CN115856287A - A test paper for rapid detection of HPV antigen and antibody - Google Patents

Abstract

The invention discloses a test paper for rapidly detecting HPV antigens and antibodies. The test paper comprises a sample pad, a colloidal gold labeling pad, a detection reaction area and a water absorption pad which are sequentially arranged along the chromatography direction, wherein the colloidal gold labeling pad contains an antibody-colloidal gold marker for resisting HPV L1 antigen. The detection reaction area is provided with a detection band and a quality control line, the detection band contains HPV L1 antigen, and the quality control line contains antibody capable of being combined with HPV antibody. The test paper is convenient and quick to use, low in cost and suitable for home self-inspection, clinical quick diagnosis and large-scale application in basic epidemiological investigation fields.

Description

A test paper for rapid detection of HPV antigen and antibody

technical field

The invention belongs to the field of immunoassay detection reagents, and relates to a test strip (card) that can be used for rapid detection of both HPV antigen and HPV antibody.

Background technique

Cervical cancer is a common gynecological malignancy, and its incidence rate ranks second among female tumors. The number of new cases in the world is about 470,000 each year, and it causes 300,000 deaths. It is second only to breast cancer. leading cause of death for women. The etiology of cervical cancer is related to many factors, among which human papillomavirus (human papillomavirus, HPV) infection is the most important factor. According to reports, 99.8% of cervical cancers are combined with HPV infection, while HPV-negative patients hardly occur cervical cancer. At present, there is no effective treatment for HPV infection. Prevention of HPV infection and regular detection of infected people are the main ways to reduce the incidence of cervical cancer.

HPV belongs to the papillomaviridae family and is a double-stranded closed circular DNA virus that phagocytizes mucous membranes and skin. So far, more than 120 kinds of HPV have been reported and discovered, of which more than 40 are related to human reproductive tract infection. Among the high-risk HPV types, HPV16, HPV18, HPV31, and HPV33 are most closely related to the pathogenesis of cervical cancer. The detection rate of HPV16 and HPV18 in cervical cancer is over 70%. HPV16 is more common in cervical squamous cell carcinoma. HPV18 is more common in cervical adenocarcinoma; low-risk HPV is mostly found in benign lesions such as condyloma.

The detection of HPV mostly focuses on histopathology or DNA detection of HPV virus. These methods have limitations such as time-consuming, professional technicians and equipment, complex operating procedures, high cost, and difficulty in popularization. It has been reported in the literature that the monoclonal antibody and polyclonal antibody of HPV16E6 protein were used to prepare colloidal gold diagnostic test strips (Hu Renjian. Development of high-risk human papillomavirus type 16 immune colloidal gold diagnostic test strips. Chongqing University of Technology, 2009), which is currently in the trial In the paper strip, the mouse anti-HPV16E6 monoclonal antibody is used as the gold-labeled antibody to coat the binding pad, and the rabbit anti-HPV16E6 polyclonal antibody is coated on the detection line. However, both of these two antibodies use HPV16E6 as the antigen, and the antigen-antibody binding site is the same epitope. Therefore, the binding ability of the sample HPV antigen combined with the gold-labeled antibody to the rabbit anti-HPV16E6 polyclonal antibody on the detection line is limited, making the test strip The detection sensitivity is limited to a certain extent. The test strip (card) of the double-antibody sandwich method in the Chinese patent CN108761091A uses the main capsid protein of HPV, that is, HPV L1 as the antigen, and uses the anti-human IgG antibody as the antigen at the same time, thereby realizing the detection of the HPV antibody in the sample. Sensitive and accurate detection of HPV L1 IgG antibodies. Although HPV antigen and HPV antibody in blood or serum are markers for in vitro diagnosis of HPV virus infection, the test strip (card) cannot detect HPV antigen in cervical cells. Therefore urgently need to propose a kind of HPV high-risk type virus rapid detection test paper that has advantages of convenience and quickness, low cost, simple and reliable, easy to observe the difference, suitable for home self-examination, and easy to popularize.

There is no report on the test strips for rapid detection of HPV virus (virus antigen and antibody) based on immunocompetitive technology.

Contents of the invention

The purpose of the present invention is to provide a test paper for rapid detection of HPV antigen and antibody, which is convenient and quick to use, accurate and reliable in results, and has high detection efficiency for high-risk HPV viruses.

To achieve the above object, the present invention adopts the following technical solutions:

A test paper for detecting HPV antigens and HPV antibodies, the test paper includes sample pads, colloidal gold marker pads, detection reaction areas and water-absorbent pads arranged in sequence along the chromatography direction, and the detection reaction area includes detection belts and quality control lines; colloid The gold marker pad includes an antibody against the HPV L1 antigen-colloidal gold marker, the detection zone includes the HPV L1 antigen, and the quality control line includes an antibody that can bind to the HPV antibody (ie, the antibody against the HPV L1 antigen).

Preferably, the detection reaction area further includes a nitrocellulose membrane, and the antibody that binds to the HPV antibody and the HPV L1 antigen are coated on the nitrocellulose membrane.

Preferably, the colloidal gold labeling pad further includes a glass fiber membrane or a polyester membrane, and the anti-HPV L1 antigen antibody-colloidal gold marker is spread and adsorbed on the glass fiber membrane or polyester membrane.

Preferably, the HPV L1 antigen is derived from HPV16 or HPV18.

Preferably, the anti-HPV L1 antigen antibody is one or more of polyclonal antibodies against HPV L1 antigen derived from mouse, rabbit, goat, horse, camel or guinea pig.

Preferably, the antibody that binds to the HPV antibody refers to an antibody that can form an immune complex by combining with the HPV antibody (ie, the antibody against HPV L1 antigen).

The preparation method of the test paper for above-mentioned detection HPV antigen and HPV antibody, comprises the following steps:

Step 1 Preparation of Colloidal Gold Labeling Pad

Use the colloidal gold solution to label the antibody against the HPV L1 antigen to obtain a colloidal gold marker (ie, the antibody-colloidal gold marker against the HPV L1 antigen) solution; coat the colloidal gold marker solution on a glass fiber membrane or a polyester membrane After drying, set aside;

Step 2: Preparation of detection reaction zone

The solution of the HPV L1 antigen solution and the antibody that can be combined with the HPV antibody (ie, the antibody against the HPV L1 antigen) is coated on two different areas on the nitrocellulose membrane and then dried to obtain a detection band and a quality control line. spare;

Step 3 Preparation of sample pads

Dip the glass fiber membrane or polyester membrane in the treatment solution containing blocking agent and then dry it for later use; or use the glass fiber membrane or polyester membrane directly without dipping and drying; the sample pad obtained after treatment with blocking agent , can inhibit non-specific binding, thereby reducing the false positive rate of the test paper.

Step 4 Install the sample pad, colloidal gold marker pad, detection reaction area and water-absorbent pad on the bottom plate in sequence, and then cut according to the specifications of the test paper (such as test strips, test paper cards).

Preferably, in the step 1, the preparation method of the colloidal gold marker solution specifically includes the following steps: 2-200mL colloidal gold solution (10-100nm in particle size) is adjusted to pH 5.5-5.5 with 0.03-0.3M potassium carbonate solution. 7.0, then mixed with 50-100 μg of anti-HPV L1 antigen antibody, then reacted at 18-30°C for 10-30 minutes, and then added BSA (bovine serum albumin) to the reaction system to a final concentration of 0.5%-1% ( g/mL), then let stand for 5-10 minutes, then centrifuge at 2-25°C, 1000-12000rpm for 5-30 minutes and collect the supernatant, then centrifuge the supernatant at 2-25°C, 5000-15000rpm for 5 ~ 60 minutes and collect the precipitate, which was dissolved in 0.5 ~ 5 mL of PBS (pH 6.0 ~ 7.5) containing 0.1% ~ 10% (g/mL) BSA.

Preferably, in the step 1, the coating condition of the colloidal gold marker solution is: dilute the colloidal gold marker solution with PBS (pH 6.0-7.5) containing 0.1%～10% (g/mL) BSA for 1～ After 2 times, spray at 40-80μL/cm ² on the glass fiber membrane or polyester membrane; the drying conditions are: 18-24 hours at 35-40°C.

Preferably, in the step 2, the concentrations of the HPV L1 antigen solution and the antibody solution that can bind to the HPV antibody are respectively 0.5-4 mg/mL, and the solvents are all PBS (pH 6.0-7.5); the drying conditions are: 35 18-24 hours at ~40°C.

A method for detecting HPV, using the test paper prepared by the above steps to detect the sample to be tested, specifically comprising the following steps:

Step 1 Sample Processing

Dilute serum or plasma 1 to 100 times with a diluent (the diluent is water, 0.9% sodium chloride solution, phosphate buffer or borate buffer) and add 50 to 150 μL to the sample pad of the test paper (i.e. sample addition); or, take 2-200 μL of whole blood sample and dilute 1-100 times immediately after sample addition (that is, add the required volume of sample buffer, such as phosphate buffer); or, the cervical sample after sampling Place the epithelial swab in 0.5-2 mL of sample preservation solution (such as phosphate buffered saline) and mix thoroughly, and take 50-150 μL of the obtained cervical epithelial sample to add the sample;

Serum sampling conditions are as follows: take 1-5 mL of whole blood in a serum collection tube, let it stand for 1-2 hours, centrifuge at 3000-5000 rpm for 10-30 minutes, and take the supernatant;

Plasma sampling conditions are as follows: take 1-5 mL of whole blood in a sodium citrate or sodium heparin anticoagulant tube, centrifuge at 1000-3000 rpm for 10-30 minutes, and take the supernatant;

Step 2: Add the treated sample to the sample pad of the test paper and let the test paper stand for 5 to 20 minutes, then observe the detection reaction area of the test paper;

Step 3 Interpretation of results

Both the test band and the quality control line show color (the color of the test band is the same or close to the quality control line, and the color is darker), and the result is negative (if it is a sample from blood, it means that the antibody is negative, if it is from cervical epithelium sample, it means antigen-negative); the test strip does not show color or shows a lighter color (compared to the quality control line) and the quality control line shows color (darker color), and the result is positive (if it is a blood-derived sample, it means Antibody positive, if it is a sample from cervical epithelium, it means antigen positive); the quality control line does not show color, indicating that the test strip is invalid.

The beneficial effects of the present invention are reflected in:

In the present invention, the test paper based on the competition method is applied to the detection of HPV antibody and HPV antigen in samples (blood-derived samples and cervical epithelial-derived samples) for the first time, and the test paper is convenient and quick to use, and the cost is low, and only one test paper is used for detection , can judge the situation of human infection with the corresponding HPV virus (virus antigen and antibody), provide rapid auxiliary diagnosis for cervical cancer screening and treatment, suitable for home self-examination and clinical rapid diagnosis of hospitals, disease control departments, and basic health units Large-scale application in the field of epidemiological investigation.

Further, the present invention uses the competition method to detect the amount of the gold-labeled antibody that is not bound to the HPV antigen in the sample (cervical epithelial sample) by detecting the band interception (binding), and then reflects the HPV antigen concentration in the sample, avoiding the use of the same antibody in the literature reports. The antigen-antibody binding reaction of the binding site limits the detection sensitivity, and makes the test paper meet the requirements for HPV antigen and antibody detection in clinical diagnosis.

Furthermore, the HPV antigen used in the present invention is HPV L1 protein, which does not contain complete virus invasion and replication components, and is not pathogenic.

Detailed ways

The present invention is described in further detail below in conjunction with embodiment. The examples are only used to explain the present invention, not to limit the protection scope of the present invention.

(1) Preparation of HPV L1 antigen and antibody

1.1 Preparation of HPV L1 antigen:

The whole gene of HPV16 L1 and the whole gene of HPV18 L1 were respectively constructed in the pQE80L plasmid by gene cloning technology, and the primers were as follows:

HPV16L1 forward primer-Sph1: CATGCATGCCAGGTGACTTTTATTTACATC

HPV16L1 reverse primer-Sac1: CGAGCTCCAGCTTACGTTTTTTGCGTTT

HPV18L1 forward primer-Sph1: CATGCATGCGCTTTGTGGCGGCCTAGT

HPV18L1 reverse primer-Sac1: CGAGCTCCTTTCTGGCACGTACACGC

Using the total DNA of the extracted human cervical cancer cell line (obtained from the China Center for Type Culture Collection) as a template, the above primers were used to amplify the target gene. The constructed recombinant plasmids were respectively expressed in the prokaryotic expression system BL21, and purified by nickel column affinity chromatography to obtain HPV16 L1 protein and HPV18 L1 protein.

1.2 Preparation of HPV L1 antibody:

Mix HPV16 L1 protein or HPV18 L1 protein at a concentration of 0.5-2 mg/mL with Freund's immune adjuvant at a volume ratio of 1:1 and immunize BALB/c mice. The HPV16 L1 protein or HPV18 L1 protein immunized in each mouse The amount of protein is 100 μg, boost immunization twice with the same dose on the 14th and 28th day after the initial immunization, collect mouse serum on the 35th day after the initial immunization, and use rProteinG affinity purification to prepare polyclones of HPV16 L1 protein respectively Antibodies (ie, antibodies against HPV16 L1 antigen) and polyclonal antibodies to HPV18 L1 protein (ie, antibodies against HPV18 L1 antigen).

(2) Detection of HPV L1 antibody titer

(1) First, 1 μg of HPV16 L1 protein and HPV18L1 protein were used to coat the 96-well ELISA plate, and the coated plate was incubated overnight at 4°C.

(2) Each well was washed 6 times with PBST washing buffer containing 0.05% Tween-20, and 100 μL of blocking solution (PBST solution containing 5% skimmed milk powder) was added to each well to block for 2 hours.

(3) Add 100 μL of corresponding HPV L1 antibody (anti-HPV16 L1 antigen antibody or anti-HPV18 L1 antigen antibody) sample diluted 200 times with PBST solution to each well, react at 37°C for 1 hour, and then wash each well with PBST solution 4 times, 5 minutes each time.

(4) Add 100 μL horseradish peroxidase-coupled goat anti-mouse IgG antibody to each well, react at 37°C for 1 hour, and then wash each well 6 times with PBST solution to remove unbound enzyme-labeled antibody.

(5) Add 100 μL of TMB color developing solution to each well, and carry out color developing reaction at room temperature for half an hour.

(6) Add 50 μL of reaction termination solution (2M sulfuric acid solution) to each well to terminate the color reaction, measure the absorbance at 450 nm wavelength with a microplate reader after 5 minutes, and record the results.

The serum antibodies of 6 mice immunized according to the method described in (1) and a negative control mouse without immunization were measured, and 3 of them were absorbed by the 450nm wavelength of serum antibodies of HPV16 L1 protein immunized mice The values were 0.726, 1.258, 0.915 respectively, the absorption values at 450nm wavelength of the serum antibodies of the three mice immunized with HPV18 L1 protein were 0.612, 0.834, 0.793, and the absorption values of the serum antibodies of the negative control mice were 0.049. It can be judged that immunizing mice with HPV L1 protein can activate the immune system of mice and produce corresponding specific antibodies to HPV16 L1 protein and HPV18 L1 protein.

(3) Preparation of rapid detection HPV antigen and antibody test strips (cards)

(1) Preparation of colloidal gold: Auric chloride (HAuCl ₄ ) is made into 1% auric acid solution, takes 1mL of the auric acid solution, adds 99mL deionized water, mixes and heats to boiling, then immediately adds 2mL of 1% The sodium citrate solution in the mixture was stirred rapidly, continued heating for 10 minutes, cooled to room temperature, added deionized water to 100mL, and obtained a colloidal gold solution (average particle diameter of 30nm) with an absorbance value greater than 0.7.

(2) Preparation of colloidal gold markers: take 10 mL of prepared colloidal gold solution, adjust the pH value to 6.5 with 0.1 M potassium carbonate solution, add 50 μg of anti-HPV16 L1 antigen antibody or anti-HPV18 L1 antigen antibody, mix Mix well, react at room temperature for 30 minutes, then add BSA to a final concentration of 0.5% (g/mL), mix and let stand for 10 minutes, centrifuge at 4°C, 2000rpm for 20 minutes, discard the precipitate, and centrifuge the supernatant at 4°C, 12000rpm for 60 The supernatant was discarded, and the precipitate was dissolved in 1 mL of PBS (pH6.5) solution (containing 0.2% BSA) to obtain a colloidal gold marker solution.

(3) Preparation of gold standard pad: Take the colloidal gold marker solution and dilute it with PBS (pH6.5) solution (containing 0.2% BSA) to 1 time, and then spray (use a sprayer) glass fiber membrane according to 60 μL/ ^cm2 , Spread the colloidal gold marker evenly on the glass fiber membrane, and then dry it at 37° C. for 18 hours under a humidity of 30%. The dried gold standard pad should be sealed with a tin foil bag (with desiccant inside), and stored at room temperature.

(4) Coating of detection band and quality control line: Dilute HPV L1 antigen (specifically HPV16 L1 protein or HPV18 L1 protein) with PBS (pH6.5) solution to a concentration of 1 mg/mL, and streak on the NC membrane (or sprayed on the NC membrane), and the corresponding HPVL1 antigen was solidified (dried at 37° C. for 18 hours) on the NC membrane as the detection zone T. At 6 mm away from the detection zone T, draw another line with 1 mg/mL (diluted in PBS solution with pH 6.5) that can bind to HPV antibody (goat anti-mouse antibody purchased from Hangzhou Xianzhi Biology) (or spray on NC film), cured (dried at 37°C for 18 hours) as quality control line C.

(5) Pretreatment of the sample pad (blocker treatment)

Another piece of glass fiber membrane was prepared, soaked in a treatment solution containing 10% rabbit serum (solvent: PBS with pH 6.5) at room temperature for 30 minutes, and dried at 37° C. for 1 hour after taking it out.

(6) Assembly of test strips (cards)

The test paper consists of sequentially connected sample pads, gold standard pads, nitrocellulose membranes (NC membranes) and additionally prepared absorbent pads (such as absorbent filter paper). The upper section is the water-absorbing pad, the middle section is the detection reaction area on the nitrocellulose membrane (including the detection band T and the quality control line C), and the gold standard pad adsorbed with colloidal gold markers is placed between the middle and lower sections; the lower section is the sample pad The whole test paper is attached to the center of the PVC board, cut into strips with a width of 4mm, and the test paper strips are obtained, sealed and dried, and stored for later use.

In addition, the test strip is put into the casing composed of the upper plate and the lower plate, the sample pad part of the test strip is facing the sample hole provided on the upper plate, and the detection reaction area part is facing the upper plate. If there is a detection window, the test paper card can be obtained.

(4) Process of sample processing, sample addition and interpretation

Serum sample: Take 1mL of whole blood in a serum collection tube, let it stand for 1 hour, centrifuge at 5000rpm for 20 minutes, and take the supernatant. According to the detection accuracy of the test strip (card), the serum sample was diluted 1 to 10 times with sample buffer (PBS solution at pH 6.5) or deionized water, and then 100 μL was added dropwise to the test strip (card) On the sample pad part, observe the color development result of the test strip (card) detection reaction zone part after standing for 10 minutes.

Plasma sample: Take 1mL of whole blood in a sodium citrate or sodium heparin anticoagulant tube, centrifuge at 2000rpm for 20 minutes, and take the supernatant. According to the detection accuracy of the test strip (card), dilute the plasma sample 1 to 10 times with sample loading buffer (PBS solution at pH 6.5) or deionized water, and then take 100 μL and add it dropwise to the test strip (card) On the sample pad part, observe the color development result of the test strip (card) detection reaction zone part after standing for 10 minutes.

Whole blood sample: Take about 50 μL of fresh blood from the fingertip or earlobe, drop it on the sample pad part of the test strip (card), and immediately add it dropwise with 50 μL sample buffer (PBS buffer at pH 6.5) or deionized water Dilute to the sample pad and observe the result after standing for 10 minutes.

Cervical epithelial sample: take a vaginal swab to take cervical epithelial tissue, then put the vaginal swab into 1mL sample preservation solution (PBS solution with pH 6.5) or deionized water, mix well (1-2 minutes), add 100μL Put it on the sample pad part of the test strip (card), and observe the color development result of the test strip (card) detection reaction zone after standing for 10 minutes.

(5) Detection principle of test strip (card)

5.1 Principle of detection of HPV antigen

Add the sample to be tested (cervical epithelial sample) to the sample pad, and then the HPV L1 antigen in the sample and the colloidal gold marker on the gold pad (colloidal gold-labeled anti-HPV16 L1 antigen antibody or anti-HPV18 L1 antigen antibody, Colloidal gold-labeled HPV L1 antibody for short) forms an immune complex. Due to the capillary effect, the immune complex continues to swim toward the water-absorbing pad, because the binding site of the colloidal gold-labeled HPV L1 antibody is blocked by the HPV L1 antigen in the sample. Occupied, so the immune complex cannot react with the corresponding HPV L1 antigen coated on the detection zone T, and the detection zone T does not show purple or light color; the immune complex continues to move forward due to capillary action Swimming, and having a specific immune reaction with the antibody coated on the quality control line C that can bind to the HPV antibody is intercepted, and gradually enriched on the quality control line C to form a darker purple band. Unbound substances continue to be chromatographed on the absorbent pad, so it is only judged as a positive result of HPV antigen of cervical cells when a colored band appears on the quality control line C; if the sample to be tested does not contain HPV L1 antigen, colloidal gold-labeled The HPV L1 antibody reacts immunologically with the HPV L1 antigen immobilized on the detection zone T, so a colored band appears on the detection zone T, and the colloidal gold-labeled HPV L1 antibody on the excess gold pad continues to swim forward. The specific immune reaction with the antibody coated on the quality control line C that can bind to the HPV antibody is intercepted, and gradually enriched on the quality control line C to form a purple band, so between the detection band T and the quality control line When color bands appear in C, it is judged that the cervical cells are negative for HPV antigen.

5.2 Principle of detection of HPV antibody

Add the sample to be tested (serum, plasma or whole blood sample) to the sample pad, and then the HPV L1 antibody in the sample and the colloidal gold marker on the gold pad (colloidal gold-labeled anti-HPV16 L1 antigen antibody or anti-HPV18 L1 The antibody to the antigen, referred to as the colloidal gold-labeled HPV L1 antibody) forms a competitive relationship. Due to the capillary effect, all the HPV L1 antibodies swim in the direction of the water-absorbing pad, and the HPV L1 antibody that forms a competitive relationship and the corresponding HPV L1 antibody that is solidified on the detection zone T Competitive binding of HPV L1 antigen, the higher the concentration of HPV L1 antibody in the sample, the more obvious its competitive advantage, and the colloidal gold-labeled HPV L1 antibody on the gold label pad will react with the corresponding HPV L1 antigen immobilized on the test strip T Weaker, the lighter the purple color of the detection band T; due to capillary action, the HPV L1 antibody labeled with colloidal gold is specific to the antibody that can bind to the HPV L1 antibody coated on the quality control line C The positive immune reaction is intercepted and gradually enriched on the quality control line C to form a darker purple-red band, and the excess unbound substances continue to be chromatographed on the water-absorbing pad, so the detection band T shows light or no color and The color-developed band on the C line of the quality control band is judged to be a positive result of HPV antibody in the blood; if the sample to be tested does not contain HPV L1 antibody, the colloidal gold-labeled HPV L1 antibody on the gold label pad and the HPV L1 antibody solidified on the test band T The corresponding HPV L1 antigen had a strong immune reaction and was intercepted, and the purple-red color of the detection band T was obvious; due to capillary action, the colloidal gold-labeled HPV L1 antibody and the coated on the quality control line C Antibodies that can bind to the HPV L1 antibody undergo a specific immune reaction and are trapped and gradually enriched on the quality control line C to form a darker purple band. Therefore, when bands appear on both the detection band T and the quality control line C It was judged as a negative result of HPV antibody in the blood.

5.3 Combined interpretation of antigen and antibody

The test strip (card) of the present invention can detect both the HPV antigen of cervical cells and the HPV antibody in the blood, that is, two important markers of human infection with HPV virus can be detected on a test paper product. The results also have more detailed clinical guidance:

(1) For the same test individual, the HPV antigen in the cervical cells and the HPV antibody in the blood are both positive, indicating that the human body is infected by HPV and the body produces corresponding antibodies;

(2) For the same test individual, the HPV antigen of the cervical cells is positive, but the HPV antibody in the blood is negative, indicating that the human body is infected by HPV and the body has not yet produced the corresponding antibody;

(3) For the same test individual, the HPV antigen of the cervical cells is negative, but the HPV antibody in the blood is positive, indicating that the human body has been infected by HPV but the antibody produced by the body has eliminated HPV;

(4) For the same test individual, the HPV antigen in the cervical cells and the HPV antibody in the blood are both negative, indicating that the human body has not been infected by HPV before and now, and the body has no corresponding antibody.

(6) False positive rate and false negative test experiment of HPV antibody detection

(1) Serum sample collection

50 cases of healthy people under the age of 15, 50 cases were injected with HPV vaccine; 1mL of whole blood was taken from each case in a serum collection tube, allowed to stand for 1 hour, centrifuged at 5000g for 10 minutes, and the supernatant was taken to obtain a serum sample.

(2) False positive rate detection

Dilute 100 μL of serum samples from healthy people under the age of 15 with 100 μL of deionized water, take 100 μL and drop it on the sample pad of the test strip (HPV16), let it stand for 10 minutes, observe the detection band T and the quality control line C; determine the presence of HPV antibodies Condition.

The judgment rules are as follows:

Negative: The test band T and the quality control line C both show purple bands, which means that the sample does not contain HPV antibodies;

Positive: Only the quality control line C shows a purple-red band (or the quality control line C shows a purple-red band and the test band T shows a lighter color), then it is determined that the sample contains HPV antibodies;

Invalid: if the quality control line C does not show a purple-red band, no matter whether the detection band T shows a purple-red band or not, the test strip is judged invalid;

The results showed that 1 serum sample from a healthy person under the age of 15 was positive, and 49 were negative, all of which were effective. The results showed that the test strips had a 2% false positive rate for HPV antibody detection.

(3) False negative rate detection

Dilute 100 μL of the serum sample of the person injected with HPV vaccine with 100 μL of deionized water, take 100 μL and drop it on the sample pad of the test strip (HPV16), let it stand for 10 minutes, observe the detection band T and the quality control line C; determine the presence of HPV antibodies .

The judgment rules are as follows:

Negative: The test band T and the quality control line C both show purple bands, which means that the sample does not contain HPV antibodies;

Positive: Only the quality control line C shows a purple-red band (or the quality control line C shows a purple-red band and the test band T shows a lighter color), then it is determined that the sample contains HPV antibodies;

Invalid: if the quality control line C does not show a purple-red band, no matter whether the detection band T shows a purple-red band or not, the test strip is judged invalid;

The results showed that 1 serum sample from the HPV vaccine recipients was negative and 49 were positive, all of which were effective. The results showed that the test strips had a 2 percent false negative rate for HPV antibody detection.

(7) False positive rate and false negative test experiment of HPV antigen detection

(1) Collection of cervical epithelial samples

50 cases of healthy people under the age of 15 and 50 cases of HPV16 patients; each case took a vaginal swab to take cervical epithelial tissue (rotate 3 to 5 circles), put it into 1mL sample preservation solution (PBS solution with pH 6.5), and mix well. Homogenize the cervical epithelial sample.

(2) False positive rate detection

Drop 100 μL of cervical epithelial samples of healthy people under the age of 15 on the sample pad of the test strip (HPV16), let it stand for 10 minutes, observe the test band T and the quality control line C; determine the carrying status of HPV antigen.

The judgment rules are as follows:

Negative: The test band T and the quality control line C both show purple bands, then it is determined that the sample does not contain HPV antigen;

Positive: Only the quality control line C shows a purple-red band (or the quality control line C shows a purple-red band and the test band T shows a lighter color), then it is determined that the sample contains HPV antigen;

Invalid: if the quality control line C does not show a purple-red band, no matter whether the detection band T shows a purple-red band or not, the test strip is judged invalid;

The results showed that 1 sample of cervical epithelium of healthy people under 15 years old was positive, and 49 samples were negative, all of which were effective. The results showed that the false positive rate of HPV antigen detection by the test strip was 2%.

(3) False negative rate detection

Drop 100 μL of the cervical epithelial sample of the HPV16 patient on the sample pad of the test strip (HPV16), let it stand for 10 minutes, observe the detection band T and the quality control line C; determine the carrying status of HPV antigen.

The judgment rules are as follows:

Negative: The test band T and the quality control line C both show purple bands, then it is determined that the sample does not contain HPV antigen;

Positive: Only the quality control line C shows a purple-red band (or the quality control line C shows a purple-red band and the test band T shows a lighter color), then it is determined that the sample contains HPV antigen;

Invalid: if the quality control line C does not show a purple-red band, no matter whether the detection band T shows a purple-red band or not, the test strip is judged invalid;

The results showed that 2 samples of cervical epithelial samples from patients with HPV16 were negative and 48 samples were positive, all of which were effective. The results showed that the test strips had a 4% false-negative rate for HPV antigen detection.

Claims (10)

1. A test paper for detecting HPV antigens and HPV antibodies is characterized in that: the test paper comprises a sample pad, a colloidal gold labeling pad, a detection reaction area and a water absorption pad which are sequentially arranged along the chromatography direction, wherein the detection reaction area comprises a detection zone and a quality control line; the colloidal gold labeled pad comprises an antibody-colloidal gold label against HPV L1 antigen, the detection zone comprises HPV L1 antigen, and the quality control line comprises an antibody combined with HPV antibody.

2. The test strip of claim 1, wherein the test strip comprises: the detection reaction area also comprises a nitrocellulose membrane, and the antibody combined with the HPV antibody and the HPV L1 antigen are respectively coated on the nitrocellulose membrane.

3. The test strip of claim 1, wherein the test strip comprises: the colloidal gold labeling pad also comprises a glass fiber membrane or a polyester membrane, and the antibody-colloidal gold labeling substance for resisting HPV L1 antigen is dispersed, laid and adsorbed on the glass fiber membrane or the polyester membrane.

4. The test strip of claim 1, wherein the test strip comprises: the HPV L1 antigen is derived from HPV16 or HPV18.

5. The test strip of claim 1, wherein the test strip comprises: the antibody for resisting the HPV L1 antigen is one or more of polyclonal antibodies of mouse source, rabbit source, sheep source, horse source, camel source or guinea pig source for resisting the HPV L1 antigen.

6. The test strip of claim 1, wherein the test strip comprises: the antibody binding to the HPV antibody means an antibody capable of forming an immune complex by binding to the HPV antibody.

7. A method for preparing a test paper for detecting HPV antigens and HPV antibodies according to claim 1, comprising: the method comprises the following steps:

step 1 preparation of colloidal gold labeled pad

Carrying out labeling treatment on the antibody of the anti-HPV L1 antigen by using a colloidal gold solution to obtain a colloidal gold label solution; coating the colloidal gold marker solution on a glass fiber membrane or a polyester membrane and then drying;

step 2 preparation of detection reaction zone

Respectively coating an HPV L1 antigen solution and an antibody solution combined with an HPV antibody on two different areas of a nitrocellulose membrane, and then drying to obtain a detection band and a quality control line;

step 3 preparation of sample pad

Soaking the glass fiber film or the polyester film in a treatment solution containing a blocking agent and then drying; or the glass fiber film or the polyester film is directly used without being dipped and dried;

and 4, sequentially mounting the sample pad, the colloidal gold labeling pad, the detection reaction area and the water absorption pad on a bottom plate, and then cutting according to the specification of the test paper.

8. The method of claim 7, wherein: in the step 1, the preparation method of the colloidal gold marker solution specifically comprises the following steps: adjusting the pH value of 2-200 mL of colloidal gold solution to 5.5-7.0 by 0.03-0.3M of potassium carbonate solution, then uniformly mixing the colloidal gold solution with 50-100 mu g of antibody against HPV L1 antigen, then reacting at 18-30 ℃ for 10-30 minutes, then adding BSA into the reaction system to the final concentration of 0.5-1%, then standing for 5-10 minutes, then centrifuging at 2-25 ℃ and 1000-12000 rpm for 5-30 minutes and collecting the supernatant, centrifuging the supernatant at 2-25 ℃ and 5000-15000 rpm for 5-60 minutes and collecting the precipitate, and dissolving the precipitate in 0.5-5 mL of PBS containing 0.1-10 BSA.

9. The method for producing according to claim 7, characterized in that: in the step 1, the coating conditions of the colloidal gold marker solution are as follows: diluting the colloidal gold-labeled solution 1 to 2 times with PBS containing 0.1 to 10% BSA, and then diluting the solution at 40 to 80. Mu.L/cm ² Spraying on glass fiber film or polyester film; the drying conditions were: at 35-40 deg.c for 18-24 hr.

10. The method of claim 7, wherein: in the step 2, the concentrations of the HPV L1 antigen solution and the antibody solution combined with the HPV antibody are respectively 0.5-4 mg/mL, and the solvents are PBS; the drying conditions were: at 35-40 deg.c for 18-24 hr.