Human saliva pepsin fluorescence immunochromatography test strip and preparation method thereof

Abstract

The invention relates to the technical field of test strips, in particular to a human saliva pepsin fluorescence immunochromatography test strip and a preparation method thereof.The human saliva pepsin fluorescence immunochromatography test strip is characterized in that a sample pad, a marking pad, a nitrocellulose membrane and a water absorption pad are pasted to the upper surface of a PVC plate, and the surface of the marking pad is sprayed with a pepsin marked antibody compound containing fluorescent microsphere marks; the fluorescence immunochromatography test strip provided by the invention can be used for specific detection of human saliva pepsin antigen, has a wide linear range, high sensitivity and high precision, can maintain the test value of the test strip to be basically unchanged for 14 days through accelerated destruction at 37 DEG C, shows good stability, and can be applied to detection of human saliva pepsin antigen. Meanwhile, the test strip is not interfered by amylase and lipase and has relatively high specificity, in addition, a method for detecting human saliva pepsin by adopting a fluorescence immunochromatography method has the advantages of operation detection, less time consumption, small matched equipment, low cost and the like, can meet the requirement of instant detection and has relatively high application value.

Description

Technical Field

[0001] The present invention relates to the technical field of test strips, and particularly to a human salivary pepsin fluorescence immunochromatographic test strip and a preparation method thereof. Background Art

[0002] The contents of laryngopharyngeal reflux can stimulate or damage the mucosa of the laryngeal region, thereby causing a series of laryngopharyngeal reflux diseases. Due to the relatively weak mucosal barrier in the laryngeal region, repeated stimulation and damage may cause chronic cough, a feeling of obstruction or even suffocation in the throat, and can lead to laryngeal cancer. However, clinically, because the symptoms of this disease are non-specific, it is often misdiagnosed as ordinary pharyngitis or respiratory diseases and not treated correctly. At present, the relatively reliable diagnostic method for laryngopharyngeal reflux disease is to insert a catheter into the nose - pharynx - esophagus for 24 - hour monitoring, and use a multi-channel intraluminal impedance - pH probe to determine whether there is gastric content refluxing into the laryngeal region. This method is obviously very painful for patients, and the cost is high, the time consumption is long, and patients cannot tolerate it. Therefore, it is necessary to develop a non-invasive, simple and highly specific detection method. Although there is a colloidal gold immunochromatographic test strip for human salivary pepsin, due to its poor sensitivity and susceptibility to interference by amylase and lipase in saliva, the present invention provides a highly sensitive and highly stable human salivary pepsin fluorescence immunochromatographic test strip and a preparation method thereof. Summary of the Invention

[0003] The purpose of the present invention is to provide a human salivary pepsin fluorescence immunochromatographic test strip and a preparation method thereof to solve the technical problems raised in the above background art.

[0004] To achieve the above purpose, the present invention provides the following technical solutions: In the first aspect, the present invention provides a human salivary pepsin fluorescence immunochromatographic test strip, including a PVC board. On the upper surface of the PVC board, a sample pad, a labeled pad, a nitrocellulose membrane, and an absorbent pad are sequentially adhered from one end to the other end. The surface of the labeled pad is sprayed with a pepsin-labeled antibody complex containing fluorescent microspheres, and the pepsin-labeled antibody complex is europium-containing polystyrene fluorescent microspheres. The surface of the nitrocellulose membrane is provided with a detection T line and a quality control C line. The detection T line is a pepsin capture antibody, and the quality control C line is goat anti-mouse IgG.

[0005] Preferably, the distance between the detection T line and the quality control C line is 0.5 cm - 1.0 cm.

[0006] Preferably, the content of the pepsin-labeled antibody in the pepsin-labeled antibody complex is 30 - 40 μg.

[0007] Preferably, the fluorescent microsphere-labeled pepsin-labeled antibody complex, pepsin capture antibody, and goat anti-mouse IgG are all sprayed using a gold-spraying and membrane-stripping instrument.

[0008] Preferably, one end of the sample pad overlaps above the labeling pad, and the overlapping length is 0.5 - 1.5 mm. One end of the labeling pad and the absorbent pad that are close to each other overlap both ends of the nitrocellulose membrane respectively, and the overlapping length is 1 - 2 mm.

[0009] Preferably, the particle size of the europium-containing polystyrene fluorescent microspheres is 300 nm.

