A fluorescent immunochromatographic test strip for detecting free IgE and a preparation method thereof

Abstract

The application provides a fluorescent immunochromatography test strip for detecting free IgE and a preparation method thereof. A second buffer solution containing a mouse anti-human IgG monoclonal antibody is used to treat a second cellulose membrane to obtain a filter membrane; a third buffer solution containing a fluorescent microsphere-labeled FcERI antigen and a fluorescent microsphere-labeled DNP-BSA is used to treat a third cellulose membrane to obtain a conjugate pad; a detection line and a quality control line are arranged on a fourth cellulose membrane to obtain a detection membrane, wherein the detection line is coated with a mouse anti-human IgE antibody, and the quality control line is coated with a DNP monoclonal antibody; a sample pad, the filter membrane, the conjugate pad, the detection membrane and water absorption material are sequentially loaded on a bottom plate to obtain the fluorescent immunochromatography test strip for detecting free IgE. The fluorescent immunochromatography test strip has the advantages of high sensitivity, high accuracy, good stability, good repeatability, simple operation and the like, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of immunofluorescence detection technology, specifically relating to a fluorescent immunochromatographic test strip for detecting free IgE and its preparation method. Background Technology

[0002] Free IgE refers to immunoglobulin E (IgE) in serum that is not bound to cells. IgE is one of the most abundant immunoglobulins in human serum and plays an important role in type I hypersensitivity reactions, also known as allergic reactions. IgE can bind to high-affinity IgE receptors (FcεRI) on the surface of effector cells (such as mast cells and basophils). When allergens re-enter the body, they can trigger these cells to release inflammatory mediators such as histamine, leading to allergic symptoms.

[0003] In the treatment of allergic diseases such as asthma, the application of anti-IgE antibodies, such as omalizumab, is of great significance. Omalizumab is a recombinant 95% humanized monoclonal anti-IgE antibody that inhibits the release of inflammatory mediators by binding to free IgE, preventing IgE from binding to high-affinity IgE receptors on the surface of effector cells, thereby achieving therapeutic goals. Furthermore, the use of omalizumab can reduce the level of free IgE and downregulate the expression of FcεRI on the surface of effector cells, thus weakening the effector effect of IgE. Therefore, the detection of free IgE helps to further understand the clinical efficacy of omalizumab and is of important guiding significance for identifying specific allergens in patients, assessing symptom severity, and guiding the clinical use of omalizumab.

[0004] Currently, enzyme-linked immunosorbent assay (ELISA) is the most common method for measuring free IgE in clinical and laboratory settings. ELISA suffers from poor precision and sensitivity linearity, and is also characterized by numerous steps and long detection times. While fluorescent immunochromatographic test strips offer advantages such as short detection time and ease of use, there are currently no reports or commercially available products specifically designed for detecting free IgE. Furthermore, patients treated with omalizumab often experience a significant decrease in blood free IgE levels, making it difficult to detect these levels once treatment begins. Therefore, there is a need to develop fluorescent immunochromatographic test strips that simultaneously guarantee the sensitivity, accuracy, and stability required for free IgE detection, and are suitable for patients treated with omalizumab. Summary of the Invention

[0005] The purpose of this invention is to provide a fluorescent immunochromatographic test strip for detecting free IgE, its preparation method, and its application in a detection system.

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0007] The first aspect of this invention provides a method for preparing a fluorescent immunochromatographic test strip for detecting free IgE, comprising the following steps:

[0008] (1) The first cellulose membrane was treated with the first buffer solution to obtain the sample pad;

[0009] (2) The second cellulose membrane was treated with a second buffer containing mouse anti-human IgG monoclonal antibody to coat the second cellulose membrane with mouse anti-human IgG monoclonal antibody to obtain a filter membrane;

[0010] (3) The third cellulose membrane was treated with a third buffer containing fluorescent microsphere-labeled FcERI antigen and fluorescent microsphere-labeled DNP-BSA to coat the third cellulose membrane with fluorescent microsphere-labeled FcERI antigen and fluorescent microsphere-labeled DNP-BSA to obtain a conjugation pad.

[0011] (4) A detection line and a control line are set on the fourth cellulose membrane to obtain a detection membrane, wherein the detection line is coated with mouse anti-human IgE antibody and the control line is coated with DNP monoclonal antibody.

[0012] (5) The sample pad, the filter membrane, the conjugate pad, the detection membrane and the absorbent material are sequentially mounted on the base plate to obtain the fluorescent immunochromatographic test strip for detecting free IgE.

[0013] According to some specific embodiments, the second buffer solution contains 0.1–1 mg / mL of mouse anti-human IgG monoclonal antibody, for example, the concentration of the mouse anti-human IgG monoclonal antibody is 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, or 1 mg / mL; or, the second buffer solution contains 0.1–1 mg / mL of biotinylated mouse anti-human IgG monoclonal antibody and 0.025–0.25 mg / mL of streptavidin conjugated with BSA. Preferably, the second buffer solution does not contain biotin and streptavidin in the amplification system. Studies have found that while the fluorescence immunochromatographic test strips containing biotin and streptavidin have good anti-interference ability, their sensitivity and repeatability are slightly worse.

