A β-Actin dominant epitope antigen, a β-Actin antibody and its application

Abstract

The present invention relates to the field of virus detection technology, and in particular to a β-Actin dominant epitope antigen, a β-Actin antibody and an application thereof. The present invention provides a β-Actin dominant epitope antigen, and the amino acid sequence of the β-Actin dominant epitope antigen is shown in SEQ ID NO: 1 and SEQ ID NO: 2. A fluorescent immunochromatographic influenza virus antigen double quality control test strip is prepared using the β-Actin dominant epitope antigen and the β-Actin antibody, which can not only detect influenza viruses, but also accurately calibrate test samples, solving the problem of lack of accurate calibration of test samples in clinical practice, and has important clinical significance for the effective prevention and treatment of diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of virus detection, and in particular to a β-Actin dominant epitope antigen, a β-Actin antibody and applications thereof. Background Art

[0002] Nasopharyngeal (NP) swabs are the reference sampling method recommended by the World Health Organization (WHO) for detecting severe respiratory viruses. Although NP swab specimens have the advantages of being easy to store and transport, they are subject to a variety of sampling errors when using NP swabs to collect samples, such as the professionalism of the sample collector, the sample content at the collection site, and the degree of cooperation of the person being collected. These factors can produce unqualified test samples, increase the missed detection rate, increase clinical false negative judgments, and hinder the detection of diseases. Therefore, the impact of test samples on the accurate diagnosis of pathogenic microorganisms cannot be ignored. Solving the problem of lack of accurate calibration of test samples in clinical practice is of great clinical significance for the effective prevention and treatment of diseases. Summary of the invention

[0003] The object of the present invention is to provide a β-Actin dominant epitope antigen, a β-Actin antibody and applications thereof, which can be applied to the preparation of a fluorescent immunochromatography influenza virus antigen dual quality control detection reagent.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The present invention provides a β-Actin dominant epitope antigen, the amino acid sequence of the β-Actin dominant epitope antigen is shown in SEQ ID NO: 1 and SEQ ID NO: 2.

[0006] The present invention also provides a β-Actin antibody, wherein the β-Actin antibody is obtained by screening a β-Actin dominant epitope antigen as a detection protein;

[0007] The β-Actin dominant epitope antigen is the β-Actin dominant epitope antigen.

[0008] The present invention also provides the use of the β-Actin dominant epitope antigen and the β-Actin antibody in the preparation of a fluorescent immunochromatography influenza virus antigen double quality control detection reagent.

[0009] The present invention also provides the use of the β-Actin dominant epitope antigen and the β-Actin antibody in preparing a fluorescent immunochromatography influenza virus antigen double quality control test strip.

[0010] The present invention also provides a test strip for double quality control detection of influenza virus antigens using fluorescent immunochromatography, the test strip comprising a sample addition pad, a binding pad, a reaction pad, a water absorption pad and a bottom plate;

[0011] The reaction pad is prepared by coating antibodies on a cellulose membrane;

[0012] The cellulose membrane is a nitrocellulose membrane or a cellulose acetate membrane;

[0013] The coated antibodies include influenza virus monoclonal antibodies, β-Actin antibodies and secondary antibodies;

[0014] The position of the influenza virus monoclonal antibody coating is the T detection line;

[0015] The position of the coated β-Actin antibody is the internal reference line A;

[0016] The position of the coated secondary antibody is the C quality control line;

[0017] The coating concentration of influenza virus monoclonal antibodies is 0.5-1.5 mg / mL;

[0018] The β-Actin antibody is the β-Actin antibody described above;

[0019] The coating concentration of β-Actin antibody was 0.5 to 1.5 mg / mL;

[0020] The coating concentration of the secondary antibody is 0.5-1.5 mg / mL.

[0021] Preferably, the preparation method of the sample loading pad is: mixing a glass fiber membrane and a treatment liquid to obtain a sample loading pad;

[0022] The treatment solution includes the following components in final concentrations: 12-13 g / L Tris, 14-16 g / L bovine serum albumin, 1-3 g / L casein, 2-4 mL / L X-Triton, and 2-3 mL / L Proclin 300 liquid biological preservative.

