CN111665365A

CN111665365A - Novel coronavirus 2019-nCoV antibody spectrum detection kit

Info

Publication number: CN111665365A
Application number: CN202010441422.8A
Authority: CN
Inventors: 吴玉章; 吴长龙
Original assignee: Wuxi Fuwei Biomedical Technology Co ltd
Current assignee: Wuxi Fuwei Biomedical Technology Co ltd
Priority date: 2020-05-22
Filing date: 2020-05-22
Publication date: 2020-09-15

Abstract

The invention relates to a novel coronavirus 2019-nCoV antibody spectrum detection kit, and belongs to the technical field of novel coronavirus 2019-nCoV detection. The detection kit provided by the invention comprises a barreled test strip, bottled sample diluent, bottled eluent, bottled detection antibodies, a bottled substrate A, a bottled substrate B, an incubation groove, tweezers, a specification and a self-sealing bag. The detection kit provided by the invention jointly detects the antibodies (IgM/IgG/IgA/IgE) in serum or plasma samples by utilizing a plurality of novel coronavirus specific recombinant antigen test strips, can be used for qualitatively detecting the novel coronavirus antibodies in human serum or plasma samples in vitro, effectively increases the sensitivity and specificity, finds early infectors in time, can be used for evaluating and diagnosing the antibodies in the bodies of rehabilitation patients to monitor the course of diseases, and can be widely used for evaluating the vaccine human body effect in the future.

Description

Novel coronavirus 2019-nCoV antibody spectrum detection kit

Technical Field

The invention relates to a novel coronavirus 2019-nCoV antibody spectrum detection kit, and belongs to the technical field of novel coronavirus 2019-nCoV detection.

Background

Immunoblotting (Western blotting) is a hybridization technique that combines high-resolution gel electrophoresis with immunochemical analysis techniques. The immunoblotting method has the advantages of large analysis capacity, high sensitivity, strong specificity and the like, is a most common method for detecting protein characteristics, expression and distribution, and is used for qualitative and quantitative detection of tissue antigens, quality determination of polypeptide molecules, detection of antibodies or antigens of viruses and the like.

Traditional immunoblotting is performed in three stages. The first stage is SDS-polyacrylamide gel electrophoresis (SDS-PAGE): protein samples such as antigens and the like are treated by SDS and carry negative charges, and the protein samples migrate from a cathode to a principle anode in polyacrylamide gel, and the smaller the molecular weight is, the faster the migration speed is. The separation effect at this stage is not visible to the naked eye (the electrophoretic zones are only apparent after staining). The second stage is electrotransfer: transferring the separated strips in the gel to a nitrocellulose membrane, and electrifying for 45min to finish the transfer by selecting low voltage and large current. The protein bands isolated at this stage remain invisible to the naked eye. The third stage is enzyme immunolocalization: after the nitrocellulose membrane (equivalent to a solid phase carrier coated with an antigen) printed with a protein band is sequentially reacted with a specific antibody and an enzyme-labeled secondary antibody, an enzyme reaction substrate capable of forming an insoluble color substance is added to stain the zone. Common HRP substrates are 3,3' -diaminobenzidine (brown) and 4-chloro-1-naphthol (blue-violet). The positive reaction bands are distinct and distinguishable, and the molecular weight of each component can be determined according to the molecular weight standard added during SDS-PAGE. The traditional immunoblotting method combines the high resolution of SDS-PAGE and the high specificity and sensitivity of ELISA, is an effective analysis means, not only can be widely used for analyzing antigen components and their immunological activity, but also can be used for diagnosing diseases. However, the method has the disadvantages of complex operation, multiple steps and complex production process, and is not easy to form commercial products.

Coronaviruses are a large family of viruses known to cause more serious diseases such as the common cold, Middle East Respiratory Syndrome (MERS), and Severe Acute Respiratory Syndrome (SARS). The novel coronavirus is a new strain of coronavirus that has not been previously discovered in humans. The world health organization named the disease caused by this new type of coronavirus as 2019 coronavirus disease (COVID-19), lethal virus severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2).

