Influenza virus detection test strip based on cellulose nanospheres and preparation method thereof

Abstract

The invention relates to an influenza virus detection test strip based on cellulose nanospheres and a preparation method thereof, and belongs to the technical field of immunodetection. Cellulose nanospheres are used for replacing a traditional colloidal gold material and used for preparing the immunochromatography test strip, a sample pad, a combination pad, a coating film and a water absorption pad are sequentially formed on a bottom plate from a sample adding end, an influenza A virus detection line and an influenza B virus detection line are arranged on the coating film, and the water absorption pad is arranged on the bottom plate. The test strip provided by the invention is applied to detection of influenza A and B virus antigens, the detection limit is as low as 0.05 ng / mL and 0.25 ng / mL, the detection test strip has lower detection limit and better specificity due to the use of the cellulose nanospheres, the preparation process is simplified, the stability of the test strip is also improved, and the test strip has wide application prospects.

Description

Technical Field

[0001] The invention relates to the technical field of immunoassay detection, in particular to an influenza virus detection test strip based on cellulose nanospheres and a preparation method thereof. Background Art

[0002] Influenza A virus, with its high degree of infectivity and rapid mutation, can easily cause a flu pandemic, infecting a large number of people, causing symptoms such as high fever and cough, and even endangering life in severe cases. In addition, influenza A may also lead to school closures and reduced business activities. Although influenza B virus has a relatively small degree of mutation, it is also contagious and can cause serious systemic symptoms, posing a greater threat to specific groups such as children and the elderly. Therefore, it is crucial to strengthen the prevention and control of influenza viruses to reduce their harm to human society.

[0003] How to quickly and accurately identify and differentiate influenza A virus from influenza B virus is of immeasurable value for formulating effective prevention and control strategies, optimizing clinical diagnosis and treatment plans, and reducing the risk of virus transmission. Traditional detection technologies, such as polymerase chain reaction (PCR), have occupied a place in the field of influenza virus detection with their high accuracy and reliability. PCR technology can effectively identify trace viral particles by amplifying specific fragments in the viral genetic material, thereby ensuring the accuracy of the test results. However, although PCR technology is accurate and error-free, its operation process is relatively complicated and requires a professional laboratory environment and technicians to operate. At the same time, the detection cycle is long. It often takes several hours or even longer from sample collection to the issuance of the final results. When faced with large-scale screening needs, it has undoubtedly become a considerable challenge.

[0004] Given the limitations of traditional detection technologies, researchers and medical workers are constantly exploring more efficient and convenient methods for influenza detection. Among them, lateral flow test strips, as a rapid detection technology based on the principle of antigen-antibody immune reaction, have shown great potential in the initial screening and on-site diagnosis of influenza viruses due to their simple operation, low cost and ability to detect immediately. The design principle of lateral flow test strips is to use the binding reaction of specific antibodies with target virus antigens to quickly indicate the test results through intuitive methods such as color changes, which greatly shortens the detection time and facilitates rapid deployment in resource-limited areas or emergency situations.

[0005] However, despite the many advantages of lateral flow test strips, most of the mainstream test strip products on the market currently use colloidal gold as a detection marker. Although colloidal gold can meet the needs of rapid detection to a certain extent, its detection performance still has some inherent limitations. Especially in terms of sensitivity and specificity, colloidal gold-labeled test strips may have difficulty capturing extremely low concentrations of virus particles, resulting in an increase in the missed detection rate; at the same time, when faced with virus mutations or cross-reactions with other pathogens, its specificity may also be affected, thereby increasing the risk of misdiagnosis. Therefore, how to further improve the detection sensitivity and specificity of lateral flow test strips while maintaining their convenience is a key issue that needs to be urgently addressed in the current development of influenza detection technology. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a test strip for influenza A and B virus detection based on cellulose nanospheres and a preparation method thereof, wherein cellulose nanospheres are used instead of traditional colloidal gold materials, and the capture antibodies of influenza A and B viruses are labeled with cellulose nanospheres for preparing immunochromatographic test strips, and a sample pad, a binding pad, a coating film and a water-absorbing pad are sequentially formed on the bottom plate from the sample application end, and an influenza A virus detection line and an influenza B virus detection line are arranged on the coating film. The influenza A and B virus antigens bind to the capture antibodies labeled with the cellulose nanospheres on the binding pad, move along the test strip under the action of capillaries, bind to the detection antibodies on the coating film, and the cellulose nanospheres aggregate to show obvious colors.

