An immunochromatographic test strip based on paired single-domain heavy chain antibodies and a method for detecting receptor binding domain protein of novel coronavirus

Abstract

The application discloses a pair of single-domain heavy chain antibody-based immunochromatographic test strip and a method for detecting a novel coronavirus receptor binding domain protein, and belongs to the field of immunochromatographic detection.The immunofluorescence test strip provided by the application comprises a PVC bottom plate, a sample pad, a binding pad, an NC membrane and a water absorption pad, donor microspheres-single chain heavy domain antibody W is spotted on the binding pad, and receptor microspheres-single domain heavy chain antibody Q and anti-His monoclonal antibody are respectively line-marked on a detection line and a quality control line; RBD protein is prepared from mammalian cells, and single-domain heavy chain antibody is prepared by prokaryotic expression; the antibodies are respectively coupled to the surfaces of the donor microspheres and the receptor microspheres, 0.01-1000 ng / mL novel coronavirus RBD protein is loaded to prepare the test strip, and the sensitivity of the test strip is evaluated.The immunofluorescence test strip provided by the application can rapidly and sensitively detect the novel coronavirus receptor binding domain protein, and compared with a commercially available colloidal gold test strip for detecting a novel coronavirus antigen, has higher sensitivity, better detection accuracy and stability and lower detection cost.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of immunochromatographic detection, in particular to a novel coronavirus receptor binding domain protein detection immunochromatographic test strip based on paired single domain heavy chain antibodies. BACKGROUND

[0002] The receptor binding domain protein of the novel coronavirus is in variation and has evolved into various mutant strains, such as Omicron, Delta, N501Y. The transmission of the novel coronavirus is generally divided into direct, indirect and aerosol transmission, and the most effective way is to cut off the transmission of the virus to prevent the virus from spreading widely among the population or environment.

[0003] The detection methods of the novel coronavirus are divided into nucleic acid detection method, immunological analysis method and virus isolation method. Virus isolation method has long cycle, complex operation and is easy to cause secondary infection, so it is rarely used in the detection of the novel coronavirus; the reverse transcription-polymerase chain reaction (RT-PCR) method in nucleic acid detection method has high detection sensitivity and large detection throughput, and is used as the gold standard for the detection of the novel coronavirus, but the RT-PCR method has complex operation, high operation requirement, needs complex instruments and difficult sample preservation, etc. Subsequently, the government issued the colloidal gold test strip method in the immunological analysis method as an auxiliary means for the detection of the novel coronavirus, such as the patent "Novel coronavirus nucleocapsid protein antibody for in vitro diagnosis" (application number: CN202111644661.4) provides an in vitro indirect detection method for the novel coronavirus antibody, which has simple operation, fast detection speed and is suitable for multiple scene detection, but has low detection sensitivity, high false negative rate and large batch difference, and still has a large improvement space.

[0004] Fluorescence resonance energy transfer is a kind of energy transfer phenomenon between two fluorescent molecules in close proximity. When two molecules are within 10 nm, a non-radioactive fluorescence energy transfer phenomenon occurs, the fluorescence quantum yield and intensity are high, and it is applied to the test strip system to effectively improve the detection sensitivity of the system. In addition, the traditional test strip, such as the patent "Recombinant protein for detecting 2019 novel coronavirus antibody by double-antigen sandwich method, test strip, preparation method and application" (authorization number: CN111303297B), uses double-antigen sandwich method to detect novel coronavirus antigen, wherein the preparation cycle of single antibody with different epitopes is long, the process is complicated, the batch difference is large, a large amount of antigen is consumed, the paired antibody screening is difficult and the screening efficiency is low. Single domain heavy chain antibody is an excellent alternative element of single antibody, which has simple preparation, low cost, short cycle and small batch difference. The present application is based on paired single domain heavy chain antibodies and combines fluorescence resonance energy transfer technology to establish a POCT detection method with high operation detection, high sensitivity, fast detection speed and strong specificity. SUMMARY

[0005] The present application is based on some deficiencies of the currently marketed RT-PCR and colloidal gold test strip detection kit, aiming to establish a fluorescent immunochromatographic test strip with high sensitivity, fast detection speed, simple operation and adaptability to multiple scene detection, which is applied to large-scale screening of new coronavirus. First, the RBD protein eukaryotic expression vector and the nanobody prokaryotic expression vector for different epitopes of RBD protein are constructed, and the protein expression is carried out by transfection into the host and the biological activity is verified; the paired nanobodies are coupled to the surface of the acceptor / donor microspheres, which are scribed or dotted on the test strip, and the RBD protein is diluted to different concentrations by PBST and added to the sample pad of the test strip, and the luminescence of the test line (T line) and the control line (C line) is observed to evaluate the detection sensitivity of the test strip, and the specific steps are as follows:

[0006] According to the codon bias of mammals, the codon of SARS-COV-2 RBD protein is optimized, and the pCMV-SARS-COV-2 RBD eukaryotic expression vector is constructed, which is transfected into HEK293 cells, and the expression culture medium supernatant is purified by NI-NAT column, and the purified protein concentration is determined by BCA kit, and the purity and biological activity of the expressed RBD protein are determined by SDS-PAGE and ELISA.

[0007] According to the spatial structure of RBD protein, the donor microspheres-single domain heavy chain antibody W and the acceptor microspheres-single domain heavy chain antibody Q for different epitopes of RBD protein are selected, the codon is optimized, the prokaryotic expression vector is constructed, and the BL21(DE3) is transformed, and the cell broken supernatant is purified by NI-NAT column, and the purified protein concentration is determined by BCA kit, and the purity and biological activity of the expressed single domain heavy chain antibody are determined by SDS-PAGE and ELISA. The sequences of the single domain heavy chain antibodies W and Q are SEQ ID NO:1 and SEQ ID NO:2 respectively.

[0008] A certain amount of donor / acceptor microspheres is diluted in MES solution, and the carboxyl groups on the surface of the microspheres are activated using EDC / NHS; the single domain heavy chain antibody W and Q are added, so that the single domain heavy chain antibody W and Q are coupled to the surface of the donor / acceptor, respectively; BSA is used to block the excess sites, and the prepared nanoprobes are resuspended in the preservation solution and stored at 4°C.

[0009] The test strip is composed of NC membrane, sample pad, binding pad, water absorption pad and PVC bottom plate; the NC membrane is placed in a vacuum drying oven for drying, the NC membrane is pasted on the bottom plate, the sample pad is pressed on the binding pad, the binding pad is pressed on the NC membrane, and the water absorption pad is pressed on the NC membrane about 2-6mm, and the vacuum drying oven is dried; the prepared acceptor microspheres-single domain heavy chain antibody Q and anti-His monoclonal antibody are scribed on the test line and the control line respectively, and after drying, the test strip is cut according to the size of the test strip, and the donor microspheres-single domain heavy chain antibody W is dotted on the binding pad, and the test strip is stored at 4°C.

[0010] The RBD protein is diluted into different concentrations by PBS buffer, and then is put into PBST of a certain concentration. A certain volume of the RBD diluted protein is spotted on the sample pad of the test strip, and the test strip is left for a period of time. Then, under the irradiation of a 365 nm ultraviolet lamp, the T and C line detection results are observed, and the detection sensitivity of the test strip is evaluated.

[0011] Further, in the above steps 1) and 2), the transfection method of the eukaryotic expression vector is liposome transfection, and the transformation method of the prokaryotic expression vector is calcium transfection.

[0012] Further, in the above step 3), the concentration of MES is 0.01-0.1 M; the concentration of EDC / NHS is 1-20 mg / mL; and the concentration of BSA is 1-3%.

[0013] Further, in the above step 4), the size of the cut test strip is 4-6*60-100 mm; the distance between the detection line and the quality control line is 2-6 mm; the concentration of the acceptor microsphere-single domain heavy chain antibody Q on the detection line is 0.1-1 mg / mL; the concentration of the anti-His monoclonal antibody on the quality control line is 0.1-1 mg / mL; and the concentration of the donor microsphere-single domain heavy chain antibody W is 0.1-1 mg / mL.

[0014] Further, in the above step 5), the dilution concentration of the RBD protein is 0.01-1000 ng / mL; the concentration of PBST is 0.01-0.1%; and the spotting volume of the diluted sample is 30-100 μL.

[0015] Beneficial effects:

[0016] 1. Compared with the gold standard RT-PCR method for detecting the novel coronavirus, the fluorescent chromatography test strip provided by the application has high sensitivity, simple operation, fast detection speed, and can be applied to multiple scenes for detection, and has a broad application prospect in the large-scale screening of the novel coronavirus in the population.

