Chair-side test paper for predicting clinical treatment prognosis effect of periodontitis as well as preparation method and application of chair-side test paper

Abstract

The invention discloses chair-side test paper for predicting the clinical treatment prognosis effect of periodontitis as well as a preparation method and application of the chair-side test paper. The test paper is coupled with ultrasonic extract of periodontitis pathogenic bacterium porphyromonas gingivalis based on a colloidal gold lateral flow chromatography technology to form a detection probe, the titer level of an anti-porphyromonas gingivalis specific sIgA antibody in saliva of a clinical patient is rapidly detected, the sensitivity of the test paper is debugged, and the specificity of the sIgA antibody in the saliva of the clinical patient is improved. And when the antibody titer is higher than 200, the detection result is positive, so that the periodontitis resistance of the patient is relatively strong, and the prognosis effect is good after periodontal basic treatment. The method can be used for self-inspection of a patient or assisting a clinician in pre-judging prognosis of the patient, so that a personalized treatment or review cycle plan is formulated, the technical breakthrough of clinical conventional diagnosis and treatment of periodontitis is realized, and the review cycle of the patient is shortened.

Description

Technical Field

[0001] This invention relates to the field of periodontitis, specifically to the prediction of the prognosis of patients with periodontal inflammation after clinical periodontal basic treatment, and particularly to chairside test strips for predicting the prognosis of clinical periodontal basic treatment for periodontitis, their preparation method and uses. Background Technology

[0002] Oral health, as an important component of overall public health, is receiving increasing attention. The damage to oral and systemic health caused by periodontitis has become one of the globally recognized health challenges. How to reduce the irreversible periodontal tissue damage caused by uncontrolled severe periodontitis and recurrent inflammation, and improve the regenerative effect of refractory periodontitis, thereby mitigating the damage of periodontitis to oral and systemic health, is a pressing clinical problem that needs to be solved.

[0003] Current clinical treatment plans for periodontitis are generic and lack personalization. Existing clinical examination technologies, including the latest periodontitis diagnostic kits, primarily function to diagnose periodontitis. The assessment of prognosis and recurrence relies entirely on the attending physician's clinical experience and frequent outpatient follow-up examinations. Patients lack the ability to objectively assess the disease's outcome after treatment, and they lack self-monitoring and objective judgment of their post-treatment outcomes. Furthermore, the relevant clinical treatment process lacks personalized and precise plans. Therefore, there is an urgent need to develop technologies that can predict the prognosis and recurrence risk of periodontitis treatment.

[0004] The information in the background section is merely intended to illustrate the general background of the invention and should not be construed as an admission or implication in any way that such information constitutes prior art known to those skilled in the art. Summary of the Invention

[0005] This invention involves collecting saliva and serum samples from 200 clinical patients with chronic periodontitis, and further detecting the periodontitis pathogen *Porphyromonas gingivalis* (abbreviated as Porphyromonas) in the clinical samples. Pg Correlation analysis was conducted between the specific antibody titer level and the degree of improvement in various clinical indicators of patients after basic periodontal treatment. The results showed that the lack of Porphyromonas gingivalis-specific sIgA antibodies in saliva was a key risk factor for poor prognosis in patients with periodontitis. This invention was completed based on at least this.

[0006] Furthermore, this invention found that subjects with better prognoses had an average titer of >200 for *Porphyromonas gingivalis*-specific sIgA antibodies in their saliva, while subjects with poor prognoses had an average titer of less than 100. Based on this, the applicant further researched a chairside test strip for predicting the clinical treatment prognosis of periodontal inflammation. Based on colloidal gold lateral flow chromatography, a detection probe is formed by coupling the probe with an ultrasonic extract of *Porphyromonas gingivalis*, a periodontal pathogen. This probe rapidly detects the titer level of *Porphyromonas gingivalis*-specific sIgA antibodies in the saliva of clinical patients. By adjusting the sensitivity of the test strip, the detection of antigen-specific sIgA antibody titers was achieved. A titer higher than 200 was defined as positive. At this point, the saliva antigen-specific sIgA antibody level of the positive patient is higher than the average of the patient population, theoretically indicating the patient has the ability to resist periodontal pathogen infection, resulting in a better prognosis and a low risk of disease recurrence after periodontal treatment. Further validation was conducted using 117 clinical periodontitis patients, demonstrating that the chairside test strip has ideal detection sensitivity and accuracy. The clinical indicators of patients who tested positive with the test strip showed significantly better improvement after basic periodontal treatment than those who tested negative, which is in line with the original design intent of this invention and the needs of clinical diagnosis.

[0007] Specifically, the present invention includes the following.

[0008] In a first aspect, based on key biomarkers obtained through screening for predicting the prognosis of periodontal inflammation treatment, a chairside test strip for predicting the prognosis of periodontal inflammation treatment is provided. The strip includes a base plate and a sample pad, a gold-labeled pad, a reaction membrane, and an absorbent pad sequentially disposed on the base plate. The gold-labeled pad includes colloidal gold particles coupled with extracts of periodontal inflammatory pathogens. The reaction membrane is provided with a detection line and a control line. The detection line is coated with an anti-human IgA antibody, and the control line is coated with an antibody selected from mouse anti-periodontal inflammatory pathogens.

[0009] In some embodiments, the chairside test strip for predicting the clinical prognosis of periodontal inflammation according to the present invention includes at least one of Porphyromonas gingivalis, Actinobacillus actinomycetii, Forsythoracica, Fusobacterium nucleatum, and Prevotella intermedius.

[0010] In some embodiments, the chairside test strip for predicting the clinical prognosis of periodontal inflammation according to the present invention comprises, wherein the test line is coated with 0.1 μg / mL-1.0 μg / mL of anti-human IgA antibody, with a coating amount of 0.5 μL / cm-2.0 μL / cm; the control line is coated with 0.5 μg / mL-1.5 μg / mL of mouse anti-pathogenic polyclonal antibody, with a coating amount of 0.5 μL / cm-2.0 μL / cm; and the gold-labeled pad is coated with 1 mg / mL-7 mg / mL of colloidal gold-labeled pathogenic bacterial extract, with a coating amount of 2 μL / cm-10 μL / cm.

[0011] In some embodiments, the chairside test strip for predicting the clinical prognosis of periodontal inflammation according to the present invention, wherein the length of the gold-labeled pad overlapping the sample pad is 1.2 mm-2.0 mm, the length of the gold-labeled pad overlapping the reaction membrane is 0.8 mm-1.5 mm, and / or the length of the absorbent pad overlapping the reaction membrane is 1.2 mm-2.0 mm.

[0012] In some embodiments, the chairside test strip for predicting the clinical prognosis of periodontal inflammation according to the present invention, wherein the sample pad is obtained by treating the sample pad buffer, the sample pad buffer containing 0.1-1% bovine serum albumin, 0.4-1% tris(hydroxymethyl)aminomethane, 2.5-5% trehalose and 0.01-0.5M phosphate buffer; The gold-labeled pad is obtained by processing with a gold-labeled pad buffer solution, which contains 0.1-1% bovine serum albumin, 0.4-1% tris(hydroxymethyl)aminomethane, 1-5% trehalose, 10-50% sucrose and 0.01-0.5M phosphate buffer.

