CN114280296A - Novel coronavirus (2019-nCOV) IgG/IgM antibody detection kit and preparation method thereof - Google Patents

Abstract

The invention discloses a novel coronavirus (2019-nCOV) IgG/IgM antibody detection kit. The test strip includes: a sample pad, a binding pad 1, a binding pad 2, a nitrocellulose membrane, a water-absorbing pad and a bottom liner The labeling process of the N protein-tracer particle complex includes: mixing the N protein with sodium caseinate and then adding it into the colloidal gold solution; the nitrocellulose membrane is provided with a detection line T2, a detection line T1 and Quality control line C, the detection line T2 is coated with mouse anti-human IgM monoclonal antibody, the detection line T1 is coated with mouse anti-human IgG monoclonal antibody, and the quality control line is coated with goat anti-chicken IgY antibody . In the process of labeling colloidal gold and N protein, the invention solves the problem that the N protein and colloidal gold solution are prone to aggregation and dead gold phenomenon by mixing N protein and sodium caseinate in advance and then adding them into the colloidal gold solution. At the same time, the detection performance of the test strip was significantly improved.

Description

A novel coronavirus (2019-nCOV) IgG/IgM antibody detection kit and preparation method thereof

technical field

The invention relates to the field of medical testing, in particular to a novel coronavirus (2019-nCOV) IgG/IgM antibody detection kit and a preparation method thereof.

Background technique

Colloidal gold immunochromatography (Gold immunichromatogra-phy assay, GICA) is a solid-phase labeled immunoassay technology that organically combines colloidal gold labeling technology, immunodetection technology, and chromatography and chromatographic analysis technology. . Its principle is: the strip-shaped fiber chromatography material is used as the solid phase, and the analyte in the sample and the receptor (antigen or antibody) on the chromatographic material for the analyte have high specificity and high affinity through capillary action. In the process of chromatography, the immune complexes are enriched or trapped in a certain area (detection zone) of the chromatography material, and the experimental phenomenon (color development) of a straight tube is obtained by using a visually detectable marker (colloidal gold). ). The potent label crosses the detection zone and is automatically separated from the bound label. This analysis technique has the advantages of simple and rapid operation, single-unit measurement, and no need for special instruments.

Colloidal gold can be combined with various biological macromolecules such as proteins. The colloidal gold particles have a layer of negative surface charges, and the positive charges on the surface of proteins are adsorbed by electrostatic induction. Therefore, the environmental pH and ionic strength are the main factors affecting adsorption. Others such as colloids The size of gold particles, protein molecular weight and protein concentration also affect protein adsorption. Although most proteins can be easily combined and adsorbed with colloidal gold, there are still some proteins that appear dead gold after adding colloidal gold solution (such as the new coronavirus N protein). Usually in this case, the pH of the colloidal gold will be adjusted first, and if it cannot be improved, the protein will be simply treated, such as dilution, dialysis, etc. However, for some proteins, especially expensive raw materials, the processing of the protein often leads to large losses, or the amount of protein is too small to be processed once. At this time, an effective solution is needed.

SUMMARY OF THE INVENTION

By adding an appropriate amount of sodium caseinate during the labeling process of colloidal gold and labeled protein, the invention can effectively solve abnormal phenomena such as aggregation and dead gold caused by the action of electric charge, and at the same time, adjust the pH value to ensure that the protein has higher labeling efficiency , which significantly improves the detection performance (including stability, sensitivity and specificity) of the test strip.

In order to achieve the above purpose, the present invention adopts the following technical means: a novel coronavirus (2019-nCOV) IgG/IgM antibody detection kit, characterized in that the kit comprises a test paper strip and a diluent, and the test paper The strip includes: a sample pad, a binding pad, a nitrocellulose membrane, a water-absorbing pad and a backing, and the sample pad, the binding pad 1, the binding pad 2, the nitrocellulose membrane and the water-absorbing pad are sequentially lapped and pasted on the backing ; The labeling pad 1 is coated with RBD protein-tracer particle complexes, and the labeling pad 2 is coated with N protein-tracer particle complexes; the nitrocellulose membrane is provided with detection lines T2, Detection line T1 and quality control line C.

