Novel coronavirus antigen detection kit based on immunochromatography
Through the novel coronavirus antigen detection kit based on immunochromatography and latex microsphere labeling process, the problem of limited sample types and low detection sensitivity in the prior art is solved, and a high sensitivity detection of multiple samples is realized, suitable for self-screening and self-detection.
Patent Information
- Application Number
- CN202311838781.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-12-28
- Publication Date
- 2025-07-01
AI Technical Summary
The existing antigen detection methods for the new coronavirus require pretreatment of samples, and the applicable types of samples are limited, especially for nasal and pharyngeal swab samples that are difficult to collect in children, and the detection sensitivity is low.
The novel coronavirus antigen detection kit based on immunochromatography and latex microsphere labeling process is used, which is suitable for nasopharyngeal swabs, pharyngeal swabs, nasal swabs, and saliva samples. The new coronavirus antibodies are labeled by latex microspheres and tested in combination with immunochromatography to simplify operations and improve sensitivity.
It realizes high sensitivity detection for a variety of sample types, especially without pretreatment for saliva samples. It is suitable for self-screening and self-detection. The sensitivity is 2-4 times higher than that of the colloidal gold method, low cost and easy to operate.
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Figure BDA0004638270890000061
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of immunochromatography, and particularly to a novel coronavirus antigen detection kit based on immunochromatography. Background Art
[0002] The novel coronavirus (hereinafter referred to as SARS-CoV-2), a betacoronavirus, has an envelope, and the particles are round or oval in shape, with a diameter of 60-140 nm. COVID-19 is a disease caused by a virus. The most common symptoms are fever, chills, and sore throat, but there are also a series of other symptoms. There are five "variants of concern" (VOC) proposed by the World Health Organization (WHO), namely Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Omicron (B.1.1.529).
[0003] To further optimize the SARS-CoV-2 detection strategy, according to the needs of epidemic prevention and control, the domestic monitoring mode of "antigen screening and nucleic acid diagnosis" is promoted, and antigen detection is added as a supplement to nucleic acid detection to improve the ability to "detect early" SARS-CoV-2 infected persons. Globally, rapid SARS-CoV-2 antigen detection has been widely recognized. As of April 7, 2022, the National Medical Products Administration (NMPA) of China has approved multiple antigen rapid detection reagents. The antigen detection sensitivity is positively correlated with the viral load of infected persons. For samples with a high viral load and strong infectivity, the detection sensitivity is higher. The SARS-CoV-2 antigen detection sensitivities of common samples from high to low are nasal swabs (83%), nasopharyngeal swabs (71%), oropharyngeal swabs (69%), and saliva (68%).
[0004] According to the markers used in the detection reagents, they can be divided into colloidal gold immunochromatography, latex immunochromatography, and fluorescence immunochromatography. The currently marketed kits are applicable to nasal swabs, nasopharyngeal swabs, oropharyngeal swabs, and saliva. The samples need to be pretreated and a sample addition operation is required. Summary of the Invention
[0005] To solve the above problems, the present invention provides a novel coronavirus antigen detection kit based on immunochromatography. The kit is a detection reagent card including a test strip, a sample collection pad, and a plastic cartridge. The test strip is prepared by sequentially pasting a coated NC membrane, a conjugate pad, a sample pad, and a blotting paper onto a PVC backplane. The kit can detect the N protein antigen of the novel coronavirus in nasopharyngeal swabs, throat swabs, nasal swabs, and saliva samples. The sample pad of the detection reagent card is pretreated with 50-200 mM, pH 9.0 Tris-HCL, 1.5-4% NaCl, 0.2%-0.4% SDS, 3-5% BSA, and 0.2-1% Tween-20; and latex microspheres are used for labeling the novel coronavirus antibody.
[0006] In one embodiment, the activation buffer for labeling the novel coronavirus antibody with latex microspheres is 25-100 mM MES buffer, the coupling buffer is 25-100 mM HEPES buffer, and the protein amount is 50 μg-100 μg / mg microspheres.
[0007] In one embodiment, the activation buffer for labeling the novel coronavirus antibody with latex microspheres is 50 mM MES buffer, the coupling buffer is 50 mM HEPES buffer, and the protein amount is 50 μg / mg microspheres.
