CN114563565B

CN114563565B - Fluorescent immunochromatography test strip and preparation method thereof

Info

Publication number: CN114563565B
Application number: CN202210134748.5A
Authority: CN
Inventors: 王恒强; 陈廷友; 丁芝; 张秀杰; 张晓刚
Original assignee: Beijing Innotech Biotechnology Co ltd
Current assignee: Beijing Innotech Biotechnology Co ltd
Priority date: 2022-02-14
Filing date: 2022-02-14
Publication date: 2024-10-15
Anticipated expiration: 2042-02-14
Also published as: CN114563565A

Abstract

The invention relates to the technical field of immunodetection, in particular to a fluorescent immunochromatography test strip and a preparation method thereof. The preparation method comprises the following steps: preparing an antigen-fluorescent microsphere complex; the preparation of the antigen-fluorescent microsphere complex comprises the following steps: diluting fluorescent microspheres by using HEPES solution, performing activation treatment and centrifugation, and then re-dissolving and precipitating by using re-dissolving solution; adding new coronavirus RBD antigen, sealing, centrifuging, and redissolving and precipitating by adopting the redissolution; the amino acid sequence of the novel coronavirus RBD antigen is shown as SEQ ID NO. 1. The preparation method of the antigen-fluorescent microsphere compound provided by the invention can effectively improve the binding level of the fluorescent microsphere conjugate on the conjugate pad, and has important significance in the field of detecting the neutralizing antibody of the novel coronavirus based on a fluorescent immunochromatography method.

Description

Fluorescent immunochromatography test strip and preparation method thereof

Technical Field

The invention relates to the technical field of immunodetection, in particular to a fluorescent immunochromatography test strip and a preparation method thereof.

Background

Neutralizing antibodies are a class of antibodies that bind to viruses and cause them to lose infectivity by stimulating the body with the envelope or capsid antigen of the outermost layer of the virus. After invasion of the virus into the human body, plasma cells produce virus-specific antibodies, only a portion of which are neutralizing antibodies.

Neutralizing antibody mechanisms of action generally involve altering the viral surface configuration; the virus epitope is combined with the virus epitope related to adsorption to prevent the virus from adsorbing, so that the virus cannot invade cells to carry out proliferation; forms immune complex with virus, is easy to be phagocytized and cleared by macrophage; after binding of the surface antigen of the enveloped virus to the neutralizing antibody, complement is activated, which can lead to lysis of the virus.

The laboratory gold standard for neutralizing antibody detection is an infection inhibition assay, which essentially includes a plaque reduction neutralization assay (plaque reduction neutralization test, PRNT) with live virus and a minimal cell neutralization assay by detecting the amount of cytopathic (cytopathic effect, CPE) assay. Both methods used quantitative live virus mixed with equal serum of different dilutions, and then inoculated with a pre-prepared monolayer of cells, and the degree of pathology of the cells was evaluated by different indices to evaluate neutralizing antibody titers.

Traditional neutralizing antibody detection is difficult to effectively meet the requirements of rapid and large-scale detection due to the factors of long time consumption, high requirements on detection conditions, high biosafety risk and the like. Thus, there is an urgent need for a simple and rapid alternative method for the assessment of protective antibodies in large-scale populations.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a quantum dot fluorescence immunochromatography test strip and application thereof, and the combination degree of antigen-quantum dots and a conjugate pad can be effectively improved through a specific quantum dot microsphere complex solution, so that the detection stability is improved.

In a first aspect, the present invention provides a method for preparing a fluorescent immunochromatographic test strip, including:

Preparing an antigen-fluorescent microsphere complex;

The preparation of the antigen-fluorescent microsphere complex comprises the following steps:

Diluting fluorescent microspheres by using HEPES solution, performing activation treatment and centrifugation, and then re-dissolving and precipitating by using re-dissolving solution;

Adding new coronavirus RBD antigen, sealing, centrifuging, and redissolving and precipitating by adopting the redissolution;

the amino acid sequence of the novel coronavirus RBD antigen is shown as SEQ ID NO. 1.

The invention improves the RBD protein of the novel coronavirus, improves the binding capacity of the RBD protein and the antibody, and further improves the sensitivity of the reagent.

