Quantum dot-based immunochromatographic assay kit for detecting pepsin in saliva and its preparation method

Abstract

This invention belongs to the field of in vitro diagnostic technology, specifically relating to a quantum dot-based immunochromatographic assay kit for detecting pepsin in saliva and its preparation method. This invention achieves adsorption of impurities in saliva samples by adding a chitosan filter pad to the sample pad of the pepsin assay kit. Simultaneously, utilizing the specific binding properties of proteins such as SPA / SPG, a dual SPA / SPG-mediated signal enhancement system is constructed at the binding pad. The T-line is first modified with SPA / SPG before being loaded with pepsin antibody 2. These improvements significantly enhance the antibody conjugation efficiency of the kit, increase the antibody conjugation efficiency at the T-line, reduce non-specific antibody binding, and significantly improve the signal intensity of the kit, ultimately significantly improving the detection sensitivity and accuracy of the kit.

Description

Technical Field

[0001] This invention belongs to the field of in vitro diagnostic technology, specifically relating to a quantum dot-based immunochromatographic assay kit for detecting pepsin in saliva and its preparation method. Background Technology

[0002] Gastroesophageal reflux disease (GERD) is a common digestive disorder characterized by the abnormal reflux of stomach contents (including stomach acid and food) into the esophagus, causing irritation and damage to the esophageal mucosa. Typical symptoms of GERD include acid reflux, heartburn, chest pain, and sore throat. Some patients may also experience difficulty swallowing, hoarseness, and cough. If left uncontrolled for a long time, it can lead to esophagitis, esophageal ulcers, Barrett's esophagus, and even esophageal cancer.

[0003] Traditional diagnostic methods for GERD mainly rely on invasive or semi-invasive techniques such as gastroscopy and 24-hour esophageal pH monitoring. These methods have limitations such as complex operation, poor patient compliance, and high testing costs, making it difficult to meet the needs of large-scale screening and long-term follow-up.

[0004] With the development of non-invasive testing technology, saliva testing has become a research hotspot in the diagnosis of gastroesophageal reflux disease due to its advantages such as convenient sample collection, low testing cost, and the ability to achieve dynamic monitoring. When gastric contents reflux, they can reflux into the esophagus and even the pharynx. Some components can enter the oral cavity with saliva secretion. By detecting specific markers in saliva, such as pepsin and pepsinogen, the frequency and severity of gastric reflux can be indirectly determined, providing a non-invasive solution for disease diagnosis.

[0005] Pepsin, as a hydrolytic enzyme that digests proteins in the stomach, is important for the early diagnosis and monitoring of digestive system diseases such as GERD.

[0006] Quantum dot fluorescence chromatography, combining the high fluorescence properties of quantum dots with the speed of chromatography, has become an ideal technique for the detection of pepsin. However, existing quantum dot fluorescent microsphere immunochromatographic kits face core technical bottlenecks in the detection of pepsin in saliva, specifically:

[0007] Firstly, when quantum dot microspheres are directly coupled with pepsin antibodies, they are easily affected by steric hindrance, which prevents the antibody from fully binding to the active sites on the surface of the microspheres, resulting in a low coupling rate. The effective antibody loading on the surface of quantum dot microspheres is insufficient, and the signal carrier capacity cannot be fully utilized. Furthermore, the binding of pepsin antibodies to quantum dot microspheres is random, and it is possible that the Fab end binds to the quantum dot microspheres. In this case, the antibody cannot bind to the antigen efficiently and specifically, leading to a decrease in the sensitivity of the kit.

[0008] Secondly, when the detection line (T line) on the NC membrane is directly coated with pepsin antibody, it relies only on the weak interaction between the hydroxyl groups on the membrane surface and the amino groups of the pepsin antibody. Furthermore, the antibody adsorption sites are relatively random, and some antibodies may be tightly adsorbed to the NC membrane at the Fab end or even the antibody hypervariable region. This results in the antibody being unable to efficiently capture low concentrations of pepsin-antibody complexes, resulting in weak signal intensity, which cannot cover the low concentration salivary pepsin detection needs of patients with early-stage gastroesophageal reflux disease in clinical practice.

[0009] Third, when T-line antibodies are directly conjugated, the antibodies are prone to exposing non-specific binding sites due to changes in spatial conformation. These sites can then bind to various impurities in saliva, such as mucin and other proteins, leading to an increase in background signal. This further masks the specific signal of low-concentration target analytes, resulting in reduced accuracy.

[0010] Fourth, if saliva samples are not filtered, the mucin will adsorb signal marker particles such as quantum dot microspheres and colloidal gold, causing the signal markers to clump together, resulting in slow or even impossible chromatography. Summary of the Invention

[0011] To address the aforementioned technical problems, this invention provides a quantum dot-based immunochromatographic assay kit for detecting pepsin in saliva and its preparation method.

