A gouty arthritis auxiliary diagnosis kit and a preparation method thereof

Abstract

This invention belongs to the field of biodetection technology, specifically relating to an auxiliary diagnostic kit for gouty arthritis and its preparation method. The auxiliary diagnostic kit includes a paper packaging box, a pre-coated 96-well plate, and detection reagents. The detection reagents include the following raw materials: detection probe A, detection probe B, streptavidin, diluent, washing solution, TMB chromogenic solution, stop solution, and standards. By designing a UOx-NAb fusion protein detection probe A that can specifically bind to the MSU crystal-ApoE complex, the fixation amount and accuracy of gouty arthritis biomarkers are improved, avoiding diagnostic errors caused by simply detecting uric acid levels. This provides an efficient, accurate, highly specific, and highly sensitive in vitro detection solution for gouty arthritis.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to an auxiliary diagnostic kit for gouty arthritis and its preparation method. Background Technology

[0002] Gouty arthritis (GA) is a metabolic inflammatory joint disease caused by elevated uric acid levels in the blood due to purine metabolism disorders, leading to the deposition of MSU (monosodium urate) crystals in the joints and surrounding tissues. It differs fundamentally from other types of arthritis such as rheumatoid arthritis and osteoarthritis in its pathological mechanism. While the latter two are mainly related to autoimmune attacks or mechanical wear and tear on the joints, the core pathogenic factor of gout lies in the supersaturation of soluble uric acid in the local tissue environment, forming MSU crystals with specific morphologies. These crystals are recognized by immune cells, activating inflammasomes and triggering a rapid and severe inflammatory response. Currently, the clinical diagnosis of this disease largely relies on typical acute exacerbations, such as sudden onset of redness, swelling, heat, and pain in a single joint at night, especially the first metatarsophalangeal joint. However, not all patients present with typical symptoms, making differential diagnosis challenging. Current auxiliary diagnostic techniques primarily rely on polarized light microscopy of joint aspiration fluid to locate intracellular MSU crystals as a diagnostic criterion. However, this method is highly dependent on the experience and skill level of the laboratory personnel, and is prone to missed detection when the number of crystals is small or their morphology is atypical. Furthermore, it is an invasive procedure with low patient acceptance and is unsuitable for early screening and dynamic monitoring. In addition, the widely used serum uric acid concentration test has poor specificity. Since most patients with hyperuricemia never experience gout attacks, and some patients during acute gout attacks may have normal serum uric acid levels, this indicator can only be used as a risk factor assessment and cannot be used as a definitive diagnosis. This lag and inadequacy in diagnostic capabilities directly leads to a persistently high rate of misdiagnosis and missed diagnosis of gouty arthritis in clinical practice, not only delaying the optimal treatment window but also affecting the accurate assessment of disease activity and the judgment of long-term management outcomes.

[0003] Therefore, finding specific biomarkers unique to gouty arthritis and developing an efficient, accurate, highly specific, and highly sensitive auxiliary diagnostic tool based on these biomarkers is of great clinical significance for achieving early detection, early diagnosis, precise intervention, and efficacy evaluation of gouty arthritis. Summary of the Invention

[0004] To address the above issues, this invention provides an auxiliary diagnostic kit for gouty arthritis and its preparation method. Through a designed fusion protein probe, it specifically identifies the hallmark components of uric acid and sodium urate crystal-apolipoprotein E complex in gouty arthritis. The signal is amplified by linking a catalytic enzyme with streptavidin, thereby enabling diagnosis of the pathological aspects of the disease. This provides a highly reliable in vitro detection solution for the precise auxiliary diagnosis of gouty arthritis.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] This invention provides an auxiliary diagnostic kit for gouty arthritis. The outer packaging of the auxiliary diagnostic kit is a paper box, and the inside of the paper box contains a pre-coated 96-well plate and a detection reagent.

[0007] Furthermore, the pre-wrapped 96-hole plate is located at the top of the paper packaging box, and there are two of them.

