Preparation method of a triple detection kit for influenza A, influenza B and new coronavirus antigens

Abstract

The present invention discloses a preparation method of a triple detection kit for influenza A, influenza B and new coronavirus antigens, and belongs to the field of in vitro detection of respiratory viruses. In the antibody labeling stage, the present invention adds a linking arm so that the antigen and the antibody can be more fully combined, which is beneficial to the improvement of the sensitivity of antigen detection. In the gold spraying stage, the new coronavirus monoclonal antibody, influenza A virus monoclonal antibody and influenza B virus monoclonal antibody labeled with latex microspheres are sprayed with gold in multiple layers at the same position in sequence with a gold sprayer, and the two antibodies are separated by trehalose solution to complete the gold spraying of three layers of antibodies isolated from each other, so as to avoid cross-reaction between different antibodies on the binding pad during storage, produce non-specific binding, and improve storage stability. The detection kit prepared by the method of the present invention can specifically detect the new coronavirus, influenza A virus and influenza B virus, and has the advantages of high sensitivity and good repeatability.

Description

Technical Field

[0001] The present invention relates to the field of in vitro detection of respiratory viruses, and in particular to a method for preparing a triple detection kit for influenza A, influenza B and novel coronavirus antigens. Background Art

[0002] Influenza A and B are respiratory infections caused by influenza A and B viruses. Respiratory infections have a great impact on people's lives. Long-term high fever caused by untimely treatment may even lead to death. The new coronavirus infection is mainly an acute infectious disease caused by SARS-CoV-2 infection. The disease is contagious. The source of infection is mainly patients infected with the new coronavirus. It is contagious during the incubation period. Therefore, the ability to quickly and accurately detect viruses that cause respiratory infections is very effective for early diagnosis.

[0003] According to data from the National Disease Control and Prevention Bureau, the number of reported cases of Class C statutory infectious diseases in 2023 reached 15.677 million, of which the number of influenza cases reached 12.528 million, a year-on-year increase of 407.7%. This data shows that the number of cases of respiratory diseases is often high, leading to a steady increase in the demand for testing. In addition, the development of multi-link detection technology is conducive to the precise diagnosis and treatment of respiratory infections, and has promoted the expansion of the testing market. The respiratory multiple pathogen detection technology on the market is mainly based on molecular biology, including qPCR, RNA constant temperature amplification-gold probe chromatography technology (RGT), double amplification technology (DAT), gene chip technology, microfluidics technology, etc. However, these methods need to be tested in hospitals or specific testing institutions, which are expensive and cumbersome to operate.

[0004] Currently, most rapid test kits for influenza A, influenza B, and novel coronavirus antigens on the market are single-test products using immunochromatography. There are no registered triple-test products in China. The commercialized respiratory combined test products usually use two or more windows for separate testing, which is not only cumbersome to operate but also inefficient. It is necessary to develop a triple-test kit that can detect influenza A, influenza B, and novel coronavirus simultaneously in the same window, reduce testing time, simplify the operating process, and provide users with a more efficient testing experience. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing a triple detection kit for influenza A, influenza B and new coronavirus antigens, so as to solve the problems existing in the above-mentioned prior art and realize the detection of three respiratory viruses at the same time with high sensitivity and strong specificity.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a method for preparing a triple detection kit for influenza A, influenza B and new coronavirus antigens, comprising the following steps:

[0008] After the latex microspheres are suspended, the pH value is adjusted, and a polyethylene glycol SMCC cross-linking agent is added for activation to obtain activated latex microspheres;

[0009] Mixing the novel coronavirus monoclonal antibody, influenza A virus monoclonal antibody, and influenza B virus monoclonal antibody with Traut's reagent, respectively, to obtain mixed solution I, mixed solution II, and mixed solution III;

[0010] The activated latex microspheres are respectively mixed with the mixed solution I, the mixed solution II and the mixed solution III to perform coupling reaction, centrifuged statically, the precipitate is collected, and the precipitate is resuspended with a resuspending solution to obtain latex microspheres labeled with a novel coronavirus monoclonal antibody, latex microspheres labeled with an influenza A virus monoclonal antibody, and latex microspheres labeled with an influenza B virus monoclonal antibody;

[0011] The latex microspheres labeled with the novel coronavirus monoclonal antibody, the latex microspheres labeled with the influenza A virus monoclonal antibody, and the latex microspheres labeled with the influenza B virus monoclonal antibody are sprayed onto the conjugate pad by a multi-layer gold spraying method;

[0012] Using a gold spray film streaking instrument, the C-line antibodies and T-line antibodies are respectively coated on the nitrocellulose membrane fixed on the bottom plate, cut into test strips and assembled with card shells to obtain influenza A, influenza B and new coronavirus antigen detection kits.

