CN117003862A

CN117003862A - New coronavirus RBD specific monoclonal antibody and application

Info

Publication number: CN117003862A
Application number: CN202311018625.6A
Authority: CN
Inventors: 韩晓建; 王应明; 胡超; 李婷婷; 王建为; 李胜龙; 金艾顺
Original assignee: Chongqing Medical University
Current assignee: Chongqing Medical University
Priority date: 2020-08-19
Filing date: 2020-08-19
Publication date: 2023-11-07
Also published as: CN116813760A; CN111909263A; CN117736313A; CN114920832A; CN111909263B; CN114920832B; CN115925896A; WO2022037616A1

Abstract

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

Description

New coronavirus RBD specific monoclonal antibody and application

The application is a divisional application of patent with application date 2022.05.23, application number 2022105649661. X and patent name of new coronavirus RBD specific monoclonal antibody and application.

Technical Field

The application 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 (RBDs) are located on the S1 subunit, whose primary function is to recognize host cell surface receptors, mediating fusion with host cells.

There is no specific drug targeted therapy against the new pathogen covd-19 at present, and vaccine development is still required. The plasma of the recently cured discharged patient contains high-concentration specific antigen neutralizing antibodies, and after the high-concentration specific antigen neutralizing antibodies are input into the patient, the novel coronavirus can be neutralized to mediate effective immune response, so that the recovery-period plasma is expected to provide effective treatment means for curing patients infected with the novel coronavirus, the death rate is reduced, and the life safety of the patient is ensured.

The chinese patent application publication No. CN111303280a discloses a fully human monoclonal antibody against SARS-CoV-2 with high neutralizing activity, which provides a fully human monoclonal antibody with a recognition region of S1 non-RBD region, but since the invasion of a new coronavirus into host cells is bound to ACE2 of the host cells by RBD, the blocking effect of the fully human monoclonal antibody obtained by the above patent against the virus is limited, and the above patent is an antibody cDNA obtained by labeling plasma cells, but the memory B cells react rapidly after activation compared to the plasma cells, so that the memory B cells can elicit a faster and stronger humoral immune response than the primary reaction, and the humoral immune response elicited by the plasma cells is limited.

Disclosure of Invention

The object of the present application is to provide a novel coronavirus RBD-specific monoclonal antibody and use directed against RBD and capable of eliciting a more intense humoral immune response.

In order to achieve the aim, the application provides a novel coronavirus RBD specific monoclonal antibody, in particular to a monoclonal antibody with the heavy chain variable region amino acid sequence shown in SEQ ID NO. 1; the amino acid sequence of the light chain variable region can be shown as SEQ ID NO. 2 (monoclonal antibody 11-CQTS 011). 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 12-CQTS 012). 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 13-CQTS 013). The amino acid sequence of the heavy chain variable region can be also shown as SEQ ID NO. 7; the light chain variable region amino acid sequence is also shown in SEQ ID NO. 8 (mab 14-CQTS 014). 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 15-CQTS 015). 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 16-CQTS 016). 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 17-CQTS 017). The amino acid sequence of the heavy chain variable region can be also shown as SEQ ID NO. 15; the light chain variable region amino acid sequence may also be as shown in SEQ ID NO. 16 (mab 18-CQTS 018). 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 (mab 19-CQTS 019). The amino acid sequence of the heavy chain variable region can also be shown as SEQ ID NO. 19; the amino acid sequence of the light chain variable region can also be shown as SEQ ID NO. 20 (mab 20-CQTS 020).

The application also provides the 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 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 application 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 monoclonal antibodies specific to RBD of the novel coronavirus are prepared by sorting RBD specific memory B cells and obtaining cDNA of the variable region of the antibody by mRNA of the RBD specific memory B cells.

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

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

(2) The monoclonal antibody provided by the application 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 application can trigger stronger humoral immune response. In addition, the application 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 the experimental results of RBD specificity.

Detailed Description

The following description of the embodiments of the present application 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 application. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.

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 RBD-specific monoclonal antibody of the present example can be used to prepare a nucleic acid molecule, or to prepare an expression cassette, recombinant vector, recombinant bacterium or transgenic cell line comprising the nucleic acid molecule, or to prepare a pharmaceutical composition comprising the novel RBD-specific monoclonal antibody of coronavirus described above and a pharmaceutically acceptable excipient, diluent or carrier.

