Bigeminy immunoassay kit for detecting HPV (human papillomavirus) antibody as well as preparation method and application of bigeminy immunoassay kit

Abstract

The invention discloses a bigeminy immunoassay kit for detecting an HPV (human papillomavirus) antibody and a preparation method of the bigeminy immunoassay kit, relates to the field of biomedicine, and is characterized in that the bigeminy immunoassay kit for detecting the HPV antibody and the preparation method of the bigeminy immunoassay kit are provided. The magnetic microspheres for detecting the neutralizing antibodies of the human papilloma viruses 16 and 18 are coated with an HPV16 antibody and an HPV18 antigen, acridinium ester is used for marking the HPV16 antigen and the HPV18 antibody, an in-vitro diagnostic kit for detecting the neutralizing antibodies of the human papilloma viruses 16 and 18 is established on the basis, and a chemiluminescence immunoassay method is combined for analysis. The bigeminy immunoassay kit for quantitative detection of the HPV antibody is high in sensitivity, good in specificity, simple and convenient to operate and appropriate in price.

Description

Technical Field

[0001] This invention relates to a detection reagent in the field of biotechnology, specifically to a dual immunoassay kit for HPV antibody detection, its preparation method, and its application. Background Technology

[0002] Human papillomavirus (HPV) is a double-stranded deoxyribonucleic acid (DNA) virus with strong epithelial phagocytic activity. It is sexually transmitted and infects human skin and mucous membranes, causing benign warts or malignant tumors. Currently, more than 200 HPV subtypes have been identified, of which more than 40 are associated with genital tract infections. Based on their carcinogenic potential, they can be broadly divided into low-risk and high-risk HPV types. Low-risk HPV includes subtypes 6, 11, and 42, which often cause benign warts and generally do not lead to cervical cancer. The World Health Organization defines 14 high-risk types, of which 12 are confirmed high-risk HPV types: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59, and 2 are suspected high-risk HPV types: 68 and 73. Persistent high-risk HPV infection is closely related to cervical cancer. Recent research indicates that HPV infection can be detected in 99.8% of cervical cancer cases, and approximately 70% of cervical cancers are caused by HPV types 16 / 18. Therefore, preventing HPV infection is one of the most important means of reducing or controlling cervical cancer. Currently, several companies both domestically and internationally have developed HPV preventative vaccines.

[0003] HPV vaccination is being gradually promoted in China, and the efficacy of HPV vaccines has become a widespread concern. Vaccine efficacy is primarily evaluated through in vitro testing. HPV vaccines exert their protective effect mainly by inducing the body to produce neutralizing antibodies against HPV. In vitro evaluation involves detecting the level of neutralizing antibodies in the immune serum after vaccination to preliminarily assess the vaccine's efficacy. Neutralizing antibodies inhibit viral infection through different mechanisms, with two classic modes of action: The first is by inhibiting viral adsorption to host cells. This antibody's effect occurs before viral adsorption; once the virus binds to the cell, the antibody loses its neutralizing ability. The second is that the virus can still adsorb to cells after binding to the antibody, but it inhibits the binding of the virus to endocytosis receptors on the cell membrane, preventing the virus from entering the cell or, if it does enter, preventing further transport, ultimately leading to infection failure.

[0004] Currently, the most common HPV vaccines on the market are bivalent, quadrivalent, and nonavalent. These three types of HPV vaccines protect against different types of the virus: the bivalent vaccine protects against HPV types 16 and 18; the quadrivalent vaccine protects against HPV types 6, 11, 16, and 18; and the nonavalent vaccine protects against HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58. All three HPV vaccines are effective against HPV types 16 and 18, which are high-risk types and the most common causes of cervical cancer. Therefore, after completing HPV vaccination, the detection of neutralizing antibodies against HPV types 16 and 18 is gaining increasing attention to understand the vaccine's efficacy. Currently, the main methods for detecting neutralizing antibodies against HPV types 16 and 18 include the pseudovirus neutralization test, enzyme-linked immunosorbent assay (ELISA), and in vitro keratinocyte culture neutralization test. However, chemiluminescent assay kits that can simultaneously detect both types of antibodies are not yet available on the market. The dual immunoassay kit for HPV detection provided by this invention can be used in fully automated measurement systems, enabling rapid, high-volume testing and making it easier to promote and apply. Summary of the Invention

[0005] The purpose of this invention is to provide a dual immunoassay kit for HPV antibody detection and its preparation method in order to solve the above-mentioned problems.

[0006] To address the problems of the prior art, the technical solution adopted by the present invention is as follows:

[0007] This invention provides a dual immunoassay kit for HPV antibody detection, comprising:

[0008] Magnetic microspheres incorporating monoclonal antibodies against HPV16 antigen;

[0009] Acridinium ester-labeled HPV16 antigen;

[0010] Acridinium ester-labeled monoclonal antibody against HPV18 antigen;

[0011] Magnetic microspheres that bind to HPV18 antigen;

[0012] Calibrators and quality control materials, wherein the calibrators and quality control materials contain known concentrations of HPV types 16 and 18 antibodies;

[0013] The kit detects the concentrations of HPV types 16 and 18 antibodies by forming immune complexes and using a chemiluminescent reaction.

[0014] Furthermore, the magnetic microspheres combining a monoclonal antibody against HPV16 antigen and the acridine ester-labeled monoclonal antibody against HPV18 antigen described in this embodiment of the invention achieve specific detection of HPV16 and 18 antibodies through the formation of immune complexes.