[0013] Optionally, the pH value is adjusted to adjust the pH of the latex microsphere suspension to 6.5-7.5. In the embodiment of the present invention, the pH value is 7.5.

[0014] Optionally, the volume ratio of the latex microsphere suspension to the PEGylated SMCC crosslinker is (10-15): (3-5); and / or the PEGylated SMCC crosslinker is SM(PEG) 4. In an embodiment of the present invention, the volume ratio of the latex microsphere suspension to the PEGylated SMCC crosslinker is 12.5:3.5.

[0015] Optionally, the molar ratio of the novel coronavirus monoclonal antibody, influenza A virus monoclonal antibody or influenza B virus monoclonal antibody to the Traut reagent is 1:20.

[0016] Optionally, the volume ratio of the activated latex microspheres to the mixed solution I, mixed solution II or mixed solution III is (15-18): 20, and the volume ratio of 16.5:20 is used for illustration in the embodiment of the present invention; and / or the coupling reaction conditions are: 37° C., 40 rpm coupling for 2-3 h. The coupling conditions used in the embodiment of the present invention are 37° C., 40 rpm coupling for 2 h.

[0017] Optionally, the C line is coated with a sheep anti-mouse IgG polyclonal antibody with a concentration of 2.5 mg / mL; the T lines include: the T1 line is coated with a new coronavirus monoclonal antibody with a concentration of 1.0 mg / mL, the T2 line is coated with an influenza A virus monoclonal antibody with a concentration of 1.0 mg / mL, and the T3 line is coated with an influenza B virus monoclonal antibody with a concentration of 1.5 mg / mL.

[0018] Optionally, the multi-layer gold spraying method is: the latex microspheres labeled with the novel coronavirus monoclonal antibody, the latex microspheres labeled with the influenza A virus monoclonal antibody, and the latex microspheres labeled with the influenza B virus monoclonal antibody are sprayed onto the same position of the conjugate pad according to the structure pattern of antibody I-trehalose-antibody II-trehalose-antibody III, wherein the antibody I is one of the latex microspheres labeled with the novel coronavirus monoclonal antibody, the latex microspheres labeled with the influenza A virus monoclonal antibody, and the latex microspheres labeled with the influenza B virus monoclonal antibody, the antibody II is one of the other two latex microspheres, and the antibody III is the remaining latex microsphere. For example, the structural pattern of novel coronavirus antibody-trehalose-influenza A virus antibody-trehalose-influenza B virus can be used for gold spraying.

[0019] The present invention also provides a test kit for detecting influenza A, influenza B and novel coronavirus antigens prepared by the preparation method.

[0020] The present invention also provides a method for detecting influenza A, influenza B and new coronavirus for non-disease diagnosis or treatment purposes, using the detection kit for detection.

[0021] The present invention discloses the following technical effects:

[0022] In the antibody labeling stage, the present invention adds a linker arm SM (PEG) 4 to enable the antigen and the antibody to be more fully combined. Traut's reagent (2-iminothiolane hydrochloride) reacts with an amidine bond at pH 7.0-10.0 to obtain a free thiol group; the thiol group is introduced into the antibody by adding Traut's reagent. SM(PEG)4 contains an amine-reactive N-hydroxysuccinimide (NHS ester) and a thiol-reactive maleimide group. The NHS ester reacts with primary amines at pH 7.0-9.0 to form a stable amide bond, and the maleimide group reacts with thiol groups at pH 6.5-7.5 to form a stable thioether bond. Maleimide groups are introduced into latex microspheres by adding SM(PEG)4 reagent. The maleimide groups carried by the activated latex microspheres react with antibodies carrying thiol groups, and pure compounds PEG (polyethylene glycol) with a certain structure and molecular weight are introduced between the antibodies and latex microspheres to increase linker arms, thereby ensuring repeatable protein modification effects, helping to maintain the solubility of the conjugates, and solving the problem of low antigen detection sensitivity of similar products currently on the market.

