CN111925442B - New coronavirus RBD specific monoclonal antibody and application - Google Patents

Abstract

The invention belongs to the technical field of monoclonal antibodies, and specifically discloses a novel coronavirus RBD-specific monoclonal antibody. The heavy chain amino acid sequence is shown in SEQ ID NO: 1; the light chain amino acid sequence is shown in SEQ ID NO: 2. The present invention also provides the application of the above-mentioned novel coronavirus RBD-specific monoclonal antibody. The invention has important scientific significance and application prospects for the prevention, clinical treatment and research and development of diagnostic reagents for diseases caused by the novel coronavirus SARS-CoV-2.

Description

Novel coronavirus RBD-specific monoclonal antibodies and applications

technical field

The invention belongs to the technical field of monoclonal antibodies, and in particular relates to novel coronavirus RBD-specific monoclonal antibodies and applications.

Background technique

Antibodies are immunoglobulin molecules composed of four polypeptide chains, including two heavy chains (H chains) and two light chains (L chains). The H chain consists of a heavy chain variable region (VH) and a heavy chain constant region, and the heavy chain constant region consists of three regions CH1, CH2 and CH3. The L chain is composed of the L chain variable region (VL) and the light chain constant region, and the light chain constant region is composed of the CL region. VH and VL can be further divided into hypervariable regions called complementarity determining regions (CDRs) and alternately conserved regions called framework regions (FRs).

Current research has found that the new coronavirus (SARS-CoV-2) has four main structural proteins, namely the spike protein (S protein), the nucleocapsid protein (N protein), the membrane protein (M protein) and the envelope protein. (E protein), in which the S protein has two subunits: S1 and S2, the receptor binding site (RBD) is located on the S1 subunit, and its main function is to recognize the host cell surface receptor and mediate fusion with the host cell .

At present, there is no specific drug-targeted treatment for the emerging pathogen COVID-19, and vaccine development will take time. The plasma of recently cured and discharged patients contains a high concentration of specific antigen-neutralizing antibodies. After infusion into the patient, it can neutralize the new crown pathogen and mediate an effective immune response. Therefore, the use of convalescent plasma is expected to provide treatment for patients infected with the new coronavirus. Effective treatment methods can reduce mortality and ensure the safety of patients' lives.

The Chinese invention patent application with the application publication number CN111303280A discloses a fully human monoclonal antibody against SARS-CoV-2 with high neutralization activity. The above patent provides a fully human monoclonal antibody whose recognition region is the S1 non-RBD region, However, since the new coronavirus invades the host cell by binding to the ACE2 of the host cell through RBD, the fully human monoclonal antibody obtained in the above-mentioned patent has limited blocking effect on the virus, and the above-mentioned patent obtains the antibody cDNA by labeling plasma cells, but Compared with plasma cells, memory B cells respond quickly after activation, so memory B cells can trigger a faster and stronger humoral immune response than the initial response, while plasma cells trigger a limited humoral immune response.

