CN117233382A - Antigen detection kit capable of simultaneously detecting low-risk type and high-risk type human papilloma viruses and application thereof - Google Patents

Abstract

The invention discloses an antigen detection kit capable of simultaneously detecting low-risk and high-risk human papilloma viruses and application thereof, and the kit comprises a gold standard strip, a sample pad and a sample diluent, and is characterized in that the gold standard strip contains an anti-HPVL 1 antibody directly marked by nano gold with the average particle size of 80-120nm, and the sample diluent contains Tween20 or tiron X-100 and SDS. The invention adopts colloidal gold with large particle size, and can effectively detect low-risk HPV6, HPV11, high-risk HPV16, HPV18, HPV31, HPV33, HPV45, HPV52 and HPV58 of human papilloma virus at the same time. In the sample diluent, the combination of Tween20 or tiron X-100 and SDS is adopted, so that the sample diluent can destroy mucus in a cervical swab sample and can also play a role in splitting viruses; thereby releasing the protein to be detected.

Description

An antigen detection kit capable of simultaneously detecting low-risk and high-risk human papillomavirus and its application

Technical Field

The invention belongs to the field of medical technology, and specifically relates to an antigen detection kit capable of simultaneously detecting low-risk and high-risk human papillomaviruses and an application thereof.

Background Art

Human papillomavirus (HPV) belongs to the genus Papillomavirus of the family Palliomaviridae. It is a type of small double-stranded circular DNA virus without an envelope and tropism for epithelial tissue. Papillomavirus can infect the skin and mucous membranes of mammals such as humans, cattle, dogs, and rabbits, causing warty hyperplasia of epithelial tissue and even benign and malignant tumors.

Natural HPV virus particles have a regular icosahedral symmetric structure, a diameter of 52nm-55nm, and are small double-stranded circular DNA viruses. The HPV genome is approximately 7.2-8kb, including the early gene region, the late gene region, and the long control region. The early gene region has six early genes (E1, E2, E4, E5, E6, and E7), which encode 6 non-structural proteins (E1, E2, E4, E5, E6, and E7), respectively. Among them, E1 and E2 are used to regulate the replication and transcription of the viral genome, E4 is related to viral escape, and E5, E6, and E7 are related to the occurrence of cancer. The non-structural proteins of certain types of HPV can also reduce the stability of the target cell genome. The late gene region (L region) encodes two structural proteins, namely the major capsid protein (L1) and the minor capsid protein (L2). The two proteins together constitute the capsid structure of the virus and are closely related to viral entry into the cell. The major capsid protein L1 is the main component of the HPV icosahedral capsid, with an apparent molecular weight of 50-60KD, accounting for 10% of the total capsid protein. Wraps the viral genome. The secondary capsid protein L2 is located on the long axis at the center of the pentamer and may play a major role in the packaging of viral genomic DNA and participate in multiple steps of viral invasion of host cells. The LCR region is also called the upstream regulatory region (URR), which contains cis-regulatory elements and is involved in regulating the replication and transcription of viral genetic material.

The core of the tertiary structure of the HPV16L1 capsid protein is a typical "jelly roll" β-fold barrel composed of amino acid residues at positions 20-382, which contains two groups of antiparallel structures composed of 4 β-sheets each. The 8 β-sheet structures are named B, C, D, E, F, G, H and I. The β-sheets are connected by B-C, C-D, D-E, E-F, F-G and H-I cyclic connecting peptides. Among them, the three cyclic connecting peptides DE, FG and HI have greater flexibility and protrude significantly from the outer surface of the β-fold barrel. They are the main distribution area of the HPV16L1 capsid protein antigen epitopes. The five L1 proteins are in close contact to form a hollow truncated cone-shaped pentamer.

