A newly developed glucagon sandwich ELISA is useful for more accurate glucagon evaluation than the currently used sandwich ELISA in subjects with elevated plasma proglucagon‐derived peptide levels

Abstract

Aims/Introduction: Glucagon, a peptide hormone produced from proglucagon, is involved in the pathophysiology of diabetes. Plasma glucagon levels are currently measured by sandwich enzyme-linked immunosorbent assay (ELISA), but the currently used sandwich ELISA cross-reacts with proglucagon-derived peptides, thereby providing incorrect results in subjects with elevated plasma proglucagon-derived peptide levels. We aimed to develop a more broadly reliable ELISA for measuring plasma glucagon levels. Materials and Methods: A new sandwich ELISA was developed using newly generated monoclonal antibodies against glucagon. After its validation, plasma glucagon levels were measured with the new ELISA and the currently used ELISA in subjects who underwent laparoscopic sleeve gastrectomy (LSG) and in outpatients with suspected glucose intolerance. The ELISA results were compared with those from liquid chromatography-high resolution mass (LC-HRMS) analysis, which we previously established as the most accurate measuring system. Results: The new ELISA has high specificity (<1% cross-reactivities) and high sensitivity (a lower range of 0.31 pmol/L). Plasma glucagon values in the subjects who underwent laparoscopic sleeve gastrectomy and some outpatients with suspected glucose intolerance differed between the new ELISA and the currently used ELISA. These subjects also showed markedly high plasma glicentin levels. Despite the elevated plasma glicentin levels, the new ELISA showed better positive correlation with LC-HRMS than did the currently used ELISA. Conclusions: The new ELISA enables more accurate measurement of plasma glucagon than the currently used ELISA, even in subjects with elevated proglucagon-derived peptide levels. It should be clinically useful in elucidating the pathophysiology of individual diabetic patients.

Glucagon: Physiological and Pharmacological Functions and Pathophysiological Significance in Type 2 Diabetes

“…We have previously reported that the Mercodia sandwich ELISA yields falsely elevated plasma glucagon levels post-pancreatectomy due to the increased presence of glicentin in patients’ plasma [ 7 ]. A similar phenomenon was observed in patients who underwent bariatric surgery (gastric sleeve resection) [ 8 ]. Therefore, although Mercodia sandwich ELISA is more accurate than the conventional competitive RIA, it may provide incorrect glucagon values after resection of the pancreas or stomach.…”

“…As shown in the bottom panel of Fig. 5 , cross-reactivity tests revealed almost no cross-reactivity with glucagon-related peptides, including glicentin, in the new sandwich ELISA, a striking contrast to the Mercodia sandwich ELISA, which showed cross-reactivity (at several percentage points) with other peptides [ 8 ]. The newly developed sandwich ELISA also showed a close correlation with LC-MS/MS ( Fig.…”

“…The newly developed sandwich ELISA also showed a close correlation with LC-MS/MS ( Fig. 5C , D ), even after bariatric surgery, whereas the Mercodia sandwich ELISA showed a poor correlation with LC-MS/MS measurements [ 8 ].…”

“…Plasma glucagon values after bariatric surgery measured by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) and compared to the values obtained by (C) the Mercodia sandwich ELISA or (D) the new sandwich ELISA. Modified from Kobayashi et al [ 8 ]. HRP, horseradish peroxidase; IgG, immunoglobulin G; RIA, radioimmunoassay; ND, not detected.…”

The Regulation and Secretion of Glucagon in Response to Nutrient Composition: Unraveling Their Intricate Mechanisms

“…However, Kobayashi et al recently revealed a significant issue with cross-reactivity in the conventional ELISA method, particularly in subjects with elevated plasma proglucagon-derived peptide levels [ 164 ]. This cross-reactivity might lead to incorrect results and, consequently, potential misinterpretation of blood glucagon levels from previous clinical data using traditional glucagon assays, which should be revisited and re-evaluated.…”