Our results demonstrated that the sandwich ELISA method described here is capable of measuring MrgX2 concentration in human whole blood. Currently, no effective clinical detection methods exist for MrgX2.
Our method has the inherent advantage of the dual antibody sandwich detection . In particular, the response intensity is directly related to an increase in MrgX2 concentration. Since two specific antibodies against MrgX2 protein are used, the detection results are accurate and reliable . From a practical viewpoint, ELISA can be performed in clinical laboratories and test results can be obtained within three hours without the need for complex equipment or highly specialized operator expertise. Another advantage of our assay is its limit of quantification at 3.125 ng/mL. This is especially important, since we observed that whole blood MrgX2 concentrations of healthy volunteers were < 10 ng/mL.
From a practical standpoint, the advantage of an ELISA over an LC-MS is that ELISA can be performed in clinical laboratories that do not have the complex equipment or the highly specialized operator expertise required to perform LC-MS type assays. In addition, unlike LC-MS, ELISA also has the potential for higher throughput and therefore provides the basis for first dual antibody sandwich immunoassay to measure MrgX2. Our results indicate that we have successfully established a dual-antibody sandwich ELISA detection method with high specificity, accuracy, reproducibility and sufficient sensitivity that can be used for detection MrgX2 in human whole blood.
Mas-related G protein‐coupled receptor‐X2 (MrgX2), expressed in mast cells, is an endogenous receptor associated with IgE‐independent activation of mast cells. MrgX2 has specific characteristics that induces degranulation of mast cells and regulates inflammatory responses [2, 12, 13]. It is well known that while mast cells are located around tissues, and leukocytes are distributed in peripheral blood , their common feature is the release of allergic mediators such as histamine through the degranulation pathway. These are the key effector cells that trigger IgE-mediated type Ⅰ allergic reactions . Mast cells and basophils are derived from bone marrow differentiation and have similar biological characteristics . For monitoring allergic diseases, blood basophils can reflect the situation in the body as comprehensively as possible . The sandwich ELISA method described here can be used clinically to further increase our understanding of the role of MrgX2 in regulating chronic urticaria.
This high-throughput method is particularly important for clinical trials to determine the concentration of MrgX2 protein in human whole blood. Based on the frequency distribution data and ROC curves of 75 healthy individuals, we determined the initial truncation value of MrgX2 to be 60.91 ng/mL (95% confidence interval). Using the established ELISA kit, the human whole blood MrgX2 concentrations were found to be higher in CU patients than in healthy controls. The results were similar in the skin MCs that express MrgX2 at higher levels in CU patients than in healthy controls . Furthermore, there was no significant difference in the MrgX2 protein expression in male and female CU patients. However, it should be noted that owing to the limited number of patients in our study, data obtained from CU patients must be interpreted with caution.
In addition to these observations, the dual antibody sandwich ELISA has several other uses. Allergic asthma, the most common phenotype of asthma, is clinically defined by the presence of allergic sensitization and a correlation between asthma symptoms and allergen exposure [18, 19]. MrgX2 may promote the development of asthma and may serve as a potential new target for regulating this chronic inflammatory disease . MrgX2 may also be as a potential biomarker for predicting treatment outcomes in allergic asthma . Mast cells are important effector cells that orchestrate the development of airway hyperresponsiveness and inflammation in asthma via their close interaction with smooth muscle cells, T cells and leukocytes in the airway [22–24]. For asthma patients, the ELISA test results may indicate whether MrgX2 levels are correlated with the disease and provide richer clinical data for clinical diagnosis and treatment. MrgX2 receptors also play an important role in pruritus and erythema-related inflammatory disorders. Our ELISA kit can be used to determine the human whole blood MrgX2 concentrations to better guide the treatment of other MrgX2 related chronic inflammatory diseases.
In summary, our MrgX2 sandwich ELISA test can help improve our understanding of the role of MrgX2 in regulating chronic urticaria. The use of the two antibodies in the sandwich format provides specificity for the active form of the protein, with a limit of quantification of 3.125 ng/mL, and a broad dynamic range for the clinical detection of MrgX2 related chronic urticaria.