Fluorescence immunochromatography test strip for detecting free IgE (Immunoglobulin E) and preparation method of fluorescence immunochromatography test strip

Abstract

The invention provides a fluorescence immunochromatography test strip for detecting free IgE (Immunoglobulin E) and a preparation method of the fluorescence immunochromatography test strip. The preparation method comprises the following steps: treating a second cellulose membrane by adopting a second buffer solution containing a mouse anti-human IgG monoclonal antibody to obtain a filter membrane; treating the third cellulose membrane with a third buffer solution containing a fluorescent microsphere labeled FcERI antigen and a fluorescent microsphere labeled DNP-BSA to obtain a conjugate pad; a detection line and a quality control line are arranged on the fourth cellulose membrane, a detection membrane is obtained, 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 combination pad, the detection membrane and a water absorption material are sequentially loaded on a bottom plate, and the fluorescence immunochromatography test strip for detecting the free IgE is obtained. The fluorescence immunochromatography test strip has the advantages of high sensitivity, high accuracy, good stability, good repeatability, simplicity, easiness in operation and the like, and has a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of immunofluorescence detection, and particularly relates to a fluorescence immunochromatographic test strip for detecting free IgE and a preparation method thereof. Background Art

[0002] Free IgE refers to immunoglobulin E (IgE) that is not bound to cells in serum. IgE is one of the immunoglobulins with the lowest content in human serum, and it plays an important role in type I allergic reactions, that is, allergic reactions. IgE can bind to the high-affinity IgE receptor (FcεRI) on the surface of effector cells (such as mast cells and basophils). When an allergen enters the body again, it can trigger the release of inflammatory mediators such as histamine by these cells, leading to allergic symptoms.

[0003] In the treatment of allergic diseases such as asthma, the application of anti-IgE antibodies such as omalizumab has important significance. Omalizumab is a recombinant 95% humanized monoclonal anti-IgE antibody. By binding to free IgE, it prevents IgE from binding to the high-affinity IgE receptor on the surface of effector cells, thereby inhibiting the release of inflammatory mediators and achieving the therapeutic purpose. In addition, the use of omalizumab can also reduce the level of free IgE and down-regulate the expression of FcεRI on the surface of effector cells, thereby weakening the effector function of IgE. Therefore, the detection of free IgE helps to further understand the clinical efficacy of omalizumab, and has important guiding significance for methods such as determining the specific allergens of patients, evaluating the severity of symptoms, and guiding the clinical use of omalizumab.

[0004] Currently, the methods for determining free IgE in clinical and laboratory settings are mostly enzyme-linked immunosorbent assays. The enzyme-linked immunosorbent assay has poor precision and linearity of sensitivity, and also has problems such as more operation steps and long detection time. The fluorescence immunochromatographic test strip has the advantages of short detection time and simple and convenient operation. However, there are currently no relevant reports or products on the fluorescence immunochromatographic test strip for detecting free IgE on the market. Moreover, in patients treated with omalizumab, the level of free IgE in the blood often decreases significantly. Therefore, once treatment begins, it becomes difficult to detect the level of free IgE in the blood. Therefore, there is a need to develop a fluorescence immunochromatographic test strip that can simultaneously ensure the performance of sensitivity, accuracy, stability, etc. for detecting free IgE and can meet the detection requirements of free IgE in patients treated with omalizumab. Summary of the Invention

[0005] The purpose of the present invention is to provide a fluorescence immunochromatographic test strip for detecting free IgE, a preparation method thereof, and an application in a detection system.

[0006] To solve the above technical problems, the present invention adopts the following technical solutions:

[0007] In a first aspect of the present invention, a method for preparing a fluorescence immunochromatographic test strip for detecting free IgE is provided, which includes the following steps:

[0008] (1) Treating a first cellulose membrane with a first buffer solution to obtain a sample pad;

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

[0010] (3) Treating a third cellulose membrane with a third buffer solution containing a fluorescence microsphere-labeled FcERI antigen and a fluorescence microsphere-labeled DNP-BSA to coat the fluorescence microsphere-labeled FcERI antigen and the fluorescence microsphere-labeled DNP-BSA on the third cellulose membrane to obtain a conjugate pad;

[0011] (4) Setting a detection line and a quality control line 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;

[0012] (5) Sequentially mounting the sample pad, the filter membrane, the conjugate pad, the detection membrane, and the absorbent material on a bottom plate to obtain the fluorescence immunochromatographic test strip for detecting free IgE.

