An influenza a / b virus antigen detection test strip and a preparation method thereof

Abstract

The application relates to an influenza A / B virus antigen detection test paper strip and a preparation method thereof. The test paper strip comprises a PVC bottom plate and a sample pad, a gold label pad, a nitrocellulose membrane and a water absorption pad which are sequentially overlapped. The application adopts a Tris buffer system to adjust the pH value of colloidal gold, so that different target proteins are simultaneously coupled with colloidal gold. Bismercapto polyethylene glycol is added in the colloidal gold labeling process to be blocked, the colloidal gold coupling agent is gathered into large particles through gold-sulfur bond combination, and the color development intensity is enhanced. Large particle coupling agents are formed by using the specific adsorption of BSA and BSA-Ab. Lignosulfonate sodium is added in the sample pad treatment liquid to reduce non-specific adsorption. The application significantly improves the detection sensitivity and specificity, and is simple to operate, does not need instruments, and is suitable for large-scale popularization and application.

Description

Technical Field

[0001] This invention belongs to the field of biomedical detection technology, specifically relating to a test strip for detecting influenza A / B virus antigens and its preparation method, and more specifically, relating to a combined test strip for detecting influenza A / B virus antigens based on colloidal gold immunochromatography technology and its preparation method. Background Technology

[0002] Influenza is an acute respiratory infectious disease caused by the influenza virus, which seriously endangers human health. Influenza viruses can be divided into four types: A, B, C, and D. Type A (H1N1 and H3N2 subtypes) and type B influenza viruses cause seasonal epidemics every year. Outbreaks can occur in densely populated places such as schools, childcare facilities, and nursing homes.

[0003] Influenza patients and asymptomatic carriers are the main sources of infection for seasonal influenza. The influenza virus is primarily transmitted through droplets produced by infected individuals' sneezing and coughing, and can also be transmitted through direct or indirect contact with mucous membranes such as the mouth, nose, and eyes. For example, touching virus-contaminated objects and then touching one's mouth or nose is a common mode of transmission. In specific places, such as crowded and poorly ventilated rooms, the influenza virus may also spread through aerosols. The incubation period for influenza is typically 1-4 days (average 2 days), and the virus is contagious from the end of the incubation period until the acute phase after the onset of symptoms.

[0004] Currently, the main detection methods for influenza A / B viruses used in clinical testing are PCR and colloidal gold assays. While fluorescent PCR offers high sensitivity, its cumbersome sample processing and time-consuming nature require specialized equipment and professionally trained operators, limiting its use by ordinary users. Colloidal gold immunochromatography, on the other hand, is widely used in point-of-care testing (POCT) due to its ease of operation, rapid detection, and lack of instrument requirements.

[0005] However, currently available commercially available colloidal gold-based detection kits for influenza A / B virus antigens generally suffer from low sensitivity or complex manufacturing processes, leading to a high likelihood of false negative results. Therefore, developing a highly sensitive, specific, and easily prepared detection kit for influenza A / B virus antigens is of significant importance and value. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a test strip for influenza A / B virus antigen detection, its preparation method, and its application. This invention significantly improves detection sensitivity and specificity by employing techniques such as blocking with dithiol polyethylene glycol, forming large-particle conjugates through specific adsorption of BSA / BSA-Ab, and adding sodium lignosulfonate to the sample pad treatment solution. Furthermore, the method is simple to operate, requires no instruments, and is suitable for large-scale application.

[0007] To solve the above-mentioned technical problems, the present invention is achieved through the following technical solution.

[0008] The first aspect of the present invention provides an influenza A / B virus antigen test strip, comprising a base plate, a sample pad, a gold label pad, a nitrocellulose membrane, and an absorbent pad; wherein the sample pad, the gold label pad, the nitrocellulose membrane, and the absorbent pad are sequentially overlapped and pasted on the base plate in the direction of chromatography;

[0009] The sample pad is treated by soaking in a sample pad treatment solution, which includes Tris (tris(hydroxymethyl)aminomethane), Casein (casein), Tween 20, S9 (Tetronic 1307), and sodium lignosulfonate.

[0010] The gold-labeled pad carries colloidal gold-labeled conjugate 1 and gold-labeled conjugate 2. Gold-labeled conjugate 1 comprises influenza A monoclonal antibody 1 and BSA (bovine serum albumin), and is blocked with dithiol polyethylene glycol. Gold-labeled conjugate 2 comprises influenza B monoclonal antibody 1 and BSA-Ab (anti-bovine serum albumin antibody), and is blocked with dithiol polyethylene glycol.

[0011] The nitrocellulose membrane is coated with a control line (C line) and a detection line (T line). The control line is coated with anti-IgG antibodies, and the detection lines include influenza A virus detection lines and influenza B virus detection lines, which are coated with influenza A monoclonal antibody 2 and influenza B monoclonal antibody 2, respectively.

[0012] Preferably, the molecular weight of the dithiol polyethylene glycol is 5,000-30,000; more preferably, the molecular weight of the dithiol polyethylene glycol is 15,000-25,000; and most preferably, the molecular weight of the dithiol polyethylene glycol is 20,000.

[0013] Preferably, the base plate is selected from PVC base plates.

[0014] Preferably, the anti-IgG antibody is a goat anti-mouse IgG antibody.

[0015] A second aspect of the present invention provides a method for preparing the above-mentioned influenza A / B virus antigen test strip, comprising the following steps:

[0016] (1) Dilute the influenza A monoclonal antibody 2 and influenza B monoclonal antibody 2 with coating solution and coat them onto the detection line of the nitrocellulose membrane. Dilute the anti-IgG antibody with coating solution and coat it onto the control line of the nitrocellulose membrane. Dry the membrane.

[0017] (2) Add Tris buffer to colloidal gold solution and mix well. Then add influenza A monoclonal antibody 1 and BSA and stir to react. Then add dithiol polyethylene glycol solution and stir to react. Then centrifuge and discard the supernatant. The precipitate is reconstituted with preservation solution to obtain gold-labeled conjugate 1.

[0018] Tris buffer was added to colloidal gold solution and mixed well. Then, influenza B monoclonal antibody 1 and BSA-Ab were added and stirred to react. Then, dithiol polyethylene glycol solution was added and stirred to react. After centrifugation, the supernatant was discarded, and the precipitate was reconstituted with preservation solution to obtain gold-labeled conjugate 2.