[0023] The present invention adopts a multi-layer gold spraying method in the gold spraying stage of the binding pad, and sprays gold with a gold spraying instrument in sequence at the same position with a new coronavirus monoclonal antibody, an influenza A virus monoclonal antibody, and an influenza B virus monoclonal antibody labeled with latex microspheres, and uses a trehalose solution as an isolation after the antibody solution is dried to complete the three-layer antibody gold spraying isolated from each other. The gold spraying mode of the present invention is different from the gold spraying method of multiple antibody mixtures of similar products on the market, which avoids cross-reactions between different antibodies on the binding pad during storage, avoids non-specific binding with other antibodies, and can improve storage stability.

[0024] The detection kit prepared by the preparation method of the present invention can specifically detect the new coronavirus, influenza A virus and influenza B virus, and has the advantages of high sensitivity and good repeatability. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0026] Figure 1 This is a schematic diagram of the structure of the influenza A, influenza B and new coronavirus antigen triple detection kit;

[0027] Figure 2 This is a schematic diagram of the structure of the test strip for influenza A, influenza B and new coronavirus antigen triple detection;

[0028] Figure 3 Schematic diagram of the reaction between Traut's reagent and the amino group of the antibody;

[0029] Figure 4 The reaction principle diagram for introducing the linker arm SM(PEG)4 in the present invention. DETAILED DESCRIPTION

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0032] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0033] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0034] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0035] The main reagents involved in the following examples are:

[0036] (1) Novel coronavirus monoclonal antibody, influenza A virus monoclonal antibody, influenza B virus monoclonal antibody, and goat anti-mouse IgG polyclonal antibody were purchased from Beijing Jianyingjia Testing Technology Co., Ltd.

[0037] (2) SMCC crosslinker SM (PEG)n The structural formula is as follows:

[0038] .

[0039] SM (PEG) 4 was used in the examples of the present invention and was purchased from Thermo Fisher Scientific with a catalog number of 22104.

[0040] (3) Latex microspheres, purchased from Suzhou Weidu Biotechnology Co., Ltd., product number DR0300CA, particle size: 300 nm, solid content: 4.0%.

[0041] (4) Traut’s reagent (2-iminothiolane hydrochloride, CAS No. 4781-83-3, molecular weight 137.63) was purchased from Thermo Fisher Scientific, catalog number 26101.

[0042] Example 1 A method for preparing a kit for detecting influenza A, influenza B and novel coronavirus antigens

[0043] (1) Preparation of latex microsphere-labeled antibodies

[0044] A1: Take MES buffer in a clean centrifuge tube, add colored microspheres with 4% solid content, mix by ultrasonication, centrifuge at 4℃ and 16000rpm for 30min, collect the precipitate, and wash twice.

[0045] A2: Add 1 mL of MES buffer to the precipitate obtained in A1 and adjust the pH to 7.5. After ultrasonic mixing, add 3.5 μL of SM(PEG)4 (the volume ratio of the latex microsphere suspension to SM(PEG)4 is (10-15): (3-5), and 12.5:3.5 was selected for this experiment) for every 12.5 μL of microspheres. After ultrasonic mixing, place on a turntable and activate at 37°C and 40 rpm for 20 min. Then adjust to 4°C and centrifuge at 16,000 rpm for 20 min. Remove the supernatant and collect the precipitate.

[0046] A3: Add MES buffer to the precipitate obtained in A2, resuspend it at 150% volume, mix it by ultrasonication, centrifuge it at 4°C and 16,000 rpm for 20 min, collect the precipitate, and wash it twice.

