CN101535811A - Method for in vitro diagnosis of PVL-producing staphylococcus aureus - Google Patents
Method for in vitro diagnosis of PVL-producing staphylococcus aureus Download PDFInfo
- Publication number
- CN101535811A CN101535811A CNA2007800385924A CN200780038592A CN101535811A CN 101535811 A CN101535811 A CN 101535811A CN A2007800385924 A CNA2007800385924 A CN A2007800385924A CN 200780038592 A CN200780038592 A CN 200780038592A CN 101535811 A CN101535811 A CN 101535811A
- Authority
- CN
- China
- Prior art keywords
- pvl
- staphylococcus aureus
- biological sample
- antibody
- sample
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 238000000034 method Methods 0.000 title claims abstract description 58
- 241000191967 Staphylococcus aureus Species 0.000 title claims abstract description 50
- 238000003745 diagnosis Methods 0.000 title claims abstract description 12
- 238000000338 in vitro Methods 0.000 title claims abstract description 6
- 239000012472 biological sample Substances 0.000 claims abstract description 26
- 230000001900 immune effect Effects 0.000 claims abstract description 18
- 238000003556 assay Methods 0.000 claims description 23
- 241000894006 Bacteria Species 0.000 claims description 13
- 230000008859 change Effects 0.000 claims description 11
- 238000010438 heat treatment Methods 0.000 claims description 8
- RJQXTJLFIWVMTO-TYNCELHUSA-N Methicillin Chemical compound COC1=CC=CC(OC)=C1C(=O)N[C@@H]1C(=O)N2[C@@H](C(O)=O)C(C)(C)S[C@@H]21 RJQXTJLFIWVMTO-TYNCELHUSA-N 0.000 claims description 5
- 229960003085 meticillin Drugs 0.000 claims description 5
- 238000001514 detection method Methods 0.000 abstract description 10
- 238000012360 testing method Methods 0.000 abstract description 7
- 108010078471 Panton-Valentine leukocidin Proteins 0.000 abstract description 5
- 230000001580 bacterial effect Effects 0.000 description 30
- 239000000523 sample Substances 0.000 description 28
- 208000015181 infectious disease Diseases 0.000 description 16
- 210000000621 bronchi Anatomy 0.000 description 14
- 239000012634 fragment Substances 0.000 description 14
- 238000002965 ELISA Methods 0.000 description 13
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 12
- 238000011534 incubation Methods 0.000 description 12
- 108090000765 processed proteins & peptides Proteins 0.000 description 12
- 239000006228 supernatant Substances 0.000 description 11
- 238000005406 washing Methods 0.000 description 11
- 238000013016 damping Methods 0.000 description 10
- 230000008569 process Effects 0.000 description 10
- 108090000623 proteins and genes Proteins 0.000 description 10
- 241000283973 Oryctolagus cuniculus Species 0.000 description 9
- 230000009870 specific binding Effects 0.000 description 9
- 239000012530 fluid Substances 0.000 description 8
- 239000001963 growth medium Substances 0.000 description 8
- 235000015097 nutrients Nutrition 0.000 description 8
- 241001465754 Metazoa Species 0.000 description 7
- 230000027455 binding Effects 0.000 description 7
- 238000005859 coupling reaction Methods 0.000 description 7
- 230000008521 reorganization Effects 0.000 description 7
- 210000002966 serum Anatomy 0.000 description 7
- IMNFDUFMRHMDMM-UHFFFAOYSA-N N-Heptane Chemical compound CCCCCCC IMNFDUFMRHMDMM-UHFFFAOYSA-N 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 6
- 238000005516 engineering process Methods 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 6
- 102000004169 proteins and genes Human genes 0.000 description 6
- 102000003992 Peroxidases Human genes 0.000 description 5
- 241000191940 Staphylococcus Species 0.000 description 5
- 239000000427 antigen Substances 0.000 description 5
- 108091007433 antigens Proteins 0.000 description 5
- 102000036639 antigens Human genes 0.000 description 5
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N biotin Natural products N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 5
- 230000008878 coupling Effects 0.000 description 5
- 238000010168 coupling process Methods 0.000 description 5
- 239000012228 culture supernatant Substances 0.000 description 5
- 239000007924 injection Substances 0.000 description 5
- 238000002347 injection Methods 0.000 description 5
- 235000013336 milk Nutrition 0.000 description 5
- 239000008267 milk Substances 0.000 description 5
- 210000004080 milk Anatomy 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 108040007629 peroxidase activity proteins Proteins 0.000 description 5
- 229920000136 polysorbate Polymers 0.000 description 5
- 239000000843 powder Substances 0.000 description 5
- 235000018102 proteins Nutrition 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 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 4
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 4
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 239000002671 adjuvant Substances 0.000 description 4
- 238000001042 affinity chromatography Methods 0.000 description 4
- 101150118925 bioM gene Proteins 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 238000000502 dialysis Methods 0.000 description 4
- 239000007928 intraperitoneal injection Substances 0.000 description 4
- 108010045069 keyhole-limpet hemocyanin Proteins 0.000 description 4
- 238000002372 labelling Methods 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 3
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 3
- 206010000269 abscess Diseases 0.000 description 3
- 229960002685 biotin Drugs 0.000 description 3
- 235000020958 biotin Nutrition 0.000 description 3
- 239000011616 biotin Substances 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- 229940041514 candida albicans extract Drugs 0.000 description 3
- 230000002950 deficient Effects 0.000 description 3
- 239000003398 denaturant Substances 0.000 description 3
- 210000004408 hybridoma Anatomy 0.000 description 3
- 230000003053 immunization Effects 0.000 description 3
- 238000002649 immunization Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 230000010355 oscillation Effects 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 229920006395 saturated elastomer Polymers 0.000 description 3
- 239000007790 solid phase Substances 0.000 description 3
- 239000003053 toxin Substances 0.000 description 3
- 231100000765 toxin Toxicity 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 239000012138 yeast extract Substances 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 2
- 206010003445 Ascites Diseases 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 208000005119 Necrotizing Pneumonia Diseases 0.000 description 2
- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 description 2
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 2
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 230000004520 agglutination Effects 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 239000002158 endotoxin Substances 0.000 description 2
- 239000002095 exotoxin Substances 0.000 description 2
- 231100000776 exotoxin Toxicity 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 235000013905 glycine and its sodium salt Nutrition 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 238000010253 intravenous injection Methods 0.000 description 2
- 210000004698 lymphocyte Anatomy 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 238000006386 neutralization reaction Methods 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 229960001019 oxacillin Drugs 0.000 description 2
- UWYHMGVUTGAWSP-JKIFEVAISA-N oxacillin Chemical compound N([C@@H]1C(N2[C@H](C(C)(C)S[C@@H]21)C(O)=O)=O)C(=O)C1=C(C)ON=C1C1=CC=CC=C1 UWYHMGVUTGAWSP-JKIFEVAISA-N 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 102000040430 polynucleotide Human genes 0.000 description 2
- 108091033319 polynucleotide Proteins 0.000 description 2
- 239000002157 polynucleotide Substances 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 210000002345 respiratory system Anatomy 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- 206010040872 skin infection Diseases 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 238000007669 thermal treatment Methods 0.000 description 2