[0012] The first aspect of the present invention is to provide a quantum dot-based immunochromatographic assay kit for detecting pepsin in saliva, comprising a PVC base plate and a sample pad, a conjugate pad, an NC membrane, and an absorbent pad fixed on the PVC base plate and sequentially overlapping each other, wherein the NC membrane is provided with T lines and C lines, and the C lines are coated with goat anti-mouse IgG antibodies.

[0013] The above-described structures are standard settings for the reagent kit and are not improvements of this invention. The main improvements of this invention to the reagent kit are as follows:

[0014] Firstly, a chitosan filter pad with a width of 15-18 mm is set 5-15 mm upstream of the sample pad. In this invention, "upstream" and "downstream" are both based on the flow direction of the sample to be tested, that is, the sample to be tested is chromatographically deposited from the "upstream" of the test strip to the "downstream".

[0015] Secondly, the pad is coated with a quantum dot microsphere-IgBPs-pepsin antibody 1 complex;

[0016] Third, the T-line is coated with a mixture of IgBPs-pepsin antibody 2 complex and IgBPs-pepsin antibody 1 complex.

[0017] In fact, in this invention, the ideal state of the T-line is that it is only coated with the IgBPs-pepsin antibody 2 complex. However, in the actual preparation process, when pepsin antibody 2 is used to bind with IgBPs, some of the active sites of IgBPs will remain. When the sample is detected, these unbound remaining active sites may specifically bind to the quantum dot microspheres-IgBPs-pepsin antibody 1 complex released on the binding pad, thus producing a weak false positive. However, after the inventors use pepsin antibody 1 to block the possible remaining active sites in IgBPs, the above problem can be completely avoided, thereby further ensuring the detection specificity of the kit.

[0018] The "IgBPs" mentioned in this invention are officially called "bacterial surface immunoglobulin binding proteins", and the IgBPs are selected from at least one of staphylococcal A protein (SPA) and streptococcal G protein (SPG).

[0019] As a further explanation of the present invention, in the above-mentioned kit, preferably, the quantum dots are 80~300 nm carboxylated quantum dot microspheres with a fluorescence emission wavelength of 620~650 nm and a fluorescence quantum yield ≥85%.

[0020] Preferably, the quantum dot microspheres are selected from quantum dot fluorescent microspheres with particle sizes of 85 nm, 100 nm, 200 nm, and 300 nm.

[0021] Preferably, the quantum dot microsphere-IgBPs-pepsin antibody 1 complex is prepared using the following method:

[0022] First, a quantum dot microsphere solution was prepared using MES buffer and activated in an EDC / NHS system. Then, IgBPs were added to the activated system, and the mixture was shaken at 30-40°C for 2-3 hours to obtain a quantum dot microsphere-IgBPs complex. After the reaction, borate buffer containing 1% bovine serum albumin (BSA) was added as a blocking solution to block the sites on the quantum dot microspheres that were not bound to IgBPs at room temperature. Finally, pepsin antibody 1 was added to the complex, and the mixture was shaken at 35-40°C for 2-4 hours. After centrifugation and washing, the quantum dot microsphere-IgBPs-pepsin antibody 1 complex was obtained. Finally, pepsin antibody 2 was added to block the active sites on the IgBPs that were not bound to pepsin antibody 1.

[0023] Preferably, the concentration of the MES buffer is 0.02~0.08 M and the pH is 6.0.

[0024] Preferably, the concentration (w / v) of the quantum dot microspheres is 0.25% to 1.5%.

[0025] Preferably, the EDC / NHS system is a MES buffer solution of EDC and NHS, wherein the concentration of EDC is 10~20 mg / mL and the concentration of NHS is 5~20 mg / mL.

[0026] Preferably, the volume ratio of the quantum dot microsphere solution to the EDC / NHS system is 1:0.02~0.3, the mass ratio of the quantum dot microspheres to IgBPs is 40~60:1, the molar ratio of IgBPs to pepsin antibody 1 is 1:1.1~2, the amount of pepsin antibody 2 added is 0.005%~0.02% (w / v), and the concentration of the borate buffer containing 1% BSA is 22~25mM, with a pH of 7.2~7.8.

[0027] A second aspect of the present invention is that a method for preparing the above-mentioned reagent kit is provided, comprising the following steps:

[0028] (1) Raw material preparation: Prepare IgBPs solution with a concentration of 0.2-0.8 mg / mL, pepsin antibody 2 solution with a concentration of 0.8-1.5 mg / mL, pepsin antibody 1 solution with a concentration of 1-2 mg / mL, and goat anti-mouse IgG antibody solution with a concentration of 0.5-1.5 mg / mL using 0.01-0.05 M phosphate (PBS) buffer with a pH of 7.1-7.8. Prepare a quantum dot microsphere-IgBPs-pepsin antibody 1 complex solution with a concentration of 0.05%-0.2% using 22-25 mM Tris-HCl buffer with a pH of 7.2-7.8 for later use;

[0029] (2) Preparation of T lines: First, IgBPs solution was sprayed onto the NC membrane and dried to form an NC membrane-IgBPs substrate. Then, T lines were drawn on the NC membrane-IgBPs substrate using pepsin antibody 2 solution. The width of the NC membrane-IgBPs substrate area minus the width of the T lines was ≤60μm. After drying again, pepsin antibody 1 solution was uniformly sprayed onto the T line area of ​​the NC membrane-IgBPs substrate. After spraying, the reaction was carried out at room temperature for 20~50 min and then dried to obtain T lines modified with “IgBPs-pepsin antibody 2 complex, IgBPs-pepsin antibody 1 complex”.

