CN114264821A - Kit for detecting novel coronavirus neutralizing antibody and preparation method and application thereof - Google Patents
Kit for detecting novel coronavirus neutralizing antibody and preparation method and application thereof Download PDFInfo
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- CN114264821A CN114264821A CN202111605091.8A CN202111605091A CN114264821A CN 114264821 A CN114264821 A CN 114264821A CN 202111605091 A CN202111605091 A CN 202111605091A CN 114264821 A CN114264821 A CN 114264821A
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Abstract
The invention relates to a kit for detecting a novel coronavirus neutralizing antibody, a preparation method and application thereof, and belongs to the technical field of biomedical detection. The kit comprises a card shell and a test strip positioned in the card shell; the test strip comprises a PVC base plate, a water absorption pad, a reaction membrane, a combination pad and a sample pad, wherein the combination pad is loaded with a goat anti-mouse IgG antibody marked by colloidal gold and a detection antigen; the reaction membrane is provided with a detection line and a quality control line; the detection line is coated with capture antigen, and the quality control line is coated with a quality control substance; the capture antigen and the detection antigen are both S-RBD protein; the nucleotide sequence for coding the S-RBD protein is shown as SEQ ID No. 1; the amino acid sequence of the S-RBD protein is shown as SEQ ID No. 2. The kit has the characteristics of simplicity, convenience, rapidness, good stability, high sensitivity, good specificity and the like, and has important significance for utility evaluation of the new coronavirus vaccine, screening of inoculated population and evaluation of neutralizing antibody level of follow-up rehabilitation population.
Description
Technical Field
The invention relates to the technical field of biomedical detection, in particular to a kit for detecting a novel coronavirus neutralizing antibody and a preparation method and application thereof.
Background
The outbreak of new coronavirus pneumonia (COVID-19) poses a serious threat to global public health. Patients with COVID-19 have symptoms similar to those of patients with severe acute respiratory syndrome in 2003 and middle east respiratory syndrome in 2012, and show a series of symptoms including dry cough, fever, headache, dyspnea and the like, and the fatality rate reaches 3-5%. By 2021, 8 and 31 days, more than 2.1 million people in the world have diagnosed COVID-19, and 453 million people die, and the disease causes serious economic loss in the world. 20/1/2020, Weijian Commission No. 1/2020, takes pneumonia infected by novel coronavirus into infectious disease B, and takes preventive and control measures for infectious disease A. Pneumonia infected by the novel coronavirus is brought into quarantine infectious disease management.
An important spinous process S protein (spike protein) in a novel coronavirus (SARS-CoV-2) has a receptor binding domain called Receptor Binding Domain (RBD) which is a site directly bound to ACE2 on human cells. Research proves that S-RBD is the main epitope of the neutralizing antibody, and the specific antibody generated aiming at the S-RBD can block the binding of the novel coronavirus and ACE2, so that the virus can not invade host cells and plays a role in neutralizing the virus, and is called as the neutralizing antibody.
The new coronavirus has formed a global pandemic, which causes severe burden to public health, social economy, etc., and an effective control means is urgently required. Vaccines have become an important means of combating new coronaviruses in countries of the world. According to the health and health commission, the new coronavirus vaccine is inoculated for 206758.9 ten thousand doses according to the national cumulative report by 2021, 8 and 31 days. The detection of the novel coronavirus neutralizing antibody can reflect the infection rate of a population, the rehabilitation condition of an individual and the effect of a vaccine, but the traditional virus neutralizing experiment needs live viruses and cells, and has higher requirements on the safety of a laboratory and the skill of an operator. The traditional neutralization detection method with long time consumption and high requirement is not suitable for the novel coronavirus with high transmission speed and wide infection coverage. Therefore, it is of great interest to establish a method for rapidly detecting neutralizing antibodies.
Disclosure of Invention
In order to solve the technical problems, the invention provides a kit for detecting a novel coronavirus neutralizing antibody, and a preparation method and application thereof. The double antigen sandwich method of the S-RBD specific binding neutralizing antibody only adopts the S-RBD as an antigen to detect the total neutralizing antibody, including IgG, IgM and IgA total antibodies aiming at the neutralizing epitope of the S-RBD, and has higher sensitivity and specificity than the detection of IgG or IgM alone.
