A kit for detecting neutralizing antibodies against novel coronavirus in saliva and its application

Abstract

The present invention discloses a simple and rapid detection kit for neutralizing antibodies against novel coronavirus in saliva. The nano-carbon immunochromatography technology is adopted, and the double-antigen sandwich method is used to detect the content of neutralizing antibodies against novel coronavirus in saliva. The test strip uses an NC membrane as the matrix, and the reagent pad uses nano-carbon-labeled S-RBD and murine IgG. The S-RBD protein and goat anti-mouse IgG are used as the test line T and the quality control line C. After the reaction is completed, the color development situation is directly observed with the naked eye for testing. By the intensity of nano-carbon color development on the test line, the content of neutralizing antibodies can be judged, and the detection method is simple. Moreover, the detection kit obtained in this application has high sensitivity, intuitive results, low infection risk, safety and non-invasiveness, and is a simple, accurate and economical on-site rapid detection method.

Description

Technical Field

[0001] The present invention relates to the field of novel coronavirus detection kits, G01N33 / 569, and particularly to a kit for detecting neutralizing antibodies against novel coronavirus in saliva and its application. Background Art

[0002] The novel coronavirus (SARS-CoV-2) belongs to the Coronaviridae family and the Coronavirus genus. It is a single-stranded positive-sense RNA virus and also a widespread zoonosis. The virus has an envelope structure, and its main structural proteins are composed of spike protein (S), matrix protein (M), envelope protein (E), and nucleocapsid protein (N). SARS CoV 2 binds to the human angiotensin-converting enzyme 2 (ACE2) receptor on the host cell through the receptor-binding domain (RBD) of its surface spike protein, enabling the virus to enter the host cell and replicate. The specific antibody against S-RBD, namely the neutralizing antibody, can block the binding of the new coronavirus to ACE2, thereby preventing the virus from invading. The neutralizing antibody has the characteristics of strong specificity and high affinity, and is quite different from the IgM, IgG, and IgA antibodies detected clinically traditionally in terms of function. It is one of the important indicators in the process of vaccine research and development and clinical evaluation. The level of neutralizing antibodies in the body is the main indicator for evaluating immune protection efficacy. Therefore, it is very meaningful to establish a method for rapidly detecting neutralizing antibodies against the new coronavirus.

[0003] Patent application CN202010218566.7 discloses a novel coronavirus IgM antibody enzyme-linked immunosorbent assay kit and its detection method. By coating the new coronavirus antigen on a coated plate to form a pre-coated plate with the new coronavirus antigen, during detection, the blood sample to be detected needs to be diluted and then dropped onto the coated plate, and then an enzyme-linked immune reaction is used for determination. This detection method requires specific instruments, and the sample is blood, which belongs to invasive sampling and is also complex in operation and not suitable for the general population.

[0004] There are mainly two types of immunoglobulins in saliva: IgA (90%-98%) and IgG (1%-10%), and the rest are extremely small amounts of IgM, IgD, and IgE. IgG and IgA in serum enter the oral cavity through gingival crevicular fluid, mucosal exudate, and ultrafiltration of salivary gland acini to detect novel coronavirus antibodies in saliva. Patent application CN202011230093.9 discloses a new coronavirus detection test strip and its preparation method and use method, which mainly includes a sample pad, a gold-labeled pad, a detection pad, and a water absorption plate. It detects by the recognition and binding of sialic acid small molecules to the S protein on the surface of the new coronavirus. This detection test strip can quickly and conveniently determine the polarity of the sample, but the gold-labeled pad has poor stability and sensitivity and is also expensive. Summary of the Invention

[0005] To solve the above problems, a first aspect of the present invention provides a simple and rapid detection kit for saliva-based COVID-19 neutralizing antibodies, comprising a test strip and a desiccant.

[0006] In some preferred embodiments, the preparation method of the detection kit is: sealing the test strip and the desiccant in a sealed bag, thus obtaining the kit.

[0007] In some preferred embodiments, the test strip comprises: a reagent strip, a liquid absorption rod, and a plastic part.

[0008] In some preferred embodiments, the reagent strip comprises: a sample pad, a reagent pad, an NC membrane, a blotting paper, and a bottom plate.

[0009] In some preferred embodiments, the liquid absorption rod is a liquid absorption rod treated with PBS buffer; preferably, the treatment method is to immerse the liquid absorption rod in PBS buffer until it is completely soaked, and then dry it overnight.

[0010] In some preferred embodiments, the present application does not specifically limit the amount of PBS buffer used; preferably, the amount of PBS buffer is 300 mL per 1000 liquid absorption rods.

[0011] In some preferred embodiments, the molar concentration of the PBS buffer is 30 - 65 mM; preferably, the molar concentration of the PBS buffer is 50 mM.

[0012] In some preferred embodiments, the PBS buffer contains 0.25 - 0.35 mg / mL of sodium dihydrogen phosphate dihydrate, 2 - 4 mg / mL of disodium hydrogen phosphate dodecahydrate, and 0.7 - 1.0% (w / v) sodium chloride; preferably, the composition of the PBS buffer comprises: 0.2968 mg / mL of sodium dihydrogen phosphate dihydrate, 2.98 mg / mL of disodium hydrogen phosphate dodecahydrate, and 0.85% (w / v) sodium chloride.

[0013] In some preferred embodiments, the sample pad is selected from one of filter paper, glass fiber, non-woven fabric, and polyester film; preferably, the sample pad is glass fiber; more preferably, the sample pad is glass fiber treated with a treatment solution.

