WO2023049890A1 - Tr-fret based assay for detection of neutralizing antibodies for viral infections - Google Patents

Tr-fret based assay for detection of neutralizing antibodies for viral infections Download PDF

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WO2023049890A1
WO2023049890A1 PCT/US2022/077006 US2022077006W WO2023049890A1 WO 2023049890 A1 WO2023049890 A1 WO 2023049890A1 US 2022077006 W US2022077006 W US 2022077006W WO 2023049890 A1 WO2023049890 A1 WO 2023049890A1
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reagent
cov
fret
assay
sars
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PCT/US2022/077006
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French (fr)
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Eric Fischer
Radoslaw Nowak
Daan OVERWIJN
Hong YUE
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Dana-Farber Cancer Institute, Inc.
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Publication of WO2023049890A1 publication Critical patent/WO2023049890A1/en

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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983Viruses
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14Hydrolases (3)
    • C12N9/48Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/485Exopeptidases (3.4.11-3.4.19)
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12YENZYMES
    • C12Y304/00Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/14Dipeptidyl-peptidases and tripeptidyl-peptidases (3.4.14)
    • C12Y304/14005Dipeptidyl-peptidase IV (3.4.14.5)
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12YENZYMES
    • C12Y304/00Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/17Metallocarboxypeptidases (3.4.17)
    • C12Y304/17023Angiotensin-converting enzyme 2 (3.4.17.23)
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011Details
    • C12N2770/20011Coronaviridae
    • C12N2770/20022New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08RNA viruses
    • G01N2333/165Coronaviridae, e.g. avian infectious bronchitis virus
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00Immunoassays for the detection of microorganisms
    • G01N2469/20Detection of antibodies in sample from host which are directed against antigens from microorganisms

Definitions

  • Coronaviruses constitute a group of phylogenetically diverse enveloped viruses that encode the largest plus strand RNA genomes and replicate efficiently in most mammals.
  • Human CoV (HCoVs-229E, OC43, NL63, and HKU1) infections typically result in mild to severe upper and lower respiratory tract disease.
  • Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) emerged in 2002-2003 causing acute respiratory distress syndrome (ARDS) with 10% mortality overall and up to 50% mortality in aged individuals.
  • Middle Eastern Respiratory Syndrome Coronavirus (MERS-CoV) emerged in the Middle East in April of 2012, manifesting as severe pneumonia, acute respiratory distress syndrome (ARDS) and acute renal failure.
  • the virus is still circulating and has been shown to have a mortality rate of about 49%.
  • Platforms for generating reagents and therapeutics are needed to detect and control the emergence of new strains, especially early in an outbreak prior to the development of type specific serologic reagents and therapeutics.
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus-2
  • CO VID-19 coronavirus disease 2019
  • Nucleic acid-based tests for the identification of infected individuals have been widely implemented (Tang, etal., J Clin Microbiol 26(58) :e00512-20 (2020)), but these tests can only detect the virus during a narrow window of acute disease.
  • Serological assays currently used to detect anti-SARS-Cov-1, CoV-2, and MERS-Cov-2 antibodies are either enzyme-linked immunosorbent assays (ELISA), quantitative suspension array technology (qSAT), flow cytometry based or commercial solutions on large diagnostics platforms (Corradini, et al., Hemasphere :e408-3 (2020); Amanat, et al., medRxivl doi: 10.1038/s41591- 020-0913-5 (2020); Premkumar, etal., Sci Immunol 5(48):eabc8413-9 (2020); Gruer, et al., BMJ 370:m2910 (2020); Malickova, et al., Scand J Gastroenterol 55:917-919 (2020); Ozcurumez, et al., J Allergy Clin Immunol 146:35-43 (2020); Theel, et al., J Clin Microbiol, 5S:e00
  • the present invention includes a rapid mix-and-read assay that may accurately detect seroconversion in patients suffering from SARS-CoV-1, SARS-CoV-2, or MERS-CoV, in very small volumes of fluid samples, and with high sensitivity and specificity.
  • the present assay addresses the important need for robust, simple implementation, and scalable serological tests.
  • FRET fluorescence resonance energy transfer
  • donor fluorophore an excited molecular fluorophore (referred to herein as the donor fluorophore) is brought into close proximity (e.g., within 10 nm) with another fluorophore (referred to herein as the acceptor fluorophore)
  • acceptor fluorophore another fluorophore
  • energy is transferred non-radiatively from the donor to the acceptor by means of intermolecular long-range dipole-dipole coupling.
  • the acceptorophore Upon excitation at a characteristic wavelength, the energy absorbed by the donor fluorophore is transferred to the acceptor, which in turn emits the energy, referred to herein as the FRET signal.
  • the assays are time-resolved (TR) as well, which provide even greater sensitivity and accuracy.
  • inventive methods are homogeneous, which allow for fast reaction times e.g., taking seconds to minutes), a single incubation of the sample and reagent(s) which may be pre-mixed, and without a solid phase or any washing steps.
  • one aspect of the present invention provides a homogeneous, time-resolved FRET (TR-FRET)-based method for detection of betacoronavirus ( ⁇ -CoVs) neutralizing antibodies in a patient fluid sample.
  • TR-FRET time-resolved FRET
  • ⁇ -CoVs betacoronavirus neutralizing antibodies
  • the first reagent a SARS-CoV-1 or SARS-CoV-2 Spike protein or a human (ACE2)- binding fragment thereof that contains the spike receptor binding domain (S-RBD).
  • the second reagent is a full length human ACE2 or an S-RBD binding fragment thereof.
  • the two reagents are labeled with a donor fluorophore and an acceptor fluorophore, respectively, or the acceptor fluorophore and the donor fluorophore, respectively.
  • the first reagent is full length SARS-CoV-1, SARS-CoV-2 Spike protein and the second reagent is a human ACE2 or an S-RBD binding fragment thereof.
  • the full-length Spike Protein comprises the amino acid sequence of SEQ ID NO: 1.
  • the first reagent comprises the amino acid sequence of SEQ ID NO: 2.
  • the first reagent comprises a fragment of a full-length SARS-CoV-1 or SARS- CoV-2 Spike protein that includes the receptor binding domain (S-RBD) that binds human ACE2.
  • the ACE2 -binding fragment of a full-length SARS-CoV-1 or SARS-CoV- 2 Spike protein that contains the receptor binding domain (S-RBD) also contains the amino acid residues of 318 to 510 of SEQ ID NO: 1 or amino acid residues 318 to 541 of SEQ ID NO: 2.
  • the first reagent contains at least one amino acid mutation relative to SEQ ID NO: 2. In some embodiments, the first reagent contains a D614G mutation relative to SEQ ID NO: 2.
  • the first reagent is a full-length MERS-CoV Spike protein and the second reagent is a human dipeptidyl-peptidase 4 (DPP4) or an S-RBD binding fragment thereof.
  • the full-length Spike Protein comprises the amino acid sequence of SEQ ID NO: 9.
  • the first reagent comprises a fragment of a full-length MERS- CoV Spike protein that includes the receptor binding domain (S-RBD) that binds human DPP4.
  • the DPP4-binding fragment contains the amino acid residues of 358 to 558 of SEQ ID NO: 9.
  • the first reagent is labeled with the donor fluorophore and the second reagent is labeled with the acceptor fluorophore. In some embodiments, the first reagent is labeled with the acceptor fluorophore and the second reagent is labeled with the donor fluorophore.
  • the body fluid sample is brought into contact with the labeled reagents in a homogeneous assay format, thus forming an assay mixture. If present in the patient sample, a betacoronavirus ( ⁇ -CoVs) neutralizing antibody will bind the first reagent, thereby preventing the first and second reagents from binding and bringing the donor and acceptor fluorophores into close proximity.
  • ⁇ -CoVs betacoronavirus
  • ⁇ -CoVs neutralizing antibody binding activity will reduce the detectable FRET signal relative to a control sample that does not contain ⁇ -CoVs neutralizing antibodies (such as a body fluid sample from an uninfected patient that does not contain ⁇ -CoVs neutralizing antibodies).
  • a reduced FRET signal indicates presence of the ⁇ -CoVs neutralizing antibodies in the body fluid sample.
  • a strong FRET signal relative to the control indicates an absence of ⁇ -CoVs neutralizing antibodies in the body fluid sample.
  • a patient fluid sample such as plasma, serum, or dried blood is contacted with a ⁇ -CoVs Spike protein or a fragment thereof that binds human ACE2 or DPP4 receptor binding domain (S-RBD) labeled with the donor or acceptor fluorophore as the first reagent and as a second reagent, a human ACE2, human DPP4, or an S-RBD binding fragment thereof with the donor or acceptor fluorophore.
  • S-RBD human ACE2 or DPP4 receptor binding domain
  • ⁇ -CoVs neutralizing antibodies Due to the multi-valent properties of antibodies in general, if ⁇ -CoVs neutralizing antibodies are present in the sample, they will bind to the first reagent, thereby preventing the donor and acceptor fluorophores from being in close proximity, resulting in generation of a reduced FRET signal relative to a control. Detection of the reduced FRET signal relative to the control indicates presence of ⁇ -CoVs neutralizing antibodies in the fluid sample.
  • a further aspect of the present invention is directed to an assay kit for homogeneous, TR- FRET-based method for detection of ⁇ -CoVs antibodies in a patient fluid sample, comprising: a) a first reagent comprising a ⁇ -CoVs Spike protein or a fragment thereof that binds human ACE2 or DPP4 receptor binding domain (S-RBD) and a second reagent comprising a human ACE2, DPP4, or S-RBD binding fragment thereof, wherein the one of the first and second reagents is labelled with a donor fluorophore and the other reagent is labelled with an acceptor fluorophore, respectively, or with the acceptor fluorophore and the donor fluorophore, respectively, and wherein the first and second reagents are disposed in the same or different containers; and b) printed instructions for using the first and second reagents in a homogeneous, TR-FRET-based method for detection of ⁇ -CoVs neutral
  • the kit contains a full-length SARS-CoV-1 Spike Protein that comprises the amino acid sequence of SEQ ID NO: 1 as a first reagent. In some embodiments, the kit contains a full-length SARS-CoV-2 Spike Protein that comprises the amino acid sequence of SEQ ID NO: 2 as a first reagent. In some embodiments, the kit contains a first reagent that comprises a fragment of a full-length SARS-CoV-1 or SARS-CoV-2 Spike protein comprising the S-RBD thereof that binds human ACE2.
  • the a fragment of a full-length SARS-CoV-1 or SARS-CoV-2 Spike protein comprising the S-RBD thereof that binds human ACE2 also contains the amino acid residues of 318 to 510 of SEQ ID NO: 1 or amino acid residues 318 to 541 of SEQ ID NO: 2.
  • the kit contains a first reagent that contains at least one amino acid mutation relative to SEQ ID NO: 2.
  • the kit contains a first reagent that contains a D614G mutation relative to SEQ ID NO: 2.
  • the kit contains a full-length MERS-CoV Spike Protein that comprises the amino acid sequence of SEQ ID NO: 9 as a first reagent.
  • the kit contains a first reagent that comprises a fragment of a full-length MERS-CoV Spike protein comprising the S-RBD thereof that binds human DPP4.
  • the fragment of a full-length MERS-CoV Spike protein comprising the S-RBD thereof that binds human DPP4 also contains the amino acid residues of 358 to 558 of SEQ ID NO: 9.
  • the present invention fulfills an urgent yet unmet need for a serological assay from numerous standpoints. For example, it provides a scalable alternative to current assay platforms.
  • antibody detection assays with signal amplification such as ELISA (Engvall, et al., Immunochemistry 5:871-74 (1971)), or digitized detection such as SIMOA (Cohen, et al., Annu Rev Anal Chem 70:345-63 (2017)), offer superior detection of low levels of analyte.
  • TR-FRET -based assay described herein offsets this by low background, allowing for sensitive, accurate detection of betacoronavirus ( ⁇ -CoVs) seroconversion.
  • the TR-FRET assay performs equivalent or superior in discriminating betacoronavirus ( ⁇ -CoVs) (Nilles, et al., medRxiv 2020.11.11.20229724).
  • ⁇ -CoVs betacoronavirus
  • the present methods are relatively simple and relatively inexpensive to implement. Reproducible results may be obtained without automated plate washers or similar liquid handling systems.
  • the relatively low cost is due in part to the miniaturization and lack of large volume wash steps. With widely available plate readers, a single operator can perform several hundred tests a day using manual multichannel pipettes without sacrificing accuracy of the results.
  • a further advantage demonstrated in a working example herein shows how this assay performs in circumstances (e.g., dried blood samples) where other types of tests fail.
  • FIG. 1 is a schematic of a betacoronavirus ( ⁇ -CoVs) virion particle.
  • FIGs. 2A-2G are a series of schematics, line graphs and scatterplots showing the assay setup and CR3022 validation.
  • FIG. 2A is a schematic showing the principle of the TR-FRET assay.
  • FIG. 2B is a schematic showing a flow-chart of the TR-FRET assay.
  • FIG. 2C is a scatterplot showing the titration of CR3022 IgG/IgM/IgAl into preformed mix of Tb-S protein and BODIPY labelled ⁇ lgG/ ⁇ lgM/ ⁇ lgA.
  • FIG. 2D is a scatterplot showing the titration of 1 : 150 dilution of negative serum into preformed mix of Tb-S protein and BODIPY labelled ⁇ lgG/ ⁇ lgM/ ⁇ lgA.
  • FIG. 2E is a scatterplot showing titration of positive and negative serum in final assay conditions of BODIPY- ⁇ lgG and Tb-S.
  • FIG. 2F is a line graph showing the TR-FRET ⁇ lgG-S assay.
  • FIG. 2G is a box and whisker plot showing the TR-FRET ⁇ lgG-S limit of detection assay.
  • FIGs. 3 A-3H are a series of scatterplots, a histogram, and a table showing the sensitivity and specificity of TR-FRET ⁇ lgG-S assay.
  • FIG. 3 A is a scatterplot showing the sensitivity and specificity of TR-FRET ⁇ lgG-S assay performed on a cohort of 68 SARS-CoV-2 PCR positive samples (CoV2+), and 100 pre-pandemic negative samples (healthy).
  • FIG. 3B is a scatterplot showing the sensitivity and specificity of ELISA IgG performed on the same cohort.
  • FIG. 3C is a scatterplot showing the correlation of TR-FRET IgG and ELISA IgG at the same concentration of serum.
  • FIG. 3 A is a scatterplot showing the sensitivity and specificity of TR-FRET ⁇ lgG-S assay performed on a cohort of 68 SARS-CoV-2 PCR positive samples (CoV2+), and 100 pre-pandemic negative samples
  • FIG. 3D is a histogram showing the age distribution in the MGB samples set from the Mass General Brigham Biobank containing 68 SARS-CoV-2 PCR positive samples (CoV2+), and 100 pre-pandemic healthy controls (healthy, CoV-).
  • FIG. 3E is a bar graph showing the gender distribution of MGB set.
  • FIG. 3F is scatter plot showing the comparison of IgG titer against S protein within different age groups.
  • FIG. 3G is a series of scatterplots showing the comparison between three independent runs of performed on different days by three different operators of a TR-FRET ⁇ lgG-S assay on a set of positive responders as well as negative control samples (68 total).
  • FIG. 3H is a table showing the calculated average repeatability across operators (CV%) and average intermediate precision (calculated across days and operators) corresponding to data in FIG. 3G.
  • FIGs. 4A-4I are a series of scatterplots and a series of histograms showing that TR-FRET is compatible with other antigens.
  • FIG. 4A is a scatter plot showing the sensitivity and specificity of TR-FRET ⁇ lgG - S protein assay performed on MassCPR set including 90 pre-pandemic negative samples and 100 SARS-CoV-2 positive.
  • FIG. 4B is a scatterplot showing the sensitivity and specificity of TR-FRET ⁇ lgG - N protein assay performed on MassCPR.
  • FIG. 4C is a scatter plot showing the correlation of ⁇ lgG - S titer in TR-FRET assay versus ELISA assay for MassCPR.
  • FIG. 4D is a scatter plot showing the correlation of IgG titer N protein in TR-FRET assay versus ELISA assay for MassCPR.
  • FIG. 4E is a histogram showing the hospital admission status of the 100 SARS-CoV-2 positive cohort. ER - Emergency Room, IP - inpatient, OP - outpatient.
  • FIG. 4F is a histogram showing that IgG titer as measured by TR-FRET assay stratified by number of days since last positive SARS-CoV-2 test.
  • FIG. 4G is a scatter plot showing that the correlation of TR-FRET ⁇ lgG-S and TR-FRET ⁇ lgG-N assays performed on MassCPR indicates diverse immune response to different antigens.
  • FIG. 4H is a scatter plot showing the cross reactivity between S proteins of SARS-CoV-2 and SARS-CoV measured by TR-FRET IgG titer on MassCPR.
  • FIG. 4H and FIG. 41 is a set of two scatter plots showing the cross reactivity between S proteins of SARS-CoV-2 vs. SARS-CoV-2 (FIG. 4H) and SARS-CoV-2 vs. MERS-CoV (FIG. 41) measured by TR-FRET IgG titer on MassCPR.
  • FIGs. 5A-5D are a series of line graph showing the TR-FRET Neutralization Assay setup and validation.
  • FIG. 5 A is a schematic showing the principle of the TR-FRET Neutralization assay.
  • FIG. 5B is a line graph showing the titration of B38, H4, SAD-S35, 40491-MM43, CR3022 and a negative control aFlag into preformed mix of btn-ACE2, Tb-SA and BODIPY-S.
  • FIG. 5C is a line graph showing the titration of serum and a negative control aFlag into preformed mix of btn- ACE2, Tb-SA and BODIPY-S.
  • FIG. 5D is a line graph showing the titration of positive and negative serum in final assay conditions for btn-ACE2, Tb-SA and BODIPY-S.
  • FIGs. 6A-6H are a series of scatter plots and a line graph showing that the TR-FRET neutralization assay correlates with cellular neutralization.
  • FIG. 6A is a scatterplot showing the sensitivity and specificity of TR-FRET ACE2-Spike neutralization assay performed on MassCPR on 90 pre-pandemic negative samples and 100 SARS-CoV-2 positive samples.
  • FIG. 6B is a scatter plot showing the sensitivity and specificity of reported cellular pseudovirus neutralization assay performed on MassCPR on 90 pre-pandemic negative samples and 100 SARS-CoV-2 positive samples.
  • FIG. 6C is a scatter plot showing the comparison between TR-FRET ACE2-S inhibition and the level of IgG S antibodies as detected by the TR-FRET.
  • FIG. 6D is a scatter plot showing the comparison of cellular neutralization NT50 against TR-FRET IgG S response.
  • FIG. 6E is a scatter plot comparison between ACE2-S inhibition TR-FRET and the level of IgG N antibodies as detected by the TR-FRET.
  • FIG. 6F is a scatter plot comparison of cellular neutralization NT50 against TR-FRET IgG N response.
  • FIG. 6G is a scatter plot comparison of cellular neutralization NT50 against TR-FRET ACE2-S inhibition.
  • FIG. 6H is a line graph showing a receiver operating characteristic curve (ROC curve) indicating the performance of detection of IgG levels for S or N using ELISA, or TR-FRET and TR-FRET ACE2-S inhibition assay.
  • ROC curve receiver operating characteristic curve
  • FIGs. 7A-7E are a series of line graphs and schematics showing the titration of BODIPY and CR3022.
  • FIG. 7A is a line graph showing the titration of BODIPY labeled CR3022 IgG antibody into Tb-labelled RBD mix.
  • FIG. 7B is a schematic showing alternative labeling strategies for the TR-FRET assay, where the donor fluorophore is located on the antigen (RBD) and the acceptor fluorophore on the detection antibody ( ⁇ lgG/ ⁇ lgM/ ⁇ lgA).
  • RBD antigen
  • FIG. 7A is a schematic showing alternative labeling strategies for the TR-FRET assay, where the donor fluorophore is located on the antigen (RBD) and the acceptor fluorophore on the detection antibody ( ⁇ lgG/ ⁇ lgM/ ⁇ lgA).
  • FIG. 7C is a schematic showing the alternative labeling strategies for the TR-FRET assay, where the donor fluorophore is located on the detection antibody ( ⁇ lgG/ ⁇ lgM/ ⁇ lgA) and the acceptor fluorophore on the antigen (RBD).
  • FIG. 7D is a line graph showing the titration of CR3022 IgG/IgM/IgAl into preformed mix of Tb-RBD protein and BODIPY labeled ⁇ lgG/ ⁇ lgM/ ⁇ lgA.
  • 7E is a line graph showing the titration of CR3022 IgG/IgM/IgAl into preformed mix of BODIPY-RBD protein and Tb-labeled ⁇ lgG/ ⁇ lgM/ ⁇ lgA.
  • FIGs. 8A-8I are a series line graphs and scatterplots showing the optimization of antigen amount, detection antibody amount, detecting antibody dilutions.
  • FIG. 8A is a line graph showing the titration of CR3022 IgG into BODIPY- ⁇ lgG Ab with varying concentrations of Tb-S protein.
  • FIG. 8B is a line graph showing the titration of CR3022 IgG into Tb-S with varying concentrations of BODIPY - ⁇ lgG.
  • FIG. 8C is a scatter plot showing the sensitivity and specificity of ELISA IgG assay.
  • FIG. 8A is a line graph showing the titration of CR3022 IgG into BODIPY- ⁇ lgG Ab with varying concentrations of Tb-S protein.
  • FIG. 8B is a line graph showing the titration of CR3022 IgG into Tb-S with varying concentrations of BODIPY - ⁇
  • FIG. 8D is a scatter plot showing the sensitivity and specificity of TR-FRET ⁇ lgG-S assay on the same cohort of samples at FIG. 8C at 1 : 100 dilution.
  • FIG. 8E is scatter plot showing the correlation of TR-FRET ⁇ lgG-S assay with serum dilution 1 : 100 and the ELISA ⁇ lgG-S at 1 : 100 serum dilution.
  • FIG. 8F is a scatter plot showing the sensitivity and specificity analysis for TR- FRET ⁇ lgG-S assay with the 1 :50 serum dilution.
  • FIG. 8G is a scatter plot showing the correlation of TR-FRET ⁇ lgG-S assay with serum dilution 1 :50 and the ELISA ⁇ lgG-S at 1 : 100 serum dilution.
  • FIG. 8H is a scatter plot showing the sensitivity and specificity analysis for TR-FRET ⁇ lgG-S assay with the 1 : 150 serum dilution.
  • FIG. 81 is a scatter plot showing the correlation of TR-FRET ⁇ lgG-S assay with serum dilution 1 : 150 and the ELISA ⁇ lgG-S at 1 : 100 serum dilution. [0028] FIGs.
  • FIG. 9A-9G are a series of line graphs and scatter plots showing the optimization of degree of labeling of Tb-S protein and TR-FRET ⁇ lgG-N protein assay.
  • FIG. 9A is a line graph showing titration of CR3022 IgG into BODIPY- ⁇ lgG Ab with Tb-S with varying degree of labeling.
  • FIG. 9B is a line graph showing the titration of positive or negative serum into BODIPY- ⁇ lgG Ab with Tb-S with varying degree of labeling.
  • FIG. 9C is a line graph showing titration of positive and negative serum into BODIPY- ⁇ lgG Ab with biotinylated N and Tb-SA.
  • FIG. 9A is a line graph showing titration of CR3022 IgG into BODIPY- ⁇ lgG Ab with Tb-S with varying degree of labeling.
  • FIG. 9B is a line graph showing the titration
  • FIG. 9D is a scatter plot showing the sensitivity and specificity of TR-FRET IgG - N assay performed on 96w_testset using N protein.
  • FIG. 9E is a scatter plot showing the correlation of TR-FRET ⁇ lgG- S to TR-FRET ⁇ lgG-N assays performed on the 96w_testset.
  • FIG. 9F is a scatter plot showing the sensitivity and specificity of ELISA IgG - S assay performed on MassCPR on 90 pre-pandemic negative samples and 100 SARS-CoV-2 positive samples.
  • FIG. 9G is a scatter plot showing the sensitivity and specificity of ELISA IgG - N assay performed on MassCPR.
  • FIGs. 10A-10F are a series of line graphs showing the optimization of the ACE2 and S displacement assay.
  • FIG. 10A is a line graph showing titration of BODIPY-S into Tb-SA and btn- ACE2 mix.
  • FIG. 10B is a line graph showing titration of two positive control antibodies (B38 and H4) or PCR CoV2+ and CoV2- serum to btn-ACE2 and BODIPY-S with Tb-Streptavidin.
  • FIG. 10C is line graph showing titration of CoV2+ serum followed by 1 hour or 4 hour pre-incubation.
  • FIG. 10D is line graph showing affinity of BODIPY-S and Tb-ACE2.
