A homogeneous method for detecting novel coronavirus receptor binding domain protein based on nanobody-mab sandwich assay

Abstract

The application discloses a kind of based on nano antibody-monoclonal sandwich detection novel coronavirus receptor binding domain protein homogeneous method, it is related to antigen detection field.The expression vector of novel coronavirus RBD protein, nano antibody is constructed to carry out protein expression, the pairing performance of nano antibody and monoclonal antibody is identified by ELISA method, nano antibody and monoclonal antibody are coupled to the surface of fluorescent microsphere and magnetic nanometer microsphere respectively, novel coronavirus RBD protein is diluted into different concentrations using PBS buffer solution, is input into the fluorescent and magnetic nano probe of target RBD protein, fluorescence value is determined after being separated by magnetic frame, and the standard curve for detecting novel coronavirus RBD protein is established, and the minimum detection line is 0.09ng / mL.The immunofluorescence homogeneous method constructed by the application is simple in operation, fast in detection speed, less in steps, low in cost, suitable for a variety of scene detection, and lays a foundation for the development of novel coronavirus rapid detection kit.

Description

Technical Field

[0001] This invention relates to the field of antigen detection, and specifically provides a homogeneous method for rapid screening and detection of receptor-binding region proteins of the novel coronavirus based on nanobodies and monoclonal antibodies. Technical Background

[0002] The novel coronavirus, belonging to the β-coronavirus genus, is the third novel virus to cause widespread human respiratory disease, following Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS). It is highly infectious and pathogenic, and can lead to COVID-19. Early detection of the novel coronavirus is the most effective way to cut off the virus transmission chain and control the epidemic.

[0003] The novel coronavirus is composed of RNA, structural proteins, and non-structural proteins. Generally, the detection targets for the novel coronavirus are the RNA chain, nucleocapsid proteins, and receptor-binding domain proteins. Currently, the national standard for novel coronavirus detection is real-time fluorescent reverse transcriptase polymerase chain reaction (RT-PCR), with colloidal gold test strips serving as a supplementary method for nucleic acid testing. RT-PCR offers high sensitivity and throughput, but it is cumbersome to operate, difficult to preserve samples, requires specialized operators and equipment, and has stringent environmental requirements. Colloidal gold test strips are simple to operate and fast, but have low sensitivity and a high false-positive / negative rate. Therefore, a simple, sensitive, and fast point-of-care testing (POCT) method for novel coronavirus detection is still needed. Immunofluorescence homogeneous assays offer advantages such as simple operation, high sensitivity, and the ability to perform in-situ detection, making them an effective and rapid method for detecting the novel coronavirus.

[0004] Traditional homogeneous immunofluorescence assays for detecting large protein molecules are based on a double monoclonal antibody sandwich method. For example, the patent "A Method for Detecting Target Proteins Using Magnetic Separation Homogeneous Immunoassay" (application number: CN201711039199.9) discloses a double monoclonal antibody sandwich method for detecting procalcitonin. However, the core detection element—the monoclonal antibody—involves a complex, time-consuming, and batch-to-batch variability preparation process with high experimental requirements, including animal experiments, significantly impacting the accuracy, stability, and precision of the detection system. Furthermore, screening paired monoclonal antibodies for different epitopes of large proteins is labor-intensive and inefficient. Therefore, finding alternative elements for monoclonal antibodies and a rapid screening method for paired antibodies is crucial. Nanobodies, also known as single-domain heavy chain antibodies, offer short preparation cycles, low cost, minimal batch-to-batch variability, and high stability, making them excellent alternatives to monoclonal antibodies. Moreover, molecular docking software can easily simulate the binding sites of nanobodies to target molecules, improving screening efficiency and antibody pairing performance compared to the random screening of traditional paired monoclonal antibodies. This invention prepares nanobodies targeting different epitopes of RBD, rapidly screens out a pair of antibody pairs with excellent pairing performance, and combines them with quantum dot microspheres that have good biocompatibility, low non-specific adsorption, high quantum yield, stable fluorescence spectrum, and broad absorption spectrum to establish a simple, sensitive, and fast POCT homogeneous immunoassay. Summary of the Invention

[0005] This invention discloses a homogeneous method for detecting the receptor-binding region protein of the novel coronavirus based on a nanobody-monoclonal antibody sandwich method. The method uses nanobodies instead of monoclonal antibodies to prepare nanobodies with different epitopes, rapidly screen and obtain antibody pairs with excellent performance, and overcomes the shortcomings of traditional double-antibody sandwich immunoassay methods, such as high cost of monoclonal antibodies, unstable detection signals, low sensitivity, time and labor consumption, difficulty in screening paired antibodies and low screening efficiency.

