HBV PreS1 antibody qualitative detection test strip, its preparation method and application

Abstract

The application discloses a kind of for HBV PreS1 antibody qualitative detection test strip and its preparation method and application, it is related to virology, immunology, biological detection technology and immunochromatography analysis technical field.The test strip is based on the specific recognition principle of antigen and antibody, the specific binding of PreS1 antigen and PreS1 antibody is realized, the rapid detection of PreS1 antibody in serum or plasma sample, it is sequentially assembled by sample pad, gold mark pad, nitrocellulose membrane (NC membrane) and water absorption pad, wherein, gold mark pad is fixed with colloidal gold labeled anti-human IgG antibody as gold mark probe, NC membrane detection line (T line) is coated HBV PreS1 antigen, for capturing PreS1 antibody in sample, NC membrane quality control line (C line) is coated HB2 antibody, for judging whether the test strip detection process is effective.The test strip in the application does not need complex instrument equipment, detection process is simple, fast and fast, and has good sensitivity and specificity, can be used for hepatitis B vaccine immune effect evaluation and large-scale screening.

Description

Technical Field

[0001] This invention relates to the fields of virology, immunology, biological detection technology and immunochromatographic analysis technology, specifically to a qualitative test strip for HBV PreS1 antibody, its preparation method and application. Background Technology

[0002] Hepatitis B, caused by hepatitis B virus (HBV) infection, remains a serious global public health problem, leading to chronic hepatitis, cirrhosis, and even hepatocellular carcinoma (HCC). In my country, hepatitis B is one of the three key infectious diseases requiring prevention and control, posing a serious threat to public health and safety. Although my country has widely implemented a hepatitis B vaccination program, resulting in a significant decrease in HBV infection rates, many challenges remain in hepatitis B prevention and control due to individual differences in vaccine efficacy, viral mutations, and the persistence of immunity.

[0003] Recent studies have shown that the pre-HBV surface protein region (PreS region) plays a crucial role in viral infection and the immune response. PreS1 protein, a key component of the HBV surface antigen, participates in the binding of the virus to hepatocyte receptors and is an important functional protein in the early stages of HBV infection. PreS1 antibodies are important immune markers produced after the body's immune system recognizes the PreS1 antigen, reflecting the level of the body's immune response to the HBV PreS1 antigen. Therefore, detecting PreS1 antibodies not only helps evaluate the immunizing efficacy of novel hepatitis B vaccines containing the PreS1 antigen but can also be used to monitor the body's immune status, making it significant in the fields of hepatitis B prevention and control and vaccine research.

[0004] Currently, HBV-related antibody detection methods mainly include enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay (CLIA), and other laboratory immunoassay techniques. Among these, ELISA is widely used for HBV antibody detection due to its high sensitivity and specificity. However, this type of detection typically requires specialized laboratory equipment such as ELISA readers and plate washers, and the detection process is relatively complex, involving multiple steps including coating, blocking, incubation, washing, and color development. The entire process usually takes 2-4 hours to complete. For primary healthcare institutions or on-site testing environments, these methods are cumbersome, time-consuming, and require stringent experimental conditions, making them unsuitable for rapid testing. Furthermore, in clinical practice, hospital resources are often strained, and complex testing procedures increase the workload of medical staff. Many primary healthcare institutions also lack adequate immunoassay equipment, hindering the widespread implementation of HBV immune status testing. For vaccine recipients or hepatitis B prevention and screening, rapid, convenient, and low-cost testing would help improve testing coverage and promote hepatitis B prevention and control efforts. Therefore, it is of great significance to develop a simple, rapid, and equipment-free method for detecting HBV PreS1 antibodies.

[0005] In recent years, colloidal gold immunochromatography (GICA) has been widely used in the rapid detection of infectious diseases as a rapid immunoassay technique. This technology utilizes the specific recognition reaction between antigens and antibodies, using colloidal gold-labeled antibodies as signal indicators to form visual test results on test strips. It offers advantages such as short testing time, simple operation, and no need for complex instruments, making it particularly suitable for point-of-care testing (POCT). Therefore, applying colloidal gold immunochromatography to HBV PreS1 antibody detection holds promise for achieving rapid and convenient assessment of immune status. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a qualitative test strip for HBV PreS1 antibodies, its preparation method, and its applications. Based on the principle of antigen-antibody specific recognition, this test strip uses PreS1 antigen as the coating antigen for the detection line. Through the specific binding of the PreS1 antigen to PreS1 antibodies in the sample, it achieves rapid detection of PreS1 antibodies in serum or plasma samples. The detection process is simple and convenient, requiring no complex equipment, and the detection time is short, completing the test within approximately 15 minutes. It is suitable for primary healthcare institutions, on-site testing, and home self-testing. It provides a rapid, convenient, and widely applicable detection tool for evaluating the immunization efficacy of hepatitis B vaccines and monitoring HBV immune status. It can be applied to the fields of viral biomarker detection, hepatitis B virus (HBV) related antibody detection and analysis, in vitro diagnostic reagent development, and point-of-care testing (POCT).