Preferably, the tracer particles are colloidal gold.

Preferably, the labeling process of the N protein-colloidal gold includes: mixing the N protein and the casein sodium, and then adding it to the colloidal gold solution, and the mass ratio of the N protein to the casein sodium is 1.25-5.

Preferably, the pH of the colloidal gold solution is 7.0-9.0, preferably 8.0.

Preferably, the detection line T2 is coated with mouse anti-human IgM monoclonal antibody, the detection line T1 is coated with mouse anti-human IgG monoclonal antibody, and the quality control line is coated with goat anti-chicken IgY antibody.

Preferably, the diluent comprises 0.2-2.0g/L buffer, 0.2-10g/L salt ion, 1-2g/L preservative; the buffer is selected from disodium hydrogen phosphate dodecahydrate, phosphoric acid At least one of potassium dihydrogen, MOPS, Tris buffer, preferably a combination of disodium hydrogen phosphate dodecahydrate and potassium dihydrogen phosphate; the salt ion is selected from at least one of sodium chloride or potassium chloride , preferably a combination of sodium chloride and potassium chloride; the preservative is selected from at least one of sodium azide or Proclin-300, preferably Proclin-300.

A kind of preparation method of novel coronavirus (2019-nCOV) IgG/IgM antibody detection kit, is characterized in that, comprises the following steps:

(1) Handling of the bonding pad 1

Add RBD protein to colloidal gold solution 1, after labeling for 10-30 minutes, add 2-10% blocking agent, stir for 10-30 minutes, centrifuge at 10,000 rpm for 20 minutes, discard the supernatant, and restore the volume with the colloidal gold labeling solution. The RBD protein gold-labeled complex was obtained and stored at 4°C;

The above-mentioned RBD protein gold-labeled complex is coated on the binding pad 1 at the concentration of OD20-OD50, and then freeze-dried to obtain the binding pad 1;

(2) Handling of the bonding pad 2

After mixing the N protein with sodium caseinate, add it to colloidal gold solution 2, mark for 10-30min, add 2-10% blocking agent, stir for 10-30min, centrifuge at 10000rpm for 20 minutes, discard the supernatant, and precipitate with The colloidal gold-labeled complex solution was restored to volume to obtain the N-protein gold-labeled complex, which was stored at 4°C;

The above-mentioned N protein gold-labeled complex is coated on the binding pad 2 at the concentration of OD10-OD40, and then freeze-dried to obtain the binding pad 2;

(3) Treatment of nitrocellulose membrane:

The test line T2, test line T1 and quality control line C are coated on the nitrocellulose membrane and then dried:

a. Preparation of detection line T2

The mouse anti-human IgM monoclonal antibody was sprayed on the nitrocellulose membrane at 1.0-3.0ul/cm near the end of the sample pad;

b. Preparation of T1 detection line:

The mouse anti-human IgG monoclonal antibody is coated on the side of the T2 detection line at 1.0-3.0ul/cm, and away from the sample pad;

Preparation of d.C quality control line:

The goat anti-chicken IgY antibody solution is coated on the side of the T1 detection line at 1.0-3.0 ul/cm and away from the sample pad;

(4) Assembly:

Paste the sample pad, nitrocellulose membrane and water-absorbing pad in sequence on the substrate, and cut into 3-5mm wide test strips.

Preferably, in step (1), the mass ratio of the N protein to sodium caseinate is 1.25-5; in steps (1) and (2), the pH of the colloidal gold solution 1 is 6.0- 8.0, preferably 7.0; the pH value of the colloidal gold solution 2 is 7.0-9.0, preferably 8.0.

Preferably, the components of the colloidal gold labeling complex solution include: 20-50mmol/buffer, 1-10g/protein, 10-20g/L surfactant, 20-60g/L protective agent, macromolecular substance 0.1- 0.5g/L, 1-2.0g/L preservative; the buffer is selected from at least one of glycine, Tris buffer, MOPS buffer; the protein is selected from at least one of casein or BSA, It is preferably a combination of casein and BSA; the surfactant is selected from at least one of Tween-80, Tween-20, Triton X-100 or A90; the macromolecular substance is selected from PVP, PEG or PVA At least one; the preservative is selected from at least one of sodium azide or Proclin-300.

