A deconstructor for affinity testing of autoantibodies in primary membranous nephropathy

Abstract

The present invention discloses a deconstructor for the affinity test of autoantibodies for primary membranous nephropathy, which belongs to the field of medicine. The method is based on an immunological method to detect the affinity of antibodies in human samples, thereby evaluating the condition of kidney disease, predicting the prognosis, and judging the therapeutic effect.

Description

Technical Field

[0001] The invention relates to a deconstructor for primary membranous nephropathy autoantibody affinity test and belongs to the medical field. Background Art

[0002] Idiopathic nephropathy (IMN) is the most common type of kidney disease, accounting for about 15-20% of all kidney diseases. Compared with other kidney diseases, the prognosis of IMN is poor, and nearly 50% of IMN eventually develop into renal failure. There is currently no effective treatment for patients who have been confirmed to have IMN. If the risk of IMN can be detected as early as possible before the symptoms of IMN appear, and preventive measures can be taken in time, the incidence of IMN and the poor prognosis of IMN will be significantly reduced.

[0003] Since Beck et al. discovered in 2009 that Pla2r is a specific marker for IMN, the diagnosis and treatment of IMN has progressed rapidly and has been one of the hot topics in the field of kidney disease diagnosis and treatment in the past decade. Clinical practice has confirmed that antibody concentration and affinity are key factors in the progression of autoimmune diseases; the same is true for IMN driven by Pla2r antibodies; the Kidney Disease: Improving Global Outcomes (KDIGO) is the most authoritative kidney disease medical organization in the world. The "Guidelines for Clinical Practice in Glomerulonephritis" issued in 2021 clearly pointed out that Pla2r antibody concentration and affinity are closely related to clinical prognosis, that is, high Pla2r antibody concentration and high affinity indicate a high risk of worsening kidney function. Pla2r plays two major roles in the management of IMN: (1) Early detection: IMN is an autoimmune disease, that is, the patient develops an immune attack against his own glomerular cells, causing renal damage. Before the onset of clinical symptoms, high concentrations of Pla2r have already appeared in the patient's serum, and the antibody affinity of Pla2r has increased. Through timely Pla2r antibody testing, positive patients can be found and immune blocking can be used to significantly reduce the incidence of IMN or to improve the prognosis of IMN to a limited extent. (2) Disease monitoring: For confirmed IMN patients, the Pla2r antibody concentration and antibody affinity in their serum are highly consistent with the recurrence of the disease. By detecting the Pla2r antibody concentration and antibody affinity in the serum, the IMN disease can be detected in real time to avoid worsening of the disease. Although the development of Pla2r antibody concentration detection kits is quite mature, a single Pla2r antibody concentration is not sufficient to represent the severity and prognosis of IMN. Using Pla2r antibody affinity for auxiliary evaluation is expected to further enhance the accuracy of IMN screening / evaluation.

[0004] The affinity of an antibody refers to the ability and tightness of the antibody to bind to an antigen; antibody affinity plays a key role in the occurrence and development of the disease; for example, the affinity measurement kits that have been on the market, the cytomegalovirus IgG antibody affinity detection kit, the Toxoplasma IgG antibody affinity detection kit, the rubella virus IgG antibody affinity detection kit, the herpes simplex virus IgG antibody affinity detection kit, etc., all reflect the severity and changes of the disease through antibody affinity.

[0005] Antibody affinity is generally determined by the secondary bonds between "antibody→antigen". The so-called secondary bonds are: hydrogen bonds, salt bonds (ionic bonds, chemical bonds formed by electrostatic effects), van der Waals forces and hydrophobic bonds; antibody affinity testing is achieved by measuring the substances or external forces required to destroy the above secondary bonds. For example, the common antibody affinity testing methods generally use enzyme-linked immunosorbent assay, using antigens for competition, or using deconstructors to examine the differences or ratios between the test group and the control group, and calculate the relative affinity constant (RAI) of the antibody; among them, the antigen competition method is complex to operate, has many interfering factors, and has poor repeatability, so it is rarely used; most of the commercialized test kits use deconstructors (such as urea, guanidine hydrochloride, etc.) as the technical route for antibody affinity testing.

[0006] The authenticity of the affinity test results must first ensure that the detection antigen is not affected, that is, the deconstructor cannot cause the antigen to undergo changes in its immunological activity. This has an extremely huge impact on some detection antigens with conformational epitopes. Secondly, even linear epitope detection antigens will be affected by the deconstructor, but not significantly.