[0023] Preferably, the binding pad is prepared by fixing a microsphere composite on a glass fiber membrane.

[0024] Preferably, the microsphere complex comprises a microsphere-influenza A antibody complex, a microsphere-influenza B antibody complex and a microsphere-β-Actin antibody complex;

[0025] The volume ratio of the microsphere-influenza A antibody complex, the microsphere-influenza B antibody complex and the microsphere-β-Actin antibody complex is 0.5-1.5:0.5-1.5:0.5-1.5;

[0026] The microsphere-influenza A antibody complex is obtained by mixing influenza A virus monoclonal antibody and fluorescent microspheres;

[0027] The mass volume ratio of the influenza A virus monoclonal antibody and the fluorescent microspheres is 5-15 μl: 5-15 g;

[0028] The microsphere-influenza B virus antibody complex is obtained by mixing influenza B virus monoclonal antibody and fluorescent microspheres;

[0029] The mass volume ratio of the influenza B virus monoclonal antibody and the fluorescent microspheres is 5-15 μl: 5-15 g;

[0030] The microsphere-β-Actin antibody complex is obtained by mixing β-Actin monoclonal antibody and fluorescent microspheres;

[0031] The mass volume ratio of the mixture of the β-Actin antibody and the fluorescent microspheres is 5-15 μl: 5-15 g;

[0032] The β-Actin antibody is the β-Actin antibody described above.

[0033] Beneficial effects of the present invention:

[0034] The present invention designs a group of dominant epitope antigens of β-Actin, and uses the dominant epitope antigens of β-Actin as immunogens to immunize animals to obtain a β-Actin antibody. The dominant epitope antigens of β-Actin and the β-Actin antibody are used to prepare a fluorescent immunochromatographic influenza virus antigen double quality control test strip, which can not only detect influenza viruses but also accurately calibrate test samples, thus solving the problem of lack of accurate calibration of test samples in clinical practice, and has important clinical significance for the effective prevention and treatment of diseases.

[0035] When the fluorescent immunochromatographic test strip of influenza virus antigen of the present invention is used, the sample to be tested is vertically dripped on the sample loading pad, and the sample to be tested transports the sample to the binding pad containing fluorescent microsphere-labeled antibodies through capillary action. When the sample to be tested contains influenza virus antigen, the influenza virus antigen forms an antigen-antibody complex with the antibody on the fluorescent microsphere. With the chromatography, the complex moves forward and first reaches the detection area T coated with the influenza virus monoclonal antibody to form an antibody-antigen-antibody sandwich complex, which gathers at the detection area T. With the chromatography, the complex moves forward. If the sample to be tested is collected qualified, β-Actin will bind to the β-Actin monoclonal antibody labeled with the microsphere and reach the internal reference line A. After β-Actin binds to another monoclonal antibody, a detection signal is generated on the A line. The unbound complex continues to move forward, and when it finally reaches the quality control line C, it binds to the sheep anti-mouse IgG antibody to form an antigen-antibody complex at the C line. The remaining fluorescent microsphere-labeled antibodies continued to move forward to the absorbent pad. The entire reaction was completed within 15 minutes. Finally, a fluorescence reader was used to detect the intensity of the fluorescence signal, and the content of the analyte in the sample was calculated based on the T value and T / C value. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 A reference diagram for determining the test results of the test strip;

[0037] Figure 2 A schematic diagram of a test strip for double quality control detection of influenza virus antigens by fluorescent immunochromatography;

[0038] Figure 3 is the standard curve of influenza A virus;

[0039] Figure 4 is the standard curve of influenza B virus;

[0040] Figure 5 is the standard curve of β-Actin;

[0041] Figure 6 A line detection threshold determination diagram;

[0042] Figure 7 Figure 3. Threshold determination for T-line detection. DETAILED DESCRIPTION

[0043] The present invention provides a β-Actin dominant epitope antigen, the amino acid sequence of the β-Actin dominant epitope antigen is shown in SEQ ID NO: 1 and SEQ ID NO: 2;

[0044] The SEQ ID NO: 1 is: REIVRDIKEKLCYVALDFEQEMTTAASSSSLEKSYEL;

[0045] The SEQ ID NO: 2 is: APSTMKIMIIAPPERKYSVWIGGSILASLS.