[0015] Furthermore, the concentrations of the calibrators and quality control samples described in this embodiment of the invention are as follows:

[0016] HPV16 antibody calibrator 1: 4.94 IU / L;

[0017] HPV16 antibody calibrator 2: 44.44 IU / L;

[0018] HPV16 antibody quality control sample 1: 3.95~5.93 IU / L;

[0019] HPV16 antibody quality control sample 2: 35.56~53.33 IU / L;

[0020] HPV18 antibody calibrator 1: 12.35 IU / L;

[0021] HPV18 antibody calibrator 2: 333.33 IU / L;

[0022] HPV18 antibody quality control sample 1: 9.88~14.82 IU / L;

[0023] HPV18 antibody quality control sample 2: 266.67~400.00IU / L.

[0024] This invention also provides a method for preparing a dual immunoassay kit for HPV antibody detection, comprising the following steps:

[0025] Prepare dialysate 1 and dialysate 2;

[0026] Preparation of acridinium ester-labeled HPV16 antigen and HPV18 antibody, including the steps of dialysis, labeling, termination and re-dialysis;

[0027] Prepare calibrators and quality control samples containing known concentrations of HPV types 16 and 18 antibodies;

[0028] The components of the kit are mixed and packaged to form the dual immunoassay kit.

[0029] The formula for dialysate 1 used for HPV antibody detection is 2L: 18g NaCl, 27.6g Na2HPO4,

[0030] 0.9g NaH2PO4·2H2O; the formula for dialysate 2 is 2L: 18g NaCl, 10.8g Na2HPO4,

[0031] 19.34g NaH2PO4·2H2O.

[0032] Further, the method for labeling HPV16 antigen with acridine ester is as follows: Prepare dialysate 1, dilute HPV16 antigen 100ug to 2mg / ml with it, place the antigen in a leak-tested dialysate tube, and dialyze overnight at 2-8℃ with low-speed stirring; take out the dialyzed antigen and bring the volume to 160ul, add 20ul of acridine ester stock solution, and incubate at room temperature with shaking for 30min; add 20ul of stop solution and incubate at room temperature with shaking for 30min; add 200ul of the acridine ester labeled product after termination to a dialysate tube, place it in dialysate 2, and dialyze overnight at 2-8℃ with low-speed stirring. Bring the dialyzed acridine ester labeled product to 1ml with acridine ester preservation solution and store at 4℃ for later use.

[0033] Further, the method for labeling HPV18 antibody with acridine ester is as follows: Prepare dialysate 1, dilute 100ug of HPV18 antibody to 2mg / ml with it, place the antibody in a leak-tested dialysate tube, and dialyze overnight at 2-8℃ with low-speed stirring; take out the dialyzed antibody and bring the volume to 160ul, add 20ul of acridine ester stock solution, and incubate at room temperature with shaking for 30min; add 20ul of stop solution and incubate at room temperature with shaking for 30min; add 200ul of the acridine ester labeled product after termination to a dialysate tube, place it in dialysate 2, and dialyze overnight at 2-8℃ with low-speed stirring. Bring the dialyzed acridine ester labeled product to 1ml with acridine ester preservation solution and store at 4℃ for later use.

[0034] Furthermore, the preparation method of the dual immunoassay kit described in this embodiment of the invention also includes the following steps:

[0035] HPV16 Program Editing:

[0036] a. Edit the sample dilution procedure to a dilution ratio of 1:9 (10-fold dilution);

[0037] b. The loading volume for edited samples, calibrators, and quality control samples is 30 μL;

[0038] c. The sample loading volume of the magnetic microsphere solution was 40 μL, and the luminescent label was 80 μL.

[0039] d. The incubation time is 20 minutes;

[0040] e. Washing is performed 4 times;

[0041] f. Pre-activation solution is 100 μL, 1 minute, activation solution is 100 μL;

[0042] g. The fitting method is double logarithmic, four-parameter fitting;

[0043] HPV18 Program Editing:

[0044] a. The loading volume of the edited samples, calibrators, and quality control samples is set to 60 microliters (60 μL).

[0045] b. The loading volume of the magnetic microsphere solution was 40 μL, and the loading volume of the luminescent label was 80 μL.

[0046] c. Set the incubation time to 20 minutes.

[0047] d. Set the number of washes to 4.

[0048] e. Set the volume of the pre-activation solution to 100 μL and the reaction time to 1 minute; f. Set the volume of the activation solution to 100 μL.

[0049] g. Set the fitting method to double logarithmic and use a four-parameter fitting method;

[0050] Calibration and quality control: Scan the master curve of the reagent kit, and test the calibrators and quality control samples according to the edited program. The test results should be within the design range.

[0051] Sample loading: Sort and number the samples to be tested, load them onto the instrument, and wait for the results.

[0052] The instrument uses a four-parameter Logistic curve fitting and data reduction method (4PLC, Y-weighted) to generate a calibration curve. A calibration curve is established by plotting the logarithm of the processed signal value of each calibrator on the x-axis and the logarithm of the concentration of each calibrator on the y-axis. The concentration of the tested sample is calculated on the standard curve based on the signal intensity of the tested sample. The time from sample loading to instrument output is no more than half an hour.