A method for labeling colloidal gold and a substance to be labeled for immunochromatographic reagents, characterized in that the labeling method includes the step of mixing the substance to be labeled with sodium caseinate and then adding it into a colloidal gold solution.

The beneficial effects of the present invention are: the present invention effectively solves the difficulty in labeling the new coronavirus N protein and colloidal gold particles by mixing the new coronavirus N protein and sodium caseinate in advance and then adding it to the colloidal gold solution, and the labeling of During the process, abnormal phenomena such as aggregation and precipitation of gold are very likely to occur. At the same time, by adjusting the pH of the colloidal gold solution to a specific range and cooperating with the reagent preparation system of the present invention, the detection performance of the reagent can be effectively improved, and the clinical detection needs can be fully met. .

Description of drawings

Fig. 1 is the structural representation of the test strip of the present invention.

The reference numbers are:

1-PVC bottom plate; 1-sample pad; 3-binding pad 1; 4-binding pad 2; 5-nitrocellulose membrane; 6-absorbent paper; 7-detection line T2; 8-detection line T1; 9-quality control line C.

specific embodiment

The following examples are included herein to illustrate the technical solutions of the present invention more clearly and explicitly by way of illustration. Those of skill in the art should, in light of the disclosure herein, appreciate that many changes can be made in the specific embodiments disclosed and still obtain a like or similar result without departing from the spirit and scope of the present invention. The specific embodiments of the present invention are only used to explain the present invention, and are not intended to limit the present invention in any way.

Example 1 Preparation method of novel coronavirus (2019-nCOV) IgG/IgM antibody detection kit (colloidal gold method)

(1) Preparation of diluent

Configure according to the following formula, stir and mix well, and store at 2-8°C.

(2) Preparation of colloidal gold labeling complex solution

Configure according to the following formula, stir and mix well, and store at 2-8°C.

(3) Preparation of colloidal gold-antibody complexes

Take 1ml of colloidal gold, adjust the pH of colloidal gold to 7.4 with 0.2mol/L K ₂ CO ₃ solution, add 20ug of RBD antigen for labeling to the pH-adjusted colloidal gold, label for 20 minutes, add 30ul of 5% casein Sodium, blocked and stirred for 20 minutes, then centrifuged at 10,000 rpm for 20 minutes, discarded the supernatant, and recovered the volume with the colloidal gold-labeled reconstitution solution to obtain the RBD protein gold-labeled complex, which was stored at 4°C.

Take 1ml of colloidal gold, adjust the pH of colloidal gold to 8.0 with 0.1mol/L NaOH solution, mix 20ug of N protein antigen for labeling and 1ul of 5% sodium caseinate and add it to the colloidal gold solution after adjusting the pH. For 20 minutes, add 30ul of 5% sodium caseinate, block and stir for 20 minutes, then centrifuge at 10,000 rpm for 20 minutes, discard the supernatant, and restore the volume with the colloidal gold-labeled solution to obtain the N-protein gold-labeled complex, which is stored at 4°C.

(4) Preparation of bonding pads

When the purified RBD protein gold-labeled complex is diluted and the concentration is OD30, it is coated on a glass fiber pad and placed in a freeze dryer to obtain a binding pad 1;

The purified N protein gold-labeled complex was diluted and then coated on a glass fiber pad at an OD15 concentration and placed in a freeze dryer to prepare a binding pad 2.

