CN118818064A - A kind of anti-phospholipase A2 receptor antibody detection kit - Google Patents

Abstract

The invention relates to the field of biochemistry, and discloses an anti-phospholipase A2 receptor antibody immunoturbidimetry detection kit, which comprises a reagent 1, a reagent 2 and a calibrator, wherein the reagent 1 comprises a buffer solution, an electrolyte, a stabilizer, an accelerator, a surfactant and a preservative; the reagent 2 comprises microsphere-PLA 2R, buffer solution, electrolyte, stabilizer, surfactant and preservative; the calibrator comprises anti-PLA2R IgG, a buffer solution, a stabilizer and a preservative. The protein-coated and Linker-modified microsphere-PLA 2R is added in the reagent 2, so that the protein load and effective antigen can be increased, the detection signal intensity can be effectively improved, the detection sensitivity and the linear range can be improved, and the accuracy of the detection result can be ensured.

Description

A kind of anti-phospholipase A2 receptor antibody detection kit

Technical Field

The present invention relates to the field of biochemistry, and in particular to a kit for detecting the concentration of anti-phospholipase A2 receptor antibodies in human serum and an application thereof.

Background Art

Membranous nephropathy (MN) is an autoimmune disease and a common pathological type of nephrotic syndrome, of which about 80% are idiopathic membranous nephrology (IMN). IMN is one of the most common pathological types of chronic kidney disease in adults. Its pathological characteristics are the deposition of subepithelial immune complexes on the outer side of the glomerular basement membrane, leading to diffuse thickening of the basement membrane. The main clinical manifestations are nephrotic syndrome or asymptomatic proteinuria.

The autoimmune mechanism of IMN was first proposed in 2009, and PLA2R (Phospholipase A2 Receptor) was determined to be the main target antigen of IMN. PLA2R is expressed on normal podocytes and exists in the immune deposits of the glomeruli of IMN patients. The level of anti-phospholipase A2 receptor antibody (anti-Phospholipase A2 Receptor IgG, anti-PLA2R IgG) in serum can be used for the diagnosis and treatment monitoring of IMN, with a detection rate of 70-80%. However, in secondary membranous nephropathy or other renal diseases, the level of anti-PLA2R IgG does not increase significantly. With the deepening of research, subsequent studies have discovered other IMN target antigens, such as THSD7A, NRLL-1, etc., but PLA2R is still the main pathogenic target antigen of IMN. Timely diagnosis and reasonable intervention are of great significance in delaying the disease progression of IMN patients and help reduce the occurrence of end-stage renal disease (ESRD).

At present, the detection methods of anti-PLA2R IgG reported in literature, patent disclosure and clinical application mainly include enzyme-linked immunosorbent assay (ELISA), fluorescent immunoassay (IFA) and chemiluminescence assay (CLIA). Among them, ELISA is the most widely used detection method in clinical practice at home and abroad. Despite its wide application, it still has problems such as low sensitivity, complex operation, long time consumption and low degree of automation. Although IFA has high sensitivity, it is complex and time-consuming to operate. Some detection reagents require manual operation and microscopic observation, and rely on the experience of the operator. Although CLIA has high sensitivity and high automation, which significantly improves the detection efficiency, its reagents and equipment costs are relatively high.

In clinical testing, the repeatability, economy and timeliness of the testing method are particularly critical. Although the existing testing methods have their own advantages, they also have certain limitations. Therefore, they need to be improved.

Summary of the invention

In order to solve the above problems, the present invention provides a microsphere-PLA2R conjugate, which comprises a protein-coated latex microsphere and a PLA2R antigen, wherein the protein-coated latex microsphere and the PLA2R antigen are covalently connected via a linker.

Furthermore, the latex microspheres are polystyrene microspheres, the PLA2R antigen is a recombinant antigen, and the protein includes any one or more of bovine serum albumin, human serum albumin and ovalbumin.

Furthermore, the diameter of the polystyrene microspheres is 50-450 nm, and the surface of the polystyrene microspheres has carboxyl functional groups.

Furthermore, the connector is any one of the following structures:

Furthermore, in the structural formula, the N-terminus is connected to the PLA2R antigen, and the S-terminus is connected to the protein-coated latex microsphere.

The second aspect of the present invention provides a method for preparing a microsphere-PLA2R conjugate, comprising the following steps:

S1: PLA2R and heterobifunctional cross-linker are added to activation buffer to react and obtain PLA2R-MA;

S2: adding polystyrene microspheres, EDC and NHS to the above activation buffer to activate the microspheres, adding carrier protein for protein coating, and obtaining microsphere-carrier protein conjugates;

S3: dispersing the microsphere-carrier protein conjugate obtained in S2 in a thiolation buffer, adding a thiolation reagent for modification, and obtaining a thiolated microsphere suspension;

S4: Add PLA2R-MA to the thiol-modified microsphere suspension in S3 to react and obtain a microsphere-PLA2R conjugate.

Furthermore, the carrier protein includes any one or more of bovine serum albumin, human serum albumin and ovalbumin.

Furthermore, the activation buffer comprises any one or more of MES, PBS and sodium bicarbonate buffer.

Furthermore, the thiol-replacing agent includes any one or more of SATP/NH ₂ OH·HCl, Traut's Reagent, TCEP, DTT and SPDP/DTT.

Furthermore, the thiolation buffer comprises any one or more of MES, PBS and sodium bicarbonate buffer, wherein 1-10 mM EDTA is added to prevent the formation of disulfide bonds.

Furthermore, the heterobifunctional cross-linking agent includes any one or more of SMCC, SMPB and NHS-PEG4-Maleimide.

The third aspect of the present invention provides an anti-phospholipase A2 receptor antibody detection kit, comprising reagent 1 and reagent 2; wherein reagent 1 comprises a buffer, an electrolyte, a stabilizer, a promoter, a surfactant and a preservative; and reagent 2 comprises the aforementioned microsphere-PLA2R conjugate, a buffer, an electrolyte, a stabilizer, a surfactant and a preservative.

Furthermore, the reagent 1 comprises 10-200 mM buffer, 0.1-1% w/w electrolyte, 0.1-15% w/w stabilizer, 0.5-1.5% w/w promoter, 0.05-1% w/w surfactant and 0.01-0.1% w/w preservative.

Furthermore, the reagent 2 includes 0.05-0.2% w/w microsphere-PLA2R, 10-200 mM buffer, 0.1-1% w/w electrolyte, 0.1-15% w/w stabilizer, 0.05-1% w/w surfactant and 0.01-0.1% w/w preservative.

Furthermore, the detection kit also includes a calibrator, which includes 0-1500 RU/mL of anti-PLA2R IgG, 10-200 mM of buffer, 0.1-15% w/w of stabilizer and 0.01-0.1% w/w of preservative.

Furthermore, the pH of the reagent 1, the reagent 2 or the calibrator is 7.4.

Furthermore, the buffer is any one or more of PBS, Tris, MES, HEPES, sodium bicarbonate buffer, borate buffer, acetate buffer and citrate buffer.

Furthermore, the electrolyte is any one or more of sodium ions, magnesium ions, potassium ions and calcium ions.

Furthermore, the stabilizer is any one or more of bovine serum albumin, human serum albumin, ovalbumin, trehalose, sucrose and mannitol.

Furthermore, the promoter is any one or more of PEG-2000, PEG-4000, PEG-6000, PEG-8000, PEG-20000, PVP and PVA.

Furthermore, the surfactant is any one or more of Tween-20, Tween-80 and TritonX-100.

Furthermore, the preservative is any one or more of NaN ₃ and ProClin 300.

Furthermore, the anti-PLA2R IgG is a monoclonal antibody or a polyclonal antibody.

The present invention also provides a method for detecting anti-phospholipase A2 receptor antibodies, using the anti-phospholipase A2 receptor antibody IgG immunoturbidimetric detection kit as described above.

Compared with the prior art, the present invention has the following beneficial effects:

(1) The present invention provides an anti-phospholipase A2 receptor antibody immunoturbidimetric detection kit, wherein microsphere-PLA2R is added to reagent 2 as a signal enhancement carrier, which can effectively improve the detection signal intensity, thereby significantly improving the detection sensitivity and linear range of the kit, and also ensuring the accuracy of the detection results.

(2) The preparation method of microsphere-PLA2R in the kit of the present invention is different from the direct coupling of the conventional method. The present invention is to coat the latex microspheres with proteins and modify the linker to increase its antigen loading capacity. At the same time, the linker of appropriate length avoids the effective epitope of the antigen from being encapsulated, reduces the steric hindrance around the antigen, and maintains the activity and specificity of the antigen. The sensitivity, linear range, precision and recovery rate of the detection kit using this microsphere-PLA2R all meet the requirements.

(3) The anti-phospholipase A2 receptor antibody immunoturbidimetric detection kit of the present invention can be used in a fully automatic biochemical analyzer, has a fast testing speed, can achieve high-throughput testing of samples, and meets clinical needs.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative labor.

FIG1 is a flow chart of the preparation of microsphere-PLA2R of Example 1 of the present invention;

FIG2 is a trend diagram of a calibration curve obtained by detecting a calibrant using the kits of Example 1 and Comparative Example 1 of the present invention;

FIG3 is a linear range of the test samples of the kit of Example 1 of the present invention;

FIG. 4 is a comparison result of the methodologies of detecting samples by the kit of Example 1 of the present invention and the ELISA control kit of Omen.

DETAILED DESCRIPTION

In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention clearer, the present invention is further described in detail below in conjunction with the embodiments and drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. The present invention is specifically introduced below in conjunction with the specific embodiments.

The embodiment of the present invention provides a microsphere-PLA2R receptor conjugate, comprising a protein-coated latex microsphere and a PLA2R antigen. The protein-coated latex microsphere and the PLA2R antigen are covalently linked by a Linker. The present invention innovatively develops an anti-PLA2R IgG detection kit based on immunoturbidimetry for the first time, which has the characteristics of rapidity, high efficiency and economy, can overcome the shortcomings of existing detection methods, and provide a more accurate and convenient tool for the diagnosis and monitoring of IMN. The present invention adopts a novel microsphere-PLA2R conjugate as a signal enhancement carrier, and the microsphere-PLA2R is protein-coated and Linker-modified to increase its antigen loading capacity. At the same time, the Linker of appropriate length avoids the effective epitope of the antigen from being encapsulated, reduces the steric hindrance around the antigen, maintains the activity and specificity of the antigen, and optimizes the immunoturbidimetry by using this novel microsphere-PLA2R conjugate, while improving the detection speed and reducing the cost, maintaining the high sensitivity, high specificity and wide linear range of the detection.

Specifically, the latex microspheres are polystyrene microspheres, the PLA2R antigen is a recombinant antigen, and the protein includes any one or more of bovine serum albumin, human serum albumin and ovalbumin.

Specifically, the diameter of the polystyrene microspheres is 50-450 nm, and the surface of the polystyrene microspheres has carboxyl functional groups.

A second aspect of an embodiment of the present invention provides a method for preparing a microsphere-PLA2R conjugate, comprising the following steps:

S1: PLA2R and heterobifunctional cross-linker are added to activation buffer for reaction, and purified by desalting column or dialysis to obtain PLA2R-MA;

S2: adding polystyrene carboxyl microspheres, EDC and NHS to the above activation buffer to activate the microspheres, adding carrier protein for protein coating, and obtaining microsphere-carrier protein conjugates;

S3: dispersing the microsphere-carrier protein conjugate obtained in S2 in a thiolation buffer, adding a thiolation reagent for modification, and obtaining thiolated microspheres;

S4: Add PLA2R-MA to the thiol-modified microspheres in S3 to react and obtain microsphere-PLA2R conjugates.

