A homogeneous method for detecting the novel coronavirus nucleocapsid protein based on a nanobody-monoclonal antibody sandwich

Abstract

A homogeneous method for detecting the nucleocapsid protein of the new coronavirus based on a nano-antibody-monoclonal antibody sandwich relates to the field of antigen detection. The present invention prepares a variety of novel coronavirus nucleocapsid protein nano-antibodies as solid-phase antibodies, nucleocapsid protein monoclonal antibodies as detection antibodies, and novel coronavirus N protein as detection targets. A group of antibody pairs with excellent detection performance are screened out by enzyme-linked immunosorbent assay, with a minimum detection line of 0.8 ng / mL. In addition, a one-step activation method couples nano-antibodies and monoclonal antibodies to the surfaces of quantum dot microspheres and magnetic nano-microspheres, respectively, and successfully prepares two probes targeting the nucleocapsid protein of the new coronavirus. It also constructs a magnetic separation immunohomogeneous method for rapid detection of the nucleocapsid protein of the new coronavirus, with a minimum detection line of 0.15 ng / mL. The present invention is simple to operate, low in cost, fast in reaction speed, can realize in situ detection, and has high efficiency in paired antibody screening. It has good application prospects in the rapid detection of novel coronaviruses.

Description

Technical Field

[0001] The present invention relates to a homogeneous immunoassay method for detecting the novel coronavirus nucleocapsid protein based on a nanoantibody-monoclonal antibody sandwich. The present invention screens and obtains antibody pairs targeting the novel coronavirus N protein, and prepares two nanoprobes that can selectively enrich and detect the novel coronavirus N protein in samples, thereby achieving rapid and sensitive detection of the novel coronavirus. Background Art

[0002] SARS-COV-2 can be transmitted through human contact, aerosols, feces, etc. It has extremely high infectivity and mortality rate. Early detection of the virus's transmission trajectory is the most effective strategy to control the epidemic. Therefore, it is very important to develop rapid and effective detection methods for the new coronavirus.

[0003] Real-time fluorescence RT-PCR is currently the gold standard for novel coronavirus detection. Colloidal gold test strips are used as a supplementary method for novel coronavirus detection. The RT-PCR method has complicated steps, high requirements for the operating environment, requires professional operators and equipment, and samples are difficult to preserve, which greatly reduces the screening efficiency of the novel coronavirus. The colloidal gold test strip method has low detection sensitivity. A simple, fast, and sensitive novel coronavirus detection method still needs to be developed. Homogeneous immunofluorescence immunoassay has the advantages of simple operation, few detection steps, and high detection sensitivity. It has also been used in the detection of new coronaviruses. For example, the patent "Magnetic Microparticle Chemiluminescence Detection Kit for New Coronavirus Neutralizing Antibodies and Its Application" (Application Number: CN202111020666.X) provides a chemiluminescence homogeneous kit for accurately and efficiently detecting neutralizing antibodies against the new coronavirus. However, the luminescent reagents of the chemiluminescence system have poor stability and low accuracy, and the detection of neutralizing antibodies cannot accurately determine the presence of the new coronavirus virus, which has great limitations. Quantum dot microspheres have good biocompatibility, low nonspecific adsorption, high quantum yield, stable fluorescence spectrum, and wide absorption spectrum. They are the preferred signal material for high-sensitivity detection and homogeneous detection analysis.

[0004] In the antigen immunoassay system of the new coronavirus, monoclonal antibodies are the most conventional detection elements. For example, “Monoclonal antibodies and antibody combinations against the new coronavirus and their application in the detection of viral antigens” (application number: CN202010463556.X) discloses the paired antibody sequences against the new coronavirus nucleocapsid protein and describes its application prospects in the detection of the new coronavirus. However, in the traditional double monoclonal antibody sandwich system for detecting macromolecules, the preparation process of paired monoclonal antibodies for different epitopes is complicated, costly, long, with large batch differences, low efficiency in paired antibody screening, and high difficulty in operation. It is of great significance to find a new paired monoclonal antibody screening method. Compared with traditional monoclonal antibodies, nanoantibodies have low preparation costs, short cycles, strong stability, and small batch differences. In addition, through molecular simulation calculations, nanoantibodies targeting different epitopes of the target can be quickly screened out and then paired with traditional monoclonal antibodies. Compared with traditional random screening, the screening efficiency and performance are greatly improved, and it is an excellent alternative element to monoclonal antibodies. At present, there are no reports on the combination of quantum dot microspheres and nanoantibodies for homogeneous immunoassay of new coronavirus antigens. Summary of the Invention

