A detection method and kit for detecting anti-nephrin antibodies

Abstract

The present application relates to the technical field of protein detection, and particularly relates to a detection method and kit for detecting anti-Nephrin antibody. The detection method comprises the following steps: using biotinylated Nephrin protein to capture anti-Nephrin antibody in a sample to be detected on a SA matrix to obtain a first complex; performing acidolysis desorption, neutralization and Protein G directed antibody immobilization on the first complex to obtain a second complex; using a biotin-streptavidin-HRP system to detect the second complex; the acidolysis desorption comprises the following steps: using an acid solution with a pH of 2-3 to treat the first complex, and treating at 35-40 DEG C and 500-800 rpm for 10-20 minutes. The method provided by the present application can accurately and quickly detect the anti-Nephrin antibody level in a sample, and has important application value.

Description

Technical Field

[0001] This invention relates to the field of protein detection technology, and in particular to a detection method and kit for detecting anti-Nephrin antibodies. Background Technology

[0002] Anti-Nephrin antibodies are antibodies against the Nephrin protein, also known as the glomerular-specific cell adhesion receptor (NPHS1, NPHN). Nephrin is highly expressed in the glomeruli and belongs to the immunoglobulin family of cell adhesion molecules. Mutations in the Nephrin gene can lead to congenital nephrotic syndrome, an autosomal recessive genetic disorder characterized by massive proteinuria during fetal development and nephropathy at birth. Recent studies have found that anti-Nephrin autoantibodies play an important role in podocyte diseases, particularly MCD and FSGS. This discovery may redefine the understanding, diagnosis, and treatment of these diseases. Anti-Nephrin autoantibodies can not only serve as a non-invasive diagnostic tool but may also be novel therapeutic targets for future kidney diseases. Currently, there are two commonly used methods for detecting anti-Nephrin antibodies: enzyme-linked immunosorbent assay (ELISA) and immunoprecipitation (IP).

[0003] Immunoprecipitation is a relatively complex technique requiring precise operational skills and condition control. It demands high standards in sample preparation, typically involving significant time and cost, making it unsuitable for large-scale sample screening. Furthermore, due to the generally low titers of anti-Nephrin antibodies and the lack of standards, inconsistencies and incomparability in results can arise between different laboratories and detection methods. Therefore, the reliability of routine immunoassays for anti-Nephrin antibodies (such as ELISA) remains controversial. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides a detection method and kit for detecting anti-Nephrin antibodies.

[0005] In a first aspect, the present invention provides a method for detecting anti-Nephrin antibodies, comprising:

[0006] The first complex was obtained by using biotinylated Nephrin protein to capture anti-Nephrin antibody in the test sample onto the SA matrix.

[0007] The first complex was subjected to acid desorption, neutralization, and Protein G-directed fixation of the antibody to obtain the second complex.

[0008] The second complex was detected using the biotin-streptavidin-HRP system;

[0009] The acid desorption process includes treating the first complex with an acid solution with a pH between 2 and 3 for 10 to 20 minutes at 35 to 40°C and 500 to 800 rpm.

[0010] The first complex of this invention is: SA-Biotin-Nephrin protein (capture protein)-anti-Nephrin antibody. The second complex of this invention is: Protein G-anti-Nephrin antibody-Biotin-Nephrin protein (detection protein).

[0011] This invention utilizes biotinylated Nephrin protein, combining acid desorption, neutralization, and Protein G-directed antibody immobilization to capture anti-Nephrin antibodies while effectively eliminating most interfering components (especially free receptors or interacting proteins). A biotin-streptavidin-HRP cascade amplification technique is then employed for the detection of anti-Nephrin antibodies. This process may introduce the antigen (Nephrin protein) into subsequent detection systems, interfering with the detection process. Therefore, this invention uses biotinylated Nephrin protein for antibody capture, employing the same reagents as the biotin-streptavidin-HRP detection method, and effectively avoids related interfering factors. The method provided by this invention significantly improves the sensitivity and specificity of detecting anti-Nephrin antibodies, ultimately achieving accurate quantitative analysis of anti-Nephrin antibodies.

[0012] This invention provides an improved affinity capture elution (ACE) ELISA method for detecting Nephrin antibody levels in serum samples. It significantly reduces nonspecific background, eliminating the need for extensive dilution of serum samples during detection (existing techniques often require significant sample dilution to reduce background), thus significantly improving detection sensitivity and anti-interference capabilities.

[0013] Further, the first complex is obtained by the following method:

[0014] The SA matrix was coated with biotinylated Nephrin protein solution and blocking solution for 60-120 minutes at 35-40℃ and 500-800 rpm.

[0015] Mix the test sample and acid solution, acidify at 35-40℃ and 500-800rpm for 15-25 minutes, neutralize and wash, and then incubate with the coated SA matrix at 35-40℃ and 500-800rpm for 50-70 minutes.

