EP4314821A1 - Coronavirus assay - Google Patents
Coronavirus assayInfo
- Publication number
- EP4314821A1 EP4314821A1 EP22714221.3A EP22714221A EP4314821A1 EP 4314821 A1 EP4314821 A1 EP 4314821A1 EP 22714221 A EP22714221 A EP 22714221A EP 4314821 A1 EP4314821 A1 EP 4314821A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- coronavirus
- protein
- antigen
- sample
- sars
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000003556 assay Methods 0.000 title description 47
- 241000004176 Alphacoronavirus Species 0.000 title 1
- 241000711573 Coronaviridae Species 0.000 claims abstract description 153
- 239000000427 antigen Substances 0.000 claims abstract description 133
- 102000036639 antigens Human genes 0.000 claims abstract description 133
- 108091007433 antigens Proteins 0.000 claims abstract description 133
- 239000000758 substrate Substances 0.000 claims abstract description 127
- 238000000034 method Methods 0.000 claims abstract description 70
- 239000004971 Cross linker Substances 0.000 claims description 80
- 239000003795 chemical substances by application Substances 0.000 claims description 68
- 230000027455 binding Effects 0.000 claims description 54
- 201000003176 Severe Acute Respiratory Syndrome Diseases 0.000 claims description 44
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 35
- 102000004169 proteins and genes Human genes 0.000 claims description 32
- 108090000623 proteins and genes Proteins 0.000 claims description 32
- 239000000203 mixture Substances 0.000 claims description 31
- 241000127282 Middle East respiratory syndrome-related coronavirus Species 0.000 claims description 30
- 241000315672 SARS coronavirus Species 0.000 claims description 30
- 125000000325 methylidene group Chemical group [H]C([H])=* 0.000 claims description 30
- 108010061994 Coronavirus Spike Glycoprotein Proteins 0.000 claims description 27
- 239000000049 pigment Substances 0.000 claims description 27
- 238000009472 formulation Methods 0.000 claims description 25
- 102000005962 receptors Human genes 0.000 claims description 22
- 108020003175 receptors Proteins 0.000 claims description 22
- 101710204837 Envelope small membrane protein Proteins 0.000 claims description 20
- 101710145006 Lysis protein Proteins 0.000 claims description 20
- 229940096437 Protein S Drugs 0.000 claims description 20
- 101710198474 Spike protein Proteins 0.000 claims description 20
- 238000000018 DNA microarray Methods 0.000 claims description 19
- 125000001174 sulfone group Chemical group 0.000 claims description 19
- 230000003472 neutralizing effect Effects 0.000 claims description 18
- JXLHNMVSKXFWAO-UHFFFAOYSA-N azane;7-fluoro-2,1,3-benzoxadiazole-4-sulfonic acid Chemical compound N.OS(=O)(=O)C1=CC=C(F)C2=NON=C12 JXLHNMVSKXFWAO-UHFFFAOYSA-N 0.000 claims description 11
- 239000004925 Acrylic resin Substances 0.000 claims description 10
- 229920000178 Acrylic resin Polymers 0.000 claims description 10
- 108010001336 Horseradish Peroxidase Proteins 0.000 claims description 10
- 239000003822 epoxy resin Substances 0.000 claims description 8
- 229920000647 polyepoxide Polymers 0.000 claims description 8
- 210000002966 serum Anatomy 0.000 claims description 8
- 101000629318 Severe acute respiratory syndrome coronavirus 2 Spike glycoprotein Proteins 0.000 claims description 7
- 239000000919 ceramic Substances 0.000 claims description 7
- 210000003296 saliva Anatomy 0.000 claims description 7
- 239000002904 solvent Substances 0.000 claims description 7
- 102100035765 Angiotensin-converting enzyme 2 Human genes 0.000 claims description 6
- 108090000975 Angiotensin-converting enzyme 2 Proteins 0.000 claims description 6
- 239000004593 Epoxy Substances 0.000 claims description 6
- 101000629313 Severe acute respiratory syndrome coronavirus Spike glycoprotein Proteins 0.000 claims description 6
- 210000004369 blood Anatomy 0.000 claims description 6
- 239000008280 blood Substances 0.000 claims description 6
- 210000002381 plasma Anatomy 0.000 claims description 6
- 239000008199 coating composition Substances 0.000 claims description 5
- 238000004519 manufacturing process Methods 0.000 claims description 5
- 102000004190 Enzymes Human genes 0.000 claims description 4
- 108090000790 Enzymes Proteins 0.000 claims description 4
- 239000000654 additive Substances 0.000 claims description 4
- 210000001175 cerebrospinal fluid Anatomy 0.000 claims description 4
- 229920000642 polymer Polymers 0.000 claims description 4
- 239000011347 resin Substances 0.000 claims description 4
- 229920005989 resin Polymers 0.000 claims description 4
- 108700002099 Coronavirus Nucleocapsid Proteins Proteins 0.000 claims description 3
- 206010036790 Productive cough Diseases 0.000 claims description 3
- 229910052794 bromium Inorganic materials 0.000 claims description 3
- 229910052801 chlorine Inorganic materials 0.000 claims description 3
- 229910052731 fluorine Inorganic materials 0.000 claims description 3
- JCXJVPUVTGWSNB-UHFFFAOYSA-N nitrogen dioxide Inorganic materials O=[N]=O JCXJVPUVTGWSNB-UHFFFAOYSA-N 0.000 claims description 3
- 150000004756 silanes Chemical class 0.000 claims description 3
- 210000003802 sputum Anatomy 0.000 claims description 3
- 208000024794 sputum Diseases 0.000 claims description 3
- 210000004243 sweat Anatomy 0.000 claims description 3
- 210000001138 tear Anatomy 0.000 claims description 3
- 210000002700 urine Anatomy 0.000 claims description 3
- 206010050337 Cerumen impaction Diseases 0.000 claims description 2
- 108700003471 Coronavirus 3C Proteases Proteins 0.000 claims description 2
- 108700002673 Coronavirus M Proteins Proteins 0.000 claims description 2
- 206010028980 Neoplasm Diseases 0.000 claims description 2
- 230000000996 additive effect Effects 0.000 claims description 2
- 210000001185 bone marrow Anatomy 0.000 claims description 2
- 239000013592 cell lysate Substances 0.000 claims description 2
- 210000002939 cerumen Anatomy 0.000 claims description 2
- 239000000284 extract Substances 0.000 claims description 2
- 239000012530 fluid Substances 0.000 claims description 2
- 231100000640 hair analysis Toxicity 0.000 claims description 2
- 210000001006 meconium Anatomy 0.000 claims description 2
- 210000005087 mononuclear cell Anatomy 0.000 claims description 2
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 claims description 2
- 102000016622 Dipeptidyl Peptidase 4 Human genes 0.000 claims 2
- 108010067722 Dipeptidyl Peptidase 4 Proteins 0.000 claims 2
- 239000000523 sample Substances 0.000 description 53
- 235000018102 proteins Nutrition 0.000 description 26
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 21
- 101710085938 Matrix protein Proteins 0.000 description 19
- 101710127721 Membrane protein Proteins 0.000 description 19
- 210000004027 cell Anatomy 0.000 description 18
- 101710141454 Nucleoprotein Proteins 0.000 description 16
- 125000003118 aryl group Chemical group 0.000 description 13
- 239000000126 substance Substances 0.000 description 13
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 12
- 102100030988 Angiotensin-converting enzyme Human genes 0.000 description 11
- 108090000882 Peptidyl-Dipeptidase A Proteins 0.000 description 11
- 238000006243 chemical reaction Methods 0.000 description 10
- 210000002845 virion Anatomy 0.000 description 10
- UUUHXMGGBIUAPW-UHFFFAOYSA-N 1-[1-[2-[[5-amino-2-[[1-[5-(diaminomethylideneamino)-2-[[1-[3-(1h-indol-3-yl)-2-[(5-oxopyrrolidine-2-carbonyl)amino]propanoyl]pyrrolidine-2-carbonyl]amino]pentanoyl]pyrrolidine-2-carbonyl]amino]-5-oxopentanoyl]amino]-3-methylpentanoyl]pyrrolidine-2-carbon Chemical compound C1CCC(C(=O)N2C(CCC2)C(O)=O)N1C(=O)C(C(C)CC)NC(=O)C(CCC(N)=O)NC(=O)C1CCCN1C(=O)C(CCCN=C(N)N)NC(=O)C1CCCN1C(=O)C(CC=1C2=CC=CC=C2NC=1)NC(=O)C1CCC(=O)N1 UUUHXMGGBIUAPW-UHFFFAOYSA-N 0.000 description 9
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 9
- 238000001514 detection method Methods 0.000 description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 8
- 108090001074 Nucleocapsid Proteins Proteins 0.000 description 8
- 125000000217 alkyl group Chemical group 0.000 description 8
- 238000012875 competitive assay Methods 0.000 description 8
- 238000002360 preparation method Methods 0.000 description 8
- 238000012360 testing method Methods 0.000 description 8
- 241001678559 COVID-19 virus Species 0.000 description 7
- 208000025370 Middle East respiratory syndrome Diseases 0.000 description 7
- HRNLUBSXIHFDHP-UHFFFAOYSA-N N-(2-aminophenyl)-4-[[[4-(3-pyridinyl)-2-pyrimidinyl]amino]methyl]benzamide Chemical compound NC1=CC=CC=C1NC(=O)C(C=C1)=CC=C1CNC1=NC=CC(C=2C=NC=CC=2)=N1 HRNLUBSXIHFDHP-UHFFFAOYSA-N 0.000 description 7
- 230000000694 effects Effects 0.000 description 7
- 230000036039 immunity Effects 0.000 description 7
- 239000002243 precursor Substances 0.000 description 7
- 108010076504 Protein Sorting Signals Proteins 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- -1 for example Proteins 0.000 description 6
- 230000028993 immune response Effects 0.000 description 6
- 239000003446 ligand Substances 0.000 description 6
- 230000001681 protective effect Effects 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- PNEYBMLMFCGWSK-UHFFFAOYSA-N Alumina Chemical compound [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 description 5
- 101000829958 Homo sapiens N-acetyllactosaminide beta-1,6-N-acetylglucosaminyl-transferase Proteins 0.000 description 5
- 102100023315 N-acetyllactosaminide beta-1,6-N-acetylglucosaminyl-transferase Human genes 0.000 description 5
- 230000004913 activation Effects 0.000 description 5
- 239000000872 buffer Substances 0.000 description 5
- 230000007246 mechanism Effects 0.000 description 5
- 230000002829 reductive effect Effects 0.000 description 5
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 4
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 4
- 108091005609 SARS-CoV-2 Spike Subunit S1 Proteins 0.000 description 4
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 4
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 4
- 239000007983 Tris buffer Substances 0.000 description 4
- 241000700605 Viruses Species 0.000 description 4
- 150000001413 amino acids Chemical group 0.000 description 4
- 238000002820 assay format Methods 0.000 description 4
- 230000008901 benefit Effects 0.000 description 4
- 230000001588 bifunctional effect Effects 0.000 description 4
- 238000011088 calibration curve Methods 0.000 description 4
- 125000000524 functional group Chemical group 0.000 description 4
- 238000010348 incorporation Methods 0.000 description 4
- 230000003993 interaction Effects 0.000 description 4
- 125000005647 linker group Chemical group 0.000 description 4
- 229910052757 nitrogen Inorganic materials 0.000 description 4
- 230000009871 nonspecific binding Effects 0.000 description 4
- 229920001343 polytetrafluoroethylene Polymers 0.000 description 4
- 239000004810 polytetrafluoroethylene Substances 0.000 description 4
- 238000011533 pre-incubation Methods 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- 238000001179 sorption measurement Methods 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 4
- 238000000108 ultra-filtration Methods 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 208000001528 Coronaviridae Infections Diseases 0.000 description 3
- 102100025012 Dipeptidyl peptidase 4 Human genes 0.000 description 3
- 101000908391 Homo sapiens Dipeptidyl peptidase 4 Proteins 0.000 description 3
- 108060003951 Immunoglobulin Proteins 0.000 description 3
- 108010052285 Membrane Proteins Proteins 0.000 description 3
- 102000018697 Membrane Proteins Human genes 0.000 description 3
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 3
- 108091006197 SARS-CoV-2 Nucleocapsid Protein Proteins 0.000 description 3
- 101000667982 Severe acute respiratory syndrome coronavirus 2 Envelope small membrane protein Proteins 0.000 description 3
- 101000953880 Severe acute respiratory syndrome coronavirus 2 Membrane protein Proteins 0.000 description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 3
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 3
- 235000001014 amino acid Nutrition 0.000 description 3
- 239000012472 biological sample Substances 0.000 description 3
- 210000004899 c-terminal region Anatomy 0.000 description 3
- 239000006229 carbon black Substances 0.000 description 3
- 108700010904 coronavirus proteins Proteins 0.000 description 3
- 239000012043 crude product Substances 0.000 description 3
- 125000004122 cyclic group Chemical group 0.000 description 3
- 238000002405 diagnostic procedure Methods 0.000 description 3
- 229940090124 dipeptidyl peptidase 4 (dpp-4) inhibitors for blood glucose lowering Drugs 0.000 description 3
- 238000003818 flash chromatography Methods 0.000 description 3
- 235000014304 histidine Nutrition 0.000 description 3
- 125000000487 histidyl group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C([H])=N1 0.000 description 3
- 102000018358 immunoglobulin Human genes 0.000 description 3
- HWYHZTIRURJOHG-UHFFFAOYSA-N luminol Chemical compound O=C1NNC(=O)C2=C1C(N)=CC=C2 HWYHZTIRURJOHG-UHFFFAOYSA-N 0.000 description 3
- 238000003032 molecular docking Methods 0.000 description 3
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 3
- 238000006386 neutralization reaction Methods 0.000 description 3
- 239000003921 oil Substances 0.000 description 3
- 150000002978 peroxides Chemical class 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 239000000741 silica gel Substances 0.000 description 3
- 229910002027 silica gel Inorganic materials 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 239000000080 wetting agent Substances 0.000 description 3
- 125000006702 (C1-C18) alkyl group Chemical group 0.000 description 2
- KUBDPQJOLOUJRM-UHFFFAOYSA-N 2-(chloromethyl)oxirane;4-[2-(4-hydroxyphenyl)propan-2-yl]phenol Chemical compound ClCC1CO1.C=1C=C(O)C=CC=1C(C)(C)C1=CC=C(O)C=C1 KUBDPQJOLOUJRM-UHFFFAOYSA-N 0.000 description 2
- HGOBHXGITKMPPL-UHFFFAOYSA-N 4-[3-(4-methylphenyl)sulfonyl-2-[(4-methylphenyl)sulfonylmethyl]propanoyl]benzoic acid Chemical compound C1=CC(C)=CC=C1S(=O)(=O)CC(C(=O)C=1C=CC(=CC=1)C(O)=O)CS(=O)(=O)C1=CC=C(C)C=C1 HGOBHXGITKMPPL-UHFFFAOYSA-N 0.000 description 2
- 208000025721 COVID-19 Diseases 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 108010047041 Complementarity Determining Regions Proteins 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 102100040004 Gamma-glutamylcyclotransferase Human genes 0.000 description 2
- 101000886680 Homo sapiens Gamma-glutamylcyclotransferase Proteins 0.000 description 2
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 2
- UFWIBTONFRDIAS-UHFFFAOYSA-N Naphthalene Chemical compound C1=CC=CC2=CC=CC=C21 UFWIBTONFRDIAS-UHFFFAOYSA-N 0.000 description 2
- 101001028244 Onchocerca volvulus Fatty-acid and retinol-binding protein 1 Proteins 0.000 description 2
- 101001024637 Severe acute respiratory syndrome coronavirus 2 Nucleoprotein Proteins 0.000 description 2
- BLRPTPMANUNPDV-UHFFFAOYSA-N Silane Chemical group [SiH4] BLRPTPMANUNPDV-UHFFFAOYSA-N 0.000 description 2
- 239000012505 Superdex™ Substances 0.000 description 2
- 239000006180 TBST buffer Substances 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 125000003545 alkoxy group Chemical group 0.000 description 2
- 239000012491 analyte Substances 0.000 description 2
- 102000023732 binding proteins Human genes 0.000 description 2
- 108091008324 binding proteins Proteins 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 239000012267 brine Substances 0.000 description 2
- 125000004432 carbon atom Chemical group C* 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000002738 chelating agent Substances 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 238000010835 comparative analysis Methods 0.000 description 2
- 230000002860 competitive effect Effects 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 239000000356 contaminant Substances 0.000 description 2
- JHIVVAPYMSGYDF-UHFFFAOYSA-N cyclohexanone Chemical compound O=C1CCCCC1 JHIVVAPYMSGYDF-UHFFFAOYSA-N 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- DOIRQSBPFJWKBE-UHFFFAOYSA-N dibutyl phthalate Chemical compound CCCCOC(=O)C1=CC=CC=C1C(=O)OCCCC DOIRQSBPFJWKBE-UHFFFAOYSA-N 0.000 description 2
- ZUOUZKKEUPVFJK-UHFFFAOYSA-N diphenyl Chemical compound C1=CC=CC=C1C1=CC=CC=C1 ZUOUZKKEUPVFJK-UHFFFAOYSA-N 0.000 description 2
- 241001493065 dsRNA viruses Species 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 238000005194 fractionation Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 238000001502 gel electrophoresis Methods 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 2
- 229910052736 halogen Inorganic materials 0.000 description 2
- 150000002367 halogens Chemical class 0.000 description 2
- 238000003306 harvesting Methods 0.000 description 2
- 108010028403 hemagglutinin esterase Proteins 0.000 description 2
- 210000005260 human cell Anatomy 0.000 description 2
- 230000002209 hydrophobic effect Effects 0.000 description 2
- 238000010191 image analysis Methods 0.000 description 2
- 238000003018 immunoassay Methods 0.000 description 2
- PQNFLJBBNBOBRQ-UHFFFAOYSA-N indane Chemical group C1=CC=C2CCCC2=C1 PQNFLJBBNBOBRQ-UHFFFAOYSA-N 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 125000002950 monocyclic group Chemical group 0.000 description 2
- 229910052759 nickel Inorganic materials 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 229920001542 oligosaccharide Polymers 0.000 description 2
- 150000002482 oligosaccharides Chemical class 0.000 description 2
- CTSLXHKWHWQRSH-UHFFFAOYSA-N oxalyl chloride Chemical compound ClC(=O)C(Cl)=O CTSLXHKWHWQRSH-UHFFFAOYSA-N 0.000 description 2
- 230000036961 partial effect Effects 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 239000013618 particulate matter Substances 0.000 description 2
- 239000013610 patient sample Substances 0.000 description 2
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 2
- 238000005498 polishing Methods 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- 229920006395 saturated elastomer Polymers 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- WTMNSNRISDDHPC-UHFFFAOYSA-N silylsulfonylsilane Chemical class [SiH3]S([SiH3])(=O)=O WTMNSNRISDDHPC-UHFFFAOYSA-N 0.000 description 2
- 229910052938 sodium sulfate Inorganic materials 0.000 description 2
- 235000011152 sodium sulphate Nutrition 0.000 description 2
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical compound O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 2
- 230000009870 specific binding Effects 0.000 description 2
- 125000001424 substituent group Chemical group 0.000 description 2
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 2
- 238000002255 vaccination Methods 0.000 description 2
- WYTZZXDRDKSJID-UHFFFAOYSA-N (3-aminopropyl)triethoxysilane Chemical compound CCO[Si](OCC)(OCC)CCCN WYTZZXDRDKSJID-UHFFFAOYSA-N 0.000 description 1
- 125000004178 (C1-C4) alkyl group Chemical group 0.000 description 1
- 125000004169 (C1-C6) alkyl group Chemical group 0.000 description 1
- DURPTKYDGMDSBL-UHFFFAOYSA-N 1-butoxybutane Chemical compound CCCCOCCCC DURPTKYDGMDSBL-UHFFFAOYSA-N 0.000 description 1
- YBYIRNPNPLQARY-UHFFFAOYSA-N 1H-indene Natural products C1=CC=C2CC=CC2=C1 YBYIRNPNPLQARY-UHFFFAOYSA-N 0.000 description 1
- JTTIOYHBNXDJOD-UHFFFAOYSA-N 2,4,6-triaminopyrimidine Chemical compound NC1=CC(N)=NC(N)=N1 JTTIOYHBNXDJOD-UHFFFAOYSA-N 0.000 description 1
- NGNBDVOYPDDBFK-UHFFFAOYSA-N 2-[2,4-di(pentan-2-yl)phenoxy]acetyl chloride Chemical compound CCCC(C)C1=CC=C(OCC(Cl)=O)C(C(C)CCC)=C1 NGNBDVOYPDDBFK-UHFFFAOYSA-N 0.000 description 1
- POAOYUHQDCAZBD-UHFFFAOYSA-N 2-butoxyethanol Chemical compound CCCCOCCO POAOYUHQDCAZBD-UHFFFAOYSA-N 0.000 description 1
- VHCSBTPOPKFYIU-UHFFFAOYSA-N 2-chloroethanesulfonyl chloride Chemical compound ClCCS(Cl)(=O)=O VHCSBTPOPKFYIU-UHFFFAOYSA-N 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- 108090001008 Avidin Proteins 0.000 description 1
- 241000282465 Canis Species 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- 102100034330 Chromaffin granule amine transporter Human genes 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 241000494545 Cordyline virus 2 Species 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 description 1
- 102100027723 Endogenous retrovirus group K member 6 Rec protein Human genes 0.000 description 1
- 101710091045 Envelope protein Proteins 0.000 description 1
- 241000282324 Felis Species 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000641221 Homo sapiens Chromaffin granule amine transporter Proteins 0.000 description 1
- 101000724418 Homo sapiens Neutral amino acid transporter B(0) Proteins 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- PWHULOQIROXLJO-UHFFFAOYSA-N Manganese Chemical class [Mn] PWHULOQIROXLJO-UHFFFAOYSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 1
- 102100028267 Neutral amino acid transporter B(0) Human genes 0.000 description 1
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 1
- 108010089430 Phosphoproteins Proteins 0.000 description 1
- 102000007982 Phosphoproteins Human genes 0.000 description 1
- 102100029812 Protein S100-A12 Human genes 0.000 description 1
- 101710110949 Protein S100-A12 Proteins 0.000 description 1
- 101710188315 Protein X Proteins 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 206010057190 Respiratory tract infections Diseases 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 208000037847 SARS-CoV-2-infection Diseases 0.000 description 1
- 108010090804 Streptavidin Proteins 0.000 description 1
- 101710172711 Structural protein Proteins 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical group [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 239000005864 Sulphur Chemical group 0.000 description 1
- 201000008754 Tenosynovial giant cell tumor Diseases 0.000 description 1
- RTAQQCXQSZGOHL-UHFFFAOYSA-N Titanium Chemical class [Ti] RTAQQCXQSZGOHL-UHFFFAOYSA-N 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- 108020000999 Viral RNA Proteins 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 230000010530 Virus Neutralization Effects 0.000 description 1
- QCWXUUIWCKQGHC-UHFFFAOYSA-N Zirconium Chemical compound [Zr] QCWXUUIWCKQGHC-UHFFFAOYSA-N 0.000 description 1
- 229920006397 acrylic thermoplastic Polymers 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 150000001336 alkenes Chemical class 0.000 description 1
- 125000003342 alkenyl group Chemical group 0.000 description 1
- 229920000180 alkyd Polymers 0.000 description 1
- 239000004411 aluminium Chemical class 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical class [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009831 antigen interaction Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000012131 assay buffer Substances 0.000 description 1
- 125000004429 atom Chemical group 0.000 description 1
- 125000003828 azulenyl group Chemical group 0.000 description 1
- 125000002619 bicyclic group Chemical group 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 235000010290 biphenyl Nutrition 0.000 description 1
- 239000004305 biphenyl Substances 0.000 description 1
- 239000001055 blue pigment Substances 0.000 description 1
- 239000001058 brown pigment Substances 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 239000011545 carbonate/bicarbonate buffer Substances 0.000 description 1
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000008614 cellular interaction Effects 0.000 description 1
- 229910010293 ceramic material Inorganic materials 0.000 description 1
- 150000005829 chemical entities Chemical class 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000010941 cobalt Substances 0.000 description 1
- 229910017052 cobalt Inorganic materials 0.000 description 1
- GUTLYIVDDKVIGB-UHFFFAOYSA-N cobalt atom Chemical class [Co] GUTLYIVDDKVIGB-UHFFFAOYSA-N 0.000 description 1
- 230000009137 competitive binding Effects 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 239000003431 cross linking reagent Substances 0.000 description 1
- 125000000753 cycloalkyl group Chemical group 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 230000001934 delay Effects 0.000 description 1
- 230000002939 deleterious effect Effects 0.000 description 1
- 239000002274 desiccant Substances 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- MTHSVFCYNBDYFN-UHFFFAOYSA-N diethylene glycol Chemical compound OCCOCCO MTHSVFCYNBDYFN-UHFFFAOYSA-N 0.000 description 1
- 208000035647 diffuse type tenosynovial giant cell tumor Diseases 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000002270 dispersing agent Substances 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 230000005670 electromagnetic radiation Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 229920002313 fluoropolymer Polymers 0.000 description 1
- 239000004811 fluoropolymer Substances 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 239000001056 green pigment Substances 0.000 description 1
- 210000004209 hair Anatomy 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 229930195733 hydrocarbon Natural products 0.000 description 1
- 150000002430 hydrocarbons Chemical class 0.000 description 1
- 230000005661 hydrophobic surface Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 125000003454 indenyl group Chemical group C1(C=CC2=CC=CC=C12)* 0.000 description 1
- 239000000976 ink Substances 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 125000000959 isobutyl group Chemical group [H]C([H])([H])C([H])(C([H])([H])[H])C([H])([H])* 0.000 description 1
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 231100000518 lethal Toxicity 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 229910052748 manganese Inorganic materials 0.000 description 1
- 239000011572 manganese Chemical class 0.000 description 1
- 230000000873 masking effect Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 239000002480 mineral oil Substances 0.000 description 1
- 235000010446 mineral oil Nutrition 0.000 description 1
- 230000000116 mitigating effect Effects 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 125000004108 n-butyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 125000001624 naphthyl group Chemical group 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 239000001053 orange pigment Substances 0.000 description 1
- 125000001181 organosilyl group Chemical group [SiH3]* 0.000 description 1
- 150000002924 oxiranes Chemical class 0.000 description 1
- 239000003209 petroleum derivative Substances 0.000 description 1
- 239000004014 plasticizer Substances 0.000 description 1
- 229920003229 poly(methyl methacrylate) Polymers 0.000 description 1
- 125000003367 polycyclic group Chemical group 0.000 description 1
- 235000013824 polyphenols Nutrition 0.000 description 1
- 238000007781 pre-processing Methods 0.000 description 1
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 description 1
- 238000002331 protein detection Methods 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 230000004850 protein–protein interaction Effects 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 239000002096 quantum dot Substances 0.000 description 1
- 239000001054 red pigment Substances 0.000 description 1
- 208000020029 respiratory tract infectious disease Diseases 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000003938 response to stress Effects 0.000 description 1
- 125000006413 ring segment Chemical group 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 125000002914 sec-butyl group Chemical group [H]C([H])([H])C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 229910000077 silane Inorganic materials 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 125000001273 sulfonato group Chemical group [O-]S(*)(=O)=O 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- ISXSCDLOGDJUNJ-UHFFFAOYSA-N tert-butyl prop-2-enoate Chemical compound CC(C)(C)OC(=O)C=C ISXSCDLOGDJUNJ-UHFFFAOYSA-N 0.000 description 1
- 208000002918 testicular germ cell tumor Diseases 0.000 description 1
- CXWXQJXEFPUFDZ-UHFFFAOYSA-N tetralin Chemical group C1=CC=C2CCCCC2=C1 CXWXQJXEFPUFDZ-UHFFFAOYSA-N 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 239000010936 titanium Chemical class 0.000 description 1
- 229910052719 titanium Chemical class 0.000 description 1
- 238000012876 topography Methods 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 239000011534 wash buffer Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 230000036642 wellbeing Effects 0.000 description 1
- 239000012463 white pigment Substances 0.000 description 1
- 239000008096 xylene Substances 0.000 description 1
- 239000001052 yellow pigment Substances 0.000 description 1
- 229910052726 zirconium Inorganic materials 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
Definitions
- the present invention relates to a method of detecting the presence of an antibody in a sample, wherein said antibody has affinity for a coronavirus antigen, for example, a SARS-CoV antigen, a SARS-CoV2 antigen or a MERS- CoV antigen.
- a coronavirus antigen for example, a SARS-CoV antigen, a SARS-CoV2 antigen or a MERS- CoV antigen.
- the present invention also relates to a substrate having immobilised on its surface said coronavirus antigen and methods for producing said substrate.
- SARS-CoV-2 coronavirus pandemic and the incipient mortality and post-infection wellbeing impact has highlighted the fundamental importance of medical healthcare.
- a burgeoning and mobile global population has presented new challenges compared to previous pandemics, and world-wide diagnostic testing and vaccination programmes have emerged as the principle means being used to combat the virus.
- Coronaviruses are a group of related RNA viruses that cause respiratory tract infections in their host, the severity of which can range from mild to lethal. Examples of viruses relating to this family, in addition to SARS-CoV-2, include SARS-CoV and MERS-CoV, both of which have been responsible for viral outbreaks in 2002 and 2012, respectively. Coronaviruses are positive sense single-stranded RNA virus.
- the virion has an average diameter of approximately 80 -120 nm and incorporates four structural proteins, the spike, envelope (“E” protein), membrane (“M” protein) and nucleocapsid (“N” protein) proteins.
- the immune system produces antibodies to various component parts of the coronavirus virion in an individual who has either been infected by the virus or who has been vaccinated for virus protection.
- diagnostic tests relating to detection of coronaviruses, such as SARS-CoV-2, including real-time polymerase chain reaction (RT-PCR), antibody or serology tests and antigen tests, there is a continued need for improved diagnostic assays that can accurately determine if an individual who is/has been infected with a coronavirus has antibodies that confer protective immunity upon the individual.
- Nab neutralising antibodies
- Bab binding antibodies
- Nab are defined as antibodies that prevent entry of the virion into the host cell whereas Bab bind to the coronavirus but do not prevent host-cell infection by the virion.
- Nab can exert their neutralising effect and prevent virion docking onto the host cell receptor, for example, ACE-2, through binding to the coronavirus and directly blocking attachment or by an ‘indirect’ mechanism by altering coronavirus conformation and disabling potential attachment.
- ACE-2 a host cell receptor
- coronavirus diagnostic assays that are amenable to high through-put processing, efficient, reliable and provide a strong indicator as to the level of protective immunity an individual may possess.
- the present invention is based on the surprising discovery that the antibody detection assay herein described provides a highly sensitive and efficacious assay. Furthermore, said assay does not require a pre-incubation step, as is the case with other known assays, and therefore provides for a rapid and energy efficient test format.
- the present invention provides for a method of detecting the presence of an antibody in a sample obtained from a subject, wherein said antibody has affinity for a coronavirus antigen, or portion thereof, and wherein said method comprises bringing the sample obtained from the subject into contact with a coronavirus antigen, or portion thereof, which is immobilised on a substrate support, in the presence of a detectably-labelled molecule that can compete with any antibody present for binding to the coronavirus antigen, or portion thereof, and detecting the binding of antibody to the coronavirus antigen, or portion thereof, by measuring the amount of binding of said molecule compared to a control, wherein the coronavirus antigen, or portion thereof, is immobilised to the substrate surface by means of a linking agent.
- the present invention provides for a substrate having immobilised on its surface a coronavirus antigen, or portion thereof, as defined herein.
- the present invention provides for a method for producing a substrate having a coronavirus antigen immobilised thereon, comprising attaching the coronavirus antigen, or portion thereof, to the substrate surface via a sulphone or sulphonate-based cross-linker.
- Figure 1 shows a schematic of an exemplary assay format in which the receptor binding domain (RBD) is spotted onto a discrete test region (DTR) on the substrate surface.
- the RBD may be covalently attached to the substrate DTR by way of a substrate linking agent and optionally a cross-linker.
- the substrate surface can be optionally covered with an ink layer on the non-DTR area, to reduce non-specific binding of reagents to the substrate.
- the upper part of the RBD comprises the receptor binding motif (RBM).
- the neutralising antibody (Nab) prevents ACE-2 protein from binding to the RBD, whereas the binding antibody (Bab) does not.
- the Nab anti-ACE-2 effect can operate through either binding directly to the RBM (so-called ‘direct’ effect) or to a non-RBM portion of the RBD (so-called ‘indirect’ effect).
- Figure 2 shows the cross-linker 4-[bis(4-methylphenylsulphonylmethyl)-1- oxoethyl] benzoic acid, which can incorporate a silane moiety prior to addition to the substrate (see Figure 7, substrate linking agent B) or can be bonded to a substrate-linking agent (such as a silane moiety) already present on the substrate surface.
- Figure 3 shows the synthesis of 4-[bis(4-methylphenylsulphonylmethyl)-1- oxoethyl] benzoic acid and subsequent addition of a silyl moiety (APTES) to form a substrate linking agent.
- Figure 4 shows calibration curves for competitive neutralisation assay using RBD on substrate and ACE-2-HRP conjugate/anti-RBD antibodies in assay buffer.
- Figure 5 shows generic assay device components for an immunoassay incorporating a sulphone or sulphonate-based cross-linker.
- Figure 6 shows a device that can be used in a SARS-CoV-2 neutralisation antibody assay. Includes the S1 spike subunit with poly-His tag, a sulphone-silyl cross-linker/substrate linking agent and optional ink layer (w/wo means with or without).
- Figure 7 shows examples of suitable sulphone substrate linking agents.
- the terms “patient” and “subject” are used interchangeably herein and refer to any animal (e.g. mammal), including, but not limited to, humans, non-human primates, canines, felines, rodents and the like, which is to be where the sample is obtained from.
- the subject or patient is a human.
- a sample includes biological samples obtained from a patient or subject, which may comprise blood, plasma, serum, urine, saliva, mucous, wax, tears, hair, sweat or sputum.
- the sample is a blood or serum sample.
- detecting refers to qualitatively analysing for the presence or absence of a substance, for example, a signal, usually above a set threshold value to account for background signal noise, indicating presence or absence of the substance.
- determining refers to quantitatively analysing for the amount of substance present.
- detectably-labelled molecule refers to a molecule that has a label covalently attached to said molecule to enable its detection.
- labels may include, but are not limited to, radionuclides, fluorophores, dyes, quantum dots, polymers or enzymes, including, for example, horse-radish peroxidase (HRP) and alkaline phosphatase.
- HRP horse-radish peroxidase
- alkaline phosphatase preferably, the label is HRP
- antibody refers to an immunoglobulin which specifically recognises an epitope on a target as determined by the binding characteristics of the immunoglobulin variable domains of the heavy and light chains (V H S and V L S), more specifically the complementarity-determining regions (CDRs).
- the term “antibody” refers to an immunoglobulin which specifically recognises an epitope on a coronavirus antigen, for example, the spike protein of SARS-CoV, SARS-CoV2 or MERS- CoV, the nucleocapsid protein (“N” protein) of SARS-CoV, SARS-CoV2 or MERS-CoV, the HE protein of SARS-CoV, SARS-CoV2 or MERS-CoV, the Plpro protein of SARS-CoV, SARS-CoV2 or MERS-CoV, the 3CLPro protein of SARS-CoV, SARS-COV2 or MERS-CoV, the E protein of SARS-CoV, SARS- CoV2 or MERS-CoV, or the M protein of SARS-CoV, SARS-CoV2 or MERS- CoV.
- the antibody may recognise an epitope on the N protein, E protein or M protein of SARS-CoV, SARS-CoV2 or MERS- CoV2
- the term “has affinity for”, in the context of antibody-coronavirus antigen interactions, refers to an interaction wherein the antibody and epitope of the coronavirus antigen associate more frequently or rapidly, or with greater duration or affinity, or with any combination of the above, than when either antibody or coronavirus antigen is substituted for an alternative substance, for example an unrelated protein, such as a non-coronavirus related protein, or a coronavirus protein other than the coronavirus antigen in question.
- reference to binding means specific recognition.
- specific binding, or lack thereof may be determined by comparative analysis with a control comprising the use of an antibody which is known in the art to specifically recognise said target and/or a control comprising the absence of, or minimal, specific recognition of said target (for example wherein the control comprises the use of a non-specific antibody).
- Said comparative analysis may be either qualitative or quantitative. It is understood, however, that an antibody or binding moiety which demonstrates exclusive specific recognition of a given target is said to have higher specificity for said target when compared with an antibody which, for example, specifically recognises both the target and a homologous protein.
- coronavirus antigen refers to any part, or portion thereof, of the coronavirus virion that is capable of producing an immune response in the host/subject i.e. the production of antibodies in response to said antigen.
- coronavirus antigen may refer to the spike protein of SARS-CoV, SARS-CoV2 or MERS-CoV, the nucleocapsid protein (“N” protein) of SARS-CoV, SARS-CoV2 or MERS-CoV, the E protein of SARS-CoV, SARS-Co /2 or MERS-CoV, the M protein of SARS-CoV, SARS-CoV2 or MERS- CoV, the HE protein of SARS-CoV, SARS-CoV2 or MERS-CoV, the Plpro protein of SARS-CoV, SARS-CoV2 or MERS-CoV, or the 3CLPro protein of SARS-CoV, SARS-CoV2 or MERS-CoV
- coronavirus spike protein and “S1 protein” are used interchangeably and refers to a glycoprotein found on the surface of all coronaviruses.
- the spike protein is known to be important for the coronavirus gaining entry to the host cell via attaching to the complementary host cell receptor, for example, the host cell receptor for the spike protein of SARS-C0V2 is the angiotensin converting enzyme (ACE-2) receptor, and subsequent fusion with the host cell membrane.
- the spike protein may have the amino acid sequence according to Uniprot accession number P0DTC2, of which the RBD section spans amino acids 319-541 and the RBM section spans amino acids 437-508.
- the spike protein may be from any coronavirus, including variants of the spike protein containing one or more sequence variations or mutations.
- the coronavirus spike protein may be in monomeric form or, it may be a homotrimer to represent its native conformation on the virion.
- the coronavirus spike protein is an RBD or RBM.
- nucleocapsid protein and “N protein” are used interchangeably and refers to a highly conserved immunogenic phosphoprotein found in all coronaviruses that binds to viral RNA and leads to the formation of the helical nucleopcapsid.
- the nucleocapsid protein is composed of two separate domains, an N-terminal domain (NTD) and a C-terminal domain (CTD). It is thought that the abundance and high hydrophilicity of the N protein are key contributors in provoking an immune response in a subject following coronavirus infection.
- M protein or “membrane protein” are used interchangeably and refers to an integral membrane protein that is known to play a crucial role in coronavirus assembly.
- M proteins have three transmembrane domains and are glycosylated in all coronaviruses, either by N-linked or by O- linked oligosaccharides. These oligosaccharides have been shown to be important for folding, structure, stability, intracellular sorting of proteins and to play a role in the generation of immune responses in a subject following coronavirus infection.
- ⁇ protein or “envelope protein” are used interchangeably and refers to a small, hydrophobic membrane protein that contains at least one alpha-helical transmembrane domain.
- the E protein is known to play an important role in the assembly of virions, as well as in the host stress response.
- control refers to a value or values previously determined or determined simultaneously during the assay to which the assay results can be assessed and quantified against.
- the control will typically provide a measure of the extent of binding between the coronavirus spike protein and the detectably-labelled molecule in the absence of antibody. This can be used to provide a measure of the amount of binding that occurs in the assay between the coronavirus spike protein and antibody.
- the signal determined in the assay will be reduced due to the competitive binding of the antibody.
- the extent of the reduction compared to the control value(s) will indicate whether antibody is present in the sample and optionally the concentration of the antibody present.
- the control can be established as a calibration, alternatively, a calibration curve can be generated using neutralizing antibody preparations at multiple concentrations (see Figure 4).
- the assay signal output generated from a sample can be applied to the calibration curve to enable quantification of the Nab activity of said sample.
- epitope refers to the portion of a target which is specifically recognised by a given antibody.
- substrate linking agent As used herein, the terms “substrate linking agent”, “linker”, “linking agent” and “linking group” are used interchangeably and refers to a chemical moiety that connects the coronavirus spike protein to the substrate surface.
- cross-linker refers to any suitable chemical entity capable of binding a substrate linking agent to a coronavirus antigen, or portion thereof, for example, the RBD or RBM of the spike protein, the N protein, the M protein or the E protein.
- competitive and “competitive assay” are used interchangeably, and in the context of the present invention refers to the detectably labelled molecule and antibodies present in a sample competing for binding to the chip on which is immobilised a coronavirus spike protein.
- Such a competitive assay format may be a simultaneous competitive assay format (for example, the sample and detectably-labelled molecule are added at the same time) or a sequential competitive assay (for example, the detectably-labelled molecule is added to the substrate subsequently to the sample). Accordingly, the skilled person will readily understand that the assay herein disclosed is considered a “competitive assay” regardless of the order or timing of the addition of the competing moieties.
- the present invention provides for an assay device by which antibodies having affinity for a coronavirus antigen, or portion thereof, can be efficiently and accurately determined.
- Such an assay allows for an effective method of diagnosing subjects or patients suffering from a coronavirus related disease, for example, COVID-19, severe acute respiratory syndrome or Middle East respiratory syndrome, as well as an additional means of identifying subjects who may now have active immunity from said coronavirus.
- the present invention provides for a method of detecting the presence of an antibody in a sample obtained from a subject, wherein said antibody has affinity for a coronavirus antigen, or portion thereof, and wherein said method comprises bringing the sample obtained from the subject into contact with a coronavirus antigen, or portion thereof, which is immobilised on a substrate support, in the presence of a detectably-labelled molecule that can compete with any antibody present for binding to the coronavirus antigen, or portion thereof, and detecting the binding of antibody to the coronavirus antigen, or portion thereof, by measuring the amount of binding of said molecule compared to a control, wherein the coronavirus antigen, or portion thereof, is immobilised to the substrate surface by means of a linking agent.
- the coronavirus antigen, or portion thereof may be any part of a coronavirus that is capable of generating antibodies in a subject.
- the coronavirus antigen, or portion thereof may be a coronavirus spike protein, a coronavirus N protein, a coronavirus E protein, a coronavirus M protein and/or combinations thereof.
- the antigen, or portion thereof is the coronavirus spike protein.
- the method herein disclosed uses a device in which the coronavirus antigen, or portion thereof, is immobilised on the surface and is brought into contact with an ex vivo sample and a detectably-labelled molecule that has affinity for the coronavirus antigen, or portion thereof. It is therefore understood that, in one format, such an assay is a competitive binding assay, with the potential antibodies in the sample competing with the detectably-labelled molecule for binding to the coronavirus antigen, or portion thereof. Accordingly, a reduction in the signal produced by the detectably-labelled molecule signifies that there is a higher level of antibodies having affinity for a coronavirus antigen in the sample, and vice versa.
- the format of the assay herein described provides for an improved assay where the sample and detectably-labelled molecule do not require a pre-incubation step, thus allowing for a more efficient assay to be provided.
- the sample and detectably-labelled molecule can be mixed immediately prior to addition to the device/substrate surface, or added at the same time.
- the assay herein disclosed may be a simultaneous competitive assay format.
- the sample may be added first to the substrate, with the detectably-labelled molecule added to the substrate in a sequential step.
- the coronavirus spike protein, or portion thereof, immobilised on the substrate support may be brought into contact with the sample prior to the addition of the detectably-labelled molecule to the substrate support.
- the assay disclosed herein may be a sequential competitive binding assay. This format provides a large reduction in the expected binding when the detectably-labelled molecule is added if said sample contains antibodies having affinity for the coronavirus antigen, or portion thereof.
- the method herein disclosed provides an assay whereby the coronavirus antigen, or portion thereof, is immobilised to the substrate surface by means of a linking agent and optionally a cross-linker.
- Such a format allows for the projection of the coronavirus antigen, or portion thereof, away from the substrate surface, exposing the binding portion of the coronavirus antigen, or portion thereof, as well as producing a greater engagement of the coronavirus antigen, or portion thereof with the sample compared to a passive adsorption, resulting in a more sensitive assay. Additionally, this particular format minimises the influence of the substrate surface, leading to improved assay kinetics. Further details of appropriate linking agents and cross-linkers are provided below.
- the method herein disclosed may be suitable for the detection of both neutralising and binding antibodies, however, it is particularly envisaged that the method may be used to detect an antibody which is a neutralising antibody.
- the presence of Nab are said to demonstrate the individual having protective immunity against said coronavirus, and therefore have obvious benefits in being able to accurately and efficiently determine both their presence and amount in which they are present.
- the present invention also provides for a method of detecting whether an individual has protective immunity against a coronavirus, for example, SARS-CoV, SARS-CoV2, and MERS-CoV. Nab exert their neutralising effect by preventing the coronavirus from docking to its corresponding host cell receptor.
- Nab can prevent the coronavirus docking to the host cell by both direct and indirect mechanisms.
- the direct mechanism may involve the Nab binding to the RBM of the coronavirus spike protein and therefore preventing any interaction between the coronavirus spike protein and the corresponding host cell receptor.
- indirect mechanisms may involve the Nab binding elsewhere on the coronavirus, thus resulting in a conformational change of the coronavirus proteins important for coronavirus-host cell interaction, and preventing binding to the corresponding host cell receptor in this manner.
- the method herein may be used to detect antibodies having affinity for any coronavirus antigen, or portion thereof.
- the coronavirus antigen, or portion thereof may be a SARS-CoV antigen, SARS-CoV2 antigen or MERS- CoV antigen.
- the coronavirus antigen, or portion thereof may be a SARS-CoV2 spike protein, N protein, M protein and/or E protein.
- it is the SARS-C0V2 spike protein.
- the method herein may be used to detect neutralising antibodies having affinity for any coronavirus antigen, or portion thereof, for example, SARS-CoV antigen, SARS-CoV2 antigen or MERS-CoV antigen.
- the method herein disclosed may be used to detect neutralising antibodies having an affinity for SARS-C0V2.
- the method herein disclosed is exemplified in relation to SARS-CoV, SARS-C0V2 and MERS-CoV, it is envisaged that said method will also be applicable to any coronavirus, including any as of yet unknown future novel coronaviruses.
- the detectably-labelled molecule may be dependent on the coronavirus in question, for example, if the coronavirus is SARS-CoV, SARS-CoV2 or MERS-CoV. Accordingly, if the method is for the detection of antibodies, for example, neutralising antibodies, having affinity for the SARS-CoV spike protein or the SARS-Co /2 spike protein, the detectably-labelled molecule can be ACE-2. However, if the coronavirus spike protein is the MERS-CoV spike protein, then the detectably labelled molecule can be DPP4.
- coronavirus antigen, or portion thereof is not a coronavirus spike protein
- other suitable molecules can be used to compete with the antibodies in the patient sample, wherein said suitable molecules have affinity for the coronavirus antigen, or portion thereof, for example, wherein said suitable molecules have affinity for an epitope present on the N protein, M protein and/or E protein of SARS-CoV, SARS-CoN/2 or MERS-CoV.
- a detectably-labelled antibody (or suitable fragment or derivative thereof) that has affinity for the coronavirus antigen, or portion thereof could be used in the competition assay format as disclosed herein. This concept applies to any other coronavirus that may be used in the context of the method herein disclosed, including any future unknown coronavirus to which the target receptor is as of yet also unknown.
- the antibodies to be detected in a sample obtained from a subject may have an affinity for any epitope on the coronavirus antigen in question, for example, the SARS-CoV spike protein, N protein, M protein, or E protein, the SARS-CoV2 spike protein, N protein, M protein or E protein, or the MERS-CoV spike protein, N protein, M protein or E protein.
- the antibody may have an affinity for the RBD of the coronavirus spike protein in question.
- the antibody may have an affinity for the RBM of the coronavirus spike protein in question.
- the antibodies to be detected in a sample obtained from a subject may have an affinity for any epitope on a coronavirus antigen, for example, the antibodies to be detected may have an affinity for one or more of the coronavirus N protein, M protein and/or E protein.
- the different epitopes to which the antibodies bind may affect the mechanism by which the antibody exerts its effect. For example, for Nab, this may directly prevent the coronavirus from binding to the host cell receptor, for example, ACE-2 or DPP4.
- a conformational change may be induced which affects the binding capability of the coronavirus, thus preventing the binding to the host cell receptor in this way, for example, a partial blocking effect may occur, in which binding of the Nab reduces the efficiency of the virion approach, for example, to ACE-2 or DPP4.
- the presence of a linking agent and optionally a cross linker on the substrate surface allows for the coronavirus antigen, or portion thereof to be projected away from the substrate surface, exposing the binding portion of the coronavirus antigen, or portion thereof and producing a greater engagement of the coronavirus antigen, or portion thereof, with the sample compared to if no linking agent was present, i.e. passive adsorption.
- the present invention provides for a method in which the coronavirus antigen, or portion thereof, may be covalently attached to the substrate. This feature inevitably results in a more sensitive assay.
- the substrate linking agent may be any suitable chemical moiety that can support/bond to the coronavirus antigen, or portion thereof, or bond to a cross linker.
- the linking agent is an epoxy silane derivative, an epoxy oligomer or an epoxy polymer. Examples include, but are not limited to, (EtO) 3 Si-(CH2)n-NH 2 (see EP0874242 for silane derivative examples) and EPON-SU8 (also referred to as Epoly-8).
- the linking agent is EPON- SU8 (CAS No. 28906-96-9, commercially available from Hexion Inc).
- the coronavirus antigen, or portion thereof engages with the substrate (i.e. is supported by the substrate) by way of covalent bonds provided by a linking agent covalently attached to the substrate surface (the substrate linking agent) prior to coronavirus antigen addition.
- the coronavirus antigen, or portion thereof, for example, a SARS-CoV, a SARS- CoV2 or a MERS-CoV antigen may bond to the substrate linking agent and optionally a cross-linker through inherent functional groups such as carboxy, amino or sulphur present in the amino acids that constitute the protein.
- the assay effectiveness may be improved via the incorporation of a chemical activating group in the coronavirus antigen, or portion thereof.
- the attachment of the coronavirus antigen, or portion thereof, to a substrate linking agent and optionally a cross-linker requires that the binding characteristics of the coronavirus antigen, or portion thereof, are not affected, and that the specificity and affinity of the coronavirus antigen, or portion thereof, following the bonding process remain fit for purpose.
- Possible deleterious effects upon the coronavirus antigen, or portion thereof, upon chemical activation and or bonding to the substrate include coronavirus antigen tertiary structure disruption leading to reduced bonding and/or specificity to the target antibody.
- the method herein disclosed may use a coronavirus antigen, or portion thereof, immobilised to the substrate surface that further comprises a protein tag.
- the method herein disclosed may use a coronavirus antigen, or portion thereof, immobilised to the substrate surface that further comprises a poly-His tag or a coronavirus antigen, or portion thereof, immobilised to the substrate surface that has been modified to have a poly-His tag.
- the SARS-CoV spike protein, N protein, M protein or E protein may further comprise a poly-His tag or be modified to have a poly-His tag
- the SARS-CoV2 spike protein, N protein, M protein or E protein may further comprise a poly-His tag or be modified to have a poly-His tag
- the MERS-CoV spike protein, N protein, M protein or E protein may further comprise a poly-His tag or be modified to have a poly-His tag. Accordingly, such a modification results in a recombinant coronavirus antigen, or portion thereof, that helps to reduce structural disruption of the coronavirus antigen, or portion thereof, when bound to the linking agent/cross-linker.
- poly-His tag preferably a poly-His tag
- recombinant DNA methods comprising inserting the DNA encoding a protein into a suitable vector encoding a His-tag (Loughran and Walls, (2011), Purification of poly-histidine-tagged proteins, Methods Mol Biol, 681 :311 -35).
- the method herein disclosed may comprise a recombinantly produced coronavirus antigen, or portion thereof, incorporating a poly-His tag attached to a substrate linking agent/cross-linker.
- the poly-His tag can be varied in the number of histidine units but it is preferable to incorporate between 2 and 10 histidines, more preferably 6, 7 or 8 histidines; a 6 unit histidine tag is most preferred.
- the method herein disclosed also discloses an assay in which the coronavirus antigen, or portion thereof, may be immobilised to the substrate surface by means of a linking agent and a cross-linker.
- the method herein disclosed may use any cross-linker that is capable of binding the substrate linking agent to the coronavirus antigen, or portion thereof, for example, a coronavirus spike protein, N protein, M protein or E protein.
- the cross-linker is a bifunctional cross-linker.
- a bifunctional cross linker we refer to a molecule that has two reactive groups, each reactive group capable of reacting with a moiety of the substrate linking agent or moiety of the coronavirus antigen, or portion thereof, such that the substrate linking agent and coronavirus antigen, or portion thereof, are bound together by the cross-linker.
- the two reactive groups may be the same or different.
- Suitable bifunctional cross-linkers are well known in the art (for example, suitable bifunctional cross linkers are disclosed in Bioconjugate Techniques, G.T. Hermanson, Third Edition 2013).
- the cross-linker may be bonded to the surface linking agent prior to their attachment to the substrate surface; alternatively, the cross-linker may be bonded to a substrate linking agent, the substrate linking agent having already been attached (covalently bonded) to the substrate surface, or the cross-linker agent may be attached to the coronavirus antigen, or portion thereof, prior to their attachment to the substrate linking agent.
- “Sulphone-based cross-linker” refers to a cross-linker comprising at least one sulphone moiety
- “sulphonate-based” refers to a cross-linker comprising at least one sulphonate moiety.
- Suitable examples of sulphone-based cross-linkers include, but are not limited to: a,b-unsaturated ketone sulphones and precursors to a,b-unsaturated ketone sulphones, such as the bis-sulphone of Figure 3. Accordingly, in a preferred embodiment, a,b- unsaturated ketone sulphones and precursors to a,b-unsaturated ketone sulphones are used.
- sulphonate-based cross-linkers include, but are not limited to: a,b- unsaturated ketone sulphonates and precursors to a,b-unsaturated ketone sulphonates.
- Such cross-linkers incorporate strong leaving groups which promote favourable reaction kinetics and bonding to the poly-His tag if present in the coronavirus spike protein. It is noted that, for this reason, sulphonate-based cross-linkers may also be used as the substrate linking agent. Without being bound by theory, it is believed that the use of sulphonate-based cross-linkers in this manner achieve this effect due to their strong leaving group properties.
- the cross-linker when the cross-linker is a sulphone-based cross-linker, preferably an a,b- unsaturated ketone sulphone or precursor thereof, the cross-linker may have a structure according to Formula wherein X is -CH 2 -;
- R 2 is selected from -Ce- ⁇ aryl-Z, -Ci-isalkyl-Z, and -C2-2oalkenyl-Z, wherein Z is selected from COR 3 , -NH 2 and -OH, and R 3 is selected from H, OH, NH 2 , -Ci- ealkyl-OH and (EtO) 3 Si-(CH 2 ) n -NH- in which n is 1-6; and R is H or is optionally substituted with one or more Ci-ealkyl, N0 2 , F, Cl or Br;.
- Ri is CH 3 -Ph
- Ph is substituted with a Ci-ealkyl group, preferably methyl.
- R 2 is selected from -Ph-Z, -CioH s -Z, -Ci-ealkyl-Z and -C 2 -ealkenyl-Z, more preferably from -Ph-Z and -CioH s -Z, and more preferably -Ph-Z.
- Z is selected from COR 3 .
- the cross-linker is a sulphone-based cross-linker, preferably an a,b- unsaturated ketone sulphone or precursor thereof, the cross-linker may have a structure according to Formula (2): wherein X is -CH 2 -;
- Ri is CH 3 or CH 3 -Ph, wherein the Ph is optionally substituted with one or more Ci-ealkyl;
- R 3 is selected from H, OH, NH 2 , -Ci-ealkyl-OH and (EtO) 3 Si-(CH 2 ) n -NH- in which n is 1-6.
- Ri is CH 3 -Ph, and Ph is substituted with a Ci-ealkyl group, preferably methyl.
- R 3 is OH, H or (EtO) 3 Si-(CH 2 )n-NH- in which n is 1-6.
- the cross-linker when the cross-linker is a sulphone-based crosslinker, preferably an a,b- unsaturated ketone sulphone or precursor thereof, the cross-linker may have a structure according to Formula (3) or Formula (4): wherein R 3 is selected from H, OH, NH 2 , -Ci-ealkyl-OH and (EtO) 3 Si-(CH 2 ) n -NH- in which n is 1-6, preferably OH, H or (EtO) 3 Si-(CH 2 ) n -NH- in which n is 1-6.
- the cross-linker is a sulphone-based cross-linker. More preferably, the cross-linker is a sulphone-based crosslinker of Formulas (1), (2), (3) or (4) as detailed above.
- Ph refers to phenyl
- CioH s refers to a naphthalene or napthyl group.
- Ci-is alkyl demotes a straight or branched saturated alkyl group having from 1 to 18 carbon atoms; For parts of the range Ci-is alkyl, all sub-groups thereof are contemplated, such as Ci-e alkyl, C 5-15 alkyl, C 5-10 alkyl, and C 1-6 alkyl.
- Examples of said C 1-4 alkyl groups include methyl, ethyl, n- propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, and tert-butyl.
- the alkyl groups may be optionally substituted with one or more functional groups, including C 1-18 alkyl groups, "Ce- 12 aryl", and "C 1-18 alkoxy", halogen, and "C 3-18 cycloalkyl".
- C 2 -isalkenyl denotes a "Ci-is alkyl” group containing some degree of unsaturation (partial unsaturation) i.e. containing one or more alkene/alkenyl moiety(s).
- Ce- 12 aryl denotes a monocyclic or polycyclic conjugated unsaturated ring system having from 6 to 12 carbon atoms.
- Ce- 12 aryl all sub-groups thereof are contemplated, such as Ce-io aryl, C 10-12 aryl, and Ce-s aryl.
- An aryl group includes condensed ring groups such as monocyclic ring groups, or bicyclic ring groups. Examples of Ce- 12 aryl groups include phenyl, biphenyl, indenyl, naphthyl or azulenyl.
- Condensed rings such as indan and tetrahydro naphthalene are also included in the Ce- 12 aryl group.
- the aryl groups may be optionally substituted with other functional groups.
- the aryl groups may be optionally substituted with one or more functional groups, including C 1-18 alkyl groups, halogen, and "C 1-18 alkoxy".
- the aryl groups may be substituted with these substituents at a single position on their unsaturated ring system, or may be substituted with these substituents at multiple positions on their unsaturated ring system.
- the term “fully saturated” refers to rings where there are no multiple bonds between ring atoms.
- the cross-linker may be subject to chemical activation prior to attachment to the substrate linking agent.
- chemical activation is meant a process whereby the cross-linker is altered such that it has increased propensity for subsequent reaction, i.e. increased propensity for bonding with the substrate linking group and/or coronavirus spike protein.
- Suitable methods of chemical activation will be well known by a skilled person, for example, the EDC method or a maleimido-group incorporation.
- Z of formula (1) is COR 3 and R 3 is OH
- R 3 of formula (2) is OH
- the EDC method or a maleimido-group incorporation may be used to activate the cross-linker.
- the present method also provides for the incorporation of a substrate linking agent in the sulphone- or sulphonate-based cross-linker prior to the addition to the substrate. This has the advantage of omitting the substrate chemical-activation step, which in turn benefits efficiency of device production.
- Figures 3 and 5 present examples of silyl-sulphone compounds.
- the method herein provides for an immunoassay in which the antibodies in a sample and a detectably-labelled molecule compete with one another to bind to the immobilised coronavirus antigen, or portion thereof, bonded to the substrate surface via the linking agent and optionally the cross-linker described above (see Figure 1). Accordingly, the detectably-labelled molecule provides a detectable and measurable signal which is reduced in the presence of antibodies, thus allowing the antibodies present in said sample to be detected and/or quantified.
- the measurable signal may be electromagnetic radiation based on, for example, phosphorescence, fluorescence, chemiluminescence (e.g. HRP/luminol/peroxide system).
- fluorescence or chemiluminescence is used.
- a detecting agent suitable for use with the present invention is the streptavidin- biotin-enzyme complex
- avidin may also be used in place of streptavidin, resulting in a complex with the molecule to be detected, for example, an ACE-2 - biotin-streptavidin-enzyme complex, a DPP4-biotin-streptavidin-enzyme complex or an antibody-(or suitable fragment or derivative thereof)-biotin-streptavidin- enzyme complex.
- the method herein disclosed may include a detectably-labelled molecule labelled with a streptavidin-biotin-enzyme complex or an avidin-biotin-enzyme complex.
- the enzyme of said complex may be HRP, which when exposed to luminol/peroxide system produces a detectable signal.
- a calibrator or standard which can be used for effecting assay calibration, is well known in the art and enables a threshold concentration or the exact or calibrator equivalent amount of analyte(s) to be determined. The determination of an exact or calibrator equivalent amount of analyte(s) usually requires the construction of a calibration curve (also known as a standard curve). The number of calibrator points vary but is preferably from 5 to 9. Alternatively, the calibrator value can be a single pre-determined threshold value.
- the surface of the substrate for use in the method of the present invention may be selectively covered with a coating composition.
- the coating composition may be any coating that would be expected to enhance or maintain the desirable properties of the surface to which the coronavirus antigen, or portion thereof, is immobilised, i.e. the DTR.
- the term “selectively covered”, as used in the context of the present invention, refers to areas of the substrate where the coronavirus antigen, or portion thereof, is immobilised.
- the coating composition may be an ink formulation.
- Ink formulations have been added to the surfaces of substrates, such as chips and biochips used in medical diagnostics, to minimise the attachment of chemical and biological components present in patient samples which can affect assay performance. It is understood that the choice of ink can be dependent upon the assay configuration of the substrate and the assay objectives and is usually characterised as being on the hydrophilic-hydrophobic continuum using the water contact angle. It has been found that the assay devices and methods of the current invention can further benefit from an increase in performance if the substrate surface supports an ink layer. This leads to yet a further increase in the sensitivity of the assay.
- the ink formulation may comprise a pigment, a resin, an ink solvent, an ink additive and/or a structuring agent.
- pigments may be selected from the group of inorganic artificial pigment, inorganic natural pigment, organic artificial pigment, organic natural pigment, black pigment, blue pigment, brown pigment, green pigment, orange pigment, red pigment, violet pigment, white pigment or yellow pigment.
- resins include, but are not limited to, acrylics, alkyds, epoxides, hydrocarbons, phenolics or fluoropolymers such as a polytetrafluoroethylene (PTFE).
- Suitable ink solvents include, but are not limited to, cyclohexanone, butoxyethanol and aromatic distillates.
- Suitable ink additives include, but are not limited to, carbon black (black pigment), mineral oil (wetting agent), petroleum distillate, dibutyl phthalate (plasticizer), salts of cobalt, manganese or zirconium (drying agent), aluminium and titanium chelator (chelating agent), antioxidants, surfactants and defoamers.
- Suitable structuring agents include, but are not limited to CERAFLOUR® 965.
- the ink formulation may comprise one or more resins, pigments, ink solvents/additives and/or structuring agents selected from the lists above, and combinations thereof.
- the ink formulation may be a composition according to those disclosed in EP3377900 A1.
- the ink formulation may comprise an epoxy or acrylic resin, a pigment and a structuring agent.
- the ink composition may comprise an acrylic resin, a pigment and a structuring agent.
- One pigment may be present or multiple pigments may be used.
- Epoxy and acrylic resin are used to increase ink viscosity, rheological properties and adhesion to the substrate.
- the pigment imparts a dark colour, preferably a black colour, and hence imparts optical opacity to the ink.
- the structuring agent provides hydrophilic/hydrophobic properties to the surface of the substrate and also help adhesion to the substrate.
- the pigment may be present in an amount of 1 to 15% w/w of the ink formulation; the epoxy resin may be present in an amount of 10 to 60 % w/w, the acrylic resin may be present in an amount of 1 to 20% w/w, and the structuring agent may be present in an amount of 10 to 60% w/w.
- the pigment preferably black pigment, is present in an amount of 1 to 8% w/w of the ink formulation; the epoxy resin is present in an amount of 15 to 50 % w/w of the ink formulation, the acrylic resin is present in an amount of 2 to 15% w/w of the ink formulation, and the structuring agent is present in an amount of 15-50% w/w of the ink formulation.
- the pigment preferably black pigment, is present in an amount of 5% w/w of the masking composition; the epoxy resin is present in an amount of 30 % w/w of the ink formulation, the acrylic resin is present in an amount of 10% w/w of the ink formulation, and the structuring agent is present in an amount of 20% w/w of the ink formulation.
- carbon black pigment is used in the ink formulation, preferably Elftex 285.
- the acrylic resin is B-67.
- the structuring agent is a PTFE wax, such as CERAFLOUR® 965.
- the ink formulation comprises an epoxy resin, preferably Epikote 1004.
- the pigment is Elftex 285, the acrylic resin is B-67, the epoxy resin is Epikote 1004 and the structuring agent is CERAFLOUR® 965.
- the ink formulation may further comprise one or more agents selected from the list of solvents, such as ethanol, propanol, xylene, diglycol, butyl ether; dispersing agents; pigment wetting agents; levelling agents; pigment wetting agents and/or crosslinking agents.
- the ink formulation has a contact angle of 20- 175°, more preferably 20-170° more preferably 90-120°, even more preferably about 1 10°.
- the measurement is taken using the following protocol: The contact angle is measured using a KSV CAM200 contact angle meter equipped with automated dispenser controlled using stepper motor, LED source and CCD camera.
- the contact angle meter is connected to a software tool for dispense controller, image grabbing and image analysis. A droplet of deionised water of 3.5 pi is dispensed on the substrate at a predefined location and the image is captured using a CCD camera. Image analysis is performed using software to estimate the contact angle of the water droplet.
- the thickness of the ink formulation applied to the substrate is 1-100 pm thick, preferably, 2-50 pm thick. This creates a discrete reaction zone that is a well having a depth of 1 -100 pm, preferably 2-50 pm, respectively. Most preferably the thickness of coating is 3 to 20 pm thick and the resulting depth of the well is 3 to 20 pm in depth.
- the ink is applied to the substrate such that there are exposed areas of the substrate that define the reaction sites.
- the areas coated with the ink are not intended to be the reaction sites and provide a typically hydrophobic surface that prevents non-specific binding from occurring and helps retain solutions at the reaction site to provide a clear image of the reaction site.
- the sample for use in the method of the present invention may be any biological sample taken from the individual in which antibodies may be detected.
- the biological samples require no pre-processing and can be used neat in the assay herein disclosed.
- the sample may be a serum sample, plasma sample, whole blood sample, urine sample, mucous sample, saliva sample, CSF sample, sputum sample, ear wax sample, hair sample, sweat sample, tear sample, meconium, skin, solid tumour extracts, peripheral blood mononuclear cells, bone marrow mononuclear cells, cerebrospinal fluid, cystic fluid or any suitable cell lysate.
- the sample is a serum or plasma sample; alternatively, it is a whole blood sample or a saliva sample.
- the sample may be obtained from the subject or patient by methods routinely used in the art, for example, via venous blood collection, swab testing or tissue biopsy.
- the determination and/or detection of antibodies, for example, neutralising antibodies may be done on one or more samples obtained from the subject.
- the substrate for use in the present invention may be of a planar conformation, such as a glass slide, microtitre plate or a chip/biochip.
- the term “biochip” refers to a chip whose use is biomedical and is made of a thin, wafer like substrate with a planar surface.
- the substrate may be a bio-chip due to its stability and adaptability.
- the biochip may be made of any suitable material, such as glass or any suitable polymer, preferably, the biochip is made of ceramic. Even more preferably, the biochip is made of aluminium oxide based ceramic and may be chemically activated.
- a ceramic substrate can be manufactured to provide a range of grain sizes.
- Typical grain sizes are 1 to 30 pm; ⁇ 10 pm is preferred as the reduced particle size imparts increased surface homogeneity which improves assay performance.
- the preferred ceramic material consists of about 94% alumina (AI 2 O 3 ) with a particle size in the range of 4-8 pm.
- the surface topography is usually withing the range of 0.6 to 0.8 pm after being ground. This can be improved through polishing to yield a surface with variation of 0.4-0.5 pm which can be further improved through by lapping and polishing to 0.05-0.1 pm.
- the present invention provides for a substrate having immobilised on its surface a coronavirus antigen, or portion thereof, as defined by any of the features herein described. Accordingly, the present invention provides for a substrate having immobilised on its surface a SARS-CoV antigen, a SARS CoV-2 antigen or a MERS-CoV spike antigen connected by a linking agent and optionally a cross-linker.
- the substrate may have immobilised on its surface a single type of coronavirus and/or coronavirus antigen, or portion thereof, for example, SARS-CoV spike protein, N protein, M protein, E protein, SARS-C0V2 spike protein, N protein, M protein, E protein or MERS-CoV spike protein, N protein, M protein or E protein.
- the substrate may have immobilised on its surface a combination of coronavirus and/or coronavirus anitgens, for example, SARS-CoV spike protein, N protein, M protein, E protein, SARS-Co /2 spike protein, N protein, M protein, E protein or MERS-CoV spike protein, N protein, M protein or E protein or a combination of variants of the coronavirus antigen.
- each detectably-labelled molecule having affinity for a specific coronavirus antigen, or portion thereof may have different labels, for example, different fluorophores, attached in order to differentiate the signal arising from different coronavirus antigens, or portions thereof.
- the substrate herein disclosed may be spatially arranged in such a manner that the same effect is achieved and the same detection labels may be used. The skilled person will recognise that being able to identify antibodies in a sample directed at multiple targets will help reduce the level of false negatives, thus resulting in a more sensitive, accurate and reliable assay.
- the present invention provides for a substrate having immobilised on its surface a SARS-CoV2 antigen, or portion thereof.
- the linking agent is EPON-SU8 and the cross-linker is a sulphone- or sulphonate-based cross-linker according to Formula (1), even more preferably, the cross-linker is a sulphone-based crosslinker according to Formula (2), (3) or (4).
- the substrate is a bio-chip, even more preferably, the substrate is a ceramic bio-chip.
- the substrate may be selectively covered with an ink formulation, as described above.
- a method for producing a substrate having a coronavirus antigen, or portion thereof, immobilised thereon, comprising attaching the coronavirus antigen, or portion thereof, to the substrate surface via a sulphone or sulphonate-based cross-linker comprising attaching the coronavirus antigen, or portion thereof, to the substrate surface via a sulphone or sulphonate-based cross-linker.
- the coronavirus antigen, or portion thereof may be a SARS-CoV antigen, or portion thereof, SARS-CoV2 antigen, or portion thereof, or a MERS-CoV antigen, or portion thereof.
- the coronavirus antigen, or portion thereof is a SARS-CoV2 antigen, or portion thereof.
- the present invention also discloses, a coronavirus antigen, or portion thereof recombinantly modified to include a poly-His tag.
- the coronavirus antigen, or portion thereof, recombinantly modified to include a poly-His tag may be a SARS-CoV antigen, or portion thereof, a SARS-CoV2 antigen, or portion thereof, or a MERS-CoV2 antigen, or portion thereof.
- the coronavirus antigen, or portion thereof, recombinantly modified to include a poly-His tag is a SARS-C0V2 antigen, or portion thereof.
- SEQ ID NO: 2 and SEQ ID NO: 6 were expressed in human cells using standard cloning of codon optimized SEQ ID NO: 4 and SEQ ID NO: 8 respectively into a constitutive high level mammalian expression vector with an artificial or native signal peptide sequence and C terminal His tag, resulting in the expression of SEQ ID NO: 2 and SEQ ID NO: 6 and after removal of the signal peptide sequence, SEQ ID NO: 3 and SEQ ID NO: 7.
- Transfected cells were cultured at 37°C and 8% CO2 in Expression Medium (Thermoscientific) containing glutamine for three to six days before harvesting.
- SEQ ID NO: 3 and SEQ ID NO: 7 comprised cell supernatant collection which was clarified by centrifugation for 30 minutes at 7,000rpm. Remaining particulate matter was removed by filtration through 1.2mM and 0.45mM low protein binding filter units respectively.
- SEQ ID NO: 3 and SEQ ID NO: 7 protein was purified from the clarified and filtered supernatant at 4°C by the Akta Avant system (Cytiva) using Nickel HisTrap Excel columns (Cytiva Lifesciences, USA) preequilibrated with 20mM Tris, 500mM Sodium chloride pH8.0.
- Contaminant non-specific binding protein was removed by a wash containing 20mM Tris, 500mM sodium chloride, 30mM imidazole and SEQ ID NO: 3 and SEQ ID NO: 7 were eluted by increasing the concentration of imidazole to 500mM. All fractions containing either SEQ ID NO: 3 or SEQ ID NO: 7 were pooled and concentrated by ultrafiltration (Vivaspin, Sartorius, Gottingen Germany) and buffer exchanged into PBS pH7.4 (Melford, UK).
- SEQ ID 3 and SEQ ID NO: 7 were further purified by fractionation on a HiLoad 26/600 Superdex 75 PG column (GE Healthcare, USA) pre-equilibrated in 2X PBS pH7.4 (Melford, UK). The column fractions were monitored for absorbance at A280nm. Selected fractions containing SEQ ID NO: 3 or SEQ ID NO: 7 were collected, pooled and concentrated by ultrafiltration (Vivaspin, Sartorius, Gottingen Germany). The final preparations of SEQ ID NO: 3 and SEQ ID NO: 7 proteins were evaluated by SDS gel electrophoresis. For spotting to the surface of the biochip, expressed protein SEQ ID NO: 3 was diluted in buffer.
- SARS-CoV-2 Nucleoprotein (Uniprot ID P0DTC9) was expressed in human cells using standard cloning methods into a constitutive high level mammalian expression vector with an artificial signal peptide sequence and C terminal His tag, resulting in the expression of SEQ ID NO: 9 after removal of the signal peptide.
- Transfected cells were cultured at 37oC and 8% C02 in Expression Medium (Thermoscientific) containing glutamine for three to six days before harvesting.
- SEQ ID NO: 9 Preparation of SEQ ID NO: 9 comprised cell supernatant collection which was clarified by centrifugation for 30 minutes at 7,000rpm. Remaining particulate matter was removed by filtration through 1.2mM and 0.45mM low protein binding filter units respectively.
- SEQ ID NO: 9 protein was purified from the clarified and filtered supernatant at 4°C by the Akta Avant system (Cytiva) using Nickel HisTrap Excel columns (Cytiva Lifesciences, USA) preequilibrated with 20mM Tris, 500mMSodium chloride pH8.0.
- Contaminant non-specific binding protein was removed by a wash containing 20mM Tris, 500mM sodium chloride, 30mM imidazole and SEQ ID NO: 9 was eluted by increasing the concentration of imidazole to 500mM. All fractions containing either SEQ ID NO:9 was pooled and concentrated by ultrafiltration (Vivaspin, Sartorius, Gottingen Germany) and buffer exchanged into PBS pH7.4 (Melford, UK). Expressed SEQ ID NO: 9 was further purified by fractionation on a Hil_oad26/600 Superdex 75 PG column (GE Healthcare, USA) pre-equilibrated in 2X PBSpH7.4 (Melford, UK).
- SEQ ID NO: 9 The column fractions were monitored for absorbance atA280nm. Selected fractions containing SEQ ID NO: 9 was collected, pooled and concentrated by ultrafiltration (Vivaspin, Sartorius, Gottingen Germany). The final preparations of SEQ ID NO: 9 proteins were evaluated by SDS gel electrophoresis. For spotting to the surface of the biochip, expressed protein SEQ ID NO: 9 was diluted in buffer.
- a ceramic substrate was washed with RBS 35 concentrate and water and plasma treated before addition of Epon SU8 (Hexion Incorporated, Ohio, US) or (OEt)3-Si-(CH 2 )3-NH 2 or a silyl-sulphone (B of Figure 7). Following stirring it was deposited on the ceramic substrate by spray coating and the chips cured for 1 hr at 140°C.
- Epon SU8 Hydrophilic Incorporated, Ohio, US
- OEt OEt3-Si-(CH 2 )3-NH 2 or a silyl-sulphone
- Substrate linking agent/Cross-linker preparation (see Figures 3 & 7) Substrate linking agent/Cross-linker A. To a cooled solution (0°C) of (3- aminopropyl) triethoxysilane (APTES) (22.131 g, 0.1 mol) and diisopropylethylamine (20.9mls, 0.12mol) in dichloromethane (300ml) under nitrogen was added dropwise a solution 2-chloroethylsulphonyl chloride (16.63g, 0.102mol) in dichloromethane (50mls). The mixture was than stirred at 0°C for two hours and then overnight at room temperature.
- APTES (3- aminopropyl) triethoxysilane
- 2-chloroethylsulphonyl chloride (16.63g, 0.102mol
- Substrate linking agent/Cross-linker B To a solution of bis-sulphone acid (2g, 4mmol) in dichloromethane was added N-hydroxysuccinimide (506mg, 1.1 eq) and dicyclohexylcarbodiimide (908mg, 1.1 eq) under nitrogen. The mixture was stirred at room temperature for 2h, the reaction was filtered to remove the urea by-product and the filtrate was evaporated to dryness in vacuo to give the crude product (2.74g) as a white solid.
- Substrate linking agent/Cross-linker C Monosul phone carboxylic acid (2.4027g, lOmmol) was dissolved in dichloromethane (40ml) containing N,N- dimethylformamide (1ml). To the suspension was added oxalyl chloride (5ml, 5.8eq) in dichloromethane (10ml) dropwise. On completion of addition the mixture was stirred at room temperature for 1h. The solvents were removed in vacuo to give the crude product (2.62g) as a solid. The crude acid chloride (2.62g, lOmmol) was dissolved in anhydrous tetrahydrofuran (50ml) under nitrogen.
- Biochips prepared in Example 2 using EPON-SU8 as the substrate linking agent, were incubated for 30 min at 37 °C in a thermoshaker at 370 rpm. The biochips were then washed with TBST wash buffer (BT020/000/UL, Randox) - 2 washes followed by 4 washes at 2 min intervals. 300mI of ACE-2-HRP conjugate was added to the appropriate discrete test region (DTR).
- DTR discrete test region
- the biochips were incubated again for 30min at 37°C in a thermoshaker at 370 rpm, followed by washing with TBST buffer (BT020/000/UL, Randox) - 2 washes followed by 4 washes at 2 min intervals.
- the biochips were developed with 250mI of a 1 :1 ratio of luminol: peroxide (EV841 , Randox) for 2 min in the dark and then imaged on the Randox Evidence Investigator.
- the S1 protein attached directly to a substrate linking agent or via the various substrate liking agents/cross-linking groups produced positive protein detection results without the need of a sample pre-incubation step. Improved detection sensitivity was achieved using EPON-SU8 or an ab-unsaturated keto-sulphone derivative; unsaturated sulphones without the ab configurations produced less sensitive assays than ab-unsaturated keto-sulphones.
- SARS-CoV-2 neutralising antibodies SARS-CoV-2 neutralising antibodies (Spike Receptor Binding Domain & Nucleocapsid Protein) were successfully detected in both sera and saliva samples from different patients using the commercially available assay EV4447 produced by Randox, which detects the two different sets of neutralising antibodies simultaneously.
- the Spike Receptor Binding Domain was detected with the assay detector ACE-2 and the nucleocapsid protein with an anti- nucleocapsid antibody (although it is understood that any other suitable binding ligands could also be used). Detection of spike RBD neutralising antibodies were particularly high.
- SEQ ID NO: 1 SARS-CoV-2 S1 Receptor Binding Domain
- SEQ ID NO: 2 SARS-CoV-2 S1 Receptor Binding Domain, C terminally his- tagged, as expressed in the cells
- SEQ ID NO: 3 SARS-CoV-2 S1 Receptor Binding Domain, C terminally his- tagged, after cleavage of the signal peptide
- SEQ ID NO: 4 SARS-CoV-2 S1 Receptor Binding Domain, nucleotide sequence encoding SEQ ID NO: 2
- SEQ ID NO: 5 Angiotensin converting enzyme-2
- SEQ ID NO: 6 Angiotensin converting enzyme-2, C terminally his-tagged, as expressed in the cells
- SEQ ID NO: 7 Angiotensin converting enzyme-2, C terminally his-tagged, after cleavage of the signal peptide
- RSRINDAFRLNDNSLEFLGIQPTLGPPNQPPVSHHHHHHHHHH SEQ ID NO: 8 Angiotensin converting enzyme-2, nucleotide sequence encoding SEQ ID NO: 6
- SEQ ID NO: 9 SARS-CoV-2 Nucleoprotein
Abstract
The present invention relates to a method of detecting the presence of an antibody in a samples obtained from a subject, wherein said antibody has affinity for a coronavirus antigen, or portion thereof. The present invention also relates to a substrate having immobilised on its surface said coronavirus antigen and methods for producing said substrate.
Description
CORONAVIRUS ASSAY
FIELD OF THE INVENTION
The present invention relates to a method of detecting the presence of an antibody in a sample, wherein said antibody has affinity for a coronavirus antigen, for example, a SARS-CoV antigen, a SARS-CoV2 antigen or a MERS- CoV antigen. The present invention also relates to a substrate having immobilised on its surface said coronavirus antigen and methods for producing said substrate.
BACKGROUND OF THE INVENTION
The current SARS-CoV-2 coronavirus pandemic and the incipient mortality and post-infection wellbeing impact has highlighted the fundamental importance of medical healthcare. A burgeoning and mobile global population has presented new challenges compared to previous pandemics, and world-wide diagnostic testing and vaccination programmes have emerged as the principle means being used to combat the virus.
Coronaviruses are a group of related RNA viruses that cause respiratory tract infections in their host, the severity of which can range from mild to lethal. Examples of viruses relating to this family, in addition to SARS-CoV-2, include SARS-CoV and MERS-CoV, both of which have been responsible for viral outbreaks in 2002 and 2012, respectively. Coronaviruses are positive sense single-stranded RNA virus. The virion has an average diameter of approximately 80 -120 nm and incorporates four structural proteins, the spike, envelope (“E” protein), membrane (“M” protein) and nucleocapsid (“N” protein) proteins.
The immune system produces antibodies to various component parts of the coronavirus virion in an individual who has either been infected by the virus or who has been vaccinated for virus protection. Whilst, there are numerous
diagnostic tests relating to detection of coronaviruses, such as SARS-CoV-2, including real-time polymerase chain reaction (RT-PCR), antibody or serology tests and antigen tests, there is a continued need for improved diagnostic assays that can accurately determine if an individual who is/has been infected with a coronavirus has antibodies that confer protective immunity upon the individual.
The immune response to a coronavirus produces so-called neutralising antibodies (Nab) and binding antibodies (Bab) in the subject. Nab are defined as antibodies that prevent entry of the virion into the host cell whereas Bab bind to the coronavirus but do not prevent host-cell infection by the virion. Nab can exert their neutralising effect and prevent virion docking onto the host cell receptor, for example, ACE-2, through binding to the coronavirus and directly blocking attachment or by an ‘indirect’ mechanism by altering coronavirus conformation and disabling potential attachment. Nab are thought to be a strong indicator that the individual in question has protective immunity.
The development of coronavirus protein diagnostic tests to identify Nab presence, after viral infection and following vaccination, is a critical component of the pandemic mitigation strategy (Wajnberg A. et al., (2020). Robust neutralising antibodies to SARS-CoV-2 infection persists for months. Science, 37: 1227- 1230). Tan et al., (2020) describes a neutralisation assay format for SARS-CoV- 2 in which ACE-2 protein is located on a substrate surface and following incubation of horseradish peroxidase (HRP)-conjugated RBD with sample and their addition to the substrate, the HRP-RBD conjugate and in-sample SARS- CoV-2 antibodies compete for binding to ACE-2 (Tan C.W. et al,. (2020). A SARS-CoV-2 surrogate virus neutralization test based on antibody-mediated blockage of ACE-2-spike protein-protein interaction. Nature Biotechnology, 38:1073-1078).
However, there are a number of disadvantages associated with this particular assay format; firstly, such a format is not as amenable to high throughput
processing, leading to a less efficient process, and secondly, such a format requires pre-incubation of the HRP-conjugated RBD with the sample prior to addition to the substrate, which requires energy input and further delays the speed at which results can be generated.
Accordingly, there is a particular need in the art for coronavirus diagnostic assays that are amenable to high through-put processing, efficient, reliable and provide a strong indicator as to the level of protective immunity an individual may possess.
SUMMARY OF THE INVENTION
The present invention is based on the surprising discovery that the antibody detection assay herein described provides a highly sensitive and efficacious assay. Furthermore, said assay does not require a pre-incubation step, as is the case with other known assays, and therefore provides for a rapid and energy efficient test format.
Accordingly, in a first aspect, the present invention provides for a method of detecting the presence of an antibody in a sample obtained from a subject, wherein said antibody has affinity for a coronavirus antigen, or portion thereof, and wherein said method comprises bringing the sample obtained from the subject into contact with a coronavirus antigen, or portion thereof, which is immobilised on a substrate support, in the presence of a detectably-labelled molecule that can compete with any antibody present for binding to the coronavirus antigen, or portion thereof, and detecting the binding of antibody to the coronavirus antigen, or portion thereof, by measuring the amount of binding of said molecule compared to a control, wherein the coronavirus antigen, or portion thereof, is immobilised to the substrate surface by means of a linking agent.
In a second aspect, the present invention provides for a substrate having immobilised on its surface a coronavirus antigen, or portion thereof, as defined herein.
In a third aspect, the present invention provides for a method for producing a substrate having a coronavirus antigen immobilised thereon, comprising attaching the coronavirus antigen, or portion thereof, to the substrate surface via a sulphone or sulphonate-based cross-linker.
DESCRIPTION OF FIGURES
Figure 1 shows a schematic of an exemplary assay format in which the receptor binding domain (RBD) is spotted onto a discrete test region (DTR) on the substrate surface. The RBD may be covalently attached to the substrate DTR by way of a substrate linking agent and optionally a cross-linker. The substrate surface can be optionally covered with an ink layer on the non-DTR area, to reduce non-specific binding of reagents to the substrate. The upper part of the RBD comprises the receptor binding motif (RBM). The neutralising antibody (Nab) prevents ACE-2 protein from binding to the RBD, whereas the binding antibody (Bab) does not. The Nab anti-ACE-2 effect can operate through either binding directly to the RBM (so-called ‘direct’ effect) or to a non-RBM portion of the RBD (so-called ‘indirect’ effect).
Figure 2 shows the cross-linker 4-[bis(4-methylphenylsulphonylmethyl)-1- oxoethyl] benzoic acid, which can incorporate a silane moiety prior to addition to the substrate (see Figure 7, substrate linking agent B) or can be bonded to a substrate-linking agent (such as a silane moiety) already present on the substrate surface.
Figure 3 shows the synthesis of 4-[bis(4-methylphenylsulphonylmethyl)-1- oxoethyl] benzoic acid and subsequent addition of a silyl moiety (APTES) to form a substrate linking agent.
Figure 4 shows calibration curves for competitive neutralisation assay using RBD on substrate and ACE-2-HRP conjugate/anti-RBD antibodies in assay buffer.
Figure 5 shows generic assay device components for an immunoassay incorporating a sulphone or sulphonate-based cross-linker.
Figure 6 shows a device that can be used in a SARS-CoV-2 neutralisation antibody assay. Includes the S1 spike subunit with poly-His tag, a sulphone-silyl cross-linker/substrate linking agent and optional ink layer (w/wo means with or without).
Figure 7 shows examples of suitable sulphone substrate linking agents.
DETAILED DESCRIPTION
In order that the present invention may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.
As used herein, the terms “patient” and “subject” are used interchangeably herein and refer to any animal (e.g. mammal), including, but not limited to, humans, non-human primates, canines, felines, rodents and the like, which is to be where the sample is obtained from. Preferably, the subject or patient is a human.
As used herein, the term “a sample” includes biological samples obtained from a patient or subject, which may comprise blood, plasma, serum, urine, saliva, mucous, wax, tears, hair, sweat or sputum. Preferably, the sample is a blood or serum sample.
As used herein, the term ‘detecting’ refers to qualitatively analysing for the presence or absence of a substance, for example, a signal, usually above a set
threshold value to account for background signal noise, indicating presence or absence of the substance.
As used herein, the term ‘determining’ refers to quantitatively analysing for the amount of substance present.
As used herein, the term “detectably-labelled molecule” refers to a molecule that has a label covalently attached to said molecule to enable its detection. Such labels may include, but are not limited to, radionuclides, fluorophores, dyes, quantum dots, polymers or enzymes, including, for example, horse-radish peroxidase (HRP) and alkaline phosphatase. Preferably, the label is HRP
As used herein, the term “antibody” refers to an immunoglobulin which specifically recognises an epitope on a target as determined by the binding characteristics of the immunoglobulin variable domains of the heavy and light chains (VHS and VLS), more specifically the complementarity-determining regions (CDRs). In the case of the present invention, the term “antibody” refers to an immunoglobulin which specifically recognises an epitope on a coronavirus antigen, for example, the spike protein of SARS-CoV, SARS-CoV2 or MERS- CoV, the nucleocapsid protein (“N” protein) of SARS-CoV, SARS-CoV2 or MERS-CoV, the HE protein of SARS-CoV, SARS-CoV2 or MERS-CoV, the Plpro protein of SARS-CoV, SARS-CoV2 or MERS-CoV, the 3CLPro protein of SARS-CoV, SARS-COV2 or MERS-CoV, the E protein of SARS-CoV, SARS- CoV2 or MERS-CoV, or the M protein of SARS-CoV, SARS-CoV2 or MERS- CoV. Preferably, the antibody may recognise an epitope on the N protein, E protein or M protein of SARS-CoV, SARS-CoV2 or MERS-CoV.
As used herein, the term “has affinity for”, in the context of antibody-coronavirus antigen interactions, refers to an interaction wherein the antibody and epitope of the coronavirus antigen associate more frequently or rapidly, or with greater duration or affinity, or with any combination of the above, than when either antibody or coronavirus antigen is substituted for an alternative substance, for
example an unrelated protein, such as a non-coronavirus related protein, or a coronavirus protein other than the coronavirus antigen in question. Generally, but not necessarily, reference to binding means specific recognition. By way of example and not limitation, specific binding, or lack thereof, may be determined by comparative analysis with a control comprising the use of an antibody which is known in the art to specifically recognise said target and/or a control comprising the absence of, or minimal, specific recognition of said target (for example wherein the control comprises the use of a non-specific antibody). Said comparative analysis may be either qualitative or quantitative. It is understood, however, that an antibody or binding moiety which demonstrates exclusive specific recognition of a given target is said to have higher specificity for said target when compared with an antibody which, for example, specifically recognises both the target and a homologous protein.
As used herein, the term “coronavirus antigen” refers to any part, or portion thereof, of the coronavirus virion that is capable of producing an immune response in the host/subject i.e. the production of antibodies in response to said antigen. Accordingly, the term “coronavirus antigen” may refer to the spike protein of SARS-CoV, SARS-CoV2 or MERS-CoV, the nucleocapsid protein (“N” protein) of SARS-CoV, SARS-CoV2 or MERS-CoV, the E protein of SARS-CoV, SARS-Co /2 or MERS-CoV, the M protein of SARS-CoV, SARS-CoV2 or MERS- CoV, the HE protein of SARS-CoV, SARS-CoV2 or MERS-CoV, the Plpro protein of SARS-CoV, SARS-CoV2 or MERS-CoV, or the 3CLPro protein of SARS-CoV, SARS-CoV2 or MERS-CoV. Additionally, the term “coronavirus antigen” may also refer to any portion of the coronavirus virion that is not listed above but may cause an immune response in the host/subject.
As used herein, the terms “coronavirus spike protein” and “S1 protein” are used interchangeably and refers to a glycoprotein found on the surface of all coronaviruses. The spike protein is known to be important for the coronavirus gaining entry to the host cell via attaching to the complementary host cell receptor, for example, the host cell receptor for the spike protein of SARS-C0V2
is the angiotensin converting enzyme (ACE-2) receptor, and subsequent fusion with the host cell membrane. The spike protein may have the amino acid sequence according to Uniprot accession number P0DTC2, of which the RBD section spans amino acids 319-541 and the RBM section spans amino acids 437-508. The spike protein may be from any coronavirus, including variants of the spike protein containing one or more sequence variations or mutations. The coronavirus spike protein may be in monomeric form or, it may be a homotrimer to represent its native conformation on the virion. Preferably, the coronavirus spike protein is an RBD or RBM.
As used herein, the terms “nucleocapsid protein” and “N protein” are used interchangeably and refers to a highly conserved immunogenic phosphoprotein found in all coronaviruses that binds to viral RNA and leads to the formation of the helical nucleopcapsid. The nucleocapsid protein is composed of two separate domains, an N-terminal domain (NTD) and a C-terminal domain (CTD). It is thought that the abundance and high hydrophilicity of the N protein are key contributors in provoking an immune response in a subject following coronavirus infection.
As used herein, the term “M protein” or “membrane protein” are used interchangeably and refers to an integral membrane protein that is known to play a crucial role in coronavirus assembly. M proteins have three transmembrane domains and are glycosylated in all coronaviruses, either by N-linked or by O- linked oligosaccharides. These oligosaccharides have been shown to be important for folding, structure, stability, intracellular sorting of proteins and to play a role in the generation of immune responses in a subject following coronavirus infection.
As used herein, the term Έ protein” or “envelope protein” are used interchangeably and refers to a small, hydrophobic membrane protein that contains at least one alpha-helical transmembrane domain. The E protein is
known to play an important role in the assembly of virions, as well as in the host stress response.
As used herein, the term “control” refers to a value or values previously determined or determined simultaneously during the assay to which the assay results can be assessed and quantified against. The control will typically provide a measure of the extent of binding between the coronavirus spike protein and the detectably-labelled molecule in the absence of antibody. This can be used to provide a measure of the amount of binding that occurs in the assay between the coronavirus spike protein and antibody. In this context, the signal determined in the assay will be reduced due to the competitive binding of the antibody. The extent of the reduction compared to the control value(s) will indicate whether antibody is present in the sample and optionally the concentration of the antibody present. The control can be established as a calibration, alternatively, a calibration curve can be generated using neutralizing antibody preparations at multiple concentrations (see Figure 4). The assay signal output generated from a sample can be applied to the calibration curve to enable quantification of the Nab activity of said sample.
As used herein, the term “epitope” refers to the portion of a target which is specifically recognised by a given antibody.
As used herein, the terms “substrate linking agent”, “linker”, “linking agent” and “linking group” are used interchangeably and refers to a chemical moiety that connects the coronavirus spike protein to the substrate surface.
As used herein, the term “cross-linker” refers to any suitable chemical entity capable of binding a substrate linking agent to a coronavirus antigen, or portion thereof, for example, the RBD or RBM of the spike protein, the N protein, the M protein or the E protein.
As used herein, the terms “competitive” and “competitive assay” are used interchangeably, and in the context of the present invention refers to the detectably labelled molecule and antibodies present in a sample competing for binding to the chip on which is immobilised a coronavirus spike protein. Such a competitive assay format may be a simultaneous competitive assay format (for example, the sample and detectably-labelled molecule are added at the same time) or a sequential competitive assay (for example, the detectably-labelled molecule is added to the substrate subsequently to the sample). Accordingly, the skilled person will readily understand that the assay herein disclosed is considered a “competitive assay” regardless of the order or timing of the addition of the competing moieties.
The present invention provides for an assay device by which antibodies having affinity for a coronavirus antigen, or portion thereof, can be efficiently and accurately determined. Such an assay allows for an effective method of diagnosing subjects or patients suffering from a coronavirus related disease, for example, COVID-19, severe acute respiratory syndrome or Middle East respiratory syndrome, as well as an additional means of identifying subjects who may now have active immunity from said coronavirus.
Accordingly, in a first aspect, the present invention provides for a method of detecting the presence of an antibody in a sample obtained from a subject, wherein said antibody has affinity for a coronavirus antigen, or portion thereof, and wherein said method comprises bringing the sample obtained from the subject into contact with a coronavirus antigen, or portion thereof, which is immobilised on a substrate support, in the presence of a detectably-labelled molecule that can compete with any antibody present for binding to the coronavirus antigen, or portion thereof, and detecting the binding of antibody to the coronavirus antigen, or portion thereof, by measuring the amount of binding of said molecule compared to a control, wherein the coronavirus antigen, or portion thereof, is immobilised to the substrate surface by means of a linking agent.
The coronavirus antigen, or portion thereof, may be any part of a coronavirus that is capable of generating antibodies in a subject. In a preferred embodiment, the coronavirus antigen, or portion thereof, may be a coronavirus spike protein, a coronavirus N protein, a coronavirus E protein, a coronavirus M protein and/or combinations thereof. In the most preferred embodiment, the antigen, or portion thereof, is the coronavirus spike protein.
The method herein disclosed uses a device in which the coronavirus antigen, or portion thereof, is immobilised on the surface and is brought into contact with an ex vivo sample and a detectably-labelled molecule that has affinity for the coronavirus antigen, or portion thereof. It is therefore understood that, in one format, such an assay is a competitive binding assay, with the potential antibodies in the sample competing with the detectably-labelled molecule for binding to the coronavirus antigen, or portion thereof. Accordingly, a reduction in the signal produced by the detectably-labelled molecule signifies that there is a higher level of antibodies having affinity for a coronavirus antigen in the sample, and vice versa. The format of the assay herein described provides for an improved assay where the sample and detectably-labelled molecule do not require a pre-incubation step, thus allowing for a more efficient assay to be provided. The sample and detectably-labelled molecule can be mixed immediately prior to addition to the device/substrate surface, or added at the same time. Accordingly, the assay herein disclosed may be a simultaneous competitive assay format. Alternatively, and preferably, the sample may be added first to the substrate, with the detectably-labelled molecule added to the substrate in a sequential step. Accordingly, the coronavirus spike protein, or portion thereof, immobilised on the substrate support may be brought into contact with the sample prior to the addition of the detectably-labelled molecule to the substrate support. As such, in one embodiment of the assay disclosed herein may be a sequential competitive binding assay. This format provides a large reduction in the expected binding when the detectably-labelled molecule is
added if said sample contains antibodies having affinity for the coronavirus antigen, or portion thereof.
Passive adsorption of binding ligands such as the coronavirus antigen, or portions thereof, to substrate surfaces results in random orientation of the binding ligand at the substrate surface, which often results in an assay of reduced sensitivity, as the specific binding portion of the ligand can be substrate facing and unable to bind the cognate ligand. This is a disadvantage that the present invention overcomes. The method herein disclosed provides an assay whereby the coronavirus antigen, or portion thereof, is immobilised to the substrate surface by means of a linking agent and optionally a cross-linker. Such a format allows for the projection of the coronavirus antigen, or portion thereof, away from the substrate surface, exposing the binding portion of the coronavirus antigen, or portion thereof, as well as producing a greater engagement of the coronavirus antigen, or portion thereof with the sample compared to a passive adsorption, resulting in a more sensitive assay. Additionally, this particular format minimises the influence of the substrate surface, leading to improved assay kinetics. Further details of appropriate linking agents and cross-linkers are provided below.
It is known that in a subject infected with a coronavirus, the immune response of the subject produces neutralising antibodies (Nab) and binding antibodies (Bab), with the generation of neutralising antibodies being particularly advantageous from a protective immunity perspective.
Accordingly, the method herein disclosed may be suitable for the detection of both neutralising and binding antibodies, however, it is particularly envisaged that the method may be used to detect an antibody which is a neutralising antibody. The presence of Nab are said to demonstrate the individual having protective immunity against said coronavirus, and therefore have obvious benefits in being able to accurately and efficiently determine both their presence and amount in which they are present. Accordingly, the present invention also
provides for a method of detecting whether an individual has protective immunity against a coronavirus, for example, SARS-CoV, SARS-CoV2, and MERS-CoV. Nab exert their neutralising effect by preventing the coronavirus from docking to its corresponding host cell receptor. It is understood that the corresponding host cell receptor is reliant upon the coronavirus in question, for example, SARS-CoV, SARS-Co /2 or MERS-CoV. Nab can prevent the coronavirus docking to the host cell by both direct and indirect mechanisms. The direct mechanism may involve the Nab binding to the RBM of the coronavirus spike protein and therefore preventing any interaction between the coronavirus spike protein and the corresponding host cell receptor. Alternatively, indirect mechanisms may involve the Nab binding elsewhere on the coronavirus, thus resulting in a conformational change of the coronavirus proteins important for coronavirus-host cell interaction, and preventing binding to the corresponding host cell receptor in this manner.
The method herein may be used to detect antibodies having affinity for any coronavirus antigen, or portion thereof. Accordingly, the coronavirus antigen, or portion thereof may be a SARS-CoV antigen, SARS-CoV2 antigen or MERS- CoV antigen. Preferably, the coronavirus antigen, or portion thereof, may be a SARS-CoV2 spike protein, N protein, M protein and/or E protein. Preferably it is the SARS-C0V2 spike protein. The method herein may be used to detect neutralising antibodies having affinity for any coronavirus antigen, or portion thereof, for example, SARS-CoV antigen, SARS-CoV2 antigen or MERS-CoV antigen. Preferably, the method herein disclosed may be used to detect neutralising antibodies having an affinity for SARS-C0V2. Whilst the method herein disclosed is exemplified in relation to SARS-CoV, SARS-C0V2 and MERS-CoV, it is envisaged that said method will also be applicable to any coronavirus, including any as of yet unknown future novel coronaviruses.
It is understood that the skilled person would readily understand that the detectably-labelled molecule may be dependent on the coronavirus in question, for example, if the coronavirus is SARS-CoV, SARS-CoV2 or MERS-CoV.
Accordingly, if the method is for the detection of antibodies, for example, neutralising antibodies, having affinity for the SARS-CoV spike protein or the SARS-Co /2 spike protein, the detectably-labelled molecule can be ACE-2. However, if the coronavirus spike protein is the MERS-CoV spike protein, then the detectably labelled molecule can be DPP4. In cases where the coronavirus antigen, or portion thereof is not a coronavirus spike protein, other suitable molecules can be used to compete with the antibodies in the patient sample, wherein said suitable molecules have affinity for the coronavirus antigen, or portion thereof, for example, wherein said suitable molecules have affinity for an epitope present on the N protein, M protein and/or E protein of SARS-CoV, SARS-CoN/2 or MERS-CoV. For example, a detectably-labelled antibody (or suitable fragment or derivative thereof) that has affinity for the coronavirus antigen, or portion thereof, could be used in the competition assay format as disclosed herein. This concept applies to any other coronavirus that may be used in the context of the method herein disclosed, including any future unknown coronavirus to which the target receptor is as of yet also unknown.
The antibodies to be detected in a sample obtained from a subject may have an affinity for any epitope on the coronavirus antigen in question, for example, the SARS-CoV spike protein, N protein, M protein, or E protein, the SARS-CoV2 spike protein, N protein, M protein or E protein, or the MERS-CoV spike protein, N protein, M protein or E protein. The antibody may have an affinity for the RBD of the coronavirus spike protein in question. The antibody may have an affinity for the RBM of the coronavirus spike protein in question. In another embodiment, the antibodies to be detected in a sample obtained from a subject may have an affinity for any epitope on a coronavirus antigen, for example, the antibodies to be detected may have an affinity for one or more of the coronavirus N protein, M protein and/or E protein. The different epitopes to which the antibodies bind may affect the mechanism by which the antibody exerts its effect. For example, for Nab, this may directly prevent the coronavirus from binding to the host cell receptor, for example, ACE-2 or DPP4. If the Nab binds elsewhere on the coronavirus, a conformational change may be induced which
affects the binding capability of the coronavirus, thus preventing the binding to the host cell receptor in this way, for example, a partial blocking effect may occur, in which binding of the Nab reduces the efficiency of the virion approach, for example, to ACE-2 or DPP4.
As previously described, the presence of a linking agent and optionally a cross linker on the substrate surface allows for the coronavirus antigen, or portion thereof to be projected away from the substrate surface, exposing the binding portion of the coronavirus antigen, or portion thereof and producing a greater engagement of the coronavirus antigen, or portion thereof, with the sample compared to if no linking agent was present, i.e. passive adsorption. Accordingly, the present invention provides for a method in which the coronavirus antigen, or portion thereof, may be covalently attached to the substrate. This feature inevitably results in a more sensitive assay. Additionally, the stronger interaction between the substrate linking agent/cross-linker and the coronavirus antigen, or portion thereof, as a result of covalent bonding as opposed to the non-bonding interaction of passive adsorption, results in a more robust assay device and method.
The substrate linking agent may be any suitable chemical moiety that can support/bond to the coronavirus antigen, or portion thereof, or bond to a cross linker. Preferably, the linking agent is an epoxy silane derivative, an epoxy oligomer or an epoxy polymer. Examples include, but are not limited to, (EtO)3Si-(CH2)n-NH2 (see EP0874242 for silane derivative examples) and EPON-SU8 (also referred to as Epoly-8). Preferably, the linking agent is EPON- SU8 (CAS No. 28906-96-9, commercially available from Hexion Inc). Thus, it is preferred that the coronavirus antigen, or portion thereof, engages with the substrate (i.e. is supported by the substrate) by way of covalent bonds provided by a linking agent covalently attached to the substrate surface (the substrate linking agent) prior to coronavirus antigen addition.
The coronavirus antigen, or portion thereof, for example, a SARS-CoV, a SARS- CoV2 or a MERS-CoV antigen may bond to the substrate linking agent and
optionally a cross-linker through inherent functional groups such as carboxy, amino or sulphur present in the amino acids that constitute the protein. However, the assay effectiveness may be improved via the incorporation of a chemical activating group in the coronavirus antigen, or portion thereof. The attachment of the coronavirus antigen, or portion thereof, to a substrate linking agent and optionally a cross-linker requires that the binding characteristics of the coronavirus antigen, or portion thereof, are not affected, and that the specificity and affinity of the coronavirus antigen, or portion thereof, following the bonding process remain fit for purpose. Possible deleterious effects upon the coronavirus antigen, or portion thereof, upon chemical activation and or bonding to the substrate include coronavirus antigen tertiary structure disruption leading to reduced bonding and/or specificity to the target antibody. Accordingly, the method herein disclosed may use a coronavirus antigen, or portion thereof, immobilised to the substrate surface that further comprises a protein tag. Preferably, the method herein disclosed may use a coronavirus antigen, or portion thereof, immobilised to the substrate surface that further comprises a poly-His tag or a coronavirus antigen, or portion thereof, immobilised to the substrate surface that has been modified to have a poly-His tag. For example, the SARS-CoV spike protein, N protein, M protein or E protein may further comprise a poly-His tag or be modified to have a poly-His tag, the SARS-CoV2 spike protein, N protein, M protein or E protein may further comprise a poly-His tag or be modified to have a poly-His tag, or the MERS-CoV spike protein, N protein, M protein or E protein may further comprise a poly-His tag or be modified to have a poly-His tag. Accordingly, such a modification results in a recombinant coronavirus antigen, or portion thereof, that helps to reduce structural disruption of the coronavirus antigen, or portion thereof, when bound to the linking agent/cross-linker. Additionally, it will be readily understood that such a modification, i.e. the inclusion of protein tag, preferably a poly-His tag, may also be used for recombinant protein purification. The skilled person will readily understand the methods by which a poly-His tag could be incorporated into a coronavirus antigen, for example, recombinant DNA methods comprising inserting the DNA encoding a protein into a suitable vector encoding a His-tag
(Loughran and Walls, (2011), Purification of poly-histidine-tagged proteins, Methods Mol Biol, 681 :311 -35).
Therefore, the method herein disclosed may comprise a recombinantly produced coronavirus antigen, or portion thereof, incorporating a poly-His tag attached to a substrate linking agent/cross-linker. The poly-His tag can be varied in the number of histidine units but it is preferable to incorporate between 2 and 10 histidines, more preferably 6, 7 or 8 histidines; a 6 unit histidine tag is most preferred.
The method herein disclosed also discloses an assay in which the coronavirus antigen, or portion thereof, may be immobilised to the substrate surface by means of a linking agent and a cross-linker.
The method herein disclosed may use any cross-linker that is capable of binding the substrate linking agent to the coronavirus antigen, or portion thereof, for example, a coronavirus spike protein, N protein, M protein or E protein. Preferably, the cross-linker is a bifunctional cross-linker. By ’’bifunctional” cross linker we refer to a molecule that has two reactive groups, each reactive group capable of reacting with a moiety of the substrate linking agent or moiety of the coronavirus antigen, or portion thereof, such that the substrate linking agent and coronavirus antigen, or portion thereof, are bound together by the cross-linker. The two reactive groups may be the same or different. Suitable bifunctional cross-linkers are well known in the art (for example, suitable bifunctional cross linkers are disclosed in Bioconjugate Techniques, G.T. Hermanson, Third Edition 2013).
In the context of the present invention, the cross-linker may be bonded to the surface linking agent prior to their attachment to the substrate surface; alternatively, the cross-linker may be bonded to a substrate linking agent, the substrate linking agent having already been attached (covalently bonded) to the substrate surface, or the cross-linker agent may be attached to the coronavirus antigen, or portion thereof, prior to their attachment to the substrate linking agent.
The cross-linker is preferably a sulphone- (-S(=0)2-) or sulphonate- (-S(=0)2-0-) based cross-linker. “Sulphone-based cross-linker” refers to a cross-linker comprising at least one sulphone moiety, whilst “sulphonate-based” refers to a cross-linker comprising at least one sulphonate moiety. Suitable examples of sulphone-based cross-linkers include, but are not limited to: a,b-unsaturated ketone sulphones and precursors to a,b-unsaturated ketone sulphones, such as the bis-sulphone of Figure 3. Accordingly, in a preferred embodiment, a,b- unsaturated ketone sulphones and precursors to a,b-unsaturated ketone sulphones are used. It has surprisingly been found that the use of these particular cross-linkers further increases the sensitivity of the assay. Suitable examples of sulphonate-based cross-linkers include, but are not limited to: a,b- unsaturated ketone sulphonates and precursors to a,b-unsaturated ketone sulphonates. Such cross-linkers incorporate strong leaving groups which promote favourable reaction kinetics and bonding to the poly-His tag if present in the coronavirus spike protein. It is noted that, for this reason, sulphonate-based cross-linkers may also be used as the substrate linking agent. Without being bound by theory, it is believed that the use of sulphonate-based cross-linkers in this manner achieve this effect due to their strong leaving group properties.
When the cross-linker is a sulphone-based cross-linker, preferably an a,b- unsaturated ketone sulphone or precursor thereof, the cross-linker may have a structure according to Formula
wherein X is -CH2-;
Y is methylidene (=CH2), or Y is selected from -CH2-S02-Ph-R , CH2-S02-Ph- CHs, -CH2-S02-CH3, , -CH2-0-S02-Ph-FU , -CH2-0-S02-Ph-CH3 or -CH2-0-S02- CH3;
Ri is CH3 or CH3-Ph, wherein the Ph is optionally substituted with one or more Ci-ealkyl; and
R2 is selected from -Ce-^aryl-Z, -Ci-isalkyl-Z, and -C2-2oalkenyl-Z, wherein Z is selected from COR3, -NH2 and -OH, and R3 is selected from H, OH, NH2, -Ci- ealkyl-OH and (EtO)3Si-(CH2)n-NH- in which n is 1-6; and R is H or is optionally substituted with one or more Ci-ealkyl, N02, F, Cl or Br;.
It will be appreciated that when Y is methylidene (=CH2) in Formula (1), a double bond is present as shown in Formula (1a) below :
It will be appreciated that when Y is methylidene (=CH2), the cross-linker is an a,b-unsaturated ketone sulphone, and when Y is selected from CH2-S02-Ph- CHs, -CH2-S02-CH3, -CH2-0-S02-Ph-CH3 or -CH2-0-S02-CH3-, the cross-linker is a precursor to a a,b-unsaturated ketone sulphone.
Preferably Y is methylidene (=CH2), CH2-S02-Ph-CH3, or -CH2-0-S02-Ph-CH3, more preferably Y is methylidene (=CH2) or CH2-S02-Ph-CH3.
Preferably, Ri is CH3-Ph, and Ph is substituted with a Ci-ealkyl group, preferably methyl.
Preferably, R2 is selected from -Ph-Z, -CioHs-Z, -Ci-ealkyl-Z and -C2-ealkenyl-Z, more preferably from -Ph-Z and -CioHs-Z, and more preferably -Ph-Z. Preferably, Z is selected from COR3.
When the cross-linker is a sulphone-based cross-linker, preferably an a,b- unsaturated ketone sulphone or precursor thereof, the cross-linker may have a structure according to Formula (2):
wherein X is -CH2-;
Y is methylidene (=CH2)or Y is selected from CH2-S02-Ph-CH3, -CH2-S02-CH3, - CH2-0-S02-Ph-CH3 or -CH2-0-S02-CH3;
Ri is CH3 or CH3-Ph, wherein the Ph is optionally substituted with one or more Ci-ealkyl; and
R3 is selected from H, OH, NH2, -Ci-ealkyl-OH and (EtO)3Si-(CH2)n-NH- in which n is 1-6.
It will be appreciated that when Y is methylidene (=CH2) in Formula 2, a double bond is present as shown in Formula (2a) below:
Preferably Y is methylidene (=CH2), CH2-S02-Ph-CH3, or -CH2-0-S02-Ph-CH3, more preferably Y is methylidene (=CH2) or CH2-S02-Ph-CH3.
Preferably, Ri is CH3-Ph, and Ph is substituted with a Ci-ealkyl group, preferably methyl.
Preferably, R3 is OH, H or (EtO)3Si-(CH2)n-NH- in which n is 1-6.
When the cross-linker is a sulphone-based crosslinker, preferably an a,b- unsaturated ketone sulphone or precursor thereof, the cross-linker may have a structure according to Formula (3) or Formula (4):
wherein R3 is selected from H, OH, NH2, -Ci-ealkyl-OH and (EtO)3Si-(CH2)n-NH- in which n is 1-6, preferably OH, H or (EtO)3Si-(CH2)n-NH- in which n is 1-6.
Preferably, the cross-linker is a sulphone-based cross-linker. More preferably, the cross-linker is a sulphone-based crosslinker of Formulas (1), (2), (3) or (4) as detailed above.
As used herein, ‘Ph’ refers to phenyl, “CioHs” refers to a naphthalene or napthyl group.
As used herein, the term "Ci-is alkyl" demotes a straight or branched saturated alkyl group having from 1 to 18 carbon atoms; For parts of the range Ci-is alkyl, all sub-groups thereof are contemplated, such as Ci-e alkyl, C5-15 alkyl, C5-10
alkyl, and C1-6 alkyl. Examples of said C1-4 alkyl groups include methyl, ethyl, n- propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, and tert-butyl. The alkyl groups may be optionally substituted with one or more functional groups, including C1-18 alkyl groups, "Ce-12 aryl", and "C1-18 alkoxy", halogen, and "C3-18 cycloalkyl".
As used herein, the term "C2-isalkenyl" denotes a "Ci-is alkyl" group containing some degree of unsaturation (partial unsaturation) i.e. containing one or more alkene/alkenyl moiety(s).
As used herein, the term "Ce-12 aryl" denotes a monocyclic or polycyclic conjugated unsaturated ring system having from 6 to 12 carbon atoms. For parts of the range Ce-12 aryl, all sub-groups thereof are contemplated, such as Ce-io aryl, C10-12 aryl, and Ce-s aryl. An aryl group includes condensed ring groups such as monocyclic ring groups, or bicyclic ring groups. Examples of Ce- 12 aryl groups include phenyl, biphenyl, indenyl, naphthyl or azulenyl. Condensed rings such as indan and tetrahydro naphthalene are also included in the Ce-12 aryl group. The aryl groups may be optionally substituted with other functional groups. The aryl groups may be optionally substituted with one or more functional groups, including C1-18 alkyl groups, halogen, and "C1-18 alkoxy". The aryl groups may be substituted with these substituents at a single position on their unsaturated ring system, or may be substituted with these substituents at multiple positions on their unsaturated ring system.
The terms “unsaturated” and “partially saturated” refer to rings wherein the ring structure(s) contains atoms sharing more than one valence bond i.e. the ring contains at least one multiple bond e.g. a C=C, CºC or N=C bond. The term “fully saturated” refers to rings where there are no multiple bonds between ring atoms.
“Optional” or “optionally” means that the subsequently described event or circumstance may but need not occur, and that the description includes
instances where the event or circumstance occurs and instances in which it does not.
In the context of the present invention, the cross-linker may be subject to chemical activation prior to attachment to the substrate linking agent. By ‘chemical activation’ is meant a process whereby the cross-linker is altered such that it has increased propensity for subsequent reaction, i.e. increased propensity for bonding with the substrate linking group and/or coronavirus spike protein. Suitable methods of chemical activation will be well known by a skilled person, for example, the EDC method or a maleimido-group incorporation. For example, when Z of formula (1) is COR3 and R3 is OH, or R3 of formula (2) is OH, the EDC method or a maleimido-group incorporation may be used to activate the cross-linker. It will be understood by a skilled person that such chemical activation is typically used to promote bonding of the cross-linker to the substrate linking group and/or coronavirus spike protein. Accordingly, the present method also provides for the incorporation of a substrate linking agent in the sulphone- or sulphonate-based cross-linker prior to the addition to the substrate. This has the advantage of omitting the substrate chemical-activation step, which in turn benefits efficiency of device production. Figures 3 and 5 present examples of silyl-sulphone compounds.
The method herein provides for an immunoassay in which the antibodies in a sample and a detectably-labelled molecule compete with one another to bind to the immobilised coronavirus antigen, or portion thereof, bonded to the substrate surface via the linking agent and optionally the cross-linker described above (see Figure 1). Accordingly, the detectably-labelled molecule provides a detectable and measurable signal which is reduced in the presence of antibodies, thus allowing the antibodies present in said sample to be detected and/or quantified.
The measurable signal may be electromagnetic radiation based on, for example, phosphorescence, fluorescence, chemiluminescence (e.g. HRP/luminol/peroxide system). Preferably, fluorescence or chemiluminescence is used. An example of
a detecting agent suitable for use with the present invention is the streptavidin- biotin-enzyme complex, avidin may also be used in place of streptavidin, resulting in a complex with the molecule to be detected, for example, an ACE-2 - biotin-streptavidin-enzyme complex, a DPP4-biotin-streptavidin-enzyme complex or an antibody-(or suitable fragment or derivative thereof)-biotin-streptavidin- enzyme complex. Accordingly, the method herein disclosed may include a detectably-labelled molecule labelled with a streptavidin-biotin-enzyme complex or an avidin-biotin-enzyme complex. Preferably, the enzyme of said complex may be HRP, which when exposed to luminol/peroxide system produces a detectable signal. A calibrator or standard, which can be used for effecting assay calibration, is well known in the art and enables a threshold concentration or the exact or calibrator equivalent amount of analyte(s) to be determined. The determination of an exact or calibrator equivalent amount of analyte(s) usually requires the construction of a calibration curve (also known as a standard curve). The number of calibrator points vary but is preferably from 5 to 9. Alternatively, the calibrator value can be a single pre-determined threshold value.
The surface of the substrate for use in the method of the present invention may be selectively covered with a coating composition. The coating composition may be any coating that would be expected to enhance or maintain the desirable properties of the surface to which the coronavirus antigen, or portion thereof, is immobilised, i.e. the DTR. The term “selectively covered”, as used in the context of the present invention, refers to areas of the substrate where the coronavirus antigen, or portion thereof, is immobilised. Preferably, the coating composition may be an ink formulation.
Ink formulations have been added to the surfaces of substrates, such as chips and biochips used in medical diagnostics, to minimise the attachment of chemical and biological components present in patient samples which can affect assay performance. It is understood that the choice of ink can be dependent upon the assay configuration of the substrate and the assay objectives and is usually characterised as being on the hydrophilic-hydrophobic continuum using
the water contact angle. It has been found that the assay devices and methods of the current invention can further benefit from an increase in performance if the substrate surface supports an ink layer. This leads to yet a further increase in the sensitivity of the assay.
The ink formulation may comprise a pigment, a resin, an ink solvent, an ink additive and/or a structuring agent. Suitable examples of pigments may be selected from the group of inorganic artificial pigment, inorganic natural pigment, organic artificial pigment, organic natural pigment, black pigment, blue pigment, brown pigment, green pigment, orange pigment, red pigment, violet pigment, white pigment or yellow pigment. Suitable examples of resins include, but are not limited to, acrylics, alkyds, epoxides, hydrocarbons, phenolics or fluoropolymers such as a polytetrafluoroethylene (PTFE). Suitable ink solvents include, but are not limited to, cyclohexanone, butoxyethanol and aromatic distillates. Suitable ink additives include, but are not limited to, carbon black (black pigment), mineral oil (wetting agent), petroleum distillate, dibutyl phthalate (plasticizer), salts of cobalt, manganese or zirconium (drying agent), aluminium and titanium chelator (chelating agent), antioxidants, surfactants and defoamers. Suitable structuring agents include, but are not limited to CERAFLOUR® 965. The ink formulation may comprise one or more resins, pigments, ink solvents/additives and/or structuring agents selected from the lists above, and combinations thereof.
Preferably, the ink formulation may be a composition according to those disclosed in EP3377900 A1. Accordingly, the ink formulation may comprise an epoxy or acrylic resin, a pigment and a structuring agent. Preferably, the ink composition may comprise an acrylic resin, a pigment and a structuring agent. One pigment may be present or multiple pigments may be used. Epoxy and acrylic resin are used to increase ink viscosity, rheological properties and adhesion to the substrate. Preferably, the pigment imparts a dark colour, preferably a black colour, and hence imparts optical opacity to the ink. The
structuring agent provides hydrophilic/hydrophobic properties to the surface of the substrate and also help adhesion to the substrate.
Preferably, the pigment may be present in an amount of 1 to 15% w/w of the ink formulation; the epoxy resin may be present in an amount of 10 to 60 % w/w, the acrylic resin may be present in an amount of 1 to 20% w/w, and the structuring agent may be present in an amount of 10 to 60% w/w.
More preferably, the pigment, preferably black pigment, is present in an amount of 1 to 8% w/w of the ink formulation; the epoxy resin is present in an amount of 15 to 50 % w/w of the ink formulation, the acrylic resin is present in an amount of 2 to 15% w/w of the ink formulation, and the structuring agent is present in an amount of 15-50% w/w of the ink formulation. Most preferably, the pigment, preferably black pigment, is present in an amount of 5% w/w of the masking composition; the epoxy resin is present in an amount of 30 % w/w of the ink formulation, the acrylic resin is present in an amount of 10% w/w of the ink formulation, and the structuring agent is present in an amount of 20% w/w of the ink formulation.
In a preferred embodiment, carbon black pigment is used in the ink formulation, preferably Elftex 285. Preferably the acrylic resin is B-67. Preferably the structuring agent is a PTFE wax, such as CERAFLOUR® 965. Preferably, the ink formulation comprises an epoxy resin, preferably Epikote 1004. Most preferably, the pigment is Elftex 285, the acrylic resin is B-67, the epoxy resin is Epikote 1004 and the structuring agent is CERAFLOUR® 965. The ink formulation may further comprise one or more agents selected from the list of solvents, such as ethanol, propanol, xylene, diglycol, butyl ether; dispersing agents; pigment wetting agents; levelling agents; pigment wetting agents and/or crosslinking agents. Preferably, the ink formulation has a contact angle of 20- 175°, more preferably 20-170° more preferably 90-120°, even more preferably about 1 10°. The measurement is taken using the following protocol: The contact angle is measured using a KSV CAM200 contact angle meter equipped with
automated dispenser controlled using stepper motor, LED source and CCD camera. The contact angle meter is connected to a software tool for dispense controller, image grabbing and image analysis. A droplet of deionised water of 3.5 pi is dispensed on the substrate at a predefined location and the image is captured using a CCD camera. Image analysis is performed using software to estimate the contact angle of the water droplet.
Preferably, the thickness of the ink formulation applied to the substrate is 1-100 pm thick, preferably, 2-50 pm thick. This creates a discrete reaction zone that is a well having a depth of 1 -100 pm, preferably 2-50 pm, respectively. Most preferably the thickness of coating is 3 to 20 pm thick and the resulting depth of the well is 3 to 20 pm in depth.
The ink is applied to the substrate such that there are exposed areas of the substrate that define the reaction sites. The areas coated with the ink are not intended to be the reaction sites and provide a typically hydrophobic surface that prevents non-specific binding from occurring and helps retain solutions at the reaction site to provide a clear image of the reaction site.
The sample for use in the method of the present invention may be any biological sample taken from the individual in which antibodies may be detected. Preferably, the biological samples require no pre-processing and can be used neat in the assay herein disclosed. Accordingly, the sample may be a serum sample, plasma sample, whole blood sample, urine sample, mucous sample, saliva sample, CSF sample, sputum sample, ear wax sample, hair sample, sweat sample, tear sample, meconium, skin, solid tumour extracts, peripheral blood mononuclear cells, bone marrow mononuclear cells, cerebrospinal fluid, cystic fluid or any suitable cell lysate. Preferably, the sample is a serum or plasma sample; alternatively, it is a whole blood sample or a saliva sample. The sample may be obtained from the subject or patient by methods routinely used in the art, for example, via venous blood collection, swab testing or tissue biopsy.
The determination and/or detection of antibodies, for example, neutralising antibodies may be done on one or more samples obtained from the subject.
The substrate for use in the present invention may be of a planar conformation, such as a glass slide, microtitre plate or a chip/biochip. As used herein, the term “biochip” refers to a chip whose use is biomedical and is made of a thin, wafer like substrate with a planar surface. Preferably, the substrate may be a bio-chip due to its stability and adaptability. Whist the biochip may be made of any suitable material, such as glass or any suitable polymer, preferably, the biochip is made of ceramic. Even more preferably, the biochip is made of aluminium oxide based ceramic and may be chemically activated. Various aspects of biochip technology are described in EP0874242. A ceramic substrate can be manufactured to provide a range of grain sizes. Typical grain sizes are 1 to 30 pm; < 10 pm is preferred as the reduced particle size imparts increased surface homogeneity which improves assay performance. The preferred ceramic material consists of about 94% alumina (AI2O3) with a particle size in the range of 4-8 pm. The surface topography is usually withing the range of 0.6 to 0.8 pm after being ground. This can be improved through polishing to yield a surface with variation of 0.4-0.5 pm which can be further improved through by lapping and polishing to 0.05-0.1 pm.
In a second aspect, the present invention provides for a substrate having immobilised on its surface a coronavirus antigen, or portion thereof, as defined by any of the features herein described. Accordingly, the present invention provides for a substrate having immobilised on its surface a SARS-CoV antigen, a SARS CoV-2 antigen or a MERS-CoV spike antigen connected by a linking agent and optionally a cross-linker. The substrate may have immobilised on its surface a single type of coronavirus and/or coronavirus antigen, or portion thereof, for example, SARS-CoV spike protein, N protein, M protein, E protein, SARS-C0V2 spike protein, N protein, M protein, E protein or MERS-CoV spike protein, N protein, M protein or E protein. Alternatively, the substrate may have immobilised on its surface a combination of coronavirus and/or coronavirus
anitgens, for example, SARS-CoV spike protein, N protein, M protein, E protein, SARS-Co /2 spike protein, N protein, M protein, E protein or MERS-CoV spike protein, N protein, M protein or E protein or a combination of variants of the coronavirus antigen. In instances where the substrate may have multiple coronavirus antigens, or portions thereof, immobilised upon its surface, it is understood that each detectably-labelled molecule having affinity for a specific coronavirus antigen, or portion thereof, may have different labels, for example, different fluorophores, attached in order to differentiate the signal arising from different coronavirus antigens, or portions thereof. Alternatively, the substrate herein disclosed may be spatially arranged in such a manner that the same effect is achieved and the same detection labels may be used. The skilled person will recognise that being able to identify antibodies in a sample directed at multiple targets will help reduce the level of false negatives, thus resulting in a more sensitive, accurate and reliable assay.
Preferably, the present invention provides for a substrate having immobilised on its surface a SARS-CoV2 antigen, or portion thereof. In a preferred embodiment the linking agent is EPON-SU8 and the cross-linker is a sulphone- or sulphonate-based cross-linker according to Formula (1), even more preferably, the cross-linker is a sulphone-based crosslinker according to Formula (2), (3) or (4). Preferably, the substrate is a bio-chip, even more preferably, the substrate is a ceramic bio-chip. The substrate may be selectively covered with an ink formulation, as described above.
In a third aspect, a method for producing a substrate having a coronavirus antigen, or portion thereof, immobilised thereon, comprising attaching the coronavirus antigen, or portion thereof, to the substrate surface via a sulphone or sulphonate-based cross-linker is provided. The coronavirus antigen, or portion thereof, may be a SARS-CoV antigen, or portion thereof, SARS-CoV2 antigen, or portion thereof, or a MERS-CoV antigen, or portion thereof. Preferably, the coronavirus antigen, or portion thereof, is a SARS-CoV2 antigen, or portion thereof.
The present invention also discloses, a coronavirus antigen, or portion thereof recombinantly modified to include a poly-His tag. The coronavirus antigen, or portion thereof, recombinantly modified to include a poly-His tag may be a SARS-CoV antigen, or portion thereof, a SARS-CoV2 antigen, or portion thereof, or a MERS-CoV2 antigen, or portion thereof. Preferably, the coronavirus antigen, or portion thereof, recombinantly modified to include a poly-His tag is a SARS-C0V2 antigen, or portion thereof.
The invention is further described with reference to the following non-limiting examples:
EXAMPLES Example 1
Protein expression & purification of SARS-CoV-2 Spike Receptor Binding Domain
SEQ ID NO: 2 and SEQ ID NO: 6 were expressed in human cells using standard cloning of codon optimized SEQ ID NO: 4 and SEQ ID NO: 8 respectively into a constitutive high level mammalian expression vector with an artificial or native signal peptide sequence and C terminal His tag, resulting in the expression of SEQ ID NO: 2 and SEQ ID NO: 6 and after removal of the signal peptide sequence, SEQ ID NO: 3 and SEQ ID NO: 7. Transfected cells were cultured at 37°C and 8% CO2 in Expression Medium (Thermoscientific) containing glutamine for three to six days before harvesting. Preparation of SEQ ID NO: 3 and SEQ ID NO: 7 comprised cell supernatant collection which was clarified by centrifugation for 30 minutes at 7,000rpm. Remaining particulate matter was removed by filtration through 1.2mM and 0.45mM low protein binding filter units respectively. SEQ ID NO: 3 and SEQ ID NO: 7 protein was purified from the clarified and filtered supernatant at 4°C by the Akta Avant system (Cytiva) using Nickel HisTrap Excel columns (Cytiva Lifesciences, USA) preequilibrated with 20mM Tris, 500mM Sodium chloride pH8.0. Contaminant non-specific binding protein was removed by a wash containing 20mM Tris, 500mM sodium chloride,
30mM imidazole and SEQ ID NO: 3 and SEQ ID NO: 7 were eluted by increasing the concentration of imidazole to 500mM. All fractions containing either SEQ ID NO: 3 or SEQ ID NO: 7 were pooled and concentrated by ultrafiltration (Vivaspin, Sartorius, Gottingen Germany) and buffer exchanged into PBS pH7.4 (Melford, UK). Expressed SEQ ID 3 and SEQ ID NO: 7 were further purified by fractionation on a HiLoad 26/600 Superdex 75 PG column (GE Healthcare, USA) pre-equilibrated in 2X PBS pH7.4 (Melford, UK). The column fractions were monitored for absorbance at A280nm. Selected fractions containing SEQ ID NO: 3 or SEQ ID NO: 7 were collected, pooled and concentrated by ultrafiltration (Vivaspin, Sartorius, Gottingen Germany). The final preparations of SEQ ID NO: 3 and SEQ ID NO: 7 proteins were evaluated by SDS gel electrophoresis. For spotting to the surface of the biochip, expressed protein SEQ ID NO: 3 was diluted in buffer.
Protein expression & purification of SARS-CoV-2 Nucleoprotein SARS-CoV-2 Nucleoprotein (Uniprot ID P0DTC9) was expressed in human cells using standard cloning methods into a constitutive high level mammalian expression vector with an artificial signal peptide sequence and C terminal His tag, resulting in the expression of SEQ ID NO: 9 after removal of the signal peptide. Transfected cells were cultured at 37oC and 8% C02 in Expression Medium (Thermoscientific) containing glutamine for three to six days before harvesting.
Preparation of SEQ ID NO: 9 comprised cell supernatant collection which was clarified by centrifugation for 30 minutes at 7,000rpm. Remaining particulate matter was removed by filtration through 1.2mM and 0.45mM low protein binding filter units respectively. SEQ ID NO: 9 protein was purified from the clarified and filtered supernatant at 4°C by the Akta Avant system (Cytiva) using Nickel HisTrap Excel columns (Cytiva Lifesciences, USA) preequilibrated with 20mM Tris, 500mMSodium chloride pH8.0. Contaminant non-specific binding protein was removed by a wash containing 20mM Tris, 500mM sodium chloride, 30mM imidazole and SEQ ID NO: 9 was eluted by increasing the concentration of
imidazole to 500mM. All fractions containing either SEQ ID NO:9 was pooled and concentrated by ultrafiltration (Vivaspin, Sartorius, Gottingen Germany) and buffer exchanged into PBS pH7.4 (Melford, UK). Expressed SEQ ID NO: 9 was further purified by fractionation on a Hil_oad26/600 Superdex 75 PG column (GE Healthcare, USA) pre-equilibrated in 2X PBSpH7.4 (Melford, UK). The column fractions were monitored for absorbance atA280nm. Selected fractions containing SEQ ID NO: 9 was collected, pooled and concentrated by ultrafiltration (Vivaspin, Sartorius, Gottingen Germany). The final preparations of SEQ ID NO: 9 proteins were evaluated by SDS gel electrophoresis. For spotting to the surface of the biochip, expressed protein SEQ ID NO: 9 was diluted in buffer.
Example 2
Substrate Preparation
A ceramic substrate was washed with RBS 35 concentrate and water and plasma treated before addition of Epon SU8 (Hexion Incorporated, Ohio, US) or (OEt)3-Si-(CH2)3-NH2 or a silyl-sulphone (B of Figure 7). Following stirring it was deposited on the ceramic substrate by spray coating and the chips cured for 1 hr at 140°C. For ink formulations and preparation of ink-coated chips see WO201 7085509. Various ink formulations can be used on the substrate as described in WO2017085509; the preferred ink substrate comprises a carbon black pigment, a PTFE structuring agent and acrylic resin. 10 nl of S1 spike subunit binding ligand incorporating a poly-His tag in carbonate/bicarbonate buffer pH 9.5 was spotted onto discrete test regions on the respective substrates using a sciflexarrayer S100. Controls (G-148-C, Biospacific) were spotted at 0.15mg/ml in the same buffer onto discrete DTRs (x3). The substrate was left to incubate at 37°C for 24 hr prior to use. After these steps, substrates were assembled into carriers of n=9 and analysed using an Evidence Investigator.
Example 3
Substrate linking agent/Cross-linker preparation (see Figures 3 & 7)
Substrate linking agent/Cross-linker A. To a cooled solution (0°C) of (3- aminopropyl) triethoxysilane (APTES) (22.131 g, 0.1 mol) and diisopropylethylamine (20.9mls, 0.12mol) in dichloromethane (300ml) under nitrogen was added dropwise a solution 2-chloroethylsulphonyl chloride (16.63g, 0.102mol) in dichloromethane (50mls). The mixture was than stirred at 0°C for two hours and then overnight at room temperature. The solution was washed (150ml) by water and brine (100ml_). The solution was dried over sodium sulphate filtered and concentrated to dryness. The crude product obtained was purified by flash chromatography on silica gel using ethyl acetate/hexane (40/60) to give a clear oil in 80% yield.
Substrate linking agent/Cross-linker B. To a solution of bis-sulphone acid (2g, 4mmol) in dichloromethane was added N-hydroxysuccinimide (506mg, 1.1 eq) and dicyclohexylcarbodiimide (908mg, 1.1 eq) under nitrogen. The mixture was stirred at room temperature for 2h, the reaction was filtered to remove the urea by-product and the filtrate was evaporated to dryness in vacuo to give the crude product (2.74g) as a white solid. The crude bis-sulphone acid N- hydroxysuccininide (2.74g, 4.59mmol) was dissolved in anhydrous tetrahydrofuran (50ml) under nitrogen. To this was added APTES (1.016g, 1eq) and the reaction stirred at room temperature overnight. Solvents were removed in vacuo and the crude purified by flash chromatography on silica gel using 20- 50% ethyl acetate in pet ether to give the title compound (2.08g, 74%) as a pale- yellow viscous oil/semi-solid
Substrate linking agent/Cross-linker C. Monosul phone carboxylic acid (2.4027g, lOmmol) was dissolved in dichloromethane (40ml) containing N,N- dimethylformamide (1ml). To the suspension was added oxalyl chloride (5ml, 5.8eq) in dichloromethane (10ml) dropwise. On completion of addition the mixture was stirred at room temperature for 1h. The solvents were removed in vacuo to give the crude product (2.62g) as a solid. The crude acid chloride (2.62g, lOmmol) was dissolved in anhydrous tetrahydrofuran (50ml) under nitrogen. To this was added APTES (2.21 g, 1eq) and triethylamine (2.02g, 2eq)
and the reaction stirred at room temperature for 2h. Dichloromethane (100ml) was added and washed with brine (50ml). Organics were dried over sodium sulphate, filtered and evaporated to dryness. The crude was purified by flash chromatography on silica gel using 50% ethyl acetate in dichloromethane to give the title compound (1.948g, 45%) as a yellow viscous oil.
Example 4
Evidence Investigator Analysis
288pl of Assay diluent (EV808), then 12mI of neat sample/neat controls were added to the appropriate biochip wells. Biochips, prepared in Example 2 using EPON-SU8 as the substrate linking agent, were incubated for 30 min at 37 °C in a thermoshaker at 370 rpm. The biochips were then washed with TBST wash buffer (BT020/000/UL, Randox) - 2 washes followed by 4 washes at 2 min intervals. 300mI of ACE-2-HRP conjugate was added to the appropriate discrete test region (DTR). The biochips were incubated again for 30min at 37°C in a thermoshaker at 370 rpm, followed by washing with TBST buffer (BT020/000/UL, Randox) - 2 washes followed by 4 washes at 2 min intervals. The biochips were developed with 250mI of a 1 :1 ratio of luminol: peroxide (EV841 , Randox) for 2 min in the dark and then imaged on the Randox Evidence Investigator.
The S1 protein attached directly to a substrate linking agent or via the various substrate liking agents/cross-linking groups produced positive protein detection results without the need of a sample pre-incubation step. Improved detection sensitivity was achieved using EPON-SU8 or an ab-unsaturated keto-sulphone derivative; unsaturated sulphones without the ab configurations produced less sensitive assays than ab-unsaturated keto-sulphones.
SARS-CoV-2 neutralising antibodies (Spike Receptor Binding Domain & Nucleocapsid Protein) were successfully detected in both sera and saliva samples from different patients using the commercially available assay EV4447 produced by Randox, which detects the two different sets of neutralising antibodies simultaneously. The Spike Receptor Binding Domain was detected
with the assay detector ACE-2 and the nucleocapsid protein with an anti- nucleocapsid antibody (although it is understood that any other suitable binding ligands could also be used). Detection of spike RBD neutralising antibodies were particularly high.
Patient 1
Table 1-SARS-CoV-2 IgG (RBD & NP) Array Serum:
Table 2-SARS-CoV-2 IgG (RBD & NP) Saliva
Patient 2
Table 3-SARS-CoV-2 IgG (RBD & NP) Array Serum:
Table 4-SARS-CoV-2 IgG (RBD & NP) Saliva
SEQUENCES FORMING PART OF THE DESCRIPTION:
SEQ ID NO: 1 (SARS-CoV-2 S1 Receptor Binding Domain)
RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSA
SFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLP
DDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPC
NGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNL
VKNKCVNF
SEQ ID NO: 2 (SARS-CoV-2 S1 Receptor Binding Domain, C terminally his- tagged, as expressed in the cells)
MFVFLVLLPLVSSQRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRIS NCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPG QTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFER DISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLH APATVCG PKKSTNLVKN KCVN F H H H H H H
SEQ ID NO: 3 (SARS-CoV-2 S1 Receptor Binding Domain, C terminally his- tagged, after cleavage of the signal peptide)
RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSA
SFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLP
DDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPC
NGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNL
VKNKCVNFHHHHHH
SEQ ID NO: 4 (SARS-CoV-2 S1 Receptor Binding Domain, nucleotide sequence encoding SEQ ID NO: 2)
ATGTTCGTGTTTCTGGTGCTGCTGCCTCTGGTGTCCAGCCAGCGGGTGCAG CCCACCG AAT CCAT CGT GCGGTT CCCCAATAT CACCAAT CT GT GCCCCTT CG GCGAGGT GTT CAATGCCACCAGATTCGCCT CT GT GTACGCCT GGAACCGGA AGCGGAT CAGCAATT GCGTGGCCGACTACT CCGTGCT GTACAACT CCGCCA
GCTT CAGCACCTT CAAGT GCTACGGCGT GT CCCCTACCAAGCT GAACGACC TGT GCTT CACAAACGT GTACGCCGACAGCTT CGT GAT CCGGGGAGAT GAAG T GCGGCAGATTGCCCCT GG ACAGACAGGCAAGAT CGCCG ACTACAACTACA AGCT GCCCGACGACTT CACCGGCT GTGT GATT GCCTGGAACAGCAACAAC CTGGACTCCAAAGTCGGCGGCAACTACAATTACCTGTACCGGCTGTTCCGG AAGTCCAATCTGAAGCCCTTCGAGCGGGACATCTCCACCGAGATCTATCAG GCCGGCAGCACCCCTT GTAACGGCGT GGAAGGCTT CAACT GCTACTT CCCA CTGCAGTCCTACGGCTTTCAGCCCACAAATGGCGTGGGCTATCAGCCCTAC AGAGT GGTGGTGCT GAGCTT CGAACT GCTGCAT GCCCCT GCCACAGT GT G CGGCCCTAAGAAAAGCACCAATCT CGT GAAGAACAAATGCGT GAACTT CCA CCAT CACCAT CACCAT
SEQ ID NO: 5 (Angiotensin converting enzyme-2)
QSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKW
SAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTM
STIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQL
RPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVE
HTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLT
VPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLT
DPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYA
AQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLK
QALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPH
DETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNS
TEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQN
KNSFVGWSTDWSPYADQSIKVRISLKSALGDKAYEWNDNEMYLFRSSVAYAMR
QYFLKVKNQMILFGEEDVRVANLKPRISFNFFVTAPKNVSDIIPRTEVEKAIRMS
RSRINDAFRLNDNSLEFLGIQPTLGPPNQPPVS
SEQ ID NO: 6 (Angiotensin converting enzyme-2, C terminally his-tagged, as expressed in the cells)
MSSSSWLLLSLVAVTAAQSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNI
TEENVQNMNNAGDKWSAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSS
VLSEDKSKRLNTILNTMSTIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNE
RLWAWESWRSEVGKQLRPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVN
GVDGYDYSRGQLIEDVEHTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAH
LLGDMWGRFWTNLYSLTVPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVS
VGLPNMTQGFWENSMLTDPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDD
FLTAHHEMGHIQYDMAYAAQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIG
LLSPDFQEDNETEINFLLKQALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKK
WWEMKREIVGVVEPVPHDETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEAL
CQAAKHEGPLHKCDISNSTEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNV
RPLLNYFEPLFTWLKDQNKNSFVGWSTDWSPYADQSIKVRISLKSALGDKAYE
WNDNEMYLFRSSVAYAMRQYFLKVKNQMILFGEEDVRVANLKPRISFNFFVTAP
KNVSDIIPRTEVEKAIRMSRSRINDAFRLNDNSLEFLGIQPTLGPPNQPPVSHHH
HHHHH
SEQ ID NO: 7 (Angiotensin converting enzyme-2, C terminally his-tagged, after cleavage of the signal peptide)
QSTIEEQAKTFLDKFNHEAEDLFYQSSLASWNYNTNITEENVQNMNNAGDKW
SAFLKEQSTLAQMYPLQEIQNLTVKLQLQALQQNGSSVLSEDKSKRLNTILNTM
STIYSTGKVCNPDNPQECLLLEPGLNEIMANSLDYNERLWAWESWRSEVGKQL
RPLYEEYVVLKNEMARANHYEDYGDYWRGDYEVNGVDGYDYSRGQLIEDVE
HTFEEIKPLYEHLHAYVRAKLMNAYPSYISPIGCLPAHLLGDMWGRFWTNLYSLT
VPFGQKPNIDVTDAMVDQAWDAQRIFKEAEKFFVSVGLPNMTQGFWENSMLT
DPGNVQKAVCHPTAWDLGKGDFRILMCTKVTMDDFLTAHHEMGHIQYDMAYA
AQPFLLRNGANEGFHEAVGEIMSLSAATPKHLKSIGLLSPDFQEDNETEINFLLK
QALTIVGTLPFTYMLEKWRWMVFKGEIPKDQWMKKWWEMKREIVGVVEPVPH
DETYCDPASLFHVSNDYSFIRYYTRTLYQFQFQEALCQAAKHEGPLHKCDISNS
TEAGQKLFNMLRLGKSEPWTLALENVVGAKNMNVRPLLNYFEPLFTWLKDQN
KNSFVGWSTDWSPYADQSIKVRISLKSALGDKAYEWNDNEMYLFRSSVAYAMR
QYFLKVKNQMILFGEEDVRVANLKPRISFNFFVTAPKNVSDIIPRTEVEKAIRMS
RSRINDAFRLNDNSLEFLGIQPTLGPPNQPPVSHHHHHHHH
SEQ ID NO: 8 (Angiotensin converting enzyme-2, nucleotide sequence encoding SEQ ID NO: 6)
AT GAGCT CCAGCAGCTGGCT GCTGCTGTCTCTGGTGGCTGT GACAGCCGC T CAAAGCACCAT CGAAGAGCAAGCCAAG ACATTT CT GGATAAGTT CAACCAC GAGGCT G AGGAT CT GTT CTACCAG AGCT CT CT GGCCAGCT GGAACTATAAC ACAAATATCACCGAGGAGAACGTGCAGAACATGAATAACGCCGGCGACAAG TGGAGCGCCTTT CT GAAAGAGCAGAGCACCCT CGCT CAGAT GTATCCTCTG CAAGAGAT CCAAAAT CT G ACAGT GAAGCT CCAGCT GCAAGCCCT CCAGCAG AACGGCAGCAGCGTGCT CAGCGAGGACAAGT CCAAG AGGCT GAACACCAT T CT GAATACCAT GT CCACCAT CTACT CCACCGGCAAGGT GT GCAACCCCG A CAACCCT CAAGAGT GT CT GCT GCTGGAACCCGGACT GAACGAGAT CAT GGC CAACT CCCT CGACTATAACGAGAGGCT GTGGGCTT GGG AG AGCT GG AGGT CCGAGGT CGGCAAGCAACT GAGACCT CT CTACG AGGAATACGTGGT GCTG AAGAACGAGATGGCTAGAGCCAACCATTACGAGGACTATGGCGACTACTGG AGAGGCGACTACGAAGTCAACGGCGTGGACGGCTATGACTACTCTAGAGGC CAGCT GAT CGAAGACGT CGAGCACACCTTT GAGGAGAT CAAGCCCCT CTAT GAG CACCT CCACG CCTACGT G AG GGCCAAG CT CAT G AACG CCTACCCCAG CTACAT CT CCCCCATT GGAT GTCT GCCCGCCCAT CT GCT GGGAGACAT GT G GGGAAGATT CTGGACCAACCT CTACAGCCT CACAGT CCCCTTT GGCCAGAA GCCCAATAT CGAT GT GACCGACGCTAT GGT GG ACCAAGCTTGGGACGCCCA GAGGATTTTTAAGG AGGCCGAGAAGTTTTT CGT CAGCGT GGG ACT GCCTAA CATGACCCAAGGCTTTTGGGAGAACAGCATGCTGACCGACCCCGGCAATGT GCAGAAAGCT GT CT GCCACCCTACAGCTT GGGAT CTGGGCAAAGGCGACTT TAGAATT CT GAT GT GCACAAAGGT CACCAT GG ACGATTTT CT GACCGCCCAC CACG AAATGGGCCACAT CCAGTACG ATATGGCCTAT GCCGCT CAACCTTTT C TGCT G AGAAACGGAGCCAACGAGGGCTT CCAT GAAGCCGT GGGCGAGAT C ATGT CCCT CAGCGCCGCCACCCCTAAACAT CT GAAAAGCAT CGGACT GCTG T CCCCCGACTT CCAAGAGG ACAACGAGACCGAAATTAACTTT CTGCT CAAG CAAGCT CT GACCAT CGT CGGCACACT GCCCTT CACCTACAT GCT GGAGAAG T GGAGATGGAT GGT GTTTAAGGGAG AGATCCCCAAGGACCAGT GGAT GAAG AAAT GGT GGGAGAT GAAG AG AGAGAT CGT GGGCGTCGT GGAACCCGTGCC CCAT G ACG AGACCTACT GCGACCCCGCCT CT CT GTTT CACGT CT CCAAT GA
CTATT CCTT CATCAGATACTACACAAGAACACT GTACCAGTT CCAGTT CCAAG AGGCT CTGT GCCAAGCT GCCAAACAT GAAGGCCCT CT GCACAAAT GCGACA T CAGCAATT CCACCGAGGCCGGCCAGAAGCT GTT CAACAT GCT GAGACT GG GCAAGT CCG AGCCTT GGACACTGGCT CT GGAAAAT GT CGT CGGCGCTAAG AACAT GAAT GT GAGGCCT CT GCT G AACTACTTT GAGCCT CT GTT CACAT GGC T CAAAGAT CAG AATAAGAACAGCTT CGT CGGCTGGAGCACAGATT GG AGCC CCTACGCT GACCAGT CCAT CAAGGT G AGGATTAGCCT CAAG AGCGCT CT CG GCGATAAGGCCTACGAGTGGAACGACAACGAGATGTATCTGTTTAGAAGCTC CGT GGCCTAT GCCAT G AGGCAGTACTT CCT CAAGGT CAAAAACCAGAT GATT CT GTT CGGCGAAG AGGAT GT GAGGGT CGCCAAT CT G AAGCCTAGGAT CAGC TTTAACTT CTT CGT CACCGCCCCCAAGAACGT CT CCGACAT CAT CCCCAGAA CCGAGGTGGAGAAGGCTATTAGAATGTCTAGATCTAGAATCAACGACGCCTT TAGACT CAACGATAACT CT CT GGAGTTT CTGGGAAT CCAGCCTACCCT CGGC CCT CCTAACCAGCCT CCCGT GT CCCACCAT CAT CACCACCAT CACCAT
SEQ ID NO: 9 (SARS-CoV-2 Nucleoprotein)
SDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASW
FTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLS
PRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVL
QLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSSRNSTPGSSRGTSPARM
AGNGGDAALALLLLDRLNQLESKMSGKGQQQQGQTVTKKSAAEASKKPRQK
RTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASA
FFGMSRIGMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYKTFPPTEP
KKDKKKKADETQALPQRQKKQQTVTLLPAADLDDFSKQLQQSMSSADSTQAH
HHHHHHH
Claims
1. A method of detecting the presence of an antibody in a sample obtained from a subject, wherein said antibody has affinity for a coronavirus antigen, or portion thereof, and wherein said method comprises bringing the sample obtained from the subject into contact with a coronavirus antigen, or portion thereof, which is immobilised on a substrate support, in the presence of a detectably-labelled molecule that can compete with any antibody present for binding to the coronavirus antigen, or portion thereof, and detecting the binding of antibody to the coronavirus antigen, or portion thereof, by measuring the amount of binding of said molecule compared to a control, wherein the coronavirus antigen, or portion thereof, is immobilised to the substrate surface by means of a linking agent.
2. The method of claim 1 , wherein the linking agent is an epoxy silane derivative, an epoxy oligomer or an epoxy polymer.
3. The method of claim 2, wherein the linking agent is EPON-SU8.
4. The method of claims 1 to 3, wherein the coronavirus antigen, or portion thereof, immobilised on the substrate support is brought into contact with the sample prior to the addition of the detectably-labelled molecule to the substrate support.
5. The method of claims 1 to 4, wherein the antibody to be detected is a neutralising antibody.
6. The method of claims 1 to 5, wherein the coronavirus antigen, or portion thereof, is a SARS-CoV antigen, or portion thereof, SARS-CoV2 antigen, or portion thereof, or MERS-CoV antigen, or portion thereof, preferably the coronavirus antigen is a SARS-CoV2 antigen, or portion thereof.
7. The method of any one of claims 1 to 6, wherein the coronavirus antigen, or portion thereof, is a coronavirus spike protein, a coronavirus nucleocapsid protein, a coronavirus E protein, a coronavirus M protein or any combination thereof,
8. The method of claim 7, wherein the antibody has affinity for the receptor binding domain (RBD) of the coronavirus spike protein and/or the receptor binding motif (RBM) of the coronavirus spike protein.
9. The method of claim 7 or 8, wherein the coronavirus spike protein is a SARS- CoV spike protein, a SARS-CoV2 spike protein or a MERS-CoV spike protein, preferably wherein the coronavirus spike protein is a SARS-CoV2 spike protein.
10. The method of claim 8, wherein the coronavirus spike protein is a SARS- CoV spike protein, or a SARS-CoV2 spike protein, and the detectably-labelled molecule is angiotensin-converting enzyme 2 (ACE2), or wherein the coronavirus spike protein is a MERS-CoV spike protein and the detectably- labelled molecule is dipeptidyl peptidase 4 (DPP4).
11. The method of any one of claims 1 to 10, wherein the coronavirus antigen further comprises a protein tag.
12. The method of claim 11 , wherein the protein tag is a poly-His tag.
13. The method of any one of claims 1 to 2, wherein the coronavirus antigen, or portion thereof, is immobilised to the substrate surface by means of a linking agent and a cross-linker.
14. The method of claim 13, wherein the cross-linker is a sulphone- or sulphonate-based cross-linker, preferably the cross-linker is a sulphone-based cross-linker.
15. The method of claim 14, wherein the cross-linker is a sulphone-based cross linker and has a structure according to Formula (1):
wherein X is -CH2-;
Y is methylidene (=CH2), or Y is selected from -CH2-S02-Ph-R , CH2-S02-Ph- CHs, -CH2-S02-CH3,-CH2-0-S02-Ph-R4,-CH2-0-S02-Ph-CH3 or -CH2-0-S02- CH3; preferably Y is methylidene (=CH2), CH2-S02-Ph-CH3, or -CH2-0-S02-Ph- CH3, more preferably Y is methylidene (=CH2) or CH2-S02-Ph-CH3;
Ri is CH3 or CH3-Ph, wherein the Ph is optionally substituted with one or more Ci-ealkyl, N02, F, Cl or Br; preferably Ri is CH3-Ph, and Ph is substituted with a Ci-ealkyl group, preferably methyl; and
R2 is selected from -Ce-^aryl-Z, -Ci-isalkyl-Z, and -C2-2oalkenyl-Z, wherein Z is selected from COR3, -NH2 and -OH, R3 is selected from H, OH, NH2, -Ci-ealkyl- OH and (EtO)3Si-(CH2)n-NH- in which n is 1-6; and R is H or is optionally substituted with one or more Ci-ealkyl, N02, F, Cl or Br; preferably R2 is selected from -Ph-Z, -CioHs-Z, -Ci-ealkyl-Z and -C2-ealkenyl-Z, more preferably from -Ph- Z and -CioHe-Z, and more preferably -Ph-Z, and preferably Z is selected from COR3.
16. The method of claim 14 or 15, wherein the cross-linker is a sulphone-based cross-linker and has a structure according to Formula (2):
wherein X is -CH2-;
Y is methylidene(=CH2) or Y is selected from CH2-S02-Ph-CH3, -CH2-S02-CH3, - CH2-0-S02-Ph-CH3 or -CH2-0-S02-CH3; preferably Y is methylidene (=CH2), CH2-S02-Ph-CH3, or -CH2-0-S02-Ph-CH3, more preferably Y is methylidene (=CH2)or CH2-S02-Ph-CH3.;
Ri is CH3 or CH3-Ph, wherein the Ph is optionally substituted with one or more Ci-ealkyl; preferably Ri is CH3-Ph, and Ph is substituted with a Ci-ealkyl group, preferably methyl; and
R3 is selected from H, OH, NH2, -Ci-ealkyl-OH and (EtO)3Si-(CH2)n-NH- in which n is 1-6, preferably R3 is OH, H or (EtO)3Si-(CH2)n-NH- in which n is 1-6.
17. The method of any of claims 14 to 16, wherein the cross-linker is a sulphone-based cross-linker and has a structure according to Formula (3) or Formula (4):
wherein R3 is selected from H, OH, NH2, -Ci-ealkyl-OH and (EtO)3Si-(CH2)n-NH- in which n is 1-6, preferably OH, H or (EtO)3Si-(CH2)n-NH- in which n is 1-6.
18. The method of any one of claims 1 to 17, wherein the detectably-labelled molecule is labelled with horseradish peroxidase (HRP), or a streptavidin-biotin- enzyme complex or an avidin-biotin-enzyme complex.
19. The method of any one of claims 1 to 18, wherein the surface of the substrate is selectively covered with a coating composition, preferably wherein the coating composition is an ink formulation.
20. The method of claim 19, wherein the ink formulation comprises a pigment, a resin, an ink solvent, an ink additive and/or a structuring agent.
21. The method of claim 20, wherein the ink formulation comprises an epoxy resin, an acrylic resin, a pigment and a structuring agent.
22. The method of any one of claims 1 to 21 , wherein the sample is a serum sample, plasma sample, whole blood sample, saliva sample, urine sample, mucous sample, CSF sample, sputum sample, ear wax sample, hair sample, sweat sample, meconium, skin, solid tumour extracts, peripheral blood mononuclear cells, bone marrow mononuclear cells, cerebrospinal fluid, cystic fluid or any suitable cell lysate tear sample, preferably the sample is a serum or plasma sample.
23. The method of any one of claims 1 to 22, wherein the substrate is a bio chip, preferably wherein said bio-chip is ceramic.
24. A substrate having immobilised on its surface a coronavirus antigen, or portion thereof, immobilised on its surface as defined in any of claims 1 to 23.
25. A method for producing a substrate having a coronavirus antigen, or portion thereof, immobilised thereon, comprising attaching the coronavirus antigen, or portion thereof, to the substrate surface via a sulphone or sulphonate-based cross-linker.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP21166455 | 2021-03-31 | ||
GBGB2104662.8A GB202104662D0 (en) | 2021-03-31 | 2021-03-31 | Coronavirus assay 2 |
PCT/EP2022/058691 WO2022207863A1 (en) | 2021-03-31 | 2022-03-31 | Coronavirus assay |
Publications (1)
Publication Number | Publication Date |
---|---|
EP4314821A1 true EP4314821A1 (en) | 2024-02-07 |
Family
ID=81074156
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP22714221.3A Pending EP4314821A1 (en) | 2021-03-31 | 2022-03-31 | Coronavirus assay |
Country Status (2)
Country | Link |
---|---|
EP (1) | EP4314821A1 (en) |
WO (1) | WO2022207863A1 (en) |
Family Cites Families (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0874242B2 (en) | 1997-04-21 | 2009-06-03 | Randox Laboratories Ltd. | Device and apparatus for the simultaneous detection of multiple analytes |
GB201520341D0 (en) | 2015-11-18 | 2015-12-30 | Randox Lab Ltd And Randox Teoranta | Improvements relating to substrates for the attachment of molecules |
KR102205028B1 (en) * | 2020-03-22 | 2021-01-20 | (주)셀트리온 | A binding molecules able to neutralize SARS-CoV-2 |
CN111484560A (en) * | 2020-03-24 | 2020-08-04 | 深圳市华启生物科技有限公司 | Coronavirus model and application thereof |
CN111983226A (en) * | 2020-03-25 | 2020-11-24 | 新加坡国立大学 | Detection of SARSr-CoV antibodies |
CN111474365B (en) * | 2020-03-27 | 2021-09-17 | 北京大学 | Biosensor and preparation method thereof, and virus detection system and method |
CN111796093A (en) * | 2020-07-18 | 2020-10-20 | 范春雷 | Novel coronavirus, MERS and influenza A/B virus four-in-one rapid detection kit |
CN112098644B (en) * | 2020-09-11 | 2022-03-08 | 江苏美克医学技术有限公司 | Kit for detecting novel coronavirus neutralizing antibody by enzyme-linked immunosorbent assay and detection method thereof |
-
2022
- 2022-03-31 WO PCT/EP2022/058691 patent/WO2022207863A1/en active Application Filing
- 2022-03-31 EP EP22714221.3A patent/EP4314821A1/en active Pending
Also Published As
Publication number | Publication date |
---|---|
WO2022207863A1 (en) | 2022-10-06 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP7402244B2 (en) | Single molecule quantitative detection method and detection system | |
JP5903492B2 (en) | Molecular diagnostic assay device and method of use | |
US6139781A (en) | Chemiluminescent electron-rich aryl-substituted 1,2-dioxetanes | |
KR20080090532A (en) | Monoclonal antibodies binding to avian influenza virus subtype h5 haemagglutinin and uses thereof | |
US20230228752A1 (en) | Kits and methods for the enrichment and detection of RNA viruses of the Coronaviridae family | |
JP2003533697A (en) | Linker system for activating surfaces for bioconjugation and methods of use thereof | |
Shojaei et al. | A review on emerging diagnostic assay for viral detection: the case of avian influenza virus | |
CN110079583B (en) | Immunochromatography detection method and detection kit for nucleic acid and application of detection kit | |
US20120149008A1 (en) | Chromatographic system for nucleic acid detection | |
US11249083B1 (en) | Covid-19 spike-ACE2 binding assay for drug and antibody screening | |
KR20110137379A (en) | Method for detecting antibody against sith-1 in biological sample | |
KR20120001741A (en) | Method for detecting substance in biological sample | |
EP4314821A1 (en) | Coronavirus assay | |
JP6734011B2 (en) | Biomolecule detection test kit, biomolecule detection method using the same, biomolecule detection test piece and biomolecule detection labeling reagent used therein | |
US9506918B2 (en) | Methods of specifically releasing a sub-group of objects | |
US20170276671A1 (en) | Receptor linked regenerated cellulose membrane and methods for producing and using the same | |
JP3008690B2 (en) | Method for measuring ligand or receptor | |
EP4314822A1 (en) | Detection assay | |
CA3125208A1 (en) | Monoclonal antibody or antigen binding fragment thereof that binds to the l protein of the human parainfluenza virus (piv); method and kit for detecting piv | |
KR20040039349A (en) | Support for ligand immobilization | |
WO2021254484A1 (en) | Efficient antibody dna-barcoding reagents for multiplexed molecular imaging | |
JP4393378B2 (en) | Method for simultaneously detecting hybridization and immune reactions and use thereof in diagnosis | |
CA1339353C (en) | Macromolecular conjugate | |
WO2023122556A1 (en) | Methods and kits for determining the presence and/or amount of a human igg3 antibody specific for a flavivirus antigen in a sample | |
WO2021176378A1 (en) | Compositions and methods for the detection of viruses in a biological sample |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: UNKNOWN |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE |
|
PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE |
|
17P | Request for examination filed |
Effective date: 20230928 |
|
AK | Designated contracting states |
Kind code of ref document: A1 Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR |