TW202317610A - Broad reactive anti-spike monoclonal antibodies for diagnosis of sars-cov-2 - Google Patents

Abstract

The present invention provides a novel conserved epitope sequence of SARS-CoV-2 Spike comprising HADQ (position 625,626,627,628) and TPW (position630,631,633), which can be used to detect the Alpha strain, Beta strain, Gamma strain, Delta strain and Omicron strain of SARS-CoV-2 variant. The two conserved antigen sequences can be applied for the biomarkers of monoclonal antibodies.

Description

Anti-spike protein monoclonal antibody for broad diagnosis of acute respiratory syndrome coronavirus 2

The invention discloses a broad-spectrum anti-spike protein monoclonal antibody for SARS-CoV-2 virus diagnosis, which can be applied to commercial SARS-CoV-2 detection reagents.

Coronavirus Disease 2019 (COVID-19) is a viral pneumonia disease that has spread rapidly around the world. It is caused by Acute Respiratory Syndrome Coronavirus 2 (Severe Acute Respiratory Syndrome Coronavirus 2, SARS-CoV-2) . SARS-CoV-2 is a new form of coronavirus similar to SARS virus (SARS-CoV) and MERS virus, also known as new coronary pneumonia virus.

Infection with SARS-CoV-2 can cause severe symptoms and possibly death. To mitigate a pandemic, effective methods of screening potentially infected persons are necessary to stop the spread of the virus.

At present, the rapid screening of potential infected persons mostly relies on reverse transcription-polymerase chain reaction (RTPCR) or real-time polymerase chain reaction (Real-time polymerase chain reaction) detection. However, the sample preprocessing steps are complex and time-consuming, and the accuracy of these tests is prone to false positives and false negatives.

In addition, the current SARS-CoV-2 virus strains continue to mutate. At present, in addition to the wild-type Wuhan virus strain, the mutant strains include the α (alpha) strain, β (beta) strain, γ (gamma) strain, δ (delta) strain, λ (Lambda) strain, μ (Mu), ο (Omicron) strain.

Therefore, in order to improve the detection accuracy of virus strains, identifying the non-variant conservative sequence of SARS-CoV-2 virus will help to develop detection reagents that are widely applicable to multiple variant strains.

In view of the foregoing technical situation, the present invention discloses a novel SARS-CoV-2 spike protein conserved epitope sequence, including HADQ (positions 625, 626, 627, 628) (SEQ ID NO: 1) and TPW (positions 630, 631, 633).

The present invention discloses an antibody or its antigen-binding fragment that specifically binds to the spike protein of acute respiratory syndrome coronavirus 2, wherein the antibody or its antigen-binding fragment is bound to SEQ ID NO: 1 or TPW amino acid sequence specific binding.

The invention discloses a detection kit for detecting acute respiratory syndrome coronavirus 2, which comprises: the antibody or its antigen-binding fragment; and a detection reagent.

The present invention discloses a method for detecting whether an individual is infected with acute respiratory syndrome coronavirus 2, which comprises the following steps: (a) providing a biological sample obtained from the individual; (b) making the biological sample and the spike protein identifiable The antibody or antigen-binding fragment thereof of the amino acid sequence of SEQ ID NO: 1 or TPW is contacted when a spike protein of acute respiratory syndrome coronavirus 2 is present in the biological sample and the spike protein comprises SEQ ID NO: 1 or TPW amino acid sequence, the antibody or antigen-binding fragment thereof forms a binding complex with the spike protein; and (c) detecting the presence of the binding complex produced in step (b), when step (b) produces When the binding complex exists, it is determined that the individual is infected with acute respiratory syndrome coronavirus 2.

Figure 1 shows the production and characterization of SARS-CoV-2 spike monoclonal antibody characteristics.

Figure 2 shows the patterns of two anti-spike monoclonal antibodies.

Figure 3 shows the binding ability of two anti-spike protein monoclonal antibodies to different variants of SARS-CoV-2 on the spike protein.

Figure 4 shows the gene sequence alignment of two anti-spike protein monoclonal antibodies.

Figure 5 shows the positions of two anti-spike monoclonal antibodies on the SARS-CoV-2 spike protein epitope.

