A novel coronavirus antigen detection test paper and preparation method thereof

Abstract

The present invention discloses a novel coronavirus antigen detection test paper and a preparation method thereof. A quality control reinforcement binding pad is added, which greatly improves the sensitivity of the detection, and at the same time has high accuracy and good specificity.

Description

Technical Field

[0001] The present invention relates to the field of test strips, and in particular to a novel coronavirus antigen test strip and a preparation method thereof. Background Art

[0002] There is no correlation between the detection markers on the conjugate pad of traditional novel coronavirus antigen test strips and the C-line quality control markers, which cannot enhance the detection signal. The detection sensitivity is low, which can easily lead to false negative results or too low detection rates due to performance problems. Summary of the invention

[0003] The purpose of the present invention is to provide a novel coronavirus antigen detection test paper with high sensitivity, high accuracy and good specificity.

[0004] To achieve the above object, the present invention provides a method for preparing a novel coronavirus antigen detection test paper, which comprises the following steps:

[0005] S1. Add K2CO3 to the nanogold and mix well, then add mouse anti-COVID-19 N antigen IgG2 for reaction. After the reaction is completed, add BSA to continue the reaction, centrifuge to remove the supernatant, add the gold solution for ultrasonic dispersion and suspension;

[0006] S2. After adding the gold solution obtained above, the total solution 1 is added to the glass fiber and evenly spread, and then dried; preferably, the ratio of the total solution to the glass fiber is 0.5-2ml / 17cm 2， , as a conjugate pad 1;

[0007] S3. Add K2CO3 to the nanogold and mix well, then add rabbit anti-mouse IgG to react, add BSA to continue the reaction after the reaction is completed, centrifuge to remove the supernatant, add gold solution and ultrasonically disperse and suspend;

[0008] S4. After adding the gold solution obtained in S3, the total solution 2 is added to the glass fiber and evenly spread, and then dried; preferably, the ratio of the total solution to the glass fiber is 0.5-2ml / 17cm 2 , as a quality control reinforcement bonding pad 2;

[0009] S5: coating, assembly;

[0010] The coating process is to draw T-line antibody and C-line antibody onto the NC membrane and then dry it;

[0011] The assembly is to assemble the absorbent paper, NC membrane, gold label pad and sample pad in order, and then cut them into the required test paper size.

[0012] Furthermore, in step S1, the usage ratio of K2CO3, nanogold, and mouse anti-new coronavirus N antigen IgG2 is: 0.2*4.5μL: 1*4 / 10,000 mL: 15μg; K2CO3 is 0.2M, and the concentration of nanogold is 4 / 10,000.

[0013] Furthermore, in step S1, 4.5 μL of 0.2M K2CO3 was added to 1 mL of 4 / 10,000 concentration of nanogold, mixed and then 15 μg of mouse anti-COVID-19 N antigen IgG2 was added, reacted for about 25-40 minutes, 10 μL of 20% BSA was added for 25-40 minutes, the supernatant was removed by centrifugation, and 0.5 mL of gold solution was added for ultrasonic dispersion and suspension.

[0014] Further, in step S2, the ratio of the total solution to the glass fiber is 1 ml / 17 cm 2， The drying step is to dry the mixture in a drying oven at 37-45°C for more than 4 hours.

[0015] Furthermore, in step S3, preferably, the usage ratio of K2CO3 to nanogold and rabbit anti-mouse IgG is: 0.2*7μL: 1*4 / 10,000 mL: 20μg; K2CO3 is 0.2M and nanogold is 4 / 10,000 concentration.

[0016] Furthermore, in step S3, 7 μL of 0.2M K2CO3 was added to 1mL of 4 / 10,000 concentration of nanogold, mixed and then 20 μg of rabbit anti-mouse IgG was added, reacted for 25-40 min, 10 μL of 20% BSA was added for 25-40 min, the supernatant was removed by centrifugation, and 0.5mL of gold plating solution was added for ultrasonic dispersion and suspension.

[0017] Furthermore, in step S4, the ratio of total solution to glass fiber is 1 ml / 17 cm 2， The drying is carried out in a drying oven at 37-45°C for more than 4 hours.

[0018] The present invention also protects the novel coronavirus antigen detection test paper prepared by the preparation method.

[0019] The structure and principle diagram of the present invention are shown in Figure 1-3 Compared with the prior art, the present invention adds a reinforced binding pad 2 to make the detection more sensitive. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a schematic diagram of the structure of the test strips of the present invention and the prior art.

[0021] Figure 2 It is a schematic diagram of the structure of the test strips of the present invention and the prior art.

[0022] Figure 3 It is a schematic diagram of the color development principle after the reaction of the test paper of the present invention and the prior art. DETAILED DESCRIPTION

[0023] Embodiments of the present invention are described in detail below, and the examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals represent the same or similar elements or elements with the same or similar functions from beginning to end. The embodiments described below by reference to the accompanying drawings are exemplary, are intended to be used to explain the present invention, and are not to be construed as limitations of the present invention. Those who do not indicate specific techniques or conditions in the embodiments are carried out according to the techniques or conditions described in the documents in this area or according to the product specification. Those who do not indicate the manufacturer of reagents or instruments used are all conventional products that can be obtained commercially.

[0024] The solution formulations used in the following examples are:

[0025] Gold solution: 35 mM tris, 0.1% PEG, 0.5% PVP, 5% trehalose, 0.2% casein-Na, 0.5 / k proclin300.

[0026] / 10,000 means 10 -4 Grams / liter: For example, a concentration of 4 / 10,000 nanogold refers to 4×10 -4 g / L of nanogold; 5 / 10,000 proclin300 means 5×10 -4 g / l of proclin 300. And so on.

[0027] Embodiment 1:

[0028] Combination Figure 1-3 To prepare.

[0029] Conjugate pad preparation:

[0030] ① Add 4.5μL 0.2M K2CO3 to 1mL 4 / 10,000 concentration of nanogold, mix well, add 15μg mouse anti-COVID-19 N antigen IgG2, react for about 30 minutes, add 10μL 20% BSA for 30 minutes, centrifuge to remove the supernatant, add 0.5mL gold solution, ultrasonically disperse and suspend, and store for later use;

[0031] ② Add 7 μL 0.2M K2CO3 to 1mL 4 / 10,000 concentration of nanogold, mix well, add 20 μg rabbit IgG, react for about 30 minutes, add 10 μL 20% BSA for 30 minutes, centrifuge to remove the supernatant, add 0.5mL gold solution for ultrasonic dispersion and suspension, and store for later use;

[0032] ③ Add 7 μL 0.2M K2CO3 to 1mL 4 / 10,000 concentration of nanogold, mix well, add 20 μg rabbit anti-mouse IgG, react for about 30 minutes, add 10 μL 20% BSA for 30 minutes, centrifuge to remove the supernatant, add 0.5mL gold solution, ultrasonically disperse and suspend, and store for later use;

[0033] ④ Mix equal volumes of ① and ② and add to the 17cm 2 On the glass fiber, spread it evenly, put it in a 37-45℃ drying oven for more than 4 hours, seal it and store it for later use. It is a conventional test strip binding pad used for control experiment 1;

[0034] ⑤ Add the gold solution from step ① to 1 mL and add to the 17 cm 2 The glass fiber is evenly spread, placed in a 37-45°C drying oven for more than 4 hours, and sealed for storage for later use, which is the bonding pad 1 of the present invention;

[0035] ⑥Add ③ to 1mL of gold solution and add to 17cm 2 On the glass fiber, evenly spread it, put it into a 37-45°C drying box for more than 4 hours, and seal it for storage for later use, which is the quality control reinforcement bonding pad 2 of the present invention;

[0036] ⑦ Mix ① and ③ and add to 17cm 2 On the glass fiber, spread it evenly, put it into a 37-45℃ drying oven for more than 4 hours, seal it and store it for use as a composite bonding pad for control experiment 2.

[0037] 2) Preparation of coated NC membrane:

[0038] Coating: Dilute the T-line antibody and C-line antibody to 0.8 mg / mL (coating solution configuration: 30 mM TRIS-HCl, 2% sucrose, 5 / 10,000 proclin300), use a film scribing instrument, according to the parameter requirement of 1.0 μL / cm, scribble onto the NC membrane, put it in a 37-45℃ drying oven to dry for more than 4 hours, and seal it for storage.

[0039] Assembly: Assemble the base plate, absorbent paper, NC membrane, conjugate pad, sample pad and other components according to Figure 1 Assemble it and then cut it into test papers of about 4mm for use. Figure 1 and Figure 2 The structure A is a schematic diagram of the structure of the detection paper of the prior art, and the structure B is a schematic diagram of the structure of the detection paper of the present invention. Figure 3 A is a schematic diagram of the color development principle after the test paper of the prior art reacts, and B is a schematic diagram of the color development principle after the test paper of the present invention reacts.

[0040] Example 2: Effect verification experiment

[0041] Samples: Samples were collected by swab. The three groups of experiments used the same tube of samples with the same sample volume.

[0042] Detection method: The sample was dripped onto the sample pad and tested using three test strips: the test strip of control experiment 1, the test strip of control experiment 2, and the test strip of the present invention. The results are shown in Table 1.

[0043] Table 1 Experimental results

[0044]

[0045] Note: N1-N10 are the numbers of 10 negative samples. Buffer is used instead of sample in negative samples.

[0046] It can be seen from the results in Table 1 that the product sensitivity of the test strips of the present invention is higher than that of the test strips of Control 1 and Control 2, and the specificity has no significant change.

[0047] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and intent of the present invention.

Claims

1. A method for preparing a novel coronavirus antigen detection test paper, characterized in that: The following steps are included: S1. Add K2CO3 to the nanogold and mix well, then add mouse anti-COVID-19 N antigen IgG2 to react. After the reaction is completed, add BSA to continue the reaction, centrifuge to remove the supernatant, add gold-plating solution and ultrasonically disperse and suspend; the gold-plating solution is 35mM tris, 0.1% PEG, 0.5% PVP, 5% trehalose, 0.2% casein-Na, 5×10 -4 g / L of proclin 300; S2. Add the gold solution to the solution obtained in S1 and add the total solution 1 obtained after adding it to the glass fiber and spread it evenly, and then dry it; as a bonding pad 1; S3. Add K2CO3 to the nanogold and mix well, then add rabbit anti-mouse IgG to react, add BSA to continue the reaction after the reaction is completed, centrifuge to remove the supernatant, add gold solution and ultrasonically disperse and suspend; S4. Add the gold solution obtained in S3 to the total solution 2, spread it evenly on the glass fiber, and then dry it; the ratio of total solution to glass fiber is 0.5-2ml / 17cm 2 , as a quality control reinforcement bonding pad 2; S5: coating, assembly; The coating is to draw the T-line antibody and the C-line antibody onto the NC membrane and then dry them; the T-line antibody is rabbit anti-COVID-19 N antigen igG1, and the C-line antibody is goat anti-rabbit antibody; The assembly is to assemble the absorbent paper, NC membrane, gold label pad and sample pad in order, and then cut them into the required test paper size.

2. The method for preparing a novel coronavirus antigen test paper according to claim 1, wherein: In step S1, the dosage ratio of K2CO3, nanogold, and mouse anti-COVID-19 N antigen IgG2 is: 0.9 μL: 0.4 μg: 15 μg; K2CO3 is 0.2M.

3. The method for preparing a novel coronavirus antigen test paper according to claim 2, wherein: In step S1, 4.5 μL of 0.2M K2CO3 was added to 1 mL of 4×10 -4 After mixing with 500 grams / liter of nanogold, add 15 μg of mouse anti-COVID-19 N antigen IgG2, react for 25-40 minutes, add 10 μL of 20% BSA for 25-40 minutes, centrifuge to remove the supernatant, and add 0.5 mL of gold solution for ultrasonic dispersion and suspension.

4. The method for preparing a novel coronavirus antigen test paper according to claim 1, wherein: In step S2, the ratio of the total solution to the glass fiber is 1 ml / 17 cm 2 The drying step is to dry the mixture in a drying oven at 37-45°C for more than 4 hours.

5. The method for preparing a novel coronavirus antigen test paper according to claim 1, wherein: In step S2, the ratio of total solution to glass fiber is 0.5-2ml / 17cm 2 .

6. The method for preparing a novel coronavirus antigen test paper according to claim 1, wherein: In step S3, the usage ratio of K2CO3 to nanogold and rabbit anti-mouse IgG is: 1.4 μL: 0.4 μg: 20 μg; K2CO3 is 0.2M.

7. The method for preparing a novel coronavirus antigen test strip according to claim 6, wherein: In step S3, 7 μL of 0.2 M K2CO3 was added to 1 mL of 4 × 10 -4 After mixing with 500 g / L of nanogold, add 20 μg of rabbit anti-mouse IgG and react for 25-40 min. Then add 10 μL of 20% BSA for 25-40 min, centrifuge to remove the supernatant, and add 0.5 mL of gold solution for ultrasonic dispersion and suspension.

8. The method for preparing a novel coronavirus antigen test paper according to claim 1, wherein: In step S4, the ratio of total solution to glass fiber is 1ml / 17cm 2 The drying step is to dry the mixture in a drying oven at 37-45°C for more than 4 hours.

9. A novel coronavirus antigen detection test paper prepared by the preparation method according to any one of claims 1 to 8.

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