Document Detail


Generation and characterization of anti-leptin antisera against synthetic peptides and recombinant protein.
MedLine Citation:
PMID:  15647625     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
The objective of this study was to generate antisera against recombinant bovine leptin and synthetic oligopeptides corresponding to the amino acid sequence 21-40 and 91-110 of bovine leptin. Recombinant bovine leptin was raised in the 293 cells and purified from 10 L of conditioned medium and utilized for immunization. The synthetic peptides were conjugated with keyhole limpet hemocyanin and inoculated into rabbits for antibody generation. Antibody titer was monitored by enzymeimmunoassay, immunoblotting and sandwich binding assay techniques. Each of the antisera, against three different antigens, was found to react with bovine leptin. The titers of anti-peptide antisera were lower than that of anti-recombinant leptin antiserum. Since anti-recombinant leptin antiserum was not neutralized by the leptin peptides 21-40 and 91-110, it is suggested that each antiserum recognizes a distinct epitope. In immunoblot analyses, all antisera exhibited cross-reactivity with human and mouse leptins. However, in the sandwich binding assay, the combination of anti-peptide antisera and anti-recombinant leptin antiserum, originated from bovine leptin, did not cross-react with either human or mouse leptin. The discrepancy of antigenic recognition between the immunoblot analyses and sandwich assay is thought to be dependent on the conformational status of leptin molecules between the species. The antisera generated in this study, which recognized distinct epitopes of bovine leptin, will provide a useful tool for studies of bovine leptin functions.
Authors:
Toru Takahashi; Kei Imai; Kazuyoshi Hashizume
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  The Journal of reproduction and development     Volume:  50     ISSN:  0916-8818     ISO Abbreviation:  J. Reprod. Dev.     Publication Date:  2004 Dec 
Date Detail:
Created Date:  2005-01-13     Completed Date:  2005-05-12     Revised Date:  2013-11-14    
Medline Journal Info:
Nlm Unique ID:  9438792     Medline TA:  J Reprod Dev     Country:  Japan    
Other Details:
Languages:  eng     Pagination:  717-24     Citation Subset:  IM    
Affiliation:
National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan. tatoru@affrc.go.jp
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MeSH Terms
Descriptor/Qualifier:
Animals
Cattle
Cell Line
Culture Media, Conditioned / pharmacology
Dose-Response Relationship, Drug
Electrophoresis, Polyacrylamide Gel
Enzyme-Linked Immunosorbent Assay
Hemocyanin / chemistry
Humans
Immunoblotting
Immunoenzyme Techniques
Leptin / chemistry,  immunology*,  metabolism
Mice
Peptides / chemistry*
Protein Binding
Rabbits
Recombinant Proteins / chemistry*
Chemical
Reg. No./Substance:
0/Culture Media, Conditioned; 0/Leptin; 0/Peptides; 0/Recombinant Proteins; 9013-72-3/Hemocyanin; FV4Y0JO2CX/keyhole-limpet hemocyanin

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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