Detection card for quantitatively detecting human total IgE antibody, preparation method, kit and application

Abstract

The invention belongs to the technical field of immunodiagnosis, and relates to a detection card for quantitatively detecting human total IgE antibodies, which comprises a card shell and a test strip, and the test strip is arranged in the card shell; the test strip comprises a sample pad, a combination pad, a reaction film and an absorption pad which are arranged on a bottom plate, wherein an animal anti-human IgE monoclonal antibody marked by lanthanide fluorescent microspheres is arranged on the combination pad; a detection line coated with an animal anti-human IgE polyclonal antibody and a quality control line coated with an animal IgG antibody are arranged on the reaction film. The invention further provides a preparation method, a kit and application of the detection card for quantitatively detecting the human total IgE antibody. The method can effectively eliminate the interference of non-specific fluorescence, greatly improves the analysis sensitivity, and is simple and convenient to operate and low in cost.

Description

Technical Field

[0001] The present application relates to the field of immunodiagnosis technology, and in particular to a detection card for quantitatively detecting total human IgE antibodies, a preparation method, a kit and applications. Background Art

[0002] IgE (Immunoglobulin E, abbreviated as IgE) is immunoglobulin E, a type of antibody that only exists in mammals, and the immunoglobulin with the lowest content in normal human serum. The molecular weight of immunoglobulin E is 160kD, and its monomer consists of two heavy chains and two light chains. IgE is mainly produced by plasma cells in the lamina propria of the respiratory and digestive tract mucosa, and its concentration in normal human serum is extremely low, about 5x10^-5mg / ml. The tail of IgE has a high affinity with basophils and mast cells, so it is also called reactin or cell-friendly antibody. When the antibody binds to the antigen, basophils and mast cells release substances such as histamine to promote the development of inflammation. This is also an antibody that triggers rapid allergic reactions. IgE can cause type I hypersensitivity (also known as allergy), and may also be related to the body's anti-parasitic immunity. When parasites and allergens invade, the content of immunoglobulin E in the serum is significantly increased. Patients with allergic constitution or hypersensitivity have significantly higher IgE in their serum than normal people. Detection of total IgE, especially IgE autoantibodies, helps to understand the role of IgE in autoimmune diseases, and more comprehensively understand the etiology and pathogenesis of AID; improve the diagnostic ability of AID, expand the clinical diagnosis and treatment vision and level, and help develop targeted drugs. A large number of POCT (Point-of-Care Testing) products are also constantly pouring into the market, and the performance of these products generally does not meet the requirements of high sensitivity, such as low detection rate, inability to monitor the development trend of the disease, and insufficient sensitivity and specificity. Therefore, a detection method and kit that can improve the detection rate and monitor the total IgE content in the patient's blood at any time is particularly important for primary hospitals or wards.

[0003] At present, the commonly used detection methods for total human IgE include radioimmunoassay and enzyme-linked immunosorbent assay, which are all based on the specific binding reaction of antigen and antibody. Radioimmunoassay is expensive, radionuclides have certain potential hazards to the human body, and the waste treatment of the test is difficult; the operation process of enzyme-linked immunosorbent assay is cumbersome, the reaction takes a long time, and the detection is slow. Summary of the invention

[0004] The purpose of the present application is to propose a test card for quantitatively detecting total human IgE antibodies, a preparation method, a kit and an application thereof, so as to solve the technical problems in the prior art of high cost, complicated operation process, long reaction time and slow detection of total human IgE antibodies.

[0005] In order to solve the above technical problems, the present application embodiment provides a test card for quantitatively detecting total human IgE antibodies, which adopts the following technical solution:

[0006] A card case and a test strip, wherein the test strip is installed in the card case; the test strip comprises a sample pad, a binding pad, a reaction membrane and an absorption pad arranged on a bottom plate;

[0007] Wherein, the binding pad is provided with animal anti-human IgE monoclonal antibody labeled with lanthanide fluorescent microspheres; the reaction membrane is provided with a detection line coated with animal anti-human IgE polyclonal antibody and a quality control line coated with animal IgG antibody.

[0008] Furthermore, the lanthanide element is selected from Eu 3+ , Tb 3+ 、Sm 3+ and Dy 3+ One of them.

[0009] Furthermore, the diameter of the lanthanide fluorescent microspheres is 290-350 nm, and the solid content is 1%.

[0010] Furthermore, the species of the animals include rabbits, goats, sheep, chickens and mice.

[0011] In order to solve the above technical problems, the present application also provides a method for preparing a test card for quantitatively detecting total human IgE antibodies, which adopts the following technical solution:

[0012] A sample pad, a conjugate pad and a reaction membrane are prepared separately, wherein the conjugate pad is provided with an animal anti-human IgE monoclonal antibody labeled with lanthanide fluorescent microspheres; and the reaction membrane is provided with a detection line coated with an animal anti-human IgE polyclonal antibody and a quality control line coated with an animal IgG antibody;

[0013] The reaction membrane is arranged on a bottom plate, one end of the conjugate pad is overlapped on one end of the reaction membrane close to the detection line, one end of the pre-prepared absorption pad is overlapped on the other end of the reaction membrane close to the quality control line, and one end of the sample pad is overlapped on the other end of the conjugate pad to form a test strip;

[0014] The test strip is installed into the card housing to obtain a test card.

