Human papillomavirus detection reagent and application thereof

Abstract

The application belongs to the technical field of detection, and relates to a human papilloma virus detection reagent and application thereof. The human papilloma virus detection reagent comprises human papilloma virus antigen coated magnetic microparticle reagent and alkaline phosphatase labeled human papilloma virus antibody reagent. The human papilloma virus antigen coated magnetic microparticle is sealed by a sealing agent which is mixed by 2-10wt% PEG6000 solution, DB1130 and 0.1-1wt% gelatin solution. The system for detecting human papilloma virus antibody adopted by the application fills the gap of domestic HPV chemiluminescence antibody detection, and has high sensitivity, accuracy and clinical value.

Description

Technical Field

[0001] The present invention belongs to the technical field of detection, and relates to a human papillomavirus detection reagent and application thereof. Background Art

[0002] Human papillomavirus (HPV) is a group of tissue-specific, double-stranded DNA tumor viruses that can cause proliferation of epithelial and mucosal tissues, primarily through direct or indirect contact with human skin and mucosal tissues. Genital warts are a common sexually transmitted disease caused by HPV infection, with a high infection rate and strong contagion. Current research suggests that infection with high-risk HPV types is essential for the development of cervical cancer. In particular, HPV16 has the highest detection rate in cervical cancer tissue, reaching 50%. HPV16 is also the predominant type of cervical cancer in women in my country. A survey conducted by the International Agency for Research on Cancer (IARC) in 22 countries focused on HPV types that cause invasive cervical cancer (ICC). Among 1,000 cases of histologically confirmed ICC, 99.7% tested positive for HPV DNA, with the primary HPV types being HPV16 (53%) and HPV18 (15%).

[0003] Cervical cancer is the second most common female malignancy, and human papillomavirus (HPV) is essential for its development. Although HPV infection is common, with infection rates as high as 20.8% among women aged 35 and older in high-risk areas for cervical cancer, over 80% of women who test positive for HPV DNA will not develop cancer or precancerous lesions. Therefore, a limitation of HPV DNA testing is its inability to distinguish between transient infections and those that can cause cervical lesions, resulting in a high false-positive rate. Numerous studies have shown that the products of HPV oncogene expression (oncoproteins) have high carcinogenic activity, and therefore these biomarkers can serve as more accurate and specific predictors of cervical cancer than HPV DNA.

[0004] In addition to HPV DNA testing and HPV oncogenic protein testing, HPV antibodies in blood (body fluids) have also been shown to be highly correlated with a history of HPV infection and cervical cancer. HPV L1 blood antibodies can be tested to provide information about HPV infection. As HPV infection progresses, HPV induces the production of anti-HPV antibodies, which can be detected in serum.

[0005] However, most HPV detection methods are currently nucleic acid detection methods, and the National Medical Products Administration has not yet obtained relevant product registration information for chemiluminescence testing of HPV antibodies. Therefore, it is of great significance to develop a kit for HPV chemiluminescence antibody detection. Summary of the Invention

[0006] The purpose of the present invention is to provide a human papillomavirus detection reagent and its application in response to the deficiencies in the prior art.

[0007] One object of the present invention is achieved through the following technical solutions:

[0008] A human papillomavirus detection reagent comprises a human papillomavirus antigen-coated magnetic particle reagent and an alkaline phosphatase-labeled human papillomavirus antibody reagent.

[0009] Preferably, the method for preparing the human papillomavirus antigen-coated magnetic microparticle reagent comprises the following steps:

[0010] Human papillomavirus antigen is added to the magnetic particle dispersion, and then a reaction catalyst is added for suspension reaction, and then the pH is adjusted to 7.0-8.0, and a blocking agent is added for blocking reaction. After washing, a diluent is added and resuspended to obtain a human papillomavirus antigen-coated magnetic particle reagent.