[0006] This invention utilizes eukaryotic and prokaryotic expression systems to prepare nanobodies targeting the receptor-binding domain (RBD) protein of the novel coronavirus and different epitopes of the RBD. Protein concentrations were determined using a Bicinchoninic Acid Assay (BCA) protein quantification kit, and the purity and biological activity of the expression materials were identified by SDS-PAGE and enzyme-linked immunosorbent assay (ELISA). The prepared nanobodies were paired with traditional monoclonal antibodies. The nanobodies were conjugated to fluorescent microspheres, and the conjugation time, nanobodies dosage, and activator dosage were optimized to prepare two nanoprobes targeting the novel coronavirus RBD protein. Different concentrations of the novel coronavirus RBD protein were diluted, probes were added, and after incubation for a period of time, magnetic microspheres were rapidly separated using a magnetic rack. The fluorescence intensity in the wells was detected, and a standard curve for the novel coronavirus RBD protein was established. The specific scheme is as follows:

[0007] 1) The coding codons of the RBD protein and RBD nanobodies were optimized, and eukaryotic expression vectors of the novel coronavirus RBD protein and prokaryotic expression vectors of anti-RBD nanobodies (R1, R2, R3, R4) were constructed. The eukaryotic expression vector of the RBD protein was transfected into human embryonic adrenal cells (HEK293), and the prokaryotic expression vector of the RBD nanobodies was transformed into E. coli BL21 (DE3) for protein expression. The culture temperature was lowered to increase the yield of the target protein. After expression, the cell culture supernatant and the E. coli lysate supernatant were purified. The concentration of purified protein was determined by BCA protein quantification kit, and the purity and biological activity of purified protein were identified by SDS-PAGE and ELISA.

[0008] 2) Coat RBD nanobodies with good purity and biological activity into enzyme-labeled wells, block with skim milk, dilute RBD protein to different concentrations with phosphate-buffered saline (PBS) buffer, set up negative and blank controls, incubate for a period of time, add anti-RBD protein monoclonal antibody and incubate again; wash away unbound monoclonal antibody and add secondary antibody, develop color with tetramethylbenzidine (TMB), and terminate with sulfuric acid; based on the color development results, determine the pairing performance of nanobodies and RBD protein monoclonal antibodies, and calculate the minimum detection limit.

[0009] 3) RBD nanobodies and RBD monoclonal antibodies were conjugated to the surfaces of fluorescent and magnetic nanospheres, respectively. Fluorescent and magnetic nanospheres were taken, and activators 1-ethyl-(3-dimethylaminopropyl)carbodiimide (ECD) and N-hydroxysuccinimide (NHS) were added to activate the carboxyl groups on the surface of the nanospheres. After activation, the nanospheres were separated by centrifugation / magnetic rack, washed once with PBS buffer, and the nanobodies / monoclonal antibodies were added and incubated at 25°C and 40 rpm for a period of time. Bovine serum albumin (BSA) was added to a final concentration of 1-2%, and the mixture was incubated at 25°C and 40 rpm for a period of time. Excess antibody and BSA were removed by centrifugation / magnetic rack separation, and the probes were washed once and stored at 4°C for later use. Based on the above steps, the conjugation time, the amount of activator, and the amount of target protein added during the conjugation process were optimized.

[0010] 4) Dilute the novel coronavirus RBD protein to different concentrations using PBS, set up negative and blank controls, add the fluorescent probe and magnetic nanoprobe synthesized in step 3), and incubate at 37°C for a period of time; separate with a magnetic rack, wash twice with PBS, measure the fluorescence value in the wells with a fluorescence microplate reader, and establish a standard curve for detecting the novel coronavirus RBD protein based on the fluorescence intensity in the wells of different protein concentrations.

[0011] Furthermore, in step 1) above, the buffer used is PBS, with an optimal concentration of 0.01-0.1 mM; the eukaryotic expression vector is pCMV, and the prokaryotic expression vector is the pET series vector.