[0007] According to one aspect of the present invention, a method for preparing a qualitative test strip for HBV PreS1 antibody is provided, comprising: S1, Preparation of colloidal gold nanoparticles: Heat 100 parts by volume of ultrapure water, add 1 part by volume of 1% chloroauric acid solution, stir and heat to boiling, then add 1.56 parts by volume of 1% sodium citrate solution, continue boiling and stirring, after the solution color turns wine red, stop heating, cool to room temperature, add ultrapure water to make up to 100 parts by volume to obtain colloidal gold solution, store at room temperature in the dark; S2, Preparation of gold-labeled antibody resuspension: Add 0.01% PVP40, 0.1% Tween-20 and 0.5% sucrose to the basal buffer to obtain the gold-labeled antibody resuspension; S3, Preparation of gold-labeled Anti-Human IgG antibody solution: Adjust the pH of the colloidal gold solution to 8.5-10 using potassium carbonate solution. Take 200 volumes of the pH-adjusted colloidal gold solution and add 20 volumes of Anti-Human IgG antibody diluted 8000-15000 times. Incubate at room temperature with shaking to allow the antibody to fully bind with the colloidal gold nanoparticles. Then add 2 volumes of BSA solution and stir well. Centrifuge and discard the supernatant. Resuspend the collected gold precipitate in the gold-labeled antibody resuspension obtained in S2. Filter through a 0.45 µm filter membrane and store at 4 °C. S4, Preparation of test strips for qualitative detection of HBV PreS1 antibody: S41, Preparation of the gold labeling pad: Glass fiber cotton was selected as the gold labeling pad. A 3% w / v BSA solution was sprayed onto the cut glass fiber cotton, and it was dried in an oven. Then, it was sprayed with 6-7 µL / cm2 The gold-labeled Anti-Human IgG antibody solution obtained by spraying S3 was dried in an oven and then sealed and stored in the dark. S42, Preparation of sample pad: Immerse the sample pad in the sample pad treatment solution, then remove it and place it in an oven to dry, and store it in a sealed container away from light; S43, Preparation of the NC membrane: Dilute the PreS1 antigen to 1-1.2 mg / mL, centrifuge to remove precipitate impurities, filter through a 0.22 µm filter membrane, and then dilute to 1-1.2 mg / mL. Use this solution to spray detection lines onto the NC membrane at a spraying rate of 1-2 µL / cm. 2 Meanwhile, HB2 antibody was sprayed onto the NC membrane as a quality control line. After spraying, it was placed in an oven to dry, then immersed in 2% w / v BSA solution for blocking, and then dried at room temperature and stored in a sealed container away from light. S44, Test strip assembly: Attach the NC membrane, gold label pad, sample pad, and absorbent pad to the backing plate in sequence, with each layer overlapping by 1-2 mm. Use a strip cutter to cut the assembled backing plate with each component attached. Finally, place it into the test strip plastic housing to complete the assembly.

[0008] Optionally, in S2, the basic buffer solution is composed of Triz-base, trisodium citrate dihydrate, sodium casein, trehalose, NaN3, and ddH2O, wherein each 1000 mL of basic buffer solution includes 2 g Triz-base, 0.7 g trisodium citrate dihydrate, 3 g sodium casein, 2.5 g trehalose, 0.05 g NaN3, and the remainder is ddH2O.

[0009] Optionally, in S3, the pH of the colloidal gold solution is adjusted to 9; the Anti-Human IgG antibody is diluted 10,000 times.

[0010] Optionally, in S3, the concentration of potassium carbonate solution used is 0.2 mol / L; the room temperature shaking incubation speed is 1000 rpm for 30 min; the BSA solution is added and stirred for 20 min; and centrifugation is performed at 10000 rpm for 35 min.

[0011] Optionally, in S41, the amount of gold-labeled Anti-Human IgG antibody solution sprayed is 6 µL / cm. 2 The first drying process involves drying in a 37°C oven for 30 minutes, and the second drying process involves drying in a 50°C oven for at least 2 hours.

[0012] Optionally, in S42, the sample pad is immersed in the sample pad treatment solution for 6 hours, and then removed and dried in a 50 °C oven for at least 2 hours.

