CN114740199A - SARS-CoV-2 neutralizing antibody reagent kit and its use - Google Patents

SARS-CoV-2 neutralizing antibody reagent kit and its use Download PDF

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CN114740199A
CN114740199A CN202210278881.8A CN202210278881A CN114740199A CN 114740199 A CN114740199 A CN 114740199A CN 202210278881 A CN202210278881 A CN 202210278881A CN 114740199 A CN114740199 A CN 114740199A
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章文羿
程焕义
黄清瑞
尚雨寒
南鹏娟
巩惠琴
赵会娟
陈瀚
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Beijing Anqi Biomedical Technology Co ltd
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Abstract

The invention provides a SARS-CoV-2 neutralizing antibody reagent box based on flow and its application, belonging to the field of biomedical detection. The invention simulates ACE2 receptor of cells by coating acceptor ACE2 protein of SARS-CoV-2 virus on fluorescent microspheres, simulates virus infection by using the strong and weak affinity of tripolymer RBD protein and ACE2, and quantitatively detects the amount of neutralizing antibody in a sample by measuring the efficiency of the neutralizing antibody for inhibiting the combination of ACE2 and tripolymer RBD, thereby being used for judging the expression level of the neutralizing antibody after vaccination and evaluating the protective power of vaccine.

Description

SARS-CoV-2 neutralizing antibody reagent kit and its use
Technical Field
The invention belongs to the field of biomedical detection, and particularly relates to a SARS-CoV-2 neutralizing antibody kit based on flow type and application thereof.
Background
Coronaviruses are a class of RNA viruses that cause respiratory and intestinal infections in animals and humans, of which the etiologies of Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS) and novel coronavirus pneumonia (COVID-19) belong to the genus coronavirus. According to the analysis of genome structure and phylogeny of coronavirus, the family coronaviridae can be divided into 4 genera of alpha, beta, gamma and delta, coronaviruses of alpha and beta generally infect mammals and humans, coronaviruses of gamma and delta mainly infect birds. Wherein SARS-CoV-2 is a novel coronavirus of beta genus, circular or elliptical, with a diameter of about 60-140nm, and has a crown-like shape under electron microscope.
Coronaviruses can cause respiratory tract infection of human beings or intestinal tract infection of animals, and the process of infecting host cells by the coronaviruses needs to involve receptors on the surface of host cell membranes. The S protein on the surface of coronaviruses recognizes and binds to a receptor and subsequently invades host cells via an endocytic pathway mediated by the receptor protein. Different coronaviruses can use different cellular receptors to accomplish invasion, for example, the receptor for HCoV-229E is aminopeptidase N (also known as CD13), the receptor for SARS-CoV is angiotensin converting enzyme 2(ACE2), and the receptor for MERS-CoV is dipeptidyl peptidase 4(DPP4, also known as CD 26). Studies have shown that neither aminopeptidase N nor DPP4 are receptors for SARS-CoV-2, but ACE2 can act as its receptor.
COVID-19 caused by SARS-CoV-2 is mainly manifested by symptoms of fever, dry cough, hypodynamia, etc., a few patients can be accompanied by watery nasal discharge, pharyngalgia and diarrhea, some patients can have dyspnea, and severe patients can rapidly progress to Acute Respiratory Distress Syndrome (ARDS), coagulation dysfunction, septic shock, etc. Besides, it has been proved that SARS-CoV-2 nucleic acid can be detected in the excrement of COVID-19 patient, and it can be used for prompting that SARS-CoV-2 can be transmitted through excrement-mouth route via digestive tract.
The prevention and treatment of infectious diseases mainly depend on means such as protecting susceptible people and blocking transmission ways. As the SRAS-CoV-2 is susceptible to various people and can be transmitted through the air, the development and inoculation of the vaccine form the immunity of the population, and the SRAS-CoV-2 becomes a key way for blocking the virus transmission. Currently, inactivated virus vaccines, adenovirus vaccines, RNA vaccines, recombinant protein vaccines, etc. have been approved for the market in succession, and how to evaluate the protective effect of the vaccines is the key to vaccine development and selection. The traditional vaccine effect mainly passes through a live virus plaque reduction neutralization test, a wild virus animal challenge test and the like, and as the new crown SARS-CoV-2 is propagated through air and needs to be completed in a BSL3 laboratory, the detection has high requirement on the laboratory, long time consumption and high cost, the preliminary evaluation of the protective capacity of the vaccine by improving the false virus neutralization test and enzyme-immune method neutralization antibody detection is gradually accepted and widely used. For example, patent CN202110065490.3 discloses a detection method and a detection kit for a SARS-CoV-2 neutralizing antibody, which are mainly based on an hACE2-RBD amplified latex enhanced turbidimetric immunoassay, and comprise first latex microspheres labeled with RBD of an S protein receptor binding domain of SARS-CoV-2 and second latex microspheres labeled with human hACE2, wherein the kit can be used for detecting SARS-CoV-2 virus and evaluating the clinical effect after inoculation of SARSCoV-2 vaccine. Patent CN202010560947.3 also discloses a SARS-CoV-2 neutralizing antibody detection kit, which comprises a solid phase carrier, an S protein antigen of SARS-CoV-2 and a competitive substance; the competitive substance is marked with a signal substance and can be specifically combined with the S protein antigen of the new coronavirus, and the detection of a neutralizing antibody can be realized through an immunodiagnosis technology to judge whether the new coronavirus is infected and whether the infection risk exists.
At present, although a kit for detecting SARS-CoV-2 neutralizing antibodies by an immunological method can determine whether or not a sample contains a neutralizing antibody by the inhibition ratio of ACE2 and RBD protein, it is difficult to quantitatively detect the amount of the neutralizing antibody in the sample. Therefore, it is desired to provide a detection kit which has high detection sensitivity and can quantitatively detect the amount of the SARS-CoV-2 neutralizing antibody.
Disclosure of Invention
Aiming at the defects, the invention provides a SARS-CoV-2 neutralizing antibody kit based on flow type and application thereof. The invention simulates ACE2 receptor of cells by coating acceptor ACE2 protein of SARS-CoV-2 virus on fluorescent microspheres, simulates virus infection by using the strong and weak affinity of tripolymer RBD protein and ACE2, and quantitatively detects the amount of neutralizing antibody in a sample by measuring the efficiency of the neutralizing antibody for inhibiting the combination of ACE2 and tripolymer RBD, thereby being used for judging the expression level of the neutralizing antibody after vaccination and evaluating the protective power of vaccine.
In order to achieve the above object, the technical solution of the present invention is as follows:
in one aspect, the invention provides an RBD protein of SARS-CoV-2, the RBD protein comprises 319-541 amino acid of SARS-CoV-2spike protein, and the 319-541 amino acid sequence of SARS-CoV-2spike protein is shown in SEQ ID NO. 7.
Specifically, the RBD protein also comprises a secretion signal peptide of IL-2, and the sequence of the secretion signal peptide is shown as SEQ ID NO. 6.
In further detail, the RBD protein can be directly expressed as an RBD monomer, an RBD dimer or assembled into a trimeric form by adding a T4 folding peptide.