[0011] Magnetic microparticles refer to highly stable colloidal composite materials formed by the combination of magnetic nanoparticles and organic or inorganic molecules, which can be uniformly dispersed in a specific base liquid. The surface of the magnetic microparticles contains one or more reactive functional groups such as tosyl, amino, carboxyl, hydroxyl, and ethylene oxide. A magnetic microparticle dispersion is formed by dispersing the magnetic microparticles in a buffer solution. The particle size of the magnetic microparticles is preferably 0.5 to 3.0 μm. The buffer solution may be Hepes, MES, boric acid, or phosphate buffer at a pH of 5.0 to 9.5 and a concentration of 0.05 to 0.3 mol / L. The concentration of the magnetic microparticles in the magnetic microparticle dispersion is preferably 5 to 20 mg / mL.

[0012] Preferably, the mass ratio of the magnetic particles to the human papillomavirus antigen is 1:5 to 1:100.

[0013] Preferably, the reaction catalyst is an ammonium sulfate solution with a pH of 9.0 to 10.0 and a concentration of 0.5 to 4 mol / L.

[0014] Preferably, the added amount of the reaction catalyst is 0.5 to 2 times the total volume of the magnetic particle dispersion and the human papillomavirus antigen.

[0015] Preferably, the suspension reaction is carried out at 20-35° C. for 20-80 h.

[0016] Preferably, the blocking agent is a mixture of 2-10 wt% PEG6000 solution, DB1130 and 0.1-1 wt% gelatin solution. TM DB1130 was purchased from MBL, Japan.

[0017] Preferably, 2-10 wt% PEG6000 solution, DB1130 and 0.1-1 wt% gelatin solution are mixed in a mass ratio of (0.8-1.2):(0.8-1.2):(1-5).

[0018] Preferably, 100 to 150 μL of blocking agent is added per 10 mg of magnetic particles.

[0019] Preferably, the blocking reaction is carried out at 20-35° C. for 15-30 h.

[0020] Preferably, the concentration of the human papillomavirus antigen-coated magnetic particle reagent obtained by resuspending after adding the diluent is 5 to 20 mg / ml (calculated based on the mass of the magnetic particles).

[0021] Preferably, the method for preparing the alkaline phosphatase-labeled human papillomavirus antibody reagent comprises the following steps:

[0022] S1. The human papillomavirus antibody is replaced with an amino-free and thiol-free buffer and concentrated. Then, dithiothreitol (DTT) or tris(2-carbonylethyl)phosphine hydrochloride (TCEP) is added to the human papillomavirus antibody for reaction. A glycine solution is added, the reaction is continued, and the antibody is passed through a desalting column and concentrated to 2-4 mg / ml (calculated based on the mass of the human papillomavirus antibody).

[0023] S2. Replace the alkaline phosphatase with an amino- and thiol-free buffer and concentrate it. Then, add the SMCC solution to react. Add the glycine solution and continue the reaction. Pass the solution through a desalting column and concentrate it to 2-4 mg / ml (calculated based on the mass of the alkaline phosphatase).

[0024] S3, mixing the product of step S1 and the product of step S2, and then adding magnesium chloride solution to react;

[0025] S4. Purify the product of step S3 and then dilute it to a final concentration of 0.2-2 μg / ml.

[0026] In the above steps S1 and S2, the amino-free and thiol-free buffer can be exemplified by borate buffer, HEPES buffer, etc., with a pH of 7.0 to 7.5 and a concentration of 0.01 to 0.05 mol / L.

[0027] In the above steps S1 and S2, the method of replacing the buffer solution with one free of amino groups and thiol groups includes dialysis or using a PD-10 desalting column. After replacing the buffer solution, the human papillomavirus antibody and alkaline phosphatase are concentrated to 2-4 mg / ml.

[0028] The time for adding dithiothreitol (DTT) or tris (2-carboxyethyl) phosphine hydrochloride (TCEP) in step S1 is 10-30 min, and the reaction is carried out at 15-40 °C.