[0010] In a second aspect, the present invention provides a preparation method of a human saliva pepsin fluorescence immunochromatographic test strip, comprising the following steps: 1), successively paste the sample pad, labeling pad, nitrocellulose membrane, and absorbent pad on a PVC board to obtain a first test strip; 2), cut the first test strip into a human oral and pharyngeal saliva pepsin fluorescence immunochromatographic test strip with a width of 3.9 mm using a high-speed continuous strip cutter; The preparation method of the labeling pad comprises the following steps: S1. Dilute the europium-containing polystyrene fluorescent microspheres with a particle size of 300 nm to a concentration of 0.5 mg / ml using 0.01 M pH 8.0 borate buffer, then add 10 mM carbodiimide and 15 mM N-hydroxysuccinimide and react at room temperature for 15 min, and then centrifuge at a rate of 15000 r / min to remove the supernatant; S2. Add pepsin-labeled antibody with a final concentration of 0.1 mg / mL to the precipitate, react at room temperature for 2 h, then add a blocking agent and treat for 90 min, centrifuge at a rate of 15000 r / min to remove the supernatant, and then add a storage solution to the precipitate to obtain a fluorescent microsphere-labeled antibody complex; S3. Dilute the fluorescent microsphere-labeled antibody complex with a resuspension solution at a ratio of 1:1 - 1:6 to obtain a dilution solution, and uniformly spray the dilution solution on a glass cellulose membrane at a spraying speed of 6 μL / cm using a gold-spraying and membrane-stripping instrument, and dry at 37°C for 240 min to obtain a labeling pad; The preparation method of the nitrocellulose membrane comprises the following steps: S1. Dilute the pepsin capture antibody solution with 0.02 M, pH = 7.2 phosphate buffer containing 0.4% trehalose to a final concentration of 1 - 2 mg / mL to obtain a pepsin antibody coating solution; S2. Dilute goat anti-mouse IgG with 0.02 M, pH = 7.2 phosphate buffer containing 0.4% trehalose to 0.5 - 1.0 mg / mL to obtain a goat anti-mouse IgG coating solution; S3. Use a gold spraying and membrane scribing instrument to evenly spray the pepsin coating solution on a part of the nitrocellulose membrane as the detection T line, and use the gold spraying and membrane scribing instrument to evenly spray the goat anti-mouse IgG coating solution on another part of the nitrocellulose coating plate as the quality control C line. Then place it in an oven at 40 - 45 °C and dry for 12 - 24 h to obtain the dried nitrocellulose membrane.

[0011] Preferably, the storage solution in the preparation method of the labeling pad is a mixed solution of 50 mM Tris-base, 3% glycine, 1% BSA, 0.1% sodium caseinate, 0.1% Tween 20, 0.87% sodium chloride + and 0.1% proclin 300, and the pH of the storage solution is 7.2; The resuspension solution in the preparation method of the labeling pad is a mixed solution of 20 mM phosphate buffer, 1% trehalose, 20% sucrose, 0.87% sodium chloride, 0.1% sodium EDTA, 1% BSA, 0.1% Tween 20 and 0.1% proclin 300, and the pH of the resuspension solution is 7.2.

[0012] Preferably, the dilution ratio of the fluorescent microsphere-labeled antibody complex to the resuspension solution in step S3 is 1:4.

[0013] Preferably, in step S1, borate buffer with a pH of 8.0 is used for dilution.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: The fluorescent immunochromatographic test strip provided by the present invention can be used for the specific detection of human salivary pepsin antigen, has a wide linear range, high sensitivity and precision, and after accelerated destruction at 37 °C, the test value of the test strip can remain basically unchanged for 14 days, showing good stability. At the same time, the test strip is not interfered by amylase and lipase and has high specificity. In addition, the method for detecting human salivary pepsin by fluorescent immunochromatography has the advantages of simple operation, less time consumption, small supporting equipment and low cost, etc., can meet the needs of point-of-care testing, and has high application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for describing the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present invention, and those of ordinary skill in the art can also obtain other drawings based on these drawings without creative efforts.

[0016] Figure 1 It is a schematic structural diagram of the test strip of the present invention.

[0017] Figure 2It is a test result diagram of Example 2 and Comparative Examples 1 and 2.