[0047] A4: Add the mixture of novel coronavirus monoclonal antibody and Traut reagent (used to prepare latex microspheres labeled with novel coronavirus monoclonal antibody), the mixture of influenza A virus monoclonal antibody and Traut reagent (used to prepare latex microspheres labeled with influenza A virus monoclonal antibody), and the mixture of influenza B virus monoclonal antibody and Traut reagent (used to prepare latex microspheres labeled with influenza B virus monoclonal antibody) to the precipitate obtained in A3, and then mix by ultrasonic for 1 minute; place the centrifuge tube on a turntable and couple at 37°C and 40rpm for 2 hours. The above mixture is obtained by vortex mixing at a molar ratio of 1:20 between the corresponding antibody and Traut; the volume ratio of activated microspheres to the corresponding mixture is (15-18): 20, and 16.5:20 is selected in this experiment;

[0048] A5: Add blocking solution to the coupling reaction system (add 12.5 μL blocking solution to every 50 μL coupling reaction system) and block at 37°C and 40 rpm for 1 hour. Centrifuge at 4°C and 16,000 rpm for 20 minutes, remove the supernatant, add washing solution, resuspend at 150% volume, mix by ultrasound, centrifuge at 16,000 rpm for 20 minutes, wash twice, remove the supernatant, dilute with preservation solution at a concentration of 0.1 μg / μL, mix by ultrasound, and obtain monoclonal antibody-labeled latex microspheres.

[0049] (2) Preparation of kit

[0050] B1. Prepare PVC board: stick nitrocellulose membrane on PVC board, and stick absorbent paper on PVC board to press the membrane 1-1.5mm (1mm is selected in this experiment).

[0051] B2. Antibody labeling: Use protein diluent to prepare C-line and T-line coating solutions, and use a film stripper to draw lines on the nitrocellulose membrane. The parameters of the film stripper are set as follows: the C-line coating solution is a 2.5 mg / mL goat anti-mouse IgG polyclonal antibody, the T1-line coating solution is a 1.0 mg / mL novel coronavirus monoclonal antibody, the T2-line coating solution is a 1.0 mg / mL influenza A virus monoclonal antibody, and the T3-line coating solution is a 1.5 mg / mL influenza B virus monoclonal antibody.

[0052] Adjust the instrument parameters and position, place the pasted PVC board on the film scribing instrument for scribing, dry, and obtain the film board.

[0053] B3, gold spraying: Take out the latex microspheres labeled with the three monoclonal antibodies, fill them up to 500μL with MES buffer and mix them well. Complete the overlapping three-layer antibody gold spraying on the conjugate pad in the mode of antibody I-trehalose-antibody II-trehalose-antibody III, in which a trehalose solution with a volume ratio of 5~10% is used as isolation, and the gold spraying instrument is used to spray 500μL / 30cm gold to the same position on the conjugate pad to complete the overlapping three-layer antibody gold spraying, and put it in a 45℃ drying oven for 2.5-3h (2.5h was selected in this experiment). In the above gold spraying process, trehalose is used as an isolation layer, mainly based on trehalose as an antioxidant, which can effectively capture free groups, reduce oxidative damage, and maintain the activity of biological analytes; and it has good moisture retention, can absorb moisture in the air and keep the relative humidity in the formula stable. This feature is particularly important for some IVD reagents that are susceptible to moisture, and effectively prevents the reagents from failing due to moisture during storage. Therefore, the present invention can enhance the binding ability of antibodies by adding a certain concentration of trehalose, thereby improving the reliability of the results.

[0054] B4. Assembly: Figure 1 As shown in the figure, the sample pad, conjugate pad, nitrocellulose membrane, and absorbent paper are assembled on the bottom plate in the direction of each other, cut into strips by a strip cutter, assembled into finished products by a PLA card shell, and sealed by a sealing machine. Figure 2 .

[0055] Example 2 Application of the kit

[0056] 1. Detection method

[0057] (1) Immediately place the swab after collecting the sample in a sampling container containing sample processing fluid, place the swab head close to the bottom and side of the collection tube, and rotate the swab 10 times (at least 30 seconds);

[0058] (2) Gently squeeze the sampling container and add 3 drops of sample to the sample well of the test card;

[0059] (3) The results should be read within 10 to 30 minutes. Results read before 10 minutes or after 30 minutes are invalid.

[0060] 2. Criteria for judging results

[0061] (1) Positive:

[0062] Influenza A positive: The C line and T2 line show red bands, indicating that the influenza A virus antigen is positive.

[0063] Influenza B positive: The C line and T3 line show red bands, indicating that the influenza B virus antigen is positive.

[0064] Positive for novel coronavirus: The C line and T1 line show red strips, indicating that the novel coronavirus antigen is positive.

[0065] Influenza A and influenza B positive: The C line, T2 line and T3 line all show red bands, indicating that influenza A virus and influenza B virus antigens are positive.

