A kit for detecting antigen myosin 1-IgG antibody

Abstract

The present invention provides a kit for detecting antigen myosin 1 IgG antibodies, which is composed of antigen protein Tropomyosin 1 (tropomyosin 1), a solid phase carrier, a standard, a positive quality control product, a negative quality control product, a labeled antibody, a substrate color developing solution, an antibody diluent, an antigen diluent, a sample dilution buffer, a washing solution, a stop solution, etc. The present invention utilizes the target antigen Tropomyosin 1 corresponding to the anti-Tropomyosin 1 IgG antibody, and uses human anti-label peptide IgG as a standard for quantitative detection to perform anti-Tropomyosin 1 IgG antibody detection on the antigen-antibody complex formed in the serum. The kit of the present invention is easy to operate, has very good reagent stability, is easy to store, and fills the gap in identifying biomarkers for patients with immune nephrotic syndrome at home and abroad.

Description

[0001] This application is a divisional application of application number 202110743541.3, application date July 1, 2021, and invention name A kit for detecting antigen myosin 1-IgG antibodies. Technical Field

[0002] The present invention belongs to the field of biotechnology and relates to a kit for detecting antigen myosin 1-IgG antibodies in serum. The present invention provides for the first time a kit for rapid, simple, and highly sensitive qualitative or quantitative analysis of antigen myosin 1-IgG levels in immune nephrotic syndrome. Background Art

[0003] The diagnosis and treatment of immune-related nephrotic syndrome (NS) remains an ongoing challenge in biomedical science. Nephrotic syndrome is a clinical syndrome characterized by a series of pathological changes resulting from increased permeability of the glomerular filtration membrane, leading to increased filtration of plasma proteins and, consequently, high levels of proteinuria. The numerous symptoms manifested can be largely attributed to a variety of causes, including congenital genetic factors, renal impairment and insufficiency, genitourinary tract infections and diseases, substance abuse, loss of appetite and malnutrition, diabetes, hypertension, infection, injury, immune deficiency, and cancer.

[0004] Early diagnosis and prognosis of nephrotic syndrome are particularly important. The annual incidence of primary nephrotic syndrome varies by race and nationality, ranging from approximately 1.15 to 16.9 per 100,000 people. Minimal change disease is the most common pathological type. While the vast majority of patients with this condition are sensitive to steroid therapy, approximately 10% to 20% develop steroid resistance. The probability of progression to end-stage renal disease within 5 years of diagnosis is 8% to 35%, and within 15 years of diagnosis is 24% to 66%.

[0005] Ali et al. observed that kidneys transplanted from patients with refractory minimal change disease returned to normal renal function and showed no proteinuria. The potential for proteinuria is primarily due to issues with the donor's internal environment. With the exception of some children whose condition stems from genetic defects, most children with primary nephrotic syndrome experience significant improvement after receiving steroids and immunosuppressants. Therefore, the development of primary nephrotic syndrome may be closely related to the patient's autoimmune status.

[0006] Antigens bind to the BCR on the surface of B cells and are subsequently transduced into B cells via Igα / Igβ, which is the first signal for B cell activation. Simultaneously, the interaction between CD154 molecules expressed on Th cells and CD40 molecules on the surface of B cells provides a second signal for effective B cell activation. This is a crucial signal for B cell proliferation, germinal center reactions, and ultimately differentiation into plasma cells. These plasma cells synthesize and secrete antibodies specific for the antigens mentioned above. Recent studies have revealed that B cell dysfunction plays a significant role in primary nephrotic syndrome. The number of B cells in the peripheral blood of patients with steroid-sensitive nephrotic syndrome increases significantly during disease relapse. High numbers of activated B cells have been detected in patients with steroid-dependent nephrotic syndrome, while the number of B cells is significantly decreased in patients with steroid-sensitive nephrotic syndrome in remission. Glucocorticoids have also been shown to be ineffective in inhibiting B cell activation in patients with either steroid-sensitive or steroid-dependent nephrotic syndrome.

[0007] Cyclophosphamide and rituximab can induce long-term remission after drug withdrawal in patients with steroid-dependent nephrotic syndrome by depleting B cells. Rituximab specifically binds to the transmembrane antigen CD20 on the surface of pre-B cells and mature B cells, depleting B cells through three pathways: complement activation, antibody-dependent cytotoxicity, and induction of apoptosis, thereby exerting its immunosuppressive effects. Furthermore, memory B cells (particularly the number of converted memory B cells) can predict relapse after rituximab treatment. Although multiple multicenter clinical studies worldwide have demonstrated the successful use of rituximab in the treatment of minimal change disease nephrotic syndrome, some studies have found that the B cell depletion effect of rituximab in steroid-dependent nephrotic syndrome lasts for approximately five months, followed by relapses after six to seven months as B cell numbers recover. This suggests that patients with primary nephrotic syndrome harbor pathological B cell clones, making the identification and precise elimination of these pathological B cell clones crucial.

[0008] To date, the target antigens of pathological B cells in children with primary nephrotic syndrome remain unclear. Pathologically, both minimal change disease and focal segmental glomerulosclerosis are considered podocyte diseases resulting from loss or alteration of podocyte function, leading to high levels of proteinuria. Podocytes are glomerular epithelial cells that adhere to the lateral surface of the glomerular basement membrane and serve as the final barrier against protein loss. Podocyte damage often manifests as high levels of proteinuria. Studies have shown that the foot processes of glomerular epithelial cells and the slit diaphragms between adjacent foot processes play a crucial role in maintaining the physiological barrier function of the glomerular filtration membrane. The maintenance of the specialized epithelial cell structure relies primarily on the actin-based cytoskeleton and foot process molecules expressed in the foot processes. Numerous molecules expressed in the foot processes and slit diaphragms of glomerular epithelial cells have been identified, and they play a crucial role in maintaining glomerular development, membrane integrity, podocyte signaling, and preventing protein leakage. Podocytes adapt to changes in glomerular filtration volume by adjusting their morphology and altering the intercellular filtration space. This function is entirely dependent on the robust podocyte cytoskeletal system. Tropomyosin 1, however, functions closely with the cytoskeleton. Tropomyosin 1, a multifunctional protein that functions as a cytoskeletal molecular motor, primarily provides force for muscle contraction. Tropomyosin 1 is a member of the tropomyosin family, which comprises many highly conserved proteins involved in the contractile systems of striated and smooth muscle, as well as the cytoskeleton of non-muscle cells. Tropomyosin 1 is composed of two α-helical chains arranged in a coiled-coil arrangement. Tropomyosin 1 is attached end-to-end to the two grooves of actin filaments, providing stability to the filaments. Tropomyosin 1 binds to actin filaments in both muscle and non-muscle cells. Tropomyosin 1 also binds to the troponin complex to regulate the calcium-dependent interaction between actin and myosin during muscle contraction. In non-muscle cells, tropomyosin 1 is associated with stabilization of cytoskeletal actin filaments. Alternatively spliced transcript variants encoding a range of isoforms have been described in smooth muscle and non-muscle cells. Tropomyosin 1 isoform 1 is expressed in adult and fetal skeletal muscle and heart tissue, with higher expression in heart tissue, whereas isoform 10 is expressed in adult and fetal heart tissue but not in skeletal muscle. Mutations in the tropomyosin 1 gene are associated with familial hypertrophic cardiomyopathy type 3.

[0009] Studies have found that when Myo1e, a molecule in Tropomyosin 1, is downregulated, experimental zebrafish develop edema, a decrease in localized glomerular cell number, and alterations in the lumen of renal tubules and pronephric ducts, as well as in endosomes of epithelial cells (a phenomenon similar to the proteinuria seen in human glomerular diseases). Mice with Myo1e knockout develop proteinuria and chronic renal damage. This suggests that Myo1e, a molecule in Tropomyosin 1, is essential for the functional integrity of podocytes and the glomerular filtration membrane. Reports suggest that low Tropomyosin 1 protein expression may be associated with the development of non-small cell lung cancer and could potentially serve as a new target for the diagnosis and treatment of non-small cell lung cancer. Other reports suggest that low Tropomyosin 1 expression is associated with the tumor stage of bladder cancer. However, research on Tropomyosin 1 protein expression and the detection of anti-Tropomyosin 1 IgG antibodies has been lacking for the diagnosis or treatment of nephrotic syndrome. The present invention makes the diagnosis, treatment and monitoring of the disease more convenient and can improve the physical and mental pain and economic burden brought to patients by the existing detection means (renal puncture).

[0010] There are studies showing the application of Tropomyosin 1 expression in urinary system diseases, but there are no studies on the presence of Tropomyosin 1 autoantibodies, especially in immune nephrotic syndrome, let alone their value. Summary of the Invention

[0011] The present invention aims to provide a kit for detecting antibodies to the antigen Tropomyosin 1 IgG, which mainly comprises the antigen protein Tropomyosin 1, a solid phase carrier, a standard substance, a positive quality control substance, a negative quality control substance, a labeled antibody (anti-human IgG labeled with an enzyme, a chemiluminescent agent, or biotin), a substrate color development solution, an antibody diluent, an antigen diluent, a sample dilution buffer, a washing solution, a stop solution, etc. Signal detection methods include chemiluminescence and photochromic methods.

[0012] The sequence of the antigen protein Tropomyosin 1 is shown in SEQ ID NO. 1 as follows: MAGSSSLEAVRRKIRSLQEQADAAEERAGTLQRELDHERKLRETAEADVAS LNRRIQLVEEELDRAQERL.

[0013] The antigen protein Tropomyosin 1 is immobilized on a solid phase carrier, which includes silicon or glass surfaces, paper, polystyrene, membranes, metals, microfluidic channels, beads (magnetic beads), column chromatography media, polyacrylamide gels, biochips, etc. Preferred solid phase carriers include: nitrocellulose membranes (NC membranes) and enzyme-labeled microplates.

[0014] The positive quality control or standard can be recombinant human anti-tag peptide immunoglobulin G or a fragment thereof, or anti-Tropomyosin 1-IgG antibody extracted from patient serum, and the serum of healthy subjects is the negative quality control.

[0015] The color developing agent can be acridinium ester, AMPPD, TMB and 4-MUP; the antigen diluent is 1x PBS pH7.4, NaCl 163mM and 1% TritonX-100; the sample dilution buffer is 0.01M PBS pH 7.4 + 10% newborn calf serum; the antibody diluent is 1M D-glucose and 2% glycerol added to 0.01M PBS containing 0.35% Tween2 and the volume is adjusted to 100ml; the washing solution is: 1x PBS pH7.4, NaCl 163mM, 1% TritonX-100 and 10% glycerol; the stop solution is: 2M hydrochloric acid.

[0016] The antigen protein Tropomyosin 1 is provided with a tag peptide, and the tag peptide includes: c-Myc tag, GST tag, Flag tag, His tag or biotin tag.

[0017] According to the present invention, the relative titer of serum autoantibodies corresponding to the antigen Tropomyosin 1 is quantitatively calculated using the specific immune reaction between a unified tag peptide and anti-tag peptide antibodies and the regression of a standard curve.

[0018] According to the present invention, the antigen protein Tropomyosin 1 can be expressed in eukaryotic or prokaryotic cells, preferably eukaryotic cells, more preferably mammalian cells, more preferably human cells such as HEK293 cells. Examples of mammalian cells include CHO, HEK293 or COS-7 cells.

[0019] According to the present invention, the antigen protein Tropomyosin 1 is purified by Ni column affinity chromatography, molecular sieve, ion exchange column and hydrophobic column.

[0020] According to the present invention, the label used in the chemiluminescence method is acridinium ester.

[0021] According to the present invention, the sample is any sample containing autoantibodies, selected from whole blood, serum, plasma, urine, lymph, pleural effusion and ascites, preferably serum, more preferably mammalian (human) serum.

[0022] This invention uses genetic engineering to successfully express and purify the protein Tropomyosin 1, which serves as the antigen protein in the kit. By using the antigen-coated solid-phase carrier, this method provides the first test kit suitable for detecting anti-Tropomyosin 1-IgG antibodies in the serum of patients with immune nephrotic syndrome. This kit is used for the qualitative or quantitative analysis of anti-Tropomyosin 1-IgG antibodies in human serum.

[0023] The method for fixing the antigen protein Tropomyosin 1 can be a direct coating method: (1) the antigen is bound to a nitrocellulose membrane or a polystyrene microplate by physical adsorption or non-covalent bonding; (2) the carboxyl magnetic particles are activated by EDC / Sulfo-NHS and covalently bonded to the antigen (amino residue) to form a stabilizer.

[0024] Based on extensive clinical and molecular mechanism studies, the present invention has detected anti-Tropomyosin 1-IgG antibodies in the serum of patients with nephrotic syndrome for the first time. The kit of the present invention can be used to detect this autoantibody, providing support for the research of autoimmune nephrotic syndrome.

[0025] The innovative features of the present invention can be summarized as follows:

[0026] (1) The present invention uses recombinant human anti-His-tagged immunoglobulin as a standard, which can specifically identify antigens with His tags, thereby improving the accuracy and specificity of the kit detection.

[0027] (2) The present invention is the first to develop a qualitative or quantitative kit for detecting autoantibodies in patients with immune nephrotic syndrome.

[0028] (3) The kit of the present invention relates to the qualitative and quantitative analysis of anti-Tropomyosin 1-IgG antibodies in human serum, wherein the advantages of the solid phase membrane immunoassay are simple operation, strong adsorption capacity of the NC membrane, close to 100%, and the trace antigen can be completely adsorbed, and the amount of reagents used is also small, which is nearly 10 times less than the traditional ELISA; the NC membrane adsorbed with antigens or antibodies or with existing results can be stored for a long time (can be stored at -20°C for half a year) without affecting its activity, which is beneficial for physical examination screening of healthy people. The chemiluminescence immunoassay adopts acridinium ester direct labeling method. The chemiluminescence quantum yield of acridinium ester is relatively high, and its labeling conditions are quite mild, the labeling rate is also high, and the separation will not be affected after labeling. At the same time, the reaction speed of the acridinium ester chemiluminescence process is extremely fast and the background is low. Even in the presence of sodium hydroxide and hydrogen peroxide in the reaction, it can still emit light. During the redox reaction process, the conjugate is decomposed, but this does not affect the luminescence of the free acridinium ester. In addition, acridinium ester chemiluminescence reagents are very stable and easy to store.

[0029] (4) Currently, there is no information on Tropomyosin 1 and anti-Tropomyosin1-IgG for patients with immune nephrotic syndrome at home and abroad. The kit of the present invention fills the gap in biomarkers for identifying patients with immune nephrotic syndrome at home and abroad. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It was shown that Tropomyosin 1 protein is the main target antigen of autoantibodies in patients with autoimmune nephrotic syndrome. Figure 1 a is the two-dimensional electrophoresis protein spot when the primary antibody is healthy human serum; Figure 1 b is the two-dimensional electrophoresis protein spot of the serum of patients with primary nephrotic syndrome when the primary antibody was used; Figure 1 c is the mass spectrometry identification of Tropomyosin 1 in patients with nephrotic syndrome. The peak indicated by the arrow is the Tropomyosin 1 protein peak.

[0031] Figure 2 The recombinantly synthesized Tropomyosin 1 protein identified by SDS-PAGE electrophoresis is shown.

[0032] Figure 3 This is a solid-phase membrane immunoassay kit for detecting anti-Tropomyosin1-IgG antibodies in the serum of patients with autoimmune nephrotic syndrome.

[0033] Figure 4 Schematic diagram of carboxyl magnetic microparticles coated with the antigen protein Tropomyosin 1.

[0034] Figure 5This is a schematic diagram of the detection principle of the chemiluminescence kit used to detect anti-Tropomyosin 1-IgG antibodies.

[0035] Figure 6 The expression of anti-Tropomyosin 1-IgG antibodies in the test samples of each group is shown.

[0036] Figure 7 The efficacy of anti-Tropomyosin 1-IgG antibodies in the differential diagnosis of autoimmune nephrotic syndrome was demonstrated. DETAILED DESCRIPTION

[0037] The present invention will be further described below with reference to the accompanying drawings and specific examples. The following examples are intended to illustrate the present invention but are not intended to limit the scope of the present invention.

[0038] Example 1

[0039] 1. Expression of Tropomyosin 1 protein antigen: Use bioengineering methods to express the corresponding antigen protein.

[0040] 1.2 Antigen protein immobilization method: The antigen coating solid phase carrier method of the present invention uses a direct coating method: (1) the antigen is bound to the polystyrene microplate or nitrocellulose membrane by physical adsorption or non-covalent bonding; (2) the antigen is bound to the magnetic particles containing carboxyl functional groups by chemical coupling.

[0041] 1.3 Positive quality control and standard substances: The positive quality control and standard substances selected in the present invention are IgG or human anti-tag peptide IgG extracted and quantified from patient serum.

[0042] 1.4 Labeled Antibody and Substrate Color Developer: The labeled antibody selected in the present invention can be acridinium ester-labeled anti-human IgG, horseradish peroxidase-labeled anti-human IgG, or biotin-labeled anti-human IgG; the color developer can be 4-MUP, TMB, or AMPPD.

[0043] 1.5 Signal Detection: Detect the light signal using an enzyme-labeled instrument, chemiluminescence analyzer, or other methods, and quantitatively analyze the anti-Tropomyosin 1–IgG antibodies in human serum by plotting a standard curve.

[0044] 1.6 Conventional reagents and solutions used in the present invention.

[0045] 1.6.1 Nitrocellulose membrane, polystyrene microplate, and magnetic beads.

[0046] 1.6.2 Prepare PBS pH 7.4: NaCl 137mM, KCl 2.7mM, Na2HPO4 8.1mM, KH2PO4 1.5mM; prepare 32.20X wash buffer: KCl 54mM, NaCl 2.74mM, KH2PO4 30mM, Na2HPO4 162mM, then filter under negative pressure using a 0.22μm filter membrane.

[0047] 1.6.3 Preparation of 5% BSA blocking solution: Dissolve 5 g of bovine serum albumin in 100 mL of 0.01 M PBS solution.

[0048] 1.6.4 Antigen diluent: 1× PBS pH 7.4, 1% Triton X-100, 163 mM NaCl.

[0049] 1.6.5 Serum / antibody dilution: Take 1M D-glucose (19.82g) and 2% glycerol (2ml) and add 0.01M PBS containing 0.35% Tween2 to make up to 100ml, then filter under negative pressure using a 0.22μm filter membrane.

[0050] 1.6.6 Washing solution: 1×PBS pH 7.4, NaCl 163 mM, 1% Triton X-100, 10% glycerol.

[0051] 1.6.7 TMB Colorimetric Reagent: Substrate Development Solution A: 1.6 g citric acid, 13.6 g sodium acetate, 0.3 ml 30% hydrogen peroxide, add distilled water to 500 ml. Substrate Development Solution B: 0.95 g citric acid, 0.2 g disodium EDTA, 50 ml glycerol, 0.15 g TMB, add distilled water to 500 ml.

[0052] 1.6.8 Stop solution: 2M hydrochloric acid.

[0053] 1.6.9 SDS-PAGE (10%) preparation.

[0054] 10% separation gel preparation:

[0055]

[0056] Example description: The present invention adsorbs the antigen onto a solid phase carrier, then adds a positive quality control or standard or a serum sample to be tested for incubation, then adds a labeled secondary antibody to carry out an antigen-antibody reaction, and finally uses a photochromic method, chemiluminescence method or fluorescence method to detect the light signal. The change in the light signal is used to achieve the purpose of qualitative or quantitative analysis of anti-Tropomyosin 1-IgG antibodies in human serum.

[0057] Example 2 Tropomyosin 1 on podocytes is one of the main target antigens of autoantibodies in patients with autoimmune nephrotic syndrome

[0058] The podocyte cell line (MPC5) was cultured, washed 2-3 times with PBS, and then fully lysed on ice using a focused ultrasound apparatus (Covaris S220, Gene) in a lysis buffer containing 30 mm Tris-HCl, 8 m urea, 4% CHAPS, and protease inhibitors (#ab65621; Abcam, 1:200 dilution). The sample was then placed in a centrifuge at 12,000 g, 4°C, and centrifuged for 30 minutes. The supernatant was collected, which was the total protein of the collected glomerular podocytes. The concentration of the total protein of the collected glomerular podocytes was determined using a BCA protein concentration assay kit to obtain purified total protein of the glomerular podocytes. Two-dimensional electrophoresis was then performed, the membrane was transferred, and the cells were incubated with the serum of the healthy control group and the patient, respectively. The secondary antibody was then added for development, and the results are shown in the table. Figure 1 a and Figure 1 b. After the above two-dimensional electrophoresis development, differential analysis of positive spots was performed, and protein spots that were strongly positive in patients with primary nephrotic syndrome and negative or weakly positive in the healthy control group on the two-dimensional electrophoresis strip were selected for mass spectrometry analysis and identification. They were separated by nano-high performance liquid chromatography technology and identified by matrix-assisted laser desorption / ionization time-of-flight tandem mass spectrometry using a mass spectrometer. The results were analyzed and processed using ProteinPilot software. It was determined that Tropomyosin 1 was the target antigen of the autoantibody. Results are shown in Figure 1 c.

[0059] Example 3 Expression of Tropomyosin 1 Antigen Protein

[0060] The expression and purification of the antigen protein Tropomyosin 1 utilizes genetic engineering methods to perform PCR amplification using the gene encoding the Tropomyosin 1 protein as a template, and then constructs an expression vector for protein expression. The antigen protein expressed by the present invention contains a His-tagged peptide. The expressed recombinant protein is purified by nickel column affinity chromatography, ion affinity chromatography, hydrophobic column, molecular sieve, etc., and finally the molecular weight of the synthesized protein Tropomyosin 1 is identified and quantified using SDS-PAGE. Figure 2 shown.

[0061] Example 4 Preparation of a Solid-Phase Membrane Immunoassay Kit for Detecting Anti-Tropomyosin 1-IgG Antibodies

[0062] 4.1 Composition of the solid-phase membrane immunoassay kit for detecting anti-Tropomyosin 1-IgG:

[0063] 1. Nitrocellulose membrane coated with Tropomyosin 1 antigen,

[0064] 2. Positive quality control (standard) human anti-His tag immunoglobulin G (purchased from Tribioscience),

[0065] 3. Negative quality control product (serum from healthy people),

[0066] 4. Horseradish peroxidase-labeled goat anti-human immunoglobulin G,

[0067] 5. Antigen diluent,

[0068] 6. Detergent,

[0069] 7. TMB color developer,

[0070] 8. Stop solution.

[0071] 4.2 Optimization of the Tropomyosin 1 Protein Coating Concentration: Positive control serum was immobilized on the carrier in serial dilutions and then incubated with undiluted serum from patients with immune nephrotic syndrome to determine the amount of the corresponding antigen-antibody complex. An orthogonal array was designed based on the Tropomyosin 1 protein antigen coating concentration (180 μg / mL, 90 μg / mL, and 45 μg / mL), reaction time (30 min, 35 min), temperature (30°C, 35°C), and optimal dilution of the enzyme-labeled secondary antibody (1:500, 1:1000, and 1:1200). Standard positive and negative sera were tested in replicates at two levels for each factor. The ratio (P / N) of the highest luminescence value (P) of the positive serum to the lowest luminescence value (N) of the negative serum was calculated. Statistical analysis determined the optimal conditions, which significantly improved the positive detection rate of the standard positive serum. Finally, we found that the optimal antigen coating concentration of the kit of the present invention was 90 μg / ml, the optimal antigen-antibody reaction time was 30 min, the most suitable temperature was 35°C, and the optimal secondary antibody working dilution was 1:1200.

[0072] 4.3 The steps for preparing antigen-coated nitrocellulose membrane and testing serum samples are as follows:

[0073] 4.3.1 Coating and blocking: Spot 5 μl of diluted antigen solution of appropriate concentration in 0.01M PBS, pH 7.4, on the nitrocellulose membrane and dry in a 37°C incubator for 30 min. Place the nitrocellulose membrane in a plate holder and add 100 μl of 5% BSA and block in a 37°C incubator for 10 min. Discard the blocking solution and wash twice with washing solution.

[0074] 4.3.2 Incubation: Add 10 μl of diluted antibody standard or serum specimen to be tested into the reaction tank, and prepare negative and positive controls at the same time. Mix thoroughly and incubate in a 37°C wet box for 30 min. Prepare three parallel wells for each sample.

[0075] 4.3.3 Secondary antibody incubation: Discard the liquid in the tank, wash with washing solution 3 times for 1 min, then add 20 μl of horseradish peroxidase-labeled goat anti-human IgG diluted in antibody diluent, mix thoroughly and react at room temperature for 30 min.

[0076] 4.3.4 Color development: discard the liquid in the tank, wash three times with washing solution for 1 minute, add 500 μl of color development solution A / B, react at room temperature for 10 minutes, add 500 μl of 2M HCl stop solution, mix well, discard the liquid in the tank after 1 minute, wash three times, take out the test strip and blow dry the membrane with a hair dryer. After natural drying, make a qualitative judgment by naked eye. The one with obvious brown spots is positive (such as Figure 3 ), or the NC membrane was placed on a "Canon 9000f markii" developer for scanning. The developer's built-in software system used the reference standard concentration as the ordinate and the reader reading value (i.e., grayscale value) as the abscissa. A double logarithmic logistic curve fitting method was used to draw a standard curve to quantitatively analyze the anti-Tropomyosin 1-IgG level in the patient's serum.

[0077] Example 5 Preparation of a Chemiluminescent Kit for Detecting Anti-Tropomyosin 1-IgG

[0078] 5.1 Composition of the chemiluminescent kit for detecting anti-Tropomyosin 1-IgG:

[0079] 1. Magnetic particle solution coated with Tropomyosin 1 antigen,

[0080] 2. Positive quality control (standard) human anti-His tag immunoglobulin G (purchased from Tribioscience) (including target value range),

[0081] 3. Acridinium ester labeled anti-human IgG solution,

[0082] 4. Sample diluent: phosphate buffered saline.

[0083] 5. Negative quality control (serum from healthy people),

[0084] 6. Pre-excitation solution (H2O2),

[0085] 7. Excitation solution (NaOH),

[0086] 8. Cleaning fluid.

[0087] 5.2 The process is as follows:

[0088] 5.2.1 Principle of Antigen Coating: The carboxyl groups on the surface of the base magnetic beads first react with the prepared EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide) solution to generate unstable amino-reactive O-acylurea intermediates. This intermediate has three-step conversion pathways: the first step is to react with water in the solution to reduce it to carboxyl magnetic beads; the second is to react with the amino groups of the antibody to directly obtain immunomagnetic beads; the third is the unstable active O-acylurea intermediate reacts with NHS (N-hydroxysuccinimide) to generate semi-stable amino-reactive NHS esters, which then react with the amino groups on the antigen to generate immunomagnetic beads. Figure 4 .

[0089] 5.2.2 EDC / NHS-activated carboxyl magnetic beads

[0090] Place the EP tube on the magnetic rack for 4 minutes, wait for the magnetic beads and solution to separate into solid and liquid, then add 50ul of 25mmol / L MES (2-morpholineethanesulfonic acid) solution with pH=5 to the remaining EP tube containing magnetic beads, and then shake and mix thoroughly for 10 minutes. After solid-liquid separation occurs, repeat the operation once; add 25ul each of the freshly prepared EDC solution and NHS solution to the washed magnetic beads, mix thoroughly, incubate at 25°C for 30 minutes, mix thoroughly, and repeat washing the magnetic beads once after solid-liquid separation.

[0091] 5.2.3 Basic steps for coupling antigen to activated magnetic beads

[0092] Add 30 μl of 1 mg / mL antigen to 30 μl of 25 mmol / L MES solution (pH 5). This solution is then added to the activated magnetic beads and vortexed to mix thoroughly. Then, add 40 μl of 5 mmol / L MES solution (pH 5) and vortex to mix thoroughly. Incubate the mixture at 25°C for 1 hour, then place the EP tube on a magnetic stand for 4 minutes. Add 100 μl of 50 mmol / L Tris solution (pH 7.4) to the magnetic beads and incubate the EP tube at 25°C for 15 minutes, gently vortexing to prevent precipitation. After 15 minutes, solid-liquid separation occurs. Remove the liquid component, place the EP tube on a vortex to mix thoroughly, and then place the EP tube on a magnetic stand for 4 minutes. Repeat this process 4-5 times. After all steps are complete, resuspend the magnetic beads in 50 μl of PBS buffer containing 0.1% BSA to obtain the antigen-conjugated magnetic beads.

[0093] 5.2.4 Labeled Antibodies and Excitation: Acridinium esters and acridinium sulfonamides are commonly used in the experiment. Acridinium esters covalently bind to anti-IgG (amino residues) under alkaline conditions (such as in the presence of carbonates). Impurities are removed by desalting to obtain a labeled antibody solution.

[0094] 5.2.4.1 In the figure, R, R', and R" represent alkyl, alkoxy, aryl, and other substituents; X, X', and X" represent coupling groups used to couple antigens or antibodies and increase the solubility of the compound.

[0095]

[0096] 5.2.4.2 Labeling reaction mechanism of acridinium ester: Under alkaline conditions, hydrogen peroxide reacts with the 9-carbon atom of acridinium. The peroxide anion formed by the addition product under alkaline conditions then nucleophilically attacks the carbonyl carbon. The leaving group leaves and further forms an unstable four-membered ring intermediate. After ring opening, an excited acridone is formed, which releases photons during its return to the ground state.

[0097]

[0098] 5.2.5 Signal Detection: Under alkaline conditions, acridinium ester molecules are attacked by hydrogen peroxide to generate ethylene dioxide. Ethylene dioxide is extremely unstable and decomposes into CO2 and electronically excited N-methylacridone. When it returns to the ground state, it emits light with a wavelength of 430nm. By collecting the luminescent signal, the concentration of anti-Tropomyosin 1-IgG antibodies in human serum can be determined. Figure 5 .

[0099] 5.3 The specific steps are as follows:

[0100] 5.3.1 Dilute the sample according to a certain ratio;

[0101] 5.3.2 Add the diluted sample to the magnetic microparticle sample diluent and incubate at 37°C for 15-20 minutes.

[0102] 5.3.3 Then wash with cleaning solution 3 times;

[0103] 5.3.4 Add acridinium ester labeled antibody and react at 37°C for 15-20 minutes

[0104] 5.3.5 Repeat washing 3 times;

[0105] 5.3.6 Finally, add the pre-excitation solution (H2O2) and the excitation solution (NaOH) to react. The acridinium ester reacts with hydrogen peroxide to generate ethylene dioxide. Ethylene dioxide is extremely unstable and decomposes to produce CO2 and electronically excited N-methylacridone. When N-methylacridone returns to its ground state, it emits light at 430 nm. The luminescence signal is collected and the anti-Tropomyosin 1-IgG antibody concentration is calculated using a calibration curve.

[0106] Example 6 Clinical Application of a Kit for Detecting Anti-Tropomyosin 1-IgG Antibodies in Serum

[0107] 6.1 Subjects: Patients diagnosed with primary nephrotic syndrome between June 2018 and June 2020; healthy controls were drawn from healthy individuals undergoing physical examinations during the same period. The control group included 195 healthy individuals, and the patient group included 168 patients with Henoch-Schönlein purpura, 137 patients with Henoch-Schönlein purpura nephritis, 133 patients with IgA nephropathy, and 466 patients with nephrotic syndrome. Serum samples were obtained from both the patient group and the healthy control group. Serum samples were collected from all subjects before immunosuppressive therapy was initiated.

[0108] 6.2 This kit was used to test the anti-Tropomyosin 1-IgG antibodies in 195 healthy subjects, including 168 patients with Henoch-Schonlein purpura, 137 patients with Henoch-Schonlein purpura nephritis, 133 patients with IgA nephropathy, and 466 patients with nephrotic syndrome. The results showed that the anti-Tropomyosin 1-IgG antibodies were positive in patients with primary nephrotic syndrome, while the anti-Tropomyosin 1-IgG antibodies were negative in patients with purpura nephritis, Henoch-Schonlein purpura, IgA nephropathy, and healthy children. Figure 6 It is obvious from the figure that only the anti-Tropomyosin 1-IgG antibody is positive in autoimmune nephrotic syndrome.

[0109] 6.3 ROC Curve Evaluation of the Value of Anti-Tropomyosin 1-IgG Antibody as a Serological Marker for the Diagnosis of Patients with Autoimmune Nephrotic Syndrome The ROC curve was used to analyze the detection results of anti-Tropomyosin 1-IgG antibodies in patients with nephrotic syndrome to determine the value of this antibody in the diagnosis of nephrotic syndrome. The results showed that anti-Tropomyosin 1-IgG antibody is a good serological marker for the diagnosis of autoimmune nephrotic syndrome. Using an anti-Tropomyosin 1-IgG antibody greater than 9.2 as the diagnostic cutoff for the diagnosis of autoimmune nephrotic syndrome, the sensitivity was 61.11%, the specificity was 81.36%, and the area under the curve was 0.735. Figure 7 . SEQUENCE LISTING <110> Zhejiang University <120> A kit for detecting antigen myosin 1-IgG antibody <130> 2022.5.10 <160> 1 <170> PatentIn version 3.5 <210> 1 <211> 70 <212> PRT <213> Artificial sequence (Unknow) <400> 1 Met Ala Gly Ser Ser Ser Leu Glu Ala Val Arg Arg Lys Ile Arg Ser 1 5 10 15 Leu Gln Glu Gln Ala Asp Ala Ala Glu Glu Arg Ala Gly Thr Leu Gln 20 25 30 Arg Glu Leu Asp His Glu Arg Lys Leu Arg Glu Thr Ala Glu Ala Asp 35 40 45 Val Ala Ser Leu Asn Arg Arg Ile Gln Leu Val Glu Glu Glu Leu Asp 50 55 60 Arg Ala Gln Glu Arg Leu 65 70

Claims

1. Use of a tropomyosin 1 antibody binding fragment capable of forming any antigen-antibody complex in contact with a sample obtained from a patient in the preparation of a nephrotic syndrome detection reagent or kit; wherein: The antigen-antibody complex includes an antigen myosin 1-IgG antibody (anti-Tropomyosin1-IgG antibody) complex; wherein the nephrotic syndrome is autoimmune nephrotic syndrome; The sequence of the tropomyosin 1 antibody binding fragment is shown in SEQ ID NO.

1.

2. The use according to claim 1, wherein the sample is serum.

3. The use according to claim 1, wherein the sample is a sample from a patient before immunotherapy.

4. Use of a tropomyosin 1 antibody binding fragment capable of forming any antigen-antibody complex in contact with a sample obtained from a patient in the preparation of a reagent or kit for specifically detecting nephrotic syndrome relative to purpura nephritis, Henoch-Schonlein purpura, or IgA nephropathy; wherein: The antigen-antibody complex includes an antigen myosin 1-IgG antibody (anti-Tropomyosin 1-IgG antibody) complex; wherein the nephrotic syndrome is autoimmune nephrotic syndrome; The sequence of the tropomyosin 1 antibody binding fragment is shown in SEQ ID NO.1.

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