A novel coronavirus (SARS-CoV-2) antigen detection kit

Abstract

The present invention belongs to the field of chemical immunoassay, and specifically relates to a novel coronavirus antigen detection kit; a novel coronavirus antigen detection kit of the present invention comprises reagent R1 and reagent R2, wherein reagent R1 contains ACES-CABS buffer, latex particle sensitizer, and iodine propynyl alcohol butyl carbamate; reagent R2 contains ACES-CABS buffer, latex particle stabilizer, latex solution coated with mouse anti-novel coronavirus N / S protein monoclonal antibody, and iodine propynyl alcohol butyl carbamate; by optimizing latex enhanced immunoturbidimetry, improving latex particles and their sensitizers and stabilizers, the stability and linear range of the kit are significantly improved, and the sensitivity and accuracy of the kit are enhanced.

Description

Technical Field

[0001] The present invention belongs to the technical field of in vitro diagnostic reagents, and specifically relates to a novel coronavirus antigen detection kit. Background Art

[0002] The main structural proteins of the new coronavirus (SARS-CoV-2) include nucleocapsid protein (N), spike protein (S), membrane protein (E) and envelope protein (M), among which N protein is the core component of the new coronavirus, synthesized in large quantities, with multiple specific antigenic determinants and high stability. Therefore, N protein is an ideal target for antigen detection, with high detection sensitivity and specificity. The S protein is composed of two functional subunits, S1 and S2. The key domains of the S1 subunit, S1-receptor binding domain (RBD) and / or S1-N-terminal domain (NTD), can bind to the ACE2 receptor of the host cell, fuse the cell membranes of the virus and the host cell, and mediate the entry of the virus into the host cell.

[0003] Currently, the detection methods for the new coronavirus (SARS-CoV-2) include molecular detection (such as PCR nucleic acid detection), antigen detection, serological (antibody) detection, and immunoturbidimetry. Among them, molecular diagnosis has high accuracy in diagnosing new coronavirus infection, but it is time-consuming, relatively costly, and requires professionals and tools; antigen detection can be detected quickly, with simple operation and low cost, but low sensitivity; serological (antibody) detection requires reliable results 2 weeks after the onset of symptoms and can be used for retrospective analysis, but there may be false positives.

[0004] There are also reports on the use of latex immunoturbidimetry for detection. For example, CN112730827A discloses a novel coronavirus antibody detection kit and a method for preparing the same. The kit comprises reagent 1 and reagent 2, each of which is independently packaged. Reagent 2 comprises at least latex particles coated with novel coronavirus protein, which are used to react with novel coronavirus antibodies in the sample to form agglutination to change the turbidity and obtain the novel coronavirus antibody content in the sample to be tested. However, there is no sensitive and stable detection method for antigen detection of both novel coronavirus S protein and N protein.

[0005] Therefore, it is an urgent problem to find an optimized latex-enhanced immunoturbidimetry novel coronavirus antigen detection kit, which can enhance the detection accuracy and good stability while giving full play to the advantages of latex-enhanced immunoturbidimetry. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a novel coronavirus antigen detection kit, which targets the N protein and S protein of the novel coronavirus (SARS-CoV-2), adopts an optimized latex-enhanced immunoturbidimetry, and improves the latex particle preparation process, uses a new latex particle sensitizer and a latex particle stabilizer, and scientifically proportions to optimize the reaction system, greatly improve the stability of the reagent, and significantly enhance the analytical sensitivity and anti-interference ability of the reagent.

[0007] Specifically, the technical solution of the present invention is as follows:

[0008] A novel coronavirus antigen detection kit, comprising reagent R1 and reagent R2;

[0009] The reagent R1 includes the following components:

[0010] Buffer A 30~50mmol / L

[0011] Latex particle sensitizer 10~20mL / L

[0012] Preservatives 0.3-0.5 g / L;

[0013] The reagent R2 includes the following components:

[0014]

[0015]

[0016] The preservative is iodopropynyl butylcarbamate.

[0017] Furthermore, the volume ratio of the R1 reagent to the R2 reagent is 3:1.

[0018] Furthermore, in reagent R1, the buffer A is an ACES-CABS buffer with a pH of 6.85 at 25°C; and in reagent R2, the buffer B is an ACES-CABS buffer with a pH of 7.05 at 25°C.

[0019] Furthermore, in reagent R1, the preparation method of the latex particle sensitizer is: add N,N-dihydroxyethyl-N-octadecylbenzenesulfonamide propyl ammonium propane sulfonate into deionized water, stir to dissolve, add potassium chloride and polyethylene glycol 10000, dissolve, cool, adjust the pH, and finally add fetal bovine serum and xylitol, dissolve and make up to volume with deionized water to obtain the latex particle sensitizer.