[0016] The volume ratio of the sample to be tested to the acid solution is 1:(7~15), and the pH of the acid solution is 2~3.

[0017] Furthermore, neutralization after acidification can be achieved by adding Tris working solution and PBS buffer.

[0018] Further, the concentration of the biotinylated Nephrin protein solution is 0.1~5 μg / mL; and / or,

[0019] The sealing liquid is a Super Block solution; and / or,

[0020] The SA substrate is an SA plate; and / or,

[0021] The acid solution is an acetic acid solution with a concentration of 0.2~2 mol / L.

[0022] Furthermore, the SA plate is a Streptavidin Coated Plate, which is commercially available.

[0023] Furthermore, the second complex is obtained by the following method:

[0024] The first complex and the acid solution were mixed and subjected to acid desorption treatment at 35-40°C and 500-800 rpm for 10-20 minutes.

[0025] After washing, it is neutralized by mixing with Tris working solution;

[0026] Incubate on Protein G-coated substrate at 35–40°C and 500–800 rpm for 50–70 minutes.

[0027] Furthermore, the concentration of the Tris working solution is 0.5~5M, and the volume ratio of the Tris working solution to the acid solution is 1:(2~5).

[0028] Furthermore, the detection using the biotin-streptavidin-HRP system includes:

[0029] The biotinylated Nephrin protein was incubated with the second complex at 35-40°C and 500-800 rpm for 55-65 minutes.

[0030] After washing, add Streptavidin-HRP working solution and incubate at 35~40℃ and 500~800rpm for 25~35 minutes;

[0031] After washing, TMB solution is added to initiate a colorimetric reaction.

[0032] Furthermore, the colorimetric reaction is carried out at 35-40°C under light-protected conditions for 5-15 minutes.

[0033] Furthermore, it also includes:

[0034] The test results are compared with the standard curve, and qualitative or quantitative detection of anti-Nephrin antibodies is achieved based on the comparison results.

[0035] The standard curve was constructed based on the test results of the standard solution using the same method.

[0036] Secondly, the present invention provides a kit for detecting anti-Nephrin antibodies, comprising:

[0037] Biotinylated Nephrin protein, blocking solution, acid solution, Protein G coating working solution, Tris working solution, Streptavidin-HRP working solution, buffer solution, and washing solution; the acid solution has a pH of 2-3.

[0038] Thirdly, the present invention provides the application of the aforementioned kit in drug screening, development of diagnostic reagents, or construction of biomarker libraries related to kidney diseases.

[0039] The present invention has the following beneficial effects:

[0040] This invention combines Protein G-directed antibody fixation, acid desorption-neutralization transfer to optimize recovery efficiency, and biotin-streptavidin-HRP cascade system to amplify the signal, resulting in a highly sensitive, specific, accurate, and precise enzyme-linked immunosorbent assay (ELISA) method that can be used for the batch and rapid detection of anti-Nephrin antibodies.

[0041] The detection kit developed based on this invention can accurately and sensitively detect anti-Nephrin antibodies in serum, with a detection limit as low as 125 U / mL. The sample pretreatment process is simple and time-saving, and it can detect a large number of samples simultaneously. The sample detection cost is far lower than that of traditional instrument detection methods.

[0042] This invention provides an accurate and non-invasive detection of the anti-Nephrin antibody biomarker, which has high clinical value for early diagnosis and monitoring to improve the prognosis of these patients. Attached Figure Description

[0043] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0044] Figure 1 This is the detection principle provided in Embodiment 1 of the present invention.

[0045] Figure 2 This is the standard curve provided in Embodiment 1 of the present invention. Detailed Implementation

[0046] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0047] Unless otherwise specified, the experimental methods involved in the following embodiments are conventional methods in the art. For example, you can refer to the experimental manual in the art or follow the conditions recommended in the manufacturer's instructions.

[0048] Unless otherwise specified, all experimental materials and reagents used in the following examples are commercially available.

[0049] Example 1

[0050] 1. Experimental subjects

[0051] Sample to be tested: Serum collected from healthy individuals.

[0052] 2. Experimental Materials

[0053] (1) Watson LIMS system, multi-functional microplate reader, microplate constant temperature shaker, Mettler LabX 2017 system, pure water system, mixer and single / multi-channel pipettes are all commercially available.

[0054] (2) PBS buffer (pH 7.3): Take one packet of PBS buffer powder and add 1000 mL of ultrapure water and mix well.

[0055] Washing buffer (PBST): Take one packet of PBS buffer powder, add 1000 mL of ultrapure water, and then add 1.0 mL of Tween20 and mix well.

[0056] Acetic acid working solution: Add 9.6 mL of acetic acid to 490.4 mL of ultrapure water and mix well.

[0057] Biotin Recombinant Human Nephrin Solution:

[0058] (1) Take one tube of Biotin powder and bring it to room temperature.