[0014] In some preferred embodiments, the size of the glass fiber is 254 mm × 300 mm.

[0015] In some preferred embodiments, the treatment with the treatment solution specifically means that each glass fiber is infiltrated with 40 mL of the sample pad treatment solution until there are no bubbles on the surface, dried overnight, and then cut into a width of 20 mm and stored in a dry state.

[0016] In some preferred embodiments, the treatment solution is an aqueous sodium tetraborate buffer solution.

[0017] The aqueous sodium tetraborate buffer solution is 0.05 - 0.2 M; preferably, the aqueous sodium tetraborate buffer solution is 0.1 M.

[0018] In some preferred embodiments, the treatment solution further contains: 0.2 - 0.7% (w / v) CaseinNa, 0.8 - 2.0% (w / v) EDTA, 0.1 - 0.3% (w / v) sodium cholate, 0.3 - 2.5% (w / v) BSA, 3 - 6% (w / v) surfactant; preferably, the treatment solution further contains: 0.5% (w / v) CaseinNa, 1% (w / v) EDTA, 0.3% (w / v) sodium cholate, 1% (w / v) BSA, 4% (w / v) surfactant.

[0019] In some preferred embodiments, the surfactant includes at least one of Tetronic 1307, S17 (surfactant S17 RHODASURF ON - 870), CHAPS (3 - [(3 - cholamidopropyl)dimethylammonio]-1 - propanesulfonate), Tritonx - 100; preferably, the surfactant is Tetronic 1307, S17, CHAPS, Tritonx - 100.

[0020] In some preferred embodiments, the mass ratio of Tetronic 1307, S17, CHAPS, Tritonx - 100 is 1:(0.7 - 1.3):(0.8 - 1.5):(0.6 - 1.1); preferably, the mass ratio of Tetronic 1307, S17, CHAPS, Tritonx - 100 is 1:1:1:1.

[0021] The glass fiber used as the sample pad has a dense and uniform structure, good hydrophilicity, and fast permeability, but the phenomenon of "backflow" of the sample is likely to occur. The applicant has found that by pretreating the sample pad material, especially when the treatment solution contains 0.2-0.7% (w / v) CaseinNa, 0.8-2.0% (w / v) EDTA, 0.1-0.3% (w / v) sodium cholate, 0.3-2.5% (w / v) BSA, 3-6% (w / v) surfactant, and 0.05-0.2M sodium tetraborate, treating the sample pad material with it can significantly improve this situation and increase the release rate of the sample. It is speculated that the possible reason is that CaseinNa contains a variety of hydrophilic and hydrophobic groups, which can act synergistically with other components to further reduce the surface tension of the sample pad, promote the wetting and dispersion of the saliva sample in the sample pad. When the sample enters the reagent, it becomes a certain gel-like state, thereby preventing the phenomenon of sample backflow, increasing the concentration of the sample on the surface of the sample pad, and also playing a role in stabilizing the sample, thereby improving the sensitivity and accuracy of the detection kit.

[0022] In some preferred embodiments, the dosage of the treatment solution is 30-50 mL per glass fiber; preferably, the dosage of the treatment solution is 40 mL per glass fiber.

[0023] In some preferred embodiments, the reagent pad is obtained by spreading the S-RBD labeled antibody or mouse IgG labeled antibody diluted with a diluent on the glass fiber and drying it overnight; preferably, the specific preparation process of the reagent pad is: diluting the S-RBD labeled antibody or mouse IgG labeled antibody 15 times with a 40% (v / v) diluent to obtain a labeled diluent, then using it to treat the glass fiber, drying it overnight, cutting it into 5 mm wide, and storing it in a dry state to obtain.

[0024] In some preferred embodiments, the 40% (v / v) diluent is obtained by diluting the diluent with water.

[0025] In some preferred embodiments, the diluent is a Tris buffer containing sucrose, trehalose, and TritonX100; preferably, the diluent is a 65-75 mM Tris buffer containing 0.8-1.3% (w / v) BSA, 8-13% (w / v) sucrose, 3-5.5% (w / v) trehalose, and 0.35-0.5% (w / v) TritonX100; preferably, the 40% (v / v) diluent is a 70 mM Tris buffer containing 1% (w / v) BSA, 0% (w / v) sucrose, 5% (w / v) trehalose, and 0.4% (w / v) TritonX100.

[0026] It should be noted that the percentage content % (w / v) of each substance in this application is the ratio of mass to volume, and the specific unit is 1 g / 100 mL.

[0027] In some preferred embodiments, the 70 mM Tris buffer is 70 mM Tris-HCl buffer.

[0028] In some preferred embodiments, the size of the glass fiber is 200 mm × 300 mm.

[0029] In some preferred embodiments, the dosage of the labeling diluent is 10 - 15 mL per glass fiber; preferably, the dosage of the labeling diluent is 12 mL per glass fiber.

[0030] In some preferred embodiments, the S-RBD labeled antibody and the mouse IgG labeled antibody are nano-carbon labeled S-RBD protein and nano-carbon labeled mouse IgG.