[0013] According to some specific embodiments, the second buffer solution contains 0.1 to 1 mg / mL of the 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, 1 mg / mL; or, the second buffer solution contains 0.1 to 1 mg / mL of a biotinylated mouse anti-human IgG monoclonal antibody and 0.025 to 0.25 mg / mL of streptavidin conjugated with BSA. Among them, it is preferred that the second buffer solution does not contain a biotin and streptavidin amplification system. It has been found through research that although the fluorescence immunochromatographic test strip with a biotin and streptavidin amplification system has good anti-interference ability, its sensitivity and repeatability are slightly poor.

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

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

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

[0017] Further, the basic buffer solutions in the first buffer solution and the third buffer solution are independently Tris buffer solution, and the basic buffer solution in the second buffer solution is PB buffer solution.

[0018] Still further, the concentration of the Tris buffer solution is 25 mmol / L to 70 mmol / L, such as 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] Still further, the concentration of the PB buffer solution is 10 mmol / L to 50 mmol / L, such as 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, such as 8.5, 8.6, 8.7, 8.8, 8.9 or 9.0.

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

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

[0023] Further, the second buffer solution is a PB buffer solution with a pH value of 7.3 to 7.5 and a concentration of 10 mmol / L to 20 mmol / L, which contains 2 wt.% to 8 wt.% of trehalose and 0.5 to 1 mg / mL of 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 evenly sprayed onto the second cellulose membrane by a sputtering instrument, and then dried to obtain the filter membrane. Among them, it is preferred to immerse the second cellulose membrane in the second buffer solution for 1 min to 3 min, and then dry it to obtain the filter membrane. This is because the second cellulose membrane can better absorb the second buffer solution by the immersion method, so that the various performances of the test strip are more excellent. If a large amount of the second buffer solution is sprayed onto the second cellulose membrane by a sputtering instrument to fully wet the second cellulose membrane, the performance of the prepared test strip is equivalent to that of the test strip prepared by the immersion method.

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

[0026] Among them, the preparation methods of the FcERI antigen labeled with fluorescent microspheres and the DNP-BSA labeled with fluorescent microspheres are not particularly limited, and conventional methods in the art can be selected.

[0027] Preferably, the preparation methods of the FcERI antigen labeled with fluorescent microspheres and the DNP-BSA labeled with fluorescent microspheres are respectively:

[0028] 1) Activation and coupling of microspheres: Add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and fluorescent microspheres into the coupling buffer, and incubate on a shaker at room temperature for 10 min to 20 min. Mix the FcERI antigen or dinitrophenyl-conjugated bovine serum albumin (DNP-BSA) with the activated microspheres to obtain a coupling system, and incubate on a shaker at room temperature for 1.5 h to 2.5 h. Control the dosage of EDC in the coupling system to be 10 - 15 μg / mL, the volume of the fluorescent microspheres to be 8% - 12% of the total volume of the coupling system, and the concentration of the FcERI antigen or DNP-BSA to be 0.05 mg / mL to 0.5 mg / mL. The coupling buffer is preferably a MES buffer with a pH value of 5.5 - 6.5 and a concentration of 0.05 mol / L to 0.5 mol / L.

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

[0030] 3) Washing of microspheres: After blocking, centrifuge to remove the supernatant, redissolve with a coupling buffer with a volume of 150% - 250% of the total volume of the coupling system, sonicate, centrifuge and discard the supernatant, and thus 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: Mix the mouse anti-human IgE antibody with a fourth buffer to obtain a first coating solution, use the first coating solution to draw a line on the fourth cellulose membrane, and dry it to form the test line; wherein, the concentration of the mouse anti-human IgE antibody in the first coating solution is 1 mg / mL to 5 mg / mL, such as 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL.

[0032] According to some specific embodiments, mix the DNP monoclonal antibody with a fourth buffer to obtain a second coating solution, use the second coating solution to draw a line on the fourth cellulose membrane, and dry it 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, such as 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL.