[0007] The affinity determination of Pla2r antibodies is not suitable for the technical route of conventional deconstructors for the following reasons: First, the antigenic epitope of Pla2r antigen, whether in the body or coated on a carrier in the detection kit, is mainly a conformational epitope rather than a linear epitope; and the deconstructors used in common affinity tests can easily destroy the conformational epitope of the Pla2r detection antigen, thereby distorting the results of the affinity test; secondly, the conformational epitope of the Pla2r antigen is mainly formed based on disulfide bonds. In addition to being affected by deconstructors, it is also easily destroyed by factors such as oxidation, thereby distorting the results of the Pla2r antibody affinity test; finally, Pla2r may produce epitope expansion at different stages of the disease to form complex polyclonal antibodies; the affinity of these polyclonal antibodies covers a variety of secondary bond structures, which places high demands on deconstructors to ensure the authenticity of the Pla2r antibody affinity test results.

[0008] In summary, given the special and complex requirements, it is currently very difficult to accurately measure the affinity of Pla2r antibodies. Screening for deconstructors suitable for detecting the affinity of Pla2r antibodies is the key to solving this technical problem. Summary of the invention

[0009] The purpose of the present invention is to overcome the shortcomings of the above-mentioned prior art and provide a deconstructor for the affinity test of primary membranous nephropathy autoantibodies, which is used to detect the affinity of phospholipase a2 receptor protein-specific antibodies (Pla2r-Ab) in human samples.

[0010] In order to achieve the above object, the present invention provides a deconstructor for affinity test of primary membranous nephropathy autoantibodies, which contains the following components: SDS, guanidine hydrochloride, water, isothiocyanate derivatives and compounds containing peroxide groups.

[0011] In one embodiment, the destructurizing agent further contains sodium chloride.

[0012] In one embodiment, the isothiocyanate derivative includes benzyl isothiocyanate or phenethyl isothiocyanate.

[0013] In one embodiment, the peroxide group-containing compound comprises potassium peroxymonosulfonate or benzoyl peroxide.

[0014] In one embodiment, in the deconstructing agent, the mass percentage of SDS is 0.5-5%; the concentration of guanidine hydrochloride is 2-4 mol / L; the mass percentage of isothiocyanate derivatives is 0.5-1%; the mass percentage of compounds containing peroxide groups is 1-2.5%; and the mass percentage of sodium chloride is 0-1%.

[0015] In one embodiment, the destructurizing agent consists of SDS, guanidine hydrochloride, water, sodium chloride, benzyl isothiocyanate and potassium peroxymonosulfonate; the mass percentage of SDS is 0.5-5%; the concentration of guanidine hydrochloride is 2-4 mol / L; the mass percentage of benzyl isothiocyanate is 0.5-1%; the mass percentage of potassium peroxymonosulfonate is 2-2.5%; and the mass percentage of sodium chloride is 0.05-1%.

[0016] In one embodiment, the destructurizing agent consists of SDS, guanidine hydrochloride, water, phenethyl isothiocyanate and benzoyl peroxide; the mass percentage of SDS is 2.5-5%; the concentration of guanidine hydrochloride is 2-3 mol / L; the mass percentage of phenethyl isothiocyanate is 0.75-1%; and the mass percentage of benzoyl peroxide is 2-2.5%.

[0017] The present invention also provides a method for detecting the affinity of a phospholipase A2 receptor protein-specific antibody using the deconstructor.

[0018] In one embodiment, the method is to combine a sample containing a phospholipase A2 receptor protein-specific antibody with a phospholipase A2 receptor protein labeled with a marker, and detect the detection signal before and after the deconstructor is added to the sample.

[0019] In one embodiment, the ratio of the detection signal after adding the destructurizing agent to that before adding the destructurizing agent is calculated; the ratio is closely related to the prognosis and therapeutic effect of primary membranous nephropathy.

[0020] In one embodiment, the ratio is greater than or equal to 0.65, corresponding to a more severe kidney disease; and the larger the ratio, the more severe the disease.

[0021] The present invention also provides the application of the deconstructing agent or the method in preparing a diagnostic reagent for primary membranous nephropathy.

[0022] Beneficial effects:

[0023] (1) The deconstructor of the primary membranous nephropathy autoantibody affinity test of the present invention can ensure the authenticity of the antibody affinity test under the premise of protecting the stability of the phospholipase A2 receptor protein antigen; the detection sensitivity reaches more than 91% and the specificity reaches more than 88%.

[0024] (2) Based on the above deconstructors, a method for affinity testing of phospholipase A2 receptor protein-specific antibodies is provided for disease assessment and monitoring of treatment effects of primary membranous nephropathy, which can effectively treat IMN patients;

[0025] (3) The detection method is versatile and can be performed quickly and conveniently.