[0014] According to some specific embodiments, the second buffer solution also contains 2 wt.% to 8 wt.% trehalose, for example, the concentration of the trehalose is 2 wt.%, 3 wt.%, 4 wt.%, 5 wt.%, 6 wt.%, 7 wt.%, or 8 wt.%.

[0015] According to some specific embodiments, the pH value of the second buffer solution is 7.0 to 7.5, for example 7.0, 7.1, 7.2, 7.3, 7.4 or 7.5.

[0016] According to some specific embodiments, the base buffers in the first buffer, the second buffer, and the third buffer are independently selected from phosphate buffer, borate buffer, MES buffer, Tris buffer, or citrate buffer.

[0017] Furthermore, the base buffers in the first buffer and the third buffer are each independently Tris buffer, and the base buffer in the second buffer is PB buffer.

[0018] Furthermore, the concentration of the Tris buffer is 25 mmol / L to 70 mmol / L, for example 25 mmol / L, 30 mmol / L, 35 mmol / L, 40 mmol / L, 45 mmol / L, 50 mmol / L, 55 mmol / L, 60 mmol / L, 65 mmol / L or 70 mmol / L.

[0019] Furthermore, the concentration of the PB buffer is 10 mmol / L to 50 mmol / L, for example, 10 mmol / L, 15 mmol / L, 20 mmol / L, 25 mmol / L, 30 mmol / L, 35 mmol / L, 40 mmol / L, 45 mmol / L, or 50 mmol / L.

[0020] According to some specific embodiments, the pH values ​​of the first buffer solution and the third buffer solution are independently 8.5 to 9.0, for example 8.5, 8.6, 8.7, 8.8, 8.9 or 9.0.

[0021] According to some more specific embodiments, the second buffer is a PB buffer with a pH of 7.0 to 7.5 and a concentration of 10 mmol / L to 50 mmol / L, containing 2 wt.% to 8 wt.% trehalose and 0.1 to 1 mg / mL mouse anti-human IgG monoclonal antibody.

[0022] Further, the second buffer is a PB buffer with a pH of 7.0 to 7.5 and a concentration of 10 mmol / L to 20 mmol / L, containing 2 wt.% to 8 wt.% trehalose and 0.1 to 1 mg / mL mouse anti-human IgG monoclonal antibody.

[0023] Furthermore, the second buffer is a PB buffer with a pH of 7.3 to 7.5 and a concentration of 10 mmol / L to 20 mmol / L, containing 2 wt.% to 8 wt.% trehalose and 0.5 to 1 mg / mL mouse anti-human IgG monoclonal antibody.

[0024] According to some specific embodiments, in step (2), the second cellulose membrane is immersed in the second buffer solution, or the second buffer solution is uniformly sprayed onto the second cellulose membrane using a gold sprayer, and then dried to obtain the filter membrane. Preferably, the second cellulose membrane is immersed in the second buffer solution for 1 min to 3 min, and then dried to obtain the filter membrane. This is because immersion allows the second cellulose membrane to better absorb the second buffer solution, resulting in a test strip with superior performance. If a large amount of the second buffer solution is sprayed onto the second cellulose membrane using a gold sprayer to fully wet the second cellulose membrane, the performance of the prepared test strip is comparable to that of the test strip prepared by immersion.

[0025] According to some specific embodiments, the specific method of step (3) is as follows: the fluorescent microsphere-labeled FcERI antigen is mixed with the third buffer to obtain an FcERI antigen-fluorescent microsphere conjugate solution with a content of 0.05 wt.% to 0.5 wt.% of the fluorescent microsphere-labeled FcERI antigen; the fluorescent microsphere-labeled DNP-BSA is mixed with the third buffer to obtain a DNP-BSA-fluorescent microsphere conjugate solution with a content of 0.05 wt.% to 0.5 wt.% of the fluorescent microsphere-labeled DNP-BSA; then the FcERI antigen-fluorescent microsphere conjugate solution and the DNP-BSA-fluorescent microsphere conjugate solution are mixed at a volume ratio of (15 to 18):1 and sprayed onto the third cellulose membrane, and dried to obtain the conjugate pad.

[0026] There are no particular limitations on the preparation methods of the fluorescent microsphere-labeled FcERI antigen and the fluorescent microsphere-labeled DNP-BSA; conventional methods in the field can be selected.

[0027] The preferred methods for preparing the fluorescent microsphere-labeled FcERI antigen and the fluorescent microsphere-labeled DNP-BSA are as follows:

[0028] 1) Activation and Coupling of Microspheres: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and fluorescent microspheres were added to a coupling buffer and incubated on a shaker at room temperature for 10-20 minutes. FcERI antigen or dinitrophenol-conjugated bovine serum albumin (DNP-BSA) was then mixed with the activated microspheres to obtain the coupling system, which was incubated on a shaker at room temperature for 1.5-2.5 hours. The amount of EDC in the coupling system was controlled at 10-15 μg / mL, the volume of fluorescent microspheres was 8%-12% of the total volume of the coupling system, and the concentration of FcERI antigen or DNP-BSA was 0.05 mg / mL-0.5 mg / mL. The preferred coupling buffer was a 0.05 mol / L-0.5 mol / L MES buffer with a pH of 5.5-6.5.

[0029] 2) Blocking of microspheres: After incubation at room temperature, add 8%–12% of blocking buffer to the coupling system, and incubate on a shaker at room temperature for 0.5–1.5 h. The blocking buffer is preferably a 5 wt.%–15 wt.% BSA buffer.