[0013] A second aspect of the present invention provides a method for preparing chairside test strips for predicting the clinical prognosis of periodontal inflammation, comprising the following steps: (1) Preparation of colloidal gold solution; (2) Add pathogenic bacteria extract to the colloidal gold solution to obtain colloidal gold-pathogenic bacteria antigen solution. Add bovine serum albumin to the colloidal gold-pathogenic bacteria antigen solution, centrifuge to obtain a precipitate, resuspend it with the colloidal gold reconstitution buffer to obtain colloidal gold-labeled pathogenic bacteria antigen solution, and incubate the gold-labeled pad with the colloidal gold-labeled pathogenic bacteria antigen solution and dry it. (3) Prepare a detection line coated with anti-human IgA antibody and a control line coated with mouse anti-periodontal inflammatory pathogen antibody on the reaction membrane; and (4) Assemble the sample pad, gold label pad, reaction membrane and absorbent pad in sequence on the base plate to form the test paper.

[0014] In some embodiments, according to the method for preparing chairside test strips for predicting the clinical prognosis of periodontal inflammation according to the present invention, the pH value of the colloidal gold solution is 7-12; and / or The concentration of the ultrasonic extract of the pathogenic bacteria in the colloidal gold solution is 1 mg / mL-7 mg / mL; and / or The pH value of the colloidal gold reconstitution buffer is 6-11; Anti-human IgA antibody was diluted with an antibody buffer containing 0.1-5% trehalose and 0.01-1M phosphate buffer. The colloidal gold reconstitution buffer contains 0.1-1% bovine serum albumin, 0.4-1% tris(hydroxymethyl)aminomethane, 1-5% trehalose, 10-50% sucrose and 0.01-0.5M phosphate buffer.

[0015] A third aspect of the invention provides the use of extracts of periodontal inflammatory pathogens in the preparation of reagents for predicting the prognosis of clinical treatment of periodontal inflammation, particularly chairside test strips. The extracts are used for the detection or quantification of specific sIgA antibody levels against periodontal inflammatory pathogens.

[0016] In some embodiments, according to the uses described in the invention, the periodontal inflammation includes various types of inflammation of the periodontal soft and hard tissues caused by bacterial infections, such as gingivitis, chronic periodontitis, and aggressive periodontitis.

[0017] In some embodiments, according to the intended use described in the invention, the test sample is a saliva sample.

[0018] A fourth aspect of the present invention provides a method for detecting anti-porphyria gingivalis-specific sIgA antibodies, comprising the step of using the chairside test strip described above for detection.

[0019] In some embodiments, according to the method for detecting anti-Porphyromonas gingivalis-specific sIgA antibodies according to the present invention, a sample from the subject is dropped onto a sample pad, timing begins as the liquid flows, and judgment is made based on the reaction results within 10 minutes.

[0020] In some embodiments, according to the method for detecting anti-Porphyromonas gingivalis-specific sIgA antibodies according to the present invention, the judgment criteria are as follows: (1) When both the test line and the control line show color, the result is positive, indicating that the sample contains anti-porphyria gingivalis-specific sIgA antibody and the antibody titer is >200. (2) If the test line does not show color and only the control line shows color, it is negative, indicating that there is no anti-porphyria gingivalis-specific sIgA antibody or the antibody titer is <100 in the sample to be tested. (3) If the quality control line does not show color, it is invalid, indicating that the operation process is incorrect or the test card has failed.

[0021] This invention is based at least in part on correlation analysis of 200 clinical samples to screen out key factors affecting the risk of periodontal inflammation and recurrence (titer level of anti-Porphyromonas gingivalis specific sIgA antibody). Based on colloidal gold lateral flow chromatography, a detection probe is formed by coupling the probe with an ultrasonic extract of periodontal pathogens. This probe is used to rapidly detect the titer level of anti-Porphyromonas gingivalis specific sIgA antibody in the saliva of clinical patients. By adjusting the sensitivity of the test strip, a positive colorimetric result is achieved when the titer of antigen-specific sIgA antibody is higher than 200. A positive result indicates that the patient has strong resistance to periodontal inflammation, low risk of developing the disease, or good prognosis after treatment.

[0022] The present invention was further validated through testing on 117 clinical patients, demonstrating that the chairside test strip has ideal detection sensitivity and accuracy. The clinical indicators of patients who tested positive by the test strip showed significantly better improvement after basic periodontal treatment than those who tested negative, which is in line with the original design intent and clinical diagnostic needs of the present invention.

[0023] This invention proposes new clinical judgment criteria for assessing and predicting the risk of periodontitis and its recurrence. It can promote the formation of an intelligent diagnosis and personalized treatment system for clinical periodontitis. Furthermore, the convenient and rapid detection method is conducive to the promotion and popularization of chairside treatment or grassroots communities, thus pushing the level of periodontitis diagnosis and treatment in my country to a new level.

[0024] Furthermore, the method for predicting or treating prognoses in this invention can lead to a new model of personalized prediction and precision medicine for periodontitis, helping clinicians quickly determine the risk type of periodontitis patients and providing personalized diagnosis, treatment plans, and follow-up appointment schedules for clinical periodontitis patients. Through the development of this product, a technological breakthrough can be achieved in the routine clinical diagnosis and treatment of periodontitis, reducing patient follow-up appointment cycles and decreasing annual treatment costs by at least approximately 50%. Attached Figure Description

[0025] Figure 1 Structure and mechanism of action of a test strip for detecting specific sIgA antibody levels against Porphyromonas gingivalis in clinical saliva samples.

[0026] Figure 2 Blood and saliva samples from 200 clinical patients Pg Correlation analysis between specific IgG, sIgA and prognosis of periodontitis.

[0027] Figure 3 Clinical follow-up tests of 15 patients confirmed that at the time of initial diagnosis, the saliva of these patients contained... PgSpecific sIgA antibody levels and saliva at 3-month follow-up visit Pg The levels of specific sIgA antibodies were not significantly different.

[0028] Figure 4 Colloidal gold labeling Pg Screening for the optimal pH of ultrasonic extracts.

[0029] Figure 5 Colloidal gold labeling Pg Screening for the optimal concentration of ultrasonic extract.

[0030] Figure 6 Optimization of colloidal gold reconstitution buffer.

[0031] Figure 7 Preparation of gold-labeled pads, reaction membranes, and test strips.

[0032] Figure 8 The sensitivity detection effect of the prepared colloidal gold test strip.

[0033] Figure 9 The specific detection effect of the prepared colloidal gold test strip.