The genome of the coronavirus encodes, in order, a spinous process protein (spike protein), an envelope protein (E protein), a Membrane protein (Membrane protein) and a Nucleocapsid protein (N protein). The spinous process protein (spike protein) is the most important surface membrane protein of coronaviruses and contains two subunits (subbunit), S1 and S2. Wherein S1 mainly contains receptor binding domain (RBD protein) responsible for recognizing cell receptors. S2 contains the basic elements required for the membrane fusion process. The spinous process protein plays a role in the combination of virus and host cell membrane receptor and membrane fusion, and is an important action site of host neutralizing antibody and a key target of vaccine design. The spinous process protein of SARS-CoV-2(2019-nCoV) interacts with human ACE2 to infect human airway epithelial cells. The nucleocapsid protein is the most abundant protein in coronaviruses. During virion assembly, the N protein binds to viral RNA and leads to the formation of a helical nucleocapsid. The nucleocapsid protein is a highly immunogenic phosphoprotein involved in viral genome replication and regulation of cellular signaling pathways.

At present, the serum antibody detection is mainly an immunochromatography method, and the method is simple to operate, has low requirements on equipment, and even does not need detection equipment; the detection time is short; the requirement on the detection environment is not high, and the result can be obtained in about 15 minutes generally. The method has the disadvantages that the lateral chromatography method uses 1 protein with 2 proteins at most for detection, has no cleaning process, and easily causes cross reaction of tests and false positive results because the blood of individual patients contains rheumatoid factors, heterophilic antibodies, autoantibodies, medicines, tumor cells and the like. There are also a few immunoblotting methods, but the preparation of the test strip needs electrophoresis and membrane transfer operation, and the requirements on instruments and operation are high.

Therefore, a novel coronavirus detection kit which is convenient to use, suitable for large-scale population screening, capable of avoiding false negative and false positive problems, high in sensitivity and specificity and capable of being used for disease course monitoring and vaccine effect evaluation is urgently needed.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a novel coronavirus 2019-nCoV antibody spectrum detection kit. The detection kit contains special membrane strips (combination scribing of various new crown antigens) required for detection, an incubation groove, tweezers, sample diluent and other liquid components, and is loaded in a container for reasonable use, the positions of the components are set to be placed, a user can manually complete in-vitro qualitative detection of novel coronavirus antibodies in human serum or plasma samples according to the operation of a specification, the detection can be efficiently completed by an automatic immunoblot analyzer, the sensitivity and the specificity can be effectively increased, early infectors can be timely found, meanwhile, the kit can be used for evaluating and diagnosing in-vivo antibodies of rehabilitation patients to monitor the course of disease, and can be widely used for evaluating the human effect of vaccines in the future.

The invention provides a novel coronavirus 2019-nCoV antibody spectrum detection kit, which has the technical scheme that:

a novel coronavirus 2019-nCoV antibody spectrum detection kit comprises a detection test strip, a sample diluent, an eluent, a detection antibody, a substrate, an incubation groove, tweezers, an instruction book and a self-sealing bag; the detection test strip comprises a nitrocellulose membrane marked with 3 or 4 detection lines and 1 quality control line, and a novel coronavirus antigen is fixed on the detection line; the novel coronavirus antigen is a recombinant S1 protein, a recombinant RBD protein and a recombinant N protein, or the novel coronavirus antigen is a recombinant S1 protein, a recombinant RBD protein and a recombinant E protein, or the novel coronavirus antigen is a recombinant S1 protein, a recombinant RBD protein, a recombinant N protein and a recombinant E protein.

Further, the substrate is obtained by mixing a substrate A and a substrate B in equal volume, wherein the substrate A is a tris solution with the pH value of 7-8; substrate B is H₂O₂And 3, 3-diaminobenzidine; the detection test strip is in a barrel made of white polypropylene; the sample diluent, the substrate A and the eluent are bottled by white polypropylene materials; the detection antibody and the substrate B are bottled by brown polypropylene.

Further, the specification is paper; the tweezers and the incubation groove are made of polypropylene materials; the valve bag is made of polyethylene.

Furthermore, goat anti-mouse IgG is fixed on the quality control line.

Further, the detection antibody is a horse radish peroxidase-labeled mouse anti-human IgG antibody or IgM antibody or IgA antibody or IgE antibody.

Further, the sample diluent is phosphate buffer solution with pH of 7-8.

Further, the eluent is phosphate buffer solution with pH of 7-8 added with Tween-20.

Further, the width of the test strip is 3.5-4.0 mm.

Furthermore, in the novel coronavirus 2019-nCOV antibody spectrum detection test strip, the length of a PVC rubber plate is 5-6cm, the length of label paper printed with serial numbers is 1.1-1.2cm, the length of a nitrocellulose membrane is 4-5cm, the length of an interactive overlapping part is 0.1-0.2cm, the label paper printed with serial numbers in the overlapping part is arranged above the nitrocellulose membrane, the interval between a quality control line and the label paper printed with serial numbers is 0.9-1.1cm, the interval between a quality control line and an adjacent detection line is 0.9-1.1cm, and the interval between each detection line is 0.4-0.6 cm.