SUMMARY OF THE INVENTION

In order to achieve the above purpose, the present invention provides a novel coronavirus RBD-specific monoclonal antibody, specifically, the heavy chain amino acid sequence of the antibody is shown in SEQ ID NO: 1; the light chain amino acid sequence is shown in SEQ ID NO: 2 (single anti-1-CQT149). The heavy chain amino acid sequence can also be shown in SEQ ID NO: 3; the light chain amino acid sequence can also be shown in SEQ ID NO: 4 (mab 2-CQT190). The heavy chain amino acid sequence can also be shown in SEQ ID NO: 5; the light chain amino acid sequence can also be shown in SEQ ID NO: 6 (mab 3-CQT191). The heavy chain amino acid sequence can also be shown in SEQ ID NO: 7; the light chain amino acid sequence can also be shown in SEQ ID NO: 8 (mab 4-CQT195). The heavy chain amino acid sequence can also be shown in SEQ ID NO: 9; the light chain amino acid sequence can also be shown in SEQ ID NO: 10 (mab 5-CQT197). The heavy chain amino acid sequence can also be shown in SEQ ID NO: 11; the light chain amino acid sequence can also be shown in SEQ ID NO: 12 (mab 6-CQT198). The heavy chain amino acid sequence can also be shown in SEQ ID NO: 13; the light chain amino acid sequence can also be shown in SEQ ID NO: 14 (mAb 7-CQT199). The heavy chain amino acid sequence can also be shown in SEQ ID NO: 15; the light chain amino acid sequence can also be shown in SEQ ID NO: 16 (mAb 8-CQT200). The heavy chain amino acid sequence can also be shown in SEQ ID NO: 17; the light chain amino acid sequence can also be shown in SEQ ID NO: 18 (mAb 9-CQT201). The heavy chain amino acid sequence can also be shown in SEQ ID NO: 19; the light chain amino acid sequence can also be shown in SEQ ID NO: 20 (mAb 10-CQT201). The heavy chain amino acid sequence can also be shown in SEQ ID NO: 21; the light chain amino acid sequence can also be shown in SEQ ID NO: 22 (mAb 11-CQT202). The heavy chain amino acid sequence can also be shown in SEQ ID NO: 23; the light chain amino acid sequence can also be shown in SEQ ID NO: 24 (mAb 12-CQT203). The heavy chain amino acid sequence can also be shown in SEQ ID NO: 25; the light chain amino acid sequence can also be shown in SEQ ID NO: 26 (mab 13-CQT206). The heavy chain amino acid sequence can also be shown in SEQ ID NO: 27; the light chain amino acid sequence can also be shown in SEQ ID NO: 28 (mab 14-CQT209). The heavy chain amino acid sequence can also be shown in SEQ ID NO: 29; the light chain amino acid sequence can also be shown in SEQ ID NO: 30 (mab 15-CQT210). The heavy chain amino acid sequence can also be shown in SEQ ID NO: 31; the light chain amino acid sequence can also be shown in SEQ ID NO: 32 (mab 16-CQT212). The heavy chain amino acid sequence can also be shown in SEQ ID NO: 33; the light chain amino acid sequence can also be shown in SEQ ID NO: 34 (mAb 17-CQT215). The heavy chain amino acid sequence can also be shown in SEQ ID NO: 35; the light chain amino acid sequence can also be shown in SEQ ID NO: 36 (mAb 18-CQT216). The heavy chain amino acid sequence can also be shown in SEQ ID NO: 37; the light chain amino acid sequence can also be shown in SEQ ID NO: 38 (mAb 19-CQT217). The heavy chain amino acid sequence can also be shown in SEQ ID NO: 39; the light chain amino acid sequence can also be shown in SEQ ID NO: 40 (mAb 20-CQT218).

The present invention also provides the application of the above-mentioned novel coronavirus RBD-specific monoclonal antibody in the preparation of reagents or vaccines or drugs for detecting or diagnosing SARS-CoV-2; and also provides a nucleic acid molecule encoding the above-mentioned novel coronavirus RBD-specific monoclonal antibody ; also provide an expression cassette, recombinant vector, recombinant bacteria or transgenic cell line containing the above-mentioned nucleic acid molecule; also provide the application of the above-mentioned expression cassette, recombinant vector, recombinant bacteria or transgenic cell line in the preparation of products.

The present invention also provides a product, including the above-mentioned new coronavirus RBD-specific monoclonal antibody; the use of the product is any of the following (b1)-(b4): (b1) combined with the new coronavirus SARS-CoV-2; (b2) Detection of binding to the new coronavirus SARS-CoV-2; (b3) binding to the S protein of the new coronavirus SARS-CoV-2; (b4) detection of the S protein of the new coronavirus SARS-CoV-2

The present invention also provides a pharmaceutical composition comprising the above-mentioned novel coronavirus RBD-specific monoclonal antibody and a pharmaceutically acceptable excipient, diluent or carrier.

Preferably, the above-mentioned novel coronavirus RBD-specific monoclonal antibody is obtained by sorting specific memory B cells and amplifying the antibody variable region cDNA by RT-PCR.

The principle and beneficial effects of the present invention are:

(1) The monoclonal antibody provided by the present invention has RBD specificity. Compared with the monoclonal antibody against the non-RBD region of S1, the monoclonal antibody provided by the present invention is combined with RBD, which is used for antibody drug screening, diagnosis, prevention and treatment. COVID-19 provides wider application value.