Further, the compound solution is prepared from 10-50 mM borate buffer solution by weight parts and comprises: 1 to 5 parts of sucrose, 0.5 to 5 parts of BSA, 0.1 to 1 part of PC, 0.1 to 0.5 part of PVP, 0.1 to 0.5 part of casein and 0.1 to 0.5 part of Triton; the pH of the complex solution is 7-9.

After the quantum dot microsphere compound solution provided by the invention is sprayed on the conjugate pad after the antigen-quantum dot conjugate and the antibody-quantum dot conjugate are dissolved, the combination level of the two quantum dot conjugates on the conjugate pad can be effectively improved.

Further, the preparing of the antigen-microsphere complex includes:

10-200 mM HEPES solution was used (5-15): 1, dissolving fluorescent microspheres in a volume ratio to obtain a fluorescent microsphere solution; adding an activating solution with the volume of 0.1-10 times that of the fluorescent microsphere solution for treatment for 10-30 minutes, and adopting the re-dissolving precipitation with the re-dissolving solution with the volume of 1-20 times that of the fluorescent microsphere solution after centrifugation;

adding new coronavirus RBD antigen, uniformly mixing, adding 5% -20% BSA solution, uniformly mixing for 30-720 min, centrifuging, and re-dissolving and precipitating by using 5-100 times of the volume of the fluorescent microsphere solution.

Further, the content ratio of the novel coronavirus RBD antigen to the fluorescent microsphere solution is (0.1-5) mg/mL;

Further, the activating solution comprises EDC and NHS with the mass ratio of 0.1-10, and the dosage of the activating solution is 0.1-10%.

And/or the usage amount of the BSA solution is 1-10 times of that of the fluorescent microsphere solution.

Further, the method further comprises the following steps:

Preparing chicken IgY antibody-fluorescent microsphere compound, pretreating a sample and conjugate integrated pad, and preparing the sample and conjugate integrated pad, a coated detection area and a quality control area.

Further, the pretreatment of the sample and conjugate pad comprises:

Treating the sample and conjugate pad with a treatment fluid; the treatment solution comprises, by weight, 0.001-50 parts of boric acid, 0.001-50 parts of sodium tetraborate, 0.001-0.2 part of erythrocyte membrane monoclonal antibody, 0.001-50 parts of BSA, 50-500 parts of sucrose, 5-10 parts of tween and 3000.001-50 parts of procrines. The sample and conjugate pad is pre-treated to reduce intra-batch and inter-batch differences in reagents.

Further, the preparing of the sample and conjugate integrated pad includes:

Placing the antigen-fluorescent microsphere compound and the chicken IgY antibody-fluorescent microsphere compound on a sample and conjugate integrated pad at the concentration of 1-10 mu L/cm, and drying for 0.5-12 hours at the temperature of 30-50 ℃ and the relative humidity of less than or equal to 30%.

The use of integrated sample and conjugate pads can reduce intra-batch and inter-batch differences in reagents. Can be used together with a blood filtering membrane to realize the detection of a whole blood sample (venous whole blood or fingertip blood).

Further, the coating detection area and the quality control area comprise:

placing the detection area solution and the quality control area solution in the detection area and the quality control area of the nitrocellulose membrane at the concentration of 0.5-2.0 mu L/cm respectively, and drying for 0.5-4 hours under the condition that the relative humidity is less than or equal to 30% at the temperature of 30-50 ℃;

the detection zone solution comprises the following components in parts by weight:

0.005-0.025 part of mouse anti-human IgG antibody, 1-50 parts of disodium hydrogen phosphate, 0.001-10 parts of trehalose and 1-50 parts of human body;

The quality control zone solution comprises: sheep anti-chicken IgY antibody and tris;

The concentration of the sheep anti-chicken IgY antibody is 0.5-2.5 mg/mL, and the mass and volume percentage content of the tris (hydroxymethyl) aminomethane is 0.1-5%.

Further, the fluorescent microspheres include one or more of quantum dot fluorescent microspheres, time resolved fluorescent microspheres, or magnetic fluorescent microspheres.

