Anti-human laminin gamma 1 IgA autoantibody detection ELISA kit and its application

Abstract

The application discloses an ELISA kit for detecting anti-human laminin gamma 1 IgA autoantibody and application thereof. The kit comprises an enzyme-labeled plate coated with human laminin gamma 1 recombinant protein. Preferably, the human laminin gamma 1 recombinant protein is deglycosylated human laminin gamma 1 recombinant protein. Further, the application also provides application of the kit in preparation of a reagent for diagnosing mucous membrane pemphigoid, in particular Laminin gamma 1 IgA type pemphigoid. The kit is the first kit for detecting human laminin gamma 1 IgA antibody by using the ELISA method, has the advantages of simple operation, short time consumption and low cost compared with traditional immunoblotting method, and adopts full-length deglycosylated laminin gamma 1 recombinant protein as an antigen, thereby solving the problem of incomplete antigen epitope coverage in the prior art, and early detection of diseases can be realized, early treatment is achieved, and the life quality of patients is improved.

Description

Technical Field

[0001] This invention relates to a diagnostic method and kit for mucosal pemphigoid, and more particularly to a method and kit for diagnosing human laminingamma 1 IgA type pemphigoid. This invention belongs to the field of pharmaceutical technology. Background Technology

[0002] Mucous membrane pemphigoid (MMP) is a subtype of pemphigoid, a group of autoimmune bullous diseases that primarily affect mucous membranes, with some types also involving the skin. The pathogenesis involves the production of autoantibodies against components of the basement membrane, disrupting the epithelial-connective tissue junctions, leading to the formation of vesicles, erosions, and ulcers. It is more common in middle-aged and elderly people, with slightly more women than men. Typical symptoms include recurrent vesicles and erosions in the oral cavity, eyes, and other mucous membranes. The disease course is chronic and progressive, with a high recurrence rate. This subtype is currently considered difficult to diagnose and treat both internationally and domestically. Diagnosis of mucous membrane pemphigoid requires at least two criteria: first, clinical evidence of oral and / or ocular mucosal lesions; and second, detection of autoantibodies binding to or circulating basement membrane protein proteins in the patient's tissues using methods such as direct immunofluorescence, indirect immunofluorescence, Western blotting, or ELISA.

[0003] Currently known mucosal pemphigoid autoantibodies include BP180, BP230, laminin 332, integrinalpha6beta4, and collagen VII [Liu W, Li H, Jin Y, Cheng L, She L, Gao Y, Zhou Z, Feng S, Qian H, Hashimoto T, Li X. Case report: Mucous membrane pemphigoid with complicated autoantibody profile indicating the necessity of comprehensive diagnostic methods and the contribution of IgA autoantibodies. Front Immunol 2023; 14:1149119. Liu W, Sun X, Gao Y, Li H, Shi L, Cheng L, Zhou Z, Li X, Qian H. A Chinese case of concurrent anti-laminin γ1pemphigoid and anti-laminin 332-type mucous membrane pemphigoid. J Dermatol2023;50(2):e69-e71. ]. In 2022, the research group of the inventors first reported that laminin gamma 1 (laminin γ1) is also one of the autoantibodies of mucosal pemphigoid [Kuang w, Qian H, Zhang Q, Li W, Hashimoto T, Zeng X, Li X. Case report: Mucous membrane pemphigoid with IgG and IgA anti-laminin γ1 antibodies and IgA anti-laminin α5 antibodies. FrontImmunol 2022;13:903174.].Following this, the inventor's research group conducted a comprehensive screening for laminin gamma 1 autoantibodies using Western blotting on serum samples collected from Chinese patients with mucosal pemphigoid since 2015 and serum samples from Japanese collaborators with mucosal pemphigoid. The results revealed that some of these patients did indeed possess laminin gamma 1 autoantibodies, particularly IgA antibodies. These results further confirm that laminin gamma 1 is one of the autoantibodies in mucosal pemphigoid. However, this also raises a serious issue: the need to further develop an easily applicable and widely disseminated method for detecting laminin gamma 1 IgA autoantibodies, such as ELISA, for clinical diagnosis.

[0004] p200 pemphigoid primarily affects the skin, commonly occurring on the trunk and flexor surfaces of the limbs. The lesions are tense bullae, leaving pigmentation after healing. Autoantibodies are predominantly IgG type, and the condition responds well to glucocorticoids. In contrast, laminin gamma 1 IgA (laminin γ1 IgA) pemphigoid mainly affects mucous membranes, especially the oral cavity, eyes, and genitals, and is prone to scarring. Autoantibodies are predominantly IgA type, making treatment more difficult and often requiring combined immunosuppressant therapy, with a relatively poorer prognosis. Patent application CN118393148A discloses an ELISA kit for detecting human lamininγ1 IgG antibodies and its application. This ELISA method mainly uses a mixture of lamininγ1 peptides as an antigen to detect lamininγ1 IgG antibodies in p200 pemphigoid. We first optimized the method to detect lamininγ1 IgA antibodies in the serum of patients with mucosal pemphigoid. The results showed that IgA autoantibodies could be detected in the serum of less than 30% of patients, indicating that the currently designed lamininγ1 peptide epitopes are insufficient to cover the epitopes recognized by IgA antibodies in the serum of patients with mucosal pemphigoid.

[0005] Dainichi T et al. disclosed an immunoblotting method for detecting laminin gamma 1, using human dermal extract as the antigen [Dainichi T, Kurono S, Ohyama B, Ishii N, Sanzen N, Hayashi M, Shimono C, Taniguchi Y, Koga H, Karashima T, Yasumoto S, Zillikens D, Sekiguchi K, Hashimoto T. Anti-laminin gamma-1 pemphigoid. Proc Natl Acad SciU SA 2009;106(8):2800-5.]. Since 2014, our research group has first used recombinant laminin gamma1 trimer proteins, such as laminin 521, as antigens in immunoblotting experiments to detect laminin gamma1 autoantibodies [Li X, Qian H, Ishii N, Yamaya M, Fukuda H, Mukai H, Hirako Y, Hashimoto T. A case of concurrent anti-laminin γ1 pemphigoid and anti-laminin 332-type mucous membrane pemphigoid. Br J Dermatol 2014;171(5):1257-9.]. However, immunoblotting methods are not easily promoted due to their high cost, complex operation, time consumption, difficulty in preparing epidermal extracts, and the need for extensive experience in result interpretation. Currently, there is no ELISA detection method for laminin gamma1 IgA autoantibodies.

[0006] In view of this, the present invention establishes an ELISA detection method using a full-length recombinant laminin gamma 1 protein containing all epitopes as an antigen to solve the problem of detecting IgA autoantibodies in the serum of patients with mucosal pemphigoid and diagnosing the disease. Summary of the Invention

[0007] The purpose of this invention is to provide a method for detecting anti-human laminin gamma 1 IgA autoantibodies and an ELISA kit to overcome the current deficiencies in the diagnosis of human laminin gamma 1 IgA type pemphigoid.

[0008] To achieve the above objectives, the present invention employs the following technical means:

[0009] The present invention discloses an ELISA kit for detecting anti-human laminin gamma 1 IgA autoantibody, wherein the kit comprises an enzyme-labeled plate coated with recombinant human laminin gamma 1 protein.

[0010] Preferably, the human laminin gamma 1 recombinant protein is a deglycosylated laminin gamma 1 recombinant protein.

[0011] Preferably, the amino acid sequence of the recombinant human laminin gamma 1 protein is shown in SEQ ID NO.2.

[0012] Preferably, the recombinant human laminin gamma 1 protein is obtained by transfecting a recombinant plasmid containing the DNA sequence encoding the recombinant laminin gamma 1 protein into CHO-S cells in suspension culture, followed by expression and purification.

[0013] Preferably, the kit further includes:

[0014] (1) Standard serum 1 (1×): 20 mM PBS containing 0.05% w / v NaN3, pH 7.4;

[0015] (2) Standard serum 2 (1×): 20 mM PBS containing 100 U / ml anti-human laminin gamma 1 IgA autoantibody and 0.05% w / v NaN3, pH 7.4;

[0016] (3) Enzyme-labeled antibody: Rabbit anti-human IgA antibody labeled with horseradish peroxidase;

[0017] (4) Reaction buffer (1×): 20 mM PBS containing 0.05% v / v Tween 20 and 0.05% w / v NaN3, pH 7.4;

[0018] (5) Washing buffer (10×): 200mM PBS containing 0.5% v / v Tween20, pH 7.4;

[0019] (6) Enzyme substrate solution: TMB colorimetric solution;

[0020] (7) Termination solution: 0.5N hydrochloric acid solution.

[0021] Preferably, when the kit is used to detect anti-human laminin gamma 1 IgA autoantibody, it includes the following steps:

[0022] (1) Reagent preparation

[0023] Before the experiment, place all test materials at room temperature and dilute an appropriate amount of washing buffer with distilled water at a volume ratio of 1:10 as needed.

[0024] (2) Sample preparation

[0025] Dilute the serum sample to be tested with reaction buffer at a volume ratio of 1:50 and mix well.

[0026] (3) Detection steps

[0027] 1) Add 100µl of diluted serum sample to the ELISA plate and incubate at room temperature for 60 minutes; add standard serum 1 and standard serum 2 as controls at the same time.

[0028] 2) Washing the microplate: Add 200 µl of diluted washing buffer to each well and wash 3 times;

[0029] 3) Add horseradish peroxidase-labeled rabbit anti-human IgA antibody at 100 µl / well and incubate at room temperature for 45 minutes;

[0030] 4) Washing the microplate: Add 200 µl of diluted washing buffer to each well and wash 3 times;

[0031] 5) Add enzyme substrate solution at 100 µl / well and incubate at room temperature for 15 minutes;

[0032] 6) Add stop solution at a rate of 100 µl / well;

[0033] 7) Read the absorbance;

[0034] 8) Result calculation and judgment:

[0035] Formula for calculating unit value (U / ml):

[0036] (A) 450 <Sample> - A 450 <Standard Serum 1>) × 100 / (A 450 <Standard Serum 2> - A 450 <Standard Serum 1>)

[0037] Judgment criteria: A value <15 is negative; a value ≥15 is positive.

[0038] Quality control:

[0039] Each test result must meet the following conditions, otherwise the result is invalid:

[0040] OD of standard serum 1 450 Less than or equal to 0.100;

[0041] OD of standard serum 2 450 Greater than or equal to 0.500.

[0042] The kit of this invention utilizes an ELISA method to qualitatively determine IgA antibodies against laminin gamma 1 in human serum. During detection, patient serum and standard serum are added to microwells coated with recombinant laminin gamma 1 protein. The IgA antibody against laminin gamma 1 binds to the antigen. After washing, unbound serum proteins are removed. Then, horseradish peroxidase-labeled anti-human IgA antibody is added to the microwells to bind with human IgA. After washing, horseradish peroxidase substrate is added to react with horseradish peroxidase. Finally, the enzyme reaction is terminated by adding acid solution. The absorbance is measured using an ELISA reader to quantify the detection results.

[0043] Furthermore, the present invention also proposes the application of the aforementioned anti-human laminin gamma 1 IgA autoantibody detection ELISA kit in the preparation of reagents for detecting anti-human laminin gamma 1 IgA autoantibodies.

[0044] And the application of the aforementioned anti-laminin gamma 1 IgA autoantibody detection ELISA kit in the preparation of reagents for diagnosing mucosal pemphigoid, preferably, the mucosal pemphigoid is laminin gamma 1 IgA type pemphigoid.

[0045] Compared with the prior art, the beneficial effects of the present invention are:

[0046] 1. This invention's kit is the first to use the ELISA method to detect human laminin gamma 1 IgA antibodies. Compared to the traditional immunoblotting method, this kit utilizes ELISA technology, offering advantages such as simple operation, short processing time, and low cost. Immunoblotting suffers from high cost, complex operation, long processing time, and strong reliance on experience in result interpretation, making it difficult to promote and apply in primary hospitals. In contrast, the standardized operating procedures and clear result interpretation criteria of this invention's ELISA kit are more suitable for large-scale clinical screening and routine diagnosis, facilitating its widespread adoption in medical institutions at all levels.

[0047] 2. This invention is the first to use full-length deglycosylated laminin gamma 1 recombinant protein as an antigen to establish an ELISA detection method, solving the problem of incomplete antigen epitope coverage in existing technologies. Previously disclosed ELISA kits for detecting human lamininγ1 IgG antibodies (CN118393148A) mainly target IgG antibodies and have limited detection rates. This invention, however, is specifically optimized for IgA antibodies, filling the gap in laminin gamma 1 IgA pemphigoid-specific detection kits.

[0048] 3. The enzyme-labeled plate of the kit of the present invention is coated with deglycosylated laminin gamma 1 recombinant protein. Using the deglycosylated full-length laminin gamma 1 recombinant protein as the coating protein of ELISA not only improves the specificity of IgA type autoantibody detection, but also improves the sensitivity of autoantibody recognition of laminin gamma 1 antigen, with a positive detection rate of up to 96%, which greatly improves the sensitivity of disease diagnosis.

[0049] 4. The kit of this invention showed negative results for other autoimmune bullous diseases (such as bullous pemphigoid and pemphigus vulgaris) and normal individual serum, with a specificity of 100%. This means that the kit can accurately distinguish Laminin gamma 1 IgA type pemphigoid from other similar diseases, effectively avoiding misdiagnosis and providing an accurate basis for clinical treatment.

[0050] 5. The kit of this invention has been verified through repeatability experiments. Six repeated tests on the same sample all showed a CV% value of less than 15%, indicating that the kit's test results have high consistency and stability. Furthermore, the kit establishes strict quality control standards (OD of standard serum 1). 450 ≤0.100, OD of standard serum 2 450 (≥0.500) to ensure the reliability of each test result and reduce experimental error.

[0051] 6. Laminin gamma 1 IgA pemphigoid is an autoimmune disease with a poor prognosis. Early diagnosis and intervention are crucial for improving patient outcomes. The high sensitivity and specificity of the kit in this invention enable early detection of the disease, helping clinicians to develop individualized treatment plans in a timely manner, avoiding or reducing serious complications such as scarring, and improving patients' quality of life. Attached Figure Description

[0052] Figure 1 SDS-PAGE analysis of purified laminin gamma 1 recombinant protein;

[0053] Where M: Protein Marker; R: Reducing; NR: Non-reducing;

[0054] Figure 2 Immunoblotting detection using recombinant laminin gamma 1 protein and deglycosylated recombinant protein as antigens;

[0055] Among them, 1 and 2: detection results of laminin gamma 1 monoclonal antibody, where 1 is recombinant laminin gamma 1 protein; 2 is deglycosylated recombinant laminin gamma 1 protein; 3 and 4: detection results of serum from patients with mucosal pemphigoid, where 3 is recombinant laminin gamma 1 protein; 4 is deglycosylated recombinant laminin gamma 1 protein. Detailed Implementation

[0056] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0057] Example 1: Preparation of deglycosylated human laminin gamma 1 recombinant protein

[0058] 1. Preparation of recombinant laminin gamma 1 protein

[0059] 1.1 Construction of laminin gamma 1 plasmid

[0060] The laminin gamma 1 DNA sequence was designed, optimized, and synthesized, as shown in SEQ ID NO.1. The synthesized DNA sequence was subcloned into the pcDNA3.4 vector. The obtained plasmids containing the laminin gamma 1 coding sequence were sequenced and identified. After successful identification, transfection-grade plasmids were prepared in large quantities for transfection of CHO-S (CHO Suspension) cells.

[0061] 1.2 Cell Culture and Plasmid Transfection

[0062] CHO-S cells were cultured in serum-free expression medium; cells were placed in Erlenmeyer flasks and cultured on a shaker at 37°C and 5% CO2; the day before transfection, cells were seeded into Erlenmeyer flasks at an appropriate density; on the day of transfection, a recombinant plasmid containing the coding sequence of laminin gamma 1 was transiently transfected into CHO-S cells in suspension culture; after transfection, enhancers and feed were added; when cell viability was below 80%, the cell culture supernatant was collected for purification.

[0063] 1.3 Purification and Analysis of Recombinant Laminin Gamma 1 Protein

[0064] After centrifugation, the cell culture medium was filtered. The filtered cell culture supernatant was loaded onto an affinity purification column at an appropriate flow rate. After washing and elution with a suitable buffer, the eluted fractions were combined, and the buffer was replaced with the final formulation buffer. The purified protein was analyzed by SDS-PAGE and SEC-HPLC to determine its molecular weight and purity. The results are as follows: Figure 1 As shown. Using A 280 Protein concentration was determined by a specific method. The amino acid sequence of the recombinant protein laminin gamma 1 is shown in SEQ ID NO.2.

[0065] 2. Laminin gamma 1 recombinant protein deglycosylation

[0066] The recombinant laminin gamma 1 protein was dissolved in PBS at a storage concentration of 0.23 mg / ml. Deglycosylation was performed using the PNGaseF kit (NEB, Beijing, China). The specific method was as follows:

[0067] 1) In a 1.5 ml centrifuge tube, mix 84 μl of laminin gamma 1 (approximately 10 μg), 10 μl of Glycoprotein Denaturing Buffer (10×), and 6 μl of water to prepare a 100 μl reaction solution;

[0068] 2) Incubate at 100℃ for 10 minutes;

[0069] 3) Cool on ice and collect samples instantly;

[0070] 4) Add 20 μl Glycobuffer 2 (10×), 20 μl 10% NP40, 50 μl water and 10 μl PNGase F to the centrifuge tube;

[0071] 5) Incubate at 37℃ for 24 hours;

[0072] 6) Then dilute with PBS to achieve a final concentration of 1 ng / μl of deglycosylated laminin gamma 1 recombinant protein, which can then be used for the next step of antigen preparation.

[0073] 3. Immunoblotting detection of the reactivity of laminin gamma 1 recombinant protein and deglycosylated recombinant protein with antibodies.

[0074] The recognition of recombinant LAD-1 protein before and after deglycosylation by laminin gamma 1 monoclonal antibody and patient serum was detected by Western blotting. Results are shown in [Figure number missing]. Figure 2 .

[0075] from Figure 2 The results showed that after deglycosylation, the large-size fraction of laminin gamma 1 protein disappeared, while the concentration of the small-size fraction increased. Figure 2 Lanes 1 and 2); Additionally, in the same patient serum, using non-glycated laminin gamma 1 recombinant protein as the antigen, no IgA autoantibody signal was detected, while using deglycated laminin gamma 1 recombinant protein, a significant IgA autoantibody signal was detected. Figure 2 Lanes 3 and 4 indicate that deglycosylation increases the reactivity of IgA autoantibodies in the serum of patients with mucosal pemphigoid with recombinant laminin gamma 1 protein, thereby improving antibody detection rate.

[0076] Example 2: Establishment of an ELISA method for detecting anti-human laminin gamma 1 IgA autoantibodies

[0077] (1) Coating of laminin gamma 1 in 48-well microplates

[0078] The coating material was deglycosylated human laminin gamma 1 recombinant protein, which was prepared according to the method in Example 1.

[0079] Deglycosylated human laminin gamma 1 recombinant protein was diluted with PBS to achieve a final concentration of 1 ng / μl. For antigen coating, 100 µl of deglycosylated human laminin gamma 1 recombinant protein (1 ng / μl) was added to each well of a 48-well microplate and incubated overnight (12-16 hours) at 4°C.

[0080] (2) Reagent preparation

[0081] Before the experiment, place all test materials at room temperature (20-30℃); dilute an appropriate amount of washing buffer (200mM PBS containing 0.5% v / v Tween20, pH 7.4) with distilled water at a volume ratio of 1:10 as needed.

[0082] (3) Sample preparation

[0083] Dilute each patient's serum at a volume ratio of 1:50: Add 500µl of reaction buffer (20mM PBS containing 0.05% v / v Tween 20 and 0.05% w / v NaN3, pH 7.4) to 10µl of serum and mix well.

[0084] (4) Detection steps

[0085] 1) Add 100µl of diluted sample (1:50) to a 48-well microplate and incubate at room temperature (20-30℃) for 60 minutes; at the same time, add standard serum 1 (20mM PBS containing 0.05% w / v NaN3, pH 7.4) and standard serum 2 (containing 100 U / ml llaminin gamma 1 IgA autoantibody and 0.05% w / v NaN3 in 20mM PBS, pH 7.4) as controls;

[0086] 2) Washing the microplate: Add 200 µl of diluted washing buffer to each well and wash 3 times;

[0087] 3) Add enzyme-labeled antibody solution (horseradish peroxidase-labeled rabbit anti-human IgA antibody) at 100µl / well and incubate at room temperature (20-30℃) for 45 minutes;

[0088] 4) Washing the microplate: Add 200 µl of diluted washing buffer to each well and wash 3 times;

[0089] 5) Add enzyme substrate solution (TMB colorimetric solution) at 100µl / well and incubate at room temperature (20-30℃) for 15 minutes;

[0090] 6) Add the stop solution (0.5N hydrochloric acid solution) at a rate of 100µl / well.

[0091] 7) Read the absorbance (450nm);

[0092] 8) Result calculation and judgment:

[0093] Formula for calculating unit value (U / ml):

[0094] (A) 450 <Sample> - A 450 <Standard Serum 1>) × 100 / (A 450 <Standard Serum 2> - A450 <Standard Serum 1>)

[0095] Judgment criteria: A value <15 is negative; a value ≥15 is positive.

[0096] Quality control:

[0097] Each test result must meet the following conditions, otherwise the result is invalid:

[0098] OD of standard serum 1 450 Less than or equal to 0.100;

[0099] OD of standard serum 2 450 Greater than or equal to 0.500.

[0100] Example 3: Sensitivity, specificity, and repeatability experiments of the ELISA detection method for anti-human laminin gamma 1 IgA autoantibodies.

[0101] 1. Sensitivity and specificity of the ELISA method for detecting anti-human laminin gamma 1 IgA autoantibodies

[0102] First, serum samples from 20 patients known to be positive for anti-laminin gamma 1 IgA autoantibodies and serum samples from 50 normal controls were selected. The first round of testing was performed according to the method in Example 2. It was found that the detection results of the patient serum and the normal control serum were highly distinguishable. At this point, the cutoff value was set to the OD value of the 50 normal control serum samples. 450 Mean + 3SD. Select OD. 450 A patient's serum with a relative value of 1.1 was used as a positive control, with a relative value set at 100, corresponding to a relative cutoff value of 15. Using the established experimental method, we further tested the serum of 200 new normal individuals, 25 patients with mucosal pemphigoid (laminin gamma 1 IgA type), 50 patients with bullous pemphigoid, and 50 patients with pemphigus vulgaris. Unglycosylated recombinant laminin gamma 1 protein was used as the antigen for detection. Specific experimental results are shown in Table 1.

[0103] Table 1

[0104]

[0105] a: ELISA using recombinant laminin gamma 1 protein as an antigen; b: ELISA using deglycosylated recombinant laminin gamma 1 protein as an antigen.

[0106] Sensitivity and specificity results showed that using non-deglycosylated laminin gamma 1 recombinant protein as an antigen only detected IgA autoantibodies in the serum of 40% of mucosal pemphigoid patients, while using deglycosylated laminin gamma 1 recombinant protein as an antigen increased the detection rate to 96%. Furthermore, no non-specific detection was observed in serum from other diseases and normal controls, indicating that this method is not only highly sensitive but also highly specific.

[0107] 2. Reproducibility of the ELISA method for detecting anti-human laminin gamma 1 IgA autoantibodies

[0108] For each of the five samples, there were six replicates, and the CV% value of each replicate was less than 15%.

[0109] 3. Detection range of the ELISA method for detecting anti-human laminin gamma 1 IgA autoantibodies

[0110] The detection range of this detection method is 10-150 U / ml.

[0111] 4. Interfering substances

[0112] It is necessary to avoid using highly hemolyzed samples, as well as high-concentration serum diluted too low for testing.

[0113] Example 4 Assembly of an ELISA kit for detecting anti-human laminin gamma 1 IgA autoantibodies

[0114] The anti-human laminin gamma 1 IgA autoantibody ELISA kit includes:

[0115] (1) Microplates (96-well or 48-well) coated with deglycosylated human laminin gamma 1 recombinant protein.

[0116] (2) Standard serum 1 (1×): 20 mM PBS containing 0.05% w / v NaN3, pH 7.4;

[0117] (3) Standard serum 2 (1×): 20 mM PBS containing 100 U / ml anti-laminin gamma 1 IgA autoantibody and 0.05% w / v NaN3, pH 7.4;

[0118] (4) Enzyme-labeled antibody: Rabbit anti-human IgA antibody labeled with horseradish peroxidase;

[0119] (5) Reaction buffer (1×): 20 mM PBS containing 0.05% v / v Tween 20 and 0.05% w / v NaN3, pH 7.4;

[0120] (6) Washing buffer (10×): 200mM PBS containing 0.5% v / v Tween20, pH 7.4;

[0121] (7) Enzyme substrate solution: TMB colorimetric solution;

[0122] (8) Termination solution: 0.5N hydrochloric acid solution.

Claims

1. An ELISA kit for detecting anti-human laminin gamma 1 IgA autoantibodies, characterized in that, The kit includes an ELISA plate coated with recombinant human laminin gamma 1 protein.

2. The ELISA kit for detecting anti-human laminin gamma 1 IgA autoantibodies as described in claim 1, characterized in that, The human laminin gamma 1 recombinant protein described is a deglycosylated laminin gamma 1 recombinant protein.

3. The ELISA kit for detecting anti-human laminin gamma 1 IgA autoantibodies as described in claim 1, characterized in that, The amino acid sequence of the recombinant human laminin gamma 1 protein is shown in SEQ ID NO.

2.

4. The ELISA kit for detecting anti-human laminin gamma 1 IgA autoantibodies as described in claim 1, characterized in that, The human laminin gamma 1 recombinant protein was obtained by transfecting a recombinant plasmid containing the DNA sequence encoding the laminin gamma 1 recombinant protein into CHO-S cells in suspension culture, followed by expression and purification.

5. The ELISA kit for detecting anti-human laminin gamma 1 IgA autoantibodies as described in any one of claims 1-4, characterized in that, The kit also includes: (1) Standard serum 1 (1×): 20 mM PBS containing 0.05% w / v NaN3, pH 7.4; (2) Standard serum 2 (1×): 20 mM PBS containing 100 U / ml anti-human laminin gamma 1 IgA autoantibody and 0.05% w / v NaN3, pH 7.4; (3) Enzyme-labeled antibody: Rabbit anti-human IgA antibody labeled with horseradish peroxidase; (4) Reaction buffer (1×): 20 mM PBS containing 0.05% v / v Tween 20 and 0.05% w / v NaN3, pH 7.4; (5) Washing buffer (10×): 200mM PBS containing 0.5% v / v Tween20, pH 7.4; (6) Enzyme substrate solution: TMB colorimetric solution; (7) Termination solution: 0.5N hydrochloric acid solution.

6. The ELISA kit for detecting anti-human laminin gamma 1 IgA autoantibodies as described in claim 5, characterized in that, When the kit is used to detect anti-human laminin gamma 1 IgA autoantibodies, the following steps are included: (1) Reagent preparation Before the experiment, place all test materials at room temperature and dilute an appropriate amount of washing buffer with distilled water at a volume ratio of 1:10 as needed. (2) Sample preparation Dilute the serum sample to be tested with reaction buffer at a volume ratio of 1:50 and mix well. (3) Detection steps 1) Add 100µl of diluted serum sample to the ELISA plate and incubate at room temperature for 60 minutes; add standard serum 1 and standard serum 2 as controls at the same time. 2) Washing the microplate: Add 200 µl of diluted washing buffer to each well and wash 3 times; 3) Add horseradish peroxidase-labeled rabbit anti-human IgA antibody at 100 µl / well and incubate at room temperature for 45 minutes; 4) Washing the microplate: Add 200 µl of diluted washing buffer to each well and wash 3 times; 5) Add enzyme substrate solution at 100 µl / well and incubate at room temperature for 15 minutes; 6) Add stop solution at a rate of 100 µl / well; 7) Read the absorbance; 8) Result calculation and judgment: Formula for calculating unit value (U / ml): (A 450 <Sample> - A450 <Standard Serum 1>) × 100 / (A 450 <Standard Serum 2> - A 450 <Standard Serum 1>) Judgment criteria: A value <15 is negative; a value ≥15 is positive. Quality control: Each test result must meet the following conditions, otherwise the result is invalid: OD of standard serum 1 450 Less than or equal to 0.100; OD of standard serum 2 450 Greater than or equal to 0.

500.

7. The use of the anti-human laminin gamma 1 IgA autoantibody detection ELISA kit according to any one of claims 1-6 in the preparation of reagents for detecting anti-human laminin gamma 1 IgA autoantibodies.

8. The use of the anti-human laminin gamma 1 IgA autoantibody detection ELISA kit according to any one of claims 1-6 in the preparation of reagents for diagnosing mucosal pemphigoid.

9. The application as described in claim 8, characterized in that, The mucosal pemphigoid described is Laminin gamma 1IgA type pemphigoid.

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