[0017] 2. Compared with the traditional double monoclonal antibody sandwich method for detecting macromolecular proteins, the paired single domain heavy chain antibody provided by the application is easy to prepare, modify, has low cost, low batch difference, and the paired antibody is easy to screen, which can effectively improve the accuracy and accuracy of the detection system, reduce the batch difference of the detection kit, and reduce the production cost of the detection kit.

[0018] 3. Compared with the colloidal gold test strip and other novel coronavirus detection fluorescent test strips on the market, the application provides a detection signal based on fluorescence energy resonance transfer for the detection system, further improves the detection sensitivity of the novel coronavirus test strip, and provides more reliable evidence for the rapid detection of the novel coronavirus. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1SDS-PAGE and ELISA verification of paired single-domain heavy chain antibodies.

[0020] Figure 2 Sandwich ELISA to identify the binding performance of paired single-domain heavy chain antibodies to novel coronavirus RBD protein.

[0021] Figure 3 Scanning electron micrograph of donor microspheres.

[0022] Figure 4 Transmission electron micrograph of acceptor microspheres.

[0023] Figure 5 Surface Zeta potential changes of donor microspheres with different single-domain heavy chain antibody W input.

[0024] Figure 6 Surface Zeta potential changes of acceptor microspheres with different single-domain heavy chain antibody Q input.

[0025] Figure 7 Establishment of standard curve for detecting novel coronavirus RBD protein by immunochromatographic test strip.

[0026] Figure 8 Verification of specificity of immunochromatographic test strip. DETAILED DESCRIPTION

[0027] In order to make the present application more easily understood, the present application is further explained and described in conjunction with the accompanying drawings and examples. It should be understood that the specific examples described herein are only used to explain the present application and do not limit the present application, i.e., the described examples are part of the embodiments of the present application, but not all embodiments.

[0028] The materials, reagents and formulations used in all embodiments of the present application are as follows:

[0029] Main experimental materials:

[0030] Acceptor microspheres, donor microspheres (Suzhou Beaver Biotechnology Co., Ltd.), anti-His tag monoclonal antibody (Abcam, UK), Chinese hamster ovary cells (CHO), acceptor binding region protein single-domain heavy chain antibody, and acceptor binding region protein were prepared by the laboratory.

[0031] Main reagents:

[0032] Liposomes 3000 (Thermo Fisher Scientific), skimmed milk powder, tetramethylbenzidine, isopropyl-beta-D-thiogalactopyranoside (Shanghai Sangon Biotech), 1-ethyl-3-dimethylaminopropyl-carbonyldiimide (EDC), N-hydroxysuccinimide (NHS), bovine serum albumin (BSA), morpholine ethanesulfonic acid (MES, Sigma, USA).

[0033] Main reagent formula:

[0034] 1. LB plate medium: weigh 25 g of LB broth and 18 g of solid agar and dissolve in 1000 mL of ultrapure water, sterilize at 121℃ for 15 min;

[0035] 2. Buffer A: weigh 29.25 g of NaCl and 4.44 g of sodium phosphate and dissolve in 900 mL of ultrapure water, ultrasonic for 30 s, then dilute to 1 L, and adjust pH to 7.2.

[0036] 3. 2M sulfuric acid (H2SO4) 4) : take 15 mL of H2SO4, slowly add ultrapure water while stirring, and dilute to 200 mL.

[0037] 4. Storage solution: weigh 0.785 g of Tris, 0.0053 g of sodium chloride (NaCl), 1 g of BSA, and 0.2% NaN3.

[0038] The specific embodiments of the present application will be described in detail below with reference to the accompanying drawings, so that the advantages and features of the present application can be more easily understood by those skilled in the art, and the protection scope of the present application can be more clearly and definitely defined.

[0039] First, the novel coronavirus RBD protein and heavy chain single domain antibody are prepared by recombinant protein expression means, and the activity of the prepared protein is verified; the single domain heavy chain antibody is coupled with the receptor and the donor microspheres, the donor microspheres-heavy chain single domain antibody W point is added to the binding pad, and the fluorescent microspheres-heavy chain single domain antibody Q and the anti-histidine (His) monoclonal antibody are uniformly drawn on the T line and the C line of the NC membrane; when the novel coronavirus RBD protein exists in the sample, the fluorescent dye on the T line receives the energy transfer from the excited state to the fluorescent microspheres, and the test strip appears two fluorescent lines, the result is positive; on the contrary, when the novel coronavirus RBD does not exist, only the quality control line shows fluorescence, at this time the test strip appears one fluorescent line, the result is negative; if the quality control line does not show fluorescence, it indicates that the test strip is invalid.