[0031] In some preferred embodiments, the preparation method of the nano-carbon labeled S-RBD protein includes the following steps:

[0032] S1. Disperse and treat the nano-carbon and then ultrasonically homogenize it;

[0033] S2. Prepare EDC solution and NHS solution respectively with MES coupling solution;

[0034] S3. Take 400 μL of the nano-carbon obtained in S1 into a 1.5 mL centrifuge tube, add the EDC solution and NHS solution prepared in S2, mix well, and let it stand at room temperature for activation for 20 - 40 min;

[0035] S4. Add the labeled antibody S-RBD protein diluted with 0.1 M MES coupling solution to S3, and couple at room temperature for 20 - 40 min;

[0036] S5. Sequentially add blocking solution A and blocking solution B to S4, mix well and block at room temperature for 25 - 35 min, then centrifuge at 12000 rpm - 16000 rpm for 10 - 20 min, and remove the supernatant;

[0037] S6. Add nano-carbon storage solution to the substance obtained in S5, ultrasonically homogenize it, then centrifuge at 12000 rpm - 16000 rpm for 10 - 20 min, and remove the supernatant;

[0038] S7. Continue to add nano-carbon storage solution to the substance obtained in S6, ultrasonically homogenize it, and store it at 4°C, then it is ready.

[0039] In S1, the specific operation of the nano-carbon dispersion treatment includes the following steps:

[0040] (1) Ultrasonically disperse the carbon nanotubes in a solvent;

[0041] (2) Clean the carbon nanotubes obtained in (1) by centrifugation, resuspension, centrifugation, resuspension, and ultrasonication;

[0042] (3) Dilute the carbon nanotubes obtained in (2) and store for later use.

[0043] Preferably, the specific operation of the carbon nanotube dispersion treatment in S1 includes the following steps:

[0044] (1) Add the carbon nanotubes into a crushing cup, add a mixed solvent composed of pure water and a 5-13% (w / v) PVP aqueous solution, and ultrasonicate in an ice-water bath for 12-24 h;

[0045] (2) After aliquoting the carbon nanotubes obtained in (1), add pure water respectively, centrifuge at 3000-7500 rpm for 8-15 min, then take the supernatant, centrifuge at 8000-12000 rpm for 0.5-1 h and remove the supernatant, resuspend with 0.05-0.2 M MES coupling solution, centrifuge at 8000-12000 rpm for 0.5-1 h and remove the supernatant, resuspend with 0.05-0.15 M MES coupling solution and ultrasonicate to complete the cleaning;

[0046] (3) Adjust the carbon nanotubes obtained in (2) with 0.05-0.2 M MES coupling solution to an A400 of 0.105 ± 0.005, and that's it. Store for later use.

[0047] In some preferred embodiments, the concentration of the carbon nanotubes in (1) is 9-10 mg / mL; preferably, the concentration of the carbon nanotubes in (1) is 9.6 mg / mL.

[0048] In some preferred embodiments, the volume ratio of pure water to 5-13% (w / v) PVP in the mixed solvent is (20-28):1; preferably, the volume ratio of pure water to 5-13% (w / v) PVP in the mixed solvent is 24:1.

[0049] In some preferred embodiments, the volume ratio of the aliquoted carbon nanotubes to pure water in (2) is 1:(4-6), the volume ratio of the aliquoted carbon nanotubes to 0.05-0.2 M MES coupling solution is 1:(4-5.5), and the volume ratio of the aliquoted carbon nanotubes to 0.05-0.15 M MES coupling solution is 1:(3-4.5); preferably, the volume ratio of the aliquoted carbon nanotubes to pure water is 1:5, the volume ratio of the aliquoted carbon nanotubes to 0.05-0.2 M MES coupling solution is 1:5, and the volume ratio of the aliquoted carbon nanotubes to 0.05-0.15 M MES coupling solution is 1:4.

[0050] In S2, the molar concentration of the MES coupling solution is 0.07 - 0.5 M, the final concentration of the EDC solution is 0.5 - 1.5 mg / mL, and the final concentration of the NHS solution is 0.5 - 1.7 mg / mL; preferably, the molar concentration of the MES coupling solution is 0.1 M, the final concentration of the EDC solution is 0.8 mg / mL, and the final concentration of the NHS solution is 0.8 mg / mL.

[0051] In S3, the volume ratio of the EDC solution to the nano-carbon is 1:(3 - 5.5), and the volume ratio of the NHS solution to the nano-carbon is 1:(3.5 - 5); preferably, the volume ratio of the EDC solution to the nano-carbon is 1:4, and the volume ratio of the NHS solution to the nano-carbon is 1:4.

[0052] In S4, the diluted concentration of the labeled antibody S-RBD protein is 0.3 - 0.5 μg / μL, and the added volume of the 0.1 M MES coupling solution is 80 - 120 μL; preferably, the diluted concentration of the labeled antibody S-RBD protein is 0.4 μg / μL, and the added volume of the 0.1 M MES coupling solution is 100 μL.

[0053] In S5, the blocking solution A is an aqueous solution of 47 - 55 mM Tris-HCl, and the blocking solution A further contains 1 - 3% BSA. The blocking solution B is 8 - 15% Tween20; preferably, the blocking solution A is an aqueous solution of 50 mM Tris-HCl, and the blocking solution A further contains 2% BSA, and the blocking solution B is 10% Tween20.

[0054] The volume ratio of the blocking solution A to the nano-carbon in S3 is (0.5 - 1):1, and the volume ratio of the blocking solution B to the nano-carbon in S3 is (0.1 - 0.4):1; preferably, the volume ratio of the blocking solution A to the nano-carbon in S3 is 0.75:1, and the volume ratio of the blocking solution B to the nano-carbon in S3 is 0.25:1.