Furthermore, the detection kit also comprises an instruction manual.

Further, the amino acid sequence of the recombinant S1 protein is shown as SEQ ID NO. 1.

Furthermore, the amino acid sequence of the recombinant RBD protein is shown as SEQ ID NO. 2.

Furthermore, the amino acid sequence of the recombinant N protein is shown as SEQ ID NO. 3.

Furthermore, the amino acid sequence of the recombinant E protein is shown as SEQ ID NO. 4.

Further, the streaked concentrations of the recombinant antigen S1 protein, the recombinant antigen RBD protein, the recombinant antigen N protein and the recombinant antigen E protein are respectively 0.2-0.5mg/mL (1 μ L/cm), 0.1-0.2mg/mL (1 μ L/cm) and 0.1-0.2mg/mL (1 μ L/cm).

Further, the goat anti-mouse IgG was streaked at a concentration of 0.2-1mg/mL (1. mu.L/cm).

The invention also provides a use method of the novel coronavirus 2019-nCoV antibody spectrum detection kit, and the technical scheme is as follows:

a use method of a novel coronavirus 2019-nCoV antibody spectrum detection kit specifically comprises the following steps:

(1) wetting: placing the test strip in a reaction tank, adding the sample diluent, and uniformly mixing for 1-2min at 15-25 ℃; the sample diluent is a phosphate buffer solution with the pH value of 7-8;

(2) primary incubation: adding human serum or plasma sample, and incubating at 15-25 deg.C for 30-60 min;

(3) cleaning: pouring out the liquid in the reaction tank, adding the eluent into the reaction tank, and incubating for 4-6min at 15-25 ℃; cleaning is repeated for one time;

(4) and (3) secondary incubation: pouring out liquid in the reaction tank, adding the detection antibody into the reaction tank, and incubating for 30-60min at 15-25 ℃; the detection antibody is a horse radish peroxidase-labeled mouse anti-human IgG antibody or IgM antibody or IgA antibody or IgE antibody;

(5) cleaning: pouring out the liquid in the reaction tank, adding the eluent into the reaction tank, and incubating for 4-6min at 15-25 ℃;

(6) cleaning: pouring out the liquid in the reaction tank, and repeatedly cleaning once;

(7) color development: pouring out the liquid in the reaction tank, draining the reaction tank, adding a substrate into the reaction tank, and reacting for 2-10min in a dark place; if more than two bands are developed, the detection result of the novel coronavirus antibody is positive; if no band develops color, the detection result of the novel coronavirus antibody is negative.

The beneficial technical effects of the invention are as follows:

the detection kit provided by the invention jointly detects the antibody in the serum or plasma sample through a plurality of novel coronavirus specific recombinant antigens, can be used for qualitatively detecting the novel coronavirus antibody in the human serum or plasma sample in vitro, effectively increases the sensitivity and specificity, finds early infected persons in time, can be used for evaluating and diagnosing the antibody in the body of a rehabilitation patient to monitor the course of disease, and can be more widely used for evaluating the effect of a vaccine human body in the future. The kit provided by the invention has the advantages of few components, simple production process, low production cost and easy amplification production; the detection process is convenient and fast, and full automation is easy to realize.

Drawings

FIG. 1 is a schematic side view of the test strip of the present invention, wherein T1, T2, T3 and T4 represent different detection lines, and different bands of coronavirus antigen are marked on the different detection lines.

Fig. 2 is a schematic view of a test strip used in embodiment 1 of the present invention.

Fig. 3 is a schematic view of a test strip used in embodiment 2 of the present invention.

Fig. 4 is a schematic view of a test strip used in embodiment 3 of the present invention.

Detailed Description

Preparation of recombinant S1 protein: synthesizing a full-length gene for coding a novel coronavirus 2019-nCOV Spike protein, and constructing the synthesized gene on an expression vector pCMV6-c.mFC of a mouse IgG FC by using a PCR (polymerase chain reaction) method to obtain a recombinant plasmid; the recombinant plasmid is transfected into Hek293 cells to secrete and express the fusion Protein, and the fusion Protein is obtained by affinity purification through Protein A. Theoretical Mw 76.5 kD.