The invention provides a kit for detecting a novel coronavirus neutralizing antibody, which comprises a card shell, and a test strip positioned in the card shell; the test strip comprises a PVC bottom plate, a water absorption pad, a reaction membrane, a combination pad and a sample pad; the combination pad is loaded with a colloidal gold labeled goat anti-mouse IgG antibody and a detection antigen; a detection line T line and a quality control line C line are arranged on the reaction membrane; the detection line is close to one side of the combination pad, and the quality control line is close to one side of the water absorption pad; the detection line is coated with capture antigen, and the quality control line is coated with a quality control substance; encoding the capture antigen and the detection antigen are both S-RBD proteins; the nucleotide sequence of the S-RBD protein is shown as SEQ ID No. 1; the amino acid sequence of the S-RBD protein is shown as SEQ ID No. 2.
The sequence of SEQ ID No.1 is as follows:
AGGGTACAACCGACGGAGTCCATCGTACGGTTTCCGAACATCACTAACTTATGCCCATTCGGAGAAGTTTTCAATGCAACGAGATTCGCATCCGTCTATGCATGGAACCGAAAGCGAATTAGCAATTGCGTGGCCGATTATAGCGTACTATACAATTCAGCTAGCTTCTCCACGTTCAAATGCTATGGGGTATCGCCCACCAAGTTGAATGACCTATGTTTTACTAACGTCTATGCCGACAGTTTTGTAATAAGAGGAGACGAGGTACGGCAAATCGCACCGGGGCAAACTGGAAAAATTGCGGATTATAACTACAAGCTTCCGGACGATTTTACGGGTTGTGTGATAGCATGGAACTCGAATAACTTAGACAGCAAGGTCGGTGGCAACTACAACTACCTTTATAGGTTGTTTCGGAAATCAAATTTAAAGCCATTCGAGAGAGACATAAGTACGGAGATATACCAAGCCGGTAGTACACCCTGTAATGGTGTTGAAGGGTTCAATTGCTATTTCCCGTTGCAGTCGTATGGTTTTCAACCAACAAACGGTGTTGGATATCAGCCGTACCGGGTGGTTGTTCTATCCTTCGAGCTCCTACACGCCCCAGCTACGGTCTGTGGTCCAAAGAAGTCGACAAACCTAGTCAAGAATAAATGTGTTAACTTT。
the sequence of SEQ ID No.2 is as follows:
RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF。
in one embodiment of the invention, the S-RBD connects the RBD sequence of the novel coronavirus S protein to a vector, the sequence and a signal peptide sequence are added to the 5 'end, the coding sequence of 6 histidines (His-tag) is added to the 3' end, and the constructed expression vector is subjected to mass extraction of plasmids to obtain high-purity plasmids, so as to form the novel coronavirus S-RBD protein.
In one embodiment of the invention, the method for preparing the novel coronavirus S-RBD protein specifically comprises the following steps,
(1) connecting an RBD sequence of the novel coronavirus S protein to a vector, adding a sequence and a signal peptide sequence to a 5 'end, and adding a coding sequence of 6 histidines (His-tag) and a translation stop codon to a 3' end;
(2) extracting the constructed expression vector to obtain high-purity plasmid through plasmid, transiently transfecting the plasmid to HEK293F cells of suspension cells, and collecting supernatant of cell culture solution in 4-6 days; mixing all cell supernatants, filtering with a filter membrane, and removing cell debris to obtain filtrate;
(3) purifying cell supernatant by nickel ion affinity chromatography, washing affinity column with imidazole buffer solution containing 20mM imidazole to remove non-specific binding protein, performing gradient elution with imidazole buffer solution containing 50, 250, 500mM imidazole, concentrating eluate containing target protein, and further purifying by gel filtration chromatography.
In one embodiment of the present invention, in step (1), the signal peptide sequence is shown as SEQ ID No. 3.
The sequence of SEQ ID No.3 is as follows:
MHSSALLCCLVLLTGVRA。
in one embodiment of the present invention, in step (2), filtration is performed using a 0.22 μm filter membrane.
In one embodiment of the present invention, in step (3), the buffer solution in imidazole buffer solution is prepared by the following method: 7.8gNa2HPO4·2H2O, 17.54g NaCl, is dissolved in a proper amount of water to be 1000mL, and the pH is adjusted to 8.0.