[0046] The present invention also provides a β-Actin antibody, wherein the β-Actin antibody is obtained by screening a β-Actin dominant epitope antigen as a detection protein;

[0047] The β-Actin dominant epitope antigen is the β-Actin dominant epitope antigen.

[0048] The present invention also provides the use of the β-Actin dominant epitope antigen and the β-Actin antibody in the preparation of a fluorescent immunochromatography influenza virus antigen double quality control detection reagent.

[0049] The present invention also provides the use of the β-Actin dominant epitope antigen and the β-Actin antibody in preparing a fluorescent immunochromatography influenza virus antigen double quality control test strip.

[0050] The present invention also provides a test strip for double quality control detection of influenza virus antigens using fluorescent immunochromatography, the test strip comprising a sample addition pad, a binding pad, a reaction pad, a water absorption pad and a bottom plate;

[0051] The reaction pad is prepared by coating antibodies on a cellulose membrane;

[0052] The cellulose membrane is a nitrocellulose membrane or a cellulose acetate membrane;

[0053] The coated antibodies include influenza virus monoclonal antibodies, β-Actin antibodies and secondary antibodies;

[0054] The position of the influenza virus monoclonal antibody coating is the T detection line;

[0055] The position of the coated β-Actin antibody is the internal reference line A;

[0056] The position of the coated secondary antibody is the C quality control line;

[0057] The package concentration of influenza virus monoclonal antibodies is 0.5-1.5 mg / mL, preferably 1.0 mg / mL;

[0058] The β-Actin antibody is the β-Actin antibody described above;

[0059] The coating concentration of β-Actin antibody is 0.5-1.5 mg / mL, preferably 1.0 mg / mL;

[0060] The coating concentration of the secondary antibody is 0.5-1.5 mg / mL, preferably 1.0 mg / mL.

[0061] In the present invention, the instrument used for preparing the reaction pad is an X, Y, Z three-dimensional dot-scratch film gold spraying instrument; the film-scratch volume set by the X, Y, Z three-dimensional dot-scratch film gold spraying instrument is 0.5-1.5μl / cm, preferably 1.0μl / cm.

[0062] In the present invention, the secondary antibody is a goat anti-mouse IgG antibody;

[0063] In the present invention, the spacing between the T test line, the A internal reference line, and the C quality control line is 4.0 mm;

[0064] In the present invention, the preparation of the reaction pad further comprises the following steps: after coating with the antibody, drying in a vacuum drying oven at 37° C. for 4 hours.

[0065] In the present invention, the preparation method of the sample loading pad is: mixing a glass fiber membrane and a treatment liquid to obtain a sample loading pad;

[0066] The treatment solution includes the following components in final concentrations: 12-13 g / L Tris, 14-16 g / L bovine serum albumin, 1-3 g / L casein, 2-4 mL / L X-Triton, and 2-3 mL / L polyethylene glycol-300; preferably 12.5 g / L Tris, 15 g / L bovine serum albumin, 2 g / L casein, 3 mL / L X-Triton, and 2.5 mL / L Proclin 300 liquid biopreservative.

[0067] In the present invention, the method for preparing the sample loading pad further comprises the following steps: mixing the glass fiber membrane and the treatment liquid, and placing the mixture in a forced air drying oven for 4 hours.

[0068] In the present invention, the binding pad is prepared by fixing a microsphere composite on a glass fiber membrane.

[0069] In the present invention, the spraying amount of the microsphere complex is 7-9 μl / cm, preferably 8 μl / cm.