[0029] In step S1, the DTT or TCEP is in the form of a solution, for example, the DTT or TCEP can be dissolved in a pH 8.0-9.0, 0.01-0.05 mol / L Hepes buffer, and the concentration of the DTT or TCEP solution is preferably 10-50 mmol / L, and 0.5-2.5 μL of the DTT or TCEP solution is added per milligram of antibody mass.

[0030] In steps S1 and S2, the pH of the glycine solution is 7.0-7.4, and the concentration is 0.5-2 mol / L. In step S1, the volume of the glycine solution added is the same as the amount of the DTT or TCEP solution added; in step S2, the volume of the glycine solution added is the same as the amount of the SMCC solution added; and the reaction is continued for 5-10 min after the glycine solution is added.

[0031] In steps S1 and S2, the desalting column is eluted with, for example, a pH 8.0-9.0, 0.01-0.05 mol / L Hepes buffer, and then concentrated to 2-4 mg / mL.

[0032] In step S2, the SMCC solution is formed by dissolving SMCC in N,N-dimethylformamide (DMF), and the concentration is preferably 5-8 mg / mL.

[0033] In step S2, 2.5-7.5 μL of the SMCC solution is added per milligram of alkaline phosphatase, and the reaction is carried out at 20-40 °C for 10-30 min.

[0034] In step S3, the mass ratio of the product of step S1 to the product of step S2 is preferably 1:(0.5-2).

[0035] In step S3, the concentration of the magnesium chloride solution is 0.1-0.5 mol / L, and 1-5 μL of the magnesium chloride solution is added per milliliter of reaction volume.

[0036] In step S3, the temperature for adding the magnesium chloride solution for the reaction is 2-8 °C, and the reaction time is 8-20 h.

[0037] In step S4, the purification step includes passing the product of step S3 through a superdex 200 molecular sieve column, and collecting according to the protein peak. The buffer used for dilution can be, for example, a pH 7.0-7.5, 0.01-0.05 mol / L Hepes buffer.

[0038] The human papilloma virus detection reagent provided by the application further comprises a human papilloma virus antibody calibrator reagent, which is obtained by diluting a human papilloma virus antibody calibrator buffer to a plurality of series concentrations. The series concentrations can include 0.2, 1.0, 5, 25, 100 pg / ml, etc.

[0039] Preferably, the calibration buffer comprises 1-10 g / L HEPES, 20-60 g / L BSA, 2-10 g / L sodium chloride, 0.5-2 mmol / L magnesium chloride, 0.05-0.2 mmol / L zinc chloride, 1-4 g / L preservative, and water, and the pH of the buffer is adjusted to 7.0-9.0.

[0040] Another object of the application is achieved by the following technical solutions.

[0041] The application of the human papilloma virus detection reagent in the determination of human papilloma virus antibodies comprises the following steps: adding human papilloma virus antigen-coated magnetic microparticle reagents and alkaline phosphatase-labeled human papilloma virus antibody reagents to a sample to be tested, mixing, and then incubating.

[0042] A magnetic field is added to make the system after incubation settle in the magnetic field, remove the supernatant, wash the precipitate with a washing solution multiple times, remove the magnetic field, and shake.

[0043] Then, a luminescent substrate is added, the mixture is fully suspended, and the relative luminescent intensity value is detected.

[0044] In the above method, the incubation is preferably carried out at 35-38°C for 6-8 min.

[0045] In the above method, the volume ratio of the sample to be tested, the human papilloma virus antigen-coated magnetic microparticle reagents, and the alkaline phosphatase-labeled human papilloma virus antibody reagents is preferably 1:0.9-1.1:0.9-1.1.

[0046] Compared with the prior art, the application has the following beneficial effects:

[0047] 1. The application uses a chemiluminescence method to quantitatively detect human papilloma virus antibodies, and uses human papilloma virus antigens to directly coat magnetic microparticles, thereby avoiding the interference of the biotin-avidin system on the detection results.