[0030] (3) Preparation of C line: Draw C line 5 mm downstream of T line with goat anti-mouse IgG antibody solution. The amount of goat anti-mouse IgG antibody solution used for drawing is 0.5~1.5 μL / cm. Dry.

[0031] (4) Preparation of sample pads: Immerse the sample pads in PBS buffer containing 0.2% Tween-20, 0.5% BSA and 0.02% ProClin300 for 20 min, then remove and dry.

[0032] (5) Preparation of chitosan filter pad: Dissolve chitosan in 0.5%~1.0% acetic acid solution to prepare a chitosan solution with a concentration of 1%~3%, and adjust the pH value to 6.0~6.5 with 1M NaOH solution. Then, coat it evenly on the glass fiber membrane, dry it at 55℃, cut it into (15~18)×300 mm filter pads and add them to the upstream of the sample pad at 8~12 mm.

[0033] (6) Preparation of the conjugate pad: The conjugate pad was fully immersed in the quantum dot microsphere-IgBPs-pepsin antibody 1 complex solution, and then dried after removal;

[0034] (7) Assembly of the kit: Attach the components prepared in the above steps to the PVC base plate in sequence, cut and package.

[0035] As a further explanation of the present invention, in the above preparation method, preferably, in (2), the spraying amount of the IgBPs solution is 1~1.5 μL / cm, the coating amount of the pepsin antibody 2 solution is 0.8~1.5 μL / cm, and the spraying amount of the pepsin antibody 1 solution is 0.5~1.0 μL / cm.

[0036] Preferably, the drying process involves drying to a moisture content of ≤5%.

[0037] The terms used in this invention and their corresponding meanings are as follows:

[0038] "NC membrane" refers to nitrocellulose membrane, "T line" is the test line, and "C line" is the quality control line;

[0039] "Bacterial surface immunoglobulin-binding proteins" are referred to as IgBPs in this invention for convenience.

[0040] “SPA” stands for Staphylococcus Protein A.

[0041] “SPG” stands for Streptococcus Protein G.

[0042] “EDC” stands for N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride;

[0043] “NHS” stands for N-hydroxysuccinimide;

[0044] "Pepsin Antibody 1" is a mouse anti-human pepsin IgG monoclonal antibody with anti-pepsin epitope A and a titer of ≥1:10000 against human pepsin.

[0045] "Pepsin Antibody 2" is a mouse anti-human pepsin IgG monoclonal antibody with anti-pepsin epitope B. Its titer against human pepsin is ≥1:10000. Here, A and B represent only two different sites of the pepsin antigenic epitope.

[0046] The advantages of this invention compared to the prior art are as follows:

[0047] (1) The present invention sets a chitosan filter pad "upstream" of the sample pad, which can adsorb macromolecules such as mucin, amylase, and lysozyme in saliva samples, and avoid these macromolecules from interfering with the target detection substance pepsin (a low molecular weight protein with a molecular weight of tens of thousands) by adsorbing particles such as viruses, bacteria, and dust, or adsorbing markers such as quantum dot microspheres.

[0048] (2) Significantly improved antibody conjugation efficiency: This invention utilizes the characteristic that IgBPs proteins can specifically bind to the Fc end of pepsin antibodies without affecting the antigen-binding activity of the Fab end to construct a "dual IgBPs-mediated" signal enhancement system, which realizes the directional and efficient loading of pepsin antibody 1 on quantum dot microspheres, ensuring that there are more active antibodies that can bind pepsin on the surface of each quantum dot microsphere; Experimental results show that IgBPs directionally bind to the Fc end of the antibody, allowing the Fab end to fully extend to the periphery of the microsphere, improving the specific binding efficiency of the antibody and the antigen, and also helping to avoid antibody conformational changes and reduce non-specific binding rate. The detection rate difference among the three batches of kits is ≤1.5%, and the stability of industrial production is good.

[0049] (3) Increased utilization rate of T-line antibody: In this invention, IgBPs are first sprayed onto the T-line position of the NC membrane to form an NC membrane-IgBPs substrate, and then the T-line is sprayed onto the IgBPs substrate. Pepsin antibody 2 is then coated on it. Finally, pepsin antibody 1 is used to saturate and bind to the remaining IgBPs active sites on the T-line. This not only fully releases the effective binding site Fab of pepsin antibody 2 by specifically binding to the Fc end of pepsin antibody 2 by IgBPs, thus improving the effectiveness of pepsin antibody 2, but also reduces non-specific reactions by pepsin antibody 1 while supplementing the T-line. The pepsin binding site forms a dual capture mode of "quantum dot microspheres-IgBPs-antibody 1-pepsin-antibody 2-IgBPs-NC membrane" and "unbound free pepsin-antibody 1-IgBPs-NC membrane". On the one hand, it enhances the signal, and on the other hand, it reduces the hook effect caused by excessive pepsin. The detection limit of salivary pepsin is reduced from the conventional 8 ng / mL to 3 ng / mL, which can cover the low concentration sample detection needs of patients with early gastroesophageal reflux disease in clinical practice. At the same time, it effectively reduces the missed detection caused by insufficient sensitivity and assists in early clinical diagnosis. Attached Figure Description

[0050] Figure 1 A side view of the reagent kit provided by the present invention;

[0051] Figure 2 The diagram shows a structural comparison between the quantum dot microsphere-IgBPs-pepsin antibody 1 complex provided by the present invention and the traditional quantum dot microsphere directly bound to pepsin antibody 1. In the diagram, A is a schematic diagram of the traditional quantum dot microsphere directly bound to pepsin antibody 1, and B is a schematic diagram of the quantum dot microsphere-IgBPs-pepsin antibody 1 complex.

[0052] Figure 3 This is a schematic diagram of the preparation process of the T-line in the kit provided by the present invention;

[0053] Figure 4 The images show the actual detection results of the reagent kits prepared in Example 1 and Comparative Example 2 of this invention. The left image shows the detection results of the test strip in Comparative Example 2, and the right image shows the detection results of the test strip in Example 1.

[0054] Figure 5 The images show the actual detection results of the kits prepared in Example 3 and Comparative Example 1 of this invention. The left image shows the detection results of the test strip box in Comparative Example 1, and the right image shows the detection results of the kit in Example 3.

[0055] Figure 6 The images show the actual detection results of the test kits prepared in Example 1 and Comparative Example 3 of the present invention. The left image shows the detection results of the test kit in Comparative Example 3, and the right image shows the detection results of the test kit in Example 1. Detailed Implementation

[0056] To enable those skilled in the art to better understand the present invention, the present invention will now be further described in conjunction with specific embodiments.

[0057] Raw materials and reagents used in this invention:

[0058] Quantum dot fluorescent microspheres: Carboxylated quantum dot microspheres with wavelengths of 80–300 nm (85 nm, 100 nm, 200 nm, 300 nm) are selected, with fluorescence emission wavelengths of 620–650 nm and fluorescence quantum yields ≥85%;

[0059] 1.0% quantum dot microsphere solution: Prepare a 1.0% (w / v) quantum dot microsphere solution using MES buffer solution with a concentration of 0.02~0.1M and a pH of 5.5~6.5;

[0060] SPA (Staphylococcal protein A, purity ≥99%)

[0061] Pepsin monoclonal antibody 1 (anti-pepsin epitope A, titer ≥1:10000).

[0062] Pepsin monoclonal antibody 2 (anti-pepsin epitope B, titer ≥1:10000).

[0063] Goat anti-mouse IgG antibody (for C-line coating, titer ≥1:8000).

[0064] Example 1

[0065] A quantum dot-based immunochromatographic assay kit for detecting pepsin in saliva, its side view is shown below. Figure 1 As shown, from the chromatographic direction of the sample being tested, from "upstream" to "downstream" are the filter pad, sample pad, conjugate pad, NC membrane (T line, C line), and absorbent pad.

[0066] The kit is prepared using the following method:

[0067] (1) Raw material preparation: 0.05M MES buffer (pH 6.0), 25mM Tris-HCl buffer (pH 7.6), 0.01M PBS buffer (pH 7.4), EDC solution: 15 mg / mL MES buffer, NHS solution: 10 mg / mL MES buffer, 1% BSA borate buffer (25mM, pH 7.4).

[0068] 0.5 mg / mL SPG solution, 1.0 mg / mL pepsin antibody 2 solution, 1.5 mg / mL pepsin antibody 1 solution, and 1.0 mg / mL goat anti-mouse IgG antibody solution were prepared using 0.01 M PBS buffer and set aside for later use.

[0069] Preparation of quantum dot microsphere-SPG-pepsin antibody 1 complex:

[0070] Quantum dot microspheres coupled with SPG: First, take 1 mL of 1.0% quantum dot fluorescent microsphere solution. The quantum dot microspheres have a particle size of 300 nm and a fluorescence emission wavelength of 625 nm. Wash three times with 0.05 M MES buffer (8000 rpm, 10 min), and resuspend to 1 mL. Add 100 μL of EDC solution and 100 μL of NHS solution, activate at room temperature for 30 min, and centrifuge to remove the supernatant (4℃, 8000 rpm, 20 min). Add 20 mM borate buffer, resuspend the quantum dot microspheres in 500 μL, add 200 μg of SPG, and react with shaking at 37℃ for 2.5 h to form a quantum dot microsphere-SPG complex. After the reaction, add borate buffer containing 1% BSA as a blocking solution to block the unbound active sites in SPG at room temperature. Wash three times with 0.01 M PBS buffer and resuspend to 1 mL.

