Use of a fucose content detection reagent for c1 esterase inhibitor in the preparation of a product for diagnosing type 2 hereditary angioedema

Abstract

The present application relates to the technical field of biomarkers, and particularly relates to application of a fucose content detection reagent of C1 esterase inhibitor in preparation of a type 2 hereditary angioedema diagnosis product.The present application finds that the fucosylation level of C1 esterase inhibitor has significant correlation with type 2 hereditary angioedema, and can be used for diagnosis of type 2 hereditary angioedema; the fucosylation level of C1 esterase inhibitor has good diagnosis performance for type 2 hereditary angioedema through ROC curve verification, and has advantages of convenient detection, short required time and the like, and can be used alone or in combination with other markers for diagnosis of type 2 hereditary angioedema, and has good application prospect in diagnosis of type 2 hereditary angioedema.

Description

Technical Field

[0001] This invention relates to the field of biomarker technology, and in particular to the application of a fucose content detection reagent for C1 esterase inhibitors in the preparation of diagnostic products for type 2 hereditary angioedema. Background Technology

[0002] Hereditary angioedema (HAE) is a rare genetic disorder with an incidence of approximately 1.1-1.6 per 100,000. It is characterized by acute, recurrent episodes of subcutaneous and / or submucosal edema, which can affect the face, extremities, upper respiratory tract, and gastrointestinal tract, and in severe cases, can be life-threatening. HAE is classified into HAE type 1 (HAE-1) and HAE type 2 (HAE-2), accounting for approximately 85% and 15% of all HAE cases, respectively. Currently, diagnosis of HAE requires a combination of family history, clinical manifestations, and laboratory tests. Due to the rarity of HAE, clinical heterogeneity, and the lack of existing diagnostic markers, patients are easily misdiagnosed and mistreated.

[0003] Laboratory tests for HAE primarily focus on C4 concentration and the concentration and function of C1 esterase inhibitor (C1-INH). However, C4 concentration has poor specificity, as it can be decreased in many diseases, thus serving only as a supplementary reference indicator. C1-INH concentration and function are specific indicators for diagnosing HAE-2. Since there is no significant difference in C1-INH concentration between HAE-2 patients and non-HAE edema patients, functional testing of C1-INH is essential for diagnosing HAE-2. However, there are currently no commercially available C1-INH functional testing products or kits in China. C1-INH is also unstable and easily degraded, making its functional testing extremely demanding in terms of sample collection, preparation, transportation, and preservation, hindering its widespread clinical application. Therefore, there is an urgent need to develop highly specific and convenient diagnostic biomarkers for HAE-2.

[0004] Protein glycosylation is a common post-translational modification of proteins, referring to the process by which glycosyl groups (glycans) are covalently linked to the amino acid side chains of proteins, forming glycoproteins. It significantly affects protein structure and function. Protein glycosylation can be classified into N-glycosylation, O-glycosylation, C-mannosylation, and glycosylphosphatidylinositol anchoring linkages, with more than half of glycoproteins being N-glycosylated. The development of glycomics technology has made in-depth research on the glycan structure of proteins possible, and glycan markers of proteins have shown remarkable capabilities in disease diagnosis, progression prediction, and prognosis assessment. Studies have found that changes in the N-glycan profile in plasma are associated with the occurrence and development of various diseases, such as lung cancer, colorectal cancer, Alzheimer's disease, type 2 diabetes, and cardiovascular diseases.