[0026] Therefore, the detection method of primary membranous nephropathy based on immunological method of the patent of the present invention is easy to use and can be used for the condition assessment and treatment effect monitoring of IMN patients. The detection results provide a strong basis for the next step of accurate treatment and are suitable for large-scale promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The spray point positions of the test and control belts;

[0028] Figure 2 This is a schematic diagram of the structure of a large card;

[0029] Figure 3 The receiver operating characteristic curve for the affinity of phospholipase A2 receptor protein-specific antibodies in diagnosing the severity of IMN symptoms;

[0030] Figure 4 The receiver operating characteristic curve for evaluating the therapeutic effect of IMN by the affinity of the antibody specific for the phospholipase A2 receptor protein;

[0031] Figure 5 The receiver operating characteristic curve for the affinity of phospholipase A2 receptor protein-specific antibodies in diagnosing the severity of IMN symptoms;

[0032] Figure 6 Receiver operating characteristic curve for the evaluation of IMN treatment efficacy by affinity of specific antibodies for phospholipase A2 receptor protein. DETAILED DESCRIPTION

[0033] In order to more clearly understand the technical content of the present invention, the following embodiments are given in detail.

[0034] The preparation process of the primary membranous nephropathy specific antibody affinity detection reagent of the present invention is briefly described as follows. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer.

[0035] The percentages involved in the following examples, unless otherwise specified, refer to mass ratios, such as 1% SDS means that 1 g of SDS is contained in 100 mL of solution in the system.

[0036] Example 1 Destructuring Agent 1

[0037] The deconstructor contains the following ingredients:

[0038] (1) SDS 1%;

[0039] (2) Guanidine hydrochloride 4 mol / L;

[0040] (3) Sodium chloride 0.85%;

[0041] (4) Benzyl isothiocyanate 0.5%;

[0042] (5) Potassium peroxymonosulfonate 1%;

[0043] (6) Purified water.

[0044] The content of each component can be adjusted within the following ranges: the mass percentage of SDS is 0.5-5%; the concentration of guanidine hydrochloride is 2-4 mol / L; the mass percentage of benzyl isothiocyanate is 0.5-1%; the mass percentage of potassium peroxymonosulfonate is 2-2.5%; and the mass percentage of sodium chloride is 0.05-1%.

[0045] Example 2 Destructuring Agent 2

[0046] The deconstructor contains the following ingredients:

[0047] (1) SDS 0.5%;

[0048] (2) Guanidine hydrochloride 2 mol / L;

[0049] (3) 1% phenylethyl isothiocyanate;

[0050] (4) Benzoyl peroxide 2%;

[0051] (5) Purified water.

[0052] The content of each component can be adjusted within the following ranges: the mass percentage of SDS is 0.5-5%; the concentration of guanidine hydrochloride is 2-3 mol / L; the mass percentage of phenethyl isothiocyanate is 0.75-1%; and the mass percentage of benzoyl peroxide is 2-2.5%.

[0053] Example 3 Destructuring Agent 3

[0054] The deconstructor contains the following ingredients:

[0055] (1) SDS 5%;

[0056] (2) Guanidine hydrochloride 2 mol / L;

[0057] (3) 1% phenylethyl isothiocyanate;

[0058] (4) Benzoyl peroxide 2.5%;

[0059] (5) Purified water.

[0060] The content of each component can be adjusted within the following ranges: the mass percentage of SDS is 0.5-5%; the concentration of guanidine hydrochloride is 2-3 mol / L; the mass percentage of phenethyl isothiocyanate is 0.75-1%; and the mass percentage of benzoyl peroxide is 2-2.5%.

[0061] Example 4 Application of the Deconstructor on the Primary Membranous Nephropathy Autoantibody Detection Test Strip

[0062] Step 1: Fluorescent microsphere labeling

[0063] Washing: Take 200 μL fluorescent microspheres (Bangs Lab, catalog number: 11233) into a centrifuge tube, add 800 μL of 0.1M MES (pH 5.0) buffer and mix well, centrifuge at 13000 rpm, 15 min, 4°C, discard the supernatant, and resuspend with 400 μL 0.1M MES (pH 5.0) buffer for use. Activation and washing: Activate the activator 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS) and fluorescent microspheres in a mass ratio of 2:1:2. The specific operation is as follows:

[0064] Weigh 2mg EDC and 1mg NHS and dissolve them in 600μL 0.1M MES (pH 5.0) buffer, quickly add them to the washed 2mg fluorescent microspheres, seal with sealing film, place on a 200rpm shaker at room temperature for 30min, take out, centrifuge at 13000rpm, 30min, 4℃ to remove the supernatant, resuspend with 1mL 0.1M MES (pH 6.5) buffer, mix by ultrasound and wash, repeat the above operation once, that is, wash twice. After centrifugation, discard the supernatant for later use.