Further, the fluorescent microsphere comprises one or more of a quantum dot fluorescent microsphere, a green fluorescent microsphere, a red fluorescent microsphere, a blue fluorescent microsphere, a time-resolved fluorescent microsphere or a magnetic fluorescent microsphere.

As a preferred embodiment, the invention provides a preparation method of a fluorescent immunochromatographic test strip, which comprises the following steps of;

1. preparation of antigen-fluorescent microsphere complexes

10-200 MM HEPES solution was used according to (8-10): 1, adding an activating solution with the volume which is 0.1-10 times of that of the fluorescent microsphere solution, treating for 10-30 minutes, centrifuging, and re-dissolving and precipitating by adopting a re-dissolving solution with the volume which is 1-20 times of that of the fluorescent microsphere solution;

Adding a new coronavirus RBD antigen, uniformly mixing, adding 5% -20% BSA solution, uniformly mixing for 30-720 minutes, centrifuging, and re-dissolving and precipitating by using a re-dissolving solution with the volume being 5-100 times that of the fluorescent microsphere solution;

the content ratio of the novel coronavirus RBD antigen to the fluorescent microsphere is (0.1-5) mg/mL; the usage amount of the BSA solution is 1-10 times of that of the fluorescent microsphere;

The compound solution is prepared by adding 1% -5% of sucrose, 0.5% -5% of BSA, 0.1% of PC300, 0.1% -0.5% of PVPK30, 0.1% -0.5% of casein and 0.1% -5% of Triton X-100 into 10mM-50mM borate buffer solution, and the pH is 7.0-8.5.

2. Preparation of chicken IgY antibody-fluorescent microsphere complex

adding chicken IgY antibody, uniformly mixing, adding 5% -20% BSA solution, uniformly mixing for 30-720 minutes, centrifuging, and re-dissolving and precipitating by using 5-100 times of the volume of the fluorescent microsphere solution;

The content ratio of the chicken IgY antibody to the fluorescent microsphere is (0.1-5) mg/mL; the usage amount of the BSA solution is 1-10 times of the volume of the fluorescent microsphere solution;

3. pretreatment of sample and conjugate pad

The treatment fluid comprises, by weight, 0.01-0.5% boric acid, 0.01-0.5% sodium tetraborate, 10-200 μg/mL erythrocyte membrane monoclonal antibody, 0.1-0.5% BSA, 0.5-5% sucrose, 0.05-0.1% Tween-20, 0.1-0.5% Proclin300, and the balance water.

Uniformly distributing the treatment liquid on the sample and conjugate integrated pad at a concentration of 10-30 mL/piece;

4. Preparation of sample and conjugate integral pad

Placing the antigen-fluorescent microsphere compound and the chicken IgY antibody-fluorescent microsphere compound on a sample and conjugate integrated pad at the concentration of 1-10 mu L/cm, and drying for 0.5-12 hours at the temperature of 30-50 ℃ under the condition that the relative humidity is less than or equal to 30%;

5. Coating detection area and quality control area

The detection zone solution comprises:

0.5-2.5 mg/mL of mouse anti-human IgG antibody, 0.1-5% of disodium hydrogen phosphate, 0.1-1% of sodium dihydrogen phosphate, 0.1-5% of trehalose;

The quality control zone solution comprises

0.5-2.5 Mg/mL sheep anti-chicken IgY antibody and 0.1-5% of tris.

Further, the method further comprises the following steps:

6. Assembly

Pasting the prepared sample and conjugate integrated pad, the blood filtering membrane and the absorption pad on the coated nitrocellulose membrane, compacting, marking the absorption water pad with different colors, cutting the strips to have a width of 3-6 mm, loading the cut detection strips into a plastic board card, and compacting the plastic board card by using a shell pressing machine; filling the compacted detection card into an aluminum foil bag, and adding 1 g/bag of drying agent; sealing by using a sealing machine at 180-200 ℃ and 4-6 m/min.

Further, the antigen-fluorescent microsphere complex and the chicken IgY antibody-fluorescent microsphere complex can be coated on the glass cellulose membrane in a spray coating, rolling coating or soaking way.

Further, the pretreatment of the sample and conjugate pad may be by spraying, coating, rolling or immersing the sample and conjugate pad with the treatment solution.