[0053] Furthermore, in the preparation process of acridine ester-labeled HPV16 antigen and HPV18 antibody according to the embodiments of the present invention, a stop solution is used to terminate the reaction. The formula of the stop solution is 100ml: 5g lysine, diluted to 100ml with dialysis solution 1.

[0054] Furthermore, the preparation method described in this embodiment of the invention also includes the preparation of an acridinium ester preservation solution, wherein the acridinium ester preservation solution has the following formula: 1L: 4g casein, 30g trehalose, 100ul Tween 20, 1ml Proclin 300, and diluted to volume with dialysis buffer 2.

[0055] The application of the dual immunoassay kit for HPV antibody detection described in this invention in a chemiluminescence analyzer for detection, wherein the analyzer can automatically complete sample dilution, mixing, incubation, washing, excitation and signal measurement.

[0056] The present invention also provides a method for detecting antibody concentration using the dual immunoassay kit for HPV antibody detection described in the present invention, comprising the following steps:

[0057] The sample, calibrator, and quality control are mixed with the components in the kit to form an immune complex;

[0058] The immune complexes are precipitated by applying an external magnetic field, and the supernatant is removed.

[0059] Wash the precipitated complex with washing solution at least 3 times;

[0060] The substrate solution is automatically pumped in, and the results of the chemiluminescence reaction are measured.

[0061] The concentrations of HPV types 16 and 18 antibodies in the sample were calculated based on the relationship between relative light units (RLUs) and antibody concentration.

[0062] Furthermore, the results of the chemiluminescence reaction described in this embodiment of the invention are analyzed using a four-parameter Logistic curve fitting data reduction method (4PLC, Y-weighted) to generate a calibration curve and calculate the sample concentration.

[0063] Further, the acridinium ester-bound HPV16 antibody was prepared with standard curves at concentration gradients of 400 IU / L, 133.33 IU / L, 44.44 IU / L, 14.81 IU / L, 4.94 IU / L, 1.65 IU / L, and 0.55 IU / L, based on its activity; the magnetic bead-bound HPV18 antibody was prepared with standard curves at concentration gradients of 1000 IU / L, 333.33 IU / L, 111.11 IU / L, 37.04 IU / L, 12.35 IU / L, 4.12 IU / L, and 1.37 IU / L, based on its activity, and its signal values ​​were measured to obtain the master curve.

[0064] In addition, the present invention also provides a method for preparing a dual immunoassay kit for HPV antibody detection, comprising the following steps:

[0065] 1) Mix the components in the neutralizing antibody kit by blowing and aspirating, and install the reagents into the corresponding positions on the instrument.

[0066] Compared with the prior art, the technical solution provided by the present invention has the following advantages:

[0067] 1. The dual immunoassay kit for HPV antibody detection provided by this invention can sensitively and rapidly determine the content of HPV type 16 and type 18 antibodies in samples, providing reliable clinical reference value for early diagnosis and early treatment of HPV-infected individuals, and achieving the goal of non-invasive detection of HPV antibodies.

[0068] 2. The HPV neutralizing antibody quantitative detection kit described in this invention is not only fast in detection and simple in operation, but also requires a small amount of blood sample. Furthermore, the detection results are highly sensitive and specific, and can directly reflect the concentration level of HPV16 and 18 neutralizing antibodies. It can effectively reflect the efficacy of HPV vaccines on the body and plays a crucial role in vaccine research and evaluation.

[0069] 3. The dual immunoassay kit for PV antibody detection provided by this invention can be used in both open semi-automatic chemiluminescence analyzers and fully automated measurement systems, enabling rapid detection in large batches and making it easier to promote and apply. Attached Figure Description

[0070] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0071] Figure 1 The equation for the detection limit of HPV16 antibody blank in the kit.

[0072] Figure 2 Linear interval graph of HPV16 antibody detection kit.

[0073] Figure 3 Scatter plot of correlation analysis of HPV16 antibody test samples.

[0074] Figure 4 Graph of blank limit equation for HPV18 antibody detection kit.

[0075] Figure 5 Linear interval graph of HPV18 antibody detection kit.

[0076] Figure 6 Correlation analysis of HPV18 antibody test samples from the kit. Specific Implementation

[0077] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention.

[0078] To facilitate understanding of the present invention, a more complete description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0079] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this invention includes any and all combinations of one or more of the associated listed items.

[0080] Unless otherwise specified, all examples are conducted under standard experimental conditions or as recommended in the manufacturer's instructions. All raw materials and reagents used in this invention are commercially available, and any biological germplasm material can be provided for scientific research. Where specific conditions are not specified, they are conducted under standard conditions or as recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0081] Example 1

[0082] Establishment of a chemiluminescent competitive assay for HPV16 and HPV1618 neutralizing antibodies

[0083] Detection Method: In this embodiment, HPV16 antibody purchased from Rebio (Guangzhou) Biotechnology Co., Ltd. (Catalog No.: 188-10878) was used as the coating antibody to coat magnetic beads, and HPV16 antigen purchased from Zhengzhou Saitukang Biotechnology Co., Ltd. (Catalog No.: CTAHPV16Ag01) was used as the recognition antigen to label acridinium ester, forming a system in which the HPV16 neutralizing antibody of the serum to be tested competes with the HPV16 antibody coated on the magnetic beads for binding to the acridinium ester-labeled HPV16 antigen; Zhengzhou Saitukang Biotechnology Co., Ltd. was used as the detection method. HPV18 antigen purchased from Zhengzhou Saitukang Biotechnology Co., Ltd. (Catalog No.: CTAHPV18Ag01) was used as the recognition antigen to coat magnetic beads, and HPV18 antibody purchased from Zhengzhou Saitukang Biotechnology Co., Ltd. (Catalog No.: CTAHPV18Ab01) was used as the competitive antibody to label acridine ester, forming a system in which the HPV18 neutralizing antibody of the serum to be tested competes with the acridine ester-labeled HPV18 antibody to bind to the HPV18 antigen coated on the magnetic beads, thus establishing a chemiluminescent competitive method for detecting HPV16 and 18 neutralizing antibodies.