[0020] Furthermore, in reagent R2, the preparation method of the latex particle stabilizer is: polyvinyl pyrrolidone K90 is added into water, stirred to dissolve, cooled, maltitol, stachyose, sodium undecenyl monoethanolamide succinate sulfonate are added, stirred to dissolve, and pH is adjusted to obtain the latex particle stabilizer.

[0021] Furthermore, in reagent R2, the preparation steps of the latex solution coated with mouse anti-COVID-19 N protein monoclonal antibody include:

[0022] (1) mixing a solution of 112-125 nm carboxyl latex particles with a solution of 266-305 nm carboxyl latex particles in a volume ratio of 3-5:1-1.5 to obtain a mixed solution A;

[0023] (2) Add ACES-CABS buffer solution with a pH value of 7.05 to the mixed solution A, stir and mix, and oscillate for 0.5 to 1 hour to obtain a mixed solution B;

[0024] (3) diluting the mouse anti-COVID-19 N protein monoclonal antibody with the antibody diluent, shaking for 12 to 15 hours to obtain the mouse anti-COVID-19 N protein monoclonal antibody diluent, and adding it to the mixed solution B to obtain the mixed solution C;

[0025] (4) Add EDC to the mixed solution C at 33-37°C and shake for 20-30 min to obtain a mixed solution D;

[0026] (5) Add the microsphere blocking agent to the mixed solution D at 20-25°C to obtain a latex solution coated with mouse anti-COVID-19 N protein monoclonal antibody.

[0027] Furthermore, in (1), a solution of 112 nm carboxyl latex particles is mixed with a solution of 305 nm carboxyl latex particles in a volume ratio of 3:1 to obtain a mixed solution A.

[0028] Furthermore, in (3), the antibody diluent comprises the following components: 0.1 mL / L HEPES, 1 g / LBSA, 9 g / L NaCl, 50 g / L trehalose, 0.01 g / L N-lauroyl-N-hydroxyethylethylenediamine propionate, and 1 mL / L PC-300; the concentration of the mouse anti-COVID-19 N protein monoclonal antibody diluent is 0.7-2.2 mg / mL.

[0029] Furthermore, in (5), the microsphere sealing agent comprises the following components: 1 mL / L ethylenediamine, 5 g / L BSA, 0.05 ml / L TWEEN20, 20 g / L mannitol, and 0.02 g / L sodium di-sec-octyl maleate sulfonate.

[0030] Furthermore, the novel coronavirus antigen detection kit of the present invention can also be used in conjunction with a fully automatic biochemical analyzer to detect the novel coronavirus, preferably a Hitachi 7180 fully automatic analyzer, with a main detection wavelength of 570 nm and an endpoint method. Those skilled in the art believe that any feasible fully automatic biochemical analyzer is within the scope of protection of the present invention.

[0031] The beneficial effects of the present invention are:

[0032] 1) The novel coronavirus antigen detection kit of the present invention adopts an optimized latex-enhanced immunoturbidimetry, specifically: a novel coupling buffer system and a closed system are adopted to improve the antibody-latex coating efficiency; for the two different structural proteins of the novel coronavirus, a novel mouse anti-novel coronavirus N protein monoclonal antibody-coated latex solution and a mouse anti-novel coronavirus S protein monoclonal antibody-coated latex solution are selected, and the components are scientifically proportioned to have good detection accuracy;

[0033] 2) The novel coronavirus antigen detection kit of the present invention can effectively detect the novel coronavirus content, and has the advantages of high sensitivity, good stability, and wide linear range. The kit prepared by latex immunoturbidimetry in the immunoassay can be applied to a fully automatic biochemical analyzer to achieve automated operation. Serum samples can be tested without further pretreatment steps, and the result is available in a short time, within 10 to 15 minutes.

[0034] 3) The latex particle sensitizer is used, and through scientific proportioning, the analytical sensitivity and anti-interference ability of the reagent are greatly enhanced;

[0035] 4) The use of latex particle stabilizers can promote and maintain antibody stability through scientific proportions, keeping the latex particles in a stable dispersed state, and significantly enhancing the stability of the reagent. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0037] Figure 1 The present invention provides a comparative graph of the stability of the novel coronavirus antigen detection kits of various embodiments and comparative examples of the present invention. DETAILED DESCRIPTION

[0038] The present invention is described below by specific embodiments. Unless otherwise specified, the technical means used in the present invention are methods known to those skilled in the art. In addition, the embodiments should be understood as illustrative rather than limiting the scope of the present invention, and the essence and scope of the present invention are limited only by the claims. For those skilled in the art, various changes or modifications to the material composition and dosage in these embodiments, without departing from the essence and scope of the present invention, also belong to the protection scope of the present invention.