[0066] Positive for novel coronavirus and influenza A: The C line, T1 line and T2 line all show red strips, indicating that the novel coronavirus and influenza A virus antigens are positive.

[0067] Positive for novel coronavirus and influenza B: The C line, T1 line and T3 line all show red strips, indicating that the novel coronavirus and influenza B virus antigens are positive.

[0068] Positive for the new coronavirus, influenza A and influenza B: The C line, T1 line, T2 line and T3 line all show red strips, indicating that the new coronavirus, influenza A virus and influenza B virus antigens are positive.

[0069] (2) Negative: Only the C line shows a red strip, indicating a negative result.

[0070] (3) Invalid: If the C line is not visible in the window after the test, the result is considered invalid. Invalid results indicate that the operation process is incorrect or the kit is invalid. It is recommended to repeat the test with a new test card.

[0071] In order to detect the effect of adding SM(PEG)4 and Traut reagent on the test results, the following comparison experiment was performed.

[0072] Comparative Example 1

[0073] Compared with Example 1, the only difference is that Sulfo-SMCC is used to replace SM(PEG)4 during the antibody labeling process, and the other method steps are the same as those in Example 1.

[0074] Comparative Example 2

[0075] Compared with Example 1, the only difference is that during the antibody labeling process, Traut reagent is added but SM (PEG) 4 reagent is not added. The rest is the same as Example 1.

[0076] Comparative Example 3

[0077] Compared with Example 1, the only difference is that during the antibody labeling process, SM (PEG) 4 reagent is added but Traut reagent is not added. The rest is the same as Example 1.

[0078] Comparative Example 4

[0079] Compared with Example 1, the only difference is that during the antibody labeling process, SM (PEG) 4 and Traut reagent are not added, and the other method steps are the same as those in Example 1.

[0080] The test results of Example 1 and Comparative Examples 1 to 4 are shown in Tables 1 to 5.

[0081] Table 1 Test results of Example 1

[0082]

[0083] Table 2 Test results of comparative example 1

[0084]

[0085] Table 3 Test results of comparative example 2

[0086]

[0087] Table 4 Test results of comparative example 3

[0088]

[0089] Table 5 Test results of comparative example 4

[0090]

[0091] Note: "+" indicates that the actual test result is positive; "-" indicates that the actual test result is negative. Sources of the above samples: All virus samples are from ATCC, and samples of different concentrations are prepared by diluting virus liquid.

[0092] The experimental results show that when SM(PEG)4 and Traut reagent are not added during antibody labeling, the sensitivity of the sample is the lowest and the minimum detection limit is the highest; when only Traut or SM(PEG)4 is added for detection, the sensitivity of the sample is low and the minimum detection limit is high; when Sulfo-SMCC and Traut reagent are added for detection during antibody labeling, the sensitivity of the sample is high and the minimum detection limit is low; when SM(PEG)4 and Traut reagent are added for detection during antibody labeling, the sensitivity of the sample is the highest and the minimum detection limit is the lowest. Therefore, adding SM(PEG)4 and Traut reagents at the same time during antibody labeling will give more accurate experimental results.

[0093] In order to detect the influence of multi-layer gold spraying on the test results, the following comparison experiment was conducted.

[0094] Comparative Example 5

[0095] Compared with Example 1, the only difference is that during gold spraying, the three labeled antibodies are mixed and then gold is sprayed once at the same position of the conjugate pad using a gold sprayer. The other method steps are the same as those in Example 1.

[0096] Table 6 Test results of Example 1 and Comparative Example 5

[0097]

[0098] Note: “+++” indicates that the actual test result is strongly positive; “++” indicates that the actual test result is moderately positive; “+” indicates that the actual test result is weakly positive; “-” indicates that the actual test result is negative.

[0099] The experimental results show that when the three antibodies are mixed and sprayed with gold, the detection background shows uneven chromatography; when the three antibodies are sprayed with gold in multiple layers, the background disappears cleanly without uneven chromatography. Therefore, it is shown that the method of spraying gold in multiple layers of the three antibodies labeled during the gold spraying of the present invention can avoid the uneven chromatography caused by the conventional gold spraying method.