[0084] 1) Coating of HPV16 antibodies

[0085] Antibody dialysis: Prepare MES dialysis buffer (2L: 21.325g MES, 200ul Triton X100, pH adjusted to 6.0-6.3), dilute HPV16 antibody 100ug to 2mg / ml with it, place the antibody in a leak-tested dialysis tube, stir at low speed and dialyze overnight at 2-8℃;

[0086] Magnetic bead activation: Take 200 μL of carboxylated magnetic beads (purchased from Rebec Biotechnology), wash twice with MES dialysis buffer, add 40 μL of EDC (50 mg / ml) and 40 μL of NHS (50 mg / ml) prepared with MES dialysis buffer and 20 μL of MES dialysis buffer respectively, shake and incubate at room temperature for 35 min, place on a magnetic rack for 40 s, remove the liquid, and wash twice with MES dialysis buffer.

[0087] Antibody coating: After dialysis overnight, the antibody was taken out and diluted to 100 μL with MES dialysis buffer. The solution was added to the activated and washed magnetic beads and incubated with shaking at room temperature for 5 h.

[0088] Magnetic bead blocking and preservation: Wash twice with blocking solution (1L: 0.2g KCl, 7.6g NaCl, 3.03g Tris, 6g casein, 30g trehalose, 20g BSA, 100ul Triton X100, 1ml Proclin 300), then add 500ul blocking solution, shake and block at room temperature for 90min, remove the liquid, add 1ml magnetic bead preservation solution (1L: 2.42g Tris, 3.6g NaCl, 4g BSA, 8g trehalose, 2g polyethylene glycol 20000, 40ul Tween 20, 400ul Proclin 300, 40ul Triton X100), and store at 4℃.

[0089] (ii) Labeling of HPV16 antigen

[0090] Antigen dialysis: Prepare dialysis solution 1 (2L: 18g NaCl, 27.6g Na2HPO4, 0.9g NaH2PO4·2H2O), dilute HPV16 antigen 100ug to 2mg / ml with it, place the antigen in a leak-tested dialysis tube, stir at low speed and dialyze overnight at 2-8℃;

[0091] Antigen labeling: Take out the dialyzed antigen and bring the volume to 160ul, add 20ul of acridinium ester stock solution, and incubate at room temperature with shaking for 30min;

[0092] Termination: Add 20 μL of termination solution (100 mL: 5 g lysine, diluted to 100 mL with dialysis buffer 1), and incubate at room temperature with shaking for 30 min;

[0093] Dialysis and storage: Add 200 μL of the terminated acridine ester label to a dialysis tube and dialyze overnight at 2–8°C with dialysis buffer 2 (2L: 18 g NaCl, 10.8 g Na₂HPO₄, 19.34 g NaH₂PO₄·2H₂O) and stir at low speed. Dilute the dialyzed acridine ester label to 1 mL with acridine ester storage solution (1L: 4 g casein, 30 g trehalose, 100 μL Tween 20, 1 mL Proclin 300, diluted to 1L with dialysis buffer 2) and store at 4°C.

[0094] Preparation of HPV16 antibody magnetic microsphere solution and HPV16 antigen luminescent labeling solution

[0095] HPV16 antibody magnetic microsphere solution: Dilute the coated HPV16 antibody with magnetic bead preservation solution at 20x and store at 4℃ for later use.

[0096] HPV16 antigen luminescent label solution: Dilute the HPV16 antigen acridine ester label with acridine ester preservation solution at 300x and store at 4℃.

[0097] (iii) Coating of HPV18 antigen

[0098] Antigen dialysis: Prepare MES dialysis buffer (2L: 21.325g MES, 200ul Triton X100, pH adjusted to 6.0-6.3), dilute HPV18 antigen 100ug to 2mg / ml with it, place the antigen in a leak-tested dialysis tube, stir at low speed and dialyze overnight at 2-8℃;

[0099] Magnetic bead activation: Take 200 μL of carboxylated magnetic beads (purchased from Rebec Biotechnology), wash twice with MES dialysis buffer, add 40 μL of EDC (50 mg / ml) and 40 μL of NHS (50 mg / ml) prepared with MES dialysis buffer and 20 μL of MES dialysis buffer respectively, shake and incubate at room temperature for 35 min, place on a magnetic rack for 40 s, remove the liquid, and wash twice with MES dialysis buffer.

[0100] Antigen coating: After dialysis overnight, the antigen was removed and brought to a final volume of 100 μL with MES dialysis buffer. The mixture was then added to activated and washed magnetic beads and incubated with shaking at room temperature for 5 hours.