[0055] In S6, the nano-carbon preservation solution is an aqueous solution of 47 - 55 mM Triss-HCl, and the nano-carbon preservation solution further contains 0.25 - 0.6% BSA. The volume ratio of the nano-carbon preservation solution to the nano-carbon in S3 is (0.8 - 1.3):1; preferably, the nano-carbon preservation solution is an aqueous solution of 50 mM Tris-HCl, and the nano-carbon preservation solution further contains 0.5% BSA, and the volume ratio of the nano-carbon preservation solution to the nano-carbon in S3 is 1:1.

[0056] In S7, the volume ratio of the nano-carbon preservation solution to the nano-carbon in S3 is (0.3 - 0.6):1; preferably, the volume ratio of the nano-carbon preservation solution to the nano-carbon in S3 is 0.5:1.

[0057] In some preferred embodiments, the concentration of the mouse IgG antibody after dilution is 0.3 - 0.5 μg / μL, and the added volume of the 0.1M MES coupling solution is 80 - 120 μL; preferably, the concentration of the mouse IgG antibody after dilution is 0.4 μg / μL, and the added volume of the 0.1M MES coupling solution is 100 μL.

[0058] This patent uses the method of labeling antibodies with nano-carbon, which is completely different from the colloidal gold labeling method. It has high detection sensitivity, good stability, and the carbon material is environmentally friendly. The nano-carbon raw material has a lower cost than colloidal gold, and the contrast between the black and white colors of nano-carbon and the NC membrane is easier to distinguish. The applicant found that by dispersing 400 μL of nano-carbon and then adding 0.3 - 0.5 μg / μL of labeled antibody S-RBD protein and mouse IgG antibody, the nano-carbon has good dispersibility, which can enable the antibody to be fully labeled by nano-carbon, fully detect the total neutralizing antibodies produced by the human body, improve the sensitivity of the detection kit, and reduce the detection limit. Especially when the concentration of the labeled antibody is 0.4 μg / μL, the binding of nano-carbon and the labeled antibody is good, the labeling stability is enhanced, and at the same time, the storage stability of nano-carbon and the resolution of the detection kit are also improved.

[0059] In some preferred embodiments, the NC membrane is coated with parallel detection line T and quality control line C. The spacing distance between the detection line T and the quality control line C is 5 mm. The detection line T is close to the reagent pad, and the quality control line C is close to the absorbent paper.

[0060] In some preferred embodiments, the preparation method of the NC membrane is as follows: stick the NC membrane to the bottom plate, then coat the T-line antibody and the C-line antibody on the NC membrane respectively, dry overnight, and seal it with a desiccant; preferably, the preparation method of the NC membrane is as follows: tear off the protective paper in the middle of the PVC bottom plate, stick the NC membrane along the lower edge of the upper protective paper, then use a gold-spraying and membrane-scratching instrument to coat the T-line antibody and the C-line antibody on the NC membrane respectively, dry overnight, and seal it with a desiccant.

[0061] In some preferred embodiments, the size of the PVC bottom plate is 60 mm × 280 mm.

[0062] In some preferred embodiments, the NC membrane is 1UN14ER100020NT.

[0063] In some preferred embodiments, the T-line antibody is the T-line coating solution obtained by diluting the coating antibody S-RBD protein with the coating diluent.

[0064] In some preferred embodiments, the C-line antibody is a C-line coating solution obtained by diluting a goat anti-mouse IgG polyclonal antibody with a coating diluent.

[0065] In some preferred embodiments, the coating diluent is a PBS buffer solution with a pH of 7.4; preferably, the coating diluent is a PBS buffer solution containing sucrose with a pH of 7.4; more preferably, the coating diluent is a 10 mM PBS buffer solution containing 10 g / L sucrose with a pH of 7.4.

[0066] In some preferred embodiments, the mass concentration of the T-line antibody is 1 - 2 mg / mL, and the mass concentration of the C-line antibody is 1.5 - 2.5 mg / mL; preferably, the mass concentration of the T-line antibody is 1.5 mg / mL, and the mass concentration of the C-line antibody is 2.0 mg / mL.

[0067] In some preferred embodiments, the liquid application amount of the gold spraying and membrane scribing instrument is 0.5 - 1.7 μL / cm, and the membrane scribing speed is 50 - 75 mm / s; preferably, the liquid application amount of the gold spraying and membrane scribing instrument is 1 μL / cm, and the membrane scribing speed is 60 mm / s.

[0068] In some preferred embodiments, the test strip is composed of a sample pad, a reagent pad, an NC membrane, and a blotting paper pasted on a bottom plate; preferably, the specific preparation operation of the test strip is: paste a 17 mm blotting paper on the bottom plate coated with the NC membrane, and then sequentially paste one reagent pad (mouse IgG-labeled antibody) with a width of 5 mm, one reagent pad (S-RBD-labeled antibody) with a width of 5 mm, and one sample pad with a width of 20 mm.

[0069] In some preferred embodiments, the preparation method of the detection plate is to cut the test strip into a width of 3 - 4 mm, install it on the bottom plate of the plastic part, add the upper cover, install the liquid absorption rod, and press the cover tightly to obtain it.

[0070] The second aspect of the present invention provides an application of a simple and rapid detection kit for salivary COVID-19 neutralizing antibodies in the detection of salivary COVID-19.

[0071] Compared with the prior art, the present invention has the following beneficial effects:

[0072] (1) The kit obtained in this application adopts the nano-carbon immunochromatography technology and uses the double-antigen sandwich method to detect the content of neutralizing antibodies against COVID-19 in saliva. The test strip uses the NC membrane as the matrix, and the reagent pad uses nano-carbon-labeled S-RBD and mouse IgG. The S-RBD protein and goat anti-mouse IgG are used as the test line T and the quality control line C. During the detection, the saliva sample will chromatograph forward under capillary action, and the nano-carbon label on the reagent pad will dissolve and then move forward together with the sample. After the sample reaches the detection area, the nano-carbon-labeled S-RBD, the neutralizing antibody in the sample, and the S-RBD coated on the test line form a complex, showing that the test line turns grayish-black. The antibody content in the sample is positively correlated with the color intensity of the test line. After the reaction, the color development is directly observed with the naked eye for testing. By the strength of the nano-carbon color development on the test line, the content of neutralizing antibodies can be judged, and the detection method is simple.

[0073] (2) The detection kit obtained in this application has high sensitivity, intuitive results, low infection risk, is safe and non-invasive, and is a simple, accurate and economical on-site rapid detection method.

[0074] (3) The detection kit obtained in this application has high resolution and is easy to judge. Moreover, the sample is saliva, which is easy for patients to collect by themselves and is acceptable. And there is no need to consider misjudgment of results caused by improper preservation, transportation, processing, etc.

[0075] (4) In this invention, the sandwich method is used to detect the total neutralizing antibodies against COVID-19. The S-RBD of the SARS-CoV-2 S protein with high specificity is selected, which can effectively detect the total neutralizing antibodies produced by the human body, including IgA, IgM and IgG, and solves the problem of low resolution and difficulty in interpretation of the colloidal gold competition method when the concentration of neutralizing antibodies is low. Description of the Drawings

[0076] Figure 1 Structural plan view of the test board obtained in Example 1.

[0077] Figure 2 Structural sectional view of the test board obtained in Example 1.

[0078] Figure 3 Test results of negative samples of the detection kit obtained in Example 3.

[0079] Figure 4 Test results of quality control product gradients of the detection kit obtained in Example 1.

[0080] Figure 5 Test results of quality control product gradients of the detection kit obtained in Example 2.

[0081] Figure 6 Test results of quality control product gradients of the detection kit obtained in Example 3.

[0082] Description of reference numerals: 1 is a liquid absorption rod; 2 is a sample pad; 3 is a reagent pad; 4 is an NC (nitrocellulose) membrane; 5 is a blotting paper; 6 is a bottom plate. Detailed implementation mode

[0083] Example 1

[0084] 1. A simple and rapid detection kit for neutralizing antibodies against SARS-CoV-2 in saliva, comprising a detection plate and a desiccant.

[0085] The preparation method of the detection kit is: sealing the detection plate and the desiccant in a sealed bag to obtain it.

[0086] The detection plate includes: a reagent strip, a liquid absorption rod and a plastic part.

[0087] The reagent strip includes: a sample pad, a reagent pad, an NC membrane, a blotting paper, and a bottom plate.

[0088] The liquid absorption rod is a liquid absorption rod treated with PBS buffer; the treatment method is to immerse the liquid absorption rod in PBS buffer until it is completely soaked, and dry it overnight at 37°C.

[0089] The dosage of the PBS buffer is 300 mL / 1000 liquid absorption rods.

[0090] The molar concentration of the PBS buffer is 50 mM PBS.

[0091] The PBS buffer contains 0.2968 mg / mL of sodium dihydrogen phosphate dihydrate, 2.98 mg / mL of disodium hydrogen phosphate dodecahydrate, and 0.85% (w / v) sodium chloride.

[0092] The liquid absorption rod is purchased from Biologix Specialty Fibers (Ningbo) Co., Ltd.

[0093] The sample pad is glass fiber treated with a treatment solution.

[0094] The size of the glass fiber is 254 mm × 300 mm (glass fiber 8964, purchased from Shanghai HanYi Biotechnology Co., Ltd.).

[0095] The specific treatment with the treatment solution is to soak each glass fiber with 40 mL of the sample pad treatment solution until there are no bubbles on the surface, dry it overnight at 37°C, then cut it into 20 mm wide, and store it in a dry state.

[0096] The treatment solution is a sodium tetraborate buffer aqueous solution.

[0097] The sodium tetraborate buffer aqueous solution is 0.1 M.

[0098] The treatment solution contains 0.5% (w / v) CaseinNa, 1% (w / v) EDTA, 0.3% (w / v) sodium cholate (C1254 purchased from sigma company), 1% (w / v) BSA, and 4% (w / v) surfactant.

[0099] The surfactant is Tetronic 1307, S17 (surfactant S17 RHODASURF ON - 870), CHAPS (3 - [(3 - cholesteryl aminopropyl) dimethylamino] - 1 - propanesulfonic acid), Tritonx - 100.

[0100] The mass ratio of Tetronic 1307, S17, CHAPS, and Tritonx - 100 is 1:1:1:1.

[0101] The dosage of the treatment solution is 40 mL per glass fiber.

[0102] The specific preparation process of the reagent pad is as follows: Dilute the S - RBD labeled antibody 15 - fold with a 40% (v / v) diluent to obtain a labeled diluent, then use it to treat the glass fiber, dry it overnight at 37°C, cut it into 5 mm wide, and store it in a dry state to obtain the reagent pad.

[0103] The 40% (v / v) diluent is obtained by diluting the diluent with water.

[0104] The diluent contains 1% (w / v) BSA, 10% (w / v) sucrose, 5% (w / v) trehalose, and 0.4% (w / v) TritonX100 in 70 mM Tris buffer.