[0013] Optionally, in S43, the PreS1 antigen concentration is 1 mg / mL, and the spraying volume is 1.5 µL / cm. 2 Centrifuge at 10,000 rpm for 35 min; after spraying, dry in an oven at 40 ℃ for 1 h.

[0014] Optionally, in S43, the line width of the detection line and the quality control line is 1 mm; in S44, the strip cutter cuts the assembled backplate with each component attached to it with a width of 3 mm.

[0015] According to another aspect of the present invention, a qualitative test strip for HBV PreS1 antibody is provided, which is prepared by the method described above for preparing a qualitative test strip for HBV PreS1 antibody. When the test strip is used, if both the test line and the control line show color, it indicates that the sample contains HBV PreS1 antibody; if the test line does not show color but the control line does, it indicates that the sample does not contain HBV PreS1 antibody; if the control line does not show color, the test strip is invalid.

[0016] According to another aspect of the present invention, the application of the HBV PreS1 antibody qualitative test strip as described above in the preparation of a hepatitis B virus PreS1 antibody detection kit is also provided.

[0017] The beneficial effects of this invention are: This invention utilizes the PreS1 antigen as a specific recognition molecule to effectively detect the level of PreS1 antibodies produced after immunization with a hepatitis B vaccine containing the PreS1 antigen, providing a rapid, convenient, and low-cost detection method for evaluating the immunization efficacy of novel hepatitis B vaccines.

[0018] Compared with traditional ELISA detection methods, this invention overcomes the shortcomings of ELISA detection, such as complex operation, long detection time, and reliance on large-scale experimental equipment, and greatly shortens the detection time, obtaining detection results within 15-20 minutes.

[0019] Meanwhile, the test strip of this invention has a simple structure and is easy to operate. It does not require professional equipment such as enzyme-linked immunosorbent assay (ELISA) readers and has low requirements for the testing environment and the professional skills of operators. It is not only suitable for medical institutions at all levels to carry out hepatitis B vaccine immunization effect evaluation and group screening, but also suitable for primary medical units and home self-testing, and has good prospects for promotion and application. Attached Figure Description

[0020] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are used to explain the invention, but do not constitute an undue limitation of the invention. In the drawings: Figure 1This is a schematic diagram of the detection principle of the HBV PreS1 antibody qualitative detection test strip in this invention, wherein (a) the test strip structure, (b) the negative sample detection result, and (c) the positive sample detection result; Figure 2 The diagram shows the synthesis and analysis of gold nanoparticles, including (a) the ultraviolet absorption diagram of gold nanoparticles and (b) the particle size distribution diagram of gold nanoparticles. Figure 3 The diagram shows the optimal pH values ​​for antibody-colloidal gold binding, where (a) shows the color of Anti-Human IgG antibody conjugation at different pH values, and (b) shows the UV absorption analysis of Anti-Human IgG antibody conjugation at different pH values. Figure 4 The diagram shows the optimal concentration of antibody-colloidal gold binding, where (a) is the color of different concentrations of Anti-Human IgG antibody conjugated, and (b) is the UV absorption analysis of different concentrations of Anti-Human IgG antibody conjugated. Figure 5 The results of screening for the amount of gold-labeled antibody sprayed are shown in the figure. Figure 6 This is a graph showing the screening results for the concentration of the coating antigen. Figure 7 The results of screening for the amount of coating antigen sprayed; Figure 8 This is a sensitivity analysis graph; Figure 9 This is a specificity analysis graph; Figure 10 Precision analysis chart; Figure 11 This is a graph showing thermal stability analysis. Figure 12 For transportation stability analysis; Figure 13 This is a real-time stability analysis graph; Figure 14 This is a graph showing the results of the serum test. Detailed Implementation

[0021] To enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present application, and not all of them. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present application. It should be noted that, unless otherwise specified, the embodiments and features in the embodiments of the present application can be combined with each other.

[0022] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0023] Unless otherwise specified, all conditions in the examples were performed under standard conditions or according to the manufacturer's recommendations. Reagents or instruments whose manufacturers are not specified are commercially available standard products. Unless otherwise stated, all technical and scientific terms herein have the meanings commonly understood by one of ordinary skill in the art.

[0024] This invention provides a qualitative test strip for HBV PreS1 antibody detection, its preparation method, and its application. This test strip can rapidly detect HBV PreS1 antibodies in serum or plasma samples. The structure of the test strip is as follows: Figure 1 As shown, it is mainly assembled from a sample pad, a gold-labeled pad, a nitrocellulose membrane (NC membrane), and an absorbent pad. The gold-labeled pad is immobilized with colloidal gold-labeled Anti-Human IgG antibody as a gold-labeled probe. The NC membrane detection line (T line) is coated with HBV PreS1 antigen to capture PreS1 antibody in the sample. The NC membrane control line (C line) is coated with HB2 antibody (HBV PreS1 antibody reference) to determine whether the test strip detection process is effective.