Papillomavirus lacks a reliable cell culture model, and its classification is mainly based on gene sequence. It is usually classified according to L1 homology, with homology between classes less than 60%, homology between species between 60% and 70%, homology between types between 70% and 90%, homology between subtypes between 90% and 98%, and homology between intra-type variations above 98%. Based on the different HPV genotypes, more than 100 HPV genders have been isolated and identified, and are numbered in the order of discovery. Depending on the site of infection, HPV can be divided into anogenital type and skin type. HPV of the portal genital type is divided into high-risk and low-risk types based on its correlation with cervical cancer. The former is closely related to the induction of cervical cancer, including HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 72, 82; the latter includes HPV6, 11, 40, 42, 43, 44, 54, 61, 70, 72, 81 and CP6180 and uncertain types 26, 53, 66. At present, the classification basis has added the gene sequences of E1 and E2 encoding early proteins. Based on the homology of E1-E2-L1, papillomavirus can be divided into 53 categories, marked with Greek letters. HPV mainly exists in alpha, beta, gamma, mu and nu5 types. Among them, alpha HPV mainly causes lesions of the mucosa, becoming mucotropic type; beta HPV mainly causes skin lesions, becoming dermatotropic type; gamma HPV can cause both mucosal and skin lesions. Worldwide, the high-risk HPVs infected in cervical cancer patients are mainly HPV16 (55.2%), HPV18 (14.2%), HPV45 (5%), HPV33 (4.2%), HPV58 (3.9%), HPV31 (3.5%), HPV52 (3.5%), HPV35 (1.7%), HPV39 (1.5%) and HPV59 (1.4%). The high-risk HPVs infected in patients with cervical cancer in my country are mainly HPV16 (59.5%), HPV18 (9.6%), HPV58 (8.2%), HPV52 (6.5%), HPV33 (3.5%), HPV31 (2.8%), HPV59 (2.6%), HPV45 (1.9%), HPV39 (1.5%) and HPV56 (1.1%). Persistent infection with high-risk HPV is the main pathogenic factor of cervical cancer. Worldwide, cervical cancer is the fourth most common malignant tumor in women, posing a major challenge to global women's health. Therefore, cervical cancer screening and HPV preventive vaccination are the most effective measures to prevent cervical cancer.

Colloidal gold labeling technology is a labeling method developed in the 1980s, following the three major labeling technologies of fluorescein, radioisotopes and enzymes. In 1971, Faulk and Taylor introduced colloidal gold into immunobiochemistry. Since then, this labeling method has been widely used in the fields of drug testing, biomedical testing, agricultural and veterinary drug testing, clinical disease testing, etc. At present, there are hundreds of commercial colloidal gold immunochromatography kits at home and abroad, such as the common ones: early pregnancy test kits, drug detection kits, fecal occult blood test kits, infectious disease detection kits, etc.

Colloidal gold labeling technology uses colloidal gold as a labeling tracer, which was initially used in immunoelectron microscopy. With the development of immunology, it has been gradually applied to animal agglutination experiments, immunoblotting, light microscopy staining, immunospots and immunochromatography. Due to the high electron density on the surface of colloidal gold, colloidal gold can be firmly combined with most proteins without destroying the activity of the proteins.

The application of colloidal gold in immunoassay mainly involves labeling antigens or antibodies with colloidal gold, and then fixing the antibodies or antigens that can react immunologically with the labeled antigens or antibodies on a matrix, so that the binding of the antigens or antibodies is displayed as a color change visible to the naked eye. The degree of binding between colloidal gold and protein, the pH of the reaction system, and the concentration of the protein in the reaction will affect the binding of protein and colloidal gold.

Colloidal gold is also called gold solution. The structure of colloidal gold particles includes a gold core and a double ion layer surrounded by an outer layer, that is, the inner layer of negative ions AuCl2- tightly surrounds the surface of the gold core and the outer layer of positive ions H+ are dispersed in the colloidal gold solution, showing a good monodisperse state. Using different types and doses of reducing agents to reduce chloroauric acid solutions of different concentrations can synthesize colloidal gold nanoparticles of different particle sizes and morphologies, that is, the particle size of colloidal gold nanoparticles can be adjusted by changing the type, content and substrate concentration of the reducing agent.

Gold sol is produced by boiling a solution of tetrachloroauric acid with a reducing agent. At the beginning of the reduction process, gold atoms are released from the chloroauric acid. The gold atoms aggregate to form microcrystals. As more chloroauric acid is reduced, the size of the microcrystals increases until all the chloroauric acid is reduced. The type of reducing agent and the concentration of the components determine the ratio of nucleation and growth, and thus the final particle size. Gold sol can be made in the laboratory, and the particle size is usually between 2 and 40 nanometers, depending on the type and concentration of the reducing agent.

