Kit for detecting treponema pallidum antibody in urine and preparation method thereof

Abstract

The invention relates to the technical field of medicine detection, in particular to a kit for detecting treponema pallidum antibodies in urine and a preparation method of the kit. The invention relates to a kit for detecting treponema pallidum antibodies in urine. The kit consists of one or more monoclonal antibodies aiming at treponema pallidum specific antigens (TpN15, Tp47 and Tp92), one or more monoclonal antibodies marked by fluorescent substances, a solid-phase carrier, a sample treatment solution, a fluorescence detection solution, a washing solution, a stop solution, a positive reference substance and a negative reference substance. By using the monoclonal antibody combination aiming at TpN15, Tp47 and Tp92 antigens, specific recognition aiming at different antigens and reduction of non-specific binding, the sensitivity and specificity of detection can be remarkably improved, so that the accuracy of the kit is improved, and a wider infection stage can be covered by utilizing different expressions of different antigens in different stages of syphilis infection.

Description

Technical Field

[0001] The invention relates to the technical field of medical detection, and in particular to a kit for detecting Treponema pallidum antibodies in urine and a preparation method thereof. Background Art

[0002] Syphilis is a chronic infectious disease caused by Treponema pallidum, with diverse clinical manifestations and complex diagnosis. At present, the commonly used syphilis detection methods in clinical practice mainly include serological testing, dark field microscopy, molecular biology testing, etc. Among them, serological testing is one of the most common screening methods, which is diagnosed by detecting Treponema pallidum antibodies in the patient's blood or urine. However, traditional serological testing methods have some limitations, especially in early diagnosis and coverage of different infection stages.

[0003] Traditional test kits usually only detect one or a few antigens. Since Treponema pallidum will produce antibodies against different antigens at different stages of infection, single antigen detection is likely to miss samples at certain infection stages, resulting in a high false negative rate. In addition, single antibody detection is easily affected by nonspecific binding, resulting in false positive results. Single antigen detection has low sensitivity, especially in the early or late stages of infection when antibody levels are low. Single antigen detection cannot accurately identify the infection status. Summary of the invention

[0004] In view of the shortcomings of the prior art, the object of the present invention is to provide a kit for detecting Treponema pallidum antibodies in urine and a preparation method thereof.

[0005] A kit for detecting Treponema pallidum antibodies in urine, comprising:

[0006] One or more monoclonal antibodies against Treponema pallidum specific antigens (TpN15, Tp47, Tp92), one or more monoclonal antibodies labeled with fluorescent substances, solid phase carriers, sample processing solutions, fluorescent detection solutions, washing solutions, stop solutions, positive controls, and negative controls;

[0007] The solid phase carrier comprises a nitrocellulose membrane (NC membrane) or a plastic microwell plate;

[0008] The sample processing solution comprises buffer A, protease inhibitor, preservative and blocking agent A;

[0009] The fluorescent detection solution comprises a buffer solution B, an enhancer, a stabilizer and a blocking agent A;

[0010] The washing solution comprises buffer B and a detergent;

[0011] The positive control contains a urine sample with known Treponema pallidum antibodies;

[0012] The negative control is a urine sample that does not contain Treponema pallidum antibodies.

[0013] Preferably, the monoclonal antibody is prepared by hybridoma technology and purified by affinity chromatography.

[0014] Preferably, the specificity of the monoclonal antibody is verified by Western blot and ELISA techniques.

[0015] Preferably, the fluorescent substance includes fluorescein isothiocyanate (FITC).

[0016] Preferably, the monoclonal antibody on the solid phase carrier is fixed by spraying or other methods, and it is ensured that the fixed antibody has good stability and activity.

[0017] Preferably, the buffer A of the sample treatment solution is Tris-HCl, the protease inhibitor is PMSF, the preservative is sodium azide, and the blocking agent A is BSA.

[0018] Preferably, the buffer B of the fluorescent detection solution is PBS pH 7.4, the enhancer is Tween-20, the stabilizer is glycerol, and the blocking agent A is BSA.

[0019] Preferably, the buffer B of the washing solution is PBS pH 7.4, and the detergent is Tween-20.

[0020] Preferably, the stop solution is 0.5 mol / L sulfuric acid.

