CN113461809A - Method for screening binding inhibitors and/or binding domains - Google Patents

Method for screening binding inhibitors and/or binding domains Download PDF

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Publication number
CN113461809A
CN113461809A CN202010691682.0A CN202010691682A CN113461809A CN 113461809 A CN113461809 A CN 113461809A CN 202010691682 A CN202010691682 A CN 202010691682A CN 113461809 A CN113461809 A CN 113461809A
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label
rbd
sars
cov
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徐晓昱
朱婷婷
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Nanjing Novozan Biotechnology Co Ltd
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Nanjing Novozan Biotechnology Co Ltd
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Priority to PCT/CN2021/106633 priority patent/WO2022012647A1/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus

Abstract

The application relates to a method for high-flux screening an inhibitor for inhibiting the combination of RBD of SARS-COV-2 and ACE-2, a method for high-flux screening a combination region of RBD specifically combined with SARS-COV-2, an inhibitor for inhibiting the combination of RBD of SARS-COV-2 and ACE-2 and a combination region of RBD specifically combined with SARS-COV-2, which are obtained according to the method.

Description

Method for screening binding inhibitors and/or binding domains
Technical Field
The application relates to the field of biological medicine, in particular to a method for screening an inhibitor for inhibiting combination of RBD (restricted-binding domain) of SARS-COV-2 and ACE-2.
Background
The novel coronavirus SARS-COV-2 is highly infectious, and its spike glycoprotein (S-protein) in the outer shell can bind with high affinity to ACE2 on the surface of host cells, and then the carried RNA is injected into the cells to cause individual diseases, so that it is known that inhibition of SARS-COV-2 binding to ACE can reduce and eliminate the pathogenicity and infectivity of the virus.
In screening for inhibitors of protein interactions, methods of Fluorescence Activated Cell Sorting (FACS) and enzyme-linked immunosorbent assay (ELISA) are generally used. FACS single cells flow through the column with very low flux. ELISA, as a conventional method, has some drawbacks that are difficult to overcome, such as: 1) the experimental steps are multiple, and the time consumption is long. The plate washing, sample adding, color development and the like are carried out for many times, and at least one day is needed; 2) high flux can not be achieved, and the problem of more drug screening samples is difficult to solve; 3) the coated protein may shield some protein sites, so that the positive is missed, and a false negative result is obtained; 4) because the steps are more, experimental errors are easy to cause, and the result repeatability is poor and unstable.
Thus, there is a need for new methods for direct screening of inhibitors of RBD and ACE-2 binding of SARS-CoV-2 at the biochemical level.
Disclosure of Invention
The application provides a method for high-flux screening of an inhibitor for inhibiting the binding of RBD of SARS-COV-2 and ACE-2, a method for high-flux screening of a binding region for specifically binding RBD of SARS-COV-2, an inhibitor for inhibiting the binding of RBD of SARS-COV-2 and ACE-2 obtained according to the method, and a binding region for specifically binding RBD of SARS-COV-2. The method has at least one of the following advantages: (1) the inhibitor and/or the binding region can be screened at the biochemical level, a cell experiment does not need to be constructed, and the method is direct and rapid; (2) greatly saves the test workload and the test time: the experiment operation is from original coating, sealing and plate washing for many times, only a sample and a detection reagent are needed to be added at present, and the experiment time is shortened from original 1 day to 1-2 hours; (3) greatly improved the flux of the experiment: the single cell column chromatography is improved to 384 holes, even 1536 holes from 96 holes of original ELISA or FACS single cell column chromatography, thereby realizing large-batch sample screening in short time; (4) the result is reliable: homogeneous detection, the antigen protein presents natural conformation in a liquid phase system, and false positive and false negative are effectively avoided; (5) and (3) signal stabilization: the fluorescence duration is long, detection can be carried out after overnight, the detection result is not influenced, the detection result of ELISA is limited by time, and the detection result is influenced by overlong time or overlong time.
In one aspect, the present application provides a method for high throughput screening of inhibitors that inhibit binding of the RBD of SARS-COV-2 to ACE-2, comprising the steps of: a) obtaining a first binding region with a first label and a second binding region with a second label, wherein the first label and the second label respectively comprise a label selected from the group consisting of: a donor label and an acceptor label, wherein upon interaction of the donor label and the acceptor label, the acceptor label can acquire energy transferred from the donor label to generate fluorescence; b) obtaining a third protein with a third tag, and a fourth protein with a fourth tag, wherein the third protein and the fourth protein are optionally each selected from the group consisting of: the RBD and ACE-2 of SARS-COV-2, wherein the third tag can specifically bind to the first binding region and the fourth tag can specifically bind to the second binding region; c) mixing the candidate inhibitor with the first binding region bearing the first tag, the second binding region bearing the second tag, the third protein bearing the third tag, and the fourth protein bearing the fourth tag; d) detecting the ratio of absorbance at the wavelength of fluorescence of the acceptor label and the donor label after the mixing.
In certain embodiments, the third protein is an RBD of SARS-COV-2 and the fourth protein is ACE-2; alternatively, the third protein is ACE-2 and the fourth protein is RBD of SARS-COV-2.
In certain embodiments, the concentration of RBD of SARS-COV-2 is about 3-120 nM.
In certain embodiments, the concentration of the RBD of SARS-COV-2 is about 5-60 nM.
In certain embodiments, the concentration of ACE-2 is about 1 to 60 nM.
In certain embodiments, the concentration of ACE-2 is about 2-30 nM.
In certain embodiments, the first tag is the donor tag and the second tag is the acceptor tag; alternatively, the first tag is the acceptor tag and the second tag is the donor tag.
In certain embodiments, the donor tag comprises a lanthanide metal or compound thereof.
In certain embodiments, the donor label comprises europium or a compound thereof, and/or terbium or a compound thereof.
In certain embodiments, the receptor tag is selected from the group consisting of: rhodamine, cyanines, arylcyanines, coumarins, proflavins, acridines, fluoresceins, nitrobenzoxadiazoles, and/or fluorescent proteins.
In certain embodiments, the third tag or the fourth tag is an IgG Fc of a mammal, wherein the mammal is selected from the group consisting of: mouse, rat, rabbit, monkey, and human.
In certain embodiments, the first binding region or the second binding region is an IgG Fc antibody of a mammal, wherein the mammal is selected from the group consisting of: mouse, rat, rabbit, monkey, and human.
