Zarei
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology*; D000915:Antibody Affinity; D000918:Antibody Specificity; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002846:Chromatography, Affinity; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006433:Hemocyanins / Q000737:chemistry / Q000276:immunology*; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D007114:Immunization; D018796:Immunoconjugates / Q000008:administration & dosage / Q000737:chemistry / Q000276:immunology*; D007700:Kinetics; D020738:Leptin / Q000008:administration & dosage / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D011247:Pregnancy; D011994:Recombinant Proteins / Q000008:administration & dosage / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D018796:Immunoconjugates; D020738:Leptin; D011994:Recombinant Proteins; D006433:Hemocyanins; C032808:keyhole-limpet hemocyanin