[0071] Saturation binding of pepsin antibody 1: 400 μg of pepsin antibody 1 solution (calculated at a molar ratio of SPG to pepsin antibody 1 of 1:1.2 to ensure saturation) was added to the obtained quantum dot microsphere-SPG complex. The mixture was shaken at 37 °C for 3 h. After the reaction, 200 μg of pepsin antibody 2 solution was added to block the unbound active sites of pepsin antibody 1. The mixture was blocked at room temperature for 1 h, centrifuged and washed twice (8000 rpm, 10 min), and resuspended in borate buffer containing 0.1% BSA to 1 mL. The mixture was then sealed and stored at 4 °C to obtain the quantum dot microsphere-SPG-pepsin antibody 1 complex. The quantum dot microsphere-IgBPs-pepsin antibody 1 complex was prepared into a 0.1% solution using 25 mM, pH 7.6 Tris-HCl buffer for later use.

[0072] The structural diagram of the quantum dot microsphere-IgBPs-pepsin antibody 1 complex obtained in this step is shown below. Figure 2 As shown in B, Figure 2 Image A is a schematic diagram of the structure of traditional quantum dot microspheres directly binding to pepsin antibody 1. (It should be noted that...) Figure 2 The structures mentioned do not refer to the actual structures of the parts, and the size or shape of each part does not represent a complete correspondence with the actual protein; they are merely for the purpose of understanding the connection relationships between the parts.

[0073] Figure 2 As shown in Figure A, the traditional quantum dot microsphere-pepsin antibody 1 complex is susceptible to steric hindrance during preparation, which prevents pepsin antibody 1 from fully binding to the active sites on the microsphere surface, resulting in a low coupling rate. Furthermore, the figure also shows that the binding of pepsin antibody 1 to quantum dot microspheres is random; it may be the Fab end of the antibody that binds to the quantum dot microspheres, or it may be the Fc end of the antibody that binds to the quantum dot microspheres. This leads to the antibody's inability to bind efficiently and specifically to the antigen, resulting in reduced sensitivity of the kit.

[0074] In this invention, IgBPs (SPG or SPA) are first coupled with quantum dot microspheres. In this embodiment, SPG protein is used for coupling. IgBPs can specifically bind to the Fc end of pepsin antibody 1 without affecting the antigen binding activity of the Fab end. This allows the Fab end of pepsin antibody 1 to fully extend to the periphery of the microsphere, reducing the decrease in antibody activity caused by non-specific binding. At the same time, it also improves the coupling rate of pepsin antibody 1, and ultimately further improves the effective loading rate of pepsin antibody 1.

[0075] (2) Preparation of T-lines, according to Figure 3 The steps are as follows: First, spray 0.5 mg / mL SPG solution onto the NC membrane at a spray volume of 1.2 μL / cm, and dry at 37°C for 3 hours to form the NC membrane-SPG substrate. Then, use 1.0 mg / mL pepsin antibody 2 solution to draw T-lines on the NC membrane-SPG substrate at a spray volume of 1.0 μL / cm. The T-lines should overlap the NC membrane-SPG substrate area as completely as possible, or the width of the T-lines should be slightly smaller than the width of the NC membrane-SPG substrate area, with a difference not exceeding 60 μm. After drying again at 37°C for 2 hours, use pepsin antibody 1 to block the remaining active sites in the T-lines that were not bound by pepsin antibody 2. For greater assurance, in this invention, 1.5 mg / mL SPG solution is used. The mg / mL pepsin antibody 1 solution was uniformly sprayed onto the T-line area, completely overlapping with or slightly covering the NC membrane-SPG substrate area. The spraying amount of pepsin antibody 1 was 0.8 μL / cm, ensuring that the molar ratio of (antibody 1 + antibody 2) to IgBPs was >1.5:1. After spraying, the reaction was carried out at room temperature for 30 min and dried at 37℃ for 2 h to obtain the T-line modified with "SPG-pepsin antibody 2, SPG-pepsin antibody 1".

[0076] (3) Preparation of C line: Draw C line 5 mm downstream of T line with 1.0 mg / mL goat anti-mouse IgG antibody solution, with a streaking volume of 1.0 μL / cm, dry at 37℃ for 3 h, and store in a sealed container.

[0077] (4) Preparation of sample pads: Immerse the sample pads in PBS buffer containing 0.2% Tween-20, 0.5% BSA and 0.02% ProClin300, and dry at 37°C for 1.5 h;

[0078] (5) Preparation of chitosan filter pad: Dissolve 2% chitosan in 0.8% acetic acid solution to prepare a uniform solution. Adjust the pH value to 6.5 with 1M NaOH solution. Then coat it evenly on the glass fiber membrane, dry at 55℃, cut into 18×300 mm filter pads and add them to the "upstream" 10 mm of the sample pad.