- YMXHPSHLTSZXKH-RVBZMBCESA-N (2,5-dioxopyrrolidin-1-yl) 5-[(3as,4s,6ar)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoate Chemical compound C([C@H]1[C@H]2NC(=O)N[C@H]2CS1)CCCC(=O)ON1C(=O)CCC1=O YMXHPSHLTSZXKH-RVBZMBCESA-N 0.000 description 1
- OTLLEIBWKHEHGU-UHFFFAOYSA-N 2-[5-[[5-(6-aminopurin-9-yl)-3,4-dihydroxyoxolan-2-yl]methoxy]-3,4-dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-3,5-dihydroxy-4-phosphonooxyhexanedioic acid Chemical compound C1=NC=2C(N)=NC=NC=2N1C(C(C1O)O)OC1COC1C(CO)OC(OC(C(O)C(OP(O)(O)=O)C(O)C(O)=O)C(O)=O)C(O)C1O OTLLEIBWKHEHGU-UHFFFAOYSA-N 0.000 description 1
- UAIUNKRWKOVEES-UHFFFAOYSA-N 3,3',5,5'-tetramethylbenzidine Chemical compound CC1=C(N)C(C)=CC(C=2C=C(C)C(N)=C(C)C=2)=C1 UAIUNKRWKOVEES-UHFFFAOYSA-N 0.000 description 1
- 102100031126 6-phosphogluconolactonase Human genes 0.000 description 1
- 108010029731 6-phosphogluconolactonase Proteins 0.000 description 1
- 208000002874 Acne Vulgaris Diseases 0.000 description 1
- 101710186708 Agglutinin Proteins 0.000 description 1
- 206010060968 Arthritis infective Diseases 0.000 description 1
- 108010001478 Bacitracin Proteins 0.000 description 1
- 206010007882 Cellulitis Diseases 0.000 description 1
- 208000014912 Central Nervous System Infections Diseases 0.000 description 1
- 101710112752 Cytotoxin Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- 206010017553 Furuncle Diseases 0.000 description 1
- 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 1
- 108010018962 Glucosephosphate Dehydrogenase Proteins 0.000 description 1
- 101710146024 Horcolin Proteins 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 102000018071 Immunoglobulin Fc Fragments Human genes 0.000 description 1
- 108010091135 Immunoglobulin Fc Fragments Proteins 0.000 description 1
- 206010062717 Increased upper airway secretion Diseases 0.000 description 1
- 101710189395 Lectin Proteins 0.000 description 1
- 108010014603 Leukocidins Proteins 0.000 description 1
- 101000735344 Lymantria dispar Pheromone-binding protein 2 Proteins 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 101710179758 Mannose-specific lectin Proteins 0.000 description 1
- 101710150763 Mannose-specific lectin 1 Proteins 0.000 description 1
- 101710150745 Mannose-specific lectin 2 Proteins 0.000 description 1
- 102000016943 Muramidase Human genes 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 241000872931 Myoporum sandwicense Species 0.000 description 1
- 201000002481 Myositis Diseases 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- RCEAADKTGXTDOA-UHFFFAOYSA-N OS(O)(=O)=O.CCCCCCCCCCCC[Na] Chemical compound OS(O)(=O)=O.CCCCCCCCCCCC[Na] RCEAADKTGXTDOA-UHFFFAOYSA-N 0.000 description 1
- 206010031252 Osteomyelitis Diseases 0.000 description 1
- 102000057297 Pepsin A Human genes 0.000 description 1
- 108090000284 Pepsin A Proteins 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 108010053210 Phycocyanin Proteins 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 206010061926 Purulence Diseases 0.000 description 1
- 206010057190 Respiratory tract infections Diseases 0.000 description 1
- 230000018199 S phase Effects 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- 108010090804 Streptavidin Proteins 0.000 description 1
- 208000033809 Suppuration Diseases 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- LDCBAHYRQATDLE-JRSVCXIZSA-N [S].C(C)(C)[C@]1(O)[C@H](O)[C@@H](O)[C@@H](O)[C@H](O1)CO Chemical compound [S].C(C)(C)[C@]1(O)[C@H](O)[C@@H](O)[C@@H](O)[C@H](O1)CO LDCBAHYRQATDLE-JRSVCXIZSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 206010000496 acne Diseases 0.000 description 1
- 239000000910 agglutinin Substances 0.000 description 1
- 102000005840 alpha-Galactosidase Human genes 0.000 description 1
- 108010030291 alpha-Galactosidase Proteins 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000002421 anti-septic effect Effects 0.000 description 1
- 238000011091 antibody purification Methods 0.000 description 1
- 210000003567 ascitic fluid Anatomy 0.000 description 1
- 150000001540 azides Chemical class 0.000 description 1
- 229960003071 bacitracin Drugs 0.000 description 1
- 229930184125 bacitracin Natural products 0.000 description 1
- CLKOFPXJLQSYAH-ABRJDSQDSA-N bacitracin A Chemical compound C1SC([C@@H](N)[C@@H](C)CC)=N[C@@H]1C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]1C(=O)N[C@H](CCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CC=2N=CNC=2)C(=O)N[C@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)NCCCC1 CLKOFPXJLQSYAH-ABRJDSQDSA-N 0.000 description 1
- 150000001615 biotins Chemical class 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 238000009640 blood culture Methods 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 108010079058 casein hydrolysate Proteins 0.000 description 1
- WZOZEZRFJCJXNZ-ZBFHGGJFSA-N cefoxitin Chemical compound N([C@]1(OC)C(N2C(=C(COC(N)=O)CS[C@@H]21)C(O)=O)=O)C(=O)CC1=CC=CS1 WZOZEZRFJCJXNZ-ZBFHGGJFSA-N 0.000 description 1
- 229960002682 cefoxitin Drugs 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000012916 chromogenic reagent Substances 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 231100000599 cytotoxic agent Toxicity 0.000 description 1
- 239000002619 cytotoxin Substances 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- CCIVGXIOQKPBKL-UHFFFAOYSA-N ethanesulfonic acid Chemical compound CCS(O)(=O)=O CCIVGXIOQKPBKL-UHFFFAOYSA-N 0.000 description 1
- 238000002795 fluorescence method Methods 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- ISNICOKBNZOJQG-UHFFFAOYSA-O guanidinium ion Chemical compound C[NH+]=C(N(C)C)N(C)C ISNICOKBNZOJQG-UHFFFAOYSA-O 0.000 description 1
- -1 haptens/antibody Proteins 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 210000003709 heart valve Anatomy 0.000 description 1
- 150000002460 imidazoles Chemical class 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 229920006008 lipopolysaccharide Polymers 0.000 description 1
- 238000002796 luminescence method Methods 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 101150008979 mecA gene Proteins 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 230000002906 microbiologic effect Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 208000026435 phlegm Diseases 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 210000001747 pupil Anatomy 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- RYVMUASDIZQXAA-UHFFFAOYSA-N pyranoside Natural products O1C2(OCC(C)C(OC3C(C(O)C(O)C(CO)O3)O)C2)C(C)C(C2(CCC3C4(C)CC5O)C)C1CC2C3CC=C4CC5OC(C(C1O)O)OC(CO)C1OC(C1OC2C(C(OC3C(C(O)C(O)C(CO)O3)O)C(O)C(CO)O2)O)OC(CO)C(O)C1OC1OCC(O)C(O)C1O RYVMUASDIZQXAA-UHFFFAOYSA-N 0.000 description 1
- 239000002510 pyrogen Substances 0.000 description 1
- 229940107700 pyruvic acid Drugs 0.000 description 1
- 108700002783 roundabout Proteins 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
- 235000011152 sodium sulphate Nutrition 0.000 description 1
- 210000004872 soft tissue Anatomy 0.000 description 1
- 235000014347 soups Nutrition 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 208000019206 urinary tract infection Diseases 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 239000000304 virulence factor Substances 0.000 description 1
- 230000007923 virulence factor Effects 0.000 description 1
Images
Classifications
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
-
- 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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Analytical Chemistry (AREA)
- Biophysics (AREA)
- Urology & Nephrology (AREA)
- Zoology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Hematology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Toxicology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The present invention relates to a method for in vitro diagnosis of Staphylococcus aureus producing Panton-Valentine Leukocidin (PVL), using a biological sample derived from an individual liable to be colonized by or infected with Staphylococcus aureus, wherein the diagnosis is carried out by detection of PVL using a routine immunological test, characterized in that said biological sample is pretreated in order to denature the PVL.
