Colloidal gold kit for detecting p16 protein as well as preparation method and application of colloidal gold kit

Abstract

The invention discloses a colloidal gold kit for detecting p16 protein as well as a preparation method and application of the colloidal gold kit, and belongs to the technical field of biological detection. The colloidal gold kit comprises a colloidal gold test strip and a sample diluent, a detection antibody and a capture antibody of the colloidal gold test strip are anti-p16 monoclonal antibodies with the clone numbers of 1F4F12 and 3G3G11 respectively, and the sample diluent comprises PBST, BSA and other reagents. The kit is used for detecting p16 protein in body fluid samples such as whole blood, serum, hydrothorax and ascites, cervical curettage, vaginal secretion and bronchus brushing, so that screening of p16 high-expression tumors is realized. The kit has the advantages of low preparation cost, simplicity in operation and convenience in use, has relatively high sensitivity and specificity, and can be used as a bedside detection kit, and a patient can autonomously complete screening of malignant tumors with high expression of the p16 protein.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and particularly relates to a colloidal gold kit for detecting p16 protein, a preparation method thereof and an application thereof. Background Art

[0002] The p16 protein, a 16-kDa protein encoded by the p16 gene (MTS1), is a crucial cell cycle regulator that plays an essential role in normal cell cycle progression. Abnormally high expression of p16 protein is closely associated with the development of HPV-related malignancies such as cervical cancer and head and neck cancer, as well as melanoma, small cell lung carcinoma, and various non-HPV-related malignancies. It serves as a broad-spectrum molecular marker for these malignancies. Therefore, detecting p16 protein levels in samples has important clinical value for the screening and early diagnosis of these malignancies.

[0003] Currently, methods for detecting p16 protein primarily include immunohistochemical staining, immunocytochemical staining, flow cytometry, enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay (CLIA), Western blot, and the colloidal gold method. While these methods are generally based on immunological principles for detecting p16 protein, each has its own unique characteristics. Immunohistochemical staining, immunocytochemical staining, and flow cytometry can be used to detect in situ p16 protein expression in tissue sections, cell specimens, or specific cells within single cell suspensions, respectively. ELISA, chemiluminescence immunoassay, and immunoblotting can be used to detect p16 protein in body fluid samples, secretions, tissues, and cell homogenates. However, the operation and interpretation of the results of the above-mentioned detection methods require high technical capabilities of both the instruments and the operators. Not only do they require professional technicians to use specialized reagents and dedicated instruments to perform standardized experimental operations, but they also need professional knowledge and experience to interpret the results. In addition, the sample preparation process is relatively complicated and the detection cost is relatively high. Moreover, the interpretation of immunohistochemistry and immunocytochemistry test results not only requires professional pathology knowledge and experience, but also has a high degree of subjectivity. This is also the biggest disadvantage and limitation of the above-mentioned methods, which has seriously affected the promotion and use of the above-mentioned methods.

[0004] In summary, these methods all have drawbacks such as complex operation, high cost, long time consumption, complicated result interpretation, and the need for specialized personnel. However, compared to other detection methods, the colloidal gold method offers relatively simple test kit preparation, low testing cost, and rapid and easy operation. It also eliminates the need for specialized personnel to operate complex instruments and equipment, or to perform tedious experimental steps and data analysis. Test results can be obtained quickly simply by visually observing the color change on the test strip. Even non-professionals, or even patients or their families, can complete sample collection and testing on their own, demonstrating the typical characteristics of point-of-care testing (POCT). Therefore, it is suitable for rapid screening and on-site testing, making it easy to promote and apply. For this reason, the colloidal gold method has been widely used as a point-of-care test reagent.

[0005] However, compared with laboratory precision detection methods, the colloidal gold method has the limitation of insufficient detection sensitivity when detecting low concentrations of p16 protein due to poor pairing between the coating antibody and the detection antibody. This leads to missed detection in the case of early tumors or trace p16 protein expression, thus affecting the early diagnosis of malignant tumors.

[0006] Therefore, developing and screening appropriate paired antibodies is an important method to improve the sensitivity of colloidal gold method. Summary of the Invention

[0007] In view of the problems of existing p16 protein detection methods such as complex operation, long detection cycle, high cost, low sensitivity and specificity, especially the limitation of insufficient sensitivity of the colloidal gold method, there is an urgent need to develop and screen suitable paired antibodies for the preparation of a colloidal gold kit for detecting p16 protein. The present invention aims to provide a colloidal gold kit for detecting p16 protein, as well as its preparation method and application.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions: The present invention provides a colloidal gold test kit for detecting p16 protein, comprising a test strip cartridge, a protective cover located outside the test strip cartridge, and a colloidal gold test strip located within the test strip cartridge; the colloidal gold test strip comprises a base plate, on which a sample pad, a colloidal gold binding pad, a nitrocellulose membrane, and a water-absorbing pad are sequentially adhered in an extended axis direction; a detection line and a quality control line are provided on the nitrocellulose membrane near the colloidal gold binding pad; The colloidal gold conjugate pad is coated with a detection antibody labeled with colloidal gold for detecting p16 protein; The detection line is coated with a capture antibody for capturing p16 protein; The quality control line is coated with goat anti-mouse IgG antibody.