[0015] Furthermore, the sample pad is prepared by the following preparation steps:

[0016] The sample pad treatment solution prepared in advance is applied on the glass fiber, and then dried to obtain the sample pad;

[0017] The conjugate pad is prepared by the following preparation steps:

[0018] Using lanthanide element fluorescent microspheres to label animal anti-human IgE monoclonal antibodies to obtain fluorescent microsphere labeled antibody solution;

[0019] The fluorescent microsphere-labeled antibody solution is uniformly sprayed on the initial binding pad at a spray volume of 3.0-5.0 μL / cm;

[0020] The sprayed initial bonding pad is placed in an environment of 35-39° C. and ≤30% humidity to dry for 16-24 hours to obtain the bonding pad; and

[0021] The reaction membrane is prepared by the following preparation steps:

[0022] Dilute the animal anti-human IgE polyclonal antibody to 1.0-1.5 mg / mL using coating diluent to obtain T-line coating working solution;

[0023] Use coating diluent to dilute animal IgG antibody to 1.0-1.5 mg / mL to obtain C-line coating working solution;

[0024] Streaking the T-line coating working solution and the C-line coating working solution on a nitrocellulose membrane to form a corresponding detection line and quality control line;

[0025] The nitrocellulose membrane after the filming is dried in an environment of 45-55° C. and humidity ≤30% to obtain a reaction membrane.

[0026] Furthermore, the fluorescent microsphere-labeled antibody solution is prepared by the following preparation steps:

[0027] Take the first centrifuge tube, add the labeling buffer, vortex-mix the lanthanide fluorescent microspheres with a solid content of 1%, add them to the labeling buffer, and vortex-mix;

[0028] Add the marking activation solution into the first centrifuge tube and mix well;

[0029] After the mixing is completed, the first centrifuge tube is placed on a rotary mixer for rotation reaction;

[0030] After the reaction is completed, centrifuge, wash with labeling buffer, and centrifuge again;

[0031] After centrifugation, take out the first centrifuge tube, aspirate the supernatant, keep the precipitate, add the labeling buffer, and completely break up the precipitate by ultrasound;

[0032] Add animal anti-human IgE monoclonal antibody into the first centrifuge tube, mix well and perform a rotation reaction;

[0033] After the rotation reaction is completed, the labeling blocking solution is added to the first centrifuge tube and the reaction is rotated;

[0034] After blocking, centrifuge;

[0035] After centrifugation, discard the supernatant, keep the precipitate, add the labeled preservation solution into the centrifuge tube, and use ultrasound to completely break up the precipitate;

[0036] After the precipitate is evenly dispersed, it is transferred to a second centrifuge tube, and the labeling preservation solution is aspirated to clean the first centrifuge tube used for labeling. After cleaning, the solution is transferred to the second centrifuge tube and mixed to obtain a fluorescent microsphere labeled antibody solution, which is stored at 2-8°C for future use.

[0037] Furthermore, the sample pad treatment solution includes bovine serum albumin, sucrose, 1 wt% anti-human red blood cell antibody, 0.1 M boric acid borax buffer and Tween-20;

[0038] The coating diluent includes sucrose and 0.05M pH 8.0 Tris buffer;

[0039] The labeling buffer comprises a MES buffer having a pH of 6.0±0.05;

[0040] The marking activation solution includes 1-ethyl-(3-dimethylaminopropyl)carbodiimide;

[0041] The labeling blocking solution includes bovine serum albumin;

[0042] The labeling preservation solution includes bovine serum albumin, sucrose, casein sodium salt, trehalose, 0.05M pH 8.0 Tris buffer and Proclin 300.

[0043] In order to solve the above technical problems, the present application also provides a kit for quantitatively detecting total human IgE antibodies, which adopts the following technical solution:

[0044] The kit comprises the above-mentioned detection card for quantitatively detecting total human IgE antibodies or the detection card prepared by the above-mentioned preparation method, a sample diluent and a chip, wherein the chip contains standard curve data.

[0045] In order to solve the above technical problems, the embodiments of the present application also provide an application of a kit for quantitatively detecting total human IgE antibodies, wherein the kit is used to detect the content of total IgE antibodies in serum, plasma or whole blood samples.

[0046] Compared with the prior art, this application has the following beneficial effects:

[0047] The detection card for quantitative detection of total human IgE antibodies provided by the present application utilizes non-isotope immunoassay technology, uses lanthanide elements for labeling, measures fluorescence using time-resolved technology based on the luminescence characteristics of lanthanide chelates, and simultaneously detects the two parameters of wavelength and time for signal resolution, which can effectively eliminate the interference of non-specific fluorescence and greatly improve the analysis sensitivity; at the same time, the decay time of lanthanide chelate fluorescence is more than a hundred times that of traditional fluorescence, which greatly reduces background fluorescence and achieves a high signal-to-noise ratio; in addition, the present application is easy to operate, low in cost, does not require large-scale biochemical instruments, and can be detected through a portable fluorescent immunochromatography quantitative analyzer, which can reflect the severity of human allergies, facilitate clinical auxiliary rapid detection of total human IgE, facilitate promotion in clinical and community families, and is easy to use widely. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] In order to more clearly illustrate the scheme in the present application, a brief introduction is given below to the drawings required for use in the description of the embodiments of the present application. Obviously, the drawings described below are some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0049] Figure 1 This is a schematic diagram of the structure of a test strip of a test card for quantitatively detecting total human IgE antibodies of the present application;

[0050] Figure 2 It is a schematic diagram of the correlation results of the detection of a kit for quantitatively detecting total human IgE antibodies of the present application and a comparative kit;

[0051] Figure 3 This is a schematic diagram of the linear results of a kit for quantitatively detecting total human IgE antibodies of the present application;

[0052] Figure 4 This is a schematic diagram of the stability results of a kit for quantitatively detecting total human IgE antibodies in the present application. DETAILED DESCRIPTION

[0053] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by technicians in the technical field of the present application; the terms used in the specification of the application herein are only for the purpose of describing specific embodiments and are not intended to limit the present application; the terms "including" and "having" and any variations thereof in the specification and claims of the present application and the above-mentioned drawings are intended to cover non-exclusive inclusions. The terms "first", "second", etc. in the specification and claims of the present application or the above-mentioned drawings are used to distinguish different objects, not to describe a specific order.