[0065] Labeling: Resuspend the activated fluorescent microspheres in 500 μL 0.1 M MES (pH 6.5) buffer, quickly add 500 μL of single-streptavidin (Panga Gene, Catalog No.: MSA-01005) with a final concentration of 0.25 mg / mL, mix well, place at room temperature, and shake on a shaker at 200 rpm for 4 hours.

[0066] Blocking: Take the labeled microspheres mentioned above and centrifuge them at 13000 rpm and 4°C for 30 min, remove the supernatant, and collect the precipitated labeled microspheres; measure the protein content (unlabeled single-streptavidin) in the supernatant using the BCA protein concentration assay, which should meet the requirements of Table 1.

[0067] Add 1 mL of blocking solution (final concentration of 2% bovine serum albumin, 1% polyethylene glycol 20000 in 0.1 M M ES (pH 6.5) solution) to the precipitated microspheres, resuspend by ultrasound, and place at room temperature, shake on a shaker at 200 rpm for 1 hour. After the reaction is completed, centrifuge at 13000 rpm, 4°C, 20 min, remove the supernatant, and collect the precipitated fluorescent microspheres.

[0068] Table 1

[0069]

[0070]

[0071] The precipitated fluorescent microspheres were placed in 0.5 mL 0.1 M MES (pH 6.5) buffer, ultrasonically blown and mixed, and biotinylated recombinant Pla2r protein (Longwei Biotechnology, catalog number: RW-TC018-AG-02) was added at a ratio of 15 μg / mg single-streptavidin labeled microspheres, and reacted at room temperature for 30 minutes. The precipitate was collected by centrifugation at 13000 rpm, 20 minutes, and 4°C in a high-speed refrigerated centrifuge.

[0072] Wash & resuspend: add 1mL resuspending solution (final concentration of 0.5% bovine serum albumin, 0.1% polyethylene glycol 20000, 1% Tween 20, 2% trehalose in 0.1M MES (pH6.5) solution), resuspend by ultrasonic blowing, centrifuge at 13000rpm, 20min, 4℃, discard the supernatant; add 200μL resuspending solution, resuspend by ultrasonic blowing, add biotinylated recombinant Pla2r protein to the labeled microsphere solution, the ratio is 15μg / mg single-chain streptavidin labeled microspheres. This is the fluorescent labeled Pla2r antigen solution.

[0073] Step 2: Spraying the bonding pad

[0074] The spraying method of the conjugate pad for membranous nephropathy detection is as follows: dilution of fluorescent labeled Pla2r antigen solution: dilute the fluorescent labeled Pla2r antigen solution prepared above by 4 times with a resuspension solution (final concentration of 0.5% bovine serum albumin, 0.1% polyethylene glycol 20000, 1% Tween 20, 2% trehalose in 0.1MMES (pH 6.5) solution) to obtain a fluorescent labeled Pla2r antigen resuspension solution; set the membrane spotter, turn on the power of the membrane spotter, set the spraying program, and the spraying volume is 8ul / cm; pipe No. 1 is the spraying channel; initialization of the membrane spotter: place pipe No. 1 in the fluorescent labeled Pla2r antigen resuspension solution. In the solution, select the initialization program and initialize 6 cycles; spraying: place the conjugate pad (Ahlstrom 8964, item number: JY-BX101) flat on the film spotter in a fixed position, press the "GO" button on the control panel to start spraying, remove it after spraying, and check the sprayed conjugate pad. The fluorescent labeled Pla2r antigen band sprayed is uniform, continuous, and a straight line running through the entire conjugate pad is a qualified spray product, and the presence of breakpoints is an unqualified spray product; for each conjugate pad placed, press the "GO" button on the control panel once to spray once (one piece); after spraying, place the sprayed conjugate pad at room temperature to dry naturally for 1 hour, and no traces of spraying should be seen on the membrane.

