CN115925902B - Novel coronavirus RBD specific monoclonal antibody and application - Google Patents

Abstract

The present invention belongs to the technical field of monoclonal antibodies, and specifically discloses a novel coronavirus RBD-specific monoclonal antibody and the application of the novel coronavirus RBD-specific monoclonal antibody. The present invention has important scientific significance and application prospects for the prevention, clinical treatment and development of diagnostic reagents for diseases caused by the novel coronavirus SARS-CoV-2.

Description

Novel coronavirus RBD specific monoclonal antibody and application

Technical Field

The invention belongs to the technical field of monoclonal antibodies, and particularly relates to a novel coronavirus RBD specific monoclonal antibody and application thereof.

Background

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 consisting of three regions CH1, CH2 and CH 3. The L chain consists of an L chain variable region (VL) and a light chain constant region consisting of a CL region. VH and VL can be further divided into hypervariable regions called Complementarity Determining Regions (CDRs) and conserved regions called Framework Regions (FR) alternating.

The current research finds that: the novel coronavirus (SARS-CoV-2) has four major structural proteins, spike protein (S protein), nucleocapsid protein (N protein), membrane protein (M protein) and envelope protein (E protein), respectively, wherein the S protein has two subunits: s1 and S2, receptor binding sites (novel coronavirus RBDs) are located on the S1 subunit, whose primary function is to recognize host cell surface receptors, mediating fusion with host cells.

The Chinese patent application publication No. CN111303280A discloses a fully human monoclonal antibody with high neutralization activity against SARS-CoV-2, and the above-mentioned patent provides a fully human monoclonal antibody whose recognition region is S1 non-novel coronavirus RBD region, but because the novel coronavirus invaded host cell is combined with ACE2 of host cell by means of novel coronavirus RBD, the blocking effect of the fully human monoclonal antibody obtained by the above-mentioned patent on virus is limited, and the above-mentioned patent is obtained by labeling plasma cell to obtain antibody cDNA, but compared with plasma cell, the memory B cell can make quick response after activation, so that the memory B cell can make more quick and strong humoral immune response than primary reaction, and the humoral immune response caused by plasma cell is limited.

Disclosure of Invention

The invention aims to provide a novel coronavirus RBD specific monoclonal antibody which aims at RBD and can trigger stronger humoral immune response and application thereof.

In order to achieve the aim, the invention provides a novel coronavirus RBD specific monoclonal antibody, and particularly, the amino acid sequence of a heavy chain variable region of the antibody is shown as SEQ ID NO. 1; the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 2 (monoclonal antibody 1-CQTS 045). The amino acid sequence of the heavy chain variable region can be also shown as SEQ ID NO. 3; the amino acid sequence of the light chain variable region can also be shown as SEQ ID NO. 4 (monoclonal antibody 2-CQTS 046). The amino acid sequence of the heavy chain variable region can be also shown as SEQ ID NO. 5; the amino acid sequence of the light chain variable region can also be shown as SEQ ID NO. 6 (monoclonal antibody 3-CQTS 048). The amino acid sequence of the heavy chain variable region can be also shown as SEQ ID NO. 7; the amino acid sequence of the light chain variable region can also be shown as SEQ ID NO. 8 (monoclonal antibody 4-CQTS 049). The amino acid sequence of the heavy chain variable region can also be shown as SEQ ID NO. 9; the amino acid sequence of the light chain variable region can also be shown as SEQ ID NO. 10 (monoclonal antibody 5-CQTS 050). The amino acid sequence of the heavy chain variable region can be also shown as SEQ ID NO. 11; the amino acid sequence of the light chain variable region can also be shown as SEQ ID NO. 12 (monoclonal antibody 6-CQTS 051). The amino acid sequence of the heavy chain variable region can also be shown as SEQ ID NO. 13; the amino acid sequence of the light chain variable region can also be shown as SEQ ID NO. 14 (monoclonal antibody 7-CQTS 052). The amino acid sequence of the heavy chain variable region can be also shown as SEQ ID NO. 15; the amino acid sequence of the light chain variable region can also be shown as SEQ ID NO. 16 (monoclonal antibody 8-CQTS 053). The amino acid sequence of the heavy chain variable region can be also shown as SEQ ID NO. 17; the amino acid sequence of the light chain variable region can also be shown as SEQ ID NO. 18 (monoclonal antibody 9-CQTS 054). The invention also provides application of the novel coronavirus RBD specific monoclonal antibody in preparing a reagent or vaccine or medicament for detecting or diagnosing SARS-CoV-2, wherein the medicament comprises the novel coronavirus RBD specific monoclonal antibody and pharmaceutically acceptable excipient, diluent or carrier; nucleic acid molecules encoding the novel coronavirus RBD-specific monoclonal antibodies described above are also provided; also provided are expression cassettes, recombinant vectors, recombinant bacteria or transgenic cell lines comprising the above nucleic acid molecules; also provides the application of the expression cassette, the recombinant vector, the recombinant bacterium or the transgenic cell line in preparing products.