[0025] The preparation method of this test strip includes the following steps: preparation of colloidal gold nanoparticles, coupling of colloidal gold with anti-human IgG antibody, preparation of gold-labeled pad, sample pad treatment, coating of PreS1 antigen and quality control antibody with NC membrane, and assembly of test strip.

[0026] The technical solution and its effects of the present invention will be fully explained below through specific implementation steps and performance analysis; The main instruments and consumables involved include: Gold standard machine (model: HGS510-1), film scribing machine (model: HGS510-2), programmable strip cutter (model: HGS201), ultraviolet spectrophotometer (model: UV-2550), 500 ml polyvinylpyrrolidone (PVP40), 33 cm × 3 m nitrocellulose membrane, 50 g kanamycin sulfate, 500 g trehalose, 100 g casein sodium salt, 500 g trisodium citrate dihydrate.

[0027] Example 1: Preparation of colloidal gold nanoparticles: Take a clean conical flask, add 100 mL of ultrapure water, stir and heat to about 90 °C, add 1 mL of 1% chloroauric acid solution (HAuCl4) to the flask, continue stirring and heating to boiling, quickly add 1.56 mL of 1% sodium citrate solution to the flask, continue boiling and stirring, stop heating after stirring for 15 min, cool to room temperature, add ultrapure water to make up to 100 mL, and store at room temperature away from light.

[0028] When preparing gold nanoparticles using the sodium citrate reduction method, the solution gradually darkened in color after about 3 minutes of adding sodium citrate, slowly changing from colorless to purple. With continued stirring, the solution color gradually lightened from purple to wine red after about 8 minutes. Direct observation of the prepared colloidal gold solution revealed it to be clear and transparent, with different particle sizes displaying different colors. Ultraviolet absorption measurements of the colloidal gold nanoparticles yielded the following results: Figure 2 As shown in (a), the ultraviolet absorption peak is 520 nm. Dynamic light scattering (DLS) was used to determine the particle size distribution, revealing a relatively concentrated size distribution of gold nanoparticles, exhibiting a normal distribution around 20 nm. These characterization results demonstrate the successful preparation of gold nanoparticles.

[0029] Example 2: Preparation of colloidal gold-labeled antibody conjugates: (1) Determination of the optimal pH value for antibody-colloidal gold binding: The pH of the colloidal gold solution was adjusted to 6, 6.5, 7, 7.5, 8, 8.5, 9, 10, 11, and 12 using potassium carbonate solution. Ten EP tubes were used, and 1000 µL of the pH-adjusted colloidal gold solution was added to each tube. Then, 100 µL of 100 µg / mL Anti-Human IgG antibody was added to each EP tube. The tubes were incubated at room temperature with shaking for 30 min to allow the antibody to fully bind to the colloidal gold nanoparticles, obtaining colloidal gold-labeled antibody conjugates. Subsequently, 100 µL of 40% NaCl solution was added to each EP tube. The color change of the solution was observed to determine whether the antibody had successfully coupled to the surface of the gold nanoparticles, thus preventing NaCl-induced aggregation of the colloidal gold particles. The coupling between colloidal gold and the antibody mainly relies on electrostatic adsorption. The surface of colloidal gold nanoparticles (10-40 nm) usually carries a negative charge, which originates from the electric double layer structure formed by stabilizers such as citrate ions. By adjusting the solution pH to near the isoelectric point of the antibody, the positively charged regions on the antibody surface can be exposed, allowing them to bind with the negatively charged regions on the colloidal gold surface via electrostatic attraction. Therefore, pH conditions have a significant impact on the coupling efficiency of colloidal gold and antibodies. Figure 3As shown in (a), when the solution pH is 8.5, the solution color remains basically stable under naked-eye observation, and no obvious aggregation phenomenon is observed. Further analysis using ultraviolet-visible spectrophotometry, such as... Figure 3 As shown in (b), the absorption peak intensity of the colloidal gold-Anti-Human IgG antibody complex (i.e., the colloidal gold-labeled antibody conjugate) reaches its maximum at pH 9, stabilizing at approximately 525 nm, indicating that the antibody-colloidal gold binding effect is optimal at this pH. Therefore, pH=9 is determined to be the optimal reaction condition for the conjugation of Anti-Human IgG antibody with colloidal gold.