[0101] Blocking and preservation of magnetic beads: Wash twice with blocking buffer (1L: 0.2g KCl, 7.6g NaCl, 3.03g Tris, 6g casein, 30g trehalose, 100ul Triton X100, 1ml Proclin 300), then add 500ul blocking buffer, shake and block at room temperature for 90min, remove the liquid, add 1ml magnetic bead preservation buffer (1L: 100g trehalose, 300g sucrose, 5g surfactant S9, 5g PVP40K, 40ml Tween 20, 1ml Proclin 300, and bring the volume to 1L with 1XPBS, and store at 4℃.

[0102] (iv) Labeling of HPV18 antibodies

[0103] Antibody dialysis: Prepare dialysis buffer 1 (2L: 18g NaCl, 27.6g Na2HPO4, 0.9g NaH2PO4·2H2O), dilute HPV18 antibody 100ug to 2mg / ml with it, place the antibody in a leak-tested dialysis tube, stir at low speed and dialyze overnight at 2-8℃;

[0104] Antibody labeling: Take out the dialyzed antibody and adjust the volume to 160ul, add 20ul of acridinium ester stock solution, and incubate at room temperature with shaking for 30min;

[0105] Termination: Add 20 μL of termination solution (100 mL: 5 g lysine, diluted to 100 mL with dialysis buffer 1), and incubate at room temperature with shaking for 30 min;

[0106] Dialysis and storage: Add 200 μL of the terminated acridine ester label to a dialysis tube and dialyze overnight at 2–8°C with dialysis buffer 2 (2L: 18 g NaCl, 10.8 g Na₂HPO₄, 19.34 g NaH₂PO₄·2H₂O) and stir at low speed. Dilute the dialyzed acridine ester label to 1 mL with acridine ester storage solution (1L: 4 g casein, 30 g trehalose, 100 μL Tween 20, 1 mL Proclin 300, diluted to 1L with dialysis buffer 2) and store at 4°C.

[0107] (v) Preparation of HPV18 antigen magnetic microsphere solution and HPV18 antibody luminescent labeling solution

[0108] HPV18 antigen magnetic microsphere solution: Dilute the coated HPV18 antigen with magnetic bead preservation solution at 20x and store at 4℃ for later use.

[0109] HPV18 antibody luminescent labeling solution: Dilute the HPV18 antibody acridine ester label with acridine ester preservation solution at 300x and store at 4℃.

[0110] Preparation of calibrators, quality control samples, and master curves: HPV18 antibody was used to prepare standard curves with concentration gradients of 1000 IU / L, 333.33 IU / L, 111.11 IU / L, 37.04 IU / L, 12.35 IU / L, 4.12 IU / L, and 1.37 IU / L, based on its activity. HPV16 antibody was used to prepare standard curves with concentration gradients of 400 IU / L, 133.33 IU / L, 44.44 IU / L, 14.81 IU / L, 4.94 IU / L, 1.65 IU / L, and 0.55 IU / L, based on its activity. The signal values ​​were measured to obtain the master curve. Based on the concentration signal values ​​of the master curve, calibrators and quality control samples of corresponding concentrations were prepared, aliquoted, and stored at 4℃ for later use.

[0111] Example 2

[0112] A chemiluminescent reagent kit for detecting HPV16 and 18 neutralizing antibodies is provided, the composition of which is as follows:

[0113] 1. HPV16 antibody magnetic microsphere solution: The volume is 5.5 ml, and its main components include casein, phosphate buffer, preservatives, and magnetic microspheres coated with HPV16 antibody.

[0114] 2. HPV16 antigen luminescent marker: The volume is 11 ml, and its main components include casein, phosphate buffer, preservatives, and HPV16 antigen labeled with acridine ester.

[0115] 3. HPV18 antigen magnetic microsphere solution: The volume is 5.5 ml, and its main components include casein, phosphate buffer, preservatives, and magnetic microspheres coated with HPV18 antigen.

[0116] 4. HPV18 antibody luminescent label: The volume is 11 ml, and its main components include casein, phosphate buffer, preservatives, and HPV18 antibody labeled with acridine ester.

[0117] 5. HPV16 calibrator 1: 0.5 ml in volume, its main components include casein, phosphate buffer, preservative, and HPV16 antibody at a concentration of 4.94 IU / L.

[0118] 6. HPV16 calibrator 2: 0.5 ml in volume, its main components include casein, phosphate buffer, preservative, and HPV16 antibody at a concentration of 44.44 IU / L.

[0119] 7. HPV16 control sample 1: 0.5 ml in volume, its main components include casein, phosphate buffer, preservatives, and HPV16 antibody with a concentration range of 3.95 to 5.93 IU / L.

[0120] 8. HPV16 control sample 2: 0.5 ml in volume, its main components include casein, phosphate buffer, preservatives, and HPV16 antibody with a concentration range of 35.56 to 53.33 IU / L.

[0121] 9. HPV18 antibody calibrator 1: 0.5 ml in volume, its main components include casein, phosphate buffer, preservatives and HPV18 antibody at a concentration of 12.35 IU / L.

[0122] 10. HPV18 antibody calibrator 2: 0.5 ml in volume, its main components include casein, phosphate buffer, preservatives and HPV18 antibody at a concentration of 333.33 IU / L.

[0123] 11. HPV18 antibody quality control sample 1: The volume is 0.5 ml, and its main components include casein, phosphate buffer, preservatives, and HPV18 antibody with a concentration range of 9.88 to 14.82 IU / L.

[0124] 12. HPV18 antibody quality control sample 2: The volume is 0.5 ml, and its main components include casein, phosphate buffer, preservatives, and HPV18 antibody with a concentration range of 266.67 to 400.00 IU / L.