The invention also provides a product comprising the novel coronavirus RBD specific monoclonal antibody; the product uses are any one of the following (b 1) - (b 4): (b 1) binding to the novel coronavirus SARS-CoV-2; (b 2) detecting the binding of the 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.

Preferably, the novel coronavirus RBD specific monoclonal antibodies are prepared by sorting RBD specific memory B cells and then obtaining antibody variable region cDNA through mRNA of the RBD specific memory B cells.

The principle and the beneficial effects of the invention are as follows:

(1) Compared with the monoclonal antibody aiming at the S1 non-RBD region, the monoclonal antibody provided by the invention combines with RBD, and provides wider application value for screening antibody medicines, diagnosing, preventing and treating novel coronavirus pneumonia.

(2) The monoclonal antibody provided by the invention is obtained by sorting RBD specific memory B cells, and compared with the prior art by sorting plasma cells, the monoclonal antibody prepared by the invention can trigger stronger humoral immune response. In addition, the invention only carries out subsequent RT-PCR, nested PCR and antibody function analysis aiming at RBD specific memory B cells, thereby greatly improving the specific binding capacity of monoclonal antibodies and RBD.

Drawings

FIG. 1 is a diagram of cell sorting using flow cytometry to analyze memory B cells;

FIG. 2 is a diagram of cell sorting using flow cytometry to analyze RBD specific memory B cells;

FIG. 3 is a gel electrophoresis diagram of a single cell antibody gene PCR product;

FIG. 4 is a diagram of agarose gel electrophoresis after PCR amplification of an antibody gene expression cassette comprising a CMV promoter, WPRE-gamma or WPRE-kappa element;

FIG. 5 is a graph of experimental results specific for the novel coronavirus RBD.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are only some, but not all embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Example 1

The embodiment provides a novel coronavirus RBD specific monoclonal antibody, and the amino acid sequence of a heavy chain variable region is shown as SEQ ID NO. 1; the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 2.

The embodiment also provides the application of the novel coronavirus RBD specific monoclonal antibody in preparing reagents or medicines for detecting or diagnosing SARS-CoV-2.

In practice, the present example can be used to prepare nucleic acid molecules using novel coronavirus RBD-specific monoclonal antibodies, or to prepare expression cassettes, recombinant vectors, recombinant bacteria or transgenic cell lines comprising the nucleic acid molecules, or to prepare pharmaceutical compositions comprising the novel coronavirus RBD-specific monoclonal antibodies and pharmaceutically acceptable excipients, diluents or carriers.

When applied, this example results in a related product prepared using the novel coronavirus RBD-specific monoclonal antibody, which may have the uses of any one of the following (b 1) - (b 4): (b 1) binding to the novel coronavirus SARS-CoV-2; (b 2) detecting the binding of the 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.

Examples 2 to 9

Examples 2-9 differ from example 1 in that: the variable region amino acid sequences of the novel coronavirus RBD specific monoclonal antibodies are different, and the variable region amino acid sequences of examples 2-9 are shown in the following table:

The novel coronavirus RBD-specific monoclonal antibodies provided in examples 1-9 above were all obtained by the following method: firstly, single RBD specific memory B cells are obtained from peripheral blood of a novel coronavirus pneumonia rehabilitation patient, mRNA of the RBD specific memory B cells is obtained, an antibody variable region gene expression cassette is constructed through RT-PCR and nested PCR, the antibody variable region gene expression cassette is transduced into 293T cells to express antibodies, the supernatant is collected, RBD specificity of the supernatant is detected by an ELISA method, and novel coronavirus RBD specific monoclonal antibodies are obtained through screening.