More specifically, the amino acid sequence of the monomer RBD protein is shown as SEQ ID NO. 8.
More specifically, the amino acid sequence of the RBD dimer protein is shown in SEQ ID NO. 9.
More specifically, the RBD trimer protein also comprises a T4 folding peptide sequence, and the T4 folding peptide sequence is shown as SEQ ID NO. 10.
More specifically, the amino acid sequence of the RBD trimer protein is shown as SEQ ID NO. 11.
More specifically, the coding nucleotide sequence of the monomer RBD protein is shown as SEQ ID NO. 12.
More specifically, the coding nucleotide sequence of the RBD dimer protein is shown as SEQ ID NO. 13.
More specifically, the coding nucleotide sequence of the RBD trimer protein is shown as SEQ ID NO. 14.
On the other hand, the invention provides the application of the RBD protein in the preparation of a SARS-CoV-2 neutralizing antibody kit.
In still another aspect, the present invention provides a SARS-CoV-2 neutralizing antibody kit, which comprises the above RBD protein.
Specifically, the kit also comprises ACE2-hFc protein, wherein the ACE2-hFc protein comprises amino acid sequences of 18-740 positions of ACE2 protein, and the amino acid sequence of 18-740 positions of the ACE2 protein is shown as SEQ ID NO: 1.
More specifically, the ACE2-hFc protein further comprises a signal peptide sequence of HSA and an Fc sequence of human IgG1, wherein the signal peptide sequence of HSA is shown as SEQ ID NO. 2, and the Fc sequence of human IgG1 is shown as SEQ ID NO. 3.
More specifically, the amino acid sequence of the ACE2-hFc protein is shown as SEQ ID NO. 4.
More specifically, the encoding nucleotide sequence of the ACE2-hFc protein is shown as SEQ ID NO. 5.
Specifically, the kit further comprises a capture fluorescent microsphere, biotin, PE-streptavidin, a novel coronavirus neutralizing antibody calibrator, a sample diluent and a 20 × washing buffer.
More specifically, the capture fluorescent microspheres are 4-10 μm, preferably 5 μm.
Further specifically, the ACE2-hFc protein is coated by capture fluorescent microspheres.
More specifically, the RBD protein is coupled with biotin, and the molar concentration of the RBD protein and the biotin is 1:20-30, preferably 1: 25.
More specifically, the sample diluent used was 10mM phosphate buffer containing 0.025% Proclin300 and 2% BSA.
More specifically, the novel coronavirus neutralizing antibody calibrator is prepared by mixing a certain concentration of novel coronavirus neutralizing antibody solution and a freeze-drying protective agent in an equal volume ratio of 1:1, wherein the freeze-drying protective agent comprises 10% of trehalose and 10% of mannitol.
More specifically, the 20 × washing buffer comprises 160.0g/L of sodium chloride, 4g/L of potassium chloride, 58g/L of disodium hydrogen phosphate dodecahydrate, 4g/L of potassium dihydrogen phosphate and 1% of Tween-20 in volume ratio.
In another aspect, the invention provides the use of the RBD protein or the kit in the detection of SARS-CoV-2 neutralizing antibody.
In still another aspect, the present invention provides a method for detecting SARS-CoV-2 neutralizing antibody, said method is a non-disease diagnosis and treatment method, said method comprises using the above-mentioned RBD protein or kit to detect SARS-CoV-2 neutralizing antibody in the sample to be tested.
Specifically, the method comprises the following steps:
(1) preparing ACE2-hFc protein capture fluorescent microspheres;
(2) coupling RBD protein biotin;
(3) incubating the fluorescent microspheres prepared in the step (1) and the biotin-coupled RBD protein prepared in the step (2) with a sample to be detected or a novel coronavirus neutralizing antibody calibrator;
(4) after the incubation in the step (3) is finished, adding PE-streptavidin for incubation;
(5) flow detection and data analysis.
In particular, the detection method can be used for evaluating the content of neutralizing antibodies after vaccine immunization.
Compared with the prior art, the invention has the advantages that:
(1) in the kit, the protein hACE2 is coated on the surface of a fluorescent microsphere with the particle size of 4-10 microns, and the ACE2 protein on the surface of the fluorescent microsphere can simulate the mechanism of the action of a cell surface receptor ACE2 protein and a new coronavirus. (2) The kit of the invention uses a trimeric RBD protein with strong affinity with ACE2 receptor to simulate the interaction of the novel coronavirus with ACE2 receptor. (3) The kit can quantitatively calculate the amount of the SARS-CoV-2 neutralizing antibody in blood plasma and blood serum by adding the standard curve of the new coronavirus neutralizing antibody. (4) The kit provided by the invention has the advantages that the biotin is coupled on the trimer RBD protein, and the signal is detected by a PE-marked streptavidin method, so that the detection sensitivity of the SARS-CoV-2 neutralizing antibody is improved. (5) The invention relates to a method for detecting SARS-CoV-2 neutralizing antibody, which is based on the immunological technology of flow liquid chip, uses the receptor ACE2 protein of SARS-CoV-2 coated on fluorescent microsphere to simulate infected cells, and simultaneously uses RBD protein trimer to simulate SARS-CoV-2 virus to detect SARS-CoV-2 neutralizing antibody in blood plasma, blood serum or vaccine, and the whole experimental process can be completed within 3 h. And the HEK293 cell with over-expressed ACE2 on the cell surface is used for verifying the pseudovirus neutralizing antibody, the culture time of 2 days is required, the cost is high, and a large amount of sample detection is difficult to carry out. In addition, based on SARS-CoV-2 neutralizing antibody detection of competitive ELISA, ACE2 protein is directly coated on an ELISA plate, and RBD monomer is mostly used for simulating virus, which is greatly different from actual cell-virus binding reaction. Therefore, the method has the advantages of simple and convenient operation and better simulation of a cell and virus reaction system, and is basically consistent with the detection result of a pseudovirus neutralizing antibody inhibition test.
Drawings
FIG. 1 is an SDS-PAGE Coomassie blue staining test result of ACE2-hFc protein, wherein, lane M: standard protein Marker, lane 1: 100mM DTT reduction treatment, lane 2: electrophoresis in the non-reduced state.
FIG. 2 is a SDS-PAGE Coomassie blue staining test result of RBD protein.
FIG. 3 is a graph showing the results of affinity detection of monomeric RBD and ACE 2.
FIG. 4 is a graph showing the affinity detection results of dimer RBD and ACE 2.
FIG. 5 is a graph showing the results of affinity detection of trimeric RBD and ACE 2.
FIG. 6 is a graph showing the results of the detection of three RBD proteins by biotin coupling Western Blot.
FIG. 7 is a graph showing the correlation between plasma neocorona neutralizing antibody concentration and pseudovirus inhibition rate.
Detailed Description
The present invention will be described in further detail with reference to specific examples, which are not intended to limit the present invention, but to illustrate the present invention. The experimental methods used in the following examples are not specifically described, and the materials, reagents and the like used in the following examples are generally commercially available under the usual conditions without specific descriptions.