In certain embodiments, the third tag or the fourth tag is a selectable marker selected from the group consisting of: his, GST, Flag, Cmyc, and MBP.
In certain embodiments, the first binding region or the second binding region specifically binds to the selectable marker.
In certain embodiments, the concentration of the first binding region is 0.05 to 2 μ g/mL.
In certain embodiments, the concentration of the second binding region is 0.5-20 μ g/mL.
In certain embodiments, the fluorescence wavelength of the donor label is 620 nm.
In certain embodiments, the acceptor label has a fluorescence wavelength of 665 nm.
In certain embodiments, the candidate inhibitors include small molecule compounds, and/or antibodies or antigen-binding fragments thereof.
In certain embodiments, the RBD of SARS-COV-2 comprises an RBD of SARS-COV-2 and/or a mutant thereof.
In certain embodiments, the ACE-2 comprises human ACE-2 and/or a functional fragment thereof.
In certain embodiments, the high flux is at least 96.
In another aspect, the present application provides an inhibitor for inhibiting binding of RBD of SARS-COV-2 to ACE-2, obtained by the method for high throughput screening of inhibitors for inhibiting binding of RBD of SARS-COV-2 to ACE-2 as described herein.
In certain embodiments, the inhibitor comprises a small molecule compound, and/or an antibody or antigen-binding fragment thereof.
In certain embodiments, the inhibitor comprises an antibody or antigen-binding portion thereof that specifically binds to the RBD of SARS-COV-2.
In certain embodiments, the inhibitor comprises an antibody or antigen-binding portion thereof that specifically binds ACE-2.
In another aspect, the present application provides a method for high throughput screening of a binding region that specifically binds to an RBD of SARS-COV-2, comprising the steps of: a) obtaining a first binding region with a first label and a second binding region with a second label, wherein the first label and the second label respectively comprise a label selected from the group consisting of: a donor label and an acceptor label, wherein upon interaction of the donor label and the acceptor label, the acceptor label can acquire energy transferred from the donor label to generate fluorescence; b) obtaining an RBD of SARS-COV-2 bearing a third tag, wherein said third tag can specifically bind to said first binding region and said second binding region can specifically bind to an RBD of SARS-COV-2; c) mixing candidate said binding region with said first tag, said second binding region with said second tag, said RBD of SARS-COV-2 with said third tag; d) detecting the ratio of absorbance at the fluorescence wavelength of the acceptor label and the donor label after mixing.
In certain embodiments, the RBD of SARS-COV-2 is at a concentration of about 0.5 to 60 nM.
In certain embodiments, the concentration of RBD of SARS-COV-2 is about 3-10 nM.
In certain embodiments, the first tag is the donor tag and the second tag is the acceptor tag; alternatively, the first tag is the acceptor tag and the second tag is the donor tag.
In certain embodiments, the donor tag comprises a lanthanide metal or compound thereof.
In certain embodiments, the donor label comprises europium or a compound thereof, and/or terbium or a compound thereof.
In certain embodiments, the receptor tag is selected from the group consisting of: rhodamine, cyanines, arylcyanines, coumarins, proflavins, acridines, fluoresceins, nitrobenzoxadiazoles, and/or fluorescent proteins.
In certain embodiments, the third tag is an IgG Fc of a mammal, wherein the mammal is selected from the group consisting of: mouse, rat, rabbit, monkey, and human.
In certain embodiments, the first binding region is an IgG Fc antibody of a mammal, wherein the mammal is selected from the group consisting of: mouse, rat, rabbit, monkey, and human.
In certain embodiments, the second binding region is an RBD antibody of SARS-COV-2 or an antigen-binding fragment thereof.
In certain embodiments, the concentration of the first binding region is 0.05 to 2 μ g/mL.
In certain embodiments, the concentration of the second binding region is 0.5-20 μ g/mL.
In certain embodiments, the fluorescence wavelength of the donor label is 620 nm.
In certain embodiments, the acceptor label has a fluorescence wavelength of 665 nm.
In another aspect, the present application provides an antibody or antigen-binding fragment thereof that specifically binds to an RBD of SARS-COV-2, obtained by the method for high-throughput screening of binding regions that specifically bind to an RBD of SARS-COV-2 described herein.
Other aspects and advantages of the present application will be readily apparent to those skilled in the art from the following detailed description. Only exemplary embodiments of the present application have been shown and described in the following detailed description. As those skilled in the art will recognize, the disclosure of the present application enables those skilled in the art to make changes to the specific embodiments disclosed without departing from the spirit and scope of the invention as it is directed to the present application. Accordingly, the descriptions in the drawings and the specification of the present application are illustrative only and not limiting.
Drawings
The specific features of the invention to which this application relates are set forth in the appended claims. The features and advantages of the invention to which this application relates will be better understood by reference to the exemplary embodiments described in detail below and the accompanying drawings. The brief description of the drawings is as follows:
FIG. 1 shows a schematic diagram of a high throughput screening method for inhibitors that inhibit the binding of RBD of SARS-COV-2 to ACE-2 as described herein.
FIG. 2 shows a schematic diagram of the high throughput screening method for binding regions that specifically bind to RBD of SARS-COV-2 as described herein.
FIG. 3 shows 665/620 values after addition of a candidate inhibitor using the methods described herein.
FIG. 4 shows the result of the systematic establishment of the method for high-throughput screening of RBD binding region specifically binding to SARS-COV-2 as described in the present application.
Detailed Description
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification.
Definition of terms
In the present application, the term "first tag" generally refers to a labeling molecule carried on the first binding region of the present application, which first tag may be the donor tag or the first tag may be the acceptor tag.
In the present application, the "second tag" generally refers to a labeling molecule carried on the second binding region of the present application, and the second tag may be the donor tag, or the second tag may be the acceptor tag.
In the present application, the term "donor label" generally refers to a substance that is capable of being excited by an external energy source (e.g., an extraneous light source) to generate energy. The energy generated by the donor label can migrate to the acceptor label.
In this application, the term "acceptor label" generally refers to a substance that is capable of receiving energy (e.g., energy from a donor label) and thereby exciting fluorescence. Energy transfer can occur between the donor label and the acceptor label. The acceptor label may receive or absorb energy from the donor label.
In the present application, the term "second binding region" generally refers to a substance capable of specifically binding to the fourth tag. The second binding region may comprise an antibody.