(2) The monoclonal antibody provided by the present invention is obtained by sorting RBD-specific memory B cells. Compared with the prior art by sorting plasma cells, the monoclonal antibody prepared by the present invention can induce stronger humoral immunity reaction. In addition, the present invention only performs subsequent RT-PCR, nested PCR and antibody function analysis for RBD-specific memory B cells, which greatly improves the specific binding ability of the monoclonal antibody to RBD.

Description of drawings

Figure 1 is a cell sorting diagram of memory B cells analyzed by flow cytometry;

Figure 2 is a cell sorting diagram of RBD-specific memory B cells analyzed by flow cytometry;

Figure 3 is a gel electrophoresis image of a single-cell antibody gene PCR product;

Figure 4 is an agarose gel electrophoresis image after PCR amplification of an antibody gene expression cassette comprising CMV promoter, WPRE-γ or WPRE-κ element;

Figure 5 is a graph showing the results of RBD-specific detection.

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

Example 1

This example provides a novel coronavirus RBD-specific monoclonal antibody, the amino acid sequence of the heavy chain is shown in SEQ ID NO: 1; the amino acid sequence of the light chain is shown in SEQ ID NO: 2.

This embodiment also provides the application of the above-mentioned novel coronavirus RBD-specific monoclonal antibody in the preparation of reagents or drugs for detecting or diagnosing SARS-CoV-2.

In actual production, the RBD-specific monoclonal antibody obtained in this example can be used to prepare a nucleic acid molecule, or an expression cassette, a recombinant vector, a recombinant bacteria or a transgenic cell line containing the nucleic acid molecule can be prepared, or a pharmaceutical composition can be prepared. The substance includes the above-mentioned novel coronavirus RBD-specific monoclonal antibody and a pharmaceutically acceptable excipient, diluent or carrier.

In application, this example obtains related products prepared by RBD-specific monoclonal antibodies, which can be used in any of the following (b1)-(b4): (b1) binding to the new coronavirus SARS-CoV-2; (b2) Detection of binding to the new coronavirus SARS-CoV-2; (b3) detection of the S protein of the new coronavirus SARS-CoV-2; (b4) detection of the S protein of the new coronavirus SARS-CoV-2.

Example 2-20

The difference between Example 2-20 and Example 1 is that the amino acid sequences of RBD-specific monoclonal antibodies are different, and the amino acid sequences of Example 2-20 are shown in the following table:

test group heavy chain amino acid sequence light chain amino acid sequence Example 1 - CQT149 SEQ ID NO: 1 SEQ ID NO: 2 Example 2 - CQT190 SEQ ID NO: 3 SEQ ID NO: 4 Example 3 - CQT191 SEQ ID NO: 5 SEQ ID NO: 6 Example 4 - CQT195 SEQ ID NO: 7 SEQ ID NO: 8 Example 5 - CQT197 SEQ ID NO: 9 SEQ ID NO: 10 Example 6 - CQT198 SEQ ID NO: 11 SEQ ID NO: 12 Example 7 - CQT199 SEQ ID NO: 13 SEQ ID NO: 14 Example 8 - CQT200 SEQ ID NO: 15 SEQ ID NO: 16 Example 9 - CQT201 SEQ ID NO: 17 SEQ ID NO: 18 Example 10 - CQT201 SEQ ID NO: 19 SEQ ID NO: 20 Example 11 - CQT202 SEQ ID NO: 21 SEQ ID NO: 22 Example 12 - CQT203 SEQ ID NO: 23 SEQ ID NO: 24 Example 13 - CQT206 SEQ ID NO: 25 SEQ ID NO: 26 Example 14 - CQT209 SEQ ID NO: 27 SEQ ID NO: 28 Example 15 - CQT210 SEQ ID NO: 29 SEQ ID NO: 30 Example 16 - CQT212 SEQ ID NO: 31 SEQ ID NO: 32 Example 17 - CQT215 SEQ ID NO: 33 SEQ ID NO: 34 Example 18 - CQT216 SEQ ID NO: 35 SEQ ID NO: 36 Example 19 - CQT217 SEQ ID NO: 37 SEQ ID NO: 38 Example 20 - CQT218 SEQ ID NO: 39 SEQ ID NO: 40

The RBD-specific monoclonal antibodies provided in the above Examples 1-20 were all obtained by the following methods: first, a single RBD-specific memory B cell was obtained from the peripheral blood of recovered patients with COVID-19, and then a single RBD-specific memory B cell was obtained. The mRNA was then constructed by RT-PCR and nested PCR to construct the antibody variable region gene expression cassette, and then the antibody variable region gene expression cassette was transfected into 293T cells to express the antibody and the supernatant was collected, and the RBD specificity of the supernatant was detected by ELISA. After screening, RBD-specific monoclonal antibodies were obtained.