[0034] Figure 10 Clinical trials involving 117 patients showed that patients who tested positive using colloidal gold test strips had better prognoses after basic periodontitis treatment than those who tested negative. Detailed Implementation

[0035] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0036] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0037] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0038] In this article, the term "periodontal inflammation" refers to a disease of the soft and hard tissues of the periodontium caused by an imbalance of the oral flora or infection by periodontal pathogens, preferably an inflammatory disease, examples of which include, but are not limited to, gingivitis, chronic periodontitis, aggressive periodontitis, and peri-implantitis.

[0039] In this article, the term "inflammation" refers to the symptoms and signs caused by oral bacterial infections. This article primarily focuses on gingival redness and swelling, pain, abscesses, which may be accompanied by bleeding, alveolar bone resorption, tooth loosening, wound pain, purulent discharge, hyperplasia, and accompanying discomfort when biting, bad breath, etc.

[0040] In this article, the term "prediction" refers to assisting patients in daily self-monitoring of their periodontitis risk or assisting doctors in predicting the prognosis trend after treatment, so as to develop personalized treatment plans and follow-up visit schedules. Its purpose is to avoid delaying treatment or wasting medical resources.

[0041] Test strips This invention provides a chairside test strip for predicting the clinical prognosis of periodontal inflammation. The test strip includes a base plate and a sample pad, a gold label pad, a reaction membrane, and an absorbent pad disposed on the base plate, wherein the sample pad, the gold label pad, the reaction membrane, and the absorbent pad are arranged sequentially.

[0042] In this invention, the reaction membrane is provided with a detection line and a control line. The detection line is coated with an anti-human IgA antibody, and the control line is coated with a mouse polyclonal antibody against periodontal inflammatory pathogens. The gold-labeled pad is coated with a colloidal gold-labeled ultrasonic extract of the pathogens. A schematic diagram of the overall structure and components of the test strip is shown below. Figure 1 As shown.

[0043] In this invention, the periodontal inflammatory pathogens include at least one of *Porphyromonas gingivalis*, *Actinomyces actinomycetii*, *Fusobacterium nucleatum*, and *Prevotella intermedius*. In a preferred embodiment, the gold pad includes colloidal gold particles coupled to an extract of *Porphyromonas gingivalis*, and the control lines on the reaction membrane are coated with an antibody selected from mouse anti-*Porphyromonas gingivalis*.

[0044] In this invention, the detection line on the reaction membrane is coated with 0.1-1.0 μg / mL, preferably 0.5-1.0 μg / mL, such as 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 μg / mL of anti-human IgA antibody, with a coating amount of 0.5-2.0 μL / cm, preferably 0.6-1.9 μL / cm, and even more preferably 0.8-1.5 μL / cm, such as 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, or 1.5 μL / cm.

[0045] In this invention, the control lines on the reaction membrane are coated with 0.5-1.5 μg / mL, preferably 1.0-1.5 μg / mL, such as 1.0, 1.1, 1.2, 1.3, 1.4, and 1.5 μg / mL of mouse anti-porphyria polyclonal antibody, with a coating amount of 0.5-2.0 μL / cm, preferably 0.6-1.9 μL / cm, and even more preferably 0.8-1.5 μL / cm, such as 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, and 1.5 μL / cm.

[0046] In this invention, the gold-labeled pad is coated with 1-7 mg / mL, preferably 2-6 mg / mL, such as 2, 3, 4, 5, 6 mg / mL, of colloidal gold-labeled Porphyromonas gingivalis extract, with a coating amount of 2-10 μL / cm, preferably 3-9 μL / cm, such as 3, 4, 5, 6, 7, 8, 9 μL / cm.

[0047] In this invention, the first end of the gold-labeled pad is positioned below the sample pad, and the second end is positioned above the first end of the reaction membrane. The absorbent pad is positioned above the second end of the reaction membrane. In a preferred embodiment, the length of the gold-labeled pad positioned on the sample pad is 1.2-2.0 mm, preferably 1.5-2.0 mm, for example, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 mm; the length of the gold-labeled pad positioned on the reaction membrane is 0.8-1.5 mm, preferably 0.9-1.5 mm, and even more preferably 1.0-1.5 mm, for example, 1.0, 1.1, 1.2, 1.3, 1.4, or 1.5 mm; and / or the length of the absorbent pad positioned on the reaction membrane is 1.2-2.0 mm, preferably 1.5-2.0 mm, for example, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 mm.

[0048] In this invention, the sample pad is obtained by treating it with a sample pad buffer solution, wherein the sample pad buffer solution contains 0.1-1% bovine serum albumin, 0.4-1% tris(hydroxymethyl)aminomethane, 2.5-5% trehalose, and 0.01-0.5M phosphate buffer. Preferably, the sample pad buffer solution contains 0.5-1% bovine serum albumin (e.g., 0.5, 0.6, 1.7, 0.8, 0.9, 1%), 0.5-0.85% (e.g., 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85%) tris(hydroxymethyl)aminomethane, 3-5% (e.g., 3, 3.5, 4, 4.5, 5%) trehalose, and 0.01-0.4M (e.g., 0.1, 0.2, 0.3, 0.4M) phosphate buffer.

[0049] In this invention, the gold-labeled pad is obtained by treatment with a gold-labeled pad buffer solution, wherein the gold-labeled pad buffer solution contains 0.1-1% bovine serum albumin, 0.4-1% tris(hydroxymethyl)aminomethane, 1-5% trehalose, 10-50% sucrose and 0.01-0.5M phosphate buffer. Preferably, the gold-labeled pad buffer contains 0.5-1% bovine serum albumin (e.g., 0.5, 0.6, 1.7, 0.8, 0.9, 1%), 0.5-0.85% (e.g., 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85%) tris(hydroxymethyl)aminomethane, 3-5% (e.g., 3, 3.5, 4, 4.5, 5%) trehalose, 10-30% sucrose (e.g., 10, 15, 20, 25, 30%), and 0.01-0.4M (e.g., 0.1, 0.2, 0.3, 0.4M) phosphate buffer.

[0050] Preparation method In one aspect, the present invention provides a method for preparing chairside test strips for predicting the clinical prognosis of periodontal inflammation, comprising: preparing colloidal gold particles, coupling them with periodontal inflammatory pathogenic bacteria antigens to form detection probes, assembling them with prepared lateral flow chromatography test strips to form test strips, and further verifying and optimizing them based on clinical samples, including exploring the safety, specificity and sensitivity of the test strips for diagnosis.

[0051] In a preferred embodiment, the preparation method of the present invention includes: (1) Preparation of colloidal gold solution; (2) Add pathogenic bacteria extract to the colloidal gold solution to obtain colloidal gold-pathogenic bacteria antigen solution. Add stabilizer to colloidal gold-pathogenic bacteria antigen solution, centrifuge to obtain precipitate, resuspend with the gold pad buffer solution to obtain colloidal gold-labeled pathogenic bacteria antigen solution, co-incubate the gold pad with the colloidal gold-labeled pathogenic bacteria antigen solution and dry. (3) Prepare a detection line coated with anti-human IgA antibody and a quality control line coated with mouse anti-periodontal inflammatory pathogen antibody on the reaction membrane; (4) Assemble the sample pad, gold label pad, reaction membrane and absorbent pad in sequence on the base plate to form the test paper.