[0030] 3) Cleaning of microspheres: After the blocking is completed, centrifuge to remove the supernatant, reconstitute with coupling buffer at 150% to 250% of the total volume of the coupling system, sonicate, centrifuge and discard the supernatant to obtain fluorescent microsphere-labeled FcERI antigen or fluorescent microsphere-labeled DNP-BSA.

[0031] According to some specific embodiments, the specific method of step (4) is as follows: the mouse anti-human IgE antibody is mixed with the fourth buffer to obtain the first coating solution, the first coating solution is used to draw lines on the fourth cellulose membrane, and the detection line is formed by drying; wherein, the concentration of the mouse anti-human IgE antibody in the first coating solution is 1 mg / mL to 5 mg / mL, for example 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL.

[0032] According to some specific embodiments, the DNP monoclonal antibody is mixed with a fourth buffer to obtain a second coating solution, and the second coating solution is used to streak lines on the fourth cellulose membrane, which are then dried to form the quality control line; wherein, the concentration of the DNP monoclonal antibody in the second coating solution is 1 mg / mL to 5 mg / mL, for example, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, or 5 mg / mL.

[0033] The fourth buffer solution is a PBS buffer with a pH of 7.0 to 7.5 (e.g., 7.0, 7.1, 7.2, 7.3, 7.4, 7.5) and a concentration of 5 mmol / L to 20 mmol / L, containing 2 wt.% to 8 wt.% trehalose and 2 wt.% to 8 wt.% methanol.

[0034] Furthermore, the fourth buffer solution is a PBS buffer with a pH of 7.0–7.5 and a concentration of 8 mmol / L–12 mmol / L, containing 4 wt.%–6 wt.% trehalose and 4 wt.%–6 wt.% methanol.

[0035] According to some specific embodiments, the first cellulose membrane is made of glass cellulose membrane.

[0036] According to some specific embodiments, the material of the second cellulose membrane is a nitrocellulose membrane.

[0037] According to some specific embodiments, the material of the third cellulose membrane is a glass cellulose membrane.

[0038] According to some specific embodiments, the fourth cellulose membrane is made of nitrocellulose membrane.

[0039] According to some specific embodiments, the base plate is made of PVC.

[0040] According to some specific embodiments, the absorbent material is absorbent paper.

[0041] According to some specific embodiments, the first buffer solution is a Tris buffer solution with a pH of 8.0 to 9.0 and a concentration of 45 mmol / L to 55 mmol / L, containing 0.5 wt.% to 1.5 wt.% Tween-20, 0.5 wt.% to 1.5 wt.% PEG20000, 3 wt.% to 8 wt.% trehalose and 0.1 wt.% to 0.6 wt.% casein.

[0042] Further, the first buffer solution is a Tris buffer solution with a pH of 8.4 to 8.6 and a concentration of 45 mmol / L to 55 mmol / L, containing 0.8 wt.% to 1.2 wt.% Tween-20, 0.8 wt.% to 1.2 wt.% PEG20000, 4 wt.% to 6 wt.% trehalose and 0.4 wt.% to 0.6 wt.% casein.

[0043] Furthermore, the first buffer solution also contains 0.1–1 mg / mL of HBR1.

[0044] According to some specific implementation methods, the specific implementation method of step (1) is as follows: immerse the first cellulose membrane in the first buffer solution for 1 min to 3 min, and then dry it to obtain the sample pad.

[0045] According to some specific embodiments, when preparing the sample pad, the filter membrane, the conjugate pad, and the detection membrane, the drying temperature is 30–60°C. Further, the drying temperature is 30°C–40°C. Even further, the drying is carried out in an environment with humidity less than 30% and a temperature of 18°C–28°C.

[0046] According to some specific embodiments, the third buffer solution is a Tris buffer solution with a pH of 8.0 to 9.0 and a concentration of 45 mmol / L to 55 mmol / L, containing 100 mmol / L to 200 mmol / L of sodium chloride, 8 wt.% to 12 wt.% of trehalose, 0.4 wt.% to 0.6 wt.% of Tween-20 and 0.4 wt.% to 0.6 wt.% of casein.

[0047] Further, the third buffer solution is a Tris buffer solution with a pH of 8.4 to 8.6 and a concentration of 45 mmol / L to 55 mmol / L, containing 140 mmol / L to 160 mmol / L of sodium chloride, 8 wt.% to 12 wt.% of trehalose, 0.4 wt.% to 0.6 wt.% of Tween-20 and 0.4 wt.% to 0.6 wt.% of casein.

[0048] According to some specific embodiments, the fluorescent microspheres are europium chelate fluorescent microspheres, each with a modifying group on its surface, the modifying group being amino and / or carboxyl groups. The fluorescent microspheres are activated by N-hydroxysuccinimide ester and / or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide. The diameter of the fluorescent microspheres is 0.1 μm to 0.3 μm, the excitation wavelength is 320 nm to 350 nm, and the emission wavelength is 600 nm to 620 nm.

[0049] A second aspect of the present invention provides a fluorescent immunochromatographic test strip for detecting free IgE prepared by the aforementioned preparation method.