Different from the conventional method of microsphere coupling in which latex microspheres and antigens are directly coupled together by covalent bonding, the present invention first coats the microspheres with proteins, then forms a linker through a bifunctional cross-linking agent, and connects the antigen through the linker to obtain a microsphere-PLA2R conjugate. The microsphere conjugate prepared by the method of the present invention has a high antigen loading capacity, and a linker of a suitable length can be obtained by selecting a suitable cross-linking agent, thereby avoiding the encapsulation of the antigen's effective epitope, reducing the steric hindrance around the antigen, and maintaining the activity and specificity of the antigen. The kit using the microsphere conjugate has good sensitivity, linear range, precision and recovery, and can meet various detection requirements.

Specifically, the carrier protein includes any one or more of bovine serum albumin, human serum albumin and ovalbumin.

Specifically, the activation buffer includes any one or more of MES, PBS and sodium bicarbonate buffer.

Specifically, the thiol-replacing agent includes any one or more of SATP/NH ₂ OH·HCl, Traut's Reagent, TCEP, DTT and SPDP/DTT.

Specifically, the thiolation buffer includes any one or more of MES, PBS and sodium bicarbonate buffer, wherein 1-10 mM EDTA is added to prevent the formation of disulfide bonds.

Specifically, the heterobifunctional cross-linking agent includes any one or more of SMCC, SMPB and NHS-PEG4-Maleimide.

The third aspect of the embodiment of the present invention provides an anti-phospholipase A2 receptor antibody immunoturbidimetric detection kit, comprising reagent 1, reagent 2 and a calibrator. The reagent 1 comprises a buffer, an electrolyte, a stabilizer, a promoter, a surfactant and a preservative; the reagent 2 comprises the aforementioned microsphere-PLA2R conjugate, a buffer, an electrolyte, a stabilizer, a surfactant and a preservative; and the calibrator comprises anti-PLA2R IgG, a buffer, a stabilizer and a preservative.

Specifically, the reagent 1 includes 10-200 mM buffer, 0.1-1% w/w electrolyte, 0.1-15% w/w stabilizer, 0.5-1.5% w/w promoter, 0.05-1% w/w surfactant and 0.01-0.1% w/w preservative.

Specifically, the reagent 2 includes 0.05-0.2% w/w microsphere-PLA2R, 10-200 mM buffer, 0.1-1% w/w electrolyte, 0.1-15% w/w stabilizer, 0.05-1% w/w surfactant and 0.01-0.1% w/w preservative.

Specifically, the calibrator includes 0-1500 RU/mL of anti-PLA2R IgG, 10-200 mM of buffer, 0.1-15% w/w of stabilizer and 0.01-0.1% w/w of preservative.

Preferably, the pH values of the reagent 1, the reagent 2 and the calibrator are 7.4 respectively.

Specifically, the buffer is any one or more of PBS, Tris, MES, HEPES, sodium bicarbonate buffer, borate buffer, acetate buffer and citrate buffer.

Specifically, the electrolyte is any one or more of sodium ions, magnesium ions, potassium ions and calcium ions.

Specifically, the stabilizer is any one or more of bovine serum albumin, human serum albumin, ovalbumin, trehalose, sucrose and mannitol.

Specifically, the promoter is any one or more of PEG-2000, PEG-4000, PEG-6000, PEG-8000, PEG-20000, PVP and PVA.

Specifically, the surfactant is any one or more of Tween-20, Tween-80 and Triton X-100.

Specifically, the preservative is any one or more of NaN ₃ and ProClin 300.

Specifically, the anti-PLA2R IgG is a monoclonal antibody or a polyclonal antibody.

The embodiment of the present invention also provides a method for detecting anti-phospholipase A2 receptor antibody IgG for non-diagnostic purposes, using the anti-phospholipase A2 receptor antibody IgG immunoturbidimetric detection kit as described above.

Specifically, the above detection method may be transmission turbidimetry or scattering turbidimetry, and the detection instrument may be a biochemical analyzer.