[0075] Step 3: Preparation of nitrocellulose membrane

[0076] The preparation method of nitrocellulose membrane (NC membrane, Sartorius, catalog number: CN140) for membranous nephropathy detection is as follows: 500ug of mouse anti-human IgG4 monoclonal antibody (Abcam, ab238320) is added to a 5ml graduated centrifuge tube, and the antibody diluent (0.1M PB (pH7.2) solution with a final concentration of 0.01% Tween 20 and 3% trehalose) is added to make the volume up to 1ml, which is the detection zone solution, and the container is marked with T. Separately, 25ul of rabbit anti-human Pla2r polyclonal antibody (OriGene, catalog number: AP55261PU-N) is added to a 5ml graduated centrifuge tube, and the antibody diluent is added to make the volume up to 1ml, which is the control zone solution, and the container is marked with C. Set the dot meter, turn on the power of the dot meter, set the dot program, and the dot volume is 1ul / cm; pipe No. 1 is the test strip dot channel, and pipe No. 2 is the control strip dot channel; dot meter initialization: place pipe No. 1 in the test strip solution, place pipe No. 2 in the control strip solution, select the initialization program, and initialize 6 cycles; dot: place the nitrocellulose membrane flat on the dot meter in a fixed position, press the "GO" button on the control panel to start dot, remove it after dot, check the nitrocellulose membrane with dot, the test strip and the control strip are two uniform, continuous and straight lines that run through the entire nitrocellulose membrane, which is a qualified dot product (the position of the test strip and the control strip, see the schematic diagram Figure 1 ), if there is a break in the two straight lines, it is an unqualified spray product; for each nitrocellulose membrane placed, press the "GO" button on the control panel once for one spray (one piece); after the spraying is completed, place the sprayed nitrocellulose membrane at room temperature to dry naturally for 1 hour, and no spray marks should be visible on the membrane.

[0077] Step 4: Film pasting and film cutting

[0078] Remove the wider protective paper on the bottom plate (Hangzhou Ruijian Technology Co., Ltd., item number: C12302), and stick the marked nitrocellulose membrane on the bottom plate along the lower edge of the upper protective paper with the control band on the top; stick the conjugate pad under the test band, slightly contacting the NC membrane; stick the sample pad (Jieyi Biological, item number: JY-BX16) under the conjugate pad, slightly contacting the conjugate pad; then remove the upper protective paper, stick the absorbent paper (Jieyi Biological, item number: H5072) on the top of the NC membrane, slightly contacting the NC membrane; stick the protective paper and indicator paper one by one on the outside of the assembled test strips to assemble into a large card (see the attached schematic diagram of the structure of the large card) Figure 2 ).

[0079] Turn on the power of the cutting machine, set the film cutting program, set the cutting width to 4mm and the length to 7.8cm; place the large-calorie qualified product flat on the cutting machine platform track with the front side facing up, press the "GO" button on the operation panel to start cutting; press the "GO" button on the operation panel once for each large-calorie qualified product placed until all large-calorie qualified products are cut; after cutting is completed, it is the primary membranous nephropathy test strip.

[0080] Step 5: Kit Assembly

[0081] The above-mentioned test strips are placed in a card box to form a test card.

[0082] Take the aluminum foil bag and desiccant; turn on the heat sealer and preheat; put the test card to be bagged and 1 bag of desiccant into the aluminum foil bag; cut the aluminum foil bag containing the test card and desiccant according to the specified length; seal the aluminum foil bag with the heat sealer; and attach a label.

[0083] Step 6: Clinical sample verification

[0084] The above test card was validated using clinical samples.

[0085] Add 80 μL of the same sample to be tested to the sample wells of test card A and test card B, respectively, and then add 50 μL of deconstructor 1 to B, and then place it in a fluorescence detector for reading; observe the correlation between the B / A ratio and the IMN condition.

[0086] The test results and clinical diagnosis results are shown in Table 2:

[0087] Table 2

[0088]

[0089]

[0090] Negative samples: For patients with mild symptoms, high-affinity antibodies should be negative;

[0091] Positive samples: For patients with more severe symptoms, high-affinity antibodies should be positive;

[0092] The ROC curve was calculated using the IBM SPSS Statistics data editor, and it was determined that when the cutoff value was 0.5, the AUC was the largest (0.9253) ( Figure 3 ), the corresponding detection results are shown in Table 3.

[0093] Table 3

[0094] Test results 48 negative samples 23 positive samples - 45 2 + 3 21

[0095] The results showed that the sensitivity of the detection using the deconstruction agent of the present invention reached 91.3% and the specificity reached 93.7%.

[0096] Sensitivity = number of positive samples with (+) test results / number of positive samples = 21 / 23 = 91.3%;

[0097] Specificity = number of negative samples with (-) test results / number of negative samples = 45 / 48 = 93.7%;

[0098] Step 7: Clinical sample verification

[0099] The test card prepared in step five was verified using clinical samples.

[0100] Add 80 μL of the same sample to be tested to the sample wells of test card A and test card B, respectively, and then add 50 μL of deconstructor 2 to B, and then place it in a fluorescence detector for reading; observe the correlation between the B / A ratio and whether the IMN treatment is effective.

[0101] The test results and clinical diagnosis results are shown in Table 4:

[0102] Table 4

[0103]

[0104] Negative samples: For patients who have been effectively treated, high-affinity antibodies should be negative;

[0105] Positive samples: Patients who have not responded to treatment, high-affinity antibodies should be positive;

[0106] The ROC curve was calculated using the IBM SPSS Statistics data editor, and it was determined that when the cutoff value was 0.5, the AUC was the largest (0.9118) ( Figure 4 ), the corresponding detection results are shown in Table 5.