[0125] The volume, composition, and concentration of each component described above are specific examples in the embodiments of this invention. Those skilled in the art can make appropriate adjustments and optimizations according to actual detection needs to achieve accurate detection of HPV16 and HPV18 neutralizing antibodies.

[0126] Example 3

[0127] Provides the operating procedure for a chemiluminescent reagent kit for detecting HPV16 and 18 neutralizing antibodies.

[0128] 1. Reagent mixing and installation

[0129] Each component in the neutralizing antibody kit was mixed sequentially by pipetting and aspirating to ensure uniform dispersion.

[0130] After mixing, accurately install the reagent into the corresponding position on the instrument according to the instrument manual to ensure the compatibility between the reagent and the instrument.

[0131] 2. Program Editing

[0132] 2.1 HPV16 Program Editing

[0133] Edit the sample dilution program and set the dilution ratio to 1:9, which is a 10-fold dilution.

[0134] The loading volume for editing samples, calibrators, and quality control samples was set to 30 microliters (30 μL).

[0135] The loading volume of the magnetic microsphere solution was 40 μL, and the loading volume of the luminescent label was 80 μL.

[0136] Set the incubation time to 20 minutes.

[0137] Set the number of washes to 4.

[0138] The volume of the pre-activation solution was set to 100 μL, and the reaction time was set to 1 minute; the volume of the activation solution was set to 100 μL.

[0139] The fitting method was set to double logarithmic, and a four-parameter fitting method was used.

[0140] 2.2 HPV18 Program Editing

[0141] The loading volume for editing samples, calibrators, and quality control samples was set to 60 microliters (60 μL).

[0142] The loading volume of the magnetic microsphere solution was 40 μL, and the loading volume of the luminescent label was 80 μL.

[0143] Set the incubation time to 20 minutes.

[0144] Set the number of washes to 4.

[0145] The volume of the pre-activation solution was set to 100 μL, and the reaction time was set to 1 minute; the volume of the activation solution was set to 100 μL.

[0146] The fitting method was set to double logarithmic, and a four-parameter fitting method was used.

[0147] 3. Calibration and Quality Control

[0148] Scan the master curve of the reagent kit and test the calibrators and quality control samples according to the program edited above.

[0149] The test results should be within the design range to ensure the accuracy and reliability of the kit.

[0150] 4. Sample loading

[0151] The samples to be tested are sorted in a certain order and numbered.

[0152] The numbered samples are fed into the machine in sequence, and the instrument is allowed to automatically complete the test and output the results.

[0153] 5. Data Processing and Result Output

[0154] The instrument uses a four-parameter Logistic curve fitting data reduction method (4PLC, Y-weighted) to generate a calibration curve.

[0155] Plot a calibration curve with the logarithm of the signal value after processing each calibrator as the x-axis (Y-axis) and the logarithm of the concentration of each calibrator as the y-axis (X-axis).

[0156] Based on the calibration curve and the signal intensity of the sample being tested, the concentration of the sample is calculated.

[0157] The entire process, from sample loading to instrument output, takes no more than half an hour.

[0158] The embodiments of the present invention achieve efficient and accurate detection of neutralizing antibodies through the above steps, and are characterized by simple operation and reliable results, making them suitable for clinical testing and related research fields.

[0159] Example 4

[0160] Quality analysis was performed using a chemiluminescence assay kit for detecting HPV16 neutralizing antibodies.

[0161] 1) Blank limit of the reagent kit

[0162] Using a zero-concentration calibrator as a sample, the test was repeated 20 times, and the RLU values ​​(relative luminescence values) of the 20 measurements were obtained. The mean (M) and standard deviation (SD) were calculated, and the RLU value corresponding to M²SD was obtained. Figure 1 As shown, a linear equation is obtained by performing a two-point regression fitting based on the concentration-RLU values ​​between the zero-concentration calibrator and adjacent calibrators. The RLU value corresponding to M2SD is then substituted into the above equation to obtain the corresponding concentration value, which is the blank limit.

[0163] Table 1: Results of Blank Limit Test

[0164]

[0165] Table 2: Calibrators

[0166]

[0167] The mean signal of the diluent test was 1196163, the standard deviation was 9274, and the M2SD value was 1177613. Substituting the M2SD value into the equation, the corresponding concentration value was 0.1834, which means the blank limit for HPV16 was 0.1834 IU / L. The result is not higher than 0.5 IU / L, which meets the requirements.

[0168] 2) Linearity of the kit

[0169] High-value samples approaching the upper limit of the linear range were diluted 3-fold to seven gradients, while low-value samples were approaching the lower limit of the linear range. Each concentration was measured at least twice, and the average value was calculated. The average concentration and the dilution ratio were then fitted to a straight line using the least squares method, and the linear correlation coefficient r was calculated. The result should be ≥0.9900 (see Table 3).

[0170] Table 3: Results of HPV16 linearity test

[0171]

[0172] like Figure 2 As shown, within the linear range of 400 IU / L to 0.55 IU / L of the kit, the linear correlation coefficient R is 0.9999, which meets the requirements.

[0173] 3) Accuracy of the reagent kit

[0174] Within the linear range of the kit, prepare two concentrations of reference material using the international standard for HPV16 and use them as samples for testing. Measure each sample three times and calculate the relative deviation. The relative deviation of the measurement results should not exceed ±10%.