(5) Assembly of immunochromatographic test strips

a. Treatment of nitrocellulose membrane

1.0mg/ml mouse anti-human IgG monoclonal antibody was sprayed on the nitrocellulose membrane at a speed of 1.0ul/cm as the first detection line T1, 1.0mg/ml mouse anti-human IgM monoclonal antibody was sprayed at 1.0 The speed of 1.0ul/cm was sprayed on the nitrocellulose membrane as the second detection line T2, and the 0.5mg/ml goat anti-chicken IgY antibody solution was sprayed on the nitrocellulose membrane at the speed of 1.0ul/cm as the quality control. Line C, placed in a drying oven at 45°C for 24h. The second detection line T2 is located on the nitrocellulose membrane near the end of the gold label pad, the quality control line C is located near the end of the water-absorbing pad, and the first detection line T1 is located between the second detection line and the quality control line.

b. Test strip assembly

Paste the sample pad, binding pad 1, binding pad 2, antibody-coated nitrocellulose membrane and water-absorbing pad on the PVC bottom plate in sequence (as shown in Figure 1), and cut the pasted intermediate with Cut the test strips into 3.7mm wide strips by machine and assemble them into the test strip cassettes to get the finished test strips.

The using method of embodiment 3 test strips

1. Mix the sample to be tested with the diluent and use it for later use;

2. Add 2 drops (more than 80ul) of the diluted sample to the sample hole of the test card

3. Observe the result after 5-15min.

Embodiment 4 Test strip performance verification

In order to verify the various properties of the test strip of the present invention, two groups of test kits are set up for performance verification:

Group A: the test strip described in Example 1 of the present invention;

Group B: Commercially available kit I

Group C: Commercially available kit II

The test strips of group A were tested according to the method of use described in Example 2, and the test strips of groups B and C were tested according to their instructions.

(1) The occurrence of the phenomenon of accumulation of dead gold

The test results showed that in the process of colloidal gold and protein labeling of the test strips of group A and group C, there was no colloidal gold aggregation phenomenon on the test strips in group A, while obvious colloidal gold aggregation appeared on the test strips in group C. phenomenon (dark particulate matter is visible to the naked eye in the marking solution).

(2) Clinical evaluation

64 clinical positive samples and 93 clinical negative samples were selected, and the above three groups of test strips were used to test the positive samples and negative samples respectively. The test results are shown in the following table:

Table 1 Clinical performance of test strips

*Note: Clinical sensitivity = (number of positive detections/number of positive samples)%; clinical specificity = (number of negative detections/number of negative samples)%; total coincidence rate = (number of correct detections/number of total samples)%.

(3) Repeatability

The above three groups of test strips were used to test one new coronavirus antibody reference material respectively, and each group of test strips was tested 10 times, of which 10 test results of group A test strips were all positive; group B test strips 10 times The test results were positive for 9 times and negative for one time; for group C, the test strips for 10 test results were positive for 7 times and negative for 3 times.

(4) Stability

When the above three groups of test strips were placed at 37°C for 0, 5, 10, 15, 20, 25 and 30 days, the same sample was tested, and the test results of group A test strips were the same within 30 days of thermal acceleration. The test results of group B test strips were deviated after 25 days of thermal acceleration, and the test results of group C test strips were deviated after 20 days of thermal acceleration.

From the above experimental results, the clinical sensitivity of the four groups of test strips were 98.44%, 85.94% and 73.44% respectively; the clinical specificity was 100%, 99% and 97% respectively; the overall coincidence rates were 99.36%, 93.63% and 87.26%; the experimental results show that the detection performance (including clinical compliance, repeatability and stability) of the test strip (group A) prepared in Example 1 of the present invention is significantly better than that of the test strips of groups B and C.

Example 5 Influence of adding sodium caseinate on the performance of test strips during labeling

In order to verify the effect of adding casein sodium on the performance of post-test strips during the process of colloidal gold and protein labeling, the following 4 groups of test strips were set up:

Group A: the test strip described in Example 1 of the present invention;

Group B: The difference between the test strip preparation method and Example 1 is that no sodium caseinate is added during the labeling process of colloidal gold and N protein;

Group C: The difference between the preparation method of the test strip and Example 1 is that sodium caseinate was not added during the labeling process of colloidal gold and N protein, but 1ul of 5% BSA was added;

Group D: The difference between the test strip preparation method and Example 1 is that sodium caseinate was not added during the labeling process of colloidal gold and N protein, but 1 ul of 5% PEG20000 was added.

(1) Accumulation of dead gold during the marking process

The test results showed that in the process of labeling the above four groups of test strips with colloidal gold and N protein, in addition to the test strips of group A and group C, the test strips of group B and group D all showed the phenomenon of colloidal gold aggregation (marking). Dark particulate matter is visible to the naked eye in the liquid).

(2) Clinical evaluation

64 clinical positive samples and 93 clinical negative samples were selected, and the above four groups of test strips were used to test the positive samples and negative samples respectively. The test results are shown in the following table:

Table 3 Clinical performance of test strips

*Note: Clinical sensitivity = (number of positive detections/number of positive samples)%; clinical specificity = (number of negative detections/number of negative samples)%; total coincidence rate = (number of correct detections/number of total samples)%.

From the above experimental results, the clinical sensitivity of the four groups of test strips were 100%, 45.31%, 59.38% and 76.56%, respectively; the clinical specificity was 100.00%, 100%, 97.00% and 98.00%, respectively; the total coincidence rates were 100%, 77.07%, 81.53% and 89.17%. The experimental results show that the clinical compliance of the test strips (group A) prepared in Example 1 of the present invention is significantly better than the test strips of groups B, C and D. According to the experimental results, it is speculated that sodium caseinate, as a protein, may have the effect of interfering with static electricity. At the same time, because sodium caseinate is in a dispersed state in the solution, it has a repulsive effect on the gold-labeled complex, which increases the degree of dispersion, thereby acting as a To disperse the skeleton of colloidal gold particles and maintain the colloidal stability of colloidal gold, so sodium caseinate can be used in the labeling process to deal with abnormal phenomena such as aggregation of dead gold caused by charge, especially for the labeling process. Adding an appropriate amount of sodium caseinate during the labeling process of the coagulated dead gold, such as the N protein of the new coronavirus, can not only effectively solve the technical problem of the coagulated dead gold, but also significantly improve the performance of the kit. The detection performance fully meets the clinical needs.

Example 5 Influence of the addition amount of sodium caseinate on the performance of the test strip during the labeling process

In order to verify the effect of the addition of casein sodium on the performance of subsequent test strips during the labeling process of colloidal gold and N protein, the following 4 groups of test strips were set up:

Group A: The only difference between the preparation method of the test strip and Example 1 is that 0.1 ul of 5% sodium caseinate was added during the labeling process of colloidal gold and N protein;

Group B: The difference between the preparation method of the test strip and Example 1 is that 0.5ul of 5% sodium caseinate is added during the labeling process of colloidal gold and N protein;

Group C: The difference between the preparation method of the test strip and Example 1 is that 2ul of 5% sodium caseinate was added during the labeling process of colloidal gold and N protein;

Group D: The difference between the preparation method of the test strip and Example 1 is that 3ul of 5% sodium caseinate is added during the labeling process of colloidal gold and N protein;

(1) Accumulation of dead gold during the marking process

The test results showed that during the labeling of colloidal gold and N protein, the above-mentioned four groups of test strips showed a small amount of colloidal gold coagulation on the test strips of group A (dark particulate matter was visible to the naked eye in the labeling solution), and group B , C group and D group of test strips did not appear colloidal gold aggregation phenomenon.

(2) Clinical evaluation

64 clinical positive samples and 93 clinical negative samples were selected, and the above four groups of test strips were used to test the positive samples and negative samples respectively. The test results are shown in the following table:

Table 3 Clinical performance of test strips

*Note: Clinical sensitivity = (number of positive detections/number of positive samples)%; clinical specificity = (number of negative detections/number of negative samples)%; total coincidence rate = (number of correct detections/number of total samples)%.

From the above experimental results, the clinical sensitivity of the four groups of test strips were 67%, 98%, 100%, and 83%, respectively; the clinical specificity was 97%, 100%, 100%, and 99%, respectively; the overall coincidence rates were 85%, 99%, 100% and 92%. The experimental results show that in the process of colloidal gold and the new coronavirus N protein labeling, when the mass ratio of casein sodium to the new coronavirus N protein is in the range of 1.25-5, the detection performance of the test strip is the best.

