An arginine peptidase quantitative detection kit, arginine peptidase qualitative detection kit and method of use thereof

Abstract

This invention discloses a quantitative detection kit for arginine peptidase, a qualitative detection kit for arginine peptidase, and their usage methods. The quantitative detection kit utilizes the specific substrate Nα-benzoyl-DL-arginine-4-ylbenzamide hydrochloride, which can be hydrolyzed by arginine peptidase to 4-ylbenzamide. 4-ylbenzamide has a maximum absorption peak at 405 nm, and the specific arginine peptidase activity can be rapidly detected at room temperature using spectrophotometry. The qualitative kit utilizes the specific substrate Nα-benzoyl-DL-arginine-β-naphthamide hydrochloride, which can be hydrolyzed by arginine peptidase to p-β-naphthamide. β-naphthamide combines with 4-(dimethylamino)cinnamaldehyde to form a compound that is visible to the naked eye as a pink color. The disulfide bond cleaving agent cuts the mucus disulfide bonds, exposing bacterial enzyme sites, and the synergistic effect of the enzyme activator enhances the reaction rate of arginine peptidase hydrolysis of the substrate, significantly improving the detection sensitivity of arginine peptidase in tongue coating samples.

Description

Technical Field

[0001] This invention relates to the field of biological and medical detection technology, specifically to a quantitative detection kit for arginine peptidase, a qualitative detection kit for arginine peptidase, and their usage methods. Background Technology

[0002] The human oral mucosa microbiota consists of countless bacterial species that typically coexist harmoniously with the host. Three types of bacteria—*Porphyromonas gingivalis* (Pg), *Treponema denticola* (Td), and *Tannerella forsythia* (Tf)—are collectively known as the Red Complex and are considered the most dangerous bacterial group causing severe periodontal disease. Clinical trials have confirmed that these Red Complex bacteria secrete lipopolysaccharides and gingival proteases that directly damage periodontal tissues, triggering a local immune response and promoting the release of large amounts of inflammatory mediators, which are associated virulence factors leading to periodontitis. These microorganisms successfully evade the host's antimicrobial defenses, utilizing fibrous cilia, polysaccharide capsules and lipopolysaccharides, hemagglutinin and hemolytic activity, the release of metabolic toxic products, outer membrane vesicles, and numerous enzymes to easily survive in harsh environments. The role of periodontal pathogens in systemic inflammation and organ dysfunction has recently been demonstrated in association with a number of systemic diseases, such as atherosclerosis, endocarditis, ischemic heart disease, stroke, diabetes, pneumonia, chronic kidney disease, meningitis, rheumatoid arthritis, Alzheimer's disease, and adverse pregnancy outcomes.

[0003] Methods for detecting bacterial infection and inflammation in periodontal disease examinations include: plaque adhesion testing, which involves rubbing the tooth surface with the tip of a dental probe or dental detector to determine plaque adhesion; periodontal bacterial testing, which involves collecting subgingival plaque samples on paper and requiring the testing institution to measure bacterial counts using methods such as DNA quantification; and antibody testing, which measures the titer of IgG antibodies in serum for periodontal bacteria. These methods are time-consuming and costly, and cannot allow for the simultaneous examination of multiple patients in a simple and rapid manner. These three types of bacteria possess arginine peptidase activity, specifically recognizing and hydrolyzing arginine sites. By analyzing enzyme activity, periodontal disease can be effectively screened in a shorter time and in a simpler way.

[0004] Currently available test kits are solid-phase assays, manufactured by Hakusui Trading Co., Ltd. of Japan, Medical Device Registration Certificate No.: 21400AMY00214000, for qualitative detection. The detection principle involves subgingival plaque arginine peptidase catalyzing the substrate Nα-benzoyl-DL-arginine β-naphthamide hydrochloride. The product β-naphthamide combines with Solid Black K salt hemichloride (zinc chloride) to form an insoluble pale blue precipitate, which appears on a nitrocellulose membrane. The reaction conditions are: continuous heating at 55°C for 15 minutes. For arginine peptidase activity, sufficient sensitivity cannot be obtained unless the optimal enzyme activity temperature of 50-60°C is reached, which is an obstacle to the widespread use of periodontal bacterial examination. Addressing the problems of low sensitivity, difficulty in obtaining subgingival plaque samples, and invasiveness associated with existing technologies, there is an urgent need to develop a non-invasive, easily obtainable, room-temperature-reacting, and highly sensitive arginine peptidase detection kit. Summary of the Invention

[0005] In view of this, the present invention proposes an arginine peptidase quantitative detection kit, an arginine peptidase qualitative detection kit, and a method for using the kit, thereby solving the above problems.