[0175] Table 4: HPV16 Accuracy Test Results

[0176]

[0177] 4) Reproducibility of the kit

[0178] Use two quality control samples at two different concentration levels to perform repeated tests 10 times each. Calculate the average and standard deviation of the 10 concentration measurements to obtain the coefficient of variation (CV). The CV should be less than 8%.

[0179] Table 5: Results of HPV16 Repeatability Tests

[0180]

[0181] 5) Application of the reagent kit

[0182] The kit described in this invention and the pseudovirus method were used to compare the detection of neutralizing antibodies against the same batch of clinical samples (40 serum samples). The positive reference value of the kit described in this invention was 5 IU / L, and the positive reference value of the pseudovirus method was 40 antibody titers. The detection results are shown in Tables 6 and 7.

[0183] Table 6: Comparative Experiments on the Application of Reagent Kits

[0184]

[0185]

[0186] Table 7. Analysis of Clinical Test Results

[0187]

[0188] The kit of the present invention showed a positive concordance rate of 97.14% and a negative concordance rate of 100% in 40 samples tested with the pseudovirus method, with an overall concordance rate of 97.5%, indicating that the kit of the present invention has a good concordance rate with the pseudovirus method.

[0189] like Figure 3 As shown in the figure, this scatter plot is drawn using the sample detection value as the X-axis and the spoofing method detection value as the Y-axis. A trend line and formula are generated, and the R-squared value is 0.9686. The results show that this kit has a good correlation with the spoofing method and requires less time (only half an hour, while the spoofing method requires more than 24 hours).

[0190] 6) Stability of the reagent kit

[0191] The stability of the kit was tested under 37°C heat for 7 days. The performance of the kit was tested, including blank limit, repeatability, accuracy and linearity. Two quality control samples were used for repeatability, with C1 concentration of 4.94 IU / L and C2 concentration of 44.44 IU / L. The results are shown in Tables 8-13.

[0192] Table 8: Performance Summary Analysis After 7 Days of Acceleration at 37℃

[0193]

[0194] Table 9: Results of Blank Limit Test

[0195]

[0196] Table 10: Results of Repeated Experiments

[0197]

[0198] Table 11: Results of HPV16 Repeatability Test

[0199]

[0200] Table 12. Results of HPV16 Accuracy Test

[0201]

[0202] Table 13: Results of HPV16 linearity test

[0203]

[0204] Example 5

[0205] Quality Analysis of Chemiluminescent Kit for Detecting HPV18 Neutralizing Antibodies

[0206] 1) Blank limit of the reagent kit

[0207] Using zero-concentration calibrators as samples, the tests were repeated 20 times to obtain the RLU (relative luminescence value) of the 20 measurements. The mean (M) and standard deviation (SD) of these 20 measurements were calculated to obtain the RLU value corresponding to M2SD. A two-point regression was performed based on the concentration-RLU values ​​between the zero-concentration calibrator and adjacent calibrators to obtain a linear equation. The RLU value corresponding to M2SD was substituted into the above equation to obtain the corresponding concentration value, which is the blank limit. The results are shown in Tables 14 and 15.

[0208] Table 14: Results of Blank Limit Test

[0209]

[0210] Table 15: Results of Repeated Experiments

[0211]

[0212] like Figure 4 As shown, the mean signal of the dilution test was 2061842, the standard deviation was 45535, and the M2SD value was 1970772. Substituting the M2SD value into the equation, the corresponding concentration value was calculated to be 0.3178, that is, the blank limit value of HPV18 was 0.3178 IU / L. The result is not higher than 1.0 IU / L, which meets the requirements.

[0213] 1) Linearity of the kit

[0214] High-value samples approaching the upper limit of the linear range are diluted 3-fold to seven gradients, while low-value samples must be close to the lower limit of the linear range. Each concentration sample is measured at least twice, and the average value is calculated. The average concentration and the dilution ratio are then fitted to a straight line using the least squares method, and the linear correlation coefficient r is calculated. The result should be ≥0.9900.

[0215] Table 16: Results of HPV18 Linearity Test

[0216]

[0217] like Figure 4 As shown, within the linear range of 1000 IU / L to 1.37 IU / L of the kit, the linear correlation coefficient R is 0.9999, which meets the requirements.

[0218] 2) Accuracy of the reagent kit

[0219] Within the linear range of the kit, prepare two concentrations of reference material using the national standard for HPV18, and use them as samples for testing. Measure each sample three times and calculate the relative deviation. The relative deviation of the measurement results should not exceed ±10%.

[0220] Table 17: HPV18 Accuracy Test Results

[0221]

[0222] 3) Reproducibility of the kit

[0223] Use two quality control samples at two different concentration levels to perform repeated tests 10 times each. Calculate the average and standard deviation of the 10 concentration measurements to obtain the coefficient of variation (CV). The CV should be less than 8%.

[0224] Table 18: Results of HPV18 Repeatability Tests

[0225]

[0226] 4) Application of the reagent kit

[0227] The kit described in this invention and the pseudovirus method for detecting neutralizing antibodies were compared in a test against the same batch of clinical samples (40 serum samples). The positive reference value for the kit described in this invention was 5 IU / L, while the positive reference value for the pseudovirus method was an antibody titer of 40. The detection results are as follows:

[0228] Table 19:

[0229]

[0230]

[0231] Table 20: Analysis of Clinical Test Results

[0232]

[0233] The kit of Example 6 of this invention showed a positive concordance rate of 96.97% and a negative concordance rate of 100% in 40 samples tested with the pseudovirus method, with an overall concordance rate of 97.5%, indicating that the kit of this invention has a good concordance rate with the pseudovirus method.

[0234] like Figure 6 As shown in the figure, this scatter plot is drawn using the sample detection value as the X-axis and the spoofing method detection value as the Y-axis. A trend line and formula are generated, and the R-squared value is 0.9588. The results show that this kit has a good correlation with the spoofing method and requires less time (only half an hour, while the spoofing method requires more than 24 hours).

[0235] 5) Stability of the reagent kit

[0236] The stability of the kit was tested at 37°C for 7 days. The performance of the kit was tested, including blank limit, repeatability, accuracy and linearity. Two quality control samples were used for repeatability, with C1 concentration of 12.35 IU / L and C2 concentration of 333.33 IU / L. The results are shown in Tables 21-26.

[0237] Table 21: Performance Summary and Analysis After 7 Days of Acceleration at 37℃

[0238]

[0239] Table 22: Results of Blank Limit Test

[0240]

[0241] Table 23: Results of Repeated Experiments

[0242]

[0243]

[0244] Table 24: Results of HPV18 Repeatability Tests

[0245]

[0246] Table 25: HPV18 Accuracy Test Results

[0247]

[0248] Table 26: Results of HPV18 Linearity Test

[0249]

Claims

1. A dual immunoassay kit for detecting HPV antibodies, characterized in that, The kit includes: Magnetic microspheres incorporating monoclonal antibodies against HPV16 antigen; Acridinium ester-labeled HPV16 antigen; Acridinium ester-labeled monoclonal antibody against HPV18 antigen; Magnetic microspheres that bind to HPV18 antigen; Calibrators and quality control materials, wherein the calibrators and quality control materials contain known concentrations of HPV types 16 and 18 antibodies; The kit detects the concentrations of HPV types 16 and 18 antibodies by forming immune complexes and using a chemiluminescent reaction.

2. The tetravalent immunoreagent of claim 1, wherein The magnetic microspheres containing a monoclonal antibody against HPV16 antigen and the acridinium ester-labeled monoclonal antibody against HPV18 antigen achieve specific detection of HPV16 and 18 antibodies through the formation of immune complexes.

3. The tetravalent immunogenic kit according to claim 1, wherein The concentrations of the calibrators and quality control samples are as follows: HPV16 antibody calibrator 1: 4.94 IU / L; HPV16 antibody calibrator 2: 44.44 IU / L; HPV16 antibody quality control sample 1: 3.95~5.93 IU / L; HPV16 antibody quality control sample 2: 35.56~53.33 IU / L; HPV18 antibody calibrator 1: 12.35 IU / L; HPV18 antibody calibrator 2: 333.33 IU / L; HPV18 antibody quality control sample 1: 9.88~14.82 IU / L; HPV18 antibody quality control sample 2: 266.67~400.00IU / L.

4. A process for the preparation of a bivalent immunization kit according to any one of claims 1 to 3, characterized in that, Includes the following steps: Prepare dialysate 1 and dialysate 2; Preparation of acridinium ester-labeled HPV16 antigen and HPV18 antibody, including the steps of dialysis, labeling, termination and re-dialysis; Prepare calibrators and quality control samples containing known concentrations of HPV types 16 and 18 antibodies; The components of the kit are mixed and packaged to form the dual immunoassay kit.

5. The preparation method according to claim 4, characterized in that, The formula for dialysate 1 is 2L: 18g NaCl, 27.6g Na2HPO4, and 0.9g NaH2PO4·2H2O; the formula for dialysate 2 is 2L: 18g NaCl, 10.8g Na2HPO4, and 19.34g NaH2PO4·2H2O.

6. The preparation method according to claim 4, characterized in that, In the preparation of the acridinium ester-labeled HPV16 antigen and HPV18 antibody, a stop solution is used to terminate the reaction. The stop solution is formulated as follows: 100 ml: 5 g lysine, diluted to 100 ml with dialysis buffer 1.

7. The preparation method according to claim 4, characterized in that, The preparation method further includes preparing an acridinium ester preservation solution, wherein the acridinium ester preservation solution is formulated as follows: 1L: 4g casein, 30g trehalose, 100ul Tween 20, 1ml Proclin 300, and diluted to volume with dialysis buffer 2.

8. The application of the dual immunoassay kit as described in any one of claims 1-3 in a chemiluminescence analyzer for detection, wherein the analyzer is capable of automatically performing sample dilution, mixing, incubation, washing, excitation, and signal measurement.

9. A method for detecting the concentration of antibodies using the tetravalent immunization kit according to any one of claims 1 to 3, characterized in that, Includes the following steps: The sample, calibrator, and quality control are mixed with the components in the kit to form an immune complex; The immune complexes are precipitated by applying an external magnetic field, and the supernatant is removed. Wash the precipitated complex with washing solution at least 3 times; The substrate solution is automatically pumped in, and the results of the chemiluminescence reaction are measured. The concentrations of HPV types 16 and 18 antibodies in the sample were calculated based on the relationship between relative light units (RLUs) and antibody concentration.

10. The method of claim 9, wherein, The results of the chemiluminescence reaction were analyzed using a four-parameter Logistic curve fitting data reduction method (4PLC, Y-weighted) to generate a calibration curve and calculate the sample concentration.

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