[0007] The first object of the present invention is to provide a method for preparing a test strip for influenza A and B virus detection based on cellulose nanospheres, comprising the following steps:

[0008] Step S1, using cellulose nanospheres to label influenza A virus capture antibodies and influenza B virus capture antibodies to obtain influenza A virus capture antibody cellulose nanosphere labeling solution and influenza B virus capture antibody cellulose nanosphere labeling solution;

[0009] The influenza A virus capture antibody cellulose nanosphere labeling solution and the influenza B virus capture antibody cellulose nanosphere labeling solution are mixed evenly, and sprayed on the conjugate pad;

[0010] Step S2, setting an influenza B virus detection line and an influenza A virus detection line on the coating membrane, spraying solutions containing influenza B virus detection antibodies and influenza A virus detection antibodies respectively, wherein the influenza B virus detection line and the influenza A virus detection line are arranged perpendicular to the direction of the test strip;

[0011] The final concentration of the influenza B virus detection antibody is 0.7-1.2 mg / mL, and the final concentration of the influenza A virus detection antibody is 0.5-0.8 mg / mL;

[0012] Step S3, assembling the sample pad, the binding pad, the coating membrane and the water absorption pad to obtain the influenza A and B virus detection test strip.

[0013] As an emerging nanomaterial, cellulose nanospheres can provide a more efficient tracer for disease detection due to their high specific surface area, good biocompatibility and easy functionalization. Compared with colloidal gold, cellulose nanospheres can significantly enhance the binding efficiency of antigens and antibodies when used as antibody markers, and at the same time, rely on higher material gloss to effectively improve the sensitivity and specificity of disease detection.

[0014] Furthermore, the method further includes pre-treating the sample pad and the conjugate pad, wherein the pre-treating comprises soaking the sample pad and the conjugate pad in a sample pad pre-treating solution and a conjugate pad pre-treating solution, respectively;

[0015] The components of the sample pad pretreatment solution include a surfactant, a preservative and a blocking agent;

[0016] The components of the bonding pad pretreatment solution include a surfactant.

[0017] Furthermore, the amount of the sample pad pretreatment solution added is 0.04-0.08 mL / cm 2 The amount of the conjugate pad pretreatment solution added is 0.04-0.08 mL / cm 2 .

[0018] Furthermore, in step S1, labeling of influenza virus capture antibodies comprises the following steps:

[0019] Step S11, preparing conjugation buffer, blocking solution, washing buffer and storage buffer;

[0020] Step S12, mixing the cellulose nanospheres and the influenza virus capture antibody in a conjugation buffer;

[0021] Step S13, adding blocking solution, centrifuging and removing supernatant;

[0022] Step S14, adding washing buffer to break up the precipitate and reselect it, and centrifuging to remove the supernatant;

[0023] Step S15, adding storage buffer to obtain influenza virus capture antibody cellulose nanosphere labeling solution.

[0024] Furthermore, in step S11, the conjugation buffer is divided into a Tris conjugation buffer and a boric acid conjugation buffer, the Tris conjugation buffer is used for labeling influenza A virus capture antibodies, and the boric acid conjugation buffer is used for labeling influenza B virus capture antibodies.

[0025] Furthermore, in step S12, the amount of influenza virus capture antibody added is 10-15 μg.

[0026] Furthermore, in step S1, the conjugate pad is sprayed with a quality control capture antibody labeled with colloidal gold.

[0027] Furthermore, the quality control capture antibody is a chicken IgY antibody.

[0028] Furthermore, the amount of the quality control capture antibody added to the colloidal gold solution is 8-12 μg.

[0029] Furthermore, in step S2, a quality control line is provided on the coating membrane, and a solution containing a quality control detection antibody is sprayed on the quality control line.

