WO2001027628A1 - Complexes antibiotique-metal utilises pour la detection de bacteries gram negatif et d'autres analytes biologiques - Google Patents
Complexes antibiotique-metal utilises pour la detection de bacteries gram negatif et d'autres analytes biologiques Download PDFInfo
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- WO2001027628A1 WO2001027628A1 PCT/US2000/028577 US0028577W WO0127628A1 WO 2001027628 A1 WO2001027628 A1 WO 2001027628A1 US 0028577 W US0028577 W US 0028577W WO 0127628 A1 WO0127628 A1 WO 0127628A1
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- 0 *CCNC(C(C(C(C(CO)O)O)O)O)O Chemical compound *CCNC(C(C(C(C(CO)O)O)O)O)O 0.000 description 3
- FYWIDDXZIOQEQU-UHFFFAOYSA-N OCC(C(C(C1=O)O)O)OC1O Chemical compound OCC(C(C(C1=O)O)O)OC1O FYWIDDXZIOQEQU-UHFFFAOYSA-N 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/50—Cyclic peptides containing at least one abnormal peptide link
- C07K7/54—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring
- C07K7/60—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring the cyclisation occurring through the 4-amino group of 2,4-diamino-butanoic acid
- C07K7/62—Polymyxins; Related peptides
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the invention relates generally to detection of biological analytes, and more particularly relates to novel complexes of antibiotics and metals that useful in the catalytic detection of gram-negative bacteria and other biological analytes.
- coliform bacteria refers to as group of bacterial genera made up of Eschericia, Klebsiella, Enter obacter, Serratia and Citrobacter bacteria. Coliform bacteria tend to be small, gram negative rods that may be either motile or non- motile. Coliform bacteria have complex membranes that include murein, lipoprotein, phospholipid, and lipopolysaccharide (LPS) components arranged in layers. A murein-LPS layer is about 20% of the total bacterial membrane and is responsible for bacterial cell rigidity. The LPS aids in preventing hydrophobic toxins from entering the coliform bacteria. The LPS is capable of releasing an endotoxin into a host once coliform bacteria infect the host. In human hosts, the endotoxin is released into the blood stream.
- murein-LPS layer is about 20% of the total bacterial membrane and is responsible for bacterial cell rigidity.
- the LPS aids in preventing hydrophobic toxins from entering the coliform bacteria.
- Streptomycetes as well as gram positive bacteria produce antibiotics.
- One particular class of lipopeptide antibiotic, polymyxin is produced by a soil microorganism, Bacillus polymyxa.
- the polymyxins are designated by the letters A, B, C, D and E. These peptides differ in a single amino acid substitution typically being diastereomeric isomers.
- the polymyxins are toxic to coliform bacteria because these antibiotics bind to the LPS in the outer membrane envelope and disrupt cellular metabolism once translocated to the inner membrane cytoplasmic membrane. In particular, the polymyxins are believed to alter the structure and osmotic properties of the outer membrane.
- Polymyxin antibiotics are capable of binding to both animal membranes and coliform bacterial membranes. Coliform bacteria as well as some fungi may cause urinary tract infections as well wound infections in the human host. Coliform bacteria may also cause pneumonia, meningitis, speticiemia and various gastrointestinal disorders in humans. It has been estimated that as many as 100,000 deaths in the United States each year are a consequence of gram negative infections such as coliform bacteria.
- endotoxin produced by coliform bacteria produces a variety of effects such as fever, fatal shock, leukocyte alterations, cytotoxicity, alterations in host response to infections, Sanarelli-Shwartzman reaction and various other undesirable metabolic changes.
- endotoxic shock plays an important role in weakening the individual. About 30%> of individuals with endotoxin in their blood will develop shock. About 40 to 90% of individuals in endotoxic shock die. Endotoxic shock is characterized by an inadequate supply of blood to vital organs of the host causing cellular hypoxia and metabolic failure. Survival of the host is directly proportional to the length of time needed to recognize development of bacteremia and adequate treatment of the coliform bacterial infection.
- the present techniques used for this type of screening involve aseptic transfer of a sample, streaking the sample having bacterial organisms on agar plates after serial dilution and colony enumeration. This is a laborious and lengthy process requiring a period of at least 24 to 48 hours for a positive result and substantially longer for a negative result.