[0009] Furthermore, the colloidal gold kit further comprises a sample diluent, which comprises 0.01 mol / L phosphate buffered saline (PBST) with a pH of 7.2-7.4 and a protein (such as BSA) reagent, for dissolving the sample to be tested.

[0010] Furthermore, the capture antibody was coated at a concentration of 0.5 mg / mL, and 1 μL / cm was coated on the NC membrane as a detection line; the goat anti-mouse IgG antibody was coated at a concentration of 0.5 mg / mL, and 1 μL / cm was coated on the NC membrane as a quality control line.

[0011] The detection antibody is an anti-p16 monoclonal antibody, the heavy chain amino acid sequence of the anti-p16 monoclonal antibody is shown in SEQ ID NO.1, and the light chain amino acid sequence is shown in SEQ ID NO.2. It is synthesized and secreted by an anti-p16 monoclonal antibody hybridoma cell line deposited in the General Microbiology Center of the China Culture Collection Administration with a deposit number of CGMCC No.46036, and the deposit date is August 14, 2024.

[0012] Furthermore, the heavy chain amino acid sequence of the anti-p16 monoclonal antibody is RLKLQQSGPGILQPSQTLSLTCSFSGFSLSTSGVIVGWIRQPSGKLLEWLAHIWWDDDRHYNPALKSRLTISKDTSTNQVFLKIASVDIADTATYYCTRLGDYLFPYWGNGTLVTVSA (as shown in SEQ ID NO. 1) The light chain amino acid sequence of the anti-p16 monoclonal antibody is DIVMTQTPPSLPVSLGDPASISCRSSRNIVHNNGITYLEWYLQKPGQSPKVLIYKVSNKFSGVPDRFSGSGSGTDWTLKISRVEAEDLVIYYCFQGSKVPPTFGAGTKLDIK (as shown in SEQ ID NO. 2) The capture antibody is an anti-p16 monoclonal antibody (clone number 3G3G11). The heavy chain amino acid sequence of the anti-p16 monoclonal antibody is shown in SEQ ID NO.3, and the light chain amino acid sequence is shown in SEQ ID NO.4. It is synthesized and secreted by an anti-p16 monoclonal antibody hybridoma cell line deposited in the General Microbiology Center of the China Culture Collection Administration with a deposit number of CGMCC No.46035, and the deposit date is August 14, 2024.

[0013] Preferably, the heavy chain amino acid sequence of the anti-p16 monoclonal antibody is: EVQLEESGPGILQPSQTLSLTCSFSGFSLSTSGLGVGWIRQPSAKGLEWLGHIWWDDDKHYNPAVKSRLTISKETSSNQVFLKIASVDTADTATWYCTRLGDYLFPYWGQVTLVTVSE (shown in SEQ ID NO.3) The light chain amino acid sequence of the anti-p16 monoclonal antibody is: DIVITQTPLSLPVSTGDPASISCRSSRNVVHMNGNTYLEWYLQRPGQSPKLLIYKVTNRFSGVPDRFSGTGSGTDFTLKICRVEAEDLGIYYCFNGSHVPPTFGGGTHLEIK (as shown in SEQ ID NO. 4).

[0014] The sample pad (2) is made of a glass fiber membrane and is treated overnight with a treatment solution containing Tris, casein sodium salt, and PVP-10.

[0015] The colloidal gold binding pad (3) is made of a glass fiber membrane and is treated with a treatment solution containing Tris, S-17, and bovine serum albumin.

[0016] The protective cover (9) is provided with a sample addition port (10) and a result observation port (11).

[0017] Another object of the present invention is to provide a method for preparing a colloidal gold kit for detecting p16 protein, which specifically comprises the following steps: Step 1, spraying a colloidal gold labeled detection antibody solution onto a glass fiber membrane and drying it to obtain a colloidal gold binding pad (3); Step 2: coating the capture antibody on the nitrocellulose membrane (6) to form a detection line (4), and coating the goat anti-mouse IgG antibody on the nitrocellulose membrane (6) to form a quality control line (5), thereby obtaining a nitrocellulose membrane (6); Step 3, sequentially attach the sample pad (2), the colloidal gold binding pad (3) obtained in step 1, the nitrocellulose membrane (6) obtained in step 2, and the absorbent pad (7) along the length direction of the adhesive base plate (1), cut them into test strips with a width of 3.5 mm ± 0.5 mm, and place them into the test strip holder (8).

[0018] In step 1, the colloidal gold-labeled detection antibody solution is prepared by adding the purified detection antibody to the colloidal gold solution, stirring evenly, blocking, and centrifuging to obtain the colloidal gold-labeled detection antibody solution.