[0033] Among them, the fourth buffer solution is a PBS buffer solution with a pH value of 7.0 to 7.5 (such as 7.0, 7.1, 7.2, 7.3, 7.4, 7.5), a concentration of 5 mmol / L to 20 mmol / L, and contains 2 wt.% to 8 wt.% of trehalose and 2 wt.% to 8 wt.% of methanol.

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

[0035] According to some specific embodiments, the material of the first cellulose membrane is a 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 material of the fourth cellulose membrane is a nitrocellulose membrane.

[0039] According to some specific embodiments, the material of the bottom plate is PVC.

[0040] According to some specific embodiments, the water-absorbing material is blotting paper.

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

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

[0043] Further, the first buffer solution further contains 0.1 to 1 mg / mL of HBR1.

[0044] According to some specific embodiments, the specific implementation method of step (1) is: immersing the first cellulose membrane in the first buffer solution for 1 min to 3 min, and then drying 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 to 60 °C. Further, the drying temperature is 30 °C to 40 °C. Still further, the drying is carried out in an environment with a humidity less than 30% and a temperature of 18 °C to 28 °C.

[0046] According to some specific embodiments, the third buffer solution is a Tris buffer solution with a pH value of 8.0 to 9.0 and a concentration of 45 mmol / L to 55 mmol / L, which contains 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 value of 8.4 to 8.6 and a concentration of 45 mmol / L to 55 mmol / L, which contains 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, which are respectively provided with modification groups on the surface, the modification groups are amino groups and / or carboxyl groups, the fluorescent microspheres are respectively fluorescent microspheres 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] The second aspect of the present invention provides a fluorescent immunochromatographic test strip for detecting free IgE prepared by the preparation method.

[0050] The third aspect of the present invention provides a kit for detecting free IgE, which includes the above-mentioned fluorescent immunochromatographic test strip and a sample diluent.

[0051] According to some specific embodiments, the sample diluent is a PBS buffer solution with a pH value 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.% of Tween-20.

[0052] The fourth aspect of the present invention provides a free IgE detection system, which includes a fluorescence immunochromatographic test strip for detecting free IgE prepared by the above preparation method.

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

[0054] According to some specific embodiments, the free IgE detection system further 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 fluorescence immunochromatographic test strip into the fluorescence immunoassay analyzer, read the calibration data and / or standard curves on the ID card, and then pull out the fluorescence immunochromatographic test strip after reading.

[0057] (2) Optionally dilute the sample to be tested with the sample diluent, drop the undiluted or diluted sample to be tested onto the fluorescence immunochromatographic test strip, and let it stand for reaction 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, the detection membrane and reaches the absorbent material in turn.

[0058] (3) Insert the fluorescence immunochromatographic test strip after standing reaction into the fluorescence immunoassay analyzer again, read the fluorescence intensities of the test line and the quality control line, and calculate and output the free IgE concentration value of the sample to be tested according to the calibration data and / or standard curves.

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

[0060] The present invention has the following advantages compared with the prior art:

[0061] The fluorescence immunochromatographic test strip prepared by the preparation method of the present invention can quickly quantify free IgE in one detection with extremely small clinical sample consumption and only one fluorescence immunoassay analyzer, and has the advantages of high sensitivity, high accuracy, good stability, good repeatability, simple and easy operation, etc., and has a wide application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 For the fluorescence immunochromatographic test strips for detecting free IgE in Examples 1 to 3;

[0063] Figure 2 The fluorescence immunochromatographic test strip for detecting free IgE of Comparative Example 1;

[0064] Wherein: 1. Sample pad; 2. Bottom plate; 3. Filter membrane; 4. Conjugate pad; 5. Detection membrane; 6. Test line; 7. Control line; 8. Absorbent paper. Detailed implementation manners

[0065] Currently in domestic laboratories and clinical settings, the enzyme-linked immunosorbent assay (ELISA) is mainly used to detect free IgE. However, this method is complex in operation and time-consuming, and it is difficult to meet the requirements of clinical quantitative detection. In theory, fluorescence immunochromatography can detect the concentration value of free IgE in only 15 minutes, and the operation is simple and convenient. However, the development difficulty is high, and there is no precedent for preparing a test strip for detecting free IgE on a fluorescence immunochromatography platform. The present invention has conducted a large number of studies and experimental verifications on how to accurately and efficiently detect free IgE, and has developed a preparation method for a fluorescence immunochromatographic test strip for detecting free IgE.