[0039] In some preferred embodiments of the present invention, a novel coronavirus antigen detection kit comprises reagent R1 and reagent R2;

[0040] The reagent R1 includes the following components:

[0041] Buffer A 36mmol / L

[0042] Latex particle sensitizer 10~20mL / L

[0043] Preservative 0.35g / L;

[0044] The reagent R2 includes the following components:

[0045]

[0046] The preservative is iodopropynyl butylcarbamate.

[0047] Preferably, when the novel coronavirus antigen detection kit is used, the ratio of reagent R1:reagent R2 is 3:1.

[0048] Buffer A in reagent R1 is ACES-CABS buffer at 25°C and pH 6.85; Buffer B in reagent R2 is ACES-CABS buffer at 25°C and pH 7.05;

[0049] The preparation method of the above two buffer solutions is as follows: weigh 6.56 g of ACES (N-(2-acetamido)-2-aminoethanesulfonic acid), dissolve it completely with 920 mL of deionized water, then add 8.47 g of CABS (4-(cyclohexylamino)-1-butanesulfonic acid), stir to dissolve, adjust the pH to 6.85 or 7.05 with an appropriate amount of ACES or sodium hydroxide, and finally make up to 1 L with deionized water.

[0050] Preferably, the preparation method of the latex particle sensitizer is as follows: weigh 5g of N,N-dihydroxyethyl-N-octadecylbenzenesulfonamide propylammonium propanesulfonate and add it to 250mL of deionized water, stir and dissolve it at 37°C for 30min, then add 25g of potassium chloride and 20g of polyethylene glycol 10000, and after complete dissolution, cool to room temperature, adjust the pH to 6.85 (25°C) with hydrochloric acid or sodium hydroxide, and finally add 5mL of fetal bovine serum and 10g of xylitol, and dilute to 300mL with deionized water after complete dissolution.

[0051] Preferably, the preparation method of the latex particle stabilizer is as follows: take 5g of polyvinyl pyrrolidone K90 and add it to 150mL of water, stir and dissolve it at 37°C for 1h, cool it to room temperature, add 20g of maltitol, 12g of stachyose, and 2g of sodium undecenyl monoethanolamide succinate sulfonate, stir it at 25°C for 2h, and adjust the pH to 7.05 with hydrochloric acid or sodium hydroxide after it is completely dissolved for use.

[0052] Preferably, the preparation method of the latex solution coated with mouse anti-COVID-19 N protein monoclonal antibody is:

[0053] ① Add 3 mL of 125 nm carboxyl latex particles and 1 mL of 266 nm carboxyl latex particles into a 250 mL conical flask;

[0054] ② Add 21 mL of 36 mmol / L ACES-CABS buffer (pH 7.05, 25°C), stir and mix, place in a 40°C shaking box, and shake for 0.5 h (speed 200 r / min);

[0055] ③ Take 10-20 mg of mouse anti-COVID-19 N protein monoclonal antibody and dilute it with 10 mL of antibody diluent (components: 0.1 mL / L (pH = 7.05) HEPES, 1 g / L BSA, 9 g / L NACL, 50 g / L trehalose, 0.01 g / L N-lauroyl-N′-hydroxyethylethylenediamine propionate, 1 mL / L PC-300), add it to the conical flask ②, adjust the shaking box temperature to 20 ° C, and shake for 12 hours (speed 100 r / min);

[0056] ④ Adjust the temperature of the oscillating box to 35°C, add 1-3.5 mg EDC to the conical flask ③, and oscillate for 22 minutes (speed 250 r / min);

[0057] ⑤ Adjust the temperature of the oscillation box to 25°C, add 5mL of microsphere sealing agent (components: 1mL / L ethylenediamine, 5g / L BSA, 0.05ml / L TWEEN20, 20g / L mannitol, 0.02g / L sodium di-sec-octyl maleate sulfonate) to ④, and oscillate for 4h (speed 200r / min). The final solution is the latex solution coated with mouse anti-COVID-19 N protein monoclonal antibody.