[0019] Furthermore, in step 1, the detection antibody is screened using a double antibody sandwich ELISA method.

[0020] Preferably, the anti-p16 monoclonal antibody with clone number 1F4F12 is selected as the detection antibody. The antibody is synthesized and secreted by the anti-p16 monoclonal antibody hybridoma cell line with the deposit number CGMCC No: 46036 deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms, and the deposit date is August 14, 2024.

[0021] Furthermore, in step 2, the capture antibody is screened by a double antibody sandwich ELISA method using an enzyme-labeled plate.

[0022] Furthermore, the capture antibody selected is an anti-p16 monoclonal antibody with clone number 3G3G11 as the capture antibody, which is synthesized and secreted by an anti-p16 monoclonal antibody hybridoma cell line with a deposit number of CGMCC No. 46035 deposited in the General Microbiology Center of the China Culture Collection Administration, and the deposit date is August 14, 2024.

[0023] The anti-p16 monoclonal antibody hybridoma cell line with the preservation number of CGMCC No: 46035 was revived and cultured in a serum-free culture. The anti-p16 monoclonal antibody with the clone number 3G3G11 was isolated and purified from the culture supernatant. After the antibody concentration was determined, a capture antibody coating solution was prepared. The capture antibody coating solution was then sprayed onto the detection line of a nitrocellulose membrane provided with a detection line and a quality control line to coat the capture antibody.

[0024] The quality control line of the nitrocellulose membrane provided with the detection line and the quality control line is sprayed and coated with the goat anti-mouse IgG antibody.

[0025] Another object of the present invention is to provide an application of a colloidal gold kit for detecting p16 protein, wherein the colloidal gold kit can be used to detect p16 protein in body fluid samples such as whole blood, serum, pleural effusion, ascites, cervical scrapings, vaginal secretions, and bronchial brushings.

[0026] The application of the colloidal gold test kit comprises the following specific steps: (1) Process the sample to be tested, place the sample to be tested in the sample diluent, shake well, obtain the processed sample to be tested, and set aside; (2) Take the treated sample to be tested and drop it into the sample adding hole (10) of the colloidal gold kit for detecting p16 protein, and observe the color development of the test line and the quality control line through the result observation port (11); Step 3, result interpretation: (3) Determine whether the sample to be tested contains p16 protein based on the colors of the quality control line and the test line.

[0027] A brown-red line appears on the quality control line, which means the test is successful and the result is reliable.

[0028] No brown-red line appeared on the quality control line, which was interpreted as a test failure and re-sampled for testing.

[0029] Brown-red lines appeared on both the quality control line and the test line, indicating that the sample contained p16 protein.

[0030] If a brown-red line appears on the quality control line but no brown-red line appears on the test line, it is judged as negative, indicating that the sample does not contain p16 protein, or the p16 protein content is lower than the detection sensitivity of the test strip.

[0031] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a colloidal gold kit for detecting p16 protein. The colloidal gold kit adopts a double antibody sandwich method, using screened detection antibodies and capture antibodies as paired antibodies, and can detect p16 protein in body fluid samples with high sensitivity and high specificity, overcoming the shortcomings of existing p16 protein detection methods with low sensitivity and specificity. Colloidal gold particles are used as markers to bind to the detection antibodies, retaining the antibody's immunological activity while imparting color development characteristics to form a color reaction visible to the naked eye. When p16 protein is present in the sample to be tested (such as serum, plasma, etc.), it reacts with the colloidal gold particles to detect the p16 protein in the body fluid sample. The gold-labeled antibody specifically binds to form a complex, providing a signal basis for subsequent detection; the capture antibody is coated on the test line and fixed to the membrane surface by electrostatic adsorption or covalent bonding. When the p16 protein antigen in the sample reaches the test line through chromatography, it specifically binds to the capture antibody to form an antibody-antigen-colloidal gold complex and aggregates to develop color, realizing visual detection. The sheep anti-mouse IgG antibody is used as the coating of the quality control line to capture free colloidal gold-labeled antibodies. The color development of the quality control line verifies the effectiveness of the kit to avoid false negative results due to operational errors or reagent failure.

[0032] Furthermore, the detection sensitivity of the colloidal gold kit is about 1 μg / mL. The sensitivity of p16 protein in serum samples, pleural effusion samples, cervical brushings and bronchial brushings is 72.11%, 75.38%, 85.63% and 70.83%, respectively. The specificity is 91.84%, 80.49%, 77.95% and 71.11%, respectively. The accuracy is 82.33%, 78.48%, 82.11% and 71.07%, respectively. The colloidal gold kit provided by the present invention is not only low in cost, accurate in results, fast in detection (10-15 minutes), high in sensitivity and good in specificity, but also easy to use, simple in operation, easy to interpret results, does not require complex instruments and equipment, and is easy to promote and apply. It can be used as a bedside detection kit, and patients can independently complete the screening and detection of malignant tumors with high expression of p16 protein.