[0054] Reference to "embodiments" herein means that a particular feature, structure, or characteristic described in conjunction with the embodiments may be included in at least one embodiment of the present application. The appearance of the phrase in various locations in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0055] In order to enable those skilled in the art to better understand the solution of the present application, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the accompanying drawings.

[0056] The present application provides a detection card for quantitatively detecting total human IgE antibodies, comprising a card shell and a test strip, wherein the test strip is installed in the card shell.

[0057] See also Figure 1 As shown, the test strip includes a sample pad 2 , a conjugate pad 3 , a reaction membrane 4 and an absorption pad 5 which are arranged on a bottom plate 1 .

[0058] The conjugate pad 3 is provided with animal anti-human IgE monoclonal antibody labeled with lanthanide fluorescent microspheres; the reaction membrane 4 is provided with a detection line 6 coated with animal anti-human IgE polyclonal antibody and a quality control line 7 coated with animal IgG antibody.

[0059] In some embodiments, the lanthanide element is selected from Eu 3+ , Tb 3+ 、Sm 3+ and Dy 3+ One of them.

[0060] Among them, the lanthanide fluorescent microspheres are embedded with rare earth-lanthanide elements, such as Eu 3+ , Tb 3+ 、Sm 3+ 、Dy 3+ Lanthanide chelates can emit special fluorescence under the excitation of ultraviolet light. Time-resolved Fluoroimmunoassay (TRFIA) uses microspheres as markers to label antigens, antibodies and other substances, and uses a time-resolved fluorescence analyzer to determine the amount of antigens and antibodies to be tested. Compared with ordinary fluorescence, the decay time of lanthanide chelate fluorescence is more than 100 times that of traditional fluorescence. It has the characteristics of large Stocks shift and long fluorescence lifetime. It can effectively avoid the background fluorescence in the sample and the influence of stray light (non-specific fluorescence) such as excitation light. Therefore, it has higher sensitivity and anti-interference ability than ordinary fluorescence.

[0061] In some embodiments, the lanthanide element is preferably Eu 3+ .

[0062] To encapsulate the rare earth element europium (Eu 3+ ) nano fluorescent microspheres are used as markers, and the results are determined by analyzing the fluorescence intensity of the detection area and the quality control area to achieve rapid quantitative analysis. The fluorescence decay time of europium is long and it can be repeatedly excited, making the relative specific activity of the fluorescent marker very high. At the same time, europium is covalently linked to macromolecules such as antigens and antibodies, which is firm and irreversible.

[0063] In some embodiments, the diameter of the lanthanide fluorescent microspheres is 290-350 nm and the solid content is 1%.

[0064] Nanoscale fluorescent microspheres have a huge specific surface area, which gives them extremely strong adsorption properties. Each microsphere can contain thousands of fluorescent molecules, greatly improving the labeling efficiency and effectively improving the sensitivity.

[0065] In some embodiments, the species of animal include rabbit, goat, sheep, chicken, and mouse.

[0066] Specifically, the animal anti-human IgE monoclonal antibody can be mouse anti-human IgE monoclonal antibody, rabbit anti-human IgE monoclonal antibody, goat anti-human IgE monoclonal antibody, etc.; the animal anti-human IgE polyclonal antibody can be rabbit anti-human IgE polyclonal antibody, goat anti-human IgE polyclonal antibody, etc.; the animal IgG antibody can be goat anti-mouse IgG, goat anti-rabbit IgG, etc.

[0067] For example, the mouse anti-human IgE monoclonal antibody labeled on the conjugate pad can react with the goat anti-mouse IgG antibody in the quality control line to develop color.

[0068] The test card for quantitative detection of total human IgE antibodies of the present application can effectively eliminate the interference of non-specific fluorescence and greatly improve the analysis sensitivity. In addition, it is easy to operate, low in cost, does not require large-scale biochemical instruments, and can be detected through a portable fluorescent immunochromatography quantitative analyzer. It can reflect the severity of human allergies, facilitate clinical auxiliary rapid detection of total human IgE, and is easy to promote in clinical and community families and easy to use widely.

[0069] Based on the above-mentioned test card for quantitatively detecting total human IgE antibodies, the present application also provides a method for preparing a test card for quantitatively detecting total human IgE antibodies, comprising the following steps:

[0070] A sample pad 2, a conjugate pad 3 and a reaction membrane 4 are prepared respectively, wherein the conjugate pad 3 is provided with an animal anti-human IgE monoclonal antibody labeled with fluorescent microspheres; a detection line 6 coated with an animal anti-human IgE polyclonal antibody and a quality control line 7 coated with an animal IgG antibody are provided on the reaction membrane 4;

[0071] The reaction membrane 4 is arranged on the bottom plate 1, one end of the conjugation pad 3 is overlapped on one end of the reaction membrane 4 close to the detection line 6, one end of the pre-prepared absorption pad 5 is overlapped on the other end of the reaction membrane 4 close to the quality control line 7, and one end of the sample pad 2 is overlapped on the other end of the conjugation pad 3 to form a test strip;

[0072] Install the test strip into the card housing to obtain a test card.

[0073] It should be noted that when the product pad 2, the binding pad 3, the reaction membrane 4 and the absorption pad 5 are arranged on the bottom plate 1, each layer of structure covers another layer of structure by 1-3 cm.