[0107] Table 5

[0108] Test results 34 negative samples 51 positive samples - 30 3 + 4 48

[0109] The results showed that the sensitivity of the detection using the deconstruction agent of the present invention reached 94.1% and the specificity reached 88.2%.

[0110] Sensitivity = number of positive samples with (+) test results / number of positive samples = 48 / 51 = 94.1%;

[0111] Specificity = number of negative samples with (-) test results / number of negative samples = 30 / 34 = 88.2%;

[0112] The results of the above examples show that the affinity detection of the IMN-specific autoantibodies of the present invention can make accurate judgments on the disease assessment and treatment effect evaluation of IMN, and meet the clinical application requirements.

[0113] Example 5 Application of Deconstructor on Primary Membranous Nephropathy Autoantibody Detection Microplate

[0114] Step 1: HRP labeling of phospholipase A2 receptor protein

[0115] Add 5 μg of biotinylated recombinant Pla2r protein to 0.1 mL of HRP-labeled streptavidin (Biyuntian, catalog number: A0303, specification: 0.2 mL / bottle), mix well, cover the bottle stopper, and incubate at room temperature in the dark for 0.5 hour to obtain the HRP-labeled Pla2r antigen.

[0116] Storage of HRP-labeled Pla2r antigen conjugate: After adding an equal amount of glycerol, divide into small portions and store at -20°C to prevent repeated freezing and thawing.

[0117] Step 2: Coating

[0118] The mouse anti-human IgG4 antibody was diluted to 1 μg / ml using 0.01M CBS (carbonate buffer) at pH 10; 100 ul of the above-mentioned coated antibody was added to each well of a 96-well microplate; the plate was incubated at 37°C for 1 h; the coating solution was discarded, 200 ul of blocking buffer (2% bovine serum albumin, pH 7.4) was added, the plate was incubated at 37°C for 1 h, and the blocking solution was discarded; the plate was naturally dried at room temperature for 12 h, a desiccant was added, and the plate was sealed in an aluminum foil bag and stored at 2-8°C for later use.

[0119] Step 3: ELISA kit assembly

[0120] The HRP-labeled antigen (from step 1) was dispensed into 100 μL / tube, and coated with the anti-IgG4 plate (from step 2), and placed in a packaging box to form an IMN detection microplate.

[0121] The primary membranous nephropathy samples were tested according to the following steps: the samples were added to the anti-IgG4 coated plate, reacted at 37°C for 30 min, washed 5 times with 1×PBST solution, HRP-labeled Pla2r antigen diluted 100 times with 1×PBST was added, reacted at 37°C for 30 min, washed 5 times with 1×PBST solution, and then the color developing solution (Biyuntian, product number: P0206) was added. After reacting at 37°C for 10 min, the OD value at 450 nm was read.

[0122] Step 4: Clinical sample verification

[0123] The above-mentioned microplate was validated using clinical samples.

[0124] 100 μL of the same sample to be tested was dripped into microwell A and microwell B respectively, and 50 μL of deconstructor 3 was dripped into microwell B, and then placed in an ELISA reader for reading; the correlation between the ratio of B / A and the condition of IMN was observed.

[0125] The test results and clinical diagnosis results are shown in Table 6:

[0126] Table 6

[0127]

[0128]

[0129] Negative samples: For patients with mild symptoms, high-affinity antibodies should be negative;

[0130] Positive samples: For patients with more severe symptoms, high-affinity antibodies should be positive;

[0131] The ROC curve was calculated using the IBM SPSS Statistics data editor, and it was determined that when the cutoff value was 0.5, the AUC was the largest (0.9470) ( Figure 5 ), the corresponding detection results are shown in Table 7.

[0132] Table 7

[0133] Test results 48 negative samples 23 positive samples - 45 1 + 3 22

[0134] The results showed that the sensitivity of the detection using the deconstruction agent of the present invention reached 95.6% and the specificity reached 93.7%.

[0135] Sensitivity = number of positive samples with test results (+) / number of positive samples = 22 / 23 = 95.6%;

[0136] Specificity = negative samples and number of test results (-) / number of negative samples = 45 / 48 = 93.7%.

[0137] Step 5: Clinical sample verification

[0138] The microplate prepared in step 3 was validated using clinical samples.

[0139] 100 μL of the same sample to be tested was added to microwell A and microwell B respectively, and 50 μL of deconstructor 3 was added to microwell B, and then placed in an ELISA reader for reading; the correlation between the B / A ratio and whether the IMN treatment is effective was observed.