The method specifically comprises the following steps:

s1, collecting peripheral blood of a plurality of novel coronavirus pneumonia rehabilitation patients, separating to obtain PBMC, and freezing in a refrigerator at-80 ℃ for later use.

S2, firstly removing Dead cells of PBMC obtained in the step S1 by adopting a Dead Dye (Dead Dye), and then adopting CD19, mIg-G, mIg-D and S-RBD to Dye and mark the memory B cells with high binding capacity and specificity to the living RBD in the PBMC, so as to screen out the memory B cells specific to the RBD; the specific memory B cells are sorted on a 96-well plate by using a flow cell sorter, and each well is internally provided with one specific memory B cell, and the specific memory B cells are frozen in a refrigerator at the temperature of minus 80 ℃ for standby.

Specifically, the preferred concentration range for the Dye of the present example is 1-2. Mu.g/mL, and the preferred concentration for the Dye of the present example is 1.5. Mu.g/mL; CD19 is a B cell marker produced by Biolegend and is stained at a concentration ranging from 1 to 2. Mu.g/mL, with a concentration of 1.5. Mu.g/mL being preferred for the staining of CD19 in this example. mIg-G is a B cell surface receptor produced by Biolegend, and the concentration range is 1-2 μg/mL when staining, and the preferred concentration of mIg-G in this example is 1.5 μg/mL when staining; mIg-D is a B cell surface receptor produced by Biolegend, and the concentration range is 1-2 μg/mL when staining, and the preferred concentration of mIg-D in this example is 1.5 μg/mL when staining; the novel coronavirus produced by S-RBD sinobiological is a protein receptor domain and is stained at a concentration ranging from 1 to 2. Mu.g/mL, with the preferred concentration of S-RBD being 1.5. Mu.g/mL in this example.

Sorting of RBD-specific memory B cells by flow cytometry cell sorting of PBMC by CD19, mIg-G, mIg-D and S-RBD cell sorting charts of B cells with specific memory for S-RBD are shown in FIGS. 1 and 2, wherein Batch ID 0428, 0505, 0522, 0528 in FIG. 2 is a screening lot. The principle of screening the memory B cells specific to RBD by adopting CD19, mIg-G, mIg-D and S-RBD in the embodiment is as follows: PBMC were stained with DedDye, B cell marker CD19, memory B cell markers mIg-G positive and mIg-D negative and RBD specific IgG expressing memory B cells, then CD19 cell populations were divided from the cell populations using a flow cytometer, RBD positive memory B cells were divided from the CD19 positive cell populations by mIg-G ⁺mIg-D^- cell populations, and RBD positive memory B cells were sorted from the mIg-G ⁺mIg-D^- cell populations by a flow cytometer.

S3, sorting to obtain mRNA of a single RBD specific memory B cell, and amplifying by RT-PCR to obtain the antibody variable region cDNA. Specifically, when the RT-PCR is used for amplifying the cDNA of the antibody variable region, a general Leader (see a primer sequence table I and a primer sequence table II) is designed at the front section of the primer designed in the embodiment, so that the amplification rate of the antibody gene is effectively improved, and the experimental result is shown in figure 3.

S4, amplifying the cDNA of the antibody variable region obtained by the S1-S3 by adopting nested PCR, and constructing an antibody variable region gene expression cassette.

S3 and S4 are performed in total by the following six parts: (1) extracting mRNA of RBD specific memory B cells; (2) single cell mRNA Reverse Transcription (RT); (3) adding a G tail (TDT); (4) first round PCR (1 st PCR); (5) a second round of PCR (2 nd PCR); (6) BCR-ORFPCR amplification to construct a gene expression cassette; (7) CMV, WPRE-gamma/kappa/l fragment amplification and CMV, BCR-V gamma/kappa/l ((6) product), WPRE-gamma/kappa/l overlap PCR (Overlap PCR) pre-ligation; (8) amplification of BCR-gamma ORF, BCR-kappa ORF, and BCR-lPCR.