[0019] The preservation solution includes Tris, Casein, and sucrose;

[0020] In step (2), Tris buffer is used to adjust the pH of colloidal gold. The Tris buffer maintains the colloidal gold system in a weakly alkaline state, with a mild buffering capacity. Different target proteins can be coupled with colloidal gold simultaneously without adjusting to the optimal pH. Simultaneously, bis(thiol) polyethylene glycol is used for blocking in this step. The thiol groups of bis(thiol) polyethylene glycol bind to colloidal gold via gold-sulfur bonds, blocking excess sites while allowing the thiol groups at both ends to connect to different colloidal gold particles, polymerizing into large particles. When captured, these particles enhance color intensity and improve product sensitivity. The good hydrophilicity of the PEG group allows the polymerized large particles to continue to disperse well in the solution without agglomeration or dead gold formation. Gold-labeled conjugate 1 and gold-labeled conjugate 2 form a large-particle polymer of colloidal gold-influenza A monoclonal antibody 1-BSA-BSA-Ab-influenza B monoclonal antibody 1-colloidal gold through the specific adsorption of BSA and BSA-Ab. When captured, this polymer significantly enhances color intensity and improves product sensitivity.

[0021] (3) After mixing the gold-labeled coupling compound 1 and gold-labeled coupling compound 2 obtained in step (2), add diluent to dilute, then soak the blank pad in it, take it out and dry it to obtain the gold-labeled pad.

[0022] The diluent includes disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, and NP-40;

[0023] (4) Tris, Casein, Tween 20, S9 and sodium lignosulfonate were placed in a solvent and mixed to prepare a sample pad treatment solution. Then, the blank pad was immersed in the sample pad treatment solution, taken out and dried to obtain the sample pad.

[0024] In step (4), sodium lignin sulfonate can adsorb hydrophobic interfering substances such as lipids and bilirubin in the sample, reduce background interference, and improve specificity.

[0025] (5) The sample pad, gold label pad, nitrocellulose membrane and absorbent pad are overlapped and pasted onto the base plate in the direction of chromatography, and then cut to obtain the final product.

[0026] Preferably, the coating solution in step (1) includes Tris and trehalose; more preferably, the coating solution includes Tris at a concentration of 1.2-2.4 g / L and trehalose at a concentration of 20-30 g / L.

[0027] Preferably, in step (1), the concentrations of the influenza A monoclonal antibody 2 (purchased from Jiangsu Shenji Biotechnology Co., Ltd.), influenza B monoclonal antibody 2 (purchased from Jiangsu Shenji Biotechnology Co., Ltd.), and goat anti-mouse IgG antibody (purchased from Jiangsu Shenji Biotechnology Co., Ltd.) after dilution are 0.8-1.0 mg / mL.

[0028] Preferably, the anti-IgG antibody in step (1) is a goat anti-mouse IgG antibody.

[0029] Preferably, the drying temperature in step (1) is 40-50°C and the drying time is 1-3 hours.

[0030] Preferably, the volume of the colloidal gold solution in step (2) is 8-12 mL; the final concentration of Tris after the Tris buffer is added to the colloidal gold solution is 5-10 mM.

[0031] Preferably, the colloidal gold solution in step (2) can be obtained commercially or prepared by the following method: 4 mL of 10 g / L chloroauric acid solution is added to 100 mL of ultrapure water, heated and stirred under reflux until boiling, 6.4 mL of 10 g / L sodium citrate solution is added, and after the solution turns wine red, it is heated under reflux for 15 min. Then the heating is stopped and the solution is allowed to cool naturally to room temperature to obtain the colloidal gold solution.

[0032] Preferably, in step (2), the mass-to-volume ratio (mg:mL) of the influenza A monoclonal antibody 1 (purchased from Jiangsu Shenji Biotechnology Co., Ltd.) to the colloidal gold solution is 1:50-120; the mass ratio of the influenza A monoclonal antibody 1 to BSA is 1:0.6-1.5; the mass-to-volume ratio (mg:mL) of the influenza A monoclonal antibody 2 (purchased from Jiangsu Shenji Biotechnology Co., Ltd.) to the colloidal gold solution is 1:50-120; and the mass ratio of the influenza A monoclonal antibody 2 to BSA-Ab is 1:0.6-1.5.

[0033] Preferably, in step (2), when preparing gold-labeled conjugate 1, the temperature for adding influenza A monoclonal antibody 1 and BSA and stirring reaction is 20-25℃ for 10-15 min, and the temperature for adding dithiol polyethylene glycol solution and stirring reaction is 20-25℃ for 10-15 min, with a volume ratio of dithiol polyethylene glycol solution to colloidal gold solution of 0.1-0.15:10; when preparing gold-labeled conjugate 2, the temperature for adding influenza B monoclonal antibody 1 and BSA-Ab and stirring reaction is 20-25℃ for 10-15 min, and the temperature for adding dithiol polyethylene glycol solution and stirring reaction is 20-25℃ for 10-15 min, with a volume ratio of dithiol polyethylene glycol solution to colloidal gold solution of 0.1-0.15:10.

[0034] Preferably, in step (2), the centrifugation temperature for preparing gold-labeled conjugate 1 is 4°C, the rotation speed is 8000-10000 rpm, and the time is 10-15 min; the centrifugation temperature for preparing gold-labeled conjugate 2 is 4°C, the rotation speed is 8000-10000 rpm, and the time is 10-15 min.

[0035] Preferably, the concentrations of Tris, Casein and sucrose in the preservation solution in step (2) are 2-3 g / L, 3-8 g / L and 80-120 g / L, respectively.

[0036] Preferably, the molecular weight of the dithiol in step (2) is 5000-30000; more preferably, the molecular weight of the dithiol is 15000-25000; and most preferably, the molecular weight of the dithiol is 20000.

[0037] Preferably, the concentration of the dithiol polyethylene glycol solution in step (2) is 30-80 g / L.

[0038] Preferably, the volume ratio of gold-labeled coupling agent 1 to gold-labeled coupling agent 2 in step (3) is 1:1-2.