[0018] Figure 3 It is a test result diagram of Example 2 and Comparative Examples 3, 4 and 5.

[0019] Figure 4 It is a test result diagram of Example 2 and Comparative Examples 6, 7 and 8.

[0020] Figure 5 It is a specific test result diagram of the test strip of Example 2.

[0021] Figure 6 It is a stability test result diagram of the test strip of Example 2.

[0022] In the attached drawings, the list of components represented by each reference numeral is as follows: 1. PVC board; 2. Sample pad; 3. Label pad; 4. Nitrocellulose membrane; 5. Absorbent pad; 6. Detection T line; 7. Quality control C line. Detailed implementation mode

[0023] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0024] Please refer to Figure 1 As shown, Embodiment 1 of the present invention is: A human saliva pepsin fluorescence immunochromatographic test strip, including a PVC board 1, on the upper surface of the PVC board 1, a sample pad 2, a label pad 3, a nitrocellulose membrane 4 and an absorbent pad 5 are sequentially connected from one end to the other end. The surface of the label pad 3 is sprayed with a pepsin-labeled antibody complex containing fluorescent microspheres, and the pepsin-labeled antibody complex is europium-containing polystyrene fluorescent microspheres. The surface of the nitrocellulose membrane 4 is provided with a detection T line 6 and a quality control C line 7. The detection T line is a pepsin capture antibody, and the quality control C line 7 is goat anti-mouse IgG.

[0025] In this embodiment, the distance between the detection T line 6 and the quality control C line 7 is 0.5 cm - 1.0 cm.

[0026] In this embodiment, the content of the pepsin-labeled antibody in the pepsin-labeled antibody complex is 30 - 40 μg.

[0027] In this embodiment, the fluorescent microsphere-labeled pepsin-labeled antibody complex, the pepsin capture antibody and the goat anti-mouse IgG are all sprayed using a gold-spraying and membrane-drawing instrument.

[0028] In this embodiment, one end of the sample pad 2 overlaps above the marker pad 3, and the overlapping length is 0.5 - 1.5 mm. One end of the marker pad 3 and the absorbent pad 5 that are close to each other overlap both ends of the nitrocellulose membrane 4 respectively, and the overlapping length is 1 - 2 mm.

[0029] In this embodiment, the particle size of the europium-containing polystyrene fluorescent microspheres is 300 nm. Using 300-nm microspheres can have more binding sites with larger-sized antibodies, making the fluorescence signal higher. At the same time, it also avoids affecting the release of the microsphere and antibody conjugate due to the larger size.

[0030] The detection process of the above test strip is as follows: This test strip is used for in vitro quantitative detection of the pepsinogen content in human lower throat saliva, applying the double-antibody sandwich fluorescence immunoassay chromatography method. During the test, the sample and the diluent are dropped onto the sample pad 2. The pepsinogen antigen in the sample first specifically binds to the pepsinogen-labeled antibody-fluorescent microsphere to form a complex during the chromatography process, and then continues to chromatograph upward. Subsequently, the complex will be bound by the solid-phase pepsinogen-coated antibody coated on the detection T line, and a solid-phase pepsinogen-coated antibody-pepsinogen antigen-pepsinogen-labeled antibody-fluorescent microsphere complex will be formed at the T line position. At the same time, as the liquid chromatographs upward, the pepsinogen-labeled antibody-fluorescent microsphere that is not bound to the pepsinogen antigen in the liquid will form a solid-phase goat anti-mouse IgG-pepsinogen-labeled antibody-fluorescent microsphere complex at the C line. The fluorescent microsphere emits a fluorescence signal under the excitation of the excitation light. The ratio of the T line signal to the C line signal (T / C) is proportional to the pepsinogen concentration in the sample. By comparing with the standard curve built in the supporting dry fluorescence immunoassay analyzer, the pepsinogen concentration in the sample can be directly read from the instrument screen.