[0033] The present invention provides a method for preparing a colloidal gold kit for detecting p16 protein. In the preparation method, screened paired antibodies are used as a detection antibody and a capture antibody. The anti-p16 monoclonal antibody with clone number 1F4F12 synthesized and secreted by an anti-p16 monoclonal antibody hybridoma cell line with a deposit number of CGMCC No. 46036 deposited in the General Microbiology Center of the China National Center for Microbiological Culture Collection is selected as the detection antibody. A colloidal gold-labeled detection antibody solution is directly sprayed onto a glass fiber membrane and dried to ensure uniform antibody loading and stable activity. The anti-p16 monoclonal antibody (clone 3G3G11), synthesized and secreted by a hybridoma cell line producing an anti-p16 monoclonal antibody and deposited with the China General Microbiology Center under the China Culture Collection Administration (CGMCC) with accession number CGMCC No. 46035, was selected as the capture antibody. A nitrocellulose membrane was coated with the capture antibody (forming the detection line), and a goat anti-mouse IgG antibody (forming the quality control line) was simultaneously coated on the nitrocellulose membrane, achieving dual-line functionality. These components work together through capillary action to ensure unidirectional sample flow from the sample application end to the water absorption end, optimizing the efficiency of the antigen-antibody reaction. This method is simple to operate and amenable to industrial mass production. The colloidal gold test strip prepared according to this method for detecting p16 protein exhibits a sensitivity of approximately 1 μg / mL.

[0034] The application provided by the present invention is that the kit can complete the detection within 10 to 15 minutes, and is suitable for bedside self-detection in emergency or primary medical settings or patients at home. It shows high sensitivity and specificity in the screening of malignant tumors with high p16 expression, especially the detection effect of cervical lesion samples is significant, indicating that it has important application value in the early diagnosis of cervical cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Schematic diagram of the structure of the colloidal gold kit of the present invention; Figure 2 Schematic diagram of the efficacy test results of the colloidal gold kit of the present invention; In the figure, 1-base plate, 2-sample pad, 3-colloidal gold binding pad, 4-detection line, 5-quality control line, 6-nitrocellulose membrane, 7-absorption pad, 8-test strip card, 9-protective cover, 10-sample port, 11-observation port 11. DETAILED DESCRIPTION

[0036] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of the present invention.

[0037] The use of patient samples involved in this invention has been approved by the Medical Ethics Committee of the Second Affiliated Hospital of Xi'an Jiaotong University School of Medicine (Ethics Approval No.: 2019159).

[0038] The present invention uses an anti-p16 monoclonal antibody hybridoma cell line (clone number 3G3G11) deposited on August 14, 2024, at the General Microbiology Center of the China Culture Collection Administration, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101, with a deposit number of CGMCC No. 46035. The proposed classification name is: Balb / c mouse monoclonal antibody hybridoma cell line; The anti-p16 monoclonal antibody hybridoma cell line (clone number 1D4F12) was deposited on August 14, 2024, at the General Microbiology Center of China Culture Collection Administration, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101, with the deposit number CGMCC No. 46036. The proposed classification name is: Balb / c mouse monoclonal antibody hybridoma cell line.

[0039] The present invention is described in further detail below with reference to the accompanying drawings: 1. Preparation method of the sample diluent: (1) Prepare 10× PBS buffer: weigh the following reagents: NaCl: 80 g, KCl: 2 g, Na2HPO4: 14.2 g, KH2PO4: 2.7 g; add the above reagents to a 1 L beaker, add about 800 mL of deionized water, and stir thoroughly to dissolve; adjust the pH to 7.4 with concentrated hydrochloric acid, then add deionized water to 1 L, sterilize under high pressure, and store at room temperature; (2) Dilute 10× PBS buffer to 1× PBS buffer concentration: Take 100 mL of 10× PBS buffer, add 900 mL of deionized water, and stir evenly.

[0040] (3) Take 1 L of 1× PBS buffer, add 0.5 mL of Tween-20 and 10 mL of BSA, and stir evenly; filter the prepared 1× PBST buffer with a 0.22 μm filter membrane and dispense it into sterile bottles to obtain sample dilution solution. Store it in a cool place at room temperature or 4°C until use, or freeze it at -20°C for a long time to avoid repeated freezing and thawing.

[0041] Example 1 See attached Figure 1 This embodiment provides a colloidal gold kit for detecting p16 protein. The colloidal gold kit for detecting p16 protein mainly consists of a colloidal gold test strip and a sample diluent (PBST buffer).

[0042] See attached Figure 1 A colloidal gold test strip for detecting p16 protein, the colloidal gold test strip is composed of a water absorbent pad 7, a nitrocellulose membrane 6, a colloidal gold binding pad 3, a sample pad 2 and a base plate 1 in sequence. The nitrocellulose membrane 6 is provided with a detection line 4 and a quality control line 5. The detection line 4 is coated with an antibody that captures p16 protein, and the quality control line is coated with a goat anti-mouse IgG antibody; the colloidal gold binding pad 3 is coated with a colloidal gold-labeled anti-p16 monoclonal antibody for detecting p16 protein.