[0030] (2) Screening for the optimal concentration of antibody-colloidal gold binding: Adjust the colloidal gold solution to the optimal pH (pH=9) using potassium carbonate solution. Take several EP tubes and add 1000 µL of the pH-adjusted colloidal gold solution to each tube. Add different dilutions of Anti-Human IgG antibody solution (e.g., 4000x, 8000x, 10000x, 15000x, 20000x, 25000x dilution) to each EP tube. Incubate at room temperature with shaking for 30 min to allow the antibody to fully bind to the colloidal gold nanoparticles. Then add 100 µL of 40% NaCl solution to each EP tube. Observe the color change of the solution in each EP tube to determine whether the antibody has successfully coupled to the surface of the gold nanoparticles, thereby preventing NaCl-induced aggregation of colloidal gold particles. If the antibody binds stably to the colloidal gold, the solution color remains wine red; if the binding is unstable, the solution color will change from red to purple or blue. Figure 4 As shown in (a), when the Anti-Human IgG antibody was diluted 15,000 times, the solution color remained essentially unchanged upon naked-eye observation, indicating that the antibody had stabilized the colloidal gold particles to a certain extent. Further analysis using ultraviolet-visible spectrophotometry, such as... Figure 4 As shown in (b), when the Anti-Human IgG antibody was diluted 10,000 times, the absorption peak of the colloidal gold-Anti-Human IgG antibody complex no longer showed a significant shift and stabilized at approximately 525 nm, while the absorption peak intensity reached its maximum value, indicating that the antibody's binding performance with colloidal gold was optimal at this point. Therefore, it was ultimately determined that a 10,000-fold diluted Anti-Human IgG antibody was used for colloidal gold conjugation to ensure conjugation efficiency and the stability of the test strip's detection signal.

[0031] Example 3: Preparation of gold-labeled Anti-Human IgG antibody solution: (1) Screening of gold-labeled antibody resuspension: Thirteen formulations were prepared at different concentrations of 0.01% PVP40, 0.05%, 0.1%, 0.5% Tween-20 (polysorbate-20), and 1% sucrose to screen gold-labeled antibody resuspensions. The quality of the formulations was evaluated based on the colorimetric intensity and flow rate of the control lines. PVP40 and Tween-20 were crucial for the flow of colloidal gold, while sucrose improved the colorimetric effect. The results are shown in Table 1, where "C" represents the colorimetric effect, "S" represents the flow rate, and "-", "+", and "++" indicate increasing intensity. Based on the experimental results, formulation 12 was ultimately selected as the gold-labeled antibody buffer, with 0.01% PVP40, 0.1% Tween-20, and 0.5% sucrose added to the original basal buffer. The composition of the original basal buffer is shown in Table 2.

[0032] Table 1. Screening with gold-labeled antibody buffer:

[0033] Table 2. Composition of the basal buffer solution:

[0034] Among them, Triz-base, or Tris base, or tris(hydroxymethyl)aminomethane; NaN3, sodium azide; and ddH2O, or double deionized water, or ultrapure water.

[0035] (2) Preparation of gold-labeled Anti-Human IgG antibody solution: Adjust 200 mL of colloidal gold solution to a suitable pH (pH=9, as above) using 0.2 mol / L potassium carbonate solution. Add the Anti-Human IgG antibody at the optimal dilution (10000 times, as above) to the colloidal gold solution while stirring at 1000 rpm for 30 min. Add 2 mL of BSA solution to the above solution and stir for 20 min. Centrifuge at 10000 rpm for 35 min. Discard the supernatant, resuspend the collected gold precipitate in the above gold-labeled antibody resuspension, filter through a 0.45 µm filter membrane to obtain the gold-labeled Anti-Human IgG antibody solution, and store at 4 °C.

[0036] Example 4: Preparation and analysis of colloidal gold test strips: (1) Screening of gold-labeled antibody spraying amount: The gold-labeled antibody (i.e., the gold-labeled Anti-Human IgG antibody solution obtained in Example 3) was added at concentrations of 1, 2, 3, 4, 5, 6, and 7 µL / cm³. 2The gold-labeled antibody was sprayed onto pretreated gold-labeled pads and then dried in an oven at 37 ℃ for 3 h. Different amounts of gold-labeled pads were assembled onto test strips for testing with samples. The optimal spraying amount was selected based on the appropriate color intensity of the C and T lines. Insufficient gold-labeled antibody spraying resulted in weak color development, while excessive spraying easily caused gold nanoparticle retention. Results are as follows... Figure 5 The gold-labeled antibody coating amount is 6 µL / cm 2 At this point, the C and T lines show the best color intensity; using too many would be wasteful.

