CN112129935A

CN112129935A - Immunochromatography test paper for rapid combined diagnosis of neocorona/alpha-flow/beta-flow and preparation method thereof

Info

Publication number: CN112129935A
Application number: CN202010851428.2A
Authority: CN
Inventors: 陆维克; 吴晓诚; 陈金树; 孙晓笛; 沈红萍
Original assignee: Hangzhou Alltest Biotech Co ltd
Current assignee: Hangzhou Alltest Biotech Co ltd
Priority date: 2020-08-21
Filing date: 2020-08-21
Publication date: 2020-12-25

Abstract

The invention relates to the field of immunodiagnosis, and provides immunochromatography test paper for quickly and jointly diagnosing new corona/alpha flow/beta flow aiming at the problems of long time consumption and single project of new corona virus detection, which comprises new corona test paper and alpha flow/beta flow test paper, wherein both the new corona test paper and the alpha flow/beta flow test paper comprise a PVC bottom plate, a modified nitrocellulose membrane, a water absorption pad and a sample pad, the new corona test paper comprises a latex label processing pad, the modified nitrocellulose membrane is provided with a detection line T coated with an anti-novel coronavirus N, S protein monoclonal antibody and a quality control line C coated with a streptavidin conjugate, the processing pad of the alpha flow/beta flow test paper is a gold-labeled probe processing pad, the modified nitrocellulose membrane is provided with a detection line A coated with an anti-influenza A virus monoclonal antibody, a detection line B coated with an anti-influenza B virus monoclonal antibody and a quality control line C coated with a goat anti-mouse IgG antibody. The detection efficiency of the antibody for detecting the antigen is high. The invention also provides a preparation method of the test paper.

Description

Immunochromatography test paper for rapid combined diagnosis of neocorona/alpha-flow/beta-flow and preparation method thereof

Technical Field

The invention relates to the technical field of immunodiagnosis, in particular to an immunochromatography test paper for quickly and jointly diagnosing neocorona/alpha-flow/B-flow and a preparation method thereof.

Background

The International Commission on viral Classification of Corona Virus research group (CSG) suggested that 2019 Novel coronavirus (Novel coronavirus pnuemonia, NCP) CODVI-19 was named "Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)" based on the systematic analysis of the relevant coronaviruses. Since the outbreak of pneumonia epidemic situation of new coronavirus infection, a large amount of PCR nucleic acid and IgM/IgG detection reagents appear in the market, and the detection aiming at antigens is almost not available. For example, a patent publication No. CN111454839A discloses an integrated apparatus for preparing and PCR detecting a novel coronavirus nucleic acid, comprising: a housing; a sample processing module for sucking a sample from the sample stage; a nucleic acid extraction module for extracting nucleic acid; a nucleic acid transfer module including a third conveyor belt and a nucleic acid extraction gun that extracts nucleic acids from the nucleic acid extraction plate; the PCR detection module comprises a PCR tube, a temperature control bottom plate, a laser emitter, a fluorescence signal detection device, a data processing and control element and a data output panel; after the nucleic acid extraction gun adds the extracted nucleic acid into the PCR tube, the laser emitter emits laser to irradiate the PCR tube, the fluorescence signal detection device detects the fluorescence signal in the PCR tube, and the data processing and control element receives the detection signal of the fluorescence signal detection device and displays the analysis result on the data output panel. However, 3 hours are required for a single sample to give a detection result by adopting a nucleic acid fluorescence PCR method, 6 hours are required for a nucleic acid sequencing method to give a detection result, and more importantly, the detection flux becomes the largest restriction factor due to the extremely high requirement of gene amplification on the laboratory environment, so that the supply capacity of the novel coronavirus rapid detection service is greatly restricted, and the prevention and control requirements of epidemic situations cannot be fully met.

IgM/IgG appears in humans relatively later than the antigen. And most of the current studies are mainly single studies aiming at the detection of pneumonia caused by new coronavirus. And the symptoms of patients caused by other common respiratory viruses such as influenza A virus, influenza B virus and the like are similar to those of patients with new coronavirus pneumonia, so that a plurality of respiratory virus detection reagents are combined to screen suspected cases in the early stage, the diseases of the patients are rapidly classified, and the method has very important significance for rapid isolation and targeted treatment.

Disclosure of Invention

The invention aims to overcome the problems of long time consumption and single detection item of the detection of the novel coronavirus, and provides immunochromatography detection test paper for quickly and jointly diagnosing the novel coronavirus, the influenza A virus and the influenza B virus and a preparation method thereof.

In order to achieve the purpose, the invention adopts the following technical scheme: the immunochromatography test paper for rapid joint diagnosis of the new corona/the first flow/the second flow comprises a new corona test paper and a first flow/the second flow test paper, wherein the new corona test paper and the first flow/the second flow test paper both comprise PVC base plates, a modified nitrocellulose membrane is arranged in the middle of the PVC base plates, microsphere probe treatment pads and water absorption pads are respectively arranged at two ends of the modified nitrocellulose membrane, two sides of the bottom surfaces of the microsphere probe treatment pads and the water absorption pads are respectively connected with the modified nitrocellulose membrane and the PVC base plate, sample pads are arranged on the upper surfaces of the connecting ends of the microsphere probe treatment pads and the PVC base plate, and two sides of the bottom surface of each sample pad are respectively connected with the microsphere probe treatment pads and the PVC base plate; the microsphere probe treatment pad of the new crown detection test paper is an emulsion labeled treatment pad, a detection line T coated with an anti-novel coronavirus N protein monoclonal antibody and an anti-novel coronavirus S protein monoclonal antibody and a quality control line C coated with a streptavidin conjugate are sequentially arranged at the non-connecting section of the modified nitrocellulose membrane from one side of the emulsion labeled treatment pad, the microsphere probe treatment pad of the A/B flow detection test paper is a gold labeled probe treatment pad, and a detection line A coated with the anti-A influenza virus monoclonal antibody, a detection line B coated with the anti-B influenza virus monoclonal antibody and a quality control line C coated with a goat anti-mouse IgG antibody are sequentially arranged at the non-connecting section of the modified nitrocellulose membrane from one side of the gold labeled probe treatment pad.

The sample containing the new corona virus antigen can be detected by using the new corona detection test strip. Detecting the sample by using a test strip, wherein when red strips appear on the detection line T and the quality control line C, the sample contains the new coronavirus; if only the quality control line C has a red strip, the sample does not contain the new coronavirus; and if the quality control line C does not have a red strip, the test strip is invalid. The A/B flow test paper can be used for detecting a sample containing an influenza A virus antigen and/or an influenza B virus. Detecting the sample by using a test strip, wherein when red strips appear on the detection line A and the quality control line C, the sample contains influenza A virus, when red strips appear on the detection line B and the quality control line C, the sample contains influenza B virus, and when red strips appear on the detection line A, the detection line B and the quality control line C, the sample contains influenza A virus and influenza B virus; if only the quality control line C II appears a red strip, the sample does not contain the influenza A virus and the influenza B virus; and if the quality control line C II does not have a red strip, the test strip is invalid.

Preferably, the latex labeling treatment pad is a labeling pad for labeling conjugates of the monoclonal antibody against the novel coronavirus N protein-latex particles, the monoclonal antibody against the novel coronavirus S protein-latex particles and the biotin-bovine serum albumin-latex particles; the gold-labeled probe treatment pad is a labeling pad for labeling anti-influenza A virus monoclonal antibody-colloidal gold, anti-influenza B virus monoclonal antibody-colloidal gold and mouse IgG-colloidal gold conjugates.

The principle applied by the test paper for detecting the new corona is an emulsion immunochromatography and a double-antibody sandwich method, when a sample contains a new corona virus antigen, the antigen can be firstly combined with the anti-new corona virus monoclonal antibody-emulsion particles in the marking pad after sample addition to form an antigen-antibody compound, and the compound can be captured by the anti-new corona virus monoclonal antibody coated on the nitrocellulose membrane along with the progress of the chromatography process to form the anti-new corona virus monoclonal antibody-new corona virus antigen-anti-new corona virus monoclonal antibody-emulsion particles, and the color is red, clear and identifiable, does not need any auxiliary equipment, and is convenient to operate. The principle applied by the A/B flow test paper is colloidal gold immunochromatography and a double-antibody sandwich method, when a sample contains influenza A virus and/or influenza B virus antigens, the antigens are firstly combined with an anti-influenza A virus monoclonal antibody-gold-labeled probe and/or an influenza B virus monoclonal antibody in a labeling pad after the sample is added to form an antigen-antibody compound, and along with the progress of the chromatography process, the compound is captured by an anti-influenza A virus monoclonal antibody and/or an influenza A virus monoclonal antibody coated on a nitrocellulose membrane to form an anti-influenza A virus monoclonal antibody-influenza A virus antigen-anti-influenza A virus monoclonal antibody-gold-labeled probe and/or an anti-influenza B virus monoclonal antibody-influenza B virus antigen-anti-influenza B virus monoclonal antibody-gold-labeled probe, the color is purple red, the color is clear and identifiable, no auxiliary equipment is needed, and the operation is convenient.

As a further preference, the latex particle diameter is 200-500nm, where the concentration of the labeled anti-novel coronavirus N protein monoclonal antibody-colloidal gold conjugate should be 0.2-0.5%, and the concentration of the labeled anti-novel coronavirus S protein monoclonal antibody-colloidal gold conjugate should be 0.1-0.4%; the diameter of the colloidal gold particles is 40-100nm, the using concentration of the labeled anti-influenza A virus monoclonal antibody-colloidal gold conjugate is OD1-OD4, and the using concentration of the labeled anti-influenza B virus monoclonal antibody-colloidal gold conjugate is OD1-OD 4. The different sizes of the colloidal gold particles can affect the performance of the product, the stability of the labeled conjugate and the color of the labeled conjugate, the color of the particles is reddish when the particle size is less than 40nm, and the sensitivity is weak; the particle size of more than 100nm is black, uneven in shape and easy to precipitate, so that the particle size of the colloidal gold particles is most suitable to be 40-100 nm. The anti-influenza A/B virus monoclonal antibody is marked into an anti-influenza A virus monoclonal antibody-colloidal gold conjugate/anti-influenza B virus monoclonal antibody-colloidal gold conjugate by using 70nm gold particles, and simultaneously, the anti-influenza A virus monoclonal antibody-colloidal gold conjugate is treated on glass fibers according to the absorbance of OD0.5, OD1.0, OD2.0, OD3.0, OD4.0 and OD5.0, and the glass fibers are placed in a vacuum freeze drying device for overnight (12 to 24 hours). The test is carried out by using 100HA/ml and 300HA/ml of positive reference substance of influenza A virus, 30HA/ml and 90HA/ml of positive reference substance of influenza B virus and negative reference substance, and the reading result of 15-20 minutes shows that the effect is best when the using concentration of the anti-influenza A virus monoclonal antibody-colloidal gold conjugate is OD1-OD4 and the using concentration of the anti-influenza B virus monoclonal antibody-colloidal gold conjugate is OD1-OD 4.

Preferably, the concentration of the coating anti-novel coronavirus N protein monoclonal antibody and the concentration of the coating anti-novel coronavirus S protein monoclonal antibody coated by the detection line T are respectively 0.5-2.0mg/ml and 0.5-2.0 mg/ml; the concentration of the anti-influenza A virus monoclonal antibody coated by the detection line A is 0.5-2.0mg/ml, and the concentration of the anti-influenza B virus monoclonal antibody coated by the detection line B is 0.5-2.0 mg/ml. When the concentration of the N protein monoclonal antibody and the S protein monoclonal antibody for the novel coronavirus used for coating is lower than 0.5mg/ml, the novel coronavirus antigen in the sample is insufficiently captured by the N protein monoclonal antibody and the S protein monoclonal antibody for the novel coronavirus in the reaction process, and the condition of missing detection appears clinically. Meanwhile, if the coated anti-novel coronavirus monoclonal antibody only has an anti-novel coronavirus N protein monoclonal antibody or an anti-novel coronavirus S protein monoclonal antibody, the condition of missing detection also can occur clinically. When the concentration of the anti-new coronavirus monoclonal antibody for coating is higher than 2.0mg/ml, the hybrid protein in the sample can be captured nonspecifically in the chromatography process, and the clinical false positive risk can appear, so the optimal concentration range of the anti-new coronavirus N protein monoclonal antibody and the anti-new coronavirus S protein monoclonal antibody for detecting line T coating is 0.5mg/ml-2.0 mg/ml. When the concentration of the anti-influenza A virus monoclonal antibody for coating is lower than 0.5mg/ml, the influenza A virus antigen in the sample is insufficiently captured by the anti-influenza A virus monoclonal antibody in the reaction process, and the condition of missed detection appears clinically. When the concentration of the anti-influenza A virus monoclonal antibody for coating is higher than 2.0mg/ml, the hybrid protein in the sample can be captured nonspecifically in the chromatography process, and the clinical risk of false positive can appear, so the optimal concentration range of the anti-influenza A virus monoclonal antibody for coating in the detection line A is 0.5mg/ml-2.0 mg/ml. When the concentration of the anti-influenza B virus monoclonal antibody for coating is lower than 0.5mg/ml, the influenza B virus antigen in the sample is insufficiently captured by the anti-influenza B virus monoclonal antibody in the reaction process, and the condition of missed detection appears clinically. When the concentration of the anti-influenza B virus monoclonal antibody for coating is higher than 2.0mg/ml, the hybrid protein in the sample can be captured nonspecifically in the chromatography process, and the clinical false positive risk can occur, so the optimal concentration range of the anti-influenza B virus monoclonal antibody for coating on the detection line B is 0.5mg/ml-2.0 mg/ml.

Preferably, the concentration of the streptavidin conjugate coated by the quality control line C is 0.5-2.0 mg/ml; the concentration of the goat anti-mouse IgG antibody coated by the quality control line C II is 1.0-3.0 mg/ml. When the concentration of the streptavidin conjugate coated on the quality control line C is lower than 0.5mg/ml, the biotin-bovine serum albumin-latex antibody conjugate in the microsphere probe treatment pad and the streptavidin conjugate are not sufficiently combined in the reaction process, so that the problem of weak quality control line clinically occurs. When the concentration of the streptavidin conjugate for coating the quality control line is higher than 2.0mg/ml, the streptavidin conjugate for coating treated on the nitrocellulose membrane can diffuse due to overflow, which affects the overall beauty. Therefore, the optimal concentration range of the streptavidin conjugate for coating the control line C is 0.5mg/ml to 2.0 mg/ml. When the concentration of the quality control line coated antibody is lower than 1.0mg/ml, the combination of the mouse IgG monoclonal antibody-colloidal gold conjugate and the goat anti-mouse polyclonal antibody in the labeling pad is insufficient in the reaction process, and the problem of weak quality control line clinically occurs. When the concentration of the quality control line coated antibody is higher than 3.0mg/ml, the sheep anti-mouse polyclonal antibody treated on the nitrocellulose membrane can generate a diffusion phenomenon due to the overflow amount, and the integral attractiveness is influenced. Therefore, the optimal concentration range of goat anti-mouse IgG polyclonal antibody for coating the quality control line C is 1.0mg/ml to 3.0 mg/ml.

The invention also provides a preparation method of the immunochromatographic test paper for rapid combined diagnosis of new corona/alpha-flow/B-flow, which comprises the following steps,

step one-preparation of new crown test paper,

1) preparing a detection line T and a quality control line C I: respectively diluting the anti-novel coronavirus N protein monoclonal antibody, mixing the anti-novel coronavirus S protein monoclonal antibody with a streptavidin conjugate solution, spraying the diluted solution on a nitrocellulose membrane at a speed of 1.0-1.2 mu l/cm to respectively serve as a detection line T and a quality control line C I, and drying at 36-38 ℃ to obtain the detection line T and the quality control line C I;

2) preparation of latex-labeled treatment pad: diluting the purified latex antibody to 0.2-0.5% and 0.1-0.4%, respectively, spraying onto a polyester cellulose membrane at a speed of 2.0-3.0 μ l/cm, and drying in an electric heating forced air drier to obtain a latex labeled pad;

3) preparing detection test paper: connecting and assembling the water absorption pad, the sample pad, the latex marking treatment pad, the modified nitrocellulose membrane coated with the antibody and the PVC bottom plate in sequence to obtain a finished product;

step two-preparation of A flow/B flow test paper,

1) preparing a detection line A, a detection line B and a quality control line C II: respectively diluting anti-influenza A virus monoclonal antibody, anti-influenza B virus monoclonal antibody and goat anti-mouse IgG solution, spraying the diluted solutions on a nitrocellulose membrane at a speed of 1.0-1.2 mu l/cm to respectively serve as a detection line A, a detection line B and a quality control line C II, and drying at 36-38 ℃ to obtain a detection line A, a detection line B and a quality control line C II;

2) preparing a gold-labeled probe treatment pad: coating the purified gold-labeled antibody with the concentration of OD1-OD4 after dilution on a glass fiber pad, and treating the glass fiber pad in a freeze dryer at the temperature of not more than 35 ℃ in the freeze-drying process to obtain a gold-labeled probe treatment pad;

3) preparing detection test paper: and sequentially connecting and assembling the water absorption pad, the sample pad, the gold-labeled probe treatment pad, the modified nitrocellulose membrane coated with the antibody and the PVC bottom plate to obtain a finished product.

The key process technology in the preparation process of the first flow/second flow detection test paper is the freeze drying treatment in the step 2), the freeze drying treatment is carried out under the vacuum condition, and compared with the conventional air drying, the colloidal gold conjugate is distributed on the carrier more uniformly, and the titer of the antigen or antibody is less influenced. However, in the freeze-drying process, the temperature in the cabin body is likely to exceed the expected temperature due to the continuous temperature rise of the equipment, so that the product performance is affected, and in order to ensure that the temperature of the cabin body is maintained in the expected range, the temperature control equipment is installed on the freeze-drying machine, and the temperature of the cabin body is strictly controlled not to exceed 35 ℃ in the freeze-drying process, so that the performance of the product is not affected.

Preferably, the preparation process of the purified latex antibody is as follows:

a. cleaning: selecting carboxylated red latex particles, and cleaning by adopting a mixed solution of 1.8-2.2mg/ml disodium hydrogen phosphate and 8.6-8.8mg/ml sodium chloride;

b. labeling reaction: adding the anti-new coronavirus monoclonal antibody for marking into the washed red latex particles at the marking ratio of 1:40-1:20, simultaneously adding an EDC activator, reacting at constant temperature for 12-24h, adding a proper amount of stop solution, centrifuging, adding a preserving solution, and performing ultrasound for 2-3min to obtain an anti-new coronavirus monoclonal antibody-latex particle conjugate; adding biotin into the washed red latex particles, adding an EDC activator at a labeling ratio of 1:40-1:20, reacting at constant temperature for 12-24h, adding a proper amount of stop solution, centrifuging, adding a preserving solution, and performing ultrasonic treatment for 2-3min to obtain a biotin-latex particle conjugate;

c. and mixing the anti-new coronavirus monoclonal antibody-latex particle conjugate and the biotin-latex particle conjugate to obtain the purified latex antibody.

The red latex particle size is generally 200-500nm, a carboxylated microsphere is adopted, the microsphere is firstly cleaned, then EDC activators (EDC and Sulfo-NHS) are added for activation, a monoclonal antibody for resisting novel coronavirus N protein or a monoclonal antibody for resisting novel coronavirus S protein to be marked is added for reaction, the active surface of the unreacted microsphere is closed, finally, the ultrasonic treatment is carried out, and the concentration of the conjugate is detected by an ultraviolet spectrophotometer and expressed by percentage. In the whole reaction stage, the latex particle conjugate plays a color development role, and in the coupling process, the combination of carboxyl and amino acid mainly reacts through chemical bonds, so that the label is more stable compared with colloidal gold, and the phenomena of dead gold, floating gold or gold mark precipitation cannot occur.

As a further preference, the concentration of EDC activator in step b is in the range from 0.5 to 1.5 mg/ml. In the process of coupling, the content of EDC activator is very important, the titer of the final marker can be influenced when the content is too much or too little, in the process of coupling the anti-novel coronavirus N protein monoclonal antibody or the anti-novel coronavirus S protein monoclonal antibody and the latex antibody, 0.1%, 0.5%, 1%, 1.5%, 2%, 3% EDC is respectively added, the final concentration of the coupling is more than 0.9%, the coupling is diluted to 0.25% by using latex diluent, the coupling is processed on a polyester membrane at the speed of 2.0 mul/cm, the polyester membrane is dried in an electric heating air blast drier overnight (12-24 hours), and the new coronavirus N protein positive reference products 100pg/ml, 10ng/ml, 100ng/ml, the new coronavirus S protein positive reference products 100pg/ml, 10ng/ml, 100ng/ml and the negative reference products are used for testing, the results of the 15-20 minute readings showed that the concentration range of EDC between 0.5-1.5mg/ml gave the best results. It is further preferred that the isothermal reaction temperature in step b is from 25 to 28 ℃. The temperature control in the reaction process needs to be very strict, because the reaction temperature range can influence the accuracy of the final test result of the detection test paper, the activation effect of the EDC needs to be in the standard temperature range, and researches find that the EDC activation effect is optimal and the coupling of latex particles and antibodies is most sufficient under the condition of 25-28 ℃. More preferably, the stop solution in step c is ethanolamine with the mass fraction of 1-1.2mol/L, and the preservation solution is a mixed solution of 5.8-6.2mg/ml Tris and 5-5.2mg/ml Casein.

Preferably, the preparation process of the purified gold-labeled antibody comprises the following steps:

d. obtaining colloidal gold: adding 1.8-2.2ml of 0.8-1.2% trisodium citrate aqueous solution into 98-102ml of boiling 0.008-0.012% chloroauric acid aqueous solution to obtain colloidal gold;

e. labeling colloidal gold: with K of 0.18-0.22mol/L₂CO₃Adjusting the pH value of the colloidal gold to 8.4-8.6, adding a labeled anti-influenza A virus monoclonal antibody or anti-influenza B virus monoclonal antibody into the colloidal gold after the pH value is adjusted, and labeling for 20-25 min;

f. purifying the gold-labeled antibody: and e, adding 8-12% of BSA aqueous solution by mass into the labeled colloidal gold solution obtained in the step e until the final concentration of BSA is 0.8-1.2%, stopping adding, continuing to stir for 25-30min, continuing to add 8-12% of PEG20000 until the final concentration of PEG20000 is 0.18-0.22%, continuing to stir for 10-15min, centrifuging, and removing the supernatant to obtain the purified gold-labeled antibody.

In the preparation process of the anti-influenza A/B virus monoclonal antibody-colloidal gold conjugate, the coupling process of colloidal gold and the antibody is particularly important, and because the particle size selected by the product is larger, the phenomena of gold floating and dead gold are easy to occur in the coupling process. In the reaction process, the phenomenon of gold bleaching and dead gold can be greatly reduced by adding the complex solution. Controlling the reaction condition of chloroauric acid through reducing agent trisodium citrate can obtain colloidal gold with the diameter of 65-75 nm.

More preferably, the volume ratio of the anti-influenza a virus monoclonal antibody for labeling to the colloidal gold after pH adjustment in step e is 1:1 to 1.2, and the volume ratio of the anti-influenza b virus monoclonal antibody for labeling to the colloidal gold after pH adjustment is 1:1 to 1.2. Respectively coupling the anti-influenza A/B virus monoclonal antibodies with chloroauric acid according to the proportion of 1:0.5, 1:1, 1:1.2, 1:1.4,1:1.6 and 1:2.0, wherein the final concentration values of the conjugates are all larger than OD100, diluting the conjugates to OD2 by using gold-labeled diluent, treating the conjugates on glass fibers, carrying out vacuum freezing and drying overnight (12-24 hours), testing by using 100HA/ml, 300HA/ml, 30HA/ml and 90HA/ml of influenza A virus positive reference substances and negative reference substances, and reading results in 15-20 minutes show that the volume ratio is 1:1-1:1.2, and the result is optimal. As a further preference, the centrifugation conditions in step f are: the centrifugation temperature is 4-6 ℃, the centrifugation rotation speed is 9900 and 10000rpm, and the centrifugation time is 30-40 min. The protein is stable under the condition of 2-8 ℃, the centrifugation speed of the colloidal gold conjugate is generally set at 12000rpm, the centrifugation time is about 0.5 hour, and the product has the particle size of the selected colloidal gold particles of about 70nm, so the centrifugation speed is controlled at 9900-10000rpm, the centrifugation time is 30-40 minutes, and the effect is optimal.

Therefore, the invention has the following beneficial effects: (1) the colloidal gold immunochromatographic test paper capable of simultaneously and rapidly detecting the N/S protein of the novel coronavirus and the influenza A virus/influenza B virus is provided, the product for detecting the antigen by using the antibody test paper is prepared, the detection time is short, only 15-20min is needed, and the requirement of field detection can be met;

(2) the accuracy of directly detecting the antigen is higher than that of detecting the antibody, the prepared antigen can accurately detect whether the novel coronavirus and the influenza A/B virus are infected, the antigen has no incubation period, the detection of the antigen can better avoid the clinical missed detection condition caused by low antibody titer in a window period, and the product is more accurate than that of detecting the antibody;

(3) the operation is simple and convenient, and other equipment and instruments are not needed; the detection result is displayed visually, can be judged by naked eyes and is suitable for personal use; the detection efficiency is high, the detection result is more direct, and the detection error caused by the antibody latency is avoided;

(4) the preparation process is simple, the test strip can be stored at normal temperature, special equipment and instruments are not needed, the test strip only needs to be kept dry, and the storage life can reach 2 years.

Drawings

FIG. 1 is a schematic diagram of the structure of the new crown detection test paper.

FIG. 2 is a schematic diagram of the structure of the A/B flow test strip.

FIG. 3 is a schematic view of the structure of the test strip detection window of the present invention.

In the figure: 1. the detection device comprises a sample pad, 2, a latex marking treatment pad, 3, detection lines T, 4, quality control lines C I and C5, a modified nitrocellulose membrane, 6, a water absorption pad, 7, a PVC bottom plate, 8, quality control lines C II and C9, a gold-labeled probe treatment pad, 10, detection lines A and 11 and a detection line B.

Detailed Description

The technical solution of the present invention is further illustrated by the following specific examples.

In the present invention, unless otherwise specified, all the raw materials and equipment used are commercially available or commonly used in the art, and the methods in the examples are conventional in the art unless otherwise specified.

An immunochromatography test paper for rapid combined diagnosis of neocorona/alpha-current/beta-current comprises a neocorona test paper and an alpha-current/beta-current test paper. The new crown detection test paper is totally called as immunochromatographic detection test paper for rapidly diagnosing a novel coronavirus antigen, and comprises a PVC base plate 7 as shown in figure 1, wherein a sample pad 1, a microsphere probe treatment pad, a modified nitrocellulose membrane 5 and a water absorption pad 6 are sequentially arranged on the upper surface of the PVC base plate 7 from left to right, the modified nitrocellulose membrane 5 is positioned in the middle of the PVC base plate 7, the microsphere probe treatment pad and the water absorption pad 6 are positioned at two ends of the modified nitrocellulose membrane 5, the bottom sides of one ends of the microsphere probe treatment pad and the water absorption pad 6 are respectively connected with two ends of the modified nitrocellulose membrane 5, and the bottom sides of the other ends of the microsphere probe treatment pad and the water absorption pad 6 are respectively connected with the PVC base plate 7 (in figure 1, the contact between the water absorption pad 6 and the PVC base plate 7 is not obvious due to the angle, the side edges of the two are, the same as in fig. 2); the upper side of one end of the microsphere probe treatment pad connected with the PVC bottom plate 7 is provided with a sample pad 1, the bottom side of one end of the sample pad 1 is connected with the upper side of the microsphere probe treatment pad, the bottom side of the other end of the sample pad is connected with the PVC bottom plate 7, the microsphere probe treatment pad of the new crown detection test paper is a latex marking treatment pad 2, and the latex marking treatment pad 2 is a marking pad for marking a conjugate of a novel coronavirus N protein monoclonal antibody-latex particle, a novel coronavirus S protein monoclonal antibody-latex particle and biotin-bovine serum albumin-latex particle. The non-connecting section of the modified nitrocellulose membrane 5 is sequentially provided with a detection line T3 coated with an anti-novel coronavirus N protein monoclonal antibody and an anti-novel coronavirus S protein monoclonal antibody and a quality control line C-4 coated with a streptavidin conjugate from one side of the microsphere probe treatment pad. The principle applied by the test paper for detecting the new corona is an emulsion immunochromatography and a double-antibody sandwich method, when a sample contains a new corona virus antigen, the antigen can be firstly combined with the anti-new corona virus monoclonal antibody-emulsion particles in the marking pad after sample addition to form an antigen-antibody compound, and the compound can be captured by the anti-new corona virus monoclonal antibody coated on the nitrocellulose membrane along with the progress of the chromatography process to form the anti-new corona virus monoclonal antibody-new corona virus antigen-anti-new corona virus monoclonal antibody-emulsion particles, and the color is red, clear and identifiable, does not need any auxiliary equipment, and is convenient to operate. The sample containing the new corona virus antigen can be detected by using the new corona detection test strip. Detecting the sample by using a test strip, wherein when a red strip appears on both the detection line T3 and the quality control line C-4, the sample contains the new coronavirus; if only the quality control line C-4 appears a red strip, the sample does not contain the new coronavirus; if the control line C-4 does not have a red band, the test strip is invalid.

The A flow/B flow detection test paper is called as immunochromatography detection test paper for rapidly diagnosing A flow/B flow antigens, and comprises a PVC (polyvinyl chloride) base plate 7 as shown in figure 2, wherein a sample pad 1, a microsphere probe treatment pad, a modified nitrocellulose membrane 5 and a water absorption pad 6 are sequentially arranged on the upper surface of the PVC base plate 7 from left to right, the modified nitrocellulose membrane 5 is positioned in the middle of the PVC base plate 7, the two ends of the modified nitrocellulose membrane 5 are respectively provided with the microsphere probe treatment pad and the water absorption pad 6, the bottom sides of one ends of the microsphere probe treatment pad and the water absorption pad 6 are respectively connected with the two ends of the modified nitrocellulose membrane 5, and the bottom side of the other end of the microsphere probe; the upper side of one end of the microsphere probe treatment pad connected with the PVC base plate 7 is provided with a sample pad 1, the bottom side of one end of the sample pad 1 is connected with the upper side of the microsphere probe treatment pad, the bottom side of the other end of the sample pad is connected with the PVC base plate 7, the microsphere probe treatment pad of the A/B flow detection test paper is a gold-labeled probe treatment pad 9, and the gold-labeled probe treatment pad 9 is a labeled pad for labeling anti-influenza A virus monoclonal antibody-colloidal gold, anti-influenza B virus monoclonal antibody-colloidal gold and mouse IgG-colloidal gold conjugates. The non-connecting section of the modified nitrocellulose membrane 5 is sequentially provided with a detection line A10 coated with an anti-influenza A virus monoclonal antibody, a detection line B11 coated with an anti-influenza B virus monoclonal antibody and a quality control line C II 8 coated with a goat anti-mouse IgG antibody from one side of the microsphere probe treatment pad. The principle applied by the A/B flow test paper is colloidal gold immunochromatography and a double-antibody sandwich method, when a sample contains influenza A virus and/or influenza B virus antigens, the antigens are firstly combined with an anti-influenza A virus monoclonal antibody-gold-labeled probe and/or an influenza B virus monoclonal antibody in a labeling pad after the sample is added to form an antigen-antibody compound, and along with the progress of the chromatography process, the compound is captured by an anti-influenza A virus monoclonal antibody and/or an influenza A virus monoclonal antibody coated on a nitrocellulose membrane to form an anti-influenza A virus monoclonal antibody-influenza A virus antigen-anti-influenza A virus monoclonal antibody-gold-labeled probe and/or an anti-influenza B virus monoclonal antibody-influenza B virus antigen-anti-influenza B virus monoclonal antibody-gold-labeled probe, the color is purple red, the color is clear and identifiable, no auxiliary equipment is needed, and the operation is convenient. The A/B flow test paper can be used for detecting a sample containing an influenza A virus antigen and/or an influenza B virus. Detecting the sample by using a test strip, wherein when red strips appear on the detection line A10 and the quality control line C II 8, the sample contains influenza A virus, when red strips appear on the detection line B11 and the quality control line C II 8, the sample contains influenza B virus, and when red strips appear on the detection line A10, the detection line B11 and the quality control line C II 8, the sample contains influenza A virus and influenza B virus; if only the quality control line C II 8 has a red strip, the sample does not contain the influenza A virus and the influenza B virus; if the control line C two 8 does not have a red strip, the test strip is invalid.

The preparation method of the anti-novel coronavirus N protein monoclonal antibody/anti-novel coronavirus S protein monoclonal antibody/influenza A virus monoclonal antibody/influenza B virus monoclonal antibody comprises the following steps: a. immunizing BALB/C mice with eight weeks old by using antigen, performing subcutaneous injection, performing injection once every three weeks for five times in total, measuring the antibody titer by ELISA (enzyme-linked immuno sorbent assay) for blood collection, and performing intensive injection three days before fusion; b. cell fusion and culture: performing superstrong immunization on a 3d mouse, removing a neck to kill the mouse, performing aseptic surgery to take out a spleen to prepare splenocytes, performing cell fusion with SP2/0 cells under the action of PEG, adding the fused cell suspension to a cell culture plate, standing at 37 ℃,5％CO₂selectively culturing in HAT and HT culture medium in an incubator; c. and (3) cell screening: screening positive holes by using indirect competition E LISA, and carrying out three times of limiting dilution cloning on strong positive holes with vigorous cell growth; d. production of ascites monoclonal antibody: six mice are taken, paraffin is injected into the abdominal cavity, each mouse is injected with 2.5ml, cloned hybridoma cell strains are injected after 10 days, ascites is extracted after 12 days, antibodies are purified by an ammonium caprylate-sulfate salting-out method, and the concentration of the antibodies is measured by an ultraviolet absorption method.

Example 1

A method for preparing immunochromatographic test paper for quickly and jointly diagnosing neocorona/alpha-flow/beta-flow comprises the following steps of preparing the neocorona test paper,

1) preparation detection line T3 and quality control line C — 4: respectively diluting the anti-novel coronavirus N protein monoclonal antibody, mixing the anti-novel coronavirus S protein monoclonal antibody with a streptavidin conjugate solution, spraying the diluted solution on a nitrocellulose membrane at a speed of 1.0 mu l/cm to respectively serve as a detection line T3 and a quality control line C I4, and drying at 36 ℃ to obtain a detection line T3 and a quality control line C I4; the concentrations of the coating anti-novel coronavirus N protein monoclonal antibody and the coating anti-novel coronavirus S protein monoclonal antibody coated by the detection line T3 are 0.5mg/ml and 0.5mg/ml respectively; the concentration of the streptavidin conjugate coated by the quality control line C-4 is 0.5 mg/ml;

2) preparation of latex-labeled treatment pad 2: spraying the purified latex antibody (the concentration of the diluted anti-novel coronavirus N protein monoclonal antibody-latex particle conjugate is 0.2% + the concentration of the diluted anti-novel coronavirus S protein monoclonal antibody-latex particle conjugate is 0.1%) on a polyester cellulose membrane at the speed of 2.0 mu l/cm, and placing the polyester cellulose membrane in an electrothermal blowing drier to prepare a latex labeling treatment pad 2; the preparation process of the purified latex antibody comprises the following steps: a. cleaning: selecting carboxylated red latex particles (purchased from Thermo Fisher), and cleaning by using a mixed solution of 1.8mg/ml disodium hydrogen phosphate and 8.6mg/ml sodium chloride; b. labeling reaction: adding a labeled anti-novel coronavirus N protein monoclonal antibody or anti-novel coronavirus S protein monoclonal antibody into washed red latex particles, wherein the labeling ratio is 1:40, adding an EDC activator at the same time, reacting in a constant temperature oven at 25 ℃ for 12 hours, adding 1mol/L ethanolamine as a stop solution, centrifuging, adding a mixed preservation solution of 5.8mg/ml Tris and 5mg/ml Casein, and performing ultrasonic treatment for 2 minutes to obtain an anti-novel coronavirus N protein-latex particle conjugate; adding biotin into the washed red latex particles, adding an EDC activator at a labeling ratio of 1:40, reacting in a constant temperature oven at 25 ℃ for 12 hours, adding 1mol/L ethanolamine as a stop solution, centrifuging, adding into a mixed preservation solution of 5.8mg/ml Tris and 5mg/ml Casein, and performing ultrasonic treatment for 2 minutes to obtain a biotin-latex particle conjugate; c. mixing the anti-new coronavirus monoclonal antibody-latex particle conjugate and the biotin-latex particle conjugate to obtain a purified latex antibody;

3) preparing detection test paper: and sequentially connecting and assembling the water absorption pad 6, the sample pad 1, the latex marking treatment pad 2, the modified nitrocellulose membrane 5 coated with the antibody and the PVC base plate 7 to obtain a finished product.

Step two-preparation of A flow/B flow test paper,

1) preparing a detection line A10, a detection line B11 and a quality control line C II 8: respectively diluting anti-influenza A virus monoclonal antibody, anti-influenza B virus monoclonal antibody and goat anti-mouse IgG solution, spraying the diluted solutions on a nitrocellulose membrane at a speed of 1.0 mu l/cm to respectively serve as a detection line A10, a detection line B11 and a quality control line C II 8, and drying at 36 ℃ to obtain a detection line A10, a detection line B11 and a quality control line C II 8; the concentration of the anti-influenza A virus monoclonal antibody for coating coated by the detection line A10 is 0.5 mg/ml; the concentration of the anti-influenza B virus monoclonal antibody for coating coated by the detection line B11 is 0.5 mg/ml; the concentration of the goat anti-mouse IgG antibody coated by the quality control line C II is 1.0 mg/ml;

2) preparation of gold-labeled probe treatment pad 9: diluting the purified gold-labeled antibody to OD1, coating the gold-labeled antibody on a glass fiber pad, and treating the glass fiber pad in a freeze dryer at a temperature not higher than 35 ℃ in the freeze-drying process to obtain a gold-labeled probe treatment pad 9; the preparation process of the purified gold-labeled antibody comprises the following steps: a. obtaining colloidal gold: to 98ml of boiling 0.008% aqueous chloroauric acid was added 1.8ml of 0.8% aqueous trisodium citrate to obtain a mixture with a diameter of 65nmColloidal gold; b. labeling colloidal gold: with 0.18mol/L of K₂CO₃Adjusting the pH value of the colloidal gold to 8.4 by using a solution, adding a 1mg/ml labeling anti-influenza A virus monoclonal antibody or anti-influenza B virus monoclonal antibody into the colloidal gold after the pH value is adjusted, wherein the volume ratio of the labeling anti-influenza A virus monoclonal antibody or anti-influenza B virus monoclonal antibody to the colloidal gold after the pH value is adjusted to be 1:1, and labeling for 20min to obtain an anti-influenza A virus monoclonal antibody-colloidal gold conjugate with the diameter of 40nm or an anti-influenza B virus monoclonal antibody-colloidal gold conjugate with the diameter of 40 nm; c. purifying the gold-labeled antibody: adding a BSA (bovine serum albumin) aqueous solution with the mass fraction of 8% into the labeled colloidal gold solution in the step B until the final concentration of the BSA is 0.8%, stopping adding, continuing to stir for 25min, continuing to add 8% PEG20000 to PEG20000 until the final concentration is 0.18%, continuing to stir for 10min, centrifuging at 9900rpm for 30min at 4 ℃, and discarding the supernatant to obtain the purified gold-labeled antibody;

3) preparing detection test paper: and sequentially connecting and assembling the water absorption pad 6, the sample pad 1, the gold-labeled probe treatment pad 9, the modified nitrocellulose membrane 5 coated with the antibody and the PVC bottom plate 7 to obtain a finished product.

And step three, integrating the new corona detection test paper and the first flow/second flow detection test paper on a bottom plate to obtain the immunochromatography detection test paper for quickly and jointly diagnosing the new corona/first flow/second flow.

Example 2

The difference from example 1 is in the step of preparation of a two-stream/b-stream test strip,

1) preparing a detection line A10, a detection line B11 and a quality control line C II 8: respectively diluting anti-influenza A virus monoclonal antibody, anti-influenza B virus monoclonal antibody and goat anti-mouse IgG antibody solutions, spraying the diluted solutions on a nitrocellulose membrane at a speed of 1.05 mu l/cm to respectively serve as a detection line A10, a detection line B11 and a quality control line C II 8, and drying at 36.5 ℃ to obtain a detection line and a quality control line; the concentration of the anti-influenza A virus monoclonal antibody for coating coated by the detection line A10 is 1.0 mg/ml; the concentration of the anti-influenza B virus monoclonal antibody for coating coated by the detection line B11 is 1.0 mg/ml; the concentration of the goat anti-mouse IgG antibody coated by the quality control line C II 8 is 1.5 mg/ml;

2) preparation of gold-labeled probe treatment pad 9: diluting the purified gold-labeled antibody to OD2, coating the purified gold-labeled antibody on a glass fiber pad, and treating the glass fiber pad by using a freeze dryer to obtain a gold-labeled probe treatment pad 9; the preparation process of the purified gold-labeled antibody comprises the following steps: a. obtaining colloidal gold: adding 1.9ml of 0.9% trisodium citrate aqueous solution into 99ml of boiling 0.009% chloroauric acid aqueous solution to obtain colloidal gold with a diameter of 68 nm; b. labeling colloidal gold: with 0.19mol/L of K₂CO₃Adjusting the pH value of the colloidal gold to 8.5 by using a solution, adding a labeled anti-influenza A virus monoclonal antibody or anti-influenza B virus monoclonal antibody with the concentration of 2mg/ml into the colloidal gold after the pH value is adjusted, wherein the volume ratio of the labeled anti-influenza A virus monoclonal antibody or anti-influenza B virus monoclonal antibody to the colloidal gold after the pH value is adjusted to be 1:1.1, and labeling for 21min to obtain an anti-influenza A virus monoclonal antibody-colloidal gold conjugate with the diameter of 50nm or an anti-influenza B virus monoclonal antibody-colloidal gold conjugate with the diameter of 40 nm; c. purifying the gold-labeled antibody: and C, adding a BSA (bovine serum albumin) aqueous solution with the mass fraction of 9% into the labeled colloidal gold solution obtained in the step B until the final concentration of BSA is 0.9%, stopping adding, continuing to stir for 26min, continuing to add 9% PEG20000 to PEG20000 until the final concentration is 0.19%, continuing to stir for 11min, centrifuging at 5 ℃ and 9920rpm for 32min, and removing the supernatant to obtain the purified gold-labeled antibody.

Example 3

1) preparing a detection line A10, a detection line B11 and a quality control line C II 8: respectively diluting anti-influenza A virus monoclonal antibody, anti-influenza B virus monoclonal antibody and goat anti-mouse IgG antibody solutions, spraying the diluted solutions on a nitrocellulose membrane at a speed of 1.1 mu l/cm to respectively serve as a detection line A10, a detection line B11 and a quality control line C II 8, and drying at 37 ℃ to obtain a detection line and a quality control line; the concentration of the anti-influenza A virus monoclonal antibody for coating coated by the detection line A10 is 1.5 mg/ml; the concentration of the anti-influenza B virus monoclonal antibody for coating coated by the detection line B11 is 1.5 mg/ml; the concentration of the goat anti-mouse IgG antibody coated by the quality control line C II 8 is 2.0 mg/ml;

2) preparation of gold-labeled probe treatment pad 9: diluting the purified gold-labeled antibody to OD3, coating the purified gold-labeled antibody on a glass fiber pad, and treating the glass fiber pad by using a freeze dryer to obtain a gold-labeled probe treatment pad 9; the preparation process of the purified gold-labeled antibody comprises the following steps: a. obtaining colloidal gold: adding 2.0ml of 1.0% trisodium citrate aqueous solution into 100ml of boiling 0.01% chloroauric acid aqueous solution to obtain colloidal gold with the diameter of 70 nm; b. labeling colloidal gold: with 0.2mol/L of K₂CO₃Adjusting the pH value of the colloidal gold to 8.5 by using a solution, adding a 3mg/ml labeling anti-influenza A virus monoclonal antibody or anti-influenza B virus monoclonal antibody into the colloidal gold after the pH value is adjusted, wherein the volume ratio of the labeling anti-influenza A virus monoclonal antibody or anti-influenza B virus monoclonal antibody to the colloidal gold after the pH value is adjusted to be 1:1.1, and labeling for 23min to obtain an anti-influenza A virus monoclonal antibody-colloidal gold conjugate with the diameter of 60nm or an anti-influenza B virus monoclonal antibody-colloidal gold conjugate with the diameter of 40 nm; c. purifying the gold-labeled antibody: and D, adding a BSA (bovine serum albumin) aqueous solution with the mass fraction of 10% into the labeled colloidal gold solution obtained in the step B until the final concentration of the BSA is 1.0%, stopping adding, continuing to stir for 28min, continuing to add 10% PEG20000 to PEG20000 until the final concentration is 0.2%, continuing to stir for 12min, centrifuging at 9950rpm for 35min at 5 ℃, and removing the supernatant to obtain the purified gold-labeled antibody.

Comparative example 1

The difference from example 3 is that the diameter of the anti-influenza a virus monoclonal antibody-colloidal gold conjugate and the anti-influenza b virus monoclonal antibody-colloidal gold conjugate is 20nm as small as possible.

Comparative example 2

The difference from example 3 is that the step of purifying the gold-labeled antibody in step 2) is omitted.

Comparative example 3

The difference from example 3 is that the colloidal gold particle size in step A is too large to be 90 mm.

Comparative example 4

The difference from example 3 is that the final centrifugation speed is reduced to 6000r/min and centrifugation is carried out for 0.5 hours.

Comparative example 5

The difference from the example 3 is that the gold-labeled antibody treated in the step 2) is placed in a 40 ℃ air-blast drying device to be dried overnight (12-24 hours).

Example 4

1) preparing a detection line A10, a detection line B11 and a quality control line C II 8: respectively diluting anti-influenza A virus monoclonal antibody, anti-influenza B virus monoclonal antibody and goat anti-mouse IgG antibody solutions, spraying the diluted solutions on a nitrocellulose membrane at a speed of 1.1 mu l/cm to respectively serve as a detection line A10, a detection line B11 and a quality control line C II 8, and drying at 37.5 ℃ to obtain a detection line and a quality control line; the concentration of the anti-influenza A virus monoclonal antibody for coating coated by the detection line A10 is 2.0 mg/ml; the concentration of the anti-influenza B virus monoclonal antibody for coating coated by the detection line B11 is 2.0 mg/ml; the concentration of the goat anti-mouse IgG antibody coated by the quality control line C II 8 is 2.5 mg/ml;

2) preparation of gold-labeled probe treatment pad 9: diluting the purified gold-labeled antibody to OD4, coating the purified gold-labeled antibody on a glass fiber pad, and treating the glass fiber pad by using a freeze dryer to obtain a gold-labeled probe treatment pad 9; the preparation process of the purified gold-labeled antibody comprises the following steps: a. obtaining colloidal gold: adding 2.1ml of 1.1% trisodium citrate aqueous solution into 101ml of boiling 0.011% chloroauric acid aqueous solution to obtain colloidal gold with the diameter of 72 nm; b. labeling colloidal gold: with 0.21mol/L of K₂CO₃Adjusting the pH value of the colloidal gold to 8.5 by using a solution, adding a labeled anti-influenza A virus monoclonal antibody or anti-influenza B virus monoclonal antibody with the concentration of 4mg/ml into the colloidal gold after the pH value is adjusted, wherein the volume ratio of the labeled anti-influenza A virus monoclonal antibody or anti-influenza B virus monoclonal antibody to the colloidal gold after the pH value is adjusted to be 1:1.1, and labeling for 24min to obtain an anti-influenza A virus monoclonal antibody-colloidal gold conjugate with the diameter of 80nm or an anti-influenza B virus monoclonal antibody-colloidal gold conjugate with the diameter of 40 nm; c. purifying the gold-labeled antibody: to step BAdding 11 mass percent BSA (bovine serum albumin) aqueous solution into the labeled colloidal gold solution until the final concentration of BSA is 1.1%, then stopping adding, continuing stirring for 29min, continuing adding 11 percent PEG20000 until the final concentration of PEG20000 is 0.21%, continuing stirring for 14min, centrifuging at 9980rpm for 38min at 5.5 ℃, and discarding the supernatant, thus obtaining the purified gold-labeled antibody.

Example 5

1) preparing a detection line A10, a detection line B11 and a quality control line C II 8: respectively diluting anti-influenza A virus monoclonal antibody, anti-influenza B virus monoclonal antibody and goat anti-mouse IgG antibody solutions, spraying the diluted solutions on a nitrocellulose membrane at a speed of 1.2 mu l/cm to respectively serve as a detection line A10, a detection line B11 and a quality control line C II 8, and drying at 38 ℃ to obtain a detection line and a quality control line; the concentration of the anti-influenza A virus monoclonal antibody for coating coated by the detection line A10 is 2.0 mg/ml; the concentration of the anti-influenza B virus monoclonal antibody for coating coated by the detection line B11 is 2.0 mg/ml; the concentration of the goat anti-mouse IgG antibody coated by the quality control line C II 8 is 3.0 mg/ml;

2) preparation of gold-labeled probe treatment pad 9: diluting the purified gold-labeled antibody to OD4, coating the purified gold-labeled antibody on a glass fiber pad, and treating the glass fiber pad by using a freeze dryer to obtain a gold-labeled probe treatment pad 9; the preparation process of the purified gold-labeled antibody comprises the following steps: a. obtaining colloidal gold: adding 2.2ml of 1.2% trisodium citrate aqueous solution into 102ml of boiling 0.012% chloroauric acid aqueous solution to obtain colloidal gold with the diameter of 75 nm; b. labeling colloidal gold: with 0.22mol/L of K₂CO₃Adjusting the pH value of the colloidal gold to 8.6 by using a solution, adding a labeled anti-influenza A virus monoclonal antibody or anti-influenza B virus monoclonal antibody with the concentration of 5mg/ml into the colloidal gold after the pH value is adjusted, wherein the volume ratio of the labeled anti-influenza A virus monoclonal antibody or anti-influenza B virus monoclonal antibody to the colloidal gold after the pH value is adjusted to be 1:1.2, and labeling for 25min to obtain an anti-influenza A virus monoclonal antibody-colloidal gold conjugate with the diameter of 100nm or an anti-influenza B virus monoclonal antibody-colloidal gold conjugate with the diameter of 40 nm; c. pureGold labeled antibody: and C, adding BSA (bovine serum albumin) aqueous solution with the mass fraction of 12% into the labeled colloidal gold solution in the step B until the final concentration of BSA is 1.2%, stopping adding, continuing stirring for 30min, continuing adding 12% PEG20000 to the final concentration of PEG20000 of 0.22%, continuing stirring for 15min, centrifuging at 6 ℃ and 10000rpm for 40min, and discarding the supernatant to obtain the purified gold-labeled antibody.

Example 6

The difference from example 1 is the step one-preparation of new crown test paper,

1) preparation detection line T3 and quality control line C — 4: respectively diluting the anti-novel coronavirus N protein monoclonal antibody, mixing the anti-novel coronavirus S protein monoclonal antibody with a streptavidin conjugate solution, spraying the diluted solution on a nitrocellulose membrane at the speed of 1.05 mu l/cm to respectively serve as a detection line T3 and a quality control line C-4, and drying at 36.5 ℃ to obtain a detection line and a quality control line; the concentrations of the coating anti-novel coronavirus N protein monoclonal antibody and the coating anti-novel coronavirus S protein monoclonal antibody coated by the detection line T3 are 1.0mg/ml and 1.0mg/ml respectively; the concentration of the streptavidin conjugate coated by the quality control line C-4 is 1.0 mg/ml;

2) preparation of latex-labeled treatment pad 2: treating the purified latex antibody (the concentration of the diluted anti-novel coronavirus N protein monoclonal antibody-latex particle conjugate is 0.3% + the concentration of the diluted anti-novel coronavirus S protein monoclonal antibody-latex particle conjugate is 0.2%) on a polyester cellulose membrane at the speed of 2.2 mu l/cm, and placing the polyester cellulose membrane in an electric heating air blast dryer for overnight to prepare a latex label treatment pad 2; the preparation process of the purified latex antibody comprises the following steps: a. cleaning: selecting carboxylated red latex particles (purchased from Thermo Fisher), and cleaning by using a mixed solution of 1.9mg/ml disodium hydrogen phosphate and 8.65mg/ml sodium chloride; b. labeling reaction: adding a labeled anti-novel coronavirus N protein monoclonal antibody or anti-novel coronavirus S protein monoclonal antibody into washed red latex particles, wherein the labeling ratio is 1:35, adding an EDC activator at the same time, reacting in a thermostat at 26 ℃ for 16 hours, adding 1.2mol/L ethanolamine as a stop solution, centrifuging, adding a mixed preservation solution of 5.9mg/ml Tris and 5.05mg/ml Casein, and performing ultrasonic treatment for 2.5 minutes to obtain an anti-novel coronavirus N protein-latex particle conjugate; adding biotin into the washed red latex particles, adding an EDC activator at a labeling ratio of 1:35, reacting at a constant temperature in a constant temperature box at 26 ℃ for 16 hours, adding 1.2mol/L ethanolamine as a stop solution, centrifuging, adding into a mixed preservation solution of 5.9mg/ml Tris and 5.05mg/ml Casein, and performing ultrasonic treatment for 2.5min to obtain a biotin-latex particle conjugate; c. and mixing the anti-new coronavirus monoclonal antibody-latex particle conjugate and the biotin-latex particle conjugate to obtain the purified latex antibody.

Example 7

1) preparation detection line T3 and quality control line C — 4: respectively diluting the anti-novel coronavirus N protein monoclonal antibody, mixing the anti-novel coronavirus S protein monoclonal antibody with a streptavidin conjugate solution, spraying the diluted solution on a nitrocellulose membrane at a speed of 1.1 mu l/cm to respectively serve as a detection line T3 and a quality control line C-4, and drying at 37 ℃ to obtain a detection line and a quality control line; the concentrations of the coating anti-novel coronavirus N protein monoclonal antibody and the coating anti-novel coronavirus S protein monoclonal antibody coated by the detection line T3 are 1.5mg/ml and 1.5mg/ml respectively; the concentration of the streptavidin conjugate coated by the quality control line C-4 is 1.5 mg/ml;

2) preparation of latex-labeled treatment pad 2: treating the purified latex antibody (the concentration of the diluted anti-novel coronavirus N protein monoclonal antibody-latex particle conjugate is 0.4% + the concentration of the diluted anti-novel coronavirus S protein monoclonal antibody-latex particle conjugate is 0.3%) on a polyester cellulose membrane at the speed of 2.5 mu l/cm, and placing the polyester cellulose membrane in an electric heating air blast dryer for overnight to prepare a latex label treatment pad 2; the preparation process of the purified latex antibody comprises the following steps: a. cleaning: selecting carboxylated red latex particles (purchased from Thermo Fisher), and cleaning by using a mixed solution of 2mg/ml disodium hydrogen phosphate and 8.7mg/ml sodium chloride; b. labeling reaction: adding a labeled anti-novel coronavirus N protein monoclonal antibody or anti-novel coronavirus S protein monoclonal antibody into washed red latex particles, wherein the labeling ratio is 1:30, adding an EDC activator at the same time, reacting in a thermostat at 26 ℃ for 18 hours, adding 1.1mol/L ethanolamine as a stop solution, centrifuging, adding a mixed preservation solution of 6mg/ml Tris and 5.1mg/ml Casein, and performing ultrasonic treatment for 2.5 minutes to obtain an anti-novel coronavirus N protein-latex particle conjugate; adding biotin into the washed red latex particles, adding an EDC activator at a labeling ratio of 1:30, reacting at a constant temperature in a constant temperature box at 26 ℃ for 18 hours, adding 1.1mol/L ethanolamine as a stop solution, centrifuging, adding a mixed preservation solution of 6mg/ml Tris and 5.1mg/ml Casein, and performing ultrasonic treatment for 2.5 minutes to obtain a biotin-latex particle conjugate; c. and mixing the anti-new coronavirus monoclonal antibody-latex particle conjugate and the biotin-latex particle conjugate to obtain the purified latex antibody.

Comparative example 6

The difference from example 7 is that the activator EDC is used at a concentration of 0.1%.

Comparative example 7

The difference from example 7 is that the reaction was left overnight in an incubator at 20 ℃.

Comparative example 8

The difference from example 7 is that the reaction is terminated and then sonicated for 30 seconds.

Example 8

preparation detection line T3 and quality control line C — 4: respectively diluting the anti-novel coronavirus N protein monoclonal antibody, mixing the anti-novel coronavirus S protein monoclonal antibody with a streptavidin conjugate solution, spraying the diluted solution on a nitrocellulose membrane at a speed of 1.15 mu l/cm to respectively serve as a detection line T3 and a quality control line C-4, and drying at 37.5 ℃ to obtain a detection line and a quality control line; the concentrations of the coating anti-novel coronavirus N protein monoclonal antibody and the coating anti-novel coronavirus S protein monoclonal antibody coated by the detection line T3 are respectively 2.0mg/ml and 2.0 mg/ml; the concentration of the streptavidin conjugate coated by the quality control line C-4 is 2.0 mg/ml;

2) preparation of latex-labeled treatment pad 2: treating the purified latex antibody (the concentration of the diluted anti-novel coronavirus N protein monoclonal antibody-latex particle conjugate is 0.5% + the concentration of the diluted anti-novel coronavirus S protein monoclonal antibody-latex particle conjugate is 0.4%) on a polyester cellulose membrane at the speed of 2.8 mu l/cm, and placing the polyester cellulose membrane in an electric heating air blast dryer for overnight to prepare a latex label treatment pad 2; the preparation process of the purified latex antibody comprises the following steps: a. cleaning: selecting carboxylated red latex particles (purchased from Thermo Fisher), and cleaning by using a mixed solution of 2.1mg/ml disodium hydrogen phosphate and 8.75mg/ml sodium chloride; b. labeling reaction: adding a labeled anti-novel coronavirus N protein monoclonal antibody or anti-novel coronavirus S protein monoclonal antibody into washed red latex particles, wherein the labeling ratio is 1:25, adding an EDC activator at the same time, reacting in a constant temperature oven at 27.5 ℃ for 22 hours, adding 1.15mol/L ethanolamine as a stop solution, centrifuging, adding a mixed preservation solution of 6.1mg/ml Tris and 5.15mg/ml Casein, and performing ultrasonic treatment for 2.8 minutes to obtain an anti-novel coronavirus N protein-latex particle conjugate; adding biotin into the washed red latex particles, adding an EDC activator at a labeling ratio of 1:25, reacting at a constant temperature of 27.5 ℃ in a constant temperature box for 22 hours, adding 1.15mol/L ethanolamine as a stop solution, centrifuging, adding into a mixed preservation solution of 6.1mg/ml Tris and 5.15mg/ml Casein, and performing ultrasonic treatment for 2.8 minutes to obtain a biotin-latex particle conjugate; c. and mixing the anti-new coronavirus monoclonal antibody-latex particle conjugate and the biotin-latex particle conjugate to obtain the purified latex antibody.

Example 9

1) preparation detection line T3 and quality control line C — 4: respectively diluting the anti-novel coronavirus N protein monoclonal antibody, mixing the anti-novel coronavirus S protein monoclonal antibody with a streptavidin conjugate solution, spraying the diluted solution on a nitrocellulose membrane at a speed of 1.2 mu l/cm to respectively serve as a detection line T3 and a quality control line C-4, and drying at 36-38 ℃ to obtain a detection line and a quality control line; the concentrations of the coating anti-novel coronavirus N protein monoclonal antibody and the coating anti-novel coronavirus S protein monoclonal antibody coated by the detection line T3 are respectively 2.0mg/ml and 2.0 mg/ml; the concentration of the streptavidin conjugate coated by the quality control line C-4 is 2.0 mg/ml;

2) preparation of latex-labeled treatment pad 2: treating the purified latex antibody (the concentration of the diluted anti-novel coronavirus N protein monoclonal antibody-latex particle conjugate is 0.5% + the concentration of the diluted anti-novel coronavirus S protein monoclonal antibody-latex particle conjugate is 0.4%) on a polyester cellulose membrane at the speed of 3 mul/cm, and putting the polyester cellulose membrane in an electric heating forced air drier overnight to prepare a latex labeling treatment pad 2; the preparation process of the purified latex antibody comprises the following steps: a. cleaning: selecting carboxylated red latex particles (purchased from Thermo Fisher), and cleaning by using a mixed solution of 2.2mg/ml disodium hydrogen phosphate and 8.8mg/ml sodium chloride; b. labeling reaction: adding a novel coronavirus N protein resistant monoclonal antibody or a novel coronavirus S protein resistant monoclonal antibody for marking into washed red latex particles, wherein the marking ratio is 1:20, adding an EDC activator at the same time, reacting in a constant temperature oven at 28 ℃ for 24 hours, adding 1.2mol/L ethanolamine as a stop solution, centrifuging, adding a mixed preservation solution of 6.2mg/ml Tris and 5.2mg/ml Casein, and performing ultrasonic treatment for 3 minutes to obtain a novel coronavirus N protein resistant monoclonal antibody-latex particle conjugate; adding biotin into the washed red latex particles, adding an EDC activator at a labeling ratio of 1:20, reacting at a constant temperature in a constant temperature box at 28 ℃ for 24 hours, adding 1.2mol/L ethanolamine as a stop solution, centrifuging, adding into a mixed preservation solution of 6.2mg/ml Tris and 5.2mg/ml Casein, and performing ultrasonic treatment for 3 minutes to obtain a biotin-latex particle conjugate; c. and mixing the anti-new coronavirus monoclonal antibody-latex particle conjugate and the biotin-latex particle conjugate to obtain the purified latex antibody.

Performance testing

(1) The test paper for the A/B flow test of examples 1 to 5 and comparative examples 1 to 5 was subjected to a performance test, and the results are shown in Table 1. 100HA/ml and 300HA/ml of influenza A virus positive reference substance, 30HA/ml and 90HA/ml of influenza B virus positive reference substance and negative reference substance are respectively dripped into the sample adding hole of the test strip for detection, each concentration is set to be repeated three times, and the detection results are completely consistent, so that the test strip provided by the invention is proved to be stable and reliable in detection result. The stability test adopts an accelerated stability test, the test strips of the same batch are respectively placed in ovens at 45 ℃ and 55 ℃, and the standard influenza A/B virus is respectively detected within the time period shown in the following table.

TABLE 1

Conclusion analysis: the colloidal gold immunochromatographic test paper with high detection sensitivity, accurate detection result and long service cycle can be prepared in the embodiments 1 to 5. Comparative examples 1 to 5 compared with example 3, the diameters of the anti-influenza a virus monoclonal antibody-colloidal gold conjugate and the anti-influenza b virus monoclonal antibody-colloidal gold conjugate in comparative example 1 were 20nm as small as possible. The coupling mode of the antibody and the colloidal gold is physical adsorption, and although the colloidal gold with undersize grain diameter is stable, the quantity of the anti-influenza A virus monoclonal antibody or anti-influenza B virus monoclonal antibody adsorbed on the surface of the colloidal gold is limited, thereby influencing the sensitivity of the product. In comparative example 2, the step of purifying the gold-labeled antibody in step 2) is omitted, and the risk of clinical false positive may occur due to interference of the hybrid protein in the labeling process. The particle size of the colloidal gold in step A of comparative example 3 was too large to be 90 mm. The colloidal gold used in the product has overlarge particle size, gold bleaching and dead gold are easy to occur in the marking process, and the marked colloidal gold conjugate is black in color and easy to generate false positive clinically. In comparative example 4, the final centrifugation speed was reduced to 6000r/min and the centrifugation was carried out for 0.5 hour. The centrifugation speed is slow, the time is short, the centrifugation is insufficient, the concentration value of the colloidal gold conjugate is low, part of effective components are not collected, and the sensitivity is weak. The treated antibody of step 1) of comparative example 5 was gold-labeled and placed in a 40 ℃ forced air drying apparatus and baked overnight (12-24 hours). The temperature of the blast drying equipment is high, molecules are easy to diffuse to the edge due to the blast effect, the dried marking pad is not uniform, and individual differences can occur clinically.

(2) The new crown test paper of example 1, examples 6 to 9 and comparative examples 6 to 8 were subjected to the performance test, and the results are shown in table 2. And (3) performing sensitivity test, namely respectively dripping 100pg/ml, 10ng/ml and 100ng/ml of the new coronavirus N protein positive reference substance and 100pg/ml, 10ng/ml, 100ng/ml and the negative reference substance into the sample adding hole of the test strip for detection, setting each concentration for three times, and completely consistent detection results, thereby proving that the test result of the test strip is stable and reliable. The stability test adopts an accelerated stability test, the test strips of the same batch are respectively placed in ovens at 45 ℃ and 55 ℃, and the new coronavirus standard substance is respectively detected in the time periods shown in the following table.

TABLE 2

Conclusion analysis: the embodiments 1 and 6-9 can prepare the new crown detection test paper for rapid diagnosis with high detection sensitivity, accurate detection result and long service cycle. Comparative example 6 compared to example 7, the activator EDC was used at a concentration of 0.1% in comparative example 6. Too low an activator can affect the combination effect of the antibody and the colored latex, and the clinical detection omission risk is easy to occur. In comparative example 7 the reaction was left to stand overnight in an incubator at 20 ℃. The activated substance EDC is sensitive to temperature, the effect of the activated substance is not obvious below 25 ℃, the combination effect of the antibody and the colored particles is influenced, and the missing detection risk is easy to appear clinically. After the reaction was terminated in step C of comparative example 8, sonication was carried out for 30 seconds. The purpose of ultrasound is to eliminate the agglutination phenomenon of latex, the ultrasound time is too short, the agglutination of latex is not sufficiently eliminated, and the risk of false positive is easy to appear clinically.

As shown in FIG. 3, after the test paper of the present invention is dropped with the positive standard, the test line of the test paper develops color, and after the negative sample is dropped, the test line of the test paper does not develop color. The new crown detection kit drops a detection sample into the hole of the S sample, new crown antigens in the sample are combined with new crown antibodies in the marking pad to form new crown antigen-antibody complexes, and the complexes flow to a detection line position T on the nitrocellulose membrane under the action of capillary and are combined and reacted with the new crown antibodies in the detection line T3. The influenza A/B virus detection kit drops a detection sample into an S sample hole, influenza A virus or/and influenza B virus antigens in the sample are combined with influenza A virus or/and influenza B virus antigen antibodies in a marking pad to form influenza A virus antigen-antibody and/or influenza B virus antigen-antibody complexes, the complexes flow to detection line positions A and B on a nitrocellulose membrane under the action of capillary, and the complexes are combined and reacted with influenza A virus antibodies in a detection line A10 and/or influenza B virus antibodies in a detection line B11.

As can be seen from the data of examples 1-9 and comparative examples 1-8, only the protocol within the scope of the claims of the present invention can satisfy the above requirements in all aspects, and an optimized protocol can be obtained, and a colloidal gold immunochromatographic test strip with optimal performance can be obtained. The change of the mixture ratio, the replacement/addition/subtraction of raw materials or the change of the feeding sequence can bring corresponding negative effects.

(3) Clinical sample validation

The inventor collects 50 samples infected with the new coronavirus, 45 samples infected with the influenza A and 30 samples infected with the influenza B, and the colloidal gold test strip prepared by the invention is used for detection, and the detection results show that the 50 samples infected with the new coronavirus, the 45 samples infected with the influenza A and the 30 samples infected with the influenza B are positive and completely consistent with the detection results of PCR experiments. The test strip provided by the invention is reliable in result, strong in specificity, simple and rapid to operate, can be used for detecting clinical samples without any equipment, and is intuitive and accurate in detection result display.

Although the present invention has been described with reference to a preferred embodiment, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

1. The immunochromatography test paper for rapid joint diagnosis of neocarvium cristatum/alpha flow/beta flow is characterized by comprising a neocarvium cristatum test paper and an alpha flow/beta flow test paper, wherein the neocarvium cristatum test paper and the alpha flow/beta flow test paper both comprise PVC base plates (7), a modified nitrocellulose membrane (5) is arranged in the middle of the PVC base plates (7), microsphere probe treatment pads and water absorption pads (6) are respectively arranged at two ends of the modified nitrocellulose membrane (5), two sides of the bottom surfaces of the microsphere probe treatment pads and the water absorption pads (6) are respectively connected with the modified nitrocellulose membrane (5) and the PVC base plates (7), a sample pad (1) is arranged on the upper surface of the connecting end of the microsphere probe treatment pads and the PVC base plates (7), and two sides of the bottom surfaces of the sample pad (1) are respectively connected with the microsphere probe treatment; the microsphere probe treatment pad of the new crown detection test paper is an emulsion labeled treatment pad (2), a detection line T (3) coated with an anti-novel coronavirus N protein monoclonal antibody and an anti-novel coronavirus S protein monoclonal antibody and a quality control line C (4) coated with a streptavidin conjugate are sequentially arranged on the non-connecting section of the modified nitrocellulose membrane (5) from one side of the emulsion labeled treatment pad (2), the microsphere probe treatment pad of the A/B flow detection test paper is a gold labeled probe treatment pad (9), and the non-connecting section of the modified nitrocellulose membrane (5) is sequentially provided with a detection line A (10) coated with an anti-A type influenza virus monoclonal antibody, a detection line B (11) coated with an anti-B type influenza virus monoclonal antibody and a quality control line C (8) coated with a goat anti-mouse IgG antibody from one side of the gold labeled probe treatment pad (9).

2. The immunochromatographic test strip for rapid combined diagnosis of neocorona/influenza A/influenza B according to claim 1, wherein the latex label treatment pad (2) is a label pad for labeling conjugates of monoclonal antibody against novel coronavirus N protein-latex particles, monoclonal antibody against novel coronavirus S protein-latex particles and biotin-bovine serum albumin-latex particles; the gold-labeled probe treatment pad (9) is a labeling pad for labeling anti-influenza A virus monoclonal antibody-colloidal gold, anti-influenza B virus monoclonal antibody-colloidal gold and mouse IgG-colloidal gold conjugates.

3. The immuno-chromatographic test paper for rapid combined diagnosis of neo-corona/first-flow/second-flow according to claim 2, wherein the diameter of the latex particle is 200-500nm, where the labeled anti-neo-coronavirus N protein monoclonal antibody-colloidal gold conjugate should be used at a concentration of 0.2-0.5%, and the labeled anti-neo-coronavirus S protein monoclonal antibody-colloidal gold conjugate should be used at a concentration of 0.1-0.4%; the diameter of the colloidal gold particles is 40-100nm, the using concentration of the labeled anti-influenza A virus monoclonal antibody-colloidal gold conjugate is OD1-OD4, and the using concentration of the labeled anti-influenza B virus monoclonal antibody-colloidal gold conjugate is OD1-OD 4.

4. The immunochromatographic test strip for rapid combined diagnosis of neocorona/influenza A/influenza B according to claim 1, wherein the concentrations of the coated anti-novel coronavirus N protein monoclonal antibody and the coated anti-novel coronavirus S protein monoclonal antibody coated in the test line T (3) are 0.5-2.0mg/ml and 0.5-2.0mg/ml respectively; the concentration of the anti-influenza A virus monoclonal antibody for coating coated by the detection line A (10) is 0.5-2.0mg/ml, and the concentration of the anti-influenza B virus monoclonal antibody for coating coated by the detection line B (11) is 0.5-2.0 mg/ml.

5. The immunochromatographic test strip for rapid combined diagnosis of neocorona/alpha-flux/beta-flux according to claim 1, wherein the concentration of the streptavidin conjugate coated with the quality control line C-4 is 0.5-2.0 mg/ml; the concentration of the goat anti-mouse IgG antibody coated by the quality control line C two (8) is 1.0-3.0 mg/ml.

6. A method for preparing the immunochromatographic test strip for rapid combined diagnosis of neocorona/influenza A/influenza B according to any one of claims 1 to 5, comprising the steps of,

step one-preparation of new crown test paper,

1) preparing a detection line T (3) and a quality control line C I (4): respectively diluting the anti-novel coronavirus N protein monoclonal antibody, the anti-novel coronavirus S protein monoclonal antibody and a streptavidin conjugate solution, respectively spraying the diluted solution on a nitrocellulose membrane at a speed of 1.0-1.2 mu l/cm to serve as a detection line T (3) and a quality control line C (4), and drying at 36-38 ℃ to obtain the detection line T (3) and the quality control line C (4);

2) preparation of latex-labeled treatment pad (2): diluting the purified latex antibody to 0.2-0.5% and 0.1-0.4% respectively, spraying onto the polyester cellulose membrane at a speed of 2.0-3.0 μ l/cm, and drying in an electric heating blower to obtain a latex labeled pad (2);

3) preparing detection test paper: sequentially connecting and assembling the water absorption pad (6), the sample pad (1), the latex marking treatment pad (2), the modified nitrocellulose membrane (5) coated with the antibody and the PVC bottom plate (7) to obtain a finished product;

step two-preparation of A flow/B flow test paper,

1) preparing a detection line A (10), a detection line B (11) and a quality control line C II (8): respectively diluting anti-influenza A virus monoclonal antibody, anti-influenza B virus monoclonal antibody and goat anti-mouse IgG solution, spraying the diluted solutions on a nitrocellulose membrane at a speed of 1.0-1.2 mu l/cm to respectively serve as a detection line A (10), a detection line B (11) and a quality control line C II (8), and drying at 36-38 ℃ to obtain the detection line A (10), the detection line B (11) and the quality control line C II (8);

2) preparation of gold-labeled probe treatment pad (9): coating the purified gold-labeled antibody with the concentration of OD1-OD4 after dilution on a glass fiber pad, and treating the glass fiber pad in a freeze dryer at the temperature of not more than 35 ℃ in the freeze-drying process to obtain a gold-labeled probe treatment pad (9);

3) preparing detection test paper: and sequentially connecting and assembling the water absorption pad (6), the sample pad (1), the gold-labeled probe treatment pad (9), the modified nitrocellulose membrane (5) coated with the antibody and the PVC bottom plate (7) to obtain a finished product.

7. The method for preparing the immunochromatographic test strip for rapid combined diagnosis of neocorona/influenza A/influenza B according to claim 6, wherein the preparation process of the purified latex antibody is as follows:

8. The method for preparing the immunochromatographic test strip for rapid combined diagnosis of neocorona/influenza A/influenza B according to claim 6, wherein the preparation process of the purified gold-labeled antibody comprises the following steps:

9. The method for preparing immunochromatographic test paper for rapid combined diagnosis of neocorona/influenza A/influenza B according to claim 8, wherein the volume ratio of the anti-influenza A virus monoclonal antibody for labeling to the colloidal gold after pH adjustment in step e is 1:1-1.2, and the volume ratio of the anti-influenza B virus monoclonal antibody for labeling to the colloidal gold after pH adjustment is 1: 1-1.2.

10. The method for preparing immunochromatographic test strip for rapid combined diagnosis of neocorona/influenza A/influenza B according to claim 8, wherein the centrifugation conditions in step f are as follows: the centrifugation temperature is 4-6 ℃, the centrifugation rotation speed is 9900 and 10000rpm, and the centrifugation time is 30-40 min.