A novel high-sensitivity electrochemiluminescence (ECL) sandwich immunoassay for the specific quantitative measurement of plasma glucagon.
OBJECTIVE
To develop a novel, dual-monoclonal sandwich immunoassay with superior sensitivity that provides a rapid and convenient method for measuring glucagon. Glucagon is a 29-amino acid polypeptide hormone produced in the pancreas by the α-cells of the islets of Langerhans. Working in concert with insulin, glucagon is involved in regulating circulating glucose concentrations.


METHODS
The immunoassay utilizes Meso Scale Discovery (MSD) electrochemiluminescence (ECL) technology and two affinity-optimized monoclonal antibodies. A series of experiments was performed to determine the linear range of the assay and to evaluate sensitivity, accuracy, recovery, precision, and linearity.


RESULTS
The sandwich assay was specific for glucagon and did not recognize the closely related peptide oxyntomodulin or other incretin peptides. The assay demonstrated excellent recovery, precision, and linearity, and a broad dynamic range of 0.14 pmol/L to 1950 pmol/L. In addition, assay results were highly correlated with those obtained using a previously described competitive RIA employing polyclonal antiserum.


CONCLUSIONS
The use of affinity-optimized monoclonal antibodies in a sandwich immunoassay format provides a robust, sensitive, and convenient method for measuring concentrations of glucagon that is highly sensitive and specific. This immunoassay should help to improve our understanding of the role of glucagon in the regulation of glucose metabolism.
Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285, USA. Sloan_John_Harrison@lilly.com
John H
JH
Sloan
Robert W
RW
Siegel
Yvelina T
YT
Ivanova-Cox
David E
DE
Watson
Mark A
MA
Deeg
Robert J
RJ
Konrad
D000911:Antibodies, Monoclonal / Q000276:immunology; D000915:Antibody Affinity / Q000276:immunology; D000918:Antibody Specificity / Q000276:immunology; D055664:Electrochemical Techniques / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay; D005934:Glucagon / Q000097:blood* / Q000276:immunology; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D007700:Kinetics; D008163:Luminescent Measurements / Q000379:methods*; D012680:Sensitivity and Specificity
D000911:Antibodies, Monoclonal; D005934:Glucagon