[0043] The sample pad 2 is made of absorbent filter paper and is used to receive and preliminarily process the sample to be tested. The sample begins to come into contact with the test strip here.

[0044] The colloidal gold binding pad 3 is coated with a colloidal gold-labeled anti-p16 monoclonal antibody. This antibody, as a specific detection antibody, can specifically bind to the p16 protein in the sample to form an antigen-antibody complex.

[0045] The test strip (width 3.0±0.2 mm) can be placed in the test strip holder 8 and fastened with a protective cover 9 , which is provided with a sample injection port 10 and a result observation port 11 .

[0046] The sample diluent includes 0.01 mol / L phosphate buffered saline (PBST) with a pH of 7.2-7.4, 0.05% Tween-20, and an appropriate amount of protein (such as 1% BSA) and other reagents, and is used to process body fluid samples such as cervical scrapings, vaginal secretions, and bronchial brushings.

[0047] Example 2 This embodiment provides a method for preparing a colloidal gold kit for detecting p16 protein, and the specific steps are as follows: 1. Preparation of Colloidal Gold Conjugate Pad 3 (1) Screening of detection antibodies and capture antibodies in colloidal gold test strips Screening for high-titer paired detection antibodies and capture antibodies is the basis for ensuring the specificity and sensitivity of the double-antibody sandwich colloidal gold test strip. This example uses the following method to screen detection antibodies and capture antibodies: Two high-titer anti-p16 monoclonal antibodies were selected. One anti-p16 monoclonal antibody was labeled and used as the detection antibody, and the other anti-p16 monoclonal antibody was used as the capture antibody to coat the ELISA plate. The double-antibody sandwich ELISA method was used to screen and determine the paired anti-p16 monoclonal antibody pair for the preparation of colloidal gold test strips.

[0048] The anti-p16 monoclonal antibody with clone number 1F4F12 is preferably used as the detection antibody. The anti-p16 monoclonal antibody is synthesized and secreted by the anti-p16 monoclonal antibody hybridoma cell line deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms with the deposit number CGMCC No. 46036. The deposit date is August 14, 2024. The recommended classification name is: Balb / c mouse monoclonal antibody hybridoma cell line.

[0049] The anti-p16 monoclonal antibody with clone number 3G3G11 is preferably used as the capture antibody. The antibody is synthesized and secreted by the anti-p16 monoclonal antibody hybridoma cell line deposited in the General Microbiology Center of the China Culture Collection Administration with the deposit number CGMCC No. 46035. The deposit date is August 14, 2024. The recommended classification name is: Balb / c mouse monoclonal antibody hybridoma cell line.

[0050] Antibody purification: 3G3G11 and 1F4F12 monoclonal hybridoma cell lines were revived and cultured in serum-free culture for antibody expression. The resulting cell culture supernatant was slowly loaded onto a Protein A / G agarose gel affinity purification column and eluted with glycine elution buffer to obtain the purified antibodies, which were immediately dialyzed against PBS overnight at 4°C. SDS-PAGE electrophoresis and Coomassie Brilliant Blue staining showed that the purity of both antibodies was over 90%. Indirect ELISA assays revealed that the titers of both antibodies against the antigen were approximately 3280.5 kDa. The concentrations of the anti-p16 monoclonal antibodies clones 3G3G11 and 1F4F12 were 1.20 mg / mL and 1.14 mg / mL, respectively, as determined by a BCA assay kit.

[0051] Detection antibody labeling: clone 1F4F12 anti-p16 monoclonal antibody was selected as the detection antibody. A certain amount of biotin was weighed and dissolved in 180 μL of pure water to a concentration of 5.5 mg / mL for every 1.0 mg of biotin. The biotin solution was then slowly added to the clone 1F4F12 anti-p16 monoclonal antibody solution. The reaction was carried out in a dark place at room temperature with rotation for 1 h. The solution was dialyzed against 1× PBS overnight. 10 μL of preservative was added to every 1 mL of label and the solution was stored at -20°C in the dark.

[0052] ELISA plate coating: dilute the anti-p16 monoclonal antibody clone 3G3G11 with phosphate buffered saline (PBS, pH 7.4) using PBS coating solution, mix well, and add 100 μL per well to the ELISA plate for coating. Refrigerate at 4°C overnight.

[0053] Blocking of the ELISA plate: After the ELISA plate is coated, discard the coating solution, wash the plate 3 times, add 200 μL of blocking solution to each well, and incubate at 37°C for 1 hour. Take out the ELISA plate, discard the inner solution, and wash the plate once. Protein reaction: HeLa cells cultured conventionally were lysed using RIPA lysis buffer to obtain the target protein and determine its concentration. An appropriate amount of protein was diluted to 1 μg / mL with phosphate buffered saline (PBS, pH 7.4). Starting at a concentration of 1 μg / mL, the protein was diluted 2-fold, with 100 μL per well added to the ELISA plate and incubated at 37°C for 1 h.