[0100] Example 3 Detection of minimum detection limit

[0101] (1) Initial LOD of the minimum detection limit

[0102] One virus sample was selected for gradient dilution with a negative nasal swab sample to obtain one sample for each concentration gradient. One batch of product was used to test each gradient sample, and three samples were tested at each concentration.

[0103] The test results of three virus samples, Flu A inactivated virus A1 (2009H1N1), Flu B inactivated virus B1 (B / Victoria) and 2019-nCoV inactivated virus L1 (new coronavirus Alpha strain), are shown in Tables 7 to 9, respectively.

[0104] Table 7 Flu A initial LOD test results

[0105]

[0106] Table 8 Flu B initial LOD test results

[0107]

[0108] Table 9 2019-nCoV initial LOD detection results

[0109]

[0110] The results of sample testing at different concentration gradients show that the initial LOD of Flu A is 2×103 TCID 50 / mL, and the initial LOD of Flu B was 5×10 2 TCID 50 / mL, and the initial LOD of 2019-nCoV was 8×10 2 TCID 50 / mL.

[0111] (2) Determination of minimum detection limit

[0112] Three inactivated Flu A viruses A1 (2009 H1N1), A2 (seasonal H1N1), and A3 (influenza A H3N2) were selected and graded diluted using negative nasal swab samples. The concentration after dilution was 5 × 10 3 TCID 50 / mL, 2.5×10 3 TCID 50 / mL, 1.25×10 3 TCID 50 / mL, 0.75×10 3 TCID 50 / mL, 0.375×10 3 TCID 50 / mL, repeated 3 times, to obtain 3 samples of each concentration gradient of each virus sample. The 3 samples obtained by repeating 3 times at each concentration were tested using 3 batches of products in turn, and each sample was tested repeatedly for 20 people. The minimum detection limit of influenza A virus was determined and the test results are shown in Table 10.

[0113] Table 10 Test results for influenza A virus minimum detection limit

[0114]

[0115] Two inactivated Flu B viruses B1 (B / Victoria) and B2 (B / Yamagata) were selected and serially diluted using negative nasal swab samples. The concentration after dilution was 1.2 × 10 3 TCID 50 / mL, 0.6×10 3 TCID 50 / mL, 0.3×10 3 TCID 50 / mL, 0.15×10 3 TCID 50 / mL, 0.075×10 3 TCID 50 / mL, repeated 3 times, to obtain 3 samples of each concentration gradient of each virus sample. The 3 samples obtained by repeating 3 times at each concentration were tested using 3 batches of products in turn, and each sample was tested repeatedly for 20 people. The minimum detection limit of influenza B virus was determined as shown in Table 11.

[0116] Table 11 Test results for determination of minimum detection limit of influenza B virus

[0117]

[0118] Three 2019-nCoV inactivated viruses, L1 (new coronavirus Alpha strain), L2 (new coronavirus Beta strain), and L3 (new coronavirus Delta strain), were selected and graded diluted using negative nasal swab samples. The diluted concentration was 2.0 × 10 3 TCID 50 / mL, 1.0×10 3 TCID 50 / mL, 0.5×10 3 TCID 50 / mL, 0.25×10 3 TCID 50 / mL, 0.125×10 3 TCID 50 / mL, repeated 3 times, to obtain 3 samples of each concentration gradient of each virus sample. The 3 samples obtained by repeating 3 times at each concentration were tested using 3 batches of products in turn, and each sample was tested repeatedly for 20 people. The results of the minimum detection limit of the new coronavirus are shown in Table 12.

[0119] Table 12 Test results of the minimum detection limit of the new coronavirus

[0120]

[0121] Flu A inactivated virus was diluted to 1.25 × 10 using negative nasal swab samples. 3 TCID 50 / mL, the positive detection rate can reach more than 95%, so 1.25×10 3 TCID 50 / mL was used as the minimum detection limit for Flu A. The inactivated Flu B virus was diluted to 0.3×10 3 TCID 50 / mL, the positive detection rate can reach more than 95%, so 0.3×10 3 TCID 50 / mL was used as the minimum detection limit of Flu B. The 2019-nCoV inactivated virus was diluted to 0.25×10 3 TCID 50 / mL, the positive detection rate can reach more than 95%, so 0.25×10 3 TCID 50 / mL was used as the minimum detection limit of 2019-nCoV.