[0066] Specifically, the present invention, by improving the structure of the fluorescence immunochromatographic test strip, especially by setting a filter membrane between the sample pad and the conjugate pad, and improving the coating substances on each membrane layer, enables all IgG antibodies in the test sample to be removed after the test sample flows through the sample pad and the filter membrane, including possible bound IgE antibodies or omalizumab that may affect the detection accuracy of free IgE antibodies, ensuring the sensitivity, accuracy, stability, and repeatability of the test strip. In addition, the free IgE fluorescence immunochromatographic test strip has a simple structure, a short production cycle, low equipment requirements, and is easy to achieve mass production. The free IgE fluorescence immunochromatographic test strip also has advantages such as a short detection time, simple operation, convenient use, low requirements for personnel quality, and a wide range of application scenarios.

[0067] The present invention will be further described below in conjunction with 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 specific usage requirements, and the implementation conditions not specified are conventional conditions in the industry. The technical features involved in each implementation manner 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 elaborated below in conjunction with specific examples and comparative examples.

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

[0070] In the following examples and comparative examples, the FcERI antigen used was the artificially synthesized FcERI antigen produced by Shanghai Fema Biotechnology Co., Ltd.; dinitrophenol-conjugated bovine serum albumin (DNP-BSA) and DNP monoclonal antibody were both from Nanjing Dawn Biotechnology Co., Ltd.; mouse anti-human IgG monoclonal antibody and biotinylated mouse anti-human IgG monoclonal antibody were purchased from Beijing Borsi Biotechnology Co., Ltd.; mouse anti-human IgE antibody was purchased from Shenzhen Shengyuan Biotechnology Co., Ltd.; the blocker HBR1 was purchased from Baodekangti (Beijing) Biotechnology Co., Ltd.; streptavidin conjugated to bovine serum albumin (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 a carboxyl group 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 product and calibration product of free IgE were both purchased from Shanghai Fema Biotechnology Co., Ltd.

[0071] In the following examples and comparative examples, the fluorescence immunoassay analyzer used in combination was the dry fluorescence immunoassay analyzer DL300 produced by Diarlebo (Zhangjiagang) Biotechnology Co., Ltd.

[0072] Example 1

[0073] This example provides a fluorescence immunochromatographic test strip for detecting free IgE, and its structure is as Figure 1 shown, including a bottom plate 2, a sample pad 1, a filter membrane 3, a conjugate pad 4, a detection membrane 5, and an absorbent paper 8 that are sequentially lapped on the bottom plate 2, and a detection line 6 and a quality control line 7 provided on the detection membrane 5. Among them, the material of the bottom plate 2 is PVC, the materials of the sample pad 1 and the conjugate pad 4 are both glass cellulose membranes, and the materials of the filter membrane 3 and the detection membrane 5 are both nitrocellulose membranes. A sample addition hole (not shown in the figure) is provided on the fluorescence immunochromatographic test strip for detecting free IgE. The assembly of the fluorescence immunochromatographic test strip and the setting of the sample addition hole are both conventional technical means in the art and will not be elaborated here.

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

[0075] 1. Prepare the sample pad 1:

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

[0077] 1.2 Immerse the glass cellulose membrane in the first buffer solution for 1 min. After taking it out, place it in an environment with a humidity less than 30% and a temperature of 18°C - 28°C, and dry it at 37°C for 24 hours. After cutting to the required specifications, it is reserved for use.

[0078] 2. Prepare filter membrane 3:

[0079] 2.1 Prepare the second buffer solution. The second buffer solution is a PB buffer solution with a pH value of 7.4 and a concentration of 10 mmol / L, which contains 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 min. After taking it out, place it in an environment with a humidity less than 30% and a temperature of 18°C - 28°C, and dry it at 37°C for 24 hours. After cutting to the required specifications, it is reserved for use.

