WO1999021580A1 - Reagents and immunoassay for the detection and quantitative determination of mycothiol and precursors thereof - Google Patents
Reagents and immunoassay for the detection and quantitative determination of mycothiol and precursors thereof Download PDFInfo
- Publication number
- WO1999021580A1 WO1999021580A1 PCT/US1998/022577 US9822577W WO9921580A1 WO 1999021580 A1 WO1999021580 A1 WO 1999021580A1 US 9822577 W US9822577 W US 9822577W WO 9921580 A1 WO9921580 A1 WO 9921580A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- mycothiol
- precursor
- antibody
- sample
- reagent
- Prior art date
Links
- MQBCDKMPXVYCGO-UHFFFAOYSA-N mycothiol Natural products CC(=O)NC(CS)C(=O)NC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(O)C1O MQBCDKMPXVYCGO-UHFFFAOYSA-N 0.000 title claims abstract description 365
- MQBCDKMPXVYCGO-FQBKTPCVSA-N mycothiol Chemical compound CC(=O)N[C@@H](CS)C(=O)N[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@H](O)[C@@H](O)[C@H](O)[C@H]1O MQBCDKMPXVYCGO-FQBKTPCVSA-N 0.000 title claims abstract description 361
- 108010074581 mycothiol Proteins 0.000 title claims abstract description 231
- 239000002243 precursor Substances 0.000 title claims abstract description 125
- 239000003153 chemical reaction reagent Substances 0.000 title claims description 123
- 238000001514 detection method Methods 0.000 title claims description 57
- 238000003018 immunoassay Methods 0.000 title description 53
- 238000000034 method Methods 0.000 claims abstract description 178
- 241000186361 Actinobacteria <class> Species 0.000 claims abstract description 52
- 238000004519 manufacturing process Methods 0.000 claims abstract description 28
- 230000001580 bacterial effect Effects 0.000 claims abstract description 23
- 238000003745 diagnosis Methods 0.000 claims abstract description 12
- 239000000523 sample Substances 0.000 claims description 113
- 239000012528 membrane Substances 0.000 claims description 61
- 238000006243 chemical reaction Methods 0.000 claims description 43
- 241000894006 Bacteria Species 0.000 claims description 33
- 210000001175 cerebrospinal fluid Anatomy 0.000 claims description 27
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 25
- 238000002372 labelling Methods 0.000 claims description 23
- 238000012360 testing method Methods 0.000 claims description 19
- 150000001412 amines Chemical class 0.000 claims description 18
- 239000007795 chemical reaction product Substances 0.000 claims description 14
- 239000013068 control sample Substances 0.000 claims description 13
- 210000002700 urine Anatomy 0.000 claims description 13
- 108020004774 Alkaline Phosphatase Proteins 0.000 claims description 11
- 102000002260 Alkaline Phosphatase Human genes 0.000 claims description 11
- 210000002966 serum Anatomy 0.000 claims description 10
- 206010036790 Productive cough Diseases 0.000 claims description 9
- 208000024794 sputum Diseases 0.000 claims description 8
- 210000003802 sputum Anatomy 0.000 claims description 8
- 210000004369 blood Anatomy 0.000 claims description 7
- 239000008280 blood Substances 0.000 claims description 7
- 206010003445 Ascites Diseases 0.000 claims description 6
- 238000001574 biopsy Methods 0.000 claims description 6
- 230000002255 enzymatic effect Effects 0.000 claims description 6
- 210000004910 pleural fluid Anatomy 0.000 claims description 6
- 230000028327 secretion Effects 0.000 claims description 6
- 210000001519 tissue Anatomy 0.000 claims description 6
- 230000002550 fecal effect Effects 0.000 claims description 5
- 108010001336 Horseradish Peroxidase Proteins 0.000 claims description 4
- 230000000241 respiratory effect Effects 0.000 claims description 4
- 230000002934 lysing effect Effects 0.000 claims description 3
- 150000003573 thiols Chemical class 0.000 claims description 3
- MEYZIGGCNFHINA-UHFFFAOYSA-N (6-aminoquinolin-2-yl) n-(2,5-dioxopyrrolidin-1-yl)-n-hydroxycarbamate Chemical compound C1=CC2=CC(N)=CC=C2N=C1OC(=O)N(O)N1C(=O)CCC1=O MEYZIGGCNFHINA-UHFFFAOYSA-N 0.000 claims description 2
- 125000000415 L-cysteinyl group Chemical group O=C([*])[C@@](N([H])[H])([H])C([H])([H])S[H] 0.000 claims description 2
- 108060001084 Luciferase Proteins 0.000 claims description 2
- 239000005089 Luciferase Substances 0.000 claims description 2
- 230000004075 alteration Effects 0.000 claims description 2
- 230000003247 decreasing effect Effects 0.000 claims description 2
- 241000186046 Actinomyces Species 0.000 claims 1
- 102000008857 Ferritin Human genes 0.000 claims 1
- 238000008416 Ferritin Methods 0.000 claims 1
- 108050000784 Ferritin Proteins 0.000 claims 1
- 108010046334 Urease Proteins 0.000 claims 1
- 229910001385 heavy metal Inorganic materials 0.000 claims 1
- 230000002285 radioactive effect Effects 0.000 claims 1
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 136
- 102100027467 Pro-opiomelanocortin Human genes 0.000 description 134
- 210000004027 cell Anatomy 0.000 description 115
- 239000003656 tris buffered saline Substances 0.000 description 63
- 125000003396 thiol group Chemical class [H]S* 0.000 description 53
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 47
- 238000004458 analytical method Methods 0.000 description 41
- 238000004128 high performance liquid chromatography Methods 0.000 description 40
- 238000002965 ELISA Methods 0.000 description 39
- 239000000243 solution Substances 0.000 description 38
- 239000000427 antigen Substances 0.000 description 36
- 102000036639 antigens Human genes 0.000 description 36
- 108091007433 antigens Proteins 0.000 description 36
- 241000187480 Mycobacterium smegmatis Species 0.000 description 35
- 238000003556 assay Methods 0.000 description 35
- 239000006180 TBST buffer Substances 0.000 description 34
- 239000000284 extract Substances 0.000 description 32
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 31
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 31
- 108090001008 Avidin Proteins 0.000 description 30
- 239000012634 fragment Substances 0.000 description 29
- 108090000623 proteins and genes Proteins 0.000 description 28
- PWKSKIMOESPYIA-BYPYZUCNSA-N L-N-acetyl-Cysteine Chemical compound CC(=O)N[C@@H](CS)C(O)=O PWKSKIMOESPYIA-BYPYZUCNSA-N 0.000 description 27
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 26
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 25
- 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 24
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 24
- 230000027455 binding Effects 0.000 description 24
- 229940098773 bovine serum albumin Drugs 0.000 description 24
- 239000008103 glucose Substances 0.000 description 24
- 229960001031 glucose Drugs 0.000 description 24
- 235000018102 proteins Nutrition 0.000 description 24
- 102000004169 proteins and genes Human genes 0.000 description 24
- 238000002474 experimental method Methods 0.000 description 23
- 239000006228 supernatant Substances 0.000 description 22
- 201000008827 tuberculosis Diseases 0.000 description 22
- 229940027941 immunoglobulin g Drugs 0.000 description 21
- 238000011534 incubation Methods 0.000 description 21
- 239000002609 medium Substances 0.000 description 21
- 239000000203 mixture Substances 0.000 description 21
- 239000012071 phase Substances 0.000 description 20
- 238000007792 addition Methods 0.000 description 19
- 239000000872 buffer Substances 0.000 description 19
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 18
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 18
- MSWZFWKMSRAUBD-UHFFFAOYSA-N beta-D-galactosamine Natural products NC1C(O)OC(CO)C(O)C1O MSWZFWKMSRAUBD-UHFFFAOYSA-N 0.000 description 18
- 238000011161 development Methods 0.000 description 17
- 230000018109 developmental process Effects 0.000 description 17
- 108090000765 processed proteins & peptides Proteins 0.000 description 17
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 description 17
- MSWZFWKMSRAUBD-IVMDWMLBSA-N 2-amino-2-deoxy-D-glucopyranose Chemical compound N[C@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O MSWZFWKMSRAUBD-IVMDWMLBSA-N 0.000 description 16
- FZHXIRIBWMQPQF-UHFFFAOYSA-N Glc-NH2 Natural products O=CC(N)C(O)C(O)C(O)CO FZHXIRIBWMQPQF-UHFFFAOYSA-N 0.000 description 16
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 16
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 16
- 108010045069 keyhole-limpet hemocyanin Proteins 0.000 description 16
- 150000001875 compounds Chemical class 0.000 description 15
- 230000012010 growth Effects 0.000 description 15
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 description 15
- -1 or other enzymatic Substances 0.000 description 15
- 238000002360 preparation method Methods 0.000 description 15
- XZKIHKMTEMTJQX-UHFFFAOYSA-N 4-Nitrophenyl Phosphate Chemical compound OP(O)(=O)OC1=CC=C([N+]([O-])=O)C=C1 XZKIHKMTEMTJQX-UHFFFAOYSA-N 0.000 description 14
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 14
- 230000001413 cellular effect Effects 0.000 description 14
- PEEHTFAAVSWFBL-UHFFFAOYSA-N Maleimide Chemical compound O=C1NC(=O)C=C1 PEEHTFAAVSWFBL-UHFFFAOYSA-N 0.000 description 13
- 241000283973 Oryctolagus cuniculus Species 0.000 description 13
- 229920001213 Polysorbate 20 Polymers 0.000 description 13
- 239000011230 binding agent Substances 0.000 description 13
- 230000006287 biotinylation Effects 0.000 description 13
- 238000007413 biotinylation Methods 0.000 description 13
- 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 13
- 239000002953 phosphate buffered saline Substances 0.000 description 13
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 13
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 13
- 239000000126 substance Substances 0.000 description 13
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 12
- 239000012148 binding buffer Substances 0.000 description 12
- 238000005119 centrifugation Methods 0.000 description 12
- 239000008188 pellet Substances 0.000 description 12
- 238000011282 treatment Methods 0.000 description 12
- 241000186367 Mycobacterium avium Species 0.000 description 11
- 239000006285 cell suspension Substances 0.000 description 11
- 201000010099 disease Diseases 0.000 description 11
- 239000000543 intermediate Substances 0.000 description 11
- AHEWZZJEDQVLOP-UHFFFAOYSA-N monobromobimane Chemical compound BrCC1=C(C)C(=O)N2N1C(C)=C(C)C2=O AHEWZZJEDQVLOP-UHFFFAOYSA-N 0.000 description 11
- 239000008363 phosphate buffer Substances 0.000 description 11
- 238000000926 separation method Methods 0.000 description 11
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 10
- AFVFQIVMOAPDHO-UHFFFAOYSA-N Methanesulfonic acid Chemical compound CS(O)(=O)=O AFVFQIVMOAPDHO-UHFFFAOYSA-N 0.000 description 10
- 229920002684 Sepharose Polymers 0.000 description 10
- 230000008859 change Effects 0.000 description 10
- 230000000694 effects Effects 0.000 description 10
- 230000005484 gravity Effects 0.000 description 10
- 239000000463 material Substances 0.000 description 10
- 102000004196 processed proteins & peptides Human genes 0.000 description 10
- 230000035945 sensitivity Effects 0.000 description 10
- 239000007787 solid Substances 0.000 description 10
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 10
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 10
- 102000004190 Enzymes Human genes 0.000 description 9
- 108090000790 Enzymes Proteins 0.000 description 9
- 108010010803 Gelatin Proteins 0.000 description 9
- 239000007983 Tris buffer Substances 0.000 description 9
- 229940088598 enzyme Drugs 0.000 description 9
- 239000008273 gelatin Substances 0.000 description 9
- 229920000159 gelatin Polymers 0.000 description 9
- 235000019322 gelatine Nutrition 0.000 description 9
- 235000011852 gelatine desserts Nutrition 0.000 description 9
- 230000004048 modification Effects 0.000 description 9
- 238000012986 modification Methods 0.000 description 9
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 9
- 229920001184 polypeptide Polymers 0.000 description 9
- 229920000053 polysorbate 80 Polymers 0.000 description 9
- 239000012089 stop solution Substances 0.000 description 9
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 8
- DIYPCWKHSODVAP-UHFFFAOYSA-N 1-[3-(2,5-dioxopyrrol-1-yl)benzoyl]oxy-2,5-dioxopyrrolidine-3-sulfonic acid Chemical compound O=C1C(S(=O)(=O)O)CC(=O)N1OC(=O)C1=CC=CC(N2C(C=CC2=O)=O)=C1 DIYPCWKHSODVAP-UHFFFAOYSA-N 0.000 description 8
- 241000283707 Capra Species 0.000 description 8
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 8
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 8
- 229940024606 amino acid Drugs 0.000 description 8
- 235000001014 amino acid Nutrition 0.000 description 8
- 150000001413 amino acids Chemical class 0.000 description 8
- 239000008346 aqueous phase Substances 0.000 description 8
- 230000008901 benefit Effects 0.000 description 8
- 238000001212 derivatisation Methods 0.000 description 8
- 229960003180 glutathione Drugs 0.000 description 8
- 230000003053 immunization Effects 0.000 description 8
- 108010087904 neutravidin Proteins 0.000 description 8
- 239000007790 solid phase Substances 0.000 description 8
- 239000003643 water by type Substances 0.000 description 8
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 7
- KIUMMUBSPKGMOY-UHFFFAOYSA-N 3,3'-Dithiobis(6-nitrobenzoic acid) Chemical compound C1=C([N+]([O-])=O)C(C(=O)O)=CC(SSC=2C=C(C(=CC=2)[N+]([O-])=O)C(O)=O)=C1 KIUMMUBSPKGMOY-UHFFFAOYSA-N 0.000 description 7
- 102000014914 Carrier Proteins Human genes 0.000 description 7
- 108010078791 Carrier Proteins Proteins 0.000 description 7
- 239000007995 HEPES buffer Substances 0.000 description 7
- 238000002649 immunization Methods 0.000 description 7
- 230000000670 limiting effect Effects 0.000 description 7
- 239000003960 organic solvent Substances 0.000 description 7
- 239000008194 pharmaceutical composition Substances 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 230000002829 reductive effect Effects 0.000 description 7
- 239000002904 solvent Substances 0.000 description 7
- 239000000758 substrate Substances 0.000 description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 6
- 241000588724 Escherichia coli Species 0.000 description 6
- 241000187722 Micromonospora echinospora Species 0.000 description 6
- 239000000020 Nitrocellulose Substances 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 6
- 229960002685 biotin Drugs 0.000 description 6
- 239000011616 biotin Substances 0.000 description 6
- 230000000903 blocking effect Effects 0.000 description 6
- 230000001419 dependent effect Effects 0.000 description 6
- 230000005284 excitation Effects 0.000 description 6
- 210000004408 hybridoma Anatomy 0.000 description 6
- 238000000338 in vitro Methods 0.000 description 6
- 208000015181 infectious disease Diseases 0.000 description 6
- 229960000367 inositol Drugs 0.000 description 6
- 238000005259 measurement Methods 0.000 description 6
- 229920001220 nitrocellulos Polymers 0.000 description 6
- KWNGAZCDAJSVLC-OSAWLIQMSA-N 3-(n-maleimidopropionyl)biocytin Chemical compound N([C@@H](CCCCNC(=O)CCCC[C@H]1[C@H]2NC(=O)N[C@H]2CS1)C(=O)O)C(=O)CCN1C(=O)C=CC1=O KWNGAZCDAJSVLC-OSAWLIQMSA-N 0.000 description 5
- 101000588395 Bacillus subtilis (strain 168) Beta-hexosaminidase Proteins 0.000 description 5
- 108010047041 Complementarity Determining Regions Proteins 0.000 description 5
- 108060003951 Immunoglobulin Proteins 0.000 description 5
- 241000699670 Mus sp. Species 0.000 description 5
- 206010062207 Mycobacterial infection Diseases 0.000 description 5
- 102000057297 Pepsin A Human genes 0.000 description 5
- 108090000284 Pepsin A Proteins 0.000 description 5
- 238000002835 absorbance Methods 0.000 description 5
- 238000001042 affinity chromatography Methods 0.000 description 5
- 239000013592 cell lysate Substances 0.000 description 5
- 238000007429 general method Methods 0.000 description 5
- 239000011521 glass Substances 0.000 description 5
- 150000002337 glycosamines Chemical group 0.000 description 5
- 102000018358 immunoglobulin Human genes 0.000 description 5
- 238000001727 in vivo Methods 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 229910001629 magnesium chloride Inorganic materials 0.000 description 5
- 229940098779 methanesulfonic acid Drugs 0.000 description 5
- 239000003068 molecular probe Substances 0.000 description 5
- 208000027531 mycobacterial infectious disease Diseases 0.000 description 5
- ZVEZMVFBMOOHAT-UHFFFAOYSA-N nonane-1-thiol Chemical compound CCCCCCCCCS ZVEZMVFBMOOHAT-UHFFFAOYSA-N 0.000 description 5
- 238000012216 screening Methods 0.000 description 5
- 239000011550 stock solution Substances 0.000 description 5
- 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 4
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 4
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical class CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 4
- 230000005526 G1 to G0 transition Effects 0.000 description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 4
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 4
- 108010058846 Ovalbumin Proteins 0.000 description 4
- 239000002033 PVDF binder Substances 0.000 description 4
- 239000012472 biological sample Substances 0.000 description 4
- 235000020958 biotin Nutrition 0.000 description 4
- 229940041514 candida albicans extract Drugs 0.000 description 4
- 239000000969 carrier Substances 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 4
- 238000003776 cleavage reaction Methods 0.000 description 4
- 238000010367 cloning Methods 0.000 description 4
- 230000021615 conjugation Effects 0.000 description 4
- 239000003431 cross linking reagent Substances 0.000 description 4
- KAKKHKRHCKCAGH-UHFFFAOYSA-L disodium;(4-nitrophenyl) phosphate;hexahydrate Chemical compound O.O.O.O.O.O.[Na+].[Na+].[O-][N+](=O)C1=CC=C(OP([O-])([O-])=O)C=C1 KAKKHKRHCKCAGH-UHFFFAOYSA-L 0.000 description 4
- 238000007654 immersion Methods 0.000 description 4
- 230000014759 maintenance of location Effects 0.000 description 4
- VWKNUUOGGLNRNZ-UHFFFAOYSA-N methylbimane Chemical compound CC1=C(C)C(=O)N2N1C(C)=C(C)C2=O VWKNUUOGGLNRNZ-UHFFFAOYSA-N 0.000 description 4
- 230000009871 nonspecific binding Effects 0.000 description 4
- 229940092253 ovalbumin Drugs 0.000 description 4
- 229940111202 pepsin Drugs 0.000 description 4
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 230000007017 scission Effects 0.000 description 4
- 239000001488 sodium phosphate Substances 0.000 description 4
- 229910000162 sodium phosphate Inorganic materials 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- 238000004448 titration Methods 0.000 description 4
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 4
- 229910021642 ultra pure water Inorganic materials 0.000 description 4
- 239000012498 ultrapure water Substances 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- 239000012138 yeast extract Substances 0.000 description 4
- CBOJBBMQJBVCMW-BTVCFUMJSA-N (2r,3r,4s,5r)-2-amino-3,4,5,6-tetrahydroxyhexanal;hydrochloride Chemical compound Cl.O=C[C@H](N)[C@@H](O)[C@H](O)[C@H](O)CO CBOJBBMQJBVCMW-BTVCFUMJSA-N 0.000 description 3
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 3
- 101000688206 Bos taurus Intestinal-type alkaline phosphatase Proteins 0.000 description 3