According to research reports, commonly used reducing agents include trisodium citrate, sodium borohydride, ascorbic acid, tannic acid, white phosphorus, hydroquinone, hydroxylamine, etc. In 2001, Jana NR, Gearheart L reported the synthesis of large-particle colloidal gold by seed growth method. By controlling the addition ratio of seeds, reducing agents and chloroauric acid, the particle size of the synthesized colloidal gold can be controlled. In the process of synthesizing colloidal gold by seed growth method, layer-by-layer growth can more effectively avoid the possibility of secondary nucleation than primary growth. Compared with the original direct reduction method, the colloidal gold synthesized by this method has uniform particle size, nearly spherical shape and good monodispersity. However, the colloidal gold synthesized by this method is more complicated and not easy to industrialize.

At present, the main clinical screening methods for cervical cancer include cytological detection methods, such as thin layer chromatography, colposcopy, histopathological methods, serological methods, nucleic acid hybridization and PCR detection methods, gene chip methods, hybridization capture methods and enzyme-linked immunosorbent assay. These methods require instruments, equipment and professional technical operators. At the same time, the screening time is relatively long and the screening cost is high.

The amount, type and duration of HPV infection have become important contents of HPV detection. A large amount of HPV virus may increase the risk of HPV-related cervical lesions, but the clinical significance of routine virus load detection is not very clear, mainly because there are great differences in the detection methodologies. The HC-2 test (semi-quantitative) or PCR method can be used for quantitative detection of HPV DNA, but there is currently no recognized optimal HPV DNA quantitative detection method. Real-time PCR will be a new technology with great application prospects. Multiple HPV type infection may be an important sign of persistent disease, multiple (relative to single) cervical lesions and low-grade to high-grade cervical precancerous lesions. However, widely used HPV detection systems such as the HC-2 test cannot distinguish between single or multiple HPV infections. PCR can detect type-specific HPV infection, so it can indicate whether there are multiple HPV infections. However, the sensitivity and repeatability of different PCR methods also vary greatly (for example, GP5+/6+ can only detect 47% of multiple HPV type infected specimens, while MY09/11 can detect 90%). Combining the advantages and disadvantages of the above methods, the combined use of multiple methods for HPV testing on the same specimen has important clinical application prospects. It has been reported that the negative predictive value of TCT and HC-2 methods used simultaneously for HPV DNA testing can reach 99.9%. The United States and Europe have used these two methods as common methods for early screening of cervical cancer.

In recent years, the detection of HPV, especially HPV-DNA detection, has made rapid progress and played an important role in the screening and prediction of cervical cancer and precancerous lesions. However, there are still some limitations of HPV-DNA detection in cervical cancer screening, mainly manifested in: A. The HPV infection rate is very high; B. HPV infection is usually transient, especially when using highly sensitive DNA detection methods; C. Only the persistent existence of carcinogenic HPV types and their integration with the host genome are valuable precancerous markers. D. The PCR detection method has a high false negative rate, cumbersome operation during typing, limited sample size for one-time detection, and inconvenient identification of unknown types, which limits its application in large-scale screening; E. The DNA chip technology developed in recent years has the advantages of high parallelism, diversity, miniaturization and automation, and has been widely used in DNA research fields such as gene positioning, DNA sequencing, mutation detection, gene screening, and gene diagnosis. The HPV detection methods currently used have common limitations: time-consuming and complex operating procedures. Therefore, it is urgent to establish a simple, efficient, specific and rapid diagnostic method for high-risk human papillomavirus suitable for use in hospitals at all levels. Some people in China have proposed to establish a rapid diagnostic method for HPV infection, but there are no reports on simple and rapid diagnosis of HPV infection at home and abroad. The high-risk human papillomavirus type 16 immune colloidal gold test strips developed in this study can achieve the purpose of rapid and specific diagnosis of high-risk human papillomavirus type 16 infection in hospitals at all levels. After the rapid detection method is established, it can be developed into a product like the early pregnancy HCG colloidal gold test strip, which can detect HPV quickly, simply, specifically, sensitively and at low cost. At that time, it can be widely used in primary hospitals and major hospitals for early screening of cervical cancer, and has good application prospects.

Therefore, the establishment of an immunoassay method based on immunochromatography can solve the above problems. Whether it is a qualitative detection kit using colloidal gold as a tracer or a quantitative detection kit using fluorescein as a tracer, both can solve the above problems.

At present, the use of chromatographic test kits in HPV screening scenarios in the domestic and foreign markets is still relatively rare. Based on the low cost of chromatographic test kits, no instruments or equipment are required, or very small instruments and equipment are required, and very professional technicians are not required to operate, and the detection time is short. Therefore, it is very suitable for HPV screening in the early stage and onset of cervical cancer. It is also suitable for the detection of other warts caused by papilloma, such as anal or genital condyloma acuminatum.

At present, as of July 2023, no similar products in China have obtained registration certificates. There are also few similar products abroad. In China, the current immunoassay products for HPV antigens or antibodies are mainly ELISA kits. Therefore, developing immunochromatographic detection products with short detection time, low cost, and easy results is a very effective method for universal or batch screening of cervical cancer.

Summary of the invention

Based on the above reasons, the present invention proposes an antigen detection kit and its application that can simultaneously detect low-risk and high-risk human papillomavirus. Specifically, in order to achieve the purpose of the present invention, the present invention intends to adopt the following technical solutions:

On one hand, the present invention relates to an antigen detection kit capable of simultaneously detecting low-risk and high-risk human papillomaviruses. The kit comprises a gold label strip, a sample pad, and a sample diluent. The kit is characterized in that the gold label strip contains an anti-HPVL1 antibody directly labeled with nanogold with an average particle size of 80-120 nm, and the sample diluent contains Tween20 or tironX-100 and SDS.

In a preferred embodiment of the present invention, the sample diluent contains 0.5-2 parts of Tween 20 and 0.3-0.7 parts of SDS per 100 parts, and the pH value of the sample diluent is 8.5-9.5.

In a preferred embodiment of the present invention, the sample diluent contains 0.5-1 part of tironX-100 and 0.3-0.7 part of SDS per 100 parts, and the pH value of the sample diluent is 8.5-9.5.

In a preferred embodiment of the present invention, the sample pad contains propylene oxide-ethylene oxide-vinyl diamine copolymer. After adding this component to the sample pad, the environment during chromatography is in a non-ionic state, which is conducive to the binding of antigen and antibody and eliminates non-specific reactions.

In a preferred embodiment of the present invention, the average particle size of the nano-gold is 80nm-100nm. Nano-gold of this particle size directly labels anti-HPVL1 antibodies, which is beneficial to improving the sensitivity of HPV antigen detection and enabling the kit to more sensitively detect HPV antigens, thereby being more effectively used for cervical cancer screening.

In a preferred embodiment of the present invention, the kit further comprises a nitrocellulose membrane, absorbent paper, a PVC base plate, a card shell, a cervical sampling swab, a sample extraction tube and/or a workbench.

Another aspect of the present invention also relates to the use of the above-mentioned kit in preparing a diagnostic reagent for diagnosing human papillomavirus.

In a preferred embodiment of the present invention, the diagnostic reagent is used to diagnose low-risk HPV6, HPV11 and high-risk HPV16, HPV18, HPV31, HPV33, HPV45, HPV52, and HPV58; preferably, the diagnostic reagent is used to simultaneously diagnose low-risk HPV6, HPV11 and high-risk HPV16, HPV18, HPV31, HPV33, HPV45, HPV52, and HPV58.

Beneficial Effects

The present invention adopts colloidal gold with large particle size, which can effectively detect low-risk HPV6, HPV11 and high-risk HPV16, HPV18, HPV31, HPV33, HPV45, HPV52 and HPV58 of human papillomavirus at the same time. It covers more high-risk human papillomaviruses that cause cervical cancer and increases the coverage of detection. At the same time, due to the use of colloidal gold with large particle size of 80nm-100nm, the detection reagent has higher sensitivity than the reagent prepared by colloidal gold with small particle size such as 40nm and 60nm. Therefore, in clinical application, it can be more widely and better applied to the screening, monitoring and postoperative monitoring of human cervical cancer. In the sample diluent of the present invention, a combination of Tween20 or tironX-100 and SDS is adopted, so that the sample diluent can not only destroy the mucus in the cervical swab sample, but also play a role in lysing the virus; thereby releasing the protein to be detected.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Visible-UV spectrophotometric spectrum of colloidal gold.

