MXPA00012583A - METHOD FOR DETERMINING ANTIBIOTICS WITH&bgr;-LACTAM CORE IN A BIOLOGICAL FLUID - Google Patents
METHOD FOR DETERMINING ANTIBIOTICS WITH&bgr;-LACTAM CORE IN A BIOLOGICAL FLUIDInfo
- Publication number
- MXPA00012583A MXPA00012583A MXPA/A/2000/012583A MXPA00012583A MXPA00012583A MX PA00012583 A MXPA00012583 A MX PA00012583A MX PA00012583 A MXPA00012583 A MX PA00012583A MX PA00012583 A MXPA00012583 A MX PA00012583A
- Authority
- MX
- Mexico
- Prior art keywords
- antibiotics
- ppb
- sensitive
- receptor
- lactam ring
- Prior art date
Links
- 239000003242 anti bacterial agent Substances 0.000 title claims abstract description 70
- 229940064005 Antibiotic throat preparations Drugs 0.000 title claims abstract description 66
- 229940083879 Antibiotics FOR TREATMENT OF HEMORRHOIDS AND ANAL FISSURES FOR TOPICAL USE Drugs 0.000 title claims abstract description 66
- 229940042052 Antibiotics for systemic use Drugs 0.000 title claims abstract description 66
- 229940042786 Antitubercular Antibiotics Drugs 0.000 title claims abstract description 66
- 229940093922 Gynecological Antibiotics Drugs 0.000 title claims abstract description 66
- 229940024982 Topical Antifungal Antibiotics Drugs 0.000 title claims abstract description 66
- 229940079866 intestinal antibiotics Drugs 0.000 title claims abstract description 66
- 229940005935 ophthalmologic Antibiotics Drugs 0.000 title claims abstract description 66
- 239000012530 fluid Substances 0.000 title claims abstract description 25
- 230000003115 biocidal Effects 0.000 claims abstract description 113
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 57
- 239000000203 mixture Substances 0.000 claims abstract description 34
- 241000194108 Bacillus licheniformis Species 0.000 claims abstract description 6
- 238000000034 method Methods 0.000 claims description 37
- 125000003460 beta-lactamyl group Chemical group 0.000 claims description 28
- 239000012528 membrane Substances 0.000 claims description 21
- 239000002245 particle Substances 0.000 claims description 21
- 239000000126 substance Substances 0.000 claims description 21
- 239000003550 marker Substances 0.000 claims description 15
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- 108090000790 Enzymes Proteins 0.000 claims description 14
- 238000002372 labelling Methods 0.000 claims description 13
- 239000007787 solid Substances 0.000 claims description 12
- 102000003992 Peroxidases Human genes 0.000 claims description 8
- 108090000437 Peroxidases Proteins 0.000 claims description 8
- 102000006635 beta-Lactamases Human genes 0.000 claims description 8
- 108020004256 beta-Lactamases Proteins 0.000 claims description 8
- 239000011324 bead Substances 0.000 claims description 6
- 230000005291 magnetic Effects 0.000 claims description 6
- 238000010521 absorption reaction Methods 0.000 claims description 5
- 230000002255 enzymatic Effects 0.000 claims description 5
- 108020004774 Alkaline Phosphatase Proteins 0.000 claims description 4
- 102000002260 Alkaline Phosphatase Human genes 0.000 claims description 4
- 108090001123 antibodies Proteins 0.000 claims description 4
- 102000004965 antibodies Human genes 0.000 claims description 4
- 230000015572 biosynthetic process Effects 0.000 claims description 3
- 238000005755 formation reaction Methods 0.000 claims description 3
- 229920000126 Latex Polymers 0.000 claims description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 2
- 229910052799 carbon Inorganic materials 0.000 claims description 2
- 229910052751 metal Inorganic materials 0.000 claims description 2
- 239000002184 metal Substances 0.000 claims description 2
- BUGBHKTXTAQXES-UHFFFAOYSA-N selenium Chemical compound [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 claims description 2
- NINIDFKCEFEMDL-UHFFFAOYSA-N sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 claims description 2
- 229910052717 sulfur Inorganic materials 0.000 claims description 2
- 239000011593 sulfur Substances 0.000 claims description 2
- 229910052714 tellurium Inorganic materials 0.000 claims description 2
- PORWMNRCUJJQNO-UHFFFAOYSA-N tellurium Chemical compound [Te] PORWMNRCUJJQNO-UHFFFAOYSA-N 0.000 claims description 2
- 239000000243 solution Substances 0.000 description 68
- 210000004080 Milk Anatomy 0.000 description 56
- 239000008267 milk Substances 0.000 description 56
- 235000013336 milk Nutrition 0.000 description 56
- 238000001514 detection method Methods 0.000 description 30
- 239000007853 buffer solution Substances 0.000 description 22
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 21
- UIIMBOGNXHQVGW-UHFFFAOYSA-M NaHCO3 Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 18
- 230000001808 coupling Effects 0.000 description 16
- 238000010168 coupling process Methods 0.000 description 16
- 238000005859 coupling reaction Methods 0.000 description 16
- 102000037240 fusion proteins Human genes 0.000 description 16
- 108020001507 fusion proteins Proteins 0.000 description 16
- LQOLIRLGBULYKD-JKIFEVAISA-N Cloxacillin 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=C1Cl LQOLIRLGBULYKD-JKIFEVAISA-N 0.000 description 14
- 229940056360 Penicillin G Drugs 0.000 description 14
- FAPWRFPIFSIZLT-UHFFFAOYSA-M sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 14
- 229940088598 Enzyme Drugs 0.000 description 13
- 229960003326 cloxacillin Drugs 0.000 description 13
- UQLLWWBDSUHNEB-CZUORRHYSA-N Cefaprin Chemical compound N([C@H]1[C@@H]2N(C1=O)C(=C(CS2)COC(=O)C)C(O)=O)C(=O)CSC1=CC=NC=C1 UQLLWWBDSUHNEB-CZUORRHYSA-N 0.000 description 12
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L MgCl2 Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 12
- 229960004350 cefapirin Drugs 0.000 description 12
- 239000011521 glass Substances 0.000 description 12
- AVKUERGKIZMTKX-NJBDSQKTSA-N Ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 11
- 229940106193 Cephapirin Drugs 0.000 description 11
- 229960003022 amoxicillin Drugs 0.000 description 11
- LSQZJLSUYDQPKJ-NJBDSQKTSA-N amoxicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=C(O)C=C1 LSQZJLSUYDQPKJ-NJBDSQKTSA-N 0.000 description 11
- 229960000723 ampicillin Drugs 0.000 description 11
- 102000004169 proteins and genes Human genes 0.000 description 11
- 108090000623 proteins and genes Proteins 0.000 description 11
- DHMQDGOQFOQNFH-UHFFFAOYSA-N glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 8
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 8
- 229910052737 gold Inorganic materials 0.000 description 8
- 239000010931 gold Substances 0.000 description 8
- CDBYLPFSWZWCQE-UHFFFAOYSA-L sodium carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 8
- 239000000758 substrate Substances 0.000 description 8
- ZBHXIWJRIFEVQY-IHMPYVIRSA-N Ceftiofur Chemical compound S([C@@H]1[C@@H](C(N1C=1C(O)=O)=O)NC(=O)\C(=N/OC)C=2N=C(N)SC=2)CC=1CSC(=O)C1=CC=CO1 ZBHXIWJRIFEVQY-IHMPYVIRSA-N 0.000 description 7
- 229940049954 Penicillin Drugs 0.000 description 7
- 229940072417 Peroxidase Drugs 0.000 description 7
- GSEJCLTVZPLZKY-UHFFFAOYSA-N Tris Chemical compound OCCN(CCO)CCO GSEJCLTVZPLZKY-UHFFFAOYSA-N 0.000 description 7
- 229960000626 benzylpenicillin Drugs 0.000 description 7
- 229960005229 ceftiofur Drugs 0.000 description 7
- 239000011780 sodium chloride Substances 0.000 description 7
- 239000000725 suspension Substances 0.000 description 6
- 230000000694 effects Effects 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 229910001629 magnesium chloride Inorganic materials 0.000 description 5
- YFAGHNZHGGCZAX-JKIFEVAISA-N Dicloxacillin 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=C(Cl)C=CC=C1Cl YFAGHNZHGGCZAX-JKIFEVAISA-N 0.000 description 4
- ZBCBWPMODOFKDW-UHFFFAOYSA-N Diethanolamine Chemical compound OCCNCCO ZBCBWPMODOFKDW-UHFFFAOYSA-N 0.000 description 4
- 239000004471 Glycine Substances 0.000 description 4
- 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 4
- LWIHDJKSTIGBAC-UHFFFAOYSA-K Tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 4
- 239000000853 adhesive Substances 0.000 description 4
- 230000001070 adhesive Effects 0.000 description 4
- HOKIDJSKDBPKTQ-GLXFQSAKSA-M cephalosporin C(1-) Chemical compound S1CC(COC(=O)C)=C(C([O-])=O)N2C(=O)[C@@H](NC(=O)CCC[C@@H]([NH3+])C([O-])=O)[C@@H]12 HOKIDJSKDBPKTQ-GLXFQSAKSA-M 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 229960001585 dicloxacillin Drugs 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 230000003287 optical Effects 0.000 description 4
- 229960001019 oxacillin Drugs 0.000 description 4
- JUJWROOIHBZHMG-UHFFFAOYSA-N pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 4
- 230000002285 radioactive Effects 0.000 description 4
- 239000001187 sodium carbonate Substances 0.000 description 4
- 229910000029 sodium carbonate Inorganic materials 0.000 description 4
- 239000011534 wash buffer Substances 0.000 description 4
- MLYYVTUWGNIJIB-BXKDBHETSA-N Cefazolin Chemical compound S1C(C)=NN=C1SCC1=C(C(O)=O)N2C(=O)[C@@H](NC(=O)CN3N=NN=C3)[C@H]2SC1 MLYYVTUWGNIJIB-BXKDBHETSA-N 0.000 description 3
- 229960001139 Cefazolin Drugs 0.000 description 3
- JKMHFZQWWAIEOD-UHFFFAOYSA-N HEPES Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 3
- GPXLMGHLHQJAGZ-JTDSTZFVSA-N Nafcillin Chemical compound C1=CC=CC2=C(C(=O)N[C@@H]3C(N4[C@H](C(C)(C)S[C@@H]43)C(O)=O)=O)C(OCC)=CC=C21 GPXLMGHLHQJAGZ-JTDSTZFVSA-N 0.000 description 3
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- 210000004369 Blood Anatomy 0.000 description 2
- DEFJQIDDEAULHB-QWWZWVQMSA-N D-alanyl-D-alanine zwitterion Chemical compound C[C@@H]([NH3+])C(=O)N[C@H](C)C([O-])=O DEFJQIDDEAULHB-QWWZWVQMSA-N 0.000 description 2
- 102100006287 FUS Human genes 0.000 description 2
- 241000193385 Geobacillus stearothermophilus Species 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N HCl Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 108060003951 Immunoglobulins Proteins 0.000 description 2
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- 239000008280 blood Substances 0.000 description 2
- 150000001780 cephalosporins Chemical class 0.000 description 2
- LVSJLTMNAQBTPE-UHFFFAOYSA-N disodium tetraborate Chemical compound [Na+].[Na+].O1B(O)O[B-]2(O)OB(O)O[B-]1(O)O2 LVSJLTMNAQBTPE-UHFFFAOYSA-N 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N edta Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
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- HFNQLYDPNAZRCH-UHFFFAOYSA-K hydrogen carbonate;carbonate Chemical compound OC([O-])=O.[O-]C([O-])=O HFNQLYDPNAZRCH-UHFFFAOYSA-K 0.000 description 2
- 230000002427 irreversible Effects 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N iso-propanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
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- 229910052708 sodium Inorganic materials 0.000 description 2
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- PXIPVTKHYLBLMZ-UHFFFAOYSA-N sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
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- YOQDYZUWIQVZSF-UHFFFAOYSA-N sodium borohydride Substances [BH4-].[Na+] YOQDYZUWIQVZSF-UHFFFAOYSA-N 0.000 description 2
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- ZWUPPJBUPHOXQV-UHFFFAOYSA-M 2-(3-hydroxy-6-oxoxanthen-9-yl)benzoic acid;cyanate Chemical compound [O-]C#N.OC(=O)C1=CC=CC=C1C1=C2C=CC(=O)C=C2OC2=CC(O)=CC=C21 ZWUPPJBUPHOXQV-UHFFFAOYSA-M 0.000 description 1
- ZTOJFFHGPLIVKC-CLFAGFIQSA-N ABTS Chemical compound S/1C2=CC(S(O)(=O)=O)=CC=C2N(CC)C\1=N\N=C1/SC2=CC(S(O)(=O)=O)=CC=C2N1CC ZTOJFFHGPLIVKC-CLFAGFIQSA-N 0.000 description 1
- 241000187362 Actinomadura Species 0.000 description 1
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- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 241000193755 Bacillus cereus Species 0.000 description 1
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- 102000005367 Carboxypeptidases Human genes 0.000 description 1
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- YVGGHNCTFXOJCH-UHFFFAOYSA-N DDT Chemical compound C1=CC(Cl)=CC=C1C(C(Cl)(Cl)Cl)C1=CC=C(Cl)C=C1 YVGGHNCTFXOJCH-UHFFFAOYSA-N 0.000 description 1
- GHVNFZFCNZKVNT-UHFFFAOYSA-N Decanoic acid Chemical compound CCCCCCCCCC(O)=O GHVNFZFCNZKVNT-UHFFFAOYSA-N 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 208000010201 Exanthema Diseases 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N Kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
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- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 239000012564 Q sepharose fast flow resin Substances 0.000 description 1
- 206010037844 Rash Diseases 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
- 241001438449 Silo Species 0.000 description 1
- 102000006463 Talin Human genes 0.000 description 1
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- 238000004458 analytical method Methods 0.000 description 1
- 230000001745 anti-biotin Effects 0.000 description 1
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- OGGXGZAMXPVRFZ-UHFFFAOYSA-M dimethylarsinate Chemical compound C[As](C)([O-])=O OGGXGZAMXPVRFZ-UHFFFAOYSA-M 0.000 description 1
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- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
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- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
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Abstract
The invention concerns a method for detecting antibiotics with&bgr;-lactam core in a biological fluid comprising the following steps:a) contacting a specific volume of said biological fluid with an amount of identifying agent and incubating the resulting mixture in conditions for forming a complex of the antibiotics possibly present in said biological fluid with the identifying agent;b) contacting the mixture resulting from step a) with at least a reference antibiotic immobilised on a support, in conditions for forming a complex of the reference antibiotic with the amount of identifying agent which has not reacted in step a);and c) determining the amount of identifying agent fixed on the support. The invention is characterised in that the identifying agent comprises a receptor sensitive to antibiotics with&bgr;-lactam core obtained from Bacillus licheniformis.
Description
PROCESS TO DETERMINE ANTIBIOTICS CONTAINING A BETA-LACTAMA RING IN A BIOLOGICAL FLUID
Field of Invention
The present invention relates to novel, rapid and sensitive processes for determining antibiotics containing a β-lactam ring in a biological fluid, using a receptor that is 0 sensitive to antibiotics containing a β-lactam ring of Bacillus licheniformis. The invention also relates to kits for carrying out these processes.
'5 Background of the Invention
Currently, antibiotics are widely used, not only as therapeutic agents in the treatment of infectious diseases caused by bacteria, but also as agents for food storage and as additives in animal feed to stimulate growth. In this way, a need is increasingly appreciated to have the ability to detect the presence of
Ref: 125880 antibiotics, even in very low concentrations, in complex biological fluids, such as milk, urine, blood, serum, saliva, meat extracts, fermentation liquids or in aqueous buffer media. •
The case of milk production is an example of this, since it is well known to use antibiotics to treat certain diseases
infectious of the farm livestock.
• However, for obvious medical reasons, milk intended for human consumption must, in principle, be free of evidence of
antibiotics. On the other hand, penicillin concentrations of 0.005 I.U./ml or lower can have detrimental effects during the manufacture of milk-based products such as cheese, yogurt, etc. 20 Several situations can be imagined. In a first case, for example to detect the presence of antibiotics in the farm before transferring them to the wagon, priority must be given to
a simple and extremely fast (less than 5 minutes) test. It is also possible to think of using such a rapid test where, for example, the antibiotic that has been used for the treatment is known and where, on the other hand, this test allows the detection of the antibiotic in question
• in the legal standards. In the second case, when the emphasis is not on speed, the importance is to detect the majority, if not all, of the antibiotics in the legal standards. 10 The reason for this is that the laws in certain
• Countries impose completely specific quality standards. For example, the authorities of the United States of America require that
concentrations in the milk of the following six antibiotics do not exceed completely specific values: penicillin, 5 ppb; ampicillin, 10 ppb; Amoxicillin, 10 ppb; cloxacillin, 10 ppb; cephapirin, 20 ppb; Ceftiofur, 50 ppb. The Union
European 20 imposes quality standards as follows: penicillin, 4 ppb; Amoxicillin, 4 ppb; ampicillin 4 ppb; Cloxacycline, 30 ppb; dicloxacillin, 30 ppb; oxacillin, 30 ppb; cephapirin, 10 ppb; ceftiofur, 100 ppb; cefquinone 20 ppb; nafcillin
300 ppb; cefazolin, 50 ppb.
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It may be advantageous to have access to a test that can allow most antibiotics to be detected. On the other hand, in the farming industry, it can be considered that, in the absence of a test that has all the characteristics of speed, sensitivity and simplicity, it will be advantageous to have a test that can allow the best combination of these three parameters, even if these do not they are totally
place settings.
• Several types of tests have already been proposed for the detection of antibiotics containing a β-lactam ring in a biological fluid. 15 These tests generally make use of detection methods that employ a recognition agent (receptor or antibody), which
• specifically recognizes the antibiotic or a
Analogous to this antibiotic, and a labeling agent (radioelement, enzyme, fluorescent agent, etc.), these agents are referred to below as detection reagents. Depending on the elements chosen, the terms are used
radioimmunoassay (RIA), radio-receptor assay
(RRA), enzyme immunoassay (EIA), etc. In its general principle, these tests use the minimum combination of the two aforementioned elements (detection reagents) that make it possible to obtain a result whose value is
• indication of the amount of antibiotic present.
It will be noted that, depending on the detection method selected, the marking agent can
alternatively be coupled to the recognition agent or to the antibiotic or the substance
• analogous to the antibiotic from the point of view of its recognition by the recognition agent. These are also processes in which the agent
Recognition or the antibiotic or the analogous substance of the antibiotic contain, intrinsically, the labeling agent (eg, a radiolabeled antibiotic).
• 20 For dairy products, the analytical screening tests that are most widely described relate to the detection of antibiotics.
TÍ r ^ t.taMBai¡ ^ M ^^^^^^ B ^^^^^ §BB ^ MM, ¡¡^ ^ ^ ^ BitMt M | a U.S. No. 4,239,852 describes a microbiological process for the detection in milk of antibiotics having a β-lactam ring. In accordance with this process, the milk sample is first incubated in the presence of cell parts of a microorganism that is highly sensitive to antibiotics, and especially Bacillus stearothermophilus, and secondarily in the presence of an antibiotic that is labeled C ("labeled"). ) with a radioactive element or with an enzyme. Incubation is conducted under conditions that allow antibiotics, if present in the sample, and labeled antibiotic to bind to the cell parts.
After incubation, the cell parts are separated from the mixture and washed. Subsequently, the amount of labeled antibiotic bound to the cell parts is determined and 0 compared to a standard. The amount of labeled antibiotic bound to the parts of the cell is inversely proportional to the concentration of antibiotic present in the milk sample analyzed.
This process requires a reasonably delicate handling, especially in the stage of separation of the cell parts of the mixture. Besides that, in its most sensitive version, that allows the detection of Penicillin G up to • 0.01 IU / ml and even up to 0.001 IU / ml in milk, this process uses an antibiotic marked with a radioactive element (14C or 125I). In this case, the determination of the amount of antibiotic 10 present or not in the milk, requires the use of a special instrument, such as a scintillation counter, for example. Furthermore, the handling of radioactive products even in very small quantities is not completely free of risks for the person conducting the analysis.
European Patent Application 593 112 describes another method that allows the detection of antibiotics in milk. This method uses a protein isolated from a microorganism sensitive to the antibiotic, such as Bacillus stearothermophilus. This protein is additionally labeled with an enzyme such as a peroxidase.
^^^^^^^^^^^^^^^^^^^^^^^^^^^ h ^ iA ?? ^ A? ife- .. «'". - * -.'- The test proceeds as follows: a milk sample is incubated in a tube in the presence of the labeled protein, after the incubation, the milk is transferred to a second tube in A reference antibiotic is immobilized on 5 walls, a second incubation is carried out, and then the contents of the tube are removed, the walls of this second tube are washed three times with a washing solution, which
removes itself, and then the residues present in the second tube are transferred to a
• piece of absorbent paper; a dye substrate is then added to the second tube, which is incubated once more, and then a solution is added that
retards color development; The coloration of the tube is compared with the staining of an identical test carried out in parallel on a standard sample of antibiotic. The amount of labeled protein immobilized in the support, and therefore
the intensity of the coloration, is inversely proportional to the amount of antibiotic present in the milk sample analyzed.
In accordance with Example 1 of this patent application, this test makes possible
Detect penicillin G at concentrations below the order of 5 ppb and make it possible to detect amoxicillin (5 ppb), ampicillin (10 ppb), cephapirin (5 ppb) and ceftiofur (5 ppb). This test does not allow the detection of penicillin,
• Amoxicillin and ampicillin up to the levels imposed by the European Regulations and, on the other hand, this test is quite complex; this does not fully meet the criteria of
sensitivity and simplicity sought in the context of the present invention. •
However, the tests are very exhaustive to carry out, especially by personnel without
ability. Indeed, this test comprises numerous steps, including the steps of transferring liquid and waste from one vessel to another, and a number of rinsing steps. Due to the
• number and type of steps required in this
test, a real result is obtained depending strongly on the experimental knowledge of the operator.
In addition, interpreting the result requires 25 two tests to be carried out in parallel, therefore
multiplying and complicating operations further.
Other types of enzymatic processes have also been described, making it possible to determine low concentrations of antibiotics in milk (JM Frere et al., Antimicrobial Agents and Chemotherapy, 18 (4), 506-510 (1980), and EP 85 667 patents). and EP 468 946), which are based on
the use of a specific enzyme, especially carboxypeptidase D-alanyl-D-alanine exocellular
• soluble, which is produced by the Actinomadura R39 (designated the 'R39 enzyme' hereafter.) The enzyme R39 has a specific activity of
hydrolyzation of the D-alanyl-D-alanine groups of various peptides and is also capable of hydrolyzing certain thioesters.
In addition, the R39 enzyme reacts with the
• 20 antibiotics that have a ß-lactam ring to very quickly form an equimolar enzyme-antibiotic complex that is inactive and substantially irreversible.
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In the most recent version of this test (EP 468 946) a predetermined volume of a sample of the liquid to be examined is incubated with a predetermined amount of the enzyme R39 under conditions that allow the β-lactam antibiotic, which may be present in the test. , react with the enzyme to form a complete equimolar antibiotic enzyme which is inactive and substantially irreversible. ID Subsequently, a predetermined amount of the substrate of the thioester type is incubated with the product obtained in the first step under conditions which allow the substrate to be hydrolyzed by the residual enzyme R39 which is not complex with the antibiotic in the course of the first incubation. The amount of mercaptoalkanoic acid formed in this way is then determined by colorimetric assay with the aid of a reagent 20 capable of producing a coloration by reaction with the free SH group of mercaptoalkanoic acid. The intensity of the coloration is compared to a standard established in the previous part of the samples containing known quantities of 25 antibiotics. The quantitative determination can
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carried out by measuring in a spectrometer; in the case of milk, it may be necessary to clarify the sample from the previous part.
In accordance with the embodiments
• of patent EP 468,946, this process makes it possible to determine, in milk, 10 ppb of penicillin G for a total incubation time of 5 minutes and about 2.5 ppb of penicillin G
for a total incubation time of 15 minutes.
• Given the criteria of speed, simplicity and sensitivity required by methods to detect antibiotics in food products,
the applicant places by himself the purpose of a novel investigation, even more effective methods to detect antibiotics in a biological fluid. In particular, the applicant places
• by itself the purpose of methods of
research to detect, in a simple test, the majority of antibiotics whose content is regulated by the European and American authorities. Additionally, the methods investigated make it possible to obtain this result
in a number of limited steps, which can
| flta | flUai ^ ^ preferably carried out by unqualified personnel. The applicant also investigates methods to accomplish these purposes with a shorter incubation time than the existing processes. •
Description of the invention
The applicant has now discovered new
processes to detect antibiotics that contain a ß-lactam ring in a biological fluid, which
• It allows to realize these objectives in a non-laborious way.
Accordingly, the present invention relates to new processes for detecting antibiotics containing a β-lactam ring in a biological fluid, comprising the following steps 20 a) placing a certain volume of said biological fluid in contact with an amount of agent of recognition and incubate the mixture in this way obtained under conditions that
allow the composition of antibiotics,
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that can be presented in said biological fluid, with the recognition agent,
b) placing the mixture obtained in step a) in contact with at least one antibiotic of
• reference immobilized on a support, under conditions that allow the composition of the reference antibiotic with the amount of recognition agent that does not react in step a), and
• c) determining the amount of recognition agent linked to the support, characterized in that the agent of
The recognition comprises a receptor that is sensitive to antibiotics containing a β-lactam ring obtained from Bacillus licheniformis. Figure 1 illustrates a type of support that can be used in accordance with the present invention, which is in the form of a test device comprising a solid support (1) in which the membranes (2), (3) and (4) are linked. The
Figure 25 is a front view and Figure Ib is a longitudinal cross-sectional view of the test device.
The exceptional performance of the processes according to the present invention is based on
• the use of a specific recognition agent, comprising a receptor that is sensitive to antibiotics containing a β-lactam ring obtained from Bacillus Licheniformis, that is, the
1C BlaR protein whose isolation and peptide sequence is described in Y. Zhu et al., J.
• Bacteriol, 1137-1141, (1990), or the BlaR-CTD polypeptide, which is the carboxyl terminal region of BlaR, whose isolation and peptide sequence is
describe in B. Joris et al., FEMS Microbiology Letters, 107-114, (1990).
The use of the BlaR or BlaR-CDT receivers of
• conformity with the present invention for the
The detection of antibiotics containing β-lactam rings has appreciable advantages over the recognition agents used therein. The reason for this is that the BlaR and BlaR-CDT receivers are capable of making a very complex
quickly from a very large number of biologicals such as milk, urine, blood, serum, saliva, meat extracts, fermentation liquids or aqueous buffer media.
In accordance with an embodiment
• preferred of the processes of the invention, the recognition agent is used in a form coupled to the marking agent. This marking agent can be of a diverse nature. He
The labeling agent can be of a particular type, such as metal colloidal particles (platinum,
• gold, silver, etc.), colloidal particles of selenium, carbon, sulfur or tellurium, or alternative colloidal particles of synthetic latexes of
color. The labeling agent can also be a fluorescent substance, such as activated fluorescein (available from Boeringher-Mannheim Biochemica), fluorescein isocyanate,
• rhodamine tetrametisocyanate or any other
fluorescent substance known to those skilled in the art. The labeling agent can also be enzymatic, for example a β-lactamase, a peroxidase, a phosphatase, etc. In this case, the BlaR or BlaR-CTD receivers are
chemically or genetically coupled to this
Enzymatic labeling agent to form a fused protein.
The recognition agent can be coupled with the marking agent according to the
• conventional methods known to those skilled in the art. The recognition agent can be linked either directly to the marking agent or through the formation of a complex
intermediary. The coupling between the recognition agent and the marking agent can take
• place at different times of the implementation of the processes of the invention. In accordance with a first embodiment, the
The coupling between the recognition agent and the marking agent takes place before the recognition agent is placed in contact with the biological fluid to be analyzed. From
• compliance with other forms of realization of the
In the processes of the invention, the coupling between the recognition agent and the marking agent can take place when or after the recognition agent is placed in contact with the biological fluid sample. Preferably,
the marking of the recognition agent takes place
before the recognition agent is placed in contact with the sample to be analyzed.
The first step a) of the processes of
• according to the invention, it consists of placing a determined volume of the biological fluid in contact with a quantity of recognition agent and incubating the mixture obtained under
1 conditions that allow the composition of antibiotics, which can occur in the fluid
• biological, with the recognition agent.
The biological fluid can be incubated with the
BlaR or BlaR-CTD receivers within a temperature range between 4 and 60 ° C. Preferably, this temperature is around 47 ° C. An increase in incubation temperature can
(B) have the effect of reducing its duration, and
contrary. It is always possible to reduce the duration of the process by increasing the temperature.
In the second step b) of the processes according to the present invention, the mixture
2 obtained in step a) is placed in contact with at least one reference antibiotic immobilized on a support.
The supports that can be used in accordance with the present invention can be very
• varied types. These can be solid supports such as tubes, trays or bars coated with an antibiotic reference preparation. This can be a test device in the
form of a solid support in which the membranes are bonded, one or more substances of
• Capture are placed in a certain detection area. This can be supported in the form of magnetic or non-magnetic beads (agarose,
polystyrene, etc.), capable of forming a gel and in which the reference antibiotic is immobilized.
fl The reference antibiotic can
immobilize in the support by methods known to those skilled in the art, for example by covalent or non-covalent absorption in the support, optionally by means of a spacer.
, & u "^ In accordance with a specific embodiment of the invention, steps a) and b) can take place simultaneously.
Step c) of the processes according to the present invention, consists of determining the receptors that are linked to the support in which the reference antibiotic is immobilized. The method used for this determination is associated
directly with the type of marking agent used. If the labeling agent is enzymatic, the
• Determination step involves a specific reaction for this enzyme, which is associated, for example, with the production of a given coloration. 15 If the marking agent is fluorescent, the determination is carried out by simply measuring the fluorescence of the support. In the case of metallic particles or colored latices, the
• Presence of receptors linked to the support
reflects by a coloration whose intensity is directly proportional to the number of receptors linked to the support. Without taking into account the type of marking agent used, the intensity of the detected signal is inversely proportional to the
amount of antibiotic present in the analized sample.
The present invention also relates to test kits for detecting the antibiotic in a biological fluid, comprising at least one recognition agent which comprises a receptor that is sensitive to the antibiotics containing a β-lactam ring obtained from Bacillus. licheniformis, and at least one reference antibiotic immobilized on a support. •
The examples that follow illustrate various aspects and methods for implementing the present invention, without, however, limiting its scope.
Example 1 Determination of antibiotics containing a β-lactam ring in milk. This example illustrates the detection in milk of antibiotics containing a β-lactam ring, which are controlled by the health authorities. The test described in this example uses the 25 BlaR-CTD receiver coupled to gold beads that serve as
^ ~. - ** átm * kt ~ * m *. > ~ - .. ~? iaa at. . - ^ t-J ^. ^ - * - ^^^. .. ^ ,, ^. ¿.. ^ - ^ .-- i-ií UHÜB marking agents, and uses a support in the form of a test device comprising a solid support to which membranes are bound.
1. 1. Coupling of BlaR-CTD to gold beads.
•
1. 1.1. Biotinylation of BlaR-CTD.
3. 79 ml of an agent solution
recognition BlaR-CTD having a concentration of 6.6 mg / ml is taken in a solution
• Sodium phosphate buffer, 20mM, pH 7. To this solution of BlaR-CTD is then added 41.71 ml of bicarbonate buffer solution
(0.1 M sodium bicarbonate, pH 9) and 2 ml of a capric 6- (biotinamido) N-hydroxysuccinimide ester solution containing 2.23 mg / ml or similar of bicarbonate buffer. This solution is stirred slowly in a shaker
• 20 LABINCO for tubes on a rotating shaft (available from VEL, Belgium) at a ratio of 2 revolutions / minute for 2 hours at rtemperature and away from light. 2.5 ml of a buffer solution of Tris, 1 M pH 8,
incubated with the reaction mixture under the same
conditions for 30 minutes. The solution thus obtained is again dialyzed with HNM buffer (100 mM Hepes, pH 8, 100 mM NaCl, 50 mM MgCl 2) for 24 hours. In this way a biotinylated BlaR-CTD solution is obtained which
• diluted in a buffer solution of HNM-BSA (500 mM Hepes, pH 8, 500 mM NaCl, 250 mM MgCl2, 10 mg / ml BSA) at a concentration of 250 mg biotinylated BlaR-CTD per ml of solution
shock absorber. This solution is stored at -20Ec.
1. 1.2 Marking agent
The marking agent used is made of
gold particles having a diameter of 40 nm in which a drop of antibiotin antibody is deposited in the form of suspensions in a 2mM aqueous sodium tetraborate solution, with
^ a pH of 7.2, stabilized by 0.1% sodium azido
(available from British Biocell (Ref. GAB40)). The optical density of these suspensions at 520 nm is about 10 and the protein concentration is about 24 mg / ml.
.k. ^. a ^ 1.1.3. Coupling of biotinylated BlaR-CTD to gold particles.
The biotinylated BlaR-CTD solution prepared in Example 3.1.1 is diluted 114.7 times with the
• HNM-BSA buffer solution (500 mM Hepes, pH 8, 500mM NaCl, 250mM MgCl2, 10mg / ml BSA). At room temperature, 22.5 parts by volume of this biotylated Blar-CTD solution is mixed
diluted, 7.5 parts by volume of HNM-BSA buffer, 9.27 parts by volume
• of the gold particle suspension used to mark the biotinylated BlaR-CTD and 6 parts by volume of the gold particle suspension of
reference (see example 3.1.4 below).
1. 1.4. Independent reference
flp In this test, use is also made of a
The reference substance that supplies a band whose intensity quickly enables the quantification of the antibiotic present in the sample.
¿^^^^^^^ i ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^^^ «^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ of anti-rabbit immunoglobin antibody. These particles are available from British Biocell 5 (Ref. GAR40) in the form of suspensions in a
• 2mM aqueous sodium tetraborate solution, with a pH of 7.2, stabilized by 0.1% sodium azide. The optical density of these suspensions at 520 nm is about 3, and the protein concentration 10 is about 6 mg / ml.
• 1.2. Test device
The test device used comprises a solid support (1) having a first and second end, to which a membrane (2) is connected successively from the first end to purify the fluid
• Analyzed, 20 - a membrane (3) in which two capture substances (reference antibiotic and substance capable of binding the independent reference) are immobilized, and an absorbent membrane (4). 25
1. 2.1. Assembly of test devices.
Cards that have a size of 300 x 76.2 mm are the first of the whole assembly using a
rolling mill type Clamshell laminator
• (available from BioDot, Inc.) in accordance with the following method:
A rectangular plastic support of the type
ArCare 8565 (available from Adhesive Research) is cut, measuring 300 x 76.2 mm (solid support)
• (1) ) . Subsequently, a rectangle of membrane Leukosorb LK4 (available from Pall Gelman Sciences) measuring 300 x 20 mm (membrane (2)), a rectangle
of Hi-Flow SX membrane (available from Millipore), measuring 300 H 25 mm (membrane (3)), a 3 mm rectangle of cellulose membrane (available from Whatman), measuring 300 x 40 mm (membrane (4)).
• 20 Subsequently, the membranes (2) and (4) and then the (3) are placed in a specific location in the lower mold of the laminator. The solid support (1), covered with adhesive, is in this part to help cover the device, with the
adhesive face exposed to air. The membranes
placed in the lower mold are conducted in contact with the adhesive support closing the laminator; the membranes are placed exactly in place by suction of air from a vacuum pump. When the vacuum breaks, it recovers
• a card consisting of the solid support (1) with, attached to it, the membranes (2), (3) and (4).
The following solutions are then deposited in the membrane (3) next size: first capture substance; band No.l; size
• distant: second capture substance; band No.2 These capture substances are deposited using a "dispenser" of the type X-Y Platform 15 Biojet Quanti-3000 from Bio Dot Inc.
The deposited solutions evaporate immediately placing the whole of the
• card for one minute under a force of air 20 hot at 60 ° C.
The cards obtained after assembly are cut into strips with the help of a guillotine-type device or with the aid of a rotating device
(available from BioDot, Kinematic or Akzo). The final strips are removed, with the other strips being ready for use.
Figure 1 illustrates such a test device.
• To keep it, the test devices are placed in a hermetically sealed container, opaque, in the presence of a desiccant (Silgelac, France). 10 1.2.2. First capture substance. Antibiotic • reference.
8 ml of a solution containing 213 mg of a human gamma globulin (G4386, Sigma) and 8.6 mg of 2-imino-iolane hydrochloride (Aldrich, 33056-6) in a sodium carbonate buffer solution (100 mM, pH 9) is incubated at 25Ec for one hour. • 20 In addition, 20 ml of a solution containing 119.8 mg of cephalosporin C and 54 mg of 4 - (N-maleimidomethyl) cycloexan-1-carboxylate of sulphosuccinimidylate (sSMCC, 22322 Pierce) are incubated in a
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Sodium carbonate buffer solution (100 mM, pH 9) at 25Ec for one hour.
The two solutions prepared above are then mixed. The pH of the resulting solution
• adjusts to 7.1 by adding 3 ml of 500 mM NaH2P04, and the solution is incubated at 25Ec for two hours. The mixture obtained after incubation is dialyzed 3 times against 1 liter of solution
1 sodium phosphate buffer (10 mM, pH 7.5). The resulting solution is filtered through a
• 0.22 mm filter, and then divided into aliquots and freeze at -20Ec until used.
At the moment of its use, the aliquots are thawed and a food dye is added to it before they are deposited on the membrane, so that it can indicate at any time
• exact position of the deposit and the quality of the
traces
The first capture substance makes it possible to fix the BlaR-CTD coupled to the gold particles in excess with respect to the amount of antibiotic present in the sample.
..S.Í.- fr ^ .. ^ A ^^^ M ^^. A- t ^? ¡? É 1.2.3. Second capture substance. Substance capable of fixing the independent reference.
The second capture substance used is made of a solution of rabbit immunoglobulin (Sigma I 5006) having an immunoglobulin concentration of 0.5 mg / ml in a buffer solution of 10 mM sodium phosphate, pH 7.5 gamma human globulin 5 mg / ml . This second capture substance stops the reference as the liquid migrated on the test device.
1. 3. Determination of antibiotics in milk
1. 3.1 3-minute test - rapid test.
Prepare 7 samples of fresh milk containing 0; 1; 2; 3; 4; 5 and 6 ppb of penicillin G, respectively. Each of these solutions is then analyzed in the following way:
An aliquot sample of 200 μl of milk and
45. 27 μl of the solution prepared in Example 1.1.3 are taken and placed in a glass flask. This mixture is incubated for one minute at 47 ° C. A test device is taken and placed vertically in the glass flask in such a way that the first end of the test device is in contact with the mixture and in such a way that
• the second end is on the wall of the glass flask. The mixture is allowed to migrate in the test device, while the assembly is incubated for two minutes at 47 ° C. 10 Table 1 below summarizes the results
• obtained by the 7 samples tested. An intensity value in the range from 0 to 10 is attributed to the detected bands, the value of 10
is given to the band with the highest intensity and the value of 0 is given to the band with the lowest intensity. In accordance with this scale, a value of 6 is assigned to the reference band. The intensity of
• the signal observed in the first band is
inversely proportional to the amount of penicillin G present in the sample.
Table 1 Penicillin G Intensity (Fa band band 2 nd band or 10 1 9 6 • 2 9 6 3 4 6 4 0 6 5 0 6 10 6 0 6
In this example, when the first band has a lower intensity than the second band, the
test is considered as positive. The results given in Table 1 show that these tests allow the detection of less than 4 ppb of penicillin G in a milk sample in three minutes. • 20 Tests are also carried out with other antibiotics that contain a β-lactam ring under the same conditions. This test is carried out in 3 minutes allowing the detection of
amoxicillin below 5 ppb, ampicillin under
^ i-- "-.y» "5 ppb, cloxacillin to less than 10 ppb, dicloxacillin to less than 20 ppb, oxacillin to less than 20 ppb and cephapirin to below 20 ppb, in a milk sample.
• 1.3.2. 5 minute test
Prepare 6 samples of fresh milk containing 0; 2; 4; 6; 8 and 10 ppb of cloxacillin,
respectively. Each of these solutions is then analyzed in the following way. •
A sample of aliquots of 200 μl of milk and 45.27 μl of the solution prepared in Example
1.1.3 is taken and placed in a glass flask. This mixture is incubated for 3 minutes at 47 ° C. A test device is taken and placed vertically in the glass flask in such a way
• that the first end of the test device
is in contact with the mixture and in such a way that the second end is on the wall of the glass flask. The mixture is allowed to migrate to the test device, while the assembly is incubated for 2 minutes at 47 ° C. 25
* áfc ^^^^? Xufo ^^^^^ ¡£ ^ & Table 2 below summarizes the results obtained by the 6 samples tested. An intensity value in the range from 0 to 10 is attributed to the detected bands, the value of 10 is given to the band of higher intensity and the
# value of 0 is given to the lower intensity band. In accordance with this scale, a value of 6 is assigned to the reference band. The intensity of the signal observed in the first band is
inversely proportional to the amount of cloxacillin present in the sample. •
Table 2 Cloxacil Intensity (ppb) 1 ra. 2nd band Band 0 10 6 2 6 6 4 5 6 6 3 6 8 3 6 10 3 6 15
In this example, when the first band has a lower intensity than the second band, the
^ jg ^ É ^ test is considered to be positive. The results given in Table 2 show that these tests allow the detection of less than 4 ppb of cloxacillin in a milk sample in 5 minutes. 5 • The tests are also carried out with other antibiotics that contain a β-lactam ring under the same conditions. This test is carried out in 5 minutes allowing the detection of 10 penicillin below 3 ppb, amoxicillin below 4 ppb, ampicillin below 4 ppb, dicloxacillin • below 8 ppb, oxacillin below 8 ppb, cefarin below 16 ppb , cephiorioride below 100 ppb, cefquinone to less than 20 ppb, 15 nafcillin below 20 ppb, and cefazolin below 60 ppb in a milk sample.
This test is particularly appropriate as a classification test before the 20 milk wagons are emptied into the silos.
1. 3.3. 9 minute test.
Prepare 6 samples of fresh milk containing 0; 4; 6; 8; 10 and 12 ppb of cephapirin,
^. «^ Aa,, - ..,. ^. ... .- * --a- "iif.M'iftinA.T.rfíiii G * - Í go" JUtir rm umi mm ^ ^^^ - mm respectively Each of these solutions is then analyzed in the following way .
An aliquot sample of 200 μl of milk and
V 5 45.27 μl of the solution prepared in Example 1.1.3 is taken and placed in a glass flask. This mixture is incubated for 7 minutes at 47 ° C. A test device is taken and placed vertically in the glass flask in such a way
that the first end of the test device is in contact with the mixture and in such a way that
• the second end is on the wall of the glass flask. The mixture is allowed to migrate to the test device, while the assembly is incubated for 2 hours.
minutes at 47 ° C.
Table 3 below summarizes the results obtained by the 6 samples tested.
• An intensity value in the range from 0 to
10 is attributed to the bands detected, the value of 10 is given to the band with the highest intensity and the value of 0 is given to the band of least intensity. In accordance with this scale, a value of 6 is assigned to the reference band. The intensity of
The signal observed in the first band is inversely proportional to the amount of cephapirin present in the sample.
Table 3 Cefapirin Intens id, ad • (ppb) Barry rash 2nd band 0 10 6 4 6 6 6 5 6 8 4 6 10 3 6 12 3 6
In this example, when the first band has a lower intensity than the second band, the test is considered positive. The results given in Table 3 show that this test
allows detection below 6 ppb of cephapirin
• in a milk sample in 9 minutes.
The tests were carried out with other antibiotics containing a ß-lactam ring
under the same conditions. This test was carried out in 9 minutes, allowing the detection of penicillin below 3 ppb, amoxicillin below 4 ppb, ampicillin below 4 ppb, cloxacillin below 4 ppb, dicloxacillin to less than 8 ppb, oxacillin to less than 8 ppb, ceftiofuro below 80 ppb, cefquinone to less than 20
• ppb, nafcillin to less than 20 ppb and cefazolin below 45 ppb in a milk sample.
This test was carried out in 9 minutes,
this way allowing the detection of all antibiotics currently controlled by the
• European authorities, below the legal limits imposed by these authorities.
1.3.4. 20 minute test.
Six samples of fresh milk containing 0 were prepared; twenty; 30; 40; 50 and 60 ppb of
• ceftiofur, respectively. Each of these
solutions was then analyzed as follows.
An aliquot sample of 200 μl of milk and 45.27 μl of a solution prepared in Example
1.1.3 were taken and placed in a glass flask. The mixture was incubated for 18 minutes at 47 ° C. A test device was taken and placed vertically in the glass flask in such a way that the first end of the test device is in contact with the mixture and in such a way that the second end is in the wall of the glass flask. The mixture was allowed to migrate in the test device, while the assembly was incubated for 2 minutes at 47 ° C. 10 Table 4 summarizes the results obtained by • the 6 samples tested. An intensity value in the range from 0 to 10 is attributed to the detected bands, the value 10 is given to the strongest band 15 and the value 0 is given to the less intense band. In accordance with this scale, a value of 6 is assigned to the reference band. The intensity of the signal observed in the first band is inversely proportional to the amount of ceftiofuro present in the sample.
^^^^^^ rt »J ^^^^^^^^^^^ gte ^^ a ^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^ Mfe ^ fiÉ ^ fe ^^^^^^^^^^^^ ¡^^^^^^^^^^^^^ jj ^ ¡^^^^ Table 4 Ceftiofuro Intensity (ppb ) Banded raid 2nd band 0 10 6 • 20 6 6 30 5 6 40 4 6 50 3 6 60
• In this example, when the first band has a lower intensity than the second band, the test is considered positive. The results given in Table 4 show that this test allows the detection of ceftiofuro below 30 ppb in a milk sample in 20 minutes. 10 This test was carried out in 20 minutes, in this way allowing the detection, in a simple test, of all the antibiotics currently controlled by the authorities.
European and American, below the legal limits imposed by these authorities.
Example 2. Determination of 6 antibiotics in milk.
This example illustrates the detection in milk of 6 antibiotics containing a β-lactam ring that are controlled by the US authorities. The test described in this example uses the BlaR-CTD receptor in the form of a fusion protein with β-lactamase and uses a
support in the form of magnetic beads.
• 2.1. BlaR-CTD-β-lactamase fusion protein
The BlaR-CTD-β-lactamase fusion protein is obtained by genetic coupling between the BlaR-CTD receptor (B. Joris et al., FEMS Microbiology Letters, 107-114, 1990) and Zn β-lactamase from Bacillus cereus (M. Hussain and collaborators,
• 1985, J. Bact., 164: 1, 223-229, 1985). 20 The coupling is carried out in the following way:
2. 1.1. Construction of the plasmid. 25
- -. «- - -. -i-1-- - • •? -. ~ T - »» 8-fa «'. ~ - .. ... ni *. ¿> B * -} »* T * aA ^ i < e.í ^ ^ - > ---. ^. < «^ - ~» -Aj¿ - • ».. J t &i *, * - ^ '._ ^ A 1/1 coupling is carried out between the BlaR-CTD and the β-lactamase polypeptide genes: the gene encoded for ß-lactamase is introduced in the phase and behind the BlaR-CTD gene. The plasmid
• conducts genetic fusion showing resistance to kanamycin. The fusion protein is referred to below as Fus 1.
2. 1.2. Production. 10 Strains: the plasmid that drives the genes
• merged is introduced in E. coli The clones that drive the recombinant plasmid are selected in LB + Km (50 μg / ml). 15 Selection: the labeling of the cell extracted with a radioactive antibiotic, followed by polyacrylamide gel electrophoresis
• denatured, shows that most of the
proteins are produced in the form of a fusion protein whose molecular mass is around 50,000. However, a post-translational proteolysis appears to dissociate a very small percentage (2%) of these molecules in two
separate activities.
^^^^^ ^^^^? ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^ ¡¿^ ^^^? ^^^^ Cultivation: 500 ml of LB + Km medium (50 μg / ml) are inoculated using recombinant cells stored at -70 ° C. The pre-culture is incubated at 37 ° C and stirred overnight at 225 rpm.
• 18 liters of LB + Kb medium (50 μg / ml) are inoculated with 500 ml of this preculture, the optical density of this at 600 nm is 4. The 18 liter culture stops when the optical density reaches a value of 6. 1C 2.1.3 Removal •
Immediately after stopping the culture, the cells are filtered and then centrifuged. 15 The supernatant of the pelleted cell lysed in a disintegrator is stored. It contains the FUS1 fusion protein.
• 2.1.4. Purification. 20 Buffer solutions used: • Buffer solution A: 20 mM, pH 8.0, Tris, 10% ethylene glycol, 50 μm DDT; • Buffer solution B: buffer solution A + NACÍ 1M.
IHnaS * »~? *? IKr * -J. . ««. Tó »» - + XL ~ > -. * &? lUéÍÉé £ SÍÍk,. &Xt2i ,. '»Toj M» i-Mg'-fc »» Ífc > '< W »• * - *» .. »The fusion protein is partially purified by ion chromatography and molecular sieves. After depositing the extract and washing on a QSFF column (Pharmacia, Upsala) in buffer A, the FUS1 is eluted with a gradient
• linear buffer solution B. The active fractions (± 0.25 M NaCl) are then combined and deposited in molecular sieves G-100 (Pharmacia, Upsala); this is eluted with buffer solution
A. The average specific activity of the recovered lot is estimated at 30%. •
2. 1.5. Inhibition of β-lactamase.
The fusion protein (volume 9/10) is incubated with 50 mm EDTA (1/10 volume) for 45 minutes at 37 ° C. Inhibition is checked using nitrocefin in buffer solution of
• 10 mM cacodylate, pH 6.0. In the presence or absence
2C of Zn, the signal will be positive or negative, respectively. After inhibition, the effective concentration, measured based on the β-lactamase activity, is 7.74 pmoles / μl.
^^ ¡¡¡¡¡¡¡^ ^ 2.2 Solid support: C cephalosporin magnetic beads (reference antibiotic).
BioMag 4100 particles are used (available from
• DRG Instrument GmbH, Marburg, Germany, under reference AM 4100 B), whose NH 2 ends have been activated with glutaraldehyde as follows: 10 a volume of the initial solution of BioMag 4100 particles is rinsed 4 times with 5
• volumes of 0.01M pyridine buffer solution, pH 6.0. The particles are taken in 2.5 volumes of 5% glutaraldehyde in solution
pyridine buffer and are stirred by rotation for 3 hours at room temperature. This is rinsed 10 times with 2 volumes of 0.01M KPI buffer, pH 7.0. The particles
• then they are resuspended in 1 volume of
C 0.1 M cephalosporin in Kpi buffer and are stirred by rotation overnight at + 4 ° C. The final rinse is carried out until the cephalosporin C has been completely removed from the 0.1 M Kpi buffer solution, pH 7.0. 25
i m-n? f? ifi-ltr? -, J ^ = ^ "- 2.3 Determination of the 6 antibiotics, penicillin G, ampicillin, amoxicillin, cloxacillin, cephapirin and certiofur, in milk.
2.3.1. Solutions used: •
Solution 1: 700 picomoles of lyophilized fusion protein in 100 mM, pH 8 Tris, 1 mg / ml BSA, 50 mM EDTA, 50 μm DTT become
hydrate with 5 ml of Milli-Q water; 50 μl of this solution are required to carry out
• one measurement.
Solution 2: 2 ml of BioMag-15 cephalosporin C particles prepared in Example 1.2 in 100% isopropanol; 20 μl is required to carry out a measurement.
• Solution 3: 10 mM, pH 6 of solution
cacodylate buffer, 1 M NaCl lyophilized and rehydrated with 500 ml of Milli-Q water.
Solution 4: 400 ml of 10 M nitrocefin in DMF are diluted to 4 > ml in solution 3; 400 μl is required for a detection.
2. 3.2 Detection process. •
50 μl of solution 1 are placed in the presence of 500 ml samples of adulterated milk and incubated at 47 ° C for 2 minutes. 20 μl is suspended
of the solution 2 in the milk, which is re-incubated for 2 minutes at 47 ° C. The particles are
• again attract the wall of the container using a paramagnetic magnet while the supernatant is emptied from the tube. The particles are rinsed two
times with solution 3, executing the same process with the magnet. Finally, 400 μl of solution 4 is incubated in the presence of the particles for 3 minutes at 47 ° C. The absorbency of
• residual solution at 482 nm is then measured with
relation to the nitrocefin solution.
This process allows the detection of the 6 antibiotics given in the American Regulations at concentrations below
standards imposed by the authorities, that is,
It was reported that 5% was penicillin at 5 ppb, ampicillin at 10 ppb, amoxicillin at 10 ppb, cloxacillin at 10 ppb, cephapirin at 20 ppb and ceftiofur at 50 ppb.
Example 3. Determination of 3 antibiotics
< § (penicillin G, cloxacillin, ceftiofur) in milk.
This example illustrates the detection in milk 10 of 3 antibiotics containing a β-lactam ring that are controlled by the authorities
• sanitary. The test described in this example uses the BlaR-CTD receptor in the form of two fusion proteins with phosphatase and alkaline peroxidase, respectively, and uses a support in the form of a microplate.
3. 1. BlaR-CTD-fosfat fusion fusion protein
• alkaline. 20 BlaR-CTD-alkaline phosphatase fusion protein is obtained by chemical coupling between the BlaR-CTD receptor (B. Joris et al.
FEMS Microbiology Letters, 107-114 (1990)) and the
activated alkaline phosphatase available from
^ fe ^^^^^^^^ S ^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ? ^^^^^^^^^^^ Boehringer-Mannheim Biochemica under the reference 1464752.
The coupling is carried out in the following manner: •
3. 1.1 Coagulation.
The BlaR-CTD and the alkaline phosphatase are dialysed in 100 mM of carbonate / sodium bicarbonate buffer at a pH of 9.8. HE
• incubate 15 nonomoles of BlaR-CTD in the presence of 100 μl of the activated alkaline phosphatase (20 mg / ml) for 2 hours at 25 ° C. 15 3.1.2. Stop the reaction.
40 μl of a solution of
• 2 mM tritanolamine, pH 8, followed by 50 μl of a
200 mM sodium borohydride solution. The mixture is incubated for 30 minutes at + 4 ° C. 25 μl of a 2 mm triethanolamine solution, pH 8 is then added, after which the mixture is again incubated for 2 hours at + 4 ° C. 25
^ 3.1.3. Stabilization of the coupling.
Add 10 μl of a 1M glycine solution, pH 7.0.
f 5 3.1.4. Transfer in the storage buffer.
The reaction mixture (about 300 μl) was
dialyzed for three time intervals of 8 hours against 0.5 liters of 50 mM triethanolamine buffer solution, pH 7.6, 150 mM NaCl, 0.5 mM ZnCl 2, 10 mM glycine at + 4 ° C.
3.1.5. Final assessment.
The final titration of the coupling is around 50 pmoles of active BlaR-CTD per μl
• from solution . 20 3.2. BlaR-CTD-peroxidase fusion protein
The fusion protein BlaR-CTD-peroxidase is obtained by chemical coupling between the receptor
BlaR-CTD (B. Joris et al., FEMS
Microbiology Letters, 107-114, (1990)) and the activated peroxidase available from Boehringer-Mannheim Biochemica under the reference 1428861.
The coupling is carried out from the
• Following way:
3. 2.1. Conjugation.
BlaR-CTD and peroxidase are dialyzed in 100 mM, pH 9.8 of buffer solution of
• carbonate / sodium bicarbonate. 40 nanomoles of BlaR-CTD are incubated in the presence of 100 μl of activated peroxidase (16 mg / ml) for 2 hours at 25 ° C.
3.2.2 Stopping the reaction
Add 40 μl of a 2 mM triethanolamine solution, pH 8, followed by 50 μl of
200 mM sodium borohydride solution. The mixture is incubated for 30 minutes at + 4 ° C. 25 μl of 2 mM triethanolamine solution, pH 8 is then added, after which the mixture is again incubated for 2 hours at + 4 ° C. 25
| ^^^ i ^^ g gi | g 3.2.3. Stabilization of the coupling.
Add 10 μl of a 1M glycine solution, pH 7.0.
3. 2.4. Transfer to the storage buffer.
The reaction mixture (about 400 μl) is dialysed for three time intervals of 8 hours against 0.5 liters of 10 mM potassium phosphate buffer, pH 7.5, 200 mM NaCl, 10 mM glycine at + 4 ° C.
3. 2.5. Final assessment.
The final titration of the coupling is around 100 pmol of active BlaR-CTD per μl of solution.
3. 3. Solid support
Microcharola-cephalosporin C.
3. 3.1. Preparation of the reference antibiotic solution.
8 ml of a solution containing 5 213 mg of human gamma-globulin (G4386, Sigma) and 8.6 mg of 2-iminotiolanol hydrochloride (Aldrich, 33056-6) are incubated in sodium carbonate buffer (100 mM). , pH 9) for one hour at 25 ° C.
Separately, 20 ml of a solution containing 119.8 mg of cephalosporin-C • and 54 mg of 4 - (n-maleimidomethyl) cyclohexane-1-sulfosuccinimidylate carboxylate (sSMCC, 22322 Pierce) are incubated in sodium carbonate buffer solution. Sodium (100 mM, pH 9) for one hour at 25 ° C.
The two solutions prepared above are then mixed together. The pH of the resulting solution is adjusted to 7.1 by adding 20 3 ml of 500 mM NaH2P04 and the mixture is incubated for 2 hours at 25 ° C. The mixture obtained after incubation is dialyzed three times against 1 liter of sodium phosphate buffer (10 mM, pH 7.5). The resulting solution is filtered through a 0.22 μm filter.
"'- ^ - * - < ~. - ^^». ^ - ^ i & »^. - ^ - ^ - ~ - - ^. Ji ^ & ~ ¡¡¡S * ~ - ^. . ~, -> - ^ ». ^. ^ -.- A -. ^^ - ^^! U? i &«! S ~ 3.3.2 Coating of the microplates with the reference antibiotic.
Polystyrene microplates with high protein absorption are used, under the trademark NUNK
• (Immunocharola: type Maxisorp) or trademark GREINER (microlon 600, reference 705071). The microplate cuvettes are washed with 150 mM, pH 7.2, of PBS buffer. TO
Next, an aliquot of the solution prepared in Example 3.3.1 is incubated. for 24
• hours at 4 ° C in the buckets. After incubation, the cuvettes are washed three times with 150 mM, pH 7.2, of PBS buffer,
0.1% Tween 20. The tube is then saturated for two hours at 20 ° C with 150 mM, pH 7.2, of saturation buffer PBS, 5% BSA. After washing three times with wash buffer, the tubes are dried and
store at 4 ° C away from moisture.
For cuvettes made for the recognition agent of BlaR-CTD-alkaline phosphatase, the wash buffer used is 1 M, pH
9.8, diethanolamine, 0.5 mM MgCl2; for the
cuvettes made for the BLR-CTD-peroxidase recognition agent, the washing buffer used is 50 mM, pH 5, of potassium phosphate.
• 3.4. Determination of the three antibiotics in milk.
1.4 picomoles of the agent are incubated
marked recognition in the presence of 100 μl of adulterated milk for 5 minutes at 47 ° C. The milk is
• transfer using a pipette in a cuvette that has been previously treated as indicated in Example 2.3. The milk is then incubated for 2
minutes at 47 ° C. After removing the milk, after two washings with the wash buffer (reference, Example 2.3.2.), 300 μl of the buffer, containing the revealing substrate, is incubated,
for 2 minutes in the cuvette (revealing substrate for the recognition agent BlaR-CTD-peroxidase: 500 mM, pH 5, potassium phosphate, 9.1 mM ABTS, 0.002% H202, revealing substrate for the recognition agent of BlaR- CTD-phosphatase
alkaline: 1M, pH 9.8, of diethanolamine, 0.5 mM
MgCl2, 10 mM 4-NPP). The tray is then placed in an automatic spectrometer for ELISA trays, the wavelength is placed at 405 nm.
• This test allows the detection of the three antibiotics: penicillin G, cloxacillin and ceftiofur at the control thresholds required by the US authorities (penicillin G)
1C to 5 ppb, cloxacillin at 10 ppb and ceftiofuro at 50 PPb). •
Example 4 Determination of the 6 antibiotics: penicillin G, ampicillin,
amoxicillin, cloxacillin, cephapirin and ceftiofuro, in milk.
This example illustrates the detection in milk of the 6 antibiotics containing a β-lactam ring that is controlled by the US authorities, below the standards currently required by these authorities. The test described in this example uses the BlaR-CTD receptor in the form of a fusion protein with
*! ¡^^^^^^ g ^ i ^ j ^ * ^^^^^^^^^^^^^^^^^^^^^^^^^^ or alkaline peroxidase, and used a shape of a coated tube.
4. 1. BlaR-CTD-alkaline phosphatase fusion protein. •
See Example 3.1.
4. 2. Solid support. 10 Tube covered with an antibiotic from
• reference
In this example, polystyrene tubes are used
with high protein absorption of the NUNK trademark (Maxisorp type), which are treated with a reference antibiotic solution as indicated in Example 3.3.2.
• 20 4.3. Determination of 6 antibiotics in milk.
7 picomoles of the recognition agent are incubated in the presence of 500 μl of milk per
5 minutes at 47 ° C in an Eppendorf tube. The milk
it is then transferred, using a pipette, into a tube treated as described in Example 3.2. The milk is then incubated for 2 minutes at 47 ° C. After removing the milk, the tube is washed twice with 1 ml of buffer solution.
• 1M diethanolamine, pH 9.8, 0.5 m MgCl2. 500 μl of buffer solution containing 1M diethanolamine, pH 9.8, 0.5 mM MgCl 2, 10 mM revealing substrate 4-NPP and the substrate were then added.
incubated for 2 minutes at 47 ° C. The absorption of the supernatant was then measured using a
• spectrophotometer whose wavelength was placed at 405 nm.
This method allows the determination of the 6 antibiotics below the standards required by the US authorities: penicillin G to less than 5 ppb; ampicillin to less than 10
• ppb; amoxicillin up to less than 10 ppb; 20 cloxacillin to less than 10 ppb; cephapirin to less than 20 ppb; ceftiofuro up to less than 50 ppb.
It is noted that in relation to this date, 25 the best method known to the applicant for
- t ^ umSlí ^ tía ^^^ lll ^ l ^ ¡á > ^^ ^ j * r < . ~~ * »- carrying out the aforementioned invention, is that which is clear from the present description of the invention.
Having described the invention as • foregoing, the content of the following is claimed as property.
•
^^^^ g ^^^^^ ji ^^^^^ ^ * Éb_ ^ ^
Claims (13)
- Claims 1. A process for detecting antibiotics containing a β-lactam ring in a biological fluid, comprising the following steps: a) placing a certain volume of the biological fluid in contact with a quantity of recognition agent and incubating the mixture 10 in this manner obtained under conditions that allow the formation of complexes of the • antibiotics, which can be present in the biological fluid, with the recognition agent, 15 b) place the mixture obtained in step a) in contact with at least one reference antibiotic immobilized on a support, under conditions that allow the formation of 20 complexes of the reference antibiotic with the amount of recognition agent that has not reacted in step a), and c) determining the amount of recognition agent linked to the support, . ^ ..- • ^^ -. • g? ^ ^ Égjg ^ g £ & characterized in that the recognition agent comprises a receptor that is sensitive to antibiotics containing a β-lactam ring obtained from Bacillus licheniformis.
- 2. The process according to claim 1, characterized in that the receptor that is sensitive to antibiotics containing a β-lactam ring is the BlaR receptor or the 10 receiver BlaR-CTD. • The process according to claim 1 or 2, characterized in that the receptor that is sensitive to antibiotics containing a β-lactam ring is coupled to a marking agent chosen from metal colloidal particles, colloidal selenium particles, carbon, sulfur or tellurium, and colloidal particles • synthetic colored latices. 4. The process according to claim 1 or 2, characterized in that the receptor that is sensitive to antibiotics containing a β-lactam ring is coupled to a Labeling agent chosen from fluorescent substances. 5. The process according to claim 1 or 2, characterized in that the • receptor that is sensitive to antibiotics containing a β-lactam ring is coupled to a labeling agent chosen from enzymes, such as alkaline phosphatase, peroxidases and β-lactamases. 10 6. The process in accordance with the • claim 5, characterized in that the receptor that is sensitive to antibiotics is coupled to the chemically enzymatic labeling agent or 15 genetically. 7. The process according to any of the rei indications 3 to 6, • characterized because the receiver that is sensitive to The antibiotics containing a β-lactam ring are coupled to the labeling agent before step a). 8. The process according to any of claims 3 to 6, Characterized in that the receptor that is sensitive to antibiotics containing a β-lactam ring is coupled to the labeling agent during or after step a). 9. The process of compliance with • any of claims 1 to 8, characterized in that steps a) or b) take place simultaneously. 10. The process according to claims 1 to 9, characterized in that • the support used in step b) is chosen from tubes, trays or bars coated with a reference antibiotic. The process according to one of claims 1 to 9, characterized in that the support used in step b) is a device • test that comprise a solid support that 20 has a first and second end, to which they are connected, successively, starting from the first end, a membrane for purifying the fluid analyzed, t ^^ H ^^ a membrane in which one or more capture substances is immobilized, and an absorption membrane. 12. The process according to any of claims 1 to 9, characterized in that the support used in step b) consists of a set of magnetic beads or not. 10 magnetic. • 13. A test kit to detect antibodies in a biological fluid, by the process in accordance with any of the 15 claims 1 to 12, characterized in that it comprises at least one recognition agent that is sensitive to antibiotics containing a β-lactam ring, obtained from Bacillus licheniformis, and at least one antibiotic from 20 reference immobilized on a support. ... Z * ^^. r ~. X.M. . . Mt ^ ÉÜJTMÉTí r
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
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BE9800485 | 1998-06-25 |
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MXPA00012583A true MXPA00012583A (en) | 2001-09-07 |
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