[0079] (6) Preparation of conjugate pad: The conjugate pad was immersed in 0.1% quantum dot microsphere-SPG-pepsin antibody 1 complex solution, dried at 37°C for 2 h, and sealed for storage to ensure that the conjugate pad was uniformly loaded with the complex.

[0080] (7) Assembly of the kit: Attach the components prepared in the above steps to the PVC base plate in sequence, cut and package.

[0081] Example 2

[0082] Unlike Example 1, SPA is used instead of SPG, while the rest of the operations and conditions are the same as in Example 1.

[0083] Example 3

[0084] A quantum dot-based immunochromatographic assay kit for detecting pepsin in saliva is prepared using the following method:

[0085] (1) Raw material preparation: 0.05M MES buffer (pH 6.0), 25mM Tris-HCl buffer (pH 7.6), 0.01M PBS buffer (pH 7.4), EDC solution: 15mg / mL MES buffer, NHS solution: 10mg / mL MES buffer, 1% BSA borate buffer (25mM, pH 7.4); SPA and SPG mixture (SPA / SPG) was used instead of SPG in Example 1. In SPA / SPG, the molar ratio of SPA:SPG was 1:1 and the concentration was 0.5 mg / mL.

[0086] Prepare 1.0 mg / mL pepsin antibody 2 solution, 1.5 mg / mL pepsin antibody 1 solution, and 1.0 mg / mL goat anti-mouse IgG antibody solution using 0.01 M PBS buffer for later use.

[0087] Preparation of quantum dot microsphere-SPA / SPG-pepsin antibody 1 complex:

[0088] Take 1 mL of quantum dot fluorescent microspheres (same as in Example 1), centrifuge and wash three times with 0.05 M MES buffer, and resuspend to 1 mL; add 100 μL of EDC solution and 100 μL of NHS solution, activate at room temperature for 30 min, centrifuge and remove supernatant; add 20 mM borate buffer, resuspend in 500 μL, add 200 μg of SPA / SPG mixed protein solution, and react with shaking at 37 °C for 2.5 h to form quantum dot microsphere-SPA / SPG complex. After the reaction, add borate buffer containing 1% BSA as blocking solution to block unbound sites at room temperature, centrifuge and wash three times with 0.01 M PBS buffer, and resuspend to 1 mL; add 400 μg of pepsin antibody 1 solution to the complex, react with shaking at 37 °C for 3 h, and after the reaction, add 200 μg of pepsin antibody 2 solution to block the unbound active sites of pepsin antibody 1, block at room temperature for 1 h, centrifuge and wash twice, and use 0.1% BSA buffer to block the unbound active sites of pepsin antibody 1. BSA was resuspended in borate buffer to 1 mL and stored at 4°C to obtain the quantum dot microsphere-SPA / SPG-pepsin antibody 1 complex. The complex was then prepared into a 0.1% solution using 25 mM Tris-HCl buffer for later use.

[0089] (2) Preparation of T line: The difference from Example 1 is that 0.5 mg / mL of SPA / SPG mixed solution was sprayed onto the NC membrane and dried to form NC membrane-SPA / SPG substrate. Other operations and conditions are the same as in Example 1.

[0090] Comparative Example 1

[0091] The difference between the reagent kit in this comparative example and that in Example 1 is that the kit was prepared differently:

[0092] (2) In the preparation of T line, the base layer is no longer sprayed on the NC membrane in advance, that is, the T line has no IgBPs base. The T line is directly drawn on the NC membrane using pepsin antibody 2. Other operations are the same as in Example 1.

[0093] Comparative Example 2

[0094] The difference between this comparative example and Example 1 is as follows:

[0095] The preparation steps of the chitosan filter pad in (5) are omitted. The sample pad is directly attached to the conjugate pad, and the saliva sample is directly dripped onto the sample pad during the test. Other steps (complex preparation, T / C line, conjugate pad) and operations are the same as in Example 1.

[0096] Comparative Example 3

[0097] The difference between this comparative example and Example 1 is as follows:

[0098] In the case of the binding pad, the quantum dot microspheres and pepsin antibody 1 bind in a different way. The quantum dot microspheres are directly coupled to pepsin antibody 1, and the other steps are the same as in Example 1.

[0099] Comparative Example 4

[0100] The reagent kit used in this comparative example is a conventional reagent kit, which differs from that in Example 1 in that:

[0101] On the binding pad, quantum dot microspheres were directly coupled with pepsin antibody 1 without the use of IgBPs for loading (same as comparative example 3).

[0102] T-line directly coated with pepsin antibody 2 (same as control 1).

[0103] No chitosan filter pad was used; all other steps were the same as in Example 1.

[0104] Application Example 1

[0105] The effectiveness of the kits prepared in the above embodiments and comparative examples was verified by the following methods:

[0106] 1.5 mL of fasting saliva was collected from the subject, centrifuged at 4000 rpm for 5 min to remove the precipitate, and 100 μL of the supernatant was added to 300 μL of diluent and mixed well. The mixture was then added to the sample wells of the kit, and the results were determined using a fluorescence immunochromatographic analyzer after 12-15 minutes. The chemiluminescence immunoassay kit from Jinan Saiji Medical Devices Co., Ltd. was used to detect the lowest detection limit of pepsin (3 ng / mL, clinical low concentration threshold) determined in the preliminary experiment. Three batches of the kit (batch numbers: 20240801, 20240802, 20240803) were used for continuous testing for 3 days, one round of testing per day. Each batch of kits was used to repeat the 3 ng / mL sample 20 times per round, resulting in 60 tests per day for a single sample (3 batches × 20 tests = 60 tests), and a total of 180 tests over 3 days. Three parallel 3 ng / mL samples were also included, resulting in a total of 540 tests (3 samples × 180 tests = 540 tests).

[0107] The experimental results are shown in Table 1 below. Figure 4 .

[0108] Table 1. Detection results of the reagent kits prepared for each experimental group

[0109] Group Total number of tests (times) Number of detections (times) Detection rate (%) Average fluorescence intensity of T-line (AU) Example 1 540 513 95.0 6800±350 Example 2 540 510 94.4 6700±300 Example 3 540 522 96.7 7000±320 Comparative Example 1 540 432 80.0 4000±250 Comparative Example 2 540 443 82.0 3950±260 Comparative Example 3 540 421 78.0 3600±270 Comparative Example 4 540 385 71.2 3100±290

[0110] The results in Table 1 above show that, when using the improved kits in the various embodiments of the present invention to detect pepsin in saliva samples compared with the kits in Comparative Examples 1-4, after 540 tests, the detection rate of the kits prepared by the methods in each embodiment for salivary pepsin samples with a detection limit of 3 ng / mL was consistently around 95%, with a maximum detection rate of 96.7%, which is 25.5% higher than the conventional kit (71.2%). The difference in detection rate among the three batches of kits was ≤1.5%, indicating that the kits obtained after the improvement of the kits by the method provided by the present invention have high detection sensitivity and good stability, and can effectively detect low concentrations of salivary pepsin.

[0111] in addition, Figures 4-6 The detection performance of the kits prepared in each embodiment and comparative example is shown.

[0112] Figure 4 In the middle, the left side shows the test strip of Comparative Example 2 without a chitosan filter pad, and the right side shows the test strip of Example 1. Figure 4 As can be seen, when testing the same sample, the quantum dot microsphere markers on the conjugate pad of the test strip with the added filter pad are released more fully, and the signals of the T line and C line are also stronger. In contrast, the quantum dot microsphere markers on the conjugate pad of the test strip without the filter pad have more residue, and the signals of the T line and C line are weaker than those of the test strip in Example 1. This fully demonstrates that non-detectable impurities in the saliva sample have a significant impact on the detection results, and the addition of the chitosan filter pad significantly improves the detection sensitivity of the test strip.

[0113] Figure 5 The left side of the image shows the kit for Comparative Example 1, and the right side shows the kit for Example 3. As can be seen, the fluorescence intensity of the kit for Example 3 is significantly stronger than that of the kit for Comparative Example 1, with a clear fluorescence intensity at the T-line. However, the fluorescence intensity of the kit for Comparative Example 1 at the T-line is extremely weak, even invisible to the naked eye. The direct cause of this phenomenon may be that during the preparation of the T-line in Comparative Example 1, SPG protein was not pre-coated onto the NC membrane as a base layer; instead, pepsin antibody was directly used for coating. On the one hand, the binding of the pepsin antibody to the membrane is weak; on the other hand, the antibody's adsorption sites are relatively random, resulting in the antibody's inability to efficiently capture low concentrations of the pepsin-antibody complex, ultimately leading to a weak signal intensity.

[0114] Figure 6The left side of the image shows the kit prepared in Comparative Example 3, and the right side shows the kit prepared using the method in Example 1. As can be seen in the image, the kit prepared in Example 1 exhibits obvious fluorescence at the T line. However, in Comparative Example 3, because the quantum dot microspheres directly bind to pepsin antibody 1, the coupling efficiency is low, resulting in insufficient effective antibody loading of pepsin on the surface of the quantum dot microspheres. Consequently, the signal carrier capacity cannot be fully utilized, and the signal intensity of the T line is not obvious.

Claims

1. A quantum dot-based immunochromatographic test kit for pepsin in saliva, comprising a PVC base plate and a sample pad, a conjugate pad, an NC membrane, and an absorbent pad fixed on the PVC base plate and sequentially overlapped with each other, wherein the NC membrane is provided with a T line and a C line, and the C line is coated with a goat anti-mouse IgG antibody, characterized in that: a chitosan filter pad with a width of 15-18 mm is arranged at 5-15 mm "upstream" of the sample pad; the conjugate pad is coated with a quantum dot microsphere-IgBPs-pepsin antibody 1 complex; and the T line is coated with a mixture of an IgBPs-pepsin antibody 2 complex and an IgBPs-pepsin antibody 1 complex. The IgBPs are bacterial surface immunoglobulin binding proteins, and the IgBPs are at least one selected from staphylococcal protein A and streptococcal protein G. The quantum dots are 80-300 nm carboxylated quantum dot microspheres with a fluorescence emission wavelength of 620-650 nm and a fluorescence quantum yield of ≥ 85%.

2. The kit of claim 1, wherein The quantum dot microsphere-IgBPs-pepsin antibody 1 complex is prepared by the following method:

3. The kit of any one of claims 1-2, wherein First, a quantum dot microsphere solution is prepared using an MES buffer, activated in an EDC / NHS system, then IgBPs are added to the activated system, and oscillation reaction is carried out at 30-40 ℃ for 2-3 h to obtain a quantum dot microsphere-IgBPs complex, after the reaction, a 1% bovine serum albumin-containing boric acid buffer is added as a blocking solution to block the sites of the quantum dot microspheres that are not combined with IgBPs at room temperature, finally, pepsin antibody 1 is added to the above complex, and oscillation reaction is carried out at a temperature of 35-40 ℃ for 2-4 h, centrifugal washing is performed, and the quantum dot microsphere-IgBPs-pepsin antibody 1 complex is obtained, and finally, pepsin antibody 2 is added to block the active sites of IgBPs that are not combined with pepsin antibody 1. The concentration of the MES buffer is 0.02-0.08 M, and the pH is 6.0; the concentration (w / v) of the quantum dot microsphere solution is 0.25%-1.5%, the EDC / NHS system is an EDC, NHS MES buffer, wherein the concentration of EDC is 10-20 mg / mL, and the concentration of NHS is 5-20 mg / mL, the volume ratio of the quantum dot microsphere solution to the EDC / NHS system is 1:0.02-0.3, the mass ratio of the quantum dot microspheres to IgBPs is 40-60:1, the molar ratio of IgBPs to pepsin antibody 1 is 1:1.1-2, the addition amount of pepsin antibody 2 is 0.005%-0.02% (w / v), the concentration of the 1% bovine serum albumin-containing boric acid buffer is 22-25 mM, and the pH is 7.2-7.

8.

4. The kit of claim 3, wherein The method comprises the following steps:

5. The method of claim 1 to 4, wherein the kit is prepared by mixing the components of the kit. ​ (1) Preparation of raw materials: 0.01-0.05 M, pH 7.1-7.8 phosphate buffer solution was used to prepare 0.2-0.8 mg / mL IgBPs solution, 0.8-1.5 mg / mL pepsin antibody 2 solution, 1-2 mg / mL pepsin antibody 1 solution, and 0.5-1.5 mg / mL goat anti-mouse IgG antibody solution; 22-25 mM, pH 7.2-7.8 Tris-HCl buffer solution was used to prepare quantum dot microsphere-IgBPs-pepsin antibody 1 complex solution with a concentration of 0.05%-0.2%, which was ready for use; (2) Preparation of T line: first, spray IgBPs solution on the NC film to form NC film-IgBPs substrate after drying, then draw T line on the NC film-IgBPs substrate using pepsin antibody 2 solution, the width of the NC film-IgBPs substrate region-T line is ≤60 μm, after drying again, uniformly spray pepsin antibody 1 solution on the T line region, after spraying, react at room temperature for 20-50 min, and dry to obtain "IgBPs-pepsin antibody 2 complex, IgBPs-pepsin antibody 1 complex" modified T line; (3) Preparation of C line: draw C line 5 mm downstream of the T line using goat anti-mouse IgG antibody solution, the amount of goat anti-mouse IgG antibody solution sprayed on the membrane is 0.5-1.5 μL / cm, and dry; (4) Preparation of sample pad: soak the sample pad in phosphate buffer solution containing 0.2% Tween-20, 0.5% BSA, and 0.02% ProClin300, take out after soaking, and dry; (5) Preparation of chitosan filter pad: dissolve chitosan in 0.5%-1.0% acetic acid solution to prepare a chitosan solution with a concentration of 1%-3%, and adjust the pH value to 6.0-6.5 with 1 M NaOH solution, then uniformly coat the solution on a glass fiber membrane, dry at 55°C, cut into (15-18) × 300 mm filter pads, and add to the sample pad 8-12 mm upstream; (6) Preparation of binding pad: soak the binding pad in quantum dot microsphere-IgBPs-pepsin antibody 1 complex solution, take out after soaking, and dry; (7) Assembly of kit: sequentially paste each component prepared in the above steps on a PVC base plate, cut, and package.

6. The production method according to claim 5, wherein In (2), the spraying amount of the IgBPs solution is 1-1.5 μL / cm, the amount of pepsin antibody 2 solution sprayed on the membrane is 0.8-1.5 μL / cm, and the spraying amount of pepsin antibody 1 solution is 0.5-1.0 μL / cm.

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