[0012] Furthermore, in step 1) above, the eukaryotic vector was transfected into HEK293 cells using the PEI transfection method, and the prokaryotic expression vector was transformed into BL21 cells using the calcium transfection method; the optimal concentration of the target protein for imidazole elution was 100-500 mM.

[0013] Furthermore, in step 2) above, the negative control is human serum and the blank control is PBS buffer.

[0014] Furthermore, in step 2) above, the concentration of nanobody is 1-5 μg / mL; the concentration of monoclonal antibody is 0.5-2 μg / mL; the concentration of secondary antibody is 1:2000-6000; the concentration of diluted RBD protein is 2-2000 pg / mL; and the incubation time is 30-60 min.

[0015] Furthermore, in step 3) above, the fluorescent microspheres are green quantum dot microspheres, the amount of EDC and NHS added is 4-8 μL, the centrifugation speed is 8000-13000 rpm, and the amount of antibody added is 80-100 μg.

[0016] Furthermore, in step 4) above, the concentration of RBD protein is diluted to 2-2500 pg / mL; the amount of fluorescent nanoprobe added is 2-6 μL, and the amount of magnetic nanoprobe added is 4-10 μL; the excitation wavelength of quantum dot microspheres is 460-490 nm, and the emission wavelength is 510-540 nm.

[0017] Beneficial effects:

[0018] 1) Compared with the gold standard (RT-PCR) for the detection of the novel coronavirus, the immunofluorescence homogeneous method established in this invention is simple to operate, fast to detect, has fewer washing and separation steps, low cost, and is suitable for detection in a variety of scenarios. Its sensitivity is higher than that of the colloidal gold test strip antigen detection kit, laying the foundation for the development of a rapid detection kit for the novel coronavirus.

[0019] 2) Compared with traditional double monoclonal antibody sandwich methods and paired monoclonal antibody screening methods, the nanobody provided by this invention is easy to prepare and modify, has small batch-to-batch variability, low cost, and high paired antibody screening efficiency. It can effectively reduce the cost of detection products, improve the accuracy and precision of detection, and reduce batch-to-batch variability of detection kits, laying a good foundation for the immunoassay of the novel coronavirus.

[0020] 3) This invention prepares two nanoprobes targeting the RBD protein of the novel coronavirus. They have high specificity and stable detection performance. The magnetic nanoprobes can effectively enrich the novel coronavirus, and the fluorescent nanoprobes can provide a strong fluorescent signal for the novel coronavirus detection system. They are excellent detection elements for the immunoassay of the novel coronavirus. Attached Figure Description

[0021] Figure 1 ELISA method was used to verify the binding performance of different concentrations of SARS-CoV-2 RBD protein to monoclonal antibodies.

[0022] Figure 2 ELISA method was used to verify the binding performance of different concentrations of novel coronavirus RBD protein to the prepared nanobodies.

[0023] Figure 3 Screening for optimal pairing of nanobodies and monoclonal antibodies

[0024] Figure 4 Transmission electron microscopy image of quantum dot microspheres

[0025] Figure 5 Scanning electron microscope image of magnetic nanospheres

[0026] Figure 6 Optimization of coupling conditions for quantum dot microspheres

[0027] Figure 7 Changes in the surface potential of target proteins of different masses coupled with quantum dot microspheres and magnetic nanospheres Figure 8 Establishment of a standard curve for homogeneous detection of novel coronavirus RBD protein based on nanobody-monoclonal antibody Detailed Implementation

[0028] The materials, reagents, and formulations used in the embodiments of this invention are as follows:

[0029] Main experimental materials:

[0030] Quantum dot microspheres (Shanghai Kundao Biotechnology Co., Ltd.), magnetic nanospheres (Suzhou Beaver Biotechnology Co., Ltd.), goat anti-mouse IgG monoclonal antibody (Genetax Biotechnology Co., Ltd., USA), HEK293 (Thermo Fisher Scientific Co., Ltd.), PEI (Sigma-Aldrich, USA), pCMV-SARS-COV-2-RBD (Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.), RBD protein, RBD monoclonal antibody, and RBD nanobody were all prepared in our laboratory.

[0031] Main reagents:

[0032] Tween-20 (Beijing Solarbio Science & Technology Co., Ltd.), Egg Albumin (Beijing Solarbio Science & Technology Co., Ltd.), Skim Milk Powder, Tetramethylbenzidine, Isopropyl-β-D-thiogalactoside (Shanghai Sangon Biotech Co., Ltd.), Ampicillin, Kanamycin (Beijing Solarbio Science & Technology Co., Ltd.), LB Broth (Qingdao Haibo Biotechnology Co., Ltd.)

