Monoclonal antibody for HPV16 type E7 protein detection and application thereof

Abstract

The invention belongs to the technical field of biological detection, and particularly relates to a monoclonal antibody for HPV16 type E7 protein detection and application of the monoclonal antibody. E7 protein serving as an antigen and a Freund's complete adjuvant are mixed, then a mouse is subjected to primary immunization, then the E7 protein and a Freund's incomplete adjuvant are mixed, then the mouse is subjected to enhanced immunization, and the HPV16 type E7 protein detection monoclonal antibody is obtained. The monoclonal antibody for detecting the HPV16 type E7 protein is prepared by taking mouse splenocytes and myeloma cells to be fused to prepare hybridoma cells, then performing clone detection on the hybridoma cells to screen the hybridoma cells aiming at the specificity of the HPV16 type E7 protein, and finally obtaining the monoclonal antibody for detecting the HPV16 type E7 protein. According to the present invention, the HPV16 type E7 antigen detection kit is developed by using the immunochromatography method, and the kit has advantages of high sensitivity, strong specificity, simple operation and fast detection speed, and can be used for rapid screening diagnosis and illness state monitoring of HPV16.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a monoclonal antibody for detecting HPV16 E7 protein and its application. Background Technology

[0002] Human papillomavirus (HPV) is a DNA virus with a diameter of 52-55 nm belonging to the Polyomavirus subfamily. HPV16 is a high-risk HPV infection, associated with the development of cervical cancer. Approximately 80% of cervical cancer patients have HPV16 in their precancerous lesions, making it a relatively serious viral infection. HPV16 has a significant impact on the global incidence of cervical cancer, therefore, preventing and controlling its spread is of paramount importance.

[0003] The early region of HPV encodes seven viral non-structural proteins: E1, E2, E1-E4, E5, E6, E7, and E8-E2. Among these, E5, E6, and E7 are considered virus-related oncogenic factors. The E7 protein binds to the tumor suppressor protein pRb via its conserved N-terminal sequence LXCXE, causing the normally pRb-bound transcription factor E2F to dissociate and inactivate the pRb protein. The dissociated E2F then activates gene transcription, promoting the transcription of numerous cell proliferation-related genes, leading to excessive cell proliferation and malignant transformation. Furthermore, the E7 protein can induce dormant cells to enter the cell cycle, promoting efficient viral genome replication using cellular DNA replication mechanisms. Therefore, the E7 protein has significant application value in the diagnosis and treatment of cervical cancer. By detecting the expression level of the E7 protein, the degree of precancerous lesions in cervical intraepithelial neoplasia can be identified and assessed, further evaluating the extent of HPV infection and disease progression. Monoclonal antibodies are highly specific and can accurately recognize and bind to specific antigenic epitopes for the quantitative analysis of antigens. Therefore, it is necessary to develop monoclonal antibodies against HPV16 E7 protein. Summary of the Invention

[0004] To address the aforementioned issues, this invention provides a monoclonal antibody for detecting HPV16 E7 protein and its application. Firstly, this invention provides a monoclonal antibody for detecting HPV16 E7 protein, and based on this antibody, prepares a detection kit for HPV16 E7 antigen, enabling sensitive, accurate, rapid, and efficient screening, diagnosis, and disease monitoring of HPV16 infection.

[0005] The first aspect of this invention provides a monoclonal antibody for detecting HPV16 E7 protein, said monoclonal antibody being prepared by the following steps: Mice were immunized three times using HPV16 E7 protein as the antigen, with each mouse receiving a single injection of 52-58 μg of antigen into the neck. The emulsified antigen solution for the primary immunization was prepared by mixing the antigen with Freund's complete adjuvant; the emulsified antigen solutions for the second and third immunizations were prepared by mixing the antigen with Freund's incomplete adjuvant. The first antigen entry into the animal triggered a primary immune response, with antigen-presenting cells processing and presenting the antigen, activating T cells, and assisting B cells to differentiate into plasma cells to produce antibodies. However, this process resulted in slow antibody production, small quantities, and short-lasting antibody effects. The second immunization triggered a secondary immune response, with memory cells rapidly recognizing the antigen and quickly differentiating into plasma cells, producing a large number of antibodies—a response that was faster, stronger, and longer-lasting than the primary immunization. During the third immunization, memory B cells and memory T cells were reactivated, not only rapidly producing large quantities of high-affinity antibodies but also further increasing the number of memory cells, strengthening immune memory, and making the animal's immune protection against this antigen more durable and effective.

[0006] Spleen cells and myeloma cells from immunized mice were mixed at a ratio of 4-6:1 for cell fusion culture. After 5-9 days of culture, positive hybridoma cells were screened. Clonal culture of positive hybridoma cells yielded a hybridoma cell line that stably secretes specific antibodies against HPV16 E7 protein. Each mouse was injected with 1×10⁻⁶ cells. 5 cell ~1×10 7 The mouse hybridoma cell line was fed normally for one week, and ascites fluid was extracted from the mice and purified to obtain the monoclonal antibody.

[0007] Furthermore, the interval between the first and second immunizations is 3 to 4 weeks, and the interval between the second and third immunizations is 2 to 3 weeks.

[0008] Furthermore, the volume of the emulsified antigen solution is 300 μL to 500 μL.

[0009] Furthermore, the myeloma cells are SP2 / 0 myeloma cells.

[0010] Furthermore, the titer of the anti-HPV16 E7 protein antibody in mouse serum needs to be detected before the spleen cells are mixed with myeloma cells.

[0011] Furthermore, the cell fusion culture requires a half-medium change every 2 to 3 days.

[0012] Furthermore, the mice are BALB / c mice, weighing 18g~22g.

[0013] A second aspect of the present invention provides the application of the monoclonal antibody described above in the preparation of an immunoassay product for detecting HPV16.

[0014] Furthermore, the testing product is a test strip or a test kit.

[0015] A third aspect of the present invention provides an HPV16 test strip, the test strip comprising a PVC base plate, wherein the base plate comprises, in order of sample flow, a sample pad, a gold label pad, an NC membrane and absorbent filter paper; the gold label pad is coated with the aforementioned monoclonal antibody labeled with colored latex microspheres, and the detection T line of the NC membrane is coated with a secondary antibody containing the monoclonal antibody to be tested.

[0016] Furthermore, the gold-labeled pad is also coated with chicken IgY antibodies labeled with colored latex microspheres.

[0017] Furthermore, the quality control C line of the NC membrane is coated with a secondary antibody containing chicken IgY antibody, wherein the secondary antibody is goat anti-chicken IgY.

[0018] Furthermore, the detection T line and control C line of the NC membrane are coated with a secondary antibody at a concentration of 1 mg / mL.

[0019] Furthermore, the gold-labeled pad is obtained by ultrasonically mixing HPV16 antibody labeled with gel microspheres and chicken IgY antibody labeled with latex microspheres and then spraying the mixture onto a gold-labeled pad that has been soaked in a treatment solution.

[0020] A fourth aspect of the present invention provides an HPV16 detection kit, characterized in that the kit comprises an HPV16 antigen detection kit assembled from the HPV16 test strip and the detection kit described above, a sample buffer, and a sampling swab.

[0021] In summary, compared with the prior art, the present invention has the following advantages and effects: This invention provides a monoclonal antibody for detecting HPV16 E7 protein and its application. The invention involves primary immunizing mice with a mixture of E7 protein (as antigen) and Freund's complete adjuvant, followed by booster immunization with a mixture of E7 protein and Freund's incomplete adjuvant. Hybridoma cells are prepared by fusing mouse spleen cells with myeloma cells. The hybridomas are then subjected to clonal detection to screen for hybridoma cells specific to HPV16 E7 protein, ultimately yielding a monoclonal antibody for detecting HPV16 E7 protein. This antibody exhibits high specificity for HPV16 E7 protein, avoiding cross-reactivity with other E7 protein types, thus achieving high sensitivity and specificity in detection. Based on this antibody, test strips and kits for detecting HPV16 have been developed, enabling sensitive, accurate, rapid, and efficient screening, diagnosis, and disease monitoring of HPV16 infection. Attached Figure Description

[0022] Figure 1The OD values ​​of monoclonal antibodies in serum after three immunizations are shown. Serum from mice 1, 2, and 3 were immunized with E7 protein. Control 1 is serum from a blank control mouse, and Control 2 is serum from PBS. *** indicates... P <0.001.

[0023] Figure 2 The results of antibody purification by SDS-PAGE electrophoresis are shown. From left to right, the corresponding samples in each lane are: protein marker, crude pure sample, roller mixer eluted sample, washed sample, flow-through sample, decolorizing shaker eluted sample, washed sample, and flow-through sample.

[0024] Figure 3 The results of antibody purification by SDS-PAGE electrophoresis are shown. From left to right, the corresponding samples in the lanes are: No. 3 (tumor-containing elution fraction), No. 3 (tumor-free elution fraction), No. 4 (elution fraction), No. 6 (elution fraction), No. 7 (elution fraction), No. 9 (elution fraction), No. 11 (elution fraction), and protein marker.

[0025] Figure 4 These are antibody-labeled microspheres. The microspheres on the left are labeled with HPV16 E7Ab-1, and the microspheres on the right are labeled with chicken anti-IgY.

[0026] Figure 5 The nitrocellulose membrane after antibody embedding.

[0027] Figure 6 This refers to the bonding pad for the gold label after gold plating and drying. Figure 6 In the diagram, A represents the gold-plated bonding pad. Figure 6 B in the figure represents the dried gold-labeled bonding pad.

[0028] Figure 7 This is a schematic diagram of the structure of a microparticle chromatography test strip.

[0029] Figure 8 This is the assembled microparticle detection plate for milk globules.

[0030] Figure 9 These are HPV16 test strips; the three strips in the image are parallel groups.

[0031] Figure 10 This is a display image of an HPV16 testing kit.

[0032] Figure 11 The results are from the HPV16 test strip in Experiment Example 2. Figure 11 In the diagram, A represents the negative test result. Figure 11 B in the table represents the positive test result. Detailed Implementation

[0033] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below in conjunction with specific embodiments and accompanying drawings.

[0034] Unless otherwise specified, all reagents used in this invention are commercially available, and all methods used are conventional techniques in the art.

[0035] Experimental animals: 24 SPF-grade BALB / c mice, weighing 18g~22g, license number: SCXK (Su) 2020-0009.

[0036] Main reagents and consumables: 1mg HPV 16 E7 protein (Fuxiao Biotechnology Co., Ltd.), Freund's complete adjuvant (FAC) and Freund's incomplete adjuvant (FAC without BCG), myeloma cells, HAT, HT, Hybridoma Feeder additive factor, 50% PEG 1450, protein G chromatography medium, and ready-to-use monoclonal antibody subclass identification enzymes were all provided by Beijing Biolong Immunotherapy Co., Ltd.; HRP labeling kit and BCA protein quantification kit were provided by Beijing MecoWander Biotechnology Co., Ltd.; ELISA kit, RPMI-1640 medium (Princella), penicillin antibodies, fetal bovine serum, single-component TMB (Beijing Solarbio Science & Technology Co., Ltd.), SDS-PAGE gel preparation kit (Zhonghui Hecai), NC membrane, sample pad, gold labeling conjugation pad, absorbent pad, and nitrocellulose membrane (NC membrane) were all provided by Hangzhou Fenghang Technology Co., Ltd.

[0037] HPV16 E7 protein is a key oncogenic protein of HPV16 (the most carcinogenic subtype among high-risk HPV types) (leading to cell carcinogenesis by binding to pRb protein), and it is expressed early in infection, but its intracellular expression level is extremely low (far lower than that of L1 protein). E7 protein has important applications in the diagnosis and treatment of cervical cancer. By detecting the expression level of E7 protein, the degree of precancerous lesions of cervical intraepithelial neoplasia can be identified and assessed, further evaluating the degree of HPV infection and disease progression. Monoclonal antibodies have high specificity and can accurately recognize and bind to specific antigenic epitopes for quantitative antigen analysis; therefore, it is necessary to develop monoclonal antibodies targeting HPV16 E7 protein.

[0038] This invention uses E7 protein as an antigen. The antigen is mixed with Freund's complete adjuvant and used for primary immunization of mice. Then, E7 protein is mixed with Freund's incomplete adjuvant and used for booster immunization. Mouse spleen cells are fused with myeloma cells to prepare hybridoma cells. Subsequently, hybridoma cells are subjected to clonal detection to screen for hybridoma cells specific to HPV16 E7 protein, ultimately obtaining a monoclonal antibody for the detection of HPV16 E7 protein. This antibody is then labeled using colored latex microspheres, and an HPV16 E7 antigen detection kit is developed using immunochromatography. This kit has the advantages of high sensitivity, strong specificity, simple operation, and fast detection speed, and can be used for rapid screening, diagnosis, and disease monitoring of HPV16.

[0039] Example 1: A monoclonal antibody for detecting HPV16 E7 protein 1. Preparation of main solutions Antigen preparation: Freund's complete adjuvant / Freund's incomplete adjuvant (600 μL) + 1 mg / mL HPV 16 E7 protein (165 μL) + PBS (435 μL), total 1.2 mL, 0.4 mL / animal, mix well on a vortex mixer after addition.

[0040] RPMI-1640 complete culture medium: Mix 89 mL RPMI-1640 + 10 mL fetal bovine serum + 1 mL double antibiotics, and store at 4℃.

[0041] Preparation of HAT medium: Mix 82 mL RPMI-1640 complete medium + 2 mL 50 × HAT concentrate + 5 mL 10% Hybridoma Feeder additive factor + 1 mL double antibiotic + 10 mL fetal bovine serum, and store at 4℃.

[0042] Preparation of HT medium: Mix 83 mL RPMI-1640 complete medium + 2 mL 50 × HT concentrate + 4 mL 10% Hybridoma Feeder additive factor + 1 mL double antibiotic + 10 mL fetal bovine serum, and store at 4℃.

[0043] 2. Experimental Procedure Immunization schedule: Four BALB / c mice were randomly divided into two groups: three mice in the experimental group and one mouse in the control group. The experimental group received an injection of 0.4 mL of antigen (French complete adjuvant / French incomplete adjuvant + HPV 16 E7 protein) per mouse, while the control group received no injection. The experimental group received a total of three immunizations, with a three-week interval between the first and second injections and a two-week interval between the second and third injections.

[0044] Serum collection: Blood was collected 3 days after the last injection using the tail-cutting blood collection method. The serum was collected by centrifugation and stored at -20°C for later use.

[0045] Enzyme-linked immunosorbent assay (ELISA): The OD value of serum at 450 nm was detected using the ELISA double antibody sandwich method.

[0046] Cell fusion: BALB / c mouse spleen cells were fused with SP2 / 0 myeloma cells at a ratio of 5:1. After fusion, the medium was changed halfway every two days. After screening and culturing for 1 week, the medium was changed to HAT medium.

[0047] Selection of hybridoma cells: The titer of the cultured cell supernatant was identified by ELISA, and positive wells were selected.

[0048] Clonal culture (limiting dilution method): Positive cells were seeded in 96-well plates and diluted to densities of 32, 16, 8, 4, 2, and 1 cells / well using the limiting dilution method, with 100 μL per well and two columns for each density. After 8 days of culture, the supernatant was collected to detect antibodies. This process was repeated twice until the antibody positivity rate of a single clonal well reached 100%. Finally, the cells were transferred to 6-well plates to prepare for subtype identification.

[0049] Antibody titer identification: The supernatant of cloned cells was collected by ELISA to detect antibody titer.

[0050] Antibody subtype detection: The antibody subtype in cell supernatant was identified using a double-antibody sandwich method. For specific procedures, please refer to the instructions of the ready-to-use kit for monoclonal antibody subtype identification.

[0051] Inducing ascites in mice: sensitization with ascites adjuvant was performed 15 days in advance. 15 days later, each cell line was injected into two mice, with each mouse receiving 1M cells. Ascites was collected after about a week of close observation starting three days later.

[0052] Antibody titer identification: Antibody titer was detected in mouse ascites fluid using ELISA.

[0053] Antibody purification: The antibody was purified using G protein media chromatography, and the purity of the purified antibody was detected by SDS-PAGE electrophoresis.

[0054] HRP-labeled antibodies: HRP is activated by sodium periodate oxidation, and the activated HRP reacts with the antibody via a Schiff base reaction to label the antibody.

[0055] Screening for paired antibodies: ELISA assay using the orthogonal checkerboard method.

[0056] 3. Experimental Results 3.1 ELISA test results After three immunizations, the OD values ​​of the blank control group and the experimental group were compared as shown in the figure. Figure 1 It can be seen that the serum of experimental mice 1, 2, and 3 were significantly different from those of the control mice and PBS, respectively. P <0.001), indicating that mice 1, 2, and 3 all produced polyclonal antibodies after three immunizations.

[0057] 3.2 Affinity chromatography separation and purification results from Figure 2 and Figure 3 As can be seen from the results, the target product, namely the purified IgG antibody, is present in the elution buffer. The gel electrophoresis results show that compared to the crude extract and the flow-through fraction, the elution fraction has only one band, indicating a single protein type, and the purification operation is complete.

[0058] 3.3 Screening results for paired antibodies Table 1. Statistics of results from the orthogonal chessboard method Note: In the table, / indicates that antibodies with the same number are not paired.

[0059] As shown in Table 1, the results of the orthogonal checkerboard method show that when paired antibodies are subjected to reciprocal crosses, antibody pairs with similar OD values ​​and all greater than 2.0 are selected, resulting in a total of 4 antibody pairs. Among them, 3 and 5 are paired antibodies; 6 and 5 are paired antibodies; 9 and 6 are paired antibodies; and 11 and 9 are paired antibodies.

[0060] Example 2: Preparation of HPV16 test strips and kits 1. Microsphere activation (1) 12.5 μL of colored latex microspheres with a concentration of 41 mg / mL (purchased from Suzhou Weidu Biotechnology Co., Ltd.) was added to an ultrasonicator and sonicated for 5 min. Then, 1 mL of 50 mM MES solution was added, and after uniform mixing, the mixture was centrifuged at 13000 rpm for 20 min. The supernatant was discarded and the precipitate was retained to complete the first cleaning of the colored latex microspheres. 1 mL of 50 mM MES solution was added again, and after uniform mixing, the mixture was centrifuged at 13000 rpm for 20 min. The supernatant was discarded and the precipitate was retained to complete the second cleaning of the colored latex microspheres.

[0061] (2) Add 200 μL of 50 mM MES solution and mix well. Sonicate for 5 min. Add 15 μL of 1% NHS prepared from 50 mM MES solution to the sonicated colored latex microspheres. Then add 2 μL of 1% EDC. Wrap the centrifuge tube with tin foil and place it in a roller mixer to react in the dark for 15 min.

[0062] Microsphere activation result detection: Remove the colored latex microspheres from the roller mixer and check for black precipitate. If there is no precipitate, it proves that the colored latex microspheres have been successfully activated.

[0063] 2. Antibody labeling (1) Take 0.1 mg of HPV16 E7 monoclonal antibody and chicken IgY prepared in Example 1 and add them to the activated colored latex microspheres above, and rotate them in the dark for 2 h.