Specifically, the detection principle of the above detection method is: the anti-phospholipase A2 receptor antibody IgG in the sample combines with the antigen in the reagent to form an antigen-antibody complex, thereby producing a turbidity change. The latex reagent (polystyrene microspheres) can specifically increase this turbidity change and increase the sensitivity of the reaction. The magnitude of the turbidity change is proportional to the content of the anti-phospholipase A2 receptor antibody in the sample. By measuring the turbidity change and comparing it with the test results of the calibration substance, the content of the anti-phospholipase A2 receptor antibody in the sample can be obtained.

The embodiment of the present invention also provides an application of using the above kit to evaluate the condition of a patient suffering from idiopathic membranous nephropathy.

Specifically, the application is achieved by using the above kit to monitor the dynamic changes of anti-phospholipase A2 receptor antibodies in the blood of patients with idiopathic membranous nephropathy.

The following is explained in conjunction with specific embodiments:

The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified, and the techniques not described in detail are all performed according to standard methods well known to those skilled in the art.

Example 1

Table 1 Preparation materials and dosage of reagent 1

Preparation method of reagent 1

Tris (6.06 g), NaCl (5.00 g), bovine serum albumin (2.00 g), PEG-4000 (7.50 g), Tween-20 (500 mg), and NaN ₃ (500 mg) were added to a container containing 800 mL of purified water, stirred at room temperature until the solid was completely dissolved, and the pH was adjusted to 7.4. The volume was adjusted to 1 L, and after mixing evenly, reagent 1 was obtained.

Table 2 Preparation materials and dosage of reagent 2

Preparation method of reagent 2

Preparation of microsphere-PLA2R, the preparation process is shown in Figure 1:

S1: PLA2R (30.0 mg) and heterobifunctional crosslinker (NHS-PEG4-Maleimide, 2.19 mg) were added to a container containing activation buffer (1×PBS, pH=8.0, 6 mL) and reacted at room temperature for 1 h. The excess heterobifunctional crosslinker was removed by purification through dialysis (dialysis solution 1×PBS, pH=7.4, 1000 mL×3 times) to obtain PLA2R-MA.

S2: Add EDC·HCl (25.0 mg) and NHS (100.0 mg) to a container containing activation buffer (50 mM MES, pH = 5.0, 22.5 mL), add polystyrene microspheres (10% w/w, 2500 μL, 290 nm) to activate the microspheres, and react at room temperature for 1 hour. After the reaction is completed, centrifuge and discard the supernatant, add activation buffer (50 mM MES, pH = 5.0, 15 mL) and ultrasonically wash, and repeat this process twice. Add activation solution (50 mM MES, pH = 5.0, 25.0 mL) and ultrasonically resuspend, add bovine serum albumin (12.5 mg), and react at room temperature for 2 hours. Add glycine solution (50 mM, 2.5 mL), and react at room temperature for 2 hours. Centrifuge and discard the supernatant, add thiolation buffer (1×PBS, pH = 8.0, 5 mM EDTA, 15 mL) and ultrasonically wash, and repeat this process twice. After centrifugation, thiolation buffer (1×PBS, pH=8.0, 5 mM EDTA, 25 mL) was added and ultrasonically resuspended to obtain microsphere-carrier protein conjugate (microsphere-BSA).

S3: Add thiol reagent (SATP, 2.32 mg) to microsphere-BSA and react at room temperature for 1 hour. Centrifuge and discard the supernatant, add thiol buffer (1×PBS, pH=8.0, 5mM EDTA, 25mL) and ultrasonically resuspend, add hydrolysis reagent (1×PBS, pH=8.0, 500mM NH ₂ OH, 25mM EDTA, 2.5mL) and react at room temperature for 2 hours. Centrifuge and discard the supernatant, add thiol buffer (1×PBS, pH=7.4, 5mM EDTA, 15mL) and ultrasonically wash, repeat this process twice. After centrifugation, add thiol buffer (1×PBS, pH=7.4, 5mM EDTA, 25mL) and ultrasonically resuspend to obtain thiol microsphere suspension.

