Detection kit for detecting calprotectin content in synovial fluid

Abstract

The invention discloses a detection kit for detecting the content of calprotectin in synovial fluid. The detection kit comprises a detection kit shell and a calprotectin immunochromatography test strip, and the calprotectin immunochromatography test strip is arranged in the detection kit shell; the calprotectin immunochromatography test strip comprises a sample pad, a glass fiber combination pad, a coated nitrocellulose membrane and a water absorption pad which are bonded in sequence, an anti-calprotectin antibody marked by colloidal gold is embedded in the glass fiber combination pad and is used for being combined with calprotectin in a sample to be detected to form a compound; the anti-calprotectin monoclonal antibody is coated on a detection line of the nitrocellulose membrane, and the anti-mouse IgG antibody is coated on a quality control line of the nitrocellulose membrane; the colloidal gold labeled anti-calprotectin antibody is embedded in the glass fiber cushion layer, and the image recognition module is used for collecting a tested test paper image and calculating a relative gray value of a detection line and a quality control line, so that a calprotectin concentration interval is judged.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medical devices and in vitro diagnostics, and in particular to a detection kit for detecting the content of calprotectin in synovial fluid, which is particularly suitable for rapid screening and risk grading of periprosthetic joint infection (PJI). BACKGROUND

[0002] Periprosthetic joint infection (PJI) is one of the most serious complications after joint replacement, which has a significant impact on the quality of life of patients and medical resources. Traditional PJI diagnosis methods include synovial fluid culture, white blood cell count and classification, C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), blood culture, histopathological examination, etc. These methods have several problems: 1. Long detection time: synovial fluid culture usually takes several days, delaying diagnosis and treatment; 2. Low sensitivity and specificity: inflammatory markers such as ESR and CRP are easily affected by other factors, leading to misjudgment; 3. Dependence on complex experimental equipment: some methods require special laboratories and professional operators, limiting their clinical application; 4. Strong subjectivity of results: some rely on manual microscopic counting or visual judgment, which is greatly influenced by the operator's experience.

[0003] Calprotectin is an inflammation-related protein derived from neutrophils and monocytes, which significantly increases in synovial fluid under infection, and has become an important biomarker reflecting local inflammatory response. The detection method based on calprotectin has high sensitivity, low sample requirement, and high correlation with PJI.

[0004] In summary, there is an urgent need for a rapid, simple, stable, and on-site diagnostic method to support timely identification and treatment decision-making for PJI. SUMMARY

[0005] In view of this, the embodiments of the present application provide a detection kit for detecting the content of calprotectin in synovial fluid.

[0006] To achieve the above-mentioned purposes, the embodiments of the present application provide a detection kit for detecting the content of calprotectin in synovial fluid, which comprises: a detection kit shell, A calprotectin immunochromatographic test strip is placed in the housing of a test kit. The calprotectin immunochromatographic test strip includes a sample pad, a glass fiber conjugate pad, a coated nitrocellulose membrane, and an absorbent pad, which are sequentially bonded together. The glass fiber conjugate pad contains a colloidal gold-labeled anti-calprotectin antibody for binding with calprotectin in the test sample to form a complex. An anti-calprotectin monoclonal antibody is coated on the detection line of the nitrocellulose membrane, and an anti-mouse IgG antibody is coated on the control line of the nitrocellulose membrane. Colloidal gold-labeled anti-calprotectin antibodies are embedded in a glass fiber pad. The image recognition module is used to acquire images of the test strip after testing and calculate the relative gray values ​​of the test line and the control line, thereby determining the concentration range of calprotectin.

[0007] Furthermore, the membrane material of the calcium protectant immunochromatographic test strip is a highly hydrophilic nitrocellulose membrane with a pore size of 8–12 μm.

[0008] Furthermore, the preparation method of the calprotectin immunochromatographic test strip includes: Anti-calprotectin monoclonal antibody and coating solution were coated onto the detection line of a nitrocellulose membrane and then dried; anti-mouse IgG antibody and coating solution were coated onto the control line of a nitrocellulose membrane and then dried. Anti-calprotectin antibody was added to a colloidal gold solution with a pH of 7.2-7.8 for labeling, and a stabilizer was added. The solution was then uniformly coated onto a glass fiber conjugate pad and dried. The sample pad, glass fiber bonding pad, coated nitrocellulose membrane, and absorbent pad are sequentially bonded to a PVC base plate to form a test strip.

[0009] Furthermore, the stabilizer is selected from polyethylene glycol.

[0010] Furthermore, the process of acquiring test strip images after testing and calculating the relative gray values ​​of the test line and control line to determine the calprotectin concentration range includes: Calprotectin concentration gradient standard solutions with concentrations of 0, 10, 50, 100, and 150 μg / mL were obtained. After dilution, the calprotectin concentration gradient standard solutions were added to the test strip. Calprotectin binds to the colloidal gold-labeled anti-calprotectin antibody to form a complex, which migrates forward to the detection line and produces a colored band. Collect test strip images after testing with calprotectin concentration gradient standard solution; perform grayscale processing on the test strip images, calculate the grayscale ratio of T line / C line, calibrate the calprotectin concentration range, and generate a standard curve; Acquire test strip images after testing the joint fluid of the subjects; The test strip image is processed in grayscale, the grayscale ratio of the T line to the C line is calculated, and the result is fitted with a standard curve to determine the calprotectin concentration range. The calprotectin concentration range is divided into <50 μg / mL, 50–150 μg / mL, and >150 μg / mL, which correspond to low risk, medium risk, and high risk levels, respectively.

[0011] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a test kit for detecting the content of calprotectin in synovial fluid. The kit can not only determine the presence of calprotectin, but also perform image analysis on the colored bands to achieve a rough classification of calprotectin concentration, thus assisting clinicians in assessing the risk of periprosthetic infection. Attached Figure Description

[0012] To more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0013] Figure 1 This is a schematic diagram of the reagent kit components provided in an embodiment of the present invention.

[0014] Figure 2 This is a structural diagram of the test strip provided in an embodiment of the present invention.

[0015] Figure 3 This figure shows the results of clinical sample testing using the test strip provided in this embodiment of the invention. The figure illustrates different results after the test strip is used, including negative (only the control line is colored), positive (both the test line and control line are colored), and invalid results (no color development or no color development at the control line). The figure also indicates the corresponding concentration ranges (e.g., <50 μg / mL, 50-150 μg / mL, >150 μg / mL).

[0016] Figure 4 The results of the clinical infection joint fluid sample test provided in this embodiment of the invention are as follows: After the joint fluid sample was diluted 50 times with sample diluent, 50 μL was added. After 10 minutes, the test results showed that all 17 joint infection samples had strong (+++) test lines, indicating a high risk (red).

[0017] Figure 5The results of the clinical non-infectious synovial fluid sample test provided in this embodiment of the invention are as follows. After the synovial fluid sample was diluted 50 times with sample diluent, 50 μL was added. After 10 minutes, the test results showed that none of the 8 non-infectious specimens of common arthritis or joint revision showed negative color on the test line. Another specimen of common arthritis (the fourth case) showed weak positive color on the test line, indicating a low risk (green).

[0018] In the figure, 1-sample pad, 2-colloidal gold binding pad, 3-detection line, 4-nitrocellulose membrane, 5-quality control line, 6-absorbent pad, 7-backing. Detailed Implementation

[0019] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] It should be noted that, unless otherwise specified, the features in the following embodiments and implementation methods can be combined with each other.

[0021] like Figure 1 and Figure 2 As shown, this embodiment of the invention provides a detection kit for detecting the content of calprotectin in synovial fluid, comprising: Test kit casing, A calprotectin immunochromatographic test strip is placed in the test kit housing. The calprotectin immunochromatographic test strip includes a sample pad 1, a colloidal gold conjugated pad 2, a coated nitrocellulose membrane 4, and an absorbent pad 6, which are sequentially bonded together. The colloidal gold conjugated pad contains a colloidal gold-labeled anti-calprotectin antibody, which is used to bind with calprotectin in the test sample to form a complex. An anti-calprotectin monoclonal antibody is coated on the detection line 3 of the nitrocellulose membrane, and an anti-mouse IgG antibody is coated on the control line 4 of the nitrocellulose membrane. Colloidal gold-labeled anti-calprotectin antibody is embedded in a colloidal gold binding pad.

[0022] The image recognition module is used to acquire images of the test strip after testing and calculate the relative gray values ​​of the test line and the control line, thereby determining the concentration range of calprotectin.

[0023] Furthermore, the membrane material of the calprotectin immunochromatographic test strip is a highly hydrophilic nitrocellulose membrane with a pore size of 8–12 μm, in order to improve sample fluidity and antibody binding efficiency.

[0024] Furthermore, the process of acquiring test strip images after testing and calculating the relative gray values ​​of the test line and control line to determine the calprotectin concentration range includes: Calprotectin concentration gradient standard solutions with concentrations of 0, 10, 50, 100, and 150 μg / mL were obtained. After dilution, the calprotectin concentration gradient standard solutions were added to the test strip. Calprotectin binds to the colloidal gold-labeled anti-calprotectin antibody to form a complex, which migrates forward to the detection line and produces a colored band. Collect test strip images after testing with calprotectin concentration gradient standard solution; perform grayscale processing on the test strip images, calculate the grayscale ratio of T line / C line, calibrate the calprotectin concentration range, and generate a standard curve; Acquire test strip images after testing the joint fluid of the subjects; The test strip image is processed in grayscale, the grayscale ratio of the T line to the C line is calculated, and the result is fitted with a standard curve to determine the calprotectin concentration range. The calprotectin concentration range is divided into <50 μg / mL, 50–150 μg / mL, and >150 μg / mL, which correspond to low risk, medium risk, and high risk levels, respectively.

[0025] Example 1: Preparation process of calprotectin immunochromatographic test strip Furthermore, the preparation method of the calprotectin immunochromatographic test strip includes the following steps: Anti-calprotectin monoclonal antibody and coating solution were coated onto the detection line of a nitrocellulose membrane and then dried; anti-mouse IgG antibody and coating solution were coated onto the control line of a nitrocellulose membrane and then dried. Anti-calprotectin antibody was added to a colloidal gold solution with a pH of 7.2-7.8 for labeling, and a stabilizer (in this example, polyethylene glycol was selected as the stabilizer) was added. The solution was then uniformly coated onto a glass fiber conjugate pad and dried. The sample pad, colloidal gold binding pad, coated nitrocellulose membrane, and absorbent pad are sequentially bonded to a PVC base plate to form a test strip.

[0026] Specifically, this embodiment details the preparation process of a test strip for detecting calprotectin content in synovial fluid, and further assembles it into a complete detection kit. The preparation method is based on colloidal gold immunochromatography technology, referencing optimized process steps to ensure the sensitivity and stability of the test strip, while facilitating the practical application of the kit. The specific steps are as follows: Step S1, nitrocellulose membrane coating: Prepare a first mixture and a second mixture for coating the nitrocellulose membrane (NC membrane). The first mixture contains human synovial fluid calprotectin A antibody (concentration adjusted to 0.5-1 mg / mL) and coating solution (phosphate buffer, pH 7.4, containing 0.1% Tween-20), and is sprayed onto the test line (T line) of the NC membrane at a rate of 1 μL / cm. The second mixture contains rabbit IgG antibody (concentration 1-2 mg / mL) and the same coating solution, and is sprayed onto the control line (C line) at a rate of 1 μL / cm. After coating, the NC membrane is placed in a 40°C drying oven for 2 hours to ensure strong antibody adhesion and enhance the stability of the test strip.

[0027] Step S2, colloidal gold-labeled antibody: A colloidal gold solution (particle size approximately 30 nm) at pH 7.4 was prepared, and anti-human synovial fluid calprotectin B antibody (concentration 0.1 mg / mL) was added. The solution was stirred at room temperature for 40 minutes to complete the labeling. After labeling, 1% bovine serum albumin (BSA) was added for blocking, and the mixture was stirred for 20 minutes to reduce non-specific binding. Subsequently, the solution was centrifuged at 10,000 rpm for 15 minutes, the supernatant was discarded, and the precipitate was resuspended in a reconstitution solution (containing 0.1% PEG as a stabilizer). This step optimized the uniformity of the colloidal gold labeling and improved the test strip's ability to detect low concentrations of calprotectin.

[0028] Step S3, bonding pad coating: The colloidal gold antibody-conjugate was mixed with a reconstitution solution to prepare a third mixture, which was then uniformly coated onto a glass fiber conjugate pad at a rate of 5 μL / cm. After coating, the strip was placed in a 30°C drying oven for 3 hours to ensure stable adhesion of the conjugate. This step enhances the test strip's adaptability to sample flow and reduces uneven color development.

[0029] Step S4, test strip assembly: The sample pad, glass fiber conjugate pad, coated NC membrane, and absorbent pad are sequentially bonded to the PVC base plate (backing 7) to form the test strip. The sample pad is used for initial sample absorption, the conjugate pad releases the labeled antibody, the NC membrane contains the T and C lines, and the absorbent pad ensures uniform flow. After assembly, the strips are cut into 3-5 mm wide individual strips and stored in a sealed environment. This structural design simplifies the operation process and improves the colorimetric consistency of the test strips.

[0030] Step S5, Reagent kit assembly: The prepared test strips are assembled with other components into a kit, including: 50 test strips, sample diluent (pH 7.4 phosphate buffer, pre-filled in dropper bottles), standards (calprotectin concentrations of 0, 10, 50, 100, and 150 μg / mL for calibration), quality control solutions (positive / negative controls), a test strip holder (plastic holder), a QR code for installing a mobile app, and an instruction manual. All components are sealed in aluminum foil bags and stored at 4–30°C for 12 months. The kit is designed for portability and is suitable for rapid point-of-care testing, with a testing time of less than 15 minutes.

[0031] Through the above steps, the detection limit of the test strip is reduced to 1-5 μg / mL, and the linear range covers 0-1000 μg / mL, meeting the clinical PJI screening needs.

[0032] Example 2: Testing of clinical synovial fluid samples This embodiment utilizes the kit prepared in Example 1 to test clinically collected synovial fluid samples, verifying its performance under different pathological conditions. The samples included synovial fluid from osteoarthritis, synovitis, infections (such as PJI), and rheumatic diseases, totaling 26 cases. While the sample size was limited, it covered the major pathological types. The test results were compared with clinical diagnoses (including bacterial culture, CRP / ESR, and white blood cell count) to evaluate the kit's effectiveness.

[0033] Sample preparation and testing process: Collect 20 μL of the patient's joint fluid sample, dilute it 1:100 with diluent, and mix well. Add 80 μL of the mixture to the sample well of the test strip and incubate at room temperature (20-25°C) for 15 minutes. Using the image of the collected test strip, calculate the relative intensity of the test line / control line through image recognition, match it with a preset standard curve, and output the calprotectin concentration range (<50 μg / mL low risk, green; 50-150 μg / mL medium risk, yellow; >150 μg / mL high risk, red), and provide PJI risk indication.

[0034] Test Results and Analysis: Twenty-six samples were tested, and the results showed a significant difference in calprotectin concentration between infected samples (such as PJI) and non-infected samples. Among the infected samples (17 cases)... Figure 4 Of the 17 cases, 150 μg / mL was high-risk (red alert), and 0 cases had concentrations between 50-150 μg / mL (medium-risk, yellow alert), consistent with positive bacterial culture results, resulting in a 100% positivity rate and reflecting a significant increase in calprotectin caused by infection. In non-infectious samples (9 cases, including osteoarthritis, synovitis, non-infectious revision surgery, and rheumatic diseases), (… Figure 5One case had an infection rate <50 μg / mL (low risk, green indicator), and zero cases had an infection rate of 50-150 μg / mL (medium risk, yellow indicator), consistent with a clinical diagnosis of no infection. This indicates that the kit can effectively distinguish between infected and non-infected states.