[0048] 2. In the human papillomavirus detection reagent of the present invention, the human papillomavirus antigen-coated magnetic particles are blocked with a blocking agent composed of a mixture of 2-10 wt% PEG6000 solution, DB1130, and 0.1-1 wt% gelatin solution. The three blocking agents have an excellent synergistic effect and have a better blocking effect than a single blocking agent or a blocking agent mixed with the two. The magnetic particles are more dispersed and not agglomerated, and the magnetic particles are in a better state. The sensitivity and accuracy are higher, which greatly improves the performance of the reagent and greatly reduces interference.

[0049] 3. The system for detecting human papillomavirus antibodies adopted by the present invention fills the gap in domestic HPV chemiluminescent antibody detection, and has high sensitivity and accuracy and high clinical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 This is a microscope photograph of human papillomavirus antigen-coated magnetic microparticles in Example 1;

[0051] Figure 2 This is a microscope photograph of magnetic microparticles coated with human papillomavirus antigens in Comparative Example 1;

[0052] Figure 3 This is a microscope photograph of magnetic microparticles coated with human papillomavirus antigens in Comparative Example 2;

[0053] Figure 4 This is a microscope photograph of magnetic microparticles coated with human papillomavirus antigens in Comparative Example 3;

[0054] Figure 5 This is a microscope photograph of magnetic microparticles coated with human papillomavirus antigens in Comparative Example 4;

[0055] Figure 6 This is a microscope photograph of magnetic microparticles coated with human papillomavirus antigens in Comparative Example 5;

[0056] Figure 7 This is a microscope photograph of magnetic microparticles coated with human papillomavirus antigens in Comparative Example 6;

[0057] Figure 8 This is a microscope photograph of magnetic microparticles coated with human papillomavirus antigens in Comparative Example 7;

[0058] Figure 9 This is a microscope photograph of magnetic microparticles coated with human papillomavirus antigens in Comparative Example 8;

[0059] Figure 10 This is a microscope photograph of magnetic microparticles coated with human papillomavirus antigens in Comparative Example 9;

[0060] Figure 11This is a microscope photograph of magnetic microparticles coated with human papillomavirus antigens according to Example 2;

[0061] Figure 12 This is a microscope photograph of human papillomavirus antigen-coated magnetic microparticles in Example 3;

[0062] Figure 13 This is a microscope photograph of human papillomavirus antigen-coated magnetic microparticles according to Example 4;

[0063] Figure 14 This is a microscope photograph of magnetic microparticles coated with human papillomavirus antigen in Example 5. DETAILED DESCRIPTION

[0064] Below by specific embodiment and accompanying drawing, technical scheme of the present invention is further described explanation, it should be understood that specific embodiment described herein is only for helping to understand the present invention, is not used for specific limitation of the present invention.And accompanying drawing used herein, is only for better illustrating the disclosure of the present invention, does not have limiting effect on protection scope.If no special instructions, the raw materials adopted in the embodiment of the present invention are all raw materials commonly used in this area, and the method adopted in the embodiment is all conventional method in this area.

[0065] Example 1

[0066] The human papillomavirus detection reagent of this embodiment includes a human papillomavirus antigen-coated magnetic particle reagent and an alkaline phosphatase-labeled human papillomavirus antibody reagent.

[0067] The preparation method of the human papillomavirus antigen-coated magnetic microparticle reagent comprises the following steps:

[0068] 1) Dilute magnetic particles with a particle size of 0.5-3.0 μm to 10 mg / mL in 0.1 M boric acid buffer (pH 9.5);

[0069] 2) adding human papillomavirus antigen at a mass ratio of magnetic particles to human papillomavirus antigen = 1:20, and adding pH 9.5, 2 mol / L ammonium sulfate solution at 1 times the total volume of magnetic particles and antibody;

[0070] 3) Suspension reaction at 25°C for 48 hours;

[0071] 4) Adjust the pH to about 7.5 and block with a mixed blocking agent. Add 125 μL of blocking agent per 10 mg of magnetic particles and incubate at 25°C for 24 hours.