[0052] In step (1) of the preparation method of the present invention, the colloidal gold solution can be prepared by methods known in the art, and there is no particular limitation thereto. For example, the colloidal gold solution can be obtained by boiling an aqueous solution of chloroauric acid and an aqueous solution of trisodium citrate under heating conditions.

[0053] Step (2) of the preparation method of the present invention is the preparation of a detection probe, which is a conjugate of *Porphyromonas gingivalis* extract and colloidal gold. The *Porphyromonas gingivalis* extract can be obtained using extraction methods known in the art, for example, by centrifuging and sonicating a *Porphyromonas gingivalis* culture. The centrifugation and sonication parameters are not particularly limited and can be adjusted as needed.

[0054] To ensure high stability and detection sensitivity of the antibody-labeled colloidal gold solution, this invention has conducted in-depth research and optimization of the preparation of the extract colloidal gold label and the reconstitution and resuspension process. In a preferred embodiment, the pH of the colloidal gold solution is adjusted to neutral or near-neutral (e.g., 6.6, 6.8, 7.0, 7.2, 7.4, etc.). A suitable buffer salt solution can be used to adjust the optimal pH for binding of the colloidal gold and the *Porphyromonas gingivalis* ultrasonic extract; this is not particularly limited. In a preferred embodiment, the concentration of the extract is 1-7 mg / mL, more preferably 2-6 mg / mL, for example, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6 mg / mL.

[0055] After mixing the extract with the colloidal gold solution, 8-12% (preferably 10%) of bovine serum albumin is added to bring the final concentration in the colloidal gold solution to 0.05-1%, preferably 0.1-0.5%, for example 0.1, 0.2, 0.3, 0.4, 0.5%. The mixture is then allowed to stand for 10-60 minutes, preferably 10-40 minutes, for example 10, 20, 30, 40 minutes. The mixture is then centrifuged to obtain a precipitate, which is resuspended using a colloidal gold reconstitution buffer. In a preferred embodiment, the colloidal gold reconstitution buffer has a neutral or near-neutral pH (e.g., 6.6, 6.8, 7.0, 7.2, 7.4, etc.) and contains 0.1-1% bovine serum albumin, 0.4-1% tris(hydroxymethyl)aminomethane, 1-5% trehalose, 10-50% sucrose, and 0.01-0.5M phosphate buffer. Preferably, the colloidal gold reconstitution buffer contains 0.5-1% bovine serum albumin (e.g., 0.5, 0.6, 1.7, 0.8, 0.9, 1%), 0.5-0.85% (e.g., 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85%) tris(hydroxymethyl)aminomethane, 3-5% (e.g., 3, 3.5, 4, 4.5, 5%) trehalose, 10-30% sucrose (e.g., 10, 15, 20, 25, 30%), and 0.01-0.4M (e.g., 0.1, 0.2, 0.3, 0.4M) phosphate buffer.

[0056] When preparing the gold-labeled pad, it can first be soaked in colloidal gold reconstitution buffer for a period of time, preferably 10-60 minutes, more preferably 10-50 minutes, and even more preferably 10-40 minutes, for example, 10, 20, 30, or 40 minutes. After drying, the treated gold-labeled pad is then soaked in the reconstituted Porphyromonas gingivalis ultrasonic extract colloidal gold-labeled solution mentioned above, and allowed to stand at room temperature for 10-60 minutes, preferably 10-50 minutes, and even more preferably 10-40 minutes, for example, 10, 20, 30, or 40 minutes. After drying, it is then sealed. The material of the gold-labeled pad is not particularly limited and can be selected from materials such as glass cellulose membrane, polyester membrane, or filter paper.

[0057] Step (3) of this invention is the preparation of a reaction membrane, which is a nitrocellulose membrane. In a preferred embodiment, the anti-human IgA antibody is diluted to 0.1-1.0 μg / mL, preferably 0.5-1.0 μg / mL, for example 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 μg / mL, using an antibody buffer. The antibody buffer contains 0.1-5% trehalose and 0.01-1M phosphate buffer. Preferably, the antibody buffer contains 1-5% (e.g., 1, 2, 3, 4, 5%) trehalose and 0.01-0.05M (e.g., 0.01, 0.02, 0.03, 0.04, 0.05M) phosphate buffer. The diluted antibody is then dropped onto the detection line on the nitrocellulose membrane to achieve the antibody coating amount described in this invention. In a preferred embodiment, the mouse anti-porphyria gingivalis antibody is diluted to 0.5-1.5 μg / mL, preferably 1.0-1.5 μg / mL, for example 1.0, 1.1, 1.2, 1.3, 1.4, 1.5 μg / mL, using the antibody buffer, and then dropped onto the control line on a nitrocellulose membrane to achieve the antibody coating amount described in this invention.

[0058] In step (4) of this invention, the sample pad, gold label pad, reaction membrane, and absorbent pad are sequentially assembled on the base plate to form a test strip. In one specific embodiment, the first end of the gold label pad is placed below the sample pad, the second end is placed above the first end of the reaction membrane, and the absorbent pad is placed above the second end of the reaction membrane. The gold label pad and the absorbent pad overlap the reaction membrane by 1-5 mm, preferably 1-4 mm, and even more preferably 1-3 mm. The sample pad overlaps the gold label pad by 1-5 mm, preferably 1-4 mm, and even more preferably 1-3 mm. The starting end of the sample pad can be aligned with the starting end of the base plate, and the end of the absorbent pad can be aligned with the end of the base plate. The detection line and control line on the reaction membrane are both strips perpendicular to the length direction of the test strip. The detection line is located on the side near the end of the sample pad, and the control line is located on the side near the beginning of the absorbent pad. The test strip is cut into strips of appropriate width and placed in a sealed bag with built-in desiccant to obtain the colloidal gold test strip.

[0059] use In one aspect, the present invention provides the use of pathogenic bacteria extracts in the preparation of a detection product for predicting the prognosis of basic clinical treatment for periodontal inflammation, or in the preparation of a product for the detection or quantification of specific sIgA antibody levels against periodontal inflammatory pathogens, wherein the product may be in the form of a test strip, preferably, the test strip may further comprise at least one component selected from the present invention: colloidal gold reconstitution buffer, antibody buffer, gold pad buffer, sample pad buffer, anti-human IgA antibody, and anti-periodontal inflammatory pathogen antibody (particularly anti-Porphyromonas gingivalis antibody).

[0060] In this invention, the sample to be tested is a saliva sample derived from the subject.