In one embodiment of the invention, in step (3), the eluent is concentrated by selecting an ultrafiltration tube with a 10kDa molecular weight cut-off.
In one embodiment of the present invention, in step (3), the nickel ion metal chelating affinity chromatography medium is Ni-NTA.
In one embodiment of the present invention, in step (3), the gel filtration chromatography column is Superdex20010/300 GL.
In one embodiment of the present invention, after step (3), protein purity is identified using SDS-PAGE.
In one embodiment of the invention, the reaction film is arranged in the middle of the PVC base plate, the water absorption pad is arranged at one end of the PVC base plate and is overlapped and lapped on one end of the reaction film, the combination pad is arranged at the other end of the PVC base plate and is overlapped and lapped on the other end of the reaction film, and the sample pad is arranged on the PVC base plate next to the combination pad and is overlapped and lapped on the combination pad.
In one embodiment of the invention, the capture antigen has a membrane-scratching concentration of 0.5-1.5 mg/mL; the gold label concentration of the detection antigen is 0.1-0.3 mg/mL.
In one embodiment of the invention, the capture antigen has a streaking concentration of 1 mg/mL; the gold label concentration of the detection antigen is 0.2 mg/mL.
In one embodiment of the invention, the quality control substance is a goat anti-mouse IgG antibody.
In one embodiment of the invention, the preparation method of the reaction membrane comprises the following steps of coating a goat anti-mouse IgG antibody on a nitrocellulose membrane to form a quality control line C line, coating an S-RBD protein on the nitrocellulose membrane to form a detection line T line, wherein the scribing flow rates of the goat anti-mouse IgG antibody and the S-RBD protein on the nitrocellulose membrane are both 0.8-1.0 muL/cm, and the quality control line C line and the detection line T line are parallel to each other and are spaced at a distance of 3-5 mm.
In one embodiment of the invention, the preparation method of the reaction membrane comprises the following steps of coating a goat anti-mouse IgG antibody on a nitrocellulose membrane to form a quality control line C line, coating an S-RBD protein on the nitrocellulose membrane to form a detection line T line, wherein the scribing flow rates of the goat anti-mouse IgG antibody and the S-RBD protein on the nitrocellulose membrane are both 0.9 mu L/cm, and the quality control line C line and the detection line T line are parallel to each other and are spaced at a distance of 4 mm.
In one embodiment of the invention, the concentration of the goat anti-mouse IgG antibody is 0.8-1.2 mg/mL; the concentration of the S-RBD protein is 1.3-1.7 mg/mL.
In one embodiment of the invention, the concentration of the goat anti-mouse IgG antibody is 1 mg/mL; the concentration of the S-RBD protein is 1.5 mg/mL.
In one embodiment of the present invention, the goat anti-mouse IgG antibody and the S-RBD protein are diluted with a phosphate buffer, respectively.
In one embodiment of the present invention, the phosphate buffer is 0.01M in size and has a pH of 7.4.
In one embodiment of the invention, in the preparation process of the reaction membrane, the goat anti-mouse IgG antibody and the S-RBD protein are sequentially and respectively scratched on the nitrocellulose membrane through a gold spraying and membrane scratching integrated machine.
In one embodiment of the present invention, the preparation method of the conjugate pad comprises the following steps of spraying the colloidal gold-labeled goat anti-mouse IgG antibody and the detection antigen onto the glass fiber pad uniformly for drying.
In one embodiment of the invention, the spraying flow rate of the goat anti-mouse IgG antibody and the detection antigen is 8-12 mu L/cm.
In one embodiment of the invention, the spraying flow rate of the goat anti-mouse IgG antibody and the detection antigen is 10 μ L/cm.
In one embodiment of the present invention, the volume ratio of the gold-labeled goat anti-mouse IgG antibody to the detection antigen on the conjugate pad is 1: 1.2-1.8.
In one embodiment of the present invention, the volume ratio of the gold-labeled goat anti-mouse IgG antibody to the detection antigen on the conjugate pad is 1: 1.5.
in one embodiment of the invention, the drying temperature is 35-40 ℃; the drying time is 10-14 h.
In one embodiment of the invention, the temperature of the drying is 37 ℃; the drying time is 12 h.
The second purpose of the invention is to provide an application of the kit in detecting the novel coronavirus neutralizing antibody, which comprises the following steps of adding 8-12 mu L of a sample to be detected to a test strip for reaction for 10-15 min.
In one embodiment of the invention, the sample may be diluted with 2-3 drops of diluent; the diluent is phosphate buffer containing 4-6% polyvinylpyrrolidone (PVP).
In one embodiment of the invention, the application of the kit in detecting the novel coronavirus neutralizing antibody comprises the following steps of adding 10 mu L of a sample to be detected into a sample adding hole of the kit, and adding 3 drops of sample diluent for reacting for 15 min.
In one embodiment of the present invention, the sample diluent is a phosphate buffer containing 5% polyvinylpyrrolidone (PVP) and having a pH of 7.4.
The detection principle is that an immune complex is formed by an antigen marked by a marker and a corresponding antibody in serum to be detected, then the immune complex is combined with a specific antigen fixed on a solid phase carrier to form a marked antigen-antibody-solid phase antigen complex, and the content of the antibody is judged by the marker. Wherein the capture antigen is an antigen bound to an immobilization carrier for capturing an antibody, and the detection antigen is an antigen labeled with a label.
Compared with the prior art, the technical scheme of the invention has the following advantages:
(1) the kit for detecting the novel coronavirus neutralizing antibody takes the novel coronavirus S-RBD protein as a capture antigen and a detection antigen, and can effectively reduce false positives in a mechanism due to the fact that the method is based on the virus-specific antigen detection antibody; compared with the method for detecting the new crown IgG or IgM antibody singly or jointly, the double-antigen sandwich method detects the total antibody containing IgM, IgG and IgA, and the sensitivity and the specificity of the method are higher than those of the method for detecting the IgG or IgM singly or jointly.
(2) The kit for detecting the novel coronavirus neutralizing antibody has the characteristics of simplicity, convenience, rapidness, good stability, high sensitivity, good specificity and the like, and has important significance for utility evaluation of a novel coronavirus vaccine, screening of inoculated population and evaluation of neutralizing antibody level of follow-up rehabilitation population.
(3) The kit for detecting the novel coronavirus neutralizing antibody is generally suitable for evaluating the level of the neutralizing antibody of inoculated people after various types of vaccination, is simple to operate, is suitable for on-site rapid detection, can rapidly obtain a result, is intuitive in result, and can accurately determine the content of an object to be detected.
Drawings
In order that the present disclosure may be more readily and clearly understood, reference is now made to the following detailed description of the present disclosure taken in conjunction with the accompanying drawings, in which:
FIG. 1 is a gel filtration chromatogram of the novel coronavirus S-RBD protein further purified by gel filtration chromatography according to the present invention.
FIG. 2 is an electrophoresis chart of protein purity identified by SDS-PAGE according to the present invention.
FIG. 3 is a diagram showing the construction of a kit for detecting a neutralizing antibody against a novel coronavirus and a determination method according to the present invention; wherein, (a) is a schematic diagram of the test strip, (b) is a product schematic diagram of the kit, and (c) is a determination method.
FIG. 4 is a color development of the double-antigen sandwich assay kit of the present invention for the detection of a serum sample after vaccination.
FIG. 5 is a color development of the double-antigen sandwich assay kit of the present invention for the detection of unvaccinated serum samples.
FIG. 6 is a color development diagram of the double-antigen sandwich detection kit for detecting quality control neutralizing antibodies with different concentrations.
Detailed Description
The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
The reagents involved in the following examples are as follows:
resuspending buffer: is prepared by dissolving 0.1% of Tween 20, 5% of sucrose, 0.5% of PEG 6000, 0.02% of sodium azide, 1% of polyvinylpyrrolidone, 1% of mannitol, 1% of sorbitol and 0.2% of bovine serum albumin in phosphate buffer (0.02M, pH 7.4).
Buffer in imidazole buffer: 7.8g Na2HPO4·2H2O, 17.54g NaCl, is dissolved in a proper amount of water to be 1000mL, and the pH is adjusted to 8.0.