Basic example 1. SARS-CoV-2 neutralizing antibody kit and method of use
1. Protein expression vector pcDNA3.1(-), purchased from Youbao Bio; cloning strain DH5 alpha competent cell (cat # TSV-A07) purchased from Beijing Biotechnology Ltd, an organism from Ogaceae; expression proteins were purchased from ATCC cell banks using HEK 293F; high fidelity PCR enzyme (cat # 10136ES08) purchased from an assist in; restriction enzymes NheI (cat # FD0973) and NotI (cat # FD0594), available from Thermo; t4 DNA ligase (cat # M0202S), purchased from NEB; 5 μm fluorescent microspheres (cat # FM5CR03) from thermo; sodium chloride, potassium chloride, disodium hydrogen phosphate, dihydrogen phosphate, concentrated sulfuric acid (national drug group), sheep serum (Beijing Solibao Biotech), horseradish peroxidase (Luoyang Bai ao Tong laboratory center), and ELISA plate (Xiamen Yunpeng science and technology Co., Ltd.).
2. The main apparatus is as follows: ETC811 type PCR apparatus (Suzhou Dongsheng Xingheng scientific instruments Co., Ltd.), high-speed centrifuge (Hunan instruments laboratory instruments development Co., Ltd.), ZHWY-103D constant temperature culture shaker (Zhicheng analytical instruments manufacturing Co., Ltd.), DYY16D electrophoresis apparatus (Beijing six Biotechnology Co., Ltd.), Bio-Rad gel imaging analysis system (U.S. Bio-Rad), plate washer (model: ZMX190401), single-function enzyme labeling apparatus (model: Cmax Plus), electrothermal constant temperature culture (DHP-9032B), refrigerator, magnetic stirrer, 10. mu.L-1000. mu.L model micropipeter, 30-300. mu.L 8-channel pipetter, and the like.
3. Method establishment and kit preparation
ACE2-hFc and RBD protein expression and assay
Construction of ACE2-hFc and RBD protein plasmids
(1) ACE2-hFc sequence design and synthesis:
the ACE2 protein sequence refers to Uniprot database Q9BYF1 to take extracellular amino acids 18-740 (SEQ ID NO:1) for expression, meanwhile, a signal peptide sequence MKWVTFISLLFLFSSAYS (SEQ ID NO:2) of HSA is added at the N end, an Fc sequence (SEQ ID NO:3) of human IgG1 is added at the C end for fusion expression, and the full-length amino acid sequence is shown as SEQ ID NO: 4. Sequence optimization is carried out according to an HEK293F expression system, the ACE2-hFc nucleic acid sequence is shown as SEQ ID NO. 5, NheI restriction enzyme cutting sites and NotI restriction enzyme cutting sites are respectively added to two ends of the sequence, and the optimized sequence is sent to a general purpose organism for sequence synthesis.
(2) Designing and synthesizing an RBD sequence:
the sequence of the RBD protein refers to the Uniprot database P0DTC2 to take 319-541 bit amino acid of SARS-CoV-2spike protein for expression, and simultaneously, a secretion signal sequence MKWVTFISLLFLFSSAYS (SEQ ID NO:6) of IL-2 is added at the N terminal. The folding state of the protein can influence the interaction force between the protein and the protein, and the invention designs three RBD proteins with different folding configurations: RBD monomer, RBD dimer, and RBD trimer. Wherein, monomeric RBD protein: adding 6 XHis tag at the C end of 319-541 site amino acid protein (SEQ ID NO:7) of SARS-CoV-2spike protein, wherein the full-length amino acid sequence is shown as SEQ ID NO: 8; dimeric RBD protein: the 319-541 site amino acid proteins (SEQ ID NO:7) of two SARS-CoV-2spike proteins are connected by using 3 XGGGS, the C end is added with 6 XHis label, and the full-length amino acid sequence is shown as SEQ ID NO: 9; trimeric RBD protein: a trimer folding peptide is used, a T4 folding peptide sequence GYIPEAPRDGQAYVRKDGEWVLLSTFLG (SEQ ID NO:10) and a 6 XHis purification tag are added to the C end of amino acid protein (SEQ ID NO:7) at 319-541 bit of SARS-CoV-2spike protein for fusion expression, and the full-length amino acid sequence is shown as SEQ ID NO: 11. Nucleotide sequences of monomer RBD protein, RBD dimer and RBD trimer after sequence optimization according to an HEK293F expression system are respectively shown as SEQ ID NO. 12, SEQ ID NO. 13 and SEQ ID NO. 14, NheI restriction enzyme cutting sites and NotI restriction enzyme cutting sites are respectively added to two ends of the nucleotide sequence, and the optimized sequence is sent to a general-purpose organism for sequence synthesis.
(3) Connecting ACE2-hFc and RBD protein plasmids for transformation: the synthesized ACE2-hFc and three RBD nucleic acid sequences are cut by NheI and NotI restriction enzymes, then connected to pcDNA3.1-CMV (-) vector, transferred to escherichia coli for plasmid amplification and screening, and confirm that ACE2-hFc is correctly connected with three RBDs respectively and amplified.
(4) And (3) plasmid amplification and extraction: escherichia coli transformed with ACE2-hFc and RBD protein plasmids was amplified at 37 ℃ for about 18 hours in a 100mL LB medium at 220rpm culture, and the amplified bacteria were subjected to mass extraction of plasmids using a plasmid extraction kit from a Tiangen organism.
Expression of ACE2-hFc and three RBD proteins in HEK293F cells
(1) Cell preparation: the HEK293F cells were revived and expanded for culture to prepare for transfection.
(2) Cell transfection: for plasmid transfection, PEI was used to transfect the plasmid in a PEI to plasmid ratio of 2. mu.g PEI per 1. mu.g plasmid. For the assay, 2 flasks of 100mL HEK293F cells were prepared and grown to a cell density of 1X 106At 1mL, 1. mu.g plasmid per 1mL of medium was used for transfection. After transfection, cells were cultured for 6 days to allow the cells to sufficiently proliferate and secrete proteins.
(3) Protein collection: the cells cultured for 6 days were removed from the cell culture chamber, the cell culture medium was introduced into a 500mL centrifuge flask, cells were removed by centrifugation at 3000rpm, and the cell culture supernatant was collected, filtered through a 0.45 μm filter, and then purified and recovered by using a nickel column or a protein A purification column.
ACE2-hFc and three RBD protein purification and assay
(1) Purifying and detecting ACE2-hFc protein: the C-terminal of ACE2 protein is expressed by fusion with human IgG1 Fc, the protein is adsorbed on the cell supernatant directly by using a ProteinA purification column purchased from GE company, the protein is dissociated and eluted by using glycine with pH2.8, and the collected protein dialysis exchange solution is stored in 10mM phosphate buffer solution for later use.
The ACE2-hFc protein with signal peptide removed contains 943 amino acids and has a theoretical molecular weight of 108.36kDa, the purified protein is subjected to OD280 concentration determination by using Nandrop 300, 2 mu g of the protein is subjected to SDS-PAGE electrophoresis separation, and the purity and the molecular weight of the protein are detected by Coomassie brilliant blue staining. The detection result is shown in fig. 1, only one band is present in the purified protein lane near 100KD, which indicates that the purity of the purified ACE2 protein reaches more than 90%, and subsequent experiments can be performed.