[0061] The test strip uses colloidal gold-labeled ultrasonic extracts of pathogenic bacteria coated on the gold pad and anti-human IgA antibodies coated on the detection line of the reaction membrane. During testing, the analyte in the sample first binds to the colloidal gold-labeled ultrasonic extract of pathogenic bacteria on the gold pad, forming a specific sIgA antibody-pathogenic ultrasonic extract colloidal gold complex. Subsequently, it binds to the anti-human IgA antibody on the detection line of the reaction membrane, forming an anti-human IgA antibody-specific sIgA antibody-pathogenic ultrasonic extract colloidal gold complex. This allows the test strip to detect sIgA antibodies against specific pathogenic bacteria, enabling rapid detection of specific sIgA antibodies in patient saliva and serum samples. The test is simple to use and suitable for clinical samples and chairside procedures.

[0062] Furthermore, throughout the labeling process of the ultrasonic extract of pathogenic bacteria with colloidal gold, pH, the amount of ultrasonic extract labeled with pathogenic bacteria, and the colloidal gold reconstitution buffer all affected the ultrasonic extract of pathogenic bacteria. The stability and sensitivity of colloidal gold-labeled solutions are affected to a certain extent. By optimizing the pH, the labeling amount of the ultrasonic extract of pathogenic bacteria, and the colloidal gold reconstitution buffer, the final colloidal gold-labeled solution of the ultrasonic extract of pathogenic bacteria can be made to have excellent stability and sensitivity.

[0063] In this invention, the specific sequence of the antibody involved is not particularly limited. For example, anti-human IgA antibody and mouse anti-porphyria gingivalis antibody can be purchased from commercially available products.

[0064] Performance testing First, in the sample PgThe assessment or differential diagnostic ability of specific sIgA levels as a biomarker for predicting the risk of periodontal inflammation or the prognosis of treatment can be performed using methods known in the art, such as, but not limited to, using patients or suspected patients as the experimental group and healthy subjects as the control group, or using patients before treatment as the control group and patients after treatment as the experimental group to generate ROC curves and determine... Pg The cut-off value, area under the curve (AUC), sensitivity, and specificity are indicators used for differential diagnosis based on specific sIgA levels.

[0065] Secondly, performance testing of the testing product, including but not limited to indicators such as the limit of detection, sensitivity, specificity, and accuracy, may be conducted using methods known in the art, without particular limitation. For example, quantitative determination of the test strip of the present invention may be performed using saliva samples from patients diagnosed with periodontal inflammation and saliva samples from normal controls, and the results of clinical or pathological diagnoses of periodontal inflammation may be compared. Appropriate positive reference values ​​may be determined using ROC curves, and the specificity, sensitivity, overall concordance rate, and correlation analysis of the test strip may be evaluated.

[0066] For example, clinical specificity can be measured by the proportion of saliva samples that test negative with the test strip out of the total number of cases clinically diagnosed as having a "high risk of periodontal inflammation or a poor treatment prognosis," assessing the test strip's ability to correctly screen patients for a high risk of periodontal inflammation or a poor treatment prognosis. Clinical sensitivity can be measured by the proportion of saliva samples that test positive with the test strip out of the total number of cases clinically diagnosed as having a "low risk of periodontal inflammation or a good treatment prognosis," assessing the test strip's ability to correctly determine whether a patient has a low risk of periodontal inflammation or a good treatment prognosis. For overall concordance rate, the sensitivity and specificity of the testing product are calculated comprehensively to reflect the consistency between the test product's results and the patient's high risk of periodontal inflammation or poor treatment prognosis. Kappa values ​​are then used for consistency analysis.

[0067] Furthermore, for clinical trial data processing and statistical analysis methods, descriptive statistics such as mean ± standard deviation are used, while count data are described using case numbers and percentages. Depending on the data type, appropriate statistical analysis methods for group designs can be selected, such as t-tests, rank-sum tests, chi-square tests, and exact probabilities. Clinical samples are measured using statistical testing products. Pg Specific sIgA levels, assessment Pg The correlation and linear regression analysis of specific sIgA with patients' periodontal inflammation risk and treatment prognosis were performed. The clinical analytical performance of the testing product was evaluated by calculating its sensitivity, specificity, and overall concordance rate, compared with existing clinical or pathological diagnostic standards for periodontal inflammation risk and treatment prognosis, such as the "gold standard."

[0068] Those skilled in the art will understand that, based on professional knowledge, the test results of each sample can be analyzed to determine the upper and lower limits of the measured values, the class intervals, and the cut-off points. A cumulative frequency distribution table can be created according to the selected class intervals, and the sensitivity (true positive rate), specificity, and false positive rate (1-specificity) for each cut-off point can be calculated. An ROC curve can be plotted with the true positive rate on the ordinate and the false positive rate on the abscissa. The area under the ROC curve (AUC) and 95% CI can be calculated to evaluate the diagnostic value and determine the cut-off reference value. Based on the determined cut-off reference value, all test samples can be judged as negative or positive, and indicators such as sensitivity, specificity, and overall concordance rate can be calculated. The Kappa test can be used to evaluate the consistency between the test results of the test strip and the clinical (or pathological) diagnosis.

[0069] The present invention will be further described in detail below with reference to specific embodiments.

[0070] Example 1 This embodiment collected body fluid samples from 200 clinical patients and screened for key antibody biomarkers that affect the prognosis of periodontitis treatment. The specific process is as follows: Two hundred young and middle-aged patients with chronic periodontitis were recruited clinically. After rigorous enrollment screening and evaluation, systematic periodontal clinical examination indicators and periodontal imaging indicators were measured. The severity of periodontitis and the prognosis at the 3-month follow-up visit were recorded, including the improvement rate of average probing depth and the improvement rate of the percentage of bleeding sites in the entire mouth. Saliva and serum samples were collected from patients at the initial visit and the 3-month follow-up visit, and ELISA was used to detect the presence of periodontal disease in the samples. Pg The levels of specific IgG and sIgA antibodies were analyzed, and further correlation analysis was performed with the percentage improvement in clinical periodontitis indicators (including probing depth PD and bleeding rate BO) after treatment in the subjects (e.g., Figure 2 As shown), key diagnostic indicators (saliva) affecting the prognosis of periodontitis treatment were screened. Pg Specific sIgA antibody) and diagnostic threshold (titer 200).

[0071] Furthermore, antibody level tests on saliva samples from 15 clinical patients at their initial visit and follow-up visit at 3 months revealed that antibody levels in the patients' saliva within 3 months... Pg The level of specific sIgA antibody did not fluctuate significantly. Figure 3 Therefore, the saliva obtained during the patient's initial consultation can be used as a sample. Pg The specific sIgA antibody level results serve as representative results of salivary antibody levels during the period before a follow-up visit after periodontal clinical treatment, and are used as a basis for evaluating the prognosis of subsequent periodontal treatment.

[0072] Example 2 This embodiment provides a method for preparing a colloidal gold test strip for detecting Porphyromonas gingivalis-specific sIgA antibodies in patients, comprising the following steps: Preparation of gold pads coated with colloidal gold markers of ultrasonic extracts of Porphyromonas gingivalis; A reaction membrane was prepared with anti-human IgA antibody and mouse anti-porphyria gingivalis antibody as the detection line and control line, respectively. Assemble the gold label pad, reaction membrane, sample pad, absorbent pad, and base plate prepared in the first two steps into a test strip.