[0031] As Figures 1-6 shown, the second embodiment of the present invention is: A preparation method of a human saliva pepsin fluorescence immunoassay chromatography test strip, comprising the following steps: 1), sequentially paste the sample pad, the marker pad, the nitrocellulose membrane and the absorbent pad on the PVC board to obtain the first test strip; 2), cut the first test strip into a human oral pharyngeal saliva pepsin fluorescence immunoassay chromatography test strip with a width of 3.9 mm by a high-speed continuous strip cutter; The preparation method of the marker pad comprises the following steps: S1. Dilute the europium-containing polystyrene fluorescent microspheres with a particle size of 300 nm to a concentration of 0.5 mg / ml using 0.01 M borate buffer at pH 8.0. Then add 10 mM carbodiimide and 15 mM N-hydroxysuccinimide, react at room temperature for 15 min, and centrifuge at 15000 r / min to remove the supernatant; S2. Add the pepsin-labeled antibody with a final concentration of 0.1 mg / mL to the precipitate, react at room temperature for 2 h, then add the blocking agent and treat for 90 min. Centrifuge at 15000 r / min to remove the supernatant, and then add the storage solution to the precipitate to obtain the fluorescent microsphere-labeled antibody complex; S3. Dilute the fluorescent microsphere-labeled antibody complex with the resuspension solution at a ratio of 1:4 to obtain a dilution solution. Use a gold-spraying and membrane-stripping instrument to evenly spray the dilution solution on the glass fiber membrane at a spraying speed of 6 μL / cm, and dry at 37 °C for 240 min to obtain the labeled pad; The preparation method of the nitrocellulose membrane includes the following steps: S1. Dilute the capture antibody solution of pepsin with 0.02 M phosphate buffer at pH = 7.2 containing 0.4% trehalose to a final concentration of 1 - 2 mg / mL to obtain the pepsin antibody coating solution; S2. Dilute goat anti-mouse IgG with 0.02 M phosphate buffer at pH = 7.2 containing 0.4% trehalose to 0.5 - 1.0 mg / mL to obtain the goat anti-mouse IgG coating solution; S3. Use a gold-spraying and membrane-stripping instrument to evenly spray the pepsin coating solution on one place of the nitrocellulose membrane as the detection T line, and evenly spray the goat anti-mouse IgG coating solution on another place of the nitrocellulose-coated plate as the quality control C line. Then place it in an oven at 40 - 45 °C and dry for 12 - 24 h to obtain the dried nitrocellulose membrane.

[0032] In this example, the storage solution in the preparation method of the labeled pad is a mixed solution of 50 mM Tris-base, 3% glycine, 1% BSA, 0.1% sodium caseinate, 0.1% Tween 20, 0.87% sodium chloride +, and 0.1% proclin 300, and the pH of the storage solution is 7.2; The resuspension solution in the preparation method of the labeled pad is a mixed solution of 20 mM phosphate buffer, 1% trehalose, 20% sucrose, 0.87% sodium chloride, 0.1% sodium EDTA, 1% BSA, 0.1% Tween 20, and 0.1% proclin 300, and the pH of the resuspension solution is 7.2; Comparative Examples 1 and 2: Test strips were prepared using europium-containing polystyrene fluorescent microspheres with diameters of 100 nm and 200 nm respectively. For the test strips in Example 2, Comparative Example 1, and Comparative Example 2, the FI values of the T and C lines of the test strips were recorded using a fluorescence immunoassay analyzer (the test results are as Figure 2 shown); As can be seen from the above: the fluorescence signal intensity of the t-line (HT), the fluorescence signal intensity of the c-line (HC), and the ratio of the fluorescence signal intensity of the t-line to the c-line (HT / HC) all increase with the increase in the size of the microspheres, reaching the maximum value when the size of the microspheres is 300 nm. This may be because the microspheres have more antibody binding sites with larger sizes, resulting in higher fluorescence signals. However, larger sizes will affect the release of the microsphere-antibody conjugate. Therefore, the size of the fluorescent microspheres was selected as 300 nm.

[0033] Comparative Examples 3, 4, and 5: Dilution was carried out using borate buffers with pH values of 6.0, 7.2, and 8.5 respectively. For the test strips in Example 2, Comparative Examples 3, 4, and 5, the FI values of the T and C lines of the test strips were recorded using a fluorescence immunoassay analyzer (the test results are as Figure 3 shown); As can be seen from the above: with the increase in the binding pH, HT, HC, and H / H first increase and then decrease, reaching the maximum value at pH 8.0. Therefore, pH 8.0 was selected as the optimal reaction pH.

[0034] Comparative Examples 6, 7, and 8: The fluorescent microsphere-labeled antibody complex was diluted with the resuspension at ratios of 1:1, 1:2, and 1:6 respectively. For the test strips in Example 2, Comparative Examples 6, 7, and 8, the accelerated degradation stability at 37 °C was observed (the test results are as Figure 4 shown); As can be seen from the above: when the ratio is 1:4, HCT / H is the most stable during the 14-day accelerated degradation process. Therefore, the ratio of 1:4 was selected as the optimal dilution ratio.

[0035] Meanwhile, the test strips prepared in this example were tested as follows: Test 1: By detecting other potential interfering substances such as pepsinogen I, pepsinogen II, and trypsin, the specificity of the test strip was evaluated, as Figure 5 shown; Conclusion: The detection results of the interfering substances by this test strip are close to the blank detection results, and the test strip has high specificity for pepsin detection.

[0036] Test 2: By measuring different concentrations of pepsin at 37 °C for 14 days, the stability of the developed test strip was explored. The test results are as Figure 6 shown.

[0037] As can be seen from the above: There is no significant difference in the pepsin concentration between the test strips stored at 37°C and those stored at room temperature, which proves that the strip has good stability at 37°C and can achieve on-site detection.

[0038] Test 3: Evaluate the repeatability of the test strip by analyzing within-batch and between-batch variability. As shown in Table 1.

[0039] Table 1

[0040] As can be seen from the above: The within-batch coefficient of variation (CV) is less than 10%, and the between-batch CV is less than 15%. These results indicate that the test strip has good repeatability.

[0041] Meanwhile, compare the method of detecting pepsin by using the test strip in this embodiment in combination with a fluorescence immunoassay analyzer with other pepsin detection methods, as shown in Table 2.

[0042] Table 2

[0043] As can be seen from the above: The detection method using this test strip in combination with a fluorescence immunoassay analyzer has the advantages of short reaction time (15 minutes), low detection limit (LOD) (1.9 ng / mL), and low cost. Compared with the colloidal gold immunochromatography method and the enzyme-linked immunosorbent assay method, this method has higher sensitivity and accurate quantification of the target analyte. In addition, this method avoids the application conditions of large instruments and meets the requirements of on-site detection.

[0044] In the description of the present invention, it should be understood that the orientation or positional relationship indicated by terms such as "coaxial", "bottom", "one end", "top", "middle", "the other end", "upper", "one side", "top", "inner", "front", "center", "both ends", etc. is the orientation or positional relationship based on the drawings, and is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be construed as a limitation to the present invention.

[0045] In the present invention, unless otherwise clearly specified and limited, terms such as "installed", "set", "connected", "fixed", "swiveling connection", etc. should be understood in a broad sense. For example, it can be a fixed connection, a detachable connection, or integrated; it can be a mechanical connection or an electrical connection; it can be directly connected or indirectly connected through an intermediate medium, and it can be the internal communication of two elements or the interaction relationship between two elements. Unless otherwise clearly limited, for those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific situations.

[0046] Although embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes may be made therein without departing from the principles and spirit of the invention, and the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A human salivary pepsin fluorescence immunochromatographic test strip, characterized in that: It includes a PVC board (1), on the upper surface of which a sample pad (2), a marking pad (3), a nitrocellulose membrane (4) and an absorbent pad (5) are adhesively connected in sequence from one end to the other end. On the surface of the marking pad (3), a pepsin-labeled antibody complex labeled with fluorescent microspheres is sprayed, and the pepsin-labeled antibody complex is europium-containing polystyrene fluorescent microspheres. On the surface of the nitrocellulose membrane (4), a detection T line (6) and a quality control C line (7) are arranged. The detection T line is a pepsin capture antibody, and the quality control C line (7) is goat anti-mouse IgG. The particle size of the europium-containing polystyrene fluorescent microspheres is 300 nm.

2. The human salivary pepsin fluorescence immunochromatographic test strip according to claim 1, wherein: The distance between the detection T line (6) and the quality control C line (7) is 0.5 cm - 1.0 cm.

3. A human salivary pepsin fluorescence immunochromatographic test strip according to claim 1, characterized in that: The content of the pepsin-labeled antibody in the pepsin-labeled antibody complex is 30 - 40 μg.

4. A human salivary pepsin fluorescence immunochromatographic test strip according to claim 1, characterized in that: The fluorescent microsphere-labeled pepsin-labeled antibody complex, the pepsin capture antibody and the goat anti-mouse IgG are all sprayed using a gold-spraying and membrane-stripping instrument.

5. The fluorescence immunochromatographic test strip for human salivary pepsin according to claim 1, wherein: One end of the sample pad (2) overlaps above the marking pad (3), and the overlapping length is 0.5 - 1.5 mm. One end of the marking pad (3) and the absorbent pad (5) that are close to each other respectively overlap at both ends of the nitrocellulose membrane (4), and the overlapping length is 1 - 2 mm.

6. A preparation method of a human salivary pepsin fluorescence immunochromatographic test strip, which is applied to the human salivary pepsin fluorescence immunochromatographic test strip described in any one of claims 1-5, and is characterized in that: It includes the following steps: 1), sequentially paste the sample pad (2), the marking pad (3), the nitrocellulose membrane (4) and the absorbent pad (5) on the PVC board (1) to obtain a first test strip; 2), cut the first test strip into an oral and pharyngeal saliva pepsin fluorescence immunochromatographic test strip with a width of 3.9 mm using a high-speed continuous strip cutter; The preparation method of the marking pad (3) includes the following steps: S1. Dilute the europium-containing polystyrene fluorescent microspheres with a particle size of 300 nm to a concentration of 0.5 mg / ml using a 0.01M borate buffer solution with a pH of 7 - 9, then add a carbodiimide with a concentration of 10 mM and an N-hydroxysuccinimide with a concentration of 15 mM and react at room temperature for 15 min, and then centrifuge at a rate of 15000 r / min to remove the supernatant; S2. Add a pepsin-labeled antibody with a final concentration of 0.1 mg / mL to the precipitate, react at room temperature for 2 h, then add a blocking agent and treat for 90 min, centrifuge at a rate of 15000 r / min to remove the supernatant, and then add a storage solution to the precipitate to obtain a fluorescent microsphere-labeled antibody complex; S3. Dilute the fluorescent microsphere-labeled antibody complex with a resuspension solution at a ratio of 1:1 - 1:6 to obtain a dilution solution, and uniformly spray the dilution solution on the glass cellulose membrane at a spraying speed of 6 μL / cm using a gold-spraying and membrane-stripping instrument, and dry at 37°C for 240 min to obtain the marking pad (3); The preparation method of the nitrocellulose membrane (4) includes the following steps: S1. Dilute the pepsin capture antibody solution with a 0.02M, pH = 7.2 phosphate buffer solution containing 0.4% trehalose to a final concentration of 1 - 2 mg / mL to obtain a pepsin antibody coating solution; S2. Dilute goat anti-mouse IgG to 0.5 - 1.0 mg / mL with 0.02 M phosphate buffer at pH = 7.2 containing 0.4% trehalose to obtain a goat anti-mouse IgG coating solution; S3. Use a gold spraying and membrane scribing instrument to evenly spray the pepsin coating solution on one place of the nitrocellulose membrane as the detection T line, and use the gold spraying and membrane scribing instrument to evenly spray the goat anti-mouse IgG coating solution on another place of the nitrocellulose coated plate as the quality control C line. Then place it in an oven at 40 - 45 °C and dry for 12 - 24 h to obtain the dried nitrocellulose membrane (4).

7. The preparation method of a human salivary pepsin fluorescence immunochromatographic test strip according to claim 6, characterized in that: The storage solution in the preparation method of the labeled pad (3) is a mixed solution of 50 mM Tris-base, 3% glycine, 1% BSA, 0.1% sodium caseinate, 0.1% Tween 20, 0.87% sodium chloride +, and 0.1% proclin 300, and the pH of the storage solution is 7.2; The resuspension solution in the preparation method of the labeled pad (3) is a mixed solution of 20 mM phosphate buffer, 1% trehalose, 20% sucrose, 0.87% sodium chloride, 0.1% sodium EDTA, 1% BSA, 0.1% Tween 20, and 0.1% proclin 300, and the pH of the resuspension solution is 7.

2.

8. The preparation method of a human salivary pepsin fluorescence immunochromatographic test strip according to claim 6, characterized in that: In step S3, the dilution ratio of the fluorescent microsphere-labeled antibody complex to the resuspension solution is 1:

4.

9. The preparation method of a human salivary pepsin fluorescence immunochromatographic test strip according to claim 6, wherein: In step S1, dilution is carried out with a borate buffer at pH 8.0.

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