(2) Three RBD protein purifications: the 6 XHis tags are added at the C tail ends of the three RBD proteins, so that the proteins can be directly purified by using a nickel column after being expressed, and the corresponding RBD proteins are collected. After the cell culture is finished, collecting culture solution supernatant fluid obtained by removing HEK293F cells through centrifugation, directly passing the collected protein supernatant fluid through a nickel column for adsorbing target proteins, fully washing the protein supernatant fluid by using 10mM phosphate buffer solution with pH7.4 after the adsorption is finished, removing impurity proteins, and finally dissociating and eluting the proteins by using imidazole. After purification, the collected RBD protein dialysis exchange solution is stored in 10mM phosphate buffer solution for later use.
The theoretical calculated molecular weight of the monomer RBD protein for cutting the signal peptide is 25.92KD, the theoretical calculated molecular weight of the dimer RBD protein for cutting the signal peptide is 51.78KD, and the theoretical molecular weight of the RBD protein for cutting the trimer of the signal peptide is 87.10 KD. And (3) purifying the RBD protein with the crude nickel column by using SEC again, taking 2 mu g of the three purified RBD proteins to perform SDS-PAGE electrophoretic separation, and staining with Coomassie brilliant blue to detect the purity and the molecular weight of the protein after the electrophoresis is finished. The detection result is shown in figure 2, the molecular weight of the monomer RBD protein purified by the nickel column combined with SEC in the gel is 25-35KD, the molecular weight of the dimer RBD protein in the gel is 48-63KD, and the molecular weight of the trimer RBD protein is 75-100KD, which indicates that the purity of the three purified RBD proteins can reach more than 90%, so that the subsequent coating and marking experiments can be carried out.
ACE2-hFc and three RBD protein affinity detection
The detection of the neutralizing antibody is completed by competing RBD protein with ACE2 through the principle of the flow-based SARS-Cov-2 new crown neutralizing antibody detection kit. After the ACE2-hFc and three types of RBD proteins are expressed and purified, the affinity analysis of monomer RBD protein, dimer RBD protein and trimer RBD protein and ACE2-hFc protein is carried out by using Biacore T200, and the detection steps are as follows:
1) ACE2-hFc protein immobilization: selecting a CM5 chip of GE as a sensing chip for protein-protein interaction detection, and fixing ACE2-hFc onto a CM5 sensing chip for later use by using an amino coupling kit of GE;
2) three RBD proteins were tested for affinity to ACE 2-hFc: three RBD proteins were diluted to concentration gradients of 1, 7.5, 15, 30, 60nM using Heps-PBS buffer, binding of three RBD proteins to ACE2-hFc was detected using single cycle kinetics, respectively, and after binding was regenerated using 0.05% sodium dodecyl sulfate solution in 10mM phosphate buffer, binding and regeneration data were recorded using a Biacore evaluation software 3.0 as 1: model 1 was subjected to kinetics fitting analysis. Affinity constants of monomer, dimer and trimer RBD and ACE2 are respectively 1.05e-8, 6.39e-9 and 1.82e-9, which shows that the expressed three RBD antigens have affinity activity with ACE2 and can be used for subsequent kit preparation, and the detection results of the combination and dissociation of ACE2 and RBD are shown in figures 3-5.
Coupling and assay of RBD protein Biotin
(1) Biotin coupling of RBD proteins:
when the protein biotin is coupled, the RBD monomer, the RBD dimer and the RBD trimer protein are reacted according to the molar concentration of the protein to the biotin (Alfaaesar, L09319) of 1:25, and the biotinylation of the protein is completed. The theoretical molecular weights of the purified monomer, dimer and trimer proteins are respectively 25.92KD, 51.78KD and 87.10KD, namely 78.8 mu g, 46.77 mu g and 23.45 mu g of N-hydroxysuccinimide biotin are needed for coupling 1mg of protein.
During reaction, weighing about 1mg of biotin, dissolving the biotin into 10mg/mL of DMSO for later use, adjusting the pH of 1mg of protein to 8.5 by using 1M sodium bicarbonate solution, respectively taking 7.8 muL, 4.6 muL and 2.3 muL of 10mg/mL of biotin into monomer, dimer and trimer RBD protein, fully and uniformly mixing, and carrying out shaking reaction at 37 ℃ in a dark place for 2 hours to complete the coupling reaction of the protein. After coupling, excess biotin was removed using a 10kD Millipore ultrafiltration tube and washed five times with PBS and centrifuged at 12000rpm for 5min, the biotin was removed sufficiently and the biotinylated protein was finally adjusted to 1mg/mL for storage using 10mM phosphate buffer.
(2) Bioconjugate quality control of three RBD proteins:
western Blot identification is carried out on three kinds of biotin-coupled RBD proteins, namely biotin-coupled RBD proteins, 200ng of the RBD proteins are subjected to SDS-PAGE electrophoresis and membrane transfer, SA-HRP is used for incubating a transfer membrane, enhanced luminescent agent and stabilizer are used for preparing color development liquid in a ratio of 1:1, imaging is carried out by a solar imager, the detection result is shown in figure 6, and the RBD trimer protein after biotin labeling is developed, which indicates that the three kinds of RBD proteins are successfully coupled with biotin.
3.3 preparation of liquid chip detection kit
A detection kit based on a flow-type liquid phase chip mainly comprises capture microspheres coated with ACE2 protein, RBD biotin protein, PE-streptavidin, a novel coronavirus neutralizing antibody calibrator, a sample diluent, 20 × washing buffer solution and a 96-well filter plate.
3.3.1 solution preparation
(1) Buffer solution: weighing MES (morpholine ethanesulfonic acid) and using ddH2O is prepared to 100mM, adjusted to pH6.1, and stored at 2-8 ℃ for later use.
(2) Activator EDAC (carbodiimide): weighing a certain amount of EDAC, and adding ddH 30min before experiment2O was dissolved to 10 mg/mL.
(3) Activator sulfo-NHS (N-hydroxythiosuccinimide): weighing a certain amount of sulfo-NHS, and adding ddH 30min before the experiment2O was dissolved to 10 mg/mL.
(4) Sealing liquid: after diluting 100mM MES buffer solution 4 times, Tween-20 and Proclin-300 were added to a final concentration of 0.025%, and BSA was added to a mass-to-volume ratio of 2%, to block the microspheres.