Hereinafter, preferred embodiments according to the present disclosure will be described in detail with reference to the accompanying drawings, and the detailed description disclosed below in conjunction with the accompanying drawings is an exemplary embodiment of the present disclosure, and does not limit the embodiments in which the present invention can be practiced. The following detailed description includes specific details to provide a thorough understanding of the present invention, however, one skilled in the art can practice the present disclosure without these specific details.

The following examples of implementation are not limiting and merely represent aspects and features of the invention.

Example 1: HEK293T cell culture

Using the pSpike-S1 plasmid with His-tag, transfected into 293T cells to express the SARS-CoV-2 spike protein S1 protein, where S1 is the main receptor binding domain (Receptor binding domain, RBD), and then use Purified by Ni-NTAf affinity chromatography, ready for subsequent animal immunization experiments.

Example 2: Animal BALB/c mouse model

First, using the SARS-CoV-2 spike protein (SARS- CoV-2 Spike, Wuhan-Hu-1 strain) protein to immunize BALB/c mice, after four supplementary doses, the spleen cells of the immunized mice were taken out and fused with bone marrow cancer cells (NS1 cells) to form a fusion Tumor cells (hybridoma), and the HAT medium was used to select stable hybrid tumor cell lines for monoculture, followed by limiting dilution (limiting dilution), combined with enzyme immunosorbent assay (Enzyme-linked immunosorbent assay, ELISA) selection The specific antibody that will be secreted is produced, and the cell line is monoclonalized, that is, the cells are evenly distributed in a 96-well culture plate by using the limiting dilution method, so that each hole contains only one cell and grows in groups Later, the antibody titer was confirmed again by enzyme-binding immunosorbent assay, and then a suitable monoclonal antibody cell line was screened out.

After the screened monoclonal antibody cell line is amplified and cultured, the cells are injected into the peritoneal cavity of the mouse. After about 1 to 2 weeks, a large amount of ascites can be obtained from the peritoneal cavity of the mouse. The obtained ascites is filtered through a 0.45um filter membrane and processed with a column. Purify, and finally, use dialysis to replace it with PBS for storage.

Example 3: Preparation of anti-spike protein

Add 2.5 μg of recombinant Spike protein to 0.25ml PBS and emulsify with an equal volume of Freund's complete adjuvant (Freund's adjuvant, Sigma Aldrich, Cat.No.F5881), and then immunize BALB/c mice by intraperitoneal injection . Two weeks later, a second intraperitoneal injection was performed with Freund's incomplete adjuvant (Sigma Aldrich, Cat. No. F5506) and the same dose of recombinant spike protein to activate the immune response. Finally, the last immunization was performed three days before sacrificing the mouse, and the spleen cells (splenocyte) of the mouse were collected at the time of sacrifice, and fused with myeloma cells (NS1) to form fusion tumor cells, followed by limiting dilution (limiting dilution) Combined with enzyme immunosorbent assay method to select monoclonal antibody.

Example 4: Enzyme combined with immunosorbent assay

As shown in Figure 1 (A), the challenge was determined using enzyme-combined immunosorbent assay. The ability of the selected anti-spike monoclonal antibody to bind to the spike protein. The positive control group (PC) is the serum sample of spike protein-immunized mice.

As shown in Figure 1 (A), the absorbance values of the two monoclonal antibodies 10-11C and 11-12H in this figure at a wavelength of 450nm read by the ELISA reader are close to those of the positive control group.

As shown in Figure 1 (B), the isotype determination of two monoclonal antibodies was identified by using enzyme-binding immunosorbent assay, in which the secondary antibodies of the two monoclonal antibodies 10-11C and 11-12H were in sequence: The light chains are kappa and lambda chains, and the heavy chains are IgG1, IgG2a, IgG2b, IgG3, IgM, and IgA, respectively. Two of the anti-spike mAbs are lambda and IgG2a.

