A kit for combined detection of CA724 and uric acid and its application

Abstract

The present invention provides a kit and application for joint detection of CA724 and uric acid, the kit includes a test card and a sample detection solution, the test card includes a test strip, the test strip includes a base plate, a sample pad, a conjugation pad, a nitrocellulose membrane and a water-absorbing pad, the sample pad, the conjugation pad, the nitrocellulose membrane and the water-absorbing pad are sequentially adhered to the base plate, the conjugation pad is adsorbed with uric acid antibody-labeled fluorescent microspheres and CA724 antibody-labeled fluorescent microspheres, the nitrocellulose membrane is sequentially provided with a T1 detection line coated with uric acid-bovine serum albumin hapten, a T2 detection line coated with CA724 monoclonal antibody and a quality control line C according to the sample flow direction, and the sample detection solution is composed of specific components. The kit of the present invention can realize simultaneous quantitative detection of CA724 and uric acid, and the result is accurate, the sensitivity is high, the operation steps are simplified, the sample dosage is reduced, the time is saved, the cost is saved, and it is conducive to early gout diagnosis and treatment.

Description

Technical Field

[0001] The invention belongs to the technical field of medical testing and relates to a kit for jointly detecting CA724 and uric acid and an application thereof. Background Art

[0002] Gout is a common inflammatory disease characterized by the deposition of monosodium urate in the synovial fluid and tissues of arthritis. Over the past few decades, the incidence of gout has continued to grow worldwide. The currently widely used clinical evaluation and diagnostic criteria for gout are based on peripheral joint manifestations and serum uric acid concentrations. However, for some patients, serum uric acid concentrations can be normal, even when arthritis attacks. In addition, serum uric acid levels are particularly sensitive to dietary influences. Arthritis attacks may be temporary and often resolve spontaneously without treatment, which can be easily overlooked by patients. Gout has mild early symptoms, and patients with normal uric acid levels often delay diagnosis. There is an urgent need to predict new biomarkers for better diagnosis of gout.

[0003] C724, ​​or carbohydrate antigen 72-4, is also known as carbohydrate antigen 72-4 and cancer antigen 72-4. It is essentially a high-molecular glycoprotein of the cell surface mucin class. It is widely distributed in the cytoplasm of epithelial cells and malignant tumors, with a molecular weight of more than 1000kDa. Carbohydrate antigen 724 (CA724) was first discovered by Colcher et al. in 1981 in a patient with liver cancer metastasis from breast cancer. It is a tumor-related antigen of the stomach, colorectum, liver, lung, pancreas and ovary. Its content in the serum of healthy people and patients with benign tumors is very low, and it has the functions of early tumor prediction, disease monitoring and prognosis judgment. In recent years, serum CA724 has become a reliable marker for early screening, monitoring and prognosis evaluation of gastric cancer. Its content is closely related to the clinical stage of gastric cancer, tumor size, lymph node involvement and infiltration and metastasis, and it has high specificity.

[0004] Bai et al. (Bai X, Sun M, He Y, et al. Serum CA72-4 is specifically elevated in gout patients and predicts flares [J]. Rheumatology (Oxford, England), 2020, 59 (10): 2872.) showed that compared with healthy subjects and patients with other arthritis diseases, the expression of serum CA724 in patients with gouty arthritis was significantly increased. Zhang et al. (Zhang M, Dou H, Yang D, et al. Retrospective analysis of glycanrelated biomarkers based on clinical laboratory data in two medical centers during the past 6 years [J]. Prog Mol Biol Transl Sci, 2019, 162: 141.) found that compared with tumor patients and healthy controls, CA724 was more significantly elevated in patients with gout and gouty arthritis, suggesting that CA724 is more closely related to gouty arthritis, and that CA724 may play an unknown but important role in inflammatory response. Therefore, it is speculated that CA724 may be used as a marker to distinguish gout from other joint diseases and has certain diagnostic significance.