[0008] In one embodiment, the latex microspheres are activated with 25 mM MES buffer for 20 minutes, the supernatant is discarded by centrifugation; an equal volume of 25 mM HEPES buffer is added for washing, and the supernatant is discarded by centrifugation; 1 / 2 volume of the original volume of 25 mM HEPES buffer is added, and the novel coronavirus antibody is added according to the ratio of 50 μg-100 μg / mg microspheres, and the reaction is carried out for 2 hours; 1 / 5 volume of the blocking solution is added for blocking for 1 hour, and the supernatant is discarded by centrifugation; 1 / 4 volume of the original volume of the reconstitution solution is added to resuspend the microspheres to obtain a latex microsphere-labeled complex.
[0009] In one embodiment, the antibody complex labeled with latex microspheres is sprayed onto the glass fiber at 1-3 μL / cm of the latex microsphere-labeled complex and dried at 37 °C for 16-18 hours to obtain a microsphere-labeled conjugate pad.
[0010] Compared with the prior art, the present invention has the following advantages:
[0011] 1) It is applicable to a wide variety of sample types, including nasopharyngeal swabs, throat swabs, nasal swabs, and saliva samples, meeting different testing requirements. When it comes to children or other groups who are difficult to collect nasopharyngeal or throat swab samples, saliva samples can be collected from the mouth for direct testing. This invention combines immunochromatography and latex microsphere labeling technology to detect the N protein antigen of the novel coronavirus in nasopharyngeal swabs, throat swabs, nasal swabs, and saliva samples, and can be used for the diagnosis of the novel coronavirus, self-screening and self-testing of suspected individuals, etc.
[0012] 2) It is easy to operate. For saliva samples, no pretreatment is required and they can be directly tested, reducing the problem of low sensitivity after pretreatment.
[0013] 3) It has high sensitivity. By means of latex microsphere labeling, a pretreated sample pad, and a large sample volume, the detection sensitivity is improved; latex microspheres are used for antibody labeling, and the detection sensitivity of latex microspheres is 2-4 times higher than that of colloidal gold.
[0014] 4) It has a relatively low cost. The required raw materials, technologies, and instruments are all relatively simple and easily available. Detailed implementation method
[0015] To enable those skilled in the art to better understand the technical solutions in this application, the following will further illustrate the present invention in combination with embodiments. Obviously, the embodiments are only examples and cannot limit the present invention. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the scope of protection of this application. The experimental methods used in the present invention are all conventional methods, and the experimental equipment, materials, reagents, etc. used can all be obtained through commercial channels.
[0016] Embodiment 1: Preparation of a sample pad capable of detecting saliva samples
[0017] 1. Preparation of the conjugate pad
[0018] Activate the latex microspheres with 25 mM MES buffer for 20 minutes, centrifuge and discard the supernatant; add an equal volume of 25 mM HEPES buffer for washing, centrifuge and discard the supernatant; add 1 / 2 of the original volume of 25 mM HEPES buffer, add the novel coronavirus antibody according to the ratio of 100 μg / mg microspheres, and react for 2 hours; add 1 / 5 of the original volume of the blocking solution for blocking for 1 hour, centrifuge and discard the supernatant; add 1 / 4 of the original volume of the reconstitution solution to resuspend the microspheres to obtain a latex microsphere-labeled complex.
[0019] Spray the labeled complex onto the glass fiber at a rate of 0.001 mL / cm of the latex microsphere-labeled complex, and dry at 37 °C for 16-18 hours to obtain a microsphere-labeled conjugate pad.
[0020] 2. Preparation of Coated NC Membrane
[0021] Coat the novel coronavirus antibody onto the nitrocellulose membrane at a concentration of 1.5 mg / mL, at 45 °C, with humidity < 20%, and a drying time of 14 - 16 hours.
[0022] 3. Preparation of Sample Pad
[0023] Sample Pad 1: Prepare a sample pad pretreatment solution containing 100 mM Tris-HCL (pH 9.0), 2% NaCl, 0.2% SDS, 2% BSA, and 0.5% Tween-20, and uniformly coat it onto the glass fiber, then dry at 37 °C for 24 hours to obtain Sample Pad 1.
[0024] Sample Pad 2: Prepare a sample pad pretreatment solution containing 100 mM Tris-HCL (pH 9.0), 0.2% SDS, 2% BSA, and 0.5% Tween-20, and uniformly coat it onto the glass fiber, then dry at 37 °C for 24 hours to obtain Sample Pad 2.
[0025] Sample Pad 3: Prepare a sample pad pretreatment solution containing 100 mM Tris-HCL (pH 9.0), 2% NaCl, 2% BSA, and 0.5% Tween-20, and uniformly coat it onto the glass fiber, then dry at 37 °C for 24 hours to obtain Sample Pad 3.
[0026] Sample Pad 4: Prepare a sample pad pretreatment solution containing 100 mM Tris-HCL (pH 9.0), 2% NaCl, 0.2% SDS, and 0.5% Tween-20, and uniformly coat it onto the glass fiber, then dry at 37 °C for 24 hours to obtain Sample Pad 4.
[0027] Sample Pad 5: Prepare a sample pad pretreatment solution containing 100 mM Tris-HCL (pH 9.0), 2% NaCl, 0.2% SDS, and 0.5% Tween-20, and uniformly coat it onto the glass fiber, then dry at 37 °C for 24 hours to obtain Sample Pad 5.
[0028] 4. Assembly and Stripping
[0029] Paste the coated NC membrane, conjugate pad, sample pad, and absorbent paper onto the PVC backplane in sequence to obtain a large novel coronavirus antigen detection board. After stripping according to the specified width, the test strip can be obtained.
[0030] 5. Assembly of Test Reagent
[0031] Assemble the test strip, plastic cartridge, and sample collection pad in sequence to obtain the test reagent card.
[0032] 6. Detection
[0033] Thirty saliva samples were selected (10 of which were positive for novel coronavirus). The test reagent cards prepared with different sample pads were inserted into the saliva samples. When the liquid chromatographed to the window position, the test reagent cards were taken out, covered with the covers of the test reagent cards, and placed flat on the tabletop to compare the test results of different sample pads.
[0034] Sample Pad Name Sample Pad 1 Sample Pad 2 Sample Pad 3 Sample Pad 4 Sample Pad 5 Positive Sample Concordance Rate 10 / 10 8 / 10 4 / 10 8 / 10 6 / 10 Negative Sample Concordance Rate 20 / 20 20 / 20 10 / 20 4 / 20 8 / 20
[0035] The test results showed that for the sample pad pretreatment solution of 100 mM Tris-HCL (pH 9.0), 2% NaCl, 0.2% SDS, 2% BSA, and 0.5% Tween-20, the lack of any component would affect the compliance rate of saliva sample detection.
[0036] Example 2 Evaluation of the sensitivity of different labeled conjugate pads
[0037] 1. Preparation of different labeled conjugate pads
[0038] a. Preparation of colloidal gold-labeled conjugate pad
[0039] Adjust the pH of the colloidal gold solution to 9.0 with 0.2 M potassium carbonate solution; (2) Add 20 μg of antibody per milliliter of colloidal gold, and place it on a magnetic stirrer for 30 min; (3) Add 10% BSA at 50 μL / mL to the solution and let it stand at room temperature for 30 min; (4) Centrifuge the colloidal gold mixed solution at 10,000 rpm for 30 minutes, discard the supernatant, and take the precipitate; (5) Dissolve the obtained precipitate at 1 mL / 100 μL in 0.02 M pH 7.4 Tris-HCl buffer (containing 2% BSA) for standby. Spray the labeled colloidal gold-antibody conjugate onto the glass fiber at a spraying volume of 3.0 μL / cm, and dry it at 37 °C for 16 - 18 hours to obtain the colloidal gold-labeled conjugate pad.
[0040] b. Preparation of latex microsphere conjugate pad
[0041] Activate the latex microspheres with 25 mM MES buffer for 20 minutes, centrifuge and discard the supernatant; add an equal volume of 25 mM HEPES buffer for washing, centrifuge and discard the supernatant; add 1 / 2 of the original volume of 25 mM HEPES buffer, add the novel coronavirus antibody according to the ratio of 100 μg / mg of microspheres, and react for 2 hours; add 1 / 5 of the original volume of the blocking solution for blocking for 1 hour, centrifuge and discard the supernatant; add 1 / 4 of the original volume of the reconstitution solution to resuspend the microspheres to obtain the latex microsphere-labeled complex.