[0030] Furthermore, the quality control detection antibody is a sheep anti-chicken IgY antibody.

[0031] Furthermore, the final concentration of the quality control detection antibody is 0.8-1.2 mg / mL.

[0032] The second object of the present invention is to provide a test strip for detecting influenza A and B viruses prepared by the above preparation method.

[0033] Furthermore, the influenza A and B virus detection test strip also includes a sample diluent.

[0034] Furthermore, the sample diluent includes sodium chloride, disodium hydrogen phosphate, dipotassium hydrogen phosphate, sodium stearate, and a preservative.

[0035] Beneficial effects of the present invention:

[0036] The invention provides an influenza A and B virus detection test strip based on cellulose nanospheres. The cellulose nanospheres are selected to replace traditional colloidal gold materials. The provided test strip is used for detecting influenza A and B virus antigens. The detection limits are as low as 0.05 ng / mL and 0.25 ng / mL. The use of the cellulose nanospheres enables the detection test strip to have a lower detection limit and better specificity. When influenza A virus antigens are added, the test strip only develops color on the influenza A virus detection line, and the influenza B virus detection line does not develop color; when influenza B virus antigens are added, the test strip only develops color on the influenza B virus detection line, and the influenza A virus detection line does not develop color. In addition, the preparation process is simplified, and the invention has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0038] Figure 1The present invention provides a schematic diagram of an influenza A and B virus antigen detection test strip, wherein 1 is a sample pad, 2 is a binding pad, 3 is a nitrocellulose membrane, 31 is an influenza B virus detection line, 32 is an influenza A virus detection line, 33 is a quality control line, 4 is an absorbent pad, and 5 is a bottom plate. DETAILED DESCRIPTION

[0039] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0040] The cellulose nanospheres used in the following examples are from NanoAct, red in color, with a concentration of 1 wt %, a particle size of 341 nm, and an OD value of 231.

[0041] Example 1: Preparation of test strips

[0042] 1. Pre-treat the sample pad

[0043] (1) The sample pad treatment solution consists of 40-55 mM Tris-HCl, 0.4-0.6 wt% BSA or casein, 0.8-1.2 wt% surfactant S9, 0.04-0.8 wt% Tween-20, 0.02-0.2 wt% ProClin300 or sodium azide, and 0.002-0.006 wt% blocking agent, and the pH value of the treatment solution is 7.5-9.0;

[0044] (2) Soak the sample pad in the sample pad treatment solution. The amount of treatment solution added is 0.04-0.08mL / cm 2 , soaking time is more than 7 minutes;

[0045] (3) The soaked sample pad is dried and dehydrated, then placed in an oven at 33-50°C until completely dry, and sealed and stored in an environment with a humidity of ≤30%.

[0046] 2. Pre-treat the conjugate pad

[0047] (1) Soak the conjugate pad in a 0.03-0.07 wt % Tween-20 solution, with the amount of Tween-20 added being 0.04-0.08 mL / cm 2 , soaking time is more than 8 minutes, ultrasonic treatment is more than 30 seconds;

[0048] (2) The soaked binding pad is dried and dehydrated, then placed in an oven at 37-50°C until completely dry, and sealed and stored in an environment with a humidity of ≤30%.

[0049] 3. Prepare conjugation buffer, blocking buffer, wash buffer, and storage buffer

[0050] Conjugation buffer: divided into Tris conjugation buffer and boric acid conjugation buffer. Tris conjugation buffer is 10mM Tris buffer, and the pH of the solution is adjusted to 7-7.5 by hydrochloric acid; boric acid conjugation buffer is 10mM boric acid, and the pH of the solution is adjusted to 8.5-9.5 by sodium hydroxide.

[0051] Blocking solution: composed of 0.8-1.2wt% casein or BSA, 100mM boric acid, and the pH of the solution was adjusted to 8-9 by sodium hydroxide;

[0052] Washing buffer: 50 mM boric acid, and the pH of the solution was adjusted to 9.5-10.5 with sodium hydroxide;

[0053] Storage buffer: It is composed of 33 mM boric acid, 0.2-0.3 wt% casein or BSA, 12-16 wt% sucrose, and the pH of the solution is adjusted to 9-10 by sodium hydroxide.

[0054] 4. Labeling of Mouse Anti-Human Influenza A Virus Capture Antibody

[0055] (1) Mix 1 wt% cellulose nanosphere suspension with a vortex mixer, and then pipette 10 μL into a centrifuge tube;

[0056] (2) Pipette 90 μL of Tris conjugation buffer into the above centrifuge tube and mix well;

[0057] (3) adding 10-15 μg of mouse anti-human influenza A virus capture antibody to the centrifuge tube of step (2), mixing thoroughly, and placing in a 37° C. water bath for 120 minutes;

[0058] (4) Add 1200 μL of blocking solution to the above centrifuge tube, mix thoroughly, and place in a 37°C water bath for 60 minutes;

[0059] (5) Centrifuge the above centrifuge tube for 20 minutes at a centrifugal force of 15000 g, and then remove the supernatant;

[0060] (6) Add 1200 μL of washing buffer to the centrifuge tube from which the supernatant was removed, and use a vortex mixer and an ultrasonic device to fully disperse and resuspend the precipitate. Then centrifuge for 20 minutes at a centrifugal force of 15,000 g, and then remove the supernatant again;

[0061] (7) Add 132 μL of storage buffer to the above centrifuge tube, and then use a vortex mixer and an ultrasonic device to thoroughly break up and resuspend the precipitate.

[0062] 5. Labeling of Mouse Anti-Human Influenza B Virus Capture Antibody

[0063] (1) Mix a 1 wt% cellulose nanosphere suspension with a vortex mixer, and then pipette 10 μL of the 1 wt% cellulose nanosphere suspension into a 2 mL centrifuge tube;

[0064] (2) Pipette 90 μL of boric acid conjugate buffer into the above centrifuge tube and mix well;

[0065] (3) adding 10-15 μg of mouse anti-human influenza B virus capture antibody to the centrifuge tube of step (2), mixing thoroughly, and placing in a 37° C. water bath for 120 minutes;

[0066] (4) Add 1200 μL of blocking solution to the above centrifuge tube, mix thoroughly, and place in a 37°C water bath for 60 minutes;

[0067] (5) Centrifuge the above centrifuge tube for 20 minutes at a centrifugal force of 15000 g, and then remove the supernatant;

[0068] (6) Add 1200 μL of washing buffer to the centrifuge tube from which the supernatant was removed, and use a vortex mixer and an ultrasonic device to fully disperse and resuspend the precipitate. Then centrifuge for 20 minutes at a centrifugal force of 15,000 g, and then remove the supernatant again;

[0069] (7) Add 132 μL of storage buffer to the above centrifuge tube, and then use a vortex mixer and an ultrasonic device to thoroughly break up and resuspend the precipitate.

[0070] 6. Labeling and reconstitution of colloidal gold labeled chicken IgY antibody

[0071] Chicken IgY antibody is used as the quality control antibody. Since the quality control line has lower requirements on color development performance than the detection line, colloidal gold can meet the performance requirements of the quality control line. In addition, the cost of colloidal gold is lower than that of cellulose nanospheres, and the labeling process is simpler and faster. Therefore, colloidal gold is used as the labeling material for the quality control of chicken IgY antibody.

[0072] (1) Pipette 1 mL of 4 wt % colloidal gold solution with a particle size of 40 nm into a centrifuge tube, then add 30 μL of 0.1 M potassium carbonate solution and mix thoroughly;

[0073] (2) Add 8-12 μg of chicken IgY antibody to the above centrifuge tube and mix for at least 10 minutes;

[0074] (3) adding bovine serum albumin (BSA) to the solution prepared in step (2) to a concentration of 0.8-1.2 wt %, and then mixing for 10-20 min;

[0075] (4) subjecting the solution prepared in step (3) to low-temperature high-speed centrifugation at a speed of 10,000 rpm and a temperature between 0 and 10° C. for at least 10 minutes;

[0076] (5) removing the supernatant of the solution obtained in step (4);

[0077] (6) Add 25-35 μL of storage buffer to the precipitate obtained in step (5) after removing the supernatant, and then break up and resuspend the precipitate.