Specifically include the following steps:

S1. Collect peripheral blood of several recovered patients with new coronary pneumonia, isolate PBMCs, and freeze them in a -80°C refrigerator for future use.

S2. First, use dead cell dye (Dead Dye) to remove dead cells of PBMCs obtained by S1, and then use CD19, mIg-G, mIg-D and S-RBD to memory specific and high binding capacity of live RBDs in PBMCs B cells are stained and marked to screen out RBD-specific memory B cells; use flow cytometry to sort specific memory B cells into 96-well plates, each well has one specific memory B cell, and it is in- Store in a freezer at 80°C for later use.

Specifically, the preferred concentration range of Dead Dye staining in this embodiment is 1-2 μg/mL, and the preferred concentration of Dead Dye staining in this embodiment is 1.5 μg/mL; CD19 is a B cell marker produced by Biolegend. The concentration range of CD19 is 1-2 μg/mL, and the preferred concentration of CD19 in this embodiment is 1.5 μg/mL. mIg-G is a B cell labeled surface receptor produced by Biolegend, and the concentration range during staining is 1-2 μg/mL. In this embodiment, the preferred concentration of mIg-G staining is 1.5 μg/mL; mIg-D is produced by Biolegend. B cell surface receptor, the concentration range of staining is 1-2 μg/mL, in this embodiment, the preferred concentration of mIg-D staining is 1.5 μg/mL; S-RBD is the new coronavirus produced by sinobiological, which is a protein receptor domain , the concentration range during staining is 1-2 μg/mL, and in this embodiment, the preferred concentration of S-RBD during staining is 1.5 μg/mL.

Sorting of RBD-specific memory B cells by flow cytometry, cell sorting of PBMCs by CD19, mIg-G, mIg-D, and S-RBD yields cell sorting of memory B cells specific for S-RBD Figures are shown in Figures 1 and 2, where Batch IDs 0428, 0505, 0522, and 0528 in Figure 2 are screening batches. In this example, the principle of using CD19, mIg-G, mIg-D and S-RBD to screen out RBD-specific memory B cells is as follows: PBMCs are treated with dead cell dye (Dead Dye), B cell marker CD19, memory B cells The cell markers mIg-G positive and mIg-D negative and memory B cells expressing RBD-specific IgG were stained, and then the CD19 cell population in the cell population was divided by flow cytometry. Divide the mIg-G ⁺ mIg-D ^- cell population, and then divide the RBD-positive memory B cells from the mIg-G ⁺ mIg-D ^- cell population, and then sort the RBD-positive memory B cells by flow cytometry. .

S3. The mRNA of a single RBD-specific memory B cell is obtained by sorting, and the variable region cDNA of the antibody is obtained by RT-PCR amplification. Specifically, when RT-PCR is used to amplify the variable region cDNA of the antibody, the primer front section of the primer designed in this example is designed with a general leader (see Primer Sequence Table 1 and Primer Sequence Table 2), which effectively improves the amplification of antibody genes. The experimental results are shown in Figure 3.

S4, using nested PCR to amplify the antibody variable region cDNA obtained from S1-S3 to construct an antibody variable region gene expression cassette.

S3 and S4 were performed in a total of six parts: (1) extraction of mRNA from RBD-specific memory B cells; (2) single cell mRNA reverse transcription (RT); (3) G-tailing (TDT); (4) first Round PCR (1st PCR); (5) Second round PCR (2nd PCR); (6) BCR-ORF PCR amplification to construct gene expression cassette; (7) CMV, WPRE-γ/κ/1 fragment amplification and CMV , BCR-Vγ/κ/l (product of (6)), WPRE-γ/κ/l overlap PCR (Overlap PCR) pre-ligation; (8) BCR-γORF, BCR-κORF, BCR-1 PCR amplification.

