A preparation method for a novel coronavirus antigen detection kit

Abstract

The present invention proposes a method for preparing a novel coronavirus antigen detection kit, wherein latex microspheres are labeled on antibody N72, and antibody 2 is N54 coated on a nitrocellulose membrane. When the test sample contains antigen, the sample is combined with antibody 1 labeled with latex microspheres through chromatography to form complex 1, and complex 1 continues to move forward and combines with antibody 2 coated on the T line to show red. The kit of the present invention has the advantages of high accuracy, etc.

Description

Technical Field

[0001] The present invention relates to the field of coronavirus detection, and in particular to a method for preparing a novel coronavirus antigen detection kit. Background Art

[0002] At present, there are two main methods for COVID-19 detection: nucleic acid and kits. The collection of nucleic acid requires a lot of manpower and material resources, and the detection results are time-consuming and have disadvantages such as false positives. Therefore, a new coronavirus antigen detection kit is introduced, and a method for preparing a new coronavirus antigen detection kit with high sensitivity is proposed. Summary of the invention

[0003] In view of this, the purpose of the present invention is to propose a method for preparing a novel coronavirus antigen detection kit to solve the above-mentioned problems.

[0004] The technical solution of the present invention is achieved in this way:

[0005] A method for preparing a novel coronavirus antigen detection kit comprises the following steps:

[0006] S1: Preparation of solvent

[0007] The solvent includes an activation buffer, an activation buffer reagent a, an activation buffer reagent b, a coupling buffer, a washing solution, a microsphere blocking solution, a microsphere preservation solution, a sample processing solution, a coating buffer, a microsphere diluent and a sample pad processing solution;

[0008] S2: Microsphere labeling

[0009] S2.1 Microsphere Activation

[0010] S2.1.1 Mix the microsphere suspension and activation buffer, centrifuge, remove the supernatant, continue to add activation buffer, centrifuge, remove the supernatant, and collect the microspheres;

[0011] S2.1.2 Mix the microspheres in S2.1.1 with the activation buffer, activation buffer reagent b, and activation buffer reagent a to prepare a microsphere mixture, incubate, centrifuge the incubated microsphere mixture, and collect the microspheres;

[0012] S2.1.3 Add the microspheres prepared in S2.1.2 to the activation buffer solution, mix, and centrifuge to obtain activated microspheres;

[0013] S2.2 Microsphere and Antibody Coupling

[0014] S2.2.1 Mix the activated microspheres and coupling buffer, resuspend and disperse by ultrasonication to prepare activated microsphere buffer;

[0015] S2.2.2 Add N72 antibody to a centrifuge tube, dilute with coupling buffer, add activation microsphere buffer, mix and incubate to obtain antibody-coupled microspheres;

[0016] S2.3 Sealing and storage of microspheres

[0017] S2.3.1 Mix the antibody-coupled microspheres and microsphere blocking solution, resuspend by ultrasonication, and incubate to obtain incubated microspheres;

[0018] S2.3.2 Centrifuge the microspheres incubated in S2.3.1 and remove the supernatant to obtain microspheres;

[0019] S2.3.3 Mix the microspheres prepared in S2.3.2 and the washing solution, centrifuge, remove the supernatant, and collect the microspheres;

[0020] S2.3.4 Mix the microspheres prepared in S2.3.3 with the microsphere preservation solution, resuspend by ultrasound, centrifuge, remove the supernatant, collect the microspheres, mix the microsphere preservation solution with the microspheres, vortex and age, and prepare the labeled microspheres;

[0021] S3. Preparation method of coating template

[0022] S3.1 Preparation of coating solution: Mix goat anti-mouse IgG and coating buffer to prepare coating solution 1; Mix N54 antibody and coating buffer to prepare coating solution 2;

[0023] S3.2 Coating template: Use a film spraying instrument to spray coating solution 1 and coating solution 2 onto the nitrocellulose membrane respectively;

[0024] S4. Preparation method of conjugate pad

[0025] S4.1 dilute the labeled microspheres prepared in S2 with a microsphere diluent, and perform sonication to obtain diluted microspheres;

[0026] S4.2 Use a film spraying instrument to spray the diluted microspheres onto the glass fiber membrane, dry it, and prepare a bonding pad;

[0027] S5, assembling, cutting, inner packaging and outer packaging to obtain the finished product.

[0028] Furthermore, the solvent preparation method described in step S1 is:

[0029] Activation buffer: 2-(N-morpholine)ethanesulfonic acid monohydrate was mixed with water, and the pH value was adjusted to 5.8-6.2 using a pH adjuster to prepare an activation buffer;

[0030] Activation buffer reagent a: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is mixed with an activation buffer to prepare an activation buffer reagent a;

[0031] Activation buffer reagent b: N-hydroxysuccinimide is mixed with activation buffer to prepare activation buffer reagent b;

[0032] Coupling buffer: 4-hydroxyethylpiperazineethanesulfonic acid was mixed with water, and the pH value was adjusted to 7.4-7.8 using a pH adjuster to prepare a coupling buffer;

[0033] Washing liquid: 4-hydroxyethylpiperazineethanesulfonic acid, sodium chloride and Tween-20 are mixed with water, and the pH value is adjusted to 7.4-7.8 with a pH adjuster to prepare a washing liquid;

[0034] Microsphere blocking solution: 4-hydroxyethylpiperazineethanesulfonic acid, bovine serum albumin and glycine are mixed with water, and the pH value is adjusted to 7.4-7.8 using a pH regulator to prepare a microsphere blocking solution;

[0035] Microsphere preservation solution: Tris(hydroxymethyl)aminomethane, sodium chloride, Tween-20, bovine serum albumin, trehalose and preservatives are mixed with water, and the pH value is adjusted to 8.4-8.8 using a pH regulator to prepare a microsphere preservation solution;

[0036] Sample treatment solution: 4-hydroxyethylpiperazineethanesulfonic acid, sodium chloride, polyethylene glycol octylphenyl ether, bovine serum albumin and a preservative are mixed with water, and the pH value is adjusted to 7.2-7.6 using a pH adjuster to prepare a sample treatment solution;

[0037] Coating buffer: Mix disodium hydrogen phosphate dodecahydrate, sodium chloride and trehalose with water, and adjust the pH value to 7.2-7.6 with a pH adjuster to prepare a coating buffer;

[0038] Microsphere diluent: tris(hydroxymethyl)aminomethane, Tween-20, bovine serum albumin, polyvinyl pyrrolidone, sucrose, trehalose and preservatives are mixed with water, and the pH value is adjusted to 8.4-8.8 with a pH adjuster to prepare a microsphere diluent;

[0039] Sample pad treatment solution: dilute disodium hydrogen phosphate dodecahydrate, sodium chloride, sodium caseinate, disodium ethylenediaminetetraacetate and Tween-20 with water, and use a pH adjuster to adjust the pH value to 7.2-7.6 to prepare a sample pad treatment solution.