[0064] (2) Add 10 μL of 0.1 M ethanolamine solution, mix well, rotate in the dark for 30 min, centrifuge at 13000 rpm for 10 min, and discard the supernatant.

[0065] (3) Add 500 μL of microsphere dilution solution (0.01 mol / L PBS + 1% BSA), mix well, sonicate for 5 min, wrap with tin foil and place in a roller mixer to react in the dark for 1 h, centrifuge at 13000 rpm for 10 min, and discard the supernatant.

[0066] (4) Add 200 μL of microsphere diluent, mix well, sonicate for 5 min, and store at 4℃.

[0067] Observe the microspheres labeled with HPV16 E7 monoclonal antibody (HPV16 E7 Ab-1) and the microspheres labeled with chicken anti-IgY after ultrasonic mixing, such as Figure 4 As shown, the solution was uniform in color and free of precipitate, indicating that HPV16 E7 monoclonal antibody-labeled microspheres and chicken anti-IgY-labeled microspheres were successfully prepared.

[0068] 3. Treatment of gold label pads and sample pads (1) Sample pad treatment Immerse the sample pad in the treatment solution (0.03 mol / L PBS + 0.5% SDS + 1% BSA) for 10 min, then remove it, dry it at 37℃ for 30 min, and store it at 4℃ for later use.

[0069] (2) Treatment of gold-plated bonding pads The gold-labeled conjugate pads were immersed in a gold-labeled conjugate pad treatment solution (0.05 mol / L Tris + 0.5% BSA + 0.1% Tween 20 + 5% trehalose) for 2 hours. After 2 hours, the pads were removed, dried at 37°C for 30 minutes, and then stored at 4°C for later use.

[0070] 4. Membrane scratching The secondary antibody (HPV16 E7 Ab-2) and the goat anti-chicken IgY antibody were diluted with antibody diluent to a final antibody concentration of 1.0 mg / mL. Then, they were streaked on the NC membrane at a speed of 1 μL / cm. After the streaking operation was completed, the membrane was dried at 37℃ for 15 min for later use.

[0071] like Figure 5 As shown, a 30 cm NC film was cut and pasted in the center of a PVC base plate. Then it was placed on a film-scribing and gold-spraying machine. The T line containing 1 mg / mL HPV16 E7 Ab-2 was placed at the longer end, and the C line containing 1 mg / mL goat anti-chicken IgY was placed at the shorter end. The scribing volume of the T line and the C line was 1 L / cm.

[0072] 5. Gold spraying (1) Take the antibody-labeled HPV16 E7 Ab-1 microspheres and chicken anti-IgY microspheres and mix them in an EP tube at a ratio of 8:3 to obtain a complex. Sonicate the complex to make it evenly mixed. Then mix the sonicated complex with gold spray diluent at a ratio of 2:3. Then adjust the pH of the mixed solution to 9 with K2CO3 to obtain the gold spray mixture.

[0073] (2) The gold sputtering mixture was sonicated for 5 min, and then gold was sputtered onto the prepared gold-labeled pad at a rate of 4 μL / cm. After drying for 1 h, it was sealed in a foil bag with desiccant at 4℃ for later use.

[0074] The above gold spray diluent formula is as follows: Gold sputtering diluent: 0.02% borate buffer, 0.2% PEG20000, 0.02% proclin300, 10% trehalose, 0.25% Tween 20, 1% BSA.

[0075] like Figure 6 As shown, the gold plating mixture on the gold-coated pads after plating and drying is relatively uniform.

[0076] 6. Assembly and Segmentation like Figure 7 As shown, the microparticle chromatography test strip is composed of a PVC substrate, an absorbent pad, an NC membrane, a gold label pad, and a sample pad stacked sequentially. The specific assembly steps are as follows: like Figure 8 The microparticle detection plate shown has a polyvinyl chloride (PVC) sheet as its bottom layer, which is divided into three parts: first, absorbent filter paper is attached to the short end; second, an NC film with C and T lines embedded between the two ends is attached; and third, a sample pad and a gold-labeled binding pad are attached to the long end. Figure 9As shown, the assembled breast microparticle detection plate is placed on a programmable slicer, and the slice is cut to a width of 0.4 cm by using the slice feeding function to closely adhere it to the incision, thus obtaining the HPV16 antigen test strip.

[0077] 7. Assembly of HPV antigen detection kit Assemble the HPV16 antigen test strip and the test kit to form the HPV16 antigen test kit. For example... Figure 10 As shown, the HPV antigen test kit consists of an HPV16 antigen test kit, 80 μL of sample buffer, and a sampling swab.

[0078] The HPV16 antigen test strips were analyzed using a 25 μg / mL HPV 16 antigen dilution solution and sample buffer.

[0079] The results are as follows Figure 11 As observed, only the C line of the sample buffer showed a color change, while the 25 μg / mL HPV 16 antigen dilution solution showed color changes in both the C and T lines of the HPV 16 antigen test strip. These results indicate that the HPV 16 antigen test strip was successfully prepared.

Claims

1. A monoclonal antibody for detecting HPV16 E7 protein, characterized in that, The monoclonal antibody was prepared by the following steps: Mice were immunized three times with HPV16 E7 protein as the antigen, with each mouse receiving a single injection of 52-58 μg of antigen into the neck. Spleen cells and myeloma cells from immunized mice were fused at a ratio of 4 to 6:1, and positive hybridoma cells were screened after 5 to 9 days of culture. Clonalized hybridoma cells were cultured to obtain hybridoma cell lines secreting antibodies against HPV16 E7 protein. Each mouse was injected with 1×10⁻⁶ cells. 5 cell ~1×10 7 The mouse hybridoma cell line was fed normally for one week, and ascites fluid was extracted from the mice and purified to obtain the monoclonal antibody.

2. The monoclonal antibody according to claim 1, characterized in that, In the three immunizations, the interval between the first and second immunizations is 3 to 4 weeks, and the interval between the second and third immunizations is 2 to 3 weeks.

3. The monoclonal antibody according to claim 1, characterized in that, Before mixing the spleen cells with myeloma cells, the titer of the anti-HPV16 E7 protein antibody in mouse serum needs to be detected.

4. The monoclonal antibody according to claim 1, characterized in that, The cell fusion culture requires a half-medium change every 2 to 3 days.

5. The use of the monoclonal antibody according to claim 1 in the preparation of an immunoassay product for detecting HPV16.

6. An HPV16 test strip, characterized in that, The test strip comprises a PVC base plate, on which, in the order of sample flow, are a sample pad, a gold-labeled pad, an NC membrane, and absorbent filter paper; the gold-labeled pad is coated with the monoclonal antibody of claim 1 labeled with colored latex microspheres, and the detection T line of the NC membrane is coated with a secondary antibody containing the monoclonal antibody to be tested.

7. The HPV16 test strip according to claim 6, characterized in that, The gold-labeled pad is also coated with chicken IgY antibody labeled with colored latex microspheres; the quality control C line of the NC membrane is coated with a secondary antibody containing chicken IgY antibody, wherein the secondary antibody is goat anti-chicken IgY.

8. The HPV16 test strip according to claim 7, characterized in that, The detection T line and quality control C line of the NC membrane are coated with secondary antibody at a concentration of 0.8 mg / mL to 1.2 mg / mL.

9. The HPV16 test strip according to claim 6, characterized in that, The gold-labeled pad is obtained by ultrasonically mixing HPV16 antibody labeled with gel microspheres and chicken IgY antibody labeled with latex microspheres and then spraying the mixture onto a gold-labeled pad that has been soaked in a treatment solution.

10. An HPV16 detection kit, characterized in that, The kit consists of an HPV16 antigen detection kit assembled from the HPV16 test strip and detection box as described in claim 6, a sample buffer, and a sampling swab.

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