[0006] The technical solution of this invention is implemented as follows:

[0007] A quantitative assay kit for arginine peptidase includes solution A; solution A comprises MES buffer, a disulfide bond cleaving agent, an enzyme activator, Nα-benzoyl-DL-arginine-4-ylbenzamide hydrochloride, and dimethyl sulfoxide; the disulfide bond cleaving agent comprises any one or more of tris(2-carboxyethyl)phosphine hydrochloride, β-mercaptoethanol, and dithiothreitol; the enzyme activator comprises any one or more of glycylglycine, glycamide, glycyl-asparagine, glycylphenylalanine, glycyl-L-proline, and glycyl-L-alanine.

[0008] Preferably, the concentration of MES buffer in solution A is 1.2-15.0 g / L, the concentration of disulfide bond cleaving agent is 1.2-25.0 g / L, and the concentration of enzyme activator is 1.3-28.0 g / L; the Nα-benzoyl-DL-arginine-4-ylbenzamide hydrochloride is pre-dissolved in dimethyl sulfoxide at a ratio of 44 mg / 1 ml; the volume ratio of the dimethyl sulfoxide solution of Nα-benzoyl-DL-arginine-4-ylbenzamide hydrochloride to the MES buffer is 0.5-5:50; and the pH of solution A is adjusted to 4.5-8.5.

[0009] The purpose of MES buffer is to provide a buffer system for enzymatic reactions. MES buffer can also be provided using Tris hydrochloride buffer, Tris maleate buffer, glycine buffer, phosphate buffer, sodium borate buffer, carbonate-bicarbonate buffer, or purified water as the buffer system.

[0010] The present invention also provides a method for using the above-mentioned arginine peptidase quantitative detection kit, wherein the above-mentioned quantitative detection kit is used for detection, and the detection sample is tongue plaque.

[0011] Preferably, the method of using the arginine peptidase quantitative detection kit includes the following steps:

[0012] S1: Scrape the tongue coating with a sampling swab 5 to 10 times, rotate it 5 to 10 times in solution A to prepare a sample solution, and immediately read the absorbance, which is recorded as A1;

[0013] S2: React the sample solution from step S1 at room temperature or 37°C for 5-15 minutes, and read the absorbance of the sample solution, which is recorded as A2.

[0014] S3: Calculate the sample reactivity = A2 - A1, substitute the sample reactivity into the formula to calculate the arginine peptidase activity in the sample; the formula is arginine peptidase activity = (standard enzyme activity × sample reactivity) / standard reactivity;

[0015] S4: Determine whether periodontitis exists based on the arginine peptidase activity obtained in step S3.

[0016] Preferably, the arginine peptidase activity in S4 is higher than 3 U / mL, indicating periodontitis.

[0017] Preferably, the main technical principle of the arginine peptidase quantitative detection kit of the present invention is as follows: Nα-benzoyl-DL-arginine-4-ylbenzamide hydrochloride can be hydrolyzed by arginine peptidase to 4-ylbenzamide, and 4-ylbenzamide has a maximum absorption peak at a wavelength of 405 nm. Quantitative determination is performed by spectrophotometry.

[0018] This invention also provides a qualitative detection kit for arginine peptidase, comprising solution B, solution C, and a detection strip; solution B comprises 1.2-15.0 g / L Tris hydrochloride buffer, 1.2-25.0 g / L disulfide cleaving agent, and 1.3-28.0 g / L enzyme activator; the disulfide cleaving agent comprises any one or more of tris(2-carboxyethyl)phosphine hydrochloride, β-mercaptoethanol, and dithiothreitol; the enzyme activator comprises glycylglycine, glycamide, and glycine... The reagent comprises any one or more of acyl-asparagine, glycylphenylalanine, glycyl-L-proline, and glycyl-L-alanine; solution C comprises 0.05%-1% by mass of 4-(dimethylamino)cinnamaldehyde and 0.5-2 mol / L hydrochloric acid; the raw materials used in the preparation of the test strip include Whatman filter paper, 1.2-15.0 g / L Tris hydrochloride buffer, Nα-benzoyl-DL-arginine-β-naphthylamide hydrochloride, and dimethyl sulfoxide.

[0019] Preferably, the preparation method of the detection strip is as follows: (1) Nα-benzoyl-DL-arginine-β-naphthamide hydrochloride is pre-dissolved in dimethyl sulfoxide at a ratio of 44 mg / 1 ml, and the dimethyl sulfoxide solution of Nα-benzoyl-DL-arginine-β-naphthamide hydrochloride is mixed with Tris hydrochloride buffer at a volume ratio of 0.5-5:50, and the pH is adjusted to 7.0-8.5; (2) Whatman filter paper is soaked in the above solution, taken out, and dried.

[0020] Tris hydrochloride buffer is used to provide a buffer system for enzyme-catalyzed reactions. Tris hydrochloride buffer can also be used as a buffer system with MES buffer, Tris maleate buffer, glycine buffer, phosphate buffer, sodium borate buffer, carbonate-bicarbonate buffer or purified water.

[0021] The present invention also provides a method for using the above-mentioned arginine peptidase qualitative detection kit, wherein the detection is performed using the above-mentioned qualitative detection kit, and the detection sample is tongue plaque.

[0022] Preferably, the method of using the arginine peptidase qualitative detection kit includes the following steps:

[0023] S1: Scrape the tongue coating with a sampling swab 5 to 10 times;

[0024] S2: Rotate the sampling swab from step S1 in solution B for 5 to 10 turns;

[0025] S3: Press the sampling swab processed in step S2 onto the Whatman filter paper of the test strip for 5 to 10 seconds, and react for 2 to 8 minutes at room temperature;

[0026] S4: Add 1 drop of solution C to the Whatman filter paper from step S3 and react for 3-5 minutes;

[0027] S5: Determine whether periodontitis exists based on the color obtained in step S4.

[0028] Preferably, the appearance of a pink color in S5 indicates periodontitis.

[0029] Preferably, the main technical principle of the arginine peptidase qualitative detection kit of the present invention is as follows: Nα-benzoyl-DL-arginine-β-naphthamide hydrochloride can be hydrolyzed by arginine peptidase to p-β-naphthamide. β-naphthamide combines with 4-(dimethylamino)cinnamaldehyde to form a compound that displays a visible pink color. This pink compound has a maximum absorption peak at a wavelength of 590 nm. It can also be quantitatively determined by spectrophotometry using a liquid reagent method.

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0031] This invention provides a quantitative detection kit for arginine peptidase. A disulfide bond cleavage agent breaks down mucus disulfide bonds, exposing bacterial enzyme sites. The synergistic effect of an enzyme activator enhances the rate at which arginine peptidase hydrolyzes the substrate. The specific substrate Nα-benzoyl-DL-arginine-4-ylbenzamide hydrochloride can be hydrolyzed by arginine peptidase to 4-ylbenzamide. 4-ylbenzamide has a maximum absorption peak at 405 nm. Using spectrophotometry, the specific arginine peptidase activity can be rapidly detected at room temperature, achieving an arginine peptidase activity of 1 U / mL, significantly improving the sensitivity of arginine peptidase detection in tongue coating samples.

[0032] This invention also provides a qualitative detection kit for arginine peptidase. A disulfide bond cleavage agent breaks the disulfide bonds in mucus, exposing the bacterial enzyme site. An enzyme activator synergistically enhances the rate at which arginine peptidase hydrolyzes the substrate. The specific substrate Nα-benzoyl-DL-arginine-β-naphthylamide hydrochloride can be hydrolyzed by arginine peptidase to p-β-naphthylamide. β-naphthylamide combines with 4-(dimethylamino)cinnamaldehyde to form a compound that is visible to the naked eye and is pink. Without the need for detection equipment, at room temperature, it can achieve a minimum sensitivity of 1 U / mL for arginine peptidase activity and a minimum bacterial concentration of 10- for *Porphyromonas gingivalis*. 2 The detection limit of cfu / mL greatly improves the sensitivity of arginine peptidase detection in tongue coating samples. Detailed Implementation

[0033] To better understand the technical content of this invention, specific embodiments are provided below to further illustrate the invention.

[0034] Unless otherwise specified, the experimental methods used in the embodiments of this invention are all conventional methods.

[0035] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available.

[0036] Main reagents:

[0037] Tris(2-carboxyethyl)phosphine hydrochloride, CAS: 51805-45-9;

[0038] 4-(dimethylamino)cinnamaldehyde, CAS: 6203-18-5;

[0039] Nα-benzoyl-DL-arginine-4-ylbenzamide hydrochloride, CAS: 911-77-3;

[0040] Nα-benzoyl-DL-arginine-β-naphthylamide hydrochloride, CAS: 913-04-2;

[0041] Glycylglycine, CAS: 556-50-3;

[0042] Glycineamide, CAS: 598-41-4;

[0043] Glycyl-asparagine, CAS: 32729-21-8;

[0044] Glycylphenylalanine, CAS: 3321-03-7;

[0045] Glycyl-L-proline, CAS: 704-15-4;

[0046] Glycyl-L-alanine, CAS: 3695-73-6;

[0047] β-Mercaptoethanol, CAS: 60-24-2;

[0048] Dithiothreitol, CAS: 3483-12-3.

[0049] Example 1

[0050] A quantitative assay kit for arginine peptidase includes solution A; solution A comprises 15.0 g / L MES buffer, 1.5 g / L tris(2-carboxyethyl)phosphonic acid hydrochloride (disulfide bond cleavage agent), 28.0 g / L glycylglycine (enzyme activator), Nα-benzoyl-DL-arginine-4-ylbenzamide hydrochloride, and dimethyl sulfoxide, with the pH adjusted to 5.5. Nα-benzoyl-DL-arginine-4-ylbenzamide hydrochloride is pre-dissolved in dimethyl sulfoxide at a ratio of 44 mg / 1 ml, and the volume ratio of the dimethyl sulfoxide solution of Nα-benzoyl-DL-arginine-4-ylbenzamide hydrochloride to the MES buffer is 2.5:50.

[0051] A method for using an arginine peptidase quantitative detection kit includes the following steps:

[0052] S1: Scrape the tongue coating with a sampling swab 5 to 10 times, rotate it 5 to 10 times in solution A to prepare a sample solution, and immediately read the absorbance, which is recorded as A1;

[0053] S2: React the sample solution from step S1 at room temperature for 5 minutes, and read the absorbance of the sample solution, which is recorded as A2;

[0054] S3: Calculate the sample reactivity = A2 - A1, substitute the sample reactivity into the formula to calculate the arginine peptidase activity in the sample; the formula is arginine peptidase activity = (standard enzyme activity × sample reactivity) / standard reactivity;

[0055] S4: Determine whether periodontitis exists based on the arginine peptidase activity obtained in step S3. If the arginine peptidase activity is higher than 3 U / mL, it is determined to be periodontitis.

[0056] Example 2

[0057] This example provides an arginine peptidase quantitative detection kit, whose composition is basically the same as that of Example 1. The difference is that the enzyme activator in Solution A in this example is replaced with glycine instead of glycamide. The other components and usage steps remain unchanged.

[0058] Example 3

[0059] This example provides an arginine peptidase quantitative detection kit, whose composition is basically the same as that of Example 1. The difference is that the enzyme activator in Solution A in this example is replaced with glycyl-glycine, while the other components and usage steps remain unchanged.

[0060] Example 4

[0061] This example provides an arginine peptidase quantitative detection kit, whose composition is basically the same as that of Example 1. The difference is that the enzyme activator in Solution A in this example is replaced with glycylglycine instead of glycylphenylalanine. The other components and usage steps remain unchanged.

[0062] Example 5

[0063] This example provides an arginine peptidase quantitative detection kit, whose composition is basically the same as that of Example 1. The difference is that in Solution A described in this example, the disulfide bond cleaving agent is replaced with tris(2-carboxyethyl)phosphine hydrochloride, and the enzyme reaction activator is replaced with glycyl-L-proline. The remaining components and usage steps remain unchanged.

[0064] Example 6

[0065] This example provides an arginine peptidase quantitative detection kit, whose composition is basically the same as that of Example 1. The difference is that in Solution A described in this example, the disulfide bond cleaving agent is replaced with tris(2-carboxyethyl)phosphonic acid hydrochloride, and the enzyme reaction activator is replaced with glycyl-L-alanine. The remaining components and usage steps remain unchanged.

[0066] Example 7

[0067] This example provides an arginine peptidase quantitative detection kit, whose composition is basically the same as that of Example 1. The difference is that in Solution A described in this example, the disulfide bond cleaving agent is replaced by tris(2-carboxyethyl)phosphine hydrochloride and tris(2-carboxyethyl)phosphine hydrochloride and β-mercaptoethanol (the mass ratio of the two is 1:1), and the enzyme reaction activator is replaced by glycylglycine and glycamide (the mass ratio of the two is 1:1). The remaining components and usage steps remain unchanged.

[0068] Comparative Example 1

[0069] This example provides an arginine peptidase quantitative detection kit, whose composition is basically the same as that of Example 1. The difference is that solution A in this example does not contain glycylglycine (enzyme activator), while the other components and usage steps remain unchanged.

[0070] Comparative Example 2

[0071] This example provides an arginine peptidase quantitative detection kit, whose composition is basically the same as that of Example 1. The difference is that the enzyme activator in Solution A in this example is replaced with L-cysteine ​​instead of glycylglycine. The other components and usage steps remain unchanged.

[0072] Example 8

[0073] A qualitative detection kit for arginine peptidase includes solution B, solution C, and a detection strip; solution B comprises 15.0 g / L Tris hydrochloride buffer, 7.5 g / L tris(2-carboxyethyl)phosphine hydrochloride (disulfide bond cleavage agent), and 15.0 g / L glycylglycine (enzyme activator); solution C comprises 0.1% by mass of 4-(dimethylamino)cinnamaldehyde and 1.0 mol / L hydrochloric acid;

[0074] The preparation method of the test strip is as follows: (1) Nα-benzoyl-DL-arginine-β-naphthamide hydrochloride is pre-dissolved in dimethyl sulfoxide at a ratio of 44 mg / 1 ml, and mixed with Tris hydrochloride buffer. The volume ratio of the dimethyl sulfoxide solution of Nα-benzoyl-DL-arginine-4-ylbenzamide hydrochloride to the Tris hydrochloride buffer is 1:50, and the pH is adjusted to 7.5; (2) Whatman filter paper is soaked in the above solution for 2 minutes, taken out, dried at 37°C for 4 hours, and cut into round pieces with a diameter of 8-10 mm.

[0075] A method for using an arginine peptidase qualitative detection kit includes the following steps:

[0076] S1: Scrape the tongue coating 5 times with a sampling swab;

[0077] S2: Rotate the sampling swab from step S1 in solution B for 5 rotations;

[0078] S3: Press the sampling swab processed in step S2 onto the Whatman filter paper of the test strip for 5 seconds and react for 5 minutes at room temperature;

[0079] S4: Add 1 drop of solution C to the Whatman filter paper from step S3 and react for 3 minutes;

[0080] S5: Determine whether periodontitis exists based on the color obtained in step S4. If pink appears, it is determined to be periodontitis.

[0081] Example 9

[0082] This example provides a qualitative detection kit for arginine peptidase, which has the same composition as in Example 8. The difference is that the enzyme activator in solution B in this example is replaced with glycine instead of glycamide. The other components and usage steps remain unchanged.

[0083] Example 10

[0084] This example provides a qualitative detection kit for arginine peptidase, which has the same composition as in Example 8. The difference is that the enzyme activator in Solution B in this example is replaced with glycyl-glycine, while the other components and usage steps remain unchanged.

[0085] Example 11

[0086] This example provides a qualitative detection kit for arginine peptidase, which has the same composition as in Example 8. The difference is that the enzyme activator in Solution B in this example is replaced with glycylglycine instead of glycylphenylalanine. The other components and usage steps remain unchanged.