[0054] Primary antibody reaction: remove the ELISA plate, discard the internal solution, wash the plate three times, add 100 μL of diluted biotin-labeled anti-p16 monoclonal antibody (clone number 1F4F12) to each well, and incubate at 37°C for 1 h.

[0055] Secondary antibody reaction: Remove the ELISA plate, discard the internal solution, wash the plate three times, and add 100 μL of the diluted enzyme conjugate (SA-HRP) to each well. Incubate at 37°C for 1 hour.

[0056] Color development: Remove the ELISA plate, discard the internal solution, wash the plate 4 times, add 100 μL TMB color development solution to each well, and determine the color development time based on the color depth, generally 15 min at 37°C.

[0057] Stop the reaction by adding 100 μL of 1M HCl solution to each well. Immediately read the plate at 450 nm on a microplate reader.

[0058] The sensitivity of the paired antibody 3G3G11 and 1D4F12-biotin for detecting p16 protein in HeLa cell lysate supernatant was determined to be 62.5 ng / mL.

[0059] (2) Coating of colloidal gold binding pad Preparation of detection antibody: The anti-p16 monoclonal antibody with the preferred clone number 1F4F12 in step (1) is used as the detection antibody, and the anti-p16 monoclonal antibody hybridoma cell line with the preservation number of CGMCC No. 46036 is revived and cultured in serum-free culture. The anti-p16 monoclonal antibody with the clone number 1F4F12 is isolated and purified from the culture supernatant.

[0060] Colloidal gold solution was prepared by trisodium citrate reduction method: 100 mL of ultrapure water was poured into a 250 mL conical flask, 1.0 mL of 1.0% chloroauric acid solution was added dropwise, the solution was placed in a magnetic stirrer and stirred continuously and heated to boiling, and then 2.0 mL of 1.0% trisodium citrate solution was quickly added under stirring. At this time, the solution gradually changed from light yellow to gray-black. Heating was continued until the solution turned orange-red and continued heating for 15 minutes; cooled to room temperature, and the pH of the solution was adjusted to about 6.5 with 0.1 mol / mL K2CO3; the volume was made up to 100 mL with ultrapure water to obtain colloidal gold solution, which was placed in a brown bottle and stored at 4°C for later use.

[0061] Detection antibody labeling: Pipette 1 mL of the prepared colloidal gold solution into a 2 mL centrifuge tube rinsed with ultrapure water. Adjust the pH to 8.2 by adding 0.2 M potassium carbonate to each tube. Add 20 μg of anti-p16 monoclonal antibody (clone 1D4F12) to the tube, mix thoroughly, and react for 30 minutes. Add 50 μL of blocking buffer and continue the reaction for 20 minutes. Centrifuge at 12,000 rpm for 20 minutes. Discard the supernatant, resuspend the pellet in 0.25 mL of gold rehydration solution (0.01% boric acid, 0.2% PEG 20000, and 0.02% NaN 3 ). Apply the labeled antibody to a 7 mm × 75 mm colloidal gold conjugate pad using a gold sprayer. Allow to dry at 37°C for 6 hours before use.

[0062] 2. Spraying and coating of test lines and quality control lines According to the detection antibody and capture antibody determined in step 1, use the capture antibody to spray and coat the test line.

[0063] Preparation of capture antibody: The anti-p16 monoclonal antibody with clone number 3G3G11 is preferably used as the capture antibody. The anti-p16 monoclonal antibody hybridoma cell line with the deposit number CGMCC No. 46035 is revived and cultured in serum-free culture. The anti-p16 monoclonal antibody with clone number 3G3G11 is isolated and purified from the culture supernatant.

[0064] Spraying and coating the test line: Prepare the anti-p16 monoclonal antibody with clone number 3G3G11 into a capture antibody coating solution with a concentration of 0.5 mg / mL. Then, spray and coat the capture antibody on the test line of the nitrocellulose membrane with the test line and quality control line at a dose of 1 μL / cm.

[0065] Spraying and coating of the quality control line: dilute the goat anti-mouse IgG antibody to 0.5 mg / mL, and then spray and coat the goat anti-mouse IgG antibody at a dose of 1 μL / cm on the quality control line of the nitrocellulose membrane with the test line and quality control line.

[0066] Drying of nitrocellulose membrane: Place the nitrocellulose membrane that has been sprayed and coated with the test line and quality control line at room temperature for 18 hours under low humidity to dry before use.

[0067] 3. Assembly of Colloidal Gold Kit After the colloidal gold conjugate pad 3 and the nitrocellulose membrane 6 with the detection line and the quality control line are sprayed and coated according to the above steps, the colloidal gold test strip is assembled according to the following method: Specifically, a sample pad 2, colloidal gold binding pad 3, nitrocellulose membrane 6 with test line 4 and quality control line 5, and absorption pad 7 are attached to the base plate 1 from left to right. The strips are then cut into 3 mm ± 0.2 mm wide test strips using a chopper. The cut test strips are then placed one by one into the test strip card holder 8 and secured with a protective cover 9 to complete the colloidal gold test strip card. The protective cover 9 is equipped with a sample port 10 for adding the test sample and an observation port 11 for observing the color development of the test line 4 and quality control line 5 on the test strip.

[0068] 4. Performance Verification of Colloidal Gold Test Strips See attached Figure 2 , take 4 prepared colloidal gold reagent cards 10 for detecting p16 protein and number them A, B, C, and D, then add 100 μL of sample diluent and 10 μg / mL, 1 μg / mL, and 0.1 μg / mL recombinant p16 protein solutions to the sample injection ports 10 of colloidal gold reagent cards A, B, C, and D, respectively. The results showed that the quality control lines of the four colloidal gold reagent cards for detecting p16 protein, numbered A, B, C, and D, all showed clear brown-red bands. The test line of test strip A, which was added with sample diluent, showed no obvious band. The test line of test strip B, which was added with 10 μg / mL recombinant p16 protein solution, showed a clear band. The test line of test strip C, which was added with 1 μg / mL recombinant p16 protein solution, showed a weak but clearly identifiable band. The test line of test strip D, which was spotted with 0.1 μg / mL antigen, showed no obvious band. These results indicate that the detection sensitivity of the colloidal gold test strip is approximately 1 μg / mL.

[0069] 100 μL of sample diluent was added to the sample port 10 of colloidal gold reagent card A as a control. At the same time, 10 μg / mL, 1 μg / mL, and 0.1 μg / mL of recombinant p16 protein solution were added to the sample ports 10 of colloidal gold reagent cards B, C, and D, respectively. The results showed that a clear brown-red band appeared on the control line 5 of the colloidal gold reagent cards A, B, C, and D. Among them, there was no obvious band on the detection line 4 of the colloidal gold test paper cards A and D, and a brown-red band appeared on the detection line 4 of the test paper cards B and C ( Figure 2 ). Thus, it can be seen that the detection sensitivity of the colloidal gold reagent card for detecting p16 protein prepared according to this embodiment is about 1 μg / mL.

[0070] Example 3 This example is based on Examples 1 and 2, and a clinical trial is conducted on the colloidal gold kit for detecting p16 protein of the present invention. The specific process is as follows: 1. Collect samples To further verify the colloidal gold kit for detecting p16 protein provided by the present invention, this example collected 950 cases that were diagnosed pathologically by biopsy or surgical resection of tissue samples at the Second Affiliated Hospital of Xi'an Jiaotong University between February 25, 2024 and December 20, 2024. All samples were pathologically diagnosed, and immunohistochemical staining was used to detect the expression of p16 protein in tumor cells. Cases with pathological diagnosis of malignancy and high expression of p16 protein were interpreted as clinically positive, and the remaining cases were clinically negative. Among the 950 cases collected, 285 cases had cervical lesions, and cervical scrapings were collected simultaneously during biopsy or surgery; 159 cases had lung lesions, and bronchial brushings were collected simultaneously during biopsy or surgery; 223 cases with pleural effusion and ascites were routinely collected; 283 cases of other tumors and non-tumor cases retained at least 100uL of the remaining serum from the patient for routine testing before treatment. For serum and pleural effusion body fluid samples, place them directly in the sample bottle and store them for future use; for cervical scraping samples and bronchial brushing samples, place the brush sample or the brush sample together with the sampling brush head into the sample bottle containing 1mL of sample diluent, close the bottle cap tightly, and shake it to dissolve the brush sample on the sampling brush head in the diluent as much as possible, and store it for future use.

[0071] 2. Testing and result interpretation The colloidal gold kit disclosed in the present invention was used to test the collected samples one by one. The specific detection method is as follows: For serum and pleural effusion body fluid samples, use a pipette to draw 100uL of serum or pleural effusion sample from the sample bottle and add it directly to the sample well 11 of the colloidal gold test paper card; for cervical scraping samples and bronchial brushing samples, use a pipette to draw 100uL of diluent containing the brushing sample from the corresponding sample bottle and add it directly to the sample well 11 of the colloidal gold test paper card.

[0072] Then, observe the color development of the test line 4 and the quality control line 5 on the test strip with the naked eye through the observation port 12, interpret the results according to the following method, and record the results: A brown-red line appears on the quality control line, which means the test is successful and the result is reliable.

[0073] No brown-red line appeared on the quality control line, which was interpreted as a test failure and re-sampled for testing.

[0074] Brown-red lines appeared on both the quality control line and the test line, indicating that the sample contained p16 protein.

[0075] If a brown-red line appears on the quality control line but no brown-red line appears on the test line, it is judged as negative, indicating that the sample does not contain p16 protein, or the p16 protein content is lower than the detection sensitivity of the test strip.

[0076] Experimental results: All sample quality control lines showed a brown-red line, indicating that all samples were successfully tested and the results were reliable. The test results showed that while the sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of the colloidal gold kit for detecting p16 protein provided by the present invention varied across samples such as serum, pleural effusion, and cervical brushings, with the exception of a negative predictive value of 67.67% for bronchial brushings, all other indicators were above 70.83% (Table 1). This demonstrates that the colloidal gold kit for detecting p16 protein provided by the present invention has the ability to screen for malignant tumors with high p16 expression.

[0077] Table 1: Results of a colloidal gold kit for detecting p16 protein in screening for malignant tumors with high p16 expression

[0078] The above content is only for explaining the technical idea of the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. A colloidal gold kit for detecting p16 protein, characterized in that: The colloidal gold test kit comprises a test strip cartridge (8), a protective cover (9) located outside the test strip cartridge (8), and a colloidal gold test strip located inside the test strip cartridge (8), wherein the colloidal gold test strip comprises a base plate (1), a sample pad (2), a colloidal gold binding pad (3), a nitrocellulose membrane (6), and a water-absorbing pad (7) are sequentially adhered to the base plate (1) in the direction of the extended axis, and a detection line (4) and a quality control line (5) are provided on the nitrocellulose membrane (6) near the colloidal gold binding pad (3); The colloidal gold conjugate pad (3) is coated with a detection antibody labeled with colloidal gold for detecting p16 protein; The detection line (4) is coated with a capture antibody for capturing p16 protein; The quality control line (5) is coated with goat anti-mouse IgG antibody.

2. The colloidal gold kit for detecting p16 protein according to claim 1, characterized in that: The detection antibody is an anti-p16 monoclonal antibody, the heavy chain amino acid sequence of which is shown in SEQ ID NO.1, and the light chain amino acid sequence of which is shown in SEQ ID NO.

2. It is synthesized and secreted by an anti-p16 monoclonal antibody hybridoma cell line deposited in the General Microbiology Center of the China Culture Collection Administration with a deposit number of CGMCC No. 46036, and the deposit date is August 14, 2024.

3. The colloidal gold kit for detecting p16 protein according to claim 1, characterized in that: The capture antibody is an anti-p16 monoclonal antibody, the heavy chain amino acid sequence of the anti-p16 monoclonal antibody is shown in SEQ ID NO.3, and the light chain amino acid sequence is shown in SEQ ID NO.

4. It is synthesized and secreted by an anti-p16 monoclonal antibody hybridoma cell line with a deposit number of CGMCC No.46035 deposited in the General Microbiology Center of the China Culture Collection Administration, and the deposit date is August 14, 2024.

4. The colloidal gold kit for detecting p16 protein according to claim 1, characterized in that: The sample pad (2) is made of a glass fiber membrane and is treated overnight with a treatment solution containing Tris, casein sodium salt, and PVP-10.

5. The colloidal gold kit for detecting p16 protein according to claim 1, characterized in that: The colloidal gold binding pad (3) is made of a glass fiber membrane and is treated with a treatment solution containing Tris, S-17, and bovine serum albumin.

6. The colloidal gold kit for detecting p16 protein according to claim 1, characterized in that: The protective cover (9) is provided with a sample addition port (10) and a result observation port (11).

7. The method for preparing the colloidal gold kit for detecting p16 protein according to any one of claims 1 to 6, characterized in that: The following steps are involved: Step 1, spraying a colloidal gold-labeled detection antibody solution onto a glass fiber membrane and drying it to obtain a colloidal gold conjugate pad (3); Step 2: coating the capture antibody on the nitrocellulose membrane (6) to form a detection line (4), and coating the goat anti-mouse IgG antibody on the nitrocellulose membrane (6) to form a quality control line (5), thereby obtaining a nitrocellulose membrane (6) provided with a detection line and a quality control line; Step 3, sequentially attach the sample pad (2), the colloidal gold binding pad (3) obtained in step 1, the nitrocellulose membrane (6) with the detection line and the quality control line obtained in step 2, and the absorbent pad (7) along the length direction of the adhesive base plate (1), cut them into test strips with a width of 3.0 mm ± 0.2 mm, and place them into the test strip holder (8).

8. The method for preparing the colloidal gold kit for detecting p16 protein according to claim 7, characterized in that: In step 1, the colloidal gold-labeled detection antibody solution is prepared by adding the purified detection antibody to the colloidal gold solution, stirring evenly, blocking, and centrifuging to obtain the colloidal gold-labeled detection antibody solution.

9. Use of the colloidal gold kit for detecting p16 protein according to any one of claims 1 to 6 in detecting p16 protein.

10. The use according to claim 9, characterized in that The sample to be tested is dropped into the colloidal gold kit for detecting p16 protein, and the color of the control line and the test line is used to determine whether the sample to be tested contains p16 protein.

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