[0105] The 70 mM Tris buffer is 70 mM Tris - HCl buffer.

[0106] The size of the glass fiber is 200 mm × 300 mm (glass fiber KB50, purchased from Shanghai HanYi Biotechnology Co., Ltd.).

[0107] The dosage of the labeled diluent is 12 mL per glass fiber.

[0108] The S - RBD labeled antibody and mouse IgG labeled antibody are nano - carbon labeled S - RBD labeled antibody and nano - carbon labeled mouse IgG antibody.

[0109] The preparation method of the nano - carbon labeled S - RBD labeled antibody includes the following steps:

[0110] S1. After dispersing the nano - carbon, ultrasonically homogenize it;

[0111] S2. Prepare EDC solution and NHS solution respectively with MES coupling solution.

[0112] S3. Take 400 μL of the nanocarbon obtained in S1 into a 1.5 mL centrifuge tube, add the EDC solution and NHS solution prepared in S2, mix well, and let it stand at room temperature for activation for 30 min.

[0113] S4. Add the labeled antibody S-RBD protein diluted with 0.1 M MES coupling solution to S3, and couple at room temperature for 30 min.

[0114] S5. Sequentially add blocking solution A and blocking solution B to S4, mix well and block at room temperature for 30 min, then centrifuge at 13000 rpm for 15 min, and remove the supernatant.

[0115] S6. Add nanocarbon preservation solution to the substance obtained in S5, ultrasonically mix evenly, then centrifuge at 13000 rpm for 15 min, and remove the supernatant.

[0116] S7. Continue to add nanocarbon preservation solution to the substance obtained in S6, ultrasonically mix evenly, and store at 4 °C, then it is ready.

[0117] In S1, the specific operation of the nanocarbon dispersion treatment includes the following steps:

[0118] (1) Add nanocarbon into a crushing cup, add a mixed solvent composed of pure water and an aqueous solution of 10% (w / v) PVP, and ultrasonically treat in an ice-water bath for 16 h.

[0119] (2) After aliquoting the nanocarbon obtained in (1), add purified water respectively, centrifuge at 600 rpm for 10 min, then take the supernatant, centrifuge at 9800 rpm for 1 h and remove the supernatant, resuspend with 0.1 M MES coupling solution, centrifuge at 9800 rpm for 1 h and remove the supernatant, resuspend with 0.1 M MES coupling solution and ultrasonically complete the washing.

[0120] (3) Adjust the nanocarbon obtained in (2) with 0.1 M MES coupling solution to A400 of 0.105 ± 0.005, then it is ready and store for later use.

[0121] The nanocarbon is purchased from Beijing Decod Island Gold Technology Co., Ltd., and the model is CNT302.

[0122] The concentration of the nanocarbon in (1) is 9.6 mg / mL.

[0123] The volume ratio of pure water and 10% (w / v) PVP in the mixed solvent is 24:1.

[0124] The volume ratio of the nanocarbon after packaging to purified water is 1:5, the volume ratio of the nanocarbon after packaging to the MES coupling solution with a concentration of 0.05 - 0.2 M is 1:5, and the volume ratio of the nanocarbon after packaging to the MES coupling solution with a concentration of 0.05 - 0.15 M is 1:4.

[0125] In S2, the molar concentration of the MES coupling solution is 0.1 M, the final concentration of the EDC solution is 0.8 mg / mL, and the final concentration of the NHS solution is 0.8 mg / mL.

[0126] In S3, the volume ratio of the EDC solution to the nanocarbon is 1:4, and the volume ratio of the NHS solution to the nanocarbon is 1:4.

[0127] In S4, the diluted concentration of the labeled antibody S-RBD protein is 0.4 μg / μL, and the added volume of the 0.1 M MES coupling solution is 100 μL.

[0128] In S5, the blocking solution A is a 50 mM Tris-HCl aqueous solution, and the blocking solution A also contains 2% BSA. The blocking solution B is 10% Tween 20 (product number 0777 purchased from VWR).

[0129] The volume ratio of the blocking solution A to the nanocarbon in S3 is 0.75:1, and the volume ratio of the blocking solution B to the nanocarbon in S3 is 0.25:1.

[0130] In S6, the nanocarbon storage solution is a 50 mM Tris-HCl aqueous solution, and the nanocarbon storage solution also contains 0.5% BSA. The volume ratio of the nanocarbon storage solution to the nanocarbon in S3 is 1:1.

[0131] In S7, the volume ratio of the nanocarbon storage solution to the nanocarbon in S3 is 0.5:1.

[0132] The specific implementation method of the preparation method of the mouse IgG labeled antibody labeled with nanocarbon is the same as that of the S-RBD labeled antibody labeled with nanocarbon.

[0133] The diluted concentration of the mouse IgG labeled antibody is 0.4 μg / μL, and the added volume of the 0.1 M MES coupling solution is 100 μL.

[0134] The NC membrane is coated with parallel detection line T and quality control line C. The spacing distance between the detection line T and the quality control line C is 5 mm. The detection line T is close to the reagent pad, and the quality control line C is close to the absorbent paper.

[0135] The preparation method of the NC membrane is as follows: Tear off the protective paper in the middle of the PVC bottom plate, paste the NC membrane along the lower edge of the upper protective paper, then use a gold spraying and scribing instrument to coat the T-line antibody and C-line antibody on the NC membrane respectively, dry overnight at 37°C, add a desiccant and seal it, then it is ready.

[0136] The size of the PVC bottom plate is 60mm×280mm (MT101200507 purchased from Quzhou Maike New Materials Co., Ltd.).

[0137] The NC membrane is 1UN14ER100020NT (Sartorius).

[0138] The T-line antibody is the T-line coating solution obtained by diluting the coating antibody S-RBD protein with a coating diluent.

[0139] The C-line antibody is the C-line coating solution obtained by diluting goat anti-mouse IgG with a coating diluent.

[0140] The coating diluent is a 10mM PBS buffer solution containing 10g / L sucrose with a pH of 7.4.

[0141] The mass concentration of the T-line antibody is 1.5mg / mL, and the mass concentration of the C-line antibody is 2.0mg / mL.

[0142] The liquid volume of the gold spraying and scribing instrument is 1μL / cm, and the scribing speed is 60mm / s.

[0143] The blotting paper is product 21050901 purchased from Shanghai Medical Devices (Group) Co., Ltd.

[0144] The test strip is composed of a sample pad, a reagent pad, an NC membrane, and a blotting paper pasted on the bottom plate; the specific preparation operation of the test strip is: paste a 17mm blotting paper on the bottom plate after the NC membrane is coated, and then paste the reagent pad and a sample pad with a width of 20mm in sequence.

[0145] The reagent pad is a mouse IgG-labeled antibody reagent pad with a width of 5mm and an S-RBD-labeled antibody reagent pad with a width of 5mm. The two reagent pads are arranged side by side horizontally. The S-RBD reagent pad is closer to the sample pad, and the mouse IgG reagent pad is closer to the NC membrane.

[0146] The lengths of the sample pad and the reagent pad are the same as the length of the PVC bottom plate.

[0147] The preparation method of the detection plate is to cut the test strip into 3-4mm wide, install it on the bottom plate of the plastic part, add the upper cover, install the liquid absorption rod, and press the cover tightly to obtain it (as shown in Figure 1 Figure 2 ). ​

[0148] 2. Application of a simple and rapid detection kit for neutralizing antibodies against SARS-CoV-2 in saliva in saliva SARS-CoV-2 detection.

[0149] Example 2

[0150] 1. A simple and rapid detection kit for neutralizing antibodies against SARS-CoV-2 in saliva, which is different from that in Example 1 in that:

[0151] The treatment solution contains: 1.0% (w / v) CaseinNa, 1% (w / v) EDTA, 0.5% (w / v) sodium cholate, 1% (w / v) BSA, and 4% (w / v) surfactant.

[0152] 2. Application of a simple and rapid detection kit for neutralizing antibodies against SARS-CoV-2 in saliva in saliva SARS-CoV-2 detection.

[0153] Example 3

[0154] 1. A simple and rapid detection kit for neutralizing antibodies against SARS-CoV-2 in saliva, which is different from that in Example 1 in that:

[0155] In the preparation method of the nano-carbon-labeled S-RBD labeled antibody,

[0156] S3. Take 200 μL of the nano-carbon obtained in S1 and put it into a 1.5 mL centrifuge tube, add the EDC solution and NHS solution prepared in S2, mix well, and let it stand at room temperature for activation for 30 min.

[0157] 2. Application of a simple and rapid detection kit for neutralizing antibodies against SARS-CoV-2 in saliva in saliva SARS-CoV-2 detection.

[0158] Performance test

[0159] The detection kits obtained in the examples and comparative examples were used for real sample detection.

[0160] (1) Negative sample: Select the saliva of unvaccinated people as the negative sample;

[0161] Operate according to the kit instructions: Place the pipette on the tongue, upper and lower jaws of the oral cavity, and the inner wall of the oral cavity and scrape 5 times, then place it in the mouth on the tongue. When liquid appears in the observation window and reaches the T position, the sampling can be ended and taken out, cover the lid, place it flat on the table, and start timing. Read the result at 15 min. The test results are as Figure 3 shown.

[0162] The test results were all negative, indicating that the quality control line was normal for negative sample testing.

[0163] (2) Quality control product gradient detection

[0164] Collect the saliva of unvaccinated people using a disposable urine cup as a diluent, and dilute the neutralizing antibody quality control product to different concentrations: 2.5 μg / mL, 0.5 μg / mL, 0.25 μg / mL, 0.1 μg / mL, 0.05 μg / mL.

[0165] Use a pipette to aspirate 450 μL of the saliva-diluted sample and directly add it to the absorbent rod of the test plate obtained in Examples 1-3. When the liquid appears in the observation window and reaches the T position, start timing for 15 minutes to read the result. The following values represent our test results (judged according to the chromaticity card): C3.5 represents weak positive, C5 represents medium positive, and C7 represents strong positive.

[0166] The test results of the test kit obtained in Example 1 are as Figure 4 shown: at 0.1 μg / mL, the test result is C3.5 (weak positive), and when it is smaller than 0.05 μg / mL, the test result C3 and below are negative, indicating that our sensitivity reaches 0.1 μg / mL. The quality control product at 0.5 μg / mL was repeatedly tested, and the results showed consistent color development and stable experimental results.

[0167] The test results of the test kit obtained in Example 2 are as Figure 5 shown: the sensitivity can only detect 0.5 μg / mL, weak positive C3.5.

[0168] The test results of the test kit obtained in Example 3 are as Figure 6 shown: the sensitivity can only detect 0.5 μg / mL, weak positive C4.

[0169] (3) Detection of real samples: Use the test kit obtained in Example 1 for determination.

[0170] Operate according to the kit instructions: Detect 54 clinical samples of vaccinated people: 31 vaccinated with Pfizer vaccine, 18 vaccinated with Moderna vaccine, and 5 vaccinated with Johnson & Johnson vaccine.

[0171] The test results showed that 45 samples were positive; 64 saliva samples of unvaccinated people against COVID-19 were detected, and the results showed all negative. It was preliminarily determined that the sensitivity of the kit > 83%, indicating that this detection method is feasible.

[0172] From the data of the test cards of the kits obtained in Examples 1-3, it shows that a sandwich method for detecting neutralizing antibodies against SARS-CoV-2 in saliva and the kit provided by this patent are simple to operate, have high sensitivity, intuitive results, low infection risk, and are safe and non-invasive. It is a simple, accurate and economical on-site rapid detection method.

Claims

1. A simple and rapid detection kit for neutralizing antibodies against novel coronavirus in saliva, characterized in that, It includes a detection plate and a desiccant; The detection plate includes: a reagent strip, a liquid absorption rod, and a plastic part; The reagent strip includes: a sample pad, a reagent pad, an NC membrane, absorbent paper, and a bottom plate; The liquid absorption rod is a liquid absorption rod treated with PBS buffer solution; The sample pad is glass fiber treated with a treatment solution; The specific operation of the treatment with the treatment solution is to immerse the glass fiber in the treatment solution for 0.5 - 1.5 h, dry it overnight, then cut it into 20 mm wide, and store it in a dry state; The treatment solution is a sodium tetraborate buffer aqueous solution; the sodium tetraborate buffer aqueous solution is 0.05 - 0.2 M; The treatment solution also contains: 0.2 - 0.7% (w / v) CaseinNa, 0.8 - 2.0% (w / v) EDTA, 0.1 - 0.3% (w / v) sodium cholate, 0.3 - 2.5% (w / v) BSA, 3 - 6% (w / v) surfactant; The surfactant is Tetronic 1307, S17, CHAPS, Tritonx - 100; the mass ratio of Tetronic 1307, S17, CHAPS, Tritonx - 100 is 1:(0.7 - 1.3):(0.8 - 1.5):(0.6 - 1.1).

2. The detection kit for simple and rapid detection of novel coronavirus neutralizing antibody in saliva according to claim 1, wherein, The reagent pad is obtained by spreading the S - RBD labeled antibody and mouse IgG labeled antibody diluted with a dilution solution on glass fiber and drying it overnight.

3. The detection kit for simple and rapid detection of SARS-CoV-2 neutralizing antibodies in saliva according to claim 2, characterized in that, The S - RBD labeled antibody and mouse IgG labeled antibody are nano - carbon labeled S - RBD labeled antibody and nano - carbon labeled mouse IgG labeled antibody.

4. A simple and rapid detection kit for saliva new coronavirus neutralizing antibody according to claim 3, characterized in that, The preparation method of the nano - carbon labeled S - RBD labeled antibody includes the following steps: S1. Disperse and treat the nano - carbon and then ultrasonically homogenize it; S2. Prepare EDC solution and NHS solution respectively with MES coupling solution; S3. Take 400 μL of the nano - carbon obtained in S1 into a 1.5 mL centrifuge tube, add the EDC solution and NHS solution prepared in S2, mix well, and let it stand at room temperature for activation for 20 - 40 min; S4. Add the labeled antibody S - RBD protein diluted with 0.1 M MES coupling solution to S3, and couple it at room temperature for 20 - 40 min; S5. Add blocking solution A and blocking solution B to S4 in sequence, mix well and block it at room temperature for 25 - 35 min, then centrifuge it at 12000 rpm - 16000 rpm for 10 - 20 min, and remove the supernatant; S6. Add nano - carbon preservation solution to the substance obtained in S5, ultrasonically homogenize it, then centrifuge it at 12000 rpm - 16000 rpm for 10 - 20 min, and remove the supernatant; S7. Continue to add nano - carbon preservation solution to the substance obtained in S6, ultrasonically homogenize it, and store it at 4°C to obtain.

5. A simple and rapid detection kit for saliva new coronavirus neutralizing antibody according to claim 4, characterized in that, The specific operation of the nano - carbon dispersion treatment in S1 includes the following steps: (1) Add the nano - carbon into a crushing cup, add a mixed solvent composed of pure water and 5 - 13% (w / v) PVP aqueous solution, and ultrasonically treat it in an ice - water bath for 12 - 24 h; (2)After the obtained nano-carbon in (1) is sub-packed, purified water is added respectively, and centrifuged at 3000 - 7500 rpm for 8 - 15 min. Then, the supernatant is taken, centrifuged at 8000 - 12000 rpm for 0.5 - 1 h and the supernatant is removed. It is resuspended with 0.05 - 0.2 M MES coupling solution, centrifuged at 8000 - 12000 rpm for 0.5 - 1 h and the supernatant is removed. It is resuspended with 0.05 - 0.15 M MES coupling solution and ultrasonicated to complete the cleaning; (3)Adjust the nano-carbon obtained in (2) with 0.05 - 0.2 M MES coupling solution until A 400 400 is 0.105 ± 0.005, then it is ready for storage for future use.

6. The detection kit for simple and rapid detection of neutralizing antibodies against novel coronavirus in saliva according to claim 1, characterized in that The preparation method of the NC membrane is as follows: The NC membrane is adhered to the bottom plate, then the T-line antibody and the C-line antibody are respectively coated on the NC membrane, dried overnight, and sealed with a desiccant. That's it.

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