Examples
A kit for detecting a novel coronavirus neutralizing antibody and a preparation method and application thereof specifically comprise the following steps:
A. the preparation of the novel coronavirus S-RBD protein specifically comprises the following steps:
(1) connecting an RBD sequence of the novel coronavirus S protein to a pCMV3-S1sp-RBD vector, adding a sequence and a signal peptide sequence to the 5 'end, and adding a coding sequence of 6 histidines (His-tag) to the 3' end;
(2) extracting the constructed expression vector to obtain high-purity plasmid through plasmid, transiently transfecting the plasmid to HEK293F cells of suspension cells, collecting cell culture solution in 5 days, and centrifuging all the culture solution to obtain supernatant;
(3) the cell supernatant was purified by nickel ion affinity chromatography, the affinity column was washed with imidazole buffer containing 20mM imidazole to remove non-specifically bound proteins, and then gradient elution was performed with imidazole buffers containing 50 mM, 250 mM, and 500mM imidazole, respectively, and the peak eluted at each stage was collected and the molecular weight size and purity of the protein of interest were determined by SDS-PAGE. Concentrating the eluate containing the target protein with 10kDa ultrafiltration tube, purifying with Superdex20010/300GL gel filtration pre-packed column by GE AKATpurifier protein purification system, sampling the collected protein, and determining protein purity with SDS-PAGE as shown in figure 1 and figure 2.
As can be seen from the attached figures 1-2, the target protein with single peak type and high purity can be obtained after affinity chromatography and gel filtration chromatography.
B. The preparation of the colloidal gold nanoparticles specifically comprises the following steps:
22.5mL of chloroauric acid solution (4g/L) is added into a conical flask filled with 277.5mL of ultrapure water, the solution is kept under magnetic stirring and heated (300 ℃), after the solution boils, 8mL of sodium citrate solution (20mg/mL) is added into the solution and kept under heating and stirring for 30min, and after the reaction is finished, the solution is naturally cooled to room temperature, and the colloidal gold nanoparticle solution is obtained.
C. The preparation method of the colloidal gold nanoparticle-labeled goat anti-mouse IgG antibody specifically comprises the following steps:
taking 5mL of the colloidal gold nanoparticle solution into a 15mL centrifuge tube, adjusting the pH value of the colloidal gold nanoparticle solution to 8.2 by using 0.1M potassium carbonate solution, adding 50 mu g of goat anti-mouse IgG antibody, uniformly mixing by vortex, standing for 45min, adding 10% (M/v) bovine serum albumin solution, uniformly mixing by vortex, standing for 2h, centrifuging for 50min at 9000rpm and 4 ℃ after the reaction is finished, and re-dissolving the bottom precipitate by using 0.5mL of re-suspension buffer solution to obtain the goat anti-mouse IgG antibody marked by the colloidal gold nanoparticles.
D. The preparation method of the colloidal gold nanoparticle-labeled detection antigen (S-RBD protein) specifically comprises the following steps:
taking 5mL of the colloidal gold nanoparticle solution into a 15mL centrifuge tube, adjusting the pH value of the colloidal gold nanoparticle solution to 8.2 by using 0.1M potassium carbonate solution, adding 75 mu g S-RBD protein, uniformly mixing by vortex, standing for 45min, adding 10% (M/v) bovine serum albumin solution, uniformly mixing by vortex, standing for 2h, centrifuging for 50min at 9000rpm and 4 ℃ after the reaction is finished, and finally re-dissolving the sediment at the bottom by using 0.5mL of re-suspension buffer solution to obtain the detection antigen (S-RBD protein) marked by the colloidal gold nanoparticles.
E. The preparation method of the kit for detecting the novel coronavirus neutralizing antibody specifically comprises the following steps:
(1) preparation of a reaction film: a detection line T line and a quality control line C line which are parallel to each other are sequentially marked on the nitrocellulose membrane, and the interval distance between the detection line T line and the quality control line C line is 4 mm; the goat anti-mouse IgG antibody and the capture antigen S-RBD protein were diluted to 1mg/mL and 1.5mg/mL with phosphate buffer (0.01M, pH 7.4), respectively; and sequentially and respectively scratching the diluted goat anti-mouse IgG antibody and the capture antigen S-RBD protein on a nitrocellulose membrane through a gold spraying and membrane scratching integrated machine, wherein the scratching flow rate is 0.9 mu L/cm, and the membrane scratching concentration of the capture antigen is 1 mg/mL.
