Application of keratin sulfate in diagnosis of chronic periodontitis

Abstract

The invention discloses an application of keratin sulfate in diagnosis of chronic periodontitis, and relates to the technical field of biological medicines, and the technical key points are as follows: a detection kit taking keratin sulfate as a molecular marker, a keratin sulfate monoclonal antibody and a human keratin sulfate (KS) enzyme-linked immunosorbent assay kit are utilized, and the application of keratin sulfate in diagnosis of chronic periodontitis is realized. According to the method, the expression level of keratin sulfate in gingival crevicular fluid is detected, clinical diagnosis of chronic periodontitis is assisted, a novel diagnosis and treatment strategy is provided for chronic periodontitis patients with increased keratin sulfate level, and a diagnostic technical foundation is laid for realizing long-acting management of chronic periodontitis by combining a targeted keratin sulfate therapy.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to the application of keratan sulfate in the diagnosis of chronic periodontitis. Background Art

[0002] Chronic periodontitis is a common oral disease, a chronic inflammation caused by the accumulation of dental plaque and calculus, mainly affecting the gums, periodontal ligament and alveolar bone. Common symptoms include swollen and red gums, bleeding, loose teeth, displacement, bad breath, etc. The main causes include poor oral hygiene habits, smoking, genetic factors, as well as unhealthy diet, systemic diseases (such as diabetes, cardiovascular diseases), etc. Treatment methods usually include professional dental cleaning, oral hygiene education, drug treatment (such as antibiotics) and surgical treatment in severe cases. If not treated in time, chronic periodontitis may lead to tooth loss, so maintaining good oral hygiene and regular check-ups are crucial.

[0003] At present, the diagnosis of chronic periodontitis mainly relies on clinical examinations and imaging-assisted examinations, such as gingival bleeding, deep periodontal pockets, tooth mobility, and the manifestations of alveolar bone resorption shown in X-ray films. However, these symptoms are usually taken seriously only when the disease is relatively severe. Therefore, exploring more convenient diagnostic methods for early detection and timely treatment is crucial for controlling the progression of the disease and maintaining oral health.

[0004] For this reason, the present invention aims to provide the application of keratan sulfate in the diagnosis of chronic periodontitis to solve the above problems. Summary of the Invention

[0005] The purpose of the present invention is to solve the above problems and provide the application of keratan sulfate in the diagnosis of chronic periodontitis. Through a detection kit with keratan sulfate as a molecular marker, using a monoclonal antibody against keratan sulfate and an enzyme-linked immunosorbent assay kit for human keratan sulfate, the expression level of keratan sulfate in gingival crevicular fluid is detected to assist in the clinical diagnosis of chronic periodontitis and provide a new diagnosis and treatment strategy for chronic periodontitis patients with elevated keratan sulfate levels.

[0006] In order to achieve the above purpose, the technical solution of the present invention is as follows:

[0007] The present invention provides the application of keratan sulfate in the diagnosis of chronic periodontitis. The keratan sulfate is used to prepare a kit for diagnosing chronic periodontitis, and the molecular marker of the kit is keratan sulfate.

[0008] The kit includes a keratan sulfate standard, a keratan sulfate-specific biotin-conjugated antibody, horseradish peroxidase-conjugated avidin, an enzyme-labeled plate pre-coated with keratan sulfate, a washing buffer, a substrate solution, and a termination solution. The termination solution is a sulfuric acid termination solution, and the substrate solution is TMB (tetramethylbenzidine).

[0009] When the concentration of the keratan sulfate is higher than 565.936 pg / ml, it is the disease state of chronic periodontitis.

[0010] Compared with the prior art, the beneficial effects of this solution are as follows:

[0011] The detection kit of the present invention uses keratan sulfate as a molecular marker. By using a monoclonal antibody against keratan sulfate and an enzyme-linked immunosorbent assay kit for human keratan sulfate (KS), the expression level of keratan sulfate in gingival crevicular fluid is detected to assist in the clinical diagnosis of chronic periodontitis, providing a new diagnosis and treatment strategy for patients with chronic periodontitis with elevated keratan sulfate levels, and laying a diagnostic technical foundation for the long-term management of chronic periodontitis by the combined targeted keratan sulfate therapy. Description of the Drawings

[0012] Figure 1 It is a schematic diagram of the content of keratan sulfate in gingival crevicular fluid (H) of clinically healthy patients and gingival crevicular fluid (P) of periodontitis patients in the embodiment of the present invention;

[0013] Figure 2 It is a schematic diagram of the random grouping of clinical gingival crevicular fluid samples in the embodiment of the present invention;

[0014] Figure 3 It is a schematic diagram of determining the optimal threshold of the concentration of keratan sulfate in gingival crevicular fluid based on the clinical samples of the training group in the embodiment of the present invention;

[0015] Figure 4 It is a schematic diagram of verifying the optimal threshold of the concentration of keratan sulfate in gingival crevicular fluid in the test group in the embodiment of the present invention. Detailed Embodiments

[0016] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solution of the present invention will be further described in detail below in combination with the embodiments and drawings of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work shall fall within the protection scope of the present invention.

[0017] It should be noted that, without conflict, the embodiments in the present invention and the features in the embodiments can be combined with each other. The present invention will be described in detail below in combination with the embodiments.

[0018] Example:

[0019] 1. Extracting gingival crevicular fluid with absorbent paper points:

[0020] (1) Clean the tooth surface (using the bass brushing method) to remove dental plaque;

[0021] (2) Use cotton rolls to isolate the buccal and lingual sides of the teeth in patients with periodontitis to avoid saliva contamination;

[0022] (3) Gently insert absorbent paper points (06 taper, size 30#) into the gingival sulcus about 1 - 2 mm (tooth positions 16 and 26), avoiding damage to the gingival tissue, and keep them in the gingival sulcus for 30 seconds to 1 minute to ensure sufficient absorption of gingival crevicular fluid. Collect 10 absorbent paper point samples for each patient;

[0023] (4) Remove the absorbent paper points and place them in a sterile microcentrifuge tube, and detect them in a timely manner or store them at low temperature (-20°C) for subsequent use.

[0024] 2. Laboratory ELISA detection of keratan sulfate (competitive inhibition method):

[0025] (1) Preparation before experiment: Prepare reagents and materials such as human keratan sulfate enzyme-linked immunosorbent assay kit, gingival crevicular fluid samples, keratan sulfate standards, enzyme-labeled plates, washing buffer, substrate solution, termination solution, etc.; Prepare instrument equipment such as enzyme-labeled immunosorbent assay reader, plate washer, pipette, incubator, etc.;

[0026] (2) Dilution of standards: Dilute the standards in the human keratan sulfate enzyme-linked immunosorbent assay kit with PBS diluent in gradients to prepare concentration gradients of 5000.00 pg / ml, 2500.00 pg / ml, 1250.00 pg / ml, 625.00 pg / ml, 312.50 pg / ml, 156.25 pg / ml, 78.13 pg / ml, 0.00 pg / ml;

[0027] (3) Cut off the front 5 mm of the absorbent paper points containing gingival crevicular fluid samples collected, place them in a new sterile microcentrifuge tube, add 200 μl of phosphate buffer for soaking, vortex for 10 seconds, then let stand at room temperature for 10 minutes, and centrifuge (5000 g, 10 minutes) to collect the sample supernatant for detection;

[0028] (4) Add samples, standards and detection antibodies: Add the sample supernatant (obtained in step 2), standards and biotin-labeled keratan sulfate antibody to the wells of the enzyme-labeled plate pre-coated with keratan sulfate, incubate at 37°C for 1 hour, wash the plate to remove unbound substances;

[0029] (5) Addition of enzyme conjugate: Add streptavidin conjugated with horseradish peroxidase, incubate at 37 °C for 1 hour, wash the plate to remove unbound enzyme conjugate;

[0030] (6) Addition of substrate solution: Add substrate solution TMB, incubate at 37 °C for 15 - 30 minutes, and the enzyme catalyzes the substrate to produce color;

[0031] (7) Termination of reaction: Add sulfuric acid termination solution to stop the reaction;

[0032] (8) Reading of results: Place the enzyme - labeled plate into an enzyme - label analyzer and read the absorbance value OD at a specific wavelength;

[0033] (9) Data analysis: Plot a standard curve based on the standard products, and calculate the concentration of keratan sulfate in the sample supernatant through the standard curve;

[0034] 3. Clinical measurement results: The content of keratan sulfate in gingival crevicular fluid of the healthy group and the periodontitis group was measured by ELISA (competitive inhibition method). The result showed that the content of keratan sulfate in the periodontitis group increased significantly.

[0035] 4. Threshold determination: Fourteen clinical samples (including 7 healthy individuals and 7 periodontitis patients) were randomly divided into a training set and a test set. Through analysis, the critical value of keratan sulfate was found to be 565.936 pg / ml. That is, when the content of keratan sulfate is higher than 565.936 pg / ml, it is already in the state of chronic periodontitis.

[0036] The above - mentioned specific embodiments are only explanations of the present invention, and they are not limitations of the present invention. After reading this specification, those skilled in the art can make modifications to the embodiments without creative contributions as needed, but as long as they are within the scope of the claims of the present invention, they are protected by the patent law.

Claims

1. Use of keratan sulfate in the diagnosis of chronic periodontitis, characterized by: The keratan sulfate is used for preparing a kit for diagnosing chronic periodontitis, and the molecular marker of the kit is keratan sulfate.

2. The use of keratan sulfate according to claim 1 in the diagnosis of chronic periodontitis, characterized in that: The kit includes a keratan sulfate standard, a keratan sulfate-specific biotin-conjugated antibody, horseradish peroxidase-conjugated avidin, an enzyme-labeled plate pre-coated with keratan sulfate, a washing buffer, a substrate solution, and a termination solution.

3. The use of keratan sulfate in the diagnosis of chronic periodontitis as described in claim 1, wherein: When the concentration of the keratan sulfate is higher than 565.936 pg / ml, it is the disease state of chronic periodontitis.

4. The use of keratan sulfate according to claim 2 in the diagnosis of chronic periodontitis, characterized in that: The termination solution is a sulfuric acid termination solution.

5. The use of keratan sulfate according to claim 2 in the diagnosis of chronic periodontitis, characterized in that: The substrate solution is TMB.

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