[0037] (2) Screening for the concentration of the coating antigen: PreS1 antigen was used as the coating antigen for PreS1 antibody test strips, diluted to 0.4, 0.6, 0.8, 1.0, and 1.2 mg / mL, and then sprayed onto NC membranes and dried in a 45 °C oven for 3 h before testing. Figure 6 As shown, the antigen concentration for the PreS1 antibody test strip was selected as 1 mg / mL, at which point the T line was clearly visible.

[0038] (3) Screening of the amount of coating antigen sprayed: The antigen coating volume for the PreS1 antibody test strips can be selected as 0.5, 1, 1.5, or 2 µL / cm. 2 Tests were conducted by Figure 7 As shown, the antigen spraying concentration for the PreS1 antibody test strip was selected as 1.5 µL / cm. 2 At this point, the T-line shows a clear color.

[0039] Example 5: Preparation of colloidal gold test strips: (1) Preparation of gold-labeled pads: The gold-labeled pad is made of glass fiber cotton. 3% BSA (w / v) solution is sprayed onto the cut glass fiber cotton and dried in an oven at 37°C for 30 min. Then, a colloidal gold nanoparticle solution labeled with antibodies (i.e., the gold-labeled Anti-Human IgG antibody solution obtained in Example 3) is sprayed onto the glass fiber cotton and dried in an oven at 50°C for at least 2 h. After that, it is sealed and stored in the dark.

[0040] (2) Preparation of the sample pad: The sample pads were immersed in the sample pad treatment solution for 6 hours, then dried in a 50 °C oven for at least 2 hours, and then sealed and stored away from light.

[0041] (3) Preparation of NC membrane: The PreS1 antigen was diluted to the optimal concentration, centrifuged at 10,000 rpm for 35 min to remove precipitate impurities, filtered through a 0.22 µm filter membrane, and then diluted again to the optimal concentration for use as the spray point on the detection line (T line) on the NC membrane. Simultaneously, HB2 antibody (HBV PreS1 antibody reference) was sprayed onto the NC membrane as the control line (C line), with a spray point width of 1 mm. After spraying, the membrane was dried in a 40 ℃ oven for 1 h, then immersed in 2% BSA (w / v) solution for complete sealing, dried at room temperature, and stored in a sealed, light-protected container.

[0042] (4) Assembly of colloidal gold test strips: Attach the NC membrane, gold label pad, sample pad, and absorbent pad to the backing plate in the order recommended by Millipore, such as... Figure 1 (a) There is an overlap of about 1-2 mm between each layer (preferably, a 2 mm overlap is selected). The assembled backing plate with each component is cut to a width of 3 mm using a strip cutter, and finally placed into the plastic housing of the test strip to complete the assembly.

[0043] (5) Interpretation of results from colloidal gold test strips: During testing, add 100 µL of sample, let it stand for 15 minutes, and then interpret the result based on the color intensity of the test line. PreS1 antibody test strip interpretation: If both the T and C lines are visible, the sample contains HBV PreS1 antibody; if the T line is not visible but the C line is visible, the sample does not contain HBV PreS1 antibody; if the C line is not visible, the test strip is invalid.

[0044] Example 6: Performance analysis of colloidal gold test strips: (1) Sensitivity analysis: 100 µL of protein standard solutions of different concentration gradients were added to the sample pad. After reacting for 15 min, the fluorescence signal intensity of the T and C lines was detected, and the T / C value was calculated for quantitative analysis. The detection limit was defined as the lowest concentration that could be clearly distinguished from the blank sample. Figure 8 As shown, based on naked-eye visibility, the PreS1 antibody test strip can be observed when the sample concentration is 0.05 µg / mL, and the test line is not visible when it is below 0.05 µg / mL. Therefore, its sensitivity is determined to be approximately 0.05 µg / mL.

[0045] (2) Specificity analysis: Negative samples, HCV patient serum, and murine IgG antibodies were added to the sample pad of the test strip for detection. After a 15-minute reaction, the fluorescence signal intensity of the T and C lines was measured, and the T / C value was calculated for result interpretation. Figure 9As shown, the PreS1 antibody test strip exhibits excellent specificity and shows no cross-reactivity with healthy human serum, HCV serum, or IgG mouse antibody.

[0046] (3) Precision (repeatability) analysis: Six batches of test strips were collected and tested under the same conditions on serum with high PreS1 antibody levels, serum with low PreS1 antibody levels, and serum from healthy individuals. The results are as follows: Figure 10 As shown, the PreS1 antibody test strip also exhibits good repeatability, accurately detecting both high and low levels of PreS1 antibody in serum, and no false positives were detected in negative serum.

[0047] (4) Thermal stability analysis: The same batch of prepared colloidal gold test strips were placed in a 37 ℃ incubator, and serum tests were performed before, 3 days after, 7 days after, and 10 days after incubation. The thermal stability of the test strips was tested and evaluated, and the results are as follows: Figure 11 As shown, the PreS1 antibody test strip retains its detection efficacy after being stored at 37 ℃ for 10 days, but its detection efficacy decreases significantly after being stored at 37 ℃ for 12 days. The shelf life of the pretest strip is approximately 1 year.

[0048] (5) Transportation stability analysis: Colloidal gold test strips prepared in the same batch were used for transport stability testing. The simulated transport test method involved shaking the strips at 60 rpm for 7 days on a rocker arm, followed by three drops from a height of 1.5 meters. Serum samples were tested before and after the simulated transport test; a clear and consistent T-line was considered indicative of good transport stability. The transport stability of the test strips was evaluated, and the results are as follows: Figure 12 As shown, the test strips produced clear and accurate results after the simulated transportation experiment, indicating that the impact of transportation on the test strips is negligible.

[0049] (6) Real-time stability analysis: Colloidal gold test strips prepared in the same batch were dried and sealed at room temperature in the dark. Standard solutions of different concentrations were tested on the day of preparation and at 3, 6, 9, and 12 months of storage. The real-time stability of the colloidal gold test strips was assessed by comparing the test results at different storage times. Results are as follows: Figure 13 As shown, the PreS1 antibody test strip can detect positive samples, but the detection efficacy of the PreS1 antibody test strip is slightly reduced, and the shelf life of the test strip is about 1 year.

[0050] (7) Analysis of serum test results: The PreS1 antibody test strip (colloidal gold method) and alpha kit (ELISA method) prepared according to this invention were used to detect HBV PreS1 antibodies in the serum of 88 HBV patients. The test results are as follows: Figure 14 73 samples were positive for HBVPreS1 antibody by both methods, 7 samples were negative, 1 sample was positive by colloidal gold method but negative by ELISA, and 7 samples were negative by colloidal gold method but positive by ELISA. The consistency between the two methods was 90.9%, indicating good consistency between colloidal gold method and ELISA method.

[0051] In summary, this invention uses the PreS1 antigen as a specific recognition molecule and, by comparing different operating conditions, has prepared a high-performance, colorimetric test strip for the qualitative detection of HBV PreS1 antibodies (also referred to as the PreS1 antibody test strip / colloidal gold test strip mentioned above, which have equivalent meanings in the specific embodiments of this invention). This test strip can be used directly with serum samples for detection without the need for complex instruments and equipment, simplifying the HBV PreS1 antibody detection process. The detection process is simple and fast, and it has good sensitivity, specificity, and stability, making it suitable for evaluating the immunization effect of hepatitis B vaccines and for large-scale screening.

[0052] The sequence numbers of the above embodiments of the present invention are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.

[0053] Those skilled in the art will understand that the purpose of this invention is to provide a relatively universal overall preparation process or technical principle, and to cover as many different applicable scenarios and conditions as possible (such as different raw material characteristics, production scale, product demand, etc.). Therefore, some specific operations can be flexibly adjusted according to the situation in actual implementation, as long as the expected or the same or similar technical effects as those in the embodiments of this invention can be achieved.

[0054] The steps in the method of this invention can be adjusted, combined, or deleted according to actual needs. The technical features can be combined arbitrarily. For the sake of brevity, not all possible combinations of the technical features in the embodiments are described. However, as long as the combinations of these technical features do not contradict each other, they should all be considered within the scope of this invention.