[0072] 5) The magnetic particles were washed with Tris buffer and diluted to 10 mg / mL with diluent to obtain a human papillomavirus antigen-coated magnetic particle reagent.

[0073] The mixed sealing agent is prepared by mixing 5 wt% PEG6000 aqueous solution, DB1130, and 0.5 wt% gelatin aqueous solution in a mass ratio of 1:1:1.

[0074] The method for preparing an alkaline phosphatase-labeled human papillomavirus antibody reagent comprises the following steps:

[0075] S1. Replace 1 mg of human papillomavirus antibody with an amino- and thiol-free buffer (pH 7.0, 0.02 mol / L Hepes buffer) using a PD-10 desalting column and concentrate to 3 mg / ml using a concentrator. Then, add 25 mmol / L DTT solution to the antibody, adding 1 μL of DTT solution per mg of antibody, and react at room temperature for 20 minutes. Add pH 7.3, 1 mol / L glycine solution in the same volume as DTT, and react at room temperature for 8 minutes. Use a PD-10 desalting column to replace the buffer (pH 8.5, 0.02 mol / L Hepes buffer) and concentrate to 3 mg / ml for later use.

[0076] S2. Replace 1 mg of alkaline phosphatase with an amino- and thiol-free buffer (pH 7.0, 0.02 mol / L Hepes buffer) using a PD-10 desalting column and concentrate to 3 mg / ml using a concentrator. Weigh SMCC and dissolve it in DMF to 6 mg / ml. Add 5 μL of SMCC solution per mg of alkaline phosphatase and react at room temperature for 20 minutes. Add a pH 7.3, 1 mol / L glycine solution in the same volume as the SMCC solution and react at room temperature for 15 minutes. Replace the buffer (pH 8.5, 0.02 mol / L Hepes buffer) using a PD-10 desalting column and concentrate to 3 mg / ml.

[0077] S3, the product of step S1 and the product of step S2 were mixed in a mass ratio of 1:1, 0.2 mol / L magnesium chloride solution was added, 2 μL magnesium chloride solution was added per milliliter of reaction volume, and the mixture was reacted at 4°C for 16 hours;

[0078] S4. Purify the product of step S3 and then dilute it to a final concentration of 1 μg / ml.

[0079] Comparative Example 1

[0080] The difference between Comparative Example 1 and Example 1 is that the sealing agent is DB1130, and the other components are the same as those of Example 1.

[0081] Comparative Example 2

[0082] The difference between Comparative Example 2 and Example 1 is that the sealing agent is a 0.5 wt % gelatin aqueous solution, and the rest is the same as Example 1.

[0083] Comparative Example 3

[0084] The difference between Comparative Example 3 and Example 1 is that the blocking agent is a 5 wt % PEG6000 aqueous solution, and the rest is the same as Example 1.

[0085] Comparative Example 4

[0086] The difference between Comparative Example 4 and Example 1 is that the sealing agent is prepared by mixing 5 wt % PEG6000 aqueous solution and DB1130 in a mass ratio of 1:1. The other aspects are the same as those of Example 1.

[0087] Comparative Example 5

[0088] The difference between Comparative Example 5 and Example 1 is that the sealing agent is prepared by mixing 5 wt % PEG6000 aqueous solution and 0.5 wt % gelatin aqueous solution in a mass ratio of 1:1. Other aspects are the same as those of Example 1.

[0089] Comparative Example 6

[0090] The difference between Comparative Example 6 and Example 1 is that the sealing agent is prepared by mixing DB1130 and 0.5 wt % gelatin aqueous solution in a mass ratio of 1:1, and the rest is the same as Example 1.

[0091] Comparative Example 7

[0092] The difference between Comparative Example 7 and Example 1 is that the blocking agent is 10% BSA, and the rest is the same as Example 1.