Figure 2: Negative test results using 80-100nm gold test paper;

Figure 3: Positive test results using 80-100nm gold test paper;

Figure 4: Negative test results using 40nm gold test paper;

Figure 5: Positive test results using 40nm gold test paper;

Figure 6: Test results without adding SDS and Tween20 to the diluent;

Figure 7: Test results when only Tween20 was added to the diluent;

Figure 8: Test results when only SDS was added to the diluent;

Figure 9: Test results of adding SDS and Tween20 to the diluent;

Figure 10: Another test result of adding SDS and Tween20 into the diluent;

Figure 11: Test results when the diluent contained TritonX-100 and SDS.

DETAILED DESCRIPTION

In order to further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments 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 creative work are within the scope of protection of the present invention.

Unless otherwise specified, the reagents involved in the embodiments of the present invention are all commercially available products and can be purchased through commercial channels.

Example 1

1 Preparation of colloidal gold

1.1 Weigh 0.2 g/mL of tetrachloroauric acid into a graduated tube, then add ultrapure water to prepare 5 mL of chloroauric acid aqueous solution.

1.2 Weigh trisodium citrate into a graduated tube at an amount of 0.161 g/mL, and then add ultrapure water to prepare 5 mL of trisodium citrate aqueous solution.

1.3 Weigh polyvinyl alcohol (PVA) in a 10% (weight-to-volume) amount into a graduated tube, and then add ultrapure water to prepare a 10 mL PVA aqueous solution.

1.4 Place a 2-liter Erlenmeyer flask on a magnetic stirrer with heating function, add 1L of ultrapure water into the Erlenmeyer flask, turn on the magnetic stirrer, and heat it.

1.5 When the bottom layer temperature of the conical flask is heated to 95-100℃ and the upper layer temperature is 90-95℃, add 1mL of chloroauric acid aqueous solution into the conical flask. Set the stirrer speed to the maximum speed. At this time, the aqueous solution in the conical flask is yellow.

1.6 Start timing after adding, heat for another 30s-60s, and quickly add 0.75mL-1mL of PVA aqueous solution.

1.7 Then quickly add 1mL + 0.2mL of trisodium citrate aqueous solution. Continue heating and stirring for 6-8 minutes; at this time, the aqueous solution in the conical flask is purple-black at first, then turns purple, and finally turns dark wine red. Stop heating immediately. Transfer the reaction-completed solution to another magnetic stirrer and cool it to room temperature in a water bath.

1.8 The addition time of PVA aqueous solution and the addition time of trisodium citrate aqueous solution should be almost simultaneous. The difference between the addition time of the two should not exceed 10 seconds. Otherwise, it is impossible to prepare a colloidal gold solution with rounded particle size and good dispersion.

1.9 Detection

After the colloidal gold solution to be prepared is cooled, 1 mL of the solution is taken, and then a micro-ultraviolet spectrophotometer (Nanodrop type) is used to scan the 200nm-800nm band spectrum. The colloidal gold solution prepared by this method has a maximum absorption peak at 558nm, and an OD value of 1.29, without any impurity peaks, as shown in Figure 1, thereby determining that the colloidal gold with an average particle size of 100nm is obtained.

2. Labeling of anti-human papillomavirus (HPV L1) antibodies

This technology uses a direct labeling method. That is, at room temperature, the anti-HPVL1 antibody (purchased from Abcam (Shanghai) Trading Co., Ltd.) is directly reacted with the colloidal gold solution, and then centrifuged to obtain the required gold label. The specific method is as follows:

2.1 Determination of labeled pH:

(1) Take 1 ml of the colloidal gold solution prepared in step 1 and put it into EP tubes, a total of 10 tubes, and then adjust the pH of the colloidal gold solution to 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, and 10.0 with 0.1 M potassium carbonate, and mark them. Then, add anti-HPV L1 marker antibodies at a volume of 10 μg/mL and vortex to mix.

(2) The above solutions were allowed to stand for 10 minutes, and 10% sodium chloride solution was added into each EP tube at a volume of 100 μl/tube, and vortexed to mix.

(3) Let it stand for 15 minutes and observe the color change.

(4) The pH of the colloidal gold solution tube that has no precipitation, purple or black color is selected as the best marking pH; that is, the pH of the solution that has not changed color and is still wine red is the best pH, that is, the best marking pH is 8.0

2.2 Determination of labeling amount

(1) Take 1 ml of the colloidal gold solution prepared in step 1 and put it into EP tubes, a total of 10 tubes, and then adjust the pH of the colloidal gold solution to 7.5 with 0.1 M potassium carbonate and mark them. Then, add HPV L1 labeled antibody in the amount of 2 μg/mL, 4 μg/mL, 6 μg/mL, 8 μg/mL, 10 μg/mL, 12 μg/mL, 14 μg/mL, 16 μg/mL, 18 μg/mL, and 20 μg/mL, respectively, and vortex to mix.

