CN112062837A

CN112062837A - Rapid detection test paper for HPV16 type virus antibody

Info

Publication number: CN112062837A
Application number: CN202010564872.6A
Authority: CN
Inventors: 彭南求; 常雪雪; 陈林军
Original assignee: Shanghai University of Medicine and Health Sciences
Current assignee: Shanghai University of Medicine and Health Sciences
Priority date: 2020-06-19
Filing date: 2020-06-19
Publication date: 2020-12-11

Abstract

The invention relates to a rapid test paper for HPV16 type virus antibody, firstly, the invention uses hybridoma technology to obtain anti-HPV 16E6 protein monoclonal antibody, and then prepares magnetic microspheres coated with the anti-HPV 16E6 protein monoclonal antibody; and then, preparing the HPV16 type virus antibody rapid detection test paper by using the magnetic micro-preparation coated with the anti-HPV 16E6 protein monoclonal antibody. The invention applies the nano magnetic immunity technology, selects the magnetic nano microspheres as a new marker, and can quantitatively analyze the concentration of the HPV16 type antibody by detecting the magnetic strength of the double-antibody sandwich complex captured by the nitrocellulose membrane in a magnetic field. Compared with the conventional detection method, the method has the advantages of high qualitative and quantitative property, high sensitivity, simple and quick operation, low detection cost and the like, so the method has good application prospect in the detection of the HPV virus antibody of the female cervical scrapings.

Description

Rapid detection test paper for HPV16 type virus antibody

Technical Field

The invention relates to the field of medical inspection, in particular to a rapid test paper for HPV16 type virus antibody.

Background

HPV virus is the abbreviation for human papillomavirus, a papillomavirus A genus of the papovavirus family of papovaviridae, a spherical DNA virus, whose infection is a sexually transmitted disease. The main types of HPV viruses are HPV1, 2, 6,11, 16, 18, 31, 33 and 35, etc., and long-term infection of HPV16 may be related to female cervical cancer. HPV virus invades cervical basement membrane, infected cells partially spread laterally, another part migrates to squamous epithelial cells, the virus integrates into squamous epithelial cells, then proliferates in a large amount, and the infected squamous epithelial cells gradually evolve into CIN1, CINII, CINIII until cervical cancer, but not all infected cells are converted into cancer cells.

At present, methods for diagnosing and detecting cervical cancer mainly include nucleic acid hybridization technology, Polymerase Chain Reaction (PCR) technology and gene chip technology. These techniques are further described below:

(1) nucleic acid hybridization techniques

The principle of base complementation is utilized, the nucleic acid of the virus is detected by using a labeled nucleic acid probe, and the virus DNA is typed. The conventional methods include dot hybridization, fluorescence in situ hybridization, and southern hybridization. The dot blot method is suitable for screening test and has the advantages of less reagent amount, high sensitivity and radioactivity. Fluorescent in situ hybridization is a method of hybridizing a nucleic acid of a known sequence, specifically labeled with a fluorescent substance, as a probe to a nucleic acid in a cell or tissue section and detecting it. Its advantages are high locating effect and low false positive rate. But the sensitivity is not high, and only 20-50 viruses/cell can be detected. Southem has high hybridization sensitivity, can detect 1 virus/cell theoretically, and is suitable for HPV typing and HPVDNA molecular weight identification. However, the steps are complicated, fresh tissue specimens are required, and the method is not suitable for large-scale clinical application at present.

(2) Polymerase Chain Reaction (PCR) technique

PCR techniques employ DNA polymerase catalysis with specific primers to selectively amplify target HPV DNA. The amplified DNA can be detected by different methods. The technology is simple and easy to implement, can be used for typing HPV by using the type-specific primers, and is the most widely applied HPV DNA detection method at present. Although PCR has been widely used in the research field, it has disadvantages such as easy contamination of samples and high false positive rate. FDA has not yet approved PCR for cervical HPV detection.

(3) Gene chip technology

The gene chip technology is developed in recent years with the development of human genome project, and known and unknown sequences in cDNA library are fixed on a glass slide, and the expression conditions of a plurality of known or unknown sequences in a biological sample are detected and compared. The method has the advantages of multi-sample parallel processing capability, high analysis speed, small required sample amount, less pollution and the like. However, the gene chip technology requires expensive sophisticated instruments, and there are some problems in software such as complicated sample preparation, difficult mastering of the technology, and low detection sensitivity.

In summary, the problems of the prior art are as follows:

the nucleic acid hybridization technique has the disadvantages of radioactivity, complicated steps, fresh tissue specimens, and is not suitable for large-scale clinical application at present. The Polymerase Chain Reaction (PCR) technique has the disadvantages of easy sample contamination, high false positive rate, etc. FDA has not yet approved PCR for cervical HPV detection. The gene chip technology requires expensive sophisticated instruments, and also has some problems in software, such as complicated sample preparation, difficult mastering of the technology, and low detection sensitivity.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a test paper for rapidly detecting HPV16 type virus antibodies.

The invention uses the hybridoma technology to lead the hybrid cells fused by the spleen cells of mice immunized by antigens and the myeloma cells of the mice to survive and propagate for a long time in HAT culture medium, thus leading the hybrid cells to be continuously cultured and secreting monoclonal antibodies of anti-HPV 16E6 protein. Particularly, the nano magnetic immunity technology is innovatively applied, the magnetic nano microspheres are selected as a new marker, and the HPV16 type antibody concentration can be quantitatively analyzed by detecting the magnetic strength of the double-antibody sandwich complex captured by the nitrocellulose membrane in a magnetic field.

The purpose of the invention can be realized by the following technical scheme:

in the first aspect of the invention, a monoclonal antibody is an anti-HPV 16E6 protein monoclonal antibody, and is obtained by long-term survival and propagation of fused cells of antigen immunized mouse spleen cells and mouse myeloma cells in HAT culture medium, and continuous culture and secretion of the fused cells.

The fusion cell is formed by fusing a mouse spleen cell immunized by an antigen and a mouse tumor cell.

During the fusion process, a cell fusion agent was used, which was selected from polyethylene glycol (PEG 1000-2000) and applied at a concentration of 40% (W/V). The cell fusion agent is used to cause a certain degree of damage to cell membranes, so that cells are easy to adhere to each other and fuse together. The optimal fusion effect should be minimal cell damage while producing the highest frequency of fusion.

The fusion cell has the genetic properties of both parents and can survive and propagate in HAT medium for a long time.

The present invention utilizes hybridoma technology to fuse two cells while maintaining the main characteristics of both. These two cells are antigen immunized mouse spleen cells and mouse myeloma cells, respectively. The main characteristic of the spleen cells of mice immunized with specific antigens is their antibody secretion function, but they cannot be cultured continuously in vitro, and mouse myeloma cells can divide and proliferate indefinitely under the culture conditions, i.e., they are so-called immortal. Under the action of the selection medium, only the hybrid cell formed by fusing spleen cell and myeloma cell can have the capacity of continuous culture, and cell clone which has both the antibody secretion function and the cell immortal maintaining property is formed.

Since the hybridoma technology is developed in the present application for the purpose of producing a monoclonal antibody specific to an antigen, it is necessary to select a B lymphocyte (simply referred to as a B cell) immunized with the antigen as a fused cell, and usually derived from a spleen cell of an immunized animal. The spleen is an important site for B cell aggregation, and no matter what immune mode stimulates, an obvious antibody response occurs in the spleen. The other side of the fused cells is to maintain the proliferation of the cells after cell fusion, and only tumor cells have the characteristic. Selection of cells in the same system increases the success rate of fusion. Multiple myeloma is a B cell line malignancy and is therefore an ideal fusion partner for splenocytes.

In a mixed cell suspension of mouse spleen cells and mouse myeloma cells, the cells will appear in various forms after fusion. Such as fused spleen cells and tumor cells, fused spleen cells and spleen cells, fused tumor cells and tumor cells, unfused spleen cells, unfused tumor cells, and multimeric forms of cells, etc. Normal splenocytes survive in the culture medium for only 5-7 days without special screening; multimeric forms of cells also die easily; unfused tumor cells need to be screened and removed. Only the fused cells have the genetic properties of both parents and can survive and propagate in HAT medium for a long period of time. Therefore, fusion of antigen-immunized mouse spleen cells and mouse myeloma cells can be achieved using techniques conventional in the art.

Screening out unfused tumor cells can also be accomplished using conventional biological manipulation means in the art.

The invention provides a method for screening fusion cells, which comprises the following steps: since there is a significant proportion of fusions of unrelated cells among the already fused cells, they need to be removed by screening. The screening process is generally carried out in two steps: the antibody screening of the fusion cells and the specific antibody screening based on the fusion cells are carried out. Diluting the fused cells sufficiently to ensure that the number of the cells distributed in each well of the culture plate is between 0 and a plurality of cells (30 percent of the wells are 0 to ensure that each well is a single cell), taking supernatant after culture, and selecting the cells with high antibody secretion by an ELISA method; this process is often conventionally referred to as cloning. Cloning the positive cells, finding out antibody positive cell strain aiming at target antigen by using ELISA coated by specific antigen, and performing cryopreservation, preservation, in vitro culture or animal abdominal cavity inoculation culture after proliferation.

The antigen is HPV16E6 protein.

In animal immunization, high purity antigens should be selected. An antigen often has multiple determinants, and the humoral immune response generated by an animal body after being stimulated by the antigen is essentially the secretion of antibodies of a plurality of B cell populations, and the B cells aiming at the target epitope only account for a very small part.

The invention also provides a method for obtaining the anti-HPV 16E6 protein monoclonal antibody, which comprises the following steps: HPV16E6 protein is selected to be injected into a mouse to be sensitized, after the antibody titer is detected to meet the requirement, the spleen cells of the mouse immunized by the antigen and the tumor cells of the mouse are fused to obtain a hybridoma cell strain for generating the monoclonal antibody of the HPV16E6 protein, and the monoclonal antibody of the HPV16E6 protein is proliferated by ascites of the mouse.

In a second aspect of the invention, magnetic microspheres coated with the anti-HPV 16E6 protein monoclonal antibody, referred to as immunomagnetic microspheres for short, or immunomagnetic beads, are provided.

The immunomagnetic microspheres can specifically bind to a target substance containing a corresponding antigen to form a new complex. When passed through a magnetic field, such complexes can be retained and separated from the other components, a process known as immunomagnetic separation. The immunomagnetic separation is simple and easy to implement, has high separation purity, retains the activity of a target substance, is efficient, rapid and low-toxic, and is widely applied to the fields of cell separation and purification, immunodetection, nucleic acid analysis and genetic engineering, targeting drug release carriers and the like.

In the invention, the monoclonal antibody of anti-HPV 16E6 protein is combined on the magnetic beads to form immunomagnetic microspheres, the immunomagnetic microspheres have different magnetic responsivity, and the compound moves mechanically under the action of magnetic force, thereby achieving the purpose of separating antigens.

The separation of the immune magnetic microspheres uses a direct method, the direct method uses monoclonal antibodies of anti-HPV 16E6 protein to coat magnetic particles, then the magnetic particles are combined with antigens to form a compound, and the compound is separated from other substances in a magnetic field.

At present, the method for marking biomolecules on the surface of latex particles with functional groups (amino, carboxyl, hydroxyl, epoxy and other functional groups) is more, and the invention adopts an EDC/NHS method to prepare the immunomagnetic microspheres for detecting HPV.

The invention provides a preparation method of the immunomagnetic microspheres, and the whole preparation process comprises four steps of activation, coupling, sealing and storage:

and (3) activation: taking a MEST solution as an activation buffer solution, adding carboxyl magnetic beads into the activation buffer solution, uniformly mixing, placing on a magnetic separation frame, and extracting supernatant after the magnetic beads are completely adsorbed; adding an activation buffer solution to wash the magnetic beads again for two times, adding the activation buffer solution into the magnetic beads, adding EDC and NHS, mixing uniformly, activating carboxyl on the surfaces of the magnetic beads at room temperature, washing with a MEST buffer solution to remove unreacted activating agents after activation, and washing the magnetic beads with the MEST buffer solution;

coupling: washing the magnetic beads by using a coupling buffer solution by using a BST solution as the coupling buffer solution; adding a coupling buffer solution to resuspend the washed magnetic beads, adding an anti-HPV 16E6 protein monoclonal antibody to enable activated carboxyl on the surfaces of the magnetic beads to react with amino of the anti-HPV 16E6 protein monoclonal antibody at room temperature, coupling the antibody on the surfaces of the magnetic beads to obtain immunomagnetic beads, collecting reaction supernatant after coupling is finished, and detecting the amount of coupled protein by using a BCA kit;

and (3) sealing: adding BSA (BSA is dissolved in borate Tween buffer solution in advance) to block activated groups which are not completely reacted on the surfaces of the immunomagnetic beads, and blocking other spatial sites through physical adsorption of the BSA to reduce the possibility of non-specific adsorption in a later test, and blocking the reaction for 30 minutes at room temperature;

and (3) storage: washing the sealed immunomagnetic beads with BST for four times (the tube needs to be replaced for the first time, the sealed immunomagnetic beads need to be washed cleanly, and the magnetic beads need to be re-suspended during each washing), and finally re-suspending the magnetic beads in a preservation solution and placing the preservation solution in an environment at 4 ℃ for use.

In one embodiment of the invention, the magnetic microspheres with the particle size of 100-200 nm are selected from the carboxyl magnetic beads.

In one embodiment of the invention, in the coupling process, the coupling amount of the monoclonal antibody against HPV16E6 protein is 80-120ug/mg based on the weight of the carboxyl magnetic beads.

In one embodiment of the present invention, the preparation method of the immunomagnetic microspheres comprises the following steps:

and (3) activation: taking an MEST solution as an activation buffer solution, putting 2mg of carboxyl magnetic beads into a 2ml centrifugal tube, adding 500 mu l of the activation buffer solution, uniformly mixing on a vortex oscillator, putting the centrifugal tube on a magnetic separation frame, and extracting a supernatant by using a micro desk vacuum pump when the magnetic beads are completely adsorbed; after adding 500. mu.l of activation buffer to wash the magnetic beads twice again, 485. mu.l of activation buffer was added to the magnetic beads, 2.5mg of EDC (0.5 g/ml original concentration) and 2.5mg of NHS (0.25 g/ml original concentration) were added, and the mixture was mixed well on a vortex shaker and the carboxyl groups on the surface of 2mg of magnetic beads were activated for 30 minutes at room temperature. After activation, unreacted activator was removed by washing with MEST buffer, the centrifuge tube was replaced, and the beads were washed twice with MEST buffer.

Coupling: using BST solution as coupling buffer solution, washing the magnetic beads twice by using 500 mu l of coupling buffer solution; adding 475 mul of coupling buffer solution to resuspend the washed magnetic beads, adding 25 mul of anti-HPV 16E6 protein monoclonal antibody of 150ug in total, reacting the activated carboxyl on the surfaces of the magnetic beads with the amino of the anti-HPV 16E6 protein monoclonal antibody for 3 hours at room temperature, and coupling the antibody on the surfaces of the magnetic beads to obtain the immunomagnetic beads. After the coupling is finished, collecting reaction supernatant and detecting the amount of the coupled protein by using a BCA kit.

And (3) sealing: the activated groups that were not fully reacted on the surface of the immunomagnetic beads were blocked by adding 500. mu.l of 1% BSA (BSA dissolved beforehand in borate Tween buffer), and other steric sites were blocked by physical adsorption of BSA to reduce the possibility of non-specific adsorption in the subsequent experiments. The reaction was blocked at room temperature for 30 minutes.

And (3) storage: the blocked immunomagnetic beads are washed four times with BST (the tube needs to be replaced for the first time, the tube must be washed cleanly, and the magnetic beads need to be resuspended in each washing), and finally the magnetic beads are resuspended in 500 ul of preservation solution and stored in a refrigerator at 4 ℃ for use.

In the process of preparing the immunomagnetic beads, the invention has the following key points: (1) EDC activates carboxyl on the surface of magnetic beads to form O-acylisourea derivatives, (2) because the generated derivatives are easy to hydrolyze in solution, NHS is added to generate stable NHS ester, and the purpose of activating carboxyl is achieved, and (3) the obtained activated magnetic beads react with amino of antibodies to generate immunomagnetic beads.

In the present invention, the carboxyl magnetic beads may be selected conventionally in the art.

In one embodiment of the present invention, the carboxyl magnetic beads are selected as superparamagnetic nanospheres. Superparamagnetic nanospheres (SPMNPs) are used as a new marker, and recent researches have started to apply the SPMNPs to an immunochromatography detection system because the SPMNPs not only have superparamagnetism and are easy to control, but also can be modified by functional groups on the surface of the SPMNPs. The basic detection principle is as follows: the method comprises the steps of selecting superparamagnetic nano microspheres as markers, detecting the magnetic strength of a double-antibody sandwich compound captured by a whole layer of a nitrocellulose membrane T line in a magnetic field through a magnetic signal reader, and quantitatively detecting the concentration of a substance to be detected.

The core part of the immunomagnetic nanosphere is paramagnetic nanoparticles (Fe)₃O₄，Fe₂O₃) Multiple polyethylene macromolecular materials are covered on the outer layer of the core, and the outermost layer is a functional layer formed by combining the immune ligand with a target substance, such as carboxyl (-COOH) and amino (-N)₂H) Hydroxyl (-OH), etc. and has the characteristic of biological specificity so as to ensure the special identification function of the magnetic nano microsphere. Because the size and the shape of the magnetic nano microsphere coated with the functional groups have good uniformity, the target substance can be quickly and effectively combined on the magnetic microsphere, and simultaneously, the magnetic responsiveness of the newly generated compound in a magnetic field is the same, and the behaviors are consistent. The immunomagnetic nano microsphere technology is a novel immunological detection and separation technology which combines the unique advantages of a solidified reagent and immunological reaction into a whole with high specificity. The principle is that the prepared immune magnetic particles carrying a certain antibody are mixed with a solution to be detected, an antigen corresponding to the antibody is combined with the antibody on the immune magnetic nanoparticles, and the combination is a specific reaction to form an immune complex containing magnetic beads. The technical toolHas the advantages of rapidness, high efficiency, good repeatability, simple operation and the like.

In a third aspect of the invention, a rapid test strip for HPV16 type virus antibody is provided.

The rapid detection test paper for the HPV16 type virus antibody comprises a sample pad, a combination pad, a PVC sheet and absorbent paper, wherein the combination pad and the absorbent paper are arranged at two ends of the PVC sheet; HPV16E6 protein is selected to be injected into a rabbit for sensitization, serum is harvested after the titer of a detection antibody meets the requirement to obtain a polyclonal antibody of HPV, the purified polyclonal antibody of the HPV16E6 protein is loaded on a nitrocellulose membrane (NC membrane) of a test strip to be used as a detection line, and the purified goat anti-mouse IgG antibody is loaded on the nitrocellulose membrane of the test strip to be used as a quality control line. When the sample with HPV16E6 protein flowed to the line, it was captured and developed. During use, the detected cervical mucus diluted liquid is dripped to the sample adding position to carry out rapid detection.

In one embodiment of the invention, the body of the magnetic lateral flow immunochromatographic strip is composed of 5 parts, including a base plate (3mm × 68mm), a sample pad (3mm × 18mm), a conjugate pad (3mm × 6mm), a nitrocellulose membrane (3mm × 30mm), and a water absorbent paper (3mm × 14 mm).

The magnetic immunochromatographic test strip to be constructed is mainly different from other conventional immunochromatographic systems in the selection of detection markers, and is also the core of the immunochromatographic technology.

In a fourth aspect of the invention, a method for rapid detection by using the rapid detection test paper based on the HPV16 type virus antibody is provided.

Taking a dry, sealed and stored rapid detection test strip based on HPV16 type virus antibody, taking out the test strip, horizontally placing the test strip on a table, dripping one drop of a sample to be detected (cervical orifice scratch diluted by physiological saline) into a sample hole on a sample pad, and gradually forming a khaki strip on a control line and a detection line along with the migration of a sample solution on the test strip. In each detection, whether the detection line is colored or not, if the quality control line is not colored, the detection result is invalid; if the quality control line and the detection line are both colored, indicating that the sample to be detected contains the HPV antibody with a certain concentration; if the quality control line is colored, the detection line is not colored (namely, invisible), which indicates that the sample to be detected does not contain HPV antibody (negative).

According to the invention, the detection sensitivity of the test strip is demonstrated by comparing the detection results of the magnetic bead nanoparticles and the colloidal gold test strip on the HPV virus antibody. Clinical tests further verify the accuracy of the magnetic immunochromatographic test strip, and the results of the electrochemical luminescence method measured by a large-scale instrument and the quantitative detection of the magnetic immunochromatographic test strip of the product are directly compared.

Compared with the prior art, the improvement part and the corresponding beneficial effect of the invention are as follows:

1. the invention applies the superparamagnetic nano-microsphere technology to the rapid detection of high-risk HPV16 type virus and the rapid detection of woman high-risk HPV16 type;

2. the invention utilizes the monoclonal antibody technology to prepare the high specificity and high titer monoclonal antibody for resisting HPV16E6 protein, and is applied to the rapid test paper for HPV16 type virus antibody, so as to carry out the research of rapid diagnosis of high risk type HPV16 type, and the like;

3. the invention provides a rapid detection test paper for HPV16 type virus antibody;

4. the optimized optimal conditions of the nano magnetic marker and the selection of the magnetic nano microspheres improve the marking efficiency, stability and repeatability to the maximum extent, and the developed test strip has the advantages of quickness, simplicity, convenience, easiness in judging results, no need of other equipment and the like. Has good market prospect.

Drawings

FIG. 1 shows the dispersion (A) and magnetic separation strength (B) of magnetic nanoparticles of different particle sizes in a magnetic sorting rack;

FIG. 2 shows the effect of the amount of antibody coupled to the surface of the magnetic spheres on the detection signal;

FIG. 3 flow chart of immunomagnetic bead preparation;

FIG. 4 is a comparison of the test times of test strips prepared from Sartourus CN 140 and Millipore HF 135;

in FIG. 4, ■ shows Sartourius CN 140 and O shows Millipore HF 135. The concentration of the detected HPV standard substance is 45.6 IU/L. The X-axis represents time after sample application and the Y-axis represents signal intensity over time.

FIG. 5 is a comparison of the intensity of the test signal from test strips prepared with Sartourus CN 140 and Millipore HF 135;

in FIG. 5, ■ shows Sartourius CN 140 and O shows Millipore HF 135. The X-axis represents the detected HPV standard concentration and the Y-axis represents the signal intensity read by the MAR instrument.

FIG. 6 is a schematic view of the detection of a sample pad after treatment with a BS solution;

FIG. 7 is a schematic representation of the detection of a sample pad after treatment with a BS solution containing 2% (w/v) Triton X-100;

FIG. 8 is a schematic structural diagram of a rapid test strip for HPV16 type virus antibody,

FIG. 9 is a diagram of a magnetic immunochromatographic strip in example 7;

FIG. 10 shows the effect of the size of magnetic nanoparticles on the chromatographic effect;

FIG. 11 shows the detection results of the magnetic bead nanoparticles and the HPV virus antibody by the colloidal gold test strip (the left colloidal gold test strip and the right magnetic immunochromatographic test strip in FIG. 11);

FIG. 12 shows a comparison of test strips and results of electrochemiluminescence assays;

FIG. 13 is a scattergram of the test strip and the results of the electrochemiluminescence quantitative measurements;

FIG. 14 is a comparison of the results of the test strip and the electrochemiluminescence method for detecting samples of different concentrations;

FIG. 15 is a diagram showing the use of the test strip.

Detailed Description

The invention provides a monoclonal antibody, which is an anti-HPV 16E6 protein monoclonal antibody and is obtained by long-term survival and propagation of fusion cells fused by antigen immunized mouse splenocytes and mouse myeloma cells in HAT culture medium and continuous culture and secretion of the fusion cells.

The fusion cell is formed by fusing a mouse spleen cell immunized by the antigen HPV16E6 protein and a mouse tumor cell.

In the fusion process, a cell fusion agent is used, and the cell fusion agent is selected from polyethylene glycol (PEG 1000-2000) and is applied at a concentration of 40% (W/V). The cell fusion agent is used to cause a certain degree of damage to cell membranes, so that cells are easy to adhere to each other and fuse together. The optimal fusion effect should be minimal cell damage while producing the highest frequency of fusion.

The invention also provides magnetic microspheres coated with the anti-HPV 16E6 protein monoclonal antibody, which are abbreviated as immunomagnetic microspheres or immunomagnetic beads.

The monoclonal antibody of anti HPV16E6 protein is combined to magnetic beads to form immune magnetic microspheres, the immune magnetic microspheres have different magnetic responsivity, and the compound moves mechanically under the action of magnetic force, so that the purpose of separating antigen is achieved.

The preparation method of the immunomagnetic microspheres comprises the following four steps of activation, coupling, sealing and storage in the whole preparation process:

The invention also provides a rapid detection test paper for the HPV16 type virus antibody. The rapid detection test paper for the HPV16 type virus antibody comprises a sample pad, a combination pad, a PVC sheet and absorbent paper, wherein the combination pad and the absorbent paper are arranged at two ends of the PVC sheet; HPV16E6 protein is selected to be injected into a rabbit for sensitization, serum is harvested after the titer of a detection antibody meets the requirement to obtain a polyclonal antibody of HPV, the purified polyclonal antibody of the HPV16E6 protein is loaded on a nitrocellulose membrane (NC membrane) of a test strip to be used as a detection line, and the purified goat anti-mouse IgG antibody is loaded on the nitrocellulose membrane of the test strip to be used as a quality control line. When the sample with HPV16E6 protein flowed to the line, it was captured and developed. During use, the detected cervical mucus diluted liquid is dripped to the sample adding position to carry out rapid detection.

The invention also provides a method for rapidly detecting the HPV16 type virus antibody-based rapid detection test paper.

The invention is described in detail below with reference to the figures and specific embodiments. The following examples illustrate exemplary embodiments of the present invention to assist those skilled in the art in understanding other objects, features, advantages and aspects of the present application. It should be understood that the following description and specific examples, while indicating the preferred embodiment of the invention, are given by way of illustration only and are not intended to limit the scope of the invention. The scope of the invention is to be determined from the following claims. Unless otherwise indicated, specific experiments in the following examples were performed according to methods and conditions conventional in the art, or in accordance with commercial specifications.

Example 1

An anti-HPV 16E6 protein monoclonal antibody is prepared from the fusion cells of mouse spleen cell immunized by antigen HPV16E6 protein and mouse tumor cell through long-term survival and reproduction in HAT culture medium, and continuous culture and secretion.

During the fusion process, a cell fusion agent was used, which was selected from polyethylene glycol (PEG 1000-2000) and applied at a concentration of 40% (W/V).

Example 2

Selection of magnetic nanospheres

The diameter of the magnetic nanoparticles has a critical influence on the speed and detection sensitivity of immunochromatography. In the invention, three magnetic nanoparticles with different diameters of 50nm, 100nm and 200nm are purchased in the previous research. The dispersibility and magnetic separation strength are shown in FIG. 1.

As can be seen from FIG. 1, the superparamagnetic nanospheres of 50nm are difficult to magnetically separate in a common magnetic separation rack due to their small particle size. Therefore, it was preliminarily suggested according to this example to use magnetic nanospheres with particle sizes of 100nm and 200nm to couple with HPV virus antibodies, respectively, to construct a magnetic immunochromatographic test strip.

Example 3

Optimization of antibody addition amount in immunomagnetic bead preparation system

The binding capacity of the magnetic nanoparticle surface antibody is related to the proportion of the antibody added in the preparation process of the immunomagnetic beads, the high and low binding rate of the magnetic nanoparticle surface antibody is closely related to the effect of immunochromatography, in addition, the specific monoclonal antibody is expensive, and the detection cost is directly determined by the adding amount of the antibody. Therefore, it is important to the experiment to discuss the optimal addition amount of the antibody in the process of preparing the immunomagnetic nanoparticles.

When the immunomagnetic nanoparticles are prepared, 1mg of carboxyl magnetic beads activated by the same method are taken, different amounts of HPV monoclonal antibodies 40 mug, 80 mug and 120 mug are added for coupling, then, an HPV quantitative detection kit is used for detecting the concentration of the antibodies in the coupled supernatant, so that the antibody coupling amount on the unit mass magnetic beads is obtained when different antibody addition amounts are obtained, 3 groups of immunomagnetic beads are prepared, test paper strips are respectively constructed by using the 3 groups of immunomagnetic beads for detection, a MAR instrument reads T-line signals, and the relation between the T-line signals and the antibody coupling amount is shown in figure 2. The test paper prepared by the immunomagnetic spheres with the antibody coupling amount of 12.05ug/mg is the weakest in the detection signal of the same serum sample, the detection signal of 53.43 ug/mg group is intermediate, and the detection signal of 96.35ug/mg group is the strongest.

In this experiment, the more the amount of the coupling antibody on the surface of the magnetic sphere, the stronger the detection signal. However, the amount of the magnetic sphere surface coupling antibody is not necessarily proportional to the detection signal. On one hand, the quantity of the coupled antibodies on the surface of the magnetic sphere is increased, the probability of combining the immunomagnetic sphere with the antigen is increased, the number of the immunomagnetic sphere-antigen complexes captured by the capture antibodies on the T line is increased, the detection signal is enhanced, and the positive effect is considered. On the other hand, however, when the same amount of antigen is recognized, the larger the amount of antibody on the surface of each immunomagnetic sphere, the smaller the number of immunomagnetic spheres required, the fewer the immunomagnetic sphere-antigen complexes captured by the capture antibody on the T line, and the weaker the detection signal, which we consider as a negative effect. When the amount of the coupled antibody on the surface of the magnetic ball is less, the amount of the surface antibody is increased, the positive effect is dominant, and the detection signal is obviously enhanced; when the amount of the magnetic ball surface antibody reaches a certain value, the amount of the magnetic ball surface coupling antibody is continuously increased, the negative effect is dominant, and the detection signal is weakened.

In this experiment, the detection signal was positively correlated with the amount of the antibody on the surface of the magnetic sphere, and the amount of the antibody on the surface of the magnetic sphere was estimated to be in the ascending portion of the bell-shaped curve. With increasing amounts of coupling, the detection signal may still tend to increase. However, the cost is increased by increasing the antibody coupling amount, the coupling amount of 96.35 mu g/mg can meet the detection requirement, and the immune magnetic sphere with the coupling amount of 96.35ug/mg is selected to construct a chromatography test strip from the economic viewpoint.

Example 4

This example provides a method for preparing immunomagnetic microspheres, comprising:

The preparation process of immunomagnetic beads is shown in FIG. 3.

Example 5

Selection and preparation of nitrocellulose membranes

For magnetic nano-microspheres with the same particle size, nitrocellulose membranes (NC membranes) of different types have certain influence on the detection time and the detection sensitivity of the magnetic immunochromatographic test strip. In the experiment, the influence of 2 NC membranes of different types, namely Sartourus CN 140 and Millipore HF 135, on the magnetic immunochromatography result is researched, the immunomagnetic nanoparticles with the optimal antibody coupling amount are prepared, the three NC membranes are respectively utilized to build a magnetic immunochromatography detection system, HPV virus antibody standard samples with the same concentration are detected, and the detection time and the corresponding magnetic signal intensity required by the stability of the T-line magnetic signal intensity are recorded.

The type of the nitrocellulose membrane is important in the test, and the nitrocellulose membrane is used as a reaction carrier to influence the success or failure of the whole test. The sources, types and quantities of the polymers and the surfactants used by different manufacturers for producing the nitrocellulose membranes are greatly different, and the properties of the produced membranes are greatly influenced, namely the pore diameters and the distribution structures of the membranes are different. The pore size of the membrane is reduced, the actual available surface area of the membrane is increased, and the amount of membrane-bound protein is increased; the smaller the pore size of the membrane is, the slower the chromatographic speed is, the longer the time for the labeled complex to pass through the T line is, and the more sufficient the reaction is; thus, the smaller the pore size of the membrane, the higher the sensitivity, but at the same time, the slower the running plate speed, and the higher the chance of nonspecific binding, i.e., the higher the false positives. Different envelope proteins have specific requirements on membranes, the invention analyzes 2 NC membranes of different models, namely Sartourus CN 140 and Millipore HF 135 according to the property comparison of HPV16E6 proteins, selects membranes with suitable pore size and distribution structure, and finds a suitable balance point to optimize the flow rate of the markers on the membranes.

Preparation of different nitrocellulose membranes:

the purified polyclonal antibody against HPV16E6 protein and the purified goat anti-mouse IgG antibody were each sprayed at 0.8ul/cm onto three different types of NC membranes (Sartourius CN 140 and Millipore HF 135) using a membrane-spraying apparatus using a 0.01M PBS solution (pH7.4) containing 1% sucrose to form a detection line (T line) and a quality control line (C line) with a distance of 5mm between the two lines. The 2 test strips with different models are placed in an oven at 37 ℃ for 2 hours for drying, and after the membrane is cut, the membrane is sealed and stored in a moisture-proof cabinet at the temperature of 25 ℃ and the humidity of 25-30% RH.

And (3) experimental comparison:

(1) examination of detection time:

two sets of test strips prepared from Sartourus CN 140 and Millipore HF 135, respectively, tested the same sample to be tested. Starting from the time when the sample is added to the sample pad, giving an initial reaction time of 2 minutes, then starting from 2 minutes, reading and recording the detection signal of the T-line by the MAR instrument, reading and recording every minute until 45 minutes after the sample is added, the change of the signal intensity with time can be obtained, and the reaction time necessary for the detection can be reflected. The detection time is defined as the point in time when the magnetic signal strength first approaches the plateau value.

The different detection times of the immunochromatographic test strips constructed from Sartourius CN 140 and Millipore HF 135 are shown in fig. 4. As can be seen in FIG. 4, there was no significant difference (within 20 minutes) in the test strip time constructed from Sartourius CN 140 and Millipore HF 135.

(2) Investigation of magnetic Signal Strength

The magnetic signal intensity is closely related to the sensitivity of the immunoassay. Two sets of test strips prepared from sartourus CN 140 and Millipore HF 135, respectively, were used to detect HPV standards at different concentration gradients: 0,22.8, 45.6,114,228 IU/L. And reading the magnetic signal intensity of the detection line on the test strip by the MAR instrument after 25 minutes.

The X-axis represents the detected HPV standard concentration and the Y-axis represents the signal intensity read by the MAR instrument.

It can be seen from figure 5 that the immunochromatographic assay constructed with Millipore HF 135 model NC membranes produced a stronger signal intensity than that constructed with sartourus CN 140 model NC membranes when detecting the same analyte (HPV).

In summary, in this experiment, the test strip constructed by Millipore HF 135 model NC membrane is recommended to be applied to the magnetic immunochromatographic detection system.

Example 6

Preparation of sample pad and conjugate pad

The sample pad is made of glass fiber, is cut and then is put into sample pad treatment liquid (prepared in advance) to be soaked for 30 minutes, and then is put into a 37 ℃ oven to be dried for 12 hours, and finally is put into a moisture-proof agent to be sealed and stored for standby; the sample pad treatment solution was 0.02M borate buffer containing 0.5% (w/v)) BSA, 2% (w/v) Triton X-100, 0.5% (w/v) NaCl. Sample pads containing Triton X-100 and Triton X-100 in the treatment solution were prepared simultaneously to explore the effect of surfactants in the sample pad treatment solution.

The bonding pad also adopts glass fiber, the glass fiber is cut and then placed into bonding pad treatment fluid (prepared in advance) to be soaked for 30 minutes, then the bonding pad is placed into a 37 ℃ oven to be dried for 12 hours, the prepared 4mg/ml immunomagnetic bead stock solution is diluted to 1.5mg/ml by BST buffer solution after being dried and is uniformly sprayed and printed on the pretreated bonding pad, the bonding pad is placed into the 37 ℃ oven to be dried for 30 minutes, and then a drying agent is placed into the bonding pad to be sealed and stored for standby; the conjugate pad treatment solution used was 0.02M BST buffer containing 10% sucrose and 2% (w/v) trehalose. Meanwhile, the combination pads of the immunomagnetic beads with the concentrations of 1mg/ml, 1.5mg/ml and 2.0mg/ml are respectively prepared, so as to explore the influence of the immunomagnetic beads with different concentrations on the release of the combination pads and the detection sensitivity.

The sample pad is the part to be loaded and is also the part of the test strip which is firstly contacted with the detection sample. In order to make the immunomagnetic beads completely released on the nitrocellulose membrane for rapid chromatography and improve the specificity, in addition to solving the problem of binding pad components, other components of the test strip are also treated, and the most common method is to pretreat the sample pad and add some effector substances on the sample pad.

When the untreated sample pad detects substances, the chromatography condition of the immunomagnetic beads is poor, and even the immunomagnetic beads cannot flow to the detection line, so that the antibodies on the detection line cannot be captured to generate a detection strip. Since immunomagnetic beads are stably present in BS from coupling to storage in a BS buffer system, a BS solution is considered as a basic processing solution for the sample pad, but it is not sufficient to treat the sample pad with only the BS solution. Therefore, it is necessary to add an effector substance that can promote magnetic bead chromatography to the sample pad treatment solution.

Triton X is a mild surfactant and can promote the rapid dissolution of immunomagnetic beads in a sample solution and the release of the immunomagnetic beads from the conjugate pad, and even smooth surging in the whole chromatography process. As shown in FIGS. 6 and 7, when the sample pad is treated and detected by the BS solution containing 2% (w/v) Triton X-100, the T/C lines are all seen to have bands, and the immunomagnetic beads can well flow on the NC membrane until the end of the NC membrane. Indicating that surfactants such as Triton X-100 can be very good at facilitating the whole immunochromatography.

The addition of the surfactant also improves the chromatography speed and shortens the detection time, which is important for rapid detection. The higher the surfactant concentration, the faster the chromatography speed. However, in the immunochromatography detection, the chromatography speed is too high, the time of immunoreaction between antigen and antibody is shortened, and the sensitivity is reduced accordingly. Therefore, an equilibrium point needs to be found according to the requirements of the experiment itself on sensitivity and detection time. In this application, a concentration of 2% (w/v) of Triton X-100 is recommended.

Example 7

The rapid detection test paper for the HPV16 virus antibody comprises a sample pad, a combination pad, a PVC sheet and absorbent paper, wherein the combination pad and the absorbent paper are arranged at two ends of the PVC sheet, the sample pad is positioned at one end of the combination pad, an NC membrane is arranged on the PVC sheet, and the immunomagnetic microspheres are sprayed and printed on the combination pad of the test paper strip to serve as a capture line; HPV16E6 protein is selected to be injected into a rabbit for sensitization, serum is harvested after the titer of a detection antibody meets the requirement to obtain a polyclonal antibody of HPV, the purified polyclonal antibody of the HPV16E6 protein is loaded on a nitrocellulose membrane (NC membrane) of a test strip to be used as a detection line, and the purified goat anti-mouse IgG antibody is loaded on the nitrocellulose membrane of the test strip to be used as a quality control line. When the sample with HPV16E6 protein flowed to the line, it was captured and developed. During use, the detected cervical mucus diluted liquid is dripped to the sample adding position to carry out rapid detection.

In this example, the immunomagnetic microspheres were prepared in example 4.

In this example, the NC membrane was Millipore HF 135 type NC membrane.

In this example, the sample pad was made of glass fiber and treated with a BS solution containing 2% (w/v) Triton X-100.

In this example, the conjugate pad was also made of glass fiber and soaked in the conjugate pad treatment solution (prepared in advance) for 30 minutes.

The method comprises the following steps of sequentially adhering a nitrocellulose membrane (with the length of 30mm), a combination pad (with the length of 6mm), a sample pad (with the length of 18mm) and absorbent paper (with the length of 14mm) on a PVC base plate, and mutually staggering and overlapping adjacent pads for 2mm, wherein the nitrocellulose membrane is pre-coated with a capture antibody (T line) for resisting HPV and a goat anti-mouse Ig G antibody (C line) to assemble a test paper plate, and the design of the staggered and overlapped structure ensures good transferability among all the parts. Cutting the test paper into test paper strips with the width of 3mm by a cutting machine after the test paper strips are assembled, putting the test paper strips into a detection clamping groove, and adding a moisture-proof agent for sealing and storing. Fig. 8 is a schematic structural diagram of a rapid test strip for HPV16 type virus antibodies, and fig. 9 is a physical diagram of the magnetic immunochromatographic test strip in this embodiment.

Example 8

Immunochromatography test strip pair detection time of magnetic nano microspheres with different particle sizes

The immunochromatographic test strip of magnetic nanospheres of different particle sizes was prepared according to the preparation method of example 7, wherein the magnetic nanospheres of 50nm, 100nm and 200nm in particle size were constructed by coupling HPV viral antibodies respectively.

The influence of the particle size of the magnetic nanoparticles on the chromatographic effect is analyzed according to the detection sensitivity and time of the system. As a result, as shown in fig. 10, it can be seen that the flow velocity of the magnetic spheres having a large particle diameter is low and the flow velocity of the magnetic spheres having a small particle diameter is high. The traction force in the magnetic immunochromatographic system is derived from the capillary force of an NC membrane and the attraction effect of absorbent paper, and the resistance generated by the movement of the magnetic spheres is proportional to the diameter of the magnetic spheres. The magnetic ball with large particle size has large resistance, low flow rate in the chromatography process and long detection time. If a truly fast detection is desired, the large size of the magnetic spheres becomes one of the important reference factors when selecting the magnetic spheres. However, it is not always preferable that the flow velocity of the magnetic ball is as high as possible, but rather, an excessively high flow velocity causes a decrease in sensitivity. This is because, in the immunochromatographic assay, the reaction time between antigen and antibody is not sufficient as in the case of the complete immunoreaction such as the ELISA, but is incomplete, and if the flow rate is too high, the immunoreaction time is too short, and thus the sensitivity is lowered. In the invention, the magnetic microspheres with the particle size of about 100-200 nm are recommended to be applied to a magnetic immunochromatographic detection system.

Example 9

Detection result of magnetic bead nanoparticles and colloidal gold test strip on HPV virus antibody

The lowest detection limit of magnetic bead nanoparticles on HPV viruses is 1: 10000-1: 1000 (1: 1000 signals are very clear, 1: 10000 signals are also seen). When the dilution concentration is 1: 100000 hours, it could not be detected; the lowest detection limit of colloidal gold on HPV virus is 1: 100, when the dilution concentration is 1: at 1000 hours, the detection can not be carried out, which shows that the detection sensitivity of the invention is 1-2 orders of magnitude higher than that of the traditional colloidal gold. The results are shown in FIG. 11, in which the left side of FIG. 11 is the colloidal gold test strip and the right side is the paramagnetic enzyme test strip.

Example 10

Clinical trial

In order to further verify the accuracy of the magnetic immunochromatographic test strip, a part of clinical samples are selected in affiliated hospitals of Shanghai health medical college to evaluate the clinical application value of the test strip.

Detection sensitivity of the magnetic immunochromatographic test strip: detecting the color development condition of the HPV virus 16 type antibody with each concentration by using the test strips assembled in the same batch under the same experimental conditions and environment; firstly, the test strips all have C strips, which indicates that the test strips are effective in detection and reliable in result; secondly, the color development of the detection line can be found to become lighter along with the reduction of the concentration, and the macroscopic color development can conveniently carry out qualitative judgment on the detection result along with the change of the concentration;

the results of the electrochemical luminescence method measured by a large-scale instrument and the quantitative detection result of the magnetic immunochromatographic test strip of the product are directly compared, and the relative errors are less than 5 percent. FIG. 12 is a comparison of test strips and results of electrochemiluminescence.

The detection results of the two methods are subjected to linear correlation analysis, the X axis is the quantitative detection result of an electrochemiluminescence method, the axis is the quantitative detection result of a test strip, statistical analysis shows that the quantitative detection results of the two methods are in positive correlation, r is 0.979, the regression equation between the two is y is 0.986X +9.206, and P is less than 0.05, so that the method has statistical significance. FIG. 13 is a scattergram of quantitative test results of the two methods.

Example 11

Comparative analysis of precision

According to the detection result of the electrochemical luminescence method, the batch repeatability experiments of the two detection methods are divided into three groups to be carried out on a lower concentration group, a middle concentration group and a high concentration group. And selecting samples with different concentrations of low, medium and high to perform repeated detection for 20 times. The results were statistically processed (t-test) to show P > 0.05, and there was no statistical difference between the two. FIG. 14 is a comparison of the results of two methods for detecting samples at different concentrations.

Example 12

Instructions for use

Taking a dry and sealed test strip, taking out the test strip, horizontally placing the test strip on a table, dropwise adding one drop of a sample to be tested (cervical orifice scrapings diluted by normal saline) into a sample hole, and gradually forming a khaki strip on a control line and a detection line along with the migration of a sample solution on the test strip. In each detection, whether the detection line is colored or not, if the quality control line is not colored, the detection result is invalid; if the quality control line and the detection line are both colored, indicating that the sample to be detected contains the HPV antibody with a certain concentration; if the quality control line is colored, the detection line is not colored (namely, invisible), which indicates that the sample to be detected does not contain HPV antibody (negative).

The magnetic immunochromatographic test strip has the advantages of qualitative detection and quantitative detection, so before information is read by a magnetic signal reader, dark and light brown bands on a T/C line on the test strip can be seen, and the magnetic microspheres are captured and gathered on the T/C line to develop color. As shown in FIG. 15, T/C lines were visible on all the strips. The T line shows bands with different depths due to different concentrations of the detection substances. And the C lines are also provided with strips, which indicates that the test strip is effective.

The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.

Claims

1. A monoclonal antibody against HPV16E6 protein, which is obtained by culturing and secreting a fused cell obtained by fusing a mouse spleen cell immunized with the HPV16E6 antigen and a mouse myeloma cell in HAT medium for a long period of time.

2. The monoclonal antibody of claim 1, wherein the fused cells of the antigen-immunized mouse spleen cells and the mouse myeloma cells are fused using a cell fusion agent selected from polyethylene glycol at a concentration of 40% W/V.

3. A magnetic microsphere coated with the anti-HPV 16E6 protein monoclonal antibody of claim 1.

4. A method for preparing the magnetic microspheres coated with the monoclonal antibodies against HPV16E6 protein according to claim 3, wherein the preparation process comprises the following four steps:

coupling: washing the magnetic beads by using a coupling buffer solution by using a BST solution as the coupling buffer solution; adding coupling buffer solution to resuspend the washed magnetic beads, adding an anti-HPV 16E6 protein monoclonal antibody to enable activated carboxyl on the surfaces of the magnetic beads to react with amino of the anti-HPV 16E6 protein monoclonal antibody at room temperature, and coupling the antibody on the surfaces of the magnetic beads to obtain immunomagnetic beads;

and (3) sealing: adding BSA (bovine serum albumin) to block the activated groups which do not completely react on the surfaces of the immunomagnetic beads, blocking other spatial sites through physical adsorption of the BSA, and carrying out blocking reaction for 30 minutes at room temperature;

and (3) storage: and washing the blocked immunomagnetic beads with BST for four times, and finally, resuspending the magnetic beads in a preservation solution and placing the solution in an environment at 4 ℃ for preservation for use.

5. The method according to claim 4, wherein the carboxyl magnetic beads are selected as superparamagnetic nanospheres.

6. The method according to claim 4, wherein the magnetic microspheres with a particle size of 100 to 200nm are selected as the carboxyl magnetic beads.

7. The preparation method of claim 4, wherein the coupling amount of the monoclonal antibody against HPV16E6 protein is 80-120ug/mg based on the weight of the carboxyl magnetic beads in the coupling process.

8. A test paper for rapidly detecting HPV16 type virus antibodies is characterized by comprising a sample pad, a combination pad, a PVC sheet and absorbent paper, wherein the combination pad and the absorbent paper are arranged at two ends of the PVC sheet, the sample pad is positioned at one end of the combination pad, an NC membrane is arranged on the PVC sheet, and magnetic microspheres coated with monoclonal antibodies against HPV16E6 protein as claimed in claim 3 are sprayed and printed on the combination pad of the test paper strip to serve as capture lines; HPV16E6 protein is selected to be injected into a rabbit for sensitization, serum is harvested after the titer of a detection antibody meets the requirement to obtain a polyclonal antibody of HPV, the purified polyclonal antibody of the HPV16E6 protein is loaded on a nitrocellulose membrane of a test strip to be used as a detection line, and the purified goat anti-mouse IgG antibody is loaded on the nitrocellulose membrane of the test strip to be used as a quality control line.

9. The rapid test paper for HPV16 type virus antibody according to claim 8, wherein the sample pad is made of glass fiber and is treated with a BS solution containing 2% (w/v) Triton X-100.

10. The method for using the rapid test paper for the HPV16 type virus antibody according to claim 8 is characterized in that a dry, sealed and preserved rapid test paper for the HPV16 type virus antibody is taken out and horizontally placed on a table, a drop of the sample to be tested is dripped into a sample hole on a sample pad, and soil yellow stripes gradually appear on a control line and a detection line along with the migration of the sample solution on the test paper, and each detection shows that the detection result is invalid no matter whether the detection line develops color or not, as long as a quality control line does not develop color; if the quality control line and the detection line are both colored, indicating that the sample to be detected contains the HPV antibody with a certain concentration; if the quality control line is colored, the detection line is not colored, and the HPV antibody is not contained in the sample to be detected.