[0005] The basis of gout is hyperuricemia, but not all hyperuricemia will develop into gout. In fact, only 10% of patients with hyperuricemia develop gout. At present, the diagnosis of gout mostly relies on clinical manifestations, routine blood and urine tests, blood uric acid determination, joint ultrasound and joint cavity puncture examinations, etc. Although they each have their own advantages, they are invasive examinations, or lack sensitivity or specificity, or have relatively high technical requirements, require rich clinical experience, and cannot meet clinical needs. At the same time, because gout not only causes joint inflammation, long-term gout can also become an important risk factor for hypertension, hyperlipidemia, cardiovascular disease and cerebrovascular disease. Therefore, it is very necessary to find one or more correlation indicators with high sensitivity and specificity for gout diagnosis and early treatment. Summary of the invention

[0006] In view of the deficiencies in the prior art, the object of the present invention is to provide a kit for the combined detection of CA724 and uric acid and its application.

[0007] To achieve this object, the present invention adopts the following technical solutions:

[0008] On the one hand, the present invention provides a kit for the combined detection of CA724 and uric acid, the kit comprising a detection card and a sample detection solution, the detection card comprising a test strip, the test strip comprising a bottom plate, a sample pad, a conjugation pad, a nitrocellulose membrane and a water absorbent pad, the sample pad, the conjugation pad, the nitrocellulose membrane and the water absorbent pad are sequentially adhered to the bottom plate, the conjugation pad is adsorbed with uric acid antibody-labeled fluorescent microspheres and CA724 antibody-labeled fluorescent microspheres, the nitrocellulose membrane is sequentially provided with a T1 detection line coated with a uric acid-bovine serum albumin hapten, a T2 detection line coated with a CA724 monoclonal antibody and a quality control line C from front to back according to the sample flow direction;

[0009] The sample detection solution includes the following components: 10-50mM (for example, 10mM, 20mM, 30mM, 40mM or 50mM) PBS buffer solution (pH7.4), 0.5-1% (for example, the mass percentage concentration is 0.5%, 0.8% or 1%) Biolipidure-206, 0.1-0.5% (for example, the mass percentage concentration is 0.1%, 0.3% or 0.5%) PEG10000, and 0.05% (mass percentage concentration) Proclin 300.

[0010] In the art, uric acid is usually detected by biochemical testing, and CA724 is usually detected by immunoassay. The present invention uses the test strip to achieve the combined detection of uric acid and CA724. Using one machine, the test results can be simultaneously obtained, and the test results are accurate.

[0011] In the present invention, the uric acid and CA724 combined detection test strip is based on time-resolved fluorescent immunochromatography technology, which is used to quantitatively determine the concentration of uric acid and CA724 in human blood samples. Uric acid is tested by the competitive method: the surface carboxyl-modified fluorescent microspheres wrapped with Eu europium chelate are covalently bound to the amino groups on the surface of mouse anti-human uric acid antibodies to form immune-labeled time-resolved fluorescent microspheres. The sample test solution and the blood sample are mixed and added to the reagent card, and the uric acid to be tested in the sample is combined with the mouse anti-human uric acid antibody nano-microsphere fluorescent probe on the marker pad to form a complex (i.e., the test object-antibody nano-microsphere fluorescent probe complex), and is chromatographed to the detection membrane (with two detection lines and one control line) through capillary action, and finally chromatographed to the absorbent pad. At this time, the antibody nanosphere fluorescent probe that has not combined with the analyte in the sample combines with the uric acid bovine serum albumin hapten (UA-BSA) coated on the T1 line detection membrane to form a complex (i.e., antigen-antibody nanosphere fluorescent probe complex), and the excess unreacted antibody nanosphere fluorescent probe continues to chromatograph to the absorbent pad. Therefore, the more analytes there are in the blood sample, the less the monoclonal antibody nanosphere fluorescent probe accumulates on the corresponding detection line.

[0012] CA724 uses the double antibody sandwich method test principle: the analyte CA724 in the sample combines with the mouse anti-human glycoprotein CA724 monoclonal antibody I nano-microsphere fluorescent probe on the marker pad to form a complex (i.e., the analyte-monoclonal antibody nano-microsphere fluorescent probe complex), and is chromatographed to the T2 detection line containing mouse anti-human glycoprotein CA724 monoclonal antibody II on the nitrocellulose membrane through capillary action, and is captured to form a double antibody sandwich complex (i.e., monoclonal antibody-analyte-monoclonal antibody nano-microsphere fluorescent probe complex). Therefore, the more CA724 in the sample, the more the double antibody sandwich complex accumulates on the detection line. The fluorescence intensity on the detection line is positively correlated with the concentration of the analyte CA724 in the sample.