[0140] The test results and clinical diagnosis results are shown in Table 8:

[0141] Table 8

[0142]

[0143]

[0144] Negative samples: For patients who have been effectively treated, high-affinity antibodies should be negative;

[0145] Positive samples: Patients who have not responded to treatment, high-affinity antibodies should be positive;

[0146] The ROC curve was calculated using the IBM SPSS Statistics data editor, and it was determined that when the cutoff value was 0.5, the AUC was the largest (0.9362) ( Figure 6 ), the corresponding test results are shown in Table 9.

[0147] Table 9

[0148] Test results 34 negative samples 51 positive samples - 31 2 + 3 49

[0149] The results showed that the sensitivity of the detection using the deconstructor of the present invention reached 96.1% and the specificity reached 91.2%.

[0150] Sensitivity = number of positive samples with (+) test results / number of positive samples = 49 / 51 = 96.1%;

[0151] Specificity = negative samples and number of test results (-) / number of negative samples = 31 / 34 = 91.2%.

[0152] The results of the above examples show that the affinity detection of the IMN-specific autoantibodies of the present invention can make accurate judgments on the disease assessment and treatment effect evaluation of IMN, and meet the clinical application requirements.

[0153] Therefore, the patent of the present invention improves the disease assessment and treatment effect evaluation of primary membranous nephropathy through immunological methods, and can carry out more targeted treatment plans based on the test results.

[0154] Comparative Example 1 Destructuring Agent 4

[0155] Specific implementation method Referring to Example 1, the difference is that benzyl isothiocyanate is not added;

[0156] The deconstructor contains the following ingredients:

[0157] (1) SDS 1%;

[0158] (2) Guanidine hydrochloride 4 mol / L;

[0159] (3) Sodium chloride 0.85%;

[0160] (4) Potassium peroxymonosulfonate 1%;

[0161] (5) Purified water.

[0162] The test card prepared in Example 4 was verified using clinical samples.

[0163] Add 80 μL of the same sample to be tested to the sample wells of test card A and test card B, respectively, and then add 50 μL of deconstructor 4 to B, and then place it in a fluorescence detector for reading; observe the correlation between the B / A ratio and whether the IMN treatment is effective.

[0164] The test results and clinical diagnosis results are shown in Table 10:

[0165] Table 10

[0166]

[0167] From the above data, it can be seen that dissociator 4 cannot effectively dissociate antibodies with different affinities, resulting in the B / A value being close to 1.0 for almost all samples - that is, the value of well B is almost the same as that of well A.

[0168] The above comparative example results show that the component of the present invention, benzyl isothiocyanate, is essential for the affinity detection of IMN-specific autoantibodies.

[0169] Comparative Example 2 Destructuring Agent 5

[0170] Specific implementation method Referring to Example 2, the difference is that no benzoyl peroxide is added; the destructurizing agent contains the following ingredients:

[0171] (1) SDS 0.5%;

[0172] (2) Guanidine hydrochloride 2 mol / L;

[0173] (3) 1% phenylethyl isothiocyanate;

[0174] (4) Purified water.

[0175] The microplate prepared in Example 5 was verified using clinical samples.

[0176] 100 μL of the same sample to be tested was added to microwell A and microwell B respectively, and 50 μL of deconstructor 5 was added to microwell B, and then placed in an ELISA reader for reading; the correlation between the B / A ratio and whether the IMN treatment is effective was observed.

[0177] The test results and clinical diagnosis results are shown in Table 11:

[0178] Table 11

[0179]

[0180] From the above data, it can be seen that dissociator 5 cannot effectively dissociate antibodies with different affinities, resulting in the B / A value being close to 1.0 for almost all samples - that is, the value of well B is almost the same as that of well A.

[0181] The above comparative example results show that the component of the present invention, benzoyl peroxide, is essential for the affinity detection of IMN-specific autoantibodies.

[0182] Comparative Example 3 Destructuring Agent 6

[0183] Specific implementation method Referring to Example 1, the difference is that benzyl benzoate is used to replace benzyl isothiocyanate; the deconstructing agent contains the following ingredients:

[0184] (1) SDS 0.5%;

[0185] (2) Guanidine hydrochloride 2 mol / L;

[0186] (3) Sodium chloride 0.85%

[0187] (4) Benzyl benzoate 1%;

[0188] (5) Potassium peroxymonosulfonate 1%;

[0189] (6) Purified water.

[0190] The microplate prepared in Example 5 was verified using clinical samples.

[0191] 100 μL of the same sample to be tested was added to microwell A and microwell B respectively, and 50 μL of deconstructor 5 was added to microwell B, and then placed in an ELISA reader for reading; the correlation between the B / A ratio and whether the IMN treatment is effective was observed.

