EP0952849A1 - Early detection of mycobacterial disease - Google Patents
Early detection of mycobacterial diseaseInfo
- Publication number
- EP0952849A1 EP0952849A1 EP97954653A EP97954653A EP0952849A1 EP 0952849 A1 EP0952849 A1 EP 0952849A1 EP 97954653 A EP97954653 A EP 97954653A EP 97954653 A EP97954653 A EP 97954653A EP 0952849 A1 EP0952849 A1 EP 0952849A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- antigen
- kda
- early
- tuberculosis
- antigens
- 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.)
- Withdrawn
Links
- 238000001514 detection method Methods 0.000 title claims abstract description 67
- 208000027531 mycobacterial infectious disease Diseases 0.000 title claims description 24
- 102000036639 antigens Human genes 0.000 claims abstract description 647
- 108091007433 antigens Proteins 0.000 claims abstract description 647
- 239000000427 antigen Substances 0.000 claims abstract description 643
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 374
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 358
- 238000000034 method Methods 0.000 claims abstract description 64
- 201000010099 disease Diseases 0.000 claims abstract description 51
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 51
- 239000000706 filtrate Substances 0.000 claims abstract description 51
- 239000000203 mixture Substances 0.000 claims abstract description 41
- 102000003886 Glycoproteins Human genes 0.000 claims abstract description 34
- 108090000288 Glycoproteins Proteins 0.000 claims abstract description 34
- 101710102499 Alanine and proline-rich secreted protein Apa Proteins 0.000 claims abstract description 31
- 238000003018 immunoassay Methods 0.000 claims abstract description 26
- 230000000890 antigenic effect Effects 0.000 claims abstract description 21
- 101150047914 mpt51 gene Proteins 0.000 claims abstract description 20
- 101000913850 Tetrahymena thermophila Citrate synthase, mitochondrial Proteins 0.000 claims abstract description 14
- 238000012216 screening Methods 0.000 claims abstract description 7
- 241000713772 Human immunodeficiency virus 1 Species 0.000 claims abstract description 4
- 201000008827 tuberculosis Diseases 0.000 claims description 150
- 238000002360 preparation method Methods 0.000 claims description 60
- 241000282414 Homo sapiens Species 0.000 claims description 38
- 241000588724 Escherichia coli Species 0.000 claims description 36
- 238000012360 testing method Methods 0.000 claims description 32
- 210000004027 cell Anatomy 0.000 claims description 28
- 208000015181 infectious disease Diseases 0.000 claims description 25
- 239000003153 chemical reaction reagent Substances 0.000 claims description 23
- 101710134638 88 kDa protein Proteins 0.000 claims description 19
- 230000012010 growth Effects 0.000 claims description 19
- 239000013060 biological fluid Substances 0.000 claims description 15
- 238000000746 purification Methods 0.000 claims description 12
- 208000024891 symptom Diseases 0.000 claims description 11
- 239000012228 culture supernatant Substances 0.000 claims description 10
- 206010062207 Mycobacterial infection Diseases 0.000 claims description 9
- 230000001580 bacterial effect Effects 0.000 claims description 9
- 210000001124 body fluid Anatomy 0.000 claims description 8
- 239000010839 body fluid Substances 0.000 claims description 8
- 206010036790 Productive cough Diseases 0.000 claims description 6
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 claims description 6
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 claims description 6
- 210000003802 sputum Anatomy 0.000 claims description 6
- 208000024794 sputum Diseases 0.000 claims description 6
- 230000001355 anti-mycobacterial effect Effects 0.000 claims description 5
- 239000003926 antimycobacterial agent Substances 0.000 claims description 5
- 101000980867 Schizosaccharomyces pombe (strain 972 / ATCC 24843) Curved DNA-binding protein Proteins 0.000 claims description 4
- 101000982319 Shallot virus X Uncharacterized ORF4 protein Proteins 0.000 claims description 4
- 210000004408 hybridoma Anatomy 0.000 claims description 4
- 238000012258 culturing Methods 0.000 claims description 2
- 238000003259 recombinant expression Methods 0.000 claims description 2
- 101710149506 28 kDa protein Proteins 0.000 claims 2
- 101710106459 29 kDa protein Proteins 0.000 claims 2
- 101710144734 48 kDa protein Proteins 0.000 claims 2
- 101710172503 Chemokine-binding protein Proteins 0.000 claims 2
- 101710137943 Complement control protein C3 Proteins 0.000 claims 2
- 101710159910 Movement protein Proteins 0.000 claims 2
- 101710138270 PspA protein Proteins 0.000 claims 2
- 102100022135 S-arrestin Human genes 0.000 claims 2
- 101710117586 S-arrestin Proteins 0.000 claims 2
- 101710152003 Suppressor of silencing P0 Proteins 0.000 claims 2
- 244000052616 bacterial pathogen Species 0.000 claims 1
- 238000011161 development Methods 0.000 abstract description 15
- 239000003550 marker Substances 0.000 abstract description 10
- 206010061598 Immunodeficiency Diseases 0.000 abstract description 4
- 241000187479 Mycobacterium tuberculosis Species 0.000 abstract description 3
- 235000018102 proteins Nutrition 0.000 description 327
- 230000009257 reactivity Effects 0.000 description 189
- 241000725303 Human immunodeficiency virus Species 0.000 description 162
- 238000004458 analytical method Methods 0.000 description 72
- 210000002966 serum Anatomy 0.000 description 72
- 108090000765 processed proteins & peptides Proteins 0.000 description 71
- 239000000499 gel Substances 0.000 description 49
- 238000002965 ELISA Methods 0.000 description 48
- 108010015899 Glycopeptides Proteins 0.000 description 43
- 102000002068 Glycopeptides Human genes 0.000 description 43
- 239000000523 sample Substances 0.000 description 42
- 238000003556 assay Methods 0.000 description 39
- 102000004196 processed proteins & peptides Human genes 0.000 description 36
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 31
- 230000013595 glycosylation Effects 0.000 description 30
- 230000000875 corresponding effect Effects 0.000 description 29
- 238000006206 glycosylation reaction Methods 0.000 description 29
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 28
- 239000000047 product Substances 0.000 description 27
- 102000003992 Peroxidases Human genes 0.000 description 24
- 230000005875 antibody response Effects 0.000 description 24
- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(1+) nitrate Chemical compound [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 description 24
- DQJCDTNMLBYVAY-ZXXIYAEKSA-N (2S,5R,10R,13R)-16-{[(2R,3S,4R,5R)-3-{[(2S,3R,4R,5S,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy}-5-(ethylamino)-6-hydroxy-2-(hydroxymethyl)oxan-4-yl]oxy}-5-(4-aminobutyl)-10-carbamoyl-2,13-dimethyl-4,7,12,15-tetraoxo-3,6,11,14-tetraazaheptadecan-1-oic acid Chemical compound NCCCC[C@H](C(=O)N[C@@H](C)C(O)=O)NC(=O)CC[C@H](C(N)=O)NC(=O)[C@@H](C)NC(=O)C(C)O[C@@H]1[C@@H](NCC)C(O)O[C@H](CO)[C@H]1O[C@H]1[C@H](NC(C)=O)[C@@H](O)[C@H](O)[C@@H](CO)O1 DQJCDTNMLBYVAY-ZXXIYAEKSA-N 0.000 description 23
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 23
- 210000002421 cell wall Anatomy 0.000 description 23
- 239000006166 lysate Substances 0.000 description 22
- 125000001429 N-terminal alpha-amino-acid group Chemical group 0.000 description 21
- 125000003275 alpha amino acid group Chemical group 0.000 description 21
- 108040007629 peroxidase activity proteins Proteins 0.000 description 21
- 230000035945 sensitivity Effects 0.000 description 21
- 238000001262 western blot Methods 0.000 description 21
- 102000016938 Catalase Human genes 0.000 description 20
- 108010053835 Catalase Proteins 0.000 description 20
- 241001529936 Murinae Species 0.000 description 20
- 230000029087 digestion Effects 0.000 description 20
- 241001467552 Mycobacterium bovis BCG Species 0.000 description 19
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 19
- 150000002500 ions Chemical class 0.000 description 19
- 239000002953 phosphate buffered saline Substances 0.000 description 19
- 238000003119 immunoblot Methods 0.000 description 18
- 239000012528 membrane Substances 0.000 description 18
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 18
- 101710088427 Diacylglycerol acyltransferase/mycolyltransferase Ag85C Proteins 0.000 description 17
- 101150013110 katG gene Proteins 0.000 description 17
- 239000002904 solvent Substances 0.000 description 17
- 239000000463 material Substances 0.000 description 15
- -1 polypropylene Polymers 0.000 description 15
- 238000012163 sequencing technique Methods 0.000 description 15
- 238000001179 sorption measurement Methods 0.000 description 15
- 239000000872 buffer Substances 0.000 description 14
- 230000018109 developmental process Effects 0.000 description 14
- 235000000346 sugar Nutrition 0.000 description 14
- 101001057129 Bacillus cereus Enterotoxin Proteins 0.000 description 13
- 102000004190 Enzymes Human genes 0.000 description 13
- 108090000790 Enzymes Proteins 0.000 description 13
- 101000700655 Mycobacterium leprae (strain TN) Serine-rich antigen Proteins 0.000 description 13
- 229940024606 amino acid Drugs 0.000 description 13
- 235000001014 amino acid Nutrition 0.000 description 13
- 229940088598 enzyme Drugs 0.000 description 13
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 12
- 238000003745 diagnosis Methods 0.000 description 12
- 230000006870 function Effects 0.000 description 12
- 229920001223 polyethylene glycol Polymers 0.000 description 12
- 230000002829 reductive effect Effects 0.000 description 12
- 229960001516 silver nitrate Drugs 0.000 description 12
- 229910001961 silver nitrate Inorganic materials 0.000 description 12
- 108090000787 Subtilisin Proteins 0.000 description 11
- 229960000190 bacillus calmette–guérin vaccine Drugs 0.000 description 11
- 239000012634 fragment Substances 0.000 description 11
- 229920002401 polyacrylamide Polymers 0.000 description 11
- 238000002560 therapeutic procedure Methods 0.000 description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 11
- 241000894006 Bacteria Species 0.000 description 10
- 206010061818 Disease progression Diseases 0.000 description 10
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 10
- 239000000020 Nitrocellulose Substances 0.000 description 10
- 102000019199 alpha-Mannosidase Human genes 0.000 description 10
- 108010012864 alpha-Mannosidase Proteins 0.000 description 10
- 150000001413 amino acids Chemical class 0.000 description 10
- 238000012512 characterization method Methods 0.000 description 10
- 230000005750 disease progression Effects 0.000 description 10
- 229920001220 nitrocellulos Polymers 0.000 description 10
- 239000000126 substance Substances 0.000 description 10
- 102100024341 10 kDa heat shock protein, mitochondrial Human genes 0.000 description 9
- 108010059013 Chaperonin 10 Proteins 0.000 description 9
- 101100509674 Mycolicibacterium smegmatis (strain ATCC 700084 / mc(2)155) katG3 gene Proteins 0.000 description 9
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 9
- 230000003902 lesion Effects 0.000 description 9
- 101150014428 mpt64 gene Proteins 0.000 description 9
- 208000031886 HIV Infections Diseases 0.000 description 8
- 241000283973 Oryctolagus cuniculus Species 0.000 description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 230000015556 catabolic process Effects 0.000 description 8
- 230000036541 health Effects 0.000 description 8
- 239000012071 phase Substances 0.000 description 8
- 238000004007 reversed phase HPLC Methods 0.000 description 8
- 241000894007 species Species 0.000 description 8
- 238000005406 washing Methods 0.000 description 8
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 7
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 7
- 108020004414 DNA Proteins 0.000 description 7
- 101710088334 Diacylglycerol acyltransferase/mycolyltransferase Ag85B Proteins 0.000 description 7
- 230000004989 O-glycosylation Effects 0.000 description 7
- 238000013459 approach Methods 0.000 description 7
- 239000011324 bead Substances 0.000 description 7
- 238000005119 centrifugation Methods 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 7
- 238000006731 degradation reaction Methods 0.000 description 7
- 230000014509 gene expression Effects 0.000 description 7
- 238000004128 high performance liquid chromatography Methods 0.000 description 7
- 210000000987 immune system Anatomy 0.000 description 7
- 238000013507 mapping Methods 0.000 description 7
- MYWUZJCMWCOHBA-VIFPVBQESA-N methamphetamine Chemical compound CN[C@@H](C)CC1=CC=CC=C1 MYWUZJCMWCOHBA-VIFPVBQESA-N 0.000 description 7
- 230000003287 optical effect Effects 0.000 description 7
- 239000007787 solid Substances 0.000 description 7
- 239000007790 solid phase Substances 0.000 description 7
- 239000000758 substrate Substances 0.000 description 7
- 150000008163 sugars Chemical class 0.000 description 7
- IDOQDZANRZQBTP-UHFFFAOYSA-N 2-[2-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol Chemical compound CC(C)(C)CC(C)(C)C1=CC=CC=C1OCCO IDOQDZANRZQBTP-UHFFFAOYSA-N 0.000 description 6
- QRXMUCSWCMTJGU-UHFFFAOYSA-N 5-bromo-4-chloro-3-indolyl phosphate Chemical compound C1=C(Br)C(Cl)=C2C(OP(O)(=O)O)=CNC2=C1 QRXMUCSWCMTJGU-UHFFFAOYSA-N 0.000 description 6
- 102100038222 60 kDa heat shock protein, mitochondrial Human genes 0.000 description 6
- 101710087887 Alkyl hydroperoxide reductase C Proteins 0.000 description 6
- 241000304886 Bacilli Species 0.000 description 6
- 108010058432 Chaperonin 60 Proteins 0.000 description 6
- 101710088335 Diacylglycerol acyltransferase/mycolyltransferase Ag85A Proteins 0.000 description 6
- 208000037357 HIV infectious disease Diseases 0.000 description 6
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 6
- 229920004929 Triton X-114 Polymers 0.000 description 6
- 229940043292 chymotrypsin / trypsin Drugs 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 210000003743 erythrocyte Anatomy 0.000 description 6
- 208000033519 human immunodeficiency virus infectious disease Diseases 0.000 description 6
- 238000011534 incubation Methods 0.000 description 6
- 238000001155 isoelectric focusing Methods 0.000 description 6
- 238000004519 manufacturing process Methods 0.000 description 6
- 239000002609 medium Substances 0.000 description 6
- 230000004048 modification Effects 0.000 description 6
- 238000012986 modification Methods 0.000 description 6
- 239000008188 pellet Substances 0.000 description 6
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 description 6
- 208000035404 Autolysis Diseases 0.000 description 5
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 5
- 241000283707 Capra Species 0.000 description 5
- 206010057248 Cell death Diseases 0.000 description 5
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 5
- 108010067306 Fibronectins Proteins 0.000 description 5
- 102000016359 Fibronectins Human genes 0.000 description 5
- 108060003951 Immunoglobulin Proteins 0.000 description 5
- 241001646725 Mycobacterium tuberculosis H37Rv Species 0.000 description 5
- 239000004793 Polystyrene Substances 0.000 description 5
- 239000007983 Tris buffer Substances 0.000 description 5
- 239000008346 aqueous phase Substances 0.000 description 5
- 230000001413 cellular effect Effects 0.000 description 5
- 210000000038 chest Anatomy 0.000 description 5
- 238000010790 dilution Methods 0.000 description 5
- 239000012895 dilution Substances 0.000 description 5
- 238000005194 fractionation Methods 0.000 description 5
- 102000018358 immunoglobulin Human genes 0.000 description 5
- 238000001727 in vivo Methods 0.000 description 5
- 230000007935 neutral effect Effects 0.000 description 5
- 229910052757 nitrogen Inorganic materials 0.000 description 5
- 230000008520 organization Effects 0.000 description 5
- 229920002223 polystyrene Polymers 0.000 description 5
- 230000028043 self proteolysis Effects 0.000 description 5
- 230000000405 serological effect Effects 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 238000010186 staining Methods 0.000 description 5
- 150000005846 sugar alcohols Chemical class 0.000 description 5
- 239000000725 suspension Substances 0.000 description 5
- 238000011282 treatment Methods 0.000 description 5
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 5
- 239000003656 tris buffered saline Substances 0.000 description 5
- 239000003643 water by type Substances 0.000 description 5
- YQZGYLVYTGIJAX-QGOAFFKASA-N 1-(tert-butylamino)-3-[(e)-(3,3,5-trimethylcyclohexylidene)amino]oxypropan-2-ol Chemical compound CC1C\C(=N/OCC(O)CNC(C)(C)C)CC(C)(C)C1 YQZGYLVYTGIJAX-QGOAFFKASA-N 0.000 description 4
- 101710127328 28 kDa antigen Proteins 0.000 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 208000031729 Bacteremia Diseases 0.000 description 4
- SHIBSTMRCDJXLN-UHFFFAOYSA-N Digoxigenin Natural products C1CC(C2C(C3(C)CCC(O)CC3CC2)CC2O)(O)C2(C)C1C1=CC(=O)OC1 SHIBSTMRCDJXLN-UHFFFAOYSA-N 0.000 description 4
- 238000012286 ELISA Assay Methods 0.000 description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerol Natural products OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 4
- 101710087104 Invasin IpaC Proteins 0.000 description 4
- 208000032420 Latent Infection Diseases 0.000 description 4
- 101001060713 Leptospira interrogans serogroup Icterohaemorrhagiae serovar Lai (strain 56601) Flagellar filament 35 kDa core protein Proteins 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 4
- 101001013152 Mycobacterium avium Major membrane protein 1 Proteins 0.000 description 4
- 101001013151 Mycobacterium leprae (strain TN) Major membrane protein I Proteins 0.000 description 4
- DKXNBNKWCZZMJT-UHFFFAOYSA-N O4-alpha-D-Mannopyranosyl-D-mannose Natural products O=CC(O)C(O)C(C(O)CO)OC1OC(CO)C(O)C(O)C1O DKXNBNKWCZZMJT-UHFFFAOYSA-N 0.000 description 4
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 4
- 101000638142 Treponema pallidum (strain Nichols) Membrane lipoprotein TmpC Proteins 0.000 description 4
- 208000036981 active tuberculosis Diseases 0.000 description 4
- 238000013019 agitation Methods 0.000 description 4
- 102000007362 alpha-Crystallins Human genes 0.000 description 4
- 108010007908 alpha-Crystallins Proteins 0.000 description 4
- 230000003698 anagen phase Effects 0.000 description 4
- 210000000628 antibody-producing cell Anatomy 0.000 description 4
- 230000008901 benefit Effects 0.000 description 4
- 238000007068 beta-elimination reaction Methods 0.000 description 4
- 229940098773 bovine serum albumin Drugs 0.000 description 4
- 210000004899 c-terminal region Anatomy 0.000 description 4
- 238000002512 chemotherapy Methods 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
- 230000009260 cross reactivity Effects 0.000 description 4
- QONQRTHLHBTMGP-UHFFFAOYSA-N digitoxigenin Natural products CC12CCC(C3(CCC(O)CC3CC3)C)C3C11OC1CC2C1=CC(=O)OC1 QONQRTHLHBTMGP-UHFFFAOYSA-N 0.000 description 4
- SHIBSTMRCDJXLN-KCZCNTNESA-N digoxigenin Chemical compound C1([C@@H]2[C@@]3([C@@](CC2)(O)[C@H]2[C@@H]([C@@]4(C)CC[C@H](O)C[C@H]4CC2)C[C@H]3O)C)=CC(=O)OC1 SHIBSTMRCDJXLN-KCZCNTNESA-N 0.000 description 4
- 208000037771 disease arising from reactivation of latent virus Diseases 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 238000001962 electrophoresis Methods 0.000 description 4
- 238000010828 elution Methods 0.000 description 4
- 239000012530 fluid Substances 0.000 description 4
- 238000004108 freeze drying Methods 0.000 description 4
- 102000005396 glutamine synthetase Human genes 0.000 description 4
- 108020002326 glutamine synthetase Proteins 0.000 description 4
- 230000028993 immune response Effects 0.000 description 4
- 230000000977 initiatory effect Effects 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 238000004949 mass spectrometry Methods 0.000 description 4
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 4
- 239000013642 negative control Substances 0.000 description 4
- 239000002245 particle Substances 0.000 description 4
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 4
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 4
- 239000013641 positive control Substances 0.000 description 4
- 230000002685 pulmonary effect Effects 0.000 description 4
- 230000007420 reactivation Effects 0.000 description 4
- 230000004044 response Effects 0.000 description 4
- 238000007894 restriction fragment length polymorphism technique Methods 0.000 description 4
- 229910052709 silver Inorganic materials 0.000 description 4
- 239000004332 silver Substances 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- 238000002255 vaccination Methods 0.000 description 4
- GUBGYTABKSRVRQ-PZPXDAEZSA-N 4β-mannobiose Chemical compound O[C@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-PZPXDAEZSA-N 0.000 description 3
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 108010077805 Bacterial Proteins Proteins 0.000 description 3
- 208000035473 Communicable disease Diseases 0.000 description 3
- 101710088194 Dehydrogenase Proteins 0.000 description 3
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- 241000282412 Homo Species 0.000 description 3
- 101710164418 Movement protein TGB2 Proteins 0.000 description 3
- 108010033276 Peptide Fragments Proteins 0.000 description 3
- 102000007079 Peptide Fragments Human genes 0.000 description 3
- 102000001708 Protein Isoforms Human genes 0.000 description 3
- 108010029485 Protein Isoforms Proteins 0.000 description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 3
- 210000001744 T-lymphocyte Anatomy 0.000 description 3
- 239000006180 TBST buffer Substances 0.000 description 3
- 101710120037 Toxin CcdB Proteins 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
- 238000003277 amino acid sequence analysis Methods 0.000 description 3
- 101150080488 apa gene Proteins 0.000 description 3
- 230000000721 bacterilogical effect Effects 0.000 description 3
- 244000309464 bull Species 0.000 description 3
- 150000001720 carbohydrates Chemical class 0.000 description 3
- 239000000969 carrier Substances 0.000 description 3
- 150000001793 charged compounds Chemical class 0.000 description 3
- 238000003759 clinical diagnosis Methods 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 239000002158 endotoxin Substances 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 239000007789 gas Substances 0.000 description 3
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 3
- 102000035122 glycosylated proteins Human genes 0.000 description 3
- 108091005608 glycosylated proteins Proteins 0.000 description 3
- 238000009396 hybridization Methods 0.000 description 3
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 3
- 238000002955 isolation Methods 0.000 description 3
- 229960003350 isoniazid Drugs 0.000 description 3
- QRXWMOHMRWLFEY-UHFFFAOYSA-N isoniazide Chemical compound NNC(=O)C1=CC=NC=C1 QRXWMOHMRWLFEY-UHFFFAOYSA-N 0.000 description 3
- 210000004698 lymphocyte Anatomy 0.000 description 3
- 230000014759 maintenance of location Effects 0.000 description 3
- 125000000311 mannosyl group Chemical group C1([C@@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 3
- 229910052751 metal Inorganic materials 0.000 description 3
- 239000002184 metal Substances 0.000 description 3
- 230000036961 partial effect Effects 0.000 description 3
- 230000008506 pathogenesis Effects 0.000 description 3
- 239000013610 patient sample Substances 0.000 description 3
- KHIWWQKSHDUIBK-UHFFFAOYSA-N periodic acid Chemical compound OI(=O)(=O)=O KHIWWQKSHDUIBK-UHFFFAOYSA-N 0.000 description 3
- 235000020030 perry Nutrition 0.000 description 3
- 230000004481 post-translational protein modification Effects 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 230000001681 protective effect Effects 0.000 description 3
- 230000017854 proteolysis Effects 0.000 description 3
- 230000002797 proteolythic effect Effects 0.000 description 3
- 208000008128 pulmonary tuberculosis Diseases 0.000 description 3
- 238000003127 radioimmunoassay Methods 0.000 description 3
- 230000010076 replication Effects 0.000 description 3
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 3
- 230000028327 secretion Effects 0.000 description 3
- 230000000392 somatic effect Effects 0.000 description 3
- 238000007619 statistical method Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- HNSDLXPSAYFUHK-UHFFFAOYSA-N 1,4-bis(2-ethylhexyl) sulfosuccinate Chemical compound CCCCC(CC)COC(=O)CC(S(O)(=O)=O)C(=O)OCC(CC)CCCC HNSDLXPSAYFUHK-UHFFFAOYSA-N 0.000 description 2
- PHLXSNIEQIKENK-UHFFFAOYSA-N 2-[[2-[5-methyl-3-(trifluoromethyl)pyrazol-1-yl]acetyl]amino]-4,5,6,7-tetrahydro-1-benzothiophene-3-carboxamide Chemical compound CC1=CC(C(F)(F)F)=NN1CC(=O)NC1=C(C(N)=O)C(CCCC2)=C2S1 PHLXSNIEQIKENK-UHFFFAOYSA-N 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- 101800000263 Acidic protein Proteins 0.000 description 2
- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 description 2
- 229910000013 Ammonium bicarbonate Inorganic materials 0.000 description 2
- 101100450144 Arabidopsis thaliana HAT3 gene Proteins 0.000 description 2
- 101000609447 Beet necrotic yellow vein virus (isolate Japan/S) Protein P25 Proteins 0.000 description 2
- 108010062580 Concanavalin A Proteins 0.000 description 2
- 108091035707 Consensus sequence Proteins 0.000 description 2
- 208000006313 Delayed Hypersensitivity Diseases 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 102100024023 Histone PARylation factor 1 Human genes 0.000 description 2
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 2
- 102000003960 Ligases Human genes 0.000 description 2
- 108090000364 Ligases Proteins 0.000 description 2
- 241000186366 Mycobacterium bovis Species 0.000 description 2
- 101001043272 Mycobacterium tuberculosis (strain ATCC 25177 / H37Ra) Lipoprotein LpqH Proteins 0.000 description 2
- 108700026244 Open Reading Frames Proteins 0.000 description 2
- 239000002033 PVDF binder Substances 0.000 description 2
- 206010057249 Phagocytosis Diseases 0.000 description 2
- 229920001213 Polysorbate 20 Polymers 0.000 description 2
- 108010076504 Protein Sorting Signals Proteins 0.000 description 2
- 206010051738 Pulmonary cavitation Diseases 0.000 description 2
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
- 101100230601 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) HBT1 gene Proteins 0.000 description 2
- 229920002684 Sepharose Polymers 0.000 description 2
- 238000012300 Sequence Analysis Methods 0.000 description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
- 102000019197 Superoxide Dismutase Human genes 0.000 description 2
- 108010012715 Superoxide dismutase Proteins 0.000 description 2
- 230000005867 T cell response Effects 0.000 description 2
- 108090000631 Trypsin Proteins 0.000 description 2
- 102000004142 Trypsin Human genes 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 238000001793 Wilcoxon signed-rank test Methods 0.000 description 2
- MCMNRKCIXSYSNV-UHFFFAOYSA-N Zirconium dioxide Chemical compound O=[Zr]=O MCMNRKCIXSYSNV-UHFFFAOYSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 230000004520 agglutination Effects 0.000 description 2
- 238000007818 agglutination assay Methods 0.000 description 2
- 125000003295 alanine group Chemical group N[C@@H](C)C(=O)* 0.000 description 2
- 230000007815 allergy Effects 0.000 description 2
- 235000012538 ammonium bicarbonate Nutrition 0.000 description 2
- 239000001099 ammonium carbonate Substances 0.000 description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical class N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
- 230000002365 anti-tubercular Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000002051 biphasic effect Effects 0.000 description 2
- 235000014633 carbohydrates Nutrition 0.000 description 2
- 210000004970 cd4 cell Anatomy 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000012875 competitive assay Methods 0.000 description 2
- 230000002860 competitive effect Effects 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 239000000470 constituent Substances 0.000 description 2
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 2
- 239000012531 culture fluid Substances 0.000 description 2
- 230000009089 cytolysis Effects 0.000 description 2
- 230000001086 cytosolic effect Effects 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 230000007812 deficiency Effects 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 239000003599 detergent Substances 0.000 description 2
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 2
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 2
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 238000012137 double-staining Methods 0.000 description 2
- 238000013399 early diagnosis Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000032050 esterification Effects 0.000 description 2
- 238000005886 esterification reaction Methods 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 101150028082 fbpA gene Proteins 0.000 description 2
- 101150020570 fbpC gene Proteins 0.000 description 2
- 239000012091 fetal bovine serum Substances 0.000 description 2
- 230000004992 fission Effects 0.000 description 2
- 238000000684 flow cytometry Methods 0.000 description 2
- 239000008103 glucose 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
- 238000004191 hydrophobic interaction chromatography Methods 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 230000003053 immunization Effects 0.000 description 2
- 238000002649 immunization Methods 0.000 description 2
- 230000000984 immunochemical effect Effects 0.000 description 2
- 230000002163 immunogen Effects 0.000 description 2
- 102000028557 immunoglobulin binding proteins Human genes 0.000 description 2
- 108091009323 immunoglobulin binding proteins Proteins 0.000 description 2
- 239000002198 insoluble material Substances 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 238000004020 luminiscence type Methods 0.000 description 2
- 108010005335 mannoproteins Proteins 0.000 description 2
- 238000005621 mannosylation reaction Methods 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 150000002739 metals Chemical class 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 230000005012 migration Effects 0.000 description 2
- 238000013508 migration Methods 0.000 description 2
- 150000002772 monosaccharides Chemical class 0.000 description 2
- 230000003647 oxidation Effects 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- 238000005192 partition Methods 0.000 description 2
- 125000001805 pentosyl group Chemical group 0.000 description 2
- 230000008782 phagocytosis Effects 0.000 description 2
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 2
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 2
- 229920000053 polysorbate 80 Polymers 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 230000003449 preventive effect Effects 0.000 description 2
- 238000009117 preventive therapy Methods 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 238000010188 recombinant method Methods 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 230000003362 replicative effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- JQWHASGSAFIOCM-UHFFFAOYSA-M sodium periodate Chemical compound [Na+].[O-]I(=O)(=O)=O JQWHASGSAFIOCM-UHFFFAOYSA-M 0.000 description 2
- 238000010561 standard procedure Methods 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 239000012536 storage buffer Substances 0.000 description 2
- 108060008226 thioredoxin Proteins 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 230000032258 transport Effects 0.000 description 2
- 239000012588 trypsin Substances 0.000 description 2
- 229960005486 vaccine Drugs 0.000 description 2
- 239000013598 vector Substances 0.000 description 2
- 230000000007 visual effect Effects 0.000 description 2
- 239000011534 wash buffer Substances 0.000 description 2
- ZLUSTGDGOTYUPV-DKWTVANSSA-N (2s)-2-aminopropanoic acid;propane-1,2,3-triol Chemical class C[C@H](N)C(O)=O.OCC(O)CO ZLUSTGDGOTYUPV-DKWTVANSSA-N 0.000 description 1
- WURBVZBTWMNKQT-UHFFFAOYSA-N 1-(4-chlorophenoxy)-3,3-dimethyl-1-(1,2,4-triazol-1-yl)butan-2-one Chemical compound C1=NC=NN1C(C(=O)C(C)(C)C)OC1=CC=C(Cl)C=C1 WURBVZBTWMNKQT-UHFFFAOYSA-N 0.000 description 1
- 101710097567 12 kDa protein Proteins 0.000 description 1
- 101710097814 13 kDa protein Proteins 0.000 description 1
- 101710154545 16 kDa protein Proteins 0.000 description 1
- BHNQPLPANNDEGL-UHFFFAOYSA-N 2-(4-octylphenoxy)ethanol Chemical compound CCCCCCCCC1=CC=C(OCCO)C=C1 BHNQPLPANNDEGL-UHFFFAOYSA-N 0.000 description 1
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 description 1
- XDWXDKYBDCMXLC-UHFFFAOYSA-N 4-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]naphthalen-1-ol Chemical compound C1=CC=C2C(OCCOCCOCCO)=CC=C(O)C2=C1 XDWXDKYBDCMXLC-UHFFFAOYSA-N 0.000 description 1
- IMCUVBSHZXQITN-UHFFFAOYSA-N 4-[[4-(4-chlorophenyl)-5-(2-methoxy-2-oxoethyl)-1,3-thiazol-2-yl]amino]-4-oxobutanoic acid Chemical compound S1C(NC(=O)CCC(O)=O)=NC(C=2C=CC(Cl)=CC=2)=C1CC(=O)OC IMCUVBSHZXQITN-UHFFFAOYSA-N 0.000 description 1
- HUDPLKWXRLNSPC-UHFFFAOYSA-N 4-aminophthalhydrazide Chemical compound O=C1NNC(=O)C=2C1=CC(N)=CC=2 HUDPLKWXRLNSPC-UHFFFAOYSA-N 0.000 description 1
- GUBGYTABKSRVRQ-TYAPZPMWSA-N 4α-mannobiose Chemical compound O[C@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-TYAPZPMWSA-N 0.000 description 1
- 102100031126 6-phosphogluconolactonase Human genes 0.000 description 1
- 108010029731 6-phosphogluconolactonase Proteins 0.000 description 1
- 208000030507 AIDS Diseases 0.000 description 1
- ITZMJCSORYKOSI-AJNGGQMLSA-N APGPR Enterostatin Chemical compound C[C@H](N)C(=O)N1CCC[C@H]1C(=O)NCC(=O)N1[C@H](C(=O)N[C@@H](CCCN=C(N)N)C(O)=O)CCC1 ITZMJCSORYKOSI-AJNGGQMLSA-N 0.000 description 1
- 108010022752 Acetylcholinesterase Proteins 0.000 description 1
- 102000012440 Acetylcholinesterase Human genes 0.000 description 1
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 1
- 108010000239 Aequorin Proteins 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 108010031025 Alanine Dehydrogenase Proteins 0.000 description 1
- 102000007698 Alcohol dehydrogenase Human genes 0.000 description 1
- 108010021809 Alcohol dehydrogenase Proteins 0.000 description 1
- 102000013142 Amylases Human genes 0.000 description 1
- 108010065511 Amylases Proteins 0.000 description 1
- 241000024188 Andala Species 0.000 description 1
- 101100450146 Arabidopsis thaliana HAT5 gene Proteins 0.000 description 1
- 208000002109 Argyria Diseases 0.000 description 1
- 206010003445 Ascites Diseases 0.000 description 1
- 108010024976 Asparaginase Proteins 0.000 description 1
- 102000015790 Asparaginase Human genes 0.000 description 1
- 108091005658 Basic proteases Proteins 0.000 description 1
- 102100026189 Beta-galactosidase Human genes 0.000 description 1
- 101000891022 Borrelia burgdorferi (strain ATCC 35210 / B31 / CIP 102532 / DSM 4680) Flagellar filament 41 kDa core protein Proteins 0.000 description 1
- 239000008001 CAPS buffer Substances 0.000 description 1
- 244000045232 Canavalia ensiformis Species 0.000 description 1
- 235000010520 Canavalia ensiformis Nutrition 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 108030002440 Catalase peroxidases Proteins 0.000 description 1
- 108010059892 Cellulase Proteins 0.000 description 1
- 101100065968 Cereibacter sphaeroides fbp1 gene Proteins 0.000 description 1
- 101100012235 Cereibacter sphaeroides fbp2 gene Proteins 0.000 description 1
- 101710098119 Chaperonin GroEL 2 Proteins 0.000 description 1
- 206010011224 Cough Diseases 0.000 description 1
- IGXWBGJHJZYPQS-SSDOTTSWSA-N D-Luciferin Chemical compound OC(=O)[C@H]1CSC(C=2SC3=CC=C(O)C=C3N=2)=N1 IGXWBGJHJZYPQS-SSDOTTSWSA-N 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- QNAYBMKLOCPYGJ-UHFFFAOYSA-N D-alpha-Ala Natural products CC([NH3+])C([O-])=O QNAYBMKLOCPYGJ-UHFFFAOYSA-N 0.000 description 1
- CYCGRDQQIOGCKX-UHFFFAOYSA-N Dehydro-luciferin Natural products OC(=O)C1=CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 CYCGRDQQIOGCKX-UHFFFAOYSA-N 0.000 description 1
- 108010053770 Deoxyribonucleases Proteins 0.000 description 1
- 102000016911 Deoxyribonucleases Human genes 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 108010067770 Endopeptidase K Proteins 0.000 description 1
- 101100055028 Escherichia coli (strain K12) afuB gene Proteins 0.000 description 1
- 241000186394 Eubacterium Species 0.000 description 1
- 241001267419 Eubacterium sp. Species 0.000 description 1
- 241000192016 Finegoldia magna Species 0.000 description 1
- BJGNCJDXODQBOB-UHFFFAOYSA-N Fivefly Luciferin Natural products OC(=O)C1CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 BJGNCJDXODQBOB-UHFFFAOYSA-N 0.000 description 1
- 108010073178 Glucan 1,4-alpha-Glucosidase Proteins 0.000 description 1
- 102100022624 Glucoamylase Human genes 0.000 description 1
- 108010015776 Glucose oxidase Proteins 0.000 description 1
- 239000004366 Glucose oxidase Substances 0.000 description 1
- 108010018962 Glucosephosphate Dehydrogenase Proteins 0.000 description 1
- 102000000587 Glycerolphosphate Dehydrogenase Human genes 0.000 description 1
- 108010041921 Glycerolphosphate Dehydrogenase Proteins 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 101150053933 HAT2 gene Proteins 0.000 description 1
- 108010004889 Heat-Shock Proteins Proteins 0.000 description 1
- 102000002812 Heat-Shock Proteins Human genes 0.000 description 1
- 241001622557 Hesperia Species 0.000 description 1
- 229920000209 Hexadimethrine bromide Polymers 0.000 description 1
- 102100021467 Histone acetyltransferase type B catalytic subunit Human genes 0.000 description 1
- 101000898976 Homo sapiens Histone acetyltransferase type B catalytic subunit Proteins 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 108010058683 Immobilized Proteins Proteins 0.000 description 1
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 description 1
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 description 1
- 102000012011 Isocitrate Dehydrogenase Human genes 0.000 description 1
- 108010075869 Isocitrate Dehydrogenase Proteins 0.000 description 1
- 102000004195 Isomerases Human genes 0.000 description 1
- 108090000769 Isomerases Proteins 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- QNAYBMKLOCPYGJ-UWTATZPHSA-N L-Alanine Natural products C[C@@H](N)C(O)=O QNAYBMKLOCPYGJ-UWTATZPHSA-N 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- 206010024227 Lepromatous leprosy Diseases 0.000 description 1
- 206010024229 Leprosy Diseases 0.000 description 1
- 108060001084 Luciferase Proteins 0.000 description 1
- 239000005089 Luciferase Substances 0.000 description 1
- DDWFXDSYGUXRAY-UHFFFAOYSA-N Luciferin Natural products CCc1c(C)c(CC2NC(=O)C(=C2C=C)C)[nH]c1Cc3[nH]c4C(=C5/NC(CC(=O)O)C(C)C5CC(=O)O)CC(=O)c4c3C DDWFXDSYGUXRAY-UHFFFAOYSA-N 0.000 description 1
- 108010047357 Luminescent Proteins Proteins 0.000 description 1
- 102000006830 Luminescent Proteins Human genes 0.000 description 1
- 208000019693 Lung disease Diseases 0.000 description 1
- 239000006391 Luria-Bertani Medium Substances 0.000 description 1
- 102000013460 Malate Dehydrogenase Human genes 0.000 description 1
- 108010026217 Malate Dehydrogenase Proteins 0.000 description 1
- 208000002720 Malnutrition Diseases 0.000 description 1
- 229920000057 Mannan Polymers 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 102000001696 Mannosidases Human genes 0.000 description 1
- 108010054377 Mannosidases Proteins 0.000 description 1
- 108010059724 Micrococcal Nuclease Proteins 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 241000186359 Mycobacterium Species 0.000 description 1
- 241000186367 Mycobacterium avium Species 0.000 description 1
- 241000545499 Mycobacterium avium-intracellulare Species 0.000 description 1
- 101100291912 Mycobacterium bovis (strain ATCC BAA-935 / AF2122/97) mpb64 gene Proteins 0.000 description 1
- 101100131080 Mycobacterium bovis (strain ATCC BAA-935 / AF2122/97) mpb70 gene Proteins 0.000 description 1
- 241000186362 Mycobacterium leprae Species 0.000 description 1
- 241000187480 Mycobacterium smegmatis Species 0.000 description 1
- OVRNDRQMDRJTHS-RTRLPJTCSA-N N-acetyl-D-glucosamine Chemical group CC(=O)N[C@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-RTRLPJTCSA-N 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- 108091006033 O-glycosylated proteins Proteins 0.000 description 1
- 101000793655 Oryza sativa subsp. japonica Calreticulin Proteins 0.000 description 1
- 101710116435 Outer membrane protein Proteins 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 102000008153 Peptide Elongation Factor Tu Human genes 0.000 description 1
- 108010049977 Peptide Elongation Factor Tu Proteins 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 1
- 108010053210 Phycocyanin Proteins 0.000 description 1
- 108010004729 Phycoerythrin Proteins 0.000 description 1
- 101000959005 Plasmodium falciparum Fructose-bisphosphate aldolase Proteins 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 239000004743 Polypropylene Substances 0.000 description 1
- 101800004937 Protein C Proteins 0.000 description 1
- 102000017975 Protein C Human genes 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 241000607142 Salmonella Species 0.000 description 1
- 101800001700 Saposin-D Proteins 0.000 description 1
- 101100450139 Schizosaccharomyces pombe (strain 972 / ATCC 24843) mis16 gene Proteins 0.000 description 1
- 102100021225 Serine hydroxymethyltransferase, cytosolic Human genes 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 101000815632 Streptococcus suis (strain 05ZYH33) Rqc2 homolog RqcH Proteins 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 230000024932 T cell mediated immunity Effects 0.000 description 1
- 241000473945 Theria <moth genus> Species 0.000 description 1
- 102100036407 Thioredoxin Human genes 0.000 description 1
- 101001009612 Toxoplasma gondii Dense granule protein 6 Proteins 0.000 description 1
- 102000004357 Transferases Human genes 0.000 description 1
- 108090000992 Transferases Proteins 0.000 description 1
- 241000499912 Trichoderma reesei Species 0.000 description 1
- 102000005924 Triose-Phosphate Isomerase Human genes 0.000 description 1
- 108700015934 Triose-phosphate isomerases Proteins 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 108010046334 Urease Proteins 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 238000006640 acetylation reaction Methods 0.000 description 1
- 229940022698 acetylcholinesterase Drugs 0.000 description 1
- 238000005903 acid hydrolysis reaction Methods 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- DZBUGLKDJFMEHC-UHFFFAOYSA-N acridine Chemical class C1=CC=CC2=CC3=CC=CC=C3N=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-N 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 238000001261 affinity purification Methods 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 229960003767 alanine Drugs 0.000 description 1
- GZCGUPFRVQAUEE-SLPGGIOYSA-N aldehydo-D-glucose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O GZCGUPFRVQAUEE-SLPGGIOYSA-N 0.000 description 1
- 208000026935 allergic disease Diseases 0.000 description 1
- 108010004469 allophycocyanin Proteins 0.000 description 1
- LHAOFBCHXGZGOR-NAVBLJQLSA-N alpha-D-Manp-(1->3)-alpha-D-Manp-(1->2)-alpha-D-Manp Chemical group O[C@H]1[C@H](O)[C@@H](CO)O[C@H](O)[C@H]1O[C@@H]1[C@@H](O)[C@@H](O[C@@H]2[C@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)O)[C@H](O)[C@@H](CO)O1 LHAOFBCHXGZGOR-NAVBLJQLSA-N 0.000 description 1
- WQZGKKKJIJFFOK-PQMKYFCFSA-N alpha-D-mannose Chemical compound OC[C@H]1O[C@H](O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-PQMKYFCFSA-N 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 229960002684 aminocaproic acid Drugs 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 235000019418 amylase Nutrition 0.000 description 1
- 229940025131 amylases Drugs 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 238000005571 anion exchange chromatography Methods 0.000 description 1
- 210000000776 antibody secreting cell Anatomy 0.000 description 1
- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 description 1
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 1
- 229960003272 asparaginase Drugs 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-M asparaginate Chemical compound [O-]C(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-M 0.000 description 1
- 238000002820 assay format Methods 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 239000000440 bentonite Substances 0.000 description 1
- 229910000278 bentonite Inorganic materials 0.000 description 1
- SVPXDRXYRYOSEX-UHFFFAOYSA-N bentoquatam Chemical compound O.O=[Si]=O.O=[Al]O[Al]=O SVPXDRXYRYOSEX-UHFFFAOYSA-N 0.000 description 1
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 1
- 108010005774 beta-Galactosidase Proteins 0.000 description 1
- AFYNADDZULBEJA-UHFFFAOYSA-N bicinchoninic acid Chemical compound C1=CC=CC2=NC(C=3C=C(C4=CC=CC=C4N=3)C(=O)O)=CC(C(O)=O)=C21 AFYNADDZULBEJA-UHFFFAOYSA-N 0.000 description 1
- 210000000941 bile Anatomy 0.000 description 1
- 239000004305 biphenyl Substances 0.000 description 1
- 235000010290 biphenyl Nutrition 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 238000009640 blood culture Methods 0.000 description 1
- NCMHKCKGHRPLCM-UHFFFAOYSA-N caesium(1+) Chemical compound [Cs+] NCMHKCKGHRPLCM-UHFFFAOYSA-N 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 238000012511 carbohydrate analysis Methods 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 238000005266 casting Methods 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 230000007969 cellular immunity Effects 0.000 description 1
- 230000008614 cellular interaction Effects 0.000 description 1
- 229940106157 cellulase Drugs 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 235000010980 cellulose Nutrition 0.000 description 1
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 1
- 239000003610 charcoal Substances 0.000 description 1
- 238000003115 checkerboard titration Methods 0.000 description 1
- 239000002738 chelating agent Substances 0.000 description 1
- 238000012412 chemical coupling Methods 0.000 description 1
- 239000003593 chromogenic compound Substances 0.000 description 1
- 238000007374 clinical diagnostic method Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 238000010835 comparative analysis Methods 0.000 description 1
- 102000006834 complement receptors Human genes 0.000 description 1
- 108010047295 complement receptors Proteins 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 230000037029 cross reaction Effects 0.000 description 1
- 210000000172 cytosol Anatomy 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 231100000517 death Toxicity 0.000 description 1
- 239000007857 degradation product Substances 0.000 description 1
- 230000034373 developmental growth involved in morphogenesis Effects 0.000 description 1
- 239000000032 diagnostic agent Substances 0.000 description 1
- 229940039227 diagnostic agent Drugs 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 238000009510 drug design Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000000132 electrospray ionisation Methods 0.000 description 1
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000006862 enzymatic digestion Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 238000001400 expression cloning Methods 0.000 description 1
- 238000010265 fast atom bombardment Methods 0.000 description 1
- 101150082784 fbpB gene Proteins 0.000 description 1
- 102000036072 fibronectin binding proteins Human genes 0.000 description 1
- ZFKJVJIDPQDDFY-UHFFFAOYSA-N fluorescamine Chemical compound C12=CC=CC=C2C(=O)OC1(C1=O)OC=C1C1=CC=CC=C1 ZFKJVJIDPQDDFY-UHFFFAOYSA-N 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 229930182830 galactose Natural products 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 229940116332 glucose oxidase Drugs 0.000 description 1
- 235000019420 glucose oxidase Nutrition 0.000 description 1
- 229930182470 glycoside Natural products 0.000 description 1
- 150000002338 glycosides Chemical class 0.000 description 1
- 125000003147 glycosyl group Chemical group 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 229960004198 guanidine Drugs 0.000 description 1
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 description 1
- 230000035931 haemagglutination Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 150000002402 hexoses Chemical class 0.000 description 1
- 125000000625 hexosyl group Chemical group 0.000 description 1
- 230000008348 humoral response Effects 0.000 description 1
- 230000003100 immobilizing effect Effects 0.000 description 1
- 230000002519 immonomodulatory effect Effects 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 208000026278 immune system disease Diseases 0.000 description 1
- 238000003365 immunocytochemistry Methods 0.000 description 1
- 238000000760 immunoelectrophoresis Methods 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 229940072221 immunoglobulins Drugs 0.000 description 1
- 230000006054 immunological memory Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 229910052738 indium Inorganic materials 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- JORABGDXCIBAFL-UHFFFAOYSA-M iodonitrotetrazolium chloride Chemical compound [Cl-].C1=CC([N+](=O)[O-])=CC=C1N1[N+](C=2C=CC(I)=CC=2)=NC(C=2C=CC=CC=2)=N1 JORABGDXCIBAFL-UHFFFAOYSA-M 0.000 description 1
- SZVJSHCCFOBDDC-UHFFFAOYSA-N iron(II,III) oxide Inorganic materials O=[Fe]O[Fe]O[Fe]=O SZVJSHCCFOBDDC-UHFFFAOYSA-N 0.000 description 1
- CYPPCCJJKNISFK-UHFFFAOYSA-J kaolinite Chemical compound [OH-].[OH-].[OH-].[OH-].[Al+3].[Al+3].[O-][Si](=O)O[Si]([O-])=O CYPPCCJJKNISFK-UHFFFAOYSA-J 0.000 description 1
- 229910052622 kaolinite Inorganic materials 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 238000011005 laboratory method Methods 0.000 description 1
- 229910052747 lanthanoid Inorganic materials 0.000 description 1
- 150000002602 lanthanoids Chemical class 0.000 description 1
- 239000004816 latex Substances 0.000 description 1
- 229920000126 latex Polymers 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 1
- MHCFAGZWMAWTNR-UHFFFAOYSA-M lithium perchlorate Chemical compound [Li+].[O-]Cl(=O)(=O)=O MHCFAGZWMAWTNR-UHFFFAOYSA-M 0.000 description 1
- 229910001486 lithium perchlorate Inorganic materials 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- HWYHZTIRURJOHG-UHFFFAOYSA-N luminol Chemical compound O=C1NNC(=O)C2=C1C(N)=CC=C2 HWYHZTIRURJOHG-UHFFFAOYSA-N 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 210000002751 lymph Anatomy 0.000 description 1
- 230000001320 lysogenic effect Effects 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 230000001071 malnutrition Effects 0.000 description 1
- 235000000824 malnutrition Nutrition 0.000 description 1
- LUEWUZLMQUOBSB-GFVSVBBRSA-N mannan Chemical class O[C@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@@H](O[C@@H]2[C@H](O[C@@H](O[C@H]3[C@H](O[C@@H](O)[C@@H](O)[C@H]3O)CO)[C@@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O LUEWUZLMQUOBSB-GFVSVBBRSA-N 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 125000000317 mannobiose group Chemical group 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 230000015654 memory Effects 0.000 description 1
- 238000001466 metabolic labeling Methods 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 230000011987 methylation Effects 0.000 description 1
- 238000007069 methylation reaction Methods 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- PJUIMOJAAPLTRJ-UHFFFAOYSA-N monothioglycerol Chemical compound OCC(O)CS PJUIMOJAAPLTRJ-UHFFFAOYSA-N 0.000 description 1
- 229940126619 mouse monoclonal antibody Drugs 0.000 description 1
- JPXMTWWFLBLUCD-UHFFFAOYSA-N nitro blue tetrazolium(2+) Chemical compound COC1=CC(C=2C=C(OC)C(=CC=2)[N+]=2N(N=C(N=2)C=2C=CC=CC=2)C=2C=CC(=CC=2)[N+]([O-])=O)=CC=C1[N+]1=NC(C=2C=CC=CC=2)=NN1C1=CC=C([N+]([O-])=O)C=C1 JPXMTWWFLBLUCD-UHFFFAOYSA-N 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 208000015380 nutritional deficiency disease Diseases 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 229920002113 octoxynol Polymers 0.000 description 1
- 229920001542 oligosaccharide Polymers 0.000 description 1
- 150000002482 oligosaccharides Chemical class 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 230000007110 pathogen host interaction Effects 0.000 description 1
- 229950000964 pepstatin Drugs 0.000 description 1
- 108010091212 pepstatin Proteins 0.000 description 1
- FAXGPCHRFPCXOO-LXTPJMTPSA-N pepstatin A Chemical compound OC(=O)C[C@H](O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)C[C@H](O)[C@H](CC(C)C)NC(=O)[C@H](C(C)C)NC(=O)[C@H](C(C)C)NC(=O)CC(C)C FAXGPCHRFPCXOO-LXTPJMTPSA-N 0.000 description 1
- 238000012510 peptide mapping method Methods 0.000 description 1
- 210000005105 peripheral blood lymphocyte Anatomy 0.000 description 1
- RXNXLAHQOVLMIE-UHFFFAOYSA-N phenyl 10-methylacridin-10-ium-9-carboxylate Chemical compound C12=CC=CC=C2[N+](C)=C2C=CC=CC2=C1C(=O)OC1=CC=CC=C1 RXNXLAHQOVLMIE-UHFFFAOYSA-N 0.000 description 1
- ZUOUZKKEUPVFJK-UHFFFAOYSA-N phenylbenzene Natural products C1=CC=CC=C1C1=CC=CC=C1 ZUOUZKKEUPVFJK-UHFFFAOYSA-N 0.000 description 1
- NMHMNPHRMNGLLB-UHFFFAOYSA-N phloretic acid Chemical compound OC(=O)CCC1=CC=C(O)C=C1 NMHMNPHRMNGLLB-UHFFFAOYSA-N 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- ZWLUXSQADUDCSB-UHFFFAOYSA-N phthalaldehyde Chemical compound O=CC1=CC=CC=C1C=O ZWLUXSQADUDCSB-UHFFFAOYSA-N 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 210000002381 plasma Anatomy 0.000 description 1
- 229920001467 poly(styrenesulfonates) Polymers 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 229920001155 polypropylene Polymers 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000003825 pressing Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 208000037920 primary disease Diseases 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- 238000002731 protein assay Methods 0.000 description 1
- 229960000856 protein c Drugs 0.000 description 1
- 230000012846 protein folding Effects 0.000 description 1
- 238000000734 protein sequencing Methods 0.000 description 1
- 210000004196 psta Anatomy 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 230000000306 recurrent effect Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 1
- 239000011435 rock Substances 0.000 description 1
- 239000012146 running buffer Substances 0.000 description 1
- 210000003296 saliva Anatomy 0.000 description 1
- 239000012723 sample buffer Substances 0.000 description 1
- 239000012047 saturated solution Substances 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 239000013605 shuttle vector Substances 0.000 description 1
- 238000001542 size-exclusion chromatography Methods 0.000 description 1
- 239000012279 sodium borohydride Substances 0.000 description 1
- 229910000033 sodium borohydride Inorganic materials 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000010183 spectrum analysis Methods 0.000 description 1
- 238000007447 staining method Methods 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000012916 structural analysis Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000031068 symbiosis, encompassing mutualism through parasitism Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 210000001138 tear Anatomy 0.000 description 1
- 229940035024 thioglycerol Drugs 0.000 description 1
- 229940094937 thioredoxin Drugs 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- 108010080050 trypsin drug combination chymotrypsin Proteins 0.000 description 1
- 238000000539 two dimensional gel electrophoresis Methods 0.000 description 1
- 238000001419 two-dimensional polyacrylamide gel electrophoresis Methods 0.000 description 1
- 108010087967 type I signal peptidase Proteins 0.000 description 1
- 230000005951 type IV hypersensitivity Effects 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 239000000304 virulence factor Substances 0.000 description 1
- 230000007923 virulence factor Effects 0.000 description 1
- 238000011179 visual inspection Methods 0.000 description 1
- 239000011800 void material Substances 0.000 description 1
- 238000010792 warming Methods 0.000 description 1
- 208000016261 weight loss Diseases 0.000 description 1
- 230000004580 weight loss Effects 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/02—Bacterial antigens
- A61K39/04—Mycobacterium, e.g. Mycobacterium tuberculosis
-
- 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
- G01N33/56911—Bacteria
- G01N33/5695—Mycobacteria
Definitions
- the invention in the fields of microbiology and medicine relates to methods for rapid early detection of mycobacterial disease in humans based on the presence of antibodies to particular "early" mycobacterial antigens which have not been previously recognized for this purpose. Assay of such antibodies on select partially purified or purified mycobacterial preparations containing such early antigens permits diagnosis of TB earlier than has been heretofore possible. Also provided is a surrogate marker for screening populations at risk for TB, in particular subjects infected with human immunodeficiency virus (HIV).
- HIV human immunodeficiency virus
- M. tuberculosis Mycobacterium tuberculosis; also abbreviated herein as "Mt"
- Mt Mycobacterium tuberculosis
- Delayed hypersensitivity measured as cutaneous immune reactivity to a purified protein derivative of Mt is the only marker available for detection of latent infection with Mt.
- PPD purified protein derivative of Mt
- the sensitivity of the PPD skin test is substantially reduced during HIV infection (Raviglione et al, 1992, supra, 1995, supra; Graham ⁇ .M.H. et al, 1991, JAMA 267:369-313; Huebner R.E. et al, 1994, Clin. Infect. Dis. 19:26-32; Huebner R.E. et al, 1992, JAMA 267:409-410; Caiaffa W.T. et al, 1995, Arch. Intern. Med. 755:2111-2117).
- U.S. Centers for Disease Control and Prevention recommends preventive isoniazid therapy for all HIV seropositive (HIV + ), PPD-positive (PPD + ) individuals.
- HIV + HIV seropositive
- PPD + PPD-positive
- Unnecessary therapy must be minimized because prolonged isoniazid treatment can have serious toxic side effects (Shafer et al. , supra). The impact of such treatment on emergence of drug resistant bacteria is still unclear.
- the use of preventive therapy in developing countries is seriously limited by the high frequency of PPD + individuals coupled with the lack of adequate medico-social infrastructure and economic resources.
- High risk populations are also found in the United States, primarily intravenous drug users, homeless people, prison inmates and residents of slum areas (Fitzgerald, J.M. et al, 1991, Chest 700:191-200; Graham, N.M.H. et al, 1992, JAMA 267:369-313; Friedman, L.N. et al, 1996, New Engl. J. Med. 55 :828-833).
- discovery of additional surrogate markers for early detection and prompt treatment of active, subclinical TB in such high risk populations is urgently required.
- Antibody responses in TB have been studied for several decades primarily for the purpose of developing serodiagnostic assays. Although some seroreactive antigens/epitopes have been identified, interest in antibody responses to M. tuberculosis has waned because of the lack of progress in simple detection of corresponding antibodies. Studies using crude antigen preparations revealed that healthy individuals possess antibodies that cross-react with several mycobacterial antigens. Such antibodies are believed to have been elicited by exposure to commensal and environmental bacteria and vaccinations (Bardana, E.J. et al, 1973, Clin. Exp. Immunol. 75:65-77; Das, S. et al, 1992, Clin. Exp. Immunol.
- MPB denotes a protein purified from M. bovis BCG followed by a number denoting its relative mobility in 7.7% polyacrylamide gels at a pH of 9.5.
- MPT denotes a protein isolated from M. tuberculosis. In proteins examined prior to this invention, no differences in the N-terminal amino acid sequence were shown between these two mycobacterial species.
- antigens 85 A, 85B and 85C also known as the "85 complex” or "85cx"
- antigen 85A, 85B, and 85C also known as the "85 complex” or "85cx”
- This complex was originally found in M. bovis BCG preparations which produced a secreted antigen comprising a complex of three closely related components, antigen 85A, 85B, and 85C (Wiker, H.G. et al 1986, Int. Arch. Allergy Appl. Immunol.
- the corresponding components of Mt are also actively secreted.
- the 85 complex is considered the major secreted protein constituent of mycobacterial culture fluids though it is also found in association with the bacterial surface.
- SDS-PAGE SDS- polyacrylamide gel electrophoresis
- the antigen 85 complex is often referred to as the "30/31 kDa doublet," although slightly different molecular mass designations have been reported.
- MPT32 45/47 kDa (found to be 38/42kDa by the present inventors)
- the mAb HBT4 reacted with the MPT51 protein.
- the 85 A, 85B, and 85C constituents cross-reacted extensively, though each had component-specific in addition to cross-reacting epitopes. These components also cross-reacted with MPT51 and MPT64. Amino acid sequence homology was shown between 85 A, 85B, 85C and MPT51. MPT64 showed less homology.
- the aligned amino acid sequences listed below illustrate the homology of a fragment of 85 A, 85B, 85C, MPT51 and MPT64. The numbers at the top correspond to the part of the sequence shown. The N-terminal sequences were determined on isolated proteins and aligned by visual inspection. The sequence from position 66 to 91 of MPT64 is the sequence deduced from the cloned gene. Q
- the N-terminal sequence of MPT51 showed 72% homology with the sequence of the
- Wiker and colleagues concluded that the future of the serology of antibody responses to antigen 85 would require investigation of antibodies to component- specific epitopes and in particular to species-specific epitopes.
- the extensive cross- reactivity of antigen 85 in different species of mycobacteria suggested to Wiker et al. (supra) that tests could attain sufficient sensitivity, though suitable mAbs were said to be essential for further development of tests for infection with Mt (and atypical mycobacteria).
- the present inventors note the deficiency in the art of analysis of antibodies at different stages of disease. This is one of the primary deficiencies addressed by this invention.
- the N-terminus of the Mt 41 kDa antigen known as MPT32 was very similar to the N-termini of the 50/55 kDa - and the 45-47 kDa proteins.
- the molecular mass of this Mt protein was deduced to be 45-47 kDa.
- Espitia et al, supra speculated about a diagnostic potential for these antigens based on their observation of antibodies in 70% of their TB patients. However, the potential of this antigen as an early diagnostic agent for TB was neither analyzed nor even suggested.
- the present inventors have systematically analyzed the reactivity of sera from TB patients with antigens from Mt to delineate the major targets of human antibody responses which occur early in the progression of the infection to disease. They observed that initial immunoadsorption of patient sera with E. coli antigens successfully reduced interference by cross-reactive antibodies, thus allowing a new approach to serological studies.
- the immunoadsorbed sera allowed identification of a number of antigens of Mt that are recognized by antibodies in a large proportion of patients, and during earlier stages of disease progression. These antigens are therefore useful tools in methods of diagnosing TB. Prominent among these antigens is a high molecular weight secreted protein of 88kDa or 85kDa (depending on conditions as will be described below).
- the present invention provides for the first time a surrogate marker that can be used in an inexpensive screening method in individuals at heightened risk for developing TB.
- This utility was discovered by applying the approach described herein to analyze antibody responses of HIV-infected TB patients (HIV/TB) to the secreted antigens of Mt during different stages of disease progression. A majority of the HIV/TB patients had detectable antibodies to the secreted antigens of Mt for months, even years, prior to the clinical manifestation of active tuberculous disease. These patients are termed "HIV/pre-TB”.
- HIV/TB patients compared to the TB patients not infected with HIV (designated "non-HIV/TB"), HIV/TB patients had significantly lower levels of antibodies which showed specificity for a restricted repertoire of Mt antigens. Antibodies to the 88 kDa antigen mentioned above were present in about 75% of the HIV/pre-TB sera patients who eventually developed clinical TB. HIV/TB patients who failed to develop anti-Mt antibodies did not differ in their lymphocyte profiles from those that were antibody-positive. These discoveries led to the invention of a serological surrogate marker for active pre-clinical TB in HIV-infected subjects as well as in any other high risk population. Thus, this invention provides for the first time a method for early detection of Mt infection in immunocompromised subjects. Exploitation of this discovery should make a significant contribution to the early detection of the tubercular disease and will permit a more rapid institution of therapy.
- the present invention is directed to a method for the early detection of the presence of a mycobacterial disease or infection in a subject, comprising: (a) before the onset of symptoms identifiable as clinical disease, obtaining a biological fluid sample from the subject; and (b) assaying the sample for the presence of antibodies specific for one or more early Mt antigens, wherein detection of the antibodies is indicative of the presence of the disease, or infection
- the early antigen may comprise a fraction of the lipoarabinomannan-free culture supernatant of Mt having one of the following groups of characteristics:
- a preferred embodiment of the above method includes, prior to step (b), the step of removing from the sample antibodies specific for antigens which are cross- reactive between Mt and other bacterial genera, such as by immunoadsorption of the sample with E. coli antigens.
- one or more of the early antigens is preferably a secreted Mt protein or glycoprotein selected from the group consisting of
- the method for the early detection of the presence of a mycobacterial disease or infection in a subject comprises:
- the subject is preferably a human.
- the subject is preferably a human infected with HIV-1 or at high risk for tuberculosis.
- the present invention also includes a method for the early detection of the presence of a mycobacterial disease or infection in a subject, comprising: (a) before the onset of symptoms identifiable as clinical disease, obtaining a biological fluid sample from the subject;
- step (c) assaying the sample of step (a) or the culture supernatant or fraction of step (b) for the presence of an early Mt antigen using an antiserum or mAb specific for the early antigen; wherein detection of the antigen is indicative of the presence of the disease or infection.
- Also provided is a method for the early detection of the presence of a mycobacterial disease or infection in a subject comprising: (a) before the onset of symptoms identifiable as clinical disease, obtaining a biological fluid sample from the subject;
- the present invention is further directed to an antigenic composition useful for early detection of Mt infection or disease comprising a mixture of two or more early Mt antigens substantially free of other proteins with which the early Mt antigens are natively admixed in a culture of Mt and which other proteins are not early Mt antigens.
- the two or more early antigens are selected from the group consisting of (a) an 88 kDa secreted protein having a pi of about 5.2 present in Mt lipoarabinomannan-free culture filtrate; (b) a protein characterized as Mt antigen 85C;
- composition (e) a 49 kDa protein having a pi of about 5.1 corresponding to a spot identified as Ref. No. 82 in Figure 15A-F, Figure 18, Table 9 or Table 11.
- the foregoing composition may be further supplemented with one or more of the following Mt antigenic proteins: (i) a 28 kDa antigen corresponding to the spot identified as Ref. No. 77 in Figure
- any one of the early Mt antigens may be a recombinant protein or glycoprotein, preferably produced in a mycobacterial or eukaryotic expression system.
- the antigenic protein may comprise either
- the present invention also provides a kit useful for early detection of Mt disease or infection comprising an antigenic composition as described above in combination with reagents necessary for detection of antibodies which bind to the early Mt antigen or antigens.
- the early Mt antigen is a recombinant protein or glycoprotein.
- the kit may further comprise one or more of the following Mt antigenic proteins:
- kits useful for early detection of an antibody specific for an early Mt antigen in a subject comprising
- this invention is directed to a method for obtaining a desired mAb useful (i) for detecting an early Mt antigen or anti-Mt antibody in a sample or (ii) for isolating an early Mt antigen or epitope, which method comprises: (a) isolating a Mt early antigen by biochemical purification or by recombinant expression;
- step (b) using the early antigen of step (a) to generate a collection of monoclonal antibodies each of which is specific for an epitope of the early antigen;
- an immunoassay for detecting an early mycobacterial antigen or an epitope thereof comprises incubating the mAb obtained as above with a sample suspected of containing the protein or epitope and measuring the binding of the mAb to the protein or epitope in the sample.
- the immunoassay comprises incubating the mAb obtained as above with a sample suspected of containing the early antibody and with a mycobacterial preparation containing an early antigen for which the early antibody is specific, and measuring the ability of the sample to compete with the mAb for binding to the early antigen.
- Figure 1 shows the reactivity of sera from TB" ei! HIV neg PPD + controls( O ); TB neB HIV ne PPD neg controls ( V ), TB neg , HIV + , asymptomatic controls ( ⁇ ); and
- Figure 2 shows the reactivity with fractions of LFCFP of sera from TB patients which were reactive with total LFCFP
- Figure 3 shows a comparison of reactivity of advanced, partially treated (black bars) and early, minimally treated (stippled bars) TB patients with LFCFP (LFCF), fraction 10 (F10) and fraction 15 (F15).
- LFCFP LFCFP
- F10 fraction 10
- F15 fraction 15
- Figure 4 shows an immunoblot analysis of total LFCFP, and fractions 10 and 15.
- Lanes 1, 5, and 9 contain molecular weight markers.
- Lanes 2, 6 and 10 contain LAM-free CFP, lanes 3, 7 and 11 contain Mt fraction 10 and lanes 4, 8 and 12 contain Mt fraction 15.
- the following antibody probes were used: lanes 1 to 4 were probed with pooled sera (1 :200) from ELISA + TB patients,; lanes 5-8 were probed with pooled sera from ELISA ne TB patients; lanes 9-12 were probed with pooled sera from PPD + , healthy controls.
- Figure 5 shows reactivity of sera from non-HIV, PPD skin test positive (PPD + ) healthy controls (non-HIV/PPD), non-HIV TB patients (non-HIV/TB) and HIV- infected TB patients (HIV/pre-TB, HIV/at-TB and HIV/post-TB) with total LFCFP of M. tuberculosis.
- the cut-off was determined by the mean optical density (OD) ⁇ 3 standard deviations, obtained with the healthy control sera.
- Figure 6 shows reactivity of sera from non-HIV, PPD + healthy controls (non- HIV/PPD), non-HIV TB patients (non-HIV/TB), asymptomatic HIV-infected individuals (HIV) and HIV-infected individuals with M. avium-intracellulare bacteremia (HIV/MAI), with total LFCFP. Cut-off was determined as for Figure 5.
- Figure 7 shows the time course of appearance of antibodies to total LAM-free culture filtrate in the sera of 6 ELISA + (A-F) and 3 ELISA nes (G) HIV-infected TB patients, and 3 ELISA neg HIV-infected individuals with Mycobacterium avium bacteremia (H). Time point '0' yr.
- Figure 8 shows reactivity of sera from 3 non-HIV TB patients (lanes 2, 10, 18); nine HIV-infected TB patients (lanes 3, 4, 11-14, 19-21); HIV-infected asymptomatic individuals (lanes 5, 6, 15, 22-24) and non-HIV, PPD + healthy controls (7, 8, 16, 25, 26) with fractionated LFCFPs. Lanes 1, 9 and 17 contain molecular weight markers (kDa).
- Figures 9A and 9B show reactivity of pooled sera from 6 non-HIV TB patients (Fig. 9A) and 6 HIV-infected TB patients (Fig. 9B) with sized fractions of LFCFPs.
- Figure 10 shows reactivity of sera from HIV/TB patients with total LFCFP and sized fraction numbers 7-14 (F7-14) by ELISA. The ⁇ O.D. values were calculated by subtracting the mean optical density of the PPD + healthy control group +3 standard deviations obtained with the antigen (LFCFP or sized fraction) from the optical density of the serum sample with the same antigen.
- Figure 11 shows reactivity of sera from HIV-infected TB patients with total LFCFP ( D ) and antigens in Fraction 14 ( B ).
- Time: '0' years refers to time of clinical diagnosis of TB, and negative values refer to the years preceding clinical TB.
- the ⁇ O.D. values were calculated by subtracting the mean OD of the PPD + healthy control group +3 S.D. obtained with the antigen (LFCFP or F14 antigens) from the OD of the serum sample with the same antigen.
- Figure 12 shows the results of PAGE of Ag85 proteins purified by hydrophobic interaction chromatography: (lane 1) molecular weight standards (lane 2) purified 85B, (lane 3) 85C, (lane 4) 85A.
- Figure 13 shows Western blot analysis of the purified Ag85 products (from
- Figure 14 shows a two dimensional PAGE of CFPs from M. tuberculosis H37Rv.
- Known proteins are designated by the mAb or polyclonal sera that they reacted to by 2-D western blot analysis.
- Unidentified proteins selected for N-terminal amino acid sequence are labeled A-K.
- Figure 15A-15F show 2-D PAGE maps of CFPs of M. tuberculosis strains H37Rv, H37Ra and Erdman.
- Silver nitrate stained 2-D polyacrylamide gels of M. tuberculosis H37Rv (Fig. 15 A), Erdman (Fig. 15C) and H37Ra (Fig. 15E) overlayed with the digitized image of proteins detected with the MicroScan 1000 2-D gel analysis software.
- Figures 15B, C and F are digitized images of the 2-D gels of M. tuberculosis H37Rv, Erdman and H37Ra, respectively. Reference numbers of individual protein spots correspond to those listed and described in Table 9. Matched proteins between strains have identical reference numbers.
- Figure 16 shows growth curves of M. tuberculosis: strains H37Rv, H37Ra and
- Figure 17 shows a Western blot analysis of 1-D fractionated M. tuberculosis LFCFP with E. coli adsorbed sera.
- Lane 1 molecular weight markers.
- Lanes 2-7 PPD negative healthy individuals (Group I); lanes 8-16: PPD positive healthy controls (Group I); lanes 17-24: antibody negative TB patients (Group II); lanes 25-35: antibody positive TB patients (lacking anti-38 kDa PstS antibodies, Group III); lanes 36-43: TB patients with anti-38 kDa antibodies (Group IV).
- Figure 18A-18D shows the results of 2-dimensional fractionation and immunoblot analysis of M. tuberculosis LFCFPs with four different serum pools comprised of 6 individual sera in each pool, (panel A) group I; (panel B) group II; (panel C) group III and (panel D) group IV.
- the vertical axis represents molecular mass and the horizontal axis represents isoelectric point (pi).
- Figure 19 is a graph showing reactivity of sera from advanced (black bars) and early (gray bars) TB patients to M. tuberculosis LFCFP, purified Ag85C or three fractions (13, 10 and 15) enriched for three early antigens (shown in parentheses below the fraction designation).
- Figure 20 is a graph showing reactivity of sera from advanced (black bars) and early (gray bars) TB patients to M. tuberculosis LFCFP, purified Ag85C or purified MPT32.
- Figure 21 shows the reactivity of recombinant 88 kDa protein with antibodies.
- Lane 1 molecular weight markers; lane 2 contains fractionated LFCFP probed with mAb IT-57; lanes 3 contains lysates from E. c ⁇ /t- ⁇ gtl 1 (IT-57), and lane 4 E. coli ⁇ gtl 1 without insert; lanes 2-4 probed with mAb IT-57.
- Lanes 5-14 contain lysates from E. c ⁇ /t- ⁇ gtl 1 (IT-57); lanes 5-10 probed with TB sera, lanes 11-14 probed with PPD positive healthy control sera.
- Figures 22A and 22B show the hybridization of ⁇ gtl 1 (IT-57) with the katG gene.
- Fig. 22A shows agarose gel electrophoresis of the DNA from ⁇ gtl 1 (IT-57) before (lane 2) and after digestion with EcoRl enzyme (lane 3); and plasmid pMD31 DNA containing the katG gene (lane 4) and after digestion with Kpnl and Xbal (lane 5).
- Fig. 22B is a nitrocellulose blot of the gel of Fig. 22 A probed with 32 P-labeled insert DNA from the ⁇ gtl 1 (IT-57). The 1 kb DNA ladder is shown in lane 1 and the sizes of the fragments are shown on the right.
- Figures 23A-23D show the reactivity of anti-catalase/peroxidase antibodies and TB patient sera with the catalase/peroxidase and with a novel 88 kDa antigen.
- the lanes in all four blots contain the same antigenic preparation.
- bovis BCG pMD31 alone (lane 6, labeled pMD31/BCG), were probed with (Fig. 23A) mAb IT-57, (Fig. 23B) IT-42, (Fig.23C) anti- catalase/peroxidase polyclonal antibody and (Fig. 23D) a TB patient serum.
- Figures 24A and 24B show reactivity of anti-catalase/peroxidase antibodies with lysates of the £ ⁇ tG-negative strain of M. tuberculosis (ATCC 35822).
- lane 1 contains molecular weight markers
- lanes 2, 4, and 6 contain the LFCFP
- lanes 3 contain the culture filtrates of the A ⁇ rtG-negative strain of M. tuberculosis.
- Lanes 2 and 3 were probed with mAb IT-57, lanes 4 and 5 with mAb
- Fig. 24B shows a Western blot analysis of 1-D fractionated KatG-deleted M. tuberculosis LFCFP with E. coli absorbed sera. Lanes 1, 6, 12 and 18: molecular weight markers.
- Lanes 2-5 sera of PPD positive healthy individuals (group I); lanes 7-11 : group II; lanes 13-17: group III; lanes 19-23: group IV.
- Figure 25 is a graph showing reactivity of individual sera with different antigens of M. tuberculosis. Reactivity of sera from 34 cavitary (black bars) and 20 non cavitary (open bars) TB patients with various antigens: purified 38 kDa antigen
- Ag85C MPT32
- MPT32 MPT32
- FR15/Ag85C MPT32
- FR15/Ag85C MPT32
- FR15/Ag85C/MPT32 FR15/Ag85C/MPT32
- Figures 26A and 26B are graphs showing the reactivity of TB sera with Fraction F13, which is enriched for antigen MPT32.
- Fig. 26 A shows reactivity with untreated F13.
- Fig. 26B shows reactivity of sera with periodate-treated F13, in which glycosylation is destroyed by oxidation. DESCRIPTION OF THE PREFERRED EMBODIMENTS
- the present invention provides a diagnostic immunoassay method to detect and/or quantitate antibodies specific for mycobacterial antigens, in particular, antibodies developing early in the progression of M. tuberculosis infection to disease and before clinical manifestations of that disease. On the basis of such an assay, it is possible to detect TB earlier than ever before and to institute appropriate therapy.
- the best antigen available prior to this invention for serodiagnosis of TB was the 38 kDa secreted protein also known as Ag 78 (see above).
- the present invention permits detection of serological reactivity in subject who lack detectable antibodies to this 38 kDa antigen.
- the immunoassay method is based upon the present inventors' discovery that certain Mt antigens induce in humans an earlier response than do other antigens which elicit antibodies only after the disease is already clinically advanced. In HIV-infected subjects with dysfunctional immune systems, antibodies to some of these antigens are detectable long before TB is clinically manifest. Five secreted proteins have been identified as early antigens with diagnostic value.
- a preferred early antigen is a 88kDa secreted protein of Mt, preferably enriched or semipurified (at least 50%) pure) or highly purified (at least 95% pure, preferably at least 99% pure).
- the present method is further based on the inventors' conception of the importance of first removing antibodies specific for cross-reactive antigens (which are not Mt-specific) prior to analyzing the antigenic reactivity and specificity of serum from patients infected with Mt on crude or semipurified antigenic preparations.
- purified antigens are provided or epitope-specific competitive EIAs are established based on this invention (see, for example, Wilkins, E. et al, 1991, Eur. J. Clin. Microbiol. Infect. Dis. 70:559-563)
- the need for such prior absorption steps should be obviated.
- the term "early” in reference to (1) Mt infection, (2) the antibody response to an Mt antigen, (3) an Mt antigen itself or (4) a diagnostic assay, is defined in terms of the stage of development of TB.
- Early disease is characterized in that the subject is asymptomatic or, more typically, has one or more of the following symptoms or findings: (a) constitutional symptoms including fever, cough and weight loss; (b) bacilli in sputum or other body fluid which can be grown in culture; or (c) radiographically evident pulmonary lesions which may include infiltration but without cavitation. Any antibody present in such early stages is termed an "early antibody” and any Mt antigen recognized by such antibodies is termed an "early antigen.”
- the term “late” or “advanced” in reference to disease, infection, antibody response, antigen, or assay is characterized in that the subject has frank clinical disease and more advanced pulmonary lesions as well as presence of Mt bacilli in smears of sputum or other body fluids.
- "Late TB” or “late mycobacterial disease” is used interchangeably with "advanced TB” or “advanced mycobacterial disease.”
- An antibody appearing after the onset of diagnostic clinical symptoms is a late antibody, and an antigen recognized by a late antibody (but not by an early antibody) is a late antigen.
- an early diagnostic assay must permit rapid diagnosis of Mt disease at a stage earlier than that which could have been diagnosed by conventional clinical diagnostic methods, namely, by radiologic examination and bacterial smear and culture or by other laboratory methods available prior to this invention.
- Culture positivity is the final confirmatory test but takes two weeks and more
- the present immunoassay typically comprises incubating a biological fluid, preferably serum, from a subject suspected of having TB in the presence of an Mt antigen-containing reagent which includes one or more Mt early antigens, and detecting the binding of antibodies in the sample to the mycobacterial antigen(s).
- biological fluid any fluid derived from the body of a normal or diseased subject which may contain antibodies, such as blood, serum, plasma, lymph, urine, saliva, sputum, tears, cerebrospinal fluid, bronchioalveolar lavage fluid, bile, ascites fluid, pus and the like. Also included within the meaning of this term as used herein is a tissue extract, or the culture fluid in which cells or tissue from the subject have been incubated.
- the preferred biological fluid for use in the present invention is serum.
- the mycobacterial antigen composition or preparation of the present invention may be a "fraction" of total M. tuberculosis secreted proteins based on a selected molecular weight range and reactivity with patient sera. Such a fraction will containing at least one, and possibly two or more, early antigen proteins (such as in the fractions exemplified in Examples I and II). A preferred molecular weight range of proteins/glycoproteins in such a fraction is between about 30 kDa and about 90 kDa.
- the selection of antigen or antigens to be included in the composition is based on reactivity of TB patient sera with the antigen (or with the fraction containing the antigen).
- the antigen composition may be a substantially purified preparation of one or more M. tuberculosis proteins.
- the antigen composition may be a partially purified or substantially pure preparation containing one or more M. tuberculosis epitopes which are capable of being bound by antibodies of a subject with TB.
- Such epitopes may be in the form of peptide fragments of the early antigen proteins or other "functional derivatives" of M. tuberculosis proteins as described below.
- a functional derivative is meant a “fragment,” “variant,” “analogue,” or “chemical derivative” of an early antigen protein, which terms are defined below.
- a functional derivative retains at least a portion of the function of the protein which permits its utility in accordance with the present invention - primarily the capacity to bind to an early antibody.
- a “fragment” refers to any subset of the molecule, that is, a shorter peptide.
- a “variant” refers to a molecule substantially similar to either the entire protein or fragment thereof.
- a variant peptide may be conveniently prepared by direct chemical synthesis or by recombinant means.
- An “analogue” of the protein or peptide refers to a non- natural molecule substantially similar to either the entire molecule or a fragment thereof.
- a "chemical derivative" of the antigenic protein or peptide contains additional chemical moieties not normally part of the peptide.
- Covalent modifications of the peptide are included within the scope of this invention. Such modifications may be introduced into the molecule by reacting targeted amino acid residues of the peptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues.
- This protein is an Mt secreted protein having an apparent molecular mass of about 85kDa or about 88 kDa (depending in which of two different laboratories of the present inventors the determination is made). This protein is further characterized by an isoelectric point of about pH 5.2. This protein reacts with mAbs IT-42 and IT-57 and is a major antigenic component of Fraction 15 (Example I) and Fraction 14
- Example II This protein corresponds to the protein spot designated Ref. No. 124 in Figure 15 A-F, Figure 18, Table 9 or Table 11. Hence, despite a small apparent difference in molecular mass, a single protein is intended (although different isoforms may be found to exist).
- This protein is referred to herein as the 88 kDa protein, to help distinguish it from the three Ag85 proteins (85A B and C), the naming of which bears no relation to size.
- Mt secreted protein having an apparent molecular weight of about 31 kDa and an isoelectric point of about pH 5.17.
- This protein is reactive with mAb IT-49 and has also been designated MPT45.
- Ag85C corresponds to the protein spot designated Ref. No. 119 in Figure 15 A-F, Figure 18, Table 9 or Table 11.
- This Mt secreted protein has an apparent molecular mass of about 27kDa and an isoelectric point of about 5.91. It is reactive with mAb IT-52. This protein corresponds to the protein spot designated Ref. No. 170 in Figure 15 A-F, Figure 18, Table 9 or Table 11.
- This glycoprotein has an apparent molecular mass (as a doublet peak) of 38 and 42 kDa (42/45 kDa according to Espitia et al. (supra)) and an isoelectric point of about pH 4.51. It is reactive with a polyclonal anti-MPT 32 antiserum. This protein is a major antigenic component of Fraction 13 (see Examples). MPT32 corresponds to the protein spot designated Ref. No. 14 in Figure 15 A-F, Figure 18, Table 9 or Table 11. (5) 49 kDa protein
- This protein has an apparent molecular mass of about 49 kDa protein and an isoelectric point of about pH 5.1. This protein reacts with mAb IT-58. This protein corresponds to a spot identified as Ref. No. 82 in Figure 15 A-F, Figure 18, Table 9 or Table 11.
- the mycobacterial antigen composition is brought in contact with, and allowed to bind to, a solid support or carrier, such as nitrocellulose or polystyrene, allowing the antigen composition to adsorb and become immobilized to the solid support. This immobilized antigen is then allowed to interact with the biological fluid sample which is being tested for the presence of anti-Mt antibodies, such that any antibodies in the sample will bind to the immobilized antigen.
- the support to which the antibody is now bound may then be washed with suitable buffers after which a detectably labeled binding partner for the antibody is introduced.
- the binding partner binds to the immobilized antibody. Detection of the label is a measure of the immobilized antibody.
- a preferred binding partner for this assay is an anti-immunoglobulin antibody ("second antibody”) produced in a different species.
- second antibody an anti-immunoglobulin antibody
- a detectably labeled goat anti-human immunoglobulin "second” antibody may be used.
- the solid phase support may then be washed with the buffer a second time to remove unbound antibody.
- the amount of bound label on the solid support may then be detected by conventional means appropriate to the type of label used (see below).
- Such a “second antibody” may be specific for epitopes characteristic of a particular human immunoglobulin isotype, for example IgM, IgG,, IgG 2a , IgA and the like, thus permitting identification of the isotype or isotypes of antibodies in the sample which are specific for the mycobacterial antigen.
- the second antibody may be specific for an idiotype of the ant-Mt antibody of the sample.
- binding partners for detection of the sample antibody other known binding partners for human immunoglobulins may be used.
- staphylococcal immunoglobulin binding proteins the best know of which is protein A.
- staphylococcal protein G or a recombinant fusion protein between protein A and protein G.
- Protein G of group G and group C streptococci binds to the Fc portion of Ig molecules as well as to IgG Fab fragment at the V H 3 domain.
- Protein C of Peptococcus magnus binds to the Fab region of the immunoglobulin molecule.
- Any other microbial immunoglobulin binding proteins for example from Streptococci, are also intended (for example, Langone, J.J., Adv. Immunol. 52:157 (1982)).
- a biological fluid suspected of containing antibodies specific for a Mt antigen may be brought into contact with a solid support or carrier which is capable of immobilizing soluble proteins.
- the support may then be washed with suitable buffers followed by treatment with a mycobacterial antigen reagent, which may be detectably labeled. Bound antigen is then measured by measuring the immobilized detectable label. If the mycobacterial antigen reagent is not directly detectably labeled, a second reagent comprising a detectably labeled binding partner for the Mt antigen, generally a second anti-Mt antibody such as a murine mAb, is allowed to bind to any immobilized antigen.
- the solid phase support may then be washed with buffer a second time to remove unbound antibody. The amount of bound label on said solid support may then be detected by conventional means.
- solid phase support any support capable of binding a proteinaceous antigen or antibody molecules or other binding partners according to the present invention.
- supports, or carriers include glass, polystyrene, polypropylene, polyethylene, polyvinylidene difluoride, dextran, nylon, magnetic beads, amylases, natural and modified celluloses, polyacrylamides, agaroses, and magnetite.
- the nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention.
- the support material may have virtually any possible structural configuration so long as it is capable of binding to an antigen or antibody.
- the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod.
- the surface may be flat such as a sheet, test strip, etc.
- Preferred supports include polystyrene beads, 96-well polystyrene microplates and test strips, all well-known in the art. Those skilled in the art will know many other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation.
- a preferred type of immunoassay to detect an antibody specific for a mycobacterial antigen according to the present invention is an enzyme-linked immunosorbent assay (ELISA) or more generically termed an enzyme immunoassay (El A).
- ELISA enzyme-linked immunosorbent assay
- El A enzyme immunoassay
- a detectable label bound to either an antibody-binding or antigen-binding reagent is an enzyme. When exposed to its substrate, this enzyme will react in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorometric or visual means.
- Enzymes which can be used to detectably label the reagents useful in the present invention include, but are not limited to, horseradish peroxidase, alkaline phosphatase, glucose oxidase, ⁇ - galactosidase, ribonuclease, urease, catalase, malate dehydrogenase, staphylococcal nuclease, asparaginase, ⁇ -5-steroid isomerase, yeast alcohol dehydrogenase, ⁇ - glycerophosphate dehydrogenase, triose phosphate isomerase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.
- the detectable label may be a radiolabel, and the assay termed a radioimmunoassay (RIA), as is well known in the art. See, for example,
- the radioisotope can be detected by a gamma counter, a scintillation counter or by autoradiography.
- Isotopes which are particularly useful for the purpose of the present invention are l25 1, 131 1, 35 S, 3 H and 14 C.
- fluorophore it is also possible to label the antigen or antibody reagents with a fluorophore.
- fluorescently labeled antibody When the fluorescently labeled antibody is exposed to light of the proper wave length, its presence can then be detected due to fluorescence of the fluorophore.
- fluorophores are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, fluorescamine or fluorescence-emitting metals such as 152 Eu or other lanthanides. These metals are attached to antibodies using metal chelators.
- the antigen or antibody reagents useful in the present invention also can be detectably labeled by coupling to a chemiluminescent compound.
- a chemiluminescent-tagged antibody or antigen is then determined by detecting the luminescence that arises during the course of a chemical reaction.
- useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
- a bioluminescent compound such as a bioluminescent protein may be used to label the antigen or antibody reagent useful in the present invention. Binding is measured by detecting the luminescence.
- Useful bioluminescent compounds include luciferin, luciferase and aequorin.
- Detection of the detectably labeled reagent according to the present invention may be accomplished by a scintillation counter, for example, if the detectable label is a radioactive gamma emitter, or by a fluorometer, for example, if the label is a fluorophore.
- the detection is accomplished by colorimetry to measure the colored product produced by conversion of a chromogenic substrate by the enzyme. Detection may also be accomplished by visual comparison of the colored product of the enzymatic reaction in comparison with appropriate standards or controls.
- the immunoassay of this invention may be a "two-site” or “sandwich” assay.
- the fluid containing the antibody being assayed is allowed to contact a solid support.
- a quantity of detectably labeled soluble antibody is added to permit detection and/or quantitation of the ternary complex formed between solid-phase antibody, antigen, and labeled antibody.
- Sandwich assays are described by Wide, Radioimmune Assay Method, Kirkham et al, Eds., E. & S. Livingstone, Edinburgh, 1970, pp 199-206.
- agglutination assays both direct and indirect, which are well known in the art.
- the agglutination of particles containing the antigen indicates the presence or absence of the corresponding antibody.
- Any of a variety of particles, including latex, charcoal, kaolinite, or bentonite, as well as microbial cells or red blood cells, may be used as agglutinable carriers (Mochida, US 4,308,026; Gupta et al, J. Immunol. Meth. 80:177-187 (1985); Castelan et al, J. Clin. Pathol.
- the present invention provides methods to detect and enumerate cells secreting an antibody specific for a mycobacterial antigen.
- a sample containing lymphocytes such as peripheral blood lymphocytes
- a reagent containing the antigen of interest As the antibody secreting cells of the sample secrete their antibodies, the antibodies react with the antigen, and the reaction is visualized in such a way that the number of antibody secreting cells (or plaque forming cells) may be determined.
- the antigen may be coupled to indicator particles, such as erythrocytes, preferably sheep erythrocytes, arranged in a layer.
- antibodies As antibodies are secreted from a single cell, they attach to the surrounding antigen- bearing erythrocytes. By adding complement components, lysis of the erythrocytes to which the antibodies have attached is achieved, resulting in a "hole” or “plaque” in the erythrocyte layer. Each plaque corresponds to a single antibody-secreting cell.
- the sample containing antibody-secreting cells is added to a surface coated with an antigen-bearing reagent, for example, a mycobacterial antigen alone or conjugated to bovine serum albumin, attached to polystyrene. After the cells are allowed to secrete the antibody which binds to the immobilized antigen, the cells are gently washed away.
- an antigen-bearing reagent for example, a mycobacterial antigen alone or conjugated to bovine serum albumin
- the present invention is also directed to a kit or reagent system useful for practicing the methods described herein.
- a kit will contain a reagent combination comprising the essential elements required to conduct an assay according to the disclosed methods.
- the reagent system is presented in a commercially packaged form, as a composition or admixture (where the compatibility of the reagents allow), in a test device configuration, or more typically as a test kit.
- a test kit is a packaged combination of one or more containers, devices, or the like holding the necessary reagents, and usually including written instructions for the performance of assays.
- the kit may include containers to hold the materials during storage, use or both.
- the kit of the present invention may include any configurations and compositions for performing the various assay formats described herein.
- kits for determining the presence of anti-Mt early antibodies may contain one or more early Mt antigens, either in immobilizable form or already immobilized to a solid support, and a detectably labeled binding partner capable of recognizing the sample anti-Mt early antibody to be detected, for example, a labeled anti -human Ig or anti -human Fab antibody.
- a kit for determining the presence of an early Mt antigen may contain an immobilizable or immobilized "capture" antibody which reacts with one epitope of an early Mt antigen, and a detectably labeled second (“detection") antibody which reacts with a different epitope of the Mt antigen than that recognized by the (capture) antibody.
- Any conventional tag or detectable label may be part of the kit, such as a radioisotope, an enzyme, a chromophore or a fluorophore.
- the kit may also contain a reagent capable of precipitating immune complexes.
- a kit according to the present invention can additionally include ancillary chemicals such as the buffers and components of the solution in which binding of antigen and antibody takes place.
- the present invention also provides an approach to the identification, isolation and characterization of early Mt antigens.
- an adsorbed patient serum or pool of sera containing antibody for one or more antigens can be used in initial stages of antigen preparation and purification, as well as in the process of cloning of a protein antigen.
- This antiserum can be further adsorbed with an Mt or other mycobacterial preparation to render it functionally monospecific or oligospecific.
- This "enriched" antiserum can be used along with standard biochemical purification techniques to assay for the presence of the antigen it recognizes in fractions obtained during the purification process.
- the antiserum can also be used in immobilized form as an immunoadsorbent in affinity purification of the antigen in accordance with standard methods in the art.
- the antiserum can be used in an expression cloning method to detect the presence of the antigen in bacterial colonies or phage plaques where the antigen is expressed.
- the antigen can be used to immunize animals to prepare high titer antisera or, preferably, to obtain a mAb specific for that antigen.
- an animal antiserum or mAb can be employed advantageously in place of the patient antiserum or in combination with a test body fluid sample in a competition immunoassay.
- the antiserum or mAb can be used for antigen production or purification, or in an immunoassay for detecting the antigen, for example as a binding partner (either the capture antibody or the detection antibody) in a sandwich immunoassay.
- the present invention provides an immunoassay for detecting the presence of an Mt early antigen in a body fluid or in a bacterial culture grown from a body fluid of a subject suspected of being infected with Mt.
- a sensitive immunoassay such as a direct sandwich EIA or a competitive EIA can detect an Mt protein (early antigen) in picogram amounts.
- a competitive assay allows detection of specific epitopes of the Mt antigen without the necessity of starting with a purified antigen preparation. Such assays permits detection of Mt in the patient sample at an earlier time than standard bacteriological analysis (i.e., appearance of colonies on agar).
- This method therefore provides a basis for clinical decisions to initiate therapy after several hours or days if the antigen can be detected in a body fluid. In any case, this is a major advantage over the conventional two to four (or more) weeks commonly needed to grow out Mt organisms from a patient sample. The earlier the stage of the infection, the lower would be the titer of Mt in a given body fluid, and the greater would be the advantage of the present assay over conventional diagnosis.
- a number of immunoassays for various Mt antigens are known in the art and can serve as the basis for development of assays for the early antigens of the present invention (Wilkins et al, supra; Verbon, 1994, supra; Benjamin, R.G. et al, 1984, J Med. Micro.
- a human antisera (or pool) or a mAb, preferably murine , serving as the capture antibody is immobilized to a solid phase, preferably a microplate.
- the test antigen preparation for example an Mt culture supernatant or extract is added to the immobilized antibody.
- a second "detection" antibody such as a murine mAb specific for the same antigen or preferably for a different epitope of the same protein, allowed to bind in the presence of a fixed amount of a mAb, preferably of murine origin, specific for the epitope of interest.
- the detection mAb may be enzyme-conjugated.
- a second step reagent such as an enzyme-labeled antibody specific for murine immunoglobulin may be used for detection of antigen which has become immobilized.
- the present invention permits isolation of an Mt early antigen which is then used to produce one or more epitope-specific mAbs, preferably in mice. Screening of these putative early Mt-specific mAbs is done using known patient sera which have been characterized for their reactivity with the early antigen of interest. The murine mAbs produced in this way are then employed in a highly sensitive epitope-specific competition immunoassay for early detection of TB. Thus, a patient sample is tested for the presence of antibody specific for an early epitope of Mt by its ability to compete with a known mAb for binding to a purified early antigen. For such an assay, the mycobacterial preparation may be less-than pure because, under the competitive assay conditions, the mAb provides the requisite specificity for detection of patient antibodies to the epitope of choice (for which the mAb is specific).
- the present invention provides a method to detect immune complexes containing early Mt antigens in a subject using an EIA as described above. Circulating immune complexes have been suggested to be of diagnostic value in TB. (See, for example, Mehta, P.K. et al, 1989, Med. Microbiol. Immunol. 178:229-233; Radhakrishnan, V.V. et al, 1992, J. Med. Microbiol. 36: 128-131). Methods for detection of immune complexes are well-known in the art. Complexes may be dissociated under acid conditions and the resultant antigens and antibodies detected by immunoassay.
- Immune complexes may be precipitated for direct analysis or for dissociation using known methods such as polyethylene glycol precipitation.
- Purified Mt early antigens as described herein are preferably produced using recombinant methods. See Example VI.
- Conventional bacterial expression systems utilize Gram negative bacteria such as E. coli or Salmonella species. However, it is believed that such systems are not ideally suited for production of Mt antigens (Burlein, J.E., In: Tuberculosis: Pathogenesis, Protection and Control, B. Bloom, ed., Amer. Soc. Microbiol, Washington, DC, 1994, pp.
- mycobacterial hosts for recombinant production of early Mt antigenic proteins or glycoproteins. Methods for such manipulation and gene expression are provided in Burlein, supra. Expression in mycobacterial hosts, in particular M. bovis (strain BCG) or M. smegmatis are well-known in the art. Two examples, one of mycobacterial genes (Rouse, D.A. et al, 1996, Mol. Microbiol.
- the study population included 58 HIV neB individuals with confirmed pulmonary TB. Of these, 16 were individuals attending the Infectious Disease Clinic at the Veterans Affairs Medical Center, New York. All patients were M. tuberculosis culture-positive, 9/16 patients were smear-negative, 14/16 showed minimal to no radiological lesions, and all were bled either prior to, or within 1 -2 weeks of initiation of chemotherapy for TB. Eight sera were obtained from Leonid Heifitz and Lory Powell (National Jewish Center, Denver, CO). An additional 20 sera were provided by J.M. Phadtare ( Grant Medical College, Bombay, India). Fourteen serum samples obtained from Lala Ram Sarup Tuberculosis Hospital, Mehrauli, New Delhi, India were provided by S. Singh.
- control populations consisted of the following groups: (a) 16 HIV ne , TB neg , PPD + healthy individuals (either recent immigrants from endemic countries or staff members involved in the care of TB patients in the VA Medical Center
- the antigen preparations were total cellular sonicate (CS), total culture filtrate (CF), lipoarabinomannan (LAM), LAM-free culture filtrate proteins (LFCFP), whole cell walls (CW), SDS-soluble cell wall proteins (SCWP), and cell wall core (CWC), all isolated from M. tuberculosis H 37 Rv.
- CS total cellular sonicate
- CF total culture filtrate
- LAM lipoarabinomannan
- LAM-free culture filtrate proteins LFCFP
- CW whole cell walls
- SCWP SDS-soluble cell wall proteins
- CWC cell wall core
- CS was obtained from M. tuberculosis grown in Middlebrook 7H9 broth (Difco Laboratories, Detroit, MI) for 2-3 weeks.
- the bacilli were harvested by centrifugation at 1000 rpm for 30 min and the pellet resuspended in phosphate buffered saline (PBS) containing PMSF, EDTA and DTT at a final concentration of ImM each.
- PBS phosphate buffered saline
- the suspension was frozen in liquid nitrogen and thawed (several times) to weaken the cell walls, following which the suspension was sonicated for 20 min at 4°C.
- the sonicate was centrifuged for 10 min at 10,000 rpm and the supernatant collected.
- M. tuberculosis was grown to mid- logarithmic phase (14 days) in glycerol-alanine-salts medium.
- the cells were removed by filtration through a 0.22 ⁇ m membrane, and the culture supernatant was concentrated by ultrafiltration using an Amicon apparatus (Beverly, MA) with a 10,000 MW cut-off membrane.
- the concentrated material (CF) was dialyzed against 100 mM ammonium bicarbonate and dried by lyophilization.
- CF was suspended (7mg/ml) in a buffer containing 50mM Tris HC1 (pH 7.4), and 150mM NaCl, following which 20% Triton X-114 was added to obtain a final concentration of 4%.
- the suspension was allowed to rock overnight at 4°C.
- a biphasic partition was set up by warming the 4% Triton X-114 suspension to 37°C for 40 minutes, followed by centrifugation at 12,000 x g.
- the aqueous phase was re-extracted twice with 4% Triton X-114 to ensure complete removal of the lipoarabinomannan, lipomannan (LM) and phosphatidyl-inositol- mannoside (PIM).
- LM lipomannan
- PIM phosphatidyl-inositol- mannoside
- the final aqueous phase was precipitated with 10 volumes of cold acetone, and the pellet washed several times with cold acetone to remove residual Triton X-114.
- the LAM-free aqueous phase CFPs were suspended in 100 mM ammonium bicarbonate, aliquoted and dried by lyophilization.
- LAM, LM and PIM were extracted from whole cells by mechanical lysis of the bacilli in PBS (pH 7.4) containing 4% Triton-X 114 in a Bead Beater (Biospec Products, Bartelsville, OK). Unbroken cells and cell wall material were removed by centrifugation at 12000g, 4°C for 15 min. The supernatant was collected and a biphasic partition set up. The detergent phase was obtained, back-extracted several times with cold PBS and the macromolecules in the final detergent phase were precipitated with 10 volumes of cold acetone. The precipitate was collected by centrifugation and allowed to air dry.
- This material (which contained the lipoglycans) was suspended in PBS and residual proteins were removed by extraction with PBS- saturated phenol. The aqueous phase was collected and, after dialyses against distilled water, the lipoglycans were lyophilized. LAM was further purified away from LM and PIM by size exclusion chromatography as previously described (Chatterjee, D. et al. , 1992, J. Biol. Chem. 269:66228-66233).
- M. tuberculosis cells were inactivated by isothermal killing at 80°C for 1 h and suspended at a concentration of 0.5g cells/ml, in a buffer containing PBS, pH 7.4, 4% Triton X-114, PMSF, pepstatin, EDTA, and DNase.
- the cells were disrupted in a Bead Beater using 0.1 mm Zirconia beads.
- the lysed cells were first centrifuged at 3000 x g for 5 min to remove unbroken cells followed by centrifugation at 27,000 x g, 4°C for 20 min. The resulting pellet was washed three times with cold PBS at room temperature. This final pellet was termed the CW.
- the SCWP were obtained by washing the CW twice with 2% SDS in PBS, pH 7.4 at room temperature.
- the tightly associated proteins were isolated by extracting the CW pellet three times with 2% SDS in PBS, pH 7.4, at 55°C.
- the 55°C, 2% SDS extract was recovered, and the SDS was removed by using an Extracti Gel column (Pierce, Rockford, IL).
- the eluate from the column was dialyzed against twice- distilled H 2 O, aliquoted and dried by lyophilization.
- the CWC (mycolyl-arabinogalactan-peptidoglycan complex) was generated as described (Daffe, M. et al , 1990, J. Biol Chem. 265:6734-6743) with minor modifications.
- the SDS-insoluble material obtained after extraction of the SCWP was suspended in PBS, 1% SDS, O.lmg/ml proteinase K and incubated for 20h at 50°C.
- the insoluble material was pelleted by centrifugation, washed twice with 2% SDS at 95°C for lh and collected by centrifugation. This was washed several times with water and 80% acetone to remove SDS.
- Fractionation of LFCFP by size was performed by using a preparative SDS- PAGE system (model 491 Prep cell, Bio-Rad, Hercules, CA).
- CFP (20-25 mg) was loaded directly onto a 30ml 10% preparative polyacrylamide tube gel containing a 6% stacking gel, that was poured in a casting tube with a 37mm internal diameter.
- the running buffer used consisted of 25mM Tris, pH 8.3, 192mM glycine, 0.1% SDS.
- the proteins were separated by electrophoresis using an increasing wattage gradient of 8W for 3.13h, 12W for 2.5h, and finally 20W for 1 l.lh.
- Proteins were eluted from the bottom of the tube gel with a constant flow of 5mM sodium phosphate, pH 6.8. The initial 65ml of eluant were collected as the void volume, after which 80 fractions of 4.2 ml were collected at a rate of 0.4ml/min. Individual fractions were assayed by one dimensional SDS-PAGE and were pooled accordingly. SDS was removed from the pooled concentrated fractions by elution through an Extracti-Gel (Pierce) column. The pooled fractions were dried and stored frozen until testing. Adsorption of sera with E. coli sonicate Overnight cultures of E.
- E. coli grown in Luria-Bertani medium were centrifuged to obtain bacterial pellets that were treated as described for the M. tuberculosis sonicate, except that sonication was performed for 30 sec.
- BSA bovine serum albumin
- HIV was inactivated by addition of Triton X-100 (1% final concentration) to each serum sample, followed by heating at 55°C for 60 min.
- Samples from non-HIV infected individuals were treated in the same manner to maintain consistency in sample preparation.
- Serum from each individual (20 ⁇ l) was diluted to 200 ⁇ l in PBS/Tween 20 (0.05%)) in a 96-well tissue culture plate.
- the diluted serum samples were transferred to the E. co/t-coated, blocked ELISA plate by using a multichannel pipetter.
- the sera samples were exposed to the bound E. coli antigens for 90 min after which they were transferred to another ELISA plate that had been coated with E. coli and blocked as above.
- the serum samples were exposed to 8 cycles of adsorption with E. coli antigens, following which they were transferred to a 96-well tissue culture plate where sodium azide (ImM final concentration) was added to each well. This protocol allows rapid and efficient processing of small volumes of multiple samples. Adsorbed serum samples were used within one week. ELISA with M. tuberculosis antigens
- the antigen-antibody binding was allowed to proceed for 90 min at 37°C, following which the plates were washed 6 times with PBST. Fifty ⁇ l of alkaline phosphatase-conjugated goat anti -human IgG (Zymed, CA), diluted 1 :2000 (in the same diluent as the serum samples) were added to each well. After 60 min the plates were washed 6 times with Tris-buffered saline (50mM Tris, 150mM NaCl) and the Gibco BRL Amplification System (Life Technologies, Gaithersburg, MD) used for development of color. The plates were read at 490 nm after stopping the reaction with 50 ⁇ l of 0.3M H 2 SO 4 . The optimal antigen and antibody concentrations for each antigen were determined by checkerboard titration with limited numbers of control and non-TB sera prior to performing the ELISA with the total serum panel.
- the ELISA with each of the sized fractions generated by preparative polyacrylamide gel electrophoresis was performed as described as above, except that antigen was coated at 2 ⁇ g/ml and the sera were tested at a final dilution of 1 :200. Forty-two TB sera and 44 non-TB controls (16 PPD + ; 7 HIV neg , PPD neg ; and 21 HIV + , asymptomatic individuals) were included in these assays.
- the "IT" designations are World Health Organization standards for its collection of anti-Mt antibodies.
- the alternative names of the mAbs, the antigens they recognize and the laboratory of origin are provided in Engers, H. et al, 1986, Infect. Immun. 57:718-720; Khanolkar- Young, S. et al, 1992, Infect. Immun. 60:3925-3925; Young et al, supra, which are incorporated by reference in their entirety.
- Antiserum to the 50/55 kDa antigen, MPT32 was obtained from the NIH, Contract l-AI-25147. The table below summarizes these antibodies and their reactivities.
- composition of the sized fractions was probed with the antibodies in an ELISA, similar to what was used for assessment of reactivity with human sera, except that 50 ⁇ l/well of each antibody defined above was used at a concentration recommended by the contributing laboratory.
- the second antibody was an alkaline phosphatase-conjugated rabbit anti-mouse IgG or goat anti-rabbit IgG (1:2000, Sigma Immunochemicals) added in a volume of 50 ⁇ l/well SDS-PAGE and immunoblotting
- HBT10 L-alanine POS POS 12 dehydrogenase
- NEG Negative
- POS Positive - OD 0.5 - 0.999
- SOD superoxide dismutase References: Andersen et al. , 1 984; 2 Andersen et al. , 1 986; 3 Andersen et al. , 1 989; Andersen et al. , 1 991 ; 5 Andersen et al. , 1 994; 6 Brennan et al.; 7 Coates et al. , 1 981 ; 8 Foumle et al.
- NS not significant. Antibodies reactive with LFCFP were detectable in 25/42 (60%) of the unadsorbed TB sera ( Figure 1). When tested postadsorption, anti -mycobacterial antibodies were detectable in 4/17 (24%) additional, previously negative sera, raising the sensitivity to 69%) ( Figure 1).
- IT-41 F15 IT-41, IT-57 73 Specificity of murine mAbs: IT-62 and IT-23 are anti-38 kDa; IT-41 is anti-71 kDa;
- IT-57 is anti-82 kDa.
- Anti-MPT32 antiserum was raised in rabbits.
- the ELISA + TB serum pool recognized at least 10 additional distinct bands in the fractionated total LFCFP ( Figure 4, lane 2).
- the molecular weights of the antigens ranged from 33kDa to 112kDa.
- the 38kDa antigen was the most dense band observed, indicating that it is the most abundant antigen recognized by this serum pool in the LFCFP. Since it is a strongly seroreactive antigen in several patients
- Fraction 10 contained large amounts of the 38 kDa antigen, which was the strongest band, and smaller amounts of other seroreactive proteins ranging from 30- 43kDa.
- fraction 15 contained several high molecular weight antigens ranging from 72-88 kDa, and a small amount of the 38kDa antigen (which was not detected by anti-38 kDa mAbs, Table 3). Strong seroreactivity with a doublet at 88- 84kDa, and weaker reactivity with 78kDa and 72kDa antigens was seen.
- LAM migrates as a broad band in the 30-40 kDa region on gels, antibodies reactive to LAM would obscure other useful antigens in this region on immunoblots.
- the LFCFP contains over two hundred different secreted proteins (Example
- Antibodies to an 88kDa Secreted Antigen of M. tuberculosis Serve as a Surrogate Marker of Pre-clinical TB in HIV-infected Subjects
- the study population included 49 HIV-infected individuals attending the Infectious Disease Clinic at the V.A. Medical Center, New York, who developed or presented with TB (HIV/TB) during the last several years. A total of 259 serum samples were available from these individuals. Of these samples:
- the diagnosis of TB was based on positive cultures for M. tuberculosis. Sera from 20 non-HIV TB patients (non-HIV/TB), 19 of whom were smear- positive, and all of whom showed radiological evidence of moderate to advanced cavitary disease, were included as positive controls. Sera from 19 non-HIV/ PPD skin test-positive individuals were included as negative controls. To rule out nonspecific reactivity, the study included (i) sera from 35 HIV-infected, asymptomatic individuals, with CD4 cell counts >800 and (ii) 48 serum samples from 16 HIV- infected subjects whose blood cultures were positive for Mycobacterium avium- intracellulare ("HIV/MAI". Of these, 28 HIV/MAI serum samples were obtained during the months preceding advent of MAI bacteremia.
- LAM-free culture filtrate proteins LFCFP
- This antigen mixture was subsequently fractionated based on the molecular weight of the proteins using a BioRad 491 Prep Cell (Hercules, CA) with a 30 ml 10%> preparative polyacrylamide tube gel containing a 6% stacking gel as above. Fractions were pooled according to molecular weights (as determined by SDS-PAGE) and dried.
- the second antibody used was alkaline-phosphatase conjugated rabbit anti- human IgG and the substrate was BCIP/NBT (Kirkegaard and Perry Laboratories, Gaithersburg, MD).
- M. tuberculosis was compared to reactivity of sera from 16 non-HIV/PPD + individuals (negative controls) and 20 non-HIV/TB patients (positive controls). Each serum sample from each subject was evaluated at least three times for presence of anti- tuberculosis antibodies. A representative ELISA assay showing the antibody levels for each of these groups is presented in Figure 5. With the cutoff set as the mean
- the specificity of the anti-M tuberculosis antibody responses in the HIV/TB patients was evaluated. Sera from 35 HIV-infected asymptomatic individuals (CD4 + cell counts >800) and 48 sera from 16 HIV/MAI patients were tested along with 19 non-HIV/PPD + healthy controls and 20 non-HIV/TB patients. The results are shown in Figure 6. Using the mean OD+3SD of the 19 non-HIV/PPD + control sera as the cutoff, 2/35 sera from the HIV- + group and 7/48 sera from the HIV/MAI group showed minimal reactivity with the M. tuberculosis secreted antigens. These results confirmed the specificity of the reactivity of HIV/TB sera with M. tuberculosis antigens.
- FIG. 7 depicts the presence of anti-M tuberculosis antibodies in multiple sera from 6 antibody- positive (panels A-F), 3 antibody-negative HIV/TB patients (panel G), and 3 HIV/MAI (panel H) patients. All 6 antibody-positive individuals had circulating antibodies for different intervals during the years preceding the clinical manifestation of TB.
- HIV/TB patients recognize only a subset. For example, antibodies to the 38 kDa antigen are not found in HIV/TB, whereas antibodies to antigens in fraction 10-fraction 14, and in particular to the 88 kDa antigen are maintained despite HIV infection.
- FIG. 10 shows the reactivity of the 42 HIV/TB patients with the antigens in fractions 7-14. Values obtained with HIV/pre-TB sera have been shown for most patients; and for patients for whom no pre-TB sera were available, HIV/at- TB sera are shown. Fifty percent (21/42) of the HIV/TB patients had antibodies to the unfractionated LFCFP. However, 74% (31/42) of the same patients showed positive reactivity with antigens in fraction 14.
- the repertoire of M tuberculosis antigens which elicit antibodies in the HIV/TB patients is limited in comparison to non-HIV/TB patients: antibodies to several antigens with molecular weights of 32-45 kDa are absent in these HIV/TB patients. Antibodies to a strongly seroreactive 38 kDa antigen, which are present in 50-60%) of non-HIV/TB TB patients, were absent from most HIV/TB patients.
- Example I shows that the 88 kDa antigen (present in Fraction 15 in that study, but present in Fraction 14 in the study of Example II) is one of the secreted antigens of M tuberculosis that elicits antibodies during early stages of disease progression (in non-HIV TB patients).
- the detection of anti-88 kDa antibodies in the high risk HIV-infected population can serve as a diagnostic test, and the antibody as a surrogate marker, for identifying individuals with active pre-clinical TB.
- PPD + Draggerald J.M. et al, Chest 700:191-200
- Delayed hypersensitivity skin test reactivity is known to be unstable in HIV + individuals. Since development of PPD reactivity and production of anti- mycobacterial antibodies do not necessarily occur simultaneously (Das, S. et al, Clin. Exp. Immunol. 1992;89:402-06; Kardjito, T. et al, Tubercle. 1988, 65:269-274; Balestrino, E. A. et al. , Bull. World Health Org. 1984, 62:755-761 ), the simultaneous use of both markers will enhance early detection and our ability to institute timely therapy in such patients.
- the 45 kDa culture filtrate protein of M tuberculosis is an example of a glycoprotein for which chemical proof of amino acid glycosylation is still lacking.
- 45 kDa protein is the same as MPT 32, a culture filtrate protein originally isolated by Nagai and colleagues (Nagai, S. et al, 1991, Infect. Immun. 59:312-382.).
- the DNA sequence of a gene designated apa encoding a 45/47 kDa M tuberculosis protein was elucidated by Laqueyrerie et al. (supra), and the deduced amino acid sequence of this gene yielded 100%> homology with the N- terminal sequence of the 45 kDa/MPT 32 protein (Dobos et al, supra).
- the apa sequence further indicated an abundance of Pro and Ala, confirming earlier amino acid compositional analysis. Dobos et al.
- N-terminal amino acid sequencing coupled with fast atom bombardment ⁇ mass spectrometry demonstrates that each contains O-glycosylated Thr residues within a newly recognized, conserved O-glycosylation motif.
- Carbohydrate and MS analysis established that the glycosylation units in each case consist of single mannose, mannobiose or mannotriose units possessing ( ⁇ l-2) linkages.
- the recent elucidation of the complete gene sequence for this protein now allows the location of all glycosylation sites within the N-and C-terminal regions of the polypeptide backbone.
- the elucidation of the complete primary structure of this unique glycoprotein enables further work on understanding its biosynthesis and physiological role, and approaches to its use as an antigen in for early diagnosis of
- M. tuberculosis strain Erdman was cultured in glycerol alanine salts medium for 14 days at 37°C with gentle agitation, conditions considered optimal for the production of culture filtrate proteins including the MPT32.
- organisms were grown in 5 1 of the medium at 37°C with gentle agitation for 5 days, at which time [U- 14 C]D-glucose (3 mCi per mmol; American Radiolabeled Chemicals, Inc., St. Louis, Mo.) was added to a final concentration of 1 mCi per liter, and the culture was incubated for an additional 8 days.
- the [ 14 C] labeled culture filtrate proteins were harvested as described by Dobos et al. ( supra).
- the purified 45-kDa glycoprotein (500 ⁇ g) was digested with either subtilisin (alkaline protease VIII; Sigma Chemical Co., St. Louis, Mo.) or a mixture of chymotrypsin/trypsin (1 :1) (Sigma). The proteolytic digestions were carried out in
- Digestion of the protein with ⁇ -mannosidase was conducted as follows.
- the radiolabeled 45-kDa glycoprotein (135 ⁇ g) or purified glycopeptides (320 ng to 3 ⁇ g) were solubilized in 100 ⁇ l of 0.05 M CH 3 COONa, pH 4.5, and incubated with 10 ⁇ l of ⁇ -mannosidase from Canavalia ensiformis, supplied as a 5 mg/ml suspension (Boehringer Mannheim, Indianapolis, Ind.), at 37°C for 8 h. An additional 10 ⁇ l of ⁇ -mannosidase was added to the reaction mixture and further incubated for 16 h at 37°C.
- the digestions were terminated by incubating at 100°C for 2 min, dried, and suspended in 5% CH 3 COOH.
- the released Man residues were separated from the 45 kDa protein by applying the products to a C 18 reversed-phase Sep Pak cartridge (Waters) and washing with 5% CH 3 COOH, followed by elution of the ⁇ -mannosidase- digested protein with n-propanol: acetic acid:water (50:5:45).
- the ⁇ -mannosidase- digested glycopeptides were separated from the released sugars in a similar manner.
- the peptides were eluted with n-propanol:acetic acid:water (25:5:70) followed by n-propanol:acetic acid:water (50:5:45).
- the radiolabeled 45-kDa protein (100 ⁇ g) was hydrolyzed with 2 M TFA at 120°C for 2 h.
- the hydrolyzed material was dried under nitrogen, solubilized in water, and applied to an HPLC column (4 x 250 mm) of CarboPac PA1 (Dionex Corp., Sunnyvale, Calif.) connected to a Dionex HPLC system equipped with an advanced gradient pump and a pulsed amperometric detector.
- Monosaccharides were eluted from the column with 10 mM NaOH at a flow rate of 1 ml per min (9), collected in 1 ml fractions, counted by liquid scintillation, and the elution profile compared to that of a mixture of known monosaccharides.
- subtilisin and chymotrypsin/trypsin digestions of the 45 kDa glycoprotein were resolved by microcapillary C 18 reversed-phase HPLC (45). Effluent was monitored by A 214 and then directly introduced into an on-line TSQ-700 triple-sector quadrapole mass spectrometer with an electrospray ionization source and collision gas as described (Swiderek, K. et al, 1995, "Trace structural analysis of proteins," p. 36. In B. Hancock et ⁇ /.(eds.), Meth. Enzymol, Academic Press, Inc., New York).
- Glycopeptides were detected by scanning for a neutral loss of 162 AMU, indicative of the loss of hexose (i.e., Man) units, or 132 AMU for loss of pentose units (Huddleston, M. et al, 1993, Anal Chem. 65:877-884.).
- Methyl esterification of selected ⁇ -mannosidase digested peptides was performed in 0.5 N methanolic-HCl at 20°C for 30 min. Upon drying under N 2 , the methyl esterified peptides were N-acetylated in methanol: pyridine: acetic anhydride (50: 1 :5) for 30 min at 20°C.
- the molecular weight of peptides, glycopeptides and ⁇ -mannosidase digested peptides was determined by FAB-MS. Individual peptides were dissolved in 5% CH 3 COOH (10 to 20 ⁇ l) and samples (1 ⁇ l) were added to the thioglycerol matrix. FAB-MS was conducted on a VG Autospec (Fission Instruments, Inc., Beverly, Mass.) fitted with a cesium ion gun operated at 25-30 kV. Data acquisition and processing were performed using the VG Analytical Opus software (Fission
- Oligosaccharides were released from purified glycopeptides by reductive ⁇ -elimination conducted as follows (Chatterjee, D. et al, 1987, J. Biol. Chem. 262:3528-3533). Glycopeptides (320 ng-3 ⁇ g) were suspended in a solution of 0.05
- GC-MS of the partially methylated alditol acetates was performed on a Hewlett-Packard 5890 gas chromatograph fitted with a (15 m x 0.25 mm ID) DB-5 capillary column (J & W Scientific, Folsom, Calif.) and connected to a Hewlett-Packard 5790 mass detector as described (McNeil et al, supra).
- microcapillary liquid chromatography-MS and m/z 162 neutral loss scanning were used to identify four glycopeptides (Srase S 3 , S 6 , and S n ) generated from a subtilisin digest of the 45 kDa glycoprotein.
- S u peptide only to be obtained in pure enough form for structural analyses.
- HPLC map of the peptides from a subtilisin digestion of the 45 kDa protein was obtained.
- the products were then subjected to microcapillary chromatography-MS and neutral loss scanning, seeking peptides that fragmented to give a daughter ion of m/z 162 or 132, indicative of loss of hexosyl or pentosyl unit(s), respectively.
- the digest yielded 50 peptides (S 2 - S 5) ), and, of these, only S 7 , S 18 , S 22 , S 29 , and S 41 produced daughter ions of m/z 162. No peptides were detected that produced daughter ions of m/z 132 , results which were in agreement with the compositional analysis that indicated only mannosylation of the 45 kDa glycoprotein.
- N-terminal amino acid sequence and masses of the individual peptides were established by automated Edman degradation and FAB-MS (Table 1).
- the (M+H) + pseudomolecular ion of the S 41 glycopeptide was observed at m/z 1515.5.
- this product was identical to the S ⁇ glycopeptide previously described as the first bonafide glycopeptide in M. tuberculosis with the structure DPEPAPPVPTTA-Man- Man [SEQ ID NO:6].
- N-terminal sequencing confirmed this identity (Table 1).
- the (M+H) + pseudomolecular ion of the S 29 glycopeptide was established as m/z 1150.5, and the corresponding amino acid sequence was established as XPVAPPPPAAA
- SEQ ID NO:7 The amino-acid sequence of the S ]8 glycopeptide (m/z 1008.6) was identical to that of the S 29 glycopeptide except for the absence of two Ala residues at the carboxyl terminus. Moreover, the difference between the (M+H) + pseudomolecular ions of the S 29 and S lg peptides (m z 142) corresponded to two Ala residues. N-terminal amino acid analysis of the S 22 glycopeptide established the sequence GEVAPTPTXPTPQ [SEQ ID NO: 8]. However, three individual (M+H) + pseudomolecular ions of m/z 1781.9, 1619.8, and 1457.6 were observed by FAB-MS for this S 22 glycopeptide.
- the difference between these three ions was m/z 162, a result indicating differing levels of glycosylation of the same peptide.
- the S 33 peptide produced an N-terminal sequence of GEVAPTPTTPTPQ [SEQ ID NO:9] and an
- CT 6 represented a larger version of the S 22 glycopeptide cluster. Additionally, as with the S 22 cluster, a naturally non-glycosylated form of CT 6 was detected, CT 7 (m/z 3291.2) (Table 5). As with the peptides from the subtilisin digest, several non-glycosylated peptides from the chymotrypsin/trypsin digest were identified by FAB-MS and N-terminal amino acid sequencing (Table 5).
- Amino acid sequences obtained by automated Edman degradation are shown in bold face type. Amino acid sequences inferred from FAB-MS analysis and alignment with the deduced amino acid sequence of the 45 kDa protein are shown in normal face type.
- Amino acid sequences obtained by automated Edman degradation are shown in bold face type. Amino acid sequences inferred from FAB-MS analysis and alignment with the deduced amino acid sequence of the 45 kDa glycoprotein are shown in normal face type.
- the broad peak corresponding to the S 22 peptide was comprised of three individual peptides, all of which contained the same amino acid sequence but differed in the extent of their glycosylation.
- This cluster of peptides was rechromatographed by reversed phase HPLC, and the largest of the three peaks was selected for further FAB-MS analysis.
- the m z difference between the (M+H) + pseudomolecular ions of the glycosylated and deglycosylated forms of the S 22 peptide was m/z 324, demonstrating that this component peptide of the S 22 cluster was glycosylated with two ⁇ -Man residues.
- N-terminal sequencing of the ⁇ -mannosidase digested form of this S 22 peptide produced a sequence identical to that obtained for the naturally occurring non-glycosylated S 33 peptide (GEVAPTPTTPTPQ; SEQ ID NO:25), confirming that the S 22 glycopeptide was O-glycosylated at the third Thr residue.
- Similar analyses of the two other glycopeptides of the S 22 cluster confirmed that one (m/z 1781.9) was glycosylated with three ⁇ -Man residues and the other (m/z 1457.6) with a single ⁇ -Man residue.
- Analysis of the (oligo)glycosyl alditols further demonstrated heterogeneous glycosylation of the peptide with ⁇ -Man, ( ⁇ l-2)- mannobiose or ( ⁇ l-2)-mannotriose.
- the S 41 glycopeptide was shown to be O-glycosylated at the position 10 Thr residue.
- ⁇ -Mannosidase digestion followed by N-acetylation, methyl esterification and FAB-MS analysis produced (M+H) + pseudomolecular ion of m/z 1297.6, indicating the loss of two ⁇ -Man residues.
- the oligoglycosylalditol released from the S 41 glycopeptide was found to be comprised of a pre-reduced 2- linked mannitol and a terminal Man (Table 6).Thus, the Thr residue at position 10 of this peptide was glycosylated with an ( ⁇ l -2)-linked mannobiose.
- Thr residues at amino acid positions 10 and 18 are O-glycosylated with the mannobiose unit ⁇ -D-Manp(l— »2)- ⁇ -D-Manp, the Thr 27 is substituted with a single ⁇ -D-Manrj unit, while Thr 277 (in the C-terminal region) may be linked to a ⁇ -D-Manp, ⁇ -D-Manp(l->2)-D-Manp or ⁇ -D-Manp(l ⁇ 2)- ⁇ -D-Manp(l ⁇ 2)-D-Manp unit.
- Such glycosylation microheterogeneity is consistent with other well described prokaryotic glycoproteins.
- Other known O-glycosylated bacterial proteins contain a
- GlcNAc unit O-linked to either Thr or Ser.
- the nature of O-glycosylation of the 45 kDa protein of M tuberculosis is more pronounced of the simpler sites of
- the gene sequence encoding the 45 kDa glycoprotein demonstrates the presence of a signal peptide similar to that of many other M. tuberculosis culture filtrate proteins (Young, D.B. et al, In: J. McFadden (ed.), Molecular Biology of the Mycobacteria. Surrey University Press, Surrey, U.K., 1990, pp. 1-35).
- Mannosylation of mycobacterial proteins may bear similarities to that of the yeast mannoproteins.
- the di- and tri-mannosyl units of the 45 kDa glycoprotein are identical to the "mannose caps" of mycobacterial LAM (the so-called Man-LAM) in absolute configuration and linkage (Chatterjee, D. et al, 1992, J. Biol. Chem. 267:6234-6239), suggesting that the enzymatic machinery is shared by both systems.
- the mannosyl units of the 45 kDa protein may share a role in the phagocytosis of M tuberculosis, analogous to that of the Man-LAM (Schlesinger, L.S. et al, 1994, J. Immunol. 752:4074-4079).
- M tuberculosis H37Ra culture filtrate proteins (the source of the Ag85 components in the context of their antigenicity; Wiker et ⁇ l., 1992, supra) were harvested from cells in mid-logarithmic growth as described above and precipitated with 40%) saturated (NH 4 ) 2 SO 4 , yielding a fraction with substantial transferase activity and containing the full complement of Ag85 components (Figure 12) as confirmed by Western blot analysis ( Figure 13).
- the column was washed with 3 vol of storage buffer at a flow rate of 1 ml/min which eluted the majority of proteins while leaving the Ag85 complex bound to the Phenyl Sepharose matrix.
- the individual proteins of the Ag85 complex were eluted with 30 ml of buffer A (10 mM Tris HC1 pH 8.6, 1 mM DTT, 1 mM EDTA) followed by a linear gradient composed of 100% buffer A to 100% buffer B
- CFPs culture filtrate proteins
- the present inventors have combined 2-D PAGE, western blot analysis, N-terminal amino acid sequencing and liquid chromatography-mass spectrometry-mass spectrometry (LC-MS-MS) to develop a detailed map of culture filtrate proteins and have obtained the partial amino acid sequences for five previously undefined, relatively abundant proteins within this fraction which are found to be useful as early antigens for serodiagnosis of TB.
- LC-MS-MS liquid chromatography-mass spectrometry-mass spectrometry
- Mt strains H37Rv (ATCC 27294) and H37Ra (ATCC 25177) were obtained from American Type Culture Collection (Rockville, MD). Mt strain Erdman (TMC) was obtained from American Type Culture Collection (Rockville, MD). Mt strain Erdman (TMC).
- each Mt strain was inoculated from a 1 ml frozen stock into 10 ml of glycerol alanine salts (GAS) media; three such cultures were prepared for each strain. After incubation at 37°C for 14 days with gentle agitation each 10 ml culture was passed two more times increasing the volume of media by ten times for each pass. The resulting one liter cultures were termed pass number four. For each Mt strain, three liters of pass number four cultures were used to inoculate 30 liters of GAS media. After 14 days of growth at 37°C with gentle agitation, the culture supernatant was removed from the cells by filtration and the CFPs concentrated and processed as described.
- GAS glycerol alanine salts
- Protein content of the concentrated culture filtrate was quantitated by the bicinchoninic acid protein assay.
- culture tubes 13 by 100 mm
- 3 ml of GAS media with 0.05% Tween 80 were inoculated with actively growing Mt cultures to an optical density of 0.1 at 600 nm. These cultures were incubated at 37°C with stirring and optical densities at A600 were obtained every 12 hours for a 22 day period.
- the mAbs IT-69 (HBT11) and IT-67 (L24.b4) were obtained from Dr. Ase B. Andersen, Statens Seruminstitut, Copenhagen, Denmark.
- the mAb A3h4 was obtained from Drs. P.K. Das and A. Rambukana, University of Amsterdam, Amsterdam, The Netherlands and mAbs F 126-2 and HYB 76-8 were obtained from
- Dr. S. Nagai provided polyclonal sera specific for MPT 32, MPT 35, MPT 46, MPT 53, and MPT 57.
- SDS-PAGE and 2-D PAGE of Culture Filtrate Proteins SDS-PAGE was performed under reducing conditions by the method of Laemmli with gels (7.5 x 10 cm x 0.75 mm) containing a 6% stack over a 15% resolving gel. Each gel was run at 10 mA for 15 min followed by 15 mA for 1.5 h. 2-D PAGE separation of proteins was achieved by the method of O'Farrell with minor modifications.
- Electrophoresis in the second dimension was carried out at 20 mA per gel for 0.3 h followed by 30 mA per gel for 1.8 h. Proteins were visualized by staining with silver nitrate. 4. Computer Aided Analysis of Two-Dimensional Gels
- Silver stained 2-D PAGE gels were imaged using a cooled CCD digitizing camera and analyzed with MicroScan 1000 2-D Gel Analysis Software for Windows 3.x (Technology Resources, Inc., Milwaukee, TN). Protein peak localization and analysis was conducted with the spot filter on, a minimum allowable peak height of 1.0, and minimum allowable peak area of 2.0.
- Proteins subjected to 2-D or SDS-PAGE, were transferred to nitrocellulose membranes (Schleicher and Schuell, Keene, NH.) which were blocked with 0.1% bovine serum albumin in 0.05 M Tris-HCl, pH 7.5, 0.15 M NaCl, and 0.05%> Tween 80 (TBST). These membranes were incubated for 2 h with specific antibodies diluted with TBST to the proper working concentrations (Table 7). After washing, the membranes were incubated for 1 h with goat anti-mouse or -rabbit alkaline phosphatase-conjugated antibody (Sigma) diluted in TBST. The substrates nitro-blue- tetrazolium and 5-bromo-4-chloro-3-indoyl phosphate (BCIP) were used for color development.
- BCIP 5-bromo-4-chloro-3-indoyl phosphate
- India ink as the secondary stain was washed three times in 0.05 M K 2 HPO 4 , pH 8.5.
- the total protein population was conjugated to digoxigenin by incubating the membrane for one hour at room temperature in a solution of 0.05 M K 2 HPO 4 , pH 8.5 containing 0.3 ng/ml digoxigenin-3-0-methylcarbonyl- ⁇ -amino-caproic acid N-hydroxysuccinimide ester and 0.01%) Nonidet-P40.
- the membranes were subsequently blocked with a solution of 3% bovine serum albumin in 0.05 M Tris-HCl, pH 7.5, 0.15 M NaCl (TBS) for 1 h followed by washing with TBS.
- CFPs 200 ⁇ g were resolved by 2-D PAGE and transferred to poly vinylidene difluoride membrane (Millipore, Milford, Mass.) by electroblotting at 50 V for 1 h, using CAPS buffer with 10% methanol. The membrane was stained with 0.1 % Coomassie brilliant blue in 10% acetic acid and destained with a solution of 50% methanol and 10% acetic acid. Immobilized proteins were subjected to automated Edman degradation on a gas phase sequencer equipped with a continuous-flow reactor. The phenylthiohydantoin amino acid derivatives were identified by on-line reversed-phase chromatography as described previously.
- CFPs were subjected to LC-MS-MS to determine the sequence of internal peptide fragments.
- CFPs 200 mg were resolved by 2-D PAGE and the gel stained with 0.1% Coomassie brilliant blue and destained as described for proteins immobilized to PVDF membranes.
- the protein of interest was excised from the gel, washed several times with distilled water to remove residual acetic acid and subjected to in-gel proteolytic digestion with trypsin.
- Peptides were eluted from the acrylamide and separated by C18 capillary reversed phase chromatography.
- the microcapillary reversed phase effluent was introduced directly into a Finnigan-MAT (San Jose, CA) TSQ-700 triple sector quadrupole mass spectrometer. Mass spectrometry and analysis of the data was performed as described by Blyn et al..
- IT-56 demonstrated reactivity to specific proteins of this preparation (Table 7).
- the mAb IT-56 is specific for the 65 kDa Mt GroEL homologue; a protein primarily associated with the cytosol. Additionally the mAb IT-7 reacted with a 14 kDa and not a 40 kDa CFP.
- N-terminal amino acid sequencing of selected CFPs The N-terminal amino acid sequences or complete gene sequences and functions of several of the CFPs of Mt, mapped with the available antibodies, are known. However, such information is lacking for the proteins that reacted with IT-42
- IT-43, IT-44, IT-45, IT-51, IT-52, IT-53, IT-57, IT-59 and IT-69 were selected and subjected to N-terminal amino acid sequencing ( Figure 14 and Table 8). Three of these proteins were found to correspond to previously defined products.
- the N-terminal amino acid sequence of the protein labeled D was identical to that of Ag85 B and C. This result was unexpected given that the IT-49 mAb failed to detect this protein and N-terminal amino acid analysis confirmed that those proteins reacting with IT-49 were members of the Ag85 complex.
- the protein labeled E had an N-terminal sequence identical to that of glutamine synthetase.
- a third protein which reacted with IT-52 was found to be identical to MPT 51.
- the protein labeled I possessed an N- terminal sequence with 72% identity to the amino terminus of an ⁇ -hydroxysteroid dehydrogenase from a Eubacterium species , and the protein labeled F was homologous to a deduced amino acid sequence for an open reading frame identified in the Mt cosmid MTCYl Al 1. Repeated attempts to sequence those proteins labeled as A, G, H, J, K, IT-43, IT-44, IT-49 and IT-57 were unsuccessful.
- Examples I and II show that a high molecular weight fraction of CFP of Mt reacted with a preponderance of sera from TB patients and that this fraction was distinguished from other native fractions in that it possessed the product reactive to mAb IT-57.
- the protein cluster (the 88 kDa protein) defined by IT-42 and IT-57 was excised from a 2-D polyacrylamide gel, digested with trypsin and the resulting peptides analyzed by LC-MS-MS.
- Ten of the peptides from the digest yielded molecular masses and fragmentation patterns consistent with those predicted for tryptic fragments of the Mt KatG catalase/peroxidase (Table 8).
- the portion of the protein not reactive with IT 57 appears to be the KatG product.
- the IT 57-reactive part of the 88 kDa protein cluster did not have sequence homology (following LC-MS-MS analysis) to an identified Mt protein.
- Strain H37Rv contained three apparently strain-specific proteins, numbered 9, 123 and 203 ( Figure 15A and 15B and Table 9). Similarly, the proteins numbered 206, 207, 208 and 209 were apparently specific for H37Ra ( Figure 15C and " 15D and Table 9) and twelve strain specific proteins, numbered 211-222, were associated with the CFP of the Erdman strain ( Figures 15E and 15F and Table 9). However, of the proteins apparently limited to Erdman, only 212, 210, 220, 221 and 222 were exclusive. The other seven were also present in H37Rv but at quantities below the preset software values for peak height and area detection levels. Several proteins were associated with two of the three type strains. Proteins numbered 8, 39, 62, 71, 108, 121, 138, 191, 197, 199, 202 and 204 were specific for H37Rv and
- Protein 151 and 210 were present in the culture filtrate of Mt H37Rv and Erdman, and H37Ra and Erdman, respectively.
- Protein 151 identified by its reactivity with mAb IT-45 and its absence from H37Ra secreted proteins, was confirmed by 2-D Western blot analysis In sum, proteins present only in one or two of the type strains were relatively minor components of the culture filtrates ( Figures 15 A-F). Their appearance and the resultant 2-D profile differences could have been caused by disparate growth rates or cellular autolysis during culture. The lack of detectable 65 kDa GroEL homologue, a marker for autolysis, in the present preparations discounted the possibility of autolysis.
- Table 9 Summary of protein spots detected by computer aided analysis of silver nitrate stained 2-D gels.
- Table 9 Summary of protein spots detected by computei * aided analysis of silver nitrate stained 2-D gels.
- Table 9 Summary of protein spots detected by computer aided analysis of silver nitrate stained 2-D gels.
- Table 9 Summary of protein spots detected by computer aided analysis of silver nitrate stained 2-D gels.
- Table 9 Summary of protein spots detected by computer aided analysis of silver nitrate stained 2-D gels.
- Table 9 Summary of protein spots detected by computer aided analysis of silver nitrate stained 2-D gels.
- Table 9 Summary of protein spots detected by computer aided analysis of silver nitrate stained 2-D gels.
- Table 9 Summary of protein spots detected by computer aided analysis of silver nitrate stained 2-D gels.
- Table 9 Summary of protein spots detected by computer aided analysis of silver nitrate stained 2-D gels.
- N-terminal sequences obtained by present inventors are in italics.
- the CFPs are well defined in terms of function, immunogenicity and composition.
- a detailed analysis of the total proteins, and the molecular definition and 2-D PAGE mapping of the majority of these CFPs has not been performed.
- Nagai and colleagues identified and mapped by 2-D PAGE the most abundant proteins filtrate harvested after five weeks of culture in Sauton medium.
- the present study used culture filtrates from mid- to late-logarithmic cultures of three Mt type strains H37Ra, H37Rv, and Erdman to provide for the first time a detailed analysis understanding of this widely studied fraction.
- a second product sequenced was a 25 kDa protein with a pi of 5.34. Its N- terminal sequence (XPVM/LVXPGXEXXQDN, [SEQ ID NO: 100]) showed homology to an internal fragment (DPVLVFPGMEIRQDN, [SEQ ID NO: 105]) corresponding to open reading frame 28c of the Mt cosmid MTCYl Al 1. Analysis of that deduced sequence revealed a signal peptidase I consensus sequence (Ala-Xaa- Ala) and an apparent signal peptide preceding the N-terminus of the 25 kDa protein sequenced above
- N-terminal sequencing of selected CFPs identified three novel products: (1) protein with 72% identity to the N-terminus of a 42 kDa ⁇ -hydroxysteroid dehydrogenase of Eubacterium sp. VPI 12708;
- this antigen is referred to as the 88 kDa protein.
- the mAb IT-57 recognized an 88 kDa band in the LFCFP (Fig. 21, lane 2), and in the lysate of E. coli lysogen of ⁇ gtl 1 (IT-57) (lane 3). No proteins in the lysate from the E. coli 1089 lysogenized with the wild type ⁇ gtl 1 reacted with the mAb
- FIG. 22a is an autoradiograph showing that the 3.2 kb insert from ⁇ gtl 1 (IT-57) hybridized with itself (lane 3), and with both the uncut pMD31 vector containing the katG gene (lane 4) and the katG insert DNA itself (2.9 kb, lane 5). Therefore, the 88 kDa antigen reactive with mAb IT-57 is in fact the catalase/peroxidase enzyme.
- the / ⁇ tG-negative BCG strain 35747 transformed with either the pMD31 :M. tuberculosis katG or with the control pMD31 plasmid (vector control) were tested.
- tuberculosis katG and the katG-negative BCG containing pMD31 were separated by SDS-PAGE polyacrylamide on 10% gels (Figure 23).
- the fractionated proteins were transferred to nitrocellulose filters and probed with an anti- catalase/peroxidase polyclonal serum (obtained from Dr. Clifton Barry, Rocky Mountain Laboratories, NIAID, Hamilton, MT) (Fig.23A), mAb IT-57 (Fig. 23B), mAb IT-42 (Fig. 23C) and serum from an advanced TB patient (Fig. 23D).
- the anti- catalase/peroxidase polyclonal serum and the mAb IT-57 reacted strongly with an 88 kDa antigen in the LFCFP, in the M. tuberculosis katG containing M. bovis BCG and in E. coli ⁇ gtl 1 (IT-57).
- MAb IT-42 reacted with the same bands in the LFCFP and the M. tuberculosis katG BCG, but not with the 88 kDa protein expressed in E. coli.
- the serum from the tuberculosis patient recognized an 88 kDa antigen in the lysates of the katG-negative BCG strain. This is evidence that the seroreactive 88 kDa antigen is a novel protein which has not been previously described.
- M. tuberculosis also contains a seroreactive 88 kDa antigen which is not the catalase/peroxidase, a / ⁇ tG-negative strain of M. tuberculosis
- the goal of this study was to determine the repertoire of antigens recognized by antibodies in TB patients in order to elucidate the human humoral response to Mt and to evaluate the potential of these antigens as candidates for serodiagnosis. This was accomplished by immunoblotting Mt H37Rv secreted antigens, which had been separated by 1 - and 2- dimensional electrophoresis, with sera (E. c ⁇ //-absorbed) from TB patients and healthy controls. Of the more than 200 secreted proteins of Mt, only 26 elicited antibodies in
- Serum samples from 33 HIV-negative individuals with confirmed pulmonary TB were included in the study. Twenty of these sera were provided by
- Example I Culture filtrates from log phase Mt H 37 Rv were used as the source of secreted antigens as described in Example I (LAM-free culture filtrate proteins or CFPs).
- the LFCFP preparation contained over 200 proteins (Example V, supra).
- Antigens were size fractionated by loading onto a preparative polyacrylamide tube gel, and proteins were separated by electrophoresis using an increasing wattage gradient (model 491 Prep Cell; Bio-Rad, Hercules, CA.). Fractions were collected, assayed by SDS-PAGE and pooled according to molecular weights. Contaminating SDS was removed as described above. Reactivity of each fraction with human sera and an extensive panel of murine mAbs to Mt antigens are described in Example I.
- Fractions containing the 38 (or 35) kDa PstS and the seroreactive 88 kDa antigen were identified by reaction with anti-38 kDa mAb IT-23 and mAbs IT-57 and IT-42, respectively.
- Immunoadsorption of sera against E. coli lysates was performed as described in Example I. All ELISA assays, described in Example I, were performed using sera previously immunoadsorbed on E. coli lysates.
- the 1-D and 2-D blots were blocked with 3% BSA in phosphate buffered saline (PBS) for 2 hrs, and washed for 1 hr with PBS/Tween 2% (wash buffer).
- Individual lanes containing fractionated LFCFPs were exposed overnight at 4°C to individual sera (diluted 1 :100 with 1% BSA in PBS).
- the blots containing the 2-D fractionated LFCFPs were probed with four different serum pools comprised of individual sera whose reactivity with the above antigen preparations were previously determined by ELISA.
- Sera were grouped according to reactivity by ELISA with total LFCFPs, or the sized fraction containing the 38 kDa PstS or the 88 kDa seroreactive protein (Table 10).
- Group I includes sera from 16 PPD + and 7 PPD neg healthy controls, none of whom were positive in ELISA with any of these antigen preparations.
- Group II includes 9 TB patients who tested antibody negative with all three antigen preparations; five of these patients were smear-positive and had cavitary disease. The remaining four patients lacked cavitary lesions, but two of these four were smear- positive.
- Group III includes thirteen patients with antibodies to both the LFCFPs and the fraction containing the 88 kDa antigen, but not the fraction containing the 38 kDa antigen. Five of these patients were smear-positive and had pulmonary cavitations. An additional four were smear-positive but lacked any cavitary lesions. The remaining four were smear negative and had no cavitations. Group IV included eleven patients, all of whom had antibodies to all three antigen preparations; 10/11 were smear-positive and all had radiological evidence of moderate to advanced cavitary disease.
- n number of individuals in each group
- the 30-32 and 65 kDa antigens were also recognized by sera of the 9 PPD + healthy controls (lanes 8-16), though only 3/9 sera in this group recognized the 26 kDa antigen (lanes 8, 13 and 14), and one serum sample recognized an additional 68 kDa antigen (lane 12).
- Lanes 17-24 show the reactivity of group II tuberculous sera, which were antibody negative with all 3 antigen preparations by ELISA. Despite some variability among individual tuberculous sera, all reacted with the 30-32 kDa and 65 kDa antigens, and 5/8 (lanes 19, 21-24) contained antibodies to the 26 kDa antigen that was also recognized by the controls. Serum from one patient (lane 21) showed strong reactivity with 46, 55 and 97 kDa antigens. Four sera, including the latter patient, showed faint reactivity with antigens of 74, 76, 88, 105 and 112 kDa antigens, and with some antigens between 46-55 kDa. Sera from patients with cavitary disease (lanes 19, 22-24) and sera from patients with no cavitations (lanes 17, 18, 20 and 21) showed no significant difference in reactivity.
- the other two antigens reactive with all serum groups had molecular weights of 55 kDa (#114, 120) and 58 kDa (#86, 96, 105) and failed to react with any murine mAb.
- the former antigen has been identified as the glutamine synthetase by N-group analysis (Example V, above). These antigens may correspond to the 65 kDa antigen that was reactive with the individual sera on 1-D blots.
- a 26 kDa antigen (#19, 29) and a 46 kDa (#51) were reactive with the control sera (group I) and antibody positive TB sera (group III, Figure 18C and group IV, Figure 18D), but failed to react with the antibody negative TB serum pool (group II, Figure 18B).
- the former antigen (26 kDa, #19, 29) was identified as MPT64 based on reactivity with the murine mAb IT- 67 and may be the 26 kDa antigen recognized by several control sera on the 1-D blots ( Figure 17, lanes 2-7, 8, 13 and 14).
- This serum pool was weakly reactive with the four antigens (29, 31, 55, and 58 kDa) to which the control group (group I) reacted, but failed to show any reactivity with the 25/26 (#19, 29) and 46 kDa (#51) antigens.
- antigens correspond to the multiple bands in the 30 to 60 kDa region on the 1-D blots.
- a 85 kDa protein (#113, 124, IT-42, IT-57) was reactive with this serum pool, but no antigens corresponding to the 74 and 76 kDa antigens seen on 1-D blots were discernible on the 2-D blot.
- the 85 kDa antigen (#113, 124) on the 2-D immunoblots corresponds to the 88 kDa antigen (Example I) and as seen in Figure 17 and in Example V, above).
- the serum pool from group IV TB patients recognized 11 of 12 antigens that were reactive with the group III serum pool (except the 28 kDa antigen, #77; Table 1 IB).
- the reactivity of the group IV serum pool however, with the 26 kDa (#170, MPT51), 31 kDa (#119, Ag 85C), 35 kDa (#66, PstS), 38/42 (#11, 14, MPT32), 49 kDa (#82; IT-58), 85 kDa (#113, 124) and the 104 kDa (#111) antigens, was stronger than with the group III serum pool.
- the group IV pool was reactive with all four isomers recognized by murine mAb IT-23 ( Figure 18D).
- Table 1 IB the group III and IV serum pools
- Table 11C and Figure 18D The antigen with a molecular weight below 30 kDa was the 13/14 kDa protein (#23, 38, IT- 12 and SA12, GroES).
- this serum pool recognized four new antigens, with the same 31 kDa molecular weight but differing in their pi values: 31 kDa (#15, 16, 22, 25), 31 kDa (#62), 31 kDa (#57) and 31 kDa (#37), and a fifth antigen of 38 kDa (#32). Of these only the 31 kDa (#15, 16, 22, 25) was reactive with the mAb IT-44, while the remaining 4 antigens have not been previously described.
- this pool reacted with a 66/72 kDa protein (#65, 79, mAb IT-40 and IT-41, DnaK), and an unidentified 79 kDa antigen (#78).
- 6 were reactive with the control sera (Table 11 A). Twelve of these 26 antigens are recognized by sera from groups III and IV (Table 1 IB). Thus, patients both with early, non cavitary TB and advanced cavitary TB have antibodies to these antigens. Of these 12 antigens, 5 are strongly recognized and consequently, are preferred antigens for a serodiagnostic assay for early TB as described herein.
- NR Not reactive
- the 2-D analysis and mapping of each antigen as described herein has allowed precise definition of antigens that appear to be critical for rational design of serodiagnosis and at least 5 secreted proteins as useful serodiagnostic agents.
- Antibodies to one of these, the 85 (or 88) kDa antigen are present in 80% of the advanced and 50% of the early TB.
- the 38/42 kDa antigen (#11, 14, MPT32) has also been suggested to have serodiagnostic potential (Espitia et al, 1995, supra) but not as an "early" antigen.
- the serodiagnostic potential of Ags 85 A (#149) and B (#81) has been evaluated by Van Vooren et al. (supra) by isoelectric focusing separation and immunoblot analysis.
- the 85A component was shown to be reactive with the TB as well as non-TB sera, whereas, 71% of the TB sera in their cohort recognized either Ag 85 B or C.
- no information on reactivity in early vs. advanced disease was provided.
- the present invention constitutes a major step in that direction and provides a basis for the identification and detection of such epitopes.
- MPT64 26 kDa, #19, 29
- group III TB sera lacking anti-38 kDa antibodies
- the early antigens identified herein may not be the only early antigens secreted during Mt growth in vivo. These antigens may be the only ones that are distinguishable because of their strongly seroreactive antigenic determinants.
- Several antigens of Mt were either up- or down-regulated when the organisms were grown intracellularly in macrophages. The present inventors propose that, in vivo, Mt organisms produce only those proteins required for survival and growth under these particular conditions which may different from the requirements during growth in culture media. It is noteworthy that several of the antigens that elicit antibodies relatively early in TB (based on reactivity with group III sera), are implicated as having a role in pathogenesis in vivo.
- Ag 85A, Ag 85C and MPT51 all belong to the family of secreted proteins which bind to fibronectin (Wiker et al, 1992, Scand. J. Immunol, supra)).
- MPT32 is homologous to a fibronectin-binding protein of M. leprae (Schorey, J.S. et al, 1995, Infect. Immun. 63:2652-2651).
- the protein bound by IT-42 has been identified as the KatG catalase- peroxidase enzyme (Example V, above). The identities of the adjacent protein reactive with IT-57 (#113) and with IT-58 (#82, 49kDa) have not been determined.
- seroreactive antigens which are useful for diagnostic assays for TB patients who are relatively early in disease progression.
- species-specific epitopes should now be defined for serodiagnostic uses.
- fraction 10 is enriched for a 38kDa antigen
- fraction 13 is enriched for MPT32
- fraction 15 is enriched for the 88 kDa antigen (also referred to as an 85 kDa antigen based on slight differences in PAGE migration between two laboratories of two of the present inventors). This 88 kDa antigen is described for the first time in the present disclosure.
- any single antigen on the 2-D blots with pooled sera may represent reactivity with only some of the individual sera comprising the pool.
- group III serum pool were assessed for antibodies to two of the antigens identified by the group III serum pool, Ag 85C and MPT32 which the present inventors had purified. Reactivities with the purified 38 kDa PstS antigen and the 88 kDa antigen (fraction 15) was also tested. A larger cohort of TB patients than above, classified as cavitary or non-cavitary TB, was tested.
Abstract
Description
Claims
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US3400396P | 1996-12-31 | 1996-12-31 | |
US34003P | 1996-12-31 | ||
PCT/US1997/024189 WO1998029132A1 (en) | 1996-12-31 | 1997-12-29 | Early detection of mycobacterial disease |
Publications (2)
Publication Number | Publication Date |
---|---|
EP0952849A1 true EP0952849A1 (en) | 1999-11-03 |
EP0952849A4 EP0952849A4 (en) | 2004-09-08 |
Family
ID=21873719
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP97954653A Withdrawn EP0952849A4 (en) | 1996-12-31 | 1997-12-29 | Early detection of mycobacterial disease |
Country Status (4)
Country | Link |
---|---|
EP (1) | EP0952849A4 (en) |
AU (1) | AU746752B2 (en) |
CA (1) | CA2276491C (en) |
WO (1) | WO1998029132A1 (en) |
Families Citing this family (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6291190B1 (en) | 1998-08-25 | 2001-09-18 | The Board Of Trustees Of The Leland Stanford Junior University | Molecular differences between species of the M. tuberculosis complex |
AU5294000A (en) * | 1999-05-28 | 2000-12-18 | Promega Corporation | Antibodies specific for mycobacterial polypeptides and uses thereof |
FR2818647B1 (en) * | 2000-12-21 | 2006-09-29 | Pasteur Institut | IMMUNOGENIC GLYCOPEPTIDES, SCREENING, PREPARATION AND APPLICATIONS |
EP1360498B1 (en) * | 2001-01-08 | 2009-10-14 | The Government of the United States of America, as represented by the Secretary, Department of Health & Human Services | Latent human tuberculosis diagnostic antigen and method of use |
CN1694725A (en) * | 2001-08-02 | 2005-11-09 | 纽约大学 | Early detection of mycobacterial disease using peptides |
WO2003040722A1 (en) * | 2001-11-09 | 2003-05-15 | Kreatech Biotechnology B.V. | Means and methods for the detection of immunoglobulin capable of binding to mycobacterium antigen |
US7820142B2 (en) | 2004-09-30 | 2010-10-26 | Institut Pasteur | Immunogenic glycopeptides, screening, preparation and uses |
US9335325B2 (en) * | 2008-04-19 | 2016-05-10 | New York University | Immunodominant Mycobacterium tuberculosis peptides from cell wall proteins for early diagnosis and immunization |
DE102008029834A1 (en) * | 2008-06-25 | 2010-01-14 | Justus-Liebig-Universität Giessen | Method for the specific detection of MAP antibodies |
FI125928B (en) | 2015-02-27 | 2016-04-15 | Tamara Tuuminen | Procedure for Detecting Lipoarabine Man (LAM) |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1990004041A1 (en) * | 1988-10-04 | 1990-04-19 | Dna Plant Technology Corporation | Bacterial detection by phage transduction of detectable phenotype |
WO1992008809A1 (en) * | 1990-11-19 | 1992-05-29 | University Of Florida | Assay device and method for antibody and antigen detection |
US5254459A (en) * | 1990-08-23 | 1993-10-19 | Patarroyo Manuel E | Nucleotide and amino acid sequences of protein MTP40 of M. tuberculosis and synthetic peptides derived therefrom |
WO1997034149A1 (en) * | 1996-03-12 | 1997-09-18 | Stefan Svenson | Method of diagnosing a mycobacterial disease and immunoassay kit |
-
1997
- 1997-12-29 AU AU59051/98A patent/AU746752B2/en not_active Ceased
- 1997-12-29 CA CA002276491A patent/CA2276491C/en not_active Expired - Fee Related
- 1997-12-29 EP EP97954653A patent/EP0952849A4/en not_active Withdrawn
- 1997-12-29 WO PCT/US1997/024189 patent/WO1998029132A1/en active IP Right Grant
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1990004041A1 (en) * | 1988-10-04 | 1990-04-19 | Dna Plant Technology Corporation | Bacterial detection by phage transduction of detectable phenotype |
US5254459A (en) * | 1990-08-23 | 1993-10-19 | Patarroyo Manuel E | Nucleotide and amino acid sequences of protein MTP40 of M. tuberculosis and synthetic peptides derived therefrom |
WO1992008809A1 (en) * | 1990-11-19 | 1992-05-29 | University Of Florida | Assay device and method for antibody and antigen detection |
WO1997034149A1 (en) * | 1996-03-12 | 1997-09-18 | Stefan Svenson | Method of diagnosing a mycobacterial disease and immunoassay kit |
Non-Patent Citations (1)
Title |
---|
See also references of WO9829132A1 * |
Also Published As
Publication number | Publication date |
---|---|
AU5905198A (en) | 1998-07-31 |
CA2276491C (en) | 2008-01-22 |
WO1998029132A1 (en) | 1998-07-09 |
AU746752B2 (en) | 2002-05-02 |
EP0952849A4 (en) | 2004-09-08 |
CA2276491A1 (en) | 1998-07-09 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US6506384B1 (en) | Early detection of mycobacterial disease | |
US6245331B1 (en) | Early detection of mycobacterial disease | |
US7776341B2 (en) | Biomarkers of tuberculosis that distinguish disease categories: use as serodiagnostic antigens | |
DK2417456T3 (en) | DIAGNOSTIC TEST Mycobacterium tuberculosis | |
Roche et al. | T-cell determinants and antibody binding sites on the major mycobacterial secretory protein MPB59 of Mycobacterium bovis | |
MXPA01004469A (en) | Tuberculosis diagnostic test. | |
Mutharia et al. | Analysis of culture filtrate and cell wall-associated antigens of Mycobacterium paratuberculosis with monoclonal antibodies | |
US7745141B2 (en) | Mycobacterial proteins as early antigens for serodiagnosis and vaccines | |
AU746752B2 (en) | Early detection of mycobacterial disease | |
De Kesel et al. | Composition and immunological properties of the protein fraction of A36, a major antigen complex of Mycobacterium paratuberculosis | |
AU2002324578B2 (en) | Early detection of mycobacterial disease using peptides | |
WO1998029132A9 (en) | Early detection of mycobacterial disease | |
AU2002324578A1 (en) | Early detection of mycobacterial disease using peptides | |
US9335325B2 (en) | Immunodominant Mycobacterium tuberculosis peptides from cell wall proteins for early diagnosis and immunization | |
US7807182B2 (en) | Early detection of mycobacterial disease using peptides | |
Le Moigne et al. | Expression, immunochemical characterization and localization of the Mycobacterium tuberculosis protein p27 | |
JP6698530B2 (en) | Diagnostic reagents for improving in vivo or in vitro cell-mediated immunological diagnosis of tuberculosis | |
US8470339B2 (en) | Antigens for paratuberculosis diagnosis and vaccination | |
Falla et al. | Identification of B-and T-cell epitopes within the MTP40 protein of Mycobacterium tuberculosis and their correlation with the disease course | |
Alsahag | Isolation, Purification and Identification of CFP29 from Mycobacterium tuberculosis H37Rv culture filtrate proteins | |
Belisle et al. | The proteome of Mycobacterium tuberculosis | |
Gilot et al. | Thermostable macromolecular antigens from mycobacteria | |
Sartain | Evaluation and characterization of tuberculosis serodiagnostic biomarkers | |
McGill | Characterization of humoral immune responses against Treponema pallidum antigens | |
EA041840B1 (en) | DIAGNOSTIC REAGENTS FOR IMPROVED IN VIVO OR IN VITRO CELL-MEDIATED IMMUNOLOGICAL DIAGNOSIS OF TUBERCULOSIS |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
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 |
|
17P | Request for examination filed |
Effective date: 19990709 |
|
AK | Designated contracting states |
Kind code of ref document: A1 Designated state(s): DE FR GB IE IT |
|
RAP1 | Party data changed (applicant data changed or rights of an application transferred) |
Owner name: COLORADO STATE UNIVERSITY RESEARCH FOUNDATION |
|
RAP1 | Party data changed (applicant data changed or rights of an application transferred) |
Owner name: COLORADO STATE UNIVERSITY RESEARCH FOUNDATION Owner name: NEW YORK UNIVERSITY |
|
A4 | Supplementary search report drawn up and despatched |
Effective date: 20040726 |
|
RIC1 | Information provided on ipc code assigned before grant |
Ipc: 7G 01N 33/569 B Ipc: 7G 01N 33/554 B Ipc: 7G 01N 33/53 B Ipc: 7C 12Q 1/00 B Ipc: 7A 61K 39/40 B Ipc: 7A 61K 39/04 A |
|
17Q | First examination report despatched |
Effective date: 20050118 |
|
RIN1 | Information on inventor provided before grant (corrected) |
Inventor name: BELISLE, JOHN, T. Inventor name: ZOLLA-PAZNER, SUSAN Inventor name: LAAL, SUMAN |
|
RIN1 | Information on inventor provided before grant (corrected) |
Inventor name: BELISLE, JOHN, T. Inventor name: ZOLLA-PAZNER, SUSAN Inventor name: LAAL, SUMAN |
|
RIC1 | Information provided on ipc code assigned before grant |
Ipc: G01N 33/569 20060101ALI20090107BHEP Ipc: A61K 39/40 20060101ALI20090107BHEP Ipc: A61K 39/04 20060101AFI20090107BHEP |
|
GRAP | Despatch of communication of intention to grant a patent |
Free format text: ORIGINAL CODE: EPIDOSNIGR1 |
|
RIN1 | Information on inventor provided before grant (corrected) |
Inventor name: BELISLE, JOHN, T. Inventor name: ZOLLA-PAZNER, SUSAN Inventor name: LAAL, SUMAN |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN |
|
18D | Application deemed to be withdrawn |
Effective date: 20090701 |