Dual-monoclonal, sandwich immunoassay specific for glucose-dependent insulinotropic peptide1-42, the active form of the incretin hormone.

BACKGROUND 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(1-42), which is rapidly degraded by dipeptidyl peptidase 4 to GIP(3-42), which is inactive. There is currently no described monoclonal antibody-based sandwich immunoassay to quantify concentrations of GIP(1-42), the active form of the peptide. METHODS To create a sandwich ELISA for GIP(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(1-42). RESULTS The sandwich ELISA was highly specific for GIP(1-42) and did not recognize GIP(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(1-42) concentrations in healthy volunteers increased dramatically in the postprandial state compared to the fasting state. GIP(1-42) values were correlated with total GIP values overall; however, there was substantial interindividual variation. CONCLUSIONS 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(1-42), the active form of the incretin hormone. This ELISA should help to improve our understanding of the role of GIP(1-42) in regulating glucose-dependent insulin secretion.