- MWNLTKCQHFZFHN-UHFFFAOYSA-N CBQCA reagent Chemical compound C1=CC(C(=O)O)=CC=C1C(=O)C1=CC2=CC=CC=C2N=C1C=O MWNLTKCQHFZFHN-UHFFFAOYSA-N 0.000 description 3
- XPDXVDYUQZHFPV-UHFFFAOYSA-N Dansyl Chloride Chemical compound C1=CC=C2C(N(C)C)=CC=CC2=C1S(Cl)(=O)=O XPDXVDYUQZHFPV-UHFFFAOYSA-N 0.000 description 3
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 3
- 239000004593 Epoxy Substances 0.000 description 3
- 241000206602 Eukaryota Species 0.000 description 3
- 108010024636 Glutathione Proteins 0.000 description 3
- 238000010268 HPLC based assay Methods 0.000 description 3
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 description 3
- 241001529936 Murinae Species 0.000 description 3
- 241000699660 Mus musculus Species 0.000 description 3
- 241000186359 Mycobacterium Species 0.000 description 3
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 3
- 238000005481 NMR spectroscopy Methods 0.000 description 3
- ZNXZGRMVNNHPCA-UHFFFAOYSA-N Pantetheine Natural products OCC(C)(C)C(O)C(=O)NCCC(=O)NCCS ZNXZGRMVNNHPCA-UHFFFAOYSA-N 0.000 description 3
- DJWYOLJPSHDSAL-UHFFFAOYSA-N Pantethine Natural products OCC(C)(C)C(O)C(=O)NCCC(=O)NCCSSCCNC(=O)CCNC(=O)C(O)C(C)(C)CO DJWYOLJPSHDSAL-UHFFFAOYSA-N 0.000 description 3
- 229930006000 Sucrose Natural products 0.000 description 3
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 3
- 230000000890 antigenic effect Effects 0.000 description 3
- 229940090047 auto-injector Drugs 0.000 description 3
- 150000001793 charged compounds Chemical class 0.000 description 3
- 239000000356 contaminant Substances 0.000 description 3
- 238000007796 conventional method Methods 0.000 description 3
- 238000013480 data collection Methods 0.000 description 3
- 235000015872 dietary supplement Nutrition 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- DEFVIWRASFVYLL-UHFFFAOYSA-N ethylene glycol bis(2-aminoethyl)tetraacetic acid Chemical compound OC(=O)CN(CC(O)=O)CCOCCOCCN(CC(O)=O)CC(O)=O DEFVIWRASFVYLL-UHFFFAOYSA-N 0.000 description 3
- 239000012737 fresh medium Substances 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 230000007946 glucose deprivation Effects 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 229940127121 immunoconjugate Drugs 0.000 description 3
- 238000013383 initial experiment Methods 0.000 description 3
- 238000004255 ion exchange chromatography Methods 0.000 description 3
- 125000005439 maleimidyl group Chemical group C1(C=CC(N1*)=O)=O 0.000 description 3
- 230000004060 metabolic process Effects 0.000 description 3
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 3
- 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 3
- ZNXZGRMVNNHPCA-VIFPVBQESA-N pantetheine Chemical compound OCC(C)(C)[C@@H](O)C(=O)NCCC(=O)NCCS ZNXZGRMVNNHPCA-VIFPVBQESA-N 0.000 description 3
- DJWYOLJPSHDSAL-ROUUACIJSA-N pantethine Chemical compound OCC(C)(C)[C@@H](O)C(=O)NCCC(=O)NCCSSCCNC(=O)CCNC(=O)[C@H](O)C(C)(C)CO DJWYOLJPSHDSAL-ROUUACIJSA-N 0.000 description 3
- 229960000903 pantethine Drugs 0.000 description 3
- 235000008975 pantethine Nutrition 0.000 description 3
- 239000011581 pantethine Substances 0.000 description 3
- 239000013610 patient sample Substances 0.000 description 3
- XOJVVFBFDXDTEG-UHFFFAOYSA-N pristane Chemical compound CC(C)CCCC(C)CCCC(C)CCCC(C)C XOJVVFBFDXDTEG-UHFFFAOYSA-N 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 239000011541 reaction mixture Substances 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 229920006395 saturated elastomer Polymers 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 238000000527 sonication Methods 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 239000005720 sucrose Substances 0.000 description 3
- 239000003826 tablet Substances 0.000 description 3
- 238000011830 transgenic mouse model Methods 0.000 description 3
- 238000003260 vortexing Methods 0.000 description 3
- BQWBEDSJTMWJAE-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 4-[(2-iodoacetyl)amino]benzoate Chemical compound C1=CC(NC(=O)CI)=CC=C1C(=O)ON1C(=O)CCC1=O BQWBEDSJTMWJAE-UHFFFAOYSA-N 0.000 description 2
- PMJWDPGOWBRILU-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 4-[4-(2,5-dioxopyrrol-1-yl)phenyl]butanoate Chemical compound O=C1CCC(=O)N1OC(=O)CCCC(C=C1)=CC=C1N1C(=O)C=CC1=O PMJWDPGOWBRILU-UHFFFAOYSA-N 0.000 description 2
- QYEAAMBIUQLHFQ-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 6-[3-(pyridin-2-yldisulfanyl)propanoylamino]hexanoate Chemical compound O=C1CCC(=O)N1OC(=O)CCCCCNC(=O)CCSSC1=CC=CC=N1 QYEAAMBIUQLHFQ-UHFFFAOYSA-N 0.000 description 2
- KCVPASSMLHHOIF-QFIPXVFZSA-N (2r)-2-acetamido-3-tritylsulfanylpropanoic acid Chemical compound C=1C=CC=CC=1C(C=1C=CC=CC=1)(SC[C@H](NC(=O)C)C(O)=O)C1=CC=CC=C1 KCVPASSMLHHOIF-QFIPXVFZSA-N 0.000 description 2
- QRXMUCSWCMTJGU-UHFFFAOYSA-L (5-bromo-4-chloro-1h-indol-3-yl) phosphate Chemical compound C1=C(Br)C(Cl)=C2C(OP([O-])(=O)[O-])=CNC2=C1 QRXMUCSWCMTJGU-UHFFFAOYSA-L 0.000 description 2
- XKSOTQXTPALQMY-UHFFFAOYSA-N 1-[3-[(4-azidophenyl)disulfanyl]propanoyloxy]-2,5-dioxopyrrolidine-3-sulfonic acid Chemical compound O=C1C(S(=O)(=O)O)CC(=O)N1OC(=O)CCSSC1=CC=C(N=[N+]=[N-])C=C1 XKSOTQXTPALQMY-UHFFFAOYSA-N 0.000 description 2
- VHYRLCJMMJQUBY-UHFFFAOYSA-N 1-[4-[4-(2,5-dioxopyrrol-1-yl)phenyl]butanoyloxy]-2,5-dioxopyrrolidine-3-sulfonic acid Chemical compound O=C1C(S(=O)(=O)O)CC(=O)N1OC(=O)CCCC1=CC=C(N2C(C=CC2=O)=O)C=C1 VHYRLCJMMJQUBY-UHFFFAOYSA-N 0.000 description 2
- YGIABALXNBVHBX-UHFFFAOYSA-N 1-[4-[7-(diethylamino)-4-methyl-2-oxochromen-3-yl]phenyl]pyrrole-2,5-dione Chemical compound O=C1OC2=CC(N(CC)CC)=CC=C2C(C)=C1C(C=C1)=CC=C1N1C(=O)C=CC1=O YGIABALXNBVHBX-UHFFFAOYSA-N 0.000 description 2
- ASNTZYQMIUCEBV-UHFFFAOYSA-N 2,5-dioxo-1-[6-[3-(pyridin-2-yldisulfanyl)propanoylamino]hexanoyloxy]pyrrolidine-3-sulfonic acid Chemical compound O=C1C(S(=O)(=O)O)CC(=O)N1OC(=O)CCCCCNC(=O)CCSSC1=CC=CC=N1 ASNTZYQMIUCEBV-UHFFFAOYSA-N 0.000 description 2
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 description 2
- 241000251468 Actinopterygii Species 0.000 description 2
- 229920000936 Agarose Polymers 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
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 2
- RGJOEKWQDUBAIZ-IBOSZNHHSA-N CoASH Chemical compound O[C@@H]1[C@H](OP(O)(O)=O)[C@@H](COP(O)(=O)OP(O)(=O)OCC(C)(C)[C@@H](O)C(=O)NCCC(=O)NCCS)O[C@H]1N1C2=NC=NC(N)=C2N=C1 RGJOEKWQDUBAIZ-IBOSZNHHSA-N 0.000 description 2
- 229920002307 Dextran Polymers 0.000 description 2
- 241000943303 Enterococcus faecalis ATCC 29212 Species 0.000 description 2
- 241001131785 Escherichia coli HB101 Species 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical group C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 2
- 108010067060 Immunoglobulin Variable Region Proteins 0.000 description 2
- 102000017727 Immunoglobulin Variable Region Human genes 0.000 description 2
- 102000003960 Ligases Human genes 0.000 description 2
- 108090000364 Ligases Proteins 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- 102000016943 Muramidase Human genes 0.000 description 2
- 108010014251 Muramidase Proteins 0.000 description 2
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- 108090000526 Papain Proteins 0.000 description 2
- RADKZDMFGJYCBB-UHFFFAOYSA-N Pyridoxal Chemical compound CC1=NC=C(CO)C(C=O)=C1O RADKZDMFGJYCBB-UHFFFAOYSA-N 0.000 description 2
- 241000700159 Rattus Species 0.000 description 2
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 2
- 241000194019 Streptococcus mutans Species 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- QPMSXSBEVQLBIL-CZRHPSIPSA-N ac1mix0p Chemical compound C1=CC=C2N(C[C@H](C)CN(C)C)C3=CC(OC)=CC=C3SC2=C1.O([C@H]1[C@]2(OC)C=CC34C[C@@H]2[C@](C)(O)CCC)C2=C5[C@]41CCN(C)[C@@H]3CC5=CC=C2O QPMSXSBEVQLBIL-CZRHPSIPSA-N 0.000 description 2
- ZSLZBFCDCINBPY-ZSJPKINUSA-N acetyl-CoA Chemical compound O[C@@H]1[C@H](OP(O)(O)=O)[C@@H](COP(O)(=O)OP(O)(=O)OCC(C)(C)[C@@H](O)C(=O)NCCC(=O)NCCSC(=O)C)O[C@H]1N1C2=NC=NC(N)=C2N=C1 ZSLZBFCDCINBPY-ZSJPKINUSA-N 0.000 description 2
- 229960004308 acetylcysteine Drugs 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 235000012538 ammonium bicarbonate Nutrition 0.000 description 2
- 239000001099 ammonium carbonate Substances 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-O ammonium group Chemical group [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 2
- 235000011130 ammonium sulphate Nutrition 0.000 description 2
- 230000003078 antioxidant effect Effects 0.000 description 2
- 239000012062 aqueous buffer Substances 0.000 description 2
- 210000003719 b-lymphocyte Anatomy 0.000 description 2
- MSWZFWKMSRAUBD-QZABAPFNSA-N beta-D-glucosamine Chemical compound N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O MSWZFWKMSRAUBD-QZABAPFNSA-N 0.000 description 2
- 229960002246 beta-d-glucopyranose Drugs 0.000 description 2
- 239000013060 biological fluid Substances 0.000 description 2
- 239000002981 blocking agent Substances 0.000 description 2
- 210000001124 body fluid Anatomy 0.000 description 2
- 239000010839 body fluid Substances 0.000 description 2
- 238000013276 bronchoscopy Methods 0.000 description 2
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 2
- 238000011088 calibration curve Methods 0.000 description 2
- 239000002775 capsule Substances 0.000 description 2
- 229940077731 carbohydrate nutrients Drugs 0.000 description 2
- 150000001720 carbohydrates Chemical class 0.000 description 2
- 235000014633 carbohydrates Nutrition 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 229940093530 coenzyme a Drugs 0.000 description 2
- 230000002860 competitive effect Effects 0.000 description 2
- 238000012937 correction Methods 0.000 description 2
- 238000004132 cross linking Methods 0.000 description 2
- 229960002433 cysteine Drugs 0.000 description 2
- 150000001944 cysteine derivatives Chemical class 0.000 description 2
- 230000020176 deacylation Effects 0.000 description 2
- 238000005947 deacylation reaction Methods 0.000 description 2
- 230000002950 deficient Effects 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 238000010830 demodification reaction Methods 0.000 description 2
- 238000011033 desalting Methods 0.000 description 2
- 238000001784 detoxification Methods 0.000 description 2
- ZBCBWPMODOFKDW-UHFFFAOYSA-N diethanolamine Chemical compound OCCNCCO ZBCBWPMODOFKDW-UHFFFAOYSA-N 0.000 description 2
- 238000009792 diffusion process Methods 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 229940042399 direct acting antivirals protease inhibitors Drugs 0.000 description 2
- 231100000676 disease causative agent Toxicity 0.000 description 2
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 2
- 229910000397 disodium phosphate Inorganic materials 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 239000006196 drop Substances 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000012377 drug delivery Methods 0.000 description 2
- 238000000295 emission spectrum Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 125000003700 epoxy group Chemical group 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 238000001917 fluorescence detection Methods 0.000 description 2
- 238000002795 fluorescence method Methods 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- 238000001215 fluorescent labelling Methods 0.000 description 2
- 238000001641 gel filtration chromatography Methods 0.000 description 2
- 229960001911 glucosamine hydrochloride Drugs 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- 230000002209 hydrophobic effect Effects 0.000 description 2
- 230000016784 immunoglobulin production Effects 0.000 description 2
- 230000008676 import Effects 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 125000003010 ionic group Chemical group 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 229960000274 lysozyme Drugs 0.000 description 2
- 239000004325 lysozyme Substances 0.000 description 2
- 235000010335 lysozyme Nutrition 0.000 description 2
- 238000004949 mass spectrometry Methods 0.000 description 2
- 239000002207 metabolite Substances 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 230000020477 pH reduction Effects 0.000 description 2
- 230000005298 paramagnetic effect Effects 0.000 description 2
- 230000036961 partial effect Effects 0.000 description 2
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 2
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- 238000003752 polymerase chain reaction Methods 0.000 description 2
- 229920000136 polysorbate Polymers 0.000 description 2
- 238000002953 preparative HPLC Methods 0.000 description 2
- QLNJFJADRCOGBJ-UHFFFAOYSA-N propionamide Chemical compound CCC(N)=O QLNJFJADRCOGBJ-UHFFFAOYSA-N 0.000 description 2
- 229940080818 propionamide Drugs 0.000 description 2
- 238000010791 quenching Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 2
- 238000004007 reversed phase HPLC Methods 0.000 description 2
- 239000001632 sodium acetate Substances 0.000 description 2
- 235000017281 sodium acetate Nutrition 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 239000012086 standard solution Substances 0.000 description 2
- KZNICNPSHKQLFF-UHFFFAOYSA-N succinimide Chemical group O=C1CCC(=O)N1 KZNICNPSHKQLFF-UHFFFAOYSA-N 0.000 description 2
- 229960002317 succinimide Drugs 0.000 description 2
- JJAHTWIKCUJRDK-UHFFFAOYSA-N succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate Chemical compound C1CC(CN2C(C=CC2=O)=O)CCC1C(=O)ON1C(=O)CCC1=O JJAHTWIKCUJRDK-UHFFFAOYSA-N 0.000 description 2
- 235000000346 sugar Nutrition 0.000 description 2
- 239000000829 suppository Substances 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 239000001974 tryptic soy broth Substances 0.000 description 2
- 108010050327 trypticase-soy broth Proteins 0.000 description 2
- BEOUGZFCUMNGOU-UHFFFAOYSA-N tuberculostearic acid Chemical compound CCCCCCCCC(C)CCCCCCCCC(O)=O BEOUGZFCUMNGOU-UHFFFAOYSA-N 0.000 description 2
- 238000005199 ultracentrifugation Methods 0.000 description 2
- 238000000825 ultraviolet detection Methods 0.000 description 2
- 239000011534 wash buffer Substances 0.000 description 2
- JWDFQMWEFLOOED-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 3-(pyridin-2-yldisulfanyl)propanoate Chemical compound O=C1CCC(=O)N1OC(=O)CCSSC1=CC=CC=N1 JWDFQMWEFLOOED-UHFFFAOYSA-N 0.000 description 1
- PVGATNRYUYNBHO-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 4-(2,5-dioxopyrrol-1-yl)butanoate Chemical compound O=C1CCC(=O)N1OC(=O)CCCN1C(=O)C=CC1=O PVGATNRYUYNBHO-UHFFFAOYSA-N 0.000 description 1
- DLMYFMLKORXJPO-FQEVSTJZSA-N (2R)-2-amino-3-[(triphenylmethyl)thio]propanoic acid Chemical compound C=1C=CC=CC=1C(C=1C=CC=CC=1)(SC[C@H](N)C(O)=O)C1=CC=CC=C1 DLMYFMLKORXJPO-FQEVSTJZSA-N 0.000 description 1
- JDTOWOURWBDELG-QHCPKHFHSA-N (2r)-2-[(2-methylpropan-2-yl)oxycarbonylamino]-3-tritylsulfanylpropanoic acid Chemical compound C=1C=CC=CC=1C(C=1C=CC=CC=1)(SC[C@H](NC(=O)OC(C)(C)C)C(O)=O)C1=CC=CC=C1 JDTOWOURWBDELG-QHCPKHFHSA-N 0.000 description 1
- DPEYHNFHDIXMNV-UHFFFAOYSA-N (9-amino-3-bicyclo[3.3.1]nonanyl)-(4-benzyl-5-methyl-1,4-diazepan-1-yl)methanone dihydrochloride Chemical compound Cl.Cl.CC1CCN(CCN1Cc1ccccc1)C(=O)C1CC2CCCC(C1)C2N DPEYHNFHDIXMNV-UHFFFAOYSA-N 0.000 description 1
- WAAXYLYXYLKHJZ-UHFFFAOYSA-N 1-[3-(1-hydroxy-2,5-dioxopyrrolidine-3-carbonyl)phenyl]pyrrole-2,5-dione Chemical compound O=C1N(O)C(=O)CC1C(=O)C1=CC=CC(N2C(C=CC2=O)=O)=C1 WAAXYLYXYLKHJZ-UHFFFAOYSA-N 0.000 description 1
- LCZVQHWMSQLWSC-UHFFFAOYSA-N 1-[4-(2,5-dioxopyrrol-1-yl)butanoyloxy]-2,5-dioxopyrrolidine-3-sulfonic acid Chemical compound O=C1C(S(=O)(=O)O)CC(=O)N1OC(=O)CCCN1C(=O)C=CC1=O LCZVQHWMSQLWSC-UHFFFAOYSA-N 0.000 description 1
- FPKVOQKZMBDBKP-UHFFFAOYSA-N 1-[4-[(2,5-dioxopyrrol-1-yl)methyl]cyclohexanecarbonyl]oxy-2,5-dioxopyrrolidine-3-sulfonic acid Chemical compound O=C1C(S(=O)(=O)O)CC(=O)N1OC(=O)C1CCC(CN2C(C=CC2=O)=O)CC1 FPKVOQKZMBDBKP-UHFFFAOYSA-N 0.000 description 1
- CULQNACJHGHAER-UHFFFAOYSA-N 1-[4-[(2-iodoacetyl)amino]benzoyl]oxy-2,5-dioxopyrrolidine-3-sulfonic acid Chemical compound O=C1C(S(=O)(=O)O)CC(=O)N1OC(=O)C1=CC=C(NC(=O)CI)C=C1 CULQNACJHGHAER-UHFFFAOYSA-N 0.000 description 1
- UUFQTNFCRMXOAE-UHFFFAOYSA-N 1-methylmethylene Chemical compound C[CH] UUFQTNFCRMXOAE-UHFFFAOYSA-N 0.000 description 1
- JDIIGWSSTNUWGK-UHFFFAOYSA-N 1h-imidazol-3-ium;chloride Chemical compound [Cl-].[NH2+]1C=CN=C1 JDIIGWSSTNUWGK-UHFFFAOYSA-N 0.000 description 1
- WZIMSXIXZTUBSO-UHFFFAOYSA-N 2-[[bis(carboxymethyl)amino]methyl-(carboxymethyl)amino]acetic acid Chemical compound OC(=O)CN(CC(O)=O)CN(CC(O)=O)CC(O)=O WZIMSXIXZTUBSO-UHFFFAOYSA-N 0.000 description 1
- PWKSKIMOESPYIA-UHFFFAOYSA-N 2-acetamido-3-sulfanylpropanoic acid Chemical compound CC(=O)NC(CS)C(O)=O PWKSKIMOESPYIA-UHFFFAOYSA-N 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 102100039702 Alcohol dehydrogenase class-3 Human genes 0.000 description 1
- 208000007848 Alcoholism Diseases 0.000 description 1
- 102000013142 Amylases Human genes 0.000 description 1
- 108010065511 Amylases Proteins 0.000 description 1
- 108010032595 Antibody Binding Sites Proteins 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 102100033620 Calponin-1 Human genes 0.000 description 1
- 244000045232 Canavalia ensiformis Species 0.000 description 1
- 235000010520 Canavalia ensiformis Nutrition 0.000 description 1
- 239000004215 Carbon black (E152) Substances 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 241000699800 Cricetinae Species 0.000 description 1
- 239000004971 Cross linker Substances 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
- 108020004414 DNA Proteins 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
- RWSOTUBLDIXVET-UHFFFAOYSA-N Dihydrogen sulfide Chemical compound S RWSOTUBLDIXVET-UHFFFAOYSA-N 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 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
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 101000945318 Homo sapiens Calponin-1 Proteins 0.000 description 1
- 101000652736 Homo sapiens Transgelin Proteins 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 102000018071 Immunoglobulin Fc Fragments Human genes 0.000 description 1
- 108010091135 Immunoglobulin Fc Fragments Proteins 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
- 239000004472 Lysine Substances 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 241000186366 Mycobacterium bovis Species 0.000 description 1
- 241001025881 Mycobacterium smegmatis str. MC2 155 Species 0.000 description 1
- 241000187479 Mycobacterium tuberculosis Species 0.000 description 1
- RJQIGSDXVAFGHJ-JUXQOAEFSA-N N-[(3R,4R,5S,6R)-2-[(2R)-2-amino-3-sulfanylpropanoyl]-2,4,5-trihydroxy-6-(hydroxymethyl)oxan-3-yl]acetamide Chemical compound CC(=O)N[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)OC1(O)C(=O)[C@@H](N)CS RJQIGSDXVAFGHJ-JUXQOAEFSA-N 0.000 description 1
- BAQMYDQNMFBZNA-UHFFFAOYSA-N N-biotinyl-L-lysine Natural products N1C(=O)NC2C(CCCCC(=O)NCCCCC(N)C(O)=O)SCC21 BAQMYDQNMFBZNA-UHFFFAOYSA-N 0.000 description 1
- HDFGOPSGAURCEO-UHFFFAOYSA-N N-ethylmaleimide Chemical compound CCN1C(=O)C=CC1=O HDFGOPSGAURCEO-UHFFFAOYSA-N 0.000 description 1
- 108091007491 NSP3 Papain-like protease domains Proteins 0.000 description 1
- ORZJVQVEOXGJLR-PIRQICBKSA-N N[C@@H](CS)C(=O)C1(O)[C@H](N)[C@@H](O)[C@H](O)[C@H](O1)CO Chemical compound N[C@@H](CS)C(=O)C1(O)[C@H](N)[C@@H](O)[C@H](O)[C@H](O1)CO ORZJVQVEOXGJLR-PIRQICBKSA-N 0.000 description 1
- 241000187654 Nocardia Species 0.000 description 1
- 206010029443 Nocardia Infections Diseases 0.000 description 1
- 108020004711 Nucleic Acid Probes Proteins 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 241000283977 Oryctolagus Species 0.000 description 1
- 241000282520 Papio Species 0.000 description 1
- 206010034016 Paronychia Diseases 0.000 description 1
- 108700019535 Phosphoprotein Phosphatases Proteins 0.000 description 1
- 102000045595 Phosphoprotein Phosphatases Human genes 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 241000276498 Pollachius virens Species 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- 239000004743 Polypropylene Substances 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 241000288906 Primates Species 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 241000191967 Staphylococcus aureus Species 0.000 description 1
- 108010090804 Streptavidin Proteins 0.000 description 1
- 241000187747 Streptomyces Species 0.000 description 1
- 241000187433 Streptomyces clavuligerus Species 0.000 description 1
- 108010034949 Thyroglobulin Proteins 0.000 description 1
- 102000009843 Thyroglobulin Human genes 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- SXEHKFHPFVVDIR-UHFFFAOYSA-N [4-(4-hydrazinylphenyl)phenyl]hydrazine Chemical compound C1=CC(NN)=CC=C1C1=CC=C(NN)C=C1 SXEHKFHPFVVDIR-UHFFFAOYSA-N 0.000 description 1
- VLSOAXRVHARBEQ-UHFFFAOYSA-N [4-fluoro-2-(hydroxymethyl)phenyl]methanol Chemical compound OCC1=CC=C(F)C=C1CO VLSOAXRVHARBEQ-UHFFFAOYSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000008351 acetate buffer Substances 0.000 description 1
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000005903 acid hydrolysis reaction Methods 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 239000000443 aerosol Substances 0.000 description 1
- 238000001261 affinity purification Methods 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 238000007605 air drying Methods 0.000 description 1
- 201000007930 alcohol dependence Diseases 0.000 description 1
- 208000026935 allergic disease Diseases 0.000 description 1
- 230000007815 allergy Effects 0.000 description 1
- 238000012870 ammonium sulfate precipitation Methods 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 239000003708 ampul Substances 0.000 description 1
- 235000019418 amylase Nutrition 0.000 description 1
- 229940025131 amylases Drugs 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 238000005349 anion exchange Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000003302 anti-idiotype Effects 0.000 description 1
- 210000000628 antibody-producing cell Anatomy 0.000 description 1
- 102000025171 antigen binding proteins Human genes 0.000 description 1
- 108091000831 antigen binding proteins Proteins 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000007900 aqueous suspension Substances 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 230000007940 bacterial gene expression Effects 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 230000000721 bacterilogical effect Effects 0.000 description 1
- 230000001588 bifunctional effect Effects 0.000 description 1
- 230000003851 biochemical process Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- BAQMYDQNMFBZNA-MNXVOIDGSA-N biocytin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)NCCCC[C@H](N)C(O)=O)SC[C@@H]21 BAQMYDQNMFBZNA-MNXVOIDGSA-N 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 238000010804 cDNA synthesis Methods 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 235000010980 cellulose Nutrition 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000012501 chromatography medium Substances 0.000 description 1
- 238000005352 clarification Methods 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- RGJOEKWQDUBAIZ-UHFFFAOYSA-N coenzime A Natural products OC1C(OP(O)(O)=O)C(COP(O)(=O)OP(O)(=O)OCC(C)(C)C(O)C(=O)NCCC(=O)NCCS)OC1N1C2=NC=NC(N)=C2N=C1 RGJOEKWQDUBAIZ-UHFFFAOYSA-N 0.000 description 1
- 239000005516 coenzyme A Substances 0.000 description 1
- 230000001332 colony forming effect Effects 0.000 description 1
- 238000002967 competitive immunoassay Methods 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 239000012468 concentrated sample Substances 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000001816 cooling Methods 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
- 239000006071 cream Substances 0.000 description 1
- 239000000287 crude extract Substances 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 125000002711 cysteinyl group Chemical group 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- KDTSHFARGAKYJN-UHFFFAOYSA-N dephosphocoenzyme A Natural products OC1C(O)C(COP(O)(=O)OP(O)(=O)OCC(C)(C)C(O)C(=O)NCCC(=O)NCCS)OC1N1C2=NC=NC(N)=C2N=C1 KDTSHFARGAKYJN-UHFFFAOYSA-N 0.000 description 1
- 230000014670 detection of bacterium Effects 0.000 description 1
- 230000001627 detrimental effect Effects 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 239000010432 diamond Substances 0.000 description 1
- 239000012470 diluted sample Substances 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 206010013023 diphtheria Diseases 0.000 description 1
- 150000002016 disaccharides Chemical group 0.000 description 1
- 239000007884 disintegrant Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 210000001671 embryonic stem cell Anatomy 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 210000000416 exudates and transudate Anatomy 0.000 description 1
- 210000003608 fece Anatomy 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 239000000706 filtrate Substances 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
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 238000002189 fluorescence spectrum Methods 0.000 description 1
- 235000003599 food sweetener Nutrition 0.000 description 1
- 108010049230 formaldehyde dehydrogenase (glutathione) Proteins 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
- 230000014509 gene expression Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 108010051015 glutathione-independent formaldehyde dehydrogenase Proteins 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 239000008240 homogeneous mixture Substances 0.000 description 1
- 238000000265 homogenisation Methods 0.000 description 1
- 239000013029 homogenous suspension Substances 0.000 description 1
- 244000052637 human pathogen Species 0.000 description 1
- 229930195733 hydrocarbon Natural products 0.000 description 1
- 150000002430 hydrocarbons Chemical class 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000000413 hydrolysate Substances 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000000984 immunochemical effect Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 239000007943 implant Substances 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 229940102223 injectable solution Drugs 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 150000004001 inositols Chemical class 0.000 description 1
- 230000037041 intracellular level Effects 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
- 238000010829 isocratic elution Methods 0.000 description 1
- 238000003771 laboratory diagnosis Methods 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 230000031852 maintenance of location in cell Effects 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 150000002739 metals Chemical class 0.000 description 1
- MYWUZJCMWCOHBA-VIFPVBQESA-N methamphetamine Chemical compound CN[C@@H](C)CC1=CC=CC=C1 MYWUZJCMWCOHBA-VIFPVBQESA-N 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- YKSIHFDRGQQOCJ-LHHMOHDTSA-N mycothione Chemical compound O([C@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1NC(=O)[C@@H](NC(C)=O)CSSC[C@H](NC(=O)C)C(=O)N[C@H]1[C@H](O[C@H](CO)[C@@H](O)[C@@H]1O)O[C@@H]1[C@@H]([C@H](O)[C@@H](O)[C@H](O)[C@H]1O)O)[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](O)[C@H]1O YKSIHFDRGQQOCJ-LHHMOHDTSA-N 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- TYQCGWOGNUOQAR-ZDUSSCGKSA-N n-[(3s)-1-chloro-5-methyl-2-oxohexan-3-yl]-4-methylbenzenesulfonamide Chemical compound CC(C)C[C@@H](C(=O)CCl)NS(=O)(=O)C1=CC=C(C)C=C1 TYQCGWOGNUOQAR-ZDUSSCGKSA-N 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 230000036963 noncompetitive effect Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000002853 nucleic acid probe Substances 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 229940055729 papain Drugs 0.000 description 1
- 235000019834 papain Nutrition 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 238000010647 peptide synthesis reaction Methods 0.000 description 1
- 238000002823 phage display Methods 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 210000002381 plasma Anatomy 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 108091033319 polynucleotide Proteins 0.000 description 1
- 102000040430 polynucleotide Human genes 0.000 description 1
- 239000002157 polynucleotide Substances 0.000 description 1
- 229920001155 polypropylene Polymers 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 239000004800 polyvinyl chloride Substances 0.000 description 1
- 229920000915 polyvinyl chloride Polymers 0.000 description 1
- 235000020004 porter Nutrition 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 125000006239 protecting group Chemical group 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 230000002797 proteolythic effect Effects 0.000 description 1
- 229960003581 pyridoxal Drugs 0.000 description 1
- 235000008164 pyridoxal Nutrition 0.000 description 1
- 239000011674 pyridoxal Substances 0.000 description 1
- 239000002516 radical scavenger Substances 0.000 description 1
- 238000003127 radioimmunoassay Methods 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 230000011506 response to oxidative stress Effects 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000012552 review Methods 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
- 238000002390 rotary evaporation Methods 0.000 description 1
- 238000010079 rubber tapping Methods 0.000 description 1
- 238000013341 scale-up Methods 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 238000002098 selective ion monitoring Methods 0.000 description 1
- 238000001542 size-exclusion chromatography Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- ULARYIUTHAWJMU-UHFFFAOYSA-M sodium;1-[4-(2,5-dioxopyrrol-1-yl)butanoyloxy]-2,5-dioxopyrrolidine-3-sulfonate Chemical compound [Na+].O=C1C(S(=O)(=O)[O-])CC(=O)N1OC(=O)CCCN1C(=O)C=CC1=O ULARYIUTHAWJMU-UHFFFAOYSA-M 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 210000004989 spleen cell Anatomy 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000012134 supernatant fraction Substances 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 238000004114 suspension culture Methods 0.000 description 1
- 239000003765 sweetening agent Substances 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 229960000814 tetanus toxoid Drugs 0.000 description 1
- WROMPOXWARCANT-UHFFFAOYSA-N tfa trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F.OC(=O)C(F)(F)F WROMPOXWARCANT-UHFFFAOYSA-N 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 238000011285 therapeutic regimen Methods 0.000 description 1
- 125000004035 thiopropyl group Chemical group [H]SC([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 125000005413 thiopyridyl group Chemical group 0.000 description 1
- 229960002175 thyroglobulin Drugs 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 238000001665 trituration Methods 0.000 description 1
- 125000002221 trityl group Chemical group [H]C1=C([H])C([H])=C([H])C([H])=C1C([*])(C1=C(C(=C(C(=C1[H])[H])[H])[H])[H])C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 description 1
- 241001446247 uncultured actinomycete Species 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/5695—Mycobacteria
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
- C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
- C07K16/1292—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Actinomyces; from Streptomyces (G)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/35—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Mycobacteriaceae (F)
Definitions
- This invention relates generally to the field of detection of bacteria, and more specifically to the diagnosis of actinomycetes infection, particularly to those caused by mycobacteria.
- the invention also relates generally to the field of diagnosis of bacterial infection, and more specifically to the diagnosis of diseases associated with actinomycetes infection such as tuberculosis.
- Human tuberculosis is caused primarily by the bacterium Mycobacte ⁇ um tuberculosis.
- M. tuberculosis is by far the most important cause of morbidity and mortality among the mycobacterial genus. It is estimated that 8.8 million new cases of tuberculosis occurred in 1995 and these numbers are projected to continue to increase.
- the laboratory diagnosis of tuberculosis has always been complicated by the slow growth of M. tuberculosis in culture. Visible growth on solid culture media can require up to 8 weeks of incubation. Presumptive diagnoses are based on finding mycobacteria by microscopic examination of a diagnostic specimen, often an expectorated sputum. These smears are either stained by acid fast stains like the Ziehl-Neelsen stain or by auramine-
- the conventional technique for detecting tuberculosis is by microscopic identification of the bacteria in patient specimens treated with special stains combined with cultivation on specific bacteriologic media. Detection by the staining techniques is nonspecific and relatively insensitive, and cultivation is time-consuming and expensive because Mycobacterium tuberculosis grows very slowly. Detection of M. tuberculosis by nucleic acid amplification is an adjunctive approach. Because of the duration of time required to establish a definitive diagnosis, many adjunctive laboratory diagnostic tests have been investigated. Serologic detection of antibody to M. tuberculosis has had very low predictive values (Daniel, T.M., and Debanne, S.M., 1987, Am. Rev. Respir. Dis. 158:678).
- Mycothiol is a recently discovered novel cysteine derivative produced only by actinomycetes. Among the actinomycetes, mycobacteria produce mycothiol in the greatest amounts. Mycobacteria are the main group of actinomycetes that infect humans. Among these infections are tuberculosis (TB), as well as other mycobacterial infections. Other genera of actinomycetes (corynebacteria, including the causative agent of diphtheria, and Nocardia species, which cause pulmonary nocardiosis) make MSH, but they are minor causes of human morbidity and mortality compared to tuberculosis (Newton et al, 1996, J. Bacteriol, 178).
- GSH glutathione
- prokaryotes and eukaryotes Fahey, R.C., and Sundquist, A.R., 1991, Adv. Enzymol Relat.
- MSH has superior antioxidant properties to GSH (Newton et al, 1995, supra) and thus may serve both as a stable intracellular storage form of cysteine and as the essential cofactor for oxidative stress-response and detoxification enzymes in a manner analogous to that of GSH in GSH-producing organisms.
- MSH is the cofactor for an NAD/"cofactor"-dependent formaldehyde dehydrogenase found in actinomycetes, where it serves in a detoxification role analogous to that of GSH in the NAD/GSH-dependent formaldehyde dehydrogenase of eukaryotes and Gram-negative bacteria (Misset-Smits, M., et al, 1997, FEBS Lett. 409:221-
- MSH-dependent protective enzymes It is likely that further examples of MSH-dependent protective enzymes will be found in the future. Thus, enzymes involved in the metabolism of MSH may represent targets for new drugs directed against tuberculosis and other mycobacterial infections (Newton et al, 1996, supra).
- mycobacteria produce the highest levels of mycothiol, ⁇ 5 million molecules per cell, and as few other actinomycetes besides mycobacteria are human pathogens, the present inventors have thus proposed that detection of MSH is a possible way to screen for tuberculosis and other mycobacterial infections.
- MSH analysis has previously relied on derivatization with thiol-specific fluorescent labeling reagents followed by HPLC analysis (Spies and Steenkamp, 1994, supra; Newton et al, 1993,
- the present invention is based on the discovery of methods for detecting mycothiol, a recently described novel cysteine derivative produced at millimolar intracellular levels by actinomycetes, including the pathogenic mycobacteria.
- a method of detecting a member of the taxa actinomycetes includes incubating a reagent that detects mycothiol or a precursor thereof with a sample for a time sufficient for said reagent to react with mycothiol or precursor thereof, and detecting the A method is provided for diagnosis of a subject having or at risk of having an Actinomycetes-associated disorder.
- the method includes contacting a sample from the subject . having or at risk of having an actinomycetes-associated disorder with a reagent that detects mycothiol or precursor thereof for a period of time sufficient for said reagent to react, and detecting the reaction of the reagent with mycothiol or precursor thereof.
- the reaction of the reagent that detects mycothiol or precursor thereof to the sample is compared to a control sample.
- a method for identifying a sample with altered production of mycothiol or a precursor thereof including contacting a test sample with a reagent that detects mycothiol or precursor thereof for a period of time sufficient for the reagent to react, and detecting the reaction of the reagent with mycothiol or precursor thereof.
- the reagent that detects mycothiol or precursor thereof to the test sample is compared with a control sample; a difference in the amount of reaction with the test sample as compared to the control sample is indicative of an alteration in the production of mycothiol or precursor thereof.
- a method for detecting mycothiol or precursor thereof in a bacterial colony including contacting a membrane to a bacteria plated on a bacterial culture plate for a time sufficient to allow the bacteria to adhere to the membrane and lysing the bacteria .
- the method also includes contacting the membrane with a reagent to detect mycothiol or precursor thereof, and detecting said the reaction of reagent with mycothiol or precursor thereof.
- a method for detecting mycothiol or precursor thereof including biotinylating mycothiol or precursor thereof to form biotinylated mycothiol or biotinylated mycothiol precursor, and contacting the biotinylated mycothiol or biotinylated mycothiol precursor to an antibody with binds mycothiol or precursor thereof to form a complex.
- the method further includes detecting the presence of said complex with a detection reagent. Kits are also disclosed which are useful for detecting the presence of mycothiol or precursor thereof in a sample.
- a method for detecting a mycothiol or a precursor thereof including partially purifying mycothiol or a precursor thereof, reacting the precusor of mycothiol with a reagent for fluroescent amine labeling to form a reaction product; and detecting the presence of the reaction product.
- Detecting may be at a pH range from about 7.5 to 9, and more likely from about 8.0 to 8.6.
- FIG. 1 shows the structure and names (including abbreviations) for mycothiol.
- FIG. 2 is a typical standard curve obtained using the ELISA procedure described in Example 4, using purified mycothiol from 0.02 - 1.0 pmol per well. Samples were run in quadruplicate; error bars indicate standard deviation from the mean.
- FIG. 3 illustrates membrane blot detection of mycothiol in bacterial colonies lifted
- FIG. 4 illustrates results from an immunoassay using the ELISA procedure described in Example 4, using different low-molecular mass thiols to test for antibody specificity. Thiols were assayed at 1 or 10 pmol per well as indicated. Values are the mean of quadruplicate determinations with error bars indicating standard deviation from the mean. The control value is for wells that received maleimide-BSA with no thiol added.
- FIG. 5 shows results from an immunoassay using the ELISA procedure described in Example 8, that demonstrated the effect of increasing the percentage by volume of acetonitrile in the buffer used for the antigen-binding step ("binding buffer"). Increasing the binding buffer's acetonitrile content from 0% to 25% by volume results in an approximate doubling of the observed signal.
- FIG. 6 shows results from an immunoassay using the ELISA procedure described in Example 8, demonstrating the increase in signal and negligible increase in background resulting from an extended (120 minute) development period.
- Biotinylated mycothiol (MPB- MSH) was applied in 25% v/v acetonitrile in TBS.
- FIG. 7 illustrates the dot blot described in Example 9. Biotinylated mycothiol samples were assayed from 0 to 0.3 pmol per well in quadruplicate.
- FIG. 9 shows results from the immunoassay described in Example 11 , assaying Mycobacterium avium cells in human urine.
- FIG. 10 shows results from the immunoassay described in Example 11, assaying Mycobacterium avium cells in either cerebrospinal fluid with no additives ("CSF") or cerebrospinal fluid with glucose and glycerol added ("enriched CSF"). Note that the addition of glycerol and glucose to the mycobacterial cell suspension roughly doubles the amount of mycothiol detected.
- FIG. 11 shows results from the immunoassay described in Example 12. Data shown are means of 2 samples.
- Mycobacterium smegmatis mc2-155 (solid circles) is a parent strain known to produce high levels of mycothiol
- strain 49 (open circles) is a mutant strain known to produce no mycothiol
- strain 164 (solid sq ⁇ ares) is a mutant strain which produces reduced levels of mycothiol.
- FIG. 12 is the calibration curves for analysis of GlcN (Sdo-4(O)) and GlcN-Ins (Sdo4( ))
- FIG. 13 shows HPLC chromatograms (method 5 A) for determination of Cys-GlcN-Ins and Cys-GlcN after labeling with mBBr.
- Fig. 13 A is several chromatograms: 1st panel, mB- Cys-GlcN-Ins standard; 2nd panel, mBBr-labeled extract of log phase M. smegmatis MC 2 155 cells; 3rd panel, NEM-treated and mBBr-labeled extract of log phase M. smegmatis Mc 2 155 cells showing nonthiol fluorescent components; 4th panel, mB-CG standard.
- R denotes peaks produced in controls and derived from the reagent (mBBr).
- 13B shows an expanded and amplified (5 Ox) portion of the HPLC chromatogram for an mBBr-labeled sample with a CGI content of 0.07 ⁇ mol/g RDW ⁇ solid /me).and the corresponding NEM control sample ⁇ dotted line).
- FIG. 14 shows HPLC chromatograms (method 1 A) for analysis of thiols.
- Top panel mixture of mBBr-labeled thiol standards (R, reagent-derived peaks); middle panel, mBBr- labeled extract of log phase M. smegmatis MC 2 155; bottom panel, NEM-blocked and mBBR- labeled extract of log phase M. smegmatis MC 2 155 showing nonthiol fluorescent components.
- FIG. 15 is a graph illustrating the variation in metabolite levels in M. smegmatis MC 2 155 as a function of growth cycle.
- FIG. 16 is a graph illustrating the effect of nutrient supplementation and glucose deprivation upon levels of MSH precursors and interaiediates in M. smegmatis MC 2 155 at 37° C.
- A cells suspended in 7H9 medium with 014% glucose and 1 mM /wy ⁇ -inositol
- B cells suspended in 7H9 medium with 0.4% glucose and 10 mM N-acetylcysteine
- C cells suspended in 7H9 medium without glucose.
- the present invention relates to mycothiol and precursors of mycothiol, and to reagents for the detection of mycothiol and precursors thereof, including antibodies and chemical reagents. These methods and reagents are useful for the diagnosis and treatment of actinomycetes-associated disorders, including mycobacteria-associated disorders such as tuberculosis.
- the invention provides methods for detecting a member of the taxa actinomycetes, by incubating a reagent that detects mycothiol or precursor thereof with a sample for a time sufficient for the reagent to react with mycothiol or precursor thereof. The reaction of the reagent with mycothiol or precursor thereof is then detected. Detection of a reaction is indicative of the presence of a member of the taxa actinomycetes.
- Mycothiol or “MSH” or “AcCys-GlcN-Ins” is 1 -D-/nyo-inosityl-2-(N-acetyl- L-cysteinyl)amido-2-deoxy- ⁇ -D-glucopyranoside.
- the structure of mycothiol is shown in FIG. 1.
- a "mycothiol precursor” is a molecule from which mycothiol can be produced.
- mycothiol precursors include ⁇ -acetyl-cysteine, 2-( ⁇ -acetyl-L cysteinyl) amido-2-deoxy-2-D- glucopyanoside, l-D-w O-inosityl-2-amino-2-deoxy- ⁇ -D-glycopyranoside (GlcN-Ins), (Cys- GlcN-Ins), l-D-mvo-inosityl-2-(L-cysteinyl) amido-2-deoxy-Dglucopyanoside and 2- (Lcysteinyl)amido-2deoxy-Dglucopyanoside (Cys-GlcN).
- ⁇ -acetyl-cysteine 2-( ⁇ -acetyl-L cysteinyl) amido-2-deoxy-2-D- glucopyanoside
- precursors of mycothiol are GlcN-Ins, Cys-GlcN-Ins, and AcCys.
- Mycothiol is produced at millimolar levels by the taxa actinomycetes.
- the genus Mycobacteria is a member of the taxa actinomycetes that colonizes humans, and are a causative agent in disease (e.g., tuberculosis). It should be noted that an assay of mycothiol that assesses the production of myocothiol in the reduced thiol form is useful in distinguishing viable actinomycetes from non-viable actinomycetes.
- a “viable” actinomycete is a cell that is metabolically active and which carries out the biochemical processes required for the cell.
- a “reagent that detects mycothiol or precursor” is any molecule that reacts with mycothiol when incubated with mycothiol.
- Reacting includes binding, such as an antibody binding an antigen, or the binding of a fluorescent molecule with mycothiol such that the fluorescent properties of a molecule are altered.
- Reacting also includes chemically reacting such that covalent bonds are modified.
- Reacting further includes reacting such that hydrogen bonds are modified.
- Incubating includes conditions which allow contact between the reagent that detects mycothiol or a precursor thereof with a sample.
- sample is any substance which might possibly contain mycothiol or a precursor thereof.
- the sample is a patient sample, such as a blood sample, a serum sample, a urine sample, a fecal sample, a tissue biopsy, a cerebrospinal fluid sample, a pleural fluid sample, an ascites sample, and a sputum sample.
- the sample is a bacterial sample, such as a culture of a bacterium in a growth medium.
- Detection is performed by any means suitable to identify the interaction of the reagent with mycothiol or precursor thereof.
- the reagent when the reagent is a chemical reagent, physical or chemical parameters of the reagent or the products of the interaction of the agent with mycothiol can be monitored.
- the reagent when the reagent is an antibody, the antibody can be detectably labeled.
- Detectable labels are well known in the art, and include radioisotopes, fluorophores, paramagnetic labels, enzymes (e.g., horseradish peroxidase), or other moieties or compounds which either emit a detectable signal (e.g., radioactivity, fluorescence, color) or emit a detectable signal after exposure of the label to its substrate.
- the reagent when the reagent is an antibody, detection can be performed using a second antibody which is detectably labeled which recognizes the antibody that binds mycothiol or precursor thereof.
- the antibody may also be biotinylated, and a second avidinated label used to determine the presence of the biotinylated reagent which detects mycothiol or precursor thereof.
- antibody stands for an immunoglobulin protein which is capable of - binding an antigen.
- Antibody as used herein is meant to include the entire antibody as well as any antibody fragments (e.g., F(ab', Fab, Fv) capable of binding the epitope, antigen or antigenic fragment of interest (see below).
- Preferred antibodies for assays of the invention are immunoreactive or immunospecific for and therefore specifically and selectively bind to mycothiol or precursor thereof.
- antibody encompasses all types of antibodies, e.g., polyclonal, monoclonal, and those produced by the phage display methodology.
- Particularly preferred antibodies of the invention are antibodies which have a relatively high degree of affinity for mycothiol or precursor thereof.
- Mycothiol or precursor thereof can be used to produce .antibodies which are immunoreactive or bind to mycothiol or precursor thereof. Antibodies which consist essentially of pooled antibodies with different epitopic specificities, as well as distinct monoclonal antibody preparations are provided. "Purified antibody” refers to that which is sufficiently free of other proteins, carbohydrates, and lipids with which it is naturally associated. Such an antibody “preferentially binds" to mycothiol or a precursor thereof, and does not substantially recognize or bind to other antigenetically unrelated molecules. By “binds specifically” is meant high avidity . and/or high affinity binding of an antibody to a specific molecule, e.g. , mycothiol or a precursor thereof. Antibody binding to its epitope on a specific molecule is preferably stronger than binding of the same antibody to any other molecule, particularly those which may be present in molecules in association with, or in the same sample.
- polyclonal antibodies The preparation of polyclonal antibodies is well-known to those skilled in the art. See, for example, Green et al, "Production of Polyclonal Antisera,” in: Immunochemical Protocols pages 1-5, Manson, ed., Humana Press 1992; Coligan et al, "Production of Polyclonal Antisera in Rabbits, Rats, Mice and Hamsters," in: Current Protocols in Immunology, section 2.4.1 , 1992. which are hereby incorporated by reference. The preparation of monoclonal antibodies likewise is conventional.
- monoclonal antibodies can be obtained by injecting mice with a composition comprising an antigen, verifying the presence of antibody production by removing a serum sample, removing the spleen to obtain B lymphocytes, fusing the B lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, selecting positive clones that produce antibodies to the antigen, and isolating the antibodies from the hybridoma cultures.
- Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well-established techniques. Such isolation techniques include affinity chromatography with Protein-A Sepharose, size-exclusion chromatography, and ion-exchange chromatography.
- Multiplication in vitro may be carried out in suitable culture media such as Dulbecco's Modified Eagle Medium or RPMI 1640 medium, optionally supplemented by a mammalian serum such as fetal calf serum or trace elements and growth-sustaining supplements such as normal mouse peritoneal exudate cells, spleen cells, thymocytes or bone marrow macrophages.
- suitable culture media such as Dulbecco's Modified Eagle Medium or RPMI 1640 medium
- a mammalian serum such as fetal calf serum or trace elements
- growth-sustaining supplements such as normal mouse peritoneal exudate cells, spleen cells, thymocytes or bone marrow macrophages.
- Multiplication in vivo may be carried out by injecting cell clones into mammals histocompatible with the parent cells, e.g., syngeneic mice, to cause growth of antibody-producing tumors.
- the animals are primed with a hydrocarbon, especially oils such as pristane (tetramethylpentadecane) prior to injection.
- pristane tetramethylpentadecane
- the desired monoclonal antibody is recovered from the body fluid of the animal.
- Therapeutic applications for antibodies disclosed herein are also part of the present invention.
- antibodies of the present dnvention may also be derived from subhuman- primate antibody.
- General techniques for raising therapeutically useful antibodies in baboons can be found, for example, in Goldenberg et al, International Patent Publication WO 91/1 1465, 1991, and Losman et al, 1990, Int. J. Cancer 46:310, which are hereby incorporated by reference.
- a therapeutically useful anti-mycothiol or anti-mycothiol precursor antibody may be derived from a "humanized" monoclonal antibody.
- Humanized monoclonal antibodies are produced by transferring mouse complementarity determining regions from heavy and light variable chains of the mouse immunoglobulin into a human variable domain, and then substituting human residues in the framework regions of the murine counterparts.
- the use of antibody components derived from humanized monoclonal antibodies obviates potential problems associated with the immunogenicity of murine constant regions. General techniques for cloning murine immunoglobulin variable domains are described, for example, by Orlandi et al, 1989, Proc. Nat'lAcad. Sci.
- Antibodies of the invention also may be derived from antibody fragments isolated from a combinatorial immunoglobulin library. See, for example, Barbas et al, 1991, in: Methods: a Companion to Methods in Enzymology. Vol. 2, page 1 19; Winter et al, 1994, Ann. Rev. Immunol. 12:433, which are hereby incorporated by reference.
- Cloning and expression vectors that are useful for producing a human immunoglobulin phage library can be obtained, for example, from STRATAGENE Cloning Systems (La Jolla, CA).
- antibodies of the present invention may be derived from a human monoclonal antibody.
- Such antibodies are obtained from transgenic mice that have been "engineered” to produce specific human antibodies in response to antigenic challenge.
- elements of the human heavy and light chain loci are introduced into strains of mice derived from embryonic stem cell lines that contain targeted disruptions of the endogenous heavy and light chain loci.
- the transgenic mice can synthesize human antibodies specific for human antigens, and the mice can be used to produce human antibody-secreting hybridomas.
- Methods for obtaining human antibodies from transgenic mice are described by Green et al, 1994, Nature Genet. 7:13; Lonberg et al, 1994, Nature 368:856; and Taylor et al, 1994, Int. Immunol. 6:579, which are hereby incorporated by reference.
- antibody as used in this invention includes intact molecules as well as fragments thereof, such as Fab, F(ab') 2 , and Fv which are capable of binding the epitopic determinant. These antibody fragments retain some ability to selectively bind with its antigen or receptor and are defined as follows:
- Fab the fragment which contains a monovalent antigen-binding fragment of an antibody molecule can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain;
- Fab' the fragment of an antibody molecule can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule;
- Fv defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains
- Single chain antibody defined as a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule.
- SCA Single chain antibody
- Methods of making these fragments are known in the art. (See for example, Harlow and Lane, 1988, Antibodies: A Laboratory Manual. C ld Spring Harbor Laboratory, New York, incorporated herein by reference).
- epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.
- Antibody fragments of the present invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli of DNA encoding the fragment.
- Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods.
- antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab') 2 .
- This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments.
- cleaving antibodies such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.
- Fv fragments comprise an association of V H and V L chains. This association may be noncovalent, as described in Inbar et al, 1972, Proc. Nat'lAcad. Sci. USA 69:2659.
- the variable chains can be linked by an intermolecular disulfide bond or crosslinked by chemicals such as glutaraldehyde. 'See, e.g., Sandhu, supra.
- the Fv fragments comprise V H and V L chains connected by a peptide linker.
- These single-chain antigen binding proteins (sFv) are prepared by constructing a structural gene comprising DNA sequences encoding the V H and V L domains connected by an oligonucleotide.
- the structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli.
- the recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains.
- Methods for producing sFvs are described, for example, by Whitlow et al, 1991, Methods: a Companion to Methods in ⁇ nzymology. Vol. 2, page 97; Bird et al, 1988, Science 242:423-426; Ladner et al, U.S. patent No. 4,946,778; Pack et al., 1993, Bio/Technology J ,: 1271 -77; and Sandhu, supra.
- CDR peptides (“minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick et al, 1991, Methods: a Companion to Methods in ⁇ nzymology. Vol. 2, page 106.
- Antibodies which bind to mycothiol or precursor thereof can be prepared using an intact polypeptide or fragments containing small peptides of interest as the immunizing antigen.
- the polypeptide or a peptide used to immunize an animal can be derived from translated cDNA or chemical synthesis which can be conjugated to a carrier protein, if desired.
- Such commonly used carriers which are chemically coupled to the peptide include keyhole limpet hemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA), and tetanus toxoid.
- KLH keyhole limpet hemocyanin
- BSA bovine serum albumin
- the coupled peptide is then used to immunize the animal ⁇ e.g., a mouse, a rat, or a rabbit).
- polyclonal or monoclonal antibodies can be further purified, for example, by binding to and elution from a matrix to which the polypeptide or a peptide to which the antibodies were raised is bound.
- “Purified antibody” refers to that which is sufficiently free of other proteins, carbohydrates, and lipids with which it is naturally associated. Such an antibody - “preferentially binds" to mycothiol or a precursor thereof, and does not substantially recognize or bind to other antigenetically unrelated molecules.
- binds specifically is meant high avidity and/or high affinity binding of an antibody to a specific molecule, e.g., mycothiol or a precursor thereof.
- Antibody binding to its epitope on a specific molecule is preferably stronger than binding of the same antibody to any other molecule, particularly those which may be present in molecules in association with, or in the same sample.
- Those of skill in the art will know of various techniques common in the immunology arts for purification and/or concentration of polyclonal antibodies, as well as monoclonal antibodies (See for example, Coligan et al, Unit 9, Current Protocols in Immunology. Wiley Interscience, 1991, incorporated by reference).
- an anti-idiotypic monoclonal antibody made to a first monoclonal antibody will have a binding domain in the hypervariable region which is the "image" of the epitope bound by the first monoclonal antibody.
- detectably labeled antibody an antibody (or antibody fragment which retains binding specificity), having an attached detectable label.
- the detectable label is normally attached by chemical conjugation, but where the label is a polypeptide, it could alternatively be attached by genetic engineering techniques. Methods for production of detectably labeled proteins are well known in the art.
- Detectable labels known in the art, but normally are radioisotopes, fluorophores, paramagnetic labels, enzymes (e.g., horseradish peroxidase), or other moieties or compounds which either emit a detectable signal (e.g., radioactivity, fluorescence, color) or emit a detectable signal after exposure of the label to its substrate.
- Various detectable label/substrate pairs e.g., horseradish peroxidase/diaminobenzidine, avidin streptavidin, luciferase/luciferin
- methods for labeling antibodies, and methods for using labeled antibodies are well known in the art (see, for example, Harlow and Lane, eds., 1988, Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).
- Another technique which may also result in greater sensitivity consists of coupling the antibodies to low molecular weight haptens.
- Thede haptens can then be specifically detected by - means of a second reaction.
- biotin which reacts with avidin, or dinitrophenyl, pyridoxal, and fluorescein, which can react with specific antihapten antibodies.
- a reagent that detects mycothiol or its precursor may be a chemical reagent.
- the chemical reagent is a reagent which specifically reacts with amines to produce reaction products that are detectable by their emission spectra.
- a reagent that specifically reacts with amines is a reagent for fluorescent amine labeling.
- a reagent for fluorescent amine labeling is a precolumn derivatization reagent that yields detectable fluorescent adducts.
- reagents include dansyl chloride, fluorescenceamine, 12-(N-methyl-N(7-nitro-2-oxa-l,3-diazol-4-yl) chloride, and CBQCA (Molecular Probes).
- the chemical reagent is dansyl chloride, fluorescenceamine, 12-(N-methyl-N(7-nitro-2-oxa-l,3-diazol-4-yl) chloride, and CBQCA (Molecular Probes).
- the chemical reagent is
- 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AccQ-Fluor) (Cohen, S. and Michaud, M.. Analytical Biochemistry, 211, 279-287, 1993; Cohen, S. and Michaud, M., 1993, Techniques in Protein Chemistry IV, 289-298, herein incorporated by reference).
- a buffer of lower pH than employed with amino acids is utilized for labeling of amino sugars.
- the lower pH provides an advantage to the amino sugars, whose ammonium forms have lower pK a values than most amino acids, and thus have a competitive advantage during labeling.
- the reaction products are then detected.
- Detection can include any physical or chemical method of detection and or/separation.
- normal phase or reversed phase chromatography When the molecular weight changes largely, the gel filtration chromatography can be utilized.
- ion exchange chromatography When the modification of ionic groups or demodification takes place, there can be used ion exchange chromatography.
- affinity chromatography When the sugar chain is added or released, affinity chromatography can be utilized.
- the reagent that detects mycothiol is a reagent for fluorescent amine labelling, the fluorescence amine spectra of the reaction products can be utilized.
- the reaction products can also be quantitated.
- high performance liquid chromatography is used for separation and measurement of the reaction products.
- the AccQ-Fluor derivatized samples were analyzed by HPLC utilizing a Waters 600E solvent delivery system equipped with a Waters WISP Model 710B autoinjector, LDC Fluorometer III, and a Nelson Model 444 data collection system. Separation was obtained on a Beckman Ultrasphere IP (250 x 4.6mm) analytical column equipped with a Brownley HPLC guard column containing an OD-GU 5mm C-l 8 cartridge using the following linear gradients: 0 min, 100% A (0.1% TFA in water), 10 min, 100% A; 50 min.
- a regent that detect mycothiol or precursor may be a chemical reagent.
- the chemical reagent is a reagent which specifically reacts with thiols to produce reaction products that are detectable by their emission spectra.
- a reagent that specifically reacts with thiols is a reagent for fluorescent thiol labeling.
- a reagent for fluorescent thiol labeling is a precolumn derivatization reagent that yields detectable fluorescent adducts.
- reagents examples include 4-bromomethyl-3,6,7- trimethyl-l,5-diazabicyclo[3.3.0]octa-3,6-diene-2,8-dione (monobromobimane, mBBr) (Kosower, N.S. and Kosower, E. M., 1987, Methods Enzymology, 143:76-84) and 7- diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarine (CPM) (Molecular Probes).
- mBBr 4-bromomethyl-3,6,7- trimethyl-l,5-diazabicyclo[3.3.0]octa-3,6-diene-2,8-dione
- CCM 7- diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarine
- Detection can include any physical or chemical method of detection and/or separation.
- hydrophilic or hydrophobic properties there can be used normal or reversed-phase chromatography.
- gel-filtration chromatography When the molecular weight changes largely, gel-filtration chromatography can be utilized.
- modification of ionic groups or demodification takes place, there can be used ion-exchange chromatography.
- affinity chromatography When a_ sugar chair is added or released, affinity chromatography can be utilized.
- the reagent that detect mycothiol is a reagent for fluorescent thiol labeling, the fluorescence spectra of the reaction products can be utilized.
- the reaction products can also be quantitated.
- high performance liquid chromatography is used for separation and measurement of the reaction products (Fahey, R. C. and Newton, G.L, 1987, Methods Enzymology, 143:85-96). Quantitative determination can further be conducted by any method known to one of skill in the art, such as fluorescence method or an ultraviolet detection method.
- the reagent that detects mycothiol or precursor thereof of the invention can be used to detect an actinomycetes-associated disorder.
- the method includes contacting a sample from a subject having or at risk of having an actinomycetes-associated disorder with a reagent that detects mycothiol or precursor thereof, and detecting the reaction of the reagent.
- the reaction of the reagent with the sample is then compared to a control.
- actinomycetes-associated disorder denotes any disease associated with the presence of actinomycetes such as mycobacteria.
- An example of an actinomycetes-associated disorder is tuberculosis, which is associated with an infection of M. tuberculosis.
- any biological sample which may contain a detectable amount of mycothiol or precursor thereof can be used.
- biological samples of use with the invention are blood, serum, plasma, urine, mucous, feces, cerebrospinal fluid, pleural fluid, ascites, and sputum samples.
- Tissue or cell samples can also be used with the subject invention. These samples can be obtained by many methods such as cellular aspiration, or by surgical removal of a biopsy sample.
- the level of mycothiol or a precursor thereof in the sample can be compared with the level in a sample not affected by the disease process.
- the sample not affected by the disease process can be taken from the same subject, or can be from a control subject not affected by the disease process, or can be from a cell line.
- the subject is human.
- the antibodies of the invention can be used in any subject in which it is desirable to administer in vitro or in vivo immunodiagnosis o ⁇ immunotherapy.
- the antibodies of the invention are suited for use, for example, in immunoassays in which they can be utilized in liquid phase or bound to a solid phase carrier.
- sandwich immunometric
- the antibodies of the invention can be bound to many different carriers, both soluble and insoluble, and used to detect the presence of an antigen comprising the polypeptide of the invention.
- carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses and magnetite.
- Those skilled in the art will know of other suitable carriers for binding antibodies, or will be able to ascertain such, using routine experimentation.
- labels and methods of labeling known to those of ordinary skill in the art. Examples of the types of labels which can be used in the present invention include enzymes, radioisotopes, fluorescent compounds, colloidal metals, chemiluminescent compounds, phosphorescent compounds, and bioluminescent compounds, as described above.
- the detectably labeled antibody is given a dose which is diagnostically effective.
- diagnostically effective means that the amount of detectably labeled monoclonal antibody is administered in sufficient quantity to enable detection of the site having the antigen comprising a polypeptide of the invention for which the monoclonal antibodies are specific.
- the monoclonal antibodies or polynucleotides of the invention can be used in vitro and in vivo to monitor the course of amelioration of an actinomycetes-associated disease in a subject.
- a particular therapeutic regimen such as an antibiotic regimen, aimed at ameliorating the actinomycetes- associated disease is effective.
- the term "ameliorate” denotes a lessening of the detrimental effect of the actinomycetes-associated disease in the subject receiving therapy.
- This invention involves administering to a subject a therapeutically effective dose of a pharmaceutical composition containing the antibodies of the present invention and a pharmaceutically acceptable carrier.
- administering the pharmaceutical composition of the present invention may be accomplished by any means known to the skilled artisan.
- subject is meant any mammal, preferably a human.
- the pharmaceutical compositions are preferably prepared and administered in dose units.
- Solid dose units are tablets, capsules and suppositories.
- different daily doses are necessary. Under certain circumstances, however, higher or lower daily doses may be appropriate.
- the administration of the daily dose can be carried out both by single administration in the form of an individual dose unit or else several smaller dose units and also by multiple administration of subdivided doses at specific intervals.
- compositions according to the invention are in general administered topically, intravenously, orally or parenterally or as implants, but even rectal use is possible in principle.
- suitable solid or liquid pharmaceutical preparation forms are, for example, granules, powders, tablets, coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, creams, aerosols, drops or injectable solution in ampule form and also preparations with protracted release of active compounds, in whose preparation excipients and additives and/or auxiliaries such as disintegrants, binders, coating agents, swelling agents, lubricants, flavorings, sweeteners or solubilizers are customarily used as described above.
- the pharmaceutical compositions are suitable for use in a variety of drug delivery systems. For a brief review of present methods for drug delivery, see Langer, 1990, Science 249:1527-1533, which is incorporated herein by reference.
- compositions according to the invention may be administered locally or systemically.
- therapeutically effective dose is meant the quantity of a compound according to the invention necessary to prevent, to cure or at least partially arrest the symptoms of the disorder and its complications. Amounts effective for this use will, of course, depend on the severity of the disease and the weight and general state of the patient. Typically, dosages used in vitro may provide useful guidance in the amounts useful for in situ administration of the pharmaceutical composition, and animal models may be used to determine effective dosages for treatment of particular disorders. Various considerations are described, e.g., in Goodman And Gilman's: The Pharmacological Bases of Therapeutics. 8th ed., Pergamon Press, 1990; and Remington's Pharmaceutical Sciences. 17th ed., Mack Publishing Co., Easton, Pa., 1990, each of which is herein incorporated by reference.
- the invention provides a method for identifying samples with altered production of mycothiol or a precursor thereof.
- a "test sample” is any sample to be evaluated for the production of mycothiol or precursor thereof.
- the sample is an isolated sample of actinomycetes, such as a mycobacteria sample.
- the sample is a biological sample with a known actinomycetes infection. Such samples may be identified by conventional methods of detecting actinomycetes, such as by histological staining or by moleuclar methods such as polymerase chain reaction.
- the method of the invention can be used in order to determine ifthe actinomycetes has altered production of mycothiol or a precursor thereof. As only viable actinomycetes produce mycothiol and its thiol precursors in the reduced thiol form, the method of the invention can futher'be used to determine ifthe actinomycetes are - viable.
- the test sample is contacted with a reagent that detects mycothiol or precursor thereof for a period of time sufficient for the reagent to react with mycothiol or precursor thereof.
- the reaction of the reagent is then detected and compared to the reaction of the reagent with a control.
- the control sample can be any sample which has a defined level of production of mycothiol or precursor thereof.
- the control is a known concentration of partially purified or purified mycothiol or precusor thereof.
- the control is generated as a standard curve.
- the production of mycothiol in the sample may be increased as compared to the control.
- mycothiol or a precursor thereof is substantially increased above the defined reference level greater than or equal to a 20% increase, preferably greater than or equal to a 50% increase, more preferably greater than or equal to a 75% increase, with the most preferred being a 100% increase above the control.
- mycothiol or a precursor thereof is substantially decreased below the defined reference level greater than or equal to a 20% decrease, preferably greater than or equal to a 50% decrease, more preferably greater than or equal to a 75% decrease, with the most preferred being a 100% decrease below the control.
- the control sample can be any sample that produces a reference standard of mycothiol or precursor thereof.
- the control is a sample of actinomycetes known to produce mycothiol or precursor thereof.
- the control may be a biological fluid or sample, known to be free of actinomycetes, into which a known quantity of actinomycetes is introduced.
- known amounts of purified mycothiol or a precursor thereof may be used as a control.
- One of skill in the art will readily be able to identify suitable controls for use with the method of the invention.
- Mycothiol production is specific to the taxa actinomycetes.
- an assay may be used to assay for the presence or absense of mycothiol or a precursor thereof.
- the organism may be obtained from any sample of interest, and in particular may be cultured from a patient sample of interest, such as a blood sample, a serum sample, a urine sample, a fecal sample, a tissue biopsy, cerebrospinal fluid sample, ascites sample, pleural fluid sample, respiratory secretions including broncial secretion samples obtained by bronchoscopy and a sputum sample.
- the bacterial culture is "clonal" or derived from one cell.
- the bacterial culture is a mixed population, containing two or more different organisms or strains.
- the method includes contacting a membrane to a bacteria plated on a bacterial culture plate for a time sufficient to allow the bacteria to adhere to the membrane.
- membranes are known to one of skill in the art for the adhesion of bacteria. Specific non-limiting examples of these membranes include nitrocellulose (Nitropure) or other membranes used in for detection of bacterial gene expression including polyvinylchloride, diazotized paper and other commericially available membranes such as Genescreen.
- the adherent bacteria are then lysed by any method known to one of skill in the art.
- a specific, non-limiting example of a method to lyse bacteria is N-acetylglucosaminidase (3.1 units in 10 ml TBS adjusted to pH 4.2 with acetic acid.
- the membrane is contacted with a reagent to detect mycothiol or a precursor thereof, and the reaction of the reagent with mycothiol or a precursor thereof is detected.
- the reagent is an antibody, such as a polyclonal antibody which specifically binds mycothiol or a precursor thereof.
- the antibody can be directly labeled, or a suitable detection scheme can be - utilized.
- a labeled secondary antibody which binds to the antibody which binds to mycothiol or a precursor thereof can be can be utilized. Labels for antibodies have been described above. Many such detection schemes for an antibody are well known to one of skill in the art (see Harlow, E. and Lane, D., Antiboides: A Laboratory Mannual, Cold Spring Harbor Press, 1988, the contents of which are herein incorporated by reference).
- kits may comprise a carrier means containing one or more container means such as vials, tubes, and the like, each of the container means comprising one of the separate elements to be used in the method.
- One of the container means may comprise a reagent to detect mycothiol or precursor thereof.
- the reagent is an antibody that specifically binds to mycothiol or a precursor thereof.
- the kit may also contain a container comprising detection means, such as a container containing a detection reagent to detect the reaction of mycothiol or precursor thereof with said reagent to detect mycothiol or precursor thereof.
- the reagents for detecting mycothiol or a precursor thereof of the present invention can be included in a kit and used for examining the presence of a actinomycetes in a sample.
- the sample may be a patient sample, such as a blood sample, a serum sample, a urine sample, a fecal sample, a tissue biopsy, cerebrospinal fluid sample, ascites sample, pleural fluid sample, respiratory secretions, including broncial secretion samples obtained by bronchoscopy and a sputum sample.
- the sample can also be a bacterial sample, either a mixed population or a clonal sample.
- kits may be directly labeled.
- the kit may also contain a container containing a reporter means, such as avidin or steptavidin, bound to a reporter molecule such as an enzymatic, fluorescent, or radionucleotide label to identify the directly labeled antibody.
- the kit can utilize antibodies that bind mycothiol or a precursor thereof that are unlabeled.
- the kit may then also contain a container containing a second antibody which binds to the antibody specific for mycothiol or a precursor thereof.
- The> second antibody can be directly labeled.
- the . kit may further include a container containing a reporter means, such as avidin or steptavidin, bound to a reporter molecule such as an enzymatic, fluorescent, or radionucleotide label to identify the second antibody.
- MSH MSH
- AcCys-GlcN-Ins ACGI
- Mycobacterium smegmatis - mc 2 -6 and mc 2 -155 were provided by J. Davies (University of British Columbia, Vancouver, British Columbia).
- Carrier proteins bovine serum albumin, ovalbumin, and keyhole limpet hemocyanin, and maleimide-activated bovine serum albumin
- crosslinking reagents maleimidobenzoyl-N-hydroxysulfosuccinimide ester, N-succinimidyl-3-(2- pyridyldithio)propionate
- desalting columns Karl-activated Sepharose 6B was obtained from Pharmacia.
- Nitrocellulose membrane (0.4 ⁇ m porosity) and Tween-20 were purchased from BioRad. NitroPure supported nitrocellulose membranes (81 mm diameter circles, 0.45 ⁇ m porosity) were purchased from MSI. 96- Well microtitre plates (Immulon-4) were purchased from Dynatech.
- Goat anti-rabbit-IgG (whole molecule) secondary antibody F(ab') 2 fragments conjugated to bovine intestinal alkaline phosphatase
- fish-skin gelatin N-acetylglucosaminidase (from jack beans)
- L-cysteine hydrochloride ⁇ -acetyl-L-cysteine
- coenzyme a sodium salt
- glucosamine hydrochloride glutathione
- pantethine purchased from Sigma Chemical Co., as were the alkaline phosphatase substrates 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium ("SigmaFAST" BCIP/ ⁇ BT) and/7-nitrophcnyl phosphate.
- Monobromobimane was obtained from Molecular Probes, Inc. High-purity, heavy-metal-free dithiothreitol (DTT) was obtained from Calbiochem.
- DTT dithiothreitol
- S-Trityl-cysteine, S-trityl- ⁇ -Boc-cysteine, and 2-(5-norbornene- 2,3-dicarboximido)-l,l,3,3-tetramethyluronium tetrafluoroborate were from Calbiochem- ⁇ ovabiochem, and diisopropylethylamine from Pierce.
- S-Trityl-N-acetyl-cysteine was prepared by the general procedure given by Stewart and Young (Stewart, J.M., and Young, J.D., 1984, “Solid Phase Peptide Synthesis,” Pierce Chemical Co., Rockford, Illinois).
- Pantetheine was prepared by DTT reduction of pantethine: 3 ⁇ mol pantethine was dissolved in 30 ⁇ l ultrapure water, and a solution of 22 ⁇ mol dithiothreitol in 220 ⁇ l ultrapure water was added. The mixture was allowed to react for 15 minutes at room temperature, after which the solution was acidified to pH ⁇ 1 by the addition of 10 ⁇ l of 5 M methanesulfonic acid. Residual dithiothreitol was removed by trituration of the reaction mixture with eight portions of water-saturated ethyl acetate (250 ⁇ l each). The aqueous phase was stored frozen until use. Immediately before use, the pantetheine content was determined by titration with DTNB (Ellman, G.L., 1959, Arch. Biochem. Biophys. 82:70).
- the Boc and/or trityl protecting groups were removed using trifluoroacetic acid containing DTT which was then diluted into water before extracting with ethyl acetate (1 vol., 10 times) to remove scavenger.
- the resulting free thiol was quantified by titration with Ellman's reagent, 5,5'-dithiobis(2-nitrobenzoic acid) (DT ⁇ B) (Ellman, 1959, supra) and was characterized as the monobromobimane derivative to be an approximately 1 :2 mixture of to ⁇ epimers by HPLC (Newton et al, 1995, supra).
- Mycobacterium smegmatis mc 2 -6 was grown to early stationary phase in Middlebrook 7H9 medium supplemented with 0.5% w/v Tween-80 and 0.4% w/v glucose. MSH content was determined by HPLC analysis of the monobromobimane-labeled derivative (Newton et al., 1995, supra).
- Crude MSH was isolated from stationary-phase cultures according to the general method of Newton & Fahey (Newton, G.L., and Fahey, R.C., 1987, Methods Enzymol, 143:96). The cultures were centrifuged to pellet the cells and the supernatant discarded. The pelleted cells (297 g wet weight) were lysed by homogenization in 2.0 1 warm (60°C) 50% v/v aqueous acetonitrile containing 25 mM methanesulfonic acid, followed by 15 minutes incubation at 60°C. During subsequent steps, extracts were kept chilled on ice.
- the crude extract was purified by reversed-phase HPLC on a preparative C 18 column, 25 mm x 250 mm (Vydac) by isocratic elution with 0.1% trifluoroacetic acid (TFA) in ultrapure water at a flow rate of 10 ml/min. Under these conditions MSH eluted as the free thiol at 8 minutes and as its disulfide (MSSM) at 12 minutes. Thiol concentration was determined by titration with DTNB. Purified MSH was stored as a concentrated (0.11 M) stock solution in 0.1%) TFA (aqueous), frozen at -70°C. Under these conditions MSH was stable as the free thiol for many months.
- TFA trifluoroacetic acid
- mycothiol is to be crosslinked by its sulfhydryl functionality to the carrier proteins in order to maximize surface presentation of mycothiol's novel disaccharide moiety, as well as to take advantage of the specific reactivity of the sulfliydryl group.
- BSA bovine serum albumin
- KLH keyhole limpet hemocyanin
- Purified MSH from Mycobacterium smegmatis was conjugated to keyhole limpet hemocyanin (KLH) by treatment with the bifunctional crosslinking reagent, maleimidobenzoyl- N-hydroxysulfosuccinimide ester (sulfo-MBS).
- KLH keyhole limpet hemocyanin
- sulfo-MBS maleimidobenzoyl- N-hydroxysulfosuccinimide ester
- the sulfo-MBS was first reacted with KLH, and then the MBS-activated KLH reacted with MSH at both a low and a high degree of coverage.
- HPLC analysis of the monobromobimane-labelled residual MSH showed good yields from the crosslinking reactions, estimated to be -90-100% (low-coverage conjugates) and -85% (high- coverage conjugates).
- the sulfo- MBS-activated KLH was divided into two equal aliquots (each containing 2 mg activated KLH in 1100 ⁇ l).
- Purified MSH (either 0.303 ⁇ mol or 1.212 ⁇ mol MSH) was added to 100 ⁇ l conjugation buffer and this solution was added to the KLH suspensions. The mixtures were reacted for 2 h at room temperature and then concentrated to -400 ⁇ l by centrifugation on Centricon ultrafilters, 2 h at 770x g at 4 °C.
- the conjugates were stored in -500 ⁇ g aliquots, frozen at -20 °C.
- the efficiency of crosslinking was determined by HPLC analysis of the monobromobimane-labeled residual MSH from the Centricon filtrates.
- the linker arm on this material is 12 atoms long, permitting a flexible presentation of the antigen to the antibodies. It is structurally distinct from the linker used in preparing the antigen (Equation 1) and was therefore expected to have lower affinity for antibodies directed to the antigen linker region.
- the chromatography was monitored by dot-blots; the purified MSH- specific antibody fractions were pooled, concentrated, and stored frozen in aliquots.
- the affinity resin was prepared as follows: 1 g epoxy-activated Sepharose 6B (Pharmacia) was suspended in 200 ml ultrapure water, fined, transferred to a sintered glass funnel, and washed under mild vacuum with 100 ml 0.1 M carbonate buffer, pH 8.5, to give 3 ml swelled gel which was transferred to a conical tube. Pure MSH (40 ⁇ mol) in 364 ⁇ l 0.1% TFA (aqueous) was added to 364 ⁇ l 0.1 M carbonate buffer and the pH of the mixture adjusted to 8.5. This was immediately added to 3 ml Sepharose, the suspension was mixed vigorously and incubated for 22 h at room temperature on a rotating mixer.
- the gel was then transferred to a sintered glass funnel, rinsed with 30 ml carbonate buffer, and transferred back to a conical tube. Determination of the unbound MSH that remained showed that a total of 35 ⁇ mol had reacted, corresponding to approximately one-third of the available epoxy groups. The remaining epoxy groups were blocked by reaction with 7 ml of 1 M ethanolamine, pH 9.5, for 4 h at room temperature.
- the MSH-Sepharose was then rinsed on a sintered glass funnel with alternate 30 ml rinses (3 each) of 0.1 M acetate buffer, pH 4.0 and 0.1 M carbonate buffer, pH 8.3 (each containing 0.5 M NaCl). Finally, the MSH-Sepharose gel was washed with 100 ml PBS, pH 7.2, 0.04%) NaN 3 , and stored at 4 °C.
- a typical affinity-purification follows: 3 ml MSH-Sepharose was slurry-packed with PBS in a 10 mm-i. d. column, degassed, and allowed to settle to give a bed height of -40 mm. All of the following steps were carried out at 4 °C. The column was washed sequentially with 30 ml each of: 10 mM Tris, pH 7.5; 100 mM glycine, pH 2.5; 10 mM Tris, pH 8.8; and 100 mM triethylamine, pH 11.5. The column was then rinsed with 60 ml PBS.
- the ammonium sulfate IgG fractions (-15 ml) were thawed and passed through the column four times at 15 ml/h. Unbound serum was saved for dot blot analysis. Next, the column was washed with 60 ml of 10 mM Tris, pH 7.5, followed by 60 ml of 10 mM Tris, pH 7.5, 0.5 M NaCl. MSH-specific IgG's were eluted with both low-pH (100 mM glycine, pH 2.5) and high-pH (100 mM triethylamine, pH 11.5) buffers.
- Eluted fractions were analyzed by dot blots for MSH-specific IgG; fractions containing the purified antibodies were combined, dialyzed against PBS/0.04% NaN 3 , and stored frozen in aliquots at -20 °C.
- the following presents an exemplary but not exhaustive list of reagents that may be used . to bind mycothiol to a carrier protein for the purpose of raising antibodies. Sulfo-MBS was used for this purpose. The following is a list of possible substitute reagents. The "Sulfo-" reagents are more water soluble than the parent reagents, but have the same chemistry. The following comes from the Pierce Chemical catalog which is a common source for such reagents.
- Sulfo-SIAB Sulfosuccinimidyl4-(N-maleimidomethyl)-cyclohexane- 1 - carboxylate sulfo-SMPB Sulfosuccinimidyl4-(p-maleimidophenyl)-butyrate
- the following reagents are examples that could be used to link mycothiol to ovalbumin for use in the dot blot assays described above to identify fractions of IgG that contain MSH antibodies elicited by KLH-BM-SM. SPDP was used in the studies described.
- Antibodies were raised in female New Zealand rabbits by immunization with the KLH- BS-SM conjugates. After pre-immune serum samples were taken, the rabbits were immunized with 500 ⁇ g of conjugate in 500 ⁇ l PBS plus 500 ⁇ l Freund's complete adjuvant. The rabbits received a second immunization (identical to the initial immunization) after three weeks. Subsequent immunizations consisted of conjugate plus Freund's incomplete adjuvant. Blood samples were taken from the rabbits three weeks after the second immunization and ten to fourteen days after each subsequent immunization.
- IgG fractions from rabbit sera were isolated by ammonium sulfate precipitation in two steps (33% and 50% saturation), followed by dialysis against PBS. IgG fractions were stored frozen at -20 °C. Each IgG ammonium sulfate preparation was tested for the presence of MSH- specific antibodies by dot blots on nitrocellulose membranes.
- the BioDot (BioRad) apparatus was used in most cases. Generally, the Tris-buffered saline (TBS, pH 7.3) pre-moistened nitrocellulose membrane was assembled in the apparatus and the test antigens (0.1 to 1 ⁇ g/well) applied to the wells.
- the membrane was blocked with 200 ⁇ l/well 1% fish skin gelatin in TBS before application of the sera. After washing unbound material through the membrane with Tris- buffered saline containing 0.05% v/v Tween-20 (TBST), 100 ⁇ l/well of the secondary antibody (goat anti-rabbit-IgG F(ab')2-alkaline phosphatase, 1:30,000 in TBS) were added. Unbound secondary antibody was washed through the membrane with TBST. The entire membrane was then removed from the apparatus, washed with TBS, and covered with a solution of alkaline phosphatase substrate ("SigmaFAST" BCIP/NBT). After spots appeared (typically in 1-2 minutes), the membrane was washed in distilled water and air-dried.
- TBST Tris- buffered saline containing 0.05% v/v Tween-20
- MSH crosslinker
- KLH carrier protein
- Sera from rabbits immunized with BSA-BM-SM was also tested by dot blots (using KLH-PS-SM as the test antigen to determine MSH-specificity). Although some specific antibody production was observed, these conjugates did not elicit as strong a response in rabbits as did the KLH-BM-SM conjugates, and the sera from these rabbits was not used.
- the molecule to be detected must be immobilized and subsequently detected.
- mycothiol there are two general methods for accomplishing these steps.
- mycothiol is bound via its thiol functionality using thiol-specific chemistry, and subsequently detected by means of an antibody specific to the non-thiol moiety of mycothiol.
- mycothiol is bound by means of an antibody specific for the non-thiol moiety of mycothiol, and the thiol funtionality subsequently utilized in the detection step (for example, by attaching to the thiol functionality a detectable substance).
- the following immunoassay architecture illustrates an example of the first general method.
- MSH is allowed to react with maleimide-activated bovine serum albumin ("mal-BSA").
- the MSH-mal-BSA conjugate is allowed to bind by non-specific absorption to a microtiter plate.
- Anti-MSH antibody is allowed to bind to the immobilized mycothiol.
- Alkaline phosphatase-labelled goat-anti-rabbit IgG (F(ab')2 fragments) is allowed to bind to the anti-MSH antibody, and pNPP is used to quantitate the amount of bound alkaline phosphatase.
- Other reporter enzyme-secondary antibody conjugates may be used.
- maleimide-activated BSA Pieris-activated BSA
- maleimide activation levels were 16-19 mol maleimide per mol BSA.
- the maleimide-activated BSA was reconstituted to 10 mg/ml and this stock solution stored at -70 °C until needed. The stock solution was diluted to a working concentration of 5 ng/ ⁇ l just before use and kept chilled on ice.
- TBS Tris-buffered saline: 0.1 M Tris, pH 7.3, 0.15 M NaCl, 0.04% NaN ⁇
- a "zero" standard consisted of TBS in place of an MSH solution.
- 168 ⁇ l of thiol standard were added to 252 ⁇ l of maleimide-activated BSA solution.
- Washes involved filling wells to the top with wash buffer followed by gentle aspiration to drain the wells.
- Primary antibody affinity-purified anti-MSH, 100 ⁇ l per well
- Secondary antibody goat anti-rabbit [whole IgG] F(ab') 2 fragments conjugated to bovine intestinal alkaline phosphatase, 100 ⁇ l per well
- the wells were drained, washed twice with TBST, twice with TBS, and drained.
- the alkaline phosphatase substrate (3.8 mM /7-nitrophenylphosphate in 0.2 M Tris, pH 9.8, 200 ⁇ l per well) was added and the plate incubated 30 minutes at room temperature. The reaction was quenched by addition of 50 ⁇ l per well stop solution (4 N NaOH). The plate was read at 405 nm on a microplate reader (Model EL3 ⁇ l 1 , Bio-Tek Instruments) that was programmed to automatically subtract blank values read from wells that received only the blocking agent, wash solutions, p-nitrophenyl phosphate, and stop solution. The difference in optical density was calculated for wells that had received known amounts (0.1 to 1.0 pmol per well) of MSH by subtracting from these values the mean optical density of wells that had received the "zero" standard.
- the ELISA protocol described above was based upon the results of assays in which the amounts of maleimide-activated BSA, primary antibody, secondary antibody, and blocking protein were varied, and upon the testing of different types of secondary antibody, blocking reagent, microtiter plates, washing protocols, and incubation times.
- the adopted protocol as described above measures pure MSH in buffer with the sensitivity and linearity illustrated in Figure 2.
- This ELISA has a useful range of about 0.1 - 1 pmol MSH, and a detection limit of at least 0.1 pmol MSH, and thus is approximately an order of magnitude more sensitive than the HPLC assay of monobromobimane-labelled MSH (Newton et al, 1993, J. Bacteriol, 175:2734 .
- Example 4 The immunoassay described in Example 4 was used in the ELISA format to analyzed cell lysates for their mycothiol content. In most experiments, parallel mycothiol determinations were made using HPLC analysis of monobromobimane-labelled cell lysates. A typical procedure follows.
- Mycobacterium smegmatis mc 2 -6 was grown in Middlebrook 7H9 medium supplemented with 0.4% w/v glucose and 0.05% v/v Tween-80.
- a conversion factor of 0.25 x 10 9 colony- forming units per mL (CFU/mL) per absorbance unit measured at 600 nm was used to determine cell density.
- Cells used in experiments were harvested at early- to mid-log phase growth. Typically, cells were diluted in phosphate-buffered saline, pH 7.2 (PBS) to a desired cell density. Cell samples were centrifuged 5 min at 12,000x G. Most of the supernatant was removed and discarded, leaving the cell pellet in a volume of about 100 ⁇ L.
- PBS phosphate-buffered saline
- a small volume of PBS or Tris-buffered saline (TBS) was added, and the cells resuspended.
- Acetonitrile was added to a final concentration of 50% v/v, the samples vortexed, and incubated 10 min at 60 °C to lyse the cells.
- the lysates were centrifuged 45 sec at 12,000x G to pellet cell debris, and an aliquot (40 ⁇ L per well) of the supernatant immediately added to a freshly-made solution of maleimide- activated BSA (60 ⁇ L per well of a 5 ng/ ⁇ L solution in TBS).
- a membrane-based assay based on the immunoassay architecture described in Example 4 was developed for detecting MSH production by bacterial colonies grown on solid media.
- the following protocol is typical for a single 100 mm Petri dish. All incubations were done at room temperature on an orbital platform shaker set to 90 rpm unless otherwise noted.
- Different bacteria including two strains of Mycobacterium smegmatis (mc -6 and mc 2 -155) and several non-MSH-producing species ⁇ Escherichia coli HB101 , Enterococcus faecalis ATCC 29212, and Streptococcus mutans ATCC 33402) were grown as separate streaks on a single agar dish.
- a supported nitrocellulose membrane circle (“NitroPure", 0.45 ⁇ m porosity, 81 mm diameter) was marked with a pencil for orientation on the plate, and pre-soaked in TBS. Excess TBS was drained from the membrane, a freshly-made solution of Pierce Imjcct maleimide-activated BSA (265 ⁇ g in 13.3 ml TBS, to give 5 ⁇ g/cm 2 loading) added, and the membrane incubated for 30 min. Excess liquid was drained from the membrane, which was then laid onto the surface of the bacterial plate with care to avoid bubbles or smearing of the bacterial colonies.
- the membrane was lifted carefully and laid bacteria-side up for 1 h in a clean glass Petri dish containing a solution of N-acetylglucosaminidase (3.1 units in 10 ml TBS adjusted to pH 4.2 with acetic acid). The membrane was next washed briefly with TBS to remove adhering cells and washed with 10 ml TBST. Excess liquid was drained and the membrane incubated in 10 ml 2% fish skin gelatin in TBS for 2 h. The membrane was drained, 10 ml affinity-purified anti-MSH IgG solution (containing ⁇ 18 ⁇ g total protein) added and incubated overnight at 4 °C on an orbital platform shaker set to 60 rpm.
- the antibody solution was aspirated and the membrane washed 3 times in TBST (10 ml and 10 min for each wash). Excess liquid was drained, 10 ml of secondary antibody (goat anti-rabbit [whole IgG] F(ab') 2 fragments conjugated to bovine intestinal alkaline phosphatase, diluted 1 :15000 in TBS) added, and the membrane incubated for 1 h. The membrane was drained, washed twice in TBST and thrice in TBS (10 ml and 5 min each wash). Development was with BCIP-NBT (SigmaFAST). After thoroughly washing in distilled water, the blot was air-dried. MSH-containing bacteria are revealed as dark purple stains; only the two strains of M. smegmatis produced positive signals (Figure 3).
- Immobilization of MSH for subsequent detection can also be achieved by taking advantage of the unique chemistry of the thiol functionality to link it to a binding agent.
- the binding agent in this example biotin, is chosen based upon the availability of a substance, in this example a modified form of avidin, having a high affinity for the binding agent.
- the binding agent is linked to a thiol-reactive group, in this example maleimide, which reacts with the thiol moiety of mycothiol.
- the following assay architecture illustrates an example of this method.
- MSH is biotinylated with a thiol-specific reagent.
- Biotin is an example of a small molecule that can be bound specifically by a protein (in this case, avidin); biotin-avidin systems offer the advantage of extremely strong non-covalent binding.
- biotinylated mycothiol is captured by avidin which has previously been attached to the microtiter plate.
- Unmodified avidin can be replaced by modified (e. g., deglycosylated) avidin products that exhibit lower non-specific binding than unmodified avidin.
- Anti-MSH antibody is allowed to bind to the immobilized biotinylated mycothiol.
- Alkaline phosphatase-labelled goat-anti-rabbit IgG (F(ab')2 fragments) is allowed to bind to the anti-MSH antibody, and pNPP is used to quantitate the amount of bound alkaline phosphatase.
- Other reporter enzyme-secondary antibody conjugates may be used.
- biotinylated MSH suitable for use as a standard, the following procedure was carried out: 5.0 ⁇ L of 3.7 mM pure MSH in 0.1% TFA was added to 12.3 ⁇ L 1.5 mM MPB in DMSO/acetonitrile/100 mM phosphate buffer, pH 7.0 (1:4:5). The mixture was allowed to react 1 hour at room temperature. To determine the extent of biotinylation, unreacted MSH was measured by titration with DTNB. The standard was stored in 1.0 ⁇ L aliquots frozen at -70 °C.
- the standard was diluted to a concentration of 1.0 ⁇ M in Tris- buffered saline, pH 7.3, containing 0.04% w/v sodium azide, 0.05% v/v Tween 20 and 0.1% w/v BSA (TBSTB); this solution was stable for several (>6) months when stored refrigerated as indicated by consistency of ELISA measurements.
- NeutrAvidin (Pierce) was diluted in TBS to a final concentration of 6 ng/ ⁇ L and 100 ⁇ L aliquots (equivalent to 600 ng NeutrAvidin) added to each well of a microtiter plate (Immulon 4). The sealed plate was incubated overnight at 4 °C.
- the wells were drained and washed once with TBST.
- the antigen biotinylated MSH, see above
- TBSTB 0.5% w/v BSA
- CH3CN 0.5% w/v BSA
- the wells were washed once with TBST. 100 ⁇ L of affinity-purified anti-MSH, diluted to a final concentration of 0.5 ng/ ⁇ L in TBSTB, was added to each well. The plate was incubated 2 hours at 37 °C. 6. The wells were washed twice with TBST. 100 ⁇ L of goat anti-rabbit IgG, alkaline phosphatase-labelled F(ab') 2 fragments (Sigma), diluted 1:1500 in TBSTB, was added to each well. The plate was incubated 1 hour at room temperature.
- the wells were washed four times in TBST.
- the plate was then developed with 200 ⁇ L per well of freshly-made pNPP (1 mg/mL in 1 M diethanolamine, pH 9.8, containing 0.4 mM MgCl 2 ). Development was stopped with the addition of 50 ⁇ L per well of 4 M NaOH. Absorbance was measured at 405 nm.
- Figure 4a shows results from an experiment that demonstrated the effect of increasing the percentage by volume of organic solvent (i. e., acetonitrile) in the buffer ("binding buffer") used for the antigen-binding step.
- acetonitrile organic solvent
- FIG. 4b shows results from an experiment that demonstrated the effect of increasing the percentage by volume of organic solvent (i. e., acetonitrile) in the buffer (“binding buffer”) used for the antigen-binding step.
- acetonitrile i. e., acetonitrile
- Increasing the binding buffer's acetonitrile content from 0% to 25% by volume results in an approximate doubling of the observed signal.
- Further experiments demonstrate that increasing the binding buffer's acetonitrile content from 25% to 50% by volume results in an additional increase in signal of approximately 170%. No increase in background noise is observed by the addition of acetonitrile to the binding buffer. Therefore, the inclusion of acetonitrile in the binding buffer is an important method for increasing
- Figure 4b shows results from an experiment that demonstrated the increase in signal and negligible increase in background resulting from an extended (120 minute) development period.
- Our initial experiments show this immunoassay method to be highly sensitive with a useful range (dependent primarily on development time) of about 5 - 300 fmol MSH, and a detection limit of at least 5 fmol MSH, that is to say, about 3 orders of magnitude more sensitive than our HPLC assay.
- Example 8 The immunoassay described in Example 8 was adapted for use in a dot blot assay. An example of a specific experimental procedure for this type of immunoassay follows.
- PVDF polyvinylidene fluoride
- the pre-wet membrane was assembled in the dot blot apparatus (BioRad BioDot) according to the manufacturer's instructions, and the membrane re-wetted by passing 50 ⁇ L/well TBS by vacuum.
- NeutrAvidin (Pierce) was diluted in TBS to a final concentration of 0.4 ⁇ g/ ⁇ L. A 50 ⁇ L aliquot (equivalent to 20 ⁇ g NeutrAvidin) followed by a wash of 20 ⁇ L TBS was added to each well and allowed to pass through the membrane by gravity.
- the membrane was blocked with 100 ⁇ L per well of a 2% (v/v) fish-skin gelatin solution in TBS, allowed to pass through by gravity.
- the membrane was washed with two 400 ⁇ L volumes per well of TBS containing 0.05% v/v Tween 20 ("TBST”), passed through by vacuum.
- the antigens biotinylated mycothiol, see Example 8) were prepared in 50% (v/v) acetonitrile in phosphate-buffered saline, pH 7.2, quadruplicate samples from 0 to 0.3 pmol applied per well in 500 ⁇ L volumes, and allowed to pass through by gravity. Blank wells received only the buffers and blocking agent.
- TBS 0.05% v/v Tween 20
- Affinity-purified anti-MSH was diluted to a final concentration of 1 ng/ ⁇ L in TBST containing 0.1% w/v BSA ("TBSTB”), and 50 ⁇ L added to each well and allowed to pass through by gravity.
- TSTB 0.1% w/v BSA
- the membrane was washed with two 400 ⁇ L volumes per well of TBS containing 0.05% v/v Tween 20 ("TBST”), passed through by vacuum.
- the membrane was washed with two 400 ⁇ L volumes per well of TBS containing 0.05% v/v Tween 20 ("TBST”), passed through by vacuum. The membrane was removed from the dot blot apparatus amd washed in three 50 mL volumes of TBS. Excess TBS was drained, and to the wet membrane was added 10 mL of a freshly-made solution of 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium ("SigmaFAST" BCIP/NBT alkaline phosphatase substrate). The purple color was allowed to develop for 3 minutes, after which excess substrate was washed from the membrane by rinsing in deionized water.
- results from this type of assay demonstrated that the assay had a detection limit of about 0.05 pmol (50 fmol) per well. Though it does not have the working range of an ELISA type assay (as described in Example 8), it is useful for qualitative detection of mycothiol, for example in the assay of bacterial cell lysates for the presence of mycothiol.
- Immobilization of mycothiol for subsequent detection can be achieved by first modifying mycothiol with a detectable label, capturing the modified MSH via an anti-MSH antibody prebound to a solid phase, and finally detecting the label.
- One specific method binding of biotinylated MSH to anti-MSH antibody prebound via protein A to a solid phase
- this assay architecture is outlined below.
- Protein A is bound to a high-binding microtiter plate by non-specific adsorption.
- MSH is biotinylated with the thiol-specific reagent 3-(N-maleimidopro ⁇ ionyl)biocytin ("MPB").
- the biotinylated MSH is captured by the anti-MSH antibody and thus immobilized on the plate.
- Alkaline phosphatase-labelled avidin is allowed to bind to the immobilized biotinylated mycothiol, and p ⁇ PP is used to quantitate the amount of bound alkaline phosphatase. (Other avidin derivatives could be used to quantitate the amount of captured MSH-biotin.)
- the wells were thoroughly drained by aspiration with gentle vacuum, and blocked with 400 ⁇ L of a 1% (v/v) solution of fish-skin gelatin (Sigma) in TBS, 1 h @ r. t.
- the wells were drained, 100 ⁇ L of primary antibody (2 ng/ ⁇ L affinity-purified rabbit anti-MSH IgG in TBSTB) added per well, the plate re-sealed and incubated at least overnight (>14 h) at 4 °C.
- the wells were drained and washed once with 400 ⁇ L/well TBS containing 0.04% w/v sodium azide and 0.05% v/v Tween 20 (TBST).
- the antigens (standards or unknowns), diluted to contain an identical percentage of CH3CN, were added to the wells, the plate sealed, and incubated 3.5 h at 37 °C.
- Wells were drained, washed once with 400 ⁇ L/well TBST, 100 ⁇ L/well of alkaline phosphatase-labelled avidin (Sigma ExtrAvidin- Alkaline Phosphatase) at a concentration of 0.5 ng/ ⁇ L in TBSTB added, and the plate sealed and incubated 1 h at r. t.
- Wells were drained, washed once with 400 ⁇ L/well TBST, then three times with 200 ⁇ L/well TBST.
- the first three wash volumes were removed by sharply tapping the plate upside down, and the last wash volume removed by aspiration.
- Two ⁇ L (1 ng) of the alkaline phosphatase-labelled avidin solution were added to two of the wells that received only the blocking solution and wash buffers.
- the plate was then developed with 200 ⁇ L per well of freshly-made pNPP (1 mg/mL in 1 M diethanolaminc, pH 9.8, containing 0.4 mM MgCl 2 ). Absorbance was measured at 405 nm. For time-point readings, the plate was read at successive time points without the addition of any stop solution. For a single end-point reading, pNPP development was stopped with the addition of 50 ⁇ L per well of 4 M NaOH.
- Figure 9 shows results from an immunoassay utilizing the above procedure.
- Wells received 600 ng protein A, 200 ng anti-MSH antibody, and 50 ng alkaline phosphatase-labelled avidin.
- Antigens were applied in 12.5% CH3CN in TBS. The data shown are from 60 min development with pNPP and no stop solution.
- a further variation utilizing the immunoassay architecture described in Example 10 involves carrying out the biotinylation reaction in a non-homogeneous mixture consisting of the aqueous cell suspension and a solution of the biotinylating reagent in an organic solvent that is immiscible with water.
- An important advantage of this method is that, because the organic solvent used for dissolving the biotinylation reagent is not miscible with water, subsequent to biotinylation, the aqueous phase (now containing the biotinylated mycothiol) can be removed from the organic phase and analyzed with no need for dilution.
- the method of capture and detection of antigen is identical to that previously described.
- the first example demonstrates detection of mycothiol from mycobacterial cells in human urine.
- the second example demonstrates detection of mycothiol from mycobacterial cells in human cerebrospinal fluid. This second example also demonstrates the effectiveness of adding nutrients such as glucose and glycerol to the sample in order to increase the mycothiol content of the bacterial cells.
- Mycobacterium avium was grown in Middlebrook 7H9 medium supplemented with 0.4% w/v glucose and 0.05% v/v Tween-80. Cells used in experiments were harvested at early- to mid-log phase growth, and diluted in fresh medium appropriately to give concentrations ranging from -3 x 10 3 cfu to 3 x 10 4 cfu in a volume of 10 ⁇ L. To each micro fuge tube was added 10 ⁇ L cell suspension and 990 ⁇ L sterile-filtered urine. The tubes were capped, vortexed, centrifuged 10 minutes at 13,000 rpm, and 990 ⁇ L of supernatant carefully removed without disturbing the pelleted cells.
- the ELISA procedure is identical to that described in Example 10 above, except that as the samples are essentially aqueous and could be applied to the microtiter wells without further dilution, the standards are prepared in 20 mM phosphate buffer, pH 8.0
- Figure 10 shows results from an immunoassay utilizing the above procedure.
- Wells received 600 ng protein A, 200 ng anti-MSH antibody, and 50 ng alkaline phosphatase-labelled avidin.
- Samples were 1.00 mL volumes of Mycobacterium avium cells in urine, biotinylated as described above, and 100 ⁇ L of the aqueous phase analyzed. The data shown are from 40 min development with pNPP and no stop solution.
- Mycothiol recovered from Mycobacterium avium cells seeded into urine using this protocol was 68% of that found by direct analysis of samples from the same stock of cells in phosphate buffer without centrifugation. Independent analysis by plating indicated a similar recovery from centrifugation and resuspension of the cells so most of the -35% loss can be attributed to the centrifugation step.
- Biotinylation of MSH from Mycobacterium avium cells in cerebrospinal fluid Mycobacterium avium was grown in Middlebrook 7H9 medium supplemented with 0.4% w/v glucose and 0.05% v/v Tween-80. Cells used in experiments were harvested at early- to mid-log phase growth, and diluted in fresh medium appropriately to give concentrations ranging from -3 x 10 3 cfu to 3 x 10 4 cfu in a volume of 10 ⁇ L.
- Sterile-filtered cerebrospinal fluid (“CSF”) was divided into two portions, one of which (referred to as "enriched CSF") received the addition of 1% (v/v) glycerol and 0.5% (w/v) glucose.
- the ELISA procedure is identical to that described immediately above for samples from human urine.
- FIG. 11 shows results from an immunoassay utilizing the above procedure.
- Wells received 600 ng protein A, 200 ng anti-MSH antibody, and 50 ng alkaline phosphatase-labelled avidin.
- Samples were 1.00 mL volumes of Mycobacterium avium cells in either cerebrospinal fluid or enriched cerebrospinal fluid, biotinylated as described above, and 100 ⁇ L of the aqueous phase analyzed.
- the data shown are from 40 min development with p ⁇ PP and no stop solution. Note that the addition of glycerol and glucose to the mycobacterial cell suspension roughly doubles the amount of mycothiol detected.
- EXAMPLE 12 shows results from an immunoassay utilizing the above procedure.
- Wells received 600 ng protein A, 200 ng anti-MSH antibody, and 50 ng alkaline phosphatase-labelled avidin.
- Samples were 1.00 mL volumes of Mycobacterium avium cells in either cerebrospinal
- the immunoassay methods described above can be adapted easily for use in screening large numbers of bacteria, for example a library of strains grown in a microtiter plate.
- the specific example given here makes use of the immunoassay architecture described in Example 10.
- the strains were grown to early logarithmic phase in Middlebrook 7H9 medium supplemented with 0.4% w/v glucose and 0.05% v/v Tween-80. Each strain was diluted in medium to an initial concentration of 10 8 cfu/mL, then further diluted in series as required. To each well of an Immulon-4 96-well microtiter plate was added a 100 ⁇ L aliquot of these serially diluted cell suspensions, so that the number of cells per well encompassed the range 0 to 10 7 cells (in duplicate) for each of the three strains. A 10 mM solution of 3-(N-maleimidopropionyl)biocytin (MPB) in DMSO was prepared shortly before use.
- MPB 3-(N-maleimidopropionyl)biocytin
- the ELISA procedure was carried out as described in Example 10.
- the ELISA plate wells were coated with protein A, gelatin, and anti-MSH antibody, and washed once with TBST as described.
- the drained ELISA wells each then received 75 ⁇ L TBS and 25 ⁇ L of the biotinylated cell extracts; each thus contained 25/260 or 9.6% of the original biotinylated cell extract volume, in a final CH3CN concentration of 12.5%. Standards were also applied in 12.5% acetonitrile in TBS. The rest of the analysis was performed as described in Example 10.
- Figure 8 shows results from an immunoassay utilizing the above procedure.
- Wells received 600 ng protein A, 200 ng anti-MSH antibody, and 50 ng alkaline phosphatase-labelled avidin. Both standards and biotinylated cell extracts were applied in a final CH3CN concentration of 12.5%. The data shown are from 60 min development with pNPP and no stop solution.
- Immobilization of MSH for subsequent detection can also be achieved by taking advantage of the unique chemistry of the thiol functionality to link it to a binding agent.
- the binding agent in this example biotin, is chosen based upon the availability of a substance, in this example a modified form of avidin, having a high affinity for the binding agent.
- the binding agent is linked to a thiol-reactive group, in this example maleimide, which reacts with the thiol moiety of mycothiol.
- the following assay architecture illustrates an example of this method.
- MSH is biotinylated with a thiol-specific reagent (see EXAMPLE???, antibody capture of biotinylated MSH).
- biotinylated mycothiol is captured by avidin which has previously been attached to the rnicrotiter plate.
- Unmodified avidin can be replaced by modified avidin products that exhibit lower non-specific binding than unmodified avidin products that exhibit lower non-specific binding than unmodified avidin.
- Anti-MSH antibody is allowed to bind to the immobilized biotinylated mycothiol. 5.
- Other reporter enzyme-secondary antibody conjugates may be used.
- NeutrAvidin (Pierce) was diluted in TBS to a final concentration of 6 ng/ ⁇ L and lOO ⁇ L aliquots (equivalent to 600 ng NeutrAvidin) added to each well of a microtiter plate (Immulon 4).
- the wells were washed four times in TBST.
- the plate was then developed with 200 ⁇ L per well of freshly-made pNPP (1 mg/mL in 1 M diethanolamine, pH 9.8, containing 0.4 mM MgCl 2 ). Development was stopped after 30 minutes with the addition of 50 ⁇ L per well of 4 M NaOH. Absorbance was measured at 405 nm.
- FIG. 6 shows results from an immunoassay utilizing the above procedure.
- This particular experiment demonstrates the effect of increasing the percentage by volume of organic solvent (i.e., acetonitrile) in the buffer (“binding buffer”) used for the antigen-binding step.
- acetonitrile organic solvent
- Increasing the binding buffer's acetonitrile content from 0% to 25% by volume results in an approximate doubling of the observed signal.
- Further experiments demonstrate that increasing the binding buffer's acetonitrile content from 25% to 50% by volume results in an additional increase in signal of approximately 170%. No increase in background noise is observed by the addition of acetonitrile to the binding buffer. Therefore, the inclusion of acetonitrile in the binding buffer is an important method for increasing the overall sensitivity of this assay.
- Our initial experiments show this method to be highly sensitive with a useful range of at least 0-300 fmol MSH, and a detection limit of at least 30 fmol MSH.
- the sample was pelleted by brief centrifugation in a microfuge at 4 C and the supernatant removed to an Eppendorf microcentrifuge tube on ice.
- a 15 ⁇ l aliquot was immediately taken from the supernatant for derivatization with AccQ-Fluor by addition of 12.5 ⁇ l of 1 M HEPES, pH 8.0, 42.5 ⁇ l of water, 23.8 ⁇ l of acetonitrile, and 31.2 ⁇ l of 10 mM AccQ-Fluor, and the mixture vortexed immediately.
- a second 30 ⁇ l aliquot of cell extract was taken at the same time and prepared in the same fashion except that the volumes of water and acetonitrile added were 35 and 16.8 ⁇ l, respectively.
- FIG. 14 Figure bottom panel illustrates the amine analysis from a cell pellet of Micromonospora echinospora. The cell pellet was dried in a tared tube to determine the residual dry weight (RDW) of the extract.
- Cellular thiol levels were analyzed by HPLC after fluorescent labeling of the thiol moiety with ⁇ iBBr (Newton et all 995 supra). Quantitative values for the cellular thiol levels were obtained after correction for any non-thiol fluorescent background identified in control samples in which thiols were blocked by reaction with NEM prior to treatment with mBBr. Standards were prepared by labeling of the commercial (Cys, N-acetyl-L-Cys) or synthetic (Cys-Glc ⁇ , N- acetyl-L-Cys-Glc ⁇ ) thiols, or of isolated MSH (Newton et al. 1995 supra).
- Cys-GlcN-Ins A standard for labeled Cys-GlcN-Ins was obtained by partial hydrolysis of MSmB, purification of CySmB- GlcN-Ins by preparative HPLC, and quantitation of the standard based upon the absorbance of the bimane label. All of these bimane derivatives could not be separated from each other, from reagent derived components, and from fluorescent cellular materials using a single HPLC protocol and many different HPLC separations were tested before two methods were found which provided analyses for the thiols of interest without major coeluting peaks in the NEM control sample.
- Fig. Fig. illustrates the analysis for Cys, N-acetyl-L-Cys, N-acetyl-L-Cys- Glc ⁇ , MSH, and H 2 S; Cys-Glc ⁇ -Ins and Cys-Glc ⁇ were only slightly retained and could not be separated under these conditions.
- the peaks for Cys and MSH have small coeluting peaks present in the ⁇ EM control (Fig. panel ) which amounted to 10 and 2%, respectively, of the Cys and MSH peaks measured in the sample (Fig. panel 2).
- 2 smegmatis mc 155 revealed the presence of a measurable GlcN-Ins content.
- Mycobacterium avium was grown in Middlebrook 7H9 medium supplemented with 0.4%) w/v glucose and 0.05% v/v Tween-80.
- Cells used in experiments were harvested at early- to mid-log phase growth, and diluted in fresh medium appropriately to give concentrations ranging from -3 x 10 cfu to 3 x 10 cfu in a volume of 10 ⁇ L.
- Sterile-filtered cerebrospinal fluid (“CSF”) was divided into two portions, one of which (referred to as "enriched CSF") received the addition of 1% (v/v) glycerol and 0.5% (w/v) glucose.
- enriched CSF Sterile-filtered cerebrospinal fluid
- To each microfuge tube was added 10 ⁇ L cell suspension and 990 ⁇ L of either CSF or enriched CSF.
- the tubes were capped, vortexed, centrifuged 10 minutes at 13,000 rpm, and 990 ⁇ L of supernatant carefully removed without disturbing the pelleted cells.
- To the residual 10 ⁇ L in each tube was added 100 ⁇ L 10 mM phosphate buffer, pH 7.2, followed by 5.4 ⁇ L 44.4 mM EGTA in 267 mM phosphate buffer, pH 9.0. The final pH of this mixture was 8.0.
- a 10 mM solution of 3-(N- maleimidopropionyl) biocytin (MPB) in DMSO was prepared shortly before use.
- the ELISA procedure is identical to that described immediately above, except that as the samples are essentially aqueous and could be applied to the microtiter wells without further dilution, the standards are prepared in 20 mM phosphate buffer, pH 8.0.
- FIG. 10 shows results from an immunoassay utilizing the above procedure.
- Wells received 600 ng protein A, 200 ng anti-MSH antibody, and 50 ng alkaline phosphatase-labelled avidin.
- Samples were 1.00 mL volumes of Mycobacterium avium cells in either cerebrospinal fluid or enriched cerebrospinal fluid, biotinylated as described above, and 100 ⁇ L of the aqueous phase analyzed. The data shown are from 40 min development with pNPP and no stop solution.
- the immunoassay methods described above can be adapted easily for use in screening large numbers of bacteria, for example a library of strains grown in a microtiter plate.
- the specific example given here makes use of the immunoassay architecture described in Example 15.
- the strains were grown to early logarithmic phase in Middlebrook 7H9 medium supplemented with 0.4%) w/v glucose and 0.05% v/v Tween-80. Each strain was diluted in medium to an initial concentration of 10 8 cfu/mL, then further diluted in series as required. To each well of an Immulon-4 96-well microtiter plate was added a 100 ⁇ L aliquot of these serially diluted cell suspensions, so that the number of cells per well encompassed the range 0 to 10 7 cells (in duplicate) for each of the three strains. A 10 mM solution of 3-(N-maleimidopropionyl) biocytin (MPB) in DMSO was prepared shortly before use.
- MPB 3-(N-maleimidopropionyl) biocytin
- Example XXX The ELISA procedure was carried out as described in Example XXX.
- the ELISA plate wells were coated with protein A, gelatin, and anti-MSH antibody, and washed once with TBST as described.
- the drained ELISA wells each then received 75 ⁇ L TBS and 25 ⁇ L of the biotinylated cell extracts; each thus contained 25/260 or 9.6% of the original biotinylated cell extract volume, in a final CH 3 CN concentration of 12.5%. Standards were also applied in 12.5% acetonitrile in TBS. The rest of the analysis was performed as described in Example XXX.
- FIG. 10 shows results from an immunoassay utilizing the above procedure.
- Wells received 600 ng protein A, 200 ng anti-MSH antibody, and 50 ng alkaline phosphatase-labelled avidin. Both standards and biotinylated cell extracts were applied in a final CH 3 CH concentration of 12.5%). The data shown are from 60 min development with pNPP and no stop solution.
- Example 14 use of mycothiol linked to a binding agent in the assay of mycothiol was adapted for use in a dot blot assay.
- An example of a specific experimental procedure for this type of immunoassay follows.
- PVDF polyvinylidene fluoride
- the pre-wet membrane was assembled in the dot blot apparatus (BioRad BioDot) according to the manufacturer's instructions, and the membrane re-wetted by passing 50 ⁇ L/well TBS by vacuum.
- NeutrAvidin (Pierce) was diluted in TBS to a final concentration of 0.4 ⁇ g/ ⁇ L .
- the membrane was washed with two 400 ⁇ L volumes per well of TBS containing 0.05% v/v Tween 20 ("TBST”), passed through by vacuum.
- the antigens were prepared in 50% (v/v) acetonitrile in phosphate-buffered saline, pH 7.2, applied to the wells in 500 ⁇ L volumes, and allowed to pass through by gravity.
- TBS 0.05% v/v Tween 20
- Affinity-purified anti-MSH was diluted to a final concentration of 1 ng/ ⁇ L in TBST containing 0.1% w/v BSA ("TBSTB"), and 50 ⁇ L added to each well and allowed to pass through by gravity.
- TSTB 0.1% w/v BSA
- the membrane was washed with two 400 ⁇ L volumes per well of TBS containing 0.05%) v/v Tween 20 ("TBST”), passed through by vacuum.
- TBS v/v Tween 20
- the membrane was removed from the dot blot apparatus and washed in three 50 mL volumes of TBS. Excess TBS was drained, and to the wet membrane was added 10 mL of a freshly-made solution of 5-bromo-4-Clair-3- indwell phosphate/nitroblue tetrazolium ("SigmaFAST" BCIP/NBT alkaline phosphatase substrate). The purple color was allowed to develop for 3 minutes after which excess substrate was washed from the membrane by rinsing in deionized water.
- Organisms and Culture Condition M. smegmatis strains MC 2 6 and MC 2 155 and Staphylococcus aureus RN450 were kindly provided by Julian Davies, Department of Microbiology and Immunology, University of British Columbia. Micromonospora echinospora 14847 was purchased from Norther Regional Research Center (NRRL B- 12180), and Escherichia coli HB101 was purchased from Promega. M. smegmatis cultures were grown in Middlebrook 7H9 broth (0.05% Tween 80 and 0.4% glucose) at 37°C. S. aureus and E. coli cultures were grown in Trypticase soy broth at 37°C. M. echinospora cultures were grown in Todd Hewitt broth (0.5%> yeast extract and 0.25%> sucrose) at 28°C. All cultures were shaken at 220 rpms.
- Methanesulfonic acid was supplied by Fluka. Cys-Glc ⁇ and AcCys-Glc ⁇ were prepared as described previously (Unson, J.D., et al, 1998, J. Immunol. Methods., in press). Middlebrook 7H9, yeast extract, and Todd Hewitt broth were purchased from Difco Laboratories, and Trypticase soy broth was purchased from BBL. All other reagents were reagent grade or higher quality.
- echinospora was suspended in 1 liter of 50%> acetonitrile-water (60°C) containing 20 mM H 2 SO 4 (pH 5 cell suspension). The suspension was adjusted to pH 2.5 with concentrated H 2 SO 4 and then disrupted using a Bransonic sonicator at -70% maximum power for 15 min without cooling. The cell extract was cooled on ice, and the cell debris was removed by centrifugation at 6000xg for 15 min at 4°C. The supernatant was reduced to 250 ml using a rotary evaporator and clarified by centrifugation as above.
- the supernatant contained 240 ⁇ mol of thiol by assay with 5,5'-dithiobis(2-nitrobenzoic acid) (Ellman, G.L., 1959, Arch. Biochem. Biophys. 82:70).
- MSH was recovered from the extract.
- DTT 250 ⁇ mol was added, and the extract was adjusted to pH 7.9 with concentrated NH 4 OH.
- the supernatant was passed over a 2-thioopyridine-activated thiolpropyl-agarose column (Newton et al, 1995, Eur. J. Biochem., 230:821-825), the column was eluted with DTT, and the mycothiol was purified by preparative HPLC after derivatization with 5,5'-dithiobis(2- nitrobenzoic acid).
- the unbound extract (310 ml) from the thiol affinity column (pH 7.6) was applied directly to a 2.5 x 18-cm Amberlite IRC50 (Mallinckrodt) column that had been prewashed with 3M NH 4 OH followed byH 2 O.
- the effluent pH was 10.3 when the sample was applied.
- the column was washed with water and eluted with 350 ml of IN NH 4 OH folowed by 550 ml of 3M NH 4 OH.
- the fractions from both effluents were found to contain GlcN-Ins as assayed by TLC with ninhydrin detection as previously reported (Maehr, 1980, supra).
- the fractions containing GlcN-Ins were combined and lyophilized. The residue was redissolved in water (pH ⁇ 9) and applied to a 1 x 12-cm AG1-X8 (Bio-Rad, 200-400 mesh) anion exchange column in the hydroxyl form. The column was eluted in water, and the GlcN-Ins-containing fractions were again pooled. The fractions containing GlcN-Ins (RF 0.33) were also contaminated by higher RF ninhydrin-positive materials.
- the GlcN-Ins-containing fractions were again combined, dried by lyophilization, and dissolved in 0.1% trifluoroacetic acid, water. This sample was applied to a 20-ml Sep Pak C18 cartridge (Waters) equilibrated in the same solvent. The GlcN-Ins was eluted with 0-5% methanol gradient in aqueous 0.1% trifluoroacetic acid. The GlcN-Ins-containing fractions gave a single ninhydrin spot on TLC, free of the igh RF contaminant.
- a 15- ⁇ l aliquot was immediately taken from the supernatant for derivatization with AccQ-Fluor by the addition of 12.5 ⁇ l of IM HEPES, pH 8.0, 42.5 ⁇ l of water, 23.8 ⁇ l of acetonitrile, and 31.2 ⁇ l of 10 mM AccQ-Fluor, and the mixture was vortexed immediately.
- a second 30- ⁇ l aliquot of cell extract was taken at the same time and prepared in the same fashion except that the volumes of water and acetonitrile added were 35 and 16.8 ⁇ l, respectively. After 1 min at room temperature, the samples were heated for 10 min at 60°C, diluted 4-fold with water, and stored at -70°C.
- the cell pellet was dried in a tared tube to determine the residual dry weight (RDW) of the extract.
- the AccQ-Fluor-derivatized samples were analyzed by HPLC utilizing a Waters 600E solvent delivery system equipped with a Waters WISP Model 710B autoinjector, LDC Fluorometer III, and a Nelson Model 444 data collection system.
- the sample was eluted in a 0.1% trifluoroacetic acid in water (mobile phase A) with a 1%/min linear gradient from 0 to 40%> B (0.1% trifluoroacetic acid in methanol) at a flow rate of 5 ml min "1 .
- the effluent was monitored by fluorescence detection with excitation at 370 nm and emission at 418-700 nm.
- CySmB-GlcN-Ins was collected at 32 min and repurified twice to minimize CySmB-GlcN contamination. The identity of this peak was confirmed with electrospray mass spectrometry, which yielded a major peak at 657 daltons (molecular ion + Na + ).
- M. smegmatis MC 2 155 was grown to mid-log phase in Middlebrook 7H9 medium with 0.4% glucose and 0.05% Tween. Cells were pelleted and washed with 50mM sodium phosphate (pH 7.5) containing 1 mM DTT and resuspended in the same buffer at a concentration of 0.25 g of wet weight/ml. Cells were lysed by sonication on ice and pelleted by ultracentrifugation at 100,000 x g for 30 min in a Beckman tabletop ultracentrifuge.
- Assay of the supernatant was conducted in a final volume of 600 ⁇ l containing 60 ⁇ l of supernatant, 50 ⁇ M GlcN-Ins, 100 ⁇ M Cys or AcCys, 100 ⁇ M sodium acetate, 1 mM ATP, ImM MgCl 2 , 50 mM sodium phosphate (pH 7.5), ImM DTT, and 35 ⁇ M each of the protease inhibitors phenylmethanesulfonyl flouride, N-tf-p-tosyl-L-phenylalanylchloromethyl ketone, and N- -p- tosyl-L-lysinechloromethyl ketone.
- the mixture was incubated at 30°C, and 100 ⁇ l samples were removed at 0 and 60 min for thiol analysis.
- One sample was mixed with 4 ⁇ l of 100 mM mBBr and allowed to react for 5 min at room temperature before acidification with 0.5 ⁇ l of 5M methanesulfonic acid to quench the reaction.
- a second control sample was reacted 5 min with 5 mM ⁇ EM before treatment with mBBr as above.
- a buffer of slightly lower pH than employed with amino acids was chosen so that the aminosugars, whose ammonium forms have lower pKa values than those of most amino acids, would have a competetive advantage during labeling of cell extracts.
- AccQ-Fluor was dissolved in acetonitrile to 10 mM as recommended by the manufacturer. Standard solutions of D-glucosamine «HCl and purified GlcN-Ins were prepared at 3.1 to 200 ⁇ M in water.
- AccQ-Fluor derivatized amines had the following retention times (min): GlcN, 12; GlcN-Ins, 24.
- Fig top panel shows the cliromatogram obtained with a standard GlcN and GI (middle panel) and Fig. presents a representative calibration curve.
- Numbers designated with ⁇ represent detection limits where no discemable peak was present. Values designated with ⁇ represent measurement of a discemable peak at the retention time for the indicated component but for which independent verification of the structure was not available; the value represents an upper limit for the content.
- M. smegmatis MC 2 6 the parent strain of M. smegmatis MC 2 155, also produced GlcN-Ins in significant amount. Cys was also found, but neither strain appeared to produce comparable - amounts of other potential intermediates of MSH metabolism, including AcCys, AcCys-GlcN. Cys-GlcN, or Cys-GlcN-Ins. It had been previously shown that S. aureus and E. coli did not produce MSH (Newton et al, 1996, supra). These were rcexamined using the present methods to ascertain whether they might produce one or more of the intermediates involved in mycothiol biosynthesis and especially GlcN-Ins. The results (Table II) indicate that neither of these bacteria produce any component of MSH at measurable levels, other than Cys and GlcN.
- MSH level remained remarkably constant through exponential growth and into stationary phase, as did the Cys level at about 1% the MSH level.
- stationary phase there did appear to be a significant drop in the GlcN and GlcN-Ins levels and an increase in the H 2 S level.
- the Cys-GlcN-Ins content was elevated 20-fold over normal immediately after the start of incubation but then fell a factor of 70 over the next 2.5 h. Least affected was the MSH content, which was elevated -40% after 2.5 h. The A ⁇ o value changed little (5%) over the 2.5 h incubation period, indicating that AcCys significantly inhibits growth under these conditions.
- M. smegmatis mc 155 was grown to mid-log phase in Middlebrook 7H9 medium with 0.4% glucose and 0.05% Tween. Cells were pelleted and washed with 50 mM sodium phosphate, pH 7.5, containing 1 mM DTT and resuspended in the same buffer at a concentration of 0.25 g wet weight per ml. Cells were lysed by sonication on ice and pelleted by ultracentrifugation at 100,000 x g for 30 min in a Beckman tabletop ultracentrifuge.
- Assay of the supernatant was conducted in a final volume of 600 ⁇ l containing 60 ⁇ l of supernatant, 50 ⁇ M GlcN-Ins, 100 ⁇ M Cys or N-acetyl-L-Cys, 100 ⁇ M sodium acetate, 1 mM ATP, 1 mM MgCl 2 , 50 mM sodium phosphate (pH 7.5), 1 mM DTT, and 35 mM each of the protease inhibitors phenylmethanesulfonyl fluoride, Nar- -tosyl-L-phenylalanyl chloromethyl ketone, and Na-p- tosyl-L-lysine chloromethyl ketone.
- the mixture was incubated at 30 °C and 100 ⁇ l samples removed at 0 and 60 min for thiol analysis.
- One sample was mixed with 4 ⁇ l 100 mM mBBr and allowed to react 5 min at room temperature before acidification with 0.5 ⁇ l of 5 M methanesulfonic acid to quench the reaction.
- a second control sample was reacted 5 min with 5 mM ⁇ EM prior to treatment with mBBr as above. All samples were subjected to HPLC conditions previously stated for CGI analysis.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Cell Biology (AREA)
- Virology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Microbiology (AREA)
- Genetics & Genomics (AREA)
- Food Science & Technology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
Claims
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US09/530,370 US6780418B1 (en) | 1997-10-27 | 1998-10-23 | Reagents and immunoassay for the detection and quantitative determination of mycothiol and precursors thereof |
AU11988/99A AU1198899A (en) | 1997-10-27 | 1998-10-23 | Reagents and immunoassay for the detection and quantitative determination of mycothiol and precursors thereof |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US6362097P | 1997-10-27 | 1997-10-27 | |
US60/063,620 | 1997-10-27 |
Publications (2)
Publication Number | Publication Date |
---|---|
WO1999021580A1 true WO1999021580A1 (en) | 1999-05-06 |
WO1999021580A9 WO1999021580A9 (en) | 1999-07-29 |
Family
ID=22050412
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1998/022577 WO1999021580A1 (en) | 1997-10-27 | 1998-10-23 | Reagents and immunoassay for the detection and quantitative determination of mycothiol and precursors thereof |
Country Status (2)
Country | Link |
---|---|
AU (1) | AU1198899A (en) |
WO (1) | WO1999021580A1 (en) |
Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2005021493A2 (en) * | 2003-08-27 | 2005-03-10 | University Of Cape Town | A method of isolating a thiol |
US11035832B2 (en) | 2016-06-21 | 2021-06-15 | Waters Technologies Corporation | Methods of electrospray ionization of glycans modified with amphipathic, strongly basic moieties |
US11061023B2 (en) | 2016-06-21 | 2021-07-13 | Waters Technologies Corporation | Fluorescence tagging of glycans and other biomolecules through reductive amination for enhanced MS signals |
US11150248B2 (en) | 2016-07-01 | 2021-10-19 | Waters Technologies Corporation | Methods for the rapid preparation of labeled glycosylamines from complex matrices using molecular weight cut off filtration and on-filter deglycosylation |
US11371996B2 (en) | 2014-10-30 | 2022-06-28 | Waters Technologies Corporation | Methods for the rapid preparation of labeled glycosylamines and for the analysis of glycosylated biomolecules producing the same |
US11448652B2 (en) | 2011-09-28 | 2022-09-20 | Waters Technologies Corporation | Rapid fluorescence tagging of glycans and other biomolecules with enhanced MS signals |
US11747310B2 (en) | 2014-11-13 | 2023-09-05 | Waters Technologies Corporation | Methods for liquid chromatography calibration for rapid labeled N-glycans |
-
1998
- 1998-10-23 AU AU11988/99A patent/AU1198899A/en not_active Abandoned
- 1998-10-23 WO PCT/US1998/022577 patent/WO1999021580A1/en active Application Filing
Non-Patent Citations (3)
Title |
---|
NEWTON G. L., ET AL.: "DISTRIBUTION OF THIOLS IN MICROORGANISMS: MYCOTHIOL IS A MAJOR THIOL IN MOST ACTINOMYCETES.", JOURNAL OF BACTERIOLOGY, AMERICAN SOCIETY FOR MICROBIOLOGY, US, vol. 178., no. 07., 1 April 1996 (1996-04-01), US, pages 1990 - 1995., XP002915440, ISSN: 0021-9193 * |
SPIER H S C, STEENKAMP D J: "THIOLS OF INTRACELLULAR PATHOGENS IDENTIFICATION OF OVOTHIOL A IN LEISHMANIA DONOVANI AND STRUCTURAL ANALYSIS OF A NOVEL THIOL FROM MYCOBACTERIUM BOVIS", EUROPEAN JOURNAL OF BIOCHEMISTRY, WILEY-BLACKWELL PUBLISHING LTD., GB, vol. 224, 1 August 1994 (1994-08-01), GB, pages 203 - 213, XP002915439, ISSN: 0014-2956, DOI: 10.1111/j.1432-1033.1994.tb20013.x * |
UNSON M. D., ET AL.: "AN IMMUNOASSAY FOR THE DETECTION AND QUANTITATIVE DETERMINATION OF MYCOTHIOL.", JOURNAL OF IMMUNOLOGICAL METHODS., ELSEVIER SCIENCE PUBLISHERS B.V.,AMSTERDAM., NL, vol. 214., 1 May 1998 (1998-05-01), NL, pages 29 - 39., XP002915441, ISSN: 0022-1759, DOI: 10.1016/S0022-1759(98)00034-9 * |
Cited By (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2005021493A2 (en) * | 2003-08-27 | 2005-03-10 | University Of Cape Town | A method of isolating a thiol |
WO2005021493A3 (en) * | 2003-08-27 | 2005-04-14 | Univ Cape Town | A method of isolating a thiol |
US7619121B2 (en) | 2003-08-27 | 2009-11-17 | University Of Cape Town | Method for isolating a thiol |
US11448652B2 (en) | 2011-09-28 | 2022-09-20 | Waters Technologies Corporation | Rapid fluorescence tagging of glycans and other biomolecules with enhanced MS signals |
US11371996B2 (en) | 2014-10-30 | 2022-06-28 | Waters Technologies Corporation | Methods for the rapid preparation of labeled glycosylamines and for the analysis of glycosylated biomolecules producing the same |
US11747310B2 (en) | 2014-11-13 | 2023-09-05 | Waters Technologies Corporation | Methods for liquid chromatography calibration for rapid labeled N-glycans |
US11035832B2 (en) | 2016-06-21 | 2021-06-15 | Waters Technologies Corporation | Methods of electrospray ionization of glycans modified with amphipathic, strongly basic moieties |
US11061023B2 (en) | 2016-06-21 | 2021-07-13 | Waters Technologies Corporation | Fluorescence tagging of glycans and other biomolecules through reductive amination for enhanced MS signals |
US11150248B2 (en) | 2016-07-01 | 2021-10-19 | Waters Technologies Corporation | Methods for the rapid preparation of labeled glycosylamines from complex matrices using molecular weight cut off filtration and on-filter deglycosylation |
Also Published As
Publication number | Publication date |
---|---|
WO1999021580A9 (en) | 1999-07-29 |
AU1198899A (en) | 1999-05-17 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US8030458B2 (en) | Monoclonal antibodies to tacrolimus and immunoassays methods for tacrolimus | |
US5437978A (en) | Detection for Staphylococcus spp. | |
US9512206B2 (en) | Anti-lipoarabinomannan antibody and immunoassay for acid-fast bacillary infection using the antibody | |
US8507214B2 (en) | Elisa kit for detecting lincomycin | |
EP0254172B1 (en) | Enzyme-labeled antibody reagent with polyalkyleneglycol linking group | |
WO1993025533A1 (en) | Methods and reagents for the determination of immunosuppressive agents | |
US7105170B2 (en) | Latent human tuberculosis model, diagnostic antigens, and methods of use | |
WO1999021580A1 (en) | Reagents and immunoassay for the detection and quantitative determination of mycothiol and precursors thereof | |
AU2002237764A1 (en) | Latent human tuberculosis model, diagnostic antigens, and methods of use | |
US6780418B1 (en) | Reagents and immunoassay for the detection and quantitative determination of mycothiol and precursors thereof | |
Unson et al. | An immunoassay for the detection and quantitative determination of mycothiol | |
EP1116031A1 (en) | DIAGNOSTIC ASSAYS FOR DETECTION OF $i(ENTAMOEBA HISTOLYTICA) | |
US9804159B2 (en) | Immunoreactive antigens of Mycoplasma haemofelis and diagnostic immunoassay | |
US10883989B2 (en) | Peptides of M. tuberculosis for a screening test for HIV positive patients at high-risk for tuberculosis | |
JP2003248002A (en) | Method and device for collecting and preparing specimen for detecting mycobacterial and antigen | |
WO1993003365A1 (en) | Immunoassay, monoclonal antibody and hybridoma | |
Aleixo et al. | A fluorescent enzyme immunoassay for Salmonella detection | |
WO2021202495A2 (en) | Anti-acinetobacter baumannii polyclonal antibody (ab-pab), and uses thereof | |
JP2007055920A (en) | Peptide, method for detecting strangles bacterium antibody using the peptide, method for extracting horse infected with strangles bacterium, and diagnostic agent for the horse infected with the strangles bacterium | |
JPH07191034A (en) | Pathogenic factor detecting material and pathogenic factor detecting method using it | |
JPH05249116A (en) | High polymer material containing immobilized antibody and quickly diagnosing method for infectious disease using same | |
JPH0712815A (en) | Method for quickly diagnosing infectious disease | |
AU2005256177A1 (en) | Novel methods of diagnosis of treatment of P. aeruginosa infection and reagents therefor |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A1 Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GE GH GM HR HU ID IL IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): GH GM KE LS MW SD SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
AK | Designated states |
Kind code of ref document: C2 Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GE GH GM HR HU ID IL IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: C2 Designated state(s): GH GM KE LS MW SD SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG |
|
COP | Corrected version of pamphlet |
Free format text: PAGES 1-83, DESCRIPTION, REPLACED BY NEW PAGES 1-72; PAGES 84-89, CLAIMS, REPLACED BY NEW PAGES 73-78; PAGES 1/18-18/18, DRAWINGS, REPLACED BY NEW PAGES 1/16-16/16 |
|
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
NENP | Non-entry into the national phase |
Ref country code: KR |
|
WWE | Wipo information: entry into national phase |
Ref document number: 09530370 Country of ref document: US |
|
REG | Reference to national code |
Ref country code: DE Ref legal event code: 8642 |
|
122 | Ep: pct application non-entry in european phase | ||
NENP | Non-entry into the national phase |
Ref country code: CA |