[0005] C1-INH is a highly glycosylated protein primarily synthesized in the liver and secreted into the plasma. It contains 500 amino acids, including a 22-amino acid signal peptide, a 112-amino acid N-terminal domain (NTD), and a 366-amino acid C-terminal domain (CTD), also known as the serine protease inhibitor (SERPIN) domain. The SERPIN domain is highly conserved, consisting of nine α-helices, three β-sheets, and a reaction center loop (RCL). Upon binding to target enzymes (such as C1r, C1s, MASP-1 / 2, plasma calcium kinase, or factor XIIa), C1-INH undergoes a conformational change, forming a covalent complex that irreversibly inhibits protease activity, preventing overactivation of downstream inflammatory pathways. Glycosylation is crucial for maintaining protein stability, solubility, plasma half-life, and interaction with target enzymes. C1-INH contains seven N-linked glycosylation sites, and N-glycosylation of its domains may play a crucial role in stabilizing the C1-INH protein, aiding in protein folding and function. However, there are currently no studies on the correlation between C1-INH fucosylation and HAE-2, or its diagnostic applications. Summary of the Invention

[0006] This invention provides the application of a fucose content detection reagent for C1 esterase inhibitors in the preparation of diagnostic products for type 2 hereditary angioedema.

[0007] This invention analyzes the differences in C1-INH protein fucosylation among HAE-2 patients, mast cell-mediated edema patients, and healthy individuals. The study found significant differences in C1-INH protein fucosylation levels between HAE-2 patients and mast cell-mediated edema patients and healthy individuals. Validation has shown that C1-INH protein fucosylation has good performance in the diagnosis of HAE-2 and can serve as a potential biomarker for HAE-2 diagnosis.

[0008] Specifically, the present invention provides the following technical solutions.

[0009] In a first aspect, the present invention provides the application of a fucose content detection reagent for C1 esterase inhibitors in the preparation of reagents for the diagnosis of type 2 hereditary angioedema.

[0010] Although existing technologies disclose changes in the characteristics of several fucose-containing glycans or derivatives such as MM, CA2, and CA3 in the total glucose / total serum glycosides of HAE plasma, it is not possible to directly infer from this that the fucosylation level of a specific protein, especially the C1 esterase inhibitor (C1-INH), will necessarily undergo the same or corresponding changes. The main reason for this is that: (1) The total plasma glycosides are essentially a mixed signal formed by multiple glycoproteins, multiple glycosylation sites, and proteins from different tissue sources. Existing research (Clerc F, Reiding KR, Jansen BC, Kammeijer GS, Bondt A, Wuhrer M. Human plasma protein N-glycosylation. Glycoconj J. 2016 Jun;33(3):309-43. doi: 10.1007 / s10719-015-9626-2. Epub 2015 Nov 10. PMID: 26555091; PMCID:PMC4891372.) clearly indicates that when analyzing the total plasma N-glycosylation at the glycan release level, the observed changes cannot directly determine whether they are due to changes in the relative abundance of proteins, changes in the glycoform composition of a specific protein, or the joint regulation of multiple glycoproteins. Another study (Ruhaak LR, Koeleman CA, Uh HW, Stam JC, van Heemst D, MaierAB, Houwing-Duistermaat JJ, Hensbergen PJ, Slagboom PE, Deelder AM, Wuhrer M. Targeted biomarker discovery by high throughput glycosylation profiling of human plasma alpha1-antitrypsin and immunoglobulin A. PLoS One. 2013 Sep 9;8(9):e73082. doi: 10.1371 / journal.pone.0073082. PMID: 24039863; PMCID:PMC3767703.) further points out that since the total glucose profile of plasma is derived from the total protein pool in plasma, its changes may originate from changes in protein concentration or from changes in protein-specific glycosylation patterns; at the same time, glycans of high-abundance proteins will dominate the overall glucose profile, while glycosylation changes of low-abundance proteins may not even be detected at the total glucose level.Other studies (Klein A, Carre Y, Louvet A, Michalski JC, Morelle W. Immunoglobulinsare the major glycoproteins involved in the modifications of total serum N-glycome in cirrhotic patients. Proteomics Clin Appl. 2010 Apr;4(4):379-93. doi: 10.1002 / prca.200900133. Epub 2010 Feb 3. PMID: 21137058.) have shown that in some diseases, the major changes in total serum N-glycome are actually mainly caused by a subset of proteins, rather than all serum glycoproteins changing synchronously. (2) Glycosylation is tissue / protein origin specific, and it cannot be assumed that proteins from different origins change synchronously. Previous studies (Sharapov, S., Timoshchuk, A., Zaytseva, O. et al. A genome-wide association study in 10,000 individuals links plasma N-glycome to liver disease and anti-inflammatory proteins. Nat Commun 16, 5525, 2025.) have shown that the genetic regulation of plasma protein glycosylation has obvious tissue specificity. Even within the same glycosyltransferase-related pathway, the regulatory mechanisms on proteins from different tissue sources may differ. Therefore, changes in total plasma glucose alone cannot predict the glycosylation changes of a specific protein. (3) In specific diseases, the results of total plasma protein N-glycosequence and specific protein N-glycosequence in plasma are often inconsistent, and the former cannot directly predict the latter. Several disease studies have shown that under the same pathological condition, total plasma glucose and specific protein glucose do not follow the same pattern, and their difference profiles, significance results, and directions of change can be significantly different.For example, in acute systemic inflammation, studies have clearly shown (Novokmet M, Lukić E, Vučković F, Ðurić Ž, Keser T, RajšlK, Remondini D, Castellani G, Gašparović H, Gornik O, Lauc G. Changes in IgG and total plasma protein glycomes in acute systemic inflammation. Sci Rep. 2014 Mar 11;4:4347. doi: 10.1038 / srep04347. PMID: 24614541; PMCID:PMC3949295.) that changes in total plasma glycosides are simultaneously affected by changes in glycosylation and changes in the concentration of different plasma glycoproteins, thus making it impossible to distinguish between the two; and unlike total plasma glycosides, changes in the glycosides of specific proteins IgG do not follow a common pattern. For example, in a study on atrial fibrillation (Plavša B, Szavits-Nossan J, Blivajs A, Rapčan B, Radovani B, Šesto I, Štambuk K, Mustapić V, Đerek L, Rudan D, Lauc G, Gudelj I. The N-Glycosylation of Total Plasma Proteins and IgG in Atrial Fibrillation. Biomolecules. 2023 Mar 28;13(4):605. doi: 10.3390 / biom13040605. PMID:37189353; PMCID: PMC10135591.), it was found that a certain clinical score was not significantly associated with total plasma protein N-glycans, but was significantly associated with several IgG N-glycan characteristics. It is worth noting that immunoglobulins are important contributing proteins to the total serum / total plasma glucose group. Even so, in existing studies, glycosylation results can still show different patterns from total plasma glycosylation. This indicates that in the same disease, glycosylation results at the total plasma level and at the level of a specific protein can be significantly inconsistent; the two cannot be substituted for each other, nor can the former directly predict the latter. Therefore, changes in total plasma glucose cannot predict changes in the fucosylation of the specific protein C1INH.

[0011] Secondly, the present invention provides the application of fucose content detection reagents and / or detection devices for C1 esterase inhibitors in the preparation of systems for the diagnosis of type 2 hereditary angioedema.

[0012] Preferably, the C1 esterase inhibitor is a plasma C1 esterase inhibitor.

[0013] The detection reagents mentioned above include ELISA detection reagents.

[0014] Preferably, the ELISA detection reagent includes a lectin for specifically binding fucose and one or more selected from the following: ELISA plate, washing buffer, blocking solution, horseradish peroxidase-labeled streptavidin, TMB chromogenic solution, and stop solution.

[0015] The lectin used for specific binding of fucose is preferably *Dictyophora indica* lectin. The blocking solution is preferably a 2%-4% BSA solution. The washing buffer is preferably PBS buffer and / or PBST buffer.

[0016] The detection reagents described above also include reagents for purifying C1 esterase inhibitors.

[0017] The reagents used for purifying C1 esterase inhibitors include C1-INH protein-adsorbing magnetic beads.

[0018] Thirdly, the present invention provides a product for diagnosing type 2 hereditary angioedema, the product comprising reagents and / or devices for detecting the fucose content of a C1 esterase inhibitor.

[0019] The reagents used to detect the fucose content of C1 esterase inhibitors include ELISA detection reagents.

[0020] Preferably, the ELISA detection reagent includes a lectin for specifically binding fucose and one or more selected from the enzyme-labeled plate, washing buffer, blocking solution, SA-HRP, TMB chromogenic solution, and stop solution.

[0021] The lectin used for specific binding of fucose is preferably *Dictyophora indica* lectin. The blocking solution is preferably a 2%-4% BSA solution. The washing buffer is preferably PBS buffer and / or PBST buffer.

[0022] Fourthly, the present invention provides a system for diagnosing type 2 hereditary angioedema, the system comprising: The detection module is used to detect the fucose content of plasma C1 esterase inhibitors; The input module is used to obtain the detection results from the detection module; The analysis module is used to compare the detection results obtained by the input module with the reference sample, and to diagnose type 2 hereditary angioedema based on the comparison results; The output module is used to output the diagnostic results.

[0023] In the above system, the ELISA method is preferably used to detect the fucose content of plasma C1 esterase inhibitors. More preferably, the fucose content of C1 esterase inhibitors is detected using a lectin that specifically binds to fucose.

[0024] The reagents used in the ELISA method include a lectin for specifically binding fucose and one or more selected from the following: ELISA plate, wash buffer, blocking solution, horseradish peroxidase-labeled streptavidin, TMB chromogenic solution, and stop solution. The lectin for specifically binding fucose is preferably *Dictyophora indica* lectin. The blocking solution is preferably a 2%-4% BSA solution. The wash buffer is preferably PBS buffer and / or PBST buffer.

[0025] In the above system, the reference samples are preferably healthy human samples and mast cell-mediated edema patient samples. The fucose content of the plasma C1 esterase inhibitor in the test sample is compared with the fucose content of the plasma C1 esterase inhibitor in the healthy human samples and mast cell-mediated edema patient samples to determine whether the test sample has type 2 hereditary angioedema or its risk of developing the disease.

[0026] In this invention, the diagnosis of type 2 hereditary angioedema is used to differentiate between type 2 hereditary angioedema and healthy individuals, or to differentiate between type 2 hereditary angioedema and mast cell-mediated edema.

[0027] In this invention, the C1 esterase inhibitor is preferably a plasma C1 esterase inhibitor.

[0028] The beneficial effects of this invention include at least the following: This invention discovers that the fucosylation level of C1 esterase inhibitors is significantly correlated with type 2 hereditary angioedema, and can be used to diagnose type 2 hereditary angioedema. ROC curve verification shows that the fucosylation level of C1 esterase inhibitors has good diagnostic performance for type 2 hereditary angioedema, and has advantages such as convenient detection and short detection time. It can be used alone or in combination with other markers for the diagnosis of type 2 hereditary angioedema, and has good application prospects in the diagnosis of type 2 hereditary angioedema. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0030] Figure 1This refers to the ELISA results of fucose content in plasma C1-INH protein in patients with type 2 hereditary angioedema, mast cell-mediated edema, and healthy controls in the discovery cohort of Example 1 of this invention; wherein, P < 0.05, ns represents no significant difference; HAE-2 represents the fucose content of plasma C1-INH protein in HAE-2 patients, MC-AE represents the fucose content of plasma C1-INH protein in mast cell-mediated edema patients, and HC represents the fucose content of plasma C1-INH protein in healthy control samples.

[0031] Figure 2 In the discovery cohort of Example 1 of this invention, the fucose content of C1-INH protein was used for the ROC curve of type 2 hereditary angioedema diagnosis.

[0032] Figure 3 This refers to the ELISA results of fucose content in plasma C1-INH protein in patients with type 2 hereditary angioedema, mast cell-mediated edema, and healthy controls in the validation cohort of Example 2 of the present invention; wherein, This means P < 0.01. P < 0.001, ns represents no significant difference; HAE-2 represents the fucose content of plasma C1-INH protein in HAE-2 patients, MC-AE represents the fucose content of plasma C1-INH protein in mast cell-mediated edema patients, and HC represents the fucose content of plasma C1-INH protein in healthy control samples.

[0033] Figure 4 In the validation cohort of Example 2 of this invention, the fucose content of C1-INH protein was used for the ROC curve of type 2 hereditary angioedema diagnosis. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0035] The samples used in the following examples were plasma samples. The inclusion criteria for patients with hereditary angioedema type 2 (HAE-2) were: (1) a history of recurrent skin swelling involving the limbs, face and external genitalia, accompanied by gastrointestinal symptoms and / or laryngeal edema. (2) multiple test results showed that the C1 inhibitor (C1-INH) antigen level was within the normal range or elevated, and the C1 inhibitor function was less than 50% of the normal value, while the C4 level was reduced. (3) no significant effect from antihistamines, glucocorticoids, omalizumab or adrenaline; and / or (4) a mutation in the SERPING1 gene encoding the C1 inhibitor (C1-INH) was confirmed by gene testing. The inclusion criteria for patients with mast cell-mediated edema (MC-AE) were: (1) recurrent angioedema symptoms that could resolve spontaneously. (2) a clear therapeutic response to antihistamines, glucocorticoids, adrenaline or omalizumab. (3) After comprehensive laboratory examination, the possibility of bradykinin-mediated edema, drug-induced edema, and other non-mast cell-related edema was ruled out. The inclusion criteria for healthy individuals (HC) were: no history of angioedema, no history of allergic diseases, no other autoimmune diseases, and no cardiovascular diseases, liver diseases, tumors, or inflammatory diseases, and no chronic diseases requiring long-term treatment. The exclusion criteria for each group were any comorbidities or previous treatment history that might interfere with the study evaluation, in addition to the conditions explicitly listed in the inclusion criteria for each group.

[0036] Example 1: Screening for N-glycan markers of HAE-2-related plasma C1-INH protein In the process of detecting the plasma C1-INH protein glycosylation in patients with hereditary angioedema type 2 (HAE-2), mast cell-mediated edema (MC-AE), and healthy individuals (HC) using mass spectrometry, it was found that the fucose modification level of C1-INH in HAE-2 patients was significantly different compared with MC-AE and HC. Based on the above findings, the diagnostic efficacy of plasma C1-INH protein fucose as a marker for HAE-2 was detected by ELISA. The sample information (discovery cohort) used is shown in Table 1. HAE is a rare disease. In China, HAE-1 accounts for 98.73% of HAE patients, while HAE-2 accounts for only 1.27% (Zhi Yuxiang, An Lixin, Lai He, et al. Expert consensus on the diagnosis and treatment of hereditary angioedema [J]. Chinese Journal of Clinical Immunology and Allergy, 2019, 13(1):4). Therefore, the number of clinical cases of HAE-2 is extremely limited. For such a rare disease, the sample population used in this invention is representative.

[0037] Table 1. Baseline Population Information for the Discovery Cohort

[0038] The fucosylation level of plasma C1-INH protein in the discovery cohort was detected using the following method: 1. Extraction of C1-INH protein from clinical plasma samples The plasma samples were thawed and centrifuged at 1,500 g for 10 minutes. The samples were then diluted with PBS and filtered through a 20 µm 96-well plate containing C1-INH protein-adsorbing magnetic beads. The samples were then washed with PBS to remove unbound protein. C1-INH protein was eluted from the filter plate into the 96-well plate using 0.1 M formic acid (pH 2.5). The solution was neutralized with 1 M ammonium bicarbonate (pH 7.8). The solution was concentrated by centrifugation at 4 °C for 4 hours. The protein stock solution was diluted with 30 µL of PBS buffer. 20 µL of the protein stock solution was used for BCA quantification. The protein stock solution was then diluted to a 0.5 µg / mL system using protein coating buffer.

[0039] 2. The fucose content of human plasma C1 esterase inhibitors was detected using an ELISA diagnostic kit. (1) Take an ELISA plate and add 100 μL of the 0.5 µg / mL protein stock solution diluted with protein coating buffer to each well of the ELISA plate. Coat at 4°C for 20 hours. (2) Remove the liquid from the microplate, add 285 μL of PBS buffer to each well of the microplate, and shake to wash 3 times, 1 min each time; (3) Add 100 μL of 3% BSA solution to each well, block at 37°C for 1 h, then add 285 μL of PBST buffer and shake to wash 3 times. (4) Add 100 μL of diluted *Dictyophora indica* lectin (final concentration 5 µg / mL) to each well, incubate at room temperature for 2 h, then add 285 μL of PBST buffer and shake to wash 5 times. (5) Add 100 μL of horseradish peroxidase-labeled streptavidin (SA-HRP) diluted 1:2500 to each well, incubate at 37°C for 30 min, then add 285 μL of PBST buffer and shake to wash 5 times. (6) Under light-protected conditions, add TMB colorimetric solution and incubate for 15 min; (7) Add 50 μL of stop solution to each well to terminate the reaction, and measure the OD within 15 min. 450nm .

[0040] The ELISA test results for each group are as follows: Figure 1As shown in the figure. The results indicated that the fucosylation level (i.e., fucose content) of plasma C1-INH protein was significantly different between HAE-2 and MC-AE and HC (the fucosylation level in the HAE-2 group was significantly higher than that in MC-AE and HC), and the fucosylation level of plasma C1-INH protein could serve as a potential diagnostic biomarker for HAE-2.

[0041] Furthermore, to obtain a potentially applicable diagnostic biomarker for HAE-2, the diagnostic efficacy of plasma C1-INH protein fucosylation levels was assessed using ROC curves in the discovery cohort. The results showed (Table 2 and...). Figure 2 With an AUC of 0.743, and at a diagnostic threshold of ≥70% specificity, the specificity and sensitivity were 0.786 and 0.583, respectively, indicating that it has good diagnostic potential for HAE-2.

[0042] Table 2. Variables of the test results in the region below the curve

[0043] Based on the above results, this invention has achieved novel biomarker screening and identification at the level of the specific protein C1-INH. The results show that although changes in total plasma glucose (MM), CA2, and CA3 are observed at the level of total plasma glucose in HAE-2 previously reported by the applicant, at the level of the specific protein C1INH, the differences that are retained and can serve as biomarkers are not all changes in total plasma glucose, but only CF (core fucose). This further demonstrates that changes in total plasma glucose cannot be directly extrapolated to changes in glucose in a specific protein, nor can it be predicted in advance which glucose characteristic will be retained in which protein and ultimately form a usable biomarker.

[0044] Example 2: Application of fucose from plasma C1-INH protein as a diagnostic biomarker for HAE-2 To further validate the diagnostic efficacy of fucose from plasma C1-INH protein as a marker for HAE-2, HAE-2, MC-AE, and HC samples were collected as a validation cohort (the samples in the validation cohort were completely different from those in the discovery cohort). Information on the validation cohort is shown in Table 3.

[0045] Table 3. Verification Cohort Population Baseline Information

[0046] The fucosylation level of plasma C1-INH protein in the validation cohort was detected using the following method: 1. Extraction of C1-INH protein from clinical plasma samples The plasma samples were thawed and centrifuged at 1,500 g for 10 minutes. The samples were then diluted with PBS and filtered through a 20 µm 96-well plate containing C1-INH protein-adsorbing magnetic beads. The samples were then washed with PBS to remove unbound protein. C1-INH protein was eluted from the filter plate into the 96-well plate using 0.1 M formic acid (pH 2.5). The solution was neutralized with 1 M ammonium bicarbonate (pH 7.8). The solution was concentrated by centrifugation at 4 °C for 4 hours. The protein stock solution was diluted with 30 µL of PBS buffer. 20 µL of the protein stock solution was used for BCA quantification. The protein stock solution was then diluted to a 0.5 µg / mL system using protein coating buffer.

[0047] 2. The fucose content of C1-INH in human plasma was detected using an ELISA diagnostic kit. (1) Take an ELISA plate and add 100 μL of the 0.5 µg / mL protein stock solution diluted with protein coating buffer to each well of the ELISA plate. Coat at 4°C for 20 hours. (2) Remove the liquid from the microplate, add 285 μL of PBS buffer to each well of the microplate, and shake to wash 3 times, 1 min each time; (3) Add 100 μL of 3% BSA solution to each well, block at 37°C for 1 h, then add 285 μL of PBST buffer and shake to wash 3 times. (4) Add 100 μL of diluted *Dictyophora indica* lectin (final concentration 5 µg / mL) to each well, incubate at room temperature for 2 h, then add 285 μL of PBST buffer and shake to wash 5 times. (5) Add 100 μL of SA-HRP diluted 1:2500 to each well, incubate at 37°C for 30 min, then add 285 μL of PBST buffer and shake to wash 5 times. (6) Under light-protected conditions, add TMB colorimetric solution and incubate for 15 min; (7) Add 50 μL of stop solution to each well to terminate the reaction, and measure the OD within 15 min. 450nm .

[0048] The ELISA test results of each group of samples are as follows: Figure 3 As shown in the figure. The results showed that, in the validation cohort, there were significant differences in the fucosylation level (i.e., fucose content) of plasma C1-INH protein between HAE-2 and MC-AE and HC (the fucosylation level in the HAE-2 group was significantly higher than that in MC-AE and HC), indicating that the fucosylation level of plasma C1-INH protein can serve as a diagnostic biomarker for HAE-2.

[0049] Furthermore, in the validation cohort, the diagnostic efficacy of plasma C1-INH protein fucosylation levels was evaluated using ROC curves, and the results showed (Table 4 and...). Figure 4 The AUC was 0.813, and the specificity and sensitivity were 0.893 and 0.583, respectively, at a diagnostic threshold of ≥70% specificity, indicating that the fucosylation level of plasma C1-INH protein has good diagnostic efficacy for HAE-2.

[0050] Table 4. Variables of the test results in the region below the curve

[0051] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of fucose content detection reagent for C1 esterase inhibitor in the preparation of reagents for the diagnosis of type 2 hereditary angioedema.

2. Application of fucose content detection reagents and / or detection equipment for C1 esterase inhibitors in the preparation of systems for the diagnosis of type 2 hereditary angioedema.

3. The application according to claim 1 or 2, characterized in that, The C1 esterase inhibitor is a plasma C1 esterase inhibitor.

4. The application according to claim 1 or 2, characterized in that, The testing reagents include ELISA testing reagents.

5. The application according to claim 4, characterized in that, The ELISA assay reagent includes a lectin for specifically binding fucose and one or more selected from the following: microplate, washing buffer, blocking solution, horseradish peroxidase-labeled streptavidin, TMB chromogenic solution, and stop solution.

6. The application according to claim 4, characterized in that, The detection reagent also includes reagents for purifying C1 esterase inhibitors.

7. A product for diagnosing type 2 hereditary angioedema, characterized in that, The product includes reagents and / or equipment for detecting the fucose content of C1 esterase inhibitors.

8. The product for diagnosing type 2 hereditary angioedema according to claim 7, characterized in that, The reagents used to detect the fucose content of C1 esterase inhibitors include ELISA assay reagents.

9. The product for diagnosing type 2 hereditary angioedema according to claim 8, characterized in that, The ELISA assay reagent includes a lectin for specifically binding fucose and one or more selected from the following: microplate, washing buffer, blocking solution, horseradish peroxidase-labeled streptavidin, TMB chromogenic solution, and stop solution.

10. A system for diagnosing type 2 hereditary angioedema, characterized in that, The system includes: The detection module is used to detect the fucose content of plasma C1 esterase inhibitors; The input module is used to obtain the detection results from the detection module; The analysis module is used to compare the detection results obtained by the input module with the reference sample, and to diagnose type 2 hereditary angioedema based on the comparison results; The output module is used to output the diagnostic results.

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