[0005] The present invention discloses a homogeneous immune method for detecting the nucleocapsid protein of the new coronavirus based on nano-antibodies, which overcomes the shortcomings of traditional immunoassays such as high cost, unstable detection signal, low sensitivity, time-consuming and labor-intensive, and multiple washing and incubation. The technical solution of the present invention includes the following steps: using an Escherichia coli expression system to express the new coronavirus N protein and the new coronavirus N protein nano-antibody, SDS-PAGE to identify the purity of the expressed protein, and ELISA to identify the biological activity of the recombinant protein; pairing the nano-antibodies prepared with different epitopes with traditional monoclonal antibodies, and quickly screening out a pair of paired antibodies with excellent detection performance; optimizing the EDC / NHS input amount, antibody input amount, and coupling time during the coupling process, and using EDC / NHS activators to activate quantum dot microspheres and magnetic nanoparticle surface carboxyl groups to prepare two probes targeting the new coronavirus N protein; putting the two probes into different concentrations of the new coronavirus N protein solution, incubating for a period of time, quickly separating the magnetic microspheres with a magnetic stand, detecting the fluorescence intensity, and establishing an immune homogeneous method for the new coronavirus nucleocapsid protein. The specific steps are as follows:

[0006] The detection mode of the present invention is also applied to the detection of other macromolecular proteins, taking the detection of the new coronavirus nucleocapsid protein as an example. In order to obtain nano antibodies to replace traditional monoclonal antibodies for immunoassay, prokaryotic expression vectors of the new coronavirus N protein and the new coronavirus N protein nano antibodies (N1, N2, N3, N4) were constructed and transformed into Escherichia coli engineered bacteria-BL21 (DE3). The bacteria were cultured to the logarithmic growth phase, and an appropriate concentration of IPTG was added to induce the bacteria to express the target protein. At the same time, the culture temperature was lowered to increase the yield of the target protein. At the end of expression, the bacteria were broken, the target protein was identified as soluble expression, the supernatant was collected for purification, and SDS-PAGE and ELISA were used to identify the prepared protein with good purity and biological activity.

[0007] The present invention provides a rapid screening method for paired antibodies. The immunoassay of the new coronavirus usually uses a double monoclonal antibody sandwich method, in which monoclonal antibodies are difficult to prepare and monoclonal antibodies targeting different epitopes of the target protein are difficult to screen. The present invention prepares a variety of low-cost nanoantibodies and monoclonal antibodies for pairing, and quickly screens and obtains a pair of paired antibodies with excellent detection performance, overcoming the shortcomings of the traditional method of difficult to obtain and difficult to screen paired monoclonal antibodies. The screening steps are: the prepared nanoantibody is coated in the enzyme-labeled well, after blocking with skim milk, different concentrations of the new coronavirus N protein are added, human serum is used as a negative control, and after a period of incubation, a certain amount of N protein monoclonal antibody is added; color is developed after adding the secondary antibody; according to the color development results, the nanoantibody and the N protein monoclonal antibody have the best pairing performance. In addition, the wells with human serum do not develop color, and the paired antibody has a strong detection specificity.

[0008] The present invention prepares two nanoprobes that can target and bind to the new coronavirus N protein. The coupling steps of nanoantibodies / monoclonal antibodies and quantum dots / magnetic microspheres are based on the instructions and improved. The steps are: take the nanomicrospheres and dissolve them in buffer, add EDC and NHS to activate the carboxyl groups on the surface of the nanomicrospheres, remove the activator after activation, and add PBS buffer to wash once; add antibodies to make the activated carboxyl groups covalently coupled to the amino groups of the protein, remove the uncoupled antibodies, add BSA (PBS dissolved) to block the excess sites, separate the microspheres and the supernatant, resuspend the microspheres in preservation solution, and place the prepared probes at 4°C for standby use. Based on the above steps, the amount of activator input, coupling time, and target protein input were optimized respectively.

[0009] Based on the above-mentioned preparation of probes, the present invention establishes a homogeneous immunoassay for the new coronavirus N protein. The method provided by the present invention is simple to operate, low-cost, and fast in reaction speed, and has good application prospects in the immunoassay of the new coronavirus. The steps are as follows: Use PBS to dilute the new coronavirus N protein to different concentrations, set negative and blank controls, add appropriate amounts of two nanoprobes, incubate for a period of time, separate the magnetic beads on a magnetic stand, wash twice, and measure the fluorescence value in the well with a fluorescence microplate reader. According to the fluorescence intensity in the wells of different protein concentrations, a standard curve for determining the new coronavirus N protein is established to obtain a linear equation.

[0010] In addition to detecting the novel coronavirus, the present invention is also applicable to the detection of other viruses, such as hepatitis B virus, norovirus, rotavirus, etc. It is also applicable to the detection of other biological macromolecules or pathogenic microorganisms, such as disease biomarkers - adiponectin protein, human soluble growth stimulating protein, etc.

[0011] Beneficial effects:

[0012] The novel coronavirus N protein nanoantibodies prepared by the present invention have the advantages of simple preparation, low cost, short cycle, strong stability, and small batch-to-batch variability compared to the detection elements in traditional immunoassays - monoclonal antibodies. They are excellent substitutes for monoclonal antibodies. The present invention breaks the traditional screening method of paired monoclonal antibodies, overcomes the shortcomings of traditional paired monoclonal antibody screening, and uses nanoantibodies to pair with monoclonal antibodies. Antibody pairs with excellent pairing performance can be screened out in a relatively short time, laying a good foundation for the immunoassay of the novel coronavirus.

[0013] The present invention prepares two probes targeting the N protein of the new coronavirus, which have the advantages of simple preparation, strong specificity, and stable detection performance, and are excellent detection elements for the N protein of the new coronavirus; the present invention establishes an immunoassay for homogeneous detection of the new coronavirus virus. Compared with the current gold standard for new coronavirus detection (RT-PCR), the method has simple operation, low cost, fast reaction speed, fewer washing and separation steps, and is suitable for detection in various scenarios, laying the foundation for the development of a rapid detection kit for the new coronavirus. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Schematic diagram of the principle of homogeneous immunoassay based on nanoantibodies to detect the nucleocapsid protein of the new coronavirus

[0015] Figure 2 ELISA method verifies the binding performance of the novel coronavirus N protein coated with different concentrations and monoclonal antibodies

[0016] Figure 3 ELISA method verifies the binding performance of the novel coronavirus N protein coated with different concentrations and the prepared nanoantibodies

[0017] Figure 4 Screening for the optimal pairing of nanobodies and monoclonal antibodies

[0018] Figure 5 Transmission electron microscopy image of quantum dot microspheres

[0019] Figure 6 Scanning electron microscope image of magnetic nanoparticles

[0020] Figure 7 Optimization of coupling conditions between quantum dots and microspheres

[0021] Figure 8 Changes in surface potential of target proteins of different masses coupled to quantum dot microspheres and magnetic nanospheres

[0022] Figure 9 Establishment of a standard curve for homogeneous detection of novel coronavirus N protein based on nanoantibodies DETAILED DESCRIPTION

[0023] The present invention provides an immune homogeneous method using novel coronavirus N protein nanoantibodies instead of traditional monoclonal antibodies; a variety of novel coronavirus N protein nanoantibodies are prepared, the best antibody pair is screened by ELISA, the antibodies and nanoantibodies are coupled to the surface of nanoparticles, and two probes targeting novel coronavirus N protein are prepared, thereby rapidly enriching and ultrasensitively detecting the novel coronavirus virus ( Figure 1 ).

[0024] Example 1, Expression of Novel Coronavirus N Protein and N Protein Nanobodies

[0025] Take E.coil BL21 (DE3) glycerol bacteria and streak them on a plate to prepare BL21 (DE3) competent cells; transfer the expression vector containing the target gene into the BL21 (DE3) competent cells, pick out the single colony that has been transformed and inoculate it into LB liquid medium, extract the plasmid with a plasmid extraction kit and perform enzyme digestion verification to ensure the successful construction of the expression vector; pick out the expression vector that has been transformed and inoculate it into LB / Amp test tube medium, culture it at 37℃ overnight; inoculate 40-60mL LB / Amp liquid medium for expansion culture, and wait for the bacteria to grow to OD 600 The pH value is approximately equal to 0.5-0.8, 0.1-0.5 mM IPTG is added, and the culture is carried out at 22°C for 12-16 hours; the bacterial solution is transferred to a centrifuge tube, centrifuged at 6000-8000 rpm for 10-15 minutes, the bacterial pellet is collected and resuspended in PBS buffer, and the resuspended bacteria are disrupted by an ultrasonic disruptor; after disruption, the disrupted liquid is purified by NI-NAT gravity column, the protein purity is detected by SDS-PAGE, the protein concentration is determined by BCA kit, and the imidazole in the purified protein is removed by ultrafiltration; Figure 2-3As shown, the expressed novel coronavirus N protein and nanoantibodies have good purity. Figure 3 This shows that the prepared nanoantibody has excellent binding properties with N protein.

[0026] Example 2, Screening of Paired Novel Coronavirus Nanobodies and Monoclonal Antibodies

[0027] Coat 2.5-5 μg / mL N protein nanoantibodies to the enzyme-labeled plate and incubate at 4°C overnight; wash the plate three times with 0.05% PBST, block with 3-5% skim milk, and incubate at 37°C for 1-2 hours; wash the plate three times with 0.05% PBST, add different concentrations of N protein (2500-20 pg / mL), diluted normal human serum (negative control), and PBS (blank control), and incubate at 37°C for 45-60 minutes; wash the plate three times with 0.05% PBST, add 0.5-1.5 μg / mL novel coronavirus N protein monoclonal antibody to each well, and incubate at 37°C for 45-60 minutes; wash the plate three times with 0.05% PBST, add goat anti-mouse IgG antibody to each well, and incubate at 37°C for 45-60 minutes; wash the plate three times with 0.05% PBST, add TMB colorimetric solution, and incubate at 37°C for 8-15 minutes; add 1-2M H2SO4 to each well, and measure OD 450nm Absorbance value, if Figure 4 As shown in the figure, when the prepared four nanoantibodies are paired with the novel coronavirus N protein monoclonal antibody, they can more sensitively detect the novel coronavirus N protein and have a good pairing effect. The lowest detection line of the N protein detected by ELISA is 800pg / mL.

[0028] Example 3, Preparation of Magnetic Nanosphere-Monoclonal Antibody and Quantum Dot Fluorescent Microsphere-Nanoantibody Probes

[0029] The surface of quantum dot microspheres and magnetic nanoparticles contains carboxyl groups (-COOH). Using N-(3-dimethylaminopropyl)-N-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) as cross-linking agents, the carboxyl groups on the surface of the nanoparticles are activated in a one-step method, making them easy to combine with the amino groups on the protein surface, thereby coupling the antibodies to the nanoparticle surface and providing a basis for subsequent N protein detection.

[0030] The coupling steps of nanoantibodies and quantum dot microspheres are based on the instructions for coupling quantum dot nanospheres (QDNBs) and are modified. The detailed steps are as follows: Take 10-20 μL QBs 620The quantum dot fluorescent microspheres were dissolved in 0.5-1 mL of MES buffer (0.05 M) and sonicated for 1-5 min; 4-6 μL of EDC and 4-6 μL of NHS were added, sonicated for 1-5 min, and incubated at 25°C on a rotary mixer for 20-30 min; centrifuged at 12000 rpm for 10-15 min, the supernatant was aspirated using a pipette, PBS buffer was added, and the mixture was washed once by centrifugation at 12000 rpm for 10-15 min; 0.08-0.1 mg of the target protein was added, the microspheres were resuspended, and the mixture was coupled at 25°C on a rotary mixer for 40-50 min; centrifuged at 8000 rpm for 5-10 min, and PBS buffer was added for washing once; 1-2% BSA (dissolved in PBS) was added, and the mixture was blocked at 25°C on a rotary mixer for 1-2 h; centrifuged at 8000 rpm for 5-10 min, the precipitate was resuspended with the preservation solution, and the prepared probe was placed at 4°C for standby use.

[0031] The coupling steps for magnetic nanoparticles are similar to those for quantum dot microspheres, except that, due to the magnetic properties of the nanoparticles, the centrifugation step is replaced with magnetic separation. Furthermore, after adding the monoclonal antibody, the optimal coupling conditions are: 6-10 μL of NHS / EDC, 90-120 μg of protein, and a coupling time of 50-70 minutes.

[0032] Optimization and characterization of coupling conditions for synthetic probes:

[0033] To determine the particle size and dispersibility of magnetic nanospheres and fluorescent microspheres, transmission electron microscopy and scanning electron microscopy were used to characterize them respectively; Figure 5 It was observed that the particle size of the fluorescent nanospheres was about 100 nm, and the particles were uniformly round or elliptical; Figure 6 It was observed that the average particle size of the magnetic nanoparticles was about 300 nm, the particles were round or elliptical, and had good dispersion. Referring to the above-mentioned probe synthesis steps, different masses of antibodies (20 μg, 40 μg, 60 μg, 80 μg, 100 μg) were added to the nanoparticles activated by EDC and NHS, and the coupling was identified by 0.6% agarose gel electrophoresis. The Zeta potential of the surface of the simple nanomaterial was measured by dynamic light scattering analyzer, as shown in FIG. Figure 7 (A) and Figure 8 As shown in the figure, when the protein input amount is 80-100 μg, the protein coupling on the surface of quantum dot microspheres reaches saturation; under the premise of determining the optimal antibody input amount, referring to the steps of the above probe synthesis, when activating the surface carboxyl groups of the nanomaterials, the input amounts of EDC and NHS (2 μL, 4 μL, 6 μL, 8 μL, 10 μL) are optimized, and the coupling situation is identified by 0.6% agarose gel electrophoresis, as shown in the figure. Figure 7As shown in (B), the best effect was achieved when the EDC and NHS input amounts were 4-6 μL. On the premise of determining the optimal antibody, EDC, and NHS input amounts, the optimal coupling time (30 min, 40 min, 50 min, and 60 min) was optimized with reference to the above probe synthesis steps. The coupling conditions were identified by 0.6% agarose gel electrophoresis. Figure 7 As shown in (C), when the activated quantum dot microspheres are coupled with the nanoantibody for 40-50 minutes, the protein coupling on the surface of the quantum dot microspheres reaches saturation.

[0034] Example 4: Homogeneous detection of novel coronavirus N protein based on nanoantibodies

[0035] The expressed novel coronavirus N protein was diluted with PBS to 2500, 1250, 625, 312, 156, 78, 39, 20, and 10 pg / mL. 4-8 μL of quantum dot microspheres-nanoantibody probes and 8-14 μL were added to 100 μL of different protein concentrations. Human serum was set as a negative control and PBS as a blank control. Three parallel controls were set for each protein concentration, negative control, and blank control. The mixture was incubated at 37°C for 20-30 min. The magnetic beads were separated using a magnetic stand, washed twice with PBS, and then resuspended. The fluorescence signal of each well was measured and a standard curve was drawn. Figure 9 As shown in the figure, the minimum detection line of the homogeneous immunoassay for the novel coronavirus N protein is 0.15 ng / mL.

[0036] The foregoing is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be conceived by a person skilled in the art within the technical scope disclosed by the present invention without inventive effort should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection defined in the claims.

[0037] SEQ ID NO: 1

[0038] catatgCACCATCATCATCATCATCACCACGAGGTGCAACTGCAAGCCTCCGGCGGCGGCCTGGTGCAAGCCGGCGGCTCCCTGAGACTGTCCTGCGCCGCCTCCGGCAGAACCGATTCCACCCAACATATGGCCTGGTTCAGACAAGCCCCTGGTAAGGAGAGAGAGTTCGTGACCGCCATCCAATGGAGAGGCGGCGGCACCTCCTACACCGATTCCGTGAAGGGCAGATTCACCATCTCCAGAGATAACGCCAAGAACACCGTGTACCTGGAGATGAACTCCCTGAAGCCTGAGGATACCGCCGTGTACTACTGCGCCACCAACTACAGATGGACCTACTTCTCCCCTACCGTGCCTGATAGATACGATTACTGGGGTCAAGGTACCCAAGTGACCGTGTCCTCCTCCTCCggtGACTACAAAGATGATGACGATAAATAATAActcgag

[0039] SEQ ID NO:2

[0040] catatgCACCATCATCATCATCATCACCACGAAGTTCAACTTCAAGCTAGCGGTGGTGGACTTGTTCAAGCAGGAGACAGCCTTAGACTTAGCTGTGTTGCAGTTAGCGGAAGAACAATTAGCACATTCGCAATGGGATGGTTCAGACAAGCACCAGGAAAAGAAAGAGAATTCGTTGCAACAATTAATTGGAGCGGAAGCAGCGCAAGATACGCAGACCCAGTTGAAGGAAGATTCACAATTAGCAGAGACGACGCAAAAAATACAGTTTACCTTGAAATGAGCAGCCTTAAACCAGGAGACAGCGCAGTTTACTACTGTGCAAGCGGAAGATACCTTGGAGGAATTACCAGCTACAGCCAAGGAGATTTCGCACCATGGGGACAAGGAACCCAAGTTACAGTTAGCAGCGAGCAGAAACTCATCTCTGAAGAGGATCTGTGATGActcgag

[0041] SEQ ID NO:3

[0042] catatgCACCATCATCATCATCATCACCACGAAGTACAGCTACAAGCTTCAGGTGGAGGGTTGGTCCAGGCGGGTGAGAGCCTGCGTCTCTCTTGTGTTGTGTCCGGCGGTGCGTTTCGTATTATGGACATGGGTTGGTTCCGCCAGGTTCCGGGTAAGCAACGTGAGGTGGTAGGTGTTATCAGCAGCGGTGGCTATACCAACTATGCGGATTCCGTGAAAGGCAGATTCACCATTTCTCGTGACAATGCTAAGCGCACGGTGTACCTGCACATGAACAGCCTGAAACCGGAAGATACCGCAGTTTACTACTGCAACATCATCCCGAAAAGCGACCAAGGTGCCGTGAATACTTGGGGCAAGGGCACCCTGGTTTCCGTGAGCAGCTACCCATACGACGTCCCAGACTACGCTTGATGActcgag

[0043] SEQ ID NO:4

[0044] catatgGATGTACAGCTACAAGCTTCAGGTGGAGGGTTGGTTCAGGCAGGCGGCAGCCTGCGTCTGAGCTGTGCTGCGAGCGCTCGCACCTTTTATACCATGGGTTTCTGGTTTAGACAGGTGCTGGGTAAGGACCGCGAGTTCGTGGGCGCAATTCGTTGGGGTGTTTACGCGACGACCCGTTATGCGGATTCTGTTAAAGGTCGTTTTTCGATCAGCCGTGATGATGCAACGAACACCGTGGCGCTGCAAATGAATAGCTTAAAACCGGAAGACACTGCCGTTTATTACTGCGCCGCGCGCGCTGGTCCGCTGGGCTTCGAGTTGTCTGCGACCTCCTCCGCGGAATACGACTACTGGCAAGGTGGGACCCAGGTCACCGTGAGCTCCCACCATCATCATCATCATCACCACTGATGActcgag

[0045] SEQ ID NO:5

[0046]

Claims

1. A homogeneous method for detecting the new coronavirus nucleocapsid protein based on a nanobody-monoclonal antibody sandwich, characterized in that: The following steps are involved: A. The SARS-CoV-2 N protein and SARS-CoV-2 N protein nanobodies N1, N2, N3, and N4 were prepared using an E. coli expression system, and the purity and biological activity of the expressed SARS-CoV-2 N protein and nanobodies were further characterized. B. Using the prepared nanobody as a coating antibody and the monoclonal antibody as a pairing antibody; C. Nanoantibodies were coupled to the surface of quantum dot microspheres to prepare fluorescent nanoprobes targeting the novel coronavirus N protein, providing detection signals for the homogeneous rapid detection system; D. Monoclonal antibodies against the novel coronavirus N protein were coupled to the surface of magnetic nanospheres to prepare magnetic nanoprobes targeting the novel coronavirus N protein, laying the foundation for the rapid separation of nucleocapsid proteins in a homogeneous system; E. The two prepared nanoprobes were added to different concentrations of the SARS-CoV-2 N protein, and an immunoassay for homogeneous detection of the SARS-CoV-2 N protein was established, with a minimum detection limit of 150 pg / mL. The sequences of the novel coronavirus N protein monoclonal antibody paired nanoantibodies N1, N2, N3, N4 and the novel coronavirus N protein are: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, respectively.