[0074] In some embodiments, assembly of the test card comprises the following steps:

[0075] (1) Lay the base plate 1 flat on a work surface at a temperature of (18-26)°C and a humidity of ≤30%. Remove the protective film at the place where the absorbent pad 5 is attached to the base plate 1, adhere the absorbent pad 5 thereon, and partially cover the other side of the absorbent pad 5 on the reaction membrane 4 by 2-3 mm. Remove the protective film at the place where the binding pad 3 and the sample pad 2 are attached to the base plate 1, adhere the binding pad 3 to its upper end, making sure that the binding pad 3 faces upward and one end of the binding pad 3 covers the reaction membrane 4 by 1-2 cm. Adhere the sample pad 2 thereon, making sure that the lower end of the sample pad 2 is aligned with the place where the sample pad of the base plate 1 is attached, and making sure that the upper end of the sample pad 2 covers the binding pad 3. Put the attached base plate into an aluminum foil bag for later use.

[0076] (2) Cut the prepared bottom plate into strips to obtain test strips, and install each test strip into the lower cover of the card shell. After installation, cover the upper cover and place it on the card press. Adjust the height of the card press to 3.5 mm so that the upper and lower covers of the card shell are tightly combined to obtain a test card.

[0077] In some embodiments, the sample pad 2 is prepared by the following preparation steps:

[0078] The pre-prepared sample pad treatment solution is coated on the glass fiber and dried to obtain the sample pad 2.

[0079] For example, set the wheel height of the paper impregnation pad press, the height of the left micrometer is 9mm, and the height of the right micrometer is 9.5mm; apply the sample treatment liquid evenly on the glass fiber at a treatment amount of 40mL / sheet (width: 25cm, length: 30cm), and dry it at 37±5℃ for 16-24h. Cut 5mm off the upper and lower long sides of the dried glass fiber, and then cut it into a size of (22±1)mm×(300±10)mm for use, and obtain sample pad 2.

[0080] The conjugate pad 3 is prepared by the following preparation steps:

[0081] Step S201, labeling animal anti-human IgE monoclonal antibodies using lanthanide fluorescent microspheres to obtain fluorescent microsphere labeled antibody solution;

[0082] Step S202, spraying the fluorescent microsphere-labeled antibody solution uniformly on the initial binding pad at a spray volume of 3.0-5.0 μL / cm;

[0083] Step S203 , placing the sprayed initial bonding pad in an environment of 35-39° C. and humidity ≤30% to dry for 16-24 hours to obtain bonding pad 3 .

[0084] For example, a fluorescent microsphere-labeled antibody solution is prepared, and the fluorescent microsphere-labeled antibody solution is ultrasonicated under 90W ultrasound, specifically, ultrasonicated for 2s, rested for 5s, and repeated 3 times; glass fiber 8964 is cut into (10±0.5) mm×(300±10) mm; the mixed fluorescent microsphere-labeled antibody solution is loaded into a gold spray scribing machine, and sprayed onto the cut initial binding pad at a spray volume of 3.0-5.0 μL / cm; the sprayed initial binding pad is placed in an environment of (37±2)°C and humidity ≤30% to dry for 16-24 hours, and after drying, it is cut into a width of 9.5-10.5 mm and a length of 290-310 mm to obtain a binding pad 3.

[0085] In some embodiments, taking 100 μL of fluorescent microspheres as an example, the fluorescent microsphere-labeled antibody solution is prepared by the following preparation steps:

[0086] Step S301, take a first centrifuge tube, add a labeling buffer, vortex-mix lanthanide fluorescent microspheres with a solid content of 1%, add them to the labeling buffer, and vortex-mix.

[0087] Specifically, a 2 mL centrifuge tube is selected as the first centrifuge tube, 1 mL of labeling buffer is added to the first centrifuge tube using a pipette, and 100 μL of lanthanide fluorescent microspheres with a solid content of 1% are added to the labeling buffer, and vortexed again to mix.

[0088] Step S302, adding the marking activation solution into the first centrifuge tube and mixing.

[0089] Specifically, 20 μL of the labeling activation solution was added to the first centrifuge tube, the lid was tightly closed, and the tube was slowly inverted upside down 10 to 20 times to mix well.

[0090] Step S303, after the mixing is completed, the first centrifuge tube is placed on a rotary mixer for rotation reaction.

[0091] The rotation reaction time is 20-40 minutes.

[0092] Step S304, after the reaction is completed, centrifugation is performed, and after washing with a labeling buffer, centrifugation is performed again.

[0093] For example, centrifugation is performed at 4°C, wherein the centrifugal speed is 15,000 rpm and the centrifugal time is 30 min. After washing once with the labeling buffer, centrifugation is performed again at 15,000 rpm and 4°C for 20-40 min.

[0094] Step S305, after the centrifugation is completed, the first centrifuge tube is taken out, the supernatant is aspirated, the precipitate is retained, and a labeling buffer is added, and the precipitate is completely broken up by ultrasound.

[0095] For example, after centrifugation, gently take out the centrifuge tube and slowly aspirate the supernatant with a pipette; retain the precipitate, use a pipette to aspirate 900 μL-1000 μL of labeling buffer and add it to the precipitate, and use an ultrasonic cell crusher to sonicate 3-5 times to completely break up the precipitate.

[0096] Step S306, adding animal anti-human IgE monoclonal antibody into the first centrifuge tube, mixing and then performing a rotation reaction.

[0097] For example, 100 μg of mouse anti-human IgE monoclonal antibody was added to the first centrifuge tube, which was then inverted 20 times to mix well, and placed on a rotary mixer to rotate for 120 minutes.

[0098] Step S307, after the rotation reaction is completed, the labeling blocking solution is added to the first centrifuge tube, the precipitate is completely broken up by ultrasonication with an ultrasonic cell disruptor, and the reaction is rotated.

[0099] For example, after the rotation reaction is completed, use a pipette to draw 100 μL of the labeled blocking solution into the first centrifuge tube, use an ultrasonic cell crusher to sonicate 3-5 times to completely break up the precipitate, place it on the rotation mixer again, and rotate the reaction for 60 minutes.