[0008] Furthermore, the detection reagent is located at the bottom of the paper packaging box, and the detection reagent comprises the following raw materials in parts by weight: 1.5-3 parts of detection probe A, 1.5-3 parts of detection probe B, 0.5-2 parts of streptavidin, 500 parts of diluent, 300 parts of washing solution, 15 parts of TMB (tetramethylbenzidine) colorimetric solution, 15 parts of stop solution, and 6 parts of standard.

[0009] Further, the diluent is 0.01 M PBS (phosphate buffer) with a pH of 7.4.

[0010] Furthermore, the diluent also contains 0.5% by mass of Tween 20 and 1% by mass of BSA (bovine serum albumin).

[0011] Furthermore, the washing solution is a concentrated solution obtained by concentrating PBS containing 0.5% Tween 20 by mass, 0.01 M molar concentration, and pH 7.4, and the concentration factor of the concentrated solution is 20 times.

[0012] Furthermore, the terminating solution is a sulfuric acid solution with a molar concentration of 2 M.

[0013] Furthermore, the detection probe B is Biotin-HRP (biotin-labeled horseradish peroxidase).

[0014] Furthermore, the standard comprises the following raw materials: sodium urate, recombinant human ApoE (apolipoprotein E), and uric acid, wherein the mass ratio of sodium urate, recombinant human ApoE, and uric acid is 10:1:4.

[0015] Furthermore, the method for preparing the standard is as follows:

[0016] S1: Dissolve sodium urate, slowly adjust the pH to 7.0 with 1 M HCl (hydrochloric acid) solution, heat at 60℃ and let stand for 48 hours to produce crystals, separate and dry the crystals to obtain MSU crystals;

[0017] S2: MSU crystals and recombinant human ApoE were co-dispersed in PBS (pH 7.4, containing 0.05% Tween 20 by mass), incubated at 37°C with shaking for 2 h and centrifuged to promote the adsorption of recombinant human ApoE on the surface of MSU crystals, mimicking the protein adsorption of monosodium urate in vivo. The precipitate was collected to obtain the MSU crystal-ApoE complex.

[0018] S3: Dissolve uric acid in PBS (pH 7.4, containing 1% BSA by mass) to obtain a uric acid solution. Resuspend the MSU crystal-ApoE complex in the uric acid solution to simulate the serum matrix of gouty arthritis and obtain the mother liquor, i.e., the standard.

[0019] Furthermore, the detection probe A is a UOx (uric acid oxidase)-NAb fusion protein, and its amino acid sequence is shown in SEQ ID NO.1.

[0020] Furthermore, the UOx-NAb fusion protein comprises a sequence-optimized UOx protein fragment, a NAb nanobody, and an Avi-tag.

[0021] Furthermore, the amino acid sequence of the UOx protein fragment is shown in SEQ ID NO.2.

[0022] Furthermore, the NAb nanobody is an antibody protein that specifically adsorbs the MSU crystal-ApoE complex, obtained by screening based on molecular docking and phage display technology, and its amino acid sequence is shown in SEQ ID NO.3.

[0023] Furthermore, the nucleotide sequence of the gene encoding the UOx-NAb fusion protein is shown in SEQ ID NO.4.

[0024] Furthermore, the preparation method of the detection probe A is as follows:

[0025] B1: After selecting the UOx protein fragment as the uric acid-binding domain and optimizing its sequence, it was combined with the amino acid sequence of the NAb nanobody. The NAb nanobody is an antibody protein that can specifically adsorb to the MSU crystal-ApoE complex, obtained by molecular docking and phage display technology. Its amino acid sequence is shown in SEQ ID NO.3. The selected protein fragment was combined, and a 6×His tag and an Avi-tag tag were introduced at the N-terminus and C-terminus, respectively, to obtain the amino acid sequence of the UOx-NAb fusion protein, as shown in SEQ ID NO.1. Gene sequence design and optimization were carried out using gene coding software to obtain the nucleotide sequence encoding the UOx-NAb fusion protein, as shown in SEQ ID NO.4.

[0026] B2: The encoding gene of the UOx-NAb fusion protein was cloned into an expression vector to construct a recombinant plasmid. The recombinant plasmid was transfected into HEK293F cells in suspension culture for protein expression, separation and collection to obtain crude protein.