[0050] A third aspect of the present invention provides a kit for detecting free IgE, comprising the above-described fluorescent immunochromatographic test strip and sample diluent.

[0051] According to some specific embodiments, the sample diluent is a PBS buffer with a pH of 7.0 to 8.0 and a concentration of 8 mmol / L to 12 mmol / L, which contains 0.4 wt.% to 0.6 wt.% Tween-20.

[0052] A fourth aspect of the present invention provides a free IgE detection system, comprising a fluorescent immunochromatographic test strip for detecting free IgE prepared by the aforementioned preparation method.

[0053] According to some specific embodiments, the free IgE detection system further includes a fluorescence immunoassay analyzer and an ID card disposed on the fluorescence immunochromatographic test strip for detecting free IgE; the ID card stores calibration data and / or standard curves, therefore, the free IgE detection system can quantitatively detect free IgE in the sample to be tested.

[0054] According to some specific embodiments, the free IgE detection system also includes a sample diluent for diluting the sample to be tested.

[0055] According to some specific embodiments, the detection method of the quantitative detection system includes the following steps:

[0056] (1) Insert the fluorescent immunochromatographic test strip into the fluorescent immunoassay analyzer, read the calibration data and / or standard curve on the ID card, and remove the fluorescent immunochromatographic test strip after reading is completed.

[0057] (2) The sample to be tested can be selectively diluted with sample diluent, and the undiluted or diluted sample to be tested is dropped onto the fluorescent immunochromatographic test strip and allowed to stand for 5 min to 15 min. The undiluted or diluted sample to be tested passes through the sample pad, the filter membrane, the conjugate pad, and the detection membrane in sequence to reach the absorbent material.

[0058] (3) Insert the fluorescent immunochromatographic test strip after static reaction back into the fluorescent immunoassay analyzer, read the fluorescence intensity of the detection line and the control line, and calculate and output the free IgE concentration value of the sample to be tested based on the calibration data and / or standard curve.

[0059] In this invention, the sample to be tested by the fluorescent immunochromatographic test strip and the quantitative detection system is a serum sample.

[0060] Compared with the prior art, the present invention has the following advantages:

[0061] The fluorescent immunochromatographic test strip prepared by the method of the present invention can quickly quantify free IgE in a single test with very small clinical sample volume and only one fluorescent immunoassay analyzer. It has the advantages of high sensitivity, high accuracy, good stability, good repeatability and simple operation, and has broad application prospects. Attached Figure Description

[0062] Figure 1 The fluorescent immunochromatographic test strips for detecting free IgE are those described in Examples 1 to 3;

[0063] Figure 2 The fluorescent immunochromatographic test strip for detecting free IgE is shown in Comparative Example 1.

[0064] The components include: 1. Sample pad; 2. Base plate; 3. Filter membrane; 4. Conjugate pad; 5. Detection membrane; 6. Detection line; 7. Quality control line; 8. Absorbent paper. Detailed Implementation

[0065] Currently, enzyme-linked immunosorbent assay (ELISA) is mainly used in domestic laboratories and clinical settings to detect free IgE. However, this method is complex and time-consuming, making it difficult to meet the requirements for quantitative clinical detection. Fluorescent immunochromatography (FIC) theoretically requires only 15 minutes to detect free IgE concentration and is simple and convenient to operate. However, its development is challenging, and there is no precedent for preparing test strips for detecting free IgE on a FIC platform. This invention addresses this by conducting extensive research and experimental verification on how to accurately and efficiently detect free IgE, and has developed a method for preparing a FIC test strip for detecting free IgE.

[0066] Specifically, this invention improves the structure of the fluorescent immunochromatographic test strip, particularly by incorporating a filter membrane between the sample pad and the conjugate pad, and by modifying the coating material on each membrane layer. This allows all IgG antibodies in the test sample to be removed after passing through the sample pad and filter membrane, including potentially conjugated IgE antibodies or omalid monoclonal antibodies that could affect the accuracy of free IgE antibody detection. This ensures the sensitivity, accuracy, stability, and repeatability of the test strip. Furthermore, the free IgE fluorescent immunochromatographic test strip has a simple structure, short production cycle, low equipment requirements, and is easy to mass-produce. It also offers advantages such as short detection time, simple operation, convenient use, low personnel skill requirements, and wide application scenarios.

[0067] The present invention will be further described below with reference to embodiments. However, the present invention is not limited to the following embodiments. The implementation conditions used in the embodiments can be further adjusted according to different requirements of specific applications, and the implementation conditions not specified are conventional conditions in the industry. The technical features involved in the various embodiments of the present invention can be combined with each other as long as they do not conflict with each other.

[0068] The technical solution of the present invention will be further described below with reference to specific embodiments and comparative examples.

[0069] Unless otherwise specified, all raw materials and reagents used in the following examples and comparative examples are commercially available products. In the following examples and comparative examples, room temperature refers to 25±5℃. In the following examples and comparative examples, unless otherwise specified, "%" refers to mass percentage.

[0070] In the following examples and comparative examples, the FcERI antigen used was a synthetic FcERI antigen produced by Shanghai Fermat Biotechnology Co., Ltd.; the dinitrophenol-conjugated bovine serum albumin (DNP-BSA) and DNP monoclonal antibody were both purchased from Nanjing Fuxiao Biotechnology Co., Ltd.; the mouse anti-human IgG monoclonal antibody and biotinylated mouse anti-human IgG monoclonal antibody were purchased from Beijing Bosi Biotechnology Co., Ltd.; the mouse anti-human IgE antibody was purchased from Shenzhen Shengyuan Biotechnology Co., Ltd.; the inhibitor HBR1 was purchased from Baodekang (Beijing) Biotechnology Co., Ltd.; the streptavidin conjugated with bovine serum albumin (BSA) (SA-BSA) was purchased from Hangzhou Boyue Biotechnology Co., Ltd.; the fluorescent microspheres were europium chelate fluorescent microspheres purchased from Thermo Fisher Scientific (China) Co., Ltd. (Thermo), with carboxyl groups as the surface modification group, a particle size of approximately 0.2 μm, an excitation wavelength of 333 nm, and an emission wavelength of 613 nm; the quality control and calibrators of free IgE were both purchased from Shanghai Fermat Biotechnology Co., Ltd.

[0071] In the following examples and comparative examples, the fluorescence immunoassay analyzer used is the DL300 dry fluorescence immunoassay analyzer manufactured by Dialabo (Zhangjiagang) Biotechnology Co., Ltd.

[0072] Example 1

[0073] This embodiment provides a fluorescent immunochromatographic test strip for detecting free IgE, the structure of which is as follows: Figure 1 As shown, the strip includes a base plate 2, a sample pad 1, a filter membrane 3, a conjugate pad 4, a detection membrane 5, and absorbent paper 8, all sequentially overlapped on the base plate 2, as well as a detection line 6 and a control line 7 set on the detection membrane 5. The base plate 2 is made of PVC, the sample pad 1 and the conjugate pad 4 are both made of glass fiber membrane, and the filter membrane 3 and the detection membrane 5 are both made of nitrocellulose membrane. The fluorescent immunochromatographic test strip for detecting free IgE has sample wells (not shown in the figure). The assembly of the fluorescent immunochromatographic test strip and the setting of the sample wells are standard techniques in this field and will not be described in detail here.

[0074] The preparation method of the free IgE fluorescent immunochromatographic test strip in this embodiment is as follows:

[0075] 1. Prepare sample pad 1:

[0076] 1.1 Prepare the first buffer solution, which is a Tris buffer solution with a pH of 8.5 and a concentration of 50 mmol / L, containing 1 wt.% Tween-20, 1 wt.% PEG20000, 0.5 mg / mL HBR1, 5 wt.% trehalose and 0.5 wt.% casein.

[0077] 1.2 Immerse the glass cellulose membrane in the first buffer solution for 1 minute, remove it and place it in an environment with humidity less than 30% and temperature of 18℃~28℃, dry it at 37℃ for 24 hours, and cut it to the required specifications for later use.

[0078] 2. Preparation of filter membrane 3:

[0079] 2.1 Prepare the second buffer solution, which is a PB buffer with a pH of 7.4 and a concentration of 10 mmol / L, containing 5 wt.% trehalose and 0.5 mg / mL mouse anti-human IgG monoclonal antibody.

[0080] 2.2 Immerse the nitrocellulose membrane in the second buffer solution for 1 minute, remove it and place it in an environment with humidity less than 30% and temperature of 18℃~28℃, dry it at 37℃ for 24 hours, and cut it to the required specifications for later use.

[0081] 3. Preparation of conjugate pad 4:

[0082] 3.1 Prepare the third buffer solution, which is a Tris buffer solution with a pH of 8.5 and a concentration of 50 mmol / L, containing 150 mmol / L NaCl, 10 wt.% trehalose, 0.5 wt.% Tween-20 and 0.5 wt.% casein.

[0083] 3.2 Preparation of FcERI antigen-fluorescent microsphere conjugate solution and DNP-BSA-fluorescent microsphere conjugate solution

[0084] 1) Activation and Coupling of Microspheres: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and fluorescent microspheres were added to the coupling buffer and incubated on a shaker at 25°C and 250 rpm for 15 min. The mixture was then centrifuged at 16000 rpm and 10°C for 15 min, and the supernatant was removed. The microspheres were reconstituted with the coupling buffer, and FcERI antigen was added to obtain the coupling system. The mixture was then incubated on a shaker at 25°C and 250 rpm for 2 h. The coupling buffer was a 0.1 mol / L MES buffer with a pH of 6. In the coupling system, the amount of EDC was 12.5 μg / mL, the volume of the fluorescent microspheres was 10% of the total volume of the coupling system, and the final concentration of the FcERI antigen was 0.1 mg / mL.

[0085] 2) Blocking of microspheres: After incubation on a shaker, add blocking buffer (10% of the total volume of the coupling system) to the coupling system, place on a shaker, and incubate at 25°C and 250 rpm for 1 hour. The blocking buffer is 10 wt.% BSA buffer.

[0086] 3) Cleaning of microspheres: After sealing, centrifuge to remove the supernatant, reconstitute with conjugation buffer at 200% of the total volume of the conjugation system, sonicate, centrifuge and discard the supernatant to obtain the FcERI antigen-fluorescent microsphere conjugate.

[0087] 4) Mix the FcERI antigen-fluorescent microsphere conjugate with the third buffer to obtain a FcERI antigen-fluorescent microsphere conjugate solution with a concentration of 0.1 wt.%.