[0073] The following is a detailed explanation: 1. Preparation of colloidal gold Take 100 mL of 1% chloroauric acid aqueous solution (purchased from Sigma) and put it into a 250 mL Erlenmeyer flask. Heat it to boiling with a constant temperature electromagnetic stirrer. While stirring, add 1 mL of 1% trisodium citrate aqueous solution (purchased from Sigma). The solution changes from golden yellow to purple-red within 2 minutes. After the solution color does not change, continue to boil for 10 minutes. After cooling, restore the volume to 100 mL with distilled water to prepare a colloidal gold solution. The liquid is pure and clear, without precipitation or floating matter. Store at 2-8℃ in the dark.

[0074] 2. Preparation of colloidal gold markers for ultrasonic extracts of Porphyromonas gingivalis *Porphyromonas gingivalis* (purchased from Shanghai Lianzu Biotechnology Co., Ltd.) was cultured anaerobically at 37°C for 5-7 days on TSA sheep blood agar (also purchased from Shanghai Lianzu Biotechnology Co., Ltd.) until the logarithmic growth phase and the formation of black colonies. Black colonies (microscopically identified as Gram-negative bacilli) were scraped and collected into 50 mL centrifuge tubes. The tubes were washed twice with phosphate-buffered saline (PFS), centrifuged at 1100 rpm for 7 minutes, and the supernatant was discarded. The tubes were resuspended in sterile deionized water and sonicated in an ice bath (30 minutes, 300 W, 5-second sonication, 5-second pause). The tubes were then centrifuged at 14000 rpm for 15 minutes at 4°C, the supernatant was discarded, the precipitate was weighed, and diluted with sterile deionized water. The solution was filtered through a 0.22 μm filter, aliquoted, and stored at -80°C for later use.

[0075] The pH of the colloidal gold solution was adjusted using 0.2 mol / L potassium carbonate solution (purchased from Beijing Solarbio Science & Technology Co., Ltd.). 3 mg of *Porphyromonas gingivalis* ultrasonic extract was added per milliliter of colloidal gold solution. After vortexing and mixing, the solution was allowed to stand for 30 minutes. Then, 10% bovine serum albumin was added to bring the final concentration in the colloidal gold solution to 0.1%, and the solution was allowed to stand for another 30 minutes. The solution was then centrifuged at 12000 rpm / min at 4°C for 15 minutes. The supernatant was discarded, and the precipitate was resuspended in colloidal gold rehydration buffer and stored at 4°C for later use.

[0076] 2.1 Optimization of ultrasonic extraction conditions for colloidal gold-labeled Porphyromonas gingivalis The pH, labeling amount of Porphyromonas gingivalis ultrasonic extract, and colloidal gold reconstitution buffer were optimized during the entire labeling process of colloidal gold-labeled antibody-gold antibody. The optimization results were determined based on the stability and sensitivity of the final antibody-gold antibody solution. 2.1.1 Screening for the optimal pH value of colloidal gold-labeled ultrasonic extracts of Porphyromonas gingivalis The pH of the colloidal gold solution was adjusted using 0.2 mol / L potassium carbonate solution. 0.5 mL of colloidal gold solution was added to each well of a 48-well plate, followed by 0, 4, 7, 11, 16, and 40 μL of potassium carbonate solution. After mixing, the resulting pH values ​​were 7, 8, 9, 10, 11, and 12, respectively, yielding colloidal gold solutions with different pH values. *Porphyromonas gingivalis* ultrasonic extract was then added to each solution, adjusting the final concentration to 3 mg / mL. After shaking and mixing, the solutions were allowed to stand at room temperature for 2 hours to obtain different colloidal gold complexes. The optimal pH for the combination of colloidal gold and *Porphyromonas gingivalis* ultrasonic extract was determined when no color change was observed in the colloidal gold and the potassium carbonate dosage was minimized.

[0077] The results are as follows Figure 4 As shown, the binding stability of the *Porphyromonas gingivalis* ultrasonic extract-colloidal gold complex was best when the amount of 0.2 mol / L potassium carbonate solution added was 0-4 μL, i.e., the colloidal gold solution pH=7-8, especially pH=7, which is the optimal pH for the binding of colloidal gold and *Porphyromonas gingivalis* ultrasonic extract.

[0078] 2.1.2 Screening for the optimal concentration of colloidal gold-labeled ultrasonic extract of Porphyromonas gingivalis Based on the optimal pH determined above, a colloidal gold solution was prepared. Different concentrations of *Porphyromonas gingivalis* ultrasonic extract were sequentially added to 48-well plates to final concentrations of 0 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, and 7 mg / mL. The solutions were thoroughly mixed, labeled, and allowed to stand at room temperature for 2 hours. The color change of the colloidal gold was then observed. The optimal amount of *Porphyromonas gingivalis* ultrasonic extract labeled was determined when no further color change and precipitation occurred. Figure 5 The results showed that the colloidal gold-labeled ultrasonic extract of Porphyromonas gingivalis was stable in color at a concentration of 3 mg / mL, and the concentration of 3 mg / mL of the ultrasonic extract of Porphyromonas gingivalis was selected as the optimal concentration for colloidal gold labeling and used in subsequent operations.

[0079] 2.1.3 Optimization of Colloidal Gold Reconstitution Buffer Six colloidal gold reconstitution buffers with pH values ​​of 6, 7, 8, 9, 10, and 11 were prepared: 1% bovine serum albumin (from Sigma), 0.85% Tris (from Sigma), 5% trehalose (from Beijing Solarbio Science & Technology Co., Ltd.), 20% sucrose (from Beijing Solarbio Science & Technology Co., Ltd.), and 0.01M phosphate buffer (from Wuhan Pronosei Life Sciences Co., Ltd.). Colloidal gold labels from the ultrasonically extracted *Porphyromonas gingivalis* strain, after being blocked and centrifuged, were reconstituted using these prepared colloidal gold reconstitution buffers at different pH values. The appropriate pH of the colloidal gold reconstitution buffer was determined based on the stability and sensitivity of the labeled substances. Figure 6 The results showed that the colloidal gold reconstitution buffer at pH 7 produced no precipitation and had the best effect.

[0080] 3. Preparation of gold-labeled pads After soaking the gold-labeled pads in colloidal gold reconstitution buffer for 30 minutes, they were dried in an oven at 45°C. Then, the treated gold-labeled pads were soaked in a reconstituted colloidal gold-labeled solution of Porphyromonas gingivalis ultrasonic extract (Porphyromonas gingivalis ultrasonic extract concentration was 3 mg / mL) and allowed to stand at room temperature for 30 minutes. After that, the gold-labeled pads were placed in an oven at 37°C for 12 hours, then sealed and dried with desiccant for storage.

[0081] 4. Preparation of the reaction membrane Anti-human IgA antibody (Abcam, ab97215) was coated onto a nitrocellulose membrane to form a test line, and mouse anti-porphyria gingivalis antibody (Creative Diagnostics, DMAB9447) was coated onto a nitrocellulose membrane to form a control line.

[0082] Coating process: Anti-human IgA antibody was diluted to 1.0 μg / mL with antibody buffer (2% trehalose, 0.01 M phosphate buffer) and added to the test line on the nitrocellulose membrane, with a coating volume of 1.0 μL / cm. Mouse anti-porphyria gingivalis antibody was diluted to 1.5 μg / mL with antibody buffer and added to the control line on the nitrocellulose membrane, with a coating volume of 1.0 μL / cm. The coated reaction membranes were dried at 37°C for 12 hours and then stored for later use.

[0083] 5. Preparation of sample pads Soak the sample pad in 0.01M phosphate buffer containing 1% bovine serum albumin, 0.85% Tris and 5% trehalose for 1 hour, bake in an oven at 37°C for 12 hours until completely dry, and store in a sealed dry packaging bag for later use.

[0084] 6. Assembly of test strips like Figure 7As shown, the reaction membrane, gold-labeled pad, sample pad, and absorbent pad are sequentially attached to the base plate. The first end of the gold-labeled pad overlaps the sample pad, and the second end overlaps the first end of the reaction membrane. The absorbent pad overlaps the second end of the reaction membrane, with a 2mm overlap between the gold-labeled pad and the reaction membrane, and a 2mm overlap between the sample pad and the gold-labeled pad. The beginning of the sample pad is aligned with the beginning of the base plate, and the end of the absorbent pad is aligned with the end of the base plate. The reaction membrane has a detection line and a control line, both of which are strips perpendicular to the length of the test strip. The detection line is located on the side near the end of the sample pad, and the control line is located on the side near the beginning of the absorbent pad. The test strip is cut into 2.5mm wide strips and placed in a sealed bag with built-in desiccant to obtain the colloidal gold test strip, which can be stored for 12 months at 4°C.

[0085] Example 3 The following describes a method for detecting anti-Porphyromonas gingivalis-specific sIgA antibodies in saliva samples using colloidal gold test strips.

[0086] 1. Saliva samples were collected from 117 patients with clinical periodontitis, and the saliva samples were analyzed using ELISA. Pg Specific sIgA antibody titer level.

[0087] 2. Based on the experimental results in 1, the subject samples (high antibody titer and low antibody titer samples) were tested using the test strip assembled in Example 2. The collected subject saliva was vertically dropped onto the sample pad, and the timing was started when the liquid flowed. The results were determined within 10 minutes of the reaction.

[0088] 3. Test results The sample was tested using test strips, and the results were analyzed within 10 minutes as follows: Positive (+): Both the test line and control line show color, indicating the presence of anti-Porphyromonas gingivalis specific sIgA antibody in the sample, and the antibody titer is >200 (e.g., ...). Figure 8 (As shown in the right figure in the image); Negative( ): If the test line does not develop color, and only the control line develops color, it indicates that the sample tested does not contain specific sIgA antibodies against Porphyromonas gingivalis or the antibody titer is <100 (e.g., ...). Figure 8 (As shown in the left figure); Invalid: The control line does not show color, indicating that the operation process is incorrect or the test card is invalid, and it needs to be retested.

[0089] Example 4 This embodiment is used to evaluate the performance of the test paper of the present invention.

[0090] 1. The sensitivity, negative reference compliance rate, positive reference compliance rate, intra-batch and inter-batch repeatability, specificity, stability, and clinical performance validation (overall compliance rate and accuracy) of the colloidal gold-labeled Porphyromonas gingivalis ultrasonic extract of Example 2 under different pH conditions were analyzed.

[0091] 2. The sensitivity, negative reference compliance rate, positive reference compliance rate, intra-batch and inter-batch repeatability, specificity, stability, and clinical performance validation (overall compliance rate and accuracy) of the colloidal gold-labeled Porphyromonas gingivalis ultrasonic extract of Example 2 under different concentration conditions were analyzed.

[0092] 3. The sensitivity, negative reference compliance rate, positive reference compliance rate, intra-batch and inter-batch repeatability, specificity, stability, and clinical performance validation (overall compliance rate and accuracy) of the different colloidal gold reconstitution buffers in Example 2 were analyzed.

[0093] Specifically, the sensitivity test is to... Pg Specific sIgA reference standards were diluted to different concentrations. Test strips from the examples and comparative studies were used for detection, with each concentration tested three times. The test solution was added to the test card, and the results were interpreted after 10 minutes. The results showed that as the pH value of the colloidal gold-labeled *Porphyromonas gingivalis* ultrasonic extract gradually increased, the stability of the test strip gradually decreased. This manifested as the test sample failing to diffuse smoothly along the reaction membrane when passing through the gold-labeled pad, resulting in no color development of the control line. When the pH value was greater than 8, the test strip stability was less than 60±3%. When the concentration of the colloidal gold-labeled *Porphyromonas gingivalis* ultrasonic extract was less than 3 mg / mL, the control line of the reaction membrane did not develop color; when the concentration was greater than 5 mg / mL, both the control line and the test line of the reaction membrane developed color, significantly interfering with the test results. Too low a trehalose content and too high a sucrose content in the colloidal gold reconstitution buffer both led to difficulty in diffusion of the test sample in the reaction membrane, resulting in no color development of the control line.

[0094] The negative reference sample compliance rate was determined by testing several negative reference samples using the test strips from the example, with each test strip testing several reference samples. The negative reference sample compliance rate of the test strips was 80.96 ± 1.32%.

[0095] The positive reference sample concordance rate was 85.76 ± 1.86% when several positive reference samples were tested using the test strips from the example.

[0096] Intra-batch and inter-batch repeatability tests involve testing different concentrations of test strips prepared in the same and different batches as described in the examples. PgSpecific sIgA standard. Each concentration was tested three times, and the repeatability of the test strip was observed. The repeatability of the test strip was 95.53 ± 1.47%.

[0097] Specific experiments are to Pg Standard solutions of specific sIgA and its related interfering substances were prepared at different concentrations and detected using test strips prepared in the examples and comparative examples. Each concentration was tested three times. The comparative examples were used... Pg Specific IgG and IgM antibodies, results as follows Figure 9 As shown, the test strip has good specificity, and only the sIgA antibody standard is positive.

[0098] The stability test involved storing the test strips prepared in the examples and comparative examples at 37°C for a certain period of time, then testing with standards of different concentrations. Each concentration was tested three times. The test solution was added to the test strip, and the results were interpreted after 10 minutes. The stability of the test strips in the examples was 88.34±0.98%, and the stability of the test strips in the comparative examples was 62.21±5.87%.