[0081] 3. Prepare conjugate pad 4:

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

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

[0084] 1) Activation and conjugation of microspheres: Add 1-(3 - dimethylaminopropyl)-3 - ethylcarbodiimide (EDC) and fluorescent microspheres to the conjugation buffer solution, place it on a shaker at 25°C and 250 r / min for 15 min. Then centrifuge at 16000 rpm and 10°C for 15 min and remove the supernatant. Re - dissolve it with the conjugation buffer solution, add FcERI antigen to obtain a conjugation system, place it on a shaker at 25°C and 250 r / min for 2 h. The conjugation buffer solution is a MES buffer solution with a pH value of 6 and a concentration of 0.1 mol / L. In the conjugation system, the dosage of EDC is 12.5 μg / mL, the volume of the fluorescent microspheres is 10% of the total volume of the conjugation system, and the final concentration of FcERI antigen is 0.1 mg / mL.

[0085] 2) Blocking of microspheres: After the incubation on the shaker ends, add a blocking buffer solution with a volume of 10% of the total volume of the conjugation system to the conjugation system, place it on a shaker at 25°C and 250 r / min for 1 h. The blocking buffer solution is a BSA buffer solution with a concentration of 10 wt.%.

[0086] 3) Washing of microspheres: After the blocking is completed, centrifuge to remove the supernatant, resuspend with coupling buffer at a volume 200% of the total volume of the coupling system, sonicate, centrifuge and discard the supernatant to obtain the FcεRI antigen-fluorescent microsphere conjugate.

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

[0088] The preparation steps of the DNP-BSA-fluorescent microsphere conjugate solution are basically the same as those of the FcεRI antigen-fluorescent microsphere conjugate solution, except that: the originally added FcεRI 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 obtain the required specifications. Mix the FcεRI antigen-fluorescent microsphere conjugate solution and the DNP-BSA-fluorescent microsphere conjugate solution in a volume ratio of 16:1 to prepare the gold-spraying solution. Use a gold-spraying instrument to evenly 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 a humidity of less than 30% and a temperature of 18°C to 28°C, and dry it at 37°C for 2 hours.

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

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

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

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

[0094] 4.3 Use a membrane scribing instrument to evenly coat the above first coating solution at a coating speed of 1 μL / cm at the position of the detection line 6, and evenly coat the above second coating solution at a coating speed of 1 μL / cm at the position of the quality control line 7, place it in an environment with a humidity of less than 30% and a temperature of 18°C to 28°C, and dry it at 37°C for 24 hours.

[0095] 4. Fluorescent immunochromatographic test strip assembly: 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 specific positions on the base plate 2. After assembly, it is cut with 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] Select calibration products of 6 concentrations of free IgE, and their concentrations are 0 IU / mL, 10 IU / mL, 50 IU / mL, 200 IU / mL, 1000 IU / mL, and 2000 IU / mL respectively. Each concentration standard product is tested 6 times. The test method is as follows: Take 80 μL of the sample to be tested and add it to the sample adding hole of the above-mentioned fluorescent immunoassay test strip. After waiting for 15 minutes, insert the fluorescent immunoassay test strip into the fluorescent immunoassay analyzer, and read the fluorescence intensity T value of the detection line 6 and the fluorescence intensity C value of the quality control line 7. Fit the standard curve with the free IgE concentration and the T / C value: y = (A - D) / [1 + (x / C^B)] + D, where A = 32.524, B = -1.08637, C = 1918.09021, D = 0.02406. In the formula, y represents the T / C value, x represents the concentration value of free IgE, r 2 = 0.99999.

[0099] Write the free IgE standard curve into the ID card respectively, and install the ID card into the fluorescent immunochromatographic test strip for detecting free IgE. The processor of the fluorescent immunoassay analyzer can read the content 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 that of Example 1, and its preparation method is basically the same as that of Example 1. The difference is that when preparing the filter membrane 3 in step 2, the formula of the second buffer solution remains unchanged, but it is used as the gold spraying solution. The prepared gold spraying solution is evenly sprayed onto the nitrocellulose membrane with a coating speed of 2 μL / cm by a gold spraying instrument, and placed in an environment with a humidity less than 30% and a temperature of 18°C to 28°C, and dried at 37°C for 2 hours.