[0093] Comparative Example 8

[0094] The difference between Comparative Example 8 and Example 1 is that the sealing agent is CE210 (Blockmaster TM ), and the rest is the same as in Example 1.

[0095] Comparative Example 9

[0096] The difference between Comparative Example 9 and Example 1 is that the sealing agent is prepared by mixing 5 wt % PEG6000 aqueous solution, CE210, and 0.5 wt % gelatin aqueous solution in a mass ratio of 1:1:1. The rest is the same as Example 1.

[0097] The human papillomavirus antigen-coated magnetic microparticles of Example 1 and Comparative Examples 1-9 were sonicated. After the sonication, the states of the magnetic microparticles were observed using a microscope, and photographed using a camera to record the results of the states of the magnetic microparticles.

[0098] Figure 1-10 These are microscope photos of magnetic particles coated with human papillomavirus antigens of Example 1 and Comparative Examples 1-9, respectively. Figure 1-10It can be seen that the human papillomavirus antigen-coated magnetic particles blocked with the mixed blocking agent of the present invention are more dispersed than those blocked with other blocking agents, and the linkers do not agglomerate, resulting in a better blocking effect.

[0099] The blank limit was simultaneously determined using the human papillomavirus detection reagents of Example 1 and Comparative Examples 1-9. The specific method was as follows: using a zero-concentration calibrator as a sample for detection, adding 50 μL of a human papillomavirus antigen-coated magnetic particle reagent and 50 μL of an alkaline phosphatase-labeled human papillomavirus antibody reagent to 25 μL of the zero-concentration calibrator, mixing, and incubating at 37° C. for 7 minutes; adding a magnetic field to allow the incubated system to settle in the magnetic field, removing the supernatant, and washing the precipitate multiple times with a cleaning solution. The magnetic field was removed and the mixture was shaken; then, a luminescent substrate was added, and after sufficient suspension, the relative luminescence intensity value was detected.

[0100] Repeat the measurement 20 times to obtain the luminescence values ​​(RLU) of the 20 measurements. Calculate the mean (M) and standard deviation (SD) to obtain the luminescence value corresponding to M+2SD. Perform a two-point regression fit based on the concentration-RLU values ​​between the zero-concentration calibrator and the adjacent calibrator to obtain a linear equation. Substitute the M+2SD luminescence value into the above equation to determine the corresponding concentration value, which is the blank limit. The results are shown in Table 1.

[0101] Table 1 Blank limit results of human papillomavirus detection reagent test for Example 1 and Comparative Examples 1-9

[0102]

[0103] The test results show that the antigen-coated magnetic particles prepared using the mixed blocking agent in Example 1 have the lowest blank limit of 0.015 pg / ml, which is significantly improved compared with other conditions.

[0104] Example 2

[0105] The difference between Example 2 and Example 1 is that the mixed sealing agent in Example 2 is prepared by mixing 5 wt % PEG6000 aqueous solution, DB1130, and 0.5 wt % gelatin aqueous solution in a mass ratio of 1:1:3.

[0106] Example 3

[0107] The difference between Example 3 and Example 1 is that the mixed sealing agent of Example 2 is prepared by mixing 5 wt % PEG6000 aqueous solution, DB1130, and 0.5 wt % gelatin aqueous solution in a mass ratio of 1:1:5.

[0108] Comparative Example 10

[0109] The difference between Comparative Example 10 and Example 1 is that the mixed blocking agent of Comparative Example 10 is mixed by 5wt% PEG6000 aqueous solution, DB1130, 0.5wt% gelatin aqueous solution with a mass ratio of 0.5:0.5:5.

[0110] Comparative Example 11

[0111] The difference between Comparative Example 11 and Example 1 is that the mixed blocking agent of Comparative Example 11 is mixed by 5wt% PEG6000 aqueous solution, DB1130, 0.5wt% gelatin aqueous solution with a mass ratio of 1:1:0.5.