The preparation and reaction conditions of each part of the reaction solution are as follows:

(1) Use DynabeadsTM mRNA ^DIRECTTM Purification Kit ⁽ ThermoFisherscientific) to extract single-cell mRNA, which specifically includes the following steps:

① Centrifugation: After taking out the 96-well plate with single RBD-specific memory B cells sorted from the -80°C refrigerator, centrifuge at 600×g for 30s to centrifuge the cells at the bottom of the well;

②Washing: Take out the Dynabeads oligo(dT) 25 microsphere bottle and mix by vortexing, suck up enough microspheres according to 2μl/well, place it on the magnet block, let it stand for 30s, discard the supernatant, resuspend it with 500μl Lysis Buffer hanging;

③Preparation: add 9μl/well Lysis Buffer into a 50mL centrifuge tube, add the above 500μl microsphere suspension into it, and blow it evenly with a gun;

④ Dispense: Dispense the microspheres with an eight-connected tube, and then add 9 μl/well of the microspheres to the cell plate using a discharge gun;

⑤Rinse: 96-well plate with film, then rinse around the tube wall, a total of 2 cycles;

⑥ Incubation: stand at room temperature for 5 minutes to fully release the mRNA of RBD-specific memory B cells and bind to the microspheres. After incubation, centrifuge briefly at 600 × g to make the microspheres centrifuge at the bottom of the well. Place the 96-well plate on the DynaMag ^TM -96sideMagnet magnetic plate, and discard the supernatant with a discharge gun;

⑦Wash A washing: Add Washing Buffer A according to 8μl/well, and run the plate back and forth 7-8 times to wash the microspheres thoroughly, and discard the supernatant;

⑧ Washing with Wash B: Add Washing Buffer B at 8 μl/well, go back and forth 7-8 times to wash the microspheres thoroughly, discard the supernatant, and then add the pre-prepared reverse transcription (RT) reaction solution at 10 μl/well. Reagent preparation and reaction conditions are described in (2) below.

(2) Reverse transcription (RT) (10 μl system)

The reagents to be prepared are shown in Table 1 below:

Reagent name volume DEPC-H₂O 4.5μl 5×primerscript Buffer 2.0μl 2.5mM dNTPs 2.0μl RNase Inhibitor 1μl Sample beads PrimerScript II RTase 0.5μl total capacity 10μl

Reaction conditions: 42°C for 60min (mix every 20min);

After the reaction, the 96-well plate was centrifuged at 600×g for a short time, and then the 96-well plate was placed on the DynaMag ^TM -96sideMagnet magnetic plate. and reaction conditions are as described in (3) below.

(3) Add G tail (TDT) (10 μl system)

The reagents to be prepared are shown in Table 2 below:

Reagent name volume H₂O 6.4μl 5×TdT buffer 2.0μl 10mM dGTP 0.5μl 0.1% BSA 1.0μl Sample beads TdT 0.1μl total capacity 10μl

Reaction conditions: 37°C for 40 min (mixing every 20 min).

After the reaction was completed, the 96-well plate was centrifuged at 600×g for a short time, and then placed on a DynaMag ^TM -96side Magnet magnetic plate. The supernatant was discarded with a row gun, and then 10 μl/well was added to the pre-prepared first-round PCR (1st PCR) The reaction solution, reagent preparation and reaction conditions are described in (4) below.

(4) 1st PCR (10 μl system) (see the primer sequence table for primer sequences)

The reagents to be prepared are shown in Table 3 below:

Reagent name volume H₂O 1.9μl 2×GC Buffer 5μl 2.5mM dNTPs 1μl FP:AP3-dC (10 μM) 0.5μl RP1:Cg-1st (10μM) 0.5μl RP2:Ck-1st (10μM) 0.5μl RP3:CI-RT (10μM) 0.5μl PrimesTAR 0.1μl sample beads total capacity 10μl

Based on the PCR principle, the experimental reaction conditions for 1st PCR are: ① 95°C pre-denaturation for 3 min; ② 95°C denaturation for 15 sec, 60°C annealing for 5 sec, 72°C extension for 1 min, 30-35 cycles, 30 cycles are preferred in this example; ③ 72°C external extension for 5 min, Store at 4°C.