(2) After standing for 10 minutes, add 5% BSA aqueous solution to each tube at a volume of 24 μL/mL and vortex to mix.

(3) Add 1% gelatin aqueous solution at a volume of 20 μL/mL and vortex to mix.

(4) After standing for 15 minutes, add 10% sodium chloride aqueous solution (1 part sodium chloride per 10 parts water) to each of the above tubes at a volume of 100 μL/mL and vortex mix.

(5) Let it stand for 2 hours and observe the sedimentation and color changes in each tube. The amount of protein in the colloidal gold solution tube that has no precipitation, purple or black is selected as the optimal labeling amount; that is, the amount of protein in the solution that has not changed color and is still wine red is the optimal labeling amount. That is, 10μg/mL.

2.3 Colloidal gold labeling of anti-human papillomavirus (HPVL 1) antibodies

(1) Measure the colloidal gold solution according to the amount of antibody per 100 parts of colloidal gold solution, for example: take 100 ml of colloidal gold solution and put it into a beaker.

(2) Adjust the pH of the colloidal gold solution to 8.0 with 0.1 M potassium carbonate solution and place it on a magnetic stirrer for stirring.

(3) Dilute the anti-HPVL1 labeled antibody to a concentration of 1.2 mg/ml using 2 mM Tris buffer at pH 7.4, and then add 1 ml of the diluted protein solution to the colloidal gold solution. Add the above solution while stirring.

Preparation of Tris buffer: weigh 2.2 parts of Tris per 100 parts of water and put it in a beaker, add 9 parts of water and then adjust the pH to 7.4 with 6M hydrochloric acid solution, and then make up to the required volume with purified water.

(4) Stir on a magnetic stirrer for 25 minutes;

(5) Add 5% BSA aqueous solution at a volume of 24 μl/ml to 100 ml of the above solution and continue stirring for 30 minutes.

(6) Add 1% gelatin solution to the above solution at a volume of 20 μl/ml and continue stirring for 1 hour.

(7) Transfer the above reaction solution into a centrifuge tube;

(8) Centrifuge at 8000-15000 rpm/min, for example, 12000 rpm/min, for 30 minutes at 4°C; remove the supernatant.

(9) The precipitate is redissolved with 1 part of 2 mM Tris buffer at pH 7.4 to obtain the labeled gold-labeled HPVL1 antibody.

42.2 Preparation of sample pad

4.2.2.1 Preparation of sample pad treatment solution

(1) Weigh 4 parts of borax per 100 parts of borax and put it into a beaker. Then add 80 parts of water into the beaker and place the beaker on a magnetic stirrer for stirring.

(2) Add 0.3 parts of ethylenediaminetetraacetate into the beaker and continue stirring;

(3) Add 1-1.5 parts of propylene oxide-ethylene oxide-vinyl diamine copolymer (purchased from Shanghai Xibao Biotechnology Co., Ltd.) to the above solution and continue stirring;

(4) Add 2 parts of Proclin 300 per 10,000 parts of the above solution and continue stirring;

(5) Add 1 part of alkylphenol polyoxyethylene ether per 10,000 parts of the above solution and continue stirring.

(6) After the solution is completely dissolved and clarified, adjust the pH of the solution to 8.2 with hydrochloric acid or sodium hydroxide, and then dilute to the required volume with purified water.

(7) Keep aside at room temperature.

4.2.2.2 Preparation of sample pad

(1) Prepare commercially available glass fiber mat (length * width: 300mm * 254mm);

(2) Soak the glass fiber mat at a rate of 1 piece per 100 parts; first soak the glass fiber mat in the solution of 2.4.1 for 2 minutes, then take it out and gently filter out excess water with a glass rod. Turn it over and soak the glass fiber mat in the solution again for 2 minutes, and gently filter out excess water with a glass rod. The sample mat is now obtained.

(3) Place the obtained sample pad in a 37°C oven and dry overnight (17-20 hours).

