2004 Fiscal Year Final Research Report Summary
Establishment of specific immunoassay for bioactive forms of glucagon-like peptide-1 and its application to development of GLP-1 analogs for treatment of diabetes.
| Project/Area Number |
15590503
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Laboratory medicine
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| Research Institution | Fukuoka University |
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Principal Investigator |
TATEISHI Kayoko Fukuoka University, School of Medicine, Associate Professor, 医学部, 助教授 (60179728)
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| Project Period (FY) |
2003 – 2004
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| Keywords | Glucagon-like peptide-1 / Bioactive GLP-1 / Site-specific antibody / Enzymeimmunoassay |
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| Research Abstract |
OBJECTIVE : Glucagon-like peptide-1(GLP-1) is an hormone enhancing postprandial insulin secretion but rapidly degraded to inactive metabolites cleaved off the two NH_2-terminal amino acids. Therefore, establishment of methods measuring the concentration of only intact biologically active GLP-1 without cross-reacting inactive metabolites was aimed to study significance of this hormone for regulation of insulin secretion. METHODS : Antibodies specific to N-terminal of active forms of GLP-1 were produced by the immunization procedure including treatments with a conjugate of GLP-1(11-25) with copolymer of D-glutamic acid and D-lysine (D-GL) before immunization with GLP-1(7-25)-KLH. RESULTS: The polyclonal antibody (R301) produced by the immunization procedure cross-reacted <0.05% with the inactive metabolites, GLP-1(9-37) and GLP-1(9-36)amide, while the control antiserum obtained without treatment with the D-GL conjugate showed 100% cross-reactivity with those inactive metabolites. Monoclonal antibodies prepared by the immunization procedure including treatments with the D-GL conjugate reacted selectively with GLP-1(7-25) rather than with GLP-1(9-25) which were coated on plates. Among various combinations of those antibodies, a sandwich enzyme-immunoassay procedure was established, in which C-terminal monoclonal antibody obtained from immunization with GLP-1(7-36)NH_2-KLH was immobilized on wells of microtiter plates and R301 as a 2nd antibody and peroxidase-labeled anti-rabbit IgG as a detection conjugate were used. Bioactive forms of GLP-1(7-36)amid and GLP-1(7-36) without cross-reaction with GLP-1(9-36)amide and GLP-1(9-37) were specifically measured by this assay with sensitivity of 0.05 ng/ml. This assay procedure will be applied to monitor plasma levels of bioactive GLP-1.
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