[0070] In the present invention, the microsphere complex includes a microsphere-influenza A antibody complex, a microsphere-influenza B antibody complex and a microsphere-β-Actin antibody complex;

[0071] The volume ratio of the microsphere-influenza A antibody complex, the microsphere-influenza B antibody complex and the microsphere-β-Actin antibody complex is 0.5-1.5:0.5-1.5:0.5-1.5, preferably 1:1:1;

[0072] The microsphere-influenza A antibody complex is obtained by mixing influenza A virus monoclonal antibody and fluorescent microspheres;

[0073] The mass volume ratio of the influenza A virus monoclonal antibody and the fluorescent microspheres is 5-15 μl:5-15 g, preferably 10 μl:10 g;

[0074] The microsphere-influenza B virus antibody complex is obtained by mixing influenza B virus monoclonal antibody and fluorescent microspheres;

[0075] The mass volume ratio of the influenza B virus monoclonal antibody and the fluorescent microspheres is 5-15 μl:5-15 g, preferably 10 μl:10 g;

[0076] The microsphere-β-Actin antibody complex is obtained by mixing β-Actin monoclonal antibody and fluorescent microspheres;

[0077] The mass volume ratio of the mixture of the β-Actin antibody and the fluorescent microspheres is 5-15 μl:5-15 g, preferably 10 μl:10 g;

[0078] The β-Actin antibody is the β-Actin antibody described above.

[0079] In the present invention, the preparation method of the microsphere-influenza A antibody complex is:

[0080] (1) Washing the microspheres: The fluorescent microspheres and purified water were shaken and mixed, centrifuged at 15000 rpm for 15 min, and the supernatant was discarded to obtain the washed microspheres;

[0081] (2) Activation of microspheres: The washed microspheres were resuspended in 1.0 mL of 20 mmol / L MES buffer and sonicated twice at 150 W for 3 s each time. 20 μl of 10 mg / mL EDC activator and 200 μl of 10 mg / mL NHS activator were added in sequence, mixed and reacted for 1 h in the dark, centrifuged at 15,000 rpm for 15 min, and the supernatant was discarded to obtain the activated microspheres.

[0082] (3) Microsphere labeling: The activated microspheres were resuspended in 1.0 mL of 20 mmol / L MES buffer and sonicated twice at 150 W for 3 s each time using an ultrasonic device; monoclonal antibodies against influenza A virus were added respectively, mixed and reacted for 2 h in the dark, and centrifuged at 15,000 rpm for 15 min, and the supernatant was discarded to obtain a precipitate;

[0083] (4) Blocking: resuspend the precipitate with 1.0 mL of blocking solution, sonicate twice with an ultrasonic device at 150 W, mix and react for 2 h in the dark, centrifuge at 15,000 rpm for 15 min, discard the supernatant, and obtain the blocked precipitate;

[0084] (5) Redissolution: Mix the blocked precipitate and protective solution, blow and beat evenly, sonicate twice with an ultrasonic device at 150W, and observe the uniform dispersion of the solution under ultraviolet light to prepare the microsphere-influenza A antibody complex.

[0085] In the present invention, the preparation method of the microsphere-influenza B antibody complex refers to the preparation method of the microsphere-influenza A antibody complex, wherein the influenza A virus monoclonal antibody in step (3) is replaced with the influenza B virus monoclonal antibody to prepare the microsphere-influenza B antibody complex.

[0086] In the present invention, the preparation method of the microsphere-β-Actin antibody complex refers to the preparation method of the microsphere-influenza A antibody complex, wherein the influenza A virus monoclonal antibody in step (3) is replaced with β-Actin antibody to prepare the microsphere-β-Actin antibody complex.

[0087] In the present invention, the fluorescent microspheres are europium-containing 3+ ) Time-resolved immunofluorescent microspheres, the excitation light wavelength of which is 365nm, and the reflected light wavelength is 610nm; the diameter of the fluorescent microspheres is 200nm.

[0088] The preparation of the binding pad in the present invention further comprises the following steps: spraying and fixing the microsphere composite on the glass fiber membrane, and then placing it in a 37° C. forced air drying oven to dry overnight.

[0089] In the present invention, the water absorbent pad is a cotton absorbent pad, a silica gel absorbent pad or a sponge absorbent pad.

[0090] In the present invention, the bottom plate is a polyvinyl chloride (PVC) plate, a polypropylene (PP) plate, a polyethylene (PE) plate or a polyurethane (PU) plate.

[0091] In the present invention, the preparation method of the test strip is as follows: a sample adding pad, a binding pad, a reaction pad, and a water absorbent pad are sequentially adhered to a bottom plate to prepare a test paper strip; wherein the sample adding pad, the binding pad, and the reaction pad overlap by 2 mm in sequence, and the water absorbent pad and the reaction pad overlap by 2 mm; the test paper strip is cut into 4 mm paper strips, which are assembled into the prepared test paper card shell, the sample adding window corresponds to the sample adding pad of the test paper strip, and the result display window corresponds to the reaction pad of the test paper strip.

[0092] In the present invention, two adjacent parts of the sample loading pad, conjugation pad, reaction pad and water absorption pad are overlapped and connected. Such an overlapping structure can ensure that the sample can flow through the sample loading pad, conjugation pad, reaction pad and water absorption pad in sequence during the sample loading chromatography process.

[0093] In the present invention, the method for determining the result of the test strip is as follows:

[0094] Negative: There are signals on the C and A lines, but no signal on the T line, which is negative;

[0095] Positive: If there are signals on the C, A, and T lines, it is positive;

[0096] Invalid: If there is no signal on line C, it is invalid;

[0097] Sample failure: If there is no signal on line A, the sample is failure.

[0098] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0099] Example 1 A fluorescent immunochromatographic test strip for dual quality control detection of influenza virus antigens

[0100] 1. Preparation of the sample pad

[0101] Cut the glass fiber membrane into 300 mm long and 16 mm wide sizes, soak the cut glass fiber membrane in the treatment solution, dry it in a blast drying oven for 4 hours, and set it aside;

[0102] The treatment solution includes the following components: 12.5 g Tris, 15 g bovine serum albumin, 2 g casein, 3 mL X-Triton, and 2.5 mL Proclin 300 liquid biopreservative are dissolved in 1 L of water to prepare the treatment solution;

[0103] 2. Preparation of conjugate pad

[0104] (1) Washing the microspheres: 10 μl of fluorescent microspheres and 1.0 mL of purified water were shaken and mixed, centrifuged at 15,000 rpm for 15 min, and the supernatant was discarded to obtain the washed microspheres;

[0105] (2) Activation of microspheres: The washed microspheres were resuspended in 1.0 mL of 20 mmol / L MES buffer and sonicated twice at 150 W for 3 s each time. 20 μl of 10 mg / mL EDC activator and 200 μl of 10 mg / mL NHS activator were added in sequence, mixed and reacted for 1 h in the dark, centrifuged at 15,000 rpm for 15 min, and the supernatant was discarded to obtain the activated microspheres.

[0106] (3) Microsphere labeling: The activated microspheres were resuspended in 1.0 mL of 20 mmol / L MES buffer and sonicated twice at 150 W for 3 s each time. 10 μg of influenza A virus monoclonal antibody was added to each microsphere, and the mixture was mixed and reacted for 2 h in the dark. The mixture was centrifuged at 15,000 rpm for 15 min, and the supernatant was discarded to obtain a precipitate.

[0107] (4) Blocking: resuspend the precipitate with 1.0 mL of blocking solution, sonicate twice with an ultrasonic device at 150 W, mix and react for 2 h in the dark, centrifuge at 15,000 rpm for 15 min, discard the supernatant, and obtain the blocked precipitate;

[0108] (5) Redissolution: Mix the blocked precipitate and the protective solution, blow and evenly mix, sonicate twice with an ultrasonic device at 150W, and observe the uniform dispersion of the solution under ultraviolet light to prepare the microsphere-influenza A antibody complex;

[0109] (6) Repeating the above steps (1) to (5), replacing the 10 μg of influenza A virus monoclonal antibody in step (3) with 10 μg of influenza B virus monoclonal antibody to prepare a microsphere-influenza B virus antibody complex;

[0110] (7) Repeating the above steps (1) to (5), replacing the 10 μg influenza A virus monoclonal antibody in step (3) with 10 μg β-Actin antibody to prepare a microsphere-β-Actin antibody complex;

[0111] (8) The microsphere-influenza A antibody complex, microsphere-influenza B antibody complex, and microsphere-β-Actin antibody complex were mixed at a volume ratio of 1:1:1, and after mixing, they were sprayed on the glass fiber membrane at a spray rate of 8 μl / cm, and dried in a 37°C forced air drying oven overnight for later use.

[0112] 3. Preparation of reaction pad

[0113] Using an X, Y, Z three-dimensional dot-speck membrane gold spraying apparatus, 1.0 mg / mL influenza virus monoclonal antibody (2.0 mg / mL influenza A virus monoclonal antibody and 2.0 mg / mL influenza B virus monoclonal antibody mixed in equal volumes), 1.0 mg / mL β-Actin antibody and 1.0 mg / mL goat anti-mouse antibody were coated on a nitrocellulose membrane at 1 μl / cm, which were used as the T test line, A internal reference line and C quality control line, respectively. The spacing between each coated line was 4.0 mm. The membrane was dried in a vacuum oven at 37°C for 4 h and set aside.

[0114] 4. Assembly

[0115] Choose absorbent cotton pad as absorbent pad and polyvinyl chloride (PVC) board as base board;

[0116] The sample pad, conjugation pad, reaction pad and water absorption pad are sequentially adhered to the bottom plate to prepare a test paper strip; wherein the sample pad, conjugation pad and reaction pad overlap by 2 mm, and the water absorption pad and reaction pad overlap by 2 mm; the test paper strip is cut into 4 mm paper strips and assembled into the prepared test paper card shell, the sample addition window corresponds to the sample addition pad of the test paper strip, and the result display window corresponds to the reaction pad of the test paper strip. Figure 2As shown, the sample pad in the figure is the sample loading pad, the microsphere pad is the binding pad, the NC membrane is the reaction pad, and the absorbent paper is the absorbent pad.

[0117] Example 2 Test strip linear range and standard curve

[0118] (1) Establishment of influenza virus standard curve

[0119] The test strips described in Example 1 were used to detect linear reference substances of different concentrations (0.5 ng / mL, 1.0 ng / mL, 2.5 ng / mL, 5.0 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL and 25 ng / mL). Each concentration of the reference substance was tested three times, and the average T / C value was calculated. As shown in Tables 1-2. The logarithmic value of the reference substance concentration was used as the horizontal axis, and the T / C value was used as the vertical axis. The log-logit function model was used to fit the relationship between the two, that is, Logit (Y) = A + B × Log (X), and a standard curve was established. The standard curve of influenza virus is shown in FIG. Figures 3-4 shown.

[0120] Table 1 T / C values ​​of linear reference products of influenza A virus

[0121] Linear reference (ng / mL) The average value of T / C value 0.5 0.155 1.0 0.318 2.5 0.654 5.0 1.449 10 2.966 15 5.189 20 9.302 25 16.646

[0122] Table 2 T / C values ​​of influenza B virus linear references

[0123] Linear reference (ng / mL) The average value of T / C value 0.5 0.147 1.0 0.296 2.5 0.571 5.0 1.350 10 2.339 15 4.469 20 7.921 25 12.438

[0124] like Figures 3-4 As shown: Influenza A virus at 2.5×10 3 TCID 50 / L~320×10 3 TCID 50 The linear relationship in the range of / L is Y=0.9712x-1.154, R 2 =0.9961, indicating a good linear range. Influenza B virus is at 6.25×10 5 TCID 50 / L~800×10 5 TCID 50 The linear relationship in the range of / L is Y=0.9309x-1.929, R 2 =0.9947, indicating that the linear range is good.

[0125] (2) Establishment of β-Actin standard curve

[0126] The test strips described in Example 1 were used to detect different concentrations of β-Actin linear reference substances (0.5 ng / mL, 1.0 ng / mL, 2.5 ng / mL, 5.0 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL and 25 ng / mL). Each concentration of the reference substance was measured three times, and the average value of its T / C value was calculated. As shown in Table 3. The relationship between the β-Actin linear reference substance concentration was fitted as the horizontal coordinate and the T / C value was used as the vertical coordinate. The least squares method was used to fit the relationship between the two, that is, the β-Actin standard curve was established. β-Actin standard curve Figure 5 shown.

[0127] Table 3 T / C values ​​of β-Actin reference

[0128] Linear reference (ng / mL) The average value of T / C value 0.5 0.091 1.0 0.195 2.5 0.477 5.0 1.088 10 2.180 15 2.567 20 3.823 25 4.596

[0129] like Figure 5 As shown, β-Actin is in the range of 0.5 ng / mL to 25 ng / mL. Y=0.1836x+0.06349, R 2 =0.9919, indicating that the linear range is good.

[0130] Example 3 Minimum detection limit and specificity detection of test strips

[0131] Minimum detection limit: The test strip described in Example 1 was tested on a negative reference substance (the negative reference substance was 0.9% saline) and the test was repeated 20 times to calibrate the uncertainty of the result and calculate the average value of T / C. and standard deviation (SD), calculate Substitute the linear standard curve to calculate the corresponding concentration value, which is the minimum detection limit concentration. The results are shown in Table 4;

[0132] During the test, the test strip result determination method is as follows (such as Figure 1 shown):

[0133] Negative: if there are signals on the C and A lines but no signal on the T line, it is negative; Positive: if there are signals on the C, A and T lines, it is positive; Invalid: if there is no signal on the C line, it is invalid; Unqualified sample: if there is no signal on the A line, the sample is unqualified.

[0134] Table 4 Results of minimum detection limit of β-Actin

[0135]

[0136] Threshold determination: 112 negative reference samples (negative reference sample is deionized water) were used to test the test strips described in Example 1. The results are as follows: Figure 6As shown, the average value of A / C is 0.1120, the standard deviation is 0.0048, and the internal reference line cut-off value of the test strip is calculated to be 0.1214; Figure 7 As shown, the average T / C value is 0.0981, the standard deviation is 0.0062, and the cut-off value of the test strip is calculated to be 0.1105.

[0137] +2SD specificity: A total of 4 respiratory pathogens including Mycoplasma pneumoniae (purchased from Nanjing Baikang Biotechnology Co., Ltd.), novel coronavirus (from Henan Bioengineering Technology Research Center), Epstein-Barr virus (purchased from Nanjing Baikang Biotechnology Co., Ltd.), and respiratory syncytial virus (purchased from Nanjing Baikang Biotechnology Co., Ltd.) were selected for specificity evaluation, and the test strips described in Example 1 were used for detection, and the test was repeated 5 times, and the T / C average value was calculated and compared with the detection threshold to evaluate the specificity of the test strips. The results are shown in Table 5.

[0138] Table 5 Specificity detection results

[0139]

[0140]

[0141] As can be seen from Table 4, the T / C values ​​of the detection results of Mycoplasma pneumoniae, new coronavirus, Epstein-Barr virus, and respiratory syncytial virus are all less than 0.1105, with no cross-reaction with influenza detection and good specificity.

[0142] It can be seen from the above embodiments that the present invention provides a β-Actin dominant epitope antigen, a β-Actin antibody and applications thereof, and uses the β-Actin dominant epitope antigen and the β-Actin antibody to prepare a fluorescent immunochromatographic influenza virus antigen dual quality control test strip, which can not only detect influenza virus, but also accurately calibrate the test sample, thereby solving the problem of lack of accurate calibration of the test sample in clinical practice, and has important clinical significance for the effective prevention and treatment of diseases.

[0143] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A β-Actin dominant epitope antigen, characterized in that: The amino acid sequence of the β-Actin dominant epitope antigen is shown in SEQ ID NO: 1 or SEQ ID NO: 2.

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