When applied, the relevant product of the RBD-specific monoclonal antibody preparation obtained in this example can have the uses as any one of the following (b 1) to (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 10

Examples 2-10 differ from example 1 in that: the variable region amino acid sequences of the RBD specific monoclonal antibodies are different, and the variable region amino acid sequences of examples 2-10 are shown in the following table:

the RBD-specific monoclonal antibodies provided in examples 1-10 above were all obtained by the following method: firstly, single RBD specific memory B cells are obtained from peripheral blood of a new crown infection 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, the RBD specificity of the supernatant is detected by an ELISA method, and the RBD specific monoclonal antibodies are obtained through screening.

The method specifically comprises the following steps:

s1, collecting peripheral blood of a plurality of new crown infection 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 is stained at a concentration ranging from 1 to 2. Mu.g/mL, with mIg-G being preferred in this example at a concentration of 1.5. Mu.g/mL; 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 is a protein receptor domain and is stained at a concentration ranging from 1 to 2. Mu.g/mL, with the preferred concentration for S-RBD staining of this example being 1.5. Mu.g/mL.

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 are screening batches. 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 memory B cells expressing RBD specific IgG, and then CD19 cell populations were divided from the cell populations using a flow cytometer, followed by dividing mIg-G from the CD19 positive cell populations ⁺ mIg-D ^- Cell populations, again from mIg-G ⁺ mIg-D ^- The cell population divides RBD positive memory B cells, and then the RBD positive memory B cells are sorted by a flow cell sorter.

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 construction of 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) By Dynabeads ^TM mRNA DIRECT ^TM Single-cell mRNA extraction is carried out by using a Purification Kit (Thermo Fisherscientific), and the method specifically comprises the following steps:

(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 allow mRNA of RBD specific memory B cell to be releasedPut and bind to the microspheres, after incubation, the microspheres were centrifuged at 600 Xg transiently and at the bottom of the wells. Place 96-well plates in DynaMag ^TM -96side Magnet magnetic plates, and removing the 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 μl system): the reagents required to be formulated are shown in table 1 below.

Reagent name	Volume of
		DEPC-H ₂ O	4.5μl
5×primerscript Buffer	2.0μl
		2.5mM dNTP	2.0μl
RNase Inhibitor	1μl
		Sample	beads
PrimerScript Ⅱ RTase	0.5μl
		Total volume of	10μl

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 in DynaMag ^TM The supernatant was pipetted off on a 96side Magnet magnetic plate, and then 10. Mu.l/well of the pre-formulated TDT reaction solution was added, and the reagent formulation and reaction conditions were as described in (3) below.

(3) Add tail G (TDT) (10. Mu.l system): the reagents required to be formulated are shown in table 2 below.

Reagent name	Volume of
		H ₂ O	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 and then placed in DynaMag ^TM The supernatant was pipetted off on a 96side Magnet magnetic plate, and then 10. Mu.l/well of the pre-formulated first round PCR (1 st PCR) reaction solution was added, 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:

reagent name	Volume of
		H ₂ O	1.9μl
2×GC Buffer	5μl
		2.5mM dNTP	1μl
FP:AP3-dC(10μM)	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 volume of	10μl

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
		H ₂ O	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 Kappa or Lamada chains were sequenced.

(6) Amplification and construction of antibody expression cassette (BCR-ORF): PCR amplified promoter region (CMV promoter), WPRE-gamma (antibody gamma chain) and WPRE-kappa (antibody kappa chain), and 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 amplifications and pre-ligation of CMV, BCR-V gamma/kappa/l, WPRE-gamma/kappa/l overlap PCR (Overlap PCR), 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, 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:

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

the PBST machine washed the plates (Thermoscientific wellwash versa) or hand washed (the machine washed plates still had 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 above 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 p-nitrophenylphosphate) at 10. Mu.l/well was used (Thermoscientific Muttiskan GO) to detect OD (450 mm) values of 5min,10 min, 15min, 20min, 25min, 30min, 35min, 40min, 45min, 50min, 55min and 60 min. 50mg PNPP powder (Thermo, prod # 34045) +40mL ddH ₂ O+10mL of Diethanol aminesubstrate Buffer (5X), PNPP was stored at 4℃in the dark.

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 application is merely illustrative, and not restrictive, of the application. 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 application as defined in the appended claims, but are still within the scope of the application.

Claims

1. The novel coronavirus RBD specific monoclonal antibody is characterized in that 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.

2. The novel coronavirus RBD specific monoclonal antibody is characterized in that 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.