CN111505316A - Production method of novel coronavirus 2019-nCoV antibody spectrum detection kit - Google Patents
Production method of novel coronavirus 2019-nCoV antibody spectrum detection kit Download PDFInfo
- Publication number
- CN111505316A CN111505316A CN202010442384.8A CN202010442384A CN111505316A CN 111505316 A CN111505316 A CN 111505316A CN 202010442384 A CN202010442384 A CN 202010442384A CN 111505316 A CN111505316 A CN 111505316A
- Authority
- CN
- China
- Prior art keywords
- protein
- recombinant
- detection
- ser
- thr
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Food Science & Technology (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Virology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Tropical Medicine & Parasitology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a production method of a novel coronavirus 2019-nCoV antibody spectrum detection kit, and belongs to the technical field of novel coronavirus 2019-nCoV detection. The detection kit obtained by the production method provided by the invention comprises barreled test paper strips, bottled sample diluent, bottled eluent, bottled detection antibodies, bottled substrates A, bottled substrates B, an incubation groove, tweezers, an instruction book and a self-sealing bag. The production method provided by the invention is different from the traditional immunoblotting method in that the detection test strip is prepared by using the recombinant immunoblotting method, protein gel electrophoresis and membrane conversion are not needed, and the streaking treatment is directly carried out on the nitrocellulose membrane, so that the production method of the detection kit is greatly simplified, the preparation time is shortened, and the detection kit can be produced in a large scale in a short time.
Description
Technical Field
The invention relates to a production method of 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.
The first stage is SDS-polyacrylamide gel electrophoresis (SDS-PAGE), in which the protein samples of antigen, etc. are treated by SDS and charged negatively, and electrophoresed from cathode to principle anode in polyacrylamide gel, the smaller the molecular weight, the faster the electrophoresis speed, and the invisible separation effect (the electrophoretic zone is shown only after dyeing) in this stage.
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 produce and 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 production method of a novel coronavirus 2019-nCoV antibody spectrum detection kit. The production method provided by the invention is different from the traditional immunoblotting method in that the detection test strip is prepared by using the recombinant immunoblotting method, protein gel electrophoresis and membrane conversion are not required, and the marking treatment is directly carried out on the nitrocellulose membrane, so that the production method of the detection kit is greatly simplified, the preparation time is shortened, and meanwhile, the production of the detection kit is completed by completing the process flows of preparation, subpackaging, packaging and the like of all liquid components.
The invention provides a production method of a novel coronavirus 2019-nCoV antibody spectrum detection kit, which adopts the following technical scheme:
a production method of a novel coronavirus 2019-nCoV antibody spectrum detection kit comprises the steps of preparing a detection test strip, a sample diluent, an eluent, a detection antibody and a substrate respectively, and subpackaging, combining and packaging the prepared detection test strip, sample diluent, eluent, detection antibody and substrate to form the detection kit; the preparation method of the detection test strip comprises the following steps:
(1) respectively scribing the novel coronavirus antigen and the goat anti-mouse IgG on a nitrocellulose membrane; 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) drying at 35-39 deg.C for 1-3 hr;
(3) completely immersing the nitrocellulose membrane dried in the step (2) in a sealing solution, and immersing for 0.5-2 hours at 15-25 ℃;
(4) drying at 35-39 deg.C for 1-3 hr;
(5) and (3) fixing the nitrocellulose membrane dried in the step (4) on a PVC (polyvinyl chloride) rubber plate, sticking label paper printed with serial numbers on one end of the short side of the PVC rubber plate, and cutting the label paper into test strips with the width of 3.5-4.0mm, namely the novel coronavirus 2019-nCOV antibody spectrum detection test strips.
Further, the drying in the step (2) and the step (4) is carried out in an environment with the humidity less than or equal to 30%.
Furthermore, the preparation processes of the detection test strip, the sample diluent, the eluent, the detection antibody and the substrate are all carried out in 10 ten thousand-level environment.
Further, the sample diluent is phosphate buffer solution with pH of 7-8, and is dispensed into a sample diluent bottle through a peristaltic pump under the environment of 10 ten thousand grades after being prepared.
Further, the eluent is phosphate buffer solution with pH of 7-8 added with Tween-20, and is prepared and subpackaged into eluent bottles by a peristaltic pump under the environment of 10 ten thousand.
Further, the detection antibody is a horse radish peroxidase-labeled mouse anti-human IgG antibody or IgM antibody or IgA antibody or IgE antibody, and is dispensed into a detection antibody bottle through a peristaltic pump under the environment of 10 ten thousand levels after being prepared.