[0112] The human papilloma virus antigen coated magnetic microparticles of Example 2-3 and Comparative Example 10-11 are subjected to ultrasonic, and after the ultrasonic is finished, the state of the magnetic microparticles is observed by microscope and photographed by camera, and the results of the state of the magnetic microparticles are recorded.

[0113] Figure 11 and 12 are microscope photos of the human papilloma virus antigen coated magnetic microparticles of Example 2-3, respectively, and it can be seen from Figure 1 , 11 and 12 that the magnetic beads are very dispersed, the linker does not agglomerate, and the blocking effect is good when the mixed blocking agent with different proportions (1:1:1, 1:1:3, 1:1:5) is used for blocking.

[0114] The blank limit of the human papilloma virus detection reagent of Example 2-3 and Comparative Example 10-11 is determined simultaneously, and the determination method is the same as above, and the blank limit results are shown in Table 2.

[0115] Table 2 Blank limit test results of the human papilloma virus detection reagent of Example 2-3 and Comparative Example 10-11

[0116] Example Example 2 Example 3 Comparative Example 10 Comparative Example 11 Blank limit (pg / ml) 0.016 0.019 0.027 0.025

[0117] It can be seen from Table 2 that the blank limit of the magnetic microparticles blocked by the mixed blocking agent with different proportions (1:1:1, 1:1:3, 1:1:5) is lower than that of the mixed blocking agent with other proportions.

[0118] Example 4

[0119] The difference between Example 4 and Example 1 is that in Example 4, 100μL of blocking agent is added to every 10mg of magnetic microparticles, and the others are the same as Example 1.

[0120] Example 5

[0121] The difference between Example 5 and Example 1 is that in Example 5, 150μL of blocking agent is added to every 10mg of magnetic microparticles, and the others are the same as Example 1.

[0122] Figure 13 and 14 respectively are microscope photos of human papillomavirus antigen coated magnetic microparticles of Example 4-5, from Figure 1 , 13 and 14 can be seen, using different volumes (100 μL / 10 mg magnetic microparticles, 125 μL / 10 mg magnetic microparticles, 150 μL / 10 mg magnetic microparticles) of mixed blocking agent (1:1:1) for blocking, the magnetic microparticles are all well dispersed, the linking agent does not have agglomeration phenomenon, and the blocking effect is good.

[0123] The blank limit was determined simultaneously using the human papillomavirus detection reagent of Example 4-5, and the determination method was the same as above. The blank limit results are shown in Table 3.

[0124] Table 3 Blank limit results of the human papillomavirus detection reagent of Example 4-5

[0125] Example Example 4 Example 5 Blank limit (pg / ml) 0.014 0.016

[0126] As can be seen from Table 3, the blank limit of the magnetic microparticles blocked using different volumes of mixed blocking agent is low.

[0127] Aspects, embodiments, features of the present application should be considered illustrative of all aspects, embodiments, features of the present application and not restrictive thereof, and the scope of the present application is only defined by the claims. Other embodiments, modifications, and uses will be apparent to those skilled in the art, without departing from the spirit and scope of the claimed application.

[0128] In the preparation method of the present application, the order of the steps is not limited to the listed order, and for those skilled in the art, the order of the steps can be changed without creative labor, which is within the protection scope of the present application. In addition, two or more steps or actions can be performed simultaneously.

[0129] Finally, it should be noted that the specific embodiments described herein are merely illustrative of the present application and are not intended to limit the embodiments of the present application. Those skilled in the art can make various modifications or supplements to the described specific embodiments or use similar ways to replace them, and here it is not necessary or possible to fully exemplify all embodiments. Any obvious changes or variations derived from the essential spirit of the present application still fall within the protection scope of the present application, and any additional limitation is contrary to the spirit of the present application.