Example 5: Western blot method

Using 1 μg of the SARS-CoV-2 spike protein (Wuhan-Hu-1 strain) produced by 293T cells as an antigen, by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and without adding SDS Gel electrophoresis (Native-PAGE), the components of different colloids were denatured (denature) and the original state (native) of the spike protein configuration, and then blotted on a PVDF membrane. The PBS of % Tween 20 was configured into 5% skimmed milk as the blocking buffer, and blocking was carried out at ambient room temperature for 1 hour, after the PBS wash of 0.1% Tween 20, the monoclonal antibody was added and left overnight at 4°C, and then 0.1% Tween was used Wash off unbound monoclonal antibody with 20 PBS, add anti-mouse IgG polyclonal antibody containing Horseradish peroxidase (HRP) and react at room temperature for 1 hour, then wash with 0.1% Tween 20 PBS, and finally use WesternBright ECL HRP The substrate is colored, and finally the image is presented using a cold light imaging system (BioSpectrum AC).

As shown in Figure 2, two anti-spike protein monoclonal antibodies (Anti-Spike mAb) can recognize denatured (denature) and original state (native) spike protein. The negative control group (NC) is the pre-immunized mouse serum sample (pre-immunized mice serum), and the positive control group (PC) is the serum sample of the spike protein immunized mice (spike-immunized mice serum).

In a specific embodiment, the two anti-spike protein monoclonal antibodies 10-11C and 11-12H can recognize denature form and non-denature form of the spike protein of SARS-CoV-2 ).

As shown in FIG. 3 , the binding ability of the two anti-spike monoclonal antibodies (Anti-Spike mAb) to the spike protein of different mutant virus strains was evaluated by enzyme-binding immunosorbent assay. Among them, the antibody to be tested was diluted 1000 and 5000 times respectively with (A) wild strain (Wuhan-hu-1), (B) B.1.1.7 (alpha), (C) B. 1.351 (beta), (D) B.1.617.2 (delta), (E) B.1.1.529 (omicron), (F) BA.2 (omicron) spike proteins were tested, and the negative control group ( NC) is the pre-immunized mouse serum sample (pre-immunized mice serum), and the positive control group (PC) is the serum sample of the spike protein-immunized mice (spike-immunized mice serum).

In a specific embodiment, the two anti-spike monoclonal antibodies 10-11C and 11-12H have binding ability to all spike proteins.

Example 5: Epitope mapping and sequencing alignment

Using phage display technology (phage display), using a phage (phage) to express a 7-mer peptide library (peptide library) for binding to monoclonal antibodies (New England Biolabs (NEB), Inc. Cat. No. E8102L), The biopanning process and operation method of Phage display are carried out according to the NEB manufacture protocol.

Next, the determined epitope was marked on the SARS-CoV-2 spike protein structure (Protein Data Base, PDB: 6XLU) to find out the amine groups of the epitope of the two anti-spike protein monoclonal antibodies acid sequence. Then use BioEdit to compare the epitope sequences recognized by the two antibodies with the Wuhan strain of SARS-CoV-2 spike protein and the sequences of common SARS-CoV-2 variant strains. The compared variants include α (alpha) strain, β (beta) strain, γ (gamma) strain, δ (delta) strain, λ (Lambda) strain, μ (Mu) and ο (Omicron) strain.

As shown in Figure 4, the spike protein comparison results show that the two epitopes recognized by the two anti-spike protein monoclonal antibodies are conserved epitopes in the current mutant strain of SARS-CoV-2. ), no mutations have occurred, so the two anti-spike protein monoclonal antibodies can detect common SARS-CoV-2 variants, including α (alpha) strain, β (beta) strain, γ (gamma) strain, δ ( delta) strain, λ (Lambda) strain, μ (Mu) and ο (Omicron).

As shown in Figure 5, Figure 5 (A) and Figure 5 (B) are the positions of the anti-spike protein monoclonal antibody 10-11C on the trimer and hexamer of the SRAS-CoV-2 spike protein, respectively. Among them, the red ball is the position of anti-spike protein monoclonal antibody.

Figure 5 (C) and Figure 5 (D) show the positions of the anti-spike monoclonal antibody 11-12H on the trimer and hexamer of the SRAS-CoV-2 spike protein, respectively. Among them, the red ball is the position of anti-spike protein monoclonal antibody.

The present invention discloses a novel SARS-CoV-2 Spike conserved epitope sequence, including HADQ (position 625, 626, 627, 628) (SEQ ID NO: 1) and TPW (position 630, 631, 633).

In a specific embodiment, the present invention discloses an antibody or an antigen-binding fragment thereof that specifically binds to the spike protein of acute respiratory syndrome coronavirus 2, wherein the anti The antibody or antigen-binding fragment thereof specifically binds to the amino acid sequence of SEQ ID NO: 1 or TPW in the spike protein.

In another specific embodiment, the present invention discloses a detection kit for detecting acute respiratory syndrome coronavirus 2, the detection kit comprising: the antibody or its antigen-binding fragment; and a detection reagent.

In another embodiment, the present invention discloses a method for detecting whether an individual suffers from acute respiratory syndrome coronavirus 2, which comprises the following steps: (a) providing a biological sample obtained from the individual; (b) making the biological The sample is in contact with the antibody or antigen-binding fragment thereof which can recognize the amino acid sequence of SEQ ID NO: 1 or TPW in the spike protein of acute respiratory syndrome coronavirus 2, when a spike of acute respiratory syndrome coronavirus 2 is present in the biological sample protein and the Spike protein comprises SEQ ID NO: 1 or TPW amino acid sequence, the antibody or antigen-binding fragment thereof forms a binding complex with the Spike protein; and (c) detecting the spike protein produced by step (b) Whether the binding complex exists, when the binding complex produced in step (b) exists, it is determined that the individual is infected with acute respiratory syndrome coronavirus 2.

In a specific embodiment, the applicable method of the present invention includes enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (Radioimmunoassay), lateral flow assay, western blotting, Reverse transcription polymerase chain reaction, or real-time polymerase chain reaction.

In a specific embodiment, the biological sample referred to in the present invention may be respiratory secretions, oral mucosal fluid, tear fluid, blood, feces, urine or a combination thereof.

The above detailed description is a specific description of the feasible embodiment of the present invention, but the embodiment is not used to limit the patent scope of the present invention, and those who do not deviate from the technical spirit of the present invention Equivalent implementation or changes shall be included in the patent scope of the present invention.

It will be readily appreciated by those skilled in the art that the present invention is well adapted to carry out these objects and attain the ends and advantages mentioned, as well as those inherent therein. The processes and methods for their production are representative of preferred embodiments, are exemplary, and are not intended to limit the scope of the invention. Modifications and other uses therein will occur to those skilled in the art. These modifications are included within the spirit of the invention and defined by the scope of the claims.

Claims (6)

An antibody or antigen-binding fragment thereof that specifically binds to the spike protein of acute respiratory syndrome coronavirus 2, wherein the antibody or antigen-binding fragment thereof binds to the amino acid of SEQ ID NO: 1 or TPW in the spike protein Sequence specific binding. A detection kit for detecting acute respiratory syndrome coronavirus 2, the detection kit comprising: the antibody or its antigen-binding fragment as described in Claim 1; and a detection reagent. A method for detecting whether an individual is infected with acute respiratory syndrome coronavirus 2, comprising the following steps: (a) provide a biological sample obtained from the individual; (b) contacting the biological sample with the antibody or antigen-binding fragment thereof as claimed in claim 1, when a spike protein of acute respiratory syndrome coronavirus 2 is present in the biological sample and the spike protein comprises SEQ ID NO: 1 or the amino acid sequence of TPW, the antibody or antigen-binding fragment thereof forms a binding complex with the Spike protein; and (c) Detecting whether the binding complex produced in step (b) exists, and when the binding complex produced in step (b) exists, it is determined that the individual is infected with acute respiratory syndrome coronavirus 2. The method according to claim 3, wherein the biological sample comprises: respiratory secretions, oral mucosal fluid, tears, blood, feces, urine or a combination thereof. The method as claimed in claim 3, wherein the acute respiratory syndrome coronavirus 2 comprises alpha virus strain, beta virus strain, gamma virus strain, delta virus strain, lambda virus strain, mu virus strain and o virus strain. The method as described in claim item 3, wherein the detection method of step (c) includes enzyme-binding immunosorbent assay, radioimmunoassay, lateral flow assay, western blot, reverse transcription polymerase chain reaction, Or real-time polymerase chain reaction.

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