Further, the sample and conjugate pad may use a glass cellulose film or a polyester cellulose film.

Further, the nitrocellulose membrane can be subjected to sealing treatment after being coated with a detection area and a quality control area so as to reduce nonspecific reaction.

The invention further provides a fluorescent immunochromatography test strip prepared by the preparation method.

In a second aspect, the invention provides a kit comprising the fluorescent immunochromatographic test strip.

Further, a sample diluent is included; the sample diluent comprises the following components in parts by weight:

0.001 to 5 parts of sodium dihydrogen phosphate, 0.5 to 10 parts of disodium hydrogen phosphate, 0.5 to 10 parts of sodium chloride, 0.001 to 1 part of casein, 0.005 to 5 parts of blocking agent and 0.0001 to 0.0005 part of Proclin.

Further, the sample diluent comprises:

1 per mill-5% of sodium dihydrogen phosphate, 0.5-10% of disodium hydrogen phosphate, 0.5-10% of sodium chloride, 0.1 per mill-1% of casein, 0.5 per mill-5% of blocking agent, 0.1 per mill-0.5% of procrin300 and the balance of water.

The invention has the following beneficial effects:

The invention improves the RBD protein of the novel coronavirus, improves the binding capacity of the RBD protein and the antibody, and effectively provides the sensitivity of the test strip. And the coating mode is improved, so that the detection stability is further improved.

The kit provided by the invention is simple and convenient to operate: the traditional neutralizing antibody detection method needs trained professionals and can be completed in a professional laboratory, and the method can complete detection only by simply training the detection personnel or automatically referring to a kit instruction book.

The kit provided by the invention has the advantages of short detection time: conventional methods for detecting neutralizing antibodies take days, and the detection process is only 15-20 minutes.

The kit provided by the invention has low biological safety risk: traditional neutralizing antibody detection methods need to be carried out in a three-stage biosafety protection laboratory, and the method only needs to be carried out in a common laboratory, so that the biosafety level is greatly reduced.

Drawings

Fig. 1 is a schematic diagram of a test strip for quantum dot fluorescence immunochromatography provided in example 1 of the present invention.

Detailed Description

The following examples are illustrative of the invention and are not intended to limit the scope of the invention.

Example 1

1. Preparation of antigen-Quantum dot fluorescent microsphere complexes

Diluting the quantum dot fluorescent microspheres by using 50mM HEPES solution with pH of 7.0 according to the volume ratio of 9:1 to obtain a quantum dot fluorescent microsphere solution, adding EDC and NHS solution which are 5% of the volume of the quantum dot fluorescent microsphere solution, and activating for 15 minutes. Centrifuge at 14000rpm at 4deg.C for 15 min, suck the supernatant, and re-dissolve with a 10-fold complex solution of fluorescent microsphere solution. Adding a novel coronavirus RBD antigen (the amino acid sequence of which is shown as SEQ ID NO. 1) into 1.8mg/mL of quantum dot fluorescent microsphere solution, uniformly mixing for 60 minutes, adding a BSA solution with the concentration of 10% which is 0.5 times of the total volume of the solution, uniformly mixing for 120 minutes, centrifuging at 14000rpm and 4 ℃ for 15 minutes, sucking the supernatant, and redissolving by using a compound solution with the volume of 10 times of the quantum dot fluorescent microsphere solution.

The components of the complex solution are as follows:

20mM borate buffer, 2% sucrose, 1% BSA, 0.1% PC300, 0.3% PVPK30, 0.15% casein 1% TritonX-100, pH 8.0.+ -. 0.1.

2. Preparation of chicken IgY antibody-quantum dot fluorescent microsphere complex

Diluting the quantum dot fluorescent microspheres by using 50mM HEPES solution with pH of 7.0 according to the volume ratio of 9:1 to obtain a quantum dot fluorescent microsphere solution, adding EDC and NHS solution which are 5% of the volume of the quantum dot fluorescent microsphere solution, and activating for 15 minutes. Centrifuge at 14000rpm at 4deg.C for 15min, suck the supernatant, and re-dissolve with 10 times of the volume of the fluorescent microsphere solution. Adding chicken IgY antibody into 1.2g/mL quantum dot fluorescent microsphere solution, mixing for 60 minutes, adding 10% BSA solution with the concentration which is 0.5 times of the total volume of the solution, mixing for 120 minutes, centrifuging at 14000rpm and 4 ℃ for 15 minutes, sucking the supernatant, and re-dissolving by using 10 times of complex solution with the volume of the quantum dot fluorescent microsphere solution.

3. Sample and conjugate integrated pad pretreatment: the treatment fluid contains 0.87 per mill boric acid, 0.57 per mill sodium tetraborate, 69.26 mu g/mL erythrocyte membrane monoclonal antibody, 1.3 per mill BSA,2.39 percent sucrose, 0.13 per mill Tween-20,1.3 per mill Proclin300. The treatment solution was rolled onto a sample and conjugate pad at 25 mL/Zhang Junyun and dried overnight at 42.+ -. 1 ℃.

4. Sample and conjugate integrated pads were prepared: the mixture of the novel coronavirus RBD antigen-quantum dot complex and the chicken IgY antibody-quantum dot complex was sprayed onto the sample and conjugate integrated pad at a distance of 23mm and at a spray pressure of 1.2kg/cm ², 8 strips each. The prepared sample and conjugate are transferred to a drying chamber and dried for 3 hours at a temperature of 42+ -1deg.C and a relative humidity of less than or equal to 30%.

5. And a coating detection area and a quality control area: the detection zone solution contained 0.5mg/mL murine anti-human IgG antibody, 0.725% disodium hydrogen phosphate, 0.74% sodium dihydrogen phosphate, 1% trehalose. The quality control zone solution contained 1.0mg/mL goat anti-chicken IgY antibody, 0.3% tris. The solution in the detection area and the solution in the quality control area are respectively distributed in the detection area and the quality control area of the nitrocellulose membrane according to the liquid amount of 1.0 mu L/cm, the nitrocellulose membrane (with a backboard) coated with the detection area and the quality control area is transferred to a drying chamber, and the nitrocellulose membrane is dried for 1 hour under the conditions that the relative humidity is less than or equal to 30 percent at 37+/-1 ℃.

6. And (3) assembling a semi-finished product: pasting the prepared sample and conjugate integrated pad, the blood filtering membrane and the absorption pad on the coated nitrocellulose membrane (with the backboard), compacting, marking the absorption water pad with different colors, cutting the strips to have the width of 4mm, loading the cut detection strips into a plastic board card, and compacting the plastic board card by using a shell pressing machine; filling the compacted detection card into an aluminum foil bag, and adding 1 g/bag of drying agent; sealing at 200deg.C and 6m/min with a sealing machine.

7. Sample diluent preparation and split charging: the sample dilutions contained 4.37% sodium dihydrogen phosphate, 2.58% disodium hydrogen phosphate, 3.51% sodium chloride, 1% casein, 1% blocker, 2% Proclin300. And (5) sub-packaging the sample diluent by using a liquid separator, wherein the sub-packaging amount of each bottle is 3mL.

8. And (3) assembling a finished product: marking the batch number and the expiration date on the finished product packaging box; the detection card, the sample diluent, the disposable blood taking needle, the suction tube, the alcohol cotton piece and the instruction book are put into a packaging box, and the packaging box is sealed.

During actual detection, a sample to be detected is dripped into the sample-adding hole of the test card, and when the sample to be detected contains the novel coronavirus neutralizing antibody and the concentration is higher than the lowest detection limit, the neutralizing antibody and the novel coronavirus RBD antigen marked by the quantum dot microsphere generate immune reaction to form an immune complex. When the immune complex is chromatographed to a detection area on the nitrocellulose membrane, the immune complex reacts with the mouse anti-human IgG monoclonal antibody pre-coated on the nitrocellulose membrane, so that the immune complex is fixed on the nitrocellulose membrane, a fluorescent strip appears on a detection line, and a fluorescent signal value is read by a fluorescent immunoassay instrument. The signal value is proportional to the content of neutralizing antibodies in the sample. Whether the sample contains a novel coronavirus neutralizing antibody or not, the chicken IgY antibody marked by the quantum dot microsphere is chromatographed to a quality control area, and can be combined with the sheep anti-chicken IgY antibody coated on a C line to form a fluorescent strip, wherein the quality control area is used as a standard for judging whether the chromatography process is normal or not.

The invention further uses the prepared quantum dot fluorescent chromatography test strip of the novel coronavirus neutralizing antibody to detect 299 cases of negative serum and 179 cases of positive serum confirmed by a micro neutralization test, and compares the test result with the test result of an ELISA reagent, and the results are shown in tables 1 and 2 respectively

TABLE 1 comparison of Quantum dot fluorescence chromatography test strips with micro-neutralization test detection results

TABLE 2 comparison of Quantum dot fluorescence chromatography test strips with ELISA reagent detection results

From the data in table 1, the specificity=91.64%, sensitivity=96.65% and total coincidence rate=93.51% of the quantum dot chromatographic test strip can be calculated, which indicates that the total coincidence rate of the detection result is high.

The data in table 2 can be used for calculating the specificity=96.89%, the sensitivity=99.47% and the total coincidence rate= 97.91% of the quantum dot chromatographic test strip, which indicates that the two detection methods have high coincidence, and the quantum dot fluorescent chromatographic test strip prepared by the method has high practicability.

Experimental example 1

The novel coronavirus RBD protein used in the example 1 of the present invention was modified according to the reported novel coronavirus RBD protein (the amino acid sequence before modification is shown as SEQ ID NO. 2), and the modified novel coronavirus RBD protein and the commercially available novel coronavirus RBD protein (purchased from Jiangsu Dong anti-biological medicine technology Co., ltd.) are further compared in this experimental example to obtain the sensitivity of antigen, and the following results are obtained by using the novel coronavirus RBD protein (the amino acid sequence is shown as SEQ ID NO. 1) related to the example 1 and the commercially available novel coronavirus RBD protein, respectively (the method is the same as that of the example 1):

TABLE 3 sensitivity comparison results of different novel coronavirus S protein RBD antigens

Dilution ratio of weak positive samples	Antigen preparation kit after transformation	Commercial antigen preparation kit
			1:2	32IU/mL	20IU/mL
1:4	18IU/mL	11IU/mL
			1:8	11IU/mL	5IU/mL
1:16	6IU/mL	3IU/mL
			1:32	4IU/mL	0IU/mL
1:64	0IU/mL	0IU/mL

Experimental example 2

The present experimental example compares the effects of different coating modes on the detection result of the test strip of the fluorescence immunochromatography, and specifically, the following experiments are respectively performed on the mode of coating the mixture of the novel coronavirus RBD antigen-quantum dot complex and the chicken IgY antibody-quantum dot complex on the sample and the conjugate integrated pad in the step 4 in the example 1:

1. Rolling and coating:

(1) Cleaning a screen window, a grate, a roller and a backing plate, and drying for standby.

(2) Preparing liquid: taking a proper clean container and a stirrer, adding 9ml of RBD antigen-quantum dot fluorescent microsphere compound and 3ml of chicken IgY antibody-quantum dot fluorescent microsphere compound into the container, adding 78ml of compound solution, and stirring uniformly at a medium speed for 5 minutes.

(3) Taking 1 glass fiber, placing the glass fiber on a backing plate, adjusting the measuring range of a 5ml liquid transferring gun to 4.5ml, adding two times of liquid according to 9 ml/piece, taking the uniformly mixed combination by a sample adding device for 2 times while stirring, dripping the combination on the glass fiber, rolling the combination along the directions of four sides of the glass fiber respectively by using a roller, rolling the combination 15 times in each direction to form a circle, repeating the circle, observing whether the color of the combination pad is uniform, if so, putting the combination pad into a blast drying box for drying for 3 hours at 42 ℃, if not uniform, rolling the combination pad again, and putting the combination pad into the blast drying box for drying for 3 hours at 42 ℃ after uniform.

2. Coating

(1) And cleaning the grate and drying for later use.

(2) Prepare 5ml of the applicator and tip.

(3) Preparing liquid: taking a proper clean container and a stirrer, adding 9ml of RBD antigen-quantum dot fluorescent microsphere compound and 3ml of chicken IgY antibody-quantum dot fluorescent microsphere compound into the container, adding 78ml of compound solution, and stirring uniformly at a medium speed for 5 minutes.

(4) And (3) taking glass fibers which are cut into 300 mm/23 mm, arranging the glass fibers on a grate, and arranging 10 bars/grate according to the required amount.

(5) 4Ml of the binder solution is taken by a sample applicator and uniformly coated on 300 x 23mm glass fibers until the required amount is reached, and the glass fibers are dried in a drying oven at 42 ℃ for 3 hours after the coating.

3. Spraying

(1) And cleaning the grate and drying for later use.

(3) 1 Glass fiber is taken and fixed on a metal spraying instrument platform, the metal spraying instrument pressure is 1.2kg/cm < 2 >, the glass fiber is sprayed with 10 mu L/cm, the interval is 23mm, 8 glass fibers are sprayed on each glass fiber until the spraying is finished, and the glass fibers are placed in a drying oven for drying for 3 hours at 42 ℃.

4. Soaking

(1) Cleaning the grate and drying the soaking tank for standby.

(3) 1 Glass fiber is taken and placed in a soaking tank, 25ml of conjugate solution is added by a liquid transfer device, soaking is carried out for 20 minutes, and the glass fiber is placed in a blast drying box for drying at 42 ℃ for 3 hours.

Preparing a kit by using conjugate pads prepared by different coating modes, comparing, and selecting a proper coating mode.

Reagent and material:

novel coronavirus neutralizing antibody quantum dot fluorescence immunochromatography test strip

Weak positive quality control (70+ -30 IU/mL), medium positive quality control (200+ -100 IU/mL).

Table 4 test results of test strips obtained by different coating methods for weak-cation quality control samples

TABLE 5 detection results of test strips obtained by different coating methods for Weak cationic quality control samples

The results show that compared with four binder liquid coating modes, the spray CV is obviously smaller than the other three modes, and the effect is better, so the preferred coating mode is spray.

Experimental example 3

The influence of different coating modes on the detection result of the fluorescent immunochromatography test strip is compared in the experimental example, and specifically, the following experiment is carried out by adopting different coating modes for pretreatment of the sample and conjugate integrated pad in step 3 in the embodiment 1:

1. Rolling and coating:

(2) Preparing liquid: a suitable clean vessel and stirrer were taken, and 0.027g of boric acid, 0.018g of sodium tetraborate, 3.6mg of erythrocyte membrane mab, 0.03g of BSA,0.6g of sucrose and 0.003gTween-20,0.03g Proclin300 were added to the vessel, 25.8ml of purified water was added, and the mixture was stirred and mixed at a medium speed for 5 minutes.

(3) Taking 1 glass fiber, placing the glass fiber on a backing plate, dripping 25ml of solution on the glass fiber by using a liquid transfer device, rolling the conjugate by using a roller along the directions of four sides of the glass fiber, rolling 15 times in each direction to obtain one circle, repeating the circle, observing whether the color of the conjugate pad is uniform, if so, putting the conjugate pad into a blast drying box for drying at 42 ℃ overnight, if not, rolling the circle again, and putting the conjugate pad into the blast drying box for drying at 42 ℃ overnight after uniformity.

2. Coating

(1) And cleaning the grate and drying for later use.

(2) Prepare 5ml of the applicator and tip.

(3) Preparing liquid: a suitable clean vessel and stirrer were taken, and 0.027g of boric acid, 0.018g of sodium tetraborate, 3.6mg of erythrocyte membrane mab, 0.03g of BSA,0.6g of sucrose and 0.003gTween-20,0.03g Proclin300 were added to the vessel, 25.8ml of purified water was added, and the mixture was stirred and mixed at a medium speed for 5 minutes.

(4) And (3) taking glass fibers which are cut into 300 mm, placing the glass fibers on a grate, and placing the glass fibers according to the required amount.

(5) 2.9Ml of the conjugate solution is taken by a sample applicator and uniformly coated on 300 x 23mm glass fibers until the required amount is coated, and the glass fibers are placed in a drying oven for drying at 42 ℃ overnight after the coating is finished.

3. Spraying

(1) And cleaning the grate and drying for later use.

(3) 1 Glass fiber is taken and fixed on a metal spraying instrument platform, the metal spraying instrument pressure is 1.2kg/cm < 2 >, the glass fiber is sprayed with 20 mu L/cm, the interval is 23mm, 8 glass fibers are sprayed on each glass fiber until the required amount is sprayed, and the glass fibers are placed in a drying oven to be dried overnight at 42 ℃.

4. Soaking

(1) Cleaning a screen window, a grate and a soaking tank, and drying for standby.

(3) 1 Glass fiber is taken and placed in a soaking tank, 30ml of soaking solution is added by a liquid transfer device, soaking is carried out for 20 minutes, and the glass fiber is placed in a blast drying box for drying at 42 ℃ overnight.

And preparing the kit by using the pretreatment pads obtained in different pretreatment coating modes, comparing, and selecting a proper coating mode for experiments.

Reagent and material:

1. Novel coronavirus neutralizing antibody quantum dot fluorescence immunochromatography test strip

2. Weak positive quality control (70+ -30 IU/mL), medium positive quality control (200+ -100 IU/mL).

TABLE 6 detection results of test strips obtained by different coating methods during pretreatment for Weak cationic quality control samples

Table 7 test results of test strips obtained by different coating methods in pretreatment for Weak cationic quality control samples

The results show that: compared with four pretreatment coating modes, the rolling coating CV is obviously smaller than that of the other three modes, the effect is better, and the preferred coating mode is rolling coating.

While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Sequence listing

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1 5 10 15

Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val

20 25 30

Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser

35 40 45

Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val

50 55 60

Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp

65 70 75 80

Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln

85 90 95

Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr

100 105 110

Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly

115 120 125

Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys

130 135 140

Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr

145 150 155 160

Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser

165 170 175

Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val

180 185 190

Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly

195 200 205

Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe

210 215 220

Claims

1. The preparation method of the fluorescent immunochromatography test strip is characterized by comprising the following steps of:

Preparing an antigen-fluorescent microsphere complex;

2. The method of preparing according to claim 1, wherein the preparing the antigen-microsphere complex comprises:

3. The method according to claim 2, wherein the ratio of the content of the novel coronavirus RBD antigen and the fluorescent microsphere solution is (0.1-5) mg/mL; and/or the activating solution comprises EDC and NHS with the mass ratio of 0.1-10, and/or the usage amount of the BSA solution is 1-10 times of that of the fluorescent microsphere solution.

4. A method of preparing as claimed in any one of claims 1 to 3, further comprising:

5. The method of claim 4, wherein the pretreatment of the sample and conjugate pad comprises:

Treating the sample and conjugate pad with a treatment fluid in the form of a roll coating; the treatment solution comprises, by weight, 0.001-50 parts of boric acid, 0.001-50 parts of sodium tetraborate, 0.001-0.2 part of erythrocyte membrane monoclonal antibody, 0.001-50 parts of BSA, 50-500 parts of sucrose, 5-10 parts of tween and 0.001-50 parts of Proclin.

6. The method of preparing a sample and conjugate pad of claim 4, comprising:

placing the antigen-fluorescent microsphere compound and the chicken IgY antibody-fluorescent microsphere compound on a sample and conjugate integrated pad at the concentration of 1-10 mu L/cm, and drying for 0.5-12 hours at the temperature of 30-50 ℃ under the condition that the relative humidity is less than or equal to 30%; and/or the number of the groups of groups,

The coating detection area and the quality control area comprise:

7. The method of claim 4, wherein the fluorescent microspheres comprise one or more of quantum dot fluorescent microspheres, green fluorescent microspheres, red fluorescent microspheres, blue fluorescent microspheres, time-resolved fluorescent microspheres, or magnetic fluorescent microspheres.

8. A fluorescent immunochromatographic test strip prepared by the method of any one of claims 1 to 7.

9. A kit comprising the fluorescent immunochromatographic test strip of claim 8.

10. The novel coronavirus RBD protein is characterized in that the amino acid sequence of the novel coronavirus RBD protein is shown as SEQ ID NO. 1.