S4: Add PLA2R-MA (25.0 mg) to the thiolated microsphere suspension, react at room temperature for 2 h, add 6-maleimidocaproic acid (DMF, 10 mg/mL, 2.5 mL) to block the excess thiol groups, and react at room temperature for 2 h. Centrifuge and discard the supernatant, add thiolation buffer (1×PBS, pH=7.4, 5 mM EDTA, 15 mL) and ultrasonically wash, repeat this process twice to obtain microsphere-PLA2R conjugates.

Preparation of Reagent 2 mother solution: Add Tris (1.51 g), NaCl (1.25 g), bovine serum albumin (0.50 g), Tween-20 (125 mg), and NaN ₃ (125 mg) into a container filled with 200 mL of purified water, stir at room temperature until the solid is completely dissolved, adjust pH to 8.0, and dilute to 250 mL to obtain Reagent 2 mother solution.

Preparation of Reagent 2: The microsphere-PLA2R was centrifuged, and then the mother solution of Reagent 2 (250 mL) was added to obtain Reagent 2 (0.1% w/w microsphere-PLA2R, 250 mL).

Table 3 Composition and dosage of calibrators

Preparation of Calibrators

NaCl (800 mg), KCl (20.0 mg), Na ₂ HPO ₄ (142 mg), KH ₂ PO ₄ (24.5 mg), bovine serum albumin (200 mg), ProClin 300 (50.0 mg) were added to a container containing 80 mL of purified water, stirred at room temperature until the solid was completely dissolved, and the pH was adjusted to 7.4 to obtain a calibration stock solution. Anti-PLA2R IgG was added to the calibration stock solution according to the required concentration to prepare calibration products with concentrations of 0, 15.00, 100.00, 500.00, 1000.00, and 1500.00 RU/mL.

Comparative Example 1

In this comparative example, the preparation of microsphere-PLA2R is to directly couple the PLA2R antigen to the latex microspheres. Other preparation methods and implementation steps are the same as those in Example 1.

Kit performance evaluation

1. Protein loading and effective antigen testing

The protein loading and effective antigen of the microsphere PLA2R prepared in Example 1 and Comparative Example 1 were tested respectively, and the specific steps were as follows:

Protein loading: The uncovalently bound PLA2R antigen content in the supernatant during the microsphere coupling step was detected by Quawell Q6000 UV-Vis Spectrophotometer to calculate the protein loading;

Effective antigen test: Take part of the microsphere-PLA2R complex, add excess anti-PLA2R IgG, react at room temperature for 1 hour, centrifuge, wash 2-4 times, collect the supernatant, and detect the content of anti-PLA2R IgG in the supernatant by BCA kit to determine the exposure of effective binding sites of PLA2R on the microsphere. The results are shown in Table 4.

Table 4 Microglobulin loading and effective antigen test

2. Calibration Curve

The calibration curves of the calibrators were tested using the anti-phospholipase A2 receptor antibody kits prepared in Example 1 and Comparative Example 1, respectively.

Testing instrument: Beckman AU680 fully automatic biochemical analyzer;

Reagent dosage: Reagent 1: 200 μL; Reagent 2: 50 μL; Sample volume: 3 μL;

Detection method: two-point endpoint method;

Reaction direction: rising;

Measurement wavelength: 660nm;

Reaction time: 8min;

Reading points: 12-27;

Calibration method:Spline.

The six-point calibration method was used to calibrate the Beckman AU680 fully automatic biochemical analyzer, and the concentrations of the calibration products were 0, 15.00, 100.00, 500.00, 1000.00 RU/mL, respectively, to draw a calibration curve. The calibration results are shown in Table 5 and Figure 2.

Table 5 Calibration results of the kit of Example 1 and Comparative Example 1

3. Analytical Sensitivity

The anti-phospholipase A2 receptor antibody kit prepared in Example 1 was subjected to an analytical sensitivity test.

The specific steps are as follows:

The serum sample with a concentration of 15±1.5RU/mL was tested, and the absorbance change (ΔOD _sample ) produced under the specified parameters of the reagent was recorded, and the absorbance change produced by the zero concentration calibrator (ΔOD _{0 value} ) was deducted, and converted to the absolute value of the absorbance change of the 15RU/mL sample (|ΔOD|). The sample (15.00RU/mL) was selected for the analytical sensitivity test, and |ΔOD|=589, and the analytical sensitivity was qualified. The results are shown in Table 6.

Table 6 Analytical sensitivity measurement results of Example 1 of the present invention

4. Linear range

The linear range test was performed on the anti-phospholipase A2 receptor antibody kit prepared in Example 1. The specific steps are as follows:

The anti-PLA2R IgG high concentration sample (1464.93RU/mL) and the low concentration sample (5.94RU/mL) were mixed according to the ratio in Table 7, and each linear sample was tested. The average value of the measured concentration was compared with the theoretical value to obtain the absolute deviation or relative deviation, and a linear regression analysis was performed to obtain the regression equation y=1.003x+1.8369, and the correlation coefficient R ² =0.9998, and the results are shown in Table 8 and Figure 3. The results show that the linear range of the anti-PLA2R IgG detection kit of the present invention can reach 6.00-1500RU/mL.

Table 7 Linear sample preparation

Table 8 Linear range measurement results of Example 1 of the present invention

5. Repeatability

The anti-phospholipase A2 receptor antibody kit prepared in Example 1 was tested for repeatability. The specific steps are as follows:

Use the kit to test quality control product 1 and quality control product 2, repeat the test 10 times each, and calculate the average value of the measured values Standard deviation (SD) and coefficient of variation (CV). The test results are shown in Table 9. The coefficient of variation of low-value samples is 3.12%, and the coefficient of variation of high-value samples is 2.26%, with good repeatability.

Table 9 Repeatability test results of Example 1 of the present invention

6. Accuracy

The accuracy test of the anti-phospholipase A2 receptor antibody kit prepared in Example 1 was performed. The specific steps are as follows:

A certain volume of standard solution (1500RU/mL) was added to human serum (6.00RU/mL), the ratio of the standard solution volume to the human serum sample volume was 1:9, and the monitoring was repeated 3 times to calculate the recovery rate. The test results are shown in Table 10, and the recovery rate was 98%, which is between 90% and 110%, meeting the measurement accuracy requirements.

Table 10 Recovery test results of Example 1 of the present invention

The above experimental results all show that the microsphere-PLA2R protein loading and effective antigen prepared by the present invention are higher than those of the conventional method, and when used in the detection kit, the signal intensity can be more effectively improved. At the same time, the sensitivity, linear range, repeatability and recovery rate of the kit of the present invention all meet the use requirements.

Methodological comparison:

A methodological comparison was performed on the anti-phospholipase A2 receptor antibody kit prepared in Example 1. The specific steps are as follows:

40 fresh serum samples were taken and tested using the European anti-PLA2R IgG (ELISA) as a control reagent, and the anti-phospholipase A2 receptor antibody kit prepared in Example 1 was used for testing. The reagent of the present invention was tested using a Beckman AU680 fully automatic biochemical analyzer, and the control reagent was tested using a Thermo Fisher Multiskan FC microplate reader. The test results are shown in Table 11. The measured values were subjected to linear regression analysis, and the regression equation y=0.9973x+3.5739 was calculated, and the correlation coefficient R ² =0.9940. The results show that the anti-PLA2R IgG detection kit (latex immunoturbidimetry) of the present invention has a good correlation with the European anti-PLA2R IgG (ELISA) test.

Table 11 Example 1 of the present invention - European ELISA methodology comparison test results

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the principles of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. A microsphere-PLA 2R conjugate comprising a protein-coated latex microsphere and a PLA2R antigen, the protein-coated latex microsphere and the PLA2R antigen being covalently linked by a Linker, the Linker having any one of the following structures:

and the N end of the structural formula is connected with the PLA2R antigen, and the S end of the structural formula is connected with the protein-coated latex microsphere.

2. The microsphere-PLA 2R conjugate of claim 1, wherein the latex microsphere is a polystyrene microsphere, the PLA2R antigen is a recombinant antigen, and the protein comprises any one or more of bovine serum albumin, human serum albumin, and ovalbumin.

3. The microsphere-PLA 2R conjugate of claim 2, wherein the polystyrene microsphere has a diameter of 50-450nm and the surface of the polystyrene microsphere has carboxyl functional groups.

4. A method of preparing a microsphere-PLA 2R conjugate according to any one of claims 1-3, comprising the steps of:

S1: adding PLA2R and heterobifunctional crosslinking agent into an activation buffer solution for reaction to obtain PLA2R-MA;

s2: adding polystyrene microsphere, EDC and NHS into the activation buffer solution, activating microsphere, adding carrier protein, and coating protein to obtain microsphere-carrier protein conjugate;

S3: dispersing the microsphere-carrier protein conjugate obtained in the step S2 in a thiolation buffer solution, and adding a thiolation reagent for modification to obtain thiolation microspheres;

S4: and adding PLA2R-MA into the sulfhydrylation microsphere in the S3 for reaction to obtain the microsphere-PLA 2R conjugate.

5. The method of preparing a microsphere-PLA 2R conjugate of claim 4, wherein the carrier protein includes any one or more of bovine serum albumin, human serum albumin and ovalbumin;

And/or the activation buffer solution comprises any one or more of MES, PBS and sodium bicarbonate buffer solution;

And/or the sulfhydrylation Reagent comprises any one or more of SATP/NH ₂ OH-HCl, traut's Reagent, TCEP, DTT and SPDP/DTT;

and/or, the sulfhydrylation buffer solution comprises any one or more of MES, PBS and sodium bicarbonate buffer solution, wherein 1-10mM EDTA is added to prevent disulfide bond formation;

And/or the heterobifunctional crosslinking agent comprises any one or more of SMCC, SMPB and NHS-PEG 4-MALEIMIDE.

6. An anti-phospholipase A2 receptor antibody immunoturbidimetry detection kit comprises a reagent 1 and a reagent 2; wherein the reagent 1 comprises a buffer, an electrolyte, a stabilizer, an accelerator, a surfactant and a preservative; the agent 2 comprises the microsphere-PLA 2R conjugate of claims 1-3, a buffer, an electrolyte, a stabilizer, a surfactant, and a preservative.

7. The kit for immunonephelometry detection of anti-phospholipase A2 receptor antibodies of claim 6, wherein the reagent 1 comprises 10-200mM buffer, 0.1-1% w/w electrolyte, 0.1-15% w/w stabilizer, 0.5-1.5% w/w accelerator, 0.05-1% w/w surfactant, and 0.01-0.1% w/w preservative;

And/or, the reagent 2 comprises 0.05-0.2% w/w microsphere-PLA 2R, 10-200mM buffer, 0.1-1% w/w electrolyte, 0.1-15% w/w stabilizer, 0.05-1% w/w surfactant, and 0.01-0.1% w/w preservative.

8. The kit for immunonephelometry detection of anti-phospholipase A2 receptor antibodies according to claim 6 or 7, wherein the pH of the reagent 1 and the reagent 2 is 7.4.

9. The kit for immunonephelometry detection of anti-phospholipase A2 receptor antibodies according to claim 8, wherein the buffer is any one or more of PBS, tris, MES, HEPES, sodium bicarbonate buffer, boric acid buffer, acetic acid buffer, and citric acid buffer;

and/or the electrolyte is any one or more of sodium ions, magnesium ions, potassium ions and calcium ions;

And/or the stabilizer is any one or more of bovine serum albumin, human serum albumin, ovalbumin, trehalose, sucrose and mannitol;

And/or the accelerator is any one or more of PEG-2000, PEG-4000, PEG-6000, PEG-8000, PEG-20000, PVP and PVA;

and/or the surfactant is any one or more of Tween-20, tween-80 and Triton X-100;

And/or the preservative is any one or more of NaN ₃ and ProClin 300.

10. A method for detecting an anti-phospholipase A2 receptor antibody of non-diagnostic interest, using the anti-phospholipase A2 receptor antibody IgG immunoturbidimetry detection kit of claim 9.