[0100] Step S308: After sealing is completed, centrifugation is performed.

[0101] The centrifugation was carried out at 4°C, with a centrifugal speed of 13,000 rpm and a centrifugal time of 20-40 min.

[0102] Step S309, after the centrifugation is completed, the supernatant is discarded, the precipitate is retained, and the labeling preservation solution is added to the first centrifuge tube, and the precipitate is completely broken up by ultrasound.

[0103] For example, use a pipette to draw 1 mL of the labeled preservation solution and add it into the first centrifuge tube, and use an ultrasonic cell disruptor to sonicate 3-5 times to completely break up the precipitate.

[0104] Step S310, after the precipitate is evenly dispersed, it is transferred to a second centrifuge tube, and the labeling preservation solution is aspirated to clean the first centrifuge tube used for labeling. After cleaning, the solution is transferred to the second centrifuge tube and mixed to obtain a fluorescent microsphere labeled antibody solution, which is stored at 2-8°C for future use.

[0105] The reaction membrane 4 is prepared by the following preparation steps:

[0106] Step S401, diluting animal anti-human IgE polyclonal antibody to 1.0-1.5 mg / mL using coating diluent to obtain T-line coating working solution.

[0107] For example, the required amount is calculated according to the production batch, and the rabbit anti-human IgE polyclonal antibody is diluted to 1.0-1.5 mg / mL with the coating diluent to obtain the T-line coating working solution; the prepared solution is stored at 2-8° C. for future use.

[0108] Preferably, the animal anti-human IgE polyclonal antibody is diluted to a concentration of 1.0 mg / mL.

[0109] Step S402, diluting the animal IgG antibody to 1.0-1.5 mg / mL using a coating diluent to obtain a C-line coating working solution.

[0110] For example, the required amount is calculated according to the production batch, and the goat anti-mouse IgG antibody is diluted to 1.0-1.5 mg / mL with the coating diluent to obtain the C-line coating working solution; the prepared solution is stored at 2-8°C for future use.

[0111] Preferably, the animal IgG antibody is diluted to a concentration of 1.0 mg / mL.

[0112] Step S403, streaking the T-line coating working solution and the C-line coating working solution on the nitrocellulose membrane respectively to form corresponding detection lines and quality control lines.

[0113] For example, a nitrocellulose membrane (NC membrane) is cut into 25 mm×(300±5) mm, the protective film on the NC membrane pasted on the bottom plate 1 is peeled off, and the NC membrane is evenly pushed from left to right to make it firm.

[0114] Turn on the gold-spraying film scribing machine, set the scribing volume to 1 μL / cm, change the scribing length to 300 mm, adjust the position of the scribing needle, the distance between the C line (quality control line) and the T line (test line) is (5±0.5) mm, the distance between the C line and the lower edge of the nitrocellulose membrane is (10±0.5) mm, and the distance between the T line and the upper edge of the nitrocellulose membrane is (10±0.5) mm.

[0115] Step S404 , drying the nitrocellulose membrane after the filming is completed in an environment of 45-55° C. and humidity ≤30% to obtain a reaction membrane 4 .

[0116] For example, after the film is cut, the NC film is placed in an electric hot air drying oven with a temperature of (50±5)°C and a humidity of ≤30% for 64 to 72 hours, and is stored at 4 to 30°C for later use under a relative humidity of ≤30%.

[0117] In some embodiments, the sample pad treatment solution includes bovine serum albumin, sucrose, 1 wt % anti-human erythrocyte antibody, 0.1 M boric acid borax buffer and Tween-20.

[0118] The sample pad treatment solution was prepared by dissolving 10 g bovine serum albumin, 10 g sucrose, and 1 wt% anti-human RBC in 1000 mL 0.1 M boric acid borax buffer (pH 8.0), and then adding 5 mL Tween-20 and stirring until all dissolved.

[0119] The preparation method of 0.1M boric acid borax buffer is as follows: 150mL of 0.05M borax solution, 350mL of 0.2M boric acid solution and 500mL of purified water are mixed, and the pH value is adjusted to (8.0±0.05) with borax solution or boric acid solution. The preparation process of 0.05M borax solution is as follows: 19.07g of sodium tetraborate is dissolved in 800mL of purified water, and the volume is adjusted to 1000mL after dissolution.

[0120] In this example, the coating diluent includes sucrose and 0.05M pH 8.0 Tris buffer.

[0121] Specifically, the coating diluent is prepared as follows: 1 g of sucrose is added to 100 mL of 0.05 M pH 8.0 Tris buffer, and the mixture is dissolved and mixed to obtain the coating diluent.

[0122] Among them, the preparation of 0.05M pH8.0 Tris buffer includes:

[0123] Dissolve 6.05 g Tris in 800 mL purified water, adjust pH to 8.0 ± 0.05 with 1 mol / L HCl or 1 mol / L NaOH, and make up to 1000 mL to obtain 0.05 M pH 8.0 Tris buffer.

[0124] In this embodiment, the labeling buffer includes a MES buffer having a pH of 6.0±0.05.

[0125] Specifically, 1.07 g of MES was dissolved in 80 mL of purified water, and after dissolution, the pH was adjusted to 6.0±0.05 with 1 mol / L HCl or 1 mol / L NaOH, and the volume was fixed to 100 mL to obtain a labeling buffer.

[0126] In this embodiment, the marking activation solution includes 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC).

[0127] Specifically, 0.05 g of EDC was dissolved in 1 mL of purified water to prepare a labeling activation solution, and the prepared labeling activation solution must be used within 10 minutes.

[0128] In this embodiment, the labeling blocking solution includes bovine serum albumin.

[0129] For example, 1 g of bovine serum albumin is dissolved in 10 mL of purified water, and after being fully dissolved, it is filtered through a microporous filter membrane with a pore size of 0.22 μm to obtain a labeling blocking solution.

[0130] In this embodiment, the labeling preservation solution includes bovine serum albumin, sucrose, casein sodium salt, trehalose, 0.05M pH 8.0 Tris buffer and Proclin 300.

[0131] Specifically, 1 g of bovine serum albumin, 10 g of sucrose, 0.5 g of casein sodium salt, and 5 g of trehalose are added to 100 mL of 0.05 M pH 8.0 Tris buffer to dissolve, and then 0.05 mL of Proclin 300 solution is added and mixed to obtain a labeled preservation solution.

[0132] In some embodiments, the preparation method of 0.05M pH8.0 Tris buffer is as follows: 6.05g Tris is dissolved in 800mL purified water, and after dissolution, the pH is adjusted to 8.0±0.05 with 1mol / L HCl or 1mol / L NaOH, and the volume is fixed to 1000mL.

[0133] The present application also provides a kit for quantitatively detecting total human IgE antibodies, which includes the detection card for quantitatively detecting total human IgE antibodies as described above, a sample diluent and a chip, wherein the chip contains standard curve data.

[0134] The sample diluent is a phosphate buffered saline (PBS buffer), specifically a 0.01M pH 7.4 PBS buffer, and the preparation process includes: dissolving 0.5 mL Proclin 300 in 1000 mL 0.01M pH 7.4 PBS buffer and mixing well to obtain the sample diluent.

[0135] In some embodiments, the sample diluent can be prepared in large quantities. After preparation, it is packaged into EP tubes according to the specification of 0.30 mL / tube, labeled on the tube wall, and stored at 4-30°C. The shelf life is 18 months.

[0136] In some embodiments, the preparation of the chip comprises the following steps:

[0137] 1) Prepare curve data:

[0138] Take the total IgE corporate calibrator, select Signal Test-Repeatability Test on the Lanbo AFS-1000 dry fluorescent immunoassay management tool, click New Reset Page, and then click Test to perform the test. Repeat the test for 3 times for each concentration of sample, and calculate the mean T / C value obtained by the test. Use the prepared concentration and the corresponding T / C mean as the curve data. Specifically, count the T / C mean of the repeated measurements of samples of each concentration, take the logarithm, and use a straight line fit, with the assigned concentration value as the horizontal axis and the T / C mean as the vertical axis, to establish the standard curve of the kit.

[0139] 2)Configure chip data

[0140] In the Lanbo AFS-1000 dry fluorescence immunoassay management tool, select project parameters and use New or Edit to set relevant parameters. After editing is complete and confirmation is correct, click Save and the project parameters are set.

[0141] In the Lanbo AFS-1000 dry fluorescence immunoassay management tool, select the test experiment, click New, select the total IgE project, fill in the experiment number, click OK, and complete the new experiment.

[0142] 3) In the Lanbo AFS-1000 dry fluorescent immunoassay management tool, select curve generation and select the experiment created in the previous step in the experiment list on the left;

[0143] Fill the curve data prepared in the first step into the table. After the data is filled in, click Auto Select - Auto Sort - Save in sequence;

[0144] After saving, select the reaction value transformation as logarithm, the number of decimal places is 4, select the concentration transformation as logarithm, the number of decimal places is 2, select the linear interpolation algorithm, and after confirming that it is correct, click Fit;

[0145] After fitting, the R value and curve will be displayed on the right side of the fitting result. After confirming that the fitting result is accurate, complete the curve preparation and obtain the standard curve data.

[0146] 4) In the Lanbo AFS-1000 dry fluorescent immunoassay management tool, select Generate ID file, select the newly created project in the project list on the left, and the parameters set in the above steps will be automatically displayed in the basic parameter area, sub-item parameter area and curve data;

[0147] After checking all parameters to ensure they are correct, set the production year, production month and batch in the basic parameter area. After the settings are completed, a barcode will be automatically generated at the barcode location to confirm whether the barcode is accurate;

[0148] After confirmation, click Generate ID File, the data transfer interface pops up, click Confirm to complete the chip standard curve data configuration.

[0149] 5) Chip firing

[0150] Open the Lanbo AFS-1000 dry fluorescent immunoassay management tool, right-click the mouse, and select Open File Location. Double-click the Item folder and find the latest generated HEX file corresponding to the barcode data in the Item file. Burn the chip data file in the programmer.

[0151] Based on the above-mentioned kit for quantitatively detecting total human IgE antibodies, the present application also provides an application thereof, wherein the kit is used to detect the content of total IgE antibodies in serum, plasma or whole blood samples.

[0152] The kit of the present application uses a time-resolved fluorescence labeling method to detect the total IgE content in human serum, plasma or whole blood. The total IgE concentration is positively correlated with the relative luminescence intensity. The total IgE antibody content in the tested sample can be obtained according to the total IgE concentration standard curve. At the same time, the total IgE antibody is labeled with a lanthanide chelate with high luminescence intensity and sensitivity, which can effectively eliminate the interference of non-specific fluorescence, reduce background fluorescence, and achieve a high signal-to-noise ratio.

[0153] The following is a more specific description of the content of the present application in conjunction with specific embodiments, and further elaboration of the present application, but these embodiments are by no means limiting the present application.

[0154] Example 1

[0155] This example describes in detail the method for using the kit for quantitatively detecting total human IgE antibodies.

[0156] First, sample collection.

[0157] (1) Applicable sample types

[0158] Serum, plasma, whole blood samples.

[0159] Note: It is not recommended to use samples with hemolysis, lipemia, or icterus.

[0160] (2) Sample collection

[0161] Whole blood collection: Collect venous blood from the subject using an anticoagulant tube containing lithium heparin, EDTA-K2 or sodium citrate, and shake the collected blood sample for later use;

[0162] Plasma collection: Collect venous blood from the subject using an anticoagulant tube containing lithium heparin, EDTA-K2 or sodium citrate. After blood collection, plasma should be separated as soon as possible to avoid hemolysis.

[0163] Serum collection: Collect venous blood from the subject using a serum tube or a rapid serum tube containing a coagulant. The serum should be separated as soon as possible after blood collection to avoid hemolysis.

[0164] (3) Sample preservation

[0165] After the sample is collected, it should be used as soon as possible. If it cannot be tested in time, serum / plasma / whole blood can be stored at room temperature (10-30°C) for 3 hours; serum / plasma can be stored at 2°C-8°C for 7 days, whole blood can be stored at 2°C-8°C for 3 days; serum / plasma can be stored at -20±5°C for 90 days. Whole blood samples cannot be frozen.

[0166] Note: Refrigerated and frozen samples must be fully dissolved and mixed before testing, and can only be tested after returning to room temperature. Do not freeze and thaw repeatedly.

[0167] After the sample collection is completed, the sample is tested. The test operation process is as follows:

[0168] Turn on the instrument and insert the chip with the same batch number as the reagent;

[0169] Use a pipette to draw 100 μL of sample, add it to the sample diluent, and mix by inverting;

[0170] Open the aluminum foil bag, take out the test card, and place it horizontally on the table;

[0171] Use a pipette to draw 100 μL of the diluted and mixed sample and add it to the sample well of the test card;

[0172] Select the sample type "Serum" on the supporting instrument;

[0173] Instant test: After 10 minutes of reaction at room temperature, put the test card into the instrument slot, select the "Instant Test" mode, and click "Test"; Standard test: Put the test card into the instrument slot, select the "Standard Test" mode, click "Test", the instrument will automatically start timing, and after the timing ends, it will automatically test and display the results;

[0174] Click "Print" to print the test result report.

[0175] It should be noted that the test should be carried out at room temperature (18-30°C).

[0176] Example 2

[0177] This example tests the performance indicators of a kit for quantitatively detecting total human IgE antibodies.

[0178] The performance index requirements of the kit of this application are as follows:

[0179] (1) Linearity: In the concentration range of [20, 200] IU / mL, the linear correlation coefficient (r) is not less than 0.9900.

[0180] (2) Accuracy: The deviation should be within ±10%.

[0181] (3) Detection limit: should not be higher than 20 IU / mL.

[0182] (4) Repeatability: The intra-batch coefficient of variation (CV) should be ≤10%.

[0183] 1. Linear detection

[0184] The kit of this application was tested for correlation with other similar products of Changjia (comparison kits), and the results are as follows Figure 2 As shown, R=0.983>0.95, and the correlation is good, indicating that the kit of the present application can be used to detect the content of total IgE antibodies in serum, plasma or whole blood samples.

[0185] The linear regression equation of the kit of this application is established as follows:

[0186] Human IgE (20, 50, 100, 150, 200 IU / mL) standard solution, each sample was tested three times, the average value was taken, the average value of the measured concentration and the theoretical concentration were fitted by the least squares method, and the linear regression equation was obtained: y = 0.944x + 2.3766, r 2 =0.9989, P<0.05, the results are as follows Figure 3 shown.

[0187] It can be seen that within the concentration range of [20, 200] IU / mL, the linear correlation coefficient (r) of the kit of the present application is greater than 0.9900, which meets the requirements.

[0188] 2. Accuracy test

[0189] The median and high values ​​of human IgE standard solutions of 50 and 150 IU / mL were prepared, and each sample was tested 3 times. The relative deviation of each sample was calculated. i) were -3.54%, 4.18%, 4.60%, respectively, and B at level 2 (150 IU / mL) i The values ​​are -1.53%, -2.88%, and -6.07% respectively. The test result of the accuracy of the kit does not exceed ±10%, indicating that the accuracy of the kit of this application meets the requirements.

[0190] 3. Precision testing

[0191] The human IgE standard solutions of 50 and 150 IU / mL were prepared at the middle and high values, and each sample was tested 10 times to calculate the coefficient of variation. The coefficient of variation (CV) of level 1 (50 IU / mL) was 4.43%, and the CV value of level 2 (150 IU / mL) was 6.58%. The test results of the precision of the kit of the present application did not exceed ±10%, indicating that the kit of the present application had good repeatability.

[0192] 4. Stability test

[0193] Prepare a batch of kits for quantitative detection of total human IgE antibodies, store them at room temperature (2-30°C) for one year, and test the same concentration of total human IgE samples every one month. The results are as follows: Figure 4 shown.

[0194] It can be seen from the results that the kit of the present application has good stability.

[0195] In summary, the kit of the present application has a wide detection range (20-200 IU / mL), high sensitivity (lower limit of detection is 20 IU / mL), high accuracy (determination of total IgE corporate reference, deviation within ±10%, short detection time (10min)), shortening the time required for clinical testing, while having high detection precision and small systematic error.

[0196] Obviously, the embodiments described above are only some embodiments of the present application, rather than all embodiments. The preferred embodiments of the present application are given in the accompanying drawings, but they do not limit the patent scope of the present application. The present application can be implemented in many different forms. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive. Although the present application is described in detail with reference to the aforementioned embodiments, for those skilled in the art, it is still possible to modify the technical solutions recorded in the aforementioned specific implementation methods, or to perform equivalent replacement of some of the technical features therein. Any equivalent structure made using the contents of the specification and drawings of this application, directly or indirectly used in other related technical fields, is similarly within the scope of patent protection of this application.