[0033] Main reagent formula:

[0034] 1. LB liquid culture medium: Weigh 25g of LB broth and dissolve it in 1000mL of ultrapure water, then autoclave at 121℃ for 15min;

[0035] 2. Buffer A: Weigh 29.25g NaCl and 4.44g sodium phosphate and dissolve them in 900mL of ultrapure water. Sonicate for 30s and then bring the volume up to 1L. Adjust the pH to 7-7.4.

[0036] 3. 10 mg / mL EDC / NHS: Weigh 0.01 g of EDC / NHS and dissolve it in 1 mL of 0.01-0.05 M MES buffer;

[0037] 4. Preservative solution: Weigh 0.785g Tris, 0.0053g sodium chloride, 1g bovine serum albumin, and 0.2% sodium azide.

[0038] This invention provides a homogeneous method for detecting the receptor-binding domain protein of the novel coronavirus using a nanobody-monoclonal antibody sandwich technique. A novel coronavirus RBD nanobody is prepared to replace the traditional RBD monoclonal antibody, and the optimal antibody pair is selected by ELISA screening. Microspheres are activated by EDC / NHS, and the antibody and nanobody are coupled to the surface of the nanoparticles to prepare two probes targeting the novel coronavirus RBD protein, achieving ultrasensitive detection of the novel coronavirus.

[0039] Example 1: Eukaryotic expression of RBD protein

[0040] 1) Preheat OPM-293 medium to 37°C in a water bath and add 4-8 mM L-glutamine; take 125 mL of cell shake flask, add 29-35 mL of OPM-293 medium, add 1 mL of revived HEK293 cells, and culture the cells at 37°C, 8% CO2, and 100-125 rpm.

[0041] 2) After 2-3 passages, remove the cell shake flasks and place them in a clean bench. Use trypan blue staining solution and a hemocytometer to determine cell viability and density. When the cell density is 2-5 × 10⁻⁶ cells / mL... 6 Replace with fresh OPM-293 medium when the cell count reaches 1 / mL.

[0042] 3) Add 600 μL of OPM-293 medium to a sterile centrifuge tube, then add 60-120 μg of pCMV-SARS-COV-2-RBD plasmid and 180-480 μL of PEI transfection reagent (1 mg / mL). Mix gently and incubate at room temperature for 10-15 min. After mixing, add the mixture to the cells and incubate at 37°C in a CO2 shaker for 3-6 days.

[0043] 4) Centrifuge at 3000-6000g for 10-20 min, collect the cell supernatant, purify the supernatant with NI-NAT, determine the concentration of eluted protein using a BCA kit, and detect the purity and biological activity of the protein using SDS-PAGE and ELISA. Ultrafiltration removes imidazole from the purified protein. Figure 1 As shown, the expressed RBD protein has good purity and binding performance.

[0044] Example 2, Expression of nanobodies

[0045] 1) Transform pET-nanoantibody plasmid into E. coil BL21(DE3) competent cells, plate them on LB plates, and incubate overnight at 37°C.

[0046] 2) Select successfully transformed single colonies and inoculate them into LB liquid medium. Extract plasmids using a plasmid mini-prep kit and verify them by enzyme digestion. Select successfully transformed expression vectors and inoculate them into LB test tubes and incubate overnight at 37°C.

[0047] 3) Inoculate 1-2% of the culture medium into 40-60 mL of LB / Amp liquid medium. When the cells reach the logarithmic growth phase, add 0.1-0.5 mM IPTG and incubate at 18°C ​​for 12-16 h.

[0048] 4) Transfer the bacterial culture to centrifuge tubes, centrifuge at 8000-10000 rpm for 10-20 min, resuspend the precipitate in PBS buffer, collect the bacterial cells, and sonicate them. After sonication, purify the lysate using a Ni-NAT gravity column, determine the concentration of the eluted protein using a BCA kit, and detect the purity and biological activity of the protein using SDS-PAGE and ELISA. Ultrafiltration is used to remove imidazole from the purified protein. Figure 2 As shown, the expressed RBD nanobody has good purity and binding performance.

[0049] Example 3: Screening of paired RBD nanobodies and monoclonal antibodies

[0050] 1) Coat 1-5 μg / mL RBD nanobody onto an ELISA plate and incubate overnight at 4°C.