[0058] In some preferred embodiments of the present invention, the method for preparing the latex solution coated with mouse anti-COVID-19 N protein or S protein monoclonal antibody is:

[0059] ① Add 5 mL of 112 nm carboxyl latex particles and 1.5 mL of 305 nm carboxyl latex particles into a 250 mL conical flask;

[0060] ② Add 25.5 mL of 36 mmol / L ACES-CABS buffer (pH 7.05, 25°C), stir and mix, place in a 40°C shaking box, and shake for 1 h (speed 200 r / min);

[0061] ③ Take 7-22 mg of mouse anti-COVID-19 N protein monoclonal antibody or mouse anti-COVID-19 S protein monoclonal antibody, dilute it with 10 mL of antibody diluent (component composition: 0.1 mL / L (pH = 7.05) HEPES, 1 g / L BSA, 9 g / L NaCl, 50 g / L trehalose, 0.01 g / L N-lauroyl-N′-hydroxyethylethylenediamine propionate, 1 mL / L PC-300), add it to the conical flask ②, adjust the shaking box temperature to 20 ° C, and shake for 15 h (speed 100 r / min);

[0062] ④ Adjust the temperature of the oscillating box to 35℃, add 2-4.5mg EDC to the conical flask ③, and oscillate for 35min (speed 250r / min);

[0063] ⑤ Adjust the temperature of the oscillation box to 25°C, add 8mL of microsphere sealing agent (components: 1mL / L ethylenediamine, 5g / L BSA, 0.05ml / L TWEEN20, 20g / L mannitol, 0.02g / L sodium di-sec-octyl maleate sulfonate) to ④, and oscillate for 5h (speed 200r / min). The final solution is the latex solution coated with mouse anti-COVID-19 S protein monoclonal antibody or the latex solution coated with mouse anti-COVID-19 N protein monoclonal antibody.

[0064] The dosage of the components of the present invention is not limited to the specific values ​​described above, and the ratios between the corresponding components are also within the protection scope of the present invention.

[0065] The method of using the above-mentioned new coronavirus antigen detection kit is: use a fully automatic biochemical analyzer to perform the measurement using the endpoint method, preferably a Hitachi 7180 fully automatic analyzer, with a main detection wavelength of 570nm.

[0066] The basic principle of the detection of the present invention is: mouse anti-COVID-19 N protein monoclonal antibodies and mouse anti-COVID-19 S protein monoclonal antibodies are respectively adsorbed on latex particles of different particle sizes, and the N protein and S protein of the new coronavirus (SARS-CoV-2) in the sample to be tested are reacted with the latex particles coated with the corresponding mouse anti-COVID-19 N protein and mouse anti-COVID-19 S protein monoclonal antibodies through incubation to form an immune complex. The absorbance change generated is proportional to the new coronavirus (SARS-CoV-2) antigen content in the test sample.

[0067] The present invention adopts optimized latex enhanced immunoturbidimetry, and the preparation method of the present invention adopts a novel coupling buffer system and a closed system, optimizes the coupling process, does not require centrifugation and ultrasound, and greatly improves the reagent coupling efficiency and analytical sensitivity. In addition, the combination of carboxyl microspheres of different particle sizes and scientific proportions further enhance the reaction sensitivity and linear range of the reagent, and the reagent has stronger repeatability and anti-interference ability.

[0068] Example 1

[0069] A novel coronavirus antigen detection kit, comprising reagent R1 and reagent R2;

[0070] The reagent R1 includes the following components:

[0071] ACES-CABS buffer (pH 6.85, 36mmol / L25℃)

[0072] Latex particle sensitizer 10mL / L

[0073] Iodopropynyl butylcarbamate 0.35 g / L;

[0074] The reagent R2 includes the following components:

[0075]

[0076] The preparation method of the above two buffer solutions is as follows: weigh 6.56 g of ACES (N-(2-acetamido)-2-aminoethanesulfonic acid), completely dissolve it with 920 mL of deionized water, then add 8.47 g of CABS (4-(cyclohexylamino)-1-butanesulfonic acid), stir to dissolve, adjust the pH to 6.85 or 7.05 with an appropriate amount of ACES or sodium hydroxide, and finally make up to 1 L with deionized water.

[0077] The preparation method of the latex particle sensitizer is as follows: weigh 5g of N, N-dihydroxyethyl-N-octadecylbenzenesulfonamide propyl ammonium propanesulfonate and add it to 250mL of deionized water, stir and dissolve it at 37°C for 30min, then add 25g of potassium chloride and 20g of polyethylene glycol 10000, after complete dissolution, cool to room temperature, adjust the pH to 6.85 (25°C) with hydrochloric acid or sodium hydroxide, and finally add 5mL of fetal bovine serum and 10g of xylitol, and dilute to 300mL with deionized water after complete dissolution.

[0078] The preparation method of the latex particle stabilizer is as follows: take 5g of polyvinyl pyrrolidone K90 and add it to 150mL of water, stir and dissolve it at 37°C for 1h, cool it to room temperature, add 20g of maltitol, 12g of stachyose, and 2g of sodium undecenyl monoethanolamide succinate sulfonate, stir it at 25°C for 2h, and adjust the pH to 7.05 with hydrochloric acid or sodium hydroxide after it is completely dissolved for use.

[0079] The preparation method of the latex solution coated with the mouse anti-COVID-19 N protein monoclonal antibody in this embodiment is as follows:

[0080] ① Add 5 mL of 112 nm carboxyl latex particles and 1.5 mL of 305 nm carboxyl latex particles into a 250 mL conical flask;

[0081] ② Add 25.5 mL of 36 mmol / L ACES-CABS buffer (pH 7.05, 25°C) to the conical flask ①, stir and mix, place in a 40°C shaking box, and shake for 1 h (speed 200 r / min);

[0082] ③ Take 15 mg of mouse anti-COVID-19 N protein monoclonal antibody and dilute it with 10 mL of antibody diluent (components: 0.1 mL / L (pH = 7.05) HEPES, 1 g / L BSA, 9 g / L NaCl, 50 g / L trehalose, 0.01 g / L N-lauroyl-N′-hydroxyethylethylenediamine propionate, 1 mL / L PC-300), add it to the conical flask in ②, adjust the shaking box temperature to 20 ° C, and shake for 15 hours (speed 100 r / min);

[0083] ④ Adjust the temperature of the oscillation box to 35°C, add 4.5 mg of EDC to the conical flask after oscillation in ③, and oscillate for 35 minutes (speed 250 r / min);

[0084] ⑤ Adjust the temperature of the oscillation box to 25°C, add 8mL of microsphere sealing agent (components: 1mL / L ethylenediamine, 5g / L BSA, 0.05ml / L TWEEN20, 20g / L mannitol, 0.02g / L sodium di-sec-octyl maleate sulfonate) to ④, and oscillate for 5h (speed 200r / min). The final solution is the latex solution coated with mouse anti-COVID-19 N protein monoclonal antibody.

[0085] The method for preparing the latex solution coated with the mouse anti-COVID-19 S protein monoclonal antibody in this embodiment is as follows:

[0086] ① Add 5 mL of 112 nm carboxyl latex particles and 1.5 mL of 305 nm carboxyl latex particles into a 250 mL conical flask;

[0087] ② Add 25.5 mL of 36 mmol / L ACES-CABS buffer (pH 7.05, 25°C) to the above solution, stir to mix, place in a 40°C shaking box, and shake for 1 h (speed 200 r / min);

[0088] ③ Take 22 mg of mouse anti-COVID-19 S protein monoclonal antibody and dilute it with 10 mL of antibody diluent (ingredients: 0.1 mL / L (pH = 7.05) HEPES, 1 g / L BSA, 9 g / L NaCl, 50 g / L trehalose, 0.01 g / L N-lauroyl-N′-hydroxyethylethylenediamine propionate, 1 mL / L PC-300), add it to the conical flask in ②, adjust the shaking box temperature to 20 ° C, and shake for 15 hours (speed 100 r / min);

[0089] ④ Adjust the temperature of the oscillation box to 35℃, add 4.5mg EDC to the conical flask ③, and oscillate for 35min (speed 250r / min);

[0090] ⑤ Adjust the temperature of the oscillation box to 25°C, add 8mL of microsphere sealing agent (ingredients: 1mL / L ethylenediamine, 5g / L BSA, 0.05ml / L TWEEN20, 20g / L mannitol, 0.02g / L sodium di-sec-octyl maleate sulfonate) to ④, and oscillate for 5h (speed 200r / min). The final solution is the latex solution coated with mouse anti-COVID-19 S protein monoclonal antibody.

[0091] ACES-CABS buffer (pH 6.85, 25°C), latex particle sensitizer, and iodopropynyl butylcarbamate are mixed according to the above-mentioned concentration ratio to obtain reagent R1, and ACES-CABS buffer (pH 7.05, 25°C), latex particle stabilizer, latex solution coated with mouse anti-new coronavirus N protein monoclonal antibody, latex solution coated with mouse anti-new coronavirus S protein monoclonal antibody, and iodopropynyl butylcarbamate are mixed according to the above-mentioned concentration ratio to obtain reagent R2.

[0092] Method of using the novel coronavirus antigen detection kit in this embodiment:

[0093] The novel coronavirus antigen detection kit of this embodiment is used with a Hitachi 7180 fully automatic analyzer with dual reagent function, and the endpoint method is used for determination. Reagent R1 and reagent R2 are placed in the corresponding reagent positions in a ratio of 3:1, and distilled water, standard products and samples are placed in the corresponding positions of the sample tray. The operation is as shown in Table 1.

[0094] Table 1 Reagent detection method table

[0095]

[0096] Calculation: 25-hydroxyvitamin D content (ng / mL) = (ΔA assay ÷ ΔA standard) × C standard.

[0097] Example 2

[0098] A novel coronavirus antigen detection kit, comprising reagent R1 and reagent R2;

[0099] The reagent R1 includes the following components:

[0100] ACES-CABS buffer (pH 6.85, 25°C) 36mmol / L

[0101] Latex particle sensitizer 15mL / L

[0102] Iodopropynyl butylcarbamate 0.35 g / L;

[0103] The reagent R2 includes the following components:

[0104]

[0105] The preparation method of the latex solution coated with the mouse anti-COVID-19 N protein monoclonal antibody in this embodiment is as follows:

[0106] ① Add 3 mL of 125 nm carboxyl latex particles and 1 mL of 266 nm carboxyl latex particles into a 250 mL conical flask;

[0107] ② Add 21 mL of 36 mmol / L ACES-CABS buffer (pH 7.05, 25°C) to the conical flask ①, stir and mix, place in a 40°C shaking box, and shake for 0.5 h (speed 200 r / min);

[0108] ③ Take 10 mg of mouse anti-COVID-19 N protein monoclonal antibody and dilute it with 10 mL of antibody diluent (components: 0.1 mL / L (pH = 7.05) HEPES, 1 g / L BSA, 9 g / L NaCl, 50 g / L trehalose, 0.01 g / L N-lauroyl-N′-hydroxyethylethylenediamine propionate, 1 mL / L PC-300), add it to the conical flask in ②, adjust the shaking box temperature to 20 ° C, and shake for 12 hours (speed 100 r / min);

[0109] ④ Adjust the temperature of the oscillation box to 35°C, add 3.5 mg of EDC to the conical flask after oscillation in ③, and oscillate for 22 minutes (speed 250 r / min);

[0110] ⑤ Adjust the temperature of the oscillation box to 25°C, add 5mL of microsphere sealing agent (components: 1mL / L ethylenediamine, 5g / L BSA, 0.05ml / L TWEEN20, 20g / L mannitol, 0.02g / L sodium di-sec-octyl maleate sulfonate) to ④, and oscillate for 4h (speed 200r / min). The final solution is the latex solution coated with mouse anti-COVID-19 N protein monoclonal antibody.

[0111] The method for preparing the latex solution coated with the mouse anti-COVID-19 S protein monoclonal antibody in this embodiment is as follows:

[0112] ① Add 5 mL of 112 nm carboxyl latex particles and 1.5 mL of 305 nm carboxyl latex particles into a 250 mL conical flask;

[0113] ② Add 25.5 mL of 36 mmol / L ACES-CABS buffer (pH 7.05, 25°C) to the above solution, stir to mix, place in a 40°C shaking box, and shake for 1 h (speed 200 r / min);

[0114] ③ Take 22 mg of mouse anti-COVID-19 S protein monoclonal antibody and dilute it with 10 mL of antibody diluent (ingredients: 0.1 mL / L (pH = 7.05) HEPES, 1 g / L BSA, 9 g / L NaCl, 50 g / L trehalose, 0.01 g / L N-lauroyl-N′-hydroxyethylethylenediamine propionate, 1 mL / L PC-300), add it to the conical flask in ②, adjust the shaking box temperature to 20 ° C, and shake for 15 hours (speed 100 r / min);

[0115] ④ Adjust the temperature of the oscillation box to 35℃, add 4.5mg EDC to the conical flask ③, and oscillate for 35min (speed 250r / min);

[0116] ⑤ Adjust the temperature of the oscillation box to 25°C, add 8mL of microsphere sealing agent (ingredients: 1mL / L ethylenediamine, 5g / L BSA, 0.05ml / L TWEEN20, 20g / L mannitol, 0.02g / L sodium di-sec-octyl maleate sulfonate) to ④, and oscillate for 5h (speed 200r / min). The final solution is the latex solution coated with mouse anti-COVID-19 S protein monoclonal antibody.

[0117] The preparation and detection methods of the remaining components are the same as those in Example 1.

[0118] Example 3

[0119] A novel coronavirus antigen detection kit, comprising reagent R1 and reagent R2;

[0120] The reagent R1 includes the following components:

[0121] ACES-CABS buffer (pH 6.85, 25°C) 36mmol / L

[0122] Latex particle sensitizer 20mL / L

[0123] Iodopropynyl butylcarbamate 0.35 g / L;

[0124] The reagent R2 includes the following components:

[0125]

[0126] The preparation and detection methods of each component are the same as those in Example 1.

[0127] Comparative Example 1

[0128] This comparative example provides a novel coronavirus antigen detection kit, including reagent R1 and reagent R2;

[0129] The reagent R1 includes the following components:

[0130] ACES-CABS buffer (pH 6.85, 25°C) 36mmol / L

[0131] Latex particle sensitizer 5mL / L

[0132] Iodopropynyl butylcarbamate 0.35 g / L;

[0133] The reagent R2 includes the following components:

[0134]

[0135] The preparation and detection methods of each component are the same as those in Example 1.

[0136] Comparative Example 2

[0137] This comparative example provides a novel coronavirus antigen detection kit, including reagent R1 and reagent R2;

[0138] The reagent R1 includes the following components:

[0139] ACES-CABS buffer (pH 6.85, 25°C) 36mmol / L

[0140] Latex particle sensitizer 25mL / L

[0141] Iodopropynyl butylcarbamate 0.35 g / L;

[0142] The reagent R2 includes the following components:

[0143]

[0144] The preparation and detection methods of each component are the same as those in Example 1.

[0145] Accuracy comparison test

[0146] Take one portion each of a high-value quality control material (target value 66.25 ng / mL) and a low-value quality control material (target value 10.32 ng / mL) with traceability, and use the reagents of Examples 1 to 3 and Comparative Examples 1 to 2, respectively, to prepare a new coronavirus antigen detection kit for control detection, each test was performed 5 times, and the average value was calculated and compared with the target value of the quality control material. The results are shown in Table 1.

[0147] Table 1 High value quality control material accuracy test data table

[0148] Example 1 Example 2 Example 3 Comparative Example 1 Comparative Example 2 Maximum 67.18 67.04 67.85 72.25 74.78 Minimum 63.83 64.1 65.58 60.25 59.15 average value 66.06 65.95 66.35 61.89 60.15

[0149] Table 2 Low value quality control material accuracy test data table

[0150]

[0151]

[0152] It can be seen from the test results in Table 2 that, compared with Comparative Examples 1 and 2, the test values ​​of the reagents prepared in Example 1, Example 2 and Example 3 are closer to the target value, and the difference between the average value and the target value is within 0.5, which has a higher accuracy. This shows that the components of the present invention synergize to effectively improve the analytical sensitivity; and the addition of latex particle sensitizers optimizes the reaction system through scientific proportions, significantly enhances the anti-interference ability of the reagents, and greatly improves the accuracy of the reagents.

[0153] Sensitivity comparison test

[0154] The traceable calibrator was diluted from low to high to obtain 6 concentration samples, and the novel coronavirus antigen detection kits prepared by Examples 1 to 3 and Comparative Examples 1 to 2 were used for control tests, and the test results were compared with the theoretical concentrations. The results are shown in Table 3.

[0155] Table 3 Sensitivity comparison test data table (ng / mL)

[0156] Theoretical concentration Example 1 Example 2 Example 3 Comparative Example 1 Comparative Example 2 5 5.02 4.98 5.08 3.78 2.82 10 10.34 10.58 10.25 9.35 8.94 50 52.13 51.18 51.1 51.05 50.08 100 100.6 100.63 100.47 100.43 100.48 150 152.74 150.75 151.77 148.25 147.25 200 200.63 202.12 201.78 176.45 168.25

[0157] It can be seen from the test results in Table 3 that when the sample concentration is as low as 5ng / mL, the detection values ​​of Comparative Examples 1 to 2 are 2.82 to 3.78ng / mL, while the reagents prepared in Examples 1, 2 and 3 can still detect the accurate value of the sample; and compared with Comparative Examples 1 and 2, the reagents prepared in Examples 1, 2 and 3 have higher accuracy when detecting low-value samples (5-10ng / mL) close to the linear lower limit. In addition, when the sample concentration is at the linear upper limit of 200ng / mL, the detection value of the comparative example is between 168.25 and 176.45ng / mL, and the test value is obviously low, while the reagents prepared in Examples 1, 2 and 3 can still detect the accurate value of the sample, which shows that the reagents prepared in Examples 1, 2 and 3 have higher analytical sensitivity, accuracy and a wider linear range. Therefore, it can be explained that the present invention effectively improves the analytical sensitivity and linear range of the reagent through the synergistic enhancement of each component; and the addition of latex particle sensitizer and latex particle stabilizer optimizes the reaction system through scientific proportioning, significantly enhances the anti-interference ability of the reagent, and greatly improves the accuracy and analytical sensitivity of the reagent. In summary, through the above measures, the reaction system is optimized, and the analytical sensitivity, accuracy and linear range of the reagent are greatly improved.

[0158] Stability comparison test

[0159] The reagents in Examples 1 to 3 and Comparative Examples 1 to 2 were evenly divided into 19 groups, each with a reagent volume of 18 mL for reagent R1 and 6 mL for reagent R2. They were placed in a refrigerator at 2 to 8°C. On the same day of each month, a group of reagents was taken out for quality control testing (target value was 66.25 ng / mL). The test results were as follows: Figure 1 As shown, the reagents of Examples 1 to 3 are more stable than those of Comparative Examples 1 and 2 under storage conditions of 2 to 8°C.

[0160] It can be seen from the test results that when stored for 19 months, the test values ​​of Comparative Examples 1 to 2 are 47.69 to 49.21, which differ from the target values ​​by 17.04 to 18.56, and the test values ​​show an obvious decreasing trend with the extension of storage time; while the test values ​​of the reagents prepared by the formulas of Examples 1, 2 and 3 are 66.34 to 68.13, which differ from the target values ​​by 0.09 to 1.88; this indicates that the reagents prepared by the formulas of Examples 1, 2 and 3 have higher stability and a shelf life of up to 1.5 years, which indicates that the present invention optimizes the preparation method of latex particles, adds latex particle sensitizers and latex particle stabilizers, and optimizes the reaction system through scientific proportioning, thereby greatly improving the stability of the reagents.

[0161] In summary, through verification, the new coronavirus antigen detection reagent provided by the present invention has high sensitivity, wide linear range, and high accuracy, and is a more stable and good new coronavirus antigen detection kit.

Claims

1. A novel coronavirus antigen detection kit, It is characterized in that comprising reagent R1 and reagent R2; The reagent R1 includes the following components: Buffer A 30~50mmol / L Latex particle sensitizer 10~20mL / L Preservatives 0.3-0.5 g / L; The reagent R2 includes the following components: The preservative is iodopropynyl butyl carbamate; The volume ratio of the reagent R1 to the reagent R2 is 3:1; In reagent R1, the preparation method of the latex particle sensitizer is as follows: N, N-dihydroxyethyl-N-octadecylbenzenesulfonamide propyl ammonium propanesulfonate is added to deionized water, stirred to dissolve, potassium chloride and polyethylene glycol 10000 are added, dissolved, cooled, pH is adjusted, and finally fetal bovine serum and xylitol are added, dissolved, and then fixed to volume with deionized water to obtain a latex particle sensitizer; In the reagent R2, the preparation method of the latex particle stabilizer is as follows: polyvinyl pyrrolidone K90 is added to water, stirred to dissolve, cooled, maltitol, stachyose, and sodium undecenyl monoethanolamide succinate sulfonate are added, stirred to dissolve, and the pH is adjusted to obtain the latex particle stabilizer; In reagent R2, the preparation steps of the latex solution coated with mouse anti-COVID-19 N protein monoclonal antibody or the latex solution coated with mouse anti-COVID-19 S protein monoclonal antibody include: (1) mixing a solution of 112-125 nm carboxyl latex particles with a solution of 266-305 nm carboxyl latex particles in a volume ratio of 3:1 to obtain a mixed solution A; (2) Add ACES-CABS buffer solution with a pH value of 7.05 to the mixed solution A, stir and mix, and oscillate for 0.5 to 1 hour to obtain a mixed solution B; (3) Dilute mouse anti-COVID-19 N protein monoclonal antibody or mouse anti-COVID-19 S protein monoclonal antibody in antibody diluent, shake for 12 to 15 hours, and add to mixed solution B to obtain mixed solution C; (4) Add EDC to the mixed solution C at 33-37°C and shake for 20-30 min to obtain a mixed solution D; (5) adding a microsphere blocking agent to the mixed solution D at 20 to 25° C. to obtain a latex solution coated with a mouse anti-COVID-19 N protein monoclonal antibody or a latex solution coated with a mouse anti-COVID-19 S protein monoclonal antibody; (3), the antibody diluent comprises the following components: 0.1 mL / L HEPES, 1 g / L BSA, 9 g / L NaCl, 50 g / L trehalose, 0.01 g / L N-lauroyl-N′-hydroxyethylethylenediamine propionate, and 1 mL / L PC-300; (5), the microsphere sealing agent comprises the following components: 1 mL / L ethylenediamine, 5 g / L BSA, 0.05 ml / L TWEEN20, 20 g / L mannitol, and 0.02 g / L sodium di-sec-octyl maleate sulfonate.

2. A novel coronavirus antigen detection kit as claimed in claim 1, It is characterized in that In reagent R1, the buffer A is an ACES-CABS buffer with a pH of 6.85 at 25°C; in reagent R2, the buffer B is an ACES-CABS buffer with a pH of 7.05 at 25°C.

3. A novel coronavirus antigen detection kit as claimed in claim 1, It is characterized in that (1), a solution of 112 nm carboxyl latex particles and a solution of 305 nm carboxyl latex particles were mixed at a volume ratio of 3:1 to obtain a mixed solution A.

4. A novel coronavirus antigen detection kit according to any one of claims 1 to 3, It is characterized in that The novel coronavirus antigen detection kit was detected by a fully automatic biochemical analyzer, the main detection wavelength was 570nm, and the detection method was the endpoint method.

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