[0040] Furthermore, the concentration of the activation buffer is 0.05 mol / L, the pH value of the activation buffer is 6.0, the concentration of the activation buffer reagent a is 10 mg / mL, the concentration of the activation buffer reagent b is 50 mg / mL, the concentration of the coupling buffer is 0.05 mol / L, the pH value of the coupling buffer is 7.6, the concentration of 4-hydroxyethylpiperazineethanesulfonic acid in the washing solution is 0.05 mol / L, the concentration of sodium chloride is 0.05 mol / L, the mass concentration of Tween-20 is 0.05%, and the pH value of the washing solution is 7.6.

[0041] Furthermore, the concentration of 4-hydroxyethylpiperazineethanesulfonic acid in the microsphere blocking solution is 0.05 mol / L, the concentration of bovine serum albumin is 10%, the concentration of glycine is 1%, the pH value of the microsphere blocking solution is 7.6, the concentration of tris(hydroxymethyl)aminomethane in the microsphere preservation solution is 0.025 mol / L, the concentration of sodium chloride is 0.15 mol / L, the concentration of Tween-20 is 0.02%, the concentration of bovine serum albumin is 1%, the concentration of trehalose is 5%, the concentration of preservative is 0.05%, the pH value of the microsphere preservation solution is 8.6, the concentration of 4-hydroxyethylpiperazineethanesulfonic acid in the sample processing solution is 0.05 mol / L, the concentration of sodium chloride is 0.15 mol / L, the concentration of polyethylene glycol octylphenyl ether is 0.5%, the concentration of bovine serum albumin is 0.1%, the concentration of preservative is 0.02%, and the pH value of the sample processing solution is 7.4.

[0042] Furthermore, the concentration of disodium hydrogen phosphate dodecahydrate in the coating buffer is 7.38 g / L, the concentration of sodium chloride is 0.15 mol / L, the mass concentration of trehalose is 2%, the pH value of the coating buffer is 7.4, the concentration of tris(hydroxymethyl)aminomethane in the microsphere diluent is 3.0285 g / L, the mass concentration of Tween-20 is 0.71%, the mass concentration of bovine serum albumin is 1%, the mass concentration of polyvinyl pyrrolidone is 1.2%, the mass concentration of sucrose is 19.5%, the mass concentration of trehalose is 2%, the mass concentration of the preservative is 0.02%, the pH value of the microsphere diluent is 8.6, the concentration of disodium hydrogen phosphate dodecahydrate in the sample pad treatment solution is 0.01 mol / L, the mass concentration of sodium chloride is 0.9%, the mass concentration of sodium caseinate is 1%, the mass concentration of disodium ethylenediaminetetraacetic acid is 0.3%, the mass concentration of Tween-20 is 0.5%, and the pH value of the sample pad treatment solution is 7.4.

[0043] Furthermore, the specific steps of the S2 microsphere labeling are:

[0044] S2.1 Microsphere Activation

[0045] S2.1.1 Take 400 μL of 400 nm microsphere suspension with a solid content of 4% and mix it with 1 mL of activation buffer, centrifuge it at 4°C, use 12500×g for 15 min, remove the supernatant, continue to add 1.5 mL of activation buffer, blow off the microsphere precipitate and mix it with a vortex mixer, centrifuge the mixed microsphere activation buffer at 4°C, use 12500×g for 15 min, remove the supernatant, and collect the microspheres;

[0046] S2.1.2 Mix the microspheres in S2.1.1 with 1.5 mL of activation buffer, blow off the microsphere precipitate, and disperse the dispersed microspheres by ultrasonic resuspending. First, add 195-200 μL of activation buffer reagent b to the dispersed microspheres and mix them, then add 100-110 μL of activation buffer reagent a to mix them to prepare a microsphere mixture, incubate at 37°C, incubate at 230-270 rpm for 25-35 min, centrifuge the incubated microsphere mixture at 4°C, centrifuge at 12500 x g for 20 min, remove the supernatant, and collect the microspheres;

[0047] S2.1.3 Mix the microspheres prepared in S2.1.2 with 1.5 mL of activation buffer, blow off the microsphere precipitate and centrifuge at 4°C and 12500 x g for 15 min, remove the supernatant, collect the microspheres, repeat the operation 1-2 times to obtain activated microspheres;

[0048] S2.2 Microsphere and Antibody Coupling

[0049] S2.2.1 Mix the activated microspheres with 1.5 mL of coupling buffer, blow off the microsphere precipitate, mix with a vortex mixer, and resuspend and disperse with ultrasound to prepare an activated microsphere buffer;

[0050] S2.2.2 Add 1.6 mg of N72 antibody to a centrifuge tube, dilute to 13.5 mL with coupling buffer, then add activation microsphere buffer, mix and incubate at 37°C, 250 rpm for 2.5-3.5 h to obtain antibody-coupled microspheres;

[0051] S2.3 Sealing and storage of microspheres

[0052] S2.3.1 Mix the antibody-coupled microspheres with 1.5 mL of microsphere blocking solution, resuspend by ultrasonication, and incubate at 37°C and 250 rpm for 1 hour to obtain incubated microspheres;

[0053] S2.3.2 centrifuge the microspheres incubated in S2.3.1 at 4°C and 12500 x g for 20 min, remove the supernatant, and obtain microspheres;

[0054] S2.3.3 Mix the microspheres prepared in S2.3.2 with 1.5 mL of washing solution, blow off the microsphere precipitate and centrifuge at 4°C and 12500 x g for 15 min, remove the supernatant, repeat the operation 1-2 times, and collect the microspheres;

[0055] S2.3.4 Mix the microspheres prepared in S2.3.3 with 1.5 mL of microsphere preservation solution, blow off the microsphere precipitate, resuspend it by ultrasonication, and centrifuge at 4°C. Centrifuge at 15000xg for 15 min. Remove the supernatant, collect the microspheres, mix the microspheres with the microsphere preservation solution, and mix them at a microsphere concentration of 2 mg / mL. Mix at 37°C, 250 rpm for 13-16 h to obtain labeled microspheres.

[0056] Furthermore, in step S4.2, the spraying parameters of the film spraying instrument are as follows: the spraying length is 300 mm, the Y-axis position is 6 mm, the bracket height is 28.5 mm, the platform speed is 75 mm / s, the spraying line spacing is 8 mm, the number of spraying lines is 5, the liquid line volume is 10 μL / cm, and the upper spiral knob is 4.00.

[0057] Furthermore, in step S3.2, the spraying parameters of the film spraying instrument are set to set the stroke length to 308 mm, the Y-axis position to 2.2 mm, the bracket height to 30 mm, the platform speed to 75 mm / s, the liquid stroke volume to 0.85 μL / cm, the upper spiral knob to 4.00, the coating liquid 2 stroke is recorded as the T line, and the coating liquid 1 stroke is recorded as the C line.

[0058] Further, in step S5, the assembling includes the following steps:

[0059] (1) Soak the sample pad in the sample pad treatment solution for 1.8-2.2 hours, use an oven to dry, the drying temperature is 55-65° C., the drying time is 50-70 minutes, and the oven-dried sample pad is placed in a 100,000-level drying room for 14-18 hours to obtain a treated sample pad. The process temperature in the drying room is 18-26° C., and the humidity is 15-29.8%;

[0060] (2) Assemble the binding pad, sample loading pad and glass fiber membrane.

[0061] Compared with the prior art, the present invention has the following beneficial effects:

[0062] The kit of the present invention adopts microsphere immunochromatography technology, and the N protein antibody of rabbit anti-2019-nCoV is coated with nitrocellulose membrane and N protein antibody labeled latex microspheres and other reagents. During detection, if 2019-nCoV is present in the sample, after the sample is processed, the latex microsphere antibody on the marker pad specifically binds to the 2019-nCoVN protein to form a complex, and the complex moves forward along the test strip due to the chromatography, and is captured by the rabbit anti-N protein antibody on the detection line to form a sandwich complex agglomeration color. If it is a negative specimen, a complex cannot be formed, and it cannot be captured by a specific antibody in the detection area, so it does not develop color. Regardless of whether 2019-nCoV is present in the sample, a colored strip will appear at the quality control line (C), which serves as an internal control standard for whether the chromatography process is normal and whether the reagent is invalid. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 SDS-PAGE image of SARS-CoV-2 N protein antibody

[0064] In the figure, 1: protein marker; 2: N72 antibody; 3. N54 antibody; 4: N28 antibody; 5: 6G9 antibody

[0065] 6.5E7 antibody; 7:5F9 antibody; 8:2B7 antibody DETAILED DESCRIPTION

[0066] In order to better understand the technical content of the present invention, specific embodiments are provided below to further illustrate the present invention.

[0067] Unless otherwise specified, the experimental methods used in the embodiments of the present invention are all conventional methods.

[0068] Unless otherwise specified, the materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial sources.

[0069]

[0070]

[0071] Preparation method of N28: E.coil BL (DE3) expressing 2019-nCoVN protein (P0DTC9) was used as an immunogen to immunize Balb / c mice, screen positive hybridoma cells, collect mouse ascites and purify by protein G to obtain antibody N28.

[0072] Example 1

[0073] Preparation of activation buffer

[0074] Production site: One hundred thousand level area operation room.

[0075] Reagent weighing:

[0076]

[0077] Preparation process: Mix 8 mL of purified water with the raw materials, use a magnetic stirrer to mix until completely dissolved, use 1 mol / L hydrochloric acid solution to adjust the pH value to 6.0, add purified water to make up to 10 mL, mix thoroughly, and filter with a 0.22 μm filter.

[0078] Preparation of activation buffer reagent a

[0079] Reagent weighing:

[0080]

[0081] Preparation process: Mix 1 mL of activation buffer with the raw materials and use a magnetic stirrer to mix until completely dissolved. It must be prepared and used immediately.

[0082] Preparation of activation buffer reagent b

[0083] Reagent weighing:

[0084]

[0085] Preparation process: Mix 1 mL of activation buffer with the raw materials and use a magnetic stirrer to mix until completely dissolved. It must be prepared and used immediately.

[0086] Coupling buffer preparation

[0087] Production site: One hundred thousand level area operation room.

[0088] Reagent weighing:

[0089]

[0090] Preparation process: Mix 15mL of purified water and the raw materials, and use a magnetic stirrer to mix until completely dissolved. Use 1mol / L hydrochloric acid solution to adjust the pH value to 7.6, add purified water to make up to 20mL, mix well and filter with a 0.22μm filter.

[0091] Preparation of washing liquid

[0092] Production site: One hundred thousand level area operation room.

[0093] Reagent weighing:

[0094]

[0095]

[0096] Preparation process: Use 4 mL of purified water and the raw materials to stir and mix on a magnetic stirrer; use 1 mol / L hydrochloric acid solution to adjust the pH value to 7.6, add purified water to make up to 5 mL, and filter with a 0.22 μm filter.

[0097] Preparation of microsphere blocking solution

[0098] Production site: One hundred thousand level area operation room.

[0099] Reagent weighing:

[0100]

[0101] Preparation process: Add 4 mL of purified water and mix with the raw materials, use a magnetic stirrer to mix thoroughly until completely dissolved; use 1 mol / L hydrochloric acid solution to adjust the pH value to 7.6, add purified water to make up to 5 mL, and filter with a 0.22 μm filter.

[0102] Preparation of microsphere storage solution

[0103] Production site: One hundred thousand level area operation room.

[0104] Reagent weighing:

[0105]

[0106]

[0107] Preparation process: Add 12mL of purified water and mix with the raw materials, stir and mix on a magnetic stirrer until completely dissolved; use 1mol / L hydrochloric acid solution to adjust the pH value to 8.6, add purified water to make up to 15mL, and filter with a 0.22μm filter.

[0108] Sample treatment solution preparation

[0109] Production site: One hundred thousand level area operation room.

[0110] Reagent weighing:

[0111]

[0112] Preparation process: Add 1L purified water and mix the raw materials, use a magnetic stirrer to mix thoroughly until completely dissolved; use 1mol / L hydrochloric acid solution to adjust the pH value to 7.4, add purified water to make up to 1.2L, and filter with a 0.22μm filter.

[0113] Coating buffer preparation

[0114] Production site: One hundred thousand level area operation room.

[0115] Reagent weighing:

[0116]

[0117] Preparation process: Add 3 mL of purified water and mix with the raw materials, use a magnetic stirrer to mix until completely dissolved; use 1 mol / L sodium hydroxide solution to adjust the pH value to 7.4, add purified water to make up to 5 mL, and filter with a 0.22 μm filter.

[0118] Preparation of microsphere diluent

[0119] Production site: One hundred thousand level area operation room.

[0120] Reagent weighing:

[0121]

[0122] Preparation process: Add 6 mL of purified water and mix with the raw materials, use a magnetic stirrer to mix until completely dissolved; use 1 mol / L sodium hydroxide solution to adjust the pH value to 8.6, add purified water to make up to 8 mL, and filter with a 0.22 μm filter.

[0123] Preparation of sample pad treatment solution

[0124] Production site: One hundred thousand level area operation room.

[0125] Reagent weighing:

[0126]

[0127] Preparation process: Add 400 mL of purified water and mix with the raw materials, use a magnetic stirrer to mix until completely dissolved; use 1 mol / L sodium hydroxide solution to adjust the pH value to 7.4, and add purified water to make up to 500 mL.

[0128] Microsphere labeling

[0129] Production site: One hundred thousand level area operation room.

[0130] Microsphere activation

[0131] (1) Mix the microspheres thoroughly with a vortex mixer, take 400 μL of the 400 nm microsphere suspension with a 4% solid content into a 5 mL centrifuge tube, add 1 mL of activation buffer, centrifuge at 4°C, 12500 x g for 15 min, and remove the supernatant;

[0132] (2) Add 1.5 mL of activation buffer to a 5 mL centrifuge tube, use a pipette to disperse the microsphere precipitate until there are no obvious large particles, then use a vortex mixer to fully mix the microspheres, centrifuge at 4°C, 12500 x g for 15 min, and remove the supernatant;

[0133] (3) Add 1.5 mL of activation buffer to a 5 mL centrifuge tube, blow off the microsphere precipitate with a pipette, resuspend the microspheres in an ultrasonic bath and evenly disperse them, first add 198 μL of activation buffer reagent b, mix thoroughly with a vortex mixer, then add 105 μL of activation buffer reagent a, mix thoroughly, seal the tube and incubate at 37°C, 250 rpm for 30 min;

[0134] (4) Centrifuge at 12,500 x g for 20 min at 4°C, and then carefully remove the supernatant with a pipette;

[0135] (5) Add 1.5 mL of activation buffer, use a pipette to disperse the microsphere precipitate, and after blowing away no obvious large particles, use a vortex mixer to fully mix the microspheres. Centrifuge at 4°C, 12,500 x g for 15 min, carefully remove the supernatant, add 1.5 mL of activation buffer, use a pipette to disperse the microsphere precipitate, and after blowing away no obvious large particles, use a vortex mixer to fully mix the microspheres. Centrifuge at 4°C, 12,500 x g for 15 min, carefully remove the supernatant, and obtain activated microspheres.

[0136] Conjugation of microspheres to antibodies

[0137] Add 1.5 mL of coupling buffer to the activated microspheres, blow off the microsphere precipitate with a pipette, mix the microspheres thoroughly with a vortex mixer, and resuspend and disperse them by ultrasonication under ice bath conditions; add 1.6 mg of N72 antibody to a 50 mL centrifuge tube, dilute to 13.5 mL with coupling buffer, add 1.5 mL of microspheres to the diluted antibody, and mix the microspheres thoroughly with a vortex mixer. After sealing, incubate at 37°C, 250 rpm for 3 hours to obtain antibody-coupled microspheres.

[0138] Sealing and storage of microspheres

[0139] (1) Add 1.5 mL of microsphere blocking solution to the tube, mix the microspheres thoroughly with a vortex mixer, resuspend the microspheres by ultrasonication under ice bath conditions, and incubate at 37°C and 250 rpm for 1 hour;

[0140] (2) Centrifuge at 12,500 x g for 20 min at 4°C in a large centrifuge and carefully remove the supernatant.

[0141] (3) Add 1.5 mL of washing solution to the tube, use a pipette to disperse and suspend the microsphere precipitate, transfer it to a 5 mL centrifuge tube, centrifuge at 4°C, 12500 x g for 15 min, carefully remove the supernatant, add 1.5 mL of washing solution, use a pipette to disperse and suspend the microsphere precipitate, transfer it to a 5 mL centrifuge tube, centrifuge at 4°C, 12500 x g for 15 min, carefully remove the supernatant. If the microspheres cannot be completely dispersed, they can be collected by ultrasound.

[0142] (4) Add 1.5 mL of microsphere preservation solution to the tube, blow off the microsphere precipitate with a pipette, resuspend the microspheres by ultrasound under ice bath conditions, centrifuge at 4°C, 15000xg for 15 min, carefully remove the supernatant, dilute the marked microspheres with 8 mL of microsphere preservation solution to a final concentration of 2 mg / mL, and vortex overnight at 37°C, 250 rpm for more than 12 h. After aging, store at 4°C in the dark for future use.

[0143] Coated film production

[0144] Production site: One hundred thousand level area operation room.

[0145] Reagent preparation:

[0146] name Final concentration Feeding amount (1.5mL) Sheep anti-mouse IgG 1.5 mg / mL 1.5mg / mL×1.5mL=2.25mg Coating fluid① / Add coating solution to 1.5 mL

[0147] name Final concentration Feeding amount (1.5mL) N54 antibody 2mg / mL 2mg / mL×1.5mL=3.0mg Coating fluid② / Add coating solution to 1.5 mL

[0148] (1) Tear off the 2.5 cm wide adhesive paper in the middle of the base, adhere the nitrocellulose membrane along the length (30 cm) of the base, tear off the narrower adhesive paper on the top, and stick 1.7 cm wide absorbent paper along the upper edge.

[0149] (2) Turn on the film spraying instrument, adjust the nozzle of pump No. 4 to the top, select the marking mode, then select the pump operation, turn on pumps 1 and 2, immerse the water inlets of the two pumps in purified water, select the number of cleaning times as 9, click Clean, and repeat once, then remove the purified water and click Clean to empty the pipeline.

[0150] (3) Select Main Interface → Parameters, set the stroke length to 308 mm, Y-axis position to 2.2 mm, support height to 30 mm, platform speed to 75 mm / s, liquid stroke volume to 0.85 μL / cm, and adjust the upper spiral knob to 4.00.

[0151] (4) After the setting is completed, return to the pump operation interface, insert the two pump lines into the diluted protein solution respectively (pump line 1 is the coating solution ② corresponding to the T line, and pump line 2 is the coating solution ① corresponding to the C line), clean the lines once with protein solution, click on the refill function, and then set the discharge to 10uL. Click on the discharge function until liquid is discharged from both needles, and remove the liquid on the needles.

[0152] (5) Align the front end of the pasted nitrocellulose membrane with the mark line on the platform, select the main interface, click to start marking, and observe the marking situation. If there are too many breakpoints or the marking position is inconsistent with the reference template, adjustments are required. If there are no abnormal conditions, you can mark the breakpoint position with a black pen and replace a new nitrocellulose membrane for marking.

[0153] (6) After coating, remove the coating working solution, drain the coating working solution in the pump, wash with purified water for 18 cycles, and then empty the pipeline. Transfer the coated membrane plate with two lines to the drying room and place it in a 37°C oven for 4 hours. After drying, place the prepared base plate in an aluminum foil bag in the drying room, add desiccant and seal it for use. Write the corresponding production time, preparer, storage and other corresponding information, store at room temperature, and clean the site.

[0154] Bonding pad production

[0155] Production site: One hundred thousand level area operation room.

[0156] (1) Take 7.7 mL of 2 mg / mL N72-labeled microspheres and dilute them to 1.333 mg / mL using 3.85 mL of microsphere diluent. After dilution, thoroughly sonicate until the mixture is evenly mixed.

[0157] (2) Turn on the film spraying instrument, adjust the nozzle of pump No. 4 to the lowest end, select the spraying mode, turn on the air compression pump under the machine, adjust the knob on the left side of the machine until the air pressure difference on the digital display shows 25, then select the pump operation, turn on only pump 4, immerse the pump inlet in purified water, select the number of cleaning times as 5, click Clean, and repeat once, then remove the purified water, use the corresponding working fluid to rinse the pipeline twice, and then click Clean to empty the pipeline.

[0158] (3) Select Main Interface → Parameters, set the spray length to 300 mm, Y-axis position to 6 mm, bracket height to 28.5 mm, platform speed to 75 mm / s, spray line spacing to 8 mm, number of spray lines to 5, liquid line volume to 10 μL / cm, and adjust the upper spiral knob to 4.00.

[0159] (4) After the setting is completed, return to the pump operation interface, insert the pump pipeline into the solution to be sprayed, clean the pipeline once with the spray solution, click on the liquid replenishment, and then set the discharge to 20uL, click on the discharge until liquid is discharged from the nozzle, and collect the liquid on the nozzle.

[0160] (5) Align the front end of the cut 6cm wide glass fiber membrane with the mark line on the platform, select the main interface, click to start spraying, and observe the spraying situation. If there are too many breakpoints or the spraying strip is too wide, adjustments are required. If there are no abnormal conditions, replace a new glass fiber membrane for gold spraying after spraying is completed.

[0161] (6) After all gold spraying is completed, clean it according to the "XYZ3020 Film Spraying Instrument Cleaning and Disinfection Standard Operating Procedures".

[0162] (7) The sprayed glass fiber membrane is transferred to a drying room and placed in a 37°C oven for drying for 30 minutes. After drying, the prepared bonding pad is placed in an aluminum foil bag in the drying room, and then a desiccant is added and sealed for later use. The corresponding information such as the preparation time, the preparer, and the storage are written and stored at room temperature.

[0163] Assembly

[0164] Production site: 100,000-level drying room.

[0165] The process temperature is 18-26°C and the humidity is <30%.

[0166] (1) Treat the sample pad in advance, soak it in the sample pad treatment solution for two hours, dry it in a 60℃ oven for 1 hour, and then transfer it to a drying room for overnight drying. After drying, put it in an aluminum foil bag, add desiccant, and seal it for later use.

[0167] (2) Cut the glass fiber membrane into 6mm narrow strips according to the spray mark, and assemble the large card in the corresponding order. The binding pad needs to overlap with the NC membrane by about 2mm, and the sample pad and the binding pad also need to overlap by about 2mm. After pasting, press it tightly with the torn self-adhesive film. After assembly, it can be sealed in an aluminum foil bag together with the drying room for standby.

[0168] Slitting

[0169] Production site: One hundred thousand level area operation room.

[0170] The process temperature is 18-26°C and the humidity is <30%.

[0171] (1) Use a strip cutter to cut the test card into strips with a strip width of 3.03 mm. Place the cut test strips on a tray.

[0172] Inner Packaging

[0173] Production site: One hundred thousand level area operation room.

[0174] The process temperature is 18-26°C and the humidity is <30%.

[0175] Before the product is packed, pick up the materials according to the instruction sheet.

[0176] (1) The test strips are loaded into the card shell according to the corresponding requirements, and then the test card and desiccant are loaded into the aluminum foil bag and the aluminum foil bag is sealed.

[0177] (2) Packaging of sample processing solution: The amount of sample processing solution to be packaged corresponds to the number of production inspection cards. The packaging specification is 0.35 ml / tube and the packaging is carried out in the packaging area of ​​the operation room.

[0178] Outer Packaging

[0179] Production location: packaging room in general production area.

[0180] Before outer packaging of the product, a packing instruction sheet should be obtained and materials should be collected according to the packing instruction sheet.

[0181] After the test card is produced, the sample processing solution, swab, test card and instruction manual shall be packaged according to the instructions.

[0182] Comparative Example 1 Comparative Test of Novel Coronavirus N Protein Antibodies

[0183] The sensitivity and specificity of the antibody combination for detecting the novel coronavirus antigen NP protein were evaluated by combining two antibodies as nitrocellulose membrane-coated antibodies and latex microsphere-labeled antibodies. The antibodies are N54, 6G9, N72, N28, 5E7, 5F9, and 2B7.

[0184] Test method:

[0185] (1) The seven monoclonal antibodies N54, 6G9, N72, N28, 5E7, 5F9, and 2B7 were paired in pairs and used as the detection line coating antibody and the latex microsphere labeled antibody, respectively. The detection line antibody coating concentration was 2 mg / mL, the latex microsphere: antibody ratio was labeled at 10:1, and the latex microsphere labeled antibody 0.1%, 10 μL / cm was sprayed on the binding pad and assembled for detection.

[0186] (2) The sample treatment solution was used to dilute the recombinant NP protein to a concentration of 900 pg / mL, 300 pg / mL, 100 pg / mL, and 0 pg / mL. Three groups of healthy human nasal swabs, N1, N2, and N3, were used as negative controls. The color intensity was observed after reacting at room temperature (18-28°C) for 15 minutes.

[0187] Table 1 Test line 3E2 (N54) package detection performance

[0188]

[0189] Table 2 Test line 5E6 (N72) package detection performance

[0190]

[0191]

[0192] Table 3 Detection performance of test line 6G9 package

[0193]

[0194] Table 4 Detection performance of test line 5E7 package

[0195]

[0196] Table 5 Test line 5F9 package detection performance

[0197]

[0198] Table 6 Test line N28 package test performance

[0199]

[0200] Table 7 Detection performance of test line 2B7 package

[0201]

[0202]

[0203] Note: "+" represents a positive result with obvious bands; "-" represents a negative result with no obvious bands

[0204] Combination Figure 1 The experimental results in Table 1-7 show that when 3E2 (N54) / 5E6 (N72) is used as the paired antibody, the membrane surface is clean after 15 minutes of observation, the sensitivity of the recombinant N protein reaches 100pg / mL, and there are no false positives in the three negative samples. That is, the mouse anti-2019-nCoVN protein monoclonal antibody 5E6 (N72) is used as the latex microsphere labeled antibody, and the rabbit anti-2019-nCoVN protein monoclonal antibody 3E2 (N54) is used as the coating antibody. The assembled test card exhibits the best performance of recombinant N protein detection sensitivity (100pg / mL) and the best detection specificity (100% specificity for detecting 10 negative samples), and can be used as a paired antibody for the preparation of test cards.

[0205] Comparative Example 2

[0206] On the basis of Example 1, the preparation process of the coupling step of microspheres and antibodies was adjusted as follows:

[0207] In the preparation of coupling buffer, the pH value of coupling buffer was 7.0.

[0208] Add 1.5 mL of coupling buffer to the activated microspheres, blow off the microsphere precipitate with a pipette, mix the microspheres thoroughly with a vortex mixer, and resuspend and disperse them by ultrasonication under ice bath conditions; add 3 mg of N72 antibody to a 50 mL centrifuge tube, dilute to 13.5 mL with coupling buffer, add 1.5 mL of microspheres to the diluted antibody, and then mix the microspheres thoroughly with a vortex mixer. After sealing, incubate at 37°C, 250 rpm for 3 hours to obtain antibody-coupled microspheres.

[0209] Test Example 1

[0210] The novel coronavirus antigen detection kit prepared in Example 1 was used for testing, with 100 samples and 100 samplers, repeated twice, and the detection time and accuracy were recorded.

[0211] Sampling: Nasal swab: When collecting samples, first blow your nose with toilet paper, carefully open the outer packaging of the nasal swab, and avoid touching the swab head with your hands. Then tilt your head slightly, hold the tail of the swab in one hand and stick it to one nostril, slowly go back along the bottom of the lower nasal cavity 1-1.5 cm (for subjects aged 2-14, go 1 cm), then stick it to the nasal cavity and rotate it for at least 4 times, stay for no less than 15 seconds, and then use the same swab to repeat the same operation on the other nasal cavity. Dip the swab head into the sampling container containing the sample processing solution that comes with the test strip.

[0212] Detection operation:

[0213] 1. Preparation: Equilibrate all reagents and samples to room temperature.

[0214] 2. Place the swab immediately after collecting the sample in the sample processing solution. The swab head should be rotated and mixed in the processing solution for at least 30 seconds. At the same time, squeeze the swab head through the outer wall of the processing solution tube by hand at least 5 times to ensure that the sample is fully eluted in the sampling container containing the sample processing solution that comes with the test strip.

[0215] 3. Use your hands to squeeze out the liquid from the swab head through the outer wall of the sample treatment liquid tube, discard the swab, and cover the treatment tube.

[0216] 4. Drip the liquid vertically into the sample hole of the test card, drip 3 drops (80uL) or use a micropipette to suck 80uL into the sample hole of the test card and start timing.

[0217] Result interpretation:

[0218] Positive: Two red bands appear. One is in the test area (T) and the other is in the quality control area (C). The color of the band in the test area (T) can be dark or light, which is a positive result.

[0219] Negative: Only one red band appears in the quality control area (C), and no band appears in the detection area (T).

[0220] Invalid: No red stripe appears in the quality control area (C), regardless of whether a stripe appears in the test area (T), indicating that the result is invalid and the test card needs to be retested.

[0221] Table 7 Test results of novel coronavirus antigen detection kit

[0222] name Detection time (min) Accuracy (%) Example 1 15.0 100 Comparative Example 2 21.6 100

[0223] The experimental results show that the novel coronavirus antigen detection kit of the present invention has a high accuracy rate. The experimental results of comparative example 2 show that the preparation method of the coupling liquid of the present invention and the coupling method of the microspheres and antibodies can make the microspheres and antibodies evenly mixed, avoiding the occurrence of antibody agglomeration, which leads to reduced detection performance of the antigen detection kit.

[0224] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a novel coronavirus antigen detection kit, characterized in that: The following steps are involved: S1: Preparation of solvent The solvent includes an activation buffer, an activation buffer reagent a, an activation buffer reagent b, a coupling buffer, a washing solution, a microsphere blocking solution, a microsphere preservation solution, a sample processing solution, a coating buffer, a microsphere diluent and a sample pad processing solution; The solvent preparation method described in step S1 is: (1) Activation buffer: 2-(N-morpholino)ethanesulfonic acid monohydrate was mixed with water, and the pH value was adjusted to 5.8-6.2 using a pH adjuster to prepare an activation buffer; (2) Activation buffer reagent a: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is mixed with an activation buffer to prepare an activation buffer reagent a; (3) Activation buffer reagent b: N-hydroxysuccinimide is mixed with an activation buffer to prepare an activation buffer reagent b; (4) Coupling buffer: 4-hydroxyethylpiperazineethanesulfonic acid was mixed with water, and the pH value was adjusted to 7.4-7.8 using a pH adjuster to prepare a coupling buffer; (5) washing liquid: 4-hydroxyethylpiperazineethanesulfonic acid, sodium chloride and Tween-20 are mixed with water, and the pH value is adjusted to 7.4-7.8 using a pH adjuster to prepare a washing liquid; (6) Microsphere blocking solution: 4-hydroxyethylpiperazineethanesulfonic acid, bovine serum albumin and glycine were mixed with water, and the pH value was adjusted to 7.4-7.8 using a pH adjuster to prepare a microsphere blocking solution; (7) Microsphere preservation solution: tris(hydroxymethyl)aminomethane, sodium chloride, Tween-20, bovine serum albumin, trehalose and preservatives are mixed with water, and the pH value is adjusted to 8.4-8.8 using a pH adjuster to prepare a microsphere preservation solution; (8) Sample treatment solution: 4-hydroxyethylpiperazineethanesulfonic acid, sodium chloride, polyethylene glycol octylphenyl ether, bovine serum albumin and a preservative are mixed with water, and the pH value is adjusted to 7.2-7.6 using a pH adjuster to prepare a sample treatment solution; (9) Coating buffer: Disodium hydrogen phosphate dodecahydrate, sodium chloride and trehalose are mixed with water, and the pH value is adjusted to 7.2-7.6 using a pH adjuster to prepare a coating buffer; (10) Microsphere diluent: tris(hydroxymethyl)aminomethane, Tween-20, bovine serum albumin, polyvinyl pyrrolidone, sucrose, trehalose and a preservative are mixed with water, and the pH value is adjusted to 8.4-8.8 using a pH adjuster to prepare a microsphere diluent; (11) Sample pad treatment solution: dilute disodium hydrogen phosphate dodecahydrate, sodium chloride, sodium caseinate, disodium ethylenediaminetetraacetic acid and Tween-20 with water, and adjust the pH value to 7.2-7.6 with a pH adjuster to prepare a sample pad treatment solution; S2: Microsphere labeling S2.1 Microsphere Activation S2.1.1 Mix the microsphere suspension and activation buffer, centrifuge, remove the supernatant, continue to add activation buffer, centrifuge, remove the supernatant, and collect the microspheres; S2.1.2 Mix the microspheres in S2.1.1 with the activation buffer, activation buffer reagent b, and activation buffer reagent a to prepare a microsphere mixture, incubate, centrifuge the incubated microsphere mixture, and collect the microspheres; S2.1.3 Add the microspheres prepared in S2.1.2 to the activation buffer solution, mix, and centrifuge to obtain activated microspheres; S2.2 Microsphere and Antibody Coupling S2.2.1 Mix the activated microspheres and coupling buffer, resuspend and disperse by ultrasonication to prepare activated microsphere buffer; S2.2.2 Add N72 antibody to a centrifuge tube, dilute with coupling buffer, add activation microsphere buffer, mix and incubate to obtain antibody-coupled microspheres; S2.3 Sealing and storage of microspheres S2.3.1 Mix the antibody-coupled microspheres and microsphere blocking solution, resuspend by ultrasonication, and incubate to obtain incubated microspheres; S2.3.2 centrifuge the microspheres incubated in S2.3.1, remove the supernatant, and obtain microspheres; S2.3.3 Mix the microspheres prepared in S2.3.2 and the washing solution, centrifuge, remove the supernatant, and collect the microspheres; S2.3.4 Mix the microspheres prepared in S2.3.3 with the microsphere preservation solution, resuspend by ultrasound, centrifuge, remove the supernatant, collect the microspheres, mix the microsphere preservation solution with the microspheres, vortex and age, and prepare the labeled microspheres; S3. Preparation method of coating template S3.1 Preparation of coating solution: Mix goat anti-mouse IgG and coating buffer to prepare coating solution 1; Mix N54 antibody and coating buffer to prepare coating solution 2; S3.2 Coating template: Use a film spraying instrument to spray coating solution 1 and coating solution 2 onto the nitrocellulose membrane respectively; S4. Preparation method of conjugate pad S4.1 dilute the labeled microspheres prepared in S2 with a microsphere diluent, and perform sonication to obtain diluted microspheres; S4.2 Use a film spraying instrument to spray the diluted microspheres onto the glass fiber membrane, dry it, and prepare a bonding pad; S5, assembling, cutting, inner packaging and outer packaging to obtain the finished product.

2. The method for preparing a novel coronavirus antigen detection kit according to claim 1, characterized in that: The concentration of the activation buffer is 0.05 mol / L, the pH value of the activation buffer is 6.0, the concentration of the activation buffer reagent a is 10 mg / mL, the concentration of the activation buffer reagent b is 50 mg / mL, the concentration of the coupling buffer is 0.05 mol / L, the pH value of the coupling buffer is 7.6, the concentration of 4-hydroxyethylpiperazineethanesulfonic acid in the washing solution is 0.05 mol / L, the concentration of sodium chloride is 0.05 mol / L, the mass concentration of Tween-20 is 0.05%, and the pH value of the washing solution is 7.

6.

3. The method for preparing a novel coronavirus antigen detection kit according to claim 1, characterized in that: The concentration of 4-hydroxyethylpiperazineethanesulfonic acid in the microsphere blocking solution is 0.05 mol / L, the concentration of bovine serum albumin is 10%, the concentration of glycine is 1%, and the pH value of the microsphere blocking solution is 7.

6. The concentration of tris(hydroxymethyl)aminomethane in the microsphere preservation solution is 0.025 mol / L, the concentration of sodium chloride is 0.15 mol / L, the concentration of Tween-20 is 0.02%, the concentration of bovine serum albumin is 1%, the concentration of trehalose is 5%, the concentration of preservative is 0.05%, and the pH value of the microsphere preservation solution is 8.

6. The concentration of 4-hydroxyethylpiperazineethanesulfonic acid in the sample processing solution is 0.05 mol / L, the concentration of sodium chloride is 0.15 mol / L, the concentration of polyethylene glycol octylphenyl ether is 0.5%, the concentration of bovine serum albumin is 0.1%, the concentration of preservative is 0.02%, and the pH value of the sample processing solution is 7.

4.

4. The method for preparing a novel coronavirus antigen detection kit according to claim 1, wherein: The concentration of disodium hydrogen phosphate dodecahydrate in the coating buffer is 7.38 g / L, the concentration of sodium chloride is 0.15 mol / L, the mass concentration of trehalose is 2%, and the pH value of the coating buffer is 7.

4. The concentration of tris(hydroxymethyl)aminomethane in the microsphere diluent is 3.0285 g / L, the mass concentration of Tween-20 is 0.71%, the mass concentration of bovine serum albumin is 1%, the mass concentration of polyvinyl pyrrolidone is 1.2%, the mass concentration of sucrose is 19.5%, the mass concentration of trehalose is 2%, the mass concentration of the preservative is 0.02%, and the pH value of the microsphere diluent is 8.

6. The concentration of disodium hydrogen phosphate dodecahydrate in the sample pad treatment solution is 0.01 mol / L, the mass concentration of sodium chloride is 0.9%, the mass concentration of sodium caseinate is 1%, the mass concentration of disodium ethylenediaminetetraacetic acid is 0.3%, the mass concentration of Tween-20 is 0.5%, and the pH value of the sample pad treatment solution is 7.

4.

5. The method for preparing a novel coronavirus antigen detection kit according to claim 1, wherein: The specific steps of S2 microsphere labeling are: S2.1.1 Take 400 μL of 400 nm microsphere suspension with a solid content of 4% and mix it with 1 mL of activation buffer, centrifuge it at 4°C, use 12500 x g for 15 min, remove the supernatant, continue to add 1.5 mL of activation buffer, blow off the microsphere precipitate and mix it with a vortex mixer, centrifuge the mixed microsphere activation buffer at 4°C, use 12500 x g for 15 min, remove the supernatant, and collect the microspheres; S2.1.2 Mix the microspheres in S2.1.1 with 1.5 mL of activation buffer, blow off the microsphere precipitate, and disperse the dispersed microspheres by ultrasonic resuspending. First, add 195-200 μL of activation buffer reagent b to the dispersed microspheres and mix them, then add 100-110 μL of activation buffer reagent a to mix them to prepare a microsphere mixture, incubate at 37°C, incubate at 230-270 rpm for 25-35 min, centrifuge the incubated microsphere mixture at 4°C, centrifuge at 12500 x g for 20 min, remove the supernatant, and collect the microspheres; S2.1.3 Mix the microspheres prepared in S2.1.2 with 1.5 mL of activation buffer, blow off the microsphere precipitate, and then use a vortex mixer to mix and centrifuge. The centrifugation temperature is 4°C, and the mixture is centrifuged at 12500 x g for 15 min. Remove the supernatant, collect the microspheres, and repeat the operation 1-2 times to obtain activated microspheres. S2.2.1 Mix the activated microspheres with 1.5 mL of coupling buffer, blow off the microsphere precipitate, mix with a vortex mixer, and resuspend and disperse with ultrasound to prepare an activated microsphere buffer; S2.2.2 Add 1.6 mg of N72 antibody to a centrifuge tube, dilute to 13.5 mL with coupling buffer, then add activation microsphere buffer, mix and incubate at 37°C, 250 rpm for 2.5-3.5 h to obtain antibody-coupled microspheres; S2.3.1 Mix the antibody-coupled microspheres with 1.5 mL of microsphere blocking solution, resuspend by ultrasonication, and incubate at 37°C and 250 rpm for 1 hour to obtain incubated microspheres; S2.3.2 centrifuge the microspheres incubated in S2.3.1 at 4°C and 12500 x g for 20 min, remove the supernatant, and obtain microspheres; S2.3.3 Mix the microspheres prepared in S2.3.2 with 1.5 mL of washing solution, blow off the microsphere precipitate and centrifuge at 4°C and 12500 x g for 15 min, remove the supernatant, repeat the operation 1-2 times, and collect the microspheres; S2.3.4 Mix the microspheres prepared in S2.3.3 with 1.5 mL of microsphere preservation solution, blow off the microsphere precipitate, resuspend it by ultrasonication, and centrifuge at 4°C. Centrifuge at 15000xg for 15 min, remove the supernatant, collect the microspheres, mix the microspheres with the microsphere preservation solution until the microsphere concentration is 2 mg / mL, and vortex at 37°C and 250 rpm for 13-16 h to obtain labeled microspheres.

6. The method for preparing a novel coronavirus antigen detection kit according to claim 1, wherein: In step S4.2, the spraying parameters of the film spraying instrument are as follows: spraying length of 300mm, Y-axis position of 6mm, bracket height of 28.5mm, platform speed of 75mm / s, spraying line spacing of 8mm, number of spraying lines of 5, liquid line volume of 10μL / cm, and upper spiral knob of 4.

00.

7. The method for preparing a novel coronavirus antigen detection kit according to claim 1, characterized in that: In step S3.2, the spraying parameters of the film spraying instrument are as follows: the stroke length is set to 308 mm, the Y-axis position is 2.2 mm, the bracket height is 30 mm, the platform speed is 75 mm / s, the liquid stroke volume is 0.85 μL / cm, the upper spiral knob is 4.00, the coating liquid 2 stroke is recorded as the T line, and the coating liquid 1 stroke is recorded as the C line.

8. The method for preparing a novel coronavirus antigen detection kit according to claim 1, wherein: In step S5, the assembling comprises the following steps: (1) Soak the sample pad in the sample pad treatment solution for 1.8-2.2 hours, use an oven to dry, the drying temperature is 55-65°C, the drying time is 50-70 minutes, and the sample pad after oven drying is placed in a 100,000-level drying room for 14-18 hours to obtain a treated sample pad. The process temperature in the drying room is 18-26°C, and the humidity is <30%; (2) Assemble the binding pad, sample loading pad and glass fiber membrane.

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