[0087] Example 12

[0088] This example provides a qualitative detection kit for arginine peptidase, which has the same composition as in Example 8. The difference is that in Solution B, the disulfide bond cleaving agent is replaced with tris(2-carboxyethyl)phosphonic acid hydrochloride, and the enzyme reaction activator is replaced with glycyl-L-proline. The remaining components and usage steps remain unchanged.

[0089] Example 13

[0090] This example provides a qualitative detection kit for arginine peptidase, which has the same composition as in Example 8. The difference is that in Solution B, the disulfide bond cleaving agent is replaced with tris(2-carboxyethyl)phosphonic acid hydrochloride, and the enzyme activator is replaced with glycyl-L-alanine. The remaining components and usage steps remain unchanged.

[0091] Example 14

[0092] This example provides a qualitative detection kit for arginine peptidase, which has the same composition as in Example 8. The difference is that in Solution B, the disulfide bond cleaving agent is replaced with tris(2-carboxyethyl)phosphine hydrochloride and tris(2-carboxyethyl)phosphine hydrochloride and β-mercaptoethanol (mass ratio of 1:1), and the enzyme activator is replaced with glycylglycine and glycamide (mass ratio of 1:1). The remaining components and usage steps remain unchanged.

[0093] Comparative Example 3

[0094] This example provides a qualitative detection kit for arginine peptidase, whose composition is basically the same as that in Example 8. The difference is that solution B in this example does not contain glycylglycine (an enzyme activator), while the other components and usage steps remain unchanged.

[0095] Comparative Example 4

[0096] This example provides a qualitative detection kit for arginine peptidase, which has the same composition as in Example 8. The difference is that the enzyme activator in solution B in this example is replaced with L-cysteine ​​instead of glycylglycine. The other components and usage steps remain unchanged.

[0097] Experimental Example 1: Lowest Detection Limit Test of Arginine Peptidase Quantitative Assay Kit

[0098] The arginine peptidase quantitative detection kits prepared in Examples 1-7 and Comparative Examples 1-2 were used to determine the limit of detection (LOD) of the kits by testing arginine peptidase samples with different enzyme activity gradients. The enzyme activity gradients for the arginine peptidase samples were set to 5000 U / mL, 500 U / mL, 100 U / mL, 20 U / mL, 10 U / mL, 1 U / mL, 0.5 U / mL, 0.3 U / mL, and 0.1 U / mL (one unit is defined as the amount of enzyme required to produce 0.001 ΔA253 per milliliter per minute using Nα-benzoyl-L-arginine ethyl ester as a substrate at pH 7.5 and 25°C). The test method was as follows:

[0099] S1: Add the arginine peptidase sample to solution A (100 μL enzyme solution + 900 μL A solution) to prepare the sample solution, and immediately read the absorbance, which is recorded as A1;

[0100] S2: React the sample solution from step S1 at room temperature for 10 minutes, and read the absorbance of the sample solution, which is recorded as A2;

[0101] S3: Calculate the sample reactivity = A2 - A1, substitute the sample reactivity into the formula to calculate the arginine peptidase activity in the sample; the formula is arginine peptidase activity = (standard enzyme activity × sample reactivity) / standard reactivity;

[0102] The lowest detectable enzyme activities in Examples 1-7 and Comparative Examples 1-2 were statistically analyzed, and the results are shown in Table 1.

[0103] Table 1. Limit of Detection for Arginine Peptidase Quantitative Assay Kit

[0104]

[0105] As shown in Table 1, the arginine peptidase detection kits provided in Examples 1-7 of this invention can achieve a minimum sensitivity of 1 U / mL, while the detection sensitivity of Comparative Example 1, without the addition of an enzyme activator, is significantly reduced. The detection sensitivity of Comparative Example 2 is also affected to some extent after the enzyme activator composition is changed.

[0106] Experimental Example 2: Lowest Detection Limit Test of Bacterial Concentration Using Arginine Qualitative Detection Kit

[0107] 1. Preparation of experimental materials:

[0108] After resuscitation, *Porphyromonas gingivalis* strain ATCC33277 was transferred to Bourg's agar medium, placed in an anaerobic bag, and incubated at 37°C to obtain 1.0 × 10⁻⁶ cells / year. 7 Bacterial culture medium of CFU / mL was serially diluted 10-fold with sterile physiological saline to prepare 1.0 × 10⁻⁶ CFU / mL solution. 6 cfu / mL, 1.0×105 cfu / mL, 1.0×10 4 cfu / mL, 1.0×10 3 cfu / mL, 1.0×10 2 Bacterial suspensions at cfu / mL and 10 cfu / mL.

[0109] 2. Testing Methods

[0110] The arginine peptidase qualitative detection kits prepared in Examples 8-14 and Comparative Examples 3-4 were used, and the test methods are as follows:

[0111] (1) Mix 40 μL of bacterial suspension with 40 μL of LB solution and drop it onto the Whatman filter paper of the test strip. Let it react for 5 minutes at room temperature. (2) Add 1 drop of C solution onto the Whatman filter paper and let it react for 3 minutes. If a pink color appears, it means that it can be detected.

[0112] The lowest detectable bacterial concentrations in Examples 8-4 and Comparative Examples 3-4 were statistically analyzed, and the results are shown in Table 2.

[0113] Table 2. Limit of Detection for Bacterial Concentration in Arginine Peptidase Qualitative Detection Kit

[0114]

[0115] As shown in Table 2, the arginine peptidase qualitative detection kit provided in Examples 8-14 of this invention can detect a minimum bacterial concentration of 1.0 × 10⁻⁶. 2 The detection sensitivity of Comparative Example 3, which did not contain an enzyme activator, was significantly reduced. The detection sensitivity of Comparative Example 4 was also affected to some extent after the enzyme activator composition was changed.

[0116] Example 3: Qualitative Detection Kit - Minimum arginine peptidase activity detection limit test

[0117] Arginine peptidase qualitative detection kits prepared in Examples 8-14 and Comparative Examples 3-4 were used to test arginine peptidase samples at different gradient dilutions. The enzyme activity gradients for the arginine peptidase samples were set to 5000 U / mL, 500 U / mL, 100 U / mL, 20 U / mL, 10 U / mL, 1 U / mL, 0.5 U / mL, 0.3 U / mL, and 0.1 U / mL (one unit is defined as the amount of enzyme required to produce 0.001 ΔA253 per milliliter per minute using Nα-benzoyl-L-arginine ethyl ester as a substrate at pH 7.5 and 25°C). The test method was as follows:

[0118] (1) Add 40 μL of arginine peptidase sample to 40 μL of solution B and mix. Then add the solution to the Whatman filter paper on the test strip and react for 5 minutes at room temperature. (2) Add 1 drop of solution C to the Whatman filter paper and react for 3 minutes. If a pink color appears, it means that it can be detected.

[0119] The lowest detectable enzyme activities in Examples 8-14 and Comparative Examples 3-4 are shown in Table 3.

[0120] Table 3. Limit of Detection for Arginine Peptidase Qualitative Assay Kit

[0121]

[0122] As shown in Table 3, the arginine peptidase qualitative detection kit provided in Examples 8-14 of this invention can achieve a minimum sensitivity of 1 U / mL, while the detection sensitivity of Comparative Example 3, without the addition of an enzyme activator, is significantly reduced. The detection sensitivity of Comparative Example 4 is also affected to some extent after the enzyme activator composition is changed.

[0123] Experiment 4: Kit Detection Accuracy Test

[0124] One patient diagnosed with mild periodontitis was selected and tested using the arginine peptidase quantitative detection kits prepared in Examples 1-7 and Comparative Examples 1-2, as well as the arginine peptidase qualitative detection kits prepared in Examples 8-14 and Comparative Examples 3-4.

[0125] The method of using the arginine peptidase quantitative detection kits prepared in Examples 1-7 and Comparative Examples 1-2 is as follows:

[0126] S1: Scrape the tongue coating with a sampling swab 5 to 10 times, rotate it 5 to 10 times in solution A to prepare a sample solution, and immediately read the absorbance, which is recorded as A1;

[0127] S2: React the sample solution from step S1 at room temperature for 5 minutes, and read the absorbance of the sample solution, which is recorded as A2;

[0128] S3: Calculate the sample reactivity = A2 - A1, substitute the sample reactivity into the formula to calculate the arginine peptidase activity in the sample; the formula is arginine peptidase activity = (standard enzyme activity × sample reactivity) / standard reactivity;

[0129] S4: Determine whether periodontitis exists based on the arginine peptidase activity obtained in step S3. If the arginine peptidase activity is higher than 3 U / mL, it is determined to be periodontitis.

