Dual-Monoclonal, Sandwich Immunoassay Specific for Glucose-Dependent Insulinotropic Peptide^sub 1-42^, the Active Form of the Incretin Hormone

• Published in: Clinical chemistry (Baltimore, Md.) Md.), 2011-06, Vol.57 (6), p.849
• Main Authors: Troutt, Jason S, Siegel, Robert W, Chen, Jinbiao, Sloan, John H, Deeg, Mark A, Cao, Guoqing, Konrad, Robert J
• Format: Article
• Language: English
• Subjects: Amino acids; Carbohydrates; Cloning; Medical research; Mutagenesis; Mutation
• ISSN: 0009-9147; 1530-8561

Glucose-dependent insulinotropic peptide (GIP) is an incretin peptide secreted by intestinal K cells that stimulates insulin secretion in a glucose-dependent manner. It is secreted as an active, intact 42-amino acid peptide GIP^sub 1-42^, which is rapidly degraded by dipeptidyl peptidase 4 to GIP^sub 3-42^, which is inactive. There is currently no described monoclonal antibody-based sandwich immunoassay to quantify concentrations of GIP^sub 1-42^, the active form of the peptide. To create a sandwich ELISA for GIP^sub 1-42^, we generated a monoclonal antibody specific for the intact N-terminus of the peptide, which was further optimized to increase its affinity. We used this antibody as a conjugate antibody in a sandwich ELISA and paired it with an anti-total GIP capture monoclonal antibody to create a dual monoclonal sandwich ELISA for GIP^sub 1-42^. The sandwich ELISA was highly specific for GIP^sub 1-42^ and did not recognize GIP^sub 3-42^. The ELISA demonstrated a broad dynamic range and a lower limit of quantification of 5 ng/L. Using the ELISA, we were able to show that GIP^sub 1-42^ concentrations in healthy volunteers increased dramatically in the postprandial state compared to the fasting state. GIP^sub 1-42^ values were correlated with total GIP values overall; however, there was substantial interindividual variation. The use of an N-terminal-specific monoclonal antibody in a sandwich ELISA format provides a robust and convenient method for measuring concentrations of GIP^sub 1-42^, the active form of the incretin hormone. This ELISA should help to improve our understanding of the role of GIP^sub 1-42^ in regulating glucose-dependent insulin secretion.