In the present application, the term "fourth tag" generally refers to a substance carried on a fourth protein that is capable of binding to the second binding domain. The fourth tag and the second binding domain may be any antigen and antibody thereof, protein and ligand thereof, protein and receptor thereof capable of specifically binding, as long as they are a pair of substances (e.g., polypeptide or protein) capable of specifically binding. In some cases, the third tag may include an antigen and/or a marker protein.
In the present application, the term "first binding region" generally refers to a substance capable of specifically binding to the third label. The first binding region may comprise an antibody.
In the present application, the term "third tag" generally refers to a substance carried on a third protein that is capable of binding to the first binding region. The third label and the first binding domain may be any antigen and antibody thereof, protein and ligand thereof, protein and receptor thereof capable of specifically binding, as long as they are a pair of substances (e.g., polypeptide or protein) capable of specifically binding. In some cases, the third tag may include an antigen and/or a marker protein.
In the present application, the term "third protein" generally refers to a protein selected from any one of the RBD and ACE-2 of SARS-COV-2. The third protein may have a third tag thereon.
In the present application, the term "fourth protein" generally refers to a protein selected from any one of both RBD and ACE-2 of SARS-COV-2. The fourth protein may carry a fourth tag thereon.
In the present application, the term "specific binding" generally refers to the binding of a polypeptide or protein to a given target rather than to the random binding of undesired substances.
In the present application, the "His" generally refers to a fusion tag protein composed of polyhistidine residues, which can be inserted into the C-terminus or N-terminus of a protein of interest.
In the present application, the "GST" is generally referred to as glutathione mercaptotransferase, a tagged protein.
In the present application, the "Flag" generally refers to a hydrophilic polypeptide encoding 8 amino acids, a tagged protein. An exemplary Flag tag protein sequence is DYKDDDDK.
In the present application, the term "Cmyc" generally refers to a tag protein. An exemplary C, myc-tagged protein has a sequence of EQKLISEEDL.
In the present application, the term "MBP" generally refers to maltose binding protein, a tagged protein. MBP is usually encoded by the malE gene of E.coli K12.
In the present application, the term "ACE-2", also known as angiotensin converting enzyme 2, generally refers to a type I transmembrane glycoprotein, a key molecule of the renin-angiotensin-aldosterone system (RAS). ACE-2 has enzymatic activity and is capable of catalyzing angiotensin II to angiotensin 1-7 (a vasodilator). The ACE-2 protein comprises an amino-terminal extracellular Peptidase Domain (PD) and a carboxy-terminal collectrin domain (CLD). Wherein the extracellular kinase domain comprises a zinc binding active site and is involved in a catalytic reaction, and the collectrin domain comprises a hydrophobic transmembrane domain and a short intracellular domain. Studies have shown that some coronaviruses are able to enter host cells by binding to the Peptidase Domain (PD) of ACE-2 via a Receptor Binding Domain (RBD) located in the S1 subunit of the S protein. The extracellular peptidase domain of ACE2 can be divided into two subdomains: subdomain I and subdomain II, the spike protein (S protein) may interact with the apical end of subdomain I. The "ACE-2" may encompass ACE2 of any vertebrate origin, e.g., mammals such as humans, monkeys, mice, rats, pigs, dogs, cats and tigers, birds, and the like. In the present application, the ACE-2 may comprise full-length unprocessed ACE-2, as well as any form of ACE-2 or functional fragment thereof that is processed or artificially modified by intracellular processing, and further comprise a naturally occurring functional variant of ACE2, e.g., an alternative splice and/or allelic variant, as long as it is capable of recognizing or binding to a virus. For example, the ACE-2 or functional variant thereof may be modified to lose peptidase catalytic activity but still bind to viral surface proteins (e.g., S proteins). For example, human ACE-2 may comprise three alternative splices, and the amino acid sequence of an exemplary human ACE-2 protein may be found in UniProt database accession number Q9BYF 1. ACE-2 as described herein may include at least a subdomain I portion of the peptidase domain thereof. ACE2 may be isolated from a variety of sources, such as mammalian (including human) tissue types or another source, or ACE-2 may be prepared by recombinant and/or synthetic methods.
In this application, the term "SARS-COV-2" generally refers to Severe Acute Respiratory Syndrome Coronavirus type 2, which is collectively referred to in English as Severe acid Respiratory Syndrome Virus Syndrome Coronavir 2. SARS-CoV-2 belongs to the family Coronaviridae (Coronaviridae) genus B coronavirus (Betaconoviridus) subfamily Sarbecoviridus (Sarbecovirus). SARS-CoV-2 is a non-segmented positive-stranded RNA virus with an envelope. It was found that the receptor for SARS-CoV-2 into the host cell is ACE 2. The surface of SARS-COV-2 has a transmembrane protein, commonly referred to as spike protein or S protein, which can mediate the entry of coronaviruses into host cells. The S protein typically comprises two functional subunits, S1 and S2, with the S1 subunit responsible for binding to host cell receptors and the S2 subunit responsible for viral membrane and cell membrane fusion. The S1 subunit can be generally divided into 2 domains: the N-terminal domain (S1-NTD) and the C-terminal domain (S1-CTD), 2 domains can be used as Receptor Binding Domains (RBD).
In the present application, the term "RBD of SARS-COV-2" generally refers to the receptor binding domain of SARS-COV-2, which is the portion of the S protein of SARS-COV-2 that binds to ACE-2. The RBD of SARS-COV-2 described herein can include various modified forms of the RBD of SARS-COV-2, for example, can include glycosylated forms, phosphorylated forms, ubiquinated forms, methylated forms, and/or acetylated forms. In the present application, the RBD of SARS-COV-2 can comprise unprocessed RBD, as well as any form of RBD processed or artificially modified by the cell or a functional fragment thereof, and also functional variants of naturally occurring RBD, as long as the fragment or variant still has the function of recognizing or binding to a cell surface receptor (e.g., ACE 2). Exemplary RBDs of SARS-COV-2 can be found in the amino acid sequence shown at positions 330 to 583 under GenBank accession No. QHR 63250.2.
In the present application, the term "functional fragment" generally refers to a fragment of a larger polypeptide or polynucleotide that retains the same or similar activity or ability as its larger counterpart. The level of activity of a functional fragment may be the same as, less than, or greater than the activity of the larger counterpart.
Detailed Description
In one aspect, the present application provides a method for high throughput screening for inhibitors that inhibit the binding of the RBD of SARS-COV-2 to ACE-2.