(5) The second round of PCR (2nd PCR) (10 μl system) (see primer sequence table 1 and primer sequence table 2 for primer sequences)

The reagents to be prepared are shown in Table 4 below:

Reagent name volume H₂O 1.5μl 2×GC Buffer 5μl 2.5mM dNTPs 1μl FP:MAC-AP3/AP3(10μM) 0.5μl RP:Cg-nest/K20/CI-nest (10μM) 0.5μl PrimesTAR 0.5μl sample 1μl total capacity 10μl

Based on the PCR principle, the experimental reaction conditions of 2nd PCR are as follows: (1) Pre-denaturation at 95°C for 3 min; (2) Denaturation at 95°C for 15sec, annealing at 60°C for 5s, extension at 72°C for 1min, 30-35cycles, in this example, 35cycles is preferred; Store at 4°C.

After PCR: take 4 μl per well for 1.5% agarose gel electrophoresis. Cell wells with Gamma chains paired with Kappa chains or Lamada chains are sent for sequencing.

(6) Amplification and construction of antibody expression cassette (BCR-ORF)

PCR amplification of the promoter region (CMV promoter), WPRE-γ (antibody gamma chain) and WPRE-κ (antibody kappa chain), the PCR amplification system is shown in Table 5 below:

PCR amplification conditions were: ① pre-denaturation at 95°C for 3 min; ② denaturation at 95°C for 15 sec, annealing at 56°C for 15 sec, extension at 72°C for 1 min, 30 cycles; ③ external extension at 72°C for 5 min, and storage at 12°C.

(7) CMV, WPRE-γ/κ/1 fragment amplification and CMV, BCR-Vγ/κ/1, WPRE-γ/κ/1 overlap PCR (Overlap PCR) pre-ligation

The experimental system is shown in Table 6 below:

PCR amplification conditions were: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 sec, annealing at 50°C for 15 sec, extension at 72°C for 1.5 min, 10 cycles; external extension at 72°C for 5 min, and storage at 12°C.

(8) PCR amplification of BCR-γORF, BCR-κORF, BCR-1

The experimental system is shown in Table 7 below:

PCR amplification program: pre-denaturation at 95°C for 3min; denaturation at 95°C for 15sec, annealing at 58°C for 15sec, extension at 72°C for 1.5min, 30 cycles; external extension at 72°C for 5min, and storage at 12°C.

After amplification, agarose gel electrophoresis was used, and gel imaging was used to analyze whether the size of the antibody variable region gene was correct. The experimental results are shown in Figure 4, the Marker is in the middle position, and the band is at 5000bp.

BCR-γORF and BCR-κ/ORF ethanol precipitation: Take 30 μl of PCR products of BCR-γORF and BCR-κORF and put them in 8-connected tubes, then add 120 μl absolute ethanol and 6 μl sodium acetate solution, mix well, -80 ℃ stand for 30 min; 10000 rpm, centrifuge for 20 min, discard the supernatant, rinse with 200 μl of 70% ethanol and anhydrous ethanol in turn, fully volatilize the ethanol at 56 ℃, add 40 μl of sterile water, shake to fully dissolve the precipitate, and detect The concentration of antibody variable region genes.

The Leader primers used in S3 and S4 are shown in the following primer sequence table 1:

The J-region primers used in S3 and S4 are shown in the following primer sequence table 2:

S5. The antibody variable region gene expression cassette obtained in S4 was transfected into 293T cells to express the antibody within 48 hours, and the supernatant was collected. The RBD specificity of the supernatant was detected by ELISA, and the RBD-specific fully human monoclonal antibody was screened.

(A) Dilute the antigen with PBS (final concentration 2 μg/mL), 10 μl/well, coat 384-well ELISA plate at 4°C overnight or at 37°C for 2 hours (preferably 4°C overnight in this example). NOTE: Centrifuge briefly after adding to ensure the liquid is at the bottom.

The experimental system is shown in Table 8 below:

Reagent name article number original concentration Final concentration dilution ratio SARS-COV-2RBD Cat: 40592-V08H 200μg/mL 2μg/mL 1:100 Goat pab to Hu IgG-ALP Cat: ab97221 1mg/mL 2μg/mL 1:500

(B) Preparation of PBST (0.05% Tween 20, Cat#TB220): 1L of PBS was added with 0.5mL of Tween 20;

PBST machine-washed plates (Thermoscientific wellwash versa) or hand-washed (the plates after machine washing still need to be tapped manually/centrifuged with a microplate centrifuge (MPC-P25) for 1 min, so that no water and air bubbles can be seen on the plate).