[0040] Example 1

[0041] The specific steps are as follows:

[0042] 1) According to the codon bias of mammals, the RBD protein sequence is optimized, and the pCDNA-RBD eukaryotic expression vector is constructed.

[0043] 2) Resuscitate CHO cells, put them into a T25 cell culture dish containing 7 mL of DMEM / F12 medium, and incubate at 37℃ in 5% carbon dioxide (CO2) for overnight.

[0044] 3) After two generations, the cells in the T25 cell culture flask were transferred to a 6-well plate, and when the cells covered 70%-90% of the bottom area of the flask, pCDNA-RBD was transfected into the CHO cells according to the instructions of liposome 3000 transfection, and blank and positive controls were set.

[0045] 4) 24 hours after transfection, the transfection efficiency was estimated by taking pictures with an inverted fluorescence microscope, and the cells were cultured for 2-4 days. The cell culture supernatant was aspirated, and the bacteria solution was centrifuged at 8000-10000 rpm overnight. The expression supernatant was purified by NI-NAT column.

[0046] 5) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), enzyme-linked immunosorbent assay (ELISA) were used to detect the purity and biological activity of the expressed protein, and BCA was used to determine the protein concentration. Imidazole molecules were removed by ultrafiltration.

[0047] Example 2

[0048] The single-domain heavy chain antibody vector was constructed and expressed in prokaryotes, and the specific steps were as follows:

[0049] 1) According to the codon preference of prokaryotes, the sequences of single-domain heavy chain antibodies W and Q were optimized, and pET-single-domain heavy chain antibody expression vectors were constructed.

[0050] 2) BL21(DE3) was streaked on LB plates and incubated overnight. A single colony was inoculated in 3-7 mL of LB medium and incubated overnight.

[0051] 3) The bacteria solution was centrifuged at 8000-10000 rpm overnight, and BL21(DE3) was treated with 0.1M CaCl2 to make it competent.

[0052] 4) The pET-single-domain heavy chain antibody vector was transformed into BL21, and LB / Amp solid plates were coated. A single colony was picked and inoculated in 3-7 mL of LB medium and incubated overnight.

[0053] 5) According to the inoculation amount of 1-3%, 80-120 mL of LB medium was inoculated. When the bacteria grew to the logarithmic growth phase, 0.1-1M isopropyl-beta-D-thiogalactoside (IPTG) was added, and the culture was incubated at 24°C overnight.

[0054] 6) The bacteria solution was centrifuged at 8000-10000 rpm overnight, and the expression bacteria were broken at 180-220w. The expression supernatant was purified by NI-NAT, and the purity and biological activity of the expressed protein were detected by SDS-PAGE and ELISA. The protein concentration was determined by BCA, and imidazole molecules were removed by ultrafiltration. As shown in the table, the expressed single-domain heavy chain antibodies W and Q had excellent biological activity. Figure 1

[0055] Example 3​

[0056] The performance of the paired single-domain heavy chain antibody for detecting RBD protein was verified by ELISA method, and the specific steps were as follows:

[0057] 1) 50-200 μL of single-domain heavy chain antibody w with a concentration of 1-3 μg / mL was taken per well to coat the enzyme-labeled plate, and was blocked at 37°C for 2 h.

[0058] 2) The plate was washed with 0.05% PBST, 3-5% skimmed milk was added for blocking, and was incubated at 37°C for 1-2 h.

[0059] 3) The plate was washed with 0.05% PBST, and 0-10 ng / mL of novel coronavirus RBD protein was added, and was incubated at 37°C for 40-60 min.

[0060] 4) The plate was washed with 0.05% PBST, 1-5 μg / mL of single-domain heavy chain antibody Q was added, and was incubated at 37°C for 40-60 min.

[0061] 5) The plate was washed with 0.05% PBST, 1:2000-5000 HRP-labeled anti-strep II monoclonal antibody was added, and was incubated at 37°C for 40-60 min.

[0062] 6) The plate was washed with 0.05% PBST, TMB color developing solution was added, and after color developing for a period of time, 2M H2SO4 was added for termination, and the OD450nm absorbance value was determined.

[0063] As shown in Figure 2 , the heavy chain single-domain antibody W and Q as paired antibodies had high detection sensitivity for detecting novel coronavirus RBD protein.