3.3.2. preparation of ACE2 Capture fluorescent microspheres
(1) Fluorescent microsphere coating
1) Washing the fluorescent microspheres: 1mL of 1% fluorescent microspheres with the size of 5 mu m is fully washed in 10mLMES buffer solution for 2 times, and detergent is removed;
2) activation of fluorescent microspheres: preparing an activation buffer solution of the microspheres according to the system shown in the following table 1, resuspending the washed fluorescent microspheres with the solution shown in the table 1, overturning the microspheres at room temperature, shaking and reacting for 30min, and fully activating the microspheres;
TABLE 1 fluorescent microsphere activation buffer system
Figure BDA0003553068540000101
Figure BDA0003553068540000111
3) Antibody coupling: centrifuging and precipitating the activated microspheres, fully washing for 2 times by using MES buffer solution, centrifuging for 5min at 6000rpm each time, fully removing the activating reaction reagent, and if the microspheres are obviously agglomerated after precipitation, performing microsphere dispersion by selecting ultrasound so as to facilitate subsequent full reaction; preparing a coupling reaction solution according to the following table 2, fully suspending fluorescent microspheres, reacting at 37 ℃, turning, oscillating and reacting for 60min to fully couple the antibody and the microspheres;
TABLE 2 coupling reaction system of microsphere and antibody
Reagent Volume (mL)
ddH2O 9.400
100mM buffer 2.500
5mg/mL antibody 0.100
Total 12.000
(2) Encapsulation of fluorescent microspheres
1) Washing: after the coupling reaction, washing for 2 times by using 25mM MES buffer solution, centrifuging for 5min at 6000rpm to remove the washing solution, and dispersing the microspheres by using ultrasonic if the microsphere agglomeration verification is carried out;
2) sealing the microspheres: using 2% BSA buffer solution, carrying out turnover oscillation reaction at 37 ℃ for 60min, fully sealing the microspheres, and after sealing, using 2% BSA solution with mass volume ratio prepared by 10mM phosphoric acid buffer solution with volume ratio of 0.025% Tween20 and 0.025% Proclin300 to resuspend and store for later use at 2-8 ℃ in a refrigeration way; after sealing, according to 5X 105Carrying out volume fixing on microspheres/mL;
3) and (3) subpackaging microspheres: according to the amount of 5000 microspheres/T, 5X 10 is adopted per tube5Packaging the microspheres, namely: each tube contains 1mL liquid microsphere.
4) The detection range and sensitivity of neutralizing antibodies of the ACE2-hFc capture microspheres and different RBD proteins are verified: adding 5000 fluorescent microspheres into each hole of a 96-hole filter plate, respectively adding 50 mu L of neutralizing antibody diluted to 10000, 3333.3, 1111.2, 370.4, 123.5, 41.1 and 13.75ng/mL and 50 mu L of biotinylated monomer, dimer and trimer RBD protein diluted to 2 mu g/mL, after the sample addition is finished, lightly oscillating, incubating at 37 ℃ for 60min, and fully washing for 5 times after the incubation is finished; PE-streptavidin was diluted to 0.25. mu.g/mL using 0.025% Tween20, 0.025% Proclin300 in 10mM phosphate buffer, loaded at 100. mu.L/well, and incubated at 37 ℃ for 30 min; after incubation, the microspheres were washed thoroughly 5 times and 200. mu.L of 10mM phosphate buffer was added to suspend the microspheres thoroughly. The 96-well plate is placed in a flow cytometer for fluorescence detection of the liquid phase chip. The results of the assay were processed using FCAP Array v3 software to analyze the linearity and sensitivity of the assay for each set of reactions, and the fluorescence values for each set after the reactions are shown in Table 3 below.
TABLE 3 MFI values measured for different RBD paired with ACE2
Figure BDA0003553068540000121
As shown in Table 3 above, the ACE2-hFc capture microspheres have a good detection range with trimeric RBD. When the fluorescence inhibition rate is 10 percent as a detection cutoff value, the detection limit of the capture microsphere of ACE2 paired with monomer RBD and dimer RBD is higher than 41.1ng/mL, and the detection limit paired with trimer RBD is lower than 41.1 ng/mL.
3.3.3. Preparation of liquid phase chip detection kit solution
Reagents were prepared as follows and dispensed in 96T/cassette.
(1) Preparation of biotinylation RBD solution and PE-streptavidin detection solution
1) Preparing and subpackaging a biotinylated RBD: diluting the coupled 1mg/mL biotinylated RBD protein by using 2% BSA and 2% casein solution prepared by using 0.025% Tween20, 0.025% Proclin300 and 10mM phosphate buffer solution according to 20 mu g/mL to obtain a biotinylated RBD binding reagent, subpackaging according to 70 mu L/tube, diluting 100 times by using sample diluent during detection, and adding the sample according to 50 mu L/hole;
2) preparing a PE-streptavidin detection reagent: diluting 1mg/mL PE-streptavidin purchased from Dake as a biological sample with 2% BSA and 2% casein solution prepared by using 10mM phosphate buffer solution of 0.025% Proclin300 to obtain 2.5. mu.g/mL PE-streptavidin detection reagent, subpackaging according to 1.2 mL/tube, diluting 10 times when in use, and adding 100. mu.L of sample into each hole for reaction;
(2) preparing a sample diluent:
a10 mM phosphate buffer containing 0.025% Proclin300 and 2% BSA protein was used as a sample diluent, and the sample was dispensed at 24 mL/tube.
(3) Lyophilized standard preparation (novel coronavirus neutralizing antibody calibrator):
1) preparing a freeze-drying protective agent: using sterile ddH2Preparing 10% of trehalose and 10% of mannitol by mass as freeze-drying protective agents;
2) and (3) packaging a neutralizing antibody: diluting the new crown neutralizing antibody to 20 mu g/mL by using a sterilized 10mM phosphate buffer solution, mixing the antibody diluent and a freeze-drying protective agent in an equal volume of 1:1, fully mixing uniformly, subpackaging according to 100 mu L/tube, namely each tube contains 1 mu g of neutralizing antibody, and placing in a refrigerator at the temperature of-80 ℃ for freezing for 12-18 h.
3) Freeze-drying of neutralizing antibody: after freezing, putting the protein subpackaging tube into a freeze dryer for vacuumizing and freeze-drying for not less than 18h, and fully freeze-drying the protein; screwing down the cover, and storing at 2-8 deg.C for use.
4)20 XWash buffer preparation
ddH was used in an amount of 160.0g/L sodium chloride, 4g/L potassium chloride, 58g/L disodium hydrogenphosphate dodecahydrate, 4g/L potassium dihydrogenphosphate and Tween-201%2O dissolved to obtain 20 Xwashing buffer, and 50 mL/bottle was used for split charging.
3.4. Kit application and Performance comparison
A SARS-CoV-2 neutralizing antibody detection kit consisting of capture microspheres coated with ACE2 protein, RBD trimer biological protein, PE-streptavidin, SARS-CoV-2 neutralizing antibody calibrator, sample diluent, 20 Xwashing buffer solution and 96-hole filter plates is used for detecting neutralizing antibodies in blood plasma of 12 cases of mice immunized with three groups of different SARS-CoV-2 recombinant RBD antigen vaccines. During the test, the following operations were carried out:
3.4.1. reagent preparation
1) Solution preparation: the 20 XWash buffer was washed with ddH2Diluting to 1 Xwashing buffer solution for later use;
2) preparing a freeze-dried standard: the standard product of the SARS-CoV-2 neutralizing antibody is freeze-dried powder, 3000g before uncovering is dissolved, and centrifugation is carried out for 30 s; add 1mL ddH2Dissolving O for 2-3min, and shaking to obtain standard substance of 10 μ g/mL; the dilution was sequentially carried out in 3-fold gradient, and 8 points were obtained in the dilution, thereby completing the gradient dilution from S8 to S1.
3) Sample preparation: the plasma sample to be tested was diluted 10-fold with the sample diluent to obtain 100. mu.L of plasma diluted sample.
3.4.2. Sample detection
1) Standard gradient and dilution plasma loading and incubation: according to the number of the standard products and the samples, corresponding detection reaction laths are taken, the fluorescent microspheres are fully and ultrasonically oscillated, suspended and uniformly mixed, 10 mu L of the fluorescent microspheres and 50 mu L of the diluted biotinylated RBD solution are added into each hole, then 50 mu L of the standard product gradient or the sample solution to be detected are sequentially added, a control hole only added with the diluent is made each time, and the samples are placed as shown in the table 4. After the sample is added, placing the 96-hole filter plate in an incubator at 37 ℃ for reacting for 60 min;
TABLE 4 Standard and sample loading layout
Figure BDA0003553068540000131
Figure BDA0003553068540000141
2) Washing: performing suction filtration, washing the solution with 1 × washing buffer solution at a concentration of 100 μ L/hole for 3 times, and absorbing the lower liquid by absorbent paper;
3) SA-PE incubation: diluting the sample diluent for 10 times in the SA-PE detection, adding 100 mu L of diluted SA-PE solution into each reaction hole, and reacting at 37 ℃ for 30 min;
4) washing: performing suction filtration, washing with 1 × washing buffer solution at a concentration of 100 μ L/hole for 3 times, and removing the liquid below with absorbent paper;
5) flow detection: and adding 200 mu L of 10mM phosphate buffer solution into the washed microspheres, fully oscillating to fully suspend the microspheres, and placing the microspheres in a flow cytometer to select a 96-well plate loading mode for loading detection.
6) After the reaction, the FCAP Array v3 software was used to process the assay results and analyze the expression level of the neutralizing antibody.
3.4.3. Analysis of detection results
The flow detection data are introduced into FCAP Array v3 to obtain the Mean Fluorescence Intensity (MFI), the concentration of the neutralizing antibody corresponding to the diluted plasma is calculated by curve fitting by ELISA Calc, and the concentration of the antibody in the plasma to be detected can be obtained by multiplying the concentration by the dilution factor of the antibody in the plasma. The MFI values of the gradients of the samples to be tested and of the standards are shown in Table 5 below, and the curve y-13403.65X is obtained by fitting^(-1.163)/(53.44*(-1.163)+X^(-1.163)) Fitting coefficient R2The antibody concentration in each sample was calculated as in table 6, 0.999.
TABLE 5 MFI values for standards and samples to be tested
Standard article MFI Sample(s) MFI Sample (I) MFI
A 4.625 13450 Control 13840 M8 3051
B 13.75 13190 M1 8066 M9 1160
C 41.125 12250 M2 8042 M10 1098
D 123.5 10970 M3 8465 M11 1431
E 370.375 8790 M4 8662 M12 1257
F 1111.125 3660 M5 3160
G 3333.375 1080 M6 3455
H 10000 910 M7 3716
TABLE 6 antibody concentration in samples to be tested
Sample(s) Concentration (ng/mL) Sample (I) Concentration (ng/mL)
M1 3747.00 M9 40543.23
M2 3771.14 M10 42689.17
M3 3362.32 M11 33201.23
M4 3183.07 M12 37580.07
M5 14691.59
M6 13268.64
M7 12181.55
M8 15280.25
3.4.4. Kit performance analysis
The test serum was evaluated for the neutralizing antibody inhibition rate of serum after immunizing mice by using HEK293 transgenic for expressing ACE2 protein for 12 cases of plasma detected by the kit. The inhibition rate of entry of pseudovirus into cells by the corresponding mouse serum is shown in Table 7 and FIG. 7. The antibody concentration detected by the kit and the false virus inhibition rate of the cell test are analyzed, the detection result of the kit has the same trend with the detection result of the false virus inhibition rate, and the kit can be preliminarily used for evaluating the antibody concentration in the blood plasma after immunization.
TABLE 7 serum pseudovirus inhibition
Sample(s) Inhibition ratio (%) Sample (I) Inhibition ratio (%)
M1 30.6 M9 84.4
M2 35.6 M10 84.8
M3 37.7 M11 85.4
M4 36.3 M12 83.7
M5 67.2
M6 65.0
M7 63.2
M8 68.0
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
SEQUENCE LISTING
<110> Beijing Angie biomedical science and technology Co., Ltd
<120> SARS-CoV-2 neutralizing antibody reagent kit and its application
<130> 20220309
<160> 14
<170> PatentIn version 3.5
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Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp Phe
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Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala Tyr
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Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys His
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Gly Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met Lys
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Phe Asn His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser
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Trp Asn Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn
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Asn Ala Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu
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Ala Gln Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu
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Gln Leu Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp
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Phe Ile Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu
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Ala Leu Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp
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Ile Ser Asn Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg
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Leu Gly Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly
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Ala Lys Asn Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu
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Phe Thr Trp Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser
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Thr Asp Trp Ser Pro Tyr Ala Asp Gln Ser Ile Lys Val Arg Ile Ser
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Leu Lys Ser Ala Leu Gly Asp Lys Ala Tyr Glu Trp Asn Asp Asn Glu
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Met Tyr Leu Phe Arg Ser Ser Val Ala Tyr Ala Met Arg Gln Tyr Phe
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Leu Lys Val Lys Asn Gln Met Ile Leu Phe Gly Glu Glu Asp Val Arg
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Val Ala Asn Leu Lys Pro Arg Ile Ser Phe Asn Phe Phe Val Thr Ala
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Pro Lys Asn Val Ser Asp Ile Ile Pro Arg Thr Glu Val Glu Lys Ala
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Ile Arg Met Ser Arg Ser Arg Ile Asn Asp Ala Phe Arg Leu Asn Asp
705 710 715 720
Asn Ser Leu Glu Phe Leu Gly Ile Gln Pro Thr Leu Gly Pro Pro Asn
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Gln Pro Pro Val Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys
740 745 750
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
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Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
770 775 780
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys
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Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
805 810 815
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
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Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
835 840 845
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
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Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
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Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
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Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
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Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
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Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
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Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
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<213> Artificial sequence (artificial sequence)
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ctagctagca tgaagtgggt gacctttatc tccctgcttt tcctgttttc ctccgcttat 60
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ctgcaggcgc tgcagcagaa cgggtcctct gtgctgtccg aagacaagag caagcggctt 360
aatacaattc tgaatactat gagtaccatc tactccacag gaaaagtctg caaccccgac 420
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tacgagcatc tgcacgccta cgtccgcgcg aagctcatga acgcctaccc cagctatatc 780
tcccctattg gctgcctgcc cgcccatctg ctgggggaca tgtggggcag attttggacc 840
aacttgtact ctttgactgt cccctttggg cagaaaccca acattgatgt caccgacgct 900
atggtcgacc aggcttggga tgcgcagagg atcttcaagg aggctgagaa gttctttgtt 960
agcgtgggct tgcctaatat gacccaagga ttctgggaga acagcatgct taccgacccc 1020
ggcaatgtgc agaaggcagt ttgccatcct acagcctggg atctgggcaa gggagacttc 1080
cggatcctga tgtgtactaa ggtgacaatg gacgatttcc tgacagccca ccatgaaatg 1140
gggcatattc agtacgacat ggcgtacgct gcacagccat ttcttctgcg gaacggcgcg 1200
aacgaggggt ttcacgaagc cgtcggtgaa attatgtctc tcagcgcggc aacgccaaag 1260
cacctgaaga gtatcggcct gctttctcct gatttccagg aggataacga gacagaaatc 1320
aacttcctgc tgaagcaagc attgacaatc gtcggcacgc ttccatttac ttatatgctg 1380
gaaaagtggc ggtggatggt gtttaaagga gagataccaa aggaccagtg gatgaagaag 1440
tggtgggaaa tgaagcggga aattgtgggg gtagttgaac cagtgcccca cgatgagaca 1500
tattgcgacc cagccagtct gtttcatgtt agcaacgact acagctttat acgatactat 1560
acccggaccc tctatcagtt ccagttccag gaagctctgt gccaggctgc aaagcatgaa 1620
ggacctctgc ataagtgcga cataagtaac tcaaccgagg ccggccagaa actcttcaac 1680
atgctgaggc ttgggaaatc cgagccatgg acgcttgcat tggagaatgt agtgggtgct 1740
aaaaacatga acgtcaggcc cctgttgaac tattttgaac cactgttcac atggctgaag 1800
gaccaaaaca aaaactcctt cgtgggctgg tccaccgact ggtccccata cgccgatcag 1860
agcattaaag tccgaatttc tctgaagtcc gccctcgggg acaaagccta cgagtggaat 1920
gataacgaaa tgtacctctt tcggagctcc gtcgcctacg ctatgcggca gtatttcctt 1980
aaagttaaga atcagatgat actgttcggc gaggaagatg tcagagtcgc taatctgaaa 2040
cctcgaataa gttttaactt ctttgtgact gccccaaaaa acgtgagcga tataattcca 2100
aggaccgaag tggagaaagc gatcaggatg agtcgcagcc ggattaatga cgcatttcgc 2160
ctgaacgata acagcctcga attcctcgga atccagccca ccctgggtcc acctaaccag 2220
cctccagtga gtgagcccaa atcatgtgac aagacccaca cttgtccacc gtgccctgcc 2280
cctgagcttt tggggggccc tagtgtgttt ctgttcccac cgaagccgaa ggacactctg 2340
atgatttcca gaactccaga agttacctgc gtggttgtgg atgtgtcaca tgaggatcct 2400
gaggtgaaat ttaactggta tgtcgatggt gtggaggtgc ataacgcgaa gactaaacca 2460
cgggaggaac agtataattc tacctaccgg gtggtgtcag tgctcacagt gctgcatcaa 2520
gattggttga acggaaaaga atataaatgt aaagttagca acaaagccct tccggccccc 2580
attgagaaga ccataagtaa ggccaagggt cagccacggg agccgcaggt ctataccctt 2640
cctccgagtc gggatgagtt gacaaaaaac caggtttccc tgacctgcct ggtgaaagga 2700
ttctacccaa gcgatatagc cgttgaatgg gagtccaatg ggcagccgga aaataattat 2760
aagaccactc ctcctgtgct ggattctgat ggaagcttct tcctctactc caaactgacc 2820
gtggataaaa gcagatggca acagggaaat gtgttcagct gttcagtcat gcatgaagcc 2880
ctgcataatc actacaccca gaagtccctt tcactgagcc ctgggaagta agtaataagc 2940
ggccgcaaat 2950
<210> 6
<211> 18
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 6
Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala
1 5 10 15
Tyr Ser
<210> 7
<211> 223
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 7
Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn
1 5 10 15
Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val
20 25 30
Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser
35 40 45
Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val
50 55 60
Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp
65 70 75 80
Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln
85 90 95
Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr
100 105 110
Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly
115 120 125
Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys
130 135 140
Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr
145 150 155 160
Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser
165 170 175
Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val
180 185 190
Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly
195 200 205
Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe
210 215 220
<210> 8
<211> 249
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 8
Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu
1 5 10 15
Val Thr Asn Ser Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro
20 25 30
Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg
35 40 45
Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val
50 55 60
Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys
65 70 75 80
Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn
85 90 95
Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile
100 105 110
Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro
115 120 125
Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp
130 135 140
Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys
145 150 155 160
Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln
165 170 175
Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe
180 185 190
Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln
195 200 205
Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala
210 215 220
Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys
225 230 235 240
Val Asn Phe His His His His His His
245
<210> 9
<211> 484
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 9
Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu
1 5 10 15
Val Thr Asn Ser Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro
20 25 30
Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg
35 40 45
Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val
50 55 60
Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys
65 70 75 80
Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn
85 90 95
Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile
100 105 110
Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro
115 120 125
Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp
130 135 140
Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys
145 150 155 160
Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln
165 170 175
Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe
180 185 190
Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln
195 200 205
Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala
210 215 220
Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys
225 230 235 240
Val Asn Phe Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Arg
245 250 255
Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu
260 265 270
Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr
275 280 285
Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val
290 295 300
Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser
305 310 315 320
Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser
325 330 335
Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr
340 345 350
Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly
355 360 365
Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly
370 375 380
Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro
385 390 395 400
Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro
405 410 415
Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr
420 425 430
Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val
435 440 445
Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro
450 455 460
Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe His His
465 470 475 480
His His His His
<210> 10
<211> 28
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 10
Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val Arg Lys
1 5 10 15
Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu Gly
20 25
<210> 11
<211> 277
<212> PRT
<213> Artificial sequence (artificial sequence)
<400> 11
Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu
1 5 10 15
Val Thr Asn Ser Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro
20 25 30
Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg
35 40 45
Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val
50 55 60
Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys
65 70 75 80
Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn
85 90 95
Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile
100 105 110
Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro
115 120 125
Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp
130 135 140
Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys
145 150 155 160
Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln
165 170 175
Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe
180 185 190
Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln
195 200 205
Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala
210 215 220
Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys
225 230 235 240
Val Asn Phe Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr
245 250 255
Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu Gly His
260 265 270
His His His His His
275
<210> 12
<211> 774
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 12
ctagctagca tgtatcggat gcaactgctt tcctgtatcg ccctgtctct ggccctcgtc 60
acaaacagtc gcgttcagcc cactgaatcc atcgttcgct ttcctaacat aaccaacctc 120
tgccccttcg gcgaggtgtt taatgccacg agattcgcct ccgtatatgc ttggaatcgg 180
aaaaggattt caaattgtgt ggccgattac tctgtgctct acaactctgc ctctttctcc 240
accttcaagt gctacggagt gtctcctact aaactcaacg atctgtgctt taccaacgtg 300
tacgccgatt ctttcgtgat ccgcggcgac gaagtgagac agattgcccc tggccagact 360
ggtaaaatcg ccgactataa ctacaagctc ccagacgact ttactggctg tgtcattgct 420
tggaacagta ataatctcga ctctaaagtc ggtgggaatt ataactacct ctaccgcctg 480
tttcggaagt ccaacctgaa gccgtttgaa agggacatat ctacagagat ctatcaggcc 540
gggagcaccc cctgtaatgg cgtggaggga tttaactgct acttcccact ccagtcttac 600
gggttccagc ccacgaacgg cgtgggatac cagccatata gagttgtcgt gttgagcttt 660
gagctgctgc atgcccctgc cacagtgtgt ggccccaaga agagcacaaa cctggtgaaa 720
aataaatgcg tgaacttcca tcaccaccac caccactaat aagcggccgc aaat 774
<210> 13
<211> 1461
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 13
ctagctagca tgtacaggat gcagctgctg agttgcatag ccttgagcct ggctctggtg 60
accaactctc gcgttcagcc tacagagagc atcgtacggt tcccaaatat aacgaatctg 120
tgtccgttcg gcgaggtgtt caacgctacc cgctttgcct cagtgtacgc ttggaataga 180
aagcgaatct ctaattgcgt cgctgattat agcgtgctgt ataactccgc aagctttagc 240
acgtttaaat gctacggcgt gtctcctaca aagctcaatg atctttgttt tactaatgtg 300
tatgctgact ctttcgtgat tcggggcgac gaggttagac agatcgctcc tggacagaca 360
ggaaaaatcg ctgattataa ctacaagctg cccgacgact ttacaggttg cgttatcgcg 420
tggaattcta ataacctgga ttcaaaggtg ggcggaaatt acaattacct ctataggctg 480
ttccggaaga gcaatctgaa gccatttgaa cgggatatca gcacggaaat ctaccaagct 540
ggcagtacac cctgtaacgg agtggaagga ttcaattgct acttcccact gcagtcctat 600
gggtttcaac caacgaacgg cgtgggttac cagccataca gggttgtggt gctgtctttc 660
gagctgctgc acgccccagc aaccgtgtgc gggccgaaaa aaagcaccaa cctcgtgaag 720
aataagtgtg tgaactttgg gggcggttcc gggggcggga gcggcggcgg gagccgcgtt 780
cagcctaccg aaagcatcgt taggttccct aacatcacta acctgtgccc atttggggaa 840
gtctttaacg ccacacgctt cgccagcgtg tatgcttgga accggaagcg aatttcaaat 900
tgcgtggcag actactctgt cctgtacaac agcgcctcct tcagcacttt caagtgttac 960
ggggtttctc caacaaagct gaacgacctg tgtttcacga atgtctacgc tgactcattc 1020
gtcatccggg gagacgaggt ccggcagatt gcacctggcc agacgggaaa aatcgccgat 1080
tataattaca aactcccaga cgacttcacg ggttgcgtga ttgcctggaa ttctaacaac 1140
ctcgacagta aggtgggagg taactacaac tacttgtatc ggctcttcag aaaatcaaac 1200
ctgaaacctt ttgagcggga tatttctacc gagatctatc aggccggttc cactccatgt 1260
aacggagtgg agggcttcaa ctgctatttc ccactccagt cctacgggtt tcagcccaca 1320
aacggcgttg gctaccagcc ttatcgggtt gtggtcctga gcttcgagtt gctgcatgct 1380
cctgctacag tctgtgggcc taagaaatct accaacctgg tcaaaaataa atgcgtgaac 1440
ttttaataag cggccgcaaa t 1461
<210> 14
<211> 858
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 14
ctagctagca tgtatcgcat gcagttgctt agctgtatag ccctctccct cgctctcgtc 60
accaattcaa gggtgcagcc aactgagtca atcgttaggt ttcctaacat taccaatctc 120
tgcccctttg gggaagtgtt caatgccact cggttcgcca gtgtctatgc gtggaacaga 180
aagcgcatta gtaactgtgt cgcagattat tccgtgctgt ataactcagc aagttttagt 240
acctttaaat gttatggcgt gtcccctacc aagctcaatg atctgtgttt cacaaatgta 300
tacgccgaca gcttcgtcat caggggcgac gaggttcggc agatcgcacc tggccagacc 360
ggaaagattg cagattataa ctataagttg cctgacgatt ttacggggtg tgtgatcgcc 420
tggaacagca ataatcttga ctccaaggtg ggcggtaatt acaactacct gtatagattg 480
tttaggaaaa gcaatctgaa gccttttgaa cgagacatct ccaccgaaat ctaccaggct 540
gggagtactc catgtaacgg cgttgagggg ttcaattgct acttccccct tcagtcctat 600
ggattccaac ccaccaatgg ggtcggttac cagccttaca gggtggtcgt gctttctttc 660
gagctgctgc atgccccagc cacagtgtgt ggccctaaga aaagcacaaa cctggtgaag 720
aataaatgcg tgaattttgg ctacatccct gaagcccccc gggacgggca ggcctatgtg 780
cgcaaggacg gcgaatgggt tctcctctcc acattcttgg gccaccatca tcaccaccac 840
taataagcgg ccgcaaat 858

Claims (10)

1.A SARS-CoV-2 neutralizing antibody kit, characterized in that: the kit comprises a new crown antigen RBD protein, wherein the RBD protein is an RBD monomer, an RBD dimer or an RBD trimer containing T4 folded peptide.
2. The kit of claim 1, wherein: the RBD protein is an RBD trimer containing T4 folded peptide.
3. The kit of claim 2, wherein: the amino acid sequence of the RBD trimer protein is shown in SEQ ID NO. 11.
4. The kit of claim 3, wherein: the coding nucleotide sequence of the RBD tripolymer protein is shown as SEQ ID NO. 14.
5. The kit of claim 1, wherein: the RBD protein is coupled to biotin to form a biotinylated RBD protein.
6. The kit of claim 1, wherein: the kit also comprises capture fluorescent microspheres coated with ACE2-hFc, PE-streptavidin, a novel coronavirus neutralizing antibody calibrator, a sample diluent and a 20 × washing buffer solution.
7. The kit of claim 6, wherein: the ACE2-hFc protein comprises amino acid sequences of 18-740 positions of ACE2 protein, and the amino acid sequence of 18-740 positions of ACE2 protein is shown as SEQ ID NO: 1; the ACE2-hFc protein further comprises a signal peptide sequence of HSA and an Fc sequence of human IgG1, wherein the signal peptide sequence of HSA is shown as SEQ ID NO. 2, and the Fc sequence of human IgG1 is shown as SEQ ID NO. 3.
8. The kit of claim 7, wherein: the amino acid sequence of the ACE2-hFc protein is shown as SEQ ID NO. 4; the encoding nucleotide sequence of the ACE2-hFc protein is shown as SEQ ID NO. 5.
9. Use of the kit of any one of claims 1 to 8 in the detection of SARS-CoV-2 neutralizing antibodies.
10. A method for detecting SARS-CoV-2 neutralizing antibodies, said method being a non-disease diagnostic and therapeutic method, characterized in that: the method comprises detecting SARS-CoV-2 neutralizing antibody in a sample to be tested by using the kit of any one of claims 1 to 8.
CN202210278881.8A 2022-03-18 2022-03-18 SARS-CoV-2 neutralizing antibody reagent kit and its use Pending CN114740199A (en)

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