[0059] (2) Take 100 μL of the reconstituted Recombinant Human Nephrin protein solution (1 mg / mL) and add it to the Biotin powder tube. Let it stand at room temperature for at least 30 min to complete the labeling.

[0060] (3) Rinse the new ultrafiltration tube: Add 100 μL of PBS to the ultrafiltration tube, centrifuge at 14,000 × g for 10 minutes at 4 degrees Celsius, and remove the liquid after centrifugation.

[0061] (4) Add 100 μL of Biotin-labeled Nephrin protein to an ultrafiltration tube, centrifuge at 14,000 × g for 30 minutes at 4 degrees Celsius, and remove the liquid after centrifugation.

[0062] (5) Add 100 μL of PBS to the ultrafiltration tube. Centrifuge at 14,000 × g for 30 minutes at 4 degrees Celsius.

[0063] (6) Take a new centrifuge tube, invert the ultrafiltration tube into the centrifuge tube, centrifuge at 1000 × g for 2 minutes at 4 degrees, collect the liquid after centrifugation, and transfer it to a -70 degree refrigerator for long-term storage.

[0064] Capture protein working solution: Take 5.0 µL of Biotin Recombinant Human Nephrin solution and add it to 10.0 mL of Super Block solution, then mix well.

[0065] Protein detection working solution: Take 1.0 µL of Biotin Recombinant Human Nephrin solution and add it to 20.0 mL of Super Block solution, then mix well.

[0066] Protein G coating working solution: Take 11.0 µL of Protein G solution and add it to 11.0 mL of PBS solution, then mix well.

[0067] SA board: Streptavidin Coated Plate, available commercially.

[0068] 1 M Tris working solution: Weigh 121.14 g Tris and add it to 1 L of ultrapure water. Add an appropriate amount of hydrochloric acid to adjust the pH to about 9.5 (pH test paper) and mix well.

[0069] Streptavidin-HRP working solution: Take 1.0 µL of Streptavidin-HRP solution, add 20.0 mL of sample diluent, and mix well to obtain a 1:20000 Streptavidin-HRP working solution.

[0070] The above preparation steps can be adjusted proportionally according to the actual required volume.

[0071] 3. Experimental Procedure

[0072] (1) Protein G coating: Take Protein G coating working solution, add 100 µL / well to a 96-well plate, cover with sealing film, and coat overnight (more than 16 h) in a refrigerator at 2~8 ℃.

[0073] (2) Washing: Manually wash Protein G coated plates with plate washing solution, 300 µL / well, let stand for at least 30 seconds each time, wash 3 times, and pat dry with absorbent paper after completion.

[0074] (3) Blocking: Add Super Block solution, 200 µL / well, to the washed Protein G coated plate, cover with sealing film, and block at 37 ℃, 700 rpm for 120 min.

[0075] (4) SA plate coating: Add capture protein working solution to the SA plate, 100 µL / well, cover with sealing membrane, 37 ℃, 700 rpm, coating for 120 min.

[0076] (5) Acidification treatment: The prepared Blank sample, standard curve sample and test sample are acidified. The specific method is as follows: take 20 μL of sample, add 180 μL of acetic acid working solution, and incubate at 37℃ and 700rpm for 20 min.

[0077] (6) Acid-base neutralization: Add 1 M Tris working solution, 60 μL / sample, to the acidified sample, and then add PBS buffer, 140 μL / sample.

[0078] (7) Washing: Manually wash the SA plate with washing solution, 300 µL / well, let it stand for at least 30 seconds each time, wash 3 times, and pat dry with absorbent paper after completion.

[0079] (8) Sample loading: According to the experimental plan, add the above-mentioned acid-base neutralized sample to the SA plate, 100 µL / well, cover the plate with the sealing film, and incubate at 37 ℃, 700 rpm for 60 min.

[0080] (9) Washing: Manually wash the SA plate with washing solution, 300 µL / well, let it stand for at least 30 seconds each time, wash 3 times, and pat dry with absorbent paper after completion.

[0081] (10) Acid desorption: Add 100 μL of acetic acid working solution to the SA plate and incubate at 37°C and 700 rpm for 15 min.

[0082] (11) Washing: Manually wash Protein G coated plates with plate washing solution, 300 µL / well, let stand for at least 30 seconds each time, wash 3 times, and pat dry with absorbent paper after completion.

[0083] (12) Sample loading: Add 1 M Tris working solution, 24 μL / well, to the Protein G coated plate, and then transfer the sample that has been desorbed by acid to the SA plate above, 80 μL / well. Cover the plate with the sealing film and incubate at 37 ℃, 700 rpm for 60 min.

[0084] (13) Washing: The Protein G coated plate was used as an experimental plate by hand washing with plate washing solution, 300 µL / well. Each washing was allowed to stand for at least 30 seconds, and the plate was washed 3 times. After washing, the plate was patted dry with absorbent paper.