(4) After drying, the sample pad is sealed and stored in a dry environment.

4.2.3 Preparation of Sheets

(1) Paste the nitrocellulose membrane onto the PVC base plate;

(2) Preparation of coating solution:

Test line: dilute the anti-HPVL1 coated antibody to 1 mg/ml with 0.01 M PB and prepare the test line at 2 μl/cm per piece.

Quality control line: Dilute goat anti-mouse IgG to 0.8 mg/ml with 0.01 M PB and prepare the quality control line at 2 μl/cm per slice.

Coating: Coat the solution onto nitrocellulose membrane using a stripping agent.

(3) Place the coated sheet in a 37°C oven and dry overnight.

(4) The dried sheets should be kept sealed and stored in a dry place.

4.2.4 Preparation of gold label

(1) Dilute the gold-labeled solution prepared in 2.3 with 0.01 M PB buffer containing 1 part bovine serum albumin and 20 parts sucrose per 100 parts. The dilution ratio is 46 parts of the gold-labeled solution and 54 parts of the above 0.01 M PB solution per 100 parts of the solution.

(2) The diluted solution was sprayed with gold label using a gold labeling machine at a volume of 60 μl per strip. The base pad for spraying was a glass fiber pad (length * width: 300 mm * 84 mm).

(3) After spraying, place the gold label in a 37°C oven and dry overnight.

(4) The dried gold label strip is sealed in a ziplock bag and then stored in a dry environment away from light.

4.2.5 Assembly of test strips

(1) Cut the prepared gold label into small strips with a length of 300 mm and a width of 8 mm, and stick them on the lower edge of the nitrocellulose membrane of the sheet, pressing 1-2 mm over the nitrocellulose membrane;

(2) Cut the sample pad into strips 300 mm long and 20 mm wide, and stick them on the lower end of the sheet; the lower edge of the sample pad should extend 3 mm beyond the lower edge of the sheet, and the upper edge of the sample pad should extend beyond the white edge of the gold label strip.

(3) Cut the absorbent filter paper into strips 300 mm long and 17 mm wide. Align the upper edge of the absorbent filter paper with the upper edge of the sheet and press the lower edge over the nitrocellulose membrane by about 2 mm.

(4) Press the attached reagent card tightly and then cut it into small strips with a width of 4.0 mm using a cutting machine.

4.2.6 Assembly of the kit

Put the cut 4.0 mm test strip into the slot of the lower plate of the plastic card case, and cover the upper card case tightly to obtain the human papillomavirus antigen detection kit.

4.2.7 Preparation of sample diluent

A solution was prepared by adding 1.2 parts of tris, 25 parts of sodium chloride, 1 part of tween 20, 0.5 parts of SDS and 5 parts of 1% Proclin 300 per 100 parts of water, and the pH of the solution was adjusted to 9.0 to obtain a sample dilution solution.

4.3 Testing:

4.3.1 Test of recombinant protein: The recombinant antigens of HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV45, HPV52 and HPV58 were diluted to 10 ng/mL and 1 ng/mL respectively with sample diluent. The above kit was tested with different types of HPV recombinant proteins at these two concentrations, and the test results were all positive. The negative test was performed with sample diluent, and the test results were all negative.

4.3.2 The clinically confirmed negative samples and HPV-positive cervical swab samples were tested and the test results were also positive.

5 Test Results

5.1 Recombinant protein test results

Table 1 Recombinant protein test results

Test items HPV6 HPV11 HPV16 HPV18 HPV31 HPV33 HPV45 HPV52 HPV58 Negative - - - - - - - - - 10 ng/mL positive + + + + + + + + + 1 ng/mL positive + + + + + + + + +

5.2 Positive sample test results

Table 2 Clinical sample test results

Test items Repeat test 1 Repeat Test 2 Repeat test 3 Negative cervical swab 1 - - - Negative cervical swab 2 - - - Negative cervical swab 3 - - - Negative cervical swab 4 - - - Negative cervical swab 5 - - - Positive cervical swab 1 + + + Positive cervical swab 2 + + + Positive cervical swab 3 + + + Positive cervical swab 4 + + + Positive cervical swab 5 + + +

The above test results show that the detection kit of the present invention can detect human papillomavirus antigens in a wider range, that is, it can detect low-risk HPV6 and HPV11 as well as high-risk HPV16, HPV18, HPV31, HPV33, HPV45, HPV52, and HPV58. It greatly expands the screening range of human papillomavirus types, making the detection of cervical cancer more effective, the test results more accurate, and the sensitivity of HPV detection higher.