[0122] (3) Verification of minimum detection limit

[0123] Three inactivated Flu A viruses A1 (2009 H1N1), A2 (seasonal H1N1), and A3 (influenza A H3N2) were selected and diluted to a minimum detection limit of 1.25 × 10 3 TCID 50 / mL. Three batches of products were used to test 20 people's diluted samples respectively, and the test results were observed, as shown in Table 13.

[0124] Table 13 Flu A virus strain minimum detection limit verification test results

[0125]

[0126] Three inactivated Flu B viruses B1 (B / Victoria), B2 (B / Yamagata), and B3 (B / Washington) were selected and diluted to a minimum detection limit of 0.3 × 10 3 TCID 50 / mL. Three batches of products were used to test 20 people's diluted samples respectively, and the test results were observed, as shown in Table 14.

[0127] Table 14 Flu B virus strain minimum detection limit verification test results

[0128]

[0129] Select three 2019-nCoV inactivated viruses, L4 (new coronavirus P.1 strain), L5 (new coronavirus Kappa strain), and L6 (new coronavirus Omicron strain), and dilute the negative nasal swab samples to the minimum detection limit of 0.25×10 3 TCID 50 / mL. Three batches of products were used to test 20 people's diluted samples respectively, and the test results were observed, as shown in Table 15.

[0130] Table 15 2019-nCoV virus strain minimum detection limit verification test results

[0131]

[0132] Flu A inactivated viruses (2009 H1N1, seasonal H1N1, influenza A H3N2) were diluted to the minimum detection limit (1.25 × 10 3 TCID 50 / mL) and the positive detection rate can reach more than 95%; Flu B inactivated virus (B / Victoria, B / Yamagata, B / Washington) is diluted to the minimum detection limit (0.3×10 3 TCID 50 / mL) and then tested, the positive detection rate can reach more than 95%; the 2019-nCoV inactivated virus (new coronavirus P.1 virus strain, new coronavirus Kappa virus strain, new coronavirus Omicron virus strain) is diluted to the minimum detection limit (0.25×10 3 TCID 50 / mL) and then tested, the positive detection rate can reach more than 95%.

[0133] Therefore, the minimum detection limit concentration of Flu A in the kit of the present invention is 1.25×10 3 TCID 50 / mL; the minimum detection limit of Flu B is 0.3×10 3 TCID 50 / mL; the minimum detection limit concentration of 2019-nCoV is 0.25×10 3 TCID 50 / mL.

[0134] Example 4 High-dose hook effect (HOOK)

[0135] Inactivated influenza A virus, influenza B virus, and novel coronavirus (2019-nCoV) cultures were diluted using nasal swab negative sample matrix. Three batches of products were randomly selected in sufficient quantity to test all diluted samples, with each sample tested 3 times. The results are shown in Tables 16-18.

[0136] Table 16 Flu A HOOK test results

[0137]

[0138] Table 17 Flu B HOOK test results

[0139]

[0140] Table 18 2019-nCoV HOOK test results

[0141]

[0142] The test results show that as the concentration of virus culture increases, the color depth increases, and Flu A reaches 1.26×10 6 TCID 50 / mL concentration, Flu B reached 7.2×10 6 TCID 50 / mL concentration, 2019-nCoV reached 3.8×10 6 TCID 50 / mL concentration, the color changed from dark to light, and no HOOK effect was observed in the reagents at this concentration.

[0143] Example 5 Cross-validation

[0144] The product is validated against common cross-reactive pathogenic microorganisms (see Table 19) that are clinically susceptible to the same and similar symptoms in the table below. The virus concentration is l×10 5 PFU / mL, the concentration of other bacteria and pathogens is l×10 6 CFU / mL, the results show that the product has good specificity for the cross-reactants in the table below. The results of the kit of the present invention detecting other pathogenic microorganisms are all negative, proving that the kit of the present invention has no cross-reaction with other pathogenic microorganisms and has strong specificity.

[0145] Table 19 Cross-reaction results

[0146]

[0147] Note: The virus strains in the table are all from ATCC.

[0148] Experimental Example 6 Interference Reaction

[0149] The interference verification of the kit prepared by the present invention was carried out by measuring the highest blood drug concentration of common clinical therapeutic drugs under normal usage and dosage, and adding the substances shown in Table 20 and Table 21 below to the minimum detection limit sample and negative matrix sample of the kit of the present invention, and testing 1 person each to perform interference verification on this product.