[0085] (14) Add the working solution for the detection protein: Add the working solution for the detection protein to the experimental plate, 100 µL / well, cover the plate with the sealing film, and incubate at 37 ℃, 700 rpm for 60 min.

[0086] (15) Washing: Manually wash the experimental plate with plate washing solution, 300 µL / well, let it stand for at least 30 seconds between each wash, wash 3 times, and pat dry with absorbent paper after completion.

[0087] (16) Add Streptavidin-HRP working solution: Add 100µL / well of Streptavidin-HRP working solution to the experimental plate, cover with sealing film, and incubate at 37 ℃, 700rpm, in the dark for 30 min.

[0088] (17) Washing: Manually wash the experimental plate with plate washing solution, 300 µL / well, let it stand for at least 30 seconds between each wash, wash 3 times, and pat dry with absorbent paper after completion.

[0089] (18) Color development: Add TMB solution, 100 µL / well, react at 37 °C in the dark for 10 min, then add 50 µL of stop solution to each well to stop the reaction.

[0090] (19) Reading: Read the value within 5 minutes after termination.

[0091] 4. Construction of Standard Curve

[0092] (1) This invention established a threshold (positive / negative) criterion for the Nephrin antibody detection method by evaluating serum samples from 40 healthy individuals (mean value of negative samples * 1.56). Using known strongly positive serum samples, a gradient dilution series of antibodies (1:20, 1:40, 1:80, 1:160, 1:320, 1:640, where the 1:80 dilution was defined as 1000 U / mL) was prepared. Standard curve samples were configured according to the table below. Simultaneously, at least 10 different sources of conventional human serum were mixed to obtain negative biological matrix samples, and a standard curve was plotted. This completed the assessment of the positivity / negativity of Nephrin antibodies in the serum samples to be tested, and defined the activity level of Nephrin antibody-positive samples.

[0093] Table 1. Sample Preparation Table for Standard Curve

[0094]

[0095] The parameters of the ELISA reader are as follows:

[0096] The instrument used was a Molecular Devices SpectraMax M5, and the operating software was Soft MaxPro 7.1.1 GxP. The Read Mode was set to ABS, the Read Type to Endpoint, the wavelength was 450 nm, and the reference wavelength was 630 nm.

[0097] Detection principle as follows Figure 1 As shown, the obtained standard curve is as follows: Figure 2 As shown in the figure, the corresponding detection results can be obtained at a concentration of 125 U / mL, which indicates that the detection method provided by the present invention has good analytical sensitivity.

[0098] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting anti-Nephrin antibodies for non-diagnostic purposes, characterized in that the steps include... include: SA plates were coated with biotinylated Nephrin protein solution and blocking solution for 60-120 minutes at 35-40°C and 500-800 rpm to obtain SA plates coated with Nephrin protein. The SA plates were streptavidin-coated plates. The sample and acid solution were acidified at 35-40℃ and 500-800rpm for 15-25 minutes, neutralized with Tris working solution, and then incubated with an SA plate coated with Nephrin protein at 35-40℃ and 500-800rpm for 50-70 minutes. The volume ratio of the sample to be tested to the acid solution is 1:(7~15), and the pH of the acid solution is 2~3; After washing, the acid solution is added, and the acid desorption treatment is carried out at 35~40℃ and 500~800rpm for 10~20 minutes to obtain the sample in the SA plate that has completed acid desorption. Add Tris working solution to Protein G coated plate, then transfer to SA plate to complete acid desorption of sample, and incubate at 35-40℃ and 500-800rpm for 50-70 minutes. Then wash, add biotinylated Nephrin protein, and incubate at 35-40°C and 500-800 rpm for 55-65 minutes; After washing, add Streptavidin-HRP working solution and incubate at 35~40℃ and 500~800rpm for 25~35 minutes; After washing, TMB solution is added to initiate a colorimetric reaction.

2. The method according to claim 1, characterized in that, The concentration of the biotinylated Nephrin protein solution is 0.1~5 μg / mL; and / or, The sealing liquid is a Super Block solution; And / or, The acid solution is an acetic acid solution with a concentration of 0.2~2 mol / L.

3. The method according to claim 1, characterized in that, The concentration of the Tris working solution is 0.5~5M, and the volume ratio of the Tris working solution to the acid solution is 1:(2~5).

4. The method according to claim 1, characterized in that, The colorimetric reaction is carried out at 35-40°C under light-protected conditions for 5-15 minutes.

5. The method according to any one of claims 1-4, characterized in that, Also includes: The test results are compared with the standard curve, and qualitative or quantitative detection of anti-Nephrin antibodies is achieved based on the comparison results. The standard curve was constructed based on the test results of the standard solution using the same method.

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