[0102] Example 3

[0103] This example provides a fluorescence immunochromatographic test strip for detecting free IgE. Its structure is the same as that of Example 1, and its preparation method is basically the same as that of Example 1, except 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 value of 7.4 and a concentration of 10 mmol / L, which contains 5 wt.% of trehalose, 0.5 mg / mL of biotinylated mouse anti-human IgG monoclonal antibody, and 0.125 mg / mL of streptavidin conjugated with BSA.

[0104] Comparative Example 1

[0105] This example provides a fluorescence immunochromatographic test strip for detecting free IgE. The difference from Example 1 is that it does not have the structure of the filter membrane 3, and its structure is shown in Figure 2 , and some components of the second buffer for treating the filter membrane 3 are incorporated into the first buffer for treating the sample pad 1. The parts that need to be adjusted in its preparation method are as follows: In step 1, the first buffer for treating the sample pad 1 is a Tris buffer with a pH value of 8.5 and a concentration of 50 mmol / L, which contains 1 wt.% of Tween-20, 1 wt.% of PEG20000, 0.5 mg / mL of HBR1, 5 wt.% of trehalose, 0.5 wt.% of casein, and 0.5 mg / mL of mouse anti-human IgG monoclonal antibody. In step 4, the fluorescence immunochromatographic test strip is assembled: The sample pad 1, the conjugate pad 4, the detection membrane 5, and the absorbent paper 8 are sequentially overlapped at specific positions on the bottom plate 2. After assembly, it is cut with a fixed width of 3.9 mm to obtain a fluorescence immunochromatographic test strip for detecting free IgE.

[0106] Performance Test

[0107] 1. Sensitivity Test

[0108] Six concentrations of free IgE calibrators are taken, and the signal intensities are verified using the free IgE fluorescence immunochromatographic test strips of the examples and comparative examples respectively. The concentrations of the free IgE calibrators are 0 IU / mL, 10 IU / mL, 50 IU / mL, 200 IU / mL, 1000 IU / mL, and 2000 IU / mL. Each concentration is detected 2 times repeatedly, and the average signal intensity is recorded. The detection method refers to the steps of the standard curve 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 quality control line.

[0113] 2. Anti-interference ability

[0114] In the detection of free IgE antibodies, in addition to excluding conventional interfering substances that can interfere with fluorescence immunochromatography products, it is most important to exclude bound IgE antibodies or omalizumab in the test sample that may affect the accuracy of free IgE antibody detection. Therefore, it is necessary to design a test scheme to verify the ability of the test strips in the examples or comparative examples to remove IgG antibodies from the sample solution. The test scheme is as follows: Prepare two free IgE quality control products with concentrations of 100 IU / mL and 1000 IU / mL respectively. During the preparation process, omalizumab with concentration gradients of 0, 0.01, 0.1, 1, 10, and 100 μg / mL is additionally added. The signal intensity verification is carried out using the free IgE fluorescence immunochromatography test strips of the examples and comparative examples respectively. Each concentration is detected 2 times repeatedly, and the average signal intensity is recorded. The detection method refers to the detection steps in Example 1 above. Substitute the recorded average signal intensity into the standard curve to calculate the free IgE concentration in the test sample, and calculate the concentration deviation. Among them, the calculation formula of the concentration deviation = (the detected value of free IgE concentration added with a specific concentration of omalizumab - the detected value of free IgE concentration without added omalizumab) / the detected value of free IgE concentration without added omalizumab × 100%. The specific test results are shown in Table 2.

[0115] Table 2

[0116]

[0117]

[0118] 3. Repeatability test

[0119] The free IgE fluorescence immunochromatography test strips of the examples and comparative examples are respectively used to detect two concentrations of quality control products 10 times repeatedly. The detection values of each example and comparative example are substituted into their respective standard curves for calculation. Calculate the average value (M), standard deviation (SD), and coefficient of variation CV (%) of the detection results. The quality control product concentrations for free IgE repeatability test are 100 IU / mL and 1000 IU / mL, and the results are shown in Table 3.