Claims

1. A human papillomavirus detection reagent, characterized in that: The human papillomavirus detection reagent comprises: a human papillomavirus antigen-coated magnetic particle reagent and an alkaline phosphatase-labeled human papillomavirus antibody reagent; The preparation method of the human papillomavirus antigen-coated magnetic particle reagent comprises the following steps: Human papillomavirus antigen is added to the magnetic microparticle dispersion, and then a reaction catalyst is added to suspend the suspension reaction, and then the pH is adjusted to 7.0-8.0, and a blocking agent is added to carry out a blocking reaction. After washing, a diluent is added and resuspended to obtain a magnetic microparticle reagent coated with human papillomavirus antigen; The sealing agent is a mixture of 2-10 wt% PEG6000 solution, DB1130 and 0.1-1 wt% gelatin solution.

2. A human papillomavirus detection reagent according to claim 1, characterized in that: The mass ratio of magnetic particles to human papillomavirus antigen is 1:5~1:

100.

3. A human papillomavirus detection reagent according to claim 1, characterized in that: The reaction catalyst is a 0.5-4 mol / L ammonium sulfate solution with a pH of 9.0-10.

0.

4. A human papillomavirus detection reagent according to claim 1, characterized in that The suspension reaction should be carried out at 20-35°C for 20-80 hours, and the blocking reaction should be carried out at 20-35°C for 15-30 hours; Alternatively, the diluent is added and the magnetic microparticle reagent coated with human papillomavirus antigen is resuspended to obtain a concentration of 5 to 20 mg / ml.

5. The human papillomavirus detection reagent according to claim 1, characterized in that: 2~10wt% PEG6000 solution, DB1130 and 0.1~1wt% gelatin solution were mixed in a mass ratio of (0.8~1.2):(0.8~1.2):(1~5).

6. A human papillomavirus detection reagent according to claim 1, characterized in that: Add 100-150 μL of blocking agent per 10 mg of magnetic particles.

7. The human papillomavirus detection reagent according to claim 1, characterized in that: The preparation method of the alkaline phosphatase-labeled human papillomavirus antibody reagent comprises the following steps: S1. The human papillomavirus antibody is replaced with an amino-free and thiol-free buffer and concentrated. Then, dithiothreitol or tris(2-carbonylethyl)phosphine hydrochloride is added to the human papillomavirus antibody for reaction. A glycine solution is added, the reaction is continued, and the antibody is passed through a desalting column and concentrated. S2. Replace alkaline phosphatase with amino- and sulfhydryl-free buffer and concentrate, then add SMCC solution to react, add glycine solution, continue the reaction, pass through a desalting column, and concentrate; S3, mixing the product of step S1 and the product of step S2, and then adding magnesium chloride solution to react; S4. Purify the product of step S3 and then dilute it to a final concentration of 0.2-2 μg / ml.

8. A human papillomavirus detection reagent according to claim 7, characterized in that: In step S1, dithiothreitol or tris(2-carbonylethyl)phosphine hydrochloride is added for a reaction time of 10 to 30 minutes at a temperature of 15 to 40° C.; and / or, after adding the SMCC solution in step S2, the reaction is carried out at 20-40° C. for 10-30 min; Alternatively, the temperature for reacting the magnesium chloride solution in step S3 is 2-8° C., and the reaction time is 8-20 h.

9. The human papillomavirus detection reagent according to claim 7, characterized in that: In step S1 and step S2, the pH of the glycine solution is 7.0-7.4, and the concentration is 0.5-2 mol / L. After adding the glycine solution, the reaction is continued for 5-10 minutes.

10. Use of a human papillomavirus detection reagent according to claim 1 in determining human papillomavirus antibodies, characterized in that: The application comprises the following steps: Adding human papillomavirus antigen-coated magnetic microparticle reagent and alkaline phosphatase-labeled human papillomavirus antibody reagent to the sample to be tested, mixing and incubating; Add a magnetic field to allow the incubated system to settle in the magnetic field, remove the supernatant, wash the precipitate several times with a cleaning solution, remove the magnetic field, and shake; Then add the luminescent substrate, suspend thoroughly and detect the relative luminescence intensity value.

Images