The method described herein may comprise a) obtaining a first binding region with a first label, and a second binding region with a second label, wherein the first label and the second label may each correspondingly comprise a label selected from the group consisting of: a donor label and an acceptor label, wherein upon interaction of the donor label and the acceptor label, the acceptor label can acquire energy transferred from the donor label to produce fluorescence.
In the present application, the donor label is capable of being excited to release energy by an external energy source. In the present application, the donor tag may comprise a lanthanide metal or a compound thereof. The lanthanide metal fluorescence is of a different duration than ordinary fluorescence. The half-life of ordinary fluorescence is nanosecond, and the half-life of lanthanide is millisecond, with 6 orders of magnitude difference. Therefore, in Time Resolved Fluorescence (TRF) detection, F has a 50us time delay, and the signal of ordinary fluorescence is almost zero after the time delay.
In the present application, the donor label may comprise europium or a compound thereof, and/or terbium or a compound thereof. For example, the donor label may include a europium chelate, a europium cryptate, a terbium chelate, and/or a terbium cryptate. In europium and/or terbium cryptates, europium and terbium are permanently embedded in a cage and the structure is very stable.
In the present application, the acceptor label is capable of accepting external energy and emitting fluorescence. In the present application, the receptor tag may be selected from any one or more of the following groups: rhodamine, cyanines, arylcyanines, coumarins, proflavins, acridines, fluoresceins, nitrobenzoxadiazoles, and/or fluorescent proteins. For example, the fluorescent protein may include cyan fluorescent protein, green fluorescent protein, yellow fluorescent protein, orange fluorescent protein, red fluorescent protein, and a protein that fluoresces in the far infrared range. For example, the fluorescent protein can include Allophycocyanin (APC).
In this application, the donor label may be excited by an external light source (e.g., a xenon lamp or a laser) to transfer energy resonance to the acceptor label, which is excited to emit light of a specific wavelength. In some cases, the donor label and the acceptor label can be bound to two interacting biomolecules, respectively, and the binding of the biomolecules can pull the donor label and the acceptor label close enough to generate energy transfer, and since the emission light of the donor label comes from the energy transfer, there is no need to separate unbound from bound molecules in the experiment, i.e. no washing step is needed.
In the present application, the first tag may be the donor tag and the second tag may be the acceptor tag. In the present application, the first tag may be the acceptor tag and the second tag may be the donor tag.
The method of the present application may comprise b) obtaining a third protein with a third tag, and a fourth protein with a fourth tag, wherein the third protein and the fourth protein may optionally be selected from the group consisting of: RBD of SARS-COV-2 and ACE-2.
In the present application, the third protein may be RBD of SARS-COV-2, and the fourth protein may be ACE-2. In the present application, the RBD of SARS-COV-2 may comprise the RBD of SARS-COV-2 and/or a mutant thereof. In the present application, the third protein may be ACE-2 and the fourth protein may be RBD of SARS-COV-2. In the present application, the ACE-2 may comprise human ACE-2 and/or functional fragments thereof.
In the present application, the third tag may specifically bind to the first binding region, and the fourth tag may specifically bind to the second binding region.
In the present application, the third tag may be an IgG Fc of a mammal, wherein the mammal may be selected from the group consisting of: mouse, rat, rabbit, monkey, and human. In this case, the first binding region may be an IgG Fc antibody of a mammal, wherein the mammal is selected from the group consisting of: mouse, rat, rabbit, monkey, and human.
In the present application, the fourth tag may be an IgG Fc of a mammal, wherein the mammal may be selected from the group consisting of: mouse, rat, rabbit, monkey, and human. In this case, the second binding region may be an IgG Fc antibody of a mammal, wherein the mammal is selected from the group consisting of: mouse, rat, rabbit, monkey, and human.
In the present application, the third tag may be a selection marker, for example, a selection marker selected from the group consisting of: his, GST, Flag, Cmyc, and MBP. At this time, the first binding region may bind to the selection marker.
In the present application, the fourth tag may be a selection marker, for example, a selection marker selected from the group consisting of: his, GST, Flag, Cmyc, and MBP. At this time, the second binding region may bind to the selection marker.
The methods described herein can include mixing a candidate inhibitor with the first binding region bearing a first tag, the second binding region bearing a second tag, the third protein bearing a third tag, and the fourth protein bearing a fourth tag.
In the present application, the RBD concentration of SARS-COV-2 can be about 3-120 nM. For example, the concentration of the RBD of SARS-COV-2 can be about 3-100nM, 3-80nM, 3-70nM, 3-60nM, 3-50nM, 3-40nM, 3-30nM, 3-20nM, 5-100nM, 5-80nM, 5-70nM, 5-60nM, 5-50nM, 5-40nM, 5-30nM, 5-20nM, 10-40nM, 10-60nM, or 10-120 nM. In the present application, the RBD concentration of SARS-COV-2 can be about 5-60nM or 3-20 nM.
In the present application, the concentration of ACE-2 may be about 1-60 nM. For example, the concentration of the RBD of SARS-COV-2 can be about 1-50nM, 1-40nM, 1-35nM, 1-30nM, 1-25nM, 1-20nM, 1-15nM, 1-10nM, 1-5nM, 2-60nM, 2-50nM, 2-40nM, 2-30nM, 2-20nM, or 2-10 nM. In one application, the concentration of ACE-2 is about 2-30 nM.
In the present application, the concentration of the first binding region may be 0.05 to 2. mu.g/mL, for example, 0.05 to 1.5. mu.g/mL, 0.05 to 1.0. mu.g/mL, 0.05 to 0.8. mu.g/mL, 0.05 to 0.6. mu.g/mL, 0.05 to 0.5. mu.g/mL, 0.08 to 0.4. mu.g/mL, 0.08 to 0.3. mu.g/mL, or 0.08 to 0.2. mu.g/mL. For example, the concentration of the first binding domain may be 0.08-0.1. mu.g/mL.
In the present application, the concentration of the first binding domain may be 0.08 to 5. mu.g/mL, for example, 0.08 to 4.5. mu.g/mL, 0.08 to 4. mu.g/mL, 0.08 to 3.5. mu.g/mL, 0.08 to 3. mu.g/mL, 0.08 to 2.5. mu.g/mL, 0.08 to 2. mu.g/mL, 0.08 to 1.5. mu.g/mL, 0.08 to 1. mu.g/mL, or 0.08 to 0.5. mu.g/mL. For example, the concentration of the first binding domain may be 0.08-0.4. mu.g/mL.