[0150] Table 20 Interference reaction test results-1

[0151]

[0152] Table 21 Interference reaction test results-2

[0153]

[0154] The substances in Table 21 will not interfere with the experimental results at their specified concentrations, which proves that the kit of the present invention has no cross-reaction with the interfering substances at the specified concentrations.

[0155] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing a triple detection kit for influenza A, influenza B and new coronavirus antigens, characterized in that: The following steps are involved: After the latex microspheres are suspended, the pH value is adjusted, and a polyethylene glycol SMCC cross-linking agent is added for activation to obtain activated latex microspheres; Mixing the novel coronavirus monoclonal antibody, influenza A virus monoclonal antibody, and influenza B virus monoclonal antibody with Traut's reagent, respectively, to obtain mixed solution I, mixed solution II, and mixed solution III; The activated latex microspheres are respectively mixed with the mixed solution I, the mixed solution II and the mixed solution III to perform coupling reaction, centrifuged statically, the precipitate is collected, and the precipitate is resuspended with a resuspending solution to obtain latex microspheres labeled with a novel coronavirus monoclonal antibody, latex microspheres labeled with an influenza A virus monoclonal antibody, and latex microspheres labeled with an influenza B virus monoclonal antibody; The latex microspheres labeled with the novel coronavirus monoclonal antibody, the latex microspheres labeled with the influenza A virus monoclonal antibody, and the latex microspheres labeled with the influenza B virus monoclonal antibody are sprayed onto the conjugate pad by a multi-layer gold spraying method; Using a gold-sprayed membrane streaking instrument, C-line antibodies and T-line antibodies are coated on nitrocellulose membranes fixed on a bottom plate, cut into test strips and assembled with card shells to obtain influenza A, influenza B and new coronavirus antigen detection kits; The pH value is adjusted to a value of 6.5 to 7.5 for the suspension of the latex microspheres; The PEGylated SMCC cross-linking agent is SM(PEG)4.

2. The preparation method according to claim 1, characterized in that The volume ratio of the latex microsphere suspension to the PEGylated SMCC crosslinker is (10-15):(3-5).

3. The preparation method according to claim 1, characterized in that: The molar ratio of the novel coronavirus monoclonal antibody, influenza A virus monoclonal antibody or influenza B virus monoclonal antibody to the Traut reagent is 1:

20.

4. The preparation method according to claim 1, characterized in that: The volume ratio of the activated latex microspheres to the mixed solution I, mixed solution II or mixed solution III is (15-18):20; and / or the coupling reaction conditions are: 37° C., 40 rpm, coupling for 2-3 hours.

5. The preparation method according to claim 1, characterized in that: The C line is coated with a sheep anti-mouse IgG polyclonal antibody with a concentration of 2.5 mg / mL; the T line includes: the T1 line is coated with a new coronavirus monoclonal antibody with a concentration of 1.0 mg / mL, the T2 line is coated with an influenza A virus monoclonal antibody with a concentration of 1.0 mg / mL, and the T3 line is coated with an influenza B virus monoclonal antibody with a concentration of 1.5 mg / mL.

6. The preparation method according to claim 1, characterized in that: The multi-layer gold spraying method is: spraying the latex microspheres labeled with the novel coronavirus monoclonal antibody, the latex microspheres labeled with the influenza A virus monoclonal antibody, and the latex microspheres labeled with the influenza B virus monoclonal antibody onto the same position of the binding pad according to the structural pattern of antibody I-trehalose-antibody II-trehalose-antibody III, wherein the antibody I is one of the latex microspheres labeled with the novel coronavirus monoclonal antibody, the latex microspheres labeled with the influenza A virus monoclonal antibody, and the latex microspheres labeled with the influenza B virus monoclonal antibody, the antibody II is one of the other two latex microspheres, and the antibody III is the remaining latex microsphere.

7. A triple detection kit for influenza A, influenza B and new coronavirus antigens prepared by the preparation method according to any one of claims 1 to 6.

8. A method for detecting influenza A, influenza B and novel coronavirus for non-disease diagnosis or treatment purposes, characterized in that: The detection is performed using the detection kit described in claim 7.

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