The HPV antigen detection kit prepared by the method can more effectively pre-check, detect and monitor the occurrence of cervical cancer and the postoperative status of cervical cancer.

7 Comparison of test samples of HPV antigen detection kits prepared with colloidal gold of different particle sizes

Comparison of HPV detection kit prepared with conventional 40nm colloidal gold and HPV detection kit prepared with 80nm-100nm colloidal gold. The preparation method is the same as above, only the particle size of the colloidal gold is different. The test results are shown in Table 3 and Figures 2-5 below.

Table 3: Effects of colloidal gold of different particle sizes on test results

The above test results show that, for the antibody of the present invention, the present invention uses colloidal gold with a larger particle size, which plays a certain role in improving the sensitivity of the product.

Comparison of test results of 8 different sample dilution formulas

According to 4.2.7, 4 kinds of reagents were prepared, each containing 100 parts of solution without SDS and Tween20; 1 part of Tween20; 0.5 parts of SDS; and 0.5 parts of SDS and 1 part of Tween20. The above 4 kinds of reagents were used as sample diluents to dilute negative and positive samples. The diluted samples were tested. The test results are shown in Table 4 and Figures 6-9 below.

Table 4: Effect of the type of surfactant in the sample diluent on the test results

It can be seen from the above results that when the sample diluent contains SDS and Tween20, the pathogens to be tested in the sample can be released better.

9. Experiments using Tritonx-100 instead of Tween20 as a diluent component

0.5%-1% Tritonx-100 was used to replace Tween20 to prepare the sample diluent. The positive samples were treated with the sample diluent. The test results are shown in Table 5 and Figures 10-11.

Table 5: Test results using Tritonx-100 instead of Tween20 as the diluent component

The above experimental results show that using TritonX-100 instead of Tween20 can also achieve good detection results, but Tween20 is more preferred because Tween20 combined with SDS can release pathogens in the sample, and Tween20 itself has the effect of promoting antigen-antibody reaction. While TritonX-100 and SDS can release pathogens in the sample, TritonX-100 has no promoting effect on antigen-antibody reaction, and when the content is too high, it will also destroy the antigen-antibody reaction.

The above describes the preferred embodiments of the present invention, but it is not intended to limit the present invention. Those skilled in the art may make improvements and changes to the embodiments disclosed herein without departing from the scope and spirit of the present invention.

Claims (9)

1. An antigen detection kit for detecting low-risk and/or high-risk human papillomaviruses. The kit includes a gold label strip, a sample pad, and a sample diluent. It is characterized in that the gold label strip contains an average of Gold nanoparticles with a particle size of 80-120 nm are directly labeled with anti-HPVL1 antibodies, and the sample diluent contains Tween20 or tironX-100 and SDS. 2. The kit according to claim 1, each 100 parts of the sample dilution contains 0.5-2 parts of Tween20 and 0.3-0.7 parts of SDS, and the pH value of the sample dilution is 8.5-9.5. 3. The kit according to claim 1, each 100 parts of the sample diluent contains 0.5-1 part of tironX-100 and 0.3-0.7 part of SDS, and the pH value of the sample diluent is 8.5-9.5 . 4. The kit according to claim 1, wherein the sample pad contains propylene oxide-ethylene oxide-vinyl diamine copolymer. 5. The kit according to claim 1, wherein the average particle size of the gold nanoparticles is 80-100 nm. 6. The kit according to claim 1, further comprising a nitrocellulose membrane, absorbent paper, PVC bottom plate, card case, cervical sampling swab, sample extraction tube and/or workbench. 7. Application of the kit according to any one of claims 1 to 6 in the preparation of diagnostic reagents for diagnosing human papillomavirus. 8. The application according to claim 7, wherein the diagnostic reagent is used to diagnose low-risk HPV6, HPV11 and high-risk HPV16, HPV18, HPV31, HPV33, HPV45, HPV52, and HPV58. 9. The application according to claim 7, wherein the diagnostic reagent is used to simultaneously diagnose low-risk HPV6, HPV11 and high-risk HPV16, HPV18, HPV31, HPV33, HPV45, HPV52, and HPV58.