In the present application, the concentration of the second binding region may be 0.4 to 20. mu.g/mL, for example, 0.4 to 15. mu.g/mL, 0.4 to 10. mu.g/mL, 0.4 to 5. mu.g/mL, 0.4 to 4. mu.g/mL, 0.4 to 3. mu.g/mL, 0.4 to 2. mu.g/mL, 1 to 10. mu.g/mL, 1 to 15. mu.g/mL, 2 to 20. mu.g/mL. For example, the concentration of the second binding region can be 0.4-2. mu.g/mL.
When the first binding region with the first label, the second binding region with the second label, the third protein with the third label, and the fourth protein with the fourth label are mixed, the first binding region and the third label can bind, and the second binding region and the fourth label can bind. And the third protein and the fourth protein are capable of binding such that four substances (i.e., the first binding region bearing the first tag, the second binding region bearing the second tag, the third protein bearing the third tag, and the fourth protein bearing the fourth tag) form a complex. In the complex, the first and second labels may interact, wherein the acceptor label may acquire energy transferred from the donor label to produce fluorescence. In the context of the present application, it is,
the methods described herein can further comprise d) detecting the ratio of absorbance at the wavelength of fluorescence of the acceptor label and the donor label after the mixing. In the present application, the ratio of the absorbance at the wavelength of the fluorescence of the acceptor label and the donor label reflects the degree of binding of the third protein and the fourth protein, and a larger ratio indicates a higher degree of binding.
When the candidate inhibitor is mixed with the first binding domain with the first tag, the second binding domain with the second tag, the third protein with the third tag, and the fourth protein with the fourth tag, if the candidate inhibitor is capable of inhibiting the binding between the third protein and the fourth protein, the ratio of absorbance at the wavelength of the fluorescence of the acceptor tag and the donor tag is lower than when the candidate inhibitor is not added. When the ratio is lower, it indicates that the inhibition of the candidate inhibitor is higher. A schematic diagram of the high throughput screening method for inhibitors that inhibit the binding of RBD of SARS-COV-2 to ACE-2 as described herein can be seen in FIG. 1.
In the present application, the above-mentioned concentrations may be the final concentrations of the first binding domain with the first tag, the second binding domain with the second tag, the third protein with the third tag and the fourth protein with the fourth tag, and the candidate inhibitor after mixing.
The fluorescence wavelength of the donor label in this application may be 620 nm. In the present application, the fluorescence wavelength of the acceptor label may be 665 nm.
When the concentrations of the first binding region with the first label, the second binding region with the second label, the third protein with the third label and the fourth protein with the fourth label are within the ranges described herein, a higher value of 665/620 can be obtained, and the concentrations of RBD and ACE2 are not too high, which has certain sensitivity and economy.
In another aspect, the present application provides an inhibitor for inhibiting binding of RBD of SARS-COV-2 to ACE-2, obtained by the method for high throughput screening of inhibitors for inhibiting binding of RBD of SARS-COV-2 to ACE-2 as described herein.
In the present application, the third protein with the third tag or the fourth protein with the fourth tag may be absent from the first binding region with the first tag, the second binding region with the second tag, the third protein with the third tag, and the fourth protein with the fourth tag. When the third protein with the third tag or the fourth protein with the fourth tag is not present, the method can be used for high-throughput screening of binding regions that specifically bind to the RBD of SARS-COV-2, or the method can be used for high-throughput screening of binding regions that specifically bind to ACE-2.
In another aspect, the present application provides a method for high throughput screening of a binding region that specifically binds to an RBD of SARS-COV-2, comprising the steps of: a) obtaining a first binding region with a first label and a second binding region with a second label, wherein the first label and the second label respectively comprise a label selected from the group consisting of: a donor label and an acceptor label, wherein upon interaction of the donor label and the acceptor label, the acceptor label can acquire energy transferred from the donor label to generate fluorescence; b) obtaining an RBD of SARS-COV-2 bearing a third tag, wherein said third tag can specifically bind to said first binding region and said second binding region can specifically bind to an RBD of SARS-COV-2; c) mixing candidate said binding region with said first tag, said second binding region with said second tag, said RBD of SARS-COV-2 with said third tag; d) detecting the ratio of absorbance at the fluorescence wavelength of the acceptor label and the donor label after mixing. The method for high throughput screening of binding regions that specifically bind to RBD of SARS-COV-2 can be as shown in FIG. 2. In the present application, the concentration may be the final concentration of the first binding region with the first tag, the second binding region with the second tag, the RBD of SARS-COV-2 with the third tag, and the candidate inhibitor mixed together.
In certain embodiments, the concentration of the RBD of SARS-COV-2 can be about 1-60 nM. In certain embodiments, the concentration of the RBD of SARS-COV-2 can be about 2-30nM, or about 60 nM.
In another aspect, the present application provides an antibody or antigen-binding fragment thereof that specifically binds to an RBD of SARS-COV-2, obtained by the method for high-throughput screening of binding regions that specifically bind to an RBD of SARS-COV-2 described herein.
When the first binding domain with the first label, the second binding domain with the second label and the third protein with the third label are within the ranges described herein, a higher value of 665/620 can be obtained without the RBD concentration being too high, resulting in certain sensitivity and economy.
In the present application, the candidate inhibitors may include small molecule compounds, and/or large molecule compounds, e.g., polynucleic acids, polypeptides, and/or proteins. For example, the candidate inhibitor may include a small molecule compound, and/or an antibody or antigen-binding fragment thereof. In the present application, the inhibitor may comprise an antibody or antigen-binding portion thereof that specifically binds to the RBD of SARS-COV-2. In the present application, the inhibitor may comprise an antibody or antigen-binding portion thereof that specifically binds to ACE-2. The antibodies may include monoclonal antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, and/or multispecific antibodies. Antigen-binding fragments of antibodies may include Fab, Fab', F (ab)2Fv fragment, F (ab')2scFv, di-scFv and/or dAb.
In the present application, the high throughput may be at least 96. For example, the high throughput may be at least 96, 384, 1536, or more.