[0027] B3: The crude protein was eluted and purified using a Ni-NTA agarose chromatography column to obtain the UOx-NAb fusion protein, which is the detection probe A.

[0028] Furthermore, the pre-coated 96-well plate is prepared by coating a 96-well plate with a capture protein.

[0029] Furthermore, the capture protein consists of a sequence-optimized UOx protein fragment and a NAb nanobody.

[0030] This invention also provides a method for preparing an auxiliary diagnostic kit for gouty arthritis, the specific steps of which are as follows:

[0031] Step 1: Dissolve detection probe A, detection probe B and streptavidin separately to prepare solutions and dispense them into reagent A, reagent B and reagent C respectively;

[0032] Step 2: After dispensing the diluent, washing solution, TMB colorimetric solution, stop solution and standard, place them together with reagent A, reagent B and reagent C and two pre-coated 96-well plates in a packaging box, seal with a coating, and obtain the gouty arthritis auxiliary diagnostic kit, which should be stored at -20℃.

[0033] The beneficial effects achieved by this invention are as follows:

[0034] The gouty arthritis auxiliary diagnostic kit provided by this invention designs and constructs a novel UOx-NAb fusion protein probe. One end of the probe specifically captures uric acid molecules through an engineered UOx module, while the other end accurately recognizes the MSU crystal-ApoE complex through a high-affinity nanobody obtained by phage display technology. This forms a core structure that specifically binds to the biomarker of gouty arthritis, breaking through the limitations of traditional single-indicator detection and realizing the simultaneous verification of pathogenic crystal formation and its metabolic background. After the UOx-NAb fusion protein specifically binds to uric acid and the MSU crystal-ApoE complex in the test sample, the capture protein in the pre-coated 96-well plate specifically captures the MSU crystal-ApoE complex bound to the UOx-NAb fusion protein. Streptavidin specifically and strongly binds to the Avi-tag in the UOx-NAb fusion protein under non-covalent forces. Streptavidin also connects to biotinylated Biotin-HRP, immobilizing it within the wells. Finally, the TMB chromogenic solution acts as the substrate for HRP enzyme catalysis, producing color and achieving the specific detection of gouty arthritis markers. When the test sample contains no uric acid or the MSU crystal-ApoE complex, the pre-coated 96-well plate is fully exposed. Since the capture protein at the bottom does not contain the Avi-tag, it cannot bind to streptavidin, and no color reaction occurs, greatly improving the accuracy of the detection. This invention addresses the issues of low accuracy and insufficient specificity in existing technologies for detecting uric acid markers in gouty arthritis. It features high sensitivity and high identification efficiency for gouty arthritis, providing new technical support for the accurate diagnosis of gouty arthritis. Attached Figure Description

[0035] Figure 1 This is a schematic diagram of the structural composition of an auxiliary diagnostic kit for gouty arthritis according to the present invention;

[0036] Figure 2 This is a schematic diagram illustrating the detection principle mechanism of an auxiliary diagnostic kit for gouty arthritis according to the present invention.

[0037] Figure 3 Diagram of the recombinant plasmid expressing the UOx-NAb fusion protein;

[0038] Figure 4 A standard curve was plotted for the standards used in an auxiliary diagnostic kit for gouty arthritis prepared in Example 4.

[0039] Figure 5 The results of detecting the content of gouty arthritis markers using the gouty arthritis auxiliary diagnostic kit prepared in Example 4 were obtained.

[0040] The components are: 1. Paper packaging box; 2. Pre-coated 96-well plate; 3. Diluent; 4. Washing solution; 5. Reagent A; 6. Reagent B; 7. Reagent C; 8. TMB colorimetric solution; 9. Stop solution; 10. Standard. Detailed Implementation

[0041] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to this invention. The preferred embodiments and materials described herein are for illustrative purposes only and do not limit the scope of this application.