(2) Preparation of the bonding pad: the method comprises the following steps of (1) mixing a goat anti-mouse IgG antibody marked by colloidal gold and a capture antigen S-RBD protein according to the volume ratio of 1: and after mixing according to the proportion of 1.5, uniformly spraying the mixture on a glass fiber pad with the width of 1cm by using a gold spraying and film scratching integrated machine, wherein the spraying flow is 10 mu L/cm, and finally drying the mixture at 37 ℃ for 12 hours to prepare the bonding pad, wherein the gold standard concentration of the detection antigen is 0.2 mg/mL.
(3) Preparation of the kit: the method comprises the steps of adhering a reaction membrane to the middle position of a PVC (polyvinyl chloride) base plate, adhering a water absorption pad to one end of the PVC base plate, overlapping and overlapping the water absorption pad on one end of the reaction membrane, adhering a combination pad to the other end of the PVC base plate, overlapping and overlapping the combination pad on the other end of the reaction membrane, adhering a sample pad to the PVC base plate, overlapping and overlapping the sample pad on the combination pad, so as to obtain a test paper plate, longitudinally cutting the test paper plate into test paper strips of 3mm, so that each test paper strip comprises the PVC base plate, the water absorption pad, the reaction membrane, the combination pad and the sample pad, and finally packaging the test paper strips into a card shell, so as to obtain the immunochromatography test paper card for detecting the novel coronavirus neutralizing antibody, wherein the structure of the immunochromatography test paper card is shown in attached figure 3 (a-b). The kit determination method comprises the following steps: the T detection line and the C quality control line both have clear visible red strips and are judged to be positive, and the deeper the T line is, the stronger the positive is; only the C quality control line appears a clear visible red strip, and the result is judged to be negative; if the C quality control line does not have a red band, the C quality control line is judged to be invalid, as shown in the attached figure 3 (C).
Application example
Adding 10 mu L of serum sample to be detected into a sample adding hole of the detection card, adding 3 drops of sample diluent, flowing through the combination pad to react with the detection antigen (S-RBD protein) marked by the colloidal gold, flowing through the reaction membrane to combine with the fixed capture antigen and the goat anti-mouse IgG antibody marked by the colloidal gold, finally flowing to the water absorption pad, reacting at room temperature for 15min, and observing the result by naked eyes. Wherein, the sample diluent is phosphate buffer solution containing 5% polyvinylpyrrolidone and having pH of 7.4, and is 3 mL/tube.
The test paper card is used for detecting 40 collected serum samples inoculated with two new crown vaccines and 20 negative serum samples not inoculated with vaccines, the results are shown in the attached figures 4-5, and the test paper card can effectively detect positive serum, and the serum samples not inoculated with vaccines are negative and have good specificity.
Test example
Diluting the quality control neutralizing antibody to 50ng/mL, 10ng/mL and 2ng/mL by using phosphate buffer solution, adding 10 mu L of diluted antibody into a sample adding hole of the detection card, adding 3 drops of sample diluent, reacting at room temperature for 15min, and observing the result by naked eyes. The result is shown in figure 6, and the detection limit of the quality control neutralizing antibody can reach 2ng/mL, and the sensitivity is higher. Wherein the sample diluent is phosphate buffer containing 5% polyvinylpyrrolidone and having pH of 7.4.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the spirit or scope of the invention.
SEQUENCE LISTING
<110> university of south of the Yangtze river
<120> kit for detecting novel coronavirus neutralizing antibody, preparation method and application thereof
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Claims (10)
1. A kit for detecting a novel coronavirus neutralizing antibody comprises a card shell and a test strip positioned in the card shell; the test strip comprises a PVC base plate, a water absorption pad, a reaction membrane, a combination pad and a sample pad, and is characterized in that a colloidal gold labeled goat anti-mouse IgG antibody and a detection antigen are loaded on the combination pad; the reaction membrane is provided with a detection line and a quality control line; the detection line is close to one side of the combination pad, and the quality control line is close to one side of the water absorption pad; the detection line is coated with capture antigen, and the quality control line is coated with a quality control substance; the capture antigen and the detection antigen are both S-RBD protein; the nucleotide sequence for coding the S-RBD protein is shown as SEQ ID No. 1; the amino acid sequence of the S-RBD protein is shown as SEQ ID No. 2.