[0192] The test results and clinical diagnosis results are shown in Table 12:

[0193] Table 12

[0194]

[0195] From the above data, it can be seen that dissociator 6 cannot effectively dissociate antibodies with different affinities, resulting in the B / A value being close to 1.0 for almost all samples - that is, the value of well B is almost the same as that of well A.

[0196] The above comparative example results show that the component of the present invention, benzyl isothiocyanate, is essential for the affinity detection of IMN-specific autoantibodies.

[0197] Comparative Example 4 Destructuring Agent 7

[0198] Specific implementation method Referring to Example 1, the difference is that potassium peroxymonosulfate is replaced by potassium hydrogen persulfate; the destructurizing agent contains the following ingredients:

[0199] (1) SDS 1%;

[0200] (2) Guanidine hydrochloride 4 mol / L;

[0201] (3) Sodium chloride 0.85%;

[0202] (4) 0.5% phenylethyl isothiocyanate;

[0203] (5) Potassium persulfate 1%;

[0204] (6) Purified water.

[0205] The microplate prepared in Example 5 was verified using clinical samples.

[0206] 100 μL of the same sample to be tested was added to microwell A and microwell B respectively, and 50 μL of deconstructor 5 was added to microwell B, and then placed in an ELISA reader for reading; the correlation between the B / A ratio and whether the IMN treatment is effective was observed.

[0207] The test results and clinical diagnosis results are shown in Table 13:

[0208] Table 13

[0209]

[0210] From the above data, it can be seen that dissociator 7 cannot effectively dissociate antibodies with different affinities, resulting in the B / A value being close to 1.0 for almost all samples - that is, the value of well B is almost the same as that of well A.

[0211] The above comparative example results show that the component of the present invention, potassium peroxymonosulfonate, is essential for the affinity detection of IMN-specific autoantibodies.

[0212] Comparative Example 5 Destructuring Agent 8

[0213] Specific implementation method Referring to Example 1, the difference is that the concentration of benzyl isothiocyanate is adjusted to 0.1%; the destructurizing agent contains the following ingredients:

[0214] (1) SDS 1%;

[0215] (2) Guanidine hydrochloride 4 mol / L;

[0216] (3) Sodium chloride 0.85%;

[0217] (4) 0.1% phenylethyl isothiocyanate;

[0218] (5) Potassium peroxymonosulfonate 1%;

[0219] (6) Purified water.

[0220] The microplate prepared in Example 5 was verified using clinical samples.

[0221] 100 μL of the same sample to be tested was added to microwell A and microwell B respectively, and 50 μL of deconstructor 5 was added to microwell B, and then placed in an ELISA reader for reading; the correlation between the B / A ratio and whether the IMN treatment is effective was observed.

[0222] The test results and clinical diagnosis results are shown in Table 14:

[0223] Table 14

[0224]

[0225] From the above data, it can be seen that dissociator 8 cannot effectively dissociate antibodies with different affinities, resulting in the B / A value being close to 1.0 for almost all samples - that is, the value of well B is almost the same as that of well A.

[0226] The above comparative example results show that the concentration of the component of the present invention, phenylethyl isothiocyanate, is necessary for the affinity detection of IMN-specific autoantibodies.

[0227] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A deconstructor for affinity testing of phospholipase A2 receptor protein-specific antibodies, characterized in that: The deconstructing agent is composed of SDS, guanidine hydrochloride, water, sodium chloride, benzyl isothiocyanate and potassium peroxymonosulfonate; the mass percentage of SDS is 0.5-5%; the concentration of guanidine hydrochloride is 2-4 mol / L; the mass percentage of benzyl isothiocyanate is 0.5-1%; the mass percentage of potassium peroxymonosulfonate is 2-2.5%; the mass percentage of sodium chloride is 0.05-1%; or, The deconstructing agent consists of SDS, guanidine hydrochloride, water, phenethyl isothiocyanate and benzoyl peroxide; the mass percentage of SDS is 0.5-5%; the concentration of guanidine hydrochloride is 2-3 mol / L; the mass percentage of phenethyl isothiocyanate is 0.75-1%; and the mass percentage of benzoyl peroxide is 2-2.5%.

2. Use of the deconstructor according to claim 1 in the preparation of a reagent for detecting affinity of a specific antibody against phospholipase A2 receptor protein.

3. The use according to claim 2, characterized in that: A sample containing a phospholipase A2 receptor protein-specific antibody is combined with the phospholipase A2 receptor protein labeled with a marker, and the detection signal before and after the deconstructor is added to the sample is detected.

4. Use of the deconstructing agent according to claim 1 in the preparation of a diagnostic reagent for primary membranous nephropathy.

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