[0051] 2) Wash the plate three times with 0.05% PBST, block with 3-5% skim milk, and incubate at 37°C for 1-2 hours.

[0052] 3) Wash the plate three times with 0.05% PBST, add different concentrations of RBD protein (2000-20 pg / mL), set up negative control and blank control, and incubate at 37℃ for 30-60 min.

[0053] 4) Wash the plate three times with 0.05% PBST, add 0.5-2 μg / mL monoclonal antibody, and incubate at 37°C for 30-60 min.

[0054] 5) Wash the plate three times with 0.05% PBST, add 1:2000-6000 goat anti-mouse IgG antibody to each well, and incubate at 37°C for 30-60 min.

[0055] 6) Wash the plate three times with 0.05% PBST, add TMB chromogenic solution, then add 1-2M H2SO4, and measure the OD. 450nm Absorbance value.

[0056] The results are as follows Figure 3 As shown, the combination of nanobodies R1, R2, R3, and R4 with RBD monoclonal antibodies has a good detection effect on the RBD protein of the novel coronavirus, among which nanobodies R4 have the best pairing effect.

[0057] Example 4: Preparation of RBD protein-targeting nanoprobes

[0058] 1) Dissolve 10-30 μL of quantum dot fluorescent microspheres in 0.5-1 mL of MES buffer (0.05 M) and sonicate for 1-5 min.

[0059] 2) Add 4-10mM EDC and NHS, sonicate for 1-5 minutes, and incubate in a rotary mixer at room temperature for 20-30 minutes.

[0060] 3) Centrifuge at 12000 rpm for 10-15 min, wash once with PBS buffer, add 0.08-0.12 mg of the target protein, and couple with a rotary mixer for 40-50 min;

[0061] 4) Add 1-2% BSA (dissolved in PBS), incubate with a rotary mixer for 1-2 hours; centrifuge at 6000-8000 rpm for 5-10 minutes, resuspend the precipitate in the preservation solution, and place the prepared probe at 4℃ for later use.

[0062] Optimization and characterization of synthetic probe coupling conditions:

[0063] Figure 4 , 5The fluorescent and magnetic nanospheres were observed to have particle sizes of approximately 100 nm and 300 nm, respectively. The particles exhibited uniform spherical or elliptical shapes and good dispersibility. Following the probe synthesis steps described above, different masses of antibody (20 μg, 40 μg, 60 μg, 80 μg, and 100 μg) were added to the nanospheres. Figure 6 (A) and Figure 7 As shown, the antibody dosage was 80-100 μg, and the surface conjugation of the microspheres reached saturation. The dosages of EDC and NHS (2 μL, 4 μL, 6 μL, 8 μL, 10 μL) were optimized. Figure 7 The optimization diagram of activator dosage shows that the optimal effect is achieved when the dosage of EDC and NHS is 4-8 μL; the optimal coupling time (30 min, 40 min, 50 min, 60 min) is as follows. Figure 7 As shown in the optimization diagram of coupling time, the coupling amount reached saturation after 40-50 min of coupling between activated quantum dot microspheres and nanobodies. Furthermore, according to the instruction manual for green quantum dot microspheres, the excitation wavelength of the quantum dots used in this study was 460-490 nm, and the emission wavelength was 510-540 nm.

[0064] Example 5: Homogeneous detection of novel coronavirus N protein based on nanobody-monoclonal antibody

[0065] 1) Dilute the novel coronavirus RBD protein to 2500, 1250, 625, 312, 156, 78, 39, 20, 10, 5, and 2 pg / mL with PBS buffer. Add 2-6 μL of quantum dot microspheres-nanobody or 4-10 μL of magnetic nanospheres-monoclonal antibody to 100 μL of different protein concentrations. Set up negative control and blank control. Set up three parallel controls for each protein concentration, negative control, and blank control. Incubate at 37℃ for 20-30 min.

[0066] 2) Separate the magnetic beads using a magnetic rack, wash twice with PBS buffer, resuspend the beads in PBS, measure the fluorescence signal in each well, and plot a standard curve, such as... Figure 8 As shown, the sensitivity of homogeneous immunoassay for detecting the N protein of the novel coronavirus was 0.09 ng / mL, obtained from the standard curve.

[0067] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be conceived by those skilled in the art within the technical scope disclosed in the present invention without creative effort should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope defined in the claims.