[0043] The structure of the gouty arthritis auxiliary diagnostic kit described in this application is as follows: Figure 1 As shown; the detection principle mechanism of the auxiliary diagnostic kit is as follows: Figure 2 As shown; the recombinant plasmid used to express the UOx-NAb fusion protein is as follows: Figure 3 As shown; unless otherwise specified, all methods in the following examples are conventional; unless otherwise specified, all materials used in the following examples and comparative examples are new materials purchased from the market; the PBS buffer used is 0.01 M, pH 7.4 PBS; the sodium urate used is sodium urate salt, purchased from Yisheng Biotechnology; the Biotin-HRP used is purchased from Beyotime and diluted at a volume ratio of 1:2000 before use; the amino acid sequence information of the UOx protein was retrieved from NCBI.

[0044] Example 1: This example provides a detection probe A, and the specific preparation method is as follows:

[0045] B1: Based on the core structural domain of the selected UOx protein and after optimization, the amino acid sequence shown in SEQ ID NO.2 was obtained. Based on the NAb nanobody with specific adsorption capacity of MSU crystal-ApoE complex that has been screened, the amino acid sequence is shown in SEQ ID NO.3. Sequence combination was performed, and 6×His tag and Avi-tag tag were modified at the N-terminus and C-terminus respectively to obtain UOx-NAb fusion protein, the amino acid sequence of which is shown in SEQ ID NO.1. Gene sequence design and optimization were performed on it to obtain the nucleotide sequence encoding UOx-NAb fusion protein, as shown in SEQ ID NO.4.

[0046] B2: A BglII restriction enzyme site was introduced at the 5' end of the coding gene sequence of the UOx-NAb fusion protein, and a BssHII restriction enzyme site was introduced at the 3' end. Using pET-28a(+) as the expression vector, the coding gene was introduced into the expression vector to construct a plasmid, which was transformed into competent Escherichia coli DH5α, plated on LB plates containing ampicillin (100 μg / mL), and single colonies were picked for amplification culture and resuspended to obtain a bacterial suspension.

[0047] B3: Recombinant plasmids were extracted from the bacterial suspension using an endotoxin-free plasmid extraction kit. The extracted recombinant plasmids were then transfected into HEK293F cells. The transfection reagent was linear polyethyleneimine. The cells were cultured at 37°C, 5% CO2 concentration, and 120 rpm for 5 days. The cell culture supernatant was then collected.

[0048] B4: Centrifuge the cell culture supernatant at 4000×g for 30 min at 4℃ to remove cell debris and obtain crude protein. Load the crude protein at a flow rate of 1 mL / min onto a Ni-NTA agarose chromatography column equilibrated with equilibration buffer (containing 20 mM sodium phosphate, 300 mM NaCl, 20 mM imidazole, pH 7.4) and wash thoroughly with 10 column volumes of equilibration buffer to remove unbound contaminants. Then, perform staged elution with elution buffer (containing 20 mM sodium phosphate, 300 mM NaCl, 250 mM imidazole, pH 7.4) and collect the elution peak fraction. Ultrafilter the elution peak fraction through a 10 kDa molecular weight cutoff ultrafiltration centrifuge tube to remove imidazole, and lyophilize to obtain UOx-NAb fusion protein, i.e., detection probe A.

[0049] Example 2: This example provides a standard, which comprises the following raw materials in parts by weight: 20 parts sodium urate, 2 parts recombinant human ApoE, and 8 parts uric acid. The specific preparation method is as follows:

[0050] S1: Dissolve 20 parts of sodium urate in 180 parts of distilled water to obtain sodium urate solution. Slowly adjust the pH to 7.0 with 1 M HCl solution. After heating at 60°C, seal the solution and let it stand at 4°C for 48 h to produce crystals. Collect the crystals by vacuum filtration and wash them three times with pre-cooled distilled water to obtain wet crystals. Dry the wet crystals under vacuum at 30°C for 12 h to obtain white needle-like MSU crystals.

[0051] S2: MSU crystals and two portions of recombinant human ApoE were co-dispersed in 1000 portions of PBS (pH 7.4, containing 0.05% Tween 20 by mass), incubated at 37°C with shaking for 2 h and centrifuged to promote the adsorption of recombinant human ApoE on the surface of MSU crystals, mimicking the protein adsorption of monosodium urate in vivo. The precipitate was collected by centrifugation at 4°C and 10000×g for 10 min to obtain the MSU crystal-ApoE complex.