[0120] Table 3

[0121]

[0122] As can be seen from Table 1, the sensitivities of Example 1, Example 2 and Comparative Example 1 are comparable, and the sensitivity of Example 3 is lower than that of the other three groups. As can be seen from Table 2, the anti-interference ability of Comparative Example 1 is very poor, the anti-interference ability of Example 2 is better than that of Comparative Example 1, and the anti-interference abilities of Example 1 and Example 3 are significantly better than that of Example 2. As can be seen from Table 3, the repeatability of Example 3 is worse than that of Example 1, Example 2 and Comparative Example 1. Therefore, considering all performances, it is found that the effect of Example 1 is the best.

[0123] 4 Sample correlation test:

[0124] The serum samples were detected using the fluorescence immunochromatographic test strip of Example 1 and the industry-recognized RECOVERY ELISA IgE / Omalizumab (RIO) kit of BioTeZ BERLIN-BUCH GmbH respectively, and the test results are shown in Table 4.

[0125] Table 4

[0126]

[0127]

[0128] In summary, the fluorescence immunochromatographic test strip of Example 1 has the best comprehensive performance, and the sample correlation test shows that the fluorescence immunochromatographic test strip of Example 1 has a good correlation with the gold standard method, and the correlation curve is y = 1.0094x - 5.8161, R 2 = 0.9856, meeting the acceptance criteria.

[0129] The above has described the present invention in detail. The purpose is to enable those skilled in this field to understand the content of the present invention and implement it. However, it should not be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit and essence of the present invention should be covered within the protection scope of the present invention.

Claims

1. A preparation method of a fluorescence immunochromatographic test strip for detecting free IgE, characterized in that: It includes the following steps: (1) Treat the first cellulose membrane with a first buffer solution to obtain a sample pad; (2) Treat the second cellulose membrane with a second buffer solution containing a mouse anti-human IgG monoclonal antibody to coat the mouse anti-human IgG monoclonal antibody on the second cellulose membrane to obtain a filter membrane; (3) Treat the third cellulose membrane with a third buffer solution containing a fluorescein microsphere-labeled FcERI antigen and a fluorescein microsphere-labeled DNP-BSA to coat the fluorescein microsphere-labeled FcERI antigen and the fluorescein microsphere-labeled DNP-BSA on the third cellulose membrane to obtain a conjugate pad; (4) Set a detection line and a quality control line on the 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; (5) Mount the sample pad, the filter membrane, the conjugate pad, the detection membrane and the absorbent material on a bottom plate in sequence to obtain the fluorescence immunochromatographic test strip for detecting free IgE.

2. The preparation method of the fluorescence immunochromatographic test strip for detecting free IgE according to claim 1, characterized in that: The second buffer solution contains 0.1-1 mg / mL of mouse anti-human IgG monoclonal antibody; alternatively, 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.

3. The preparation method of the fluorescence immunochromatographic test strip for detecting free IgE according to claim 2, characterized in that: The second buffer solution further contains 2 wt.% - 8 wt.% of trehalose.

4. The preparation method of the fluorescence immunochromatographic test strip for detecting free IgE according to claim 2 or 3, characterized in that: The pH value of the second buffer solution is 7.0-7.

5.

5. The preparation method of the fluorescence immunochromatographic test strip for detecting free IgE according to claim 1, characterized in that: The second buffer solution is a PB buffer solution with a pH value of 7.0-7.5 and a concentration of 10 mmol / L - 50 mmol / L, which contains 2 wt.% - 8 wt.% of trehalose and 0.1-1 mg / mL of mouse anti-human IgG monoclonal antibody.

6. The preparation method of the fluorescence immunochromatographic test strip for detecting free IgE according to claim 1, characterized in that: In step (2), immerse the second cellulose membrane in the second buffer solution, or evenly spray the second buffer solution onto the second cellulose membrane through a gold spraying instrument, and then dry it to obtain the filter membrane.

7. The preparation method of the fluorescence immunochromatographic test strip for detecting free IgE according to claim 1, characterized in that: Immerse the second cellulose membrane in the second buffer solution for 1 min - 3 min, and then dry it to obtain the filter membrane.

8. The preparation method of the fluorescence immunochromatographic test strip for detecting free IgE according to claim 1, wherein: The specific method of step (3) is: mix the fluorescein microsphere-labeled FcERI antigen with the third buffer solution to obtain an FcERI antigen-fluorescein microsphere conjugate solution with the content of the fluorescein microsphere-labeled FcERI antigen being 0.05 wt.% - 0.5 wt.%; Mix the fluorescein microsphere-labeled DNP-BSA with the third buffer solution to obtain a DNP-BSA-fluorescein microsphere conjugate solution with the content of the fluorescein microsphere-labeled DNP-BSA being 0.05 wt.% - 0.5 wt.%; then mix the FcERI antigen-fluorescein microsphere conjugate solution and the DNP-BSA-fluorescein microsphere conjugate solution in a volume ratio of (15-18):1, and spray the mixture onto the third cellulose membrane, and dry it to obtain the conjugate pad.

9. The preparation method of the fluorescence immunochromatographic test strip for detecting free IgE according to claim 1, characterized in that: The specific method of step (4) is as follows: Mix the murine anti-human IgE antibody with a fourth buffer solution to obtain a first coating solution, and use the first coating solution to draw a line on the fourth cellulose membrane, and dry it to form the test line; Among them, the concentration of the murine anti-human IgE antibody in the first coating solution is 1 mg / mL to 5 mg / mL; Mix the DNP monoclonal antibody with a fourth buffer solution to obtain a second coating solution, and use the second coating solution to draw a line on the fourth cellulose membrane, and dry it to form the quality control line; among them, the concentration of the DNP monoclonal antibody in the second coating solution is 1 mg / mL to 5 mg / mL; Among them, the fourth buffer solution is a PBS buffer solution with a pH value of 7.0 to 7.5 and a concentration of 5 mmol / L to 20 mmol / L, which contains 2 wt.% to 8 wt.% of trehalose and 2 wt.% to 8 wt.% of methanol.

10. The preparation method of the fluorescence immunochromatographic test strip for detecting free IgE according to claim 1, characterized in that: The material of the first cellulose membrane is a glass cellulose membrane; and / or, The material of the second cellulose membrane is a nitrocellulose membrane; and / or, The material of the third cellulose membrane is a glass cellulose membrane; and / or, The material of the fourth cellulose membrane is a nitrocellulose membrane; and / or, The first buffer solution is a Tris buffer solution with a pH value of 8.0 to 9.0 and a concentration of 45 mmol / L to 55 mmol / L, which contains 0.5 wt.% to 1.5 wt.% of Tween-20, 0.5 wt.% to 1.5 wt.% of PEG20000, 3 wt.% to 8 wt.% of trehalose and 0.1 wt.% to 0.6 wt.% of casein; and / or, The third buffer solution is a Tris buffer solution with a pH value of 8.0 to 9.0 and a concentration of 45 mmol / L to 55 mmol / L, which contains 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; and / or, The fluorescent microspheres are europium chelate fluorescent microspheres, with modification groups on the surface respectively. The modification groups are amino groups and / or carboxyl groups. The fluorescent microspheres are respectively fluorescent microspheres 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.

11. The preparation method of the fluorescence immunochromatographic test strip for detecting free IgE according to claim 1, wherein: The basic buffer solutions in the first buffer solution, the second buffer solution and the third buffer solution are independently selected from phosphate buffer solution, borate buffer solution, MES buffer solution, Tris buffer solution or citrate buffer solution.

12. A fluorescence immunochromatographic test strip for detecting free IgE prepared by the preparation method according to any one of claims 1 to 11.

13. A free IgE detection system, characterized in that: It includes a fluorescence immunochromatographic test strip for detecting free IgE prepared by the preparation method according to any one of claims 1 to 11.

14. The free IgE detection system according to claim 13, wherein: The free IgE detection system further includes a fluorescence immunoassay analyzer and an ID card disposed on the fluorescence immunoassay chromatographic test strip for detecting free IgE; calibration data and / or a standard curve are stored in the ID card.

15. The free IgE detection system according to claim 13, characterized in that: The free IgE detection system further includes a sample diluent for diluting a sample to be tested.

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