In the present application, there is also provided the following embodiments:
1. a method for high throughput screening of inhibitors that inhibit binding of RBD of SARS-COV-2 to ACE-2, comprising the steps of:
a) obtaining a first binding region with a first label and a second binding region with a second label, wherein the first label and the second label respectively comprise a label selected from the group consisting of: a donor label and an acceptor label, wherein the donor label and the acceptor label are different,
wherein upon interaction of the donor label and the acceptor label, the acceptor label can acquire energy transferred from the donor label to produce fluorescence;
b) obtaining a third protein with a third tag, and a fourth protein with a fourth tag, wherein the third protein and the fourth protein are optionally each selected from the group consisting of: the RBD and ACE-2 of SARS-COV-2, wherein the third tag can specifically bind to the first binding region and the fourth tag can specifically bind to the second binding region;
c) mixing the candidate inhibitor with the first binding region bearing the first tag, the second binding region bearing the second tag, the third protein bearing the third tag, and the fourth protein bearing the fourth tag;
d) detecting the ratio of absorbance at the wavelength of fluorescence of the acceptor label and the donor label after the mixing.
2. The method of embodiment 1, wherein the third protein is an RBD of SARS-COV-2 and the fourth protein is ACE-2; alternatively, the third protein is ACE-2 and the fourth protein is RBD of SARS-COV-2.
3. The method of any one of embodiments 1-2, wherein the concentration of the RBD of SARS-COV-2 is about 3-120 nM.
4. The method of any one of embodiments 1-3, wherein the concentration of the RBD of SARS-COV-2 is about 5-60 nM.
5. The method according to any one of embodiments 1-4, wherein the concentration of ACE-2 is about 1-60 nM.
6. The method according to any one of embodiments 1-5, wherein the concentration of ACE-2 is about 2-30 nM.
7. The method of any one of embodiments 1-6, wherein the first tag is the donor tag and the second tag is the acceptor tag; alternatively, the first tag is the acceptor tag and the second tag is the donor tag.
8. The method of any one of embodiments 1-7, wherein the donor tag comprises a lanthanide metal or compound thereof.
9. The method of any one of embodiments 1-8, wherein the donor label comprises europium or a compound thereof, and/or terbium or a compound thereof.
10. The method according to any one of embodiments 1-9, wherein the receptor tag is selected from the group consisting of: rhodamine, cyanines, arylcyanines, coumarins, proflavins, acridines, fluoresceins, nitrobenzoxadiazoles, and/or fluorescent proteins.
11. The method according to any one of embodiments 1-10, wherein the third or fourth tag is an IgG Fc of a mammal, wherein the mammal is selected from the group consisting of: mouse, rat, rabbit, monkey, and human.
12. The method according to any one of embodiments 1-11, wherein the first binding region or the second binding region is an IgG Fc antibody of a mammal, wherein the mammal is selected from the group consisting of: mouse, rat, rabbit, monkey, and human.
13. The method according to any one of embodiments 1-12, wherein the third or fourth tag is a selectable marker selected from the group consisting of: his, GST, Flag, Cmyc, and MBP.
14. The method of embodiment 13, wherein the first binding region or the second binding region specifically binds to the selectable marker.
15. The method according to any one of embodiments 1-14, wherein the concentration of the first binding region is 0.05-2 μ g/mL.
16. The method according to any one of embodiments 1-15, wherein the concentration of the second binding region is 0.5-20 μ g/mL.
17. The method of any one of embodiments 1-16, wherein the fluorescence wavelength of the donor label is 620 nm.
18. The method according to any one of embodiments 1-17, wherein the acceptor label has a fluorescence wavelength of 665 nm.
19. The method of any one of embodiments 1-18, wherein the candidate inhibitor comprises a small molecule compound, and/or an antibody or antigen-binding fragment thereof.
20. The method of any one of embodiments 1-19, wherein the RBD of SARS-COV-2 comprises an RBD of SARS-COV-2 and/or a mutant thereof.
21. The method according to any one of embodiments 1-20, wherein the ACE-2 comprises human ACE-2 and/or a functional fragment thereof.
22. The method of any one of embodiments 1-21, wherein the high flux is at least 96.
23. An inhibitor of binding of RBD of SARS-COV-2 to ACE-2 obtained by the method of any one of embodiments 1-22.
24. The inhibitor of embodiment 23, comprising a small molecule compound, and/or an antibody or antigen-binding fragment thereof.
25. The inhibitor according to any one of embodiments 23-24, comprising an antibody or antigen-binding portion thereof that specifically binds to an RBD of SARS-COV-2.
26. The inhibitor according to any one of embodiments 23-25 comprising an antibody or antigen-binding portion thereof that specifically binds ACE-2.
27. A method for high throughput screening of binding regions that specifically bind to RBD of SARS-COV-2, comprising the steps of:
a) obtaining a first binding region with a first label and a second binding region with a second label, wherein the first label and the second label respectively comprise a label selected from the group consisting of: a donor label and an acceptor label, wherein the donor label and the acceptor label are different,
wherein upon interaction of the donor label and the acceptor label, the acceptor label can acquire energy transferred from the donor label to produce fluorescence;
b) obtaining an RBD of SARS-COV-2 bearing a third tag, wherein said third tag can specifically bind to said first binding region and said second binding region can specifically bind to an RBD of SARS-COV-2;
c) mixing candidate said binding region with said first tag, said second binding region with said second tag, said RBD of SARS-COV-2 with said third tag;
d) detecting the ratio of absorbance at the fluorescence wavelength of the acceptor label and the donor label after mixing.
28. The method of embodiment 27, wherein the concentration of the SARS-COV-2 RBD is about 0.5-60 nM.
29. The method of any one of embodiments 27-28, wherein the concentration of the RBD of SARS-COV-2 is about 3-10 nM.
30. The method of any one of embodiments 27-29, wherein the first tag is the donor tag and the second tag is the acceptor tag; alternatively, the first tag is the acceptor tag and the second tag is the donor tag.
31. The method of any one of embodiments 27-30, wherein the donor tag comprises a lanthanide metal or compound thereof.
32. The method of any one of embodiments 27-31, wherein the donor label comprises europium or a compound thereof, and/or terbium or a compound thereof.
33. The method according to any one of embodiments 27-32, wherein the receptor tag is selected from the group consisting of: rhodamine, cyanines, arylcyanines, coumarins, proflavins, acridines, fluoresceins, nitrobenzoxadiazoles, and/or fluorescent proteins.
34. The method of any one of embodiments 27-33, wherein the third tag is an IgG Fc of a mammal, wherein the mammal is selected from the group consisting of: mouse, rat, rabbit, monkey, and human.
35. The method according to any one of embodiments 27-34, wherein the first binding region is a mammalian IgGFc antibody, wherein the mammal is selected from the group consisting of: mouse, rat, rabbit, monkey, and human.