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

(1) Single-cell mRNA extraction was performed using Dynabeads ^TMmRNA DIRECT^TM Purification Kit (Thermo Fisherscientific), specifically comprising the steps of:

① And (3) centrifuging: taking out the 96-well plate with single RBD specific memory B cells from the refrigerator at-80 ℃, and centrifuging at 600 Xg for 30s to make the cells centrifuged at the bottom of the well;

② Cleaning: taking out Dynabeads oligo (dT) 25 microsphere bottles, uniformly vortex and mix, sucking enough microspheres according to 2 μl/hole, placing the microspheres on a magnet block, standing for 30s, discarding the supernatant, and re-suspending with 500 μl of Lysis Buffer;

③ Preparing: adding 9 μl/well of Lysis Buffer into a 50mL centrifuge tube, adding the 500 μl microsphere suspension, and blowing with a gun;

④ And (5) subpackaging: dispensing the microspheres with eight tubes, then adding them to the cell plate at 9 μl/well using a lance;

⑤ And (3) rinsing: pasting a film on a 96-hole plate, and then rinsing the periphery of the pipe wall for 2 cycles;

⑥ Incubation: standing at room temperature for 5min to fully release mRNA of RBD specific memory B cells and combine the mRNA with the microsphere, and centrifuging at 600 Xg instant after incubation, so that the microsphere is centrifuged at the bottom of the hole. Placing the 96-well plate on DynaMag ^TM -96side Magnet magnetic plate, and removing supernatant with a gun;

⑦ Wash A cleaning: adding a Washing Buffer A according to 8 μl/well, removing the plate 7-8 times, washing the microspheres thoroughly, and discarding the supernatant;

⑧ Wash B cleaning: wash Buffer B was added at 8. Mu.l/well and the plate was run back and forth 7-8 times to allow the microspheres to wash well, the supernatant was discarded, and then a pre-prepared Reverse Transcription (RT) reaction was added at 10. Mu.l/well. Reagent formulation and reaction conditions are described in (2) below.

(2) Reverse Transcription (RT) (10. Mu.l System)

The reagents required to be formulated are shown in table 1 below:

Reaction conditions: 42 ℃ for 60min (mixing every 20 min);

After completion of the reaction, the 96-well plate was subjected to instantaneous centrifugation at 600 Xg, and then the 96-well plate was placed on DynaMag ^TM -96side Magnet magnetic plate, the supernatant was aspirated by a discharge gun, and then a previously prepared TDT reaction solution was added at 10. Mu.l/well, and the reagent preparation and reaction conditions were as described in (3) below.

(3) Add G tail (TDT) (10 μl System)

The reagents required to be formulated are shown in table 2 below:

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

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

At the end of the reaction, the 96-well plate was transiently centrifuged at 600 Xg, then placed on DynaMag ^TM -96side Magnet magnetic plate, the supernatant was pipetted off with a discharge gun, and then the pre-formulated first round PCR (1 st PCR) reaction solution was added at 10. Mu.l/well, and the reagent formulation and reaction conditions were as described in (4) below.

(4) 1St PCR (10. Mu.l System) (primer sequence see primer sequence Listing)

The reagents required to be formulated are shown in table 3 below:

Based on the PCR principle, the experimental reaction conditions of the 1st PCR are as follows: ① Pre-denaturation at 95℃for 3min; ② Denaturation at 95℃for 15sec, annealing at 60℃for 5sec, extension at 72℃for 1min,30-35cycles, 30cycles being preferred in this example; ③ The extension was carried out at 72℃for 5 minutes and stored at 4 ℃.

(5) Second round PCR (2 nd PCR) (10. Mu.l System) (primer sequence see primer sequence Listing first and primer sequence Listing second)

The reagents required to be formulated are shown in table 4 below:

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

Based on the PCR principle, the experimental reaction conditions of 2nd PCR are as follows: ① Pre-denaturation at 95℃for 3min; ② Denaturation at 95℃for 15sec, annealing at 60℃for 5s, extension at 72℃for 1min,30-35cycles, 35cycles being preferred in this example; the extension was carried out at 72℃for 5 minutes and stored at 4 ℃.

After the PCR is finished: mu.l of each well was subjected to 1.5% agarose gel electrophoresis. The cell wells paired with either the Kappa or Lamada strands were sequenced.

(6) Amplification and construction of antibody expression cassettes (BCR-ORFs)

PCR amplified promoter region (CMV promoter), WPRE-gamma (antibody gamma chain) and WPRE-kappa (antibody kappa chain), PCR amplified system is shown in Table 5 below:

The PCR amplification conditions were: ① Pre-denaturation at 95℃for 3min; ② Denaturation at 95℃for 15sec, annealing at 56℃for 15sec, extension at 72℃for 1min,30cycles; ③ The extension was carried out at 72℃for 5 minutes and stored at 12 ℃.

(7) CMV, WPRE-gamma/kappa/l fragment amplification and CMV, BCR-V gamma/kappa/l, WPRE-gamma/kappa/l overlap PCR (Overlap PCR) pre-ligation

The experimental system is shown in table 6 below:

The PCR amplification conditions were: pre-denaturation at 95℃for 3min; denaturation at 95℃for 15sec, annealing at 50℃for 15sec, extension at 72℃for 1.5min,10cycles; the extension was carried out at 72℃for 5 minutes and stored at 12 ℃.

(8) BCR-gamma ORF, BCR-kappa ORF, and BCR-l PCR amplification

The experimental system is shown in table 7 below:

PCR amplification procedure: pre-denaturation at 95℃for 3min; denaturation at 95℃for 15sec, annealing at 58℃for 15sec, extension at 72℃for 1.5min,30cycles; the extension was carried out at 72℃for 5 minutes and stored at 12 ℃.

After amplification, agarose gel electrophoresis is adopted, gel imaging analysis is carried out to obtain whether the size of the antibody variable region gene is correct, the experimental result is shown in figure 4, the Marker is at the middle position, and the band is at 5000 bp.

BCR-gamma ORF and BCR-kappa/ORF ethanol precipitation: respectively taking 30 mu l of PCR products of the BCR-gamma ORF and the BCR-kappa ORF, placing the PCR products in 8 connecting tubes, adding 120 mu l of absolute ethyl alcohol and 6 mu l of sodium acetate solution, fully and uniformly mixing, and standing at-80 ℃ for 30min;10000rpm, centrifuging for 20min, discarding supernatant, sequentially rinsing with 200 μl of 70% ethanol and absolute ethanol, volatilizing at 56 deg.C, adding 40 μl of sterile water, shaking, dissolving precipitate, and detecting antibody variable region gene concentration.

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

the J-region primers used in S3 and S4 are described in the following primer sequence Listing II:

primer ID	sequence
		IGHJ_01	GATGGGCCCTTGGTGGAGGGTGAGGAGACGGTGACCAGGGTGCCCTGGCCCCAGT
IGHJ_02	GATGGGCCCTTGGTGGAGGGTGAGGAGACAGTGACCAGGGTGCCACGGCCCCAGA
		IGHJ_03	GATGGGCCCTTGGTGGAGGGTGAAGAGACGGTGACCATTGTCCCTTGGCCCCAGA
IGHJ_04	GATGGGCCCTTGGTGGAGGGTGAGGAGACGGTGACCGTGGTCCCTTGCCCCCAGA
		IGKJ_01	GATGGTGCAGCCACAGTTCGTTTGATTTCCACCTTGGTCCCTTGGCCGAACGTCC
IGKJ_02	GATGGTGCAGCCACAGTTCGTTTGATTTCCACCTTGGTCCCTTGGCCGAACGTCC
		IGKJ_03	GATGGTGCAGCCACAGTTCGTTTGATATCCACTTTGGTCCCAGGGCCGAAAGTGA
IGKJ_04	GATGGTGCAGCCACAGTTCGTTTGATCTCCACCTTGGTCCCTCCGCCGAAAGTGA
		IGKJ_05	GATGGTGCAGCCACAGTTCGTTTAATCTCCAGTCGTGTCCCTTGGCCGAAGGTGA
IGLJ_01	GGGGCAGCCTTGGGCTGACCTAGGACGGTGACCTTGGTCCCAGTTCCGAAGACAT
		IGLJ_02	GGGGCAGCCTTGGGCTGACCTAGGACGGTCAGCTTGGTCCCTCCGCCGAATACCA
IGLJ_03	GGGGCAGCCTTGGGCTGACCTAAAATGATCAGCTGGGTTCCTCCACCAAATACAA
		IGLJ_04	GGGGCAGCCTTGGGCTGACCTAGGACGGTCAGCTCGGTCCCCTCACCAAACACCC
IGLJ_05	GGGGCAGCCTTGGGCTGACCTAGGACGGTCAGCTCCGTCCCCTCACCAAACACCC
		IGLJ_06	GGGGCAGCCTTGGGCTGACCGAGGACGGTCACCTTGGTGCCACTGCCGAACACAT
IGLJ_07	GGGGCAGCCTTGGGCTGACCGAGGACGGTCAGCTGGGTGCCTCCTCCGAACACAG
		IGLJ_08	GGGGCAGCCTTGGGCTGACCGAGGGCGGTCAGCTGGGTGCCTCCTCCGAACACAG