Example 6 Influence of pH value on test strip performance in marking process

In order to verify the effect of pH value on the performance of subsequent test strips during the labeling process of colloidal gold and N protein, the following five groups of test strips were set up:

Group A: The difference between the preparation method of the test strip and Example 1 is that the pH value of the colloidal gold solution in the marking process is 6.0;

Group B: The only difference between the preparation method of the test strip and Example 1 is that the pH value of the colloidal gold solution in the marking process is 7.0;

Group C: The difference between the preparation method of the test strip and Example 1 is that the pH value of the colloidal gold solution in the marking process is 9.0;

Group D: The difference between the preparation method of the test strip and Example 1 is that the pH value of the colloidal gold solution in the marking process is 10.0;

(1) Accumulation of dead gold during the marking process

The test results show that in the process of colloidal gold and protein labeling of the above four groups of test strips, there is no colloidal gold aggregation phenomenon in the four groups of test strips.

(2) Clinical evaluation

Select 53 positive samples that have been injected with the new crown vaccine and 119 negative samples that have not been vaccinated against the new crown vaccine, and use the above three sets of test strips to test the positive samples and negative samples respectively. The test results are shown in the following table:

Table 3 Clinical performance of test strips

*Note: Positive samples are vaccinated samples; negative samples are unvaccinated samples; Clinical Sensitivity = (Number of Positive Tests/Number of Positive Samples)%; Clinical Specificity = (Number of Negative Tests/Number of Negative Samples)%; Rate = (number of correct detections/number of total samples) %.

From the above experimental results, the clinical sensitivity of the four groups of test strips were 83%, 97%, 98%, and 77%, respectively; the clinical specificity was 98%, 100%, 100%, and 96%, respectively; the overall coincidence rates were 92%, 99%, 99% and 88%. The experimental results show that in the process of labeling N protein and colloidal gold solution, the pH value is in the range of 7.0-9.0, and the detection performance is the best.

Although the embodiments of the present invention have been shown and described above, it should be understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and those of ordinary skill in the art will not depart from the principles and spirit of the present invention Variations, modifications, substitutions, and alterations to the above-described embodiments are possible within the scope of the present invention without departing from the scope of the present invention.

Claims (10)

1. A novel coronavirus (2019-nCOV) IgG/IgM antibody detection kit is characterized by comprising a test strip and a diluent, wherein the test strip comprises: the test paper comprises a sample pad, a combination pad, a nitrocellulose membrane, a water absorption pad and a bottom lining, wherein the sample pad, the combination pad 1, the combination pad 2, the nitrocellulose membrane and the water absorption pad are sequentially overlapped and stuck on the bottom lining; the labeling pad 1 is coated with RBD protein-tracer particle complexes, and the labeling pad 2 is coated with N protein-tracer particle complexes; and the cellulose nitrate membrane is provided with a detection line T2, a detection line T1 and a quality control line C.

2. The novel coronavirus (2019-nCOV) IgG/IgM antibody detection kit of claim 1, wherein the tracer particle is colloidal gold.

3. The novel coronavirus (2019-nCOV) IgG/IgM antibody detection kit of claim 2, wherein the labeling process of N protein-colloidal gold comprises: and uniformly mixing the N protein and the sodium caseinate, and then adding the mixture into the colloidal gold solution, wherein the mass ratio of the N protein to the sodium caseinate is 1.25-5.

4. The novel coronavirus (2019-nCOV) IgG/IgM antibody detection kit according to claim 3, wherein the colloidal gold solution has a pH of 7.0-9.0, preferably 8.0.

5. The novel coronavirus (2019-nCOV) IgG/IgM antibody detection kit according to claim 4, wherein a mouse anti-human IgM monoclonal antibody is coated on the detection line T2, a mouse anti-human IgG monoclonal antibody is coated on the detection line T1, and a goat anti-chicken IgY antibody is coated on the quality control line.