36. The method of any one of embodiments 27-35, wherein the second binding region is an RBD antibody of SARS-COV-2 or an antigen-binding fragment thereof.
37. The method according to any one of embodiments 27-36, wherein the concentration of the first binding region is 0.05-2 μ g/mL.
38. The method according to any one of embodiments 27-37, wherein the concentration of the second binding region is 0.5-20 μ g/mL.
39. The method of any one of embodiments 27-38, wherein the fluorescence wavelength of the donor label is 620 nm.
40. The method of any one of embodiments 27-39, wherein the acceptor label has a fluorescence wavelength of 665 nm.
41. An antibody or antigen-binding fragment thereof that specifically binds to the RBD of SARS-COV-2 obtained by the method of any one of embodiments 27-40.
Without intending to be bound by any theory, the following examples are merely intended to illustrate the fusion proteins, preparation methods, uses, etc. of the present application, and are not intended to limit the scope of the invention of the present application.
Examples
Reagents and consumables:
buffer solution: detection buffer (50mM PBS,400mM KF, 0.1% BSA, 0.1% Tween20, pH7.0); dilution buffer (50mM PBS, 0.1% BSA, 0.1% Tween20, pH7.0); 1 × PBS buffer (pH 7.4); g-25 desalting column; EDC (107-06-2); NHS (6066-82-6); ultra-dry dimethyl sulfoxide (DMSO).
Sample preparation:
third protein with third tag: RBD-his of SARS-COV-2 (self-made, 293 cell expression, Ni-plated)2+Affinity, ion exchange column purification to obtain purity>90% protein)
Fourth protein with fourth tag: ACE-2 protein ACE-2-mFc with mouse Fc (self-prepared, 293 cell expression, proA purification to obtain antibody with purity > 95%)
Donor label Donor: and Eu.
Receptor tag Acceptor: cy 5.
Antibody: anti-mouse Fc antibody Anti-mFc (self-prepared, 293 cell expressed, pro a purified to > 95% antibody), Anti-human Fc antibody Anti-hFc (self-prepared, 293 cell expressed, pro a purified to > 95% antibody) and Anti-His antibody Anti-His (self-prepared, 293 cell expressed, pro a purified to > 95% antibody).
The instrument comprises the following steps: a 384-hole whiteboard; microplate reader (manufacturer, model); a one-ten-thousandth balance; a vortex oscillation instrument; a palm centrifuge;
Figure BDA0002589600620000161
a/SCG purifier.
Example 1 Donor tag Donor-labeled antibody Anti-mFc
Preparing EDC and NHS solution, adding Donor (Eu) and activating for 2h at room temperature. Adding Anti-mFc antibody to n(antibodies)：n(Donor)Activating for 2h at room temperature, wherein the ratio is 1: 8-1: 14. G25 purified the antibody to remove excess Donor.
The second binding moiety-Donor-Anti-mFc with the second tag was obtained.
The antibody was changed to Anti-His, and Donor-Anti-His was prepared in the same manner.
And measuring the labeling efficiency (DOL), calculating the concentration of the Donor and the concentration of the antibody through A280 and A304, wherein the Donor concentration/the concentration of the antibody is the labeling rate, and the labeling rate is 3-8 in the following examples.
Example 2 receptor-tag Acceptor-tagged antibody Anti-His
An NHS solution is prepared, Acceptor (cy5) is added, and activation is carried out for 2h at room temperature. Adding Anti-His antibody to n(antibodies)：n(Accepotr)Activating for 2h at room temperature under the condition of 1: 2-1: 10. G25 purifies the antibody and removes the excess Acceptor.
Obtaining a first binding moiety with a first label: Acceptor-Anti-His.
The antibody is replaced by Anti-mFc or Anti-hFc, and the Acceptor-Anti-mFc or Acceptor-Anti-hFc is prepared by the same method.
And (3) measuring the labeling efficiency (DOL), calculating the concentration of the Acceptor and the antibody through A280 and A304, wherein the concentration of the Acceptor/the concentration of the antibody is the labeling rate, and the labeling rate is 1-5 in the following embodiments.
EXAMPLE 3 establishment of RBD and ACE-2 binding System
The ACE-2 protein with murine Fc (ACE-2-mFc) was diluted to different concentrations with dilution buffer and the RBD-his of SARS-COV-2 was diluted to different concentrations. The Donor-Anti-mFc obtained in example 1 was diluted to 0.4. mu.g/ml with detection buffer; Acceptor-Anti-His obtained in example 2 was diluted to 2. mu.g/ml.
Adding 4 μ l of ACE-2-mFc with different concentrations into a 384-well white plate, adding 4 μ l of RBD-his with different concentrations into each well, and incubating at room temperature for 15 min.
The donor and acceptor were added in a 1:1 mixture and allowed to react at room temperature for 2 h. The ratio of the 665nm and 620nm emissions was calculated using a microplate reader equipped with an HTRF module with an excitation light of 320nm and two wavelengths (665nm and 620nm) of emission light (665/620). The results are shown in Table 1. The signal-to-noise ratio was the 665/620 value for each experimental group divided by the 665/620 value for the blank group.
TABLE 1665 nm and 620nm emission values
Final concentration of RBD-his (nM) ACE-2-mFc final concentration (nM) 665/620
0 0 0.0820
2.5 60 0.2177
2.5 120 0.1078
5 1 0.6823
5 2 0.8181
5 120 0.2645
10 3 1.086
20 3 1.1348
20 30 1.0274
30 5 1.1785
30 60 1.0095
60 0.5 0.5187
60 1 0.8447
60 3 0.9818
120 0.5 0.4311
120 5 0.9075
120 120 0.5374
The results show that 665/620 values are greater and the signal-to-noise ratio is greater than 10 when the concentration of RBD2-his is 3-120nM and the concentration of ACE-2-mFc is 1-60 nM. When the concentration is outside this range, 665/620 will be lower if it is too high or too low. The final ACE-2-mFc concentration of 3nM and the final RBD2-his concentration of 20nM were selected for subsequent inhibitor screening.
Example 4 screening of cell expression supernatants of antibodies capable of inhibiting RBD and ACE-2 binding
Diluting ACE-2-mFc to 15nM with dilution buffer; RBD2-his was diluted to 100 nM. Donor-Anti-mFc was diluted to 0.4. mu.g/ml with detection buffer, and Acceptor-Anti-His was diluted to 2. mu.g/ml. Adding 4 μ l diluted ACE-2-mFc into 384-well white plate, adding 4 μ l serum of convalescent coronary pneumonia patient, adding 4 μ l diluted RBD-his, and incubating at room temperature for 15 min. Finally 8. mu.l of 1:1 mixed donor and acceptor were added and incubated for 2h at room temperature. The final concentration of ACE-2-mFc is 3 nM; RBD2-his final concentration was 20 nM; the final concentration of the Donor-Anti-mFc is 0.08 mu g/ml; Acceptor-Anti-His was used at a final concentration of 0.4. mu.g/ml.
Setting a negative and positive control, wherein the positive control is as follows: mu.l ACE-2-mFC + 4. mu.l dilution buffer + 4. mu.l RBD-His of SARS-CoV2 + 8. mu.l donor and recipient mixed 1: 1. Negative controls were: mu.l dilution buffer + 8. mu.l 1:1 mixed donor and acceptor.
The value of 665/620 was calculated by measuring the excitation light at 320nm using a microplate reader equipped with an HTRF module and measuring the emitted light at two wavelengths (665nm and 620 nm).
Calculation of degree of inhibition: the degree of inhibition is (positive value-detection value)/positive value × 100%. FIG. 3 shows the 665/620 values of different antibodies in patient sera as a function of concentration. The results show that the system of the present application can be used to screen for inhibitors that inhibit the binding of RBD and ACE 2.
EXAMPLE 5 establishment of RBD and Anti-RBD binding System
The Donor-Anti-His was diluted to 2. mu.g/ml with detection buffer and the receptor Acceptor-Anti-hFc was diluted to 0.4. mu.g/ml. RBD-His was diluted with a dilution buffer to various concentrations, and the supernatant of positive antibody that can react with RBD was diluted with a dilution buffer to various concentrations. The positive antibody Anti-RBD is obtained by re-expressing and purifying the antibody which can block the combination of RBD and ACE2 and is obtained by screening in example 4.
Mu.l of gradient diluted Anti-RBD, 5. mu.l RBD-His, and 10. mu.l of 1:1 mixed donor and acceptor were added to a 384-well white plate and reacted at room temperature for 2 hours. The value of 665/620 was calculated by measuring the excitation light at 320nm using a microplate reader equipped with an HTRF module and measuring the emitted light at two wavelengths (665nm and 620 nm). The results are shown in table 2 and fig. 4.
The result shows that the detection linear range is wider when the final concentration of RBD-His is 60nM, the signal-to-noise ratio is not low, and the concentration is finally selected for screening subsequent positive antibodies.
TABLE 2665 nm and 620nm emission values
Figure BDA0002589600620000181
Example 6 screening of antibody cells expressing supernatant capable of binding to RBD of SARS-COV-2
RBD-His was diluted to 240nM with dilution buffer. The Donor Donor-Anti-His was diluted to 0.4. mu.g/ml with detection buffer and the receptor Acceptor-Anti-hFc was diluted to 2. mu.g/ml. Add 5. mu.l of diluted RBD-His to 384-well plates and add 5. mu.l of the test supernatant. Finally 10. mu.l of 1:1 mixed donor and acceptor were added and incubated for 2h at room temperature. Setting a negative control: mu.l dilution buffer + 10. mu.l 1:1 mixed donor and acceptor. The value of 665/620 was calculated by measuring the excitation light at 320nm using a microplate reader equipped with an HTRF module and measuring the emitted light at two wavelengths (665nm and 620 nm). And (3) judging standard: 665/620 greater than 2 are positive and less than 2 are negative.

Claims (10)

1. A method for high throughput screening of inhibitors that inhibit binding of RBD of SARS-COV-2 to ACE-2, comprising the steps of:
a) obtaining a first binding region with a first label and a second binding region with a second label, wherein the first label and the second label respectively comprise a label selected from the group consisting of: a donor label and an acceptor label, wherein the donor label and the acceptor label are different,
wherein upon interaction of the donor label and the acceptor label, the acceptor label can acquire energy transferred from the donor label to produce fluorescence;
b) obtaining a third protein with a third tag, and a fourth protein with a fourth tag, wherein the third protein and the fourth protein are optionally each selected from the group consisting of: the RBD and ACE-2 of SARS-COV-2, wherein the third tag can specifically bind to the first binding region and the fourth tag can specifically bind to the second binding region;
c) mixing the candidate inhibitor with the first binding region bearing the first tag, the second binding region bearing the second tag, the third protein bearing the third tag, and the fourth protein bearing the fourth tag;
d) detecting the ratio of absorbance at the wavelength of fluorescence of the acceptor label and the donor label after the mixing.
2. The method of claim 1, wherein the third protein is an RBD of SARS-COV-2 and the fourth protein is ACE-2; alternatively, the third protein is ACE-2 and the fourth protein is RBD of SARS-COV-2.
3. The method of any one of claims 1-2, wherein the concentration of the RBD of SARS-COV-2 is about 3-120 nM.
4. The method of any one of claims 1-3, wherein the concentration of the RBD of SARS-COV-2 is about 5-60 nM.
5. The method of any one of claims 1-4, wherein the concentration of ACE-2 is about 1-60 nM.
6. The method of any one of claims 1-5, wherein the concentration of ACE-2 is about 2-30 nM.
7. The method of any one of claims 1-6, wherein the first tag is the donor tag and the second tag is the acceptor tag; alternatively, the first tag is the acceptor tag and the second tag is the donor tag.
8. A method for high throughput screening of binding regions that specifically bind to RBD of SARS-COV-2, comprising the steps of:
a) obtaining a first binding region with a first label and a second binding region with a second label, wherein the first label and the second label respectively comprise a label selected from the group consisting of: a donor label and an acceptor label, wherein the donor label and the acceptor label are different,
wherein upon interaction of the donor label and the acceptor label, the acceptor label can acquire energy transferred from the donor label to produce fluorescence;
b) obtaining an RBD of SARS-COV-2 bearing a third tag, wherein said third tag can specifically bind to said first binding region and said second binding region can specifically bind to an RBD of SARS-COV-2;
c) mixing candidate said binding region with said first tag, said second binding region with said second tag, said RBD of SARS-COV-2 with said third tag;
d) detecting the ratio of absorbance at the fluorescence wavelength of the acceptor label and the donor label after mixing.
9. The method of claim 8, wherein the concentration of the SARS-COV-2 RBD is about 0.5-60 nM.
10. The method of any one of claims 8-9, wherein the concentration of the RBD of SARS-COV-2 is about 3-10 nM.
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