[0064] Example 4

[0065] The single-domain heavy chain antibody was coupled to the surface of the microsphere, and the specific steps were as follows:

[0066] 1) As shown in Figures 3-4 , high-resolution transmission electron microscopy (TEM) showed that the particle size of the donor microsphere and the acceptor microsphere was 200 nm. 20-40 μL of donor / acceptor microspheres was taken, and was ultrasonically cleaned for 1-5 min, 500-1000 μL of MES solution was added, 5-10 μL of EDC and 5-10 μL of NHS were added, and was reacted at 10-40 rpm and 25°C for 20-40 min in a rotary mixer.

[0067] 2) Centrifugation was performed at 15000-20000 rpm for 20-30 min, and after washing once with PBS buffer, it was resuspended, 100-150 μg of single-domain heavy chain antibody W and Q was added to the acceptor / donor microspheres, respectively, and was reacted at 10-40 rpm and 25°C for 20-40 min in a rotary mixer.

[0068] 3) Weigh 0.01-0.03 g of BSA and dissolve it in the acceptor / donor microspheres for blocking.

[0069] 4) Centrifuge at 15000-20000 rpm for 20-30 min, rinse twice with PBS buffer, and resuspend with 1 mL of the preservation solution. After coupling, measure the zeta potential of the material surface using a dynamic light scattering instrument. As shown in Figures 5-6 the zeta potential of the material surface decreases after coupling, indicating that the single-chain variable fragment antibodies are successfully coupled to the material surface.

[0070] Example 5

[0071] Based on the donor microspheres-single-chain variable fragment antibody W and the acceptor microspheres-single-chain variable fragment antibody Q, an immunofluorescence test strip for detecting novel coronavirus RBD protein is constructed, and the specific steps are as follows:

[0072] 1) Dry the NC membrane first, and assemble the test strip in the order of PVC bottom plate, sample pad, conjugate pad, NC membrane, and water absorption pad from left to right.

[0073] 2) Use the gold spraying and membrane drawing instrument to draw the acceptor microspheres-single-chain variable fragment antibody Q and the anti-His monoclonal antibody on the T line and the C line respectively, and dry at 37°C for 1-3 h.

[0074] 3) Cut the test strip with a test strip cutter, and cut the test strip according to the 2-6 mm width specification. Dot the donor microspheres-single-chain variable fragment antibody W on the conjugate pad of the test strip, and dry for 1-3 h.

[0075] 4) Dilute the novel coronavirus RBD protein to 0.01-1000 ng / mL with PBS buffer, and add 50-100 μL of the diluent to the sample pad of the assembled test strip. Add human serum, BSA, and OVA as negative controls, and PBS as a blank control. Wait for 10-20 min, and observe the fluorescence of the detection line and the quality control line under the excitation light of 680 nm according to the instructions of the microspheres. As shown in Figures 7-8 the test strip based on the heavy-chain single-domain antibody has a minimum detection line of 0.1 ng / mL for detecting novel coronavirus RBD protein, and presents good detection specificity.

[0076] The above-described examples are the implementation methods of the present application. For those skilled in the art, without departing from the concept of the present application, a number of applications and improvements can be made, but all belong to the protection scope of the present application.

[0077] SEQ ID NO: 1

[0078] ccatggatCACCATCATCATCATCATCACCACCAAGTACAGCTAGTTGAATCAGGTGGAGGGTTGGTTCAGGCAGGCGGCTCGCTGCGTTTGAGCTGTGCGGTGTCCGGTGCCGGTGCGCACCGTGTTGGTTGGTTTCGTCGCGCACCGGGTAAAGAGAGAGAATTCGTGGCGGCGATCGGCGCGAGCGGCGGTATGACCAACTATCTGGACAGCGTGAAGGGCCGTTTCACCATTAGCCGTGACAACGCCAAGAACACCATCTACCTGCAAATGAATTCTCTGAAACCGCAGGATACCGCAGTCTATTACTGCGCTGCGCGCGATATTGAGACGGCTGAATATATCTACTGGGGTCAAGGTACTCAGGTTACCGTAAGCTCCGACTACAAAGATGATGACGATAAATAATAActcgag

[0079] SEQ ID NO: 2

[0080] ccatggatCACCATCATCATCATCATCACCACCACGTACAACTAGTTGAATCAGGTGGAGGGTTGGTTCAGGCGGGTGGCAGCCTGCGTCTGAGCTGTGCTGCGAGCGGTGGTACGTTCACCACCTTCCGCATGGCATGGTTTCGCCAGACCCCGGGCAAAGAAAGAGAGTTCGTGGCCGCGTCCTCTTGGGGCTTTGTTAATTATGCGGATCCGGTAAAAGGTCGTTTTACTATCAGCCGTGATAACGCAAAGAACACCGTCTACCTGGAAATGAGCTCCCTCAAGACCGAGGACACCGCTGTGTATTACTGCGCCGCGCGTAACCCGGGCACGGGTCAATATGACTACTGGGGCCAAGGTACACAGGTTACCGTGTCTTCGGACTACAAAGATGATGACGATAAATAATAActcgag.