[0021] A method for preparing a kit for detecting Treponema pallidum antibodies in urine comprises the following steps:

[0022] (1) Preparation of monoclonal antibodies: Balb / c mice were selected and injected with immunogens containing single or combined antigens of TpN15, Tp47, and Tp92 for immunization;

[0023] Extract spleen cells from immunized mice and fuse them with myeloma cells to form hybridoma cells;

[0024] Hybridoma cells that can secrete high-yield and high-specificity antibodies were screened by ELISA;

[0025] Purify monoclonal antibodies using affinity chromatography;

[0026] (2) Fluorescently labeled monoclonal antibodies:

[0027] Fluorescein isothiocyanate (FITC) was used;

[0028] The purified monoclonal antibody is coupled with a fluorescent substance (such as FITC);

[0029] The labeling efficiency was determined by UV absorption;

[0030] (3) Prepare solid phase carrier and fix detection antibody:

[0031] Choose nitrocellulose membrane (NC membrane) or plastic microplate as the solid phase carrier;

[0032] Immobilize the detection antibody on a solid phase carrier using a spray method or other suitable method;

[0033] (4) preparing sample treatment solution and fluorescence detection solution;

[0034] (5) Prepare washing solution and stop solution;

[0035] (6) Preparation of positive and negative controls:

[0036] (7) Assemble the kit: Add the prepared monoclonal antibody (1 mg), fluorescently labeled monoclonal antibody (1 mg), solid phase carrier (NC membrane or plastic microplate), sample treatment solution (100 mL), washing solution (200 mL), fluorescent detection solution (100 mL), stop solution (50 mL), positive control (1 mL) and negative control (1 mL) into the kit in proportion and order, and seal for storage.

[0037] TpN15 is located in the outer membrane of Treponema pallidum and is an important antigen of Treponema pallidum; Tp47 is related to the pathogenicity of Treponema pallidum and is another important antigen; Tp92 is closely related to the infection process of Treponema pallidum.

[0038] The test kit utilizes monoclonal antibodies against Treponema pallidum-specific antigens (TpN15, Tp47, Tp92). Since different antigens are expressed differently at different stages of infection, through multiple antigen recognition, multi-target detection can cover a wider range of infection stages, improve detection coverage, and reduce false negative results.

[0039] Using fluorescein isothiocyanate (FITC)-labeled antibodies to quantitatively detect the presence of Treponema pallidum antibodies through the intensity of the fluorescent signal can accurately distinguish between positive and negative samples and reduce background interference.

[0040] Fluorescent detection fluid is used to enhance fluorescent signals, stabilize labeled antibodies, and reduce background signals, thereby improving detection sensitivity and specificity and enhancing signal stability.

[0041] The beneficial effects achieved by the present invention are as follows: 1. The present invention uses a combination of monoclonal antibodies against TpN15, Tp47, and Tp92 antigens to specifically recognize different antigens and reduce nonspecific binding, which can significantly improve the sensitivity and specificity of the detection, thereby improving the accuracy of the kit and facilitating the detection of syphilis infection at different stages.

[0042] 2. The present invention utilizes the different expressions of different antigens at different stages of syphilis infection. The use of antibodies corresponding to multiple antigens can cover a wider range of infection stages and improve the comprehensiveness of detection.

[0043] 3. The present invention can overcome the problem that a single antigen test may miss certain infected samples, while multi-antigen testing can reduce false negative results and improve the reliability of testing.

[0044] 4. The present invention ensures that the concentration and quality of each antibody are consistent, and maintains the consistency and stability of the combination through purification and labeling methods, thereby improving the repeatability and reliability of the kit.

[0045] 5. The present invention can simplify the actual detection process and make the operation of the kit easier and faster by rationally designing the sample treatment solution, fluorescent detection solution, washing solution and stop solution, and selecting the solid phase carrier and fixation method. DETAILED DESCRIPTION

[0046] The technical solution of the present invention is further described below in conjunction with specific implementation methods.

[0047] Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0048] In the present invention, unless otherwise specified, all equipment and raw materials can be purchased from the market or are commonly used in the industry. The methods in the following embodiments, unless otherwise specified, are conventional methods in the art.

[0049] Table 1 Composition of the kit for detecting Treponema pallidum antibodies in urine

[0050]

[0051]

[0052] A kit for detecting Treponema pallidum antibodies in urine, as shown in Table 1, is composed of the following:

[0053] One or more monoclonal antibodies against Treponema pallidum specific antigens (TpN15, Tp47, Tp92), one or more monoclonal antibodies labeled with fluorescent substances, solid phase carriers, sample processing solutions, fluorescent detection solutions, washing solutions, stop solutions, positive controls, and negative controls;

[0054] Solid phase carrier: nitrocellulose membrane (NC membrane) or plastic microplate;

[0055] Sample treatment solution: 10 mM Tris-HCl (buffer A) pH 7.4, 1 mM PMSF (protease inhibitor), 0.05% sodium azide (preservative), 1% BSA (bovine serum albumin), total volume 100 mL;

[0056] Fluorescence detection solution: 1x PBS (buffer B) pH 7.4, 0.05% Tween-20 (enhancer), 1% glycerol (stabilizer), 1% BSA, total volume 100 mL;

[0057] Washing solution: 1x PBS (buffer B) pH 7.4, 0.05% Tween-20 (detergent), total volume 200 mL;

[0058] Stop solution: 0.5 mol / L sulfuric acid, total volume 50 mL.

[0059] Positive control: contains a urine sample that has been confirmed by standard testing methods to contain antibodies to known Treponema pallidum;

[0060] Negative control: a urine sample that does not contain Treponema pallidum antibodies confirmed by standard testing methods;

[0061] Fluorescent substance: fluorescein isothiocyanate (FITC).

[0062] Example 1: A method for preparing a kit for detecting Treponema pallidum antibodies in urine, comprising the steps of:

[0063] (1) Preparation of monoclonal antibodies: Healthy Balb / c mice were selected and injected with immunogens containing TpN15 antigen for immunization;

[0064] The spleen cells of the immunized mice were extracted and fused with myeloma cells (SP2 / 0 cell line) to form hybridoma cells;

[0065] Hybridoma cells that can secrete high-yield and high-specificity antibodies were screened by ELISA;

[0066] Monoclonal antibodies were purified using affinity chromatography (Protein A / G affinity chromatography column);

[0067] (2) Fluorescently labeled monoclonal antibodies: Purified monoclonal antibodies are coupled with fluorescent substances (FITC);

[0068] (3) Preparing a solid phase carrier and fixing the detection antibody: Selecting a nitrocellulose membrane (NC membrane) as a solid phase carrier, and using a spray method to fix the detection antibody on the solid phase carrier;

[0069] (4) preparing sample treatment solution and fluorescence detection solution;

[0070] (5) Prepare washing solution and stop solution;

[0071] (6) preparing positive control and negative control;

[0072] (7) Assemble the kit: Add the prepared monoclonal antibody (1 mg), fluorescently labeled monoclonal antibody (1 mg), solid phase carrier (NC membrane), sample treatment solution (100 mL), washing solution (200 mL), fluorescent detection solution (100 mL), stop solution (50 mL), positive control (1 mL) and negative control (1 mL) into the kit in proportion and order, and store in a sealed container to prevent contamination.

[0073] Example 2: A method for preparing a kit for detecting Treponema pallidum antibodies in urine, comprising the steps of:

[0074] (1) Preparation of monoclonal antibodies: Healthy Balb / c mice were selected and injected with an immunogen containing equal amounts of TpN15 and Tp47 antigens for immunization;

[0075] The spleen cells of the immunized mice were extracted and fused with myeloma cells (SP2 / 0 cell line) to form hybridoma cells;

[0076] Hybridoma cells that can secrete high-yield and high-specificity antibodies were screened by ELISA;

[0077] Monoclonal antibodies were purified using affinity chromatography (Protein A / G affinity chromatography column);

[0078] (2) Fluorescently labeled monoclonal antibodies: Purified monoclonal antibodies are coupled with fluorescent substances (FITC);

[0079] (3) Preparing a solid phase carrier and fixing the detection antibody: Select a plastic microplate as a solid phase carrier and fix the detection antibody on the solid phase carrier by spraying;

[0080] (4) preparing sample treatment solution and fluorescence detection solution;

[0081] (5) Prepare washing solution and stop solution;

[0082] (6) preparing positive control and negative control;

[0083] (7) Assemble the kit: Add the prepared monoclonal antibody (1 mg), fluorescently labeled monoclonal antibody (1 mg), solid phase carrier (plastic microplate), sample treatment solution (100 mL), washing solution (200 mL), fluorescent detection solution (100 mL), stop solution (50 mL), positive control (1 mL) and negative control (1 mL) into the kit in proportion and order, and store in a sealed container to prevent contamination.

[0084] Example 3: A method for preparing a kit for detecting Treponema pallidum antibodies in urine, comprising the steps of:

[0085] (1) Preparation of monoclonal antibodies: Healthy Balb / c mice were selected and injected with an immunogen containing equal amounts of TpN15, Tp47, and Tp92 antigens for immunization;

[0086] The spleen cells of the immunized mice were extracted and fused with myeloma cells (SP2 / 0 cell line) to form hybridoma cells;

[0087] Hybridoma cells that can secrete high-yield and high-specificity antibodies were screened by ELISA;

[0088] Monoclonal antibodies were purified using affinity chromatography (Protein A / G affinity chromatography column);

[0089] (2) Fluorescently labeled monoclonal antibodies: Purified monoclonal antibodies are coupled with fluorescent substances (FITC);

[0090] (3) Preparing a solid phase carrier and fixing the detection antibody: Selecting a nitrocellulose membrane (NC membrane) as a solid phase carrier, and using a spray method to fix the detection antibody on the solid phase carrier;

[0091] (4) preparing sample treatment solution and fluorescence detection solution;

[0092] (5) Prepare washing solution and stop solution;

[0093] (6) preparing positive control and negative control;

[0094] (7) Assemble the kit: Add the prepared monoclonal antibody (1 mg), fluorescently labeled monoclonal antibody (1 mg), solid phase carrier (NC membrane), sample treatment solution (100 mL), washing solution (200 mL), fluorescent detection solution (100 mL), stop solution (50 mL), positive control (1 mL) and negative control (1 mL) into the kit in proportion and order, and store in a sealed container to prevent contamination.

[0095] Comparative Example: Conventional kit, using Tp15 single antigen;

[0096] Operation steps: Add the urine sample to be tested into the sample treatment solution to remove interfering substances; add sample, wash, develop color, and terminate; display the results through the color developing solution, and record the test results through specific instruments or naked eye observation.

[0097] Test Example: Using the kits of Examples 1-3 and the comparative example to detect Treponema pallidum antibodies in urine;

[0098] Test samples: known positive urine samples and known negative urine samples;

[0099] 1. Example 1-Example 3 Kit Detection Steps:

[0100] Mix the urine sample with a sample treatment solution to remove interfering substances in the sample;

[0101] The treated sample was added to the solid phase carrier and incubated for 30 minutes to ensure that the antibody in the sample was bound to the antibody on the solid phase carrier;

[0102] Wash the solid support with a washing solution to remove unbound material;

[0103] Add fluorescent detection solution, incubate for 15 min, and observe the color change;

[0104] Adding a stop solution to stop the color development reaction, the test result is displayed by a fluorescent signal, that is, the intensity of the fluorescent signal is read by a fluorescence detector to determine whether there is a Treponema pallidum antibody in the sample;

[0105] 2. Comparative Example Conventional kit detection steps:

[0106] Add the urine sample to be tested into the sample treatment solution and follow the instructions to remove interfering substances;

[0107] Add the treated sample to the solid phase carrier and incubate according to the instructions;

[0108] Wash the solid support with a washing solution to remove unbound material;

[0109] Add color developing solution and observe the color change; add stop solution to stop the color development reaction; use an enzyme-labeled instrument to record the test results.

[0110] The results of Example 1 show that the fluorescence signal intensity of the positive sample is significantly higher than that of the negative control sample.

[0111] Positive sample: A known positive urine sample with a fluorescence signal intensity of 180AU.

[0112] Negative control: a urine sample with a known negative fluorescence signal intensity of 10AU.

[0113] The results of Example 2 show that the fluorescence signal intensity of the positive sample is significantly higher than that of the negative control sample.

[0114] Positive sample: A known positive urine sample with a fluorescence signal intensity of 220AU.

[0115] Negative control: A urine sample with a known negative fluorescence signal intensity of 12AU.

[0116] The results of Example 3 show that the fluorescence signal intensity of the positive sample is significantly higher than that of the negative control sample.

[0117] Positive sample: A known positive urine sample with a fluorescence signal intensity of 235AU.

[0118] Negative control: A urine sample with a known negative fluorescence signal intensity of 15AU.

[0119] The comparative results showed that the absorbance value of the positive sample was significantly higher than that of the negative control sample.

[0120] Positive sample: A known positive urine sample with an absorbance value of 0.55 OD.

[0121] Negative control: a urine sample with a known negative absorbance of 0.15 OD.

[0122] The positive threshold was 100 AU or 0.4 OD.

[0123] Compared with the conventional test kit of the comparative example, the test kit of the present invention can cover different stages of Treponema pallidum infection and improve the sensitivity and specificity of detection by using a combination of multiple monoclonal antibodies. In particular, Example 3 (multiple antigens) and Example 2 (mixed antigens) show better performance in detecting Treponema pallidum antibodies in urine and are more suitable for clinical diagnosis and screening. Although Example 1 (single antigen) performs better in specificity, its sensitivity is slightly lower and is not comprehensive enough in some cases.

[0124] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A kit for detecting Treponema pallidum antibodies in urine, characterized in that: It is composed of: One or more monoclonal antibodies against TpN15, Tp47, and Tp92 Treponema pallidum specific antigens, one or more monoclonal antibodies labeled with fluorescent substances, a solid phase carrier, a sample treatment solution, a fluorescent detection solution, a washing solution, a stop solution, a positive control substance, and a negative control substance; The solid phase carrier comprises a nitrocellulose membrane or a plastic microwell plate; the sample treatment solution comprises buffer A, a protease inhibitor, a preservative and a blocking agent A; the fluorescent detection solution comprises buffer B, an enhancer, a stabilizer and a blocking agent A; the washing solution comprises buffer B and a detergent; the positive control contains a urine sample with known Treponema pallidum antibodies; and the negative control does not contain a urine sample with Treponema pallidum antibodies.

2. A kit for detecting Treponema pallidum antibodies in urine according to claim 1, characterized in that: The monoclonal antibodies were prepared by hybridoma technology and purified by affinity chromatography.

3. A kit for detecting Treponema pallidum antibodies in urine according to claim 1, characterized in that: The specificity of the monoclonal antibody was verified by Western blot and ELISA techniques.

4. A kit for detecting Treponema pallidum antibodies in urine according to claim 1, characterized in that: The fluorescent substance includes fluorescein isothiocyanate.

5. A kit for detecting Treponema pallidum antibodies in urine according to claim 1, characterized in that: The monoclonal antibody on the solid phase carrier is fixed by spraying or other methods.

6. A kit for detecting Treponema pallidum antibodies in urine according to claim 1, characterized in that: The buffer A of the sample treatment solution is Tris-HCl, the protease inhibitor is PMSF, the preservative is sodium azide, and the blocking agent A is BSA.

7. A kit for detecting Treponema pallidum antibodies in urine according to claim 1, characterized in that: The buffer B of the fluorescent detection solution is PBS pH 7.4, the enhancer is Tween-20, the stabilizer is glycerol, and the blocking agent A is BSA.

8. A kit for detecting Treponema pallidum antibodies in urine according to claim 1, characterized in that: The buffer B of the washing solution is PBS pH 7.4, and the detergent is Tween-20.

9. A kit for detecting Treponema pallidum antibodies in urine according to claim 1, characterized in that: The stop solution is 0.5 mol / L sulfuric acid.

10. A method for preparing a kit for detecting Treponema pallidum antibodies in urine, characterized in that: The method comprises the following steps: (1) preparing monoclonal antibodies: selecting Balb / c mice, injecting immunogens containing single or combined antigens of TpN15, Tp47, and Tp92 for immunization; Extract spleen cells from immunized mice and fuse them with myeloma cells to form hybridoma cells; Hybridoma cells that can secrete high-yield and high-specificity antibodies were screened by ELISA; Purify monoclonal antibodies using affinity chromatography; (2) Fluorescently labeled monoclonal antibodies: Purified monoclonal antibodies were conjugated with fluorescein using fluorescein isothiocyanate, and the labeling efficiency was determined by UV absorption. (3) Preparing a solid phase carrier and fixing the detection antibody: Selecting a nitrocellulose membrane or a plastic microplate as a solid phase carrier; using a spray method or other suitable method to fix the detection antibody on the solid phase carrier; (4) preparing sample treatment solution and fluorescence detection solution; (5) Prepare washing solution and stop solution; (6) Preparation of positive and negative controls: (7) Assembling the kit: Add the prepared monoclonal antibody, labeled antibody, solid phase carrier, sample treatment solution, washing solution, fluorescent detection solution, stop solution, positive control and negative control into the kit in proportion and order, and seal for storage.

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