The main role is the specificity of ELISA kits
Mercodia’s two new glucagon tests provide a high quality solution by offering excellent specificity, optimal sensitivity and low sample volume requirements. These tests are easy to use, do not require any laborious and time-consuming cleaning or pre-treatment, and do not involve the use of radioactivity. They can be used in basic, preclinical and clinical studies.
Accuracy plays a key role
Glucagon is, due to its ability to stimulate glucose production in the liver, an essential regulator of glycemic regulation. A number of studies have shown that glucagon dysregulation occurs in all forms of diabetes. Interest in this hormone has grown significantly in recent years, and with advances in drug development, long-term analytical barriers to its determination need to be addressed. Glucagon is a peptide hormone that is processed from a much larger proglucanone precursor.  Expression and activity of prohormone convertase 2 (PC2 / PCSK2) in pancreatic alpha cells results in the formation of a 29 amino acid glucagon polypeptide. Glucagon can be partially degraded by endopeptidase, resulting in a truncated form known as glucagon 19-29 or miniglucagon. Miniglucagon is present in islets and target tissues of glucagon in very low concentrations, but appears to is a highly effective regulator of physiological processes in these tissues. It has also been found to have opposite effects to glucagon. [2, 3, 4] Cleavage of PC2 is not the only way proglucagon is processed. The proconvertase enzyme PC1 / 3 (PCSK1 / 3) cleaves proglucagon into a number of proteins, including glycinin, oxyntomodulin, GLP-1 and GLP-2 (picture below).
Therefore, post-translational processing of proglucagon should be considered when determining glucagon. The end products are not all significantly different proteins with completely unique sequences. Many proteins contain the complete sequence of glucagon, such as proglucagon. The complete amino acid sequence of glucagon also forms a large part of glycitin and oxyntomodulin. While the biological effect of glucagon may be unique, its sequences are not, which has so far led to assay errors, as antibodies used in glucagon assays can recognize epitopes shared by the above hormones.
Circulating levels of homologous proteins that may elicit a particularly problematic response in the glucagon test, especially glycitin, should also be considered. Proglucagon 1-61 represents only a negligible fraction of immunoreactive glucagon, although samples from pancreatectomy patients or patients with renal failure may contain elevated levels. 
Oxyntomodulin circulates at detectable but low levels.  Glycintine circulates at much higher levels than glucagon, as demonstrated using new glucagon and glycitin specific assays (Fig. 3). These values are regulated differently, which means that “exposure” or background readings may not result in a correction of the glucagon test values. Inaccurate glucagon measurements can have huge negative effects on research and clinical decisions. (Fig. 4)
Dynamic changes in both insulin and glucagon are essential for the development of diabetes, and therefore the determination of the insulin: glucagon ratio has been used for many years in research as a more comprehensive metabolic index for measuring insulin alone. [8, 9,10, 11, 12] This ratio looks at the storage and utilization of nutrients in both normal and catabolic conditions, such as infection, trauma, cancer, etc.  The actual insulin: glucagon ratio cannot be determined if you use tests (for each biomarker) that are not very specific.
The fact that proglucagon 1-61, oxyntomodulin and glicentin contain the full glucagon sequence and extensions at the N-terminus and / or C-terminus can be used for specific determinations (Figures 1 and 2). Mercodia solves this problem by having two antibodies in its tests, targeting both ends of glucagon. This strategy was also chosen with regard to the presence of shorter forms of glucagon – miniglucagon, or glucagon 3-29 and 5-29, which are produced by sequential degradation (and inactivation) by dipeptidyl peptidase IV (DPPIV). [13, 14]
The highly specific murine monoclonal antibodies used are extensively characterized by ELISA and Biacore methods. These two antibodies are used in both Mercodia ELISA assays for glucagon, and cross-reactivity assays revealed an excellent specificity profile (Fig. 6).
Not only is it important to examine the increase in glucagon level, but equally important is the ability to detect a subsequent decrease in glucagon level. Glucagon circulates at low concentrations around 10 pmol / l (35 pg / ml) or less and may increase to 2020-30 pmol / l (70 to 105 pg / ml) during hypoglycaemic conditions and may decrease to 1-2 pmol / l. l (3.48 – 7 pg / ml) during hyperglycemia [16,17] Commercially available assays do not provide sufficient sensitivity to detect glucagon under various physiological conditions. The most sensitive tests had a sensitivity of approximately 10 pmol / l (35 pg / ml), but the results around this concentration were not reliable. According to the authors of a study comparing glucagon tests, tests with a sensitivity of> 5 pmol / l are unsuitable for complete characterization of glucagon secretion. In addition, some tests had poor plasma and / or buffer recovery,
Mercodia glucagon assays for human (10-1271-01) and animal samples (10-1281-01) have a sensitivity of 1.5 pmol / l (5 pg / ml) and 2 pmol / l (7 pg / ml). The sensitivity of these tests, along with a wide dynamic range, allows scientists to measure physiologically relevant glucagon concentrations in various experimental paradigms.
Overcoming sample volume limitations
Sample volume requirements also significantly limited the use of glucagon assays. Most commercially available methods require at least 50-100 μl of plasma. This has direct implications for the amount of analytes that can be measured, the number of time points that can be examined, and thus the range of conclusions.
Mercodia ELISA tests for glucagon require only 25 μl of sample for human samples and 10 μl for animal samples, thus providing a significant advantage and allowing scientists to measure glucagon in various experimental models.
Implications for translation research
A “bench-to-bedside” approach is important for understanding physiological and pathophysiological processes, as well as for developing therapeutic strategies. Mercodia’s two ELISAs provide analytical solutions for studies that cover the spectrum of translational research based on extensive work done to characterize how these methods work with different types of samples. The glucagon sequence is highly conserved within species (Fig. 7), but validation of different sample types is essential because sequence homology is not the only factor that affects antibody-antigen binding in the immunoassay. For example, matrix interferences (common in animal samples) can lead to falsely elevated or falsely low concentrations. Mercodia tests contain a unique blocking solution that prevents or minimizes matrix interference,
The 10 μl Mercodia Glucagon ELISA has been validated using mouse, rat, pig and primate samples (data available in the instructions for use), making it an excellent choice for various small and large preclinical animal studies. Both glucagon assays can be used with samples from cultured cells, providing accurate glucagon measurement capabilities for in vitro researchers. (eg conversion of alpha to beta cells and vice versa, human embryonic stem cells to alpha or beta cells, production of islet-like clusters).
In addition to the already mentioned study of various glucagon tests , the Mercodia test was included in another study of three new available glucagon tests, which concluded that the Mercodia test has the best results in terms of specificity, accuracy and sensitivity. The test results are reproducible and the test has been identified as suitable for measuring glucagon concentration.
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