Blocking: 80 μl of 5% BSA (BioFroxx, Cat. NO: 4240GR100) (prepared with PBST) was added to the above washed plate and placed in a 37° C. incubator for 1 h. Machine wash plates in PBST or hand wash.

(C) Sample addition and standard. Among them, the standard: 10μl/well original concentration 1μg/mL, the gradient dilution is 250ng/mL, 125ng/mL, 62.5ng/mL, 31.25ng/mL, 15.63ng/mL, 7.81ng/mL, 3.9ng/mL and 1.95ng/mL. (dilution in blocking solution); sample: supernatant of cells transfected with antibody gene. Negative control/blank well: 10 μl/well of blocking solution.

Incubate for 30 min at 37°C. Machine wash plates in PBST or hand wash.

(D) Secondary antibody was added at a concentration of 10 μl/well, and then incubated at 37°C for 30 min.

The experimental system is shown in Table 9 below:

Secondary antibody name article number original concentration Final concentration dilution ratio goat-anti-human IgG-ALP A18808 1.5mg/ml 0.3μg/ml 1:5000 Goat pab to Hu IgG-ALP Ab98532 0.5mg/ml 0.25μg/ml 1:2000

Machine wash plates in PBST or hand wash. 10μl/well of PNPP (disodium p-nitrophenylphosphate), using (Thermoscientific Muttiskan GO) to detect OD (450mm) for 5min, 10min, 15min, 20min, 25min, 30min, 35min, 40min, 45min, 50min, 55min and 60min value. 50 mg of PNPP powder (Thermo, Prod #34045) + 40 mL of ddH ₂ O + 10 mL of Diethanol aminesubstrate Buffer (5X), PNPP was stored at 4°C in the dark.

The experimental results are shown in Figure 5, and the OD value greater than 0.1 is positive.

The above descriptions are only preferred embodiments of the present invention, which are only illustrative rather than restrictive for the present invention; those of ordinary skill in the art will understand that the invention can be modified within the spirit and scope defined by the claims of the present invention. Numerous changes, modifications, and even equivalent changes can be made, but will fall within the protection scope of the present invention.

Claims (7)

1. The monoclonal antibody with the specificity of the new coronavirus RBD is characterized in that the amino acid sequence of a heavy chain is shown as SEQ ID NO 1; the light chain amino acid sequence is shown as SEQ ID NO. 2.

2. The monoclonal antibody with the specificity of the new coronavirus RBD is characterized in that the amino acid sequence of a heavy chain is shown as SEQ ID NO. 19; the light chain amino acid sequence is shown as SEQ ID NO. 20.

3. The monoclonal antibody specific to RBD of the novel coronavirus according to any one of claims 1-2, wherein the antibody variable region cDNA is obtained from the mRNA of RBD-specific memory B cells by sorting the RBD-specific memory B cells.

4. Use of the monoclonal antibody specific to the novel coronavirus RBD according to any one of claims 1-2 for the preparation of a reagent or a medicament for the detection or diagnosis of SARS-CoV-2, wherein the medicament comprises the monoclonal antibody specific to the novel coronavirus RBD according to any one of claims 1-2, and a pharmaceutically acceptable excipient, diluent or carrier.

5. Nucleic acid molecule encoding the novel coronavirus RBD-specific monoclonal antibody of any one of claims 1-2.

6. An expression cassette, recombinant vector, recombinant bacterium or transgenic cell line comprising the nucleic acid molecule of claim 5.

7. The use of the expression cassette, recombinant vector, recombinant strain or transgenic cell line of claim 6 in the preparation of a product for any of the following uses (b 1) to (b 4): (b 1) binds to novel coronavirus SARS-CoV-2; (b 2) detecting a novel coronavirus SARS-CoV-2; (b 3) binding to the S protein of the novel coronavirus SARS-CoV-2; (b 4) detecting the S protein of the novel coronavirus SARS-CoV-2.

Images