[0035] High concentrations (>150 μg / mL) in infected samples were associated with neutrophil activation, with a red risk level indicating a recommendation for further antibiotic treatment or surgical intervention. Low concentrations (<50 μg / mL) in non-infected samples were consistent with chronic inflammation or a sterile state, reducing the risk of misdiagnosis. Although the sample size was limited, the results showed a high correlation between infected samples and clinical diagnosis, validating the kit's potential in PJI risk assessment. Further optimization of the threshold and image algorithm could be achieved by increasing the sample size.

[0036] In summary, this invention provides a test kit for detecting the content of calprotectin in synovial fluid. This kit can not only determine the presence of calprotectin, but also perform image analysis on the colored bands to achieve a rough classification of calprotectin concentration, thus assisting clinicians in assessing the risk of periprosthetic infection.

[0037] The above embodiments are only used to illustrate the design concept and features of the present invention, and their purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly. The protection scope of the present invention is not limited to the above embodiments. Therefore, all equivalent changes or modifications made based on the principles and design ideas disclosed in the present invention are within the protection scope of the present invention.

Claims

1. A test kit for detecting the content of calprotectin in synovial fluid, characterized in that, include: Test kit casing, A calprotectin immunochromatographic test strip is placed in the housing of a test kit. The calprotectin immunochromatographic test strip includes a sample pad, a glass fiber conjugate pad, a coated nitrocellulose membrane, and an absorbent pad, which are sequentially bonded together. The glass fiber conjugate pad contains a colloidal gold-labeled anti-calprotectin antibody for binding with calprotectin in the test sample to form a complex. An anti-calprotectin monoclonal antibody is coated on the detection line of the nitrocellulose membrane, and an anti-mouse IgG antibody is coated on the control line of the nitrocellulose membrane. Colloidal gold-labeled anti-calprotectin antibodies are embedded in a glass fiber pad. The image recognition module is used to acquire images of the test strip after testing and calculate the relative gray values ​​of the test line and the control line, thereby determining the concentration range of calprotectin.

2. The detection kit for detecting the content of calprotectin in synovial fluid according to claim 1, characterized in that, The membrane material of the calprotectin immunochromatographic test strip is a highly hydrophilic nitrocellulose membrane with a pore size of 8–12 μm.

3. The detection kit for detecting the content of calprotectin in synovial fluid according to claim 1, characterized in that, The preparation method of the calprotectin immunochromatographic test strip includes: Anti-calprotectin monoclonal antibody and coating solution were coated onto the detection line of a nitrocellulose membrane and then dried; anti-mouse IgG antibody and coating solution were coated onto the control line of a nitrocellulose membrane and then dried. Anti-calprotectin antibody was added to a colloidal gold solution with a pH of 7.2-7.8 for labeling, and a stabilizer was added. The solution was then uniformly coated onto a glass fiber conjugate pad and dried. The sample pad, glass fiber bonding pad, coated nitrocellulose membrane, and absorbent pad are sequentially bonded to a PVC base plate to form a test strip.

4. The detection kit for detecting the content of calprotectin in synovial fluid according to claim 3, characterized in that, The stabilizer is polyethylene glycol.

5. A test kit for detecting the content of calprotectin in synovial fluid according to claim 1, characterized in that, The process of acquiring test strip images after testing and calculating the relative gray values ​​of the test line and control line to determine the calprotectin concentration range includes: Calprotectin concentration gradient standard solutions with concentrations of 0, 10, 50, 100, and 150 μg / mL were obtained. After dilution, the calprotectin concentration gradient standard solutions were added to the test strip. Calprotectin binds to the colloidal gold-labeled anti-calprotectin antibody to form a complex, which migrates forward to the detection line and produces a colored band. Collect test strip images after testing with calprotectin concentration gradient standard solution; perform grayscale processing on the test strip images, calculate the grayscale ratio of T line / C line, calibrate the calprotectin concentration range, and generate a standard curve; Acquire test strip images after testing the joint fluid of the subjects; The test strip image is processed in grayscale, the grayscale ratio of the T line to the C line is calculated, and the result is fitted with a standard curve to determine the calprotectin concentration range. The calprotectin concentration range is divided into <50 μg / mL, 50–150 μg / mL, and >150 μg / mL, which correspond to low risk, medium risk, and high risk levels, respectively.

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