2. The kit for detecting the neutralizing antibody against the novel coronavirus according to claim 1, wherein the reaction membrane is disposed at a middle position of the PVC base plate, the water absorption pad is disposed at one end of the PVC base plate and overlapped on one end of the reaction membrane, the binding pad is disposed at the other end of the PVC base plate and overlapped on the other end of the reaction membrane, and the sample pad is disposed on the PVC base plate next to the binding pad and overlapped on the binding pad.
3. The kit for detecting the neutralizing antibody against the novel coronavirus according to claim 1, wherein the capture antigen has a streaking concentration of 0.5 to 1.5 mg/mL; the gold label concentration of the detection antigen is 0.1-0.3 mg/mL.
4. The kit for detecting the novel coronavirus neutralizing antibody of claim 1, wherein the quality control substance is a goat anti-mouse IgG antibody.
5. The kit for detecting the neutralizing antibody against the novel coronavirus as claimed in claim 1, wherein the preparation method of the reaction membrane comprises the steps of coating goat anti-mouse IgG antibody on a nitrocellulose membrane to form a quality control line and coating S-RBD protein on the nitrocellulose membrane to form a detection line, wherein the streaking flow rates of the goat anti-mouse IgG antibody and the S-RBD protein on the nitrocellulose membrane are both 0.8-1.0 μ L/cm, and the separation distance between the quality control line and the detection line is 3-5 mm.
6. The kit for detecting the novel coronavirus neutralizing antibody of claim 5, wherein the concentration of the goat anti-mouse IgG antibody is 0.8-1.2 mg/mL; the concentration of the S-RBD protein is 1.3-1.7 mg/mL.
7. The kit for detecting the neutralizing antibody against the novel coronavirus according to claim 1, wherein the preparation method of the conjugate pad comprises the step of uniformly spraying the colloidal gold-labeled goat anti-mouse IgG antibody and the detection antigen on a glass fiber pad and drying the pad.
8. The kit for detecting the neutralizing antibody against the novel coronavirus as claimed in claim 7, wherein the spraying flow rate of the goat anti-mouse IgG antibody and the detection antigen is 8-12 μ L/cm.
9. The kit for detecting the neutralizing antibody against the novel coronavirus according to claim 7, wherein the drying is performed at 35-40 ℃ for 10-14 h.
10. The use of the kit of any one of claims 1 to 9 for the detection of neutralizing antibodies against a novel coronavirus, comprising the step of adding 8 to 12 μ L of a sample to be tested to a test strip and reacting for 10 to 15 min.
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| CN202111605091.8A CN114264821B (en) | 2021-12-24 | Kit for detecting novel coronavirus neutralizing antibody, and preparation method and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202111605091.8A CN114264821B (en) | 2021-12-24 | Kit for detecting novel coronavirus neutralizing antibody, and preparation method and application thereof |
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| US20160024477A1 (en) * | 2013-03-15 | 2016-01-28 | The Research Foundation For The State University Of New York | Attenuated influenza viruses and vaccines |
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| CN113419061A (en) * | 2020-06-19 | 2021-09-21 | 南京金斯瑞生物科技有限公司 | Magnetic particle chemiluminescence kit for detecting SARS-CoV-2 virus neutralizing antibody and application thereof |
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| US20090075378A1 (en) * | 2007-02-20 | 2009-03-19 | Anaptysbio, Inc. | Somatic hypermutation systems |
| US20160024477A1 (en) * | 2013-03-15 | 2016-01-28 | The Research Foundation For The State University Of New York | Attenuated influenza viruses and vaccines |
| WO2021188906A1 (en) * | 2020-03-19 | 2021-09-23 | Nature's Toolbox, Inc. | Novel mrna-based covid-19 multi-valent vaccine and methods of scaled production of the same |
| CN113419061A (en) * | 2020-06-19 | 2021-09-21 | 南京金斯瑞生物科技有限公司 | Magnetic particle chemiluminescence kit for detecting SARS-CoV-2 virus neutralizing antibody and application thereof |
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