[0099] Clinical performance validation involved collecting saliva samples from individuals with confirmed strong resistance to periodontal inflammation, low risk of developing the disease, or good prognosis after treatment, as well as saliva samples from healthy individuals. Test strips prepared using the examples and comparative samples were used to test the samples. The total positive / identification positive rate, the total negative / identification negative rate, and the overall concordance rate were calculated. A total of 117 clinical samples were tested. The total positive / identification positive rate of the example test strips was 82.14±1.95%, and the total negative / identification negative rate was 72.73±1.24%.

[0100] Furthermore, follow-up evaluation of these 117 patients after basic periodontal treatment revealed that, among them, patients who tested positive using the example test strip showed an improvement rate of >20% in both the average probing depth and the percentage of bleeding sites three months after treatment. Their treatment prognosis was more ideal and significantly better than that of patients who tested negative using the example test strip. This validated the conclusions as being consistent with the predictive results of the example test strip. Figure 10 ).

[0101] Although the invention has been described with reference to exemplary embodiments, it should be understood that the invention is not limited to the disclosed exemplary embodiments. Various adjustments or changes may be made to the exemplary embodiments described in this specification without departing from the scope or spirit of the invention. The scope of the claims should be interpreted in the broadest possible sense to cover all modifications and equivalent structures and functions.

Claims

1. A chairside test strip for predicting the clinical treatment prognosis of periodontal inflammation, characterized in that, It includes a base plate and a sample pad, a gold label pad, a reaction membrane, and an absorbent pad arranged sequentially on the base plate. The gold label pad includes colloidal gold particles coupled with extracts of periodontal inflammatory pathogens. The reaction membrane is provided with a detection line and a control line. The detection line is coated with anti-human IgA antibody, and the control line is coated with anti-periodontal inflammatory pathogen antibodies.

2. The chairside test strip for predicting the clinical prognosis of periodontal inflammation according to claim 1, characterized in that, The periodontal inflammatory pathogens include at least one of Porphyromonas gingivalis, Actinobacillus actinomycetii, Forsythoracis, Fusobacterium nucleatum, and Prevotella intermedius.

3. The chairside test strip for predicting the clinical prognosis of periodontal inflammation according to claim 1, characterized in that, The detection line is coated with 0.1 μg / mL-1.0 μg / mL anti-human IgA antibody, with a coating amount of 0.5 μL / cm-2.0 μL / cm; the quality control line is coated with 0.5 μg / mL-1.5 μg / mL mouse anti-pathogenic polyclonal antibody, with a coating amount of 0.5 μL / cm-2.0 μL / cm; and the gold-labeled pad is coated with 1 mg / mL-7 mg / mL colloidal gold-labeled pathogenic bacterial extract, with a coating amount of 2 μL / cm-10 μL / cm.

4. The chairside test strip for predicting the clinical prognosis of periodontal inflammation according to claim 1, characterized in that, The length of the gold-labeled pad overlapping the sample pad is 1.2mm-2.0mm, the length of the gold-labeled pad overlapping the reaction membrane is 0.8mm-1.5mm, and / or the length of the absorbent pad overlapping the reaction membrane is 1.2mm-2.0mm.

5. The chairside test strip for predicting the clinical prognosis of periodontal inflammation according to claim 1, characterized in that, The sample pad is obtained by treating it with a sample pad buffer solution, wherein the sample pad buffer solution contains 0.1-1% bovine serum albumin, 0.4-1% tris(hydroxymethyl)aminomethane, 2.5-5% trehalose and 0.01-0.5M phosphate buffer. The gold-labeled pad is obtained by processing with a gold-labeled pad buffer solution, wherein the gold-labeled pad buffer solution contains 0.1-1% bovine serum albumin, 0.4-1% tris(hydroxymethyl)aminomethane, 1-5% trehalose, 10-50% sucrose and 0.01-0.5M phosphate buffer.

6. The method for preparing chairside test strips for predicting the clinical prognosis of periodontal inflammation according to any one of claims 1-5, characterized in that, Includes the following steps: (1) Preparation of colloidal gold solution; (2) Add pathogenic bacteria extract to the colloidal gold solution to obtain colloidal gold-pathogenic bacteria antigen solution. Add stabilizer to colloidal gold-pathogenic bacteria antigen solution, centrifuge to obtain precipitate, resuspend with colloidal gold reconstitution buffer to obtain colloidal gold-labeled pathogenic bacteria antigen solution, co-incubate gold-labeled pad with colloidal gold-labeled pathogenic bacteria antigen solution and dry. (3) Prepare a detection line coated with anti-human IgA antibody and a quality control line coated with mouse anti-periodontal inflammatory pathogen antibody on the reaction membrane; and (4) Assemble the sample pad, gold label pad, reaction membrane and absorbent pad in sequence on the base plate to form the test paper.

7. The preparation method according to claim 6, characterized in that, The pH value of the colloidal gold solution is 7-12; Preferably, the concentration of the ultrasonic extract of the pathogenic bacteria in the colloidal gold solution is 1 mg / mL to 7 mg / mL; Preferably, the pH value of the colloidal gold reconstitution buffer is 6-11; Preferably, the anti-human IgA antibody is diluted with an antibody buffer containing 0.1-5% trehalose and 0.01-1M phosphate buffer. Preferably, the colloidal gold reconstitution buffer contains 0.1-1% bovine serum albumin, 0.4-1% tris(hydroxymethyl)aminomethane, 1-5% trehalose, 10-50% sucrose and 0.01-0.5M phosphate buffer. Preferably, the stabilizer is selected from at least one of bovine serum albumin, ovalbumin, polyethylene glycol, and gelatin.

8. The use of extracts of periodontal inflammatory pathogens in the preparation of reagents for predicting the clinical prognosis of periodontal inflammation treatment, among which, The extract of periodontal inflammatory pathogens can be used to detect or quantify the level of specific sIgA antibodies against periodontal inflammatory pathogens.

9. The use according to claim 8, characterized in that, The periodontal inflammation mentioned includes various types of inflammation of the soft and hard tissues of the periodontium caused by bacterial infections, including gingivitis, chronic periodontitis, and aggressive periodontitis. Preferably, the sample to be tested is a saliva sample.

10. A method for detecting anti-Porphyromonas gingivalis-specific sIgA antibodies, characterized in that, Includes the step of using chairside test strips according to any one of claims 1-5 for testing; Preferably, it comprises: The sample from the subject is dropped onto the sample pad, and the timing begins when the liquid flows. The result is judged based on the reaction within 10 minutes. Preferably, the judgment criterion is: (1) When both the test line and the control line show color, the result is positive, indicating that the sample contains anti-porphyria gingivalis-specific sIgA antibody and the antibody titer is >200. (2) If the test line does not show color and only the control line shows color, it is negative, indicating that there is no anti-porphyria gingivalis-specific sIgA antibody or the antibody titer is <100 in the sample to be tested. (3) If the quality control line does not show color, it is invalid, indicating that the operation process is incorrect or the test card has failed.

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