S5, transferring the antibody variable region gene expression cassette obtained in the S4 into 293T cells for 48 hours to express the antibody, collecting supernatant, detecting RBD specificity of the supernatant by an ELISA method, and screening RBD specific fully human monoclonal antibodies.

(A) Antigen was diluted with PBS (final concentration 2. Mu.g/mL), 10. Mu.l/well, coated 384 well ELISA plates overnight at 4℃or coated for 2h at 37℃in this example, preferably overnight at 4 ℃. NOTE: after the addition, the liquid was kept at the bottom by instantaneous centrifugation.

The experimental system is shown in table 8 below:

Reagent name	Goods 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) PBST (0.05% Tween 20, cat#tb220) was formulated: 1L of PBS was added with 0.5mL of Tween 20;

PBST machine (thermo scientific WELLWASH VERSA) or hand washing (machine washed plates still need to be manually clapped/centrifuged for 1min using a microplate centrifuge (MPC-P25)) to make the plates invisible with water and air bubbles.

Closing: mu.l of 5% BSA (BioFroxx, cat.NO:4240GR 100) (PBST formulation) was added to the washed plates and incubated in an incubator at 37℃for 1h. PBST machine washing the plates or hand washing.

(C) And (5) adding samples and standard substances. Wherein, standard substance: the 10. Mu.l/well stock concentration was 1. Mu.g/mL, and the gradient dilutions were 250ng/mL, 125ng/mL, 62.5ng/mL, 31.25ng/mL, 15.63ng/mL, 7.81ng/mL, 3.9ng/mL, and 1.95ng/mL. (blocking fluid dilution); sample: cell supernatants transfected with antibody genes. Negative control/blank wells: blocking solution 10. Mu.l/well.

Incubate at 37℃for 30min. PBST machine washing the plates or hand washing.

(D) The secondary antibody was added at a concentration of 10. Mu.l/well and then incubated at 37℃for 30min.

The experimental system is shown in table 9 below:

Second antibody name	Goods 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

PBST machine washing the plates or hand washing. PNPP (disodium paranitrophenylphosphate) at 10. Mu.l/well was used (Thermoscientific Muttiskan GO) to detect OD (450 mm) values of 5min, 10min, 15min, 20min, 25min, 30min, 35min, 40min, 45min, 50min, 55min and 60 min. 50mg of PNPP powder (Thermo, prod # 34045) +40mL of ddH ₂ O+10mL of Diethanol aminesubstrate Buffer (5X), PNPP is stored protected from light at 4 ℃.

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

The foregoing description of the preferred embodiments of the present invention is merely illustrative, and not restrictive, of the invention. It will be appreciated by those skilled in the art that many variations, modifications and even equivalent changes may be made thereto within the spirit and scope of the invention as defined in the appended claims, but are still within the scope of the invention.

Claims (2)

1. A novel RBD specific monoclonal antibody of coronavirus SARS-COV-2 is characterized in that the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 1; the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 2.

2. A novel RBD specific monoclonal antibody of coronavirus SARS-COV-2 is characterized in that the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 3; the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 4.