Claims

1. A fluorescence energy resonance transfer immunochromatographic test strip based on a paired single-domain heavy chain antibody, characterized in that, The test strip comprises a PVC base plate, a sample pad, a binding pad, an NC film, a water absorption pad, a donor microsphere-single domain heavy chain antibody W is spotted on the binding pad, a receptor microsphere-single domain heavy chain antibody Q, and an anti-His monoclonal antibody is respectively scribed on the detection line and the quality control line. The target is a novel coronavirus receptor binding region protein; The sequences of the single domain heavy chain antibodies W and Q are respectively SEQ ID NO: 1 and SEQ ID NO:

2.

2. The method for preparing an immunochromatographic test strip according to claim 1, characterized in that, The method comprises the following steps: 1) According to the codon bias of mammals, a pCDNA-RBD eukaryotic expression vector is constructed, transfected into CHO cells, and the supernatant of the expression culture medium is purified by a NI-NAT column, the purified protein concentration is determined by a BCA kit, and the purity and biological activity of the protein are identified by SDS-PAGE and ELISA; 2) According to the spatial structure of the RBD protein, single domain heavy chain antibodies-single domain heavy chain antibody W and single domain heavy chain antibody Q which are directed against different epitopes of the RBD protein are selected, a prokaryotic expression vector is constructed and transformed into BL21, the supernatant of cell crushing is purified by a NI-NAT column, the purified protein concentration is determined by a BCA kit, and the purity and biological activity of the single domain heavy chain antibody are identified by SDS-PAGE and ELISA; 3) A certain amount of donor / receptor microspheres is diluted in MES solution, the carboxyl groups on the surface of the microspheres are activated by EDC / NHS, and the single domain heavy chain antibodies W and Q are put in so that the single domain heavy chain antibodies W and Q are coupled to the surface of the donor / receptor, respectively; BSA is used to block the excess sites, the prepared nanoprobes are resuspended in a preservation solution, and the nanoprobes are stored at 4 DEG C; 4) The NC film is placed in a vacuum drying oven for drying, the NC film is pasted on the base plate, the sample pad is pressed against the binding pad, the binding pad is pressed against the NC film, the water absorption pad is pressed against the NC film with a thickness of 2-6 mm, and the vacuum drying oven is used for drying; the prepared receptor microsphere-single domain heavy chain antibody Q and the anti-His monoclonal antibody are respectively scribed on the detection line and the quality control line, after drying, the test strip is cut according to the size of the test strip, the donor microsphere-single domain heavy chain antibody W is spotted on the binding pad, and the test strip is stored at 4 DEG C; 5) The prepared RBD protein is diluted into different concentrations by using a PBS buffer, a certain volume of the diluted RBD protein is spotted on the sample pad of the test strip, the test strip is left for a period of time, under the irradiation of a 680 nm ultraviolet lamp, the detection results of the T and C lines are observed, and the detection sensitivity of the test strip is evaluated.

3. The preparation method according to claim 2, characterized in that, The transfection method of the eukaryotic expression vector is a liposome transfection method, and the transformation method of the prokaryotic expression vector is a calcium transfection method.

4. The preparation method according to claim 2, characterized in that, The concentration of MES is 0.01-0.1 M; the concentration of EDC / NHS is 1-20 mg / mL; and the concentration of BSA is 1-3 %.

5. The preparation method according to claim 2, characterized in that, The distance between the detection line and the quality control line is 2-6 mm; the scribed concentration of the receptor microsphere-single domain heavy chain antibody Q on the detection line is 0.1-1 mg / mL; the concentration of the anti-His monoclonal antibody on the quality control line is 0.1-1 mg / mL; and the concentration of the donor microsphere-single domain heavy chain antibody W is 0.1-1 mg / mL.

6. The preparation method according to claim 2, characterized in that, The dilution concentration of the RBD protein is 0.01-1000 ng / mL; and the spotting volume of the diluted sample is 30-100 μL.

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