[0130] The method of using the arginine peptidase qualitative detection kits prepared in Examples 8-14 and Comparative Examples 3-4 is as follows:

[0131] S1: Scrape the tongue coating with a sampling swab 5 to 10 times;

[0132] S2: Rotate the sampling swab from step S1 in solution B for 5 to 10 turns;

[0133] S3: Press the sampling swab processed in step S2 onto the Whatman filter paper of the test strip for 5 seconds and react for 5 minutes at room temperature;

[0134] S4: Add 1 drop of solution C to the Whatman filter paper from step S3 and react for 3 minutes;

[0135] S5: Determine whether periodontitis exists based on the color obtained in step S4. If pink appears, it is determined to be periodontitis.

[0136] The assay kits used in Examples 1-7 and Comparative Examples 1-2 detected arginine peptidase activity, and the results are shown in Table 4.

[0137] Table 4. Results of arginine peptidase activity detection in patients with mild periodontitis.

[0138]

[0139] The specific detection kits used in Examples 8-14 and Comparative Examples 3-4 detected arginine peptidase activity, and the results are shown in Table 5.

[0140] Table 5. Results of arginine peptidase activity detection in patients with mild periodontitis.

[0141]

[0142]

[0143] As can be seen from the results in Tables 4 and 5, the arginine peptidase quantitative detection kits prepared in Examples 1-7 and the arginine peptidase qualitative detection kits prepared in Examples 8-14 of this invention provide accurate and reliable detection results.

[0144] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An arginine peptidase quantitative test kit, characterized by, consists of A liquid; the A liquid consists of MES buffer, a disulfide bond cleavage agent, an enzymatic reaction activator, Nα benzoyl DL arginine 4 benzoyl amide hydrochloride and dimethyl sulfoxide; the disulfide bond cleavage agent is tris(2 carboxyethyl)phosphine hydrochloride, beta mercaptoethanol, dithiothreitol; the enzymatic reaction activator is glycylglycine; The concentration of MES buffer in the A liquid is 15.0 g / L, the concentration of disulfide bond cleavage agent is 1.5 g / L, and the concentration of enzymatic reaction activator is 28.0 g / L; the concentration of Nα benzoyl DL arginine 4 benzamide hydrochloride is pre-dissolved in dimethyl sulfoxide at a ratio of 44 mg / 1 ml; the concentration of Nα benzoyl DL arginine 4 dimethyl sulfoxide solution of benzamide hydrochloride and MES buffer is 2.5:50; the pH of the A liquid is adjusted to 5.

5.

2. An arginine peptidase qualitative test kit, characterized by, consists of 15.0 g / L Tris hydrochloride buffer, 7.5 g / L disulfide bond cleavage agent, 15.0 g / L enzymatic reaction activator; the disulfide bond cleavage agent is tris(2 carboxyethyl)phosphine hydrochloride, beta mercaptoethanol, dithiothreitol; the enzymatic reaction activator is glycylglycine; the C liquid is 0.05% 1% of 4 (dimethylamino)cinnamaldehyde, 0.5 2 mol / L hydrochloric acid; the raw material used in the production of the detection sheet is whatman filter paper, 1.2 15.0 g / L Tris hydrochloride buffer, N benzoyl DL arginine beta naphthalenamide hydrochloride, dimethyl sulfoxide.

3. The arginine peptidase qualitative test kit according to claim 2, characterized in that, The detection sheet preparation method is: (1) Nα benzoyl DL arginine β Naphthylamide hydrochloride is pre-dissolved in dimethyl sulfoxide at a ratio of 44 mg / 1 ml, Nα benzoyl DL arginine β The dimethyl sulfoxide solution of naphthylamide hydrochloride is mixed with Tris hydrochloride buffer at a volume ratio of 0.5 5:50, adjust pH = 7.0 8.5; (2) Soak whatman filter paper in the above solution, take out and dry.

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