[0088] The preparation steps of DNP-BSA-fluorescent microsphere conjugate solution are basically the same as those of FcERI antigen-fluorescent microsphere conjugate solution, except that the original FcERI antigen with a final concentration of 0.1 mg / mL is replaced with DNP-BSA with a final concentration of 0.1 mg / mL.

[0089] 3.3 Cut the glass cellulose membrane to the required specifications. Prepare a gold spraying solution by mixing the FcERI antigen-fluorescent microsphere conjugate solution and the DNP-BSA-fluorescent microsphere conjugate solution at a volume ratio of 16:1. Use a gold spraying apparatus to uniformly spray the prepared gold spraying solution onto the glass cellulose membrane at a coating speed of 2 μL / cm, and place it in an environment with humidity less than 30% and temperature of 18℃~28℃, and dry it at 37℃ for 2 hours.

[0090] 4. Preparation of the detection membrane 5:

[0091] 4.1 Prepare the fourth buffer solution, which is a PBS buffer with a pH of 7.4 and a concentration of 10 mmol / L, containing 5 wt.% trehalose and 5 wt.% methanol.

[0092] 4.2 Take an appropriate amount of the fourth buffer solution, add mouse anti-human IgE antibody and mix to obtain the first coating solution. The concentration of mouse anti-human IgE antibody in the first coating solution is 2 mg / mL.

[0093] Take an appropriate amount of the fourth buffer solution, add DNP monoclonal antibody and mix to obtain the second coating solution. The concentration of DNP monoclonal antibody in the second coating solution is 2 mg / mL.

[0094] 4.3 Using a coating apparatus, the first coating solution is uniformly coated on the detection line 6 at a coating speed of 1 μL / cm, and the second coating solution is uniformly coated on the quality control line 7 at a coating speed of 1 μL / cm. The mixture is then placed in an environment with humidity less than 30% and temperature of 18℃~28℃ and dried at 37℃ for 24 hours.

[0095] 4. Assembly of fluorescent immunochromatographic test strip: The sample pad 1, the first nitrocellulose membrane 3, the conjugate pad 4, the detection membrane 5, and the absorbent paper 8 are sequentially overlapped at a specific position on the base plate 2. After assembly, the strip is cut to a fixed width of 3.9 mm to obtain a fluorescent immunochromatographic test strip for detecting free IgE.

[0096] 5. ID card preparation:

[0097] 5.1 Free IgE Standard Curve:

[0098] Six calibrators of free IgE at concentrations of 0 IU / mL, 10 IU / mL, 50 IU / mL, 200 IU / mL, 1000 IU / mL, and 2000 IU / mL were selected. Each concentration was tested six times. The test method was as follows: 80 μL of the sample to be tested was added to the sample well of the above-mentioned fluorescent immunoassay strip. After waiting for 15 minutes, the fluorescent immunoassay strip was inserted into the fluorescent immunoassay analyzer. The fluorescence intensity T value of the detection line 6 and the fluorescence intensity C value of the control line 7 were read. The standard curve was fitted with the free IgE concentration and the T / C value: y = (AD) / [1 + (x / C^B)] + D, where A = 32.524, B = -1.08637, C = 1918.09021, D = 0.02406, where y represents the T / C value, x represents the concentration of free IgE, and r 2 =0.99999.

[0099] The free IgE standard curve is written into the ID card, and the ID card is inserted into the fluorescent immunochromatographic test strip used to detect free IgE. The processor of the fluorescent immunochromatographic analyzer can read the contents of the ID card and process the sample test results.

[0100] Example 2

[0101] This example provides a fluorescent immunochromatographic test strip for detecting free IgE. Its structure is the same as in Example 1, and its preparation method is basically the same as in Example 1. The difference is that in step 2, when preparing filter membrane 3, the formula of the second buffer solution remains unchanged, but it is used as the gold spraying solution. The prepared gold spraying solution is uniformly sprayed onto the nitrocellulose membrane at a coating speed of 2 μL / cm using a gold spraying instrument, and then placed in an environment with humidity less than 30% and temperature of 18℃~28℃, and dried at 37℃ for 2 hours.

[0102] Example 3

[0103] This example provides a fluorescent immunochromatographic test strip for detecting free IgE. Its structure is the same as in Example 1, and its preparation method is basically the same as in Example 1. The difference is that when preparing the filter membrane 3 in step 2, a biotin-streptavidin amplification system is introduced. Specifically, the second buffer is adjusted to a PBS buffer with a pH of 7.4 and a concentration of 10 mmol / L, which contains 5 wt.% trehalose, 0.5 mg / mL biotinylated mouse anti-human IgG monoclonal antibody, and 0.125 mg / mL streptavidin conjugated with BSA.

[0104] Comparative Example 1

[0105] This example provides a fluorescent immunochromatographic test strip for detecting free IgE. The difference from Example 1 is that it lacks the filter membrane 3 structure, the structure of which is shown below. Figure 2 Furthermore, a portion of the second buffer solution used to process filter membrane 3 is incorporated into the first buffer solution used to process sample pad 1. The adjustments required in the preparation method are as follows: In step 1, the first buffer solution used to process sample pad 1 is a Tris buffer with a pH of 8.5 and a concentration of 50 mmol / L, containing 1 wt.% Tween-20, 1 wt.% PEG20000, 0.5 mg / mL HBR1, 5 wt.% trehalose, 0.5 wt.% casein, and 0.5 mg / mL mouse anti-human IgG monoclonal antibody. In step 4, the fluorescent immunochromatographic test strip is assembled: Sample pad 1, conjugate pad 4, detection membrane 5, and absorbent paper 8 are sequentially overlapped at specific positions on the base plate 2. After assembly, it is cut to a fixed width of 3.9 mm to obtain the fluorescent immunochromatographic test strip for detecting free IgE.

[0106] Performance testing

[0107] 1. Sensitivity Test

[0108] Six concentrations of free IgE calibrators were taken, and their signal intensity was verified using the free IgE fluorescent immunochromatographic test strips from the examples and comparative examples. The concentrations of the free IgE calibrators were 0 IU / mL, 10 IU / mL, 50 IU / mL, 200 IU / mL, 1000 IU / mL, and 2000 IU / mL. Each concentration was tested twice, and the mean signal intensity was recorded. The detection method is the same as the standard curve procedure in Example 1 above. The specific test results are shown in Table 1.

[0109] Table 1

[0110]

[0111]

[0112] Note: In Table 1, the T value represents the fluorescence intensity of free IgE, and the C value represents the fluorescence intensity of the control line.

[0113] 2. Anti-interference capability

[0114] In the detection of free IgE antibodies, besides excluding common interfering substances that can interfere with fluorescent immunochromatographic products, it is most important to exclude the presence of bound IgE antibodies or omalid monoclonal antibodies in the test sample that may affect the accuracy of free IgE antibody detection. Therefore, an experimental protocol needs to be designed to verify the ability of the test strips in the examples or comparative examples to remove IgG antibodies from the sample solution. The experimental protocol is as follows: Prepare two free IgE quality control samples with concentrations of 100 IU / mL and 1000 IU / mL, respectively. During the preparation process, additional concentrations of omalid monoclonal antibody at concentration gradients of 0, 0.01, 0.1, 1, 10, and 100 μg / mL are added. The signal intensity is verified using the free IgE fluorescent immunochromatographic test strips in the examples and comparative examples, respectively. Each concentration is tested twice, and the mean signal intensity is recorded. The detection method is the same as the detection steps in Example 1 above. The average recorded signal intensity was substituted into the standard curve to calculate the free IgE concentration in the test sample, and the concentration deviation was calculated. The formula for calculating the concentration deviation is: (the detected free IgE concentration with added omalizumab - the detected free IgE concentration without added omalizumab) / the detected free IgE concentration without added omalizumab × 100%. The specific test results are shown in Table 2.

[0115] Table 2

[0116]

[0117]

[0118] 3. Repeatability testing

[0119] The free IgE fluorescent immunochromatographic test strips of the examples and comparative examples were used to repeatedly test the two concentrations of quality control samples 10 times each. The test values ​​of each example and comparative example were substituted into their respective standard curves for calculation. The mean (M), standard deviation (SD), and coefficient of variation (CV) of the test results were calculated. The concentrations of the quality control samples used for the free IgE repeatability test were 100 IU / mL and 1000 IU / mL. The results are shown in Table 3.

[0120] Table 3

[0121]

[0122] As shown in Table 1, the sensitivity of Examples 1, 2, and Comparative Example 1 is comparable, while the sensitivity of Example 3 is lower than the other three groups. As shown in Table 2, Comparative Example 1 has very poor anti-interference capability, Example 2 has better anti-interference capability than Comparative Example 1, and Examples 1 and 3 have significantly better anti-interference capability than Example 2. As shown in Table 3, the repeatability of Example 3 is worse than that of Examples 1, 2, and Comparative Example 1. Therefore, considering all performance aspects, Example 1 shows the best performance.

[0123] 4-sample correlation test:

[0124] Serum samples were tested using the fluorescent immunochromatographic test strip from Example 1 and the industry-recognized RECOVERYELISA IgE / Omalizumab (RIO) kit from BioTeZ BERLIN-BUCH GmbH. The results are shown in Table 4.

[0125] Table 4

[0126]

[0127]

[0128] In summary, the fluorescent immunochromatographic test strip of Example 1 exhibits the best overall performance, and the sample correlation test shows that the fluorescent immunochromatographic test strip of Example 1 has a good correlation with the gold standard method, with a correlation curve y = 1.0094x - 5.8161 and R0. 2 =0.9856, which meets the acceptance criteria.

[0129] The present invention has been described in detail above, with the aim of enabling those skilled in the art to understand and implement the invention. However, this description should not be construed as limiting the scope of protection of the invention. All equivalent changes or modifications made in accordance with the spirit and essence of the invention should be included within the scope of protection of the invention.

Claims

1. A method for preparing a fluorescent immunochromatographic test strip for detecting free IgE, characterized in that: Includes the following steps: (1) The first cellulose membrane was treated with the first buffer solution to obtain the sample pad; (2) The second cellulose membrane is immersed in a second buffer containing mouse anti-human IgG monoclonal antibody for 1 min to 3 min to coat the second cellulose membrane with mouse anti-human IgG monoclonal antibody, and then dried to obtain a filter membrane; wherein, the second buffer is PB buffer with pH value of 7.3 to 7.5 and concentration of 10 mmol / L to 20 mmol / L, which contains 2 wt.% to 8 wt.% trehalose and 0.5 to 1 mg / mL mouse anti-human IgG monoclonal antibody; (3) The third cellulose membrane was treated with a third buffer containing fluorescent microsphere-labeled FcεRI antigen and fluorescent microsphere-labeled DNP-BSA to coat the third cellulose membrane with fluorescent microsphere-labeled FcεRI antigen and fluorescent microsphere-labeled DNP-BSA to obtain a conjugation pad. (4) A detection line and a control line are set on the fourth cellulose membrane to obtain a detection membrane, wherein the detection line is coated with mouse anti-human IgE antibody and the control line is coated with DNP monoclonal antibody. (5) The sample pad, the filter membrane, the conjugate pad, the detection membrane and the absorbent material are sequentially mounted on the base plate to obtain the fluorescent immunochromatographic test strip for detecting free IgE.

2. The method for preparing the fluorescent immunochromatographic test strip for detecting free IgE according to claim 1, characterized in that: The specific method of step (3) is as follows: the fluorescent microsphere-labeled FcεRI antigen is mixed with the third buffer to obtain an FcεRI antigen-fluorescent microsphere conjugate solution with a content of 0.05 wt.%~0.5 wt.%; the fluorescent microsphere-labeled DNP-BSA is mixed with the third buffer to obtain a DNP-BSA-fluorescent microsphere conjugate solution with a content of 0.05 wt.%~0.5 wt.%; then the FcεRI antigen-fluorescent microsphere conjugate solution and the DNP-BSA-fluorescent microsphere conjugate solution are mixed at a volume ratio of 15~18:1 and sprayed onto the third cellulose membrane, and dried to obtain the conjugate pad.

3. The method for preparing the fluorescent immunochromatographic test strip for detecting free IgE according to claim 1, characterized in that: The specific method of step (4) is as follows: the mouse anti-human IgE antibody is mixed with the fourth buffer to obtain the first coating solution, the first coating solution is used to draw lines on the fourth cellulose membrane, and the detection line is formed after drying; wherein, the concentration of the mouse anti-human IgE antibody in the first coating solution is 1 mg / mL to 5 mg / mL; The DNP monoclonal antibody is mixed with a fourth buffer to obtain a second coating solution. The second coating solution is used to streak lines on the fourth cellulose membrane, and the membrane is dried to form the control line. The concentration of the DNP monoclonal antibody in the second coating solution is 1 mg / mL to 5 mg / mL. The fourth buffer solution is a PBS buffer with a pH of 7.0-7.5 and a concentration of 5 mmol / L-20 mmol / L, containing 2 wt.%-8 wt.% trehalose and 2 wt.%-8 wt.% methanol.

4. The method for preparing the fluorescent immunochromatographic test strip for detecting free IgE according to claim 1, characterized in that: The first cellulose membrane is made of glass cellulose membrane; and / or, The second cellulose membrane is made of nitrocellulose membrane; and / or, The third cellulose membrane is made of glass cellulose membrane; and / or, The fourth cellulose membrane is made of nitrocellulose membrane; and / or, The first buffer solution is a Tris buffer with a pH of 8.0–9.0 and a concentration of 45 mmol / L–55 mmol / L, containing 0.5 wt.%–1.5 wt.% Tween-20, 0.5 wt.%–1.5 wt.% PEG20000, 3 wt.%–8 wt.% trehalose, and 0.1 wt.%–0.6 wt.% casein; and / or, The third buffer solution is a Tris buffer with a pH of 8.0–9.0 and a concentration of 45 mmol / L–55 mmol / L, containing 100 mmol / L–200 mmol / L sodium chloride, 8 wt.%–12 wt.% trehalose, 0.4 wt.%–0.6 wt.% Tween-20, and 0.4 wt.%–0.6 wt.% casein; and / or, The fluorescent microspheres are europium chelate fluorescent microspheres, each with a modifying group on its surface, the modifying group being amino and / or carboxyl groups. The fluorescent microspheres are activated by N-hydroxysuccinimide ester and / or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide. The diameter of the fluorescent microspheres is 0.1 μm to 0.3 μm, the excitation wavelength is 320 nm to 350 nm, and the emission wavelength is 600 nm to 620 nm.

5. The method for preparing the fluorescent immunochromatographic test strip for detecting free IgE according to claim 1, characterized in that: The base buffers in the first buffer and the third buffer are independently selected from phosphate buffer, borate buffer, MES buffer, Tris buffer or citrate buffer, respectively.

6. A fluorescent immunochromatographic test strip for detecting free IgE, prepared by the method according to any one of claims 1 to 5.

7. A free IgE detection system, characterized in that: Includes fluorescent immunochromatographic test strips for detecting free IgE prepared by the preparation method according to any one of claims 1 to 5.

8. The free IgE detection system according to claim 7, characterized in that: The free IgE detection system also includes a fluorescence immunoassay analyzer and an ID card mounted on the fluorescence immunochromatographic test strip used for detecting free IgE; the ID card stores calibration data and / or standard curves.

9. The free IgE detection system according to claim 7, characterized in that: The free IgE detection system also includes a sample diluent for diluting the sample to be tested.

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