[0052] S3: Dissolve 8 portions of uric acid in PBS (pH 7.4, containing 1% BSA by mass) to obtain a uric acid solution. Resuspend the MSU crystal-ApoE complex in the uric acid solution to simulate the serum matrix of gouty arthritis and obtain the mother liquor, i.e., the standard.

[0053] Example 3: This example provides a pre-coated 96-well plate, which is made by coating a capture protein in a 96-well plate. The capture protein is composed of a sequence-optimized UOx protein fragment and a NAb nanobody.

[0054] The method for preparing the capture protein is the same as in Example 1, but the C-terminus is not modified with an Avi-tag.

[0055] The method for preparing the pre-coated 96-well plate is as follows:

[0056] N1: Dissolve the capture protein in 0.05 M carbonate-sodium bicarbonate buffer (pH 9.6) to prepare a 5 μg / mL capture protein solution. Add 100 μL / well of the capture protein solution to a 96-well plate using a multichannel pipette. After sealing with a sealing membrane, incubate at 4°C for 16 h to obtain the incubation plate.

[0057] N2: Carefully aspirate the liquid from the wells of the incubation plate. Add 200 μL of PBS (0.01 M, 0.05% Tween 20, pH 7.4) to each well for washing to remove capture proteins that have not bound to the bottom of the plate. Then add PBS (0.01 M, pH 7.4) containing 3% BSA (200 μL / well) for blocking. Seal and incubate for 2 h to block the blank sites at the bottom of the plate that have not been occupied by capture proteins. Discard the liquid, air dry at room temperature for 2 h, and dry in a 37°C incubator for 30 min. Cover with the 96-well plate cap to obtain a pre-coated 96-well plate.

[0058] Example 4: This example provides an auxiliary diagnostic kit for gouty arthritis. The outer packaging of the auxiliary diagnostic kit is a paper box, and the paper box contains two pre-coated 96-well plates and detection reagents.

[0059] The detection reagent comprises the following raw materials in parts by weight: 1.5 parts of detection probe A, 3 parts of detection probe B, 0.5 parts of streptavidin, 500 parts of diluent, 300 parts of washing solution, 15 parts of TMB colorimetric solution, 15 parts of stop solution, and 6 parts of standard.

[0060] This embodiment also provides a method for preparing an auxiliary diagnostic kit for gouty arthritis, which specifically includes the following steps:

[0061] Step 1: Dissolve 1.5 parts by weight of detection probe A, 3 parts by weight of detection probe B, and 0.5 parts by weight of streptavidin in PBS (0.01 M, pH 7.4, containing 1% BSA and 0.05% Proclin-300) to prepare solutions with concentrations of 15 μg / mL, 30 μg / mL, and 5 μg / mL, respectively, to obtain reagent A, reagent B, and reagent C. Each reagent is dispensed in 2 mL portions.

[0062] Step 2: Dispense 500 parts by weight of diluent, 300 parts by weight of washing solution, 15 parts by weight of TMB colorimetric solution, 15 parts by weight of stop solution and 6 parts by weight of standard, and place them together with reagent A, reagent B and reagent C and two pre-coated 96-well plates in a packaging box, seal with a film, and obtain the gouty arthritis auxiliary diagnostic kit, and store at -20℃.

[0063] Example 5: This example provides an auxiliary diagnostic kit for gouty arthritis. The outer packaging of the auxiliary diagnostic kit is a paper box, and the inside of the paper box contains two pre-coated 96-well plates and detection reagents.

[0064] The detection reagent comprises the following raw materials in parts by weight: 2.5 parts of detection probe A, 2 parts of detection probe B, 1 part of streptavidin, 500 parts of diluent, 300 parts of washing solution, 15 parts of TMB colorimetric solution, 15 parts of stop solution, and 6 parts of standard.