[0055] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a qualitative test strip for HBV PreS1 antibody, characterized in that, include: S1, Preparation of colloidal gold nanoparticles: Heat 100 parts by volume of ultrapure water, add 1 part by volume of 1% chloroauric acid solution, stir and heat to boiling, then add 1.56 parts by volume of 1% sodium citrate solution, continue boiling and stirring, after the solution color turns wine red, stop heating, cool to room temperature, add ultrapure water to make up to 100 parts by volume to obtain colloidal gold solution, store at room temperature in the dark; S2, prepare gold-labeled antibody resuspension; Add 0.01% PVP40, 0.1% Tween-20 and 0.5% sucrose to the basal buffer to obtain the gold-labeled antibody resuspension; S3, prepare gold-labeled Anti-Human IgG antibody solution; Adjust the pH of the colloidal gold solution to 8.5-10 using potassium carbonate solution. Take 200 volumes of the pH-adjusted colloidal gold solution and add 20 volumes of Anti-Human IgG antibody diluted 8000-15000 times. Incubate at room temperature with shaking to allow the antibody to fully bind with the colloidal gold nanoparticles. Then add 2 volumes of BSA solution and stir well. Centrifuge and discard the supernatant. Resuspend the collected gold precipitate in the gold-labeled antibody resuspension obtained in S2. Filter through a 0.45 µm filter membrane and store at 4 °C. S4, Prepare test strips for qualitative detection of HBV PreS1 antibody; S41, Preparation of the gold labeling pad: Glass fiber cotton was selected as the gold labeling pad. A 3% w / v BSA solution was sprayed onto the cut glass fiber cotton, dried in an oven, and then sprayed with 6-7 µL / cm 2 The gold-labeled Anti-Human IgG antibody solution obtained by spraying S3 was dried in an oven and then sealed and stored in the dark. S42, Preparation of sample pad: Immerse the sample pad in the sample pad treatment solution, then remove it and place it in an oven to dry, and store it in a sealed container away from light; S43, Preparation of the NC membrane: Dilute the PreS1 antigen to 1-1.2 mg / mL, centrifuge to remove precipitate impurities, filter through a 0.22 µm filter membrane, and then dilute to 1-1.2 mg / mL. Use this solution to spray detection lines onto the NC membrane at a spraying rate of 1-2 µL / cm. 2 Meanwhile, HB2 antibody was sprayed onto the NC membrane as a quality control line. After spraying, it was placed in an oven to dry, then immersed in 2% w / v BSA solution for blocking, and then dried at room temperature and stored in a sealed container away from light. S44, Test strip assembly: Attach the NC membrane, gold label pad, sample pad, and absorbent pad to the backing plate in sequence, with each layer overlapping by 1-2mm. Use a strip cutter to cut the assembled backing plate with each component attached. Finally, place it into the test strip plastic housing to complete the assembly.

2. The method for preparing the HBV PreS1 antibody qualitative detection test strip according to claim 1, characterized in that, In S2, the basic buffer consists of Triz-base, trisodium citrate dihydrate, sodium casein, trehalose, NaN3, and ddH2O. Each 1000 mL of basic buffer contains 2 g Triz-base, 0.7 g trisodium citrate dihydrate, 3 g sodium casein, 2.5 g trehalose, 0.05 g NaN3, and the remainder is ddH2O.

3. The method for preparing the HBV PreS1 antibody qualitative detection test strip according to claim 1, characterized in that, In S3, the pH of the colloidal gold solution was adjusted to 9; the Anti-Human IgG antibody was diluted 10,000 times.

4. The method for preparing the HBV PreS1 antibody qualitative detection test strip according to claim 1, characterized in that, In S3, the concentration of potassium carbonate solution used was 0.2 mol / L; the incubation was carried out at room temperature with shaking at 1000 rpm for 30 min; the BSA solution was added and stirred for 20 min; and centrifugation was carried out at 10000 rpm for 35 min.

5. The method for preparing the HBV PreS1 antibody qualitative detection test strip according to claim 1, characterized in that, In S41, the spraying volume of the gold-labeled Anti-Human IgG antibody solution was 6 µL / cm. 2 The first drying process involves drying in a 37°C oven for 30 minutes, and the second drying process involves drying in a 50°C oven for at least 2 hours.

6. The method for preparing the HBV PreS1 antibody qualitative detection test strip according to claim 1, characterized in that, In S42, the sample pad is immersed in the sample pad treatment solution for 6 hours, and then removed and dried in a 50 ℃ oven for at least 2 hours.

7. The method for preparing the HBV PreS1 antibody qualitative detection test strip according to claim 1, characterized in that, In S43, the PreS1 antigen concentration was 1 mg / mL, and the spraying volume was 1.5 µL / cm. 2 Centrifuge at 10,000 rpm for 35 min; after spraying, dry in an oven at 40 ℃ for 1 h.

8. The method for preparing the HBV PreS1 antibody qualitative detection test strip according to claim 1, characterized in that, In S43, the line width of the detection line and the quality control line is 1 mm; in S44, the strip cutter cuts the assembled backplate with each component attached at a width of 3 mm.

9. A test strip for qualitative detection of HBV PreS1 antibody, characterized in that, The test strip for qualitative detection of HBV PreS1 antibody is prepared using the preparation method described in any one of claims 1 to 8. When the test strip for qualitative detection of HBV PreS1 antibody is used, if both the test line and the control line are colored, it indicates that the sample contains HBV PreS1 antibody; if the test line is not colored but the control line is colored, it indicates that the sample does not contain HBV PreS1 antibody; if the control line is not colored, the test strip is invalid.

10. The use of the HBV PreS1 antibody qualitative test strip as described in claim 9 in the preparation of a hepatitis B virus PreS1 antibody detection kit.

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