[0078] 7. Preparation of mouse anti-human influenza A virus detection antibody, mouse anti-human influenza B virus detection antibody and goat anti-chicken IgY antibody coating solution

[0079] (1) First prepare a coating diluent consisting of 8-12 mM PBS and 1.5-3 wt% sucrose;

[0080] (2) diluting the mouse anti-human influenza A virus detection antibody to 0.5-0.8 mg / mL using a coating diluent to obtain a mouse anti-human influenza A virus detection antibody coating solution;

[0081] (3) diluting the mouse anti-human influenza B virus detection antibody to 0.7-1.2 mg / mL using a coating diluent to obtain a mouse anti-human influenza B virus detection antibody coating solution;

[0082] (4) Dilute the goat anti-chicken IgY antibody to 0.8-1.2 mg / mL using a coating diluent to obtain a goat anti-chicken IgY antibody coating solution.

[0083] 8. Assembly of test strips

[0084] (1) 30 μL of chicken IgY antibody gold-labeled solution, 105 μL of mouse anti-human influenza A virus capture cellulose nanosphere labeling solution, and 105 μL of mouse anti-human influenza B virus capture antibody cellulose nanosphere labeling solution were fully mixed, and then the mixed solution was heated at 8 μL / cm 2 The treated bonding pad is sprayed at a speed of 1000 g / cm2, and then the sprayed bonding pad is placed in a temperature of 37-50°C for baking until it is completely dry, and then sealed and stored in an environment with a humidity of ≤30%;

[0085] (2) spraying mouse anti-human influenza A virus detection antibody coating solution, mouse anti-human influenza B virus detection antibody coating solution and goat anti-chicken IgY antibody coating solution onto the nitrocellulose membrane at a rate of 1-3 μL / cm, respectively, to form an influenza A virus detection line, an influenza B virus detection line and a quality control line on the nitrocellulose membrane, then baking the treated nitrocellulose membrane at a temperature of 37-50° C. until it is completely dry, and then sealing and storing the dried nitrocellulose membrane in an environment with a humidity of ≤30%;

[0086] (3) The positional relationship between the sample pad, conjugate pad, nitrocellulose membrane, water-absorbing pad and bottom plate after assembly is as follows: the sample pad, conjugate pad, water-absorbing pad and nitrocellulose membrane are all bonded to the bottom plate, wherein the nitrocellulose membrane is located near the middle of the bottom plate, one end of the nitrocellulose membrane is bonded to the water-absorbing pad with an overlap of 1-3 mm, and the other end is bonded to the conjugate pad with an overlap of 1-3 mm, and the other end of the conjugate pad is bonded to the sample pad with an overlap of 1-3 mm, and the bottom plate is located at the bottom to support the entire test strip.

[0087] Embodiment 2: Test strip detection method

[0088] The test strip for influenza A and B virus antigen detection based on cellulose nanospheres of the present invention has the following detection method:

[0089] (1) preparing a sample diluent, comprising 4-6 wt % NaCl, 0.5-0.8 wt % Tris, 0.03-0.06 wt % anhydrous disodium hydrogen phosphate, 0.8-1.4 wt % BSA, 0.03-0.06 wt % anhydrous dipotassium hydrogen phosphate, 0.08-0.12 wt % sodium stearate, and 0.020.05 wt % ProClin300 or sodium azide, with a solution pH of 7.5-8;

[0090] (2) Using sample diluent, dilute the influenza A virus antigen standard to 23000 ng / mL, 230 ng / mL, 10 ng / mL, 5 ng / mL, 1 ng / mL, 0.25 ng / mL, 0.1 ng / mL, and 0.05 ng / mL; dilute the influenza B virus antigen standard to 6000 ng / mL, 60 ng / mL, 10 ng / mL, 5 ng / mL, 1 ng / mL, 0.5 ng / mL, and 0.25 ng / mL;

[0091] (3) Take 80 μL of the 8 concentrations of influenza A virus antigen standard solution and the 7 concentrations of influenza B virus antigen standard solution prepared in step (1) and add them to the test strip prepared in Example 1. Due to capillary action, the sample will move along the test strip to the conjugate pad and nitrocellulose membrane until it flows to the absorbent pad; observe the display result for 10 minutes (Note: color development is invalid after 15 minutes);