[0042] Spray the labeled complex of the latex microsphere-labeled complex onto the glass fiber at 1 μL / cm, and dry it at 37 °C for 16 - 18 hours to obtain the microsphere-labeled conjugate pad.
[0043] 2. Preparation of Coated NC Membrane
[0044] Coat the novel coronavirus antibody onto the nitrocellulose membrane at a concentration of 1.5 mg / mL, at 45 °C, with humidity < 20%, and the drying time is 14 - 16 hours.
[0045] 3. Preparation of Sample Pad
[0046] Prepare a sample pad pretreatment solution containing 100 mM Tris-HCL (PH9.0), 2% NaCl, 0.2% SDS, 2% BSA, and 0.5% Tween-20, and uniformly coat it onto the glass fiber, then dry at 37 °C for 24 hours to obtain the sample pad.
[0047] 4. Assembly and Cutting
[0048] Paste the coated NC membrane, conjugate pad, sample pad, and absorbent paper onto the PVC backboard in sequence to obtain a large novel coronavirus antigen detection board. After cutting it into strips according to the specified width, the test reagent strip can be obtained.
[0049] 5. Assembly of Test Reagent
[0050] Assemble the test reagent strip, plastic cartridge, and sample collection pad in sequence to obtain the test reagent card.
[0051] 6. Preparation of Sample Extract
[0052] Prepare a sample extract containing 100 mM Tris-HCL (PH9.0), 2% NaCl, 0.2% SDS, 2% BSA, and 0.5% Tween-20 as the sample extract, and dispense it into double-ear tubes, which is suitable for the pre-treatment of nasal swab, throat swab, and nasopharyngeal swab samples for detection.
[0053] 7. Sensitivity Evaluation
[0054] Dilute the novel coronavirus N protein at different concentrations with the sample extract, and use two conjugate pads for detection. The detection results are as follows. The sensitivity of the microsphere conjugate pad for detecting antigens is higher than that of colloidal gold, and a concentration of 50 pg / mL can be detected.
[0055] Concentration Colloidal Gold Conjugate Pad Microsphere Conjugate Pad 1 ng / mL 3 / 3(+ / +) 3 / 3(+ / +) 500 pg / mL 3 / 3(+ / +) 3 / 3(+ / +) 250 pg / mL 1 / 3(+ / +) 3 / 3(+ / +) 100 pg / mL 0 / 3(+ / +) 3 / 3(+ / +) 50 pg / mL 0 / 3(+ / +) 3 / 3(+ / +) 25 pg / mL 0 / 3(+ / +) 1 / 3(+ / +)
[0056] Results of Comparison of Novel Coronavirus Antigen Detection Method and Kit for Detecting Samples in Example 3
[0057] 1. Preparation of Microsphere Conjugate Pad
[0058] The latex microspheres were activated with 25 mM MES buffer for 20 minutes, and the supernatant was discarded by centrifugation; an equal volume of 25 mM HEPES buffer was added for washing, and the supernatant was discarded by centrifugation; 1 / 2 volume of the original volume of 25 mM HEPES buffer was added, and the novel coronavirus antibody was added according to the ratio of 50 μg - 100 μg / mg microspheres and reacted for 2 hours; 1 / 5 volume of the blocking solution was added for blocking for 1 hour, and the supernatant was discarded by centrifugation; 1 / 4 volume of the original volume of the reconstitution solution was added to resuspend the microspheres to obtain the latex microsphere-labeled complex.
[0059] The labeled complex was sprayed onto the glass fiber at 0.001 mL / cm of the latex microsphere-labeled complex and dried at 37 °C for 16 - 18 hours to obtain the microsphere-labeled conjugate pad.
[0060] 2. Preparation of the coated NC membrane
[0061] The novel coronavirus antibody was coated onto the nitrocellulose membrane at a concentration of 1.5 mg / mL, at 45 °C, humidity < 20%, and the drying time was 14 - 16 hours.
[0062] 3. Preparation of the sample pad
[0063] The sample pad pretreatment solution containing 100 mM Tris-HCL (PH9.0), 2% NaCl, 0.2% SDS, 2% BSA, and 0.5% Tween-20 was prepared and uniformly coated onto the glass fiber and dried at 37 °C for 24 hours to obtain the sample pad.
[0064] 4. Assembly and strip cutting
[0065] The coated NC membrane, conjugate pad, sample pad, and absorbent paper were sequentially pasted onto the PVC backplate to obtain the novel coronavirus antigen detection large plate, and after strip cutting according to the specified width, the test reagent strip could be obtained.
[0066] 5. Assembly of the test reagent
[0067] The test reagent strip, plastic cartridge, and sample collection pad were assembled in sequence to obtain the test reagent card.
[0068] 6. Preparation of the sample extraction solution
[0069] A liquid containing 100 mM Tris-HCL (PH9.0), 2% NaCl, 0.2% SDS, 2% BSA, and 0.5% Tween-20 was prepared as the sample extraction solution.
[0070] 7. Detection of clinical samples
[0071] 340 swab samples and 340 saliva samples were detected using the novel coronavirus antigen detection method and kit provided by the invention, and compared with the nucleic acid detection method to compare the consistency of the detection results.
[0072]
[0073] From the results, the consistency between the novel coronavirus antigen detection method and kit of the invention for detecting samples with different Ct values and the gold standard nucleic acid fluorescence PCR method can reach more than 90%; the coincidence rate of negative samples can reach 99.5%, and the result consistency is relatively high.
[0074] It should be understood that the disclosed invention is not limited to the specific methods, protocols and substances described, as these can vary. It should also be understood that the terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention, which is limited only by the appended claims.
[0075] Those skilled in the art will also recognize, or be able to ascertain, many equivalents to the specific embodiments of the invention described herein using no more than routine experimentation. These equivalents are also included in the appended claims.
Claims
1. A novel coronavirus antigen detection kit based on immunochromatography, characterized in that, The kit is a detection reagent card including a detection reagent strip, a sample collection pad and a plastic cartridge. The detection reagent strip is prepared by sequentially pasting a coated NC membrane, a conjugate pad, a sample pad and absorbent paper onto a PVC backplate. The kit is a kit for detecting the N protein antigen of the novel coronavirus in nasopharyngeal swabs, throat swabs, nasal swabs and saliva samples. The sample pad of the detection reagent card is pretreated with 50-200 mM, pH 9.0 Tris-HCL, 1.5-4% NaCl, 0.2%-0.4% SDS, 3-5% BSA, 0.2-1% Tween-20; and the antibody of the novel coronavirus is labeled with latex microspheres.
2. The kit according to claim 1, characterized in that, The activation buffer for labeling the antibody of the novel coronavirus with latex microspheres is 25-100 mM MES buffer, the coupling buffer is 25-100 mM HEPES buffer, and the protein amount is 50 μg-100 μg / mg microspheres.
3. The kit according to claim 2, characterized in that, The activation buffer for labeling the antibody of the novel coronavirus with latex microspheres is 50 mM MES buffer, the coupling buffer is 50 mM HEPES buffer, and the protein amount is 50 μg / mg microspheres.
4. The kit according to claim 3, wherein The latex microspheres are activated with 25 mM MES buffer for 20 minutes, and the supernatant is discarded by centrifugation; an equal volume of 25 mM HEPES buffer is added for washing, and the supernatant is discarded by centrifugation; 1 / 2 volume of 25 mM HEPES buffer of the original volume is added, and the antibody of the novel coronavirus is added according to the ratio of 50 μg-100 μg / mg microspheres, and the reaction is carried out for 2 hours; 1 / 5 volume of the blocking solution is added for blocking for 1 hour, and the supernatant is discarded by centrifugation; 1 / 4 volume of the reconstitution solution of the original volume is added to resuspend the microspheres to obtain a latex microsphere-labeled complex.
5. The kit according to claim 4, characterized in that, The antibody complex labeled with latex microspheres is sprayed on the glass fiber at 1-3 μL / cm of the latex microsphere-labeled complex and dried at 37°C for 16-18 hours to obtain a microsphere-labeled conjugate pad.