[0065] This embodiment also provides a method for preparing an auxiliary diagnostic kit for gouty arthritis, which specifically includes the following steps:

[0066] Step 1: Dissolve 2.5 parts by weight of detection probe A, 2 parts by weight of detection probe B, and 1 part by weight of streptavidin in PBS (0.01 M, pH 7.4, containing 1% BSA and 0.05% Proclin-300) to prepare solutions with concentrations of 25 μg / mL, 20 μg / mL, and 10 μg / mL, respectively, to obtain reagent A, reagent B, and reagent C. Each reagent is dispensed in 2 mL portions.

[0067] Step 2: Dispense 500 parts by weight of diluent, 300 parts by weight of washing solution, 15 parts by weight of TMB colorimetric solution, 15 parts by weight of stop solution and 6 parts by weight of standard, and place them together with reagent A, reagent B and reagent C and two pre-coated 96-well plates in a packaging box, seal with a film, and obtain the gouty arthritis auxiliary diagnostic kit, and store at -20℃.

[0068] Example 6: This example provides an auxiliary diagnostic kit for gouty arthritis. The outer packaging of the auxiliary diagnostic kit is a paper box, and the paper box contains two pre-coated 96-well plates and detection reagents.

[0069] The detection reagent comprises the following raw materials in parts by weight: 3 parts of detection probe A, 1.5 parts of detection probe B, 2 parts of streptavidin, 500 parts of diluent, 300 parts of washing solution, 15 parts of TMB colorimetric solution, 15 parts of stop solution, and 6 parts of standard.

[0070] This embodiment also provides a method for preparing an auxiliary diagnostic kit for gouty arthritis, which specifically includes the following steps:

[0071] Step 1: Dissolve 3 parts by weight of detection probe A, 1.5 parts by weight of detection probe B, and 2 parts by weight of streptavidin in PBS (0.01 M, pH 7.4, containing 1% BSA and 0.05% Proclin-300) to prepare solutions with concentrations of 30 μg / mL, 15 μg / mL, and 20 μg / mL, respectively, to obtain reagent A, reagent B, and reagent C. Each reagent is dispensed in 2 mL portions.

[0072] Step 2: Dispense 500 parts by weight of diluent, 300 parts by weight of washing solution, 15 parts by weight of TMB colorimetric solution, 15 parts by weight of stop solution and 6 parts by weight of standard, and place them together with reagent A, reagent B and reagent C and two pre-coated 96-well plates in a packaging box, seal with a film, and obtain the gouty arthritis auxiliary diagnostic kit, and store at -20℃.

[0073] Sample selection: Serum, urine, and joint effusion samples were collected from 40 patients clinically diagnosed with GA (gouty arthritis) as the GA group, including 12 patients in the early stage of GA. Inclusion criteria: GA confirmed by relevant imaging and ultrasound examinations; presence of microcrystals or tophi; complete medical history and high cooperation. Exclusion criteria: Concomitant rheumatic or other types of arthritis; mental illness. Serum, urine, and joint effusion samples were collected from 67 patients clinically diagnosed with RA (rheumatic or rheumatoid arthritis) as the control group, including 43 patients diagnosed with rheumatic arthritis. Serum, urine, and joint effusion samples were collected from 20 healthy patients as the healthy group.

[0074] Detection Method: The gouty arthritis auxiliary diagnostic kit prepared in Example 4 was used. The standard solution was serially diluted with washing buffer to obtain a series of standard concentrations (c) based on uric acid concentration: 600, 300, 150, 75, 30, 3, and 0.3 μg / mL. The absorbance (A) was measured at 450 nm using an ELISA reader to establish a standard curve. 90 μL of each sample from the GA group, control group, and healthy group was mixed with 10 μL of reagent A, incubated for 3 min, and then added to a pre-coated 96-well plate. The plate was incubated with shaking at 100 rpm for 5 min. The liquid was carefully aspirated, and the washing buffer was diluted 20 times with distilled water to obtain a diluted washing buffer. 100 μL of the diluted washing buffer was added to the pre-coated 96-well plate, gently shaken, and then aspirated. This process was repeated three times. After washing, 10 μL of reagent B, 10 μL of reagent C, and 80 μL of diluent were added to the pre-coated 96-well plate, and the plate was allowed to stand for 2 minutes. After 10 min, aspirate the liquid, invert and pat dry. Wash twice with diluted washing buffer. Add 10 μL of TMB chromogenic solution and 80 μL of diluent. Incubate in the dark for 10 min. Add 10 μL of stop solution, gently shake for 1 min, and measure absorbance at 450 nm using a microplate reader. Calculate the concentration based on the standard curve (see [standard curve image]). Figure 4 The sample test results are shown below. Figure 5 .