Claims

1. A test card for quantitatively detecting total human IgE antibodies, characterized in that: include: A card case and a test strip, wherein the test strip is installed in the card case; the test strip comprises a sample pad, a binding pad, a reaction membrane and an absorption pad arranged on a bottom plate; Wherein, the binding pad is provided with animal anti-human IgE monoclonal antibody labeled with lanthanide fluorescent microspheres; the reaction membrane is provided with a detection line coated with animal anti-human IgE polyclonal antibody and a quality control line coated with animal IgG antibody.

2. The test card for quantitatively detecting total human IgE antibodies according to claim 1, characterized in that: The lanthanide element is selected from Eu 3+ , Tb 3+ 、Sm 3+ and Dy 3+ One of them.

3. The test card for quantitatively detecting total human IgE antibodies according to claim 1, characterized in that: The diameter of the lanthanide fluorescent microspheres is 290-350 nm, and the solid content is 1%.

4. The test card for quantitatively detecting total human IgE antibodies according to claim 1, characterized in that: The species of animals include rabbits, goats, sheep, chickens and mice.

5. A method for preparing a test card for quantitatively detecting total human IgE antibodies, characterized in that: The following steps are involved: A sample pad, a conjugate pad and a reaction membrane are prepared separately, wherein the conjugate pad is provided with an animal anti-human IgE monoclonal antibody labeled with lanthanide fluorescent microspheres; and the reaction membrane is provided with a detection line coated with an animal anti-human IgE polyclonal antibody and a quality control line coated with an animal IgG antibody; The reaction membrane is arranged on a bottom plate, one end of the conjugate pad is overlapped on one end of the reaction membrane close to the detection line, one end of the pre-prepared absorption pad is overlapped on the other end of the reaction membrane close to the quality control line, and one end of the sample pad is overlapped on the other end of the conjugate pad to form a test strip; The test strip is installed into the card housing to obtain a test card.

6. The method for preparing a test card for quantitatively detecting total human IgE antibodies according to claim 5, characterized in that: The sample pad is prepared by the following preparation steps: The sample pad treatment solution prepared in advance is applied on the glass fiber, and then dried to obtain the sample pad; The conjugate pad is prepared by the following preparation steps: Using lanthanide element fluorescent microspheres to label animal anti-human IgE monoclonal antibodies to obtain fluorescent microsphere labeled antibody solution; The fluorescent microsphere-labeled antibody solution is uniformly sprayed on the initial binding pad at a spray volume of 3.0-5.0 μL / cm; The sprayed initial bonding pad is placed in an environment of 35-39° C. and ≤30% humidity to dry for 16-24 hours to obtain the bonding pad; and The reaction membrane is prepared by the following preparation steps: Dilute the animal anti-human IgE polyclonal antibody to 1.0-1.5 mg / mL using coating diluent to obtain T-line coating working solution; Use coating diluent to dilute animal IgG antibody to 1.0-1.5 mg / mL to obtain C-line coating working solution; Streaking the T-line coating working solution and the C-line coating working solution on a nitrocellulose membrane to form a corresponding detection line and quality control line; The nitrocellulose membrane after the filming is dried in an environment of 45-55° C. and humidity ≤30% to obtain a reaction membrane.

7. The method for preparing a test card for quantitatively detecting total human IgE antibodies according to claim 6, characterized in that: The fluorescent microsphere-labeled antibody solution is prepared by the following preparation steps: Take the first centrifuge tube, add the labeling buffer, vortex-mix the lanthanide fluorescent microspheres with a solid content of 1%, add them to the labeling buffer, and vortex-mix; Add the marking activation solution into the first centrifuge tube and mix well; After the mixing is completed, the first centrifuge tube is placed on a rotary mixer for rotation reaction; After the reaction is completed, centrifuge, wash with labeling buffer, and centrifuge again; After centrifugation, take out the first centrifuge tube, aspirate the supernatant, keep the precipitate, add the labeling buffer, and completely break up the precipitate by ultrasound; Add animal anti-human IgE monoclonal antibody into the first centrifuge tube, mix well and perform a rotation reaction; After the rotation reaction is completed, the labeling blocking solution is added to the first centrifuge tube and the reaction is rotated; After blocking, centrifuge; After centrifugation, discard the supernatant, keep the precipitate, add the labeled preservation solution into the centrifuge tube, and use ultrasound to completely break up the precipitate; After the precipitate is evenly dispersed, it is transferred to a second centrifuge tube, and the labeling preservation solution is aspirated to clean the first centrifuge tube used for labeling. After cleaning, the solution is transferred to the second centrifuge tube and mixed to obtain a fluorescent microsphere labeled antibody solution, which is stored at 2-8°C for future use.

8. The method for preparing a test card for quantitatively detecting total human IgE antibodies according to claim 7, characterized in that: The sample pad treatment solution includes bovine serum albumin, sucrose, 1wt% anti-human red blood cell antibody, 0.1M boric acid borax buffer and Tween-20; The coating diluent includes sucrose and 0.05M pH 8.0 Tris buffer; The labeling buffer comprises a MES buffer having a pH of 6.0±0.05; The marking activation solution includes 1-ethyl-(3-dimethylaminopropyl)carbodiimide; The labeling blocking solution includes bovine serum albumin; The labeling preservation solution includes bovine serum albumin, sucrose, casein sodium salt, trehalose, 0.05M pH 8.0 Tris buffer and Proclin 300.

9. A kit for quantitatively detecting total human IgE antibodies, characterized in that: The kit comprises a test card for quantitatively detecting total human IgE antibodies as described in any one of claims 1 to 4 or a test card prepared by the preparation method as described in any one of claims 5 to 8, a sample diluent and a chip, wherein the chip contains standard curve data.

10. Use of the kit for quantitatively detecting total human IgE antibodies according to claim 9, characterized in that: The kit is used to detect the content of total IgE antibodies in serum, plasma or whole blood samples.

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