[0039] Preferably, the concentrations of disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, and NP-40 in the diluent in step (3) are 5-6 g / L, 1-1.5 g / L, 8-12 g / L, and 8-12 g / L, respectively; and the dilution factor is 15-25 times.

[0040] Preferably, the drying temperature in step (3) is 35-40°C.

[0041] Preferably, the concentrations of Tris, Casein, Tween 20, S9 and sodium lignosulfonate in the sample pad treatment solution in step (4) are 6-12 g / L, 3-7 g / L, 3-7 g / L, 3-7 g / L and 0.1-0.2 g / L, respectively.

[0042] Preferably, the drying temperature in step (4) is 35-40°C.

[0043] Preferably, the base plate in step (5) is selected from PVC base plates.

[0044] A third aspect of the present invention provides an influenza A / B virus antigen detection kit, comprising the above-mentioned influenza A / B virus antigen detection test strip or the influenza A / B virus antigen detection test strip prepared according to the above preparation method, and one or more of the following: sample extraction solution, pharyngeal swab, sample extraction tube, and pipette.

[0045] A fourth aspect of this invention provides a method for detecting influenza A / B virus antigens, using the aforementioned influenza A / B virus antigen detection kit, comprising the following steps:

[0046] Add the sample to be tested to the sample extraction solution for treatment. Add the treated sample to the sample pad of the above-mentioned influenza A / B virus antigen test strip. Observe the results after chromatography. If both the test line (T line) and the control line (C line) are red, the result is considered positive. If only the control line (C line) is red and the test line (T line) is not colored, the result is considered negative. If the control line (C line) is not colored, the result is considered invalid.

[0047] Preferably, the chromatography time is 10-15 minutes.

[0048] This invention employs a double-antibody sandwich method for the qualitative detection of influenza A / B virus antigens in human nasal and oropharyngeal swabs. During detection, when influenza A / B virus antigens are present in the sample, they react with colloidal gold-labeled antibodies (gold-labeled influenza A monoclonal antibody 1 and gold-labeled influenza B monoclonal antibody 1 conjugate) on a gold-labeled pad to form a labeled antibody-antigen complex. This complex is then chromatographically ascended via capillary action and captured by the detection line (T-line) antibodies (influenza A monoclonal antibody 2 and influenza B monoclonal antibody 2) coated on a nitrocellulose membrane, resulting in a red band. The complex continues to chromatographically ascend and is captured by the control line (C-line) antibody (goat anti-mouse IgG antibody) coated on a nitrocellulose membrane, also resulting in a red band. When the analyte concentration in the sample is below the limit of detection, the detection line (T-line) remains colorless.

[0049] Compared with the prior art, the present invention has the following advantages:

[0050] (1) This invention uses di-thiol polyethylene glycol (Di-SH-PEG) as a colloidal gold blocking agent. The thiol groups at both ends of the PEG bind to different colloidal gold particles via gold-sulfur bonds, causing the colloidal gold conjugates to aggregate into large particles, resulting in a significantly enhanced color intensity upon capture. Simultaneously, the specific adsorption between BSA and BSA-Ab further promotes the formation of large polymer particles. Experiments show that the detection limit for H1N1 (2009) using this invention can reach 0.675 × 10⁻⁶. 5 The concentration of U / mL is superior to that of the comparative product, and the sensitivity is significantly improved.

[0051] (2) In this invention, sodium lignosulfonate is added to the sample pad treatment solution, which can effectively adsorb hydrophobic interfering substances such as lipids and bilirubin in the sample and reduce background interference. Validated by a large number of negative clinical samples, the specificity is as high as 100%.

[0052] (3) The present invention uses colloidal gold immunochromatography technology, with colloidal gold as a carrier, to detect nasal swab and oropharyngeal swab samples without the aid of instruments and equipment, and the test results can be reported in 10 minutes.

[0053] (4) The present invention uses Tris to adjust the pH value of colloidal gold. The Tris buffer can maintain the colloidal gold system in a weakly alkaline environment with mild buffering capacity. Target proteins with different isoelectric points can be coupled with colloidal gold at the same time without needing to be adjusted to their respective optimal pH values, which simplifies the preparation process.

[0054] (5) The PEG segments of dithiol polyethylene glycol have good hydrophilicity, which allows the large particles after polymerization to be well dispersed in the solution without aggregation and precipitation (dead gold), thus ensuring the batch-to-batch stability and storage stability of the test strip. Attached Figure Description

[0055] Figure 1 This is a schematic diagram of the test strip structure of the present invention.

[0056] Figure 2 This is a schematic diagram illustrating the detection and result interpretation when the test strip of this invention is used for influenza detection. Detailed Implementation

[0057] To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0058] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0059] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention. Unless otherwise specified, all raw materials, reagents, instruments, and equipment used in this invention are commercially available or can be prepared by existing methods.

[0060] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary. The terms "comprising," "including," "having," "containing," etc., as used herein are open-ended, meaning they include but are not limited to.

[0061] Example 1

[0062] A test strip for detecting influenza A / B virus antigens, the preparation method of which specifically includes the following steps:

[0063] (1) Influenza A monoclonal antibody 2 and influenza B monoclonal antibody 2 were diluted to 1 mg / mL with coating solution and coated onto the detection line of nitrocellulose membrane using a membrane scrubbing instrument with a parameter of 1 μL / cm. Goat anti-mouse IgG was diluted to 1 mg / mL with coating solution and coated onto the control line of nitrocellulose membrane using a membrane scrubbing instrument with a parameter of 1 μL / cm. The coated membrane was then placed in a 45℃ oven and dried for 2 h.

[0064] The specific preparation method of the coating solution is as follows: Weigh 2.4g Tris and 20g trehalose into a beaker, add 1L of purified water, stir and mix well, adjust the pH to 8.0 with HCl solution, and store at 4℃ for later use.

[0065] (2) Add 50 μL of 1M Tris buffer to 10 mL of colloidal gold solution and mix well. Then add 100 μg of influenza A monoclonal antibody 1 and 100 μg of BSA and stir at 25 °C for 15 min. Then add 0.1 mL of 50 g / L dithiol polyethylene glycol (MW 20000) solution and stir at 25 °C for 15 min. Then centrifuge at 4 °C and 8000 rpm for 15 min and discard the supernatant. Add 1 mL of preservation solution to the precipitate for reconstitution to obtain gold-labeled conjugate 1.

[0066] Add 50 μL of 1M Tris buffer to 10 mL of colloidal gold solution and mix well. Then add 100 μg of influenza B monoclonal antibody 1 and 100 μg of BSA-Ab, and stir at 25 °C for 15 min. Then add 0.1 mL of 50 g / L dithiol polyethylene glycol (MW 20000) solution and stir at 25 °C for 15 min. Then centrifuge at 4 °C and 8000 rpm for 15 min, discard the supernatant, and reconstitute the precipitate with 1 mL of preservation solution to obtain gold-labeled conjugate 2.

[0067] The specific preparation method of the 1M Tris buffer is as follows: Weigh 12g Tris into a beaker, add 100mL of purified water, stir well, and store at 4℃ for later use.

[0068] The specific preparation method of the preservation solution is as follows: Weigh 2.4g Tris, 5g Casein, and 100g sucrose into a beaker, add 1L of purified water, stir and mix well, adjust the pH to 8.0 with HCl solution, and store at 4℃ for later use.

[0069] (3) Mix the gold-labeled conjugate 1 and gold-labeled conjugate 2 obtained in step (2) at a volume ratio of 1:1, then add diluent to dilute 20 times to obtain diluent. Then immerse the blank pad in it, take it out and dry it at 37°C to obtain the gold-labeled pad.

[0070] The specific preparation method of the diluent is as follows: Weigh 5.75g of disodium hydrogen phosphate, 1.14g of sodium dihydrogen phosphate, 9g of sodium chloride, and 10g of NP-40 into a beaker, add 1L of purified water, stir and mix well, and store at room temperature for later use.

[0071] (4) Immerse the blank pad in the sample pad treatment solution, take it out and dry it at 37°C to obtain the sample pad; the specific preparation method of the sample pad treatment solution is as follows: weigh 6g Tris, 5g Casein, 5g Tween 20, 5g S9 and 0.1g sodium lignosulfonate into a beaker, add 1L of purified water, stir and mix well, adjust the pH to 8.0 with HCl solution, and store at 4°C for later use.

[0072] (5) The sample pad, gold label pad, nitrocellulose membrane and absorbent pad are overlapped and pasted onto the PVC base plate in the direction of chromatography, and then cut into test strips with a width of 3mm.

[0073] A schematic diagram of the test strip structure is shown below. Figure 1 As shown; the detection and result interpretation are as follows. Figure 2 As shown.

[0074] Example 2

[0075] A test strip for detecting influenza A / B virus antigens, the preparation method of which specifically includes the following steps:

[0076] (1) Influenza A monoclonal antibody 2 and influenza B monoclonal antibody 2 were diluted to 0.8 mg / mL with coating solution and coated onto the detection line of nitrocellulose membrane using a membrane scrubbing instrument with a parameter of 1 μL / cm. Goat anti-mouse IgG was diluted to 1 mg / mL with coating solution and coated onto the control line of nitrocellulose membrane using a membrane scrubbing instrument with a parameter of 1 μL / cm. The coated membrane was then placed in a 45℃ oven and dried for 2 h.

[0077] The specific preparation method of the coating solution is as follows: Weigh 2.4g Tris and 20g trehalose into a beaker, add 1L of purified water, stir and mix well, adjust the pH to 8.0 with HCl solution, and store at 4℃ for later use.

[0078] (2) Add 100 μL of 1M Tris buffer to 10 mL of colloidal gold solution and mix well. Then add 150 μg of influenza A monoclonal antibody 1 and 150 μg of BSA and stir at 25 °C for 10 min. Then add 0.15 mL of 50 g / L dithiol polyethylene glycol (MW 20000) solution and stir at 25 °C for 10 min. Then centrifuge at 4 °C and 10000 rpm for 10 min and discard the supernatant. Add 1 mL of preservation solution to the precipitate for reconstitution to obtain gold-labeled conjugate 1.

[0079] 100 μL of 1M Tris buffer was added to 10 mL of colloidal gold solution and mixed well. Then, 150 μg of influenza B monoclonal antibody 1 and 150 μg of BSA-Ab were added, and the mixture was stirred at 25 °C for 10 min. Next, 0.15 mL of 50 g / L dithiol polyethylene glycol (MW 20000) solution was added, and the mixture was stirred at 25 °C for 10 min. Then, the mixture was centrifuged at 4 °C and 10000 rpm for 10 min, the supernatant was discarded, and the precipitate was reconstituted in 1 mL of preservation solution to obtain gold-labeled conjugate 2.

[0080] The specific preparation method of the 1M Tris buffer is as follows: Weigh 12g Tris into a beaker, add 100mL of purified water, stir well, and store at 4℃ for later use.

[0081] The specific preparation method of the preservation solution is as follows: Weigh 2.4g Tris, 5g Casein, and 100g sucrose into a beaker, add 1L of purified water, stir and mix well, adjust the pH to 8.0 with HCl solution, and store at 4℃ for later use.

[0082] (3) Mix the gold-labeled conjugate 1 and gold-labeled conjugate 2 obtained in step (2) at a volume ratio of 1:2, then add diluent to dilute 20 times to obtain diluent. Then immerse the blank pad in it, take it out and dry it at 37°C to obtain the gold-labeled pad.

[0083] The specific preparation method of the diluent is as follows: Weigh 5.75g of disodium hydrogen phosphate, 1.14g of sodium dihydrogen phosphate, 9g of sodium chloride, and 10g of NP-40 into a beaker, add 1L of purified water, stir and mix well, and store at room temperature for later use.

[0084] (4) Immerse the blank pad in the sample pad treatment solution, take it out and dry it at 37°C to obtain the sample pad; the specific preparation method of the sample pad treatment solution is as follows: weigh 12g Tris, 5g Casein, 5g Tween 20, 5g S9 and 0.1g sodium lignosulfonate into a beaker, add 1L of purified water, stir and mix well, adjust the pH to 8.0 with HCl solution, and store at 4°C for later use.

[0085] (5) The sample pad, gold label pad, nitrocellulose membrane and absorbent pad are overlapped and pasted onto the PVC base plate in the direction of chromatography, and then cut into test strips with a width of 3mm.

[0086] Example 3

[0087] A test strip for detecting influenza A / B virus antigens, the preparation method of which specifically includes the following steps:

[0088] (1) Influenza A monoclonal antibody 2 and influenza B monoclonal antibody 2 were diluted to 0.8 mg / mL with coating solution and coated onto the detection line of nitrocellulose membrane using a membrane scrubbing instrument with a parameter of 1 μL / cm. Goat anti-mouse IgG was diluted to 0.8 mg / mL with coating solution and coated onto the control line of nitrocellulose membrane using a membrane scrubbing instrument with a parameter of 1 μL / cm. The coated membrane was then placed in a 45℃ oven and dried for 2 h.

[0089] The specific preparation method of the coating solution is as follows: Weigh 1.2g Tris and 30g trehalose into a beaker, add 1L of purified water, stir and mix well, adjust the pH to 8.0 with HCl solution, and store at 4℃ for later use.

[0090] (2) Add 50 μL of 1M Tris buffer to 10 mL of colloidal gold solution and mix well. Then add 100 μg of influenza A monoclonal antibody 1 and 120 μg of BSA and stir at 25 °C for 15 min. Then add 0.12 mL of 50 g / L dithiol polyethylene glycol (MW 20000) solution and stir at 25 °C for 10 min. Then centrifuge at 4 °C and 8500 rpm for 15 min and discard the supernatant. Add 1 mL of preservation solution to the precipitate for reconstitution to obtain gold-labeled conjugate 1.

[0091] Add 50 μL of 1M Tris buffer to 10 mL of colloidal gold solution and mix well. Then add 120 μg of influenza B monoclonal antibody 1 and 100 μg of BSA-Ab, and stir at 25 °C for 10 min. Then add 0.12 mL of 50 g / L dithiol polyethylene glycol (MW 20000) solution and stir at 25 °C for 15 min. Then centrifuge at 4 °C and 8500 rpm for 15 min, discard the supernatant, and reconstitute the precipitate with 1 mL of preservation solution to obtain gold-labeled conjugate 2.

[0092] The specific preparation method of the 1M Tris buffer is as follows: Weigh 12g Tris into a beaker, add 100mL of purified water, stir well, and store at 4℃ for later use.

[0093] The specific preparation method of the preservation solution is as follows: Weigh 2.4g Tris, 5g Casein, and 100g sucrose into a beaker, add 1L of purified water, stir and mix well, adjust the pH to 8.0 with HCl solution, and store at 4℃ for later use.

[0094] (3) Mix the gold-labeled conjugate 1 and gold-labeled conjugate 2 obtained in step (2) at a volume ratio of 1:2, then add diluent to dilute 20 times to obtain diluent. Then immerse the blank pad in it, take it out and dry it at 37°C to obtain the gold-labeled pad.

[0095] The specific preparation method of the diluent is as follows: Weigh 5.75g of disodium hydrogen phosphate, 1.14g of sodium dihydrogen phosphate, 9g of sodium chloride, and 10g of NP-40 into a beaker, add 1L of purified water, stir and mix well, and store at room temperature for later use.

[0096] (4) Immerse the blank pad in the sample pad treatment solution, take it out and dry it at 37°C to obtain the sample pad; the specific preparation method of the sample pad treatment solution is as follows: weigh 6g Tris, 5g Casein, 5g Tween 20, 5g S9 and 0.2g sodium lignosulfonate into a beaker, add 1L of purified water, stir and mix well, adjust the pH to 8.0 with HCl solution, and store at 4°C for later use.

[0097] (5) The sample pad, gold label pad, nitrocellulose membrane and absorbent pad are overlapped and pasted onto the PVC base plate in the direction of chromatography, and then cut into test strips with a width of 3mm.

[0098] Comparative Example 1

[0099] The difference between this comparative example and Example 1 is that potassium carbonate was used to adjust the pH value. Since the optimal pH values ​​of influenza B monoclonal antibody 1 and BSA-Ab are different, and the addition of potassium carbonate caused the gold to die, the gold could not be used in the gold labeling system. Therefore, this method was not used in subsequent tests.

[0100] Comparative Example 2

[0101] A test strip for detecting influenza A / B virus antigens, the preparation method of which specifically includes the following steps:

[0102] (1) Influenza A monoclonal antibody 2 and influenza B monoclonal antibody 2 were diluted to 1 mg / mL with coating solution and coated onto the detection line of nitrocellulose membrane using a membrane scrubbing instrument with a parameter of 1 μL / cm. Goat anti-mouse IgG was diluted to 1 mg / mL with coating solution and coated onto the control line of nitrocellulose membrane using a membrane scrubbing instrument with a parameter of 1 μL / cm. The coated membrane was then placed in a 45℃ oven and dried for 2 h.

[0103] The specific preparation method of the coating solution is as follows: Weigh 2.4g Tris and 20g trehalose into a beaker, add 1L of purified water, stir and mix well, adjust the pH to 8.0 with HCl solution, and store at 4℃ for later use.

[0104] (2) Add 50 μL of 1M Tris buffer to 10 mL of colloidal gold solution and mix well. Then add 100 μg of influenza A monoclonal antibody 1 and 100 μg of BSA and stir at 25 °C for 15 min. Then add 1 mL of 5% Casein and stir at 25 °C for 15 min. Then centrifuge at 4 °C and 8000 rpm for 15 min and discard the supernatant. Add 1 mL of preservation solution to the precipitate for reconstitution to obtain gold-labeled conjugate 1.

[0105] Add 50 μL of 1M Tris buffer to 10 mL of colloidal gold solution and mix well. Then add 100 μg of influenza B monoclonal antibody 1 and 100 μg of BSA-Ab, and stir at 25 °C for 15 min. Add 1 mL of 5% Casein and stir at 25 °C for 15 min. Then centrifuge at 4 °C and 8000 rpm for 15 min, discard the supernatant, and reconstitute the precipitate with 1 mL of preservation solution to obtain gold-labeled conjugate 2.

[0106] The specific preparation method of the 1M Tris buffer is as follows: Weigh 12g Tris into a beaker, add 100mL of purified water, stir well, and store at 4℃ for later use.

[0107] The specific preparation method of the preservation solution is as follows: Weigh 2.4g Tris, 5g Casein, and 100g sucrose into a beaker, add 1L of purified water, stir and mix well, adjust the pH to 8.0 with HCl solution, and store at 4℃ for later use.

[0108] (3) Mix the gold-labeled conjugate 1 and gold-labeled conjugate 2 obtained in step (2) at a volume ratio of 1:1, then add diluent to dilute 20 times to obtain diluent. Then immerse the blank pad in it, take it out and dry it at 37°C to obtain the gold-labeled pad.

[0109] The specific preparation method of the diluent is as follows: Weigh 5.75g of disodium hydrogen phosphate, 1.14g of sodium dihydrogen phosphate, 9g of sodium chloride, and 10g of NP-40 into a beaker, add 1L of purified water, stir and mix well, and store at room temperature for later use.

[0110] (4) Immerse the blank pad in the sample pad treatment solution, take it out and dry it at 37°C to obtain the sample pad; the specific preparation method of the sample pad treatment solution is as follows: weigh 6g Tris, 5g Casein, 5g Tween 20, 5g S9 and 0.1g sodium lignosulfonate into a beaker, add 1L of purified water, stir and mix well, adjust the pH to 8.0 with HCl solution, and store at 4°C for later use.

[0111] (5) The sample pad, gold label pad, nitrocellulose membrane and absorbent pad are overlapped and pasted onto the PVC base plate in the direction of chromatography, and then cut into test strips with a width of 3mm.

[0112] Comparative Example 3

[0113] A test strip for detecting influenza A / B virus antigens, the preparation method of which specifically includes the following steps:

[0114] (1) Influenza A monoclonal antibody 2 and influenza B monoclonal antibody 2 were diluted to 1 mg / mL with coating solution and coated onto the detection line of nitrocellulose membrane using a membrane scrubbing instrument with a parameter of 1 μL / cm. Goat anti-mouse IgG was diluted to 1 mg / mL with coating solution and coated onto the control line of nitrocellulose membrane using a membrane scrubbing instrument with a parameter of 1 μL / cm. The coated membrane was then placed in a 45℃ oven and dried for 2 h.

[0115] The specific preparation method of the coating solution is as follows: Weigh 2.4g Tris and 20g trehalose into a beaker, add 1L of purified water, stir and mix well, adjust the pH to 8.0 with HCl solution, and store at 4℃ for later use.

[0116] (2) Add 50 μL of 1M Tris buffer to 10 mL of colloidal gold solution and mix well. Then add 100 μg of influenza A monoclonal antibody 1 and stir at 25 °C for 15 min. Then add 0.1 mL of 50 g / L dithiol polyethylene glycol (MW 20000) solution and stir at 25 °C for 15 min. Then centrifuge at 4 °C and 8000 rpm for 15 min and discard the supernatant. Add 1 mL of preservation solution to the precipitate for reconstitution to obtain gold-labeled conjugate 1.

[0117] Add 50 μL of 1M Tris buffer to 10 mL of colloidal gold solution and mix well. Then add 100 μg of influenza B monoclonal antibody 1 and stir at 25 °C for 15 min. Add 0.1 mL of 50 g / L dithiol polyethylene glycol (MW 20000) solution and stir at 25 °C for 15 min. Then centrifuge at 4 °C and 8000 rpm for 15 min, discard the supernatant, and reconstitute the precipitate with 1 mL of preservation solution to obtain gold-labeled conjugate 2.

[0118] The specific preparation method of the 1M Tris buffer is as follows: Weigh 12g Tris into a beaker, add 100mL of purified water, stir well, and store at 4℃ for later use.

[0119] The specific preparation method of the preservation solution is as follows: Weigh 2.4g Tris, 5g Casein, and 100g sucrose into a beaker, add 1L of purified water, stir and mix well, adjust the pH to 8.0 with HCl solution, and store at 4℃ for later use.

[0120] (3) Mix the gold-labeled conjugate 1 and gold-labeled conjugate 2 obtained in step (2) at a volume ratio of 1:1, then add diluent to dilute 20 times to obtain diluent. Then immerse the blank pad in it, take it out and dry it at 37°C to obtain the gold-labeled pad.

[0121] The specific preparation method of the diluent is as follows: Weigh 5.75g of disodium hydrogen phosphate, 1.14g of sodium dihydrogen phosphate, 9g of sodium chloride, and 10g of NP-40 into a beaker, add 1L of purified water, stir and mix well, and store at room temperature for later use.

[0122] (4) Immerse the blank pad in the sample pad treatment solution, take it out and dry it at 37°C to obtain the sample pad; the specific preparation method of the sample pad treatment solution is as follows: weigh 6g Tris, 5g Casein, 5g Tween 20, 5g S9 and 0.1g sodium lignosulfonate into a beaker, add 1L of purified water, stir and mix well, adjust the pH to 8.0 with HCl solution, and store at 4°C for later use.

[0123] (5) The sample pad, gold label pad, nitrocellulose membrane and absorbent pad are overlapped and pasted onto the PVC base plate in the direction of chromatography, and then cut into test strips with a width of 3mm.

[0124] Comparative Example 4

[0125] A test strip for detecting influenza A / B virus antigens, the preparation method of which specifically includes the following steps:

[0126] (1) Influenza A monoclonal antibody 2 and influenza B monoclonal antibody 2 were diluted to 1 mg / mL with coating solution and coated onto the detection line of nitrocellulose membrane using a membrane scrubbing instrument with a parameter of 1 μL / cm. Goat anti-mouse IgG was diluted to 1 mg / mL with coating solution and coated onto the control line of nitrocellulose membrane using a membrane scrubbing instrument with a parameter of 1 μL / cm. The coated membrane was then placed in a 45℃ oven and dried for 2 h.

[0127] The specific preparation method of the coating solution is as follows: Weigh 2.4g Tris and 20g trehalose into a beaker, add 1L of purified water, stir and mix well, adjust the pH to 8.0 with HCl solution, and store at 4℃ for later use.

[0128] (2) Add 50 μL of 1M Tris buffer to 10 mL of colloidal gold solution and mix well. Then add 100 μg of influenza A monoclonal antibody 1 and 100 μg of BSA and stir at 25 °C for 15 min. Then add 0.1 mL of 50 g / L dithiol polyethylene glycol (MW 20000) solution and stir at 25 °C for 15 min. Then centrifuge at 4 °C and 8000 rpm for 15 min and discard the supernatant. Add 1 mL of preservation solution to the precipitate for reconstitution to obtain gold-labeled conjugate 1.

[0129] Add 50 μL of 1M Tris buffer to 10 mL of colloidal gold solution and mix well. Then add 100 μg of influenza B monoclonal antibody 1 and 100 μg of BSA-Ab, and stir at 25 °C for 15 min. Then add 0.1 mL of 50 g / L dithiol polyethylene glycol (MW 20000) solution and stir at 25 °C for 15 min. Then centrifuge at 4 °C and 8000 rpm for 15 min, discard the supernatant, and reconstitute the precipitate with 1 mL of preservation solution to obtain gold-labeled conjugate 2.

[0130] The specific preparation method of the 1M Tris buffer is as follows: Weigh 12g Tris into a beaker, add 100mL of purified water, stir well, and store at 4℃ for later use.

[0131] The specific preparation method of the preservation solution is as follows: Weigh 2.4g Tris, 5g Casein, and 100g sucrose into a beaker, add 1L of purified water, stir and mix well, adjust the pH to 8.0 with HCl solution, and store at 4℃ for later use.

[0132] (3) Mix the gold-labeled conjugate 1 and gold-labeled conjugate 2 obtained in step (2) at a volume ratio of 1:1, then add diluent to dilute 20 times to obtain diluent. Then immerse the blank pad in it, take it out and dry it at 37°C to obtain the gold-labeled pad.

[0133] The specific preparation method of the diluent is as follows: Weigh 5.75g of disodium hydrogen phosphate, 1.14g of sodium dihydrogen phosphate, 9g of sodium chloride, and 10g of NP-40 into a beaker, add 1L of purified water, stir and mix well, and store at room temperature for later use.

[0134] (4) Immerse the blank pad in the sample pad treatment solution, take it out and dry it at 37°C to obtain the sample pad; the specific preparation method of the sample pad treatment solution is as follows: weigh 6g Tris, 5g Casein, 5g Tween 20 and 5g S9 into a beaker, add 1L of purified water, stir and mix well, adjust the pH to 8.0 with HCl solution, and store at 4°C for later use.

[0135] (5) The sample pad, gold label pad, nitrocellulose membrane and absorbent pad are overlapped and pasted onto the PVC base plate in the direction of chromatography, and then cut into test strips with a width of 3mm.

[0136] Verification Example 1: Limit of Detection Test

[0137] The lowest detection limit reference standard of the second-generation influenza A / B virus antigen detection reagent (370117-202301) was used to detect Examples 1-3 and Comparative Examples 2-4. The results are shown in Tables 1-6 below.

[0138] Table 1. Results of detection at the lowest detection limit (L1)

[0139] Table 2 Results of L2 detection (lowest detection limit)

[0140] Table 3. Detection results of the lowest detection limit (L3)

[0141] Table 4. Detection results of the lowest detection limit (L4)

[0142] Table 5. Detection results of the lowest detection limit (L5)

[0143] Table 6. Detection results of L6 (Limit of Detection)

[0144] Note: "+" indicates a positive result, and "-" indicates a negative result.

[0145] As shown in Tables 1-6, the sensitivity of Examples 1-3 is higher than that of Comparative Examples 2 and 3, indicating that the use of bis-thiol polyethylene glycol for blocking and the preparation of large-particle conjugates by the specific adsorption of BSA and BSA-Ab can significantly improve the product sensitivity.

[0146] Validation Example 2: Specificity Detection

[0147] The negative reference material of the second-generation influenza A / B virus antigen detection reagent (370117-202301) was used to test Examples 1-3 and Comparative Examples 2-4. The test results are shown in Table 7 below.

[0148] Table 7 Negative Reference Sample Test Results

[0149] Subsequently, 200 patient samples that were confirmed negative by fluorescence PCR were collected, including 120 nasal swab samples and 80 oropharyngeal swab samples. These samples were then processed in diluent and tested. The test results are shown in Tables 8-9 below.

[0150] Table 8 Specificity test results of reagents prepared in Examples 1-3

[0151] Table 9. Specificity test results of reagents used in comparative examples 2-4

[0152] As can be seen from the comparison in Table 7-9, the specificity of Comparative Example 4 is significantly reduced, indicating that sodium lignosulfonate can reduce non-specific adsorption and improve the specificity of strong products.

[0153] Verification Example 3: Detection of Positive Samples

[0154] One hundred oropharyngeal and nasal swab samples from patients confirmed positive for influenza A by fluorescent PCR and 120 oropharyngeal and nasal swab samples from patients confirmed positive for influenza B by fluorescent PCR were collected. After being processed in diluent, they were tested using reagents prepared in Examples 1-3 and Comparative Examples 2-4. The test results are shown in Tables 10-13 below.

[0155] Table 10 Detection results of positive samples prepared by reagents in Examples 1-3

[0156] Table 11 Detection results of positive samples prepared by reagents in Examples 1-3

[0157] Table 12 Detection results of positive samples prepared by reagents in Comparative Examples 2-4

[0158] Table 13 Detection results of positive samples prepared by reagents in Comparative Examples 2-4

[0159] As can be seen from the comparison in Tables 10-13, the positive detection rates of Examples 1-3 are significantly higher than those of Comparative Examples 2 and 3, indicating that the use of bis-thiol polyethylene glycol blocking and the preparation of large-particle conjugates by the specific adsorption of BSA and BSA-Ab in this invention can significantly improve the positive detection rate of the product.

[0160] As shown in Tables 8, 10, and 11, 200 negative samples and 220 positive samples were included in the study. Statistical analysis of the detection results of this embodiment of the invention was performed, and the consistency analysis between the detection results of the embodiment and the clinical diagnostic results is as follows:

[0161] Table 14

[0162] Table 15

[0163] The statistical results above show that the positive concordance rate between the test results of the embodiments of the present invention and the clinical diagnostic results is 90.91% (95% CI: 86.38%-94.04%), the negative concordance rate is 100% (95% CI: 98.12%-100.00%), and the overall concordance rate is 95.24% (95% CI: 92.76%-96.90%). A kappa test was performed on the test results, and the kappa value was 0.872, indicating a high degree of consistency between the two test results.

[0164] The above detailed embodiments provide a specific description of the analytical methods involved in this invention. It should be noted that the above description is only intended to help those skilled in the art better understand the methods and ideas of this invention, and is not intended to limit the scope of the invention. Without departing from the principles of this invention, those skilled in the art can make appropriate adjustments or modifications to this invention, and such adjustments and modifications should also fall within the protection scope of this invention.

Claims

1. A test strip for detecting influenza A / B virus antigens, characterized in that, It includes a base plate, a sample pad, a gold label pad, a nitrocellulose membrane, and an absorbent pad; the sample pad, the gold label pad, the nitrocellulose membrane, and the absorbent pad are sequentially overlapped and adhered to the base plate in the direction of chromatography; The sample pad is treated by immersion in a sample pad treatment solution; the sample pad treatment solution includes Tris, Casein, Tween 20, S9, and sodium lignosulfonate. The gold-labeled pad carries colloidal gold-labeled conjugate 1 and gold-labeled conjugate 2. Gold-labeled conjugate 1 includes influenza A monoclonal antibody 1 and BSA, which is blocked with dithiol polyethylene glycol. Gold-labeled conjugate 2 includes influenza B monoclonal antibody 1 and BSA-Ab, which is blocked with dithiol polyethylene glycol. The nitrocellulose membrane is coated with a control line (C line) and a detection line (T line). The control line is coated with anti-IgG antibodies, and the detection lines include influenza A virus detection lines and influenza B virus detection lines, which are coated with influenza A monoclonal antibody 2 and influenza B monoclonal antibody 2, respectively.

2. The test strip according to claim 1, characterized in that, The base plate is selected from PVC base plates.

3. The test strip according to claim 1, characterized in that, The anti-IgG antibody is a goat anti-mouse IgG antibody.

4. The method for preparing the influenza A / B virus antigen test strip according to any one of claims 1-3, characterized in that, Includes the following steps: (1) Dilute the influenza A monoclonal antibody 2 and influenza B monoclonal antibody 2 with coating solution and coat them onto the detection line of the nitrocellulose membrane. Dilute the anti-IgG antibody with coating solution and coat it onto the control line of the nitrocellulose membrane. Dry the membrane. (2) Add Tris buffer to colloidal gold solution and mix well. Then add influenza A monoclonal antibody 1 and BSA and stir to react. Then add dithiol polyethylene glycol solution and stir to react. Then centrifuge and discard the supernatant. The precipitate is reconstituted with preservation solution to obtain gold-labeled conjugate 1. Tris buffer was added to colloidal gold solution and mixed well. Then, influenza B monoclonal antibody 1 and BSA-Ab were added and stirred to react. Then, dithiol polyethylene glycol solution was added and stirred to react. After centrifugation, the supernatant was discarded, and the precipitate was reconstituted with preservation solution to obtain gold-labeled conjugate 2. The preservation solution includes Tris, Casein, and sucrose; (3) After mixing the gold-labeled coupling compound 1 and gold-labeled coupling compound 2 obtained in step (2), add diluent to dilute, then soak the blank pad in it, take it out and dry it to obtain the gold-labeled pad. The diluent includes disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, and NP-40; (4) Tris, Casein, Tween 20, S9 and sodium lignosulfonate were placed in a solvent and mixed to prepare a sample pad treatment solution. Then, the blank pad was immersed in the sample pad treatment solution, taken out and dried to obtain the sample pad. (5) The sample pad, gold label pad, nitrocellulose membrane and absorbent pad are overlapped and pasted onto the base plate in the direction of chromatography, and then cut to obtain the final product.

5. The preparation method according to claim 4, characterized in that, The coating solution in step (1) includes Tris and trehalose.

6. The preparation method according to claim 4, characterized in that, In step (2), when preparing gold-labeled conjugate 1, influenza A monoclonal antibody 1 and BSA were added and the reaction was stirred at a temperature of 20-25℃ for 10-15 min. When adding dithioglycol solution, the reaction was stirred at a temperature of 20-25℃ for 10-15 min. The volume ratio of dithioglycol solution to colloidal gold solution was 0.1-0.15:

10. When preparing gold-labeled conjugate 2, influenza B monoclonal antibody 1 and BSA-Ab were added and the reaction was stirred at a temperature of 20-25℃ for 10-15 min. When adding dithioglycol solution, the reaction was stirred at a temperature of 20-25℃ for 10-15 min. The volume ratio of dithioglycol solution to colloidal gold solution was 0.1-0.15:

10.

7. The preparation method according to claim 4, characterized in that, In step (2), the centrifugation temperature for preparing gold-labeled conjugate 1 is 4℃, the centrifugation speed is 8000-10000rpm, and the centrifugation time is 10-15min; the centrifugation temperature for preparing gold-labeled conjugate 2 is 4℃, the centrifugation speed is 8000-10000rpm, and the centrifugation time is 10-15min.

8. The preparation method according to claim 4, characterized in that, The concentrations of Tris, Casein, Tween 20, S9 and sodium lignosulfonate in the sample pad treatment solution in step (4) are 6-12 g / L, 3-7 g / L, 3-7 g / L, 3-7 g / L and 0.1-0.2 g / L, respectively.

9. A type A / type B influenza virus antigen detection kit, characterized in that, Includes the influenza A / B virus antigen test strip according to any one of claims 1-3 and / or the influenza A / B virus antigen test strip prepared by the preparation method according to any one of claims 4-8, as well as one or more of sample extraction solution, pharyngeal swab, sample extraction tube, and pipette.

10. A method for detecting influenza A / B virus antigens, characterized in that, The detection using the influenza A / B virus antigen detection kit according to claim 9 includes the following steps: Add the sample to be tested to the sample extraction solution for treatment. Add the treated sample to the sample pad of the influenza A / B virus antigen test strip. Observe the results after chromatography. If both the test line (T line) and the control line (C line) are red, the result is considered positive. If only the control line (C line) is red and the test line (T line) is not colored, the result is considered negative. If the control line (C line) is not colored, the result is considered invalid.

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