CN111303297A - Recombinant protein for detecting 2019 novel coronavirus antibody by double-antigen sandwich method, test strip, preparation method and application - Google Patents
Recombinant protein for detecting 2019 novel coronavirus antibody by double-antigen sandwich method, test strip, preparation method and application Download PDFInfo
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Abstract
The invention relates to a recombinant protein for detecting 2019 novel coronavirus antibody by a double-antigen sandwich method, a test strip, a preparation method and application, and belongs to the technical field of virus detection. The amino acid sequence of the 2019 novel coronavirus antibody recombinant protein detected by the double-antigen sandwich method is shown as SEQ ID NO. 1. The recombinant protein is a fusion protein of a plurality of dominant epitopes of 2019-nCoV, can be used for preparing the antigen double-sandwich method 2019-nCoV virus antibody detection reagent, can be stored at normal temperature, can be detected at any time by a single person, is high in sensitivity, high in flux and low in instrument cost, is simple and convenient to operate, and can greatly improve the clinical use simplicity.
Description
Technical Field
The invention relates to the technical field of virus detection, and particularly relates to a recombinant protein for detecting 2019 novel coronavirus (2019-nCoV) antibody by a double-antigen sandwich method, a test strip, a preparation method and application.
Background
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 not been found in human before, and the 2019-nCoV is a seventh coronavirus isolated from human. According to related disease monitoring, a plurality of viral pneumonia cases are found, and are diagnosed as viral pneumonia/lung infection.
The novel coronavirus generally has a latent period of 3-14 days (the longest period is reported by individual cases to be 24 days) after infecting a human body, and no clinical symptoms exist in the latent period, so that the novel coronavirus is difficult to find. Current evidence indicates that latent infected individuals are also infectious, increasing the difficulty of blocking viral transmission. The clinical symptoms of the novel coronavirus infection are mainly nonspecific symptoms such as fever, cough, hypodynamia and the like, are difficult to distinguish from common cold, and the definite diagnosis of the novel coronavirus infection depends on laboratory diagnosis technology.
The diagnosis of infection by the present novel coronavirus mainly relies on detection of virus-specific gene sequences based on Polymerase Chain Reaction (PCR) or nucleic acid sequencing. However, nucleic acid diagnosis needs a special laboratory (requiring 4 physically and completely independent regions) and dozens of supporting devices and professional operators (requiring authentication) to complete, and the detection process is complex in operation and can be completed in hours; the method is easy to generate aerosol pollution to cause false positive, and has the condition of missing detection caused by improper sampling, so that the nucleic acid detection is not suitable for large-scale field development.
The immunological diagnosis is mainly made by detecting the specific antibody produced after the human body is infected with virus through antigen-antibody immunoreaction. The immunological diagnosis method has the advantages of simple operation, high detection speed, no need of special fields and (complex) instruments and equipment, high sensitivity and the like, and is suitable for carrying out novel coronavirus infection screening and diagnosis work on site in a large scale. The reported methodology of immunological diagnosis mainly includes colloidal gold method, ELISA, etc. to detect virus specific IgM and IgG antibody in blood, and belongs to indirect method. The double-antigen sandwich method has high sensitivity and strong specificity, but no relatively ideal raw material for the double-antigen sandwich of 2019-nCoV is reported in the market and the literature at present.
Disclosure of Invention
The invention aims to provide a recombinant protein for detecting 2019 novel coronavirus antibody by a double-antigen sandwich method, a test strip, a preparation method and application. The recombinant protein is a fusion protein of a plurality of dominant epitopes of 2019-nCoV, can be used for preparing the antigen double-sandwich method 2019-nCoV virus antibody detection reagent, can be stored at normal temperature, can be detected at any time by a single person, is high in sensitivity, high in flux and low in instrument cost, is simple and convenient to operate, and can greatly improve the clinical use simplicity.
The invention provides a recombinant protein for detecting 2019 novel coronavirus antibody by a double-antigen sandwich method, wherein the amino acid sequence of the recombinant protein is shown as SEQ ID No. 1.
The invention also provides a test strip for detecting the 2019 novel coronavirus antibody by using the double-antigen sandwich method, which comprises a bottom plate, and a sample absorption pad, a fluorescent microsphere pad, a nitrocellulose membrane and a water absorption pad which are sequentially overlapped and pasted on the bottom plate, wherein the fluorescent microsphere pad is sprayed with 2019 novel coronavirus multi-dominant epitope fusion protein marked by fluorescent microspheres, a detection area and a quality control area are fixed on the nitrocellulose membrane, the detection area is sprayed with the 2019 novel coronavirus multi-dominant epitope fusion protein, and the quality control area is sprayed with an anti-N protein antibody; the 2019 novel coronavirus multi-dominant epitope fusion protein is a recombinant protein described in the technical scheme.