[0068] SEQ ID NO:1

[0069] catatgGAAGTACAATTAGTAGAAAGCGGAGGAGGACTAGTACAACCAGGAGGAAGCCTAAGACTAAGCTGCGCAGCAAGCGGATTCACATTCAGCAGCTACGCAATGAGCTGGGTAAGACAAGCACCAGGAAAGGGAAGAGAATGGGTAGCAGGAATAACACCAGGAAGCGGAACATTCTACGCAGACAGCGTAAAGGGAAGATTCACAATAAGCAGAGACAACGCAAAGAACACACTAAGCCTAGAAATAAACAGCCTAAAGCCAGAAGACACAGCACTATACTACTGCGCAAAGTGCAGACAAGAATTCAGCTGGGACTTCAGCAGCAGAGACCCAGACGACTTCGACTACTGGGGACAAGGAACACAAGTAACAGTAAGCAGCCACCATCATCATCATCATCACCACTGATGActcgag

[0070] SEQ ID NO:2

[0071] catatgCACCATCATCATCATCATCACCACCATGTACAATTAGTAGAAAGCGGAGGAGGATTAGTACAAGCAGGAGGAAGCCTAAGACTAAGCTGCGCAGTAAGCGGAAGAACATTCAGCACATACGGAATGGCATGGTTCAGACAAGCACCAGGAAAGGAAAGAGACTTCGTAGCAACAATAACAAGAAGCGGAGAAACAACACTATACGCAGACAGCGTAAAGGGAAGATTCACAATAAGCAGAGACAACGCAAAGAACACAGTATACCTACAAATGAACAGCCTAAAGATAGAAGACACAGCAGTATACTACTGCGCAGTAAGAAGAGACAGCAGCTGGGGATACAGCAGAGACCTATTCGAATACGACTACTGGGGACAAGGAACACAAGTAACAGTAAGCAGCTACCCATACGACGTCCCAGACTACGCTTGATGActcgag

[0072] SEQ ID NO:3

[0073] ccatggatCACCATCATCATCATCATCACCACCACGTACAACTAGTTGAATCAGGTGGAGGGTTGGTTCAGGCGGGTGGCAGCCTGCGTCTGAGCTGTGCTGCGAGCGGTGGTACGTTCACCACCTTCCGCATGGCATGGTTTCGCCAGACCCCGGGCAAAGAAAGAGAGTTCGTGGCCGCGTCCTCTTGGGGCTTTGTTAATTATGCGGATCCGGTAAAAGGTCGTTTTACTATCAGCCGTGATAACGCAAAGAACACCGTCTACCTGGAAATGAGCTCCCTCAAGACCGAGGACACCGCTGTGTATTACTGCGCCGCGCGTAACCCGGGCACGGGTCAATATGACTACTGGGGCCAAGGTACACAGGTTACCGTGTCTTCGGACTACAAAGATGATGACGATAAATAATAActcgag

[0074] SEQ ID NO:4

[0075] ccatggatCACCATCATCATCATCATCACCACCAAGTTCAGCTAGTAGAATCAGGTGGAGGGCTCGTGCAGACCGGTGGTTCGCTGCGCCTGAGCTGCGCGGCTTCAGGCTCGACCCGTAGCATTTGGTACTCCGGCTGGTATCGCCAGGCACCGGGTAAGCAAAGAGAGCTGGTAGCCTCTTTGACCAGCGGCGGTACTACCTATTACCCGGATAGCATGAAAGGTCGTTTCACCATCAGCGGCTCTAGCGGCCTGAATATGGTTTATCTGCAAATGAACAGCTTGCGTCCGGAAGACACGGCGGTGTACAGCTGTAACGTGCGTACCGGTTCCGGCGTCTACTGGGGTCAAGGTACGCAGGTTACCGTTAGCTCCGACTACAAAGATGATGACGATAAATAATAActcgag

[0076] SEQ ID NO:5

[0077] GCCACCATGTACCGCATGCAGCTGCTGAGCTGCATCGCCCTGAGCCTGGCCCTGGTGACCAACAGCggatccAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCCACCCGCTTCGCCAGCGTGTACGCCTGGAACCGCAAGCGCATCAGCAACTGCGTGGCCGACTACAGCGTGCTGTACAACAGCGCCAGCTTCAGCACCTTCAAGTGCTACGGCGTGAGCCCCACCAAGCTGAACGACCTGTGCTTCACCAACGTGTACGCCGACAGCTTCGTGATCCGCGGCGACGAGGTGCGCCAGATCGCCCCCGGCCAGACCGGCAAGATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATCGCCTGGAACAGCAACAACCTGGACAGCAAGGTGGGCGGCAACTACAACTACCTGTACCGCCTGTTCCGCAAGAGCAACCTGAAGCCCTTCGAGCGCGACATCAGCACCGAGATCTACCAGGCCGGCAGCACCCCCTGCAACGGCGTGGAGGGCTTCAACTGCTACTTCCCCCTGCAGAGCTACGGCTTCCAGCCCACCAACGGCGTGGGCTACCAGCCCTACCGCGTGGTGGTGCTGAGCTTCGAGCTGCTGCACGCCCCCGCCACCGTGgaattcCACCACCACCACCACCACGGCGGCGGCGGCAGCCTCGAGGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGACTACAAGGATGACGATGACAAGTAG

Claims

1. A nanoprobe targeting the SARS-CoV-2 RBD protein, comprising a fluorescent nanoprobe targeting the SARS-CoV-2 RBD protein and a magnetic nanoprobe targeting the SARS-CoV-2 RBD protein, characterized in that, The nanoprobe was obtained through the following method: A. The PEI transfection method was used to transfect mammalian cells with the RBD eukaryotic expression plasmid to prepare the SARS-CoV-2 RBD protein. The purity and biological activity were identified by SDS-PAGE and ELISA. B. RBD protein nanobodies R1, R2, R3, and R4 were prepared using the Escherichia coli expression system. The purity and biological activity were identified by SDS-PAGE and ELISA. C. Nanobodies are used as coating antibodies, and monoclonal antibodies are used as detection antibodies. ELISA is used to identify the pairing performance of nanobodies and monoclonal antibodies. D. Quantum dot microspheres were activated with an activator, and nanoantibodies were coupled to their surface to prepare fluorescent nanoprobes targeting the RBD protein of COVID-19, providing detection signals for a homogeneous rapid detection system; E. Activating magnetic nanospheres with activators and coupling RBD monoclonal antibodies to their surface, a magnetic nanoprobe targeting the SARS-CoV-2 RBD protein was prepared, laying the foundation for rapid separation of nucleocapsid protein in a homogeneous system. F. The prepared nanoprobes were added to different concentrations of SARS-CoV-2 RBD protein to establish a homogeneous immunoassay for the detection of SARS-CoV-2 RBD protein; The sequences of the nanobodies R1, R2, R3, R4 and the RBD protein paired with the monoclonal antibody against the SARS-CoV-2 RBD protein are SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, respectively.

2. The nanoprobe targeting the SARS-CoV-2 RBD protein according to claim 1, characterized in that, The detection target is the SARS-CoV-2 RBD protein.

3. The nanoprobe targeting the SARS-CoV-2 RBD protein according to claim 1, characterized in that, The concentration of the COVID-19 RBD protein administered is 2-2000 pg / mL.

4. The nanoprobe targeting the SARS-CoV-2 RBD protein according to claim 1, characterized in that, The amount of quantum dot microspheres used for conjugating nanobodies is 2-6 μL, and the amount of magnetic nanospheres used is 4-10 μL.

5. The nanoprobe targeting the SARS-CoV-2 RBD protein according to claim 1, characterized in that, In step C, the coating concentration of the nanobody is 1-5 μg / mL, and the amount of monoclonal antibody added is 0.5-2 μg / mL.

6. The nanoprobe targeting the SARS-CoV-2 RBD protein according to claim 1, characterized in that, The quantum dot microspheres obtained in step D have a particle size of approximately 100 nm, an excitation wavelength of 460-490 nm, and an emission wavelength of 510-540 nm; the magnetic nanospheres obtained in step E have a particle size of approximately 300 nm.

7. The nanoprobe targeting the SARS-CoV-2 RBD protein according to claim 1, characterized in that, The coupling conditions for quantum dot microspheres were: NHS / EDC input of 4-8 μL, protein input of 80-100 μg, and coupling time of 40-50 min.

8. The nanoprobe targeting the SARS-CoV-2 RBD protein according to claim 1, characterized in that, The magnetic microsphere coupling conditions were as follows: NHS / EDC input amount was 4-8 μL, protein input amount was 80-100 μg, and coupling time was 40-50 min.

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