6. The novel coronavirus (2019-nCOV) IgG/IgM antibody detection kit according to claim 5, wherein the diluent comprises 0.2-2.0g/L buffer solution, 0.2-10g/L salt ions, 1-2g/L preservative; the buffer solution is selected from at least one of disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, MOPS and Tris buffer solution, and is preferably the combination of the disodium hydrogen phosphate dodecahydrate and the potassium dihydrogen phosphate; the salt ions are selected from at least one of sodium chloride or potassium chloride, and preferably the combination of sodium chloride and potassium chloride; the preservative is selected from at least one of sodium azide and Proclin-300, and is preferably Proclin-300.

7. The method for preparing a novel coronavirus (2019-nCOV) IgG/IgM antibody detection kit according to any one of claims 1 to 5, comprising the steps of:

(1) treatment of the bonding pad 1

Adding RBD protein into colloidal gold solution 1, labeling for 10-30min, adding 2-10% blocking agent, stirring for 10-30min, 10000rpm, centrifuging for 20 min, removing supernatant, precipitating with colloidal gold labeled compound solution to recover volume, and storing at 4 deg.C;

coating the RBD protein gold-labeled compound on the binding pad 1 at the concentration of OD20-OD50, and then carrying out freeze drying treatment to obtain the binding pad 1;

(2) treatment of the bonding pad 2

Uniformly mixing N protein and casein sodium, adding into the colloidal gold solution 2, marking for 10-30min, adding 2-10% of a sealing agent, stirring for 10-30min, centrifuging at 10000rpm for 20 min, removing supernatant, precipitating, and recovering volume of the colloidal gold-marked complex solution to obtain an N protein gold-marked complex, and storing at 4 ℃;

coating the N protein gold-labeled compound on a binding pad 2 at the concentration of OD10-OD40, and then carrying out freeze drying treatment to obtain the binding pad 2;

(3) treatment of nitrocellulose membrane:

coating a detection line T2, a detection line T1 and a quality control line C on a nitrocellulose membrane, and then drying:

a. preparation of detection line T2

Spraying the mouse anti-human IgM monoclonal antibody on the nitrocellulose membrane at a rate of 1.0-3.0ul/cm, which is close to the end of the sample pad;

b, preparing a T1 detection line:

coating the mouse anti-human IgG monoclonal antibody at the side of the T2 detection line by 1.0-3.0ul/cm, and keeping away from the sample pad end; (ii) a

d.C preparation of quality control line:

coating the goat anti-chicken IgY antibody solution at 1.0-3.0ul/cm on the side of the T1 detection line, and keeping away from the end of the sample pad;

(4) assembling:

sequentially adhering a sample pad, a nitrocellulose membrane and a water absorption pad on a bottom lining, and cutting into test strips with the width of 3-5 mm.

8. The method for preparing the novel coronavirus (2019-nCOV) IgG/IgM antibody detection kit according to claim 7, wherein in the step (1), the mass ratio of the N protein to the sodium caseinate is 1.25-5; in the step (1) and the step (2), the pH value of the colloidal gold solution 1 is 6.0-8.0, preferably 7.0; the pH value of the colloidal gold solution 2 is 7.0-9.0, preferably 8.0.

9. The method for preparing the novel coronavirus (2019-nCOV) IgG/IgM antibody detection kit according to claim 8, wherein the colloidal gold labeled complex solution comprises the following components: 20-50 mmol/buffer solution, 1-10 g/protein, 10-20g/L surfactant, 20-60g/L protective agent, 0.1-0.5g/L macromolecular substance and 1-2.0g/L preservative; the buffer solution is at least one selected from glycine, Tris buffer solution and MOPS buffer solution; the protein is selected from at least one of casein or BSA, preferably the combination of casein and BSA; the surfactant is selected from at least one of Tween-80, Tween-20, Triton X-100 or A90; the macromolecular substance is at least one of PVP, PEG or PVA; the preservative is selected from at least one of sodium azide or Proclin-300.

10. A method for marking colloidal gold and a substance to be marked for an immunochromatography reagent is characterized by comprising the step of uniformly mixing the substance to be marked and sodium caseinate and then adding the mixture into a colloidal gold solution.

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