[0092] (4) Observation of results: The minimum detection limit of influenza A virus antigen of this test strip is 0.05 ng / mL, that is, when the influenza A virus antigen concentration is ≥0.05 ng / mL, the influenza A virus detection line will show color, indicating a positive influenza A virus test result; when the influenza A virus antigen concentration is <0.05 ng / mL, the influenza A virus detection line will not show color, indicating a negative influenza A virus test result; the minimum detection limit of influenza B virus antigen of this test strip is 0.25 ng / mL, that is, when the influenza B virus antigen concentration is ≥0.25 ng / mL, the influenza B virus detection line will show color, indicating a positive influenza B virus test result; when the influenza B virus antigen concentration is <0.25 ng / mL, the influenza B virus detection line will not show color, indicating a negative influenza B virus test result; the color of the quality control line indicates a valid test result, and the absence of the quality control line indicates an invalid test result.

[0093] The test strips showed good specificity when tested with the above 8 concentrations of influenza A virus antigen standard solutions and 7 concentrations of influenza B virus antigen standard solutions, namely: when only the above concentrations of influenza A virus antigen standard solutions were added, the test strips only showed color on the influenza A virus detection line, but not on the influenza B virus detection line; when only the above concentrations of influenza B virus antigen standard solutions were added, the test strips only showed color on the influenza B virus detection line, but not on the influenza A virus detection line.

[0094] Example 3: Performance testing of test strips

[0095] The test strip for influenza A and B virus antigen detection based on cellulose nanospheres described in the present invention is compared with the commercially available test strips:

[0096] Take the test strips prepared in Example 1 and the commercially available test strips of Control Examples 1-3, and test 0.05 ng / mL, 0.1 ng / mL, 1 ng / mL, 5 ng / mL, and 10 ng / mL of influenza A virus antigen standard solutions and 0.25 ng / mL, 0.5 ng / mL, 1 ng / mL, 5 ng / mL, and 10 ng / mL of influenza B virus antigen standard solutions, respectively. The results are shown in Table 1 and Table 2, respectively.

[0097] Table 1 Comparison of influenza A virus antigen standard solution detection

[0098] Influenza A virus antigen concentration 0.05ng / mL 0.1ng / mL 1ng / mL 5ng / mL 10 ng / mL Example 1 Positive Positive Positive Positive Positive Comparative Example 1 Negative Negative Positive Positive Positive Comparative Example 2 Negative Negative Positive Positive Positive Comparative Example 3 Negative Negative Positive Positive Positive

[0099] Table 2 Comparison of influenza B virus antigen standard solution detection

[0100] Influenza B virus antigen concentration 0.25 ng / mL 0.5ng / mL 1ng / mL 5ng / mL 10 ng / mL Example 1 Positive Positive Positive Positive Positive Comparative Example 1 Negative Negative Negative Positive Positive Comparative Example 2 Negative Negative Negative Positive Positive Comparative Example 3 Negative Negative Negative Positive Positive

[0101] Example 4: Adjusting the detection antibody and capture antibody loading

[0102] Reduce the amount of marking solution sprayed:

[0103] The test strip was prepared according to the method described in Example 1, except that: in step 8, in the assembly of the test strip, the evenly mixed mouse anti-human influenza A virus capture antibody cellulose nanosphere labeling solution, mouse anti-human influenza B virus capture antibody cellulose nanosphere labeling solution, and chicken IgY antibody gold labeling solution were heated at 5 μL / cm 2 Spray the coating amount on the treated bonding pad;

[0104] The influenza A virus antigen and influenza B virus antigen standard solutions were detected according to the method described in Example 2, and the minimum detection limits were 0.1 ng / mL and 0.5 ng / mL, respectively.

[0105] Reduce the amount of labeling solution sprayed and the concentration of capture antibody used at the same time:

[0106] The test strip was prepared according to the method described in Example 1, except that: in step 7, in the dilution of the coated antibody, the mouse anti-human influenza A virus detection antibody was diluted to 0.3 mg / mL, and the mouse anti-human influenza B virus detection antibody was diluted to 0.5 mg / mL; in step 8, in the assembly of the test strip, the evenly mixed mouse anti-human influenza A virus capture antibody cellulose nanosphere labeling solution, mouse anti-human influenza B virus capture antibody cellulose nanosphere labeling solution, and chicken IgY antibody gold labeling solution were heated at 5 μL / cm 2 Spray the coating amount on the treated bonding pad;

[0107] The influenza A virus antigen and influenza B virus antigen standard solutions were detected according to the method described in Example 2, and the minimum detection limits were 0.2 ng / mL and 0.8 ng / mL, respectively.

[0108] It can be seen from this that reducing the corresponding detection antibody and capture antibody loading on the conjugate pad and the nitrocellulose membrane on the test strip alone or simultaneously will reduce the detection performance of the test strip.

[0109] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A method for preparing a test strip for influenza A and B virus detection based on cellulose nanospheres, characterized in that: The following steps are involved: Step S1, using cellulose nanospheres to label influenza A virus capture antibodies and influenza B virus capture antibodies to obtain influenza A virus capture antibody cellulose nanosphere labeling solution and influenza B virus capture antibody cellulose nanosphere labeling solution; The influenza A virus capture antibody cellulose nanosphere labeling solution and the influenza B virus capture antibody cellulose nanosphere labeling solution are mixed evenly, and sprayed on the conjugate pad; Step S2, setting an influenza B virus detection line and an influenza A virus detection line on the coating membrane, spraying solutions containing influenza B virus detection antibodies and influenza A virus detection antibodies respectively, wherein the influenza B virus detection line and the influenza A virus detection line are arranged perpendicular to the direction of the test strip; The final concentration of the influenza B virus detection antibody is 0.7-1.2 mg / mL, and the final concentration of the influenza A virus detection antibody is 0.5-0.8 mg / mL; Step S3, assembling the sample pad, the binding pad, the coating membrane and the water absorbent pad to obtain the influenza A and B virus detection test strip.

2. The preparation method according to claim 1, characterized in that: The method also includes pre-treating the sample pad and the conjugate pad, wherein the pre-treating comprises soaking the sample pad and the conjugate pad in a sample pad pre-treating solution and a conjugate pad pre-treating solution, respectively; The components of the sample pad pretreatment solution include a surfactant, a preservative and a blocking agent; The components of the bonding pad pretreatment solution include a surfactant.

3. The preparation method according to claim 2, characterized in that: The amount of sample pad pretreatment solution added is 0.04-0.08 mL / cm 2 The amount of the conjugate pad pretreatment solution added is 0.04-0.08 mL / cm 2 .

4. The preparation method according to claim 1, characterized in that: In step S1, the labeling of influenza virus capture antibodies comprises the following steps: Step S11, preparing conjugation buffer, blocking solution, washing buffer and storage buffer; Step S12, mixing the cellulose nanospheres and the influenza virus capture antibody in a conjugation buffer; Step S13, adding blocking solution, centrifuging and removing supernatant; Step S14, adding washing buffer to break up the precipitate and reselect it, and centrifuging to remove the supernatant; Step S15, adding storage buffer to obtain influenza virus capture antibody cellulose nanosphere labeling solution.

5. The preparation method according to claim 4, characterized in that: In step S12, the influenza virus capture antibody is added in an amount of 10-15 μg.

6. The preparation method according to claim 1, characterized in that: In step S1, the conjugate pad is sprayed with a quality control capture antibody labeled with colloidal gold.

7. The preparation method according to claim 6, characterized in that: The amount of the quality control capture antibody added to the colloidal gold solution is 8-12 μg.

8. The preparation method according to claim 6, characterized in that: In step S2, a quality control line is provided on the coating membrane, and a solution containing a quality control detection antibody is sprayed on the quality control line.

9. The preparation method according to claim 8, characterized in that: The final concentration of the quality control detection antibody is 0.8-1.2 mg / mL.

10. A test strip for detecting influenza A and B viruses prepared by the preparation method according to any one of claims 1 to 9.

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