  • FIG. 10E is a line graph showing comparison between btn-ACE2 - Tb-SA, and covalently labelled Tb-ACE2 assay systems.
  • FIG. 1 OF is a line graph showing the affinity of recombinant purified H4 antibody and Tb-ACE2.
  • FIGs. 11A-11H are a series of scatterplots, a line graph, and a series of schematics showing the TR-FRET neutralization assay.
  • FIG. 11 A is a scatter plot showing the correlation of total Spike specific IgG levels and neutralization activity.
  • FIG. 1 IB is a scatter plot showing the correlation of total Spike specific IgG levels and neutralization activity scaled by the size of dots of IgM titer.
  • FIG. 11C is a scatter plot showing that the neutralization discriminates CoV2+ from healthy individuals in the 96w_testset.
  • FIG. 11 A is a scatter plot showing the correlation of total Spike specific IgG levels and neutralization activity.
  • FIG. 1 IB is a scatter plot showing the correlation of total Spike specific IgG levels and neutralization activity scaled by the size of dots of IgM titer.
  • FIG. 11C is a scatter plot showing that the neutralization discriminates CoV2+ from healthy individuals in the 96w_testset.
  • FIG. 1 ID is a line graph showing the comparison of the receiver operator curve (ROC) between ELISA Spike-IgG, TR-FRET Spike-IgG and TR-FRET ACE2-Spike.
  • FIG. 1 IE is a schematic showing the flow chart of automated ELISA processing.
  • FIG. 1 IF is a schematic showing the flow chart of automated TR-FRET processing.
  • FIG. 11G is a schematic showing the estimated buffer consumption in ELISA or TR-FRET assay required for testing of 200,000 samples.
  • FIG. 11H is a bar graph showing the estimated quantities of recombinant protein and detection reagents required for ELISA or TR-FRET assay for testing of 200,000 samples.
  • FIGs. 12A-12E are a series of scatterplots, a line graph, and a series of schematics showing the TR-FRET neutralization assay accepts multiple sample types.
  • FIG. 12A is a scatter plot showing correlation of ELISA IgG-S response between the matched set of whole dried blood self-collection samples (Neoteryx® kit) and serum samples from the same donors collected within 2 weeks.
  • FIG. 12B is a scatter plot showing correlation of TR-FRET IgG-S assay between the matched set of whole dried blood self-collection samples and serum samples from the same donors collected within 2 weeks.
  • FIG. 12A is a scatter plot showing correlation of ELISA IgG-S response between the matched set of whole dried blood self-collection samples (Neoteryx® kit) and serum samples from the same donors collected within 2 weeks.
  • FIG. 12B is a scatter plot showing correlation of TR-FRET IgG-S assay between the matched set of whole dried blood self-collection samples and
  • FIG. 12C is a scatter plot showing correlation between TR-FRET and ELISA responses in the IgG-S assay on the self-collection samples.
  • FIG. 12D is a scatter plot showing the response of ELISA or TR-FRET assay compared between a set of SARS-CoV-2 negative samples.
  • FIG. 12E is a scatter plot showing the response of ELISA or TR-FRET assay compared between a set of SARS-CoV-2 positive samples.
  • FIGs. 13A-13E are a series of scatterplots and a series of histograms showing that TR- FRET can be applied for total IgG amount testing and validation.
  • FIG. 13 A is a series of scatter plots showing that correlation between TR-FRET and ELISA responses in the IgG-S assay on IMPACT study samples.
  • FIG. 13B is a series of scatter plots showing that correlation between ELISA in the IgG-S assay and total IgG on IMPACT study samples.
  • FIG. 13C is a series of scatter plots showing that correlation between TR-FRET in the IgG-S assay and total IgG on IMPACT study samples.
  • FIG. 13 A is a series of scatter plots showing that correlation between TR-FRET and ELISA responses in the IgG-S assay on IMPACT study samples.
  • FIG. 13B is a series of scatter plots showing that correlation between ELISA in the IgG-S assay and total IgG on IMPACT
  • FIG. 13D is a series of scatter plots showing that correlation of ELISA and TR- FRET with total IgG with difference total IgG levels.
  • Total IgG ⁇ 2,500 mg/dL is labeled as blue.
  • Total IgG in 2,500 -3,000 mg/dL is labeled as red.
  • Total IgG > 2,500 mg/dL is labeled as yellow.
  • FIG. 13E is a series of histograms showing that Histogram of total IgG levels in the IMPACT study.
  • FIGs. 14A-14I are a series of scatterplots, a line graph, and a series of schematics showing the principle of TR-FRET total IgG assay.
  • FIG. 14A a series of schematics showing that nanobodies recognizing human IgG are labeled with AF488.
  • Immunoglobulin-binding protein G is labeled with CoraFluor-1 (Tb).
  • Tb CoraFluor-1
  • the light pulse at 337 nm excites CoraFluor-1 chelate protein G and emits light at 490 nm which in turn triggers energy transfer to AF488-labeled nanobodies found in proximity induced by analyte generating a TR-FRET signal detected at 520 nm.
  • FIG. 14B a series of a line graph showing that titration of CR3022 IgG into preformed mix of Tb-Protein G (25 nM final) and AF488 labelled Nanobodies (25 nM final).
  • Methods described herein are designed to detect the presence of neutralizing antibodies (Abs) in a body fluid sample that neutralize members of the Betacoronavirus (P-coronavirus, P- CoV) genus, specifically, SARS-CoV-1, SARS-CoV-2 and MERS-CoV.
  • P-coronavirus Abs detectable by the present assay methods bind the S-RBD, and, therefore, compete with the Spike- ACE2 (SARS-CoV-l/SARS-CoV-2) or Spike-DPP4 (MERS-CoV) interaction which is the primary mechanism of P-CoV neutralization.
  • these antibodies are referred to herein as P- CoV neutralizing antibodies.
  • FIG. 1 A schematic of a SARS-CoV/MERS-CoV virion particle is illustrated in FIG. 1.
  • the spike proteins are the visible protrusions on the surface of SARS-CoV-1, SARS-CoV- 2, and MERS-CoV, giving these viruses their characteristic, crown-like appearance.
  • These homotrimeric proteins are heavily glycosylated, with each comprising two distinct subunits: SI and S2.
  • the role of Spike is to act as a molecular key. This mechanism is achieved by recognizing and binding to specific cellular membrane protein receptors (locks).
  • SARS-CoV-1 and SARS- CoV-2 utilize the ACE2 cell-surface receptors
  • MERS-CoV utilizes the DPP4 cell-surface receptor present on the surface of mammalian (e.g., human) cells, via the SI receptor-binding domain (S-RBD).
  • S-RBD SI receptor-binding domain
  • Spike proteins are exposed to recognition by the immune system due to their projection into the external environment. This makes Spike the immunodominant coronavirus antigen, causing it to elicit a strong neutralizing antibody response (See, Ju, et al., Nature 584'.115-19 (2020)).
  • the first labelled reagent is a SARS-CoV-1 or SARS-CoV-2 Spike protein or a receptor binding domain thereof (S-RBD or Sl-RBD) thereof.
  • the inventive methods and reagents employ a full-length SARS- CoV-1 Spike protein.
  • An exemplary SARS-CoV-1 spike protein amino acid sequence provided at UniProtKB-P59594, is herein incorporated by reference and is set forth below (SEQ ID NO: 1
  • inventive methods and reagents employ a full-length SARS-Coactivated protein
  • SARS-CoV-2 spike amino acid sequence provided at UniProtKB-P0DTC2, is herein incorporated by reference and is reproduced below (SEQ ID NO:
  • SARS-CoV-2 Spike proteins that may be useful reagents in the practice of the present assay methods and reagents are known in the art (e.g., available from the NCBI virus database, accession numbers QMT50797, QMT51409, QMT51505, QMT51865, QMT52129, QMT52237, QMT522 49, QMT52393, QMT52561, QMT52741, QMT52765, QMT53017, QMT53041, QMT53053, Q MT53065, QMT53089, QMT53101, QMT53149, QMT53173, QMT53197, QMT53221, QMT5 3233, QMT53245, QMT55880, QMT57260, QMT57332, QMT57572, QMT57584, QMT57608, QMT57644, QMT57656, QMT57692, QMT94108, QMT94756, QMT94780, QMT95200, QM T95308, QMT
  • mutated versions of the Spike protein may be useful as reagents in the practice of the present assay methods. Mutation sites and mutation types observed in human SARS-CoV-2 spike proteins according to geographical locations are set forth in Guruprasad, Proteins vol. 89,5 (2021): 569-576 in Table 3. Guruprasad found Spike proteins having from 1 to 16 mutations.
  • Spike proteins useful as reagents may have one or more mutations including mutation(s) in the Sl-RBD.
  • a Spike protein having the mutation D614G (referring to SEQ ID NO: 2) may be used as a reagent.
  • a Spike protein having the mutation N501Y mutation (referring to SEQ ID NO: 2) may be used as a reagent.
  • a Spike protein having the mutations K417N, E484K, N501Y (referring to SEQ ID NO: 2) may be used as a reagent.
  • a Spike protein having the RBD mutations K417T, E484K, and N501 Y (referring to SEQ ID NO: 2) may be used as a reagent.
  • a Spike protein having a mutation in one or more of the 49, 77, 78, 118, 139, 144, 147, 193, 227, 239, 244, 261, 311, 344, 360, 426, 437, 472, 480, 487, 501, 577, 605, 607, 608, 609, 613, 665, 701, 743, 754, 804, 860-861, 894, 999, 1001, 1132, 1148, and/or 1163 amino acid positions (referring to SEQ ID NO: 1) may be used as a reagent.
  • a Spike protein having a mutation in one or more of the 18, 69-70, 80, 144, 215, 246, 417, 484, 601, 570, 614, 681, 701, 716, 982, and/or 1118 amino acid positions may be used as reagent.
  • the first reagent is a fragment of a full-length SARS-CoV-1 or SARS-CoV-2 Spike protein that contains an SI -receptor binding domain (Sl-RBD).
  • SARS-CoV-1 Sl-RBD is located at residues 318 to 510.
  • SARS-CoV-2 Sl-RBD is located at residues 318 to 541.
  • mutated versions of Sl-RBD fragments may also be used.
  • an Sl-RBD fragment has a mutation at any one of positions 344 (e.g., A344S), 477 (e.g., S477N), 483 (e.g., V483A) and 501 (e.g., N501Y).
  • an Sl-RBD fragment has any one of the following mutations: S477N, V483A, A344S, and N501Y/T.
  • an Sl-RBD fragment has any one of the following mutations: K417N/T, E484K, and N501Y.
  • an Sl-RBD fragment has a mutation at any one of positions Y453 (e.g., Y453F), G476 (e.g., G476S), F486 (e.g., F486L), and T500 (e.g., T500I).
  • Y453F e.g., Y453F
  • G476 e.g., G476S
  • F486 e.g., F486L
  • T500 e.g., T500I
  • the second reagent useful for detecting presence of SARS CoV-1 and -2 neutralizing antibodies is a human ACE2 or an S-RBD-binding fragment thereof.
  • ACE2 acts as a viral receptor and is expressed on the surface of several pulmonary and extra-pulmonary cell types, including cardiac, renal, intestinal, and endothelial cells.
  • the ACE2 receptor acts as the receptor-binding protein for the SAR-CoV-2 virus spike complex.
  • conformational rearrangements occur that cause SI subunit shedding, cleavage of S2 subunit by host proteases, and exposure of a fusion peptide adjacent to the S2' proteolysis site.
  • ACE2 The sequence and structure of ACE2 are known in the art (See, e.g., Guy, et al., Biochemistry 42: 13185-92 (2003); Yan, et al., Science 376: 1444-48 (2020)). It exists in six isoforms, all of which bind SARS-CoV-2 (Blume, et al., Nature Genetics 53:205-14 (2021)). ACE2 is commercially available from multiple sources (e.g., Sigma-Aldrich SAE0064; Sino Biological 10108-H08H; Aero Biosystems AC2-H82E6). ACE2 fragments that bind the RBD are known in the art.
  • a fragment of human ACE2 that binds S-RBD may be used.
  • a representative example of such a fragment is an N-terminal fragment of human ACE2 that contains amino acid residues 21-119 of SEQ ID NO: 3 reproduced below (SEQ ID NO: 4):
  • Isoforms of ACE2 may be useful as reagents in the present assay methods provided that they contain a SARS-CoV-2 spike high-affinity binding site and an entry point in airway epithelial cells for SARS-CoV-2 by triggering viral fusion with the cell plasma membrane, resulting in viral
  • RNA genome delivery into the host An exemplary amino acid sequence of the ACE2 isoform 1 precursor is provided at NCBI Accession No. NP_001358344, version NP_001358344.1, incorporated herein by reference, and reproduced below (SEQ ID NO: 5):
  • An exemplary amino acid sequence of the ACE2 isoform 2 precursor is provided atNCBI Accession No. NP_001373188, version NP_001373188.1, incorporated herein by reference, and reproduced below (SEQ ID NO: 6): 1 ms ss swllls Ivavtaaqst ieeqaktfld kfnheaedl f yqs slaswny ntniteenvq 61 nmnnagdkws aflkeqstla qmyplqeiqn Itvklqlqal qqngs svlse dks krlntil 121 ntmstiystg kvcnpdnpqe clllepglne imansldyne rlwaweswrs evgkqlrply 181 eeyvvlk
  • the first reagent is a full length ACE2 protein.
  • the first reagent is ACE2 isoform 1, ACE2 isoform 2, ACE2 isoform 3, or ACE2 isoform 4.
  • the first reagent is a truncated or a fragment of an ACE2 isoform.
  • the truncated ACE2 comprises amino acid residues 21-119 (SEQ ID NO: 4), when numbered according to SEQ ID NO: 3.
  • the truncated ACE2 comprises amino acids 18-615.
  • the second reagent useful for detecting presence of SARS CoV- 2 neutralizing antibodies is a human DPP4 or an S-RBD-binding fragment thereof. See, Yi, et al., iScience 23(6): 101160-8 (2020).
  • DPP4 is a 110 kDa glycoprotein, which is ubiquitously expressed on the surface of a variety of cells. This exopeptidase selectively cleaves N-terminal dipeptides from a variety of substrates, including cytokines, growth factors, neuropeptides, and the incretin hormones. DPP4 plays a major role in glucose metabolism. It is responsible for the degradation of incretins such as GLP-1.
  • DPP4 acts as the receptor-binding domain for the MERS virus spike complex.
  • DPP4 acts as the receptor-binding domain for the MERS virus spike complex.
  • DPP4 Upon engagement of DPP4 by a receptor binding domain in SI subunit of the Spike Protein, conformational rearrangements occur that cause SI subunit shedding, cleavage of S2 subunit by host proteases, and exposure of a fusion peptide adjacent to the S2' proteolysis site.
  • DPP4 The sequence and structure of DPP4 are known in the art (e.g., Aertgeerts, et al., Protein Sci 73:412-21 (2004); Nojima, et al., BMC Struc Biol 76:11-14 (2016); Deacon et al., Front Endocrinol. 70:80-14; Wang, et al., Cell Research 23:986-93 (2013)). It exists in 4 isoforms, all of which bind MERS-CoV. DPP4 is commercially available from Aero Biosystems (DP4-H5211). [0056] An exemplary amino acid sequence of the DPP4 isoform 1 is provided at NCBI Accession No. NP_001926, version NP_001926.2, incorporated herein by reference, and reproduced below (SEQ ID NO: 9):
  • DPP4 isoform 3 An exemplary amino acid sequence of the DPP4 isoform 3 is provided at NCBI Accession No. NP_001366534, version NP_001366534.1, incorporated herein by reference, and reproduced below (SEQ ID NO: 11):
  • the first reagent is a full length DPP4 protein. In some embodiments, the first reagent is DPP4 isoform 1, DPP4isoform 2, DPP4isoform 3, or DPP4 isoform 4. In some embodiments, the first reagent is a truncated or a fragment of an DPP4 isoform.
  • the reagents and assays described herein are for the detection of MERS.
  • the labelled first reagent is a full-length MERS-CoV Spike protein.
  • the labelled first reagent is a fragment of the MERS-CoV Spike protein.
  • the labelled first reagent is the Sl-RBD of the MERS-CoV Spike protein
  • SEQ ID NO: 13 An exemplary MERS-CoV spike protein amino acid sequence, provided at UniProtKB-R9uQ53, is herein incorporated by reference and produced below (SEQ ID NO: 13), where the Sl-RBD is located at residues 358 to 558 (bolded):
  • TR-FRET Labels Donor and Acceptor Fluorophores
  • TRF time- resolved fluorescence
  • FRET FRET
  • TRF reduces background fluorescence by delaying reading the fluorescent signal, for example, by about 10 nano seconds. Following this delay (i.e., the gating period), the longer-lasting fluorescence in the sample is measured.
  • TR-FRET interfering background fluorescence due to interfering substances in the sample, for example, is not co-detected. Only the fluorescence generated or suppressed by the energy transfer is measured. The resulting fluorescence of the TR-FRET system is determined by means of appropriate measuring devices.
  • time-resolved detection systems use, for example, pulsed laser diodes, light emitting diodes (LEDs), or pulsed dye lasers as the excitation light source.
  • the measurement occurs after an appropriate time delay, i.e., after the interfering background signals have decayed.
  • Devices and methods for determining time-resolved FRET signals are described in the art and in Example 1.
  • TR-FRET requires that the signal of interest must correspond to a compound with a long fluorescent lifetime. Criteria for selecting an appropriate A TR-FRET donor and acceptor pair include one or more of the following: (1) the emission spectrum of the FRET energy donor should overlap with the excitation spectrum of the FRET energy acceptor; (2) the emission spectra of the FRET partners i.e., the FRET energy donor and the FRET energy acceptor, commonly referred to as the donor and the acceptor) should show non-overlapping fluorescence; (3) the FRET quantum yield (i.e., the energy transferred from the FRET donor to the FRET acceptor) should be as high as possible (for example, FRET should have about a 1-100%, e.g., a 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, and 99% efficiency over a measured distance, of 1-20 nm, e.g., 5-10 nm); (4) the FRET signal (i.e., fluorescence) must be distinguish
  • Donor / acceptor fluorophore pairs for use in TR-FRET-based assays are known in the art. See, e.g., Joseph R. Lakowicz (Principles of fluorescence spectroscopy, 2nd edition, Kluwer academic/plenum publishers, NY (1999)).
  • Donor fluorophores advantageously emit long-lived fluorescence, typically in the order of >0.1 milliseconds (ms), preferably between 0.5 and 6 ms.
  • excitation of the donor fluorophore by a pulsed light source such as a flash lamp
  • FRET signal measurement known in the art as a counting window
  • This property enables the assay to be conducted in a time-resolved manner which reduces background (signal-to-noise ratios) and in turn, enhances sensitivity and accuracy.
  • donor fluorophores include lanthanide metals and complexes thereof, including chelates and cryptates.
  • lanthanides include terbium (Tb), europium (Eu), dysprosium (Dy), samarium (Sm), neodymium (Nd), ytterbium (Yb), erbium (Er), and their respective 3+ complexes.
  • complexes include cryptates and chelates, representative examples of which are described, for example in U.S. Patent Application Publication 2015/0198602 Al.
  • the donor fluorophore is terbium or Europium, or a cryptate or chelate thereof, examples of which are described the ‘602 Patent Publication.
  • These donor fluorophores are commercially available, e.g., from Cisbio. Eu3+, for example, has a fluorescent lifetime in the order of milliseconds.
  • acceptor fluorophores include allophycocyanins (tradename XL665); luminescent organic molecules, such as rhodamines, cyanines (e.g., Cy5), squaraines, coumarins, proflavins, acridines, fluoresceins, boron-dipyrromethene derivatives (commercially available under the tradename "BODIPY”), fluorophores known under the name "Atto”, fluorophores known under the name "DY”, compounds known under the name "Alexa”, and nitrob enzoxadi azol e .
  • allophycocyanins tradename XL665
  • luminescent organic molecules such as rhodamines, cyanines (e.g., Cy5), squaraines, coumarins, proflavins, acridines, fluoresceins, boron-dipyrromethene derivatives (commercially available under the tradename
  • the "Alexa” compounds are commercially available, e.g., from Invitrogen; the “Atto” compounds are commercially available from Atto-tec; the “DY” compounds are commercially available from Dyomics; and the “Cy” compounds are commercially available from Amersham Biosciences.
  • Table 1 lists representative examples of donor/ acceptor pairs for TR-FRET/HTRF 1 , while Table 2 lists excitation and emission (nm) of known FRET fluorophores.
  • FRET Fluorescence Activated fluorescent energy transfer
  • Alexa Alexa dyes
  • TR-FRET Cysbio 2 Ro is the distance at which FRET efficiency is 50%.
  • the excitation and emission of various donor and acceptor fluorophores that may be useful in practicing the present invention are described in U.S. Patent Application Publication 2018/0356411 Al. *Any acceptor fluorophore which matches the excitation spectra range of the donor fluorophore can be used (e.g., Alexa647). ** Any acceptor fluorophore which matches the excitation spectra of the donor fluorophore can be used e.g., FITC, BODIPY).
  • the donor fluorophore is Tb or Eu, or a cryptate or chelate thereof
  • the fluorophore acceptor is an organoboron fluorescent dye, e.g.. boron-dipyrromethene (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene)(commercially available under the tradename BODIPYTM), sodium 6-amino-9-(5-((aminomethyl)carbamoyl)-2- carboxyphenyl)-3-iminio-3H-xanthene-4,5-disulfonate (commercially available under the tradename Alexa488TM, and 2-[5-[3,3-dimethyl-5-sulfo-l-(3-sulfopropyl)indol-l-ium-2-yl]penta- 2,4-dienylidene]-3-methyl-3-[5-oxo
  • organoboron fluorescent dye e.g
  • the fluorophore donor/acceptor pair is Tb and BODIPY. In some embodiments, the fluorophore donor/acceptor pair is Eu and ALEXA647, respectively.
  • the first reagent is labelled with Tb and the second reagent is labelled with BODIPY. In some embodiments, the first reagent is labelled with BODIPY and the second reagent is labelled with Tb.
  • kits are also available for this purpose.
  • kits commercially available from Cisbio and Perkin Elmer allow for labeling peptides, proteins and oligonucleotides with Terbium cryptate, which includes N-hydroxysuccinimide-activated Terbium-Trisbipyridine (TBP).
  • TBP N-hydroxysuccinimide-activated Terbium-Trisbipyridine
  • the respective molar concentrations for any given pair of fluorophore donor and acceptor in an inventive TR-FRET assay are determined to enhance the FRET signal and facilitate its detection.
  • the molar concentrations may vary, depending upon any given pair of fluorophore donor and acceptors, and the proteinaceous reagents (e.g., SARS-CoV-l/SARS-CoV- 2 Spike Protein and ACE2 or MERS-CoV Spike Protein and DPP4).
  • Determining the relative molar concentrations of the labels for use with the SARS-CoV-l/SARS-CoV-2 Spike Protein and ACE2 or MERS-CoV Spike Protein and DPP4 so as to optimize the FRET signal and minimize background noise is within the level of skill in the art.
  • the working examples illustrate optimization of these molar concentrations using techniques known in the art within certain constraints (e.g., volume, reaction container).
  • a concentration of the reagent labelled with the donor fluorophore such as Terbium or Europium within the range of about 0.1 nM to 50 nM, or about 0.5 nM to 30 nM, or about 1.75 nM to 30 nM (relative to a TR-FRET assay volume of 15 pL) may be useful. Concentrations outside this range, both lower and higher, may also be useful.
  • the concentration of the reagent labelled with the donor fluorophore such as Tb or Eu is about 0.5 nM to about 4 nM, or about 7.5 nM to about 15 nM. In some embodiments, the concentration of the reagent labelled with the donor fluorophore such as Tb or Eu is about 1 nM, about 2 nM, about 4 nM, about 7.5 nM or about 15 nM.
  • a concentration of the other reagent labelled with the acceptor fluorophore such as BODIPY (e.g., when used with Tb as the fluorophore donor) of about 8 nM- 1 pM (relative to a TR-FRET assay volume of 15 pL) may be useful. Concentrations outside this range, both lower and higher, may also be useful.
  • the concentration of reagent labelled with the acceptor fluorophore, e.g., BODIPY is about 8 nM, and in other embodiments, the concentration is 250 nM (relative to a TR-FRET assay volume of 15 pL).
  • the optimal molar concentrations of the fluorophore donor and acceptor relative to one another may depend on Degree of Labeling (DoL).
  • DoL is the average number of labels (which in this case are the fluorophore donor and acceptor) coupled to a protein molecule (which in this case are the SARS-CoV-l/SARS-CoV-2 Spike Protein and ACE2 and MERS-CoV Spike Protein and DPP4).
  • the DoL may vary.
  • the DoL e.g., with respect to Tb, is generally in the range of about 1.0 to about 3.8. In some embodiments, the DoL is within the range of about 1.8 to about 3.8. In some embodiments, the DoL is about 3.8. DoL values outside this range, both lower and higher, may also be useful. However, a DoL of about 8 (and higher) for Tb might be disadvantageous in that the FRET signal is too strong to be practical.
  • the working examples illustrate optimization of a DoL for Tb using techniques known in the art. As demonstrated in the working examples, DoL may be determined in accordance with standard techniques.
  • the present methods entail testing body fluid samples obtained from individuals.
  • the samples include whole blood or a component thereof such as serum and plasma, saliva, and tears.
  • the body fluid sample is serum or plasma.
  • Practice of the invention is not limited to any subpopulations of individuals. Samples may be obtained from any individual (patient), and not just individuals who exhibited symptoms of the infection. Individuals who desire, believe to be in need of, who have been required to be tested for SARS-CoV-1, SARS-CoV-2, and MERS-CoV and/or are asymptomatic may be tested.
  • a fluid sample of about 0.2 pL to about 10 pL e.g., from about 0.5 pL to about 5 pL, may be useful.
  • the fluid sample is about 0.5 pL, 1.0 pL, 2.0 pL, 3 pL, 4 pL or 5 pL.
  • the fluid e.g., serum samples
  • the fluid may be diluted with physiologically acceptable buffer to a dilution factor that may generally range from about 1 :25 to about 1 :300.
  • the serum samples may be diluted from about 1 : 50 to 1 : 150 with physiologically acceptable buffer.
  • the fluid sample, or a portion thereof is mixed (i.e., contacted with) the first and the second reagents.
  • the mixture of the fluid sample, first, and second reagent is called the assay mixture.
  • the components of the assay mixture may be added together in any order.
  • the first and second reagents are premixed.
  • the term “assay mixture” as used herein refers to the combination of the labeled reagent (i.e., the first and second reagents) with the body fluid sample or dilution thereof.
  • the body fluid sample is a homogenous mixture, such that it has a uniform composition throughout.
  • the assay method includes a wash, typically after one or more reagents have been immobilize or otherwise fixed such that they, the desired reagents are not washed away.
  • the assay is washed 10 times.
  • the assay is washed 5 times.
  • the assay is washed 3 times.
  • the assay comprises no washes. The inventive assays and methods described herein do not require washes to achieve the same result as other methods that do require washes, enabling a faster and more efficient assays and methods.
  • FRET signal refers to any measurable signal representative of FRET between the fluorescent donor compound and the acceptor compound.
  • a FRET signal may therefore be a change in the intensity or lifetime of luminescence of the fluorescent donor compound or of the acceptor compound.
  • Any of a variety of light-emitting and light-detecting instruments can be used to initiate FRET e.g., excite the donor fluorophore or excite a reagent capable of exciting the donor fluorophore) and/or detect the emission produced.
  • the light emissions produced by donor and acceptor fluorophores, /. ⁇ ., the FRET signal can be detected or measured visually, photographically, actinometrically, spectrophotometrically, or by any other convenient means, such as with the use of a fluorometer. See, e.g., Saraheimo, supra.
  • the binding of the P-CoV s neutralizing antibodies to the first reagent e.g. , Spike protein can be determined qualitatively in that neutralizing antibodies present in the fluid sample will competitively bind the Spike protein (or the S-RBC-containing fragment) and reduce the TR- FRET signal, relative to a control (e.g., a same volume/dilution bodily fluid sample that does not contain neutralizing antibodies). That is, reduction of the FRET signal indicates ⁇ -CoVs neutralizing antibodies binding to the first reagent.
  • the reduction of a FRET signal is defined by a certain threshold, i.e., after deduction of any background signal.
  • the background signal is usually determined by performing the FRET assay with all assay reagents except for the labelled reagents. However, the background signal may also be determined by measuring the minimal FRET signal achieved by performing the FRET assay with control antibodies (See infra) or a positive control. Depending on the concentration of the neutralizing antibodies that may be present in the fluid sample (and indicative of the severity of ⁇ -CoVs infection), the reduction of the TR-FRET signal may be at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99% relative to the positive control.
  • the positive control may either be existing serum with a known neutralization propensity as determined previously by TR-FRET or by another assay (e.g., a cellular neutralization assay), or a recombinant purified antibody (i.e., B38 (Wu, et al., Science 365: 1274-78 (2020)), H4 (Wu, et al., Science 365:1274-78 (2020)), SAD-S35 (AS35; Aero Biosystems), and 40491-MM43 (Sino Biological, China).
  • a negative control may be any suitable buffer or solution that resembles the body fluid sample at its final dilution (e.g., same ionic conditions).
  • the present assay methods detect ⁇ -CoVs neutralizing antibodies of different serotypes, including, for example, IgG (which may be responsible for the majority of activity), IgA, and IgM.
  • the present assay methods may detect amounts of ⁇ -CoVs neutralizing antibodies as low as 1.22 ng/mL in absence of serum and about 39 ng/mL in presence of the serum. These limits of detection (LoD) are within the range of common ELIS As. See, e.g., McDade, el al., medRxiv 2020.04.28.20081844.
  • the disclosed reagents may be conveniently packaged in an assay kit to facilitate practice of the homogeneous, TR-FRET-based method for detection of ⁇ -CoVs neutralizing antibodies in a patient fluid sample.
  • the kit may include: a) a first reagent comprising a ⁇ -CoVs Spike protein or a human ACE2 or DPP4-binding fragment thereof that contains the receptor binding domain (S-RBD) and a second reagent comprising a human ACE2, a human DPP4, and/or S-RBD binding fragment thereof, wherein the first and second reagents are labelled with a donor fluorophore and an acceptor fluorophore, and wherein the first and second reagents are disposed in the same or different containers; and b) printed instructions for using the reagents in the homogeneous, TR-FRET-based method for detection of ⁇ -CoVs neutralizing antibodies in a patient fluid sample.
  • the full-length Spike protein of SARS-CoV-2 (S protein prefusion stabilized with furin site removed, expressed in TunaCHO cells) was purchased from LakePharma (Cat. 46328), RBD of SARS-CoV-2 was purchased from LakePharma (Cat. 46438), and full- length biotinylated N protein of SARS-CoV-2 (construct 1-419 with N terminal His-Avi tag) was purchased from Aero biosystem (Cat. NUN-C81Q6).
  • Full-length Spike protein of SARS-CoV (Cat. 40634-V08B) and MERS-CoV (Cat. 40069-V08B) were purchased from Sino Biological.
  • the clarified media was filtered with a 0.45 pm filter before binding to either protein G (GE, GE17-0405-01) for IgG, protein L (GE, GE17-5478-15) for IgM or peptide M (InvivoGen, gel-pdm-5) for IgAl columns pre-equilibrated with binding buffer (PBS, 10 mM Na2HPO4, 1.8 mM KH2PO4, 137 mM NaCl, 2.7 mM KC1 at pH 7.4).
  • binding buffer PBS, 10 mM Na2HPO4, 1.8 mM KH2PO4, 137 mM NaCl, 2.7 mM KC1 at pH 7.4
  • the beads were washed with 20-50 column volumes (CV) of binding buffer.
  • the protein was eluted from the beads with 6-15 CV of 0.1 M glycine pH 3.0 elution buffer and immediately quenched using a 10: 1 ratio of 1 M Tris-HCl pH 8.0.
  • the proteincontaining fractions were pooled and flash-frozen in liquid nitrogen at 0.1-1.5 mg/mL.
  • the antibodies were stored at -80 °C until further use. Concentrations were estimated using Bradford assay.
  • CR3022 IgG was labeled with BODIPY-NHS as described below.
  • B38 and H4 Fab fragments were constructed using the CR3022 Fc regions.
  • the Fab fragment sequence was taken from Wu, et al., Science 365: 1274-78 (2020).
  • B38 and H4 antibodies were expressed in Expi293 and purified as described above for CR3022.
  • a truncated ACE2 (amino acids 18-615) without a signaling peptide was cloned and expressed in Hi-5 insect cells by using baculoviruses with C-terminal StrepII and avi fusion tags.
  • the full-length N protein was cloned and expressed in insect cells with N-term Strep-Avi-Tev fusion tag.
  • cells were lysed by sonication (in 50 mM Tris pH 8.0, 200 mM NaCl, 0.1% Triton X-100, 1 mM PMSF and 1 tablet of complete protease inhibitor cocktail Roche Applied Science), lysate cleared by high-speed centrifugation, and the supernatant passed over StrepTactin-XT HC affinity resin (IB A).
  • Target protein was eluted using biotin and subjected to Poros50HQ ion exchange chromatography. Purification was completed using size exclusion chromatography with a 26/60 Superdex S200 column (GE Healthcare) in 50 mM HEPES pH 7.4, 200 mM NaCl and 2 mM TCEP.
  • the purified avi tagged ACE2 protein was biotinylated in presence of BirA enzyme, 10 mM MgCh, 2 mM biotin, 20 mM ATP. Biotinylation was confirmed by mass spectrometry.
  • the protein-containing fractions were pooled and flash-frozen in liquid nitrogen at 0.9 mg/mL for ACE2 and 1.6 mg/mL for N protein. The proteins were stored at -80 °C until further use. Concentrations were estimated using Bradford assay.
  • Serum samples Serum/plasma samples used in this study were obtained through the Ragon Institute and Dana-Farber Cancer Institute (DFCI). Institutional IRB approval was obtained, and all samples were collected after subjects provided signed informed consent. Six groups of consented subjects were included: 1) hospitalized patients (MGH and BWH) with a SARS-CoV- 2 confirmed RNA tests; 2) convalescents patients (MGH) with a confirmed prior SARS-CoV-2 RNA+ and two repeat RNA-negative tests after 2 weeks of isolation; 3) pre-pandemic healthy controls with samples collected prior to December 1, 2019 (MGB Biobank); 4) a group of low- risk community members (Ragon); 5) Self-collection samples from DFCI employees (DFCI IRB #20-260); 6) The IMPACT study (DFCI IRB #20-332) patients samples with or without vaccination. Total IgG levels of IMPACT study samples were available from medical records. Samples were heat inactivated at 60 °C for 1 hour.
  • Protein labeling with NCP311-Tb or BODIPY Protein labeling with NCP311-Tb or BODIPY.
  • Antibodies ahs-IgG (Bethyl, A80- 104 A), ahs-IgM (Bethyl, A80-100A), ahs-IgA (Bethyl, A80-102A), S protein (LakePharma, 46328)), protein G (Life, 21193), or RBD protein (LakePharma, 46438) in a volume of 2.5 mL each at a concentration of 1 mg/mL or SARS-CoV (Cat. 40634-V08B) and MERS-CoV (Cat.
  • the reaction mixture was briefly vortexed and allowed to stand at room temperature for 1 hour.
  • the labeling reaction was buffer exchanged into 50 mM sodium phosphate buffer pH 7.4, 137 mM NaCl, 0.05% TWEEN-20 detergent using PD-10 desalting columns following manufacturer protocol using 0.5 mL elution fractions. Protein containing fractions were pooled and flash-frozen in liquid nitrogen at 0.4-0.6 mg/mL concentration and stored at -80 °C.
  • the corrected A 280 value (A 280 ,corr) of protein conjugate was determined via Nanodrop (0.1 cm path length) by measuring A 280 and A340, using equation 1 : where c/is the correction factor for the Th complex contribution to A 280 and is equal to 0.157.
  • the concentration of protein conjugate, c ab (M) was determined using equation 2: where 8 is the antibody extinction coefficient at A 280 , equal to 210,000 M -1 cm -1 for IgG class anti
  • IgG/IgM/IgA Ab 240,000 M -1 cm -1 for S protein, 80,200 M -1 cm -1 for RBD and b is path length in cm (0.1 cm).
  • Tb complex, cib (M) covalently bound to the proteins was determined using equation 3 : where 8 is the complex extinction coefficient at A340, equal to 22,000 M -1 cm -1 and b is path length in cm (0.1 cm).
  • TR-FRET assay for RBD Titration of CR3022 IgG/IgM/IgAl antibody or dilution of tested human serum samples was added to assay mix with final concentrations of 15 nM Tb-labeled RBD, 250 nM BODIPY-labeled ⁇ lgG/ ⁇ lgM/ ⁇ lgA in a buffer containing PBS, 0.05% Tween-20 (Sigma Aldrich P9416). Serum samples were diluted in the buffer containing 50 mM Tris pH 8.0, 140 mM NaCl, 0.05% Tween-20 and 1% BSA (Cell Signaling Technology 9998S).
  • TR-FRET assays were performed in 384-well microplate (Coming, 4514) with 15 pL final assay volume. Before TR-FRET measurements were conducted, the reactions were incubated for 1 hour at room temperature (RT). After excitation of terbium fluorescence at 337 nm, emission at 490 nm (terbium) and 520 nm (BODIPY) were recorded with a 70 ps delay over 130 ps to reduce background fluorescence and the reaction was followed over >20 or >100 second cycles of each data point using a PHERAstar FS microplate reader (BMG Labtech). The TR-FRET signal of each data point was extracted by calculating the 520/490 nm ratio.
  • TR-FRET assay for Spike protein Titration of CR3022 IgG/IgM/IgAl antibody or dilution of tested human serum samples was added to assay mix with final concentrations of 7.5 nM Tb-labeled S protein of SARS-CoV-2, SARS-CoV or MERS-CoV, 250 nM BODIPY-labeled ⁇ lgG/ ⁇ lgM/ ⁇ lgA in a buffer containing PBS, 0.05% Tween-20 (Sigma Aldrich P9416).
  • Serum samples were diluted in the buffer containing 50 mM Tris pH 8.0, 140 mM NaCl, 0.05% Tween- 20 and 1% BSA (Cell Signaling Technology 9998S).
  • TR-FRET assays were performed in 384- well microplate (Corning, 4514) with 15 pL final assay volume. Before TR-FRET measurements were conducted, the reactions were incubated for 1 hour at RT.
  • terbium fluorescence After excitation of terbium fluorescence at 337 nm, emission at 490 nm (terbium) and 520 nm (BODIPY) were recorded with a 70 microseconds (ps) delay over 130 ps to reduce background fluorescence and the reaction was followed over >20 or >100 second cycles of each data point using a PHERAstar FS microplate reader (BMG Labtech). The TR-FRET signal of each data point was extracted by calculating the 520/490 nm ratio.
  • TR-FRET assay for N protein Diluted tested human serum samples were added to assay mix with final concentrations of 20 nM biotinylated N protein, 2 nM Streptavidin-Tb and 250 nM BODIPY-labeled ⁇ lgG in a buffer containing PBS, 0.05% Tween-20 (Sigma Aldrich P9416). Serum samples were diluted in the buffer containing 50 mM Tris pH 8.0, 140 mM NaCl, 0.05% Tween-20 and 1% BSA (Cell Signaling Technology 9998S). TR-FRET assays were performed in 384-well microplates (Coming, 4514) with 15 pL final assay volume.
  • Biotinylated N protein and Streptavidin-Tb were premixed and incubated for 10 minutes at RT. Before TR-FRET measurements were conducted, the reactions were incubated for 1 hour at RT. After excitation of terbium fluorescence at 337 nm, emission at 490 nm (terbium) and 520 nm (BODIPY) were recorded with a 70 ps delay over 130 ps to reduce background fluorescence and the reaction was followed over >20 or >100 second cycles of each data point using a PHERAstar FS microplate reader (BMG Labtech). The TR-FRET signal of each data point was extracted by calculating the 520/490 nm ratio.
  • TR-FRET ACE2-Spike neutralization assay Diluted human serum samples were added to assay mix with final concentrations of 8 nM Biotinylated ACE2 protein, 2 nM Streptavidin-Tb and 8 nM BODIPY-labeled Spike in a buffer containing PBS, 0.05% Tween-20 (Sigma Aldrich P9416). Serum samples were diluted in the buffer containing 50 mM Tris pH 8.0, 140 mM NaCl, 0.05% Tween-20 and 1% BSA (Cell Signaling Technology 9998S). TR-FRET assays were performed in 384-well microplates (Coming, 4514) with 15 pL final assay volume.
  • Biotinylated ACE2 protein and Streptavidin-Tb (mix 1) were premixed and incubated for 10 minutes at RT.
  • BODIPY-Spike protein and antibody, or serum samples (mix 2) were pre-incubated for 30 minutes at RT.
  • Mix 1 and 2 were added together and before TR-FRET measurements were conducted, the reactions were incubated for 1 to 4 hours at RT.
  • terbium fluorescence After excitation of terbium fluorescence at 337 nm, emission at 490 nm (terbium) and 520 nm (BODIPY) were recorded with a 70 ps delay over 130 ps to reduce background fluorescence and the reaction was followed over >20 or >100 second cycles of each data point using a PHERAstar FS microplate reader (BMG Labtech).
  • the TR-FRET signal of each data point was extracted by calculating the 520/490 nm ratio.
  • the AUC was calculated as the sum of the averages of duplicate individual 8-point dose response data points.
  • TR-FRET assay for total IgG Diluted tested human serum samples were added to assay mix with final concentrations of 25 nM CoraFluor-1 -Protein G and 25 nM AF488 labeled Nanobodies in a buffer containing PBS, 0.05% Tween-20 (Sigma Aldrich P9416). Serum samples were diluted in the buffer containing 50 mM Tris pH 8.0, 140 mM NaCl, 0.05% Tween-20 and 1% BSA (Cell Signaling Technology 9998S). TR-FRET assays were performed in 384-well microplates (Coming, 4514) with 15 pL final assay volume.
  • Biotinylated N protein and Streptavidin-Tb were premixed and incubated for 10 minutes at RT. Before TR-FRET measurements were conducted, the reactions were incubated for 1 hour at RT. After excitation of terbium fluorescence at 337 nm, emission at 490 nm (terbium) and 520 nm (BODIPY) were recorded with a 70 ps delay over 130 ps to reduce background fluorescence and the reaction was followed over >20 or >100 second cycles of each data point using a PHERAstar FS microplate reader (BMG Labtech). The TR-FRET signal of each data point was extracted by calculating the 520/490 nm ratio.
  • ELISA assay for RBD protein ELISA plates (384 well; Thermo Fisher #464718) were coated with 50 pL/well of 500 ng/mL SARS-CoV-2-RBD in coating buffer (1 capsule of carbonate-bicarbonate buffer (Sigma #C3041100CAP) per 100 mL Milli-Q H 2 O) for 30 minutes at room temperature. Plates were then washed 3 times with 100 pL/well of wash buffer (0.05% Tween-20, 400 mM NaCl, 50 mM Tris (pH 8.0) in Milli-Q H2O) using a Tecan automated plate washer.
  • coating buffer 1 capsule of carbonate-bicarbonate buffer (Sigma #C3041100CAP) per 100 mL Milli-Q H 2 O) for 30 minutes at room temperature. Plates were then washed 3 times with 100 pL/well of wash buffer (0.05% Tween-20, 400 mM NaCl, 50 mM Tris (pH 8.0) in Mill
  • Plates were blocked by adding lOOpL/well of blocking buffer (1% BSA, 140 mM NaCl, 50 mM Tris (pH 8.0) in Milli-Q H2O) for 30 minutes at room temperature. Plates were then washed as described above. Diluted samples (50 p in dilution buffer; 1% BSA, 0.05% Tween-20, 140 mM NaCl, 50 mM Tris (pH 8.0) in Milli-Q H2O) were added to the wells and incubated for 30 minutes at 37 °C. Plates were then washed 5 times as described above.
  • blocking buffer 1% BSA, 140 mM NaCl, 50 mM Tris (pH 8.0) in Milli-Q H2O
  • Diluted detection antibody solution 50 pL/well; HRP-anti human IgG, IgA or IgM; Bethyl Laboratory #A80-104P, A80-100P, A80-102P
  • TMB peroxidase substrate 40 pL/well; Thermo Fisher #34029
  • OD were read at 450 nm and 570 nm on a Pherastar FSX plate reader.
  • ELISA assay for S or N protein ELISA plates (384-well; ThermoFisher #464718) were coated with 50 pL/well of 500 ng/mL SARS-CoV-2 S protein or SARS-CoV-2 N protein in coating buffer (1 capsule of carbonate-bicarbonate buffer (Sigma #C3041100CAP) per 100 mL Milli-Q H2O) for 30 minutes at room temperature. Plates were then washed 3 times with 100 pL/well of wash buffer (0.05% Tween-20, 400 mM NaCl, 50 mM Tris pH 8.0 in Milli-Q H2O) using a Tecan automated plate washer.
  • Plates were blocked by adding 100 pL/well of blocking buffer (1% BSA, 140 mM NaCl, 50 mM Tris pH 8.0 in Milli-Q H2O) for 30 minutes at room temperature. Plates were then washed as described above. Diluted samples (50 pL in dilution buffer; 1% BSA, 0.05% Tween-20, 140 mM NaCl, 50 mM Tris (pH 8.0) in Milli-Q H2O) were added to the wells and incubated for 30 minutes at 37 °C. Plates were then washed 5 times as described above.
  • blocking buffer 1% BSA, 140 mM NaCl, 50 mM Tris pH 8.0 in Milli-Q H2O
  • Diluted detection antibody solution 50 pL/well; HRP-anti human IgGBethyl Laboratory #A80-104P was added to the wells and incubated for 30 minutes at room temperature. Plates were then washed 5 times as described above. 40 pL/well of TMB peroxidase substrate (Thermo Fisher #34029) was then added to the wells and incubated at room temperature for 3 minutes ( ⁇ lgG). The reaction was stopped by adding 40 pL/well of stop solution (1 M H2SO4 in Milli-Q H2O) to each well. OD was read at 450 nm and 570 nm on a Pherastar FSX plate reader. The final data used in the analysis was calculated by subtracting 570 nm background from 450 nm signal.
  • Lentiviral particles pseudotyped with SARS-CoV-2 spike protein were produced by transient transfection of 293T cells and titered by flow cytometry on 293T-ACE2 cells.
  • Neutralization assays were performed on a Fluent Automated Workstation (Tecan) using 384-well plates (Grenier, 781090). Following an initial 12-fold dilution, the liquid handler performed serial three-fold dilutions (ranging from 1 : 12 to 1 :8,748) of each patient serum and/or purified antibody in 20 ul followed by addition of 20 pL of pseudovirus containing 125 infectious units and incubation for 1 h at room temperature.
  • 293T-ACE2 cells (10,000) in 20 pL cell media containing 15 pg/mL polybrene were added to each well and incubated at 37 °C for 60-72 hours.
  • cells were lysed using a modified form of a previously described assay buffer containing a final concentration of 20 mM Tris-HCl, 100 pM EDTA, 1.07 mM MgCh, 2.67-26.7 mM MgSCU, 17 mM dithiothreitol (DTT), 250 pM ATP, and 125-250 pM D-luciferin, 1% Triton-X and shaken for five minutes prior to quantitation of luciferase expression within Ih of buffer addition using a Spectramax L luminometer (Molecular Devices).
  • Percent neutralization was determined by subtracting background luminescence measured in cell control wells (cells only) from sample wells and dividing by virus control wells (virus and cells only). Data was analyzed using Graphpad Prism. NT50 values were calculated by taking the inverse of the 50% inhibitory concentration value for all samples with a neutralization value of 80% or higher at the highest concentration of serum or antibody.
  • FIG. 2A Described herein is the development and validation of a homogenous serological assay platform for the detection of SARS-CoV-2 antibodies in human plasma/serum (FIG. 2A).
  • the direct detection of a ternary complex between antigen and serum antibodies using a TR-FRET readout allowed for a simple mix and read protocol that lends itself to scalable automation (FIG. 2B and FIGs. 11A-11E).
  • the assay conditions were optimized to minimize the signal-to-noise ratio as follows. It was established that the TR-FRET assay format can detect the binding of immunoglobulin variants IgG, IgM and IgAl to SARS-CoV-2 antigens (FIG. 2C).
  • SARS-CoV-2 spike protein S protein
  • the SARS-CoV-2 spike protein (S protein) is responsible for binding to the host receptor ACE2 to mediate virus entry upon infection (Li, et al., Nature 426:450-54 (2003)) and most neutralizing antibodies have been found to target the S protein (Chen, etal., Cellular & Molecular Immunology 17 :647 -649(2020)).
  • S protein and the receptor binding domain of the SARS-CoV-2 spike protein was used for assay development.
  • S protein and S-RBD expressed and purified from Chinese hamster ovarian cells (CHO), were labeled with terbium or BODIPY (see Example 1).
  • Detection antibodies ⁇ lgG, ⁇ lgM, ⁇ lgAl were commercially obtained and also labeled with either terbium or BODIPY.
  • SARS-1 IgG antibody CR3022 As positive control, recombinantly expressed SARS-1 IgG antibody CR3022 (ter Meulen, et al., PLoS Med 3: 1071-1079 (2006)) that has been shown to cross-react with the S-RBD of SARS-CoV-2 (Kd of 9.1 ⁇ 0.7 nM, FIG. 7A) and IgM and IgAl antibodies engineered to contain the CR3022 variable region was used (Tian, et al., Emerg Microbes Infect 9:382-85 (2020)).
  • titrations of CR3022 into a mix of labeled S-RBD and labeled detection antibody were performed while varying the position of donor and acceptor (either on S-RBD or detection antibody) (FIGs. 7B-7C). While it was found that all combinations lead to a functional readout (FIGs. 7D-7E), it was observed that terbium conjugation to the antigen results in optimal performance when used with serum/plasma samples. Therefore, the antigen was labeled with terbium and the detection antibody was labeled with BODIPY, resulting in quantitative binding curves for CR3022 (IgG/IgM/IgAl) (FIG.
  • the LoD for the TR-FRET assay was determined to be 1.22 ng/mL in absence of serum, and 39 ng/mL in presence of the serum, which is in the range of common ELISA LoD (McDade, et al., 2020 PLoS ONE 75(8):e0237833-8 (2020)).
  • Example 4 Homogenous TR-FRET assay can detect IgG in patient serum
  • the 96w_testset was profiled with the TR-FRET assay at an initial serum dilution of 1 :100 to match the exact ELISA concentration (FIG. 8D). It was found that using a 3 standard deviations (SD) cutoff away from the healthy control mean achieved a TR-FRET in comparable sensitivity and slightly improved specificity as compared to the ELISA results (FIGs. 8C-8D).
  • SD standard deviations
  • the 96w_testset was collected during the first weeks of the pandemic and may contain false positives and therefore was not used to formally establish assay performance but rather served for optimization. A strong correlation was observed between the TR-FRET and ELISA assays (FIG. 8E).
  • the 96w_testset was re-assayed using dilution factors of 1 :150 or 1 :50. It was observed that increasing the serum concentration improves signal strength without compromising background noise (FIGs. 8F-8I). Lowering the serum concentration to 1 :150 only marginally reduces performance (FIG. 8H). During the analysis of this initial 96w_testset, several CoV2+ samples had an unexpected low response. In addition to the above-mentioned limitations of the 96w_testset, this might be the result of epitope masking since the TR-FRET assay utilizes covalent labeling of the antigen with terbium.
  • DOL Degree of Labelling
  • the TR-FRET assay was used to detect seroconversion in a larger set of samples from the Mass General Brigham Biobank containing 68 SARS-CoV-2 PCR positive samples (CoV2+), and 100 pre-pandemic healthy controls (healthy, CoV2-) (hereafter referred to as MGB set; See Table 3suprd) that was again profiled using the established ELISA assay for reference, in addition to the additional profiling with different commercial and academic assays (Nilles, et al., medRxiv 2020.11.11.20229724).
  • the gender distribution was 35 females and 33 males in the CoV2+ cohort and 30 females and 70 males in the pre-pandemic health control group (FIG. 3E). It was observed that the IgG titers against S antigen were higher in the younger age group (e.g., 30s). The significant variability of the IgG levels indicated diverse response within the tested population (FIG. 3F).
  • S protein or S-RBD were the most widely used antigens in serological assays for SARS-CoV-2, but there are other SARS-CoV-2 proteins that are highly immunogenic (Dutta, etal., J Virol 94(73):e00647-20 (2020)), such as the abundant nucleocapsid protein (N protein), which binds to viral RNA inside the virion (Lu, et al., Lancet 395:565-74 (2020); Narayanan, et al., J Virol 77:2922-27 (2003)).
  • N TR-FRET N protein TR-FRET IgG detection assay
  • FIGs. 4C-4D The clinical admission status of the MassCPR sample cohort indicated 19 patients were admitted to the emergency room (ER), 76 as inpatients (IP) and 5 as outpatients (OP). A significant difference was not observed in the IgG S antibody titers between the groups (FIG. 4E). The number of days since the last positive SARS-CoV-2 test was recorded and within the 14-30 day period IgG levels varied without significant trends (FIG.
  • the spike protein has high sequence similarity between SARS-CoV-2 and SARS-CoV and to lesser extent MERS-CoV which can result in cross reactivity in the antibody response (Li, etal., Annual Review of Virology 3:237- 61 (2016); Cueno, et al., Frontiers in Medicine 7: 1089-10 (2020); Hatmal, et al., Cells 9:2638-37 (2020)).
  • SARS-CoV-2 S TR-FRET assay S based IgG detection assays for SARS-CoV and MERS-CoV were established and tested with the MassCPR set of samples. As expected, cross-reactivity between SARS-CoV-2 and SARS-CoV (FIG.
  • a limitation of antibody detection assays such as ELISA or the TR-FRET test developed here is that they do not discriminate antibodies based on the ability to neutralize the virus.
  • the detection of neutralizing antibodies (nAB’s) is commonly conducted using either live SARS-CoV- 2 virus assays (requiring BSL3 labs) or pseudotyped virus assays (requiring BSL2 labs), which both are limited in throughput and availability (Hoffmann, et al.. Cell 757(e278/271-80 (2020); Nie, et al., Emerg Microbes Infect 9:680-86 (2020)).
  • the ACE2-spike assay tolerated different labeling strategies such as btn-ACE2 with Tb-SA or direct labeling of ACE2 with terbium (Tb-ACE2) combined with various concentrations of BODIPY-S (FIGs. 10B-10F).
  • Example 11 TR-FRET neutralization assay discriminates CoV2+ patient samples [00118] Having established that the TR-FRET ACE2-Spike assay can accurately detect the ability of recombinant purified antibodies to compete with the interaction critical for viral infection, as well as in patient sera, it was then determined whether the assay is able to discriminate Cov2+ patient serum samples from healthy individuals. Using the 96w_testset and the MassCPR set described above, the samples were profiled in the TR-FRET ACE2-Spike assay for neutralizing activity (FIGs. 6A-6H and FIGs. 11 A-l 1G).
  • FIGs. 6E-6F A similar trend was also observed for the anti-N protein IgG antibody titers (FIGs. 6E-6F). While the TR-FRET ACE2-S assay is in principle sensitive to neutralization by any serotype (IgM or IgA), the majority of activity in these samples appears to result from IgG levels (FIG. 1 IB). Importantly, it was found that the neutralization assay by itself can successfully discriminate CoV2+ from healthy individuals in the 96w_testset cohort (FIG. 11C) as well as in the Mass CPR set (FIG. 6A), consistent with what has been observed using other neutralization assays (Garcia-Beltran, et al., Cell 754:476-488 (2021)).

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Abstract

The present invention relates to a homogeneous, time resolved, Forster resonance energy transfer (TR-FRET)-based method for detection of betacoronavirus ( -CoVs) neutralizing antibodies in a patient fluid sample. The method involves obtaining a body fluid sample from a patient; contacting the body fluid sample with a first reagent and a second reagent, thus forming an assay mixture; and detecting a FRET signal. The FRET signal determines the level of betacoronavirus present in the sample that was analyzed.

Description

TR-FRET ASSAY FOR DETECTION OF NEUTRALIZING ANTIBODIES
FOR VIRAL INFECTIONS
RELATED APPLICATIONS
[0001] This application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No: 63/248,814, filed September 27, 2021, which is incorporated herein by reference in its entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on September 15, 2022, is named 52095_708001WO_ST.xml and is 23 KB bytes in size.
BACKGROUND OF THE INVENTION
[0003] Coronaviruses (CoVs) constitute a group of phylogenetically diverse enveloped viruses that encode the largest plus strand RNA genomes and replicate efficiently in most mammals. Human CoV (HCoVs-229E, OC43, NL63, and HKU1) infections typically result in mild to severe upper and lower respiratory tract disease. Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) emerged in 2002-2003 causing acute respiratory distress syndrome (ARDS) with 10% mortality overall and up to 50% mortality in aged individuals. Middle Eastern Respiratory Syndrome Coronavirus (MERS-CoV) emerged in the Middle East in April of 2012, manifesting as severe pneumonia, acute respiratory distress syndrome (ARDS) and acute renal failure. The virus is still circulating and has been shown to have a mortality rate of about 49%. Platforms for generating reagents and therapeutics are needed to detect and control the emergence of new strains, especially early in an outbreak prior to the development of type specific serologic reagents and therapeutics.
[0004] SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2) is a human betacoronavirus that has caused a global pandemic of coronavirus disease 2019 (CO VID-19) (Munster, et al., N Engl J Med 352:692-94 (2020)). To better understand the spread of the disease, manage public health responses, and assess the efficacy of vaccine rollout, rapid and ideally, population wide screening, is essential. Nucleic acid-based tests for the identification of infected individuals have been widely implemented (Tang, etal., J Clin Microbiol 26(58) :e00512-20 (2020)), but these tests can only detect the virus during a narrow window of acute disease. Robust serological assays are necessary to identify individuals who have previously been infected and developed antibodies to SARS-CoV-2. Such serological assays for SARS-CoV-2 antibodies are also critical to identify individuals with asymptomatic disease (Corradini, et al.. Hemasphere 4:e408-3 (2020); Rago, et al., Orv Hetil 767:854-60 (2020)). Serological testing can establish the spread of the virus within a population, at the pre-vaccine stage, helps epidemiologists to accurately model the prevalence of CO VID-19, and with the increasing percentage of vaccinated population it becomes an important socio-economic tool to measure both disease and vaccine acquired immunity. Important to the large number of efforts during the development SARS-CoV-2 vaccines, robust serological testing assays provide means to monitor level and duration of response in clinical trials as well as trace serological response in the general population post-vaccination. Serological testing is now often part of cancer center protocols and becoming integral part of many clinical study designs in oncology and other indications.
[0005] Serological assays currently used to detect anti-SARS-Cov-1, CoV-2, and MERS-Cov-2 antibodies are either enzyme-linked immunosorbent assays (ELISA), quantitative suspension array technology (qSAT), flow cytometry based or commercial solutions on large diagnostics platforms (Corradini, et al., Hemasphere :e408-3 (2020); Amanat, et al., medRxivl doi: 10.1038/s41591- 020-0913-5 (2020); Premkumar, etal., Sci Immunol 5(48):eabc8413-9 (2020); Gruer, et al., BMJ 370:m2910 (2020); Malickova, et al., Scand J Gastroenterol 55:917-919 (2020); Ozcurumez, et al., J Allergy Clin Immunol 146:35-43 (2020); Theel, et al., J Clin Microbiol, 5S:e00797-20 (2020); Thornton, etal., Mult SclerRelat Disord 44:102341-3 (2020); Infantino, etal., JMed Virol 92: 1671-1675 (2020)). However, reports show that these strategies have several critical limitations (Norman, et al., medRxiv 2020.04.28.20083691; Woolley, et al., Bioanalysis 5:245-64 (2013); Lin, et al., Eur J Clin Microbiol Infect Dis 39:2271-2277 (2020); Liu, et al., J Med Virol doi: 10.1002/jmv.26241 (2020); Lou, et al., Eur Respir J 93: 144-148 (2020)). ELISA assays suffer from limited scalability, mainly due to multi-step protocols with extensive wash steps that lead to the need for specialized equipment and automation. Other available assays are not quantitative and require specialized analytical laboratory platforms that are not widely available, or in a typical hospital environment compete with other clinical tests for resources.
SUMMARY OF THE INVENTION
[0006] The present invention includes a rapid mix-and-read assay that may accurately detect seroconversion in patients suffering from SARS-CoV-1, SARS-CoV-2, or MERS-CoV, in very small volumes of fluid samples, and with high sensitivity and specificity. The present assay addresses the important need for robust, simple implementation, and scalable serological tests.
[0007] The present invention exploits a phenomenon known as Forster resonance energy transfer, also known as fluorescence resonance energy transfer (FRET). FRET is a distancedependent physical process. When an excited molecular fluorophore (referred to herein as the donor fluorophore) is brought into close proximity (e.g., within 10 nm) with another fluorophore (referred to herein as the acceptor fluorophore), energy is transferred non-radiatively from the donor to the acceptor by means of intermolecular long-range dipole-dipole coupling. Upon excitation at a characteristic wavelength, the energy absorbed by the donor fluorophore is transferred to the acceptor, which in turn emits the energy, referred to herein as the FRET signal. The nature of the signal, and means for detecting or measuring it, are known in the art. As explained in more detail herein, the assays are time-resolved (TR) as well, which provide even greater sensitivity and accuracy. Further, the inventive methods (assays) are homogeneous, which allow for fast reaction times e.g., taking seconds to minutes), a single incubation of the sample and reagent(s) which may be pre-mixed, and without a solid phase or any washing steps.
[0008] Accordingly, one aspect of the present invention provides a homogeneous, time-resolved FRET (TR-FRET)-based method for detection of betacoronavirus (β-CoVs) neutralizing antibodies in a patient fluid sample. To test a body fluid sample from a patient, the inventive methods employ two reagents.
[0009] The first reagent a SARS-CoV-1 or SARS-CoV-2 Spike protein or a human (ACE2)- binding fragment thereof that contains the spike receptor binding domain (S-RBD). The second reagent is a full length human ACE2 or an S-RBD binding fragment thereof. The two reagents are labeled with a donor fluorophore and an acceptor fluorophore, respectively, or the acceptor fluorophore and the donor fluorophore, respectively. [0010] In some embodiments, the first reagent is full length SARS-CoV-1, SARS-CoV-2 Spike protein and the second reagent is a human ACE2 or an S-RBD binding fragment thereof. In some embodiments, the full-length Spike Protein comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the first reagent comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the first reagent comprises a fragment of a full-length SARS-CoV-1 or SARS- CoV-2 Spike protein that includes the receptor binding domain (S-RBD) that binds human ACE2. In some embodiments, the ACE2 -binding fragment of a full-length SARS-CoV-1 or SARS-CoV- 2 Spike protein that contains the receptor binding domain (S-RBD) also contains the amino acid residues of 318 to 510 of SEQ ID NO: 1 or amino acid residues 318 to 541 of SEQ ID NO: 2.
[0011] In some embodiments, the first reagent contains at least one amino acid mutation relative to SEQ ID NO: 2. In some embodiments, the first reagent contains a D614G mutation relative to SEQ ID NO: 2.
[0012] In some embodiments, the first reagent is a full-length MERS-CoV Spike protein and the second reagent is a human dipeptidyl-peptidase 4 (DPP4) or an S-RBD binding fragment thereof. In some embodiments, the full-length Spike Protein comprises the amino acid sequence of SEQ ID NO: 9. In some embodiments, the first reagent comprises a fragment of a full-length MERS- CoV Spike protein that includes the receptor binding domain (S-RBD) that binds human DPP4. In some embodiments, the DPP4-binding fragment contains the amino acid residues of 358 to 558 of SEQ ID NO: 9.
[0013] In some embodiments, the first reagent is labeled with the donor fluorophore and the second reagent is labeled with the acceptor fluorophore. In some embodiments, the first reagent is labeled with the acceptor fluorophore and the second reagent is labeled with the donor fluorophore. [0014] The body fluid sample is brought into contact with the labeled reagents in a homogeneous assay format, thus forming an assay mixture. If present in the patient sample, a betacoronavirus (β-CoVs) neutralizing antibody will bind the first reagent, thereby preventing the first and second reagents from binding and bringing the donor and acceptor fluorophores into close proximity. Therefore, β-CoVs neutralizing antibody binding activity will reduce the detectable FRET signal relative to a control sample that does not contain β-CoVs neutralizing antibodies (such as a body fluid sample from an uninfected patient that does not contain β-CoVs neutralizing antibodies). A reduced FRET signal indicates presence of the β-CoVs neutralizing antibodies in the body fluid sample. Conversely, a strong FRET signal relative to the control indicates an absence of β-CoVs neutralizing antibodies in the body fluid sample.
[0015] Accordingly, in some embodiments, a patient fluid sample such as plasma, serum, or dried blood is contacted with a β-CoVs Spike protein or a fragment thereof that binds human ACE2 or DPP4 receptor binding domain (S-RBD) labeled with the donor or acceptor fluorophore as the first reagent and as a second reagent, a human ACE2, human DPP4, or an S-RBD binding fragment thereof with the donor or acceptor fluorophore. The body fluid sample is brought into contact with the labeled reagents in a homogeneous assay format. Due to the multi-valent properties of antibodies in general, if β-CoVs neutralizing antibodies are present in the sample, they will bind to the first reagent, thereby preventing the donor and acceptor fluorophores from being in close proximity, resulting in generation of a reduced FRET signal relative to a control. Detection of the reduced FRET signal relative to the control indicates presence of β-CoVs neutralizing antibodies in the fluid sample.
[0016] A further aspect of the present invention is directed to an assay kit for homogeneous, TR- FRET-based method for detection of β-CoVs antibodies in a patient fluid sample, comprising: a) a first reagent comprising a β-CoVs Spike protein or a fragment thereof that binds human ACE2 or DPP4 receptor binding domain (S-RBD) and a second reagent comprising a human ACE2, DPP4, or S-RBD binding fragment thereof, wherein the one of the first and second reagents is labelled with a donor fluorophore and the other reagent is labelled with an acceptor fluorophore, respectively, or with the acceptor fluorophore and the donor fluorophore, respectively, and wherein the first and second reagents are disposed in the same or different containers; and b) printed instructions for using the first and second reagents in a homogeneous, TR-FRET-based method for detection of β-CoVs neutralizing antibodies in a patient fluid sample.
[0017] In some embodiments, the kit contains a full-length SARS-CoV-1 Spike Protein that comprises the amino acid sequence of SEQ ID NO: 1 as a first reagent. In some embodiments, the kit contains a full-length SARS-CoV-2 Spike Protein that comprises the amino acid sequence of SEQ ID NO: 2 as a first reagent. In some embodiments, the kit contains a first reagent that comprises a fragment of a full-length SARS-CoV-1 or SARS-CoV-2 Spike protein comprising the S-RBD thereof that binds human ACE2. In some embodiments, the a fragment of a full-length SARS-CoV-1 or SARS-CoV-2 Spike protein comprising the S-RBD thereof that binds human ACE2 also contains the amino acid residues of 318 to 510 of SEQ ID NO: 1 or amino acid residues 318 to 541 of SEQ ID NO: 2. In some embodiments, the kit contains a first reagent that contains at least one amino acid mutation relative to SEQ ID NO: 2. In some embodiments, the kit contains a first reagent that contains a D614G mutation relative to SEQ ID NO: 2. In some embodiments, the kit contains a full-length MERS-CoV Spike Protein that comprises the amino acid sequence of SEQ ID NO: 9 as a first reagent. In some embodiments, the kit contains a first reagent that comprises a fragment of a full-length MERS-CoV Spike protein comprising the S-RBD thereof that binds human DPP4. In some embodiments, the fragment of a full-length MERS-CoV Spike protein comprising the S-RBD thereof that binds human DPP4 also contains the amino acid residues of 358 to 558 of SEQ ID NO: 9.
[0018] The present invention fulfills an urgent yet unmet need for a serological assay from numerous standpoints. For example, it provides a scalable alternative to current assay platforms. In theory, antibody detection assays with signal amplification such as ELISA (Engvall, et al., Immunochemistry 5:871-74 (1971)), or digitized detection such as SIMOA (Cohen, et al., Annu Rev Anal Chem 70:345-63 (2017)), offer superior detection of low levels of analyte. The present, TR-FRET -based assay described herein offsets this by low background, allowing for sensitive, accurate detection of betacoronavirus (β-CoVs) seroconversion. The lack of signal amplification biases the assay towards superior specificity due to very stable background signal, and results in robust reproducibility and repeatability (CV < 5%) along with an extended dynamic range. When compared to common commercial or ELISA assays on the same samples, the TR-FRET assay performs equivalent or superior in discriminating betacoronavirus (β-CoVs) (Nilles, et al., medRxiv 2020.11.11.20229724). The scalability is also enhanced due to a lack of need for washing steps or sequential manipulations, leading to simple automation using widely available robotic platforms for hundred thousand tests a day.
[0019] Further, the present methods are relatively simple and relatively inexpensive to implement. Reproducible results may be obtained without automated plate washers or similar liquid handling systems. The relatively low cost is due in part to the miniaturization and lack of large volume wash steps. With widely available plate readers, a single operator can perform several hundred tests a day using manual multichannel pipettes without sacrificing accuracy of the results. A further advantage demonstrated in a working example herein shows how this assay performs in circumstances (e.g., dried blood samples) where other types of tests fail.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1 is a schematic of a betacoronavirus (β-CoVs) virion particle.
[0021] FIGs. 2A-2G are a series of schematics, line graphs and scatterplots showing the assay setup and CR3022 validation. FIG. 2A is a schematic showing the principle of the TR-FRET assay. FIG. 2B is a schematic showing a flow-chart of the TR-FRET assay. FIG. 2C is a scatterplot showing the titration of CR3022 IgG/IgM/IgAl into preformed mix of Tb-S protein and BODIPY labelled αlgG/αlgM/αlgA. FIG. 2D is a scatterplot showing the titration of 1 : 150 dilution of negative serum into preformed mix of Tb-S protein and BODIPY labelled αlgG/αlgM/αlgA. FIG. 2E is a scatterplot showing titration of positive and negative serum in final assay conditions of BODIPY-αlgG and Tb-S. FIG. 2F is a line graph showing the TR-FRET αlgG-S assay. FIG. 2G is a box and whisker plot showing the TR-FRET αlgG-S limit of detection assay.
[0022] FIGs. 3 A-3H are a series of scatterplots, a histogram, and a table showing the sensitivity and specificity of TR-FRET αlgG-S assay. FIG. 3 A is a scatterplot showing the sensitivity and specificity of TR-FRET αlgG-S assay performed on a cohort of 68 SARS-CoV-2 PCR positive samples (CoV2+), and 100 pre-pandemic negative samples (healthy). FIG. 3B is a scatterplot showing the sensitivity and specificity of ELISA IgG performed on the same cohort. FIG. 3C is a scatterplot showing the correlation of TR-FRET IgG and ELISA IgG at the same concentration of serum. FIG. 3D is a histogram showing the age distribution in the MGB samples set from the Mass General Brigham Biobank containing 68 SARS-CoV-2 PCR positive samples (CoV2+), and 100 pre-pandemic healthy controls (healthy, CoV-). FIG. 3E is a bar graph showing the gender distribution of MGB set. FIG. 3F is scatter plot showing the comparison of IgG titer against S protein within different age groups. FIG. 3G is a series of scatterplots showing the comparison between three independent runs of performed on different days by three different operators of a TR-FRET αlgG-S assay on a set of positive responders as well as negative control samples (68 total). FIG. 3H is a table showing the calculated average repeatability across operators (CV%) and average intermediate precision (calculated across days and operators) corresponding to data in FIG. 3G. [0023] FIGs. 4A-4I are a series of scatterplots and a series of histograms showing that TR-FRET is compatible with other antigens. FIG. 4A is a scatter plot showing the sensitivity and specificity of TR-FRET αlgG - S protein assay performed on MassCPR set including 90 pre-pandemic negative samples and 100 SARS-CoV-2 positive. FIG. 4B is a scatterplot showing the sensitivity and specificity of TR-FRET αlgG - N protein assay performed on MassCPR. FIG. 4C is a scatter plot showing the correlation of αlgG - S titer in TR-FRET assay versus ELISA assay for MassCPR. FIG. 4D is a scatter plot showing the correlation of IgG titer N protein in TR-FRET assay versus ELISA assay for MassCPR. FIG. 4E is a histogram showing the hospital admission status of the 100 SARS-CoV-2 positive cohort. ER - Emergency Room, IP - inpatient, OP - outpatient. FIG. 4F is a histogram showing that IgG titer as measured by TR-FRET assay stratified by number of days since last positive SARS-CoV-2 test. FIG. 4G is a scatter plot showing that the correlation of TR-FRET αlgG-S and TR-FRET αlgG-N assays performed on MassCPR indicates diverse immune response to different antigens. FIG. 4H is a scatter plot showing the cross reactivity between S proteins of SARS-CoV-2 and SARS-CoV measured by TR-FRET IgG titer on MassCPR. FIG. 4H and FIG. 41 is a set of two scatter plots showing the cross reactivity between S proteins of SARS-CoV-2 vs. SARS-CoV-2 (FIG. 4H) and SARS-CoV-2 vs. MERS-CoV (FIG. 41) measured by TR-FRET IgG titer on MassCPR.
[0024] FIGs. 5A-5D are a series of line graph showing the TR-FRET Neutralization Assay setup and validation. FIG. 5 A is a schematic showing the principle of the TR-FRET Neutralization assay. FIG. 5B is a line graph showing the titration of B38, H4, SAD-S35, 40491-MM43, CR3022 and a negative control aFlag into preformed mix of btn-ACE2, Tb-SA and BODIPY-S. FIG. 5C is a line graph showing the titration of serum and a negative control aFlag into preformed mix of btn- ACE2, Tb-SA and BODIPY-S. FIG. 5D is a line graph showing the titration of positive and negative serum in final assay conditions for btn-ACE2, Tb-SA and BODIPY-S.
[0025] FIGs. 6A-6H are a series of scatter plots and a line graph showing that the TR-FRET neutralization assay correlates with cellular neutralization. FIG. 6A is a scatterplot showing the sensitivity and specificity of TR-FRET ACE2-Spike neutralization assay performed on MassCPR on 90 pre-pandemic negative samples and 100 SARS-CoV-2 positive samples. FIG. 6B is a scatter plot showing the sensitivity and specificity of reported cellular pseudovirus neutralization assay performed on MassCPR on 90 pre-pandemic negative samples and 100 SARS-CoV-2 positive samples. FIG. 6C is a scatter plot showing the comparison between TR-FRET ACE2-S inhibition and the level of IgG S antibodies as detected by the TR-FRET. FIG. 6D is a scatter plot showing the comparison of cellular neutralization NT50 against TR-FRET IgG S response. FIG. 6E is a scatter plot comparison between ACE2-S inhibition TR-FRET and the level of IgG N antibodies as detected by the TR-FRET. FIG. 6F is a scatter plot comparison of cellular neutralization NT50 against TR-FRET IgG N response. FIG. 6G is a scatter plot comparison of cellular neutralization NT50 against TR-FRET ACE2-S inhibition. FIG. 6H is a line graph showing a receiver operating characteristic curve (ROC curve) indicating the performance of detection of IgG levels for S or N using ELISA, or TR-FRET and TR-FRET ACE2-S inhibition assay.
[0026] FIGs. 7A-7E are a series of line graphs and schematics showing the titration of BODIPY and CR3022. FIG. 7A is a line graph showing the titration of BODIPY labeled CR3022 IgG antibody into Tb-labelled RBD mix. FIG. 7B is a schematic showing alternative labeling strategies for the TR-FRET assay, where the donor fluorophore is located on the antigen (RBD) and the acceptor fluorophore on the detection antibody (αlgG/αlgM/αlgA). FIG. 7C is a schematic showing the alternative labeling strategies for the TR-FRET assay, where the donor fluorophore is located on the detection antibody (αlgG/αlgM/αlgA) and the acceptor fluorophore on the antigen (RBD). FIG. 7D is a line graph showing the titration of CR3022 IgG/IgM/IgAl into preformed mix of Tb-RBD protein and BODIPY labeled αlgG/αlgM/αlgA. FIG. 7E is a line graph showing the titration of CR3022 IgG/IgM/IgAl into preformed mix of BODIPY-RBD protein and Tb-labeled αlgG/ αlgM/αlgA.
[0027] FIGs. 8A-8I are a series line graphs and scatterplots showing the optimization of antigen amount, detection antibody amount, detecting antibody dilutions. FIG. 8A is a line graph showing the titration of CR3022 IgG into BODIPY-αlgG Ab with varying concentrations of Tb-S protein. FIG. 8B is a line graph showing the titration of CR3022 IgG into Tb-S with varying concentrations of BODIPY -αlgG. FIG. 8C is a scatter plot showing the sensitivity and specificity of ELISA IgG assay. FIG. 8D is a scatter plot showing the sensitivity and specificity of TR-FRET αlgG-S assay on the same cohort of samples at FIG. 8C at 1 : 100 dilution. FIG. 8E is scatter plot showing the correlation of TR-FRET αlgG-S assay with serum dilution 1 : 100 and the ELISA αlgG-S at 1 : 100 serum dilution. FIG. 8F is a scatter plot showing the sensitivity and specificity analysis for TR- FRET αlgG-S assay with the 1 :50 serum dilution. FIG. 8G is a scatter plot showing the correlation of TR-FRET αlgG-S assay with serum dilution 1 :50 and the ELISA αlgG-S at 1 : 100 serum dilution. FIG. 8H is a scatter plot showing the sensitivity and specificity analysis for TR-FRET αlgG-S assay with the 1 : 150 serum dilution. FIG. 81 is a scatter plot showing the correlation of TR-FRET αlgG-S assay with serum dilution 1 : 150 and the ELISA αlgG-S at 1 : 100 serum dilution. [0028] FIGs. 9A-9G are a series of line graphs and scatter plots showing the optimization of degree of labeling of Tb-S protein and TR-FRET αlgG-N protein assay. FIG. 9A is a line graph showing titration of CR3022 IgG into BODIPY-αlgG Ab with Tb-S with varying degree of labeling. FIG. 9B is a line graph showing the titration of positive or negative serum into BODIPY- αlgG Ab with Tb-S with varying degree of labeling. FIG. 9C is a line graph showing titration of positive and negative serum into BODIPY-αlgG Ab with biotinylated N and Tb-SA. FIG. 9D is a scatter plot showing the sensitivity and specificity of TR-FRET IgG - N assay performed on 96w_testset using N protein. FIG. 9E is a scatter plot showing the correlation of TR-FRET αlgG- S to TR-FRET αlgG-N assays performed on the 96w_testset. FIG. 9F is a scatter plot showing the sensitivity and specificity of ELISA IgG - S assay performed on MassCPR on 90 pre-pandemic negative samples and 100 SARS-CoV-2 positive samples. FIG. 9G is a scatter plot showing the sensitivity and specificity of ELISA IgG - N assay performed on MassCPR.
[0029] FIGs. 10A-10F are a series of line graphs showing the optimization of the ACE2 and S displacement assay. FIG. 10A is a line graph showing titration of BODIPY-S into Tb-SA and btn- ACE2 mix. FIG. 10B is a line graph showing titration of two positive control antibodies (B38 and H4) or PCR CoV2+ and CoV2- serum to btn-ACE2 and BODIPY-S with Tb-Streptavidin. FIG. 10C is line graph showing titration of CoV2+ serum followed by 1 hour or 4 hour pre-incubation. FIG. 10D is line graph showing affinity of BODIPY-S and Tb-ACE2. FIG. 10E is a line graph showing comparison between btn-ACE2 - Tb-SA, and covalently labelled Tb-ACE2 assay systems. FIG. 1 OF is a line graph showing the affinity of recombinant purified H4 antibody and Tb-ACE2.
[0030] FIGs. 11A-11H are a series of scatterplots, a line graph, and a series of schematics showing the TR-FRET neutralization assay. FIG. 11 A is a scatter plot showing the correlation of total Spike specific IgG levels and neutralization activity. FIG. 1 IB is a scatter plot showing the correlation of total Spike specific IgG levels and neutralization activity scaled by the size of dots of IgM titer. FIG. 11C is a scatter plot showing that the neutralization discriminates CoV2+ from healthy individuals in the 96w_testset. FIG. 1 ID is a line graph showing the comparison of the receiver operator curve (ROC) between ELISA Spike-IgG, TR-FRET Spike-IgG and TR-FRET ACE2-Spike. FIG. 1 IE is a schematic showing the flow chart of automated ELISA processing. FIG. 1 IF is a schematic showing the flow chart of automated TR-FRET processing. FIG. 11G is a schematic showing the estimated buffer consumption in ELISA or TR-FRET assay required for testing of 200,000 samples. FIG. 11H is a bar graph showing the estimated quantities of recombinant protein and detection reagents required for ELISA or TR-FRET assay for testing of 200,000 samples.
[0031] FIGs. 12A-12E are a series of scatterplots, a line graph, and a series of schematics showing the TR-FRET neutralization assay accepts multiple sample types. FIG. 12A is a scatter plot showing correlation of ELISA IgG-S response between the matched set of whole dried blood self-collection samples (Neoteryx® kit) and serum samples from the same donors collected within 2 weeks. FIG. 12B is a scatter plot showing correlation of TR-FRET IgG-S assay between the matched set of whole dried blood self-collection samples and serum samples from the same donors collected within 2 weeks. FIG. 12C is a scatter plot showing correlation between TR-FRET and ELISA responses in the IgG-S assay on the self-collection samples. FIG. 12D is a scatter plot showing the response of ELISA or TR-FRET assay compared between a set of SARS-CoV-2 negative samples. FIG. 12E is a scatter plot showing the response of ELISA or TR-FRET assay compared between a set of SARS-CoV-2 positive samples. Data are represented as means ± SD (n = 2) on a cohort of 140 CoV2+ and 35 CoV2- samples.
[0032] FIGs. 13A-13E are a series of scatterplots and a series of histograms showing that TR- FRET can be applied for total IgG amount testing and validation. FIG. 13 A is a series of scatter plots showing that correlation between TR-FRET and ELISA responses in the IgG-S assay on IMPACT study samples. FIG. 13B is a series of scatter plots showing that correlation between ELISA in the IgG-S assay and total IgG on IMPACT study samples. FIG. 13C is a series of scatter plots showing that correlation between TR-FRET in the IgG-S assay and total IgG on IMPACT study samples. FIG. 13D is a series of scatter plots showing that correlation of ELISA and TR- FRET with total IgG with difference total IgG levels. Total IgG < 2,500 mg/dL is labeled as blue. Total IgG in 2,500 -3,000 mg/dL is labeled as red. Total IgG > 2,500 mg/dL is labeled as yellow. FIG. 13E is a series of histograms showing that Histogram of total IgG levels in the IMPACT study.
[0033] FIGs. 14A-14I are a series of scatterplots, a line graph, and a series of schematics showing the principle of TR-FRET total IgG assay. FIG. 14A a series of schematics showing that nanobodies recognizing human IgG are labeled with AF488. Immunoglobulin-binding protein G is labeled with CoraFluor-1 (Tb). The light pulse at 337 nm excites CoraFluor-1 chelate protein G and emits light at 490 nm which in turn triggers energy transfer to AF488-labeled nanobodies found in proximity induced by analyte generating a TR-FRET signal detected at 520 nm. FIG. 14B a series of a line graph showing that titration of CR3022 IgG into preformed mix of Tb-Protein G (25 nM final) and AF488 labelled Nanobodies (25 nM final). FIG. 14C a series of a line graph showing that Titration of positive and negative serum in final assay condition 25 nM Tb-Protein G, 25 nM AF488-Nanobody. Data are represented as means ± SD (n = 2). FIGs. 14D - FIG. 141 are a series of a scatterplots showing that Correlation of total IgG measured by TR-FRET and ELISA on a set of 39 samples at dilutions of 1 :5,210, 1 : 10,240, 1 :20,480, 1 :40,960, 1 :81,920, and 1 : 163,840. Data are presented as n=l for ELISA and n=2 for TR-FRET.
DETAILED DESCRIPTION OF THE INVENTION
[0034] Methods described herein are designed to detect the presence of neutralizing antibodies (Abs) in a body fluid sample that neutralize members of the Betacoronavirus (P-coronavirus, P- CoV) genus, specifically, SARS-CoV-1, SARS-CoV-2 and MERS-CoV. The P-coronavirus Abs detectable by the present assay methods bind the S-RBD, and, therefore, compete with the Spike- ACE2 (SARS-CoV-l/SARS-CoV-2) or Spike-DPP4 (MERS-CoV) interaction which is the primary mechanism of P-CoV neutralization. Hence, these antibodies are referred to herein as P- CoV neutralizing antibodies.
SARS-CoV/MERS-CoV Virion Particle
[0035] A schematic of a SARS-CoV/MERS-CoV virion particle is illustrated in FIG. 1. As shown, the spike proteins are the visible protrusions on the surface of SARS-CoV-1, SARS-CoV- 2, and MERS-CoV, giving these viruses their characteristic, crown-like appearance. These homotrimeric proteins are heavily glycosylated, with each comprising two distinct subunits: SI and S2. The role of Spike is to act as a molecular key. This mechanism is achieved by recognizing and binding to specific cellular membrane protein receptors (locks). SARS-CoV-1 and SARS- CoV-2 utilize the ACE2 cell-surface receptors, while MERS-CoV utilizes the DPP4 cell-surface receptor present on the surface of mammalian (e.g., human) cells, via the SI receptor-binding domain (S-RBD). When Sl-RBD binds to ACE2 or DPP4, the Spike protein undergoes dramatic structural changes, altering the mammalian receptor’s (ACE2 or DPP4) conformation, and mediating entry of the virus into the host cell.
[0036] Spike proteins are exposed to recognition by the immune system due to their projection into the external environment. This makes Spike the immunodominant coronavirus antigen, causing it to elicit a strong neutralizing antibody response (See, Ju, et al., Nature 584'.115-19 (2020)).
[0037] In some embodiments, the first labelled reagent is a SARS-CoV-1 or SARS-CoV-2 Spike protein or a receptor binding domain thereof (S-RBD or Sl-RBD) thereof.
[0038] In some embodiments, the inventive methods and reagents employ a full-length SARS- CoV-1 Spike protein. An exemplary SARS-CoV-1 spike protein amino acid sequence, provided at UniProtKB-P59594, is herein incorporated by reference and is set forth below (SEQ ID NO: 1
), with the Sl-RBD located at residues 318 to 510 (bolded):
Figure imgf000015_0001
[0039] In some embodiments, the inventive methods and reagents employ a full-length SARS-
CoV-2 Spike Protein. An exemplary SARS-CoV-2 spike amino acid sequence, provided at UniProtKB-P0DTC2, is herein incorporated by reference and is reproduced below (SEQ ID NO:
2), with the Sl-RBD located at residues 318 to 541 (bolded):
Figure imgf000016_0001
[0040] Yet other SARS-CoV-2 Spike proteins that may be useful reagents in the practice of the present assay methods and reagents are known in the art (e.g., available from the NCBI virus database, accession numbers QMT50797, QMT51409, QMT51505, QMT51865, QMT52129, QMT52237, QMT522 49, QMT52393, QMT52561, QMT52741, QMT52765, QMT53017, QMT53041, QMT53053, Q MT53065, QMT53089, QMT53101, QMT53149, QMT53173, QMT53197, QMT53221, QMT5 3233, QMT53245, QMT55880, QMT57260, QMT57332, QMT57572, QMT57584, QMT57608, QMT57644, QMT57656, QMT57692, QMT94108, QMT94756, QMT94780, QMT95200, QM T95308, QMT95356, QMT95368, QMT95452, QMT95488, QMT95560), and five from Asia (QLL26046, QLI49781, QLF98260, QKY60061, and QKV26077). SARS CoV Spike proteins and their respective receptor binding domains are also commercially available.
[0041] It has been reported that nearly one-third of the spike protein sequence is associated with mutations. Accordingly, mutated versions of the Spike protein (and antigenic, ACE2-binding fragments thereof) may be useful as reagents in the practice of the present assay methods. Mutation sites and mutation types observed in human SARS-CoV-2 spike proteins according to geographical locations are set forth in Guruprasad, Proteins vol. 89,5 (2021): 569-576 in Table 3. Guruprasad found Spike proteins having from 1 to 16 mutations. Referring to SEQ ID NO: 2, Spike proteins having a mutation at any one or more of residues L5, L18, T19, T20, deletion of L24 to P26 (L24_P26del), P26, A27, L54, A67, deletion of H69 to V70 (H69_V70del), D80, T95, D138, G142, deletion of V143 to Y145 (V143_Y145del), deletion of G142 to Y144 (G142_Y144del), deletion of Y144 (Y144del), Y145, K147, W152, deletion of E156 to F157 (E156_F157del), R158, R190, 1210, N211, L212, V213, insertion of EPE at 214 (214EPE), D215, A222, S221, deletion of L241 to A243 (L241_A243del), G257, W258, 339, S371, S373, S375, T376, D405, R408, K417, N440, G446, L452, N460, S477, L452, S477, T478, V483, E484, F486, Q493, G496, Q498, N501, Y505, T547, A570, D614, H655, N679, P681, A701, S704, T716, N764, D769, T859, A845, N856, D950, Q954, N969, L981, S982, T1027, DI 118, VI 176, and Pl 263 may be useful as reagents. Therefore, Spike proteins useful as reagents may have one or more mutations including mutation(s) in the Sl-RBD. By way of representative example, in some embodiments, a Spike protein having the mutation D614G (referring to SEQ ID NO: 2) may be used as a reagent. In some embodiments, a Spike protein having the mutation N501Y mutation (referring to SEQ ID NO: 2) may be used as a reagent. In some embodiments, a Spike protein having the mutations K417N, E484K, N501Y (referring to SEQ ID NO: 2) may be used as a reagent. In some embodiments, a Spike protein having the RBD mutations K417T, E484K, and N501 Y (referring to SEQ ID NO: 2) may be used as a reagent.
[0042] In some embodiments, a Spike protein having a mutation in one or more of the 49, 77, 78, 118, 139, 144, 147, 193, 227, 239, 244, 261, 311, 344, 360, 426, 437, 472, 480, 487, 501, 577, 605, 607, 608, 609, 613, 665, 701, 743, 754, 804, 860-861, 894, 999, 1001, 1132, 1148, and/or 1163 amino acid positions (referring to SEQ ID NO: 1) may be used as a reagent. In some embodiments, a Spike protein having a mutation in one or more of the 18, 69-70, 80, 144, 215, 246, 417, 484, 601, 570, 614, 681, 701, 716, 982, and/or 1118 amino acid positions (referring to SEQ ID NO: 2) may be used as reagent.
[0043] In some embodiments, the first reagent is a fragment of a full-length SARS-CoV-1 or SARS-CoV-2 Spike protein that contains an SI -receptor binding domain (Sl-RBD). Referring to SEQ ID NO: 1, the SARS-CoV-1 Sl-RBD is located at residues 318 to 510. Referring to SEQ ID NO: 2, the SARS-CoV-2 Sl-RBD is located at residues 318 to 541. [0044] As in the case of SARS-CoV-2 full-length Spike proteins, mutated versions of Sl-RBD fragments may also be used. Forty-four (44) distinct mutation sites in the Sl-RBD have been reported; the mutations are located at positions 337, 344, 345, 348, 354, 357, 367, 368, 379, 382, 384, 393, 395, 403, 407, 408, 411, 413, 441, 453, 457, 458, 468, 471, 476, 477, 479, 483, 484, 485, 486, 491, 493, 494, 498, 500, 501, 506, 507, 508, 518, 519, 520, and 522 (all referring to SEQ ID NO: 2). See, Guruprasad, supra. In some embodiments, an Sl-RBD fragment has a mutation at any one of positions 344 (e.g., A344S), 477 (e.g., S477N), 483 (e.g., V483A) and 501 (e.g., N501Y). In some embodiments, an Sl-RBD fragment has any one of the following mutations: S477N, V483A, A344S, and N501Y/T. In some embodiments, an Sl-RBD fragment has any one of the following mutations: K417N/T, E484K, and N501Y. In some embodiments, an Sl-RBD fragment has a mutation at any one of positions Y453 (e.g., Y453F), G476 (e.g., G476S), F486 (e.g., F486L), and T500 (e.g., T500I).
[0045] In some embodiments, the second reagent useful for detecting presence of SARS CoV-1 and -2 neutralizing antibodies is a human ACE2 or an S-RBD-binding fragment thereof. ACE2 acts as a viral receptor and is expressed on the surface of several pulmonary and extra-pulmonary cell types, including cardiac, renal, intestinal, and endothelial cells. The ACE2 receptor acts as the receptor-binding protein for the SAR-CoV-2 virus spike complex. Upon engagement of ACE2 by a receptor binding domain in the S 1 subunit of the Spike Protein, conformational rearrangements occur that cause SI subunit shedding, cleavage of S2 subunit by host proteases, and exposure of a fusion peptide adjacent to the S2' proteolysis site.
[0046] The sequence and structure of ACE2 are known in the art (See, e.g., Guy, et al., Biochemistry 42: 13185-92 (2003); Yan, et al., Science 376: 1444-48 (2020)). It exists in six isoforms, all of which bind SARS-CoV-2 (Blume, et al., Nature Genetics 53:205-14 (2021)). ACE2 is commercially available from multiple sources (e.g., Sigma-Aldrich SAE0064; Sino Biological 10108-H08H; Aero Biosystems AC2-H82E6). ACE2 fragments that bind the RBD are known in the art. See, e.g., Renzi, et al., BioRXiv doi: 10.1101/2020.04.06.028647 (2020); Kuznetsov, et al, bioRxiv 2020.12.29.424682.
[0047] A representative amino acid sequence of the full length human ACE2 (Basit, et al., J Biol Struct Dyn 39:3605-3614(2020)) is provided at RCSB Protein Data Bank 6M17, incorporated herein by reference, and reproduced below (SEQ ID NO: 3):
Figure imgf000019_0001
[0048] In some embodiments, a fragment of human ACE2 that binds S-RBD may be used. A representative example of such a fragment is an N-terminal fragment of human ACE2 that contains amino acid residues 21-119 of SEQ ID NO: 3 reproduced below (SEQ ID NO: 4):
Figure imgf000019_0003
[0049] Isoforms of ACE2 may be useful as reagents in the present assay methods provided that they contain a SARS-CoV-2 spike high-affinity binding site and an entry point in airway epithelial cells for SARS-CoV-2 by triggering viral fusion with the cell plasma membrane, resulting in viral
RNA genome delivery into the host. An exemplary amino acid sequence of the ACE2 isoform 1 precursor is provided at NCBI Accession No. NP_001358344, version NP_001358344.1, incorporated herein by reference, and reproduced below (SEQ ID NO: 5):
Figure imgf000019_0002
[0050] An exemplary amino acid sequence of the ACE2 isoform 2 precursor is provided atNCBI Accession No. NP_001373188, version NP_001373188.1, incorporated herein by reference, and reproduced below (SEQ ID NO: 6): 1 ms ss swllls Ivavtaaqst ieeqaktfld kfnheaedl f yqs slaswny ntniteenvq 61 nmnnagdkws aflkeqstla qmyplqeiqn Itvklqlqal qqngs svlse dks krlntil 121 ntmstiystg kvcnpdnpqe clllepglne imansldyne rlwaweswrs evgkqlrply 181 eeyvvlknem aranhyedyg dywrgdyevn gvdgydys rg qliedvehtf eeikplyehl 241 hayvraklmn aypsyispig clpahllgdm wgrfwtnlys Itvpfgqkpn idvtdamvdq 301 awdaqri fke aekffvsvgl pnmtqgfwen smltdpgnvq kavchptawd Igkgdfrilm 361 ctkvtmddfl tahhemghiq ydmayaaqpf llrnganegf heavgeimsl saatpkhlks 421 igllspdfqe dneteinfll kqaltivgtl pftymlekwr wmvfkgeipk dqwmkkwwem 481 kreivgvvep vphdetycdp asl fhvsndy s firyytrtl yqfqfqealc qaakhegplh 541 kcdisnstea gqkl fnmlrl gksepwtlal envvgaknmn vrpllnyfep I ftwlkdqnk 601 ns fvgwstdw spyadqsikv rislksalgd kayewndnem yl frs svaya mrqyflkvkn 661 qmil fgeedv rvanlkpris fnffvtapkn vsdiiprtev ekairms rsr indafrlndn 721 sleflgiqpt Igppnqppvs iwlivfgvvm gvivvgivil i ftgirdrkk ptpllgkswl 781 tailkd
[0051] An exemplary amino acid sequence of the ACE2 isoform 3 precursor is provided atNCBI
Accession NP_001373189, version NP_001373189.1, incorporated herein by reference, and reproduced below (SEQ ID NO: 7):
1 ms s s swllls Ivavtaaqst ieeqaktfld kfnheaedl f yqs slaswny ntniteenvq 61 nmnnagdkws aflkeqstla qmyplqeiqn Itvklqlqal qqngs svlse dks krlntil 121 ntmstiystg kvcnpdnpqe clllepglne imansldyne rlwaweswrs evgkqlrply 181 eeyvvlknem aranhyedyg dywrgdyevn gvdgydys rg qliedvehtf eeikplyehl 241 hayvraklmn aypsyispig clpahllgdm wgrfwtnlys Itvpfgqkpn idvtdamvdq 301 awdaqri fke aekffvsvgl pnmtqgfwen smltdpgnvq kavchptawd Igkgdfrilm 361 ctkvtmddfl tahhemghiq ydmayaaqpf llrnganegf heavgeimsl saatpkhlks 421 igllspdfqe dneteinfll kqaltivgtl pftymlekwr wmvfkgeipk dqwmkkwwem 481 kreivgvvep vphdetycdp asl fhvsndy s firyytrtl yqfqfqealc qaakhegplh 541 kcdisnstea gqklleedvr vanlkpris f nffvtapknv sdiiprteve kairms rs ri 601 ndafrlndns leflgiqptl gppnqppvsi wlivfgvvmg vivvgivili ftgirdrkkk 661 nkarsgenpy asidis kgen npgfqntddv qts f
[0052] An exemplary amino acid sequence of the ACE2 isoform 4 is provided at NCBI
Accession No. NP_001375381, version NP_001375381.1, incorporated herein by reference, and reproduced below (SEQ ID NO: 8):
1 mreagwdkgg rilmctkvtm ddfltahhem ghiqydmaya aqpfllrnga negfheavge
61 imslsaatpk hlksigllsp dfqednetei nfllkqalti vgtlpftyml ekwrwmvfkg 121 eipkdqwmkk wwemkreivg vvepvphdet ycdpasl fhv sndys firyy trtlyqfqfq 181 ealcqaakhe gplhkcdisn steagqkl fn mlrlgksepw tlalenvvga knmnvrplln 241 yfepl ftwlk dqnkns fvgw stdwspyadq sikvrislks algdkayewn dnemyl frs s 301 vayamrqyfl kvknqmil fg eedvrvanlk pris fnffvt apknvsdiip rtevekairm 361 s rs rindafr Indnsleflg iqptlgppnq ppvsiwlivf gvvmgvivvg ivili ftgir 421 drkkknkars genpyasidi s kgennpgfq ntddvqts f
[0053] In some embodiments, the first reagent is a full length ACE2 protein. In some embodiments, the first reagent is ACE2 isoform 1, ACE2 isoform 2, ACE2 isoform 3, or ACE2 isoform 4. In some embodiments, the first reagent is a truncated or a fragment of an ACE2 isoform. In some embodiments, the truncated ACE2 comprises amino acid residues 21-119 (SEQ ID NO: 4), when numbered according to SEQ ID NO: 3. In some embodiments, the truncated ACE2 comprises amino acids 18-615.
[0054] In some embodiments, the second reagent useful for detecting presence of SARS CoV- 2 neutralizing antibodies is a human DPP4 or an S-RBD-binding fragment thereof. See, Yi, et al., iScience 23(6): 101160-8 (2020). DPP4 is a 110 kDa glycoprotein, which is ubiquitously expressed on the surface of a variety of cells. This exopeptidase selectively cleaves N-terminal dipeptides from a variety of substrates, including cytokines, growth factors, neuropeptides, and the incretin hormones. DPP4 plays a major role in glucose metabolism. It is responsible for the degradation of incretins such as GLP-1. Furthermore, it appears to work as a suppressor in the development of some tumors. DPP4 acts as the receptor-binding domain for the MERS virus spike complex. Upon engagement of DPP4 by a receptor binding domain in SI subunit of the Spike Protein, conformational rearrangements occur that cause SI subunit shedding, cleavage of S2 subunit by host proteases, and exposure of a fusion peptide adjacent to the S2' proteolysis site.
[0055] The sequence and structure of DPP4 are known in the art (e.g., Aertgeerts, et al., Protein Sci 73:412-21 (2004); Nojima, et al., BMC Struc Biol 76:11-14 (2016); Deacon et al., Front Endocrinol. 70:80-14; Wang, et al., Cell Research 23:986-93 (2013)). It exists in 4 isoforms, all of which bind MERS-CoV. DPP4 is commercially available from Aero Biosystems (DP4-H5211). [0056] An exemplary amino acid sequence of the DPP4 isoform 1 is provided at NCBI Accession No. NP_001926, version NP_001926.2, incorporated herein by reference, and reproduced below (SEQ ID NO: 9):
1 mktpwkvllg llgaaalvti itvpvvllnk gtddatads r ktytltdylk ntyrlklysl
61 rwisdheyly kqennilvfn aeygns svfl enstfdefgh sindysispd gqfilleyny
121 vkqwrhsyta sydiydlnkr qliteeripn ntqwvtwspv ghklayvwnn diyvkiepnl
181 psyritwtgk ediiyngitd wvyeeevfsa ysalwwspng tflayaqfnd tevplieys f
241 ysdeslqypk tvrvpypkag avnptvkffv vntdsls svt natsiqitap asmligdhyl
301 cdvtwatqer islqwlrriq nysvmdicdy des sgrwncl varqhiemst tgwvgrfrps
361 ephftldgns fykiisneeg yrhicyfqid kkdctfitkg twevigieal tsdylyyisn
421 eykgmpggrn lykiqlsdyt kvtcls celn percqyysvs fs keakyyql rcsgpglply
481 tlhs svndkg Irvlednsal dkmlqnvqmp s kkldfiiln etkfwyqmil pphfdks kky
541 pllldvyagp csqkadtvfr Inwatylast eniivas fdg rgsgyqgdki mhainrrlgt
601 fevedqieaa rqfs kmgfvd nkriaiwgws yggyvtsmvl gsgsgvfkcg iavapvs rwe
661 yydsvytery mglptpednl dhyrnstvms raenfkqvey llihgtaddn vhfqqsaqis
721 kalvdvgvdf qamwytdedh gias stahqh iythmshfik qcfslp [0057] An exemplary amino acid sequence of the DPP4 isoform 2 is provided at NCBI
Accession No. NP_001366533, version NP_001366533.1, incorporated herein by reference, and reproduced below (SEQ ID NO: 10):
1 mktpwkvllg llgaaalvti itvpvvllnk gndatads rk tytltdylkn tyrlklyslr
61 wisdheylyk qennilvfna eygns svfle nstfdefghs indysispdg qfilleynyv 121 kqwrhsytas ydiydlnkrq liteeripnn tqwvtwspvg hklayvwnnd iyvkiepnlp 181 syritwtgke diiyngitdw vyeeevfsay salwwspngt flayaqfndt evplieys fy 241 sdeslqypkt vrvpypkaga vnptvkffvv ntdsls svtn atsiqitapa smligdhylc 301 dvtwatqeri slqwlrriqn ysvmdicdyd es sgrwnclv arqhiemstt gwvgrfrpse 361 phftldgns f ykiisneegy rhicyfqidk kdctfitkgt wevigiealt sdylyyisne 421 ykgmpggrnl ykiqlsdytk vtcls celnp ercqyysvs f s keakyyqlr csgpglplyt 481 Ihs svndkgl rvlednsald kmlqnvqmps kkldfiilne tkfwyqmilp phfdks kkyp 541 llldvyagpc sqkadtvfrl nwatylaste niivas fdgr gsgyqgdkim hainrrlgtf 601 evedqieaar qfs kmgfvdn kriaiwgwsy ggyvtsmvlg sgsgvfkcgi avapvs rwey 661 ydsvyterym glptpednld hyrnstvms r aenfkqveyl lihgtaddnv hfqqsaqis k 721 alvdvgvdfq amwytdedhg ias stahqhi ythmshfikq cfslp
[0058] An exemplary amino acid sequence of the DPP4 isoform 3 is provided at NCBI Accession No. NP_001366534, version NP_001366534.1, incorporated herein by reference, and reproduced below (SEQ ID NO: 11):
1 mktpwkvllg llgaaalvti itvpvvllnk ddatads rkt ytltdylknt yrlklyslrw
61 isdheylykq ennilvfnae ygns svflen stfdefghsi ndysispdgq filleynyvk
121 qwrhsytasy diydlnkrql iteeripnnt qwvtwspvgh klayvwnndi yvkiepnlps
181 yritwtgked iiyngitdwv yeeevfsays alwwspngtf layaqfndte vplieys fys
241 deslqypktv rvpypkagav nptvkffvvn tdsls svtna tsiqitapas mligdhylcd
301 vtwatqeris Iqwlrriqny svmdicdyde s sgrwnclva rqhiemsttg wvgrfrpsep
361 hftldgns fy kiisneegyr hicyfqidkk dctfitkgtw evigiealts dylyyisney
421 kgmpggrnly kiqlsdytkv tcls celnpe rcqyysvs fs keakyyqlrc sgpglplytl
481 hs svndkglr vlednsaldk mlqnvqmps k kldfiilnet kfwyqmilpp hfdks kkypl
541 lldvyagpcs qkadtvfrln watylasten iivas fdgrg sgyqgdkimh ainrrlgtfe
601 vedqieaarq fs kmgfvdnk riaiwgwsyg gyvtsmvlgs gsgvfkcgia vapvs rweyy
661 dsvyterymg Iptpednldh yrnstvms ra enfkqveyll ihgtaddnvh fqqsaqis ka
721 Ivdvgvdfqa mwytdedhgi as stahqhiy thmshfikqc fslp
[0059] An exemplary amino acid sequence of the DPP4 isoform 4 is provided at NCBI Accession No. NP_001366535, version NP_001366535.1, incorporated herein by reference, and reproduced below (SEQ ID NO: 12):
1 mktpwkvllg llgaaalvti itvpvvllnk gtddatads r ktytltdylk ntyrlklysl
61 rwisdheyly kqennilvfn aeygns svfl enstfdefgh sindysispd gqfilleyny 121 vkqwrhsyta sydiydlnkr qliteeripn ntqwvtwspv ghklayvwnn diyvkiepnl 181 psyritwtgk ediiyngitd wvyeeevfsa ysalwwspng tflayaqfnd tevplieys f 241 ysdeslqypk tvrvpypkag avnptvkffv vntdsls svt natsiqitap asmligdhyl 301 cdvtwatqer islqwlrriq nysvmdicdy des sgrwncl varqhiemst tgwvgrfrps 361 ephftldgns fykiisneeg yrhicyfqid kkdctfitkg twevigieal tsdyliqlsd 421 ytkvtcls ce Inpercqyys vs fs keakyy qlrcsgpglp lytlhs svnd kglrvledns 481 aldkmlqnvq mps kkldfii Inetkfwyqm ilpphfdks k kypllldvya gpcsqkadtv
Figure imgf000023_0002
[0060] In some embodiments, the first reagent is a full length DPP4 protein. In some embodiments, the first reagent is DPP4 isoform 1, DPP4isoform 2, DPP4isoform 3, or DPP4 isoform 4. In some embodiments, the first reagent is a truncated or a fragment of an DPP4 isoform.
[0061] Reagents for detection of MERS neutralizing antibodies.
[0062] In some embodiments, the reagents and assays described herein are for the detection of MERS. In some embodiments, the labelled first reagent is a full-length MERS-CoV Spike protein. In some embodiments, the labelled first reagent is a fragment of the MERS-CoV Spike protein. In some embodiments, the labelled first reagent is the Sl-RBD of the MERS-CoV Spike protein An exemplary MERS-CoV spike protein amino acid sequence, provided at UniProtKB-R9uQ53, is herein incorporated by reference and produced below (SEQ ID NO: 13), where the Sl-RBD is located at residues 358 to 558 (bolded):
Figure imgf000023_0001
TR-FRET Labels: Donor and Acceptor Fluorophores [0063] Time-resolved Forster resonance energy transfer (TR-FRET) is a combination of time- resolved fluorescence (TRF) and FRET. TRF reduces background fluorescence by delaying reading the fluorescent signal, for example, by about 10 nano seconds. Following this delay (i.e., the gating period), the longer-lasting fluorescence in the sample is measured. Using TR-FRET, interfering background fluorescence due to interfering substances in the sample, for example, is not co-detected. Only the fluorescence generated or suppressed by the energy transfer is measured. The resulting fluorescence of the TR-FRET system is determined by means of appropriate measuring devices. Such time-resolved detection systems use, for example, pulsed laser diodes, light emitting diodes (LEDs), or pulsed dye lasers as the excitation light source. The measurement occurs after an appropriate time delay, i.e., after the interfering background signals have decayed. Devices and methods for determining time-resolved FRET signals are described in the art and in Example 1.
[0064] TR-FRET requires that the signal of interest must correspond to a compound with a long fluorescent lifetime. Criteria for selecting an appropriate A TR-FRET donor and acceptor pair include one or more of the following: (1) the emission spectrum of the FRET energy donor should overlap with the excitation spectrum of the FRET energy acceptor; (2) the emission spectra of the FRET partners i.e., the FRET energy donor and the FRET energy acceptor, commonly referred to as the donor and the acceptor) should show non-overlapping fluorescence; (3) the FRET quantum yield (i.e., the energy transferred from the FRET donor to the FRET acceptor) should be as high as possible (for example, FRET should have about a 1-100%, e.g., a 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, and 99% efficiency over a measured distance, of 1-20 nm, e.g., 5-10 nm); (4) the FRET signal (i.e., fluorescence) must be distinguishable from fluorescence produced by the sample, e.g., autofluorescence; and (5) the FRET donor and the FRET acceptor should have half-lives that allow detection of the FRET signal (e.g., FRET can be bright and can occur on a timescale ranging from 10-9 seconds to 10-4 seconds).
[0065] Donor / acceptor fluorophore pairs for use in TR-FRET-based assays are known in the art. See, e.g., Joseph R. Lakowicz (Principles of fluorescence spectroscopy, 2nd edition, Kluwer academic/plenum publishers, NY (1999)).
[0066] Donor fluorophores advantageously emit long-lived fluorescence, typically in the order of >0.1 milliseconds (ms), preferably between 0.5 and 6 ms. In this fashion, excitation of the donor fluorophore by a pulsed light source (such as a flash lamp), followed by a delay and then FRET signal measurement (known in the art as a counting window) allows short-lived fluorescence to subside before the measurement is made. This property enables the assay to be conducted in a time-resolved manner which reduces background (signal-to-noise ratios) and in turn, enhances sensitivity and accuracy.
[0067] Representative examples of types of donor fluorophores include lanthanide metals and complexes thereof, including chelates and cryptates. Exemplary lanthanides include terbium (Tb), europium (Eu), dysprosium (Dy), samarium (Sm), neodymium (Nd), ytterbium (Yb), erbium (Er), and their respective 3+ complexes. Such complexes include cryptates and chelates, representative examples of which are described, for example in U.S. Patent Application Publication 2015/0198602 Al. In some embodiments, the donor fluorophore is terbium or Europium, or a cryptate or chelate thereof, examples of which are described the ‘602 Patent Publication. These donor fluorophores are commercially available, e.g., from Cisbio. Eu3+, for example, has a fluorescent lifetime in the order of milliseconds.
[0068] Representative examples of acceptor fluorophores include allophycocyanins (tradename XL665); luminescent organic molecules, such as rhodamines, cyanines (e.g., Cy5), squaraines, coumarins, proflavins, acridines, fluoresceins, boron-dipyrromethene derivatives (commercially available under the tradename "BODIPY"), fluorophores known under the name "Atto", fluorophores known under the name "DY", compounds known under the name "Alexa", and nitrob enzoxadi azol e .
[0069] The "Alexa" compounds are commercially available, e.g., from Invitrogen; the "Atto" compounds are commercially available from Atto-tec; the "DY" compounds are commercially available from Dyomics; and the "Cy" compounds are commercially available from Amersham Biosciences.
[0070] Table 1 lists representative examples of donor/ acceptor pairs for TR-FRET/HTRF1, while Table 2 lists excitation and emission (nm) of known FRET fluorophores.
Table 1 : donor/acceptor pairs for TR-FRET/HTRF
Figure imgf000026_0001
Adapted from Invitrogen.com (FRET; Alexa dyes) and Cysbio (TR-FRET); 2Ro is the distance at which FRET efficiency is 50%. The excitation and emission of various donor and acceptor fluorophores that may be useful in practicing the present invention are described in U.S. Patent Application Publication 2018/0356411 Al. *Any acceptor fluorophore which matches the excitation spectra range of the donor fluorophore can be used (e.g., Alexa647). ** Any acceptor fluorophore which matches the excitation spectra of the donor fluorophore can be used e.g., FITC, BODIPY).
Table 2: FRET fluorophore excitation and emission
Figure imgf000026_0002
Figure imgf000027_0001
See, U.S. Patent Application Publication No. 2018/0356411 Al. *Any acceptor fluorophore which matches the excitation spectra range of the donor fluorophore can be used (e.g., Alexa647). ** Any acceptor fluorophore which matches the excitation spectra of the donor fluorophore can be used (e.g., FITC, BODIPY).
[0071] In some embodiments of the present invention, the donor fluorophore is Tb or Eu, or a cryptate or chelate thereof, and the fluorophore acceptor is an organoboron fluorescent dye, e.g.. boron-dipyrromethene (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene)(commercially available under the tradename BODIPY™), sodium 6-amino-9-(5-((aminomethyl)carbamoyl)-2- carboxyphenyl)-3-iminio-3H-xanthene-4,5-disulfonate (commercially available under the tradename Alexa488™, and 2-[5-[3,3-dimethyl-5-sulfo-l-(3-sulfopropyl)indol-l-ium-2-yl]penta- 2,4-dienylidene]-3-methyl-3-[5-oxo-5-(6-phosphonooxyhexylamino)pentyl]-l-(3- sulfopropyl)indole-5-sulfonic acid (commercially available under the tradename Alexa647™). [0072] In some embodiments, the fluorophore donor/acceptor pair is Tb and BODIPY. In some embodiments, the fluorophore donor/acceptor pair is Eu and ALEXA647, respectively. In some embodiments, the first reagent is labelled with Tb and the second reagent is labelled with BODIPY. In some embodiments, the first reagent is labelled with BODIPY and the second reagent is labelled with Tb.
[0073] Methods of labeling proteinaceous entities with donor and acceptor fluorophores are known in the art. See, e.g., the ‘602 Patent Publication. Commercially available kits are also available for this purpose. For example, kits commercially available from Cisbio and Perkin Elmer allow for labeling peptides, proteins and oligonucleotides with Terbium cryptate, which includes N-hydroxysuccinimide-activated Terbium-Trisbipyridine (TBP).
[0074] The respective molar concentrations for any given pair of fluorophore donor and acceptor in an inventive TR-FRET assay are determined to enhance the FRET signal and facilitate its detection. As such, the molar concentrations may vary, depending upon any given pair of fluorophore donor and acceptors, and the proteinaceous reagents (e.g., SARS-CoV-l/SARS-CoV- 2 Spike Protein and ACE2 or MERS-CoV Spike Protein and DPP4). Determining the relative molar concentrations of the labels for use with the SARS-CoV-l/SARS-CoV-2 Spike Protein and ACE2 or MERS-CoV Spike Protein and DPP4 so as to optimize the FRET signal and minimize background noise is within the level of skill in the art. The working examples illustrate optimization of these molar concentrations using techniques known in the art within certain constraints (e.g., volume, reaction container).
[0075] In some embodiments, for example, a concentration of the reagent labelled with the donor fluorophore such as Terbium or Europium (e.g, as a label for a SARS-CoV-1, SARS-CoV-2, or MERS-CoV Spike Protein) within the range of about 0.1 nM to 50 nM, or about 0.5 nM to 30 nM, or about 1.75 nM to 30 nM (relative to a TR-FRET assay volume of 15 pL) may be useful. Concentrations outside this range, both lower and higher, may also be useful. In some embodiments, the concentration of the reagent labelled with the donor fluorophore such as Tb or Eu is about 0.5 nM to about 4 nM, or about 7.5 nM to about 15 nM. In some embodiments, the concentration of the reagent labelled with the donor fluorophore such as Tb or Eu is about 1 nM, about 2 nM, about 4 nM, about 7.5 nM or about 15 nM.
[0076] In some embodiments, a concentration of the other reagent labelled with the acceptor fluorophore, such as BODIPY (e.g., when used with Tb as the fluorophore donor) of about 8 nM- 1 pM (relative to a TR-FRET assay volume of 15 pL) may be useful. Concentrations outside this range, both lower and higher, may also be useful. In some embodiments, the concentration of reagent labelled with the acceptor fluorophore, e.g., BODIPY, is about 8 nM, and in other embodiments, the concentration is 250 nM (relative to a TR-FRET assay volume of 15 pL).
[0077] The optimal molar concentrations of the fluorophore donor and acceptor relative to one another may depend on Degree of Labeling (DoL). As is known in the art, the DoL is the average number of labels (which in this case are the fluorophore donor and acceptor) coupled to a protein molecule (which in this case are the SARS-CoV-l/SARS-CoV-2 Spike Protein and ACE2 and MERS-CoV Spike Protein and DPP4). As in the case of the molar concentrations, the DoL may vary. Determining the relative DoLs of the labels for use with SARS-CoV-l/SARS-CoV-2 Spike Protein and ACE2 and MERS-CoV Spike Protein and DPP4 so as to optimize the FRET signal and minimize background noise is within the level of skill in the art. In the present methods, the DoL, e.g., with respect to Tb, is generally in the range of about 1.0 to about 3.8. In some embodiments, the DoL is within the range of about 1.8 to about 3.8. In some embodiments, the DoL is about 3.8. DoL values outside this range, both lower and higher, may also be useful. However, a DoL of about 8 (and higher) for Tb might be disadvantageous in that the FRET signal is too strong to be practical. The working examples illustrate optimization of a DoL for Tb using techniques known in the art. As demonstrated in the working examples, DoL may be determined in accordance with standard techniques.
Patients and Patient Samples
[0078] The present methods entail testing body fluid samples obtained from individuals. The samples include whole blood or a component thereof such as serum and plasma, saliva, and tears. In some embodiments, the body fluid sample is serum or plasma. Practice of the invention is not limited to any subpopulations of individuals. Samples may be obtained from any individual (patient), and not just individuals who exhibited symptoms of the infection. Individuals who desire, believe to be in need of, who have been required to be tested for SARS-CoV-1, SARS-CoV-2, and MERS-CoV and/or are asymptomatic may be tested.
Practice of the Assay using FRET Signals and Measurement/Detection Thereof
[0079] In practice, a fluid sample of about 0.2 pL to about 10 pL, e.g., from about 0.5 pL to about 5 pL, may be useful.
[0080] In some embodiments, the fluid sample is about 0.5 pL, 1.0 pL, 2.0 pL, 3 pL, 4 pL or 5 pL. The fluid, e.g., serum samples, may be diluted with physiologically acceptable buffer to a dilution factor that may generally range from about 1 :25 to about 1 :300. In some embodiments, the serum samples may be diluted from about 1 : 50 to 1 : 150 with physiologically acceptable buffer. Once the fluid sample is sufficiently diluted, the fluid sample, or a portion thereof, is mixed (i.e., contacted with) the first and the second reagents. The mixture of the fluid sample, first, and second reagent is called the assay mixture. The components of the assay mixture may be added together in any order. In some embodiments, the first and second reagents are premixed. The term “assay mixture” as used herein refers to the combination of the labeled reagent (i.e., the first and second reagents) with the body fluid sample or dilution thereof. Typically, the body fluid sample is a homogenous mixture, such that it has a uniform composition throughout.
[0081] In some embodiments, the assay method includes a wash, typically after one or more reagents have been immobilize or otherwise fixed such that they, the desired reagents are not washed away. In some embodiments, the assay is washed 10 times. In some embodiments, the assay is washed 5 times. In some embodiments, the assay is washed 3 times. In some embodiments, the assay comprises no washes. The inventive assays and methods described herein do not require washes to achieve the same result as other methods that do require washes, enabling a faster and more efficient assays and methods.
[0082] The term “FRET signal” as used herein refers to any measurable signal representative of FRET between the fluorescent donor compound and the acceptor compound. A FRET signal may therefore be a change in the intensity or lifetime of luminescence of the fluorescent donor compound or of the acceptor compound. Any of a variety of light-emitting and light-detecting instruments can be used to initiate FRET e.g., excite the donor fluorophore or excite a reagent capable of exciting the donor fluorophore) and/or detect the emission produced. The light emissions produced by donor and acceptor fluorophores, /.< ., the FRET signal, can be detected or measured visually, photographically, actinometrically, spectrophotometrically, or by any other convenient means, such as with the use of a fluorometer. See, e.g., Saraheimo, supra.
[0083] The binding of the P-CoV s neutralizing antibodies to the first reagent e.g. , Spike protein) can be determined qualitatively in that neutralizing antibodies present in the fluid sample will competitively bind the Spike protein (or the S-RBC-containing fragment) and reduce the TR- FRET signal, relative to a control (e.g., a same volume/dilution bodily fluid sample that does not contain neutralizing antibodies). That is, reduction of the FRET signal indicates β-CoVs neutralizing antibodies binding to the first reagent. Usually, the reduction of a FRET signal is defined by a certain threshold, i.e., after deduction of any background signal. The background signal is usually determined by performing the FRET assay with all assay reagents except for the labelled reagents. However, the background signal may also be determined by measuring the minimal FRET signal achieved by performing the FRET assay with control antibodies (See infra) or a positive control. Depending on the concentration of the neutralizing antibodies that may be present in the fluid sample (and indicative of the severity of β-CoVs infection), the reduction of the TR-FRET signal may be at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99% relative to the positive control. The positive control may either be existing serum with a known neutralization propensity as determined previously by TR-FRET or by another assay (e.g., a cellular neutralization assay), or a recombinant purified antibody (i.e., B38 (Wu, et al., Science 365: 1274-78 (2020)), H4 (Wu, et al., Science 365:1274-78 (2020)), SAD-S35 (AS35; Aero Biosystems), and 40491-MM43 (Sino Biological, China). A negative control may be any suitable buffer or solution that resembles the body fluid sample at its final dilution (e.g., same ionic conditions).
[0084] The present assay methods detect β-CoVs neutralizing antibodies of different serotypes, including, for example, IgG (which may be responsible for the majority of activity), IgA, and IgM. [0085] The present assay methods may detect amounts of β-CoVs neutralizing antibodies as low as 1.22 ng/mL in absence of serum and about 39 ng/mL in presence of the serum. These limits of detection (LoD) are within the range of common ELIS As. See, e.g., McDade, el al., medRxiv 2020.04.28.20081844.
Kits
[0086] The disclosed reagents may be conveniently packaged in an assay kit to facilitate practice of the homogeneous, TR-FRET-based method for detection of β-CoVs neutralizing antibodies in a patient fluid sample. Broadly, the kit may include: a) a first reagent comprising a β-CoVs Spike protein or a human ACE2 or DPP4-binding fragment thereof that contains the receptor binding domain (S-RBD) and a second reagent comprising a human ACE2, a human DPP4, and/or S-RBD binding fragment thereof, wherein the first and second reagents are labelled with a donor fluorophore and an acceptor fluorophore, and wherein the first and second reagents are disposed in the same or different containers; and b) printed instructions for using the reagents in the homogeneous, TR-FRET-based method for detection of β-CoVs neutralizing antibodies in a patient fluid sample.
[0087] Practice of embodiments would entail use of the first reagent labeled with the donor or acceptor fluorophore but not both. Providing them in separate containers provides flexibility of the kit. Likewise, ACE2 and/or DPP4 labelled with the donor fluorophore and the acceptor fluorophore are not used in the same assay. Hence, providing the ACE2 labeled with the donor fluorophore or with the acceptor fluorophore in separate containers also provides flexibility such that the kit provides the minimum necessary reagents to practice the disclosed methods throughout their entire scope.
EXAMPLES
[0088] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention.
Example 1 : Methods
[0089] Antigen production. The full-length Spike protein of SARS-CoV-2 (S protein prefusion stabilized with furin site removed, expressed in TunaCHO cells) was purchased from LakePharma (Cat. 46328), RBD of SARS-CoV-2 was purchased from LakePharma (Cat. 46438), and full- length biotinylated N protein of SARS-CoV-2 (construct 1-419 with N terminal His-Avi tag) was purchased from Aero biosystem (Cat. NUN-C81Q6). Full-length Spike protein of SARS-CoV (Cat. 40634-V08B) and MERS-CoV (Cat. 40069-V08B) were purchased from Sino Biological.
[0090] Constructs and protein purification. Plasmids encoding CR3022 light chain and IgG, IgM, IgAl heavy chains were a gift from Galit Alter, MGH, Boston, USA. Antibodies were expressed in Expi293T cells following manufacturer protocol (Thermo Fischer Scientific, A14525) with transfection ratios of 1 : 1 or 2: 1 of heavy to light chain. The cell suspension was cleared using centrifugation, 15 minutes at 46,500 ref (Ti45, Beckman Coulter). The clarified media was filtered with a 0.45 pm filter before binding to either protein G (GE, GE17-0405-01) for IgG, protein L (GE, GE17-5478-15) for IgM or peptide M (InvivoGen, gel-pdm-5) for IgAl columns pre-equilibrated with binding buffer (PBS, 10 mM Na2HPO4, 1.8 mM KH2PO4, 137 mM NaCl, 2.7 mM KC1 at pH 7.4). The beads were washed with 20-50 column volumes (CV) of binding buffer. The protein was eluted from the beads with 6-15 CV of 0.1 M glycine pH 3.0 elution buffer and immediately quenched using a 10: 1 ratio of 1 M Tris-HCl pH 8.0. The proteincontaining fractions were pooled and flash-frozen in liquid nitrogen at 0.1-1.5 mg/mL. The antibodies were stored at -80 °C until further use. Concentrations were estimated using Bradford assay. CR3022 IgG was labeled with BODIPY-NHS as described below.
[0091] Neutralizing antibodies B38 and H4 Fab fragments were constructed using the CR3022 Fc regions. The Fab fragment sequence was taken from Wu, et al., Science 365: 1274-78 (2020). B38 and H4 antibodies were expressed in Expi293 and purified as described above for CR3022.
[0092] A truncated ACE2 (amino acids 18-615) without a signaling peptide was cloned and expressed in Hi-5 insect cells by using baculoviruses with C-terminal StrepII and avi fusion tags. The full-length N protein was cloned and expressed in insect cells with N-term Strep-Avi-Tev fusion tag. For both purifications cells were lysed by sonication (in 50 mM Tris pH 8.0, 200 mM NaCl, 0.1% Triton X-100, 1 mM PMSF and 1 tablet of complete protease inhibitor cocktail Roche Applied Science), lysate cleared by high-speed centrifugation, and the supernatant passed over StrepTactin-XT HC affinity resin (IB A). Target protein was eluted using biotin and subjected to Poros50HQ ion exchange chromatography. Purification was completed using size exclusion chromatography with a 26/60 Superdex S200 column (GE Healthcare) in 50 mM HEPES pH 7.4, 200 mM NaCl and 2 mM TCEP. The purified avi tagged ACE2 protein was biotinylated in presence of BirA enzyme, 10 mM MgCh, 2 mM biotin, 20 mM ATP. Biotinylation was confirmed by mass spectrometry. The protein-containing fractions were pooled and flash-frozen in liquid nitrogen at 0.9 mg/mL for ACE2 and 1.6 mg/mL for N protein. The proteins were stored at -80 °C until further use. Concentrations were estimated using Bradford assay.
[0093] Serum samples. Serum/plasma samples used in this study were obtained through the Ragon Institute and Dana-Farber Cancer Institute (DFCI). Institutional IRB approval was obtained, and all samples were collected after subjects provided signed informed consent. Six groups of consented subjects were included: 1) hospitalized patients (MGH and BWH) with a SARS-CoV- 2 confirmed RNA tests; 2) convalescents patients (MGH) with a confirmed prior SARS-CoV-2 RNA+ and two repeat RNA-negative tests after 2 weeks of isolation; 3) pre-pandemic healthy controls with samples collected prior to December 1, 2019 (MGB Biobank); 4) a group of low- risk community members (Ragon); 5) Self-collection samples from DFCI employees (DFCI IRB #20-260); 6) The IMPACT study (DFCI IRB #20-332) patients samples with or without vaccination. Total IgG levels of IMPACT study samples were available from medical records. Samples were heat inactivated at 60 °C for 1 hour.
[0094] Protein labeling with NCP311-Tb or BODIPY. Antibodies (ahs-IgG (Bethyl, A80- 104 A), ahs-IgM (Bethyl, A80-100A), ahs-IgA (Bethyl, A80-102A), S protein (LakePharma, 46328)), protein G (Life, 21193), or RBD protein (LakePharma, 46438) in a volume of 2.5 mL each at a concentration of 1 mg/mL or SARS-CoV (Cat. 40634-V08B) and MERS-CoV (Cat. 40069-V08B) at concentration of 0.25 mg/mL was buffer exchanged into 100 mM sodium carbonate buffer at pH 8.5, 0.05% TWEEN-20 detergent using PD-10 Desalting Columns (Sigma, GE17-0851-01) according to the manufacturer’ s protocol with 0.5 mL per fraction elution. Protein containing fractions were pooled at 0.5 -1 mg/mL and the appropriate volume of either NCP311- Tb (1 mM in dimethylacetamide (DMAc)) or BODIPY-NHS (10 mM in DMSO) was added to achieve a molar ratio of approximately 4-5x NCP311-Tb or 6x BODIPY. The reaction mixture was briefly vortexed and allowed to stand at room temperature for 1 hour. To purify the labeled conjugates, the labeling reaction was buffer exchanged into 50 mM sodium phosphate buffer pH 7.4, 137 mM NaCl, 0.05% TWEEN-20 detergent using PD-10 desalting columns following manufacturer protocol using 0.5 mL elution fractions. Protein containing fractions were pooled and flash-frozen in liquid nitrogen at 0.4-0.6 mg/mL concentration and stored at -80 °C. The corrected A280 value (A280,corr) of protein conjugate was determined via Nanodrop (0.1 cm path length) by measuring A280 and A340, using equation 1 :
Figure imgf000035_0001
where c/is the correction factor for the Th complex contribution to A280 and is equal to 0.157.
The concentration of protein conjugate, cab (M) was determined using equation 2:
Figure imgf000035_0002
where 8 is the antibody extinction coefficient at A280, equal to 210,000 M-1cm-1 for IgG class anti
IgG/IgM/IgA Ab, 240,000 M-1cm-1 for S protein, 80,200 M-1cm-1 for RBD and b is path length in cm (0.1 cm).
The concentration of Tb complex, cib (M) covalently bound to the proteins was determined using equation 3 :
Figure imgf000035_0003
where 8 is the complex extinction coefficient at A340, equal to 22,000 M-1cm-1 and b is path length in cm (0.1 cm).
The degree of labeling (DOE) was calculated using equation 4:
Figure imgf000035_0004
[0095] TR-FRET assay for RBD. Titration of CR3022 IgG/IgM/IgAl antibody or dilution of tested human serum samples was added to assay mix with final concentrations of 15 nM Tb-labeled RBD, 250 nM BODIPY-labeled αlgG/αlgM/αlgA in a buffer containing PBS, 0.05% Tween-20 (Sigma Aldrich P9416). Serum samples were diluted in the buffer containing 50 mM Tris pH 8.0, 140 mM NaCl, 0.05% Tween-20 and 1% BSA (Cell Signaling Technology 9998S). TR-FRET assays were performed in 384-well microplate (Coming, 4514) with 15 pL final assay volume. Before TR-FRET measurements were conducted, the reactions were incubated for 1 hour at room temperature (RT). After excitation of terbium fluorescence at 337 nm, emission at 490 nm (terbium) and 520 nm (BODIPY) were recorded with a 70 ps delay over 130 ps to reduce background fluorescence and the reaction was followed over >20 or >100 second cycles of each data point using a PHERAstar FS microplate reader (BMG Labtech). The TR-FRET signal of each data point was extracted by calculating the 520/490 nm ratio.
[0096] TR-FRET assay for Spike protein. Titration of CR3022 IgG/IgM/IgAl antibody or dilution of tested human serum samples was added to assay mix with final concentrations of 7.5 nM Tb-labeled S protein of SARS-CoV-2, SARS-CoV or MERS-CoV, 250 nM BODIPY-labeled αlgG/αlgM/αlgA in a buffer containing PBS, 0.05% Tween-20 (Sigma Aldrich P9416). Serum samples were diluted in the buffer containing 50 mM Tris pH 8.0, 140 mM NaCl, 0.05% Tween- 20 and 1% BSA (Cell Signaling Technology 9998S). TR-FRET assays were performed in 384- well microplate (Corning, 4514) with 15 pL final assay volume. Before TR-FRET measurements were conducted, the reactions were incubated for 1 hour at RT. After excitation of terbium fluorescence at 337 nm, emission at 490 nm (terbium) and 520 nm (BODIPY) were recorded with a 70 microseconds (ps) delay over 130 ps to reduce background fluorescence and the reaction was followed over >20 or >100 second cycles of each data point using a PHERAstar FS microplate reader (BMG Labtech). The TR-FRET signal of each data point was extracted by calculating the 520/490 nm ratio.
[0097] TR-FRET assay for N protein. Diluted tested human serum samples were added to assay mix with final concentrations of 20 nM biotinylated N protein, 2 nM Streptavidin-Tb and 250 nM BODIPY-labeled αlgG in a buffer containing PBS, 0.05% Tween-20 (Sigma Aldrich P9416). Serum samples were diluted in the buffer containing 50 mM Tris pH 8.0, 140 mM NaCl, 0.05% Tween-20 and 1% BSA (Cell Signaling Technology 9998S). TR-FRET assays were performed in 384-well microplates (Coming, 4514) with 15 pL final assay volume. Biotinylated N protein and Streptavidin-Tb were premixed and incubated for 10 minutes at RT. Before TR-FRET measurements were conducted, the reactions were incubated for 1 hour at RT. After excitation of terbium fluorescence at 337 nm, emission at 490 nm (terbium) and 520 nm (BODIPY) were recorded with a 70 ps delay over 130 ps to reduce background fluorescence and the reaction was followed over >20 or >100 second cycles of each data point using a PHERAstar FS microplate reader (BMG Labtech). The TR-FRET signal of each data point was extracted by calculating the 520/490 nm ratio.
[0098] TR-FRET ACE2-Spike neutralization assay. Diluted human serum samples were added to assay mix with final concentrations of 8 nM Biotinylated ACE2 protein, 2 nM Streptavidin-Tb and 8 nM BODIPY-labeled Spike in a buffer containing PBS, 0.05% Tween-20 (Sigma Aldrich P9416). Serum samples were diluted in the buffer containing 50 mM Tris pH 8.0, 140 mM NaCl, 0.05% Tween-20 and 1% BSA (Cell Signaling Technology 9998S). TR-FRET assays were performed in 384-well microplates (Coming, 4514) with 15 pL final assay volume. Biotinylated ACE2 protein and Streptavidin-Tb (mix 1) were premixed and incubated for 10 minutes at RT. BODIPY-Spike protein and antibody, or serum samples (mix 2) were pre-incubated for 30 minutes at RT. Mix 1 and 2 were added together and before TR-FRET measurements were conducted, the reactions were incubated for 1 to 4 hours at RT. After excitation of terbium fluorescence at 337 nm, emission at 490 nm (terbium) and 520 nm (BODIPY) were recorded with a 70 ps delay over 130 ps to reduce background fluorescence and the reaction was followed over >20 or >100 second cycles of each data point using a PHERAstar FS microplate reader (BMG Labtech). The TR-FRET signal of each data point was extracted by calculating the 520/490 nm ratio. The AUC was calculated as the sum of the averages of duplicate individual 8-point dose response data points.
[0099] TR-FRET assay for total IgG. Diluted tested human serum samples were added to assay mix with final concentrations of 25 nM CoraFluor-1 -Protein G and 25 nM AF488 labeled Nanobodies in a buffer containing PBS, 0.05% Tween-20 (Sigma Aldrich P9416). Serum samples were diluted in the buffer containing 50 mM Tris pH 8.0, 140 mM NaCl, 0.05% Tween-20 and 1% BSA (Cell Signaling Technology 9998S). TR-FRET assays were performed in 384-well microplates (Coming, 4514) with 15 pL final assay volume. Biotinylated N protein and Streptavidin-Tb were premixed and incubated for 10 minutes at RT. Before TR-FRET measurements were conducted, the reactions were incubated for 1 hour at RT. After excitation of terbium fluorescence at 337 nm, emission at 490 nm (terbium) and 520 nm (BODIPY) were recorded with a 70 ps delay over 130 ps to reduce background fluorescence and the reaction was followed over >20 or >100 second cycles of each data point using a PHERAstar FS microplate reader (BMG Labtech). The TR-FRET signal of each data point was extracted by calculating the 520/490 nm ratio.
[00100] ELISA assay for RBD protein. ELISA plates (384 well; Thermo Fisher #464718) were coated with 50 pL/well of 500 ng/mL SARS-CoV-2-RBD in coating buffer (1 capsule of carbonate-bicarbonate buffer (Sigma #C3041100CAP) per 100 mL Milli-Q H2O) for 30 minutes at room temperature. Plates were then washed 3 times with 100 pL/well of wash buffer (0.05% Tween-20, 400 mM NaCl, 50 mM Tris (pH 8.0) in Milli-Q H2O) using a Tecan automated plate washer. Plates were blocked by adding lOOpL/well of blocking buffer (1% BSA, 140 mM NaCl, 50 mM Tris (pH 8.0) in Milli-Q H2O) for 30 minutes at room temperature. Plates were then washed as described above. Diluted samples (50 p in dilution buffer; 1% BSA, 0.05% Tween-20, 140 mM NaCl, 50 mM Tris (pH 8.0) in Milli-Q H2O) were added to the wells and incubated for 30 minutes at 37 °C. Plates were then washed 5 times as described above. Diluted detection antibody solution (50 pL/well; HRP-anti human IgG, IgA or IgM; Bethyl Laboratory #A80-104P, A80-100P, A80-102P) was added to the wells and incubated for 30 minutes at room temperature. Plates were then washed 5 times as described above. TMB peroxidase substrate (40 pL/well; Thermo Fisher #34029) was then added to the wells and incubated at room temperature for 3 minutes (αlgG) and 5 minutes (αlgA and αlgM). The reaction was stopped by adding 40 pL/well of stop solution (1 M H2SO4 in Milli-Q H2O) to each well. OD were read at 450 nm and 570 nm on a Pherastar FSX plate reader.
[00101] ELISA assay for S or N protein. ELISA plates (384-well; ThermoFisher #464718) were coated with 50 pL/well of 500 ng/mL SARS-CoV-2 S protein or SARS-CoV-2 N protein in coating buffer (1 capsule of carbonate-bicarbonate buffer (Sigma #C3041100CAP) per 100 mL Milli-Q H2O) for 30 minutes at room temperature. Plates were then washed 3 times with 100 pL/well of wash buffer (0.05% Tween-20, 400 mM NaCl, 50 mM Tris pH 8.0 in Milli-Q H2O) using a Tecan automated plate washer. Plates were blocked by adding 100 pL/well of blocking buffer (1% BSA, 140 mM NaCl, 50 mM Tris pH 8.0 in Milli-Q H2O) for 30 minutes at room temperature. Plates were then washed as described above. Diluted samples (50 pL in dilution buffer; 1% BSA, 0.05% Tween-20, 140 mM NaCl, 50 mM Tris (pH 8.0) in Milli-Q H2O) were added to the wells and incubated for 30 minutes at 37 °C. Plates were then washed 5 times as described above. Diluted detection antibody solution (50 pL/well; HRP-anti human IgGBethyl Laboratory #A80-104P) was added to the wells and incubated for 30 minutes at room temperature. Plates were then washed 5 times as described above. 40 pL/well of TMB peroxidase substrate (Thermo Fisher #34029) was then added to the wells and incubated at room temperature for 3 minutes (αlgG). The reaction was stopped by adding 40 pL/well of stop solution (1 M H2SO4 in Milli-Q H2O) to each well. OD was read at 450 nm and 570 nm on a Pherastar FSX plate reader. The final data used in the analysis was calculated by subtracting 570 nm background from 450 nm signal.
[00102] Cellular neutralization assay. Lentiviral particles pseudotyped with SARS-CoV-2 spike protein were produced by transient transfection of 293T cells and titered by flow cytometry on 293T-ACE2 cells. Neutralization assays were performed on a Fluent Automated Workstation (Tecan) using 384-well plates (Grenier, 781090). Following an initial 12-fold dilution, the liquid handler performed serial three-fold dilutions (ranging from 1 : 12 to 1 :8,748) of each patient serum and/or purified antibody in 20 ul followed by addition of 20 pL of pseudovirus containing 125 infectious units and incubation for 1 h at room temperature. Finally, 293T-ACE2 cells (10,000) in 20 pL cell media containing 15 pg/mL polybrene were added to each well and incubated at 37 °C for 60-72 hours. Following transduction, cells were lysed using a modified form of a previously described assay buffer containing a final concentration of 20 mM Tris-HCl, 100 pM EDTA, 1.07 mM MgCh, 2.67-26.7 mM MgSCU, 17 mM dithiothreitol (DTT), 250 pM ATP, and 125-250 pM D-luciferin, 1% Triton-X and shaken for five minutes prior to quantitation of luciferase expression within Ih of buffer addition using a Spectramax L luminometer (Molecular Devices). Percent neutralization was determined by subtracting background luminescence measured in cell control wells (cells only) from sample wells and dividing by virus control wells (virus and cells only). Data was analyzed using Graphpad Prism. NT50 values were calculated by taking the inverse of the 50% inhibitory concentration value for all samples with a neutralization value of 80% or higher at the highest concentration of serum or antibody.
[00103] Statistics. Statistical calculations were performed using Prism 8.0.2 and R v3.6.1; packages ggplot2. The correlation plots include geometrical smoothing using R v3.6.1 geom smooth function with generalized linear model calculated (glm method) confidence intervals. The samples in ELISA IgG or TR-FRET IgG were classified as positive if the value exceeded the mean (healthy) + 3 SD (healthy) threshold. Example 2: Development of a TR-FRET assay to detect SARS-CoV-2 antibodies
[00104] Described herein is the development and validation of a homogenous serological assay platform for the detection of SARS-CoV-2 antibodies in human plasma/serum (FIG. 2A). The direct detection of a ternary complex between antigen and serum antibodies using a TR-FRET readout allowed for a simple mix and read protocol that lends itself to scalable automation (FIG. 2B and FIGs. 11A-11E). The proximity of the donor (terbium) and acceptor (BODIPY) fluorophores induced by presence of serum antibodies results in a positive TR-FRET signal that was read out as a 520 nm (acceptor) / 490 nm (donor) ratio and allowed for accurate quantification of serum antibodies in an isotype specific manner (FIG. 2A).
[00105] To enable sensitive detection, the assay conditions were optimized to minimize the signal-to-noise ratio as follows. It was established that the TR-FRET assay format can detect the binding of immunoglobulin variants IgG, IgM and IgAl to SARS-CoV-2 antigens (FIG. 2C). The SARS-CoV-2 spike protein (S protein) is responsible for binding to the host receptor ACE2 to mediate virus entry upon infection (Li, et al., Nature 426:450-54 (2003)) and most neutralizing antibodies have been found to target the S protein (Chen, etal., Cellular & Molecular Immunology 17 :647 -649(2020)). S protein and the receptor binding domain of the SARS-CoV-2 spike protein (S-RBD) was used for assay development. S protein and S-RBD, expressed and purified from Chinese hamster ovarian cells (CHO), were labeled with terbium or BODIPY (see Example 1). Detection antibodies (αlgG, αlgM, αlgAl) were commercially obtained and also labeled with either terbium or BODIPY. As positive control, recombinantly expressed SARS-1 IgG antibody CR3022 (ter Meulen, et al., PLoS Med 3: 1071-1079 (2006)) that has been shown to cross-react with the S-RBD of SARS-CoV-2 (Kd of 9.1 ± 0.7 nM, FIG. 7A) and IgM and IgAl antibodies engineered to contain the CR3022 variable region was used (Tian, et al., Emerg Microbes Infect 9:382-85 (2020)). To identify ideal labeling positions, titrations of CR3022 (IgG, IgM, IgAl) into a mix of labeled S-RBD and labeled detection antibody were performed while varying the position of donor and acceptor (either on S-RBD or detection antibody) (FIGs. 7B-7C). While it was found that all combinations lead to a functional readout (FIGs. 7D-7E), it was observed that terbium conjugation to the antigen results in optimal performance when used with serum/plasma samples. Therefore, the antigen was labeled with terbium and the detection antibody was labeled with BODIPY, resulting in quantitative binding curves for CR3022 (IgG/IgM/IgAl) (FIG. 2C). [00106] The binding curves exhibit the characteristic bell-shape due to the prozone effect (Ha, et al., Cell Rep 76:2047 (2016)), which can be accurately accounted for by mathematical models (Douglass, et al., J Am Chem Soc 735:6092-99 (2013)). Next, it was established that CR3022 can similarly be detected in human serum (FIG. 2D), and while the signal was reduced, the low background level allowed for accurate quantification. During the optimization of assay conditions, it was seen that replacing RBD with the full-length SARS-CoV-2 Spike protein (S protein) significantly reduced background particularly in the presence of serum. This was likely due to 1) the trimeric nature of the full-length S protein leading to avidity effects; and 2) the exposure of otherwise shielded RBD surfaces that may cause unspecific interactions. Therefore, further validation used S protein to subsequently optimize the concentrations of antigen and detection antibody (FIGs. 8A-8B). With optimized concentrations, convalescent serum was validated to result in a dose dependent response in TR-FRET signal (FIG. 2E).
Example 3 : TR-FRET assay analytical limit of detection
[00107] In order to assess the limit of detection (LoD) of the TR-FRET assay, a titration of control antibody CR3022 IgG in the presence and absence of negative control serum at 1 : 150 serum to buffer dilution was first performed (FIG. 2F). The prozone effect was clearly visible at higher concentrations of the antibody and signal intensity is reduced in the presence of serum. Next, the lowest concentrations of CR3022 IgG antibody where signal was higher than mean + 3 SD were selected and 20 replicates were compared against the 20 replicates of blank control, both in presence and absence of serum (FIG. 2G). Based on this, the LoD for the TR-FRET assay was determined to be 1.22 ng/mL in absence of serum, and 39 ng/mL in presence of the serum, which is in the range of common ELISA LoD (McDade, et al., 2020 PLoS ONE 75(8):e0237833-8 (2020)).
Example 4: Homogenous TR-FRET assay can detect IgG in patient serum
[00108] To test the detection of antibodies in serum obtained from positive and negative controls, a set of 48 PCR tested positive (CoV2+) patients from Mass General and Brigham and Women’s Hospital, 28 PCR tested negative (healthy, CoV2-) patients from Mass General Hospital, as well as 19 pre-pandemic serum samples from Mass General Brigham Biobank (CoV2-) or community volunteers (CoV2-) was assembled (hereafter referred to as 96w_testset) (See Table 3). Table 3: Summary of Sample Cohorts
Figure imgf000042_0001
[00109] Adapting established protocols (Roy, et al., J Immunol Methods 484-485'.112832-12 (2020)), an ELISA assay was performed using S protein as reference (FIG. 8C). The 96w_testset was profiled with the TR-FRET assay at an initial serum dilution of 1 :100 to match the exact ELISA concentration (FIG. 8D). It was found that using a 3 standard deviations (SD) cutoff away from the healthy control mean achieved a TR-FRET in comparable sensitivity and slightly improved specificity as compared to the ELISA results (FIGs. 8C-8D). The 96w_testset was collected during the first weeks of the pandemic and may contain false positives and therefore was not used to formally establish assay performance but rather served for optimization. A strong correlation was observed between the TR-FRET and ELISA assays (FIG. 8E). While the discrimination between CoV2+ and CoV2- was comparable between TR-FRET and ELISA, it was found that the ELISA had stronger signal compared to TR-FRET for low responders. This can be explained by the signal amplification in the ELISA compared to equilibrium binding of the TR- FRET assay. However, the lower signal is offset by the low background noise of the TR-FRET, and additionally the TR-FRET has a larger dynamic range without the ceiling of signal at higher antibody concentrations seen with ELISA (FIG. 8E). Nevertheless, without wishing to be bound by theory, the question remained whether signal could further be boosted without compromising background noise by increasing the serum concentration, and how the assay would react to changes in serum dilution. The 96w_testset was re-assayed using dilution factors of 1 :150 or 1 :50. It was observed that increasing the serum concentration improves signal strength without compromising background noise (FIGs. 8F-8I). Lowering the serum concentration to 1 :150 only marginally reduces performance (FIG. 8H). During the analysis of this initial 96w_testset, several CoV2+ samples had an unexpected low response. In addition to the above-mentioned limitations of the 96w_testset, this might be the result of epitope masking since the TR-FRET assay utilizes covalent labeling of the antigen with terbium. Therefore, minimal epitope masking was ensured, and the Degree of Labelling (DOL) was further optimized (FIGs. 9A-9B). A DOL of approximately 3.8 resulted in no detectable epitope masking with an optimal signal. Therefore, a DOL of approximately 3.8 was used for all further validation experiments.
Example 5: TR-FRET assay can accurately detect seroconversion
[00110] With optimized conditions and DOL, the TR-FRET assay was used to detect seroconversion in a larger set of samples from the Mass General Brigham Biobank containing 68 SARS-CoV-2 PCR positive samples (CoV2+), and 100 pre-pandemic healthy controls (healthy, CoV2-) (hereafter referred to as MGB set; See Table 3suprd) that was again profiled using the established ELISA assay for reference, in addition to the additional profiling with different commercial and academic assays (Nilles, et al., medRxiv 2020.11.11.20229724). In line with previous observations, the standard deviation of the healthy controls was very low, and accurate discrimination between CoV2+ and healthy samples was achieved with 100% specificity and 100% sensitivity using a cut-off based on 3 standard deviations of the healthy control (FIG. 3 A). The results are comparable to the established ELISA assay on the same sample set (FIG. 3B), and the response of individual samples between TR-FRET and ELISA assays was well correlated (Pearson correlation coefficient of 0.9) (FIG. 3C), again showing increased dynamic range for the TR-FRET as opposed to ELISA assay. The MGB set contained samples from patients of age between 30-70 years for the CoV2+ cohort and 20-70 years in CoV2- group (FIG. 3D). The gender distribution was 35 females and 33 males in the CoV2+ cohort and 30 females and 70 males in the pre-pandemic health control group (FIG. 3E). It was observed that the IgG titers against S antigen were higher in the younger age group (e.g., 30s). The significant variability of the IgG levels indicated diverse response within the tested population (FIG. 3F).
Example 6: Assessing Intra- and inter-assay precision for the TR-FRET assay
[00111] Eliminating the wash steps and reducing the overall number of sample handling steps should result in high reproducibility and repeatability. To assess the intra- and inter-assay precision of the TR-FRET assay, a set of positive responders as well as negative control samples were selected (68 total). The assay was performed with three operators on three different days (FIGs. 3G-3H). The correlation between operators was above 99.6% with average repeatability of 4.31% and overall precision across days and operators of 5.72%, which well exceeds the commonly desired range for serological assays.
Example 7: TR-FRET assay can rapidly be extended to additional antigens
[00112] Having established a serological assay for S protein, it was next assessed whether the TR-FRET setup was compatible with other antigens. S protein or S-RBD were the most widely used antigens in serological assays for SARS-CoV-2, but there are other SARS-CoV-2 proteins that are highly immunogenic (Dutta, etal., J Virol 94(73):e00647-20 (2020)), such as the abundant nucleocapsid protein (N protein), which binds to viral RNA inside the virion (Lu, et al., Lancet 395:565-74 (2020); Narayanan, et al., J Virol 77:2922-27 (2003)). An N protein TR-FRET IgG detection assay (thereafter named N TR-FRET) was established. The same TR-FRET setup was utilized as before, with the donor fluorophore on the antigen and the acceptor fluorophore on the αlgG antibody. The commercially obtained N protein was expressed from insect cells, biotinylated and terbium-streptavidin (Tb-SA) conjugate was used to label the antigen. To validate the assay setup, a titration of convalescent CoV2+ serum into biotinylated N protein, Tb-SA and BODIPY- αlgG was performed. A dose response was observed with strong signal present at dilution (1 : 150), consistent with the S-protein TR-FRET (FIG. 9C). An N TR-FRET was performed on the 96w_testset, which resulted in a sensitivity of 80.0% and specificity of 96.6% (FIG. 9D).
[00113] In order to further assess performance of the established IgG S and N TR-FRET assays a larger sample set from the Mass CPR consortium with 100 SARS-CoV-2 RT-PCR positive samples (CoV2+), as well as 90 pre-pandemic controls from the Dana-Faber Cancer Institute Bio Bank (heathy, CoV2-), thereafter named MassCPR set, was utilized (See Table 3, Supra). Using the established mean (healthy) + 3 SD (healthy) cutoff the TR-FRET assay performance was established with 97.1% sensitivity and 97.8% specificity, respectively, for the S antigen and 95.2% sensitivity and 98.9% specificity for the N antigen (FIGs. 4A-4B). The analogous results using the ELISA resulted in 95.2% sensitivity and 97.8% specificity for the S antigen and 94.3% sensitivity and 98.9% specificity for the N antigen (FIGs. 9F-9G). In both S and N assays TR-FRET showed improved sensitivity over ELISA (97.1% for S TR-FRET, 95.2% for S ELISA, and 95.2% for N TR-FRET, 94.3% for N ELISA) with identical specificity.
[00114] As seen before with the MGB set, a ‘ceiling’ of signal with the ELISA readout was observed and increased dynamic range for the TR-FRET assay (FIGs. 4C-4D). The clinical admission status of the MassCPR sample cohort indicated 19 patients were admitted to the emergency room (ER), 76 as inpatients (IP) and 5 as outpatients (OP). A significant difference was not observed in the IgG S antibody titers between the groups (FIG. 4E). The number of days since the last positive SARS-CoV-2 test was recorded and within the 14-30 day period IgG levels varied without significant trends (FIG. 4F), in line with previously reported longitudinal studies where the S IgG level stabilizes 14 days post infection (Seow, et al., Nature Microbiology 5:1598-1607 (2020). Comparing the S and N TR-FRET readouts on the MassCPR and 96w_test set resulted in a Pearson correlation coefficient of 0.37 for 96w_testset (FIG. 9E) and 0.22 for MassCPR (FIG. 4G) indicating that the two assays are partially orthogonal and likely provide additive information on serological status when combined, which is in accordance with what has been found in other studies (Zohar, etal., Cell 753:1508-19 (2020)) (FIG. 4G and FIG. 9E). The spike protein has high sequence similarity between SARS-CoV-2 and SARS-CoV and to lesser extent MERS-CoV which can result in cross reactivity in the antibody response (Li, etal., Annual Review of Virology 3:237- 61 (2016); Cueno, et al., Frontiers in Medicine 7: 1089-10 (2020); Hatmal, et al., Cells 9:2638-37 (2020)). In analogous fashion to the SARS-CoV-2 S TR-FRET assay, S based IgG detection assays for SARS-CoV and MERS-CoV were established and tested with the MassCPR set of samples. As expected, cross-reactivity between SARS-CoV-2 and SARS-CoV (FIG. 4H) was observed, but very limited cross-reactivity with MERS-CoV (FIG. 41). This again is in line with previous observations using these antigens and demonstrates that the TR-FRET assay behaves similarly to ELISA and other formats and that cross-reactivity or sensitivity is largely determined by the choice of antigen (Zhu, et al., Science Advances 6:eabc9999-10 (2020)). Samples with high titer of IgG antibodies against MERS-CoV S were identified in ~6% of CoV2+ samples tested.
Example 8: TR-FRET assay is compatible with diverse sample types
[00115] A key distinction of research assays over diagnostics platforms is the ability to be rapidly deployed and modified to varying needs given the lower degree of integration. This also often includes diverse sampling methods that are determined by access to the targeted cohort. Specifically self-collection is an important tool for field studies or sample collection in remote locations. Therefore, the performance of TR-FRET based serology was explored using selfcollection of whole dried blood samples using Neoteryx® kits in a controlled manner by having paired serum samples within a short time interval of each other. Strikingly, it was observed that the TR-FRET assay exhibits low variability of the background signal across both serum and whole blood sample types (Neoteryx®). While ELISA measurements saw significant background increases in variability for the whole blood sample (FIGs. 12A-12E). The increased background noise in the hemolysed samples leads to reduced signal to noise and Z’ in the ELISA assay, while the performance of the TR-FRET assay is not altered. This study demonstrates how this assay can perform in circumstances where other tests fail. In this specific case the whole dried blood samples lead to very high background in ELISA type assays while the TR-FRET readout as an ‘in solution’ assay is unaffected. Example 10: TR-FRET ACE2-S assay detects neutralizing antibodies
[00116] A limitation of antibody detection assays such as ELISA or the TR-FRET test developed here is that they do not discriminate antibodies based on the ability to neutralize the virus. The detection of neutralizing antibodies (nAB’s) is commonly conducted using either live SARS-CoV- 2 virus assays (requiring BSL3 labs) or pseudotyped virus assays (requiring BSL2 labs), which both are limited in throughput and availability (Hoffmann, et al.. Cell 757(e278/271-80 (2020); Nie, et al., Emerg Microbes Infect 9:680-86 (2020)). Since the dominant neutralizing mechanism for SARS-CoV-2 is to block the binding between the SARS-CoV-2 Spike protein and the human ACE2 receptor, it was hypothesized that this interaction can be leveraged to build a surrogate neutralization assay. When built on the TR-FRET platform developed for antibody testing, such a test should be similarly rapid, scalable, and easy to implement (FIG. 5A). To test this, it was first established that the TR-FRET readout can accurately detect and quantify binding between BOD IP Y labeled Spike protein (BODIPY-S) and biotinylated recombinant human ACE2 (btn- ACE2) in presence of Tb-SA (FIG. 10A).
[00117] To establish whether the assay can accurately detect the presence of nAB’s, increasing concentrations of four well characterized recombinant human and mouse nAB’s targeting the Spike protein (B38 (Wu, et al., Science 368: 1274-78 (2020)), H4 (Wu, et al., Science 365:1274- 78 (2020)), SAD-S35 (Aero Biosystems, USA), and 40491-MM43 (Sino Biological, China)) were titrated with the S protein binding but non-neutralizing antibody CR3022 and a non-binding control antibody See Example 1) to btn-ACE2 (at 8 nM), BODIPY-S (at 8 nM) and Tb-SA (at 2 nM). Consistent with the literature reported efficacies, all four nAB’s were able to effectively compete with the ACE2-Spike interaction and scored as neutralizing in our assay in buffer (FIG. 5B) or in presence of serum (FIG. 5C). Consistent with these findings, titration of either negative (pre-pandemic control) or positive (CoV2+, PCR and IgG positive) patient sera resulted in a dose dependent signal only for the CoV2+ serum (FIG. 5D; B38 and H4 nAB’s included for reference). The robustness of the assay setup was also assessed, and similar to the TR-FRET serological assay, the ACE2-spike assay tolerated different labeling strategies such as btn-ACE2 with Tb-SA or direct labeling of ACE2 with terbium (Tb-ACE2) combined with various concentrations of BODIPY-S (FIGs. 10B-10F).
Example 11 : TR-FRET neutralization assay discriminates CoV2+ patient samples [00118] Having established that the TR-FRET ACE2-Spike assay can accurately detect the ability of recombinant purified antibodies to compete with the interaction critical for viral infection, as well as in patient sera, it was then determined whether the assay is able to discriminate Cov2+ patient serum samples from healthy individuals. Using the 96w_testset and the MassCPR set described above, the samples were profiled in the TR-FRET ACE2-Spike assay for neutralizing activity (FIGs. 6A-6H and FIGs. 11 A-l 1G). While none of the healthy control samples exhibited any detectable neutralizing activity, most of the CoV2+ samples had varying levels of neutralizing activity (FIG. 6A). The neutralization status of the MassCPR set was assessed in a cellular pseudovirus neutralization assay (Garcia-Beltran, et al.. Cell 184:476-488 (2021)) (FIG. 6B).
[00119] The response of the TR-FRET ACE2-S inhibition as measured by the AUC was correlated to the NT50 values reported by the cellular neutralization assay (FIG. 6G). In line with what has been observed with pseudotyped virus assays (Garcia-Beltran, et al., Cell 184:476-488 (2021)), and other surrogate neutralization assays (Tan, etal., Nat Biotechnol 38: 1073-78 (2020)), a correlation of total Spike specific IgG levels and neutralization activity of either the TR-FRET assay or the cellular neutralization assay (FIGs. 6C-6D; FIG. 11 A) was observed. A similar trend was also observed for the anti-N protein IgG antibody titers (FIGs. 6E-6F). While the TR-FRET ACE2-S assay is in principle sensitive to neutralization by any serotype (IgM or IgA), the majority of activity in these samples appears to result from IgG levels (FIG. 1 IB). Importantly, it was found that the neutralization assay by itself can successfully discriminate CoV2+ from healthy individuals in the 96w_testset cohort (FIG. 11C) as well as in the Mass CPR set (FIG. 6A), consistent with what has been observed using other neutralization assays (Garcia-Beltran, et al., Cell 754:476-488 (2021)). When Receiver-Operating Characteristic (ROC) analysis was performed (FIG. 6H and FIG. 1 ID), it was found that the ACE2-Spike assay performs comparable to the TR-FRET and ELISA serological assays in discriminating CoV2+ patient samples from healthy control samples (FIGs. 2F-2G, FIG. 6H, and FIGs. 11C-11D).
[00120] All patent publications and non-patent publications are indicative of the level of skill of those skilled in the art to which this invention pertains.
All these publications are herein incorporated by reference to the same extent as if each individual publication were specifically and individually indicated as being incorporated by reference. [00121] Although the invention herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present invention. It is therefore to be understood that numerous modifications may be made to the illustrative embodiments and that other arrangements may be devised without departing from the spirit and scope of the present invention as defined by the appended claims.

Claims

What is claimed is:
1. A homogeneous, TR-FRET -based method for detection of betacoronavirus (β-CoVs) neutralizing antibodies in a patient fluid sample, comprising: obtaining a body fluid sample from a patient; contacting the body fluid sample with a first reagent and a second reagent, thus forming an assay mixture; and detecting a FRET signal, wherein a reduced FRET signal relative to a control sample that does not contain β-CoVs neutralizing antibodies indicates presence of the β-CoVs neutralizing antibodies in the body fluid sample; wherein the first reagent comprises a β-CoVs Spike protein or a fragment thereof that binds human ACE2 or human Dipeptidyl-peptidase 4 (DPP4) (receptor binding domain (S- RBD)), and the second reagent comprises human ACE2, human DPP4 or an S-RBD binding fragment thereof; and wherein the first and second reagents are labeled with a donor fluorophore and an acceptor fluorophore, respectively, or the acceptor fluorophore and the donor fluorophore, respectively.
2. The method of claim 1, wherein the first reagent is a full-length Spike protein specific to SARS-CoV-1 or SARS-CoV-2.
3. The method of claim 2, wherein the full-length Spike protein comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.
4. The method of claim 3, wherein the first reagent contains at least one amino acid mutation relative to the full-length Spike protein of SEQ ID NO: 2.
5. The method of claim 4, wherein the at least one amino acid mutation is a D614G mutation.
6. The method of claim 1, wherein the first reagent comprises a fragment of a full-length SARS-CoV-1 or SARS-CoV-2 Spike protein comprising the S-RBD thereof that binds human ACE2.
7. The method of claim 6, wherein the first reagent contains amino acid residues of 318 to 510 of SEQ ID. NO: 1.
8. The method of claim 6, wherein the first reagent contains amino acid residues of 318 to 541 of SEQ ID. NO: 2.
9. The method of any one of claims 2-8, wherein the second reagent is full length human ACE2.
10. The method of claim 1, wherein the first reagent is a full-length Spike protein specific to MERS-CoV.
11. The method of claim 10, wherein the full-length Spike protein comprises the amino acid sequence of SEQ ID NO: 9.
12. The method of claim 1, wherein the first reagent comprises a fragment of a full-length MERS-CoV Spike protein comprising the S-RBD thereof that binds human DPP4.
13. The method of claim 12, wherein the first reagent contains amino acid residues of 358 to 558 of SEQ ID. NO: 9.
14. The method of any one of claims 10-13, wherein the second reagent is full length human DPP4.
15. The method of claim 1, wherein the body fluid sample comprises serum, plasma, or dried blood.
16. The method of claim 1, wherein the donor fluorophore is a lanthanide metal, or a complex thereof.
17. The method of claim 16, wherein the lanthanide metal is terbium (Tb) or europium (Eu).
18. The method of claim 17, wherein the donor fluorophore is a chelate or cryptate of terbium or europium.
19. The method of claim 18, wherein the donor fluorophore is terbium.
20. The method of claim 1, wherein the acceptor fluorophore is selected from the group consisting of organoboron fluorescent dyes, allophycocyanins, rhodamines, cyanines, squaraines, coumarins, proflavins, acridines, fluoresceins, boron-dipyrromethene derivatives, sodium 6- amino-9-(5-((aminomethyl)carbamoyl)-2-carboxyphenyl)-3-iminio-3H-xanthene-4,5-di sulfonate,
2-[5-[3,3-dimethyl-5-sulfo-l-(3-sulfopropyl)indol-l-ium-2-yl]penta-2,4-dienylidene]-3-methyl-
3-[5-oxo-5-(6-phosphonooxyhexylamino)pentyl]-l-(3-sulfopropyl)indole-5-sulfonic acid (ALEXA647), and nitrob enzoxadi azole.
21. The method of claim 20, wherein the acceptor fluorophore is an organoboron dye.
22. The method of claim 21, wherein the organoboron dye is boron-dipyrromethene (BODIPY).
23. The method of claim 1, wherein the first reagent is labeled with the donor fluorophore and the second reagent is labeled with the acceptor fluorophore.
24. The method of claim 1, wherein the first reagent is labeled with the acceptor fluorophore and the second reagent is labeled with the donor fluorophore.
25. The method of claim 1, wherein the first reagent is labeled with terbium and the second reagent is labeled with BODIPY.
26. The method of claim 25, wherein the Terbium-labelled first reagent is present in a concentration of about 0.1 nM to 50 nM and the BODIPY-labelled second reagent is present in a concentration of about 8 nM to about 250 pM.
27. The method of claim 26, wherein the concentration of the Terbium-labelled first reagent is about 0.5 nM to about 4 nM.
28. The method of claim 27, wherein the concentration of the Terbium-labelled first reagent is about 7.5 nM to about 15 nM.
29. The method of claim 28, wherein the concentration of the Terbium-labelled first reagent is about 7.5 nM.
30. The method of claim 26, wherein the concentration of the BODIPY-labelled second reagent is about 8 nM to about 1 pM.
31. The method of claim 30, wherein the concentration of the BODIPY-labelled second reagent is about 8 nM.
32. The method of any one of claims 26-31, wherein the assay mixture comprises a volume of 15 pL.
33. The method of claim 1, wherein the donor fluorophore is Eu and the acceptor fluorophore is 2-[5-[3,3-dimethyl-5-sulfo-l-(3-sulfopropyl)indol-l-ium-2-yl]penta-2,4-dienylidene]-3- methyl-3-[5-oxo-5-(6-phosphonooxyhexylamino)pentyl]-l-(3-sulfopropyl)indole-5-sulfonic acid (ALEXA647).
34. An assay kit for homogeneous, TR-FRET-based method for detection of betacoronavirus (β-CoVs) neutralizing antibodies in a patient fluid sample, comprising: a first reagent comprising a betacoronavirus (β-CoVs) Spike protein or a fragment thereof that binds human ACE2 or human DPP4 (receptor binding domain (S-RBD)) and a second reagent comprising human ACE2, a human DPP4, or S-RBD binding fragment thereof, wherein the first and the second reagent is labelled with a donor fluorophore and an acceptor fluorophore respectively, or with the acceptor fluorophore and the donor fluorophore, respectively, and wherein the first and second reagents are disposed in the same or different containers; and printed instructions for using the first and second reagents in a homogeneous, TR-FRET- based method for detection of betacoronavirus (β-CoVs) neutralizing antibodies in a patient fluid sample.
35. The assay kit of claim 34, wherein the first reagent is a full-length Spike protein specific to SARS-CoV-1 or SARS-CoV-2.
36. The assay kit of claim 35, wherein the full-length Spike protein comprises the amino acid sequence of SEQ ID NO: 2.
37. The assay kit of claim 36, wherein the first reagent contains at least one amino acid mutation relative to the full-length Spike protein of SEQ ID NO: 2.
38. The assay kit of claim 37, wherein the at least one amino acid mutation is a D614G mutation.
39. The assay kit of claim 34, wherein the first reagent comprises a fragment of a full-length SARS-CoV-1 or SARS-CoV-2 Spike protein comprising the S-RBD thereof that binds human ACE2.
40. The assay kit of claim 39, wherein the first reagent contains amino acid residues of 318 to 510 of SEQ ID. NO: 1.
41. The assay kit of claim 39, wherein the first reagent contains amino acid residues of 318 to 541 of SEQ ID. NO: 2.
42. The assay kit of any one of claims 34-41, wherein the second reagent is full length human ACE2.
43. The assay kit of claim 34, wherein the first reagent is a full-length Spike protein specific to MERS-CoV.
44. The assay kit of claim 43, wherein the full-length Spike protein comprises the amino acid sequence of SEQ ID NO: 9.
45. The assay kit of claim 34, wherein the first reagent comprises a fragment of a full-length MERS-CoV Spike protein comprising the S-RBD thereof that binds human DPP4.
46. The assay kit of claim 45, wherein the first reagent contains amino acid residues of 358 to 558 of SEQ ID. NO: 9.
47. The assay kit of claim 43, wherein the second reagent is full length human DPP4.
48. The assay kit of claim 34, wherein the donor fluorophore is a lanthanide metal, or a complex thereof.
49. The assay kit of claim 48, wherein the lanthanide metal is terbium or europium.
50. The assay kit of claim 49, wherein the donor fluorophore is a chelate or cryptate of terbium or europium.
51. The assay kit of claim 50, wherein the donor fluorophore is terbium.
52. The assay kit of claim 34, wherein the acceptor fluorophore is selected from the group consisting of organoboron fluorescent dyes, allophycocyanins, rhodamines, cyanines, squaraines, coumarins, proflavins, acridines, fluoresceins, boron-dipyrromethene derivatives, sodium 6- amino-9-(5-((aminomethyl)carbamoyl)-2-carboxyphenyl)-3-iminio-3H-xanthene-4,5-di sulfonate, and nitrob enzoxadi azole, and 2-[5-[3,3-dimethyl-5-sulfo-l-(3-sulfopropyl)indol-l-ium-2- yl]penta-2,4-dienylidene]-3-methyl-3-[5-oxo-5-(6-phosphonooxyhexylamino)pentyl]-l-(3- sulfopropyl)indole-5-sulfonic acid (ALEXA647).
53. The assay kit of claim 52, wherein the acceptor fluorophore is an organoboron dye.
54. The assay kit of claim 53, wherein the organoboron dye is BODIPY.
55. The assay kit of claim 34, wherein the first and second reagents are disposed in the same containers.
56. The assay kit of claim 34, wherein the first and second reagents are disposed in different containers.
57. The assay kit of claim 34, wherein the first reagent is labeled with terbium and the second reagent is labelled with BODIPY.
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