- test solutions containing enteric bacteria use carbohydrate and acid based indicators to demonstrate carbohydrate fermentation.
- Lactose and lactose analogues are the carbohydrates most frequently used in bacteria testing. This is because the majority of organisms of the genera Eschericia, Enterobacter and Klebsiella, the enteric organisms present in greatest numbers in fecal material, ferment this carbohydrate while other intestinal pathogens usually do not.
- Some media may also contain iron salts for the detection of hydrogen sulfide production to aid in the identification of Salmonella colonies. This approach to bacterial testing also requires a lengthy incubation time to grow enough bacteria for testing, at least 24 to 48 hours.
- analyte tests require an organism to digest a detectable material, such as fluorescein.
- an antibody specific for an antigen on the target bacteria is labeled with fluorescein to make a fluorescent antibody.
- Another approach involves use of a visualization polymer coupled to a detecting agent that binds the target organism, wherein the visualization polymer is made up of detectable visualization units, such as multiple enzymes or labeled polyolefins, which are directly or indirectly bonded together (see, e.g., U.S. Patent No. 4,687,732 to Ward et al.).
- PMB polymyxin B
- HRP horseradish peroxidase
- Polymyxin B is a cyclic decapeptide having a high percentage of 2,4-diaminobutyric acid (also “ ⁇ , ⁇ -diaminobutyric acid” or "DAB”), a fatty acid and a mixture of D- and L-amino acids.
- Polymyxin B has the following structure: ⁇ NH 2
- the present invention is directed to a novel antibiotic derivative that takes the form of a bound complex comprising a cyclic antibiotic and a metal.
- the cyclic antibiotic is preferably a polymyxin, a colistin, or an analog or fragment thereof.
- the polymyxin or colistin can be a decapeptide ⁇ as illustrated in Formula 1 , with respect to Polymyxin B— or an enzymatically derived fragment thereof, preferably a nonapeptide as described by Danner et al. (1989) Antimicrob. Agent Chemother. 33:1428-1434.
- the aforementioned nonapeptide is readily formed by removal of the 6-methyloctanoic acid moiety from Polymyxin B, for example by digestion with a standard proteolytic enzyme. As disclosed in U.S. Patent No. 5,750,357 to Olstein et al., cited supra, and Vaara et al. (1983) Nature 303:526, the cyclic nonapeptide constituent may then be easily separated from the lipid constituent and purified by a method such as liquid/liquid extraction.
- a preferred polymyxin or colistin fragment is one that retains an intact molecular cleft, as described below, retains bacterial binding activity and is preferably less toxic to mammalian tissues than naturally occurring polymyxins.
- the metal in the complex is a transition metal or a lanthanide metal; more preferably it is copper, cobalt, iron, manganese, chromium, nickel, zinc, terbium, gadolinium, europium, or technicium.
- the polymyxin is polymyxin B (PMB).
- PMB polymyxin B
- the polymyxin-metal complex is preferably a chelated metal complex containing one metal atom.
- the metal may be coordinated at four, five or six sites.
- the metal binding site is within the cleft formed by the cyclized amino acids.
- this conformation may least inhibit the electrostatic interactions of the side chain amino groups of the diaminobutyric acid moieties with the anionic charged groups in lipopolysaccharide (LPS), since these interactions are crucial to LPS binding and anti-microbial activity. Any covalent modification of the side chain amino groups abolishes biological activity of the antibiotic
- the polymyxin-metal complex of the invention is unique in that it allows detection of gram negative bacteria by covalently linking the polymyxin to a detectable label.
- Polymyxin-metal complexes can directly catalyze peroxide-driven chemiluminescent reactions (for example, reactions involving luminol, its aromatic derivatives, lucigenin, penicillin, luciferin and other polyaromatic phthalylhydrazides) without the use of an enzyme catalyst such as horseradish peroxidase or microperoxidase.
- an enzyme catalyst such as horseradish peroxidase or microperoxidase.
- most organic complexes of polymyxin do not retain their anti-microbial activity (again, as shown in Table 1 , Example 4, infra), the polymyxin-metal complexes retain substantially full anti-microbial activity.
- polymyxin-metal complex can be readily purified using standard chromatographic techniques such as gel filtration or dialysis procedures because it can be followed visually with either visible absorbance or fluorescence depending on the type of complex.
- Polymyxin B has been documented to bind tightly to immobilized LPS, and this has in fact been a method for removing endotoxins from pharmaceuticals and biologicals (Issekutz (1983) J. Immunol. Method 61 :275-281).
- the invention further provides a method for detecting gram-negative bacteria comprising adding a polymyxin-metal complex to a sample suspected of containing gram- negative bacteria, washing away the unbound complex, adding a chemiluminescent agent such as luminol or lucigenin, and then measuring the resulting luminescence using a luminometer or suitably configured photo-detection device.
- a chemiluminescent agent such as luminol or lucigenin
- the polymyxin-metal complexes of the invention can be freeze- dried or spray dried and are preferably stored in the dark, then reconstituted in water or aqueous buffers, buffered from pH 4.5 to 7.0, for later use.
- Dried PMB-metal complex can be stored indefinitely with or without refrigeration, preferably stored in the dark and in the absence of molecular oxygen.
- the polymyxin-metal complexes of the invention are capable of numerous and varied diagnostic and therapeutic uses. For example, gram negative bacteria can rapidly be detected in any environment, e.g., in quality control efforts in food processing and medical device sterilization.
- Polymyxin-metal complexes can be used to label monoclonal antibodies by cross-linking the complex to an antibody by any number of conventional cross-linking protocols.
- Paramagnetic complexes containing gadolinium as the chelated metal could be cross-linked to anti-tumor antibodies for use in medical magnetic imaging applications. Gadolinium, an element with a very large magnetic moment and high magnetic susceptability, is the preferred label for magnetic resonance imaging in high magnetic field strength instruments because of its superior spatial resolution in modern MRI equipment.
- polymyxin-metal Mab complex is also envisioned, either as a targeted bifunctional imaging/therapeutic agent or as a purely therapeutic agent.
- Preferred complexes are comprised of enzymatic fragments of polymyxin, such as the polymyxin nonapeptide, because of their lower mammalian toxicity.
- Complexes of polymyxin with radioactive technicium ( "Tc) attached to an Mab or other delivery /carrier molecule also have potential as targeted therapeutic agents.
- Tc radioactive technicium
- These diagnostic and therapeutic uses have great promise in the fields of cancer and AIDS treatment.
- Peptide-metal complexes like the polymyxin-metal complexes of the present invention are preferred over protein-metal complexes for these uses because they are less likely to be involved with non-specific interactions, thereby reducing detection background levels.
- Figure 1 shows the UV-visible spectrum of an isolated iron-polymyxin B complex at 1.5 mg/mL in phosphate buffer, pH 6.5.
- Figure 2 shows the UV-visible spectrum of an isolated cobalt-polymyxin B complex at 1.5 mg/mL in phosphate buffer, pH 6.5.
- Figure 3 shows the UV-visible spectrum of an isolated copper-polymyxin B complex at 1.5 mg/mL in phosphate buffer, pH 6.5.
- Figure 4 shows the overlayed UV-visible spectra obtained for terbium, iron and copper polymyxin B complexes.
- Figure 5 illustrates in graph form the results obtained upon chemiluminescent titration of E.coli O157:H7, signal corrected/background ratios versus serial dilutions of cell stock.
- Figure 6 illustrates in graph form the results obtained upon chemiluminescent titration of Salmonella enteriditis, signal corrected/background ratios versus serial dilutions of cell stock.
- the present invention provides for a novel antibiotic-metal chelate constituting a new class of chemiluminescent cell labels useful for rapid detection of gram negative pathogens as well as non-pathogenic bacteria, and chemical residues of these bacteria.
- the invention further provides for a method of the labels' manufacture and a method for use in a rapid detection assay for bacterial pathogens.
- antibiotics of the polymyxin and colistin type tightly bind a range of metals in aqueous solution, a finding unreported in the literature.
- Antibiotics particularly cationic antibiotics including polymyxins, colistins and aminoglycosides, will spontaneously chelate a range of transition metals or lanthanides in aqueous solution.
- the binding interaction is sufficiently tight to permit isolation of the antibiotic complex by gel filtration or dialysis, which would ordinarily separate high molecular weight compounds from simple metal salts.
- the strong absorbance bands exhibited by the antibiotic-metal complex permit the absorbance of the peptide chromophore at 270 nm and the visible absorbance bands at 400 nm to be used to follow purification of the complex.
- the efficient chelation of metals is presumably due to formation of a cleft within the structure of the antibiotic, providing both carbonyl oxygens and amide nitrogens to contribute electron density for orbital overlap in the outer electron orbitals of a metal atom.
- aminoglycosides A major constituent of the aminoglycosides are amino sugars, which permit several covalent modifications to be made to the amino groups on the antibiotics.
- the aminoglycoside kanamycin (depicted in Formula 2) is a preferred aminoglycoside, although other aminoglycosides may also be used, e.g., amikacin, streptomycin, paromomycin and gentamycin.
- the monocarboxaldehyde of 2,2'-dipyridine, salicylaldehyde or protocatechualdehyde would produce a suitable metal binding cavity in the aminoglycoside molecule to chelate several transition metals such as copper, nickel, zinc, technetium, and preferably cobalt, iron, manganese, or chromium.
- the aforementioned ligands including 2,2'- dipyridyl monocarboxlic acid, salicylic acid, and protocatechuic acid, could alternatively be grafted onto the antibiotic through an amide linkage as preformed, isolated N- hydroxysuccinimide esters.
- the ligands could either be used pre-loaded with the metals as reactive chelates, or optionally, chelated after the conjugates are formed.
- a further embodiment of the present invention relates to the use of enzymatically or chemically derived fragments of the polymyxins and colistins in these metal complexes.
- An example of this application is to prepare an imaging diagnostic reagent by cross-linking the nonapeptide fragment to an anti-tumor monoclonal antibody using a hetero-bifunctional reagent, such as N-hydroxysuccinimide-activated N- propionylmaleimide.
- the malylated peptide would then react with a native sulfhydryl on the antibody or a sulfhydryl introduced by treatment with a thiolating reagent such as iminothiolane.
- a thiolating reagent such as iminothiolane.
- the preferred metals of the present invention include the transition metals and the lanthanides.
- the transition metals are particularly preferred because of their high oxidation-reduction activity in neutral aqueous media. It is likely that these metals catalyze the process of oxidizing chemiluminescent substrates, such as luminol by hydrogen peroxide, Scheme 1 (Rost et al. (1998) J. Biolumin. Chemilumin. 13:355-363).
- the metal catalyzes the formation of superoxide and other radical oxygen species which are reactants in the chemiluminescent reaction of Scheme 1.
- Factors influencing the efficiency of individual metals include pH, ionic strength and oxidation state. Chelation chemistries that would alter the oxidation state or steric availability of the metals during catalysis could also influence the optimum catalytic activity as sensed by the time dependent emission of photons.
- Preferred transition metals measured in the antibiotic-metal complex of the present include iron, copper, cobalt, chromium, nickel, manganese, zinc and technicium. The most preferred metals, iron, cobalt, manganese and chromium, yield the most catalytically active complexes on a molar basis.
- Another preferred class of metal chelates of the present invention comprise heavy metals in the lanthanide series, including terbium, europium, gadolinium and lutitium.
- a unique and useful aspect of terbium and europium complexes is that neither the metal salts nor the antibiotic are fluorescent; however, some of the chelates are fluorescent.
- a blue fluorescent emission can be observed at 400-450 nm when illuminated with 330 nm light.
- the polymyxin B-terbium complex is also useful as an epifluorescence microscopy label for E.coli and Salmonella cells.
- Preferred methods for the preparation of the antibiotic-metal complex of the present invention can be readily ascertained by those skilled in the art.
- Complexes are readily prepared in either water or dilute buffers, preferably volatile buffers, such as acetic acid, ammonium acetate, and ammonium bicarbonate.
- Crystalline or powdered antibiotic is dissolved in aqueous medium, in concentrated solution, greater than 0.5 M, and water soluble metal salts are added to provide a slight molar excess over antibiotic.
- Chelates formed in solution can be isolated by dialysis in narrow-pore molecular weight cut-off tubing (e.g., as available from Spectro-Por) or by gel filtration on GPC media such as Sephadex G-25.
- the effluent carrying the antibiotic complex can be dried preