A-068 Development of GIP ELISAs: The Next Level of DiabetesManagement

Abstract

Abstract Background To develop sensitive and specific ELISAs for the quantitative measurement of total and intact GIP in human plasma, and other biological fluids. Glucose-dependent insulinotropic polypeptide (GIP) is a peptide hormone consisting of 42 amino acids (1-42aa). This undergoes processing by a dipeptidyl peptidase-4 (DPP-4) and produces GIP (3-42aa) and GIP (3-30aa) fragments. The processed forms have higher affinity and very efficiently inhibits GIP receptor (GIPR) activity with no intrinsic activity. It has also been observed that the GIP response is dependent not only on meal size but also on meal composition. GIP has been found to have physiological and pharmacological relevance in the development of obesity and the pathogenesis of cardiovascular disease besides its involvement in type 2 diabetic pathophysiology. GIP acts in the entero-insular axis as an anabolic hormone that increases insulin levels, which in return increases glycogen and fatty acid synthesis and inhibits the breakdown of fat. GIP also has extra-pancreatic functions as well as roles in the stomach to reduce acid secretion by the parietal cells. On the bone, GIP has a dual effect as it causes the proliferation of osteoblasts as well as inhibits osteoclastic bone resorption. The widespread expression of GIP-R in the brain suggests that GIP might play an essential function in neuro-signaling mechanisms. Methods Highly specific and reproducible total GIP (AL-1013) and Intact GIP (AL-1022) ELISAs have been developed using specific monoclonal antibodies to help estimate the total and intact GIP concentrations in plasma in the respective immunoassays. Results Total and intact GIP ELISAs have been validated using well-characterized sample set and the peptide preparations. The total GIP ELISA detects 1-42aa, 3-42aa, 1-30aa, and 3-30aa in the sample. The intact GIP assay detects GIP (1-42aa) and the most abundant 3-42aa fragment equally in the human plasma. The intact GIP assay does not detect truncated C-terminal fragments(1-30aa). These assays did not cross-react to GRPP, Glucagon, Oxyntomodulin, GLP-1, and GLP-2. Method comparison between intact and total GIP assays using 388 samples yielded a slope of 0.8154 (r=0.958, p < 0.0001). Both the assays are highly reproducible with the total coefficient of variation less than 10%. GIP concentrations were measured in both assays in lean and obese subjects on fat-rich diet at 30-minute intervals. Average concentrations in intact GIP assay at 0 minutes (baseline) in lean and obese subjects (n=10) were 115.6 and 88.5 pg/mL, respectively. Average concentrations in total GIP assay at 0 minutes (baseline) in lean and obese subjects (n=10) were 151.5 and 125.9 pg/mL, respectively. Both total and intact GIP had steep increase in concentrations up to 90 minutes and then flattened up to 180 minutes. The GIP results have been also compared to the mean plasma concentrations of Glucagon, GLP-1, C-Peptide between lean and obese subjects on fat-rich meal. Conclusions Highly sensitive and specific total and intact GIP ELISAs have been developed to reliably quantify these important endocrine and local regulators in physiological and pathophysiological studies for metabolic disorders.