Preparing a recombinant RBD protein: synthesizing genes encoding 319-Arg to 541-Phe of a novel coronavirus 2019-nCOV Spike protein, and constructing the synthesized genes on an expression vector pCMV6-c.mFC of a mouse IgG FC by using a PCR (polymerase chain reaction) method to obtain a recombinant plasmid; the recombinant plasmid is transfected into Hek293 cells to secrete and express the fusion Protein, and the fusion Protein is obtained by affinity purification through Protein A. Theoretical Mw 52 kD.

Preparation of recombinant N protein: synthesizing a complete gene for encoding a novel coronavirus 2019-nCOV nucleocapsid protein, and constructing the synthesized gene on an expression vector of pET28A-N-His by using a PCR (polymerase chain reaction) method to obtain a recombinant plasmid; transferring the recombinant plasmid into escherichia coli, expressing recombinant protein, and performing affinity purification through a nickel column to obtain the recombinant protein. Theoretical Mw 46.5 kD.

Preparing recombinant E protein: the complete gene for encoding the envelope protein of the novel coronavirus 2019-nCOV is synthesized, and the synthesized gene is constructed on an expression vector of HIS-C-pET28A-N-GST by utilizing a PCR method. And E, converting the plasmid with correct sequencing into escherichia coli after preparation, expressing the recombinant protein, and performing affinity purification through a nickel column to obtain the recombinant protein. Theoretical Mw 36.8 kD.

And (3) selecting the protein streaking concentration of the test strip: the color depth is indicated by "+"; "+ + + +" represents the strongest.

The detection method comprises the following steps: and after the protein is scribed to prepare a detection test strip, adding a colloidal gold platform false positive sample, a detection antibody and a substrate, and observing the color development intensity of the strip. As shown in tables 1-5.

Table 1 effect of different goat anti-mouse IgG antibody streaking concentrations on color; unit: mg/mL (1. mu.L/cm)

	Positive sample	False positive sample 1#	False positive sample 2#	False positive sample No. 3#
					1mg/mL	+++	+++	+++	+++
0.5mg/mL	+++	+++	+++	+++
					0.2mg/mL	++	++	++	++
0.1mg/mL	+	+	+	+

Table 2 effect of streaking concentration of different recombinant S1 proteins on color; unit: mg/mL (1. mu.L/cm)

	Positive sample	False positive sample 1#	False positive sample 2#	False positive sample No. 3#
					1mg/mL	+++	+	-	-
0.5mg/mL	+++	-	-	-
					0.2mg/mL	++	-	-	-
0.1mg/mL	-	-	-	-

Table 3 effect of streaking concentration of different recombinant RBD proteins on color; unit: mg/mL (1. mu.L/cm)

	Positive sample	False positive sample 1#	False positive sample 2#	False positive sample No. 3#
					1mg/mL	+++	-	-	+
0.5mg/mL	+++	-	-	-
					0.2mg/mL	++	-	-	-
0.1mg/mL	-	-	-	-

Table 4 effect of streaking concentration of different recombinant N proteins on colour; unit: mg/mL (1. mu.L/cm)

Table 5 effect of streaking concentration of different recombinant E proteins on colour; unit: mg/mL (1. mu.L/cm)

	Positive sample	False positive sample 1#	False positive sample 2#	False positive sample No. 3#
					1mg/mL	+++	+	-	-
0.5mg/mL	+++	-	-	-
					0.2mg/mL	+++	-	-	-
0.1mg/mL	++	-	-	-

The scribe line concentrations were determined as follows:

0.2-1mg/mL (1. mu.L/cm) of goat anti-mouse IgG antibody;

0.2-0.5mg/mL (1. mu.L/cm) of recombinant S1 protein;

0.2-0.5mg/mL (1 μ L/cm) of recombinant RBD protein;

0.1-0.2mg/mL (1. mu.L/cm) of recombinant N protein;

0.1-0.2mg/mL (1. mu.L/cm) of recombinant E protein.

Selection of detection time and temperature:

at room temperature, setting the sample incubation time to be 30min, 40min and 1 h; the incubation time for detecting the antibody is set to be 30min, 40min and 1 h;

according to different incubation times, the detection color development intensity of the positive sample is determined to be that the appropriate time for detection incubation is as follows: sample preparation: 40min, detection of antibody: and (4) 40 min.

The temperature was set at 4 ℃, 22 ℃, 37 ℃ and the incubation time was set as sample: 40min, detection of antibody: 40 min;

according to different temperatures, the detection color development intensity of the positive sample is determined to be the following suitable temperature for detection incubation: room temperature (15-25 deg.C)

The method for detecting the novel coronavirus 2019-nCoV antibody by using the detection kit provided by the invention comprises the following steps:

(1) wetting: placing a detection test strip into a reaction tank, adding 1mL of sample diluent, and uniformly mixing for 1min at room temperature (15-25 ℃);

(2) primary incubation: adding 1mL of sample (human plasma or serum), and incubating for 40min at room temperature (15-25 ℃);

(3) cleaning: pouring out liquid in the reaction tank, adding 1mL of eluent into the reaction tank, and incubating for 5min at room temperature (15-25 ℃); cleaning is repeated for one time;

(4) and (3) secondary incubation: pouring out liquid in the reaction tank, adding 1mL of detection antibody into the reaction tank, and incubating for 40min at room temperature (15-25 ℃);

(5) cleaning: pouring out liquid in the reaction tank, adding 1mL of eluent into the reaction tank, and incubating for 5min at room temperature (15-25 ℃);

(7) color development: and (3) pouring out the liquid in the reaction tank, draining the reaction tank, adding 200 mu L of substrate into the reaction tank, and reacting for 5min in a dark place.

And observing whether the strip is colored or not and the depth of the strip. When two or more than two bands develop color, the detection result of the novel coronavirus antibody is positive, and the deeper the development is, the higher the antibody content is; when all the strips are not developed, the detection result of the novel coronavirus antibody is negative.

The present invention will be described in detail with reference to examples.

The width of the detection test strip in the following embodiment is 3.5-4.0mm, the length of a PVC rubber plate is 5-6cm, the length of the label paper printed with serial numbers is 1.1-1.2cm, the length of the nitrocellulose membrane is 4-5cm, the interactive overlapping part is 0.1-0.2cm, the label paper printed with serial numbers in the overlapping part is on, the nitrocellulose membrane is under, the interval between the quality control line and the label paper printed with serial numbers is 0.9-1.1cm, the interval between the quality control line and the adjacent detection line is 0.9-1.1cm, and the interval between each detection line is 0.4-0.6 cm.

Example 1

Preparing a detection test strip:

1. preparation of a reagent:

1.1 protein dilution: weighing 55.8g of disodium hydrogen phosphate, 5.5g of sodium dihydrogen phosphate and 9g of sodium chloride; adding 999mL of pure water to dissolve, sucking 1mL of proClin300 into the solution, stirring and mixing uniformly, and fixing the volume to 1000 mL. Standing at normal temperature for later use.

1.2 preparation of sealing liquid: 55.8g of disodium hydrogen phosphate, 5.5g of sodium dihydrogen phosphate, 9g of sodium chloride and 10g of bovine serum albumin are weighed, 900mL of pure water is added for dissolution, and NaOH solution is added for adjusting the pH value to 7.4. Sucking 1mL of proClin300 into the solution, stirring uniformly, and diluting to 1000 mL. Standing at 2-8 deg.C for use.

2. The preparation method comprises the following steps:

2.1 cutting the nitrocellulose membrane into a size of 31cm × 5cm in length and width, scribing 4 high-purity specific recombinant S1 proteins, recombinant RBD proteins, recombinant N proteins and recombinant E proteins on the nitrocellulose membrane respectively to obtain 4 detection lines (the scribing concentrations are 0.2mg/mL, 0.1mg/mL and 0.1mg/mL respectively, and the scribing amounts are all 1 μ L/cm), as shown in fig. 1 and fig. 2 (the distribution sequence of each detection line on the nitrocellulose membrane does not have a significant effect on the detection results);

2.2, carrying out scribing on the goat anti-mouse IgG on a membrane plate (quality control line, C line; scribing concentration is 0.5mg/mL, and scribing amount is 1 muL/cm);

drying for 2 hours at the temperature of 2.337 ℃;

2.4 completely immersing the dried nitrocellulose membrane in the sealing solution for 1 hour at room temperature (15-25 ℃);

drying for 2 hours at the temperature of 2.537 ℃;

and 2.6 sequentially sticking the nitrocellulose membrane dried for the second time and the label paper printed with serial numbers on a PVC (polyvinyl chloride) rubber plate, and cutting the nitrocellulose membrane and the label paper into pieces with the width of 3.5-4.0mm, thereby obtaining the novel coronavirus 2019-nCOV antibody spectrum detection test paper strip.

Preparing a sample diluent: 0.1M, pH 7.4.4 phosphate buffer: weighing 55.8g of disodium hydrogen phosphate, 5.5g of sodium dihydrogen phosphate and 9g of sodium chloride; adding 999mL of pure water to dissolve, sucking 1mL of proClin300 into the solution, stirring and mixing uniformly, and fixing the volume to 1000 mL. Standing at normal temperature for later use.