2. The homogeneous method for detecting the new coronavirus nucleocapsid protein based on the nanobody-monoclonal antibody sandwich according to claim 1, characterized in that The detection target is the new coronavirus N protein.

3. The homogeneous method for detecting the new coronavirus nucleocapsid protein based on the nanobody-monoclonal antibody sandwich according to claim 1 is characterized in that The paired antibody screening method is: the nanobody is used as the solid-phase antibody and the monoclonal antibody is used as the detection antibody; or the monoclonal antibody is used as the solid-phase antibody and the nanobody is used as the detection antibody.

4. The homogeneous method for detecting the new coronavirus nucleocapsid protein based on the nanobody-monoclonal antibody sandwich according to claim 1, characterized in that The input amount of quantum dot microspheres coupled to nanoantibodies is 4-6 μL, and the input amount of magnetic nanospheres is 8-14 μL.

5. The homogeneous method for detecting the new coronavirus nucleocapsid protein based on the nanobody-monoclonal antibody sandwich according to claim 3 is characterized in that, In the paired antibody screening method, the coating concentration of the nanobody is 2.5-5 μg / mL, and the input amount of the monoclonal antibody is 0.5-1.5 μg / mL.

6. The homogeneous method for detecting the new coronavirus nucleocapsid protein based on the nanobody-monoclonal antibody sandwich according to claim 1, characterized in that Step C is achieved by the following method, wherein the average particle size of the quantum dot microspheres is 100 nm: C1. Dissolve the quantum dot microspheres in a buffer solution, sonicate for 1-5 minutes, and add EDC and NHS to activate the carboxyl groups on the surface of the nanospheres. C2. Centrifuge to remove the activator, wash once with PBS buffer, add nanoantibodies N1, N2, N3, and N4, couple for 40-50 minutes, and then centrifuge to remove uncoupled antibodies; C3. Add 1-2% BSA dissolved in PBS to block the microspheres, separate the microspheres and supernatant, wash twice with PBS, resuspend the microspheres in preservation solution, and place the prepared probe at 4°C for later use.

7. The homogeneous method for detecting the new coronavirus nucleocapsid protein based on the nanobody-monoclonal antibody sandwich according to claim 6, characterized in that The coupling conditions are as follows: NHS / EDC input volume is 4-6 μL, protein input volume is 80-100 μg, and coupling time is 40-50 min.

8. The homogeneous method for detecting the new coronavirus nucleocapsid protein based on the nanobody-monoclonal antibody sandwich according to claim 1, characterized in that Step D is achieved by the following method, wherein the average particle size of the magnetic nanoparticles is 300 nm: D1. Dissolve the magnetic nanospheres in a buffer solution, sonicate for 1-5 minutes, and add EDC and NHS to activate the carboxyl groups on the surface of the nanospheres. D2. Separate the microspheres and activator using a magnetic rack, wash once with PBS buffer, add monoclonal antibody, couple for 50-70 minutes, and separate the beads using a magnetic rack to remove uncoupled antibody. D3. Add 1-2% BSA dissolved in PBS to block the microspheres, separate the microspheres and supernatant on a magnetic rack, wash twice with PBS, resuspend the microspheres in preservation solution, and place the prepared probe at 4°C for later use.

9. The homogeneous method for detecting the new coronavirus nucleocapsid protein based on the nanobody-monoclonal antibody sandwich according to claim 8, characterized in that The coupling conditions are as follows: NHS / EDC input volume is 6-10 μL, protein input volume is 90-120 μg, and coupling time is 50-70 min.

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