CN111624349A - Novel coronavirus 2019-nCoV antibody spectrum detection test strip - Google Patents
Novel coronavirus 2019-nCoV antibody spectrum detection test strip Download PDFInfo
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Abstract
The invention relates to a novel coronavirus 2019-nCoV antibody spectrum detection test strip, and belongs to the technical field of novel coronavirus 2019-nCoV detection. The invention provides a preparation method of the test strip, which directly marks a novel coronavirus antigen with high purity and specificity on a nitrocellulose membrane, and then carries out drying, sealing and drying treatment; and finally, sticking the nitrocellulose membrane on a PVC rubber plate, sticking label paper printed with serial numbers, and cutting into a proper size to obtain the detection test strip. The detection test strip is prepared by using the recombinant immunoblotting method, is different from the traditional immunoblotting method, does not need protein gel electrophoresis and membrane transfer, and directly performs scribing treatment on the nitrocellulose membrane, thereby greatly simplifying the preparation process of the detection test strip, shortening the preparation time and being easy for mass production.
Description
Technical Field
The invention relates to a novel coronavirus 2019-nCoV antibody spectrum detection test strip, 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 by using low voltage and large current to finish the transfer. 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 dye 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 which has never been found in human body before, and the disease caused by the virus is named as 2019 coronavirus disease (COVID-19) and deadly virus severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) by the world health organization.
The coronavirus genome 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 receptor. 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.
Currently, there are two methods for detecting novel coronaviruses: nucleic acid detection and serum antibody detection. Nucleic acid detection is a detection method recommended by world health organization, but the detection method has extremely high requirements on detection environment and extremely strong operation speciality and is difficult to carry out rapid large-scale test. Furthermore, heterogeneity of breath sample material and differences in sample collection locations, such as throat swabs, saliva, or endotracheal aspirates, can affect the sensitivity of 2019-nCoV virus nucleic acid detection.
The serum antibody detection mainly utilizes an immunochromatography method, and the method has simple operation and low requirement 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 defects of high false positive rate, and the blood of individual patients contains rheumatoid factors, heterophilic antibodies, autoantibodies, medicines, tumor cells and the like, so that cross reaction of tests is easily caused, and false positive results appear. Therefore, the serum antibody detection is only used as a supplementary detection for a suspected case of the new coronavirus nucleic acid detection, and cannot be used as a diagnostic index for screening alone.
Therefore, a novel coronavirus detection test strip which has low requirement on detection environment, is suitable for large-scale population screening, can avoid the problems of false negative and false positive, has high sensitivity and specificity and can be 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 test strip for detecting an antibody spectrum of a coronavirus 2019-nCoV. The detection test strip is prepared by using the recombinant immunoblotting method, is different from the traditional immunoblotting method, does not need protein gel electrophoresis and membrane transfer, and directly performs scribing treatment on the nitrocellulose membrane, thereby greatly simplifying the preparation process of the detection test strip, shortening the preparation time and being easy for mass production.
The invention provides a novel coronavirus 2019-nCoV antibody spectrum detection test strip, which adopts the technical scheme as follows:
a novel test strip for detecting an antibody spectrum of a coronavirus 2019-nCOV is characterized in that a nitrocellulose membrane is used as a solid phase carrier, and 3 or 4 detection lines (T lines) and 1 quality control line (C line) are marked on the nitrocellulose membrane; 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; the detection paper strip comprises label paper printed with serial numbers and a PVC (polyvinyl chloride) rubber plate, and the nitrocellulose membrane and the label paper printed with the serial numbers are sequentially fixed on the PVC rubber plate.
Furthermore, goat anti-mouse IgG is fixed on the quality control line.
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/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 preparation method of the novel coronavirus 2019-nCOV antibody spectrum detection test strip, and the technical scheme is as follows:
the preparation method of the novel coronavirus 2019-nCOV antibody spectrum 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) sequentially sticking the nitrocellulose membrane dried in the step (4) and the label paper printed with serial numbers on a PVC (polyvinyl chloride) adhesive plate to obtain the novel coronavirus 2019-nCOV antibody spectrum detection test paper strip.
Further, the width of the novel coronavirus 2019-nCOV antibody spectrum detection test strip is 3.5-4.0 mm.
Furthermore, the detection test strip is divided into an upper layer structure and a lower layer structure, the nitrocellulose membrane and the label paper printed with serial numbers are of the upper layer structure, and the PVC rubber plate is of the lower layer structure; the label paper printed with serial numbers is fixed at one end of the short edge of the PVC rubber plate.
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 on the upper part, the nitrocellulose membrane is arranged on the lower part, a quality control line is 0.9-1.1cm away from an adjacent detection line, and the interval of each detection line is 0.4-0.6 cm.
The beneficial technical effects of the invention are as follows:
the detection test strip is prepared by using the recombinant immunoblotting method, is different from the traditional immunoblotting method, does not need protein gel electrophoresis and membrane transfer, and directly performs scribing treatment on the nitrocellulose membrane, thereby greatly simplifying the preparation process of the detection test strip, shortening the preparation time and being easy for mass production. The specificity and the sensitivity of the detection test strip provided by the invention are both more than 99.5%.
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.
Detailed Description
Preparing a sample diluent: 0.1M, ph7.4 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 use.
Preparation of a substrate A: trimethylolaminomethane (1.21 g) was weighed, dissolved in pure water (900 mL), and pH was adjusted to pH7.5 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 is weighed, 900mL of pure water is added for dissolution, and NaOH solution is added for adjusting the pH value to 7.5. Sucking 1mL of proClin300 into the solution, stirring and mixing uniformly, and fixing the volume to 1000 mL. Standing at 2-8 deg.C for use.
The preparation method of the detection antibody comprises the following steps:
the Horse Radish Peroxidase (HRP) marked mouse anti-human IgG antibody is diluted by using an antibody diluent to ensure that the final concentration is 0.5 mu g/mL, and the antibody is uniformly mixed and placed for standby at the temperature of 2-8 ℃.
And (3) selecting the marking concentration of the protein on the test strip: the color depth is indicated by "+"; "+ + + +" represents the strongest.
The detection method comprises the following steps: after the protein is scribed to prepare the detection test strip, a colloidal gold platform false positive sample and a positive sample, a detection antibody and a substrate are added, and the color development intensity of the strip is observed. 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)
Table 4 effect of streaking concentration of different recombinant N 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 | ++ | - | - | - |
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, 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 ℃).
The method for detecting the novel coronavirus 2019-nCoV antibody by using the detection test strip 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 ℃);
(6) cleaning: pouring out the liquid in the reaction tank, and repeatedly cleaning once;
(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 bands are developed, 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 test strip used in the following examples has a width of 3.5-4.0mm, a PVC offset plate length of 5-6cm, a label paper length of 1.1-1.2cm printed with serial numbers, a nitrocellulose membrane length of 4-5cm, an overlapping portion of 0.1-0.2cm, a label paper with serial numbers printed on the overlapping portion, a nitrocellulose membrane below, a quality control line spaced 0.9-1.1cm from the adjacent test line, and test lines spaced 0.4-0.6cm from each other.
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, scribing the goat anti-mouse IgG on the nitrocellulose membrane (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.
Example 2
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: 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 pH7.4 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, scribing the goat anti-mouse IgG on the nitrocellulose membrane (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 ℃;
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.
Example 3
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: 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 pH7.4 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 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 ℃;
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.
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 test strip prepared in the embodiment 1 are 100% and 100% respectively; 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 test strips prepared in examples 2 and 3 also achieve these test 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 test strip
<160>4
<170>PatentIn version 3.3
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Lys Ser Leu Ser His Ser Pro Gly Lys
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<210>3
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<212>PRT
<213> Artificial sequence
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Met His His His His His His Ser Asp Asn Gly Pro Gln Asn Gln Arg
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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
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Thr Arg Arg Ile Arg Gly Gly Asp Gly Lys Met Lys Asp Leu Ser Pro
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Arg Trp Tyr Phe Tyr Tyr Leu Gly Thr Gly Pro Glu Ala Gly Leu Pro
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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
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Ser Gly Lys Gly Gln Gln Gln Gln Gly Gln Thr Val Thr Lys Lys Ser
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290 295 300
Lys His Trp Pro Gln Ile Ala Gln Phe Ala Pro Ser Ala Ser Ala Phe
305 310 315 320
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325 330 335
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340 345 350
Lys Asp Gln Val Ile Leu Leu Asn Lys His Ile Asp Ala Tyr Lys Thr
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Phe Pro Pro Thr Glu Pro Lys Lys Asp Lys Lys Lys Lys Ala Asp Glu
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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
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Met Tyr Ser Phe Val Ser Glu Glu Thr Gly Thr Leu Ile Val Asn Ser
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20 25 30
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His
Claims (10)
1. A novel test strip for detecting an antibody spectrum of a coronavirus 2019-nCOV is characterized in that a nitrocellulose membrane is taken as a solid phase carrier, and 3 or 4 detection lines and 1 quality control line are marked on the nitrocellulose membrane; 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; the detection test strip further comprises label paper printed with serial numbers and a PVC rubber plate, and the nitrocellulose membrane and the label paper printed with the serial numbers are sequentially fixed on the PVC rubber plate.
2. The novel test strip for detecting the antibody spectrum of the coronavirus 2019-nCOV according to claim 1, wherein goat anti-mouse IgG is fixed on the quality control line.
3. The novel test strip for detecting the antibody profile of the coronavirus 2019-nCOV according to claim 1, wherein the amino acid sequence of the recombinant S1 protein is shown as SEQ ID No. 1.
4. The novel test strip for detecting the antibody profile of the coronavirus 2019-nCOV according to claim 1, wherein the amino acid sequence of the recombinant RBD protein is shown as SEQ ID No. 2.
5. The novel test strip for detecting the antibody profile of the coronavirus 2019-nCOV according to claim 1, wherein the amino acid sequence of the recombinant N protein is shown as SEQ ID No. 3.
6. The novel test strip for detecting the antibody profile of the coronavirus 2019-nCOV according to claim 1, wherein the amino acid sequence of the recombinant E protein is shown as SEQ ID No. 4.
7. The novel test strip for detecting the antibody spectrum of the coronavirus 2019-nCOV according to claim 1, wherein the streaking 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, 0.1-0.2mg/mL and 0.1-0.2 mg/mL.
8. The novel test strip for detecting the antibody spectrum of the coronavirus 2019-nCOV according to claim 2, wherein the streak concentration of the goat anti-mouse IgG is 0.2-1 mg/mL.
9. The method for preparing the novel coronavirus 2019-nCOV antibody spectrum detection test strip of any one of claims 1-8, which is characterized by comprising the following steps of:
(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, and pasting label paper printed with serial numbers, namely the novel coronavirus 2019-nCOV antibody spectrum detection test paper strip.
10. The method of claim 9, wherein the width of the novel coronavirus 2019-nCOV antibody spectrum detection test strip is 3.5-4.0 mm.
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Application publication date: 20200904 |