Further, before the streaking in the step (1), a protein diluent is used for diluting the novel coronavirus antigen and the goat anti-mouse IgG, and the protein diluent is prepared by weighing 55.8g of disodium hydrogen phosphate, 5.5g of sodium dihydrogen phosphate and 9g of sodium chloride, adding 999m L of pure water for dissolving, absorbing 1m L proClin300 into the solution, stirring and uniformly mixing, fixing the volume to 1000m L, and standing at normal temperature for later use.
Further, the preparation method of the sealing liquid in the step (3) comprises the steps of weighing 55.8g of disodium hydrogen phosphate, 5.5g of sodium dihydrogen phosphate and 9g of sodium chloride, weighing 10g of bovine serum albumin, adding 900m of pure water L for dissolution, adding NaOH solution for adjusting the pH to 7.4, absorbing 1m of L proClin300 into the solution, uniformly stirring, fixing the volume to 1000m of L, and standing for later use at the temperature of 2-8 ℃.
Furthermore, the preparation method of the sample diluent comprises the steps of weighing 55.8g of disodium hydrogen phosphate, 5.5g of sodium dihydrogen phosphate and 9g of sodium chloride, adding 999m of pure water L to dissolve, sucking 1m of L proClin300 into the solution, stirring and uniformly mixing, fixing the volume to 1000m of L, and standing at normal temperature for later use.
Further, the eluent is prepared by weighing 55.8g of disodium hydrogen phosphate, 5.5g of sodium dihydrogen phosphate and 9g of sodium chloride, adding 997.5m of pure water L to dissolve, respectively absorbing 1m of L proClin300 and 1.5m of L Tween-20 into the solution, uniformly stirring, fixing the volume to 1000m of L, and standing at normal temperature for later use.
Further, the substrate contains trihydroxymethyl aminomethane and H2O2And 3, 3-diaminobenzidine.
Further, the preparation method of the substrate is to mix the substrate A and the substrate B in equal volume, the preparation method of the substrate A is to weigh 1.21g of tris (hydroxymethyl) aminomethane, add 900m L of pure water for dissolution, add HCl to adjust the pH to pH7.5, fix the volume to 1000m L, place at normal temperature for standby, the preparation method of the substrate B is to weigh 30% H2O20.1m L, weighing 0.01g of 3, 3-diaminobenzidine, diluting to 100m L, and standing at 2-8 ℃ for later use.
Furthermore, the detection antibody is prepared by diluting a Horse Radish Peroxidase (HRP) -labeled mouse anti-human IgG antibody with an antibody diluent to a final concentration of 0.5 mu g/m L, uniformly mixing, standing at 2-8 ℃ for later use, wherein the antibody diluent is prepared by weighing 55.8g of disodium hydrogen phosphate, 5.5g of sodium dihydrogen phosphate and 9g of sodium chloride, weighing 20g of bovine serum albumin, adding 900m L of pure water for dissolution, adding NaOH solution for adjusting the pH to 7.5, sucking 1m L proClin300 into the solution, uniformly mixing by stirring, and keeping the volume to 1000m L, and standing at 2-8 ℃ for later use.
Further, the width of the test strip is 3.5-4.0 mm.
Furthermore, in the test strip, the length of the 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 arranged above the nitrocellulose membrane, the quality control line is spaced from the adjacent test line by 0.9-1.1cm, and the spacing between each test line is 0.4-0.6 cm.
Furthermore, the detection kit also comprises an instruction book, tweezers, an incubation groove and a self-sealing bag.
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 preparation method of the recombinant S1 protein comprises the following steps: synthesizing a full-length gene for coding a novel coronavirus 2019-nCOVSpike 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 affinity purification is carried out through ProteinA to obtain the recombinant plasmid. Theoretical Mw 76.5 kD.
Further, the preparation method of the recombinant RBD protein comprises the following steps: 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 affinity purification is carried out through ProteinA to obtain the recombinant plasmid. Theoretical Mw 52 kD.
Further, the preparation method of the recombinant N protein comprises the following steps: 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.
Further, the preparation method of the recombinant E protein comprises the following steps: 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.
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/m L (1 mu L/cm), 0.2-0.5mg/m L (1 mu L/cm), 0.1-0.2mg/m L (1 mu L/cm) and 0.1-0.2mg/m L (1 mu L/cm).
Further, the streaked concentration of the goat anti-mouse IgG was 0.2-1mg/m L (1. mu. L/cm).
The beneficial technical effects of the invention are as follows:
the production method provided by the invention is different from the traditional immunoblotting method in that the detection test strip is prepared by using the recombinant immunoblotting method, protein gel electrophoresis and membrane conversion are not needed, and the streaking treatment is directly carried out on the nitrocellulose membrane, so that the production method of the detection kit is greatly simplified, the preparation time is shortened, and the detection kit can be produced in a large scale in a short time. The detection kit produced by the production method 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 infectors 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 novel coronavirus 2019-nCoV antibody spectrum detection test strip of the present invention, wherein T1, T2, T3 and T4 respectively represent different detection lines, and different novel coronavirus antigen bands are marked on the different detection lines.
Fig. 2 is a schematic diagram of a novel coronavirus 2019-nCoV antibody spectrum detection test strip in embodiment 1 of the invention.
Fig. 3 is a schematic diagram of a novel coronavirus 2019-nCoV antibody spectrum detection test strip in embodiment 2 of the invention.
Fig. 4 is a schematic diagram of a novel coronavirus 2019-nCoV antibody spectrum detection test strip in embodiment 3 of the invention.
FIG. 5 is a flow chart of a production process of the novel coronavirus 2019-nCoV antibody spectrum detection kit disclosed by the invention.
Detailed Description
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 in mg/m L (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 concentrations of different recombinant S1 proteins on color in mg/m L (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 concentrations of different recombinant RBD proteins on color in mg/m L (1. mu. L/cm)
TABLE 4 Effect of streaking concentrations of different recombinant N proteins on color in mg/m L (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 5 Effect of streaking concentrations of different recombinant E proteins on color in mg/m L (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:
goat anti-mouse IgG antibody at 0.2-1mg/m L (1. mu. L/cm);
0.2-0.5mg/m L (1 μ L/cm) of recombinant S1 protein;
0.2-0.5mg/m L (1 μ L/cm) of recombinant RBD protein;
0.1-0.2mg/m L (1 μ L/cm) of recombinant N protein;
0.1-0.2mg/m L (1 μ 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, detecting the detection color development intensity of the test strip on the positive sample, and determining the appropriate time for detecting incubation 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 the detection color development intensity of the detection test strip on the positive sample at different temperatures, the appropriate detection incubation temperature is determined as follows: at room temperature (15-25 ℃).
Taking 48-person kits as an example, the reagents and consumables contained in the kit are shown in table 6.
TABLE 6 novel coronavirus (2019-nCOV) antibody profile detection kit
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 on the bottom, the quality control line is 0.9-1.1cm away from the adjacent detection line, and the interval of each detection line is 0.4-0.6 cm.
Example 1
1. Preparing a detection test strip:
1.1 preparation of protein dilutions and blocking solutions
The protein diluent is prepared by weighing 55.8g of disodium hydrogen phosphate, 5.5g of sodium dihydrogen phosphate and 9g of sodium chloride, adding 999m of pure water L to dissolve, sucking 1m of L proClin300 into the solution, stirring and mixing uniformly, fixing the volume to 1000m of L, and standing at normal temperature for later use.
Weighing 55.8g of disodium hydrogen phosphate, 5.5g of sodium dihydrogen phosphate, 9g of sodium chloride and 10g of bovine serum albumin, adding 900m of pure water L to dissolve, adding NaOH solution to adjust the pH value to 7.4, sucking 1m of L proClin300 into the solution, stirring and uniformly mixing, fixing the volume to 1000m of L, and standing at the temperature of 2-8 ℃ for later use.
1.2 preparation of test paper strip
(1) Preparing a quality control line solution C, namely diluting the goat anti-mouse IgG antibody to 0.5mg/m L by using a protein diluent;
(2) preparing a detection line solution T1, namely diluting the recombinant S1 protein into 0.2mg/m L by using a protein diluent;
(3) preparing a detection line solution T2, namely diluting the recombinant RBD protein into 0.2mg/m L by using a protein diluent;
(4) preparing a detection line solution T3, namely diluting the recombinant N protein into 0.1mg/m L by using a protein diluent;
(5) preparing a detection line solution T4, namely diluting the recombinant E protein into 0.1mg/m L by using a protein diluent;
(6) cutting a film: cutting the nitrocellulose membrane into membranes with the length of 310mm +/-5 mm;
(7) scribing, namely scribing and coating by using a metal spraying scribing instrument, wherein the line concentration of the coating of the detection line and the quality control line is 1 mu L/cm as shown in figure 2;
(8) drying, namely drying the coated nitrocellulose membrane at 37 +/-2 ℃ for 120min (the drying process is carried out in an environment with the humidity less than or equal to 30%);
(9) and (3) sealing: soaking the dried nitrocellulose membrane in a sealing solution for 30 min;
(10) drying, namely drying the sealed nitrocellulose membrane again at 37 +/-2 ℃ for 120min (the drying process is carried out in an environment with the humidity less than or equal to 30%);
(11) film pasting: tearing off the surface film of the middle part of the PVC rubber plate, and sticking the prepared nitrocellulose membrane;
(12) cutting the label paper printed with the serial number into 30cm × 1.1.1 cm by using a cutting machine;
(13) as shown in fig. 1, label paper printed with serial numbers is stuck to one end of the short edge of the PVC rubber plate, and the label paper is assembled;
(14) and vertically cutting the combined test paper strip into test paper strips with the width of 3.5mm by using a cutting machine.
2. Preparation of sample diluent:
0.1M phosphate buffer solution with the pH value of 7.4, weighing 55.8g of disodium hydrogen phosphate, 5.5g of sodium dihydrogen phosphate and 9g of sodium chloride, adding 999M of pure water L to dissolve, sucking 1M of L proClin300 into the solution, stirring and uniformly mixing, fixing the volume to 1000M of L, and standing at normal temperature for later use.
3. Preparation of eluent:
weighing 55.8g of disodium hydrogen phosphate, 5.5g of sodium dihydrogen phosphate and 9g of sodium chloride, adding 997.5m of pure water L to dissolve, respectively sucking 1m L proClin300 and 1.5m L Tween-20 into the solution, stirring and uniformly mixing, fixing the volume to 1000m L, and standing at normal temperature for later use.
4. Preparation of detection antibody:
a mouse anti-human IgG antibody labeled with horseradish peroxidase (HRP) was diluted with an antibody diluent to a final concentration of 0.5. mu.g/m L, mixed well, and left at 2 to 8 ℃ for further use.
5. Preparation of the substrate:
preparation of substrate A, weighing 1.21g of tris (hydroxymethyl) aminomethane, adding 900m of pure water L to dissolve, adding HCl to adjust pH to 7.5, metering to 1000m of L, and standing at normal temperature for later use.
Preparation of a substrate B: measuring 30% H2O20.1m L, weighing 0.01g of 3, 3-diaminobenzidine, diluting to 100m L, and standing at 2-8 ℃ for later use.
Preparation of a substrate: substrate A and substrate B were mixed in equal volumes. It is used as it is.
The preparation process is carried out in a hundred thousand grade environment.
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
1. Preparing a detection test strip:
1.1 preparation of protein dilutions and blocking solutions
The protein diluent is prepared by weighing 55.8g of disodium hydrogen phosphate, 5.5g of sodium dihydrogen phosphate and 9g of sodium chloride, adding 999m of pure water L to dissolve, sucking 1m of L proClin300 into the solution, stirring and mixing uniformly, fixing the volume to 1000m of L, and standing at normal temperature for later use.
Weighing 55.8g of disodium hydrogen phosphate, 5.5g of sodium dihydrogen phosphate, 9g of sodium chloride and 10g of bovine serum albumin, adding 900m of pure water L to dissolve, adding NaOH solution to adjust the pH value to 7.4, sucking 1m of L proClin300 into the solution, stirring and uniformly mixing, fixing the volume to 1000m of L, and standing at the temperature of 2-8 ℃ for later use.
1.2 preparation of test paper strip
(1) Preparing a quality control line solution C, namely diluting the goat anti-mouse IgG antibody to 0.2mg/m L by using a protein diluent;
(2) preparing a detection line solution T1, namely diluting the recombinant S1 protein into 0.3mg/m L by using a protein diluent;
(3) preparing a detection line solution T2, namely diluting the recombinant RBD protein into 0.3mg/m L by using a protein diluent;
(4) preparing a detection line solution T3, namely diluting the recombinant N protein into 0.2mg/m L by using a protein diluent;
(5) cutting a film: cutting the nitrocellulose membrane into membranes with the length of 310mm +/-5 mm;
(6) scribing, namely scribing and coating by using a metal spraying scribing instrument, wherein the line concentration of the coating of the detection line and the quality control line is 1 mu L/cm as shown in figure 3;
(7) drying, namely drying the coated nitrocellulose membrane at 37 +/-2 ℃ for 90min (the drying process is carried out in an environment with the humidity less than or equal to 30%);
(8) and (3) sealing: soaking the dried nitrocellulose membrane in a sealing solution for 40 min;
(9) drying, namely drying the sealed nitrocellulose membrane again at the temperature of 37 +/-2 ℃ for 90 min. (the drying process is carried out in the environment with the humidity less than or equal to 30 percent);
(10) film pasting: tearing off the surface film of the middle part of the PVC rubber plate, and sticking the prepared nitrocellulose membrane;
(11) cutting the label paper printed with the serial number into 30cm × 1.1.1 cm by using a cutting machine;
(12) label paper printed with serial numbers is stuck to one end of the short edge of the PVC rubber plate and combined;
(13) and vertically cutting the combined test paper strip into test paper strips with the width of 3.6mm by using a cutting machine.
2. Preparation of sample diluent:
0.1M, pH 7.4.4 phosphate buffer solution, namely weighing 55.8g of disodium hydrogen phosphate, 5.5g of sodium dihydrogen phosphate and 9g of sodium chloride, adding pure water 999m L for dissolution, sucking 1m L proClin300 into the solution, stirring and uniformly mixing, fixing the volume to 1000m L, and standing at normal temperature for later use.
3. Preparation of eluent:
weighing 55.8g of disodium hydrogen phosphate, 5.5g of sodium dihydrogen phosphate and 9g of sodium chloride, adding 997.5m of pure water L to dissolve, respectively sucking 1m L proClin300 and 1.5m L Tween-20 into the solution, stirring and uniformly mixing, fixing the volume to 1000m L, and standing at normal temperature for later use.
4. Preparation of detection antibody:
a mouse anti-human IgG antibody labeled with horseradish peroxidase (HRP) was diluted with an antibody diluent to a final concentration of 0.5. mu.g/m L, mixed well, and left at 2 to 8 ℃ for further use.
5. Preparation of the substrate:
preparation of substrate A, weighing 1.21g of tris (hydroxymethyl) aminomethane, adding 900m of pure water L to dissolve, adding HCl to adjust pH to 7.5, metering to 1000m of L, and standing at normal temperature for later use.
The preparation of the substrate B comprises the steps of measuring 30% of H2O20.1m L, weighing 0.01g of 3, 3-diaminobenzidine, and placing for later use under the condition of constant volume to 100m L.2-8 ℃.
Preparation of a substrate: substrate A and substrate B were mixed in equal volumes. It is used as it is.
The preparation process is carried out in a hundred thousand grade environment.
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 3
1. Preparing a detection test strip:
1.1 preparation of protein dilutions and blocking solutions
The protein diluent is prepared by weighing 55.8g of disodium hydrogen phosphate, 5.5g of sodium dihydrogen phosphate and 9g of sodium chloride, adding 999m of pure water L to dissolve, sucking 1m of L proClin300 into the solution, stirring and mixing uniformly, fixing the volume to 1000m of L, and standing at normal temperature for later use.
Weighing 55.8g of disodium hydrogen phosphate, 5.5g of sodium dihydrogen phosphate, 9g of sodium chloride and 10g of bovine serum albumin, adding 900m of pure water L to dissolve, adding NaOH solution to adjust the pH value to 7.4, sucking 1m of L proClin300 into the solution, stirring and uniformly mixing, fixing the volume to 1000m of L, and standing at the temperature of 2-8 ℃ for later use.
1.2 preparation of test paper strip
(1) Preparing a quality control line solution C, namely diluting the goat anti-mouse IgG antibody into 1mg/m L by using a protein diluent;
(2) preparing a detection line solution T1, namely diluting the recombinant S1 protein into 0.5mg/m L by using a protein diluent;
(3) preparing a detection line solution T2, namely diluting the recombinant RBD protein into 0.5mg/m L by using a protein diluent;
(4) preparing a detection line solution T3, namely diluting the recombinant E protein into 0.1mg/m L by using a protein diluent;
(5) cutting a film: cutting the nitrocellulose membrane into membranes with the length of 310mm +/-5 mm;
(6) scribing, namely scribing and coating by using a metal spraying scribing instrument, wherein the line concentration of the coating of the detection line and the quality control line is 1 mu L/cm as shown in figure 4;
(7) drying, namely drying the coated nitrocellulose membrane at 37 +/-2 ℃ for 100min (the drying process is carried out in an environment with the humidity less than or equal to 30%);
(8) and (3) sealing: soaking the dried nitrocellulose membrane in a sealing solution for 50 min;
(9) drying, namely drying the sealed nitrocellulose membrane again at the temperature of 37 +/-2 ℃ for 100 min. (the drying process is carried out in the environment with the humidity less than or equal to 30 percent);
(10) film pasting: tearing off the surface film of the middle part of the PVC rubber plate, and sticking the prepared nitrocellulose membrane;
(11) cutting the label paper printed with the serial number into 30cm × 1.1.1 cm by using a cutting machine;
(12) label paper printed with serial numbers is stuck to one end of the short edge of the PVC rubber plate and combined;
(13) and vertically cutting the combined test paper strip into test paper strips with the width of 4.0mm by using a cutting machine.
2. Preparation of sample diluent:
0.1M, pH 7.4.4 phosphate buffer solution, namely weighing 55.8g of disodium hydrogen phosphate, 5.5g of sodium dihydrogen phosphate and 9g of sodium chloride, adding pure water 999m L for dissolution, sucking 1m L proClin300 into the solution, stirring and uniformly mixing, fixing the volume to 1000m L, and standing at normal temperature for later use.
3. Preparation of eluent:
weighing 55.8g of disodium hydrogen phosphate, 5.5g of sodium dihydrogen phosphate and 9g of sodium chloride, adding 997.5m of pure water L to dissolve, respectively sucking 1m L proClin300 and 1.5m L Tween-20 into the solution, stirring and uniformly mixing, fixing the volume to 1000m L, and standing at normal temperature for later use.
4. Preparation of detection antibody:
a mouse anti-human IgG antibody labeled with horseradish peroxidase (HRP) was diluted with an antibody diluent to a final concentration of 0.5. mu.g/m L, mixed well, and left at 2 to 8 ℃ for further use.
5. Preparation of the substrate:
preparation of substrate A, weighing 1.21g of tris (hydroxymethyl) aminomethane, adding 900m of pure water L to dissolve, adding HCl to adjust pH to 7.5, metering to 1000m of L, and standing at normal temperature for later use.
The preparation of the substrate B comprises the steps of measuring 30% of H2O20.1m L, weighing 0.01g of 3, 3-diaminobenzidine, and placing for later use under the condition of constant volume to 100m L.2-8 ℃.
Preparation of a substrate: substrate A and substrate B were mixed in equal volumes. It is used as it is.
The preparation process is carried out in a hundred thousand grade environment.
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.
The production process flow chart of the novel coronavirus 2019-nCoV antibody spectrum detection kit is shown in figure 5.
Test example
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, namely putting a test strip into a reaction tank, adding a sample diluent of 1m L, and uniformly mixing for 1min at room temperature (15-25 ℃);
(2) a step of primary incubation, in which a sample (human plasma or serum) 1m L is added, and incubation is carried out for 40min at room temperature (15-25 ℃);
(3) cleaning, pouring out liquid in the reaction tank, adding 1m L eluent into the reaction tank, incubating for 5min at room temperature (15-25 deg.C), and repeatedly cleaning once;
(4) secondary incubation, namely pouring out liquid in the reaction tank, adding 1m L detection antibody into the reaction tank, and incubating for 40min at room temperature (15-25 ℃);
(5) washing, namely pouring out liquid in the reaction tank, adding 1m L eluent into the reaction tank, and incubating for 5min at room temperature (15-25 ℃);
(6) cleaning: pouring out the liquid in the reaction tank, and repeatedly cleaning once;
(7) and (3) color development, namely pouring out liquid in the reaction tank, draining the reaction tank, adding a substrate of 200 mu L 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.
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> production method of novel coronavirus 2019-nCoV antibody spectrum detection kit
<160>4
<170>PatentIn version 3.3
<210>1
<211>681
<212>PRT
<213> Artificial sequence
<400>1
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
50 55 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
210 215 220
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
370 375 380
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
530 535 540
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
145 150 155 160
Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser
165 170 175
Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val
180 185 190
Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly
195 200 205
Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe 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
305 310 315 320
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
305 310 315 320
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 (9)
1. A production method of a novel coronavirus 2019-nCoV antibody spectrum detection kit is characterized in that the production method comprises the steps of preparing a detection test strip, a sample diluent, an eluent, a detection antibody and a substrate respectively, and subpackaging, combining and packaging the prepared detection test strip, the sample diluent, the eluent, the detection antibody and the substrate to form the detection kit; the preparation method of the detection test strip comprises the following steps:
(1) respectively scribing the novel coronavirus antigen and the goat anti-mouse IgG on a nitrocellulose membrane; 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) drying at 35-39 deg.C for 1-3 hr;
(3) completely immersing the nitrocellulose membrane dried in the step (2) in a sealing solution, and immersing for 0.5-2 hours at 15-25 ℃;
(4) drying at 35-39 deg.C for 1-3 hr;
(5) fixing the nitrocellulose membrane dried in the step (4) on a PVC (polyvinyl chloride) rubber plate, sticking label paper printed with serial numbers on one end of the short side of the PVC rubber plate, and cutting the label paper into test strips with the width of 3.5-4.0mm, namely the novel coronavirus 2019-nCOV antibody spectrum detection test strips.
2. The production method according to claim 1, wherein the drying in the steps (2) and (4) is performed in an environment with a humidity of 30% or less.
3. The method of claim 1, wherein the test strip, the sample diluent, the eluent, the detection antibody and the substrate are all prepared in a 10 ten thousand environment.
4. The method of claim 1 or 3, wherein the sample diluent is a phosphate buffer solution having a pH of 7 to 8.
5. The production method according to claim 1 or 3, wherein the eluent is a phosphate buffer solution of pH 7-8 to which Tween-20 is added.
6. The production method according to claim 1 or 3, wherein the detection antibody is a horseradish peroxidase-labeled mouse anti-human IgG antibody or IgM antibody or IgA antibody or IgE antibody.
7. The production method according to claim 1 or 3, wherein the substrate contains tris, H2O2And 3, 3-diaminobenzidine.
8. The method of claim 1 or 3, wherein the test strip has a width of 3.5-4.0 mm.
9. The method for producing goat anti-mouse IgG, according to claim 1 or 3, wherein in step (1), 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 0.2-0.5mg/m L, 0.2-0.5mg/m L, 0.1-0.2mg/m L and 0.1-0.2mg/m L, respectively, and the streaked concentration of the goat anti-mouse IgG is 0.2-1mg/m L.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010442384.8A CN111505316A (en) | 2020-05-22 | 2020-05-22 | Production method of novel coronavirus 2019-nCoV antibody spectrum detection kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010442384.8A CN111505316A (en) | 2020-05-22 | 2020-05-22 | Production method of novel coronavirus 2019-nCoV antibody spectrum detection kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111505316A true CN111505316A (en) | 2020-08-07 |
Family
ID=71873434
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010442384.8A Pending CN111505316A (en) | 2020-05-22 | 2020-05-22 | Production method of novel coronavirus 2019-nCoV antibody spectrum detection kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111505316A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112180084A (en) * | 2020-10-10 | 2021-01-05 | 上海臻格生物技术有限公司 | Novel coronavirus enzyme-linked immunoassay kit based on microfluidics |
| CN112540174A (en) * | 2020-12-02 | 2021-03-23 | 泰州泽成生物技术有限公司 | Novel coronavirus IgG detection kit and preparation method thereof |
Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1590409A (en) * | 2003-09-05 | 2005-03-09 | 马杰 | Antibody pointed at SARS coronavirus N protein antigen and its use in detecting SARS coronavirus or its antigen |
| CN1806175A (en) * | 2003-06-10 | 2006-07-19 | 新加坡科技研究局 | Method of diagnosing SARS corona virus infection |
| CN204330777U (en) * | 2014-05-19 | 2015-05-13 | 江苏金标世纪生物科技有限公司 | People's myeloperoxidase (MPO) fast quantification immunochromatographytest test kit |
| CN109232736A (en) * | 2017-07-10 | 2019-01-18 | 洛阳普莱柯万泰生物技术有限公司 | Specifically bind monoclonal antibody, pharmaceutical composition, kit and its application of transmissible gastro-enteritis virus |
| CN109232734A (en) * | 2017-07-10 | 2019-01-18 | 洛阳普莱柯万泰生物技术有限公司 | Specifically bind monoclonal antibody, pharmaceutical composition, kit and its application of hepatitis infectiosa canis virus |
| CN109609695A (en) * | 2018-12-29 | 2019-04-12 | 华南农业大学 | RPA-LED Visualization Kit for Detection of Porcine Epidemic Diarrhea Virus |
| CN110894217A (en) * | 2019-12-16 | 2020-03-20 | 中国农业大学 | Bovine coronavirus chimeric antigen and colloidal gold immunochromatography test paper card for detecting bovine coronavirus antibody |
| CN111089962A (en) * | 2020-03-25 | 2020-05-01 | 中山生物工程有限公司 | Colloidal gold kit for joint detection of novel coronavirus IgM/IgG antibody and preparation method |
| CN111122864A (en) * | 2020-03-25 | 2020-05-08 | 中山生物工程有限公司 | Novel coronavirus IgG antibody enzyme-linked immunosorbent assay kit and detection method thereof |
| CN111157723A (en) * | 2020-02-16 | 2020-05-15 | 广西中医药大学附属瑞康医院 | Novel rapid detection kit for coronavirus |
-
2020
- 2020-05-22 CN CN202010442384.8A patent/CN111505316A/en active Pending
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1806175A (en) * | 2003-06-10 | 2006-07-19 | 新加坡科技研究局 | Method of diagnosing SARS corona virus infection |
| CN1590409A (en) * | 2003-09-05 | 2005-03-09 | 马杰 | Antibody pointed at SARS coronavirus N protein antigen and its use in detecting SARS coronavirus or its antigen |
| CN204330777U (en) * | 2014-05-19 | 2015-05-13 | 江苏金标世纪生物科技有限公司 | People's myeloperoxidase (MPO) fast quantification immunochromatographytest test kit |
| CN109232736A (en) * | 2017-07-10 | 2019-01-18 | 洛阳普莱柯万泰生物技术有限公司 | Specifically bind monoclonal antibody, pharmaceutical composition, kit and its application of transmissible gastro-enteritis virus |
| CN109232734A (en) * | 2017-07-10 | 2019-01-18 | 洛阳普莱柯万泰生物技术有限公司 | Specifically bind monoclonal antibody, pharmaceutical composition, kit and its application of hepatitis infectiosa canis virus |
| CN109609695A (en) * | 2018-12-29 | 2019-04-12 | 华南农业大学 | RPA-LED Visualization Kit for Detection of Porcine Epidemic Diarrhea Virus |
| CN110894217A (en) * | 2019-12-16 | 2020-03-20 | 中国农业大学 | Bovine coronavirus chimeric antigen and colloidal gold immunochromatography test paper card for detecting bovine coronavirus antibody |
| CN111157723A (en) * | 2020-02-16 | 2020-05-15 | 广西中医药大学附属瑞康医院 | Novel rapid detection kit for coronavirus |
| CN111089962A (en) * | 2020-03-25 | 2020-05-01 | 中山生物工程有限公司 | Colloidal gold kit for joint detection of novel coronavirus IgM/IgG antibody and preparation method |
| CN111122864A (en) * | 2020-03-25 | 2020-05-08 | 中山生物工程有限公司 | Novel coronavirus IgG antibody enzyme-linked immunosorbent assay kit and detection method thereof |
Non-Patent Citations (2)
| Title |
|---|
| TARLAN MAMEDOV 等: "Sequence analysis and amino acid variations of structural proteins deduced from novel coronavirus SARS-CoV-2 strains, isolated in different countries" * |
| XIAOMEI ZHANG 等: "Proteome-wide analysis of differentially-expressed SARS-CoV-2 antibodies in early COVID-19 infection" * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112180084A (en) * | 2020-10-10 | 2021-01-05 | 上海臻格生物技术有限公司 | Novel coronavirus enzyme-linked immunoassay kit based on microfluidics |
| CN112540174A (en) * | 2020-12-02 | 2021-03-23 | 泰州泽成生物技术有限公司 | Novel coronavirus IgG detection kit and preparation method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111624348A (en) | Method for detecting novel coronavirus 2019-nCoV through multi-protein joint inspection combination | |
| CN111624349A (en) | Novel coronavirus 2019-nCoV antibody spectrum detection test strip | |
| CN111398603B (en) | Test strip for detecting novel coronavirus antibody, preparation method and application thereof | |
| CN111647054B (en) | Reagent for detecting novel coronavirus SARS-CoV-2 antibody and its application | |
| CN111505316A (en) | Production method of novel coronavirus 2019-nCoV antibody spectrum detection kit | |
| CN111665365A (en) | Novel coronavirus 2019-nCoV antibody spectrum detection kit | |
| CN112326973B (en) | Kit for detecting novel coronavirus antibody and application thereof | |
| CN113092776B (en) | A SARS-CoV-2 neutralizing antibody detection kit | |
| CN107663235A (en) | The related PLA2R recombinant proteins of idiopathic membranous nephropathy and its application | |
| CN101526520A (en) | Method and device for biological sample detection | |
| CN108226494A (en) | Porcine reproductive and respiratory syndrome virus ELISA antibody assay kits | |
| CN112946260A (en) | Fluorescence immunochromatography reagent for detecting new coronavirus antibody and preparation method thereof | |
| US6902903B1 (en) | Helicobacter pylori diagnostics | |
| EP0946874B1 (en) | Helicobacter pylori diagnostics | |
| CN114891091B (en) | NMDAR encephalitis resistant recombinant antigen and application thereof | |
| CN113024640A (en) | Epitope peptide antigen detection neutralizing antibody kit based on screening of binding domain of new coronavirus RBD and ACE2 receptors | |
| CN213423207U (en) | Novel coronavirus 2019-nCoV antibody spectrum detection test strip | |
| CN108333369B (en) | A kind of porcine reproductive and respiratory syndrome virus antibody detection method and its dedicated test card | |
| CN113912719B (en) | Monoclonal antibody for detecting mouse interleukin 6 and preparation method and application thereof | |
| CN112745390B (en) | Binding protein containing NT-proBNP antigen binding structural domain | |
| CN112707948B (en) | Polypeptide sequence combined with classical swine fever virus E0 protein and application thereof | |
| CN213423206U (en) | Novel coronavirus 2019-nCoV antibody spectrum detection test strip | |
| CN114720691A (en) | Kit for detecting biomarkers and preparation method and application thereof | |
| CN213423205U (en) | Novel coronavirus 2019-nCoV antibody spectrum detection test strip | |
| CN115703837B (en) | Recombinant antibody for resisting growth stimulus expression gene 2 protein |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20200807 |