Preferably, the sample detected by the test strip comprises saliva, urine, serum, plasma and whole blood samples.
Preferably, the test strip further comprises a sample diluent; the sample diluent comprises a PB solution and Tween-20; the concentration of the PB solution is 0.008-0.012 mol/L; the volume content of the Tween-20 in the sample diluent is 0.4-0.6%.
Preferably, the fluorescent microsphere is a microsphere with rare earth ions Eu + wrapped by polystyrene, and the surface of the fluorescent microsphere is provided with carboxyl groups.
Preferably, the diameter of the fluorescent microsphere is 100-300 nm.
The invention also provides a preparation method of the test strip in the technical scheme, which comprises the following steps:
1) mixing and coupling the activated fluorescent microspheres and the recombinant protein in the technical scheme to obtain 2019 novel coronavirus multi-dominant epitope fusion protein marked by the fluorescent microspheres;
2) spraying the 2019 novel coronavirus multi-dominant epitope fusion protein marked by the fluorescent microspheres onto a glass fiber membrane to obtain a fluorescent microsphere pad;
3) respectively spraying 2019 novel coronavirus multi-dominant epitope fusion protein and an anti-N protein antibody on a detection region and a quality control region of a nitrocellulose membrane to obtain the nitrocellulose membrane fixed with the detection region and the quality control region;
4) and sequentially bonding a sample absorption pad, a fluorescent microsphere pad, a nitrocellulose membrane and a water absorption pad which are fixed with a detection area and a quality control area on the bottom plate in a lap joint manner to obtain the test strip.
Preferably, the preparation method of the activated fluorescent microsphere comprises the following steps: and mixing the fluorescent microspheres with a cross-linking agent and an activation buffer solution, and oscillating for 20-40 min to obtain the activated fluorescent microspheres.
The invention also provides application of the recombinant protein or the test strip in the technical scheme in preparation of a 2019 novel coronavirus detection reagent.
The invention provides a recombinant protein for detecting 2019 novel coronavirus antibody by a double-antigen sandwich method. The recombinant protein is a multi-dominant antigen epitope fusion protein, can be specifically combined with a novel coronavirus 2019-nCoV antibody, can better and completely capture the antibody, and can be used for early diagnosis on whether the novel coronavirus is infected by detecting the content of an antiviral antibody in a sample. The double-antigen sandwich test strip prepared from the recombinant protein can quickly and accurately detect a novel coronavirus 2019-nCoV antibody, can be used for detecting saliva, urine, serum, plasma and whole blood samples, has the detection time of less than 15min and higher specificity, can more accurately and sensitively detect an antiviral antibody, and is favorable for prevention, control and treatment of the novel coronavirus.
Compared with the prior art, the invention adopts a brand-new methodology to realize the detection of the 2019 novel coronavirus antibody, has the advantages of low cost of a detection instrument, simple and convenient operation and high speed, the protein can be stored and transported at normal temperature, is suitable for various samples (saliva, urine, serum, plasma and whole blood), has good stability (nucleic acid is a cold storage reagent), high specificity (compared with a double-antigen sandwich method for measuring virus antibodies by an indirect method, the double-antigen sandwich method is not influenced by a large amount of irrelevant Ig in the samples, so the specificity is high), high sensitivity (compared with a conventional indirect method adopting N protein or N + S protein, the prepared 2019nCoV protein multi-dominant epitope fusion protein can detect most of antibodies aiming at 2019-nCoV virus, so the sensitivity is higher), and early screening is realized (high sensitivity can detect low-concentration 2019-nCoV virus antibodies at early stage).
Compared with an RT-PCR method, the method has the advantages of low detection environment requirement, low detection instrument cost, avoidance of complex pretreatment process, rapid detection for 15min, high sample collection fault-tolerant rate, realization of high-flux detection, single-person packaging, good stability, high repeatability, simple operation and no need of professional personnel.
Compared with a sequencing method, the method has the advantages of low cost of the detection instrument, capability of storing the reagent at normal temperature, single-person packaging, good stability, high repeatability, simplicity in operation, no need of professional personnel and quickness in detection for 15 min.