[0013] The test strip of the present invention can complete the simultaneous detection of CA724 and uric acid, and the result is accurate, the sensitivity is high, and the detection is fast.

[0014] The sample pad is a sample pad obtained by soaking a glass cellulose membrane in a treatment solution and then drying it, wherein the treatment solution is a 20-100 mM Tris buffer solution containing 2-5% S9 by mass percentage, 0.5-2 mg / mL blocking agent, and 0.1-0.2 mg / mL RBC antibody, and its pH is 8.0;

[0015] Preferably, the blocking agent is a murine heterophilic antibody blocking agent.

[0016] Preferably, the absorbent pad is a polymer composite cotton linter pulp filter paper, ordinary fiber filter paper, a silica gel absorbent pad or a sponge absorbent pad, preferably a polymer composite cotton linter pulp filter paper.

[0017] In the present invention, the positions of the T1 detection line and the T2 detection line cannot be swapped, and such a setting of the detection line sequence can ensure fast and accurate detection.

[0018] Preferably, the concentration of the uric acid-bovine serum albumin hapten coated on the T1 test line is 0.5-2 mg / mL (e.g., 0.5 mg / mL, 0.8 mg / mL, 1 mg / mL, 1.5 mg / mL, 1.8 mg / mL or 2 mg / mL), and the volume of the line is 20-45 μL / 30cm (e.g., 20 μL / 30cm, 25 μL / 30cm, 30 μL / 30cm, 35 μL / 30cm, 40 μL / 30cm or 45 μL / 30cm); The concentration of CA724 monoclonal antibody used for drawing the T2 test line is 0.5-3 mg / mL (e.g., 0.5 mg / mL, 0.8 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, 2.5 mg / mL or 3 mg / mL), and the volume of the drawing is 20-45 μL / 30cm (e.g., 20 μL / 30cm, 25 μL / 30cm, 30 μL / 30cm, 35 μL / 30cm, 40 μL / 30cm or 45 μL / 30cm).

[0019] Preferably, when forming the conjugate pad, the concentration of the diluted uric acid antibody labeled fluorescent microsphere solution is 20-100 μg / mL (e.g., 20 μg / mL, 25 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL or 100 μg / mL), the concentration of the diluted CA724 antibody labeled fluorescent microsphere solution is 10-50 μg / mL (e.g., 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL or 50 μg / mL), and the amount of the mixed solution of uric acid and CA724 antibody labeled fluorescent microspheres sprayed is 5-10 μL / cm 2 (e.g. 5 μL / cm 2 , 6μL / cm 2 , 7μL / cm 2 , 8μL / cm 2 , 9μL / cm 2 or 10 μL / cm 2 ).

[0020] In the present invention, the uric acid antibody-labeled fluorescent microspheres are formed by covalently bonding the surface carboxyl-modified fluorescent microspheres coated with Eu chelates to the amino groups on the surface of mouse anti-human uric acid antibodies.

[0021] In the present invention, the CA724 antibody labeled fluorescent microspheres are formed by covalently bonding the surface carboxyl-modified fluorescent microspheres coated with Eu europium chelate to the amino groups on the surface of mouse anti-human glycoprotein CA724 monoclonal antibody I.

[0022] Preferably, the quality control line C is a quality control line coated with chicken immunoglobulin G (IgG).

[0023] Preferably, the concentration of sheep and chicken immunoglobulin G used for drawing the quality control line C is 0.5-2 mg / mL (for example, 0.5 mg / mL, 0.8 mg / mL, 1 mg / mL, 1.5 mg / mL, 1.8 mg / mL or 2 mg / mL), and the volume of the drawing is 20-45 μL / 30cm (for example, 20 μL / 30cm, 25 μL / 30cm, 30 μL / 30cm, 35 μL / 30cm, 40 μL / 30cm or 45 μL / 30cm).

[0024] Preferably, the fluorescent microspheres are surface carboxylated polystyrene fluorescent microspheres or polymethyl methacrylate-based phosphorescent microspheres.

[0025] Preferably, the particle size of the fluorescent microspheres is 300 nm.

[0026] Preferably, the bottom plate is a PVC bottom plate.

[0027] Preferably, the absorbent paper is any one of cellulose containing a polymer absorbent resin, absorbent cotton, a silica absorbent pad or a sponge absorbent pad.

[0028] Preferably, the detection card further comprises a card housing.

[0029] Preferably, the card shell includes an upper card shell and a lower card shell that are locked to each other, the inner surface of the lower card shell is provided with a card slot for placing the test strip, the upper card shell is provided with a sample addition port corresponding to the sample pad of the test strip, and an observation port is provided corresponding to the nitrocellulose membrane of the test strip, and the T1 test line, T2 test line and quality control line C on the nitrocellulose membrane are all exposed at the observation port.

[0030] In the present invention, the jam not only protects the test strip, avoids its damage and pollution, but also plays a fixing role, so that the test strip is not easy to slide, affecting the determination. The upper and lower jams can fix and compress the test strip, and the labeled antibody and sample diluent on the CP pad can run synchronously to ensure that the liquid flow rate is uniform, thereby further reducing the CV coefficient, improving precision and accuracy, and easy to operate at the same time, and can be quickly tested by laying flat and directly adding samples, without rigid technical requirements for operators. In addition, the jam can be used in conjunction with the detection equipment, and the jam is inserted into the corresponding channel of the detection equipment (immunofluorescence detector produced by Guangjing Biotechnology (Suzhou) Co., Ltd., LTRIC-600), and then the sample delivery test is automatically and stably performed, further improving the accuracy of the test results, and the content determination value is more accurate.

[0031] In the present invention, the reagent strip and the test card can be prepared by the following preparation method:

[0032] (1) Preparation of NC membrane

[0033] From the sample pad to the absorbent pad, the T1 detection line, T2 detection line and quality control line C are arranged at equal intervals on the NC membrane (nitrocellulose membrane).

[0034] The T1 test line is coated with uric acid-bovine serum albumin hapten, the T2 test line is coated with CA724 monoclonal antibody, and the quality control line C is coated with chicken immunoglobulin G.

[0035] First, a solution of UA-BSA hapten (self-prepared) with a concentration of 1 mg / mL, a solution of CA724 monoclonal antibody with a concentration of 1 mg / mL, and a solution of chicken immunoglobulin G with a concentration of 1 mg / mL were obtained, and the solvent in each solution was phosphate buffer (pH 7.4). The analytical membrane material was drawn with a spraying amount of 0.67 to 1.5 μL / cm on a film dispenser.

[0036] (2) Preparation of conjugate pad

[0037] The conjugate pad is adsorbed with uric acid antibody-labeled fluorescent microspheres and CA724 antibody-labeled fluorescent microspheres.

[0038] Preparation method of uric acid and CA724 combined detection reagent strip conjugate pad: The specific preparation steps include:

[0039] Antibody labeling: Antibody labeling fluorescent microspheres: Microsphere activation: 5 mg EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide) and 5 mg NHS (N-hydroxysuccinimide) dissolved in 50 mM, pH 7.0 MES (2-(N-morpholino)ethanesulfonic acid monohydrate) were added to 1 mg / mL fluorescent microspheres, activated for 60 min, centrifuged at 15000 rpm for 30 min, the supernatant was discarded, and the precipitate was resuspended with 50 mM PBS (phosphate buffer) at pH 7.4 to obtain a 1 mg / mL activated fluorescent microsphere solution; Antibody coupling: 500 μg uric acid antibody and 300 μg CA724 monoclonal antibody were added to 2 mL of 1 mg / mL activated fluorescent microsphere solution, mixed at room temperature for 1 h, centrifuged at 15000 rpm for 30 min, the supernatant was discarded, and the precipitate was resuspended with 50 mM PBS (phosphate buffer) at pH 7.4 containing 5% BSA (bovine serum albumin). Block with PBS (phosphate buffered saline) solution for 1 h and stir to mix;

[0040] Centrifuge at 15000 rpm for 10 min, and store the collected precipitate in 20 mM Tris buffer at pH 7.0 containing 0.5% BSA (bovine serum albumin) to obtain a 0.1 mg / mL uric acid antibody labeled fluorescent microsphere solution and a 0.1 mg / mL CA724 antibody labeled fluorescent microsphere solution;

[0041] Preparation of binding pad: Spray the uric acid antibody marker and CA724 antibody marker obtained in step 1) on the binding membrane material, dilute the uric acid antibody labeled fluorescent microsphere solution on the binding pad to a concentration of 80 μg / mL, dilute the CA724 antibody labeled fluorescent microsphere solution to a concentration of 40 μg / mL, and spray the uric acid and CA724 antibody labeled fluorescent microsphere solution in an amount of 5 μL / cm 2 .

[0042] (3) Preparation of sample pad

[0043] Treatment solution: The treatment solution is a 50 mM Tris buffer solution containing 2% S9, 1 mg / mL heterophilic antibody blocker, and 0.1 mg / mL RBC antibody at a mass percentage concentration, and its pH is 8.0.

[0044] The glass cellulose membrane was soaked in the treatment solution and then dried to prepare the sample pad. The sample pad prepared by this method has the best use effect, and the positive and negative control effects are good. When measured using the detection equipment, there are obvious signal values ​​at the cutoff value of UA and CA724, the minimum detection limit of the negative control meets the requirements, and the signal value test results of the positive standard with different concentration gradients have obvious gradient differences.

[0045] (4) Assembly

[0046] The sample pad, conjugate pad, NC membrane and absorbent pad are sequentially overlapped on the base plate and partially overlapped to obtain the UA and CA724 combined detection reagent strip; the PVC base plate with the sample pad, conjugate pad, analysis membrane and absorbent pad is cut into paper strips with a length of 6.5 cm and a width of 4 mm, and is loaded into a card shell and packaging to obtain the UA and CA724 combined detection test card.

[0047] Preferably, the kit further comprises a calibration card.

[0048] Preferably, the calibration card has a UA standard curve and a CA724 standard curve.

[0049] In the present invention, the detection operation of the kit is as follows: drop the sample to be tested into the sample injection port of the test card, react at room temperature for 15 minutes, use an immunofluorescence detector (LTRIC-600) to detect the fluorescence signal intensity values ​​of the T1 and T2 detection lines, and then calculate according to the standard curve of the calibration card to obtain the concentration of UA and CA724 in the sample to be tested.

[0050] When the sample concentration exceeds the upper limit of the detection range, dilute the sample with sample test solution and add it to the test card for detection. The target substance concentration will be calculated based on the standard curve to obtain the result.

[0051] On the other hand, the present invention provides use of the above-mentioned kit for the combined detection of CA724 and uric acid in the preparation of gout and / or hyperuricemia detection materials or devices.

[0052] Compared with the prior art, the present invention has the following beneficial effects:

[0053] (1) The kit of the present invention can detect CA724 and uric acid simultaneously, and the detection results are accurate and rapid. Both results can be read within 20 minutes, realizing the simultaneous application of the competitive method and the double antibody sandwich method in the immunochromatography technology. The operation is simple and fast, convenient for clinical rapid diagnosis, conducive to early diagnosis and early treatment of gout, and can be used for auxiliary diagnosis of gout and hyperuricemia and monitoring of treatment effects.

[0054] (2) The present invention can realize one sample, one sample addition, and one test, and can quickly perform quantitative detection of uric acid and CA724 at the same time. The test results are highly accurate and sensitive, and the operation steps are simplified, the sample usage is reduced, time is saved, and cost is saved.

[0055] (3) Formula optimization is performed in the sample test solution. Through compounding, non-specific blocking effects can be performed to eliminate non-specific adsorption of proteins and improve the impact of sample matrix; stabilization effects can be performed to improve protein stability and protein marker stability; sensitization effects can be performed to increase the sensitivity of immune responses. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 This is a linear correlation diagram of the test results of the kit of the present invention and the comparative reagent on CA724;

[0057] Figure 2 This is a linear correlation diagram of the test results of uric acid by the kit of the present invention and the comparative reagent. DETAILED DESCRIPTION

[0058] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0059] Example 1

[0060] In this embodiment, a reagent kit is provided, which includes a test card, a sample test solution and a calibration card, wherein the test card includes a card shell and a reagent strip, and the reagent strip includes a PVC base plate and a sample pad, a binding pad, an NC membrane and a water-absorbing pad sequentially attached to the PVC base plate. The adjacent ends of the sample pad, the binding pad, the NC membrane and the water-absorbing pad overlap each other. There are test lines T1, T2 and a quality control line C on the NC membrane.

[0061] The card shell includes an upper card shell and a lower card shell which are locked to each other. The inner surface of the lower card shell is provided with a card slot for placing the test strip. The upper card shell is provided with a sample addition port corresponding to the sample pad of the test strip and an observation port corresponding to the NC membrane of the test strip. The T1 test line, T2 test line and quality control line C on the NC membrane are all exposed at the observation port.

[0062] Preparation of reagent strips

[0063] (1) Preparation of NC membrane

[0064] From the sample pad to the absorbent pad, the T1 detection line, T2 detection line and quality control line C are arranged at equal intervals on the NC membrane (nitrocellulose membrane).

[0065] The T1 test line is coated with uric acid-bovine serum albumin hapten, the T2 test line is coated with CA724 monoclonal antibody, and the quality control line C is coated with chicken immunoglobulin Y.

[0066] First, a solution of UA-BSA hapten (self-prepared) with a concentration of 1 mg / mL, a solution of CA724 monoclonal antibody with a concentration of 1 mg / mL, and a coating solution of chicken immunoglobulin Y monoclonal antibody with a concentration of 1 mg / mL were obtained, and lines were drawn on the analytical membrane material with a spraying amount of 1.0 μL / cm on a film dispenser.

[0067] (2) Preparation of conjugate pad

[0068] Preparation method of uric acid and CA724 combined detection reagent strip conjugate pad: The specific preparation steps include:

[0069] Antibody labeling: Antibody labeling fluorescent microspheres: Microsphere activation: 5 mg EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide) and 5 mg NHS (N-hydroxysuccinimide) dissolved in 50 mM, pH 7.0 MES (2-(N-morpholino)ethanesulfonic acid monohydrate) were added to 1 mg / mL 300 nm phosphorescent microspheres of polymethyl methacrylate (refer to the phosphorescent microspheres of polymethyl methacrylate prepared in the example of CN101787276A), activated for 60 min, centrifuged at 15000 rpm for 30 min, discarded the supernatant, and the precipitate was resuspended in 50 mM PBS (phosphate buffer) at pH 7.4 to obtain a 1 mg / mL activated fluorescent microsphere solution; Antibody coupling: 500 μg uric acid antibody and 300 μg CA724 monoclonal antibody was added to 2 mL of 1 mg / mL activated fluorescent microsphere solution, mixed at room temperature for 1 h, centrifuged at 15000 rpm for 30 min, the supernatant was discarded, and the precipitate was blocked with 50 mM pH 7.4 PBS (phosphate buffer) solution containing 5% BSA (bovine serum albumin) for 1 h, and stirred to mix;

[0070] Centrifuge at 15000 rpm for 30 min, and store the collected precipitate in 20 mM Tris buffer at pH 7.0 containing 0.5% BSA (bovine serum albumin) to obtain a 0.1 mg / mL uric acid antibody labeled fluorescent microsphere solution and a 0.1 mg / mL CA724 antibody labeled fluorescent microsphere solution;

[0071] Preparation of binding pad: The uric acid antibody marker and CA724 antibody marker obtained in step 1) were diluted with microsphere diluent and then sprayed on the binding membrane material. The concentration of the diluted uric acid antibody labeled fluorescent microsphere solution on the binding pad was 80 μg / mL, and the concentration of the diluted CA724 antibody labeled fluorescent microsphere solution was 40 μg / mL. The amount of the sprayed uric acid and CA724 antibody labeled fluorescent microsphere solution was 5 μL / cm 2 , dry and set aside.

[0072] (3) Preparation of sample pad

[0073] The sample pad was obtained by soaking the glass cellulose membrane in a treatment solution and then drying it. The treatment solution was a 50 mM Tris buffer solution containing 2% S9 by mass, 1 mg / mL heterophilic antibody blocker, and 0.1 mg / mL RBC antibody, and its pH was 8.0.

[0074] (4) Assembly

[0075] The sample pad, conjugate pad, NC membrane and absorbent pad are sequentially overlapped on the base plate and partially overlapped to obtain the UA and CA724 combined detection reagent strip; the PVC base plate with the sample pad, conjugate pad, analysis membrane and absorbent pad is cut into paper strips with a length of 6.5 cm and a width of 4 mm, and is loaded into a card shell and packaging to obtain the UA and CA724 combined detection test card.

[0076] (5) Preparation of sample test solution

[0077] The sample test solution comprises 10 mM PBS buffer solution (pH 7.4), 0.5% Biolipidure-206, 0.1% PEG10000, and 0.05% Proclin 300.

[0078] The calibration card comes with UA standard curve and CA724 standard curve.

[0079] Example 2

[0080] The difference from Example 1 is the step of preparing the conjugate pad. The antibody-coupled microspheres are 300 nm polystyrene fluorescent microspheres with carboxyl groups on the surface (purchased from bangslab, item number: FCEU003). The implementation process is the same as that of Example 1.

[0081] Comparative Example 1

[0082] The only difference from Example 1 is that the sample detection solution is 10 mM PBS buffer solution (pH 7.4) and 0.05% Proclin 300.

[0083] Comparative Example 2

[0084] The only difference from Example 1 is that the sample detection solution is 10 mM PBS buffer solution (pH 7.4), 0.5% Tween-20, and 0.05% Proclin 300.

[0085] Comparative Example 3

[0086] The only difference from Example 2 is that the sample pad treatment solution is a 50 mM Tris buffer solution containing 1 mg / mL of heterophilic antibody blocking agent and 0.1 mg / mL of RBC antibody in a mass percentage concentration, and its pH is 8.0.

[0087] The reagent strips assembled in the embodiment and the comparative example were tested with an immunofluorescence analyzer (LTRIC-600), and the intensity of the signal ratio (T / C) was detected with the prepared calibration products Level 1-Level 5 (the uric acid concentration values ​​were: 200 μmol / L, 400 μmol / L, 600 μmol / L, 800 μmol / L, 1200 μmol / L; the CA724 concentration values ​​were: 5 IU / mL, 25 IU / mL, 125 IU / mL, 250 IU / mL, 500 IU / mL) and the precision was compared with the enterprise reference product (the uric acid concentration value was 400 μmol / L, the CA724 concentration value was 125 IU / mL). To evaluate the performance of the reagent strips. The comparison results are shown in Tables 1 and 2 below:

[0088] Table 1

[0089]

[0090] Table 2

[0091]

[0092] The results showed that the optimized sample detection solution and sample pad were beneficial to improving the detection sensitivity and chromatographic precision.

[0093] Application Examples

[0094] In this application example, the kit of Example 1 is used to test the sample.

[0095] During the test, drop the sample to be tested into the sample injection port of the test card. After reacting at room temperature for 15 minutes, use an immunofluorescence detector (LTRIC-600) to detect the fluorescence signal intensity values ​​of the T1 and T2 test lines, and then calculate according to the standard curve of the calibration card to obtain the concentration of UA and CA724 in the sample to be tested.

[0096] When the sample concentration exceeds the upper limit of the detection range, dilute the sample with diluent and add it to the test card for detection. The target substance concentration will be calculated based on the standard curve.

[0097] According to the standard of YY / T 1304.2-2015 Time-Resolved Fluorescence Immunoassay System Part 2: Time-Resolved Fluorescence Immunoassay Quantitative Determination Reagent (Kit), by testing 20 blank samples and calculating the mean plus twice the standard deviation, the detection limit of the kit of the present invention for UA is 60 μmol / L, and the detection limit of CA724 is 2 IU / mL.

[0098] Serum samples from 120 gout patients and 40 healthy subjects were collected, and the uric acid and CA724 concentrations in the samples were detected using the kit of Example 1 of the present invention. The uric acid concentration in the samples was detected using the Beckman Coulter biochemical analysis system (AU5800), and the CA724 concentration in the samples was measured using the Roche Cobas e601 fully automatic electrochemiluminescence immunoassay analyzer. The data correlation between the two was compared, and the results are shown in Table 3 (Y in the table indicates yes, and N indicates no).

[0099] Table 3

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106] The test results corresponding to Table 3 are plotted as a linear correlation diagram of CA724 and uric acid test results and the comparison reagent, Figure 1 and Figure 2 As can be seen from the data in Table 3, the kit of the present invention can simultaneously detect and obtain the quantitative results of CA724 and uric acid, the test results are highly accurate, and one sample, one addition, and one test can achieve the measurement results of two indicators, simplifying the operation steps, reducing the sample dosage, saving time, and saving costs.

[0107] The applicant declares that the present invention illustrates the kit and application for the combined detection of CA724 and uric acid through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A kit for the combined detection of CA724 and uric acid, characterized in that: The kit comprises a test card and a sample test solution, wherein the test card comprises a test strip, wherein the test strip comprises a bottom plate, a sample pad, a conjugation pad, a nitrocellulose membrane and a water-absorbing pad, wherein the sample pad, the conjugation pad, the nitrocellulose membrane and the water-absorbing pad are sequentially adhered to the bottom plate, wherein the conjugation pad is adsorbed with uric acid antibody-labeled fluorescent microspheres and CA724 antibody-labeled fluorescent microspheres, and the nitrocellulose membrane is sequentially provided with a T1 test line coated with uric acid-bovine serum albumin hapten, a T2 test line coated with CA724 monoclonal antibody and a quality control line C from front to back according to the sample flow direction; The sample detection solution includes the following components: 10-50 mM pH 7.4 PBS buffer solution, 0.5-1% Biolipidure-206, 0.1-0.5% PEG10000 and 0.05% Proclin 300; The sample pad is a sample pad obtained by soaking a glass cellulose membrane in a treatment solution and then drying it. The treatment solution is a 20-100 mM Tris buffer solution containing 2-5% S9 by mass percentage, 0.5-2 mg / mL blocking agent, and 0.1-0.2 mg / mL RBC antibody, and its pH is 8.

0.

2. The kit for combined detection of CA724 and uric acid according to claim 1, characterized in that: The blocking agent is a mouse heterophilic antibody blocking agent.

3. The kit for combined detection of CA724 and uric acid according to claim 1, characterized in that: The water-absorbing pad is a polymer composite cotton linter pulp filter paper, a common fiber filter paper, a silica gel water-absorbing pad or a sponge water-absorbing pad.

4. The kit for combined detection of CA724 and uric acid according to claim 3, characterized in that: The water-absorbing pad is a polymer composite cotton linter pulp filter paper.

5. The kit for combined detection of CA724 and uric acid according to claim 1, characterized in that: The concentration of uric acid-bovine serum albumin hapten used for marking the T1 test line is 0.5-2 mg / mL, and the volume of the marking line is 20-45 μL / 30 cm; the amount of CA724 monoclonal antibody used for marking the T2 test line is 0.5-3 mg / mL, and the volume of the marking line is 20-45 μL / 30 cm.

6. The kit for combined detection of CA724 and uric acid according to claim 1, characterized in that: When forming the conjugate pad, the concentration of the diluted uric acid antibody labeled fluorescent microsphere solution is 20-100 μg / mL, the concentration of the diluted CA724 antibody labeled fluorescent microsphere solution is 10-50 μg / mL, and the amount of the sprayed mixed solution of uric acid and CA724 antibody labeled fluorescent microspheres is 5-10 μL / cm 2 .

7. The kit for combined detection of CA724 and uric acid according to claim 1, characterized in that: The quality control line C is a quality control line coated with chicken immunoglobulin G.

8. The kit for combined detection of CA724 and uric acid according to claim 1, characterized in that: The concentration of goat and chicken immunoglobulin G used for streaking the quality control line C is 0.5-2 mg / mL, and the streaking volume is 20-45 μL / 30 cm.

9. The kit for combined detection of CA724 and uric acid according to claim 1, characterized in that: The fluorescent microspheres are surface carboxylated polystyrene fluorescent microspheres or polymethyl methacrylate-based phosphorescent microspheres.

10. The kit for combined detection of CA724 and uric acid according to claim 1, characterized in that: The bottom plate is a PVC bottom plate.

11. The kit for combined detection of CA724 and uric acid according to claim 1, characterized in that: The detection card also includes a card housing.

12. The kit for combined detection of CA724 and uric acid according to claim 11, characterized in that: The card shell includes an upper card shell and a lower card shell that are locked to each other. The inner surface of the lower card shell is provided with a card slot for placing the test strip. The upper card shell is provided with a sample addition port corresponding to the sample pad of the test strip and an observation port corresponding to the nitrocellulose membrane of the test strip. The T1 test line, T2 test line and quality control line C on the nitrocellulose membrane are all exposed at the observation port.

13. The kit for combined detection of CA724 and uric acid according to claim 1, characterized in that: The kit also includes a calibration card.

14. The kit for combined detection of CA724 and uric acid according to claim 13, characterized in that: The calibration card has a UA standard curve and a CA724 standard curve.

15. Use of a kit for the combined detection of CA724 and uric acid according to any one of claims 1 to 14 in the preparation of gout and / or hyperuricemia detection materials or devices.

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