[0075] Figure 4 The results showed that the standard curve was A = 0.0036c + 0.1076, and R0 was... 2 =0.9991, indicating good linearity.

[0076] Figure 5 The results showed that the gouty arthritis auxiliary diagnostic kit prepared in Example 4 had high detection sensitivity for gouty arthritis. The difference between the control group and the healthy group was small, while the content of GA in the GA group was significantly increased. This indicates that the kit prepared in this application has a specific effect on gouty arthritis and can be used for the auxiliary diagnosis of gouty arthritis, reducing the probability of misdiagnosis.

[0077] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

[0078] The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present invention. The actual application is not limited to this. In conclusion, if those skilled in the art are inspired by this description and design similar methods and embodiments without departing from the spirit of the present invention, they should all fall within the protection scope of the present invention.

Claims

1. A gouty arthritis auxiliary diagnostic kit, the outer package of which is a paper packaging box, characterized in that, The paper packaging box contains a pre-coated 96-well plate and a detection reagent inside; The detection reagent comprises the following raw materials by weight: detection probe A 1.5-3 parts, detection probe B 1.5-3 parts, streptavidin 0.5-2 parts, diluent 500 parts, washing solution 300 parts, TMB developing solution 15 parts, termination solution 15 parts, and standard 6 parts; The detection probe A is a UOx-NAb fusion protein, and the amino acid sequence is shown as SEQ ID NO. 1; The nucleotide sequence of the coding gene of the UOx-NAb fusion protein is shown as SEQ ID NO. 4; The preparation method of the detection probe A is as follows: B1: Determine the amino acid sequence of the UOx-NAb fusion protein and the nucleotide sequence of its coding gene; B2: Construct a recombinant plasmid based on the coding gene of the UOx-NAb fusion protein and transfect culture, isolate, and obtain a crude protein; B3: Purify the crude protein to obtain the UOx-NAb fusion protein, i.e., the detection probe A; The detection probe B is Biotin-HRP.

2. The diagnostic kit for gouty arthritis according to claim 1, wherein The UOx-NAb fusion protein comprises a sequence-optimized UOx protein fragment, a NAb nanobody, and an Avi-tag label; The amino acid sequence of the UOx protein fragment is shown as SEQ ID NO. 2; The amino acid sequence of the NAb nanobody is shown as SEQ ID NO.

3.

3. The diagnostic kit for gouty arthritis according to claim 1, wherein The standard comprises the following raw materials: sodium urate, recombinant human ApoE, and uric acid; The preparation method of the standard is as follows: S1: Dissolve sodium urate and adjust the pH and temperature to produce crystals, separate and dry the crystals to obtain MSU crystals; S2: Incubate the MSU crystals with recombinant human ApoE, centrifuge, and collect the precipitate to obtain an MSU crystal-ApoE complex; S3: Dissolve the uric acid and mix it with the MSU crystal-ApoE complex to obtain the standard.

4. The diagnostic kit for gouty arthritis according to claim 2, wherein The pre-coated 96-well plate is prepared by coating a capture protein in a 96-well plate; The capture protein is composed of a sequence-optimized UOx protein fragment and a NAb nanobody.

5. A method for preparing a diagnostic kit for gouty arthritis according to any one of claims 1 to 4, characterized in that, The specific preparation method is as follows: Step 1: Dissolve the detection probe A, the detection probe B, and the streptavidin respectively to prepare solutions, and obtain reagent A, reagent B, and reagent C respectively; Step 2: After dispensing the diluent, the washing solution, the TMB developing solution, the termination solution, and the standard, place them together with reagent A, reagent B, and reagent C and two pre-coated 96-well plates in the packaging box, and coat and seal it to obtain a gout auxiliary diagnostic kit.

Images