Description
The field of the invention relate to the infection relevant with there being the bacterium belong to staphylococcus and with have generation P-VL (Panton-Valentine Leukocidin, the relevant infection of staphylococcus aureus PVL) (Staphylococcus aureus).More specifically, theme of the present invention is to use conventional immunologic assay method to determine the method for PVL-producing staphylococcus aureus in the biological sample that may contain staphylococcus aureus.
The infection that the PVL-producing staphylococcus aureus causes mainly causes community to infect.Staphylococcus aureus is expressed multiple virulence factor, and one of them is P-VL (PVL), and it is a kind of cytotoxin that causes disorganization
1This toxin of PVL is common in the acquired methicillin-resistant staphylococcus aureus of community (CA-MRSA) bacterial strain, and the latter is widely distributed in the whole world
2
PVL and other leukocidin are the protein that belongs to the Synergohymenotropic toxin family.All toxin of this family constitute by having synergistic two polypeptide fractions, are called S and F.PVL is lukF-PV and lukS-PV coding by two contiguous genes of corotation record.PVL is an exotoxin because no matter cell whether cleaved its all secreted to extracellular medium, this is different from endotoxin or lipopolysaccharides, they only just discharge when their Gram-negative bacteria of secretion is destroyed.
The PVL-producing staphylococcus aureus is a gram-positive bacteria, it causes relevant specific infection for example skin infection or subcutaneous infection, bone joint infection and the serious necrotizing pneumonia of furuncle, abscess, cellulitis and myositis type the mankind, the latter mainly involves children and youth, and mortality ratio is about 70%.
Though its pathogenesis it be unclear that, have some evidence prompting PVL in the Pathological Physiology of the infection that the PVL-producing staphylococcus aureus is caused, to play a significant role:
I) there is EPDML strong relevance between the clinical manifestation of the staphylococcus aureus separator of synthetic PVL and infection,
Ii) low leucocyte mass formed by blood stasis (one of known effect of PVL) occurrence rate height,
Iii) respiratory tract gangrenosum acne pathology, with cause for rabbit intracutaneous injection PVL downright bad similar,
The PVL that iv) has the target polymorphonuclear cell in the lung,
V) have only the bacterial strain of PVL-producing or the PVL of purifying just can induce animal used as test necrotizing pneumonia to occur
7
What detection PVL-producing staphylococcus aureus was adopted at present is the determination method that detects nucleic acid, and particularly detects lukF-PV and lukS-PV by PCR
3
The defective of this type of determination method is that they are round-about ways, because they can not determine whether gene is that function and/or expression are arranged, owing to need special equipment so expense costliness, and neither be very quick, and it is big to implement difficulty.In addition, just use PCR, pollution problems may occur.
Sometimes also use polyclonal antibody to detect PVL by SRID
4, but this technology is made way for genetic test very soon, and this is because SRID is difficult for implementing and being difficult to standardization, so it does not become the conventional good tool that adopts.
Because the seriousness of some infection of causing of PVL-producing staphylococcus aureus, therefore need badly a kind of easy to implement and can overcome the rapid test method of defective of the determination method of currently used detection PVL-producing staphylococcus aureus.
The routine immunization determination method that patented claim EP 597 110A disclose by using anti-PVL monoclonal antibody detects MRSA.But, although these determination methods can overcome above-mentioned defective, its detection specificity is not high enough.
Beyond expectation is that the applicant now confirms, although PVL is the same with other exotoxins very responsive to physical chemical factor such as temperature
5, but by biological sample is carried out pre-service so that the PVL sex change, can improve the specificity that adopts conventional immunologic assay method to detect the PVL-producing staphylococcus aureus.
Therefore, theme of the present invention is to use that staphylococcus aureus is built the group or the biological sample in-vitro diagnosis of the individuality that infects produces the method for the staphylococcus aureus of P-VL (PVL) from being easy to take place, wherein carry out described diagnosis, it is characterized in that described biological sample is carried out pre-service so that the PVL sex change by adopting conventional immunologic assay method to detect PVL.
Term ＂ PVL-producing staphylococcus aureus ＂ refers to the staphylococcus aureus strains that all can produce PVL, has both comprised that methicillin-sensitivity bacterial strain (also claiming MSSA) also comprises methicillin-resistant bacterial strain (also claiming MRSA).
Term ＂ is easy to take place staphylococcus aureus and builds group's individual ＂ and refer to those and have the individuality of risk that possibility becomes the healthy carrier of this bacterium, for example medical personnel.The individual ＂ that term ＂ is easy to take place infection of staphylococcus aureus refers to the patient who shows the infection of staphylococcus aureus symptom.
Term ＂ infection of staphylococcus aureus ＂ refers to any infection that exists this bacterium to cause because of in the biosome, and what no matter infect is PVL-producing staphylococcus aureus or the staphylococcus aureus that does not have this ability.Can mention infection and muscle and bone infection in pyogenic infection, respiratory tract infection, central nervous system infection, urinary tract infections, cardiac valves and the blood vessel of skin and soft tissue as the example of this type of infection.This type of infection and relevant symptom thereof are that those skilled in the art are known and can be specifically referring to Pr é cis de Bact é riologie Clinique
5[Handbook of Clinical Bacteriology].
Staphylococcus aureus is built the group to term ＂ or the biological sample ＂ of the individuality that infects refers to any sample of the PVL that is easy to contain these bacteriums or secretion, for example purulence, respiratory tract sample, nose sample, urine or blood culture thing from being easy to take place.
The biological sample that is used for method of the present invention can be the sample of unmodified, and perhaps it can be the culture that is derived from the bacterium of described sample.Can be for example in the double dish or in culture broth, cultivate at the solid phase nutrient culture media, be appreciated that, the colony of described sample or the staphylococcus aureus that separates from described biological sample in advance can be inoculated in the culture broth, the method that forms colony is well known in the art, for example by inoculating in double dish.Can directly carry out immunologic assay then at the solid phase nutrient culture media or in meat soup.Interested bacterium or sample were cultivated about 24 hours usually.Be noted that, which kind of training method no matter, nutrient culture media will contain the factor that promotes that PVL produces, CCY nutrient culture media (casein hydrolysate and yeast extract medium) for example, can use or not use the molecule that can make that the PVL greater amount produces, for example the Oxacillin of inferior inhibition concentration and bacitracin.
Term " immunologic assay method " refer to use can specificity in conjunction with any immunologic assay method of the binding partners of PVL, described PVL can be the LukS fragment of LukF fragment, PVL of PVL or both.
Term " conventional immunologic assay method " refers to any immunologic assay method that is widely used in the laboratory routine work.For example, Chang Gui immunologic assay method can be the sandwich assay and particle (granules of polystyrene) the aggegation test of ELISA or immunochromatographic method (also claiming lateral flow) form.All these determination methods all are well known in the art.Can in one or more steps, carry out sandwich assay, promptly not have washing step or one or more washing step is arranged.
For example, the PVL specific binding partner can be antibody, antibody fragment, acceptor, mimotope and any can be in conjunction with the molecule of PVL.
Binding partners antibody for example is polyclonal antibody or monoclonal antibody.
Can use PVL, PVL fragment or PVL peptide immune animal and collect the serum of described animal subsequently and obtain polyclonal antibody.The antibody that is used for method of the present invention can be purifying or unpurified.The purifying of polyclonal antibody for example can be by getting described animal serum and described antibody separated with other serum composition carries out, particularly undertaken by affinity chromatography, antigen, particularly PVL that described antibody can specific recognition have wherein been adhered on the chromatographic column.
Can obtain monoclonal antibody by hybridoma technology, hereinafter summarize its cardinal rule.
In the first step, with PVL, PVL fragment or PVL peptide immune animal, mouse (if external immunity then is a cultured cells) normally, the bone-marrow-derived lymphocyte of this mouse just can produce the antibody at this antigen thus.The lymphocyte that subsequently these is produced antibody and " immortality " myeloma cell (be mouse among the embodiment) fusion is so that the generation hybridoma.Screen with the heterogeneous cell mixture that so obtains then and can produce specific antibodies and cell that can infinite multiplication.All with clone's form propagation, each clone produces the monoclonal antibody of a kind of specific recognition PVL to each hybridoma.Used antibody can be purifying or unpurified in the method for the present invention.Can be according to aforesaid affinity chromatography technology monoclonal antibody purification.
Monoclonal antibody can also be the recombinant antibodies that adopts genetic engineering technology well known in the art to obtain.
Term " is used for antibody fragment of the present invention " and refers to F (ab ') 2, Fab, Fab ' or the scFv matrix section of natural antibody, and they have kept the ability of specificity in conjunction with PVL.
Preferably, adopt following feature to carry out method of the present invention, described feature can adopt separately or combination mutually:
It is sandwich method that-described routine immunization is learned determination method,
-use LukS-PV-specific binding partner or LukF-PV-specific binding partner,
-use antibody fragment and particularly F (ab ') 2 fragments.
With regard to the sandwich assay of using two kinds of PVL-specific binding partners, it can use for example two kinds of monoclonal antibodies, two kinds of polyclonal antibodies or their fragment, or a kind of monoclonal antibody and a kind of polyclonal antibody or their fragment.For the particle agglutination determination method, can use single a kind of PVL-specific binding partner, for example monoclonal antibody or polyclonal antibody, they can be or not be the forms of fragment.
According to a concrete embodiment, described immunologic assay method is used at least a anti-PVL monoclonal antibody.
For the particle agglutination determination method, use the PVL-specific binding partner of acquisition mode.For sandwich assay, then use the PVL-specific binding partner of catching form and test format.
If use binding partners as detectable, it will be labeled so that find combination between the PVL/ binding partners.
On referring to and connect, term " mark binding partners " can directly or indirectly produce the label of detectable signal.The limiting examples of these labels comprises:
For example produce the enzyme of detectable signal by colourimetry, fluorescence method or luminescence method, for example horseradish peroxidase, alkaline phosphatase, alpha-galactosidase or glucose-6-phosphate dehydrogenase,
Chromophore, for example fluorescence, luminous or dye composition,
Geigers, for example
32P,
35S or
125I, and
Fluorescence molecule, for example Alexa or phycocyanin.
Also can use the indirect detection system, for example can with the part of anti-part reaction.Part/anti-part for example can be following collocation: the complementary series of biotin/streptavidin, haptens/antibody, antigen/antibody, peptide/antibody, sugar/agglutinin, polynucleotide/these polynucleotide to being well known in the art.In this case, part is loaded with binding partners.Can directly detect anti-part by aforesaid label, perhaps anti-part itself is detectable with the form of part/anti-part.
In some cases, the indirect detection system can make that signal is amplified.The amplification of signal technology is well known in the art, for example can be referring to J.Histochem.Cytochem
6.45:481-491,1997.
According to used type, those skilled in the art can add reagent mark is developed the color.
For " competition " method, the method mark PVL of mark binding partners as described above.
If what use is acquisition mode, can use methods known in the art that the PVL-specific binding partner directly or indirectly is immobilized onto solid phase.
To the The pretreatment that is used for method of the present invention can be any protein denaturation disposal route well known in the art.This processing can be passed through, and for example, changes pH, uses chemical denaturant such as urea, lauryl sodium sulfate (SDS) or guanidinium ion or contain by heating and remain the sample of denatured protein and carry out.
According to an embodiment, the temperature that sample pretreatment is included between 60 to 100 ℃ heated 10 minutes at least.Preferably, the temperature between 80 to 100 ℃ heats, the more preferably heating of the temperature between 90 to 100 ℃.
Certainly, learn mensuration, can carry out sample pretreatment described sample itself if on sample, carry out described routine immunization, otherwise, if on culture, carry out described immunologic assay method, then culture is carried out sample pretreatment.
Method of the present invention also can comprise the additional step that has staphylococcus aureus in the described biological sample of checking, and this step can shift to an earlier date or carry out simultaneously.The method that detects these bacteriums is that those skilled in the art are known, for example, can use to contain this bacterium is had specific chromogenic reagent, for example the patented claim WO 02/079486 that submits to referring to one of applicant of the application.
Similarly, method of the present invention can comprise that the staphylococcus aureus of determining in the described biological sample is the additional step of methicillin-resistant bacterium (MRSA) or methicillin-sensitivity bacterium (MSSA), has constituted a specific embodiment of the present invention thus.
The step of described definite methicillin-sensitivity (MRSA or MSSA) can adopt method well known in the art to carry out, for example immunologic assay method (as using PBP2 ' albumen is had specific binding partners, this protein is only by the MRSA expressed protein), the determination method that detects nucleic acid or other microbiological assays (for example using the nutrient culture media that contains microbiotic such as Oxacillin or cynnematin such as Cefoxitin).
Following non-limiting example and appended Fig. 1 to 3 will help to be expressly understood the present invention more, wherein:
-Fig. 1 has provided to be used for detecting and has contained PVL-producing (PVL+) or the ELISA measurement result of the PVL of the biological sample of the staphylococcus aureus strains of PVL-producing (PVL-) (OD of each bacterial strain) not,
-Fig. 2 has provided to be used for detecting and has contained PVL-producing (PVL+) or the ELISA measurement result of the PVL of the biological sample of the staphylococcus aureus strains of PVL-producing (PVL-) (OD of each bacterial strain) not, described sample through heating pretreatment so as to make the PVL sex change and
-Fig. 3 has provided to be used for detecting and has contained PVL-producing staphylococcus aureus strains (LY990084, A92007, LUG855) or not staphylococcus aureus strains (the LY990333 of PVL-producing, LY991321, RN6911) the ELISA measurement result of the PVL in the biological sample (OD of each bacterial strain), described sample is through heating pretreatment (handling 1), use chemical denaturant pre-service (handling 2) or use denaturant and heating pretreatment (handling 3), and state 0 is equivalent to sample not carried out any pre-service.
Embodiment 1: prepare anti-PVL antibody
1. produce reorganization PVL
LukS His-Tag and LukF His-Tag protein that sequence is respectively SEQ ID No.1 and SEQ ID No.2 have been produced, use carrier pIVEX2.4d (Roche) transformed into escherichia coli (Escherichia coli) bacterial strain BL21star (DE3) pLys (Invitrogen), 37 ℃, 2 liters cultivating systems shake; (isopropyl-β-D-galactose sulphur pyranoside Eurobio) induces 3 hours at 37 ℃, and then cultivated 5 hours with 1mM IPTG.Culture is divided in 6 different pipes.
4000g removed supernatant in centrifugal 20 minutes, reclaimed each cell mass.Use the explanation purification of recombinant proteins matter of QIAexpressionist kit according to manufacturer (Qiagen).The non-sex change cracked solution (Qiagen) of using 20ml is spent the night in-80 ℃ of preservations then 4 ℃ of cell lysis groups.Handle at 4 ℃ with 1mg/ml lysozyme (Eurobio) after the freeze thawing and continued cracking in 1 hour, use Vibracell (Bioblock) subsequently in 100 watts of sonicated 2 minutes, every circulation 6 seconds.
Solution centrifugal 30 minutes with 10000g, 4 ℃.Supernatant is contacted 3 hours with agarose-NiNTA (Qiagen) of 5ml, and 4 ℃, soft annular is stirred.Then agarose is placed (Biorad) on the 20ml post.With 200ml lavation buffer solution (Qiagen) washing column, the imidazoles with 250mM carries out wash-out as elution buffer then.Finally on monoSP ion exchange column (Amersham), finish purifying.
Go up concentrated recombinant protein at Centricon (Vivaspin), dialyse with 50mM MES (2-(N-morpholino) ethyl sulfonic acid) damping fluid, then with NaCl gradient (0 to 1M) wash-out.And then dialyse with the sterilized water of no pyrogen.
2. generation monoclonal antibody
Obtained following monoclonal antibody:
-anti-LukF:10D1A10,16A10A3,6H10E5 and
-anti-LukS:2H2H12,3D9D12,7C1F9,7F8D7,18A4E10
Process is as follows:
Anti-LukF monoclonal antibody: 10D1A10,16A10A3,6H10E5
According to following scheme immune mouse: at reorganization Luk F albumen and the complete Freund's adjuvant of the 0th day (D0) intraperitoneal injection 10 μ g.At the 14th day (D14), the 28th day (D28), the reorganization Luk F albumen and the incomplete Freund's adjuvant of the same amount of further intraperitoneal injection.Merged preceding 4 days, intravenous injection is with the Luk F antigen of 50 μ g of physiological saline dilution.
By 1600 portions of supernatants of indirect ELISA technology screening.The concentration that is dissolved in the PBS damping fluid (pH7.2) with 100 μ l is antigen (reorganization Luk F albumen) " bag quilt " culture plate of 1 μ g/ml." the bag quilt " plate is incubated overnight 18-22 ℃ temperature.Plate spends the night and 37 °+/-2 ℃ incubations 1 hour with the PBS-1% milk powder of 200 μ l is saturated.Add 100 μ l and be diluted in the supernatant or the ascites of PBS damping fluid-0.05% polysorbas20, then with plate 37 °+/-2 ℃ incubations 1 hour.Add 1/2000 of 100 μ l and be diluted in the polyclonal antibody in the goat anti-mouse Ig (H+L) of alkaline phosphatase (JacksonImmunoresearch ref:115-055-062) of puting together among PBS damping fluid-1% BSA, plate is subsequently 37 °+/-2 ℃ incubations 1 hour.What add 100 μ l concentration and be 2mg/ml is dissolved in DEA-HCl (bioM é rieux ref.60002989) PNPP (bioM é rieux ref.60002990) (pH=9.8).Plate was 37 °+/-2 ℃ incubations 30 minutes.By adding the 1N NaOH blocking reaction of 100 μ l.Between each step with the washing of the PBS-0.05% polysorbas20 of 300 μ l 3 times.Before adding PNPP, also to wash with distilled water.
Indirect ELISA is found 72 parts of positive supernatant, OD〉0.6.After the specificity test, aforesaid 3 kinds of antibody have been produced.
Anti-Luk S monoclonal antibody: 2H2H12,3D9D12,7C1F9,7F8D7,18A4E10
According to following scheme immune mouse: at reorganization Luk S albumen and the complete Freund's adjuvant of the 0th day (D0) intraperitoneal injection 10 μ g.At D14, D28, the reorganization Luk S albumen and the incomplete Freund's adjuvant of the same amount of further intraperitoneal injection.Merged preceding 4 days, intravenous injection is with the Luk S albumen of 50 μ g of physiological saline dilution.
By 1800 portions of supernatants of indirect ELISA technology screening.The concentration that is dissolved in the PBS damping fluid (pH7.2) with 100 μ l is reorganization Luk S albumen " bag quilt " culture plate of 1 μ g/ml." the bag quilt " plate is incubated overnight 18-22 ℃ temperature.Plate spends the night and 37 °+/-2 ℃ incubations 1 hour with the PBS-1% milk powder of 200 μ l is saturated.Add 100 μ l and be diluted in the supernatant or the ascites of PBS damping fluid-0.05% polysorbas20, then with plate 37 °+/-2 ℃ incubations 1 hour.Add 1/2000 of 100 μ l and be diluted in the polyclonal antibody in the goat anti-mouse Ig (H+L) of alkaline phosphatase (JacksonImmunoresearch ref:115-055-062) of puting together among PBS damping fluid-1% BSA, plate is subsequently 37 °+/-2 ℃ incubations 1 hour.What add 100 μ l concentration and be 2mg/ml is dissolved in DEA-HCl (bioM é rieux ref.60002989) PNPP (bioM é rieux ref.60002990) (pH=9.8).Plate was 37 °+/-2 ℃ incubations 30 minutes.By adding the 1N NaOH blocking reaction of 100 μ l.Between each step with the washing of the PBS-0.05% polysorbas20 of 300 μ l 3 times.Before adding PNPP, also to wash with distilled water.
Indirect ELISA is found 51 parts of positive supernatant, OD〉0.6.After the specificity test, aforesaid 5 kinds of antibody have been produced.
3. generation polyclonal antibody
3.1.
Produce anti-Luk S polyclonal antibody
Obtained anti-LukS polyclonal antibody No.173/89 and 176/89, process is as follows:
At D0, give the synthetic peptide of LukS-PV of rabbit (New Zealand White) intracutaneous injection 200 μ g, its sequence is CSGHDPNLFVGYKPYSQN (SEQ ID No.3), the N coupling KLH (keyhole limpet hemocyanin (Keyhole Limpet Hemocyanin)) (Agro-Bio).At D14, D28, D42 and D81, further give the synthetic peptide that is coupled to KLH of the same amount of rabbit hypodermic injection.Get the serum of animal at D0, D49 and D89.The rabbit anteserum that purifying D89 is got by carrying out affinity chromatography with described synthetic peptide.For this reason, the explanation according to manufacturer (Amersham-Pharmacia) is coupled to described peptide on the 1ml Hi-Trap Ago-Gel post.Serum 50/50 is diluted in the PBS damping fluid, and pH 7.4, is then injected on the post, and speed is 1ml/ minute.PBS (pH 7.4) washing back is with glycocoll/HCl potpourri pH3 wash-out of 50mM, then immediately in and the wash-out composition, use 0.15M PBS (pH 7.4) dialysis subsequently.
3.2.
Produce anti-Luk F polyclonal antibody
Obtained anti-LukF polyclonal antibody, process is as follows:
At D0, give the synthetic peptide of LukF-PV of rabbit (New Zealand White) intracutaneous injection 200 μ g, its sequence is CNFNWIGNNYKDENRATHTS (SEQ ID No.4), the N coupling KLH (keyhole limpet hemocyanin (Keyhole Limpet Hemocyanin)) (Agro-Bio).At D14, D28, D42 and D81, further give the synthetic peptide that is coupled to KLH of the same amount of rabbit hypodermic injection.Get the serum of animal at D0, D49 and D89.The rabbit anteserum that purifying D89 is got by carrying out affinity chromatography with described synthetic peptide.For this reason, the explanation according to manufacturer (Amersham-Pharmacia) is coupled to described peptide on the 1ml Hi-Trap Ago-Gel post.Serum 50/50 is diluted in the PBS damping fluid, and pH 7.4, is then injected on the post, and speed is 1ml/ minute.PBS (pH7.4) washing back is with glycocoll/HCl potpourri pH3 wash-out of 50mM, then immediately in and the wash-out composition, use 0.15M PBS (pH 7.4) dialysis subsequently.
4. the generation of antibody fragment and coupling
With the anti-LukS polyclonal antibody 173/89 of the pepsin digestion that is incorporated into agarose and 176/891 hour 30 minutes, 3.5,37 ℃ of pH were so that remove its Fc fragment and obtain F (ab ') 2 fragments.
With these fragments of peroxidase labelling, process is as follows: with the carbonate buffer solution of pH 9.6 dialysis F (ab ') 2 fragments, and with use NaIO in advance
4The peroxidase of oxidation (Roche) was 18-25 ℃ of coupling 2 hours, and ratio is: and the F of 1mol (ab ') the 2:2mol peroxidase.Use NaBH
4Handle 1 hour with the blocking-up coupling reaction at 2-8 ℃, product is dialysed with the PBS damping fluid that contains antiseptic.
Use biotin labeling subsequently, process is as follows: with the carbonate buffer solution of pH 8.3 dialysis F (ab ') 2 fragments, and with biotin-NHS (Roche) 18-25 ℃ of coupling 2 hours, ratio is: the F of 1mol (ab ') 2:10mol biotin.Handle 20 minutes with the blocking-up coupling reaction with lysine at 18-25 ℃, product is dialysed with the PBS+ azide.
Embodiment 2: the diagnosis of PVL-producing staphylococcus aureus bacterial strain
1. preparation biological sample
At GP agar (Difco:10g/l peptone, 5g/l yeast extract, 17g/l agar; Sigma:5g/l NaCl, 1g/l glucose) go up and cultivate from clinical staphylococcus aureus strains in advance.Cultivate after 18 hours for 37 ℃ and verify purity, then with some colonies (about 10
7CFU/ml) be seeded to 25ml glass taper culture flask interior 5ml CCY nutrient culture media (Difco:30g/l yeast extract, 20g/l casamino acid; Sigma:3.11g/l Na
2HPO
42H
2O, 0.41g/lKH
2PO
4, the 23g/l pyruvic acid) in.37 ℃ of wave and culture are after 18 hours, and centrifugal (8000g, 4 ℃, 10 minutes) collect culture supernatants, and it is standby that short-term is kept at 4 ℃ or-20 ℃ then.
Each bacterial strain is equipped with identifier (A or LY add 6 bit digital), and if bacterial strain PVL-producing then be labeled as "+", if PVL-producing then be labeled as "-" not.
2.ELISA measure
Wrap by elisa plate hole (Greiner, 100 μ l/ holes) with anti-LukS antibody 18A4E10, for this reason, usefulness PBS (Sigma PBS, pH 7.4; 0.1% sodium azide) dilute this antibody to 10 μ g/ml, with plate environment temperature (about 22 ℃) incubation 18 hours.After PBS polysorbas20 (BioradImmuno Wash 1575 machine) washing 5 times, with plate with PBS tween (0.05%)/10% milk powder/0.5% BSA (Sigma) in saturated 2 hours of environment temperature (150 μ l/ hole).
After PBS tween (0.05%) washing 5 times, 37 ℃ of incubation culture supernatants 75 minutes.
After PBS tween (0.05%) washing 5 times, with the 1/100 rabbit antibody 176-89 that is diluted in PBS-T5% milk powder, 37 ℃ of incubations of the F of peroxidase labelling (ab ' 2) fragment solution 75 minutes.Add after the PBS-tween washing 5 times 75 μ l TMB solution (3,3 ', 5,5 '-tetramethyl benzidine substrate, Sigma), plate lucifuge incubation 30 minutes.By adding the 1N H of 75 μ l
2SO
4Cessation reaction.Use the reading (OD) of MicroPlate MP Reader 680 (Biorad) in the 450nm assay plate.
3. result
The results are shown in Fig. 1, this figure has provided the OD of each bacterial strain.
This result shows, has 12 strains clearly to be detected in 15 parts of PVL+ bacterial strains, and in 14 parts of PVL-bacterial strains, have 3 parts to obtain false positive results.
These results clearly illustrate that, adopt conventional immunologic assay method can distinguish PVL+ bacterial strain and PVL-bacterial strain, but for latter's shortage specificity.
Embodiment 3: diagnosis of PVL-producing staphylococcus aureus bacterial strain behind the heating pretreatment biological sample
1. sample preparation
Biological sample as embodiment 2 preparations is carried out heat treated at 95 ℃, and the time is 10 to 30 minutes, determines OD according to embodiment 2 described schemes.
Table 1 has provided OD result, and its display process is more than 10 minutes, so that the PVL sex change.
Table 1
| Strain number | PVL | Do not heat | Heated 10 minutes | Heated 20 minutes | Heated 30 minutes |
| LY990179 | + | 0.839 | 2.126 | 2.76 | >3 |
| A960420 | + | 1.73 | 2.844 | >3 | >3 |
| A990438 | - | 1.545 | 0.852 | 0.25 | 0 |
| A980642 | + | 0.458 | 1.364 | 2.325 | >3 |
| LY990301 | - | 2.123 | 0.105 | 0 | 0 |
| HT20020275 | + | 0.729 | 2.269 | >3 | >3 |
| LY990333 | - | 0.615 | 0.202 | 0 | 0 |
| HT20040540 | + | 1.178 | >3 | >3 | >3 |
2.ELISA measure
Repeat the 2nd described process of the foregoing description 2, difference is to use is culture supernatants at pretreated 29 bacterial strains of 95 ℃ of heating 1 hour (15 parts of PVL+, 14 parts of PVL-).
3. result
The results are shown in Fig. 2, wherein provided the OD of each bacterial strain.
These results show that method of the present invention has identified all PVL+ bacterial strains, and your pupil of its OD level is to differentiate mutually with the PVL-bacterial strain.
Therefore, pre-service can improve the specificity of method of the present invention.
Embodiment 4: adopt diagnosis of PVL-producing staphylococcus aureus bacterial strain behind polytype modification treatment pre-service biological sample
1. sample preparation
Used from 3 parts of clinical PVL+ bacterial strains (LY990084, A92007, LUG855) and 3 parts of PVL-bacterial strains (LY990333, LY991321 RN6911), handle as follows:
-non-processor (0),
-95 ℃ of heat denatured 1 hour (1),
-sour sex change is with the KH of final concentration 0.1M
2PO
4Oscillation treatment 30 minutes adds NaOH neutralization (2) then,
-combined degeneration is with the KH of final concentration 0.1M
2PO
4Oscillation treatment was carried out sour sex change in 10 minutes, and 95 ℃ were boiled 10 minutes then, added NaOH neutralization (3) then.
2.ELISA measure
Repeat as embodiment 2 described processes, difference is to use is to select pretreated bacterial strain and with the 1/1000 rabbit antibody 176-89 that is diluted in PBS-tween 5% milk powder, the F of peroxidase labelling (ab ' 2) fragment solution according to the above-mentioned the 1st.
3. result
The results are shown in Fig. 3, wherein provided the OD of each bacterial strain.
These results show no matter how sample is handled, and the OD value of all PVL-bacterial strains is between 0.09 and 0.181.
For the PVL+ bacterial strain, the OD value after the processing is higher than the PVL-bacterial strain.Increase by chemical treatment, thermal treatment and the resulting OD of both combined treatment have significance,statistical (be respectively p=0.024, p＜0.001, p=0.018), and thermal treatment is seemingly effective under these conditions.
Therefore, pre-service can improve the specificity of conventional immunologic assay method.
Embodiment 5: but method early detection PVL-producing staphylococcus aureus of the present invention
1. preparation biological sample
Cultivate from 4 parts of clinical staphylococcus aureus strains (2 parts of PVL+ bacterial strains and 2 parts of PVL-bacterial strains) in advance according to embodiment 2 described methods.Then colony is used in the CCY nutrient culture media of the 5ml of 25ml glass taper culture flask, carrying out the enrichment inoculation.Immediately nutrient culture media is sampled after the inoculation, 37 ℃ of shaken cultivation, and at postvaccinal 1h, 2h, 3h and 18h continuous sampling.Centrifugal (8000g, 4 ℃, 10 minutes) collect culture supernatants, and it is standby that short-term is stored in 4 ℃ or-20 ℃.As heat treated sample as described in the embodiment 3.
2.ELISA measure
Repeat the 2nd described process of the foregoing description 2, what difference was to use is the culture supernatants of above-mentioned the 1st pretreated bacterial strain.
3. result
Table 2 has provided OD result, and it shows that method of the present invention can detect PVL (began in 2 hours and can clearly detect from cultivating) very apace.
Table 2
Embodiment 6: but the PVL in the method early detection biological samples of the present invention of sampling
1. preparation biological sample
Clinical samples is broncho-pulmonary aspirate with patient of staphylococcus aureus PVL+ bacterial strain or PVL-strain infection, bronchovesicular liquid and from the fester of dermapostasis or from the fester of deep suppuration focus.These samples are stored in-20 ℃ before use method of the present invention is analyzed.Room temperature made it the sample vortex oscillation homogenize in 15 minutes after melting.Through centrifugal 10 minutes (4000rpm, 15 ℃), supernatant is transferred to Eppendorf pipe, 95 ℃ of sex change are 1 hour then.
For the unusual sample of thickness, earlier sample 50/50 is diluted in PBS, pH7.4, vortex oscillation is 15 minutes then.Through centrifugal 10 minutes (4000rpm, 15 ℃), supernatant is transferred to Eppendorf pipe, 95 ℃ of sex change are 1 hour then.
For the supernatant of thickness, it 50/50 is diluted in normal heptane (Merck), and then vortex oscillation 10 minutes.Through centrifugal 10 minutes (4000rpm, 15 ℃), thoroughly remove the upper strata phase that is equivalent to normal heptane, and with lower floor's phase transfer to another Eppendorf pipe, 95 ℃ of sex change are 1 hour then.
2.ELISA measure
Repeat the 2nd described process of the foregoing description 2, what difference was to use is above-mentioned the 1st pretreated biological samples.
3. result
Table 3 and 4 has provided the PVL measurement result, and these results show, regardless of the methicillin-sensitivity of bacterial strain, all can directly detect the PVL in the biological samples, and does not have false positive (be PVL-bacterial strain sample none be detected as contain PVL).In addition, do not influence detecting PVL with normal heptane pre-service sample.
Table 3
| Sample | The sample type | The sample pre-service | Bacterial strain * | The amount of PVL (μ g/ml) |
| 17 | Bronchoalveolar lavage fluid | Do not have | PVL+ | <0.05 |
| 13 | The bronchus aspirate | Do not have | PVL- | <0.05 |
| 18 | The little lavation of bronchovesicular | Do not have | PVL- | <0.05 |
| 22 | The bronchus aspirate | Do not have | PVL- | <0.05 |
| 11 | The bronchus aspirate | Do not have | PVL- | <0.05 |
| 8 | The bronchus aspirate | The PBS+ normal heptane | PVL- | <0.05 |
| 16 | The bronchus aspirate | Do not have | PVL- | <0.05 |
| 10 | The bronchus aspirate | Do not have | PVL- | <0.05 |
| 19 | Phlegm | Do not have | PVL- | <0.05 |
| 14 | The bronchus aspirate | Do not have | PVL- | <0.05 |
| 15 | The bronchus aspirate | Do not have | PVL- | <0.05 |
| 12 | The bronchus aspirate | Do not have | PVL- | <0.05 |
| 7 | The bronchus aspirate | Do not have | PVL- | <0.05 |
| 5 | The bronchus aspirate | Do not have | PVL- | <0.05 |
| 23 | Joint fluid | Do not have | PVL- | <0.05 |
| 20 | Peritoneal fluid | Do not have | PVL- | <0.05 |
| 4 | Drainage liquid | Do not have | PVL+ | <0.05 |
| 2 | Abscess | Do not have | PVL+ | 1 |
| 3 | Abscess | Do not have | PVL- | <0.05 |
| 1 | Drainage liquid | Do not have | PVL- | <0.05 |
| 5 | The bronchus aspirate | PBS | PVL+ | 1.2 |
| 5 | The bronchus aspirate | The PBS+ normal heptane | PVL+ | 1.2 |
| 6 | The bronchus aspirate | The PBS+ normal heptane | PVL+ | 1.2 |
*According to Vandenesch et al.
2Whether described method is by existing the LukS-PV of the PVL that encodes and LukF-PV gene to determine that described bacterial strain is PVL-or PVL+ in the PCR detection staphylococcus aureus separator.
Table 4
*According to Vandenesch et al.
2Whether described method is by existing the LukS-PV of the PVL that encodes and LukF-PV gene to determine that described bacterial strain is PVL-or PVL+ in the PCR detection staphylococcus aureus separator.
*According to Vandenesch et al.
2Described method detects the mecA gene of coding methicillin resistance in the staphylococcus aureus separator.
Document
1：Ward?PD，Turner?WD，1980，Infect.Immun.，28(2)：393-397
2：Vandenesch?et?al.，2003，Emerg?Infect?Dis，9(8)：978-984
3：Dufour?P?et?al.，2002，Clin?Infect?Dis，35：819-824
4：Cribier?B?et?al，1992，Dermatology，185：175-185
5：Freney?J?et?al.，2000，Précis?de?Bactériologie?Clinique[Handbook?of?clinicalbacteriology]，40：298?and?793-794
6：J.Histochem.Cytochem.45：481-491，1997
7：Labandeira-Rey?M?et?al，Science?2007，in?press
Sequence table
＜110〉Biomerieux SA
National Health and Medicine Inst.
Clo Derby nail (unit of length)-University Lyon 1-Claude Bernard
＜120〉be used for the method for in-vitro diagnosis PVL-producing staphylococcus aureus
<130>PVL?STAPH
<160>4
<170>PatentIn?version?3.3
<210>1
<211>309
<212>PRT
<213>Staphylococcus?aureus
<400>1
<210>2
<211>323
<212>PRT
<213>Staphylococcus?aureus
<400>2
<210>3
<211>18
<212>PRT
<213>Staphylococcus?aureus
<400>3
<210>4
<211>20
<212>PRT
<213>Staphylococcus?aureus
<400>4
Claims (5)
1, uses that staphylococcus aureus is built the group or the biological sample in-vitro diagnosis of the individuality that infects produces the method for the staphylococcus aureus of P-VL (PVL) from being easy to take place, wherein carry out described diagnosis, it is characterized in that described biological sample is carried out pre-service so that the PVL sex change by adopting conventional immunologic assay method to detect PVL.
2, the method for claim 1 is characterized in that the immunologic assay method of described routine is a sandwich method.
3, claim 1 or 2 method is characterized in that described immunologic assay method uses at least a anti-PVL monoclonal antibody.
4, each method in the claim 1 to 3 is characterized in that described pre-service is the heating of the temperature between 60 to 100 ℃ at least 10 minutes.
5, each method in the claim 1 to 4 is characterized in that it comprises that the staphylococcus aureus of determining to be present in the described biological sample is the additional step of methicillin-resistant bacterium (MRSA) or methicillin-sensitivity bacterium (MSSA).
Applications Claiming Priority (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR0654336 | 2006-10-18 | ||
| FR0654336 | 2006-10-18 | ||
| FR0753081 | 2007-02-06 | ||
| FR0753081A FR2912219A1 (en) | 2007-02-06 | 2007-02-06 | In vitro diagnostic process of Staphylococcus aureus producers of panton velentine leukocidin by biological sample, comprises detecting panton velentine leukocidin by routine immunological test using anti-PVL monoclonal antibody |
| PCT/FR2007/052165 WO2008050041A1 (en) | 2006-10-18 | 2007-10-16 | Method for in vitro diagnosis of pvl-producing staphylococcus aureus |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101535811A true CN101535811A (en) | 2009-09-16 |
| CN101535811B CN101535811B (en) | 2016-05-04 |
Family
ID=38009743
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200780038592.4A Expired - Fee Related CN101535811B (en) | 2006-10-18 | 2007-10-16 | For the method for in-vitro diagnosis PVL-producing staphylococcus aureus |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101535811B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103025352A (en) * | 2010-05-05 | 2013-04-03 | 纽约大学 | Staphylococcus aureus leukocidins, therapeutic compositions, and uses thereof |
| CN104185790A (en) * | 2011-11-11 | 2014-12-03 | 美艾利尔圣地亚哥公司 | Devices and methods for detection of panton-valentine leukocidin (pvl) |
| CN105473613A (en) * | 2013-05-21 | 2016-04-06 | 阿尔萨尼斯生物科学有限责任公司 | Generation of highly potent antibodies neutralizing the lukgh (lukab) toxin of staphylococcus aureus |
| CN114990237A (en) * | 2021-10-29 | 2022-09-02 | 成都中医药大学 | Primer, kit and method for detecting staphylococcus aureus |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1425919A (en) * | 2003-01-30 | 2003-06-25 | 中国人民解放军军事医学科学院卫生学环境医学研究所 | Immune chip for detecting staphylococcal enterotoxin and papaverine and its preparing method |
-
2007
- 2007-10-16 CN CN200780038592.4A patent/CN101535811B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1425919A (en) * | 2003-01-30 | 2003-06-25 | 中国人民解放军军事医学科学院卫生学环境医学研究所 | Immune chip for detecting staphylococcal enterotoxin and papaverine and its preparing method |
Non-Patent Citations (1)
| Title |
|---|
| KASAI S ET AL: "IMMUNOSSAY OF THE MRSA-RELATED TOXIC PROTEIN, LEUKOCIDIN, WITH SCANNING ELECTROCHEMICAL MICROSCOPY.", 《ANALYTICAL CHEMISTRY》 * |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103025352A (en) * | 2010-05-05 | 2013-04-03 | 纽约大学 | Staphylococcus aureus leukocidins, therapeutic compositions, and uses thereof |
| CN103025352B (en) * | 2010-05-05 | 2017-07-11 | 纽约大学 | Staphylococcus aureus leukocidin and its therapeutic combination and purposes |
| US9783582B2 (en) | 2010-05-05 | 2017-10-10 | New York University | Staphylococcus aureus leukocidins, therapeutic compositions, and uses thereof |
| CN107286224A (en) * | 2010-05-05 | 2017-10-24 | 纽约大学 | Staphylococcus aureus leukocidin and its therapeutic combination and purposes |
| US10316067B2 (en) | 2010-05-05 | 2019-06-11 | New York University | Staphylococcus aureus leukocidins, therapeutic compositions, and uses thereof |
| US11584782B2 (en) | 2010-05-05 | 2023-02-21 | New York University | Staphylococcus aureus leukocidins, therapeutic compositions, and uses thereof |
| CN104185790A (en) * | 2011-11-11 | 2014-12-03 | 美艾利尔圣地亚哥公司 | Devices and methods for detection of panton-valentine leukocidin (pvl) |
| CN104185790B (en) * | 2011-11-11 | 2016-09-28 | 美艾利尔圣地亚哥公司 | Detect the apparatus and method of cytocidal toxin (PVL) |
| CN106841594A (en) * | 2011-11-11 | 2017-06-13 | 美艾利尔圣地亚哥公司 | The toxin of leucocyte is killed in detection（PVL）Apparatus and method |
| CN105473613A (en) * | 2013-05-21 | 2016-04-06 | 阿尔萨尼斯生物科学有限责任公司 | Generation of highly potent antibodies neutralizing the lukgh (lukab) toxin of staphylococcus aureus |
| CN114990237A (en) * | 2021-10-29 | 2022-09-02 | 成都中医药大学 | Primer, kit and method for detecting staphylococcus aureus |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101535811B (en) | 2016-05-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Bascomb et al. | Use of enzyme tests in characterization and identification of aerobic and facultatively anaerobic gram-positive cocci | |
| Kay et al. | Isolation and identification of Vibrio cholerae O1 from fecal specimens | |
| JP5712140B2 (en) | Method for detecting microorganisms belonging to Mycoplasma pneumoniae and / or Mycoplasma genitalium | |
| Kawatsu et al. | Development and evaluation of immunochromatographic assay for simple and rapid detection of Campylobacter jejuni and Campylobacter coli in human stool specimens | |
| Birkeland et al. | Quantitative studies of heat-stable proteinase from Pseudomonas fluorescens P1 by the enzyme-linked immunosorbent assay | |
| Pongsunk et al. | Rapid identification of Burkholderia pseudomallei in blood cultures by a monoclonal antibody assay | |
| Woo et al. | Enterococcus cecorum empyema thoracis successfully treated with cefotaxime | |
| CN101535811A (en) | Method for in vitro diagnosis of PVL-producing staphylococcus aureus | |
| Bayat et al. | Development of IgY‐Based Sandwich ELISA as a Robust Tool for Rapid Detection and Discrimination of Toxigenic Vibrio cholerae | |
| Fawcett et al. | Helicobacter pylori can be induced to assume the morphology of Helicobacter heilmannii | |
| Gessler et al. | Sensitive detection of botulinum neurotoxin types C and D with an immunoaffinity chromatographic column test | |
| JP5183635B2 (en) | In vitro diagnostic method for PVL-producing Staphylococcus aureus | |
| CN114152748A (en) | Double-antibody sandwich ELISA diagnostic kit for detecting African swine fever virus and method thereof | |
| Chaicumpa et al. | Rapid diagnosis of cholera caused by Vibrio cholerae O139 | |
| CN104673817B (en) | A kind of prokaryotic expression of lipase and application and process for fixation | |
| EP2098865A1 (en) | Procedure for the detection of legionella spp using polyclonal antibodies | |
| Lomholt et al. | Immunoglobulin A1 protease activity in Gemella haemolysans | |
| CN101805723A (en) | Anti-glutamate dehydrogenase protein monoclonal antibody, preparation method thereof and use thereof | |
| Sakata et al. | Development of a rapid immunochromatographic assay to detect contamination of raw oysters with enteropathogenic Vibrio parahaemolyticus | |
| Qian et al. | Expression and purification of two major outer membrane proteins from Vibrio alginolyticus | |
| Smith | Clostridium botulinum genomes and genetic diversity | |
| Ehrenberg | Establishment of an ELISA and a lateral flow device for detection of European and American foulbrood including genotype-differentiation of the American foulbrood causing agent (ERIC I & ERIC II) in honey bees | |
| JPWO2002065131A1 (en) | Methods and reagents for detecting Vibrio bacteria and antibodies used therefor | |
| AU2007339423B2 (en) | Method for detecting pathogens | |
| RU2575075C2 (en) | Method of detection of microorganisms belonging to species of mycoplasma pneumoniae mind and/or mycoplasma genitalium |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20160504 |