Compared with the colloidal gold method and the ELISA method, the method has the advantages that: 1) the contrast method mainly adopts an indirect method for determination, and a large amount of endogenous irrelevant Ig compete with the specific Ig for the binding site of the anti-human Ig secondary antibody, so that the sensitivity is reduced, and the non-specific binding is more; the comparison method can only detect the antibodies of IgM and IgG subtypes, but the invention can detect all types of antibodies (including IgA, IgM, IgG and IgE) combined with the virus antigen, has higher sensitivity and can detect the existence of the virus antibody earlier; the contrast method mainly adopts the N protein of the virus or the mixture of the N protein and the S protein, the invention prepares the multi-dominant epitope fusion protein (from the N protein, the S protein and the M protein) of the 2019-nCoV virus, avoids the need of optimizing the mixing ratio of different antigen proteins in the contrast method, and can furthest improve the sensitivity of detecting the virus antibody by covering more epitopes.
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FIG. 1 shows the determination of a reference value of a double-antigen sandwich method detection 2019 novel coronavirus antibody detection reagent provided by the invention.
Detailed Description
The invention provides a recombinant protein for detecting 2019 novel coronavirus antibody by a double-antigen sandwich method, wherein the amino acid sequence of the recombinant protein is shown as SEQ ID NO. 1:
DQVILLNKHIDAYKTFPPTEPKKDKKKKADETQALPQRQKKQQTVTLLPA ADLDDFSKQGGGSDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGV GYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLT ESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTS NQVAVLYQDVNCTEVPVAIHAGGGSTLAILTALRLCAYCCNIVNVSLVKPS FYVYSRVKNLNSSRVPDLLV are provided. The dominant antigen epitopes of the novel coronavirus 2019-nCoVN protein, S protein and M protein are screened by calculation and prediction and are connected together by flexible polypeptide (GGGS) to form the 2019-nCoV virus multi-dominant epitope fusion protein. The preparation method of the recombinant virus is not particularly limited, and the recombinant virus can be synthesized by a method well known by a person skilled in the art, for example, the recombinant virus is produced and prepared by entrusted Beijing Deolping biotechnology limited, and the purity of the recombinant virus is preferably more than 95%. When the novel coronavirus 2019-nCoV invades a human body, the immune system of the body can generate immune response aiming at the dominant epitope of the virus protein. The recombinant protein (multi-dominant epitope fusion protein) can be specifically combined with a novel coronavirus 2019-nCoV antibody; therefore, whether or not the sample is infected with the novel coronavirus can be diagnosed at an early stage by detecting the content of the antiviral antibody in the sample.
The invention also provides a test strip for detecting the 2019 novel coronavirus antibody by using the double-antigen sandwich method, which comprises a bottom plate, and a sample absorption pad, a fluorescent microsphere pad, a nitrocellulose membrane and a water absorption pad which are sequentially overlapped and pasted on the bottom plate, wherein the fluorescent microsphere pad is sprayed with 2019 novel coronavirus multi-dominant epitope fusion protein marked by fluorescent microspheres, a detection area and a quality control area are fixed on the nitrocellulose membrane, the detection area is sprayed with the 2019 novel coronavirus multi-dominant epitope fusion protein, and the quality control area is sprayed with an anti-N protein antibody; the 2019 novel coronavirus multi-dominant epitope fusion protein is a recombinant protein described in the technical scheme. The invention provides a double-antigen sandwich method time-resolved immunofluorescence test strip capable of quickly and accurately detecting a novel coronavirus 2019-nCoV antibody, wherein the detection time of the test strip is less than 15min, the test strip adopts a novel coronavirus multi-dominant antigen epitope, fusion expression is carried out through a genetic engineering technology, the antibody can be captured better and more completely, and the double-antigen sandwich method has higher specificity, so that the antiviral antibody can be detected more accurately and sensitively, and the prevention, control and treatment work of the novel coronavirus is facilitated.
The fluorescent microsphere pad of the test strip is sprayed with 2019 novel coronavirus multi-dominant epitope fusion protein marked by fluorescent microspheres, namely the recombinant protein of the technical scheme. The fluorescent microsphere-labeled recombinant protein (recombinant antigen) is used for capturing an anti-2019-nCoV antibody in a sample, and the recombinant antigen contains various dominant epitopes in viruses and removes irrelevant sequences, so that the antiviral antibody in the sample can be captured efficiently, and possible non-specific reaction of the irrelevant sequence protein is avoided. In the invention, the fluorescent microsphere is preferably a microsphere with rare earth ions Eu + wrapped by polystyrene, and the surface of the fluorescent microsphere is provided with carboxyl groups. In the invention, the diameter of the fluorescent microsphere is preferably 100-300 nm. The present invention preferably couples the recombinant protein and the fluorescent microspheres using a cross-linking agent, which preferably comprises EDC, DCC or NHS, more preferably EDC. In the invention, the spraying concentration of the recombinant protein marked by the fluorescent microspheres is preferably 5-15 mug/mL, and more preferably 10 mug/mL. In the invention, the spraying is preferably realized by a gold-dot film spraying instrument. The source of the fluorescent microspheres is not particularly limited in the present invention, and conventional fluorescent microspheres known to those skilled in the art may be used as a commercially available source.
A detection area and a quality control area are fixed on a nitrocellulose membrane of the test strip, the detection area is sprayed with a 2019 novel coronavirus multi-dominant epitope fusion protein, and the quality control area is sprayed with an anti-N protein antibody. In the present invention, the source of the anti-N protein antibody is NP protein rabbit serum against 2019-nCoV virus, preferably produced by beijing de oxepin biotechnology limited. In the invention, the spraying concentration of the 2019 novel coronavirus multi-dominant epitope fusion protein is preferably 0.8-1.2 mg/mL, and more preferably 1 mg/mL; the amount of sprayed film is preferably 1.0 to 1.3. mu.L/cm, more preferably 1.2. mu.L/cm. The spraying concentration of the N protein antibody is preferably 0.4-0.6 mg/mL, and more preferably 0.5 mg/mL; the amount of sprayed film is preferably 1.0 to 1.3. mu.L/cm, more preferably 1.2. mu.L/cm.
The base plate, the sample absorption pad and the absorbent pad are not particularly limited, and the base plate, the sample absorption pad and the absorbent pad used in the conventional test strip in the field can be adopted.
In the present invention, the sample detected by the test strip preferably includes saliva, urine, serum, plasma and whole blood samples.
In the present invention, the test strip further comprises a sample diluent; the sample diluent comprises a PB solution and Tween-20; the concentration of the PB solution is 0.008-0.012 mol/L, and more preferably 0.01 mol/L; the volume content of the Tween-20 in the sample diluent is 0.4-0.6%, and more preferably 0.5%. Before the sample is detected, a sample diluent is preferably used for dilution, and the dilution is preferably 8-12 times, and more preferably 10 times.
The invention also provides a preparation method of the test strip in the technical scheme, which comprises the following steps:
1) mixing and coupling the activated fluorescent microspheres and the recombinant protein in the technical scheme to obtain 2019 novel coronavirus multi-dominant epitope fusion protein marked by the fluorescent microspheres;
2) spraying the 2019 novel coronavirus multi-dominant epitope fusion protein marked by the fluorescent microspheres onto a glass fiber membrane to obtain a fluorescent microsphere pad;
3) respectively spraying 2019 novel coronavirus multi-dominant epitope fusion protein and an anti-N protein antibody on a detection region and a quality control region of a nitrocellulose membrane to obtain the nitrocellulose membrane fixed with the detection region and the quality control region;
4) and sequentially bonding a sample absorption pad, a fluorescent microsphere pad, a nitrocellulose membrane and a water absorption pad which are fixed with a detection area and a quality control area on the bottom plate in a lap joint manner to obtain the test strip.
According to the invention, activated fluorescent microspheres are mixed and coupled with the recombinant protein in the technical scheme to obtain 2019 novel coronavirus multi-dominant epitope fusion protein marked by the fluorescent microspheres. In the present invention, the preparation method of the activated fluorescent microsphere preferably comprises the following steps: and mixing the fluorescent microspheres with a cross-linking agent and an activation buffer solution, and oscillating for 20-40 min to obtain the activated fluorescent microspheres. In the invention, the activation buffer is preferably 20-200 mmol/L MES, more preferably 100mmol/L MES, and the pH value is 6.0. In the present invention, the crosslinking agent preferably includes EDC, DCC or NHS, and more preferably EDC. In the invention, the ratio of the fluorescent microspheres, the cross-linking agent and the activation buffer is preferably 100 μ L to 2mg to 400 μ L. In the present invention, the time of the oscillation is preferably 30 min. The temperature for activating the fluorescent microspheres is not particularly limited, and the temperature can be room temperature, such as 20-25 ℃. After mixing and shaking, the invention preferably carries out centrifugal operation, abandons the supernatant and collects the activated fluorescent microspheres; the rotating speed of the centrifugation is preferably 8000-11000 rpm, more preferably 10000rpm, and the centrifugation is carried outThe time of the core is preferably 7-12 min, more preferably 10min, and the temperature of the centrifugation is preferably 2-5 ℃, more preferably 4 ℃. In the invention, the coupling is specifically to mix the activated fluorescent microspheres, the recombinant protein and a coupling buffer solution, and perform oscillation coupling. In the invention, the ratio of the fluorescent microspheres to the recombinant protein is preferably 90-110 muL to 2 mug, and more preferably 100 muL to 2 mug, based on the volume of the fluorescent microspheres before activation. In the invention, the time for coupling by shaking is preferably 25-35 min, and more preferably 30 min. The temperature of the oscillating coupling is preferably 22-27 ℃, and more preferably 25 ℃. In the invention, the coupling buffer solution is preferably a PB solution, and the concentration of the PB solution is preferably 90-120 mmol/L, and more preferably 100 mmol/L; the pH value of the PB solution is preferably 6.8-7.2, and more preferably 7.0. After the oscillation coupling, the invention preferably further comprises oscillation sealing operation; preferably, the vibration blocking is performed by adding a BSA solution into the vibration coupling system; the mass concentration of the BSA solution is preferably 0.9-1.3%, and more preferably 1.0%; the volume ratio of the BSA solution to the fluorescent microspheres before activation is preferably (0.9-1.1): 1, and the equal volume is more preferred. In the invention, the oscillation sealing time is preferably 10-14 h, and more preferably 12 h. The present invention is preferably centrifuged, resuspended and stored after the concussion is closed. In the invention, the rotation speed of the centrifugation is preferably 9000-12000 rpm, more preferably 10000rpm, the time of the centrifugation is preferably 8-11 min, more preferably 10min, and the temperature of the centrifugation is preferably 3-5 ℃, more preferably 4 ℃. In the present invention, the resuspension storage buffer resuspension solution preferably comprises 0.01% by mass of NaN3And a PB solution with the mass fraction of 0.1% BSA, wherein the pH value of the storage buffer solution is preferably 7.2-7.6, and more preferably 7.4. In the present invention, it is preferable that the storage buffer is washed and then stored, and the number of washing is preferably 1 to 2. In the invention, the preservation temperature is preferably 3-5 ℃, and more preferably 4 ℃; the preservation is preferably carried out in the absence of light.
After the 2019 novel coronavirus multi-dominant epitope fusion protein marked by the fluorescent microspheres is obtained, the 2019 novel coronavirus multi-dominant epitope fusion protein marked by the fluorescent microspheres is sprayed on a glass fiber membrane to obtain the fluorescent microsphere pad. In the invention, the 2019 novel coronavirus multi-dominant epitope fusion protein marked by the fluorescent microspheres is preferably diluted before spraying. In the present invention, the storage buffer is preferably used for dilution, and the concentration after dilution is preferably 5 to 15. mu.g/mL, more preferably 10. mu.g/mL. The invention preferably uses a gold-marking film gold spraying instrument for spraying, and the spraying speed is preferably 0.8-1.2 mu L/mL, and more preferably 1 mu L/mL. In the invention, the spraying is preferably followed by drying, the drying time is preferably 35-39 ℃, more preferably 37 ℃, and the drying time is preferably 2.5-3.5 h, more preferably 3 h.
Respectively spraying the 2019 novel coronavirus multi-dominant epitope fusion protein and the anti-N protein antibody on a detection region and a quality control region of a nitrocellulose membrane to obtain the nitrocellulose membrane fixed with the detection region and the quality control region. Before spraying, the 2019 novel coronavirus multi-dominant epitope fusion protein is preferably diluted to 0.8-1.2 mg/mL, and more preferably to 1 mg/mL. The invention preferably uses a PB buffer solution for dilution, the concentration of the PB buffer solution is preferably 0.03-0.06 mol/L, more preferably 0.05mol/L, and the pH value is preferably 7.0-7.3, more preferably 7.2. The invention preferably uses a gold-marking film gold spraying instrument to spray the 2019 novel coronavirus multi-dominant epitope fusion protein on a detection area (T) on an NC film, and the film spraying amount is preferably 1.0-1.3 mu L/cm, and more preferably 1.2 mu L/cm. The N protein antibody is preferably diluted by using the same PB buffer solution before spraying, preferably to be 0.4-0.6 mg/mL, and more preferably to be 0.5 mg/mL. According to the invention, the N protein antibody is sprayed on the detection area (C) on the NC membrane by using a gold-dot membrane gold spraying instrument, and the membrane spraying amount is preferably 1.0-1.3 muL/cm, more preferably 1.2 muL/cm. After spraying, drying is preferably carried out, the drying time is preferably 35-39 ℃, more preferably 37 ℃, and the drying time is preferably 4-6 hours, more preferably 5 hours.
And sequentially bonding a sample absorption pad, a fluorescent microsphere pad, a nitrocellulose membrane and a water absorption pad which are fixed with a detection area and a quality control area on the bottom plate in a lap joint manner to obtain the test strip. Specifically, the sample absorption pad, the glass fiber pad, the nitrocellulose membrane and the water absorption pad are preferably overlapped and stuck and fixed on the bottom plate from left to right, the tail end of the sample absorption pad is connected with the initial section of the glass fiber pad, the tail end of the glass fiber pad is connected with the initial end of the nitrocellulose membrane, the tail end of the nitrocellulose membrane is connected with the initial end of the water absorption pad, the initial end of the sample absorption pad is aligned with the initial end of the bottom plate, and the tail end of the water absorption pad is aligned with the tail end of the bottom plate. The test paper strip is preferably cut by a machine, preferably into small strips with the width of 3.8-4.0 mm, more preferably 3.96mm, and the small strips are arranged in a special plastic card to form the test paper card.
The detection principle of the invention is as follows: after a sample is added into the detection hole, the sample moves under the action of capillary action, a novel coronavirus antibody in the sample is combined with a novel coronavirus multi-dominant epitope fusion protein marked by a fluorescent microsphere to form a microsphere-antigen-antibody complex, the immune complex is chromatographed to a detection area (T) along a nitrocellulose membrane and is combined with the novel pre-coated coronavirus multi-dominant epitope fusion protein, and the fluorescence intensity of the immune complex is in direct proportion to the content of the novel coronavirus antibody in the sample; and (3) carrying out chromatography on the novel coronavirus multi-dominant epitope fusion protein which is not combined with the T line and marked by the microsphere to a quality control region (C), and combining the protein with the pre-coated rabbit anti-N protein antibody.
In the present invention, the method for using the test strip preferably comprises the following steps: and adding a sample to be detected into a sample adding hole of the test strip, acting for 15min at the temperature of 20-25 ℃, and detecting by using a fluorescence detector. And the fluorescence detector obtains the content of the corresponding anti-virus antibody through software processing and calculation according to the measured fluorescence signal intensity.
The invention also provides application of the recombinant protein or the test strip in the technical scheme in preparation of a 2019 novel coronavirus detection reagent.
The recombinant protein, the test strip, the preparation method and the application for detecting 2019 novel coronavirus antibody by using the double-antigen sandwich method are further described in detail in the following embodiments, and the technical scheme of the invention includes but is not limited to the following embodiments.
Example 1
Preparing a reagent strip for detecting a novel coronavirus 2019-nCoV antibody by a double-antigen sandwich method.
1. Preparation of novel coronavirus 2019-nCoV multi-dominant epitope fusion recombinant protein:
dominant antigen epitopes of novel coronavirus 2019-nCoVN proteins, S proteins and M proteins are screened by calculation and prediction and are connected together by flexible polypeptide (GGGS) to form the 2019-nCoV virus multi-dominant epitope fusion protein, and the amino acid sequence is shown as sequence SEQ ID NO. 1. The product is prepared by Beijing Deolping biotechnology limited company, and the purity is more than 95 percent.
DQVILLNKHIDAYKTFPPTEPKKDKKKKADETQALPQRQKKQQTVT LLPAADLDDFSKQGGGSDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPT NGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGT GVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPG TNTSNQVAVLYQDVNCTEVPVAIHAGGGSTLAILTALRLCAYCCNIVNVSL VKPSFYVYSRVKNLNSSRVPDLLV
2. Preparation of fluorescent microsphere labeled novel coronavirus 2019-nCoV multi-dominant epitope fusion antigen
(1) And (3) activation: suspending 100 μ L of microsphere suspension with embedded fluorescent dye and modified carboxyl functional group in 400 μ L of activating buffer (100mmol/L MES pH6.0), adding 2mg EDC, mixing, and activating at room temperature for 30 min;
(2) coupling: centrifuging the suspension of the step (1) at 4 ℃ and 10000rpm for 10min, then discarding the supernatant, suspending the suspension in a coupling buffer solution (100mmol/L PB pH7.0), adding 2 mu g of novel coronavirus 2019-nCoV dominant epitope fusion protein, uniformly mixing, and then performing oscillation coupling at 25 ℃ for 30 min;
(3) and (3) sealing: adding 100 mu L of 1% BSA solution into the suspension obtained in the step (2), uniformly mixing, and then oscillating and sealing at room temperature overnight;
(4) and (3) storage: centrifuging the suspension of (3) at 4 ℃ and 10000rpm for 10min, discarding the supernatant, resuspending in storage buffer (0.01% NaN3, 0.1% BSA, pH 7.4 PB buffer), washing the microspheres 1 time by this method, mixing uniformly, and storing at 4 ℃ in the dark.
3. Preparation of glass fiber mats
Diluting the stored fluorescent microsphere-marked novel coronavirus 2019-nCoV multi-dominant epitope fusion protein with a storage buffer solution to a concentration of 10 mu g/mL, spraying by using a gold-labeled dot-film gold spraying instrument at a spraying speed of 1 mu L/mL, drying for 3h at 37 ℃, taking out, sealing and storing.
4. Preparation of cellulose Nitrate (NC) membranes
Diluting the novel coronavirus 2019-nCoV multi-dominant epitope fusion protein to 1mg/mL by using 0.05mol/L, pH 7.2.2 PB buffer solution, and spraying the protein to a detection area (T) on an NC membrane by using a gold-labeled dot membrane gold spraying instrument, wherein the membrane spraying amount is 1.2 mu L/cm; diluting the rabbit anti-2019-nCoVN protein antibody to 0.5mg/mL by using 0.05mol/L PB buffer solution with pH7.2, and spraying the rabbit anti-2019-nCoVN protein antibody to a detection area (C) on an NC membrane by using a gold-labeled dot membrane gold spraying instrument, wherein the membrane spraying amount is 1.2 mu L/cm; drying at 37 deg.C for 5 h.
5. Assembly of test strips
A sample absorption pad, a glass fiber pad, a nitrocellulose membrane and a water absorption pad are sequentially overlapped and stuck and fixed on a bottom plate from left to right, the tail end of the sample absorption pad is connected with the initial section of the glass fiber pad, the tail end of the glass fiber pad is connected with the initial end of the nitrocellulose membrane, the tail end of the nitrocellulose membrane is connected with the initial end of the water absorption pad, the initial end of the sample absorption pad is aligned with the initial end of the bottom plate, the tail end of the water absorption pad is aligned with the tail end of the bottom plate, then the sample absorption pad is cut into small strips with the width of 3.96mm by a machine and the small strips are arranged in a special plastic card to form.
Example 2
Application of novel coronavirus 2019-nCoV antibody detection reagent strip adopting double-antigen sandwich method
1. Sample pretreatment
2. Detection with test strips
Accurately sucking 80 mu L of sample solution to be detected into a sample adding hole of a test strip by using a micropipettor, and acting for 15min at room temperature (20-25 ℃); inserting the test paper card into a carrier of a fluorescence detector, selecting an item to be detected through a touch display screen, pressing a 'start detection' button, and automatically performing scanning test on the test paper card by the fluorescence detector; and reading or printing the detection result on a display screen of the instrument.
3. Analysis of detection results
Quantitative detection
After the test is finished, the instrument obtains the ratio of the time-resolved fluorescence intensity of the detection area on the test paper card to the time-resolved fluorescence intensity of the quality control area, and obtains the content of the novel coronavirus 2019-nCoV antibody in the sample to be tested based on the relation curve between the ratio of the time-resolved fluorescence intensity of the detection area on the test paper card to the time-resolved fluorescence intensity of the quality control area and the novel coronavirus 2019-nCoV antibody which are built in advance.
The curve equation adopts four-parameter fitting, y ═ A-D)/(1 + (x/C) ^ B ] + D
Wherein: 2734.54812, 0.83893, 113356.11891, 0-0051
If the quality control area does not detect the intensity of the fluorescence signal, the incorrect operation process or the failure of the test paper card is indicated.
Example 3
Interference immunity detection
Positive whole blood samples were prepared by simulating the addition of 1% (V/V) murine anti 2019-nCoV N protein serum to normal human whole blood. Human IgG and human IgM with different concentrations are added into normal human whole blood and the simulated positive sample, the numerical values before and after the addition are tested, and whether endogenous irrelevant IgG and IgM have influence on the determination result is observed.
As can be seen from the table, the relative deviation before and after addition of the interfering substance was less than 5%, and the measurement results were hardly affected. The double-antigen sandwich method is not influenced by the content of non-virus specific antibodies in a sample, and has high specificity.
Example 4
Determination of a reference value (cutoff value) for a clinical sample
60 healthy people (without 2019 novel coronavirus infection) are selected as clinical samples, the results are shown in figure 1, and the cutoff value of the healthy people is 0.1 when the time-resolved immunochromatographic test strip for determining the 2019 novel coronavirus infection antibody is used for determining.
The embodiment shows that the 2019 novel coronavirus multi-dominant epitope fusion protein, namely the recombinant protein, provided by the invention realizes double-antigen sandwich detection of an antiviral antibody by fusing a plurality of dominant epitopes of a novel coronavirus structural protein, and is not interfered by irrelevant Ig in a sample, so that the detection specificity and the sensitivity are higher. Meanwhile, the test strip is suitable for saliva, urine and blood samples, is flexible and convenient, is beneficial to popularization of medical institutions, particularly basic medical treatment, and can effectively assist 2019 in prevention and control of novel coronavirus.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
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BEIJING DIAGREAT BIOTECHNOLOGY Co.,Ltd.
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Claims (9)
1. A recombinant protein for detecting 2019 novel coronavirus antibody by a double-antigen sandwich method is disclosed, wherein an amino acid sequence of the recombinant protein is shown as SEQ ID NO. 1.
2. A test strip for detecting 2019 novel coronavirus antibodies by a double-antigen sandwich method comprises a base plate, and a sample absorption pad, a fluorescent microsphere pad, a nitrocellulose membrane and a water absorption pad which are sequentially overlapped and pasted on the base plate, and is characterized in that the fluorescent microsphere pad is sprayed with 2019 novel coronavirus multi-dominant epitope fusion proteins marked by fluorescent microspheres, a detection area and a quality control area are fixed on the nitrocellulose membrane, the detection area is sprayed with 2019 novel coronavirus multi-dominant epitope fusion proteins, and the quality control area is sprayed with anti-N protein antibodies; the 2019 novel coronavirus multi-dominant epitope fusion protein is the recombinant protein as claimed in claim 1.
3. The test strip of claim 2, wherein the sample detected by the test strip comprises saliva, urine, serum, plasma, and whole blood samples.
4. The test strip of claim 2, further comprising a sample diluent; the sample diluent comprises a PB solution and Tween-20; the concentration of the PB solution is 0.008-0.012 mol/L; the volume content of the Tween-20 in the sample diluent is 0.4-0.6%.
5. The test strip of claim 2, wherein the fluorescent microsphere is a polystyrene-coated rare earth ion Eu +, and the surface of the fluorescent microsphere has carboxyl groups.
6. The test strip of claim 2 or 5, wherein the fluorescent microspheres have a diameter of 100-300 nm.
7. A method for preparing the test strip of any one of claims 2 to 6, comprising the steps of:
1) mixing and coupling the activated fluorescent microspheres with the recombinant protein of claim 1 to obtain 2019 novel coronavirus multi-dominant epitope fusion protein marked by the fluorescent microspheres;
2) spraying the 2019 novel coronavirus multi-dominant epitope fusion protein marked by the fluorescent microspheres onto a glass fiber membrane to obtain a fluorescent microsphere pad;
3) respectively spraying 2019 novel coronavirus multi-dominant epitope fusion protein and an anti-N protein antibody on a detection region and a quality control region of a nitrocellulose membrane to obtain the nitrocellulose membrane fixed with the detection region and the quality control region;
4) and sequentially bonding a sample absorption pad, a fluorescent microsphere pad, a nitrocellulose membrane and a water absorption pad which are fixed with a detection area and a quality control area on the bottom plate in a lap joint manner to obtain the test strip.
8. The method of claim 7, wherein the activated fluorescent microspheres are prepared by the method comprising the steps of: and mixing the fluorescent microspheres with a cross-linking agent and an activation buffer solution, and oscillating for 20-40 min to obtain the activated fluorescent microspheres.
9. Use of the recombinant protein of claim 1 or the test strip of any one of claims 2 to 6 in the preparation of a 2019 novel coronavirus detection reagent.
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