Preparation of an eluent: weighing 55.8g of disodium hydrogen phosphate, 5.5g of sodium dihydrogen phosphate and 9g of sodium chloride; adding 997.5mL of pure water to dissolve, sucking 1mL of proClin300 and 1.5mL of Tween-20 into the solution respectively, stirring uniformly, and diluting to 1000 mL. Standing at normal temperature for later use.

Preparation of a substrate A: trimethylolaminomethane (1.21 g) was weighed, dissolved in pure water (900 mL), and adjusted to pH 7.5 with HCl. The volume is up to 1000 mL. Standing at normal temperature for later use.

Preparation of a substrate B: measuring 30% H₂O₂0.1mL, 0.01g of 3, 3-diaminobenzidine was weighed out to 100 mL. Standing at 2-8 deg.C for use.

Preparation of a substrate: substrate A and substrate B were mixed in equal volumes. It is used as it is.

Preparing an antibody diluent: weighing 55.8g of disodium hydrogen phosphate, 5.5g of sodium dihydrogen phosphate and 9g of sodium chloride; 20g of bovine serum albumin was weighed, dissolved in 900mL of pure water, and adjusted to pH 7.5 with NaOH solution. Sucking 1mL of proClin300 into the solution, stirring uniformly, and diluting to 1000 mL. Standing at 2-8 deg.C for use.

The preparation method of the detection antibody comprises the following steps:

a mouse anti-human IgG antibody labeled with horseradish peroxidase (HRP) is diluted with an antibody diluent to a final concentration of 0.5 mu g/mL, mixed uniformly, and placed at 2-8 ℃ for later use.

Combining and packaging the detection test strip, the sample diluent, the detection antibody, the eluent, the substrate A, the substrate B, the incubation groove and the self-sealing bag according to the dosage not less than a certain detection frequency to form a kit, and putting the tweezers and the instruction together into the kit. Wherein, the detection test strip is in a barrel made of white polypropylene; the sample diluent, the substrate A and the eluent are bottled by white polypropylene materials; the detection antibody and the substrate B are bottled by using a brown polypropylene material; the specification is paper; the tweezers and the incubation groove are made of polypropylene materials; the valve bag is made of polyethylene.

Example 2

Preparing a detection test strip:

1. preparation of a reagent:

1.2 preparation of sealing liquid: weighing 55.8g of disodium hydrogen phosphate, 5.5g of sodium dihydrogen phosphate and 9g of sodium chloride; 10g of bovine serum albumin was weighed, dissolved in 900mL of pure water, and adjusted to pH 7.4 by adding NaOH solution. Sucking 1mL of proClin300 into the solution, stirring uniformly, and diluting to 1000 mL. Standing at 2-8 deg.C for use.

2. The preparation method comprises the following steps:

2.1, cutting the nitrocellulose membrane into a size of 31cm × 5cm in length and width, and scribing 3 high-purity specific recombinant S1 proteins, recombinant RBD proteins and recombinant N proteins on the nitrocellulose membrane respectively to obtain 3 detection lines (the scribing concentrations are 0.3mg/mL, 0.3mg/mL and 0.2mg/mL respectively, and the scribing amounts are all 1 μ L/cm), as shown in FIG. 3 (the distribution sequence of each detection line on the nitrocellulose membrane does not have a significant influence on the detection results);

2.2, carrying out scribing on the goat anti-mouse IgG on a membrane plate (quality control line, C line; scribing concentration is 0.2mg/mL, and scribing amount is 1 muL/cm);

drying for 2 hours at the temperature of 2.337 ℃;

drying for 2 hours at the temperature of 2.537 ℃;

The preparation method of the detection antibody comprises the following steps:

Example 3

Preparing a detection test strip:

1. preparation of a reagent:

2. The preparation method comprises the following steps:

2.1, cutting the nitrocellulose membrane into a size of 31cm × 5cm in length and width, and scribing 3 high-purity specific recombinant S1 proteins, recombinant RBD proteins and recombinant E proteins on the nitrocellulose membrane respectively to obtain 3 detection lines (the scribing concentrations are 0.5mg/mL, 0.5mg/mL and 0.5mg/mL respectively, and the scribing amounts are all 1 μ L/cm), as shown in FIG. 4 (the distribution sequence of each detection line on the nitrocellulose membrane does not have a significant influence on the detection results);

2.2, scribing the goat anti-mouse IgG on the nitrocellulose membrane (quality control line, C line; scribing concentration is 1mg/mL, and scribing amount is 1 muL/cm);

drying for 2 hours at the temperature of 2.337 ℃;

drying for 2 hours at the temperature of 2.537 ℃;

The preparation method of the detection antibody comprises the following steps:

Test example

200 negative serum samples (including false positive samples detected by 9 colloidal gold kits; including 5 hepatitis B, positive hepatitis A and hepatitis B antibody and 2 hepatitis B antigen positive samples and the like) and 20 positive serum samples are detected, and the prediction sensitivity and specificity of the detection kit prepared in the embodiment 1 are respectively 100% and 100%; the detection results of 200 samples can be obtained within 2h by using a full-automatic immunoassay analyzer. The operation process is convenient and quick, and the detection result is accurate. The detection kits prepared in example 2 and example 3 can also achieve these detection effects.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

<110> Fuweier biomedical science and technology Co., Ltd, Wuxi, City

<120> novel coronavirus 2019-nCoV antibody spectrum detection kit

<160>4

<170>PatentIn version 3.3

<210>1

<211>681

<212>PRT

<213> Artificial sequence

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Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser

1 5 10 15

Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val

20 25 30

Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr

35 40 45

Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe

5055 60

Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr

65 70 75 80

Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp

85 90 95

Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val

100 105 110

Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val

115 120 125

Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val

130 135 140

Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe

145 150 155 160

Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu

165 170 175

Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His

180 185 190

Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu

195 200 205

Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln

210215 220

Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser

225 230 235 240

Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln

245 250 255

Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp

260 265 270

Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu

275 280 285

Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg

290 295 300

Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375380

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

385 390 395 400

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

405 410 415

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

420 425 430

Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe

530 535540

Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala

545 550 555 560

Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser

565 570 575

Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln

580 585 590

Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala

595 600 605

Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly

610 615 620

Ser Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His

625 630 635 640

Val Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys

645 650 655

Ala Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg Ala His

660 665 670

His His His His His His His His His

675 680

<210>2

<211>457

<212>PRT

<213> Artificial sequence

<400>2

Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn

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 150155 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 Ala

210 215 220

Asp Asp Asp Asp Lys Ala Val Pro Arg Asp Ser Gly Cys Lys Pro Cys

225 230 235 240

Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys

245 250 255

Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val

260 265 270

Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe

275 280 285

Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu

290 295 300

Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met His

305 310315 320

Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala

325 330 335

Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg

340 345 350

Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met

355 360 365

Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro

370 375 380

Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn

385 390 395 400

Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val

405 410 415

Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr

420 425 430

Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr Glu

435 440 445

Lys Ser Leu Ser His Ser Pro Gly Lys

450 455

<210>3

<211>425

<212>PRT

<213> Artificial sequence

<400>3

Met His His His His His His Ser Asp Asn Gly Pro Gln Asn Gln Arg

1 5 10 15

Asn Ala Pro Arg Ile Thr Phe Gly Gly Pro Ser Asp Ser Thr Gly Ser

20 25 30

Asn Gln Asn Gly Glu Arg Ser Gly Ala Arg Ser Lys Gln Arg Arg Pro

35 40 45

Gln Gly Leu Pro Asn Asn Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln

50 55 60

His Gly Lys Glu Asp Leu Lys Phe Pro Arg Gly Gln Gly Val Pro Ile

65 70 75 80

Asn Thr Asn Ser Ser Pro Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala

85 90 95

Thr Arg Arg Ile Arg Gly Gly Asp Gly Lys Met Lys Asp Leu Ser Pro

100 105 110

Arg Trp Tyr Phe Tyr Tyr Leu Gly Thr Gly Pro Glu Ala Gly Leu Pro

115 120 125

Tyr Gly Ala Asn Lys Asp Gly Ile Ile Trp Val Ala Thr Glu Gly Ala

130 135 140

Leu Asn Thr Pro Lys Asp His Ile Gly Thr Arg Asn Pro Ala Asn Asn

145 150 155 160

Ala Ala Ile Val Leu Gln Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly

165 170 175

Phe Tyr Ala Glu Gly Ser Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser

180 185 190

Ser Ser Arg Ser Arg Asn Ser Ser Arg Asn Ser Thr Pro Gly Ser Ser

195 200 205

Arg Gly Thr Ser Pro Ala Arg Met Ala Gly Asn Gly Gly Asp Ala Ala

210 215 220

Leu Ala Leu Leu Leu Leu Asp Arg Leu Asn Gln Leu Glu Ser Lys Met

225 230 235 240

Ser Gly Lys Gly Gln Gln Gln Gln Gly Gln Thr Val Thr Lys Lys Ser

245 250 255

Ala Ala Glu Ala Ser Lys Lys Pro Arg Gln Lys Arg Thr Ala Thr Lys

260 265 270

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

275 280 285

Gln Gly Asn Phe Gly Asp Gln Glu Leu Ile Arg Gln Gly Thr Asp Tyr

290 295 300

Lys His Trp Pro Gln Ile Ala Gln Phe Ala Pro Ser Ala Ser Ala Phe

305 310315 320

Phe Gly Met Ser Arg Ile Gly Met Glu Val Thr Pro Ser Gly Thr Trp

325 330 335

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

340 345 350

Lys Asp Gln Val Ile Leu Leu Asn Lys His Ile Asp Ala Tyr Lys Thr

355 360 365

Phe Pro Pro Thr Glu Pro Lys Lys Asp Lys Lys Lys Lys Ala Asp Glu

370 375 380

Thr Gln Ala Leu Pro Gln Arg Gln Lys Lys Gln Gln Thr Val Thr Leu

385 390 395 400

Leu Pro Ala Ala Asp Leu Asp Asp Phe Ser Lys Gln Leu Gln Gln Ser

405 410 415

Met Ser Ser Ala Asp Ser Thr Gln Ala

420 425

<210>4

<211>81

<212>PRT

<213> Artificial sequence

<400>4

Met Tyr Ser Phe Val Ser Glu Glu Thr Gly Thr Leu Ile Val Asn Ser

1 5 10 15

Val Leu Leu Phe Leu Ala Phe Val Val Phe Leu Leu Val Thr Leu Ala

20 25 30

Ile Leu Thr Ala Leu Arg Leu Cys Ala Tyr Cys Cys Asn Ile Val Asn

35 40 45

Val Ser Leu Val Lys Pro Ser Phe Tyr Val Tyr Ser Arg Val Lys Asn

50 55 60

Leu Asn Ser Ser Arg Val Pro Asp Leu Leu Val His His His His His

65 70 75 80

His

Claims

1. A novel coronavirus 2019-nCoV antibody spectrum detection kit is characterized in that the detection kit comprises a detection test strip, a sample diluent, an eluent, a detection antibody, a substrate, an incubation groove, tweezers, an instruction book and a self-sealing bag; the detection test strip comprises a nitrocellulose membrane marked with 3 or 4 detection lines and 1 quality control line, and a novel coronavirus antigen is fixed on the detection line; the novel coronavirus antigen is a recombinant S1 protein, a recombinant RBD protein and a recombinant N protein, or the novel coronavirus antigen is a recombinant S1 protein, a recombinant RBD protein and a recombinant E protein, or the novel coronavirus antigen is a recombinant S1 protein, a recombinant RBD protein, a recombinant N protein and a recombinant E protein.

2. The novel coronavirus 2019-nCoV antibody spectrum detection kit as claimed in claim 1, wherein the substrate is obtained by mixing a substrate A and a substrate B in equal volume, wherein the substrate A is a tris solution with pH of 7-8; substrate B is H₂O₂And 3, 3-diaminobenzidine; the detection test strip is in a barrel made of white polypropylene; the sample diluent, the substrate A and the eluent are bottled by white polypropylene materials; the detection antibody and the substrate B are bottled by brown polypropylene.

3. The novel coronavirus 2019-nCoV antibody spectrum detection kit as claimed in claim 1, wherein the instruction is paper; the tweezers and the incubation groove are made of polypropylene materials; the valve bag is made of polyethylene.

4. The novel coronavirus 2019-nCoV antibody spectrum detection kit as claimed in claim 1, wherein goat anti-mouse IgG is fixed on a quality control line of the detection test strip.

5. The novel coronavirus 2019-nCoV antibody spectrum detection kit as claimed in claim 1, wherein the detection antibody is a horseradish peroxidase-labeled mouse anti-human IgG antibody or IgM antibody or IgA antibody or IgE antibody.

6. The novel coronavirus 2019-nCoV antibody spectrum detection kit according to claim 1, wherein the sample diluent is a phosphate buffer solution with pH of 7-8.

7. The novel coronavirus 2019-nCoV antibody spectrum detection kit according to claim 1, wherein the eluent is phosphate buffer solution with pH 7-8 added with Tween-20.

8. The novel coronavirus 2019-nCoV antibody spectrum detection kit as claimed in claim 1, wherein the width of the detection test strip is 3.5-4.0 mm.

9. The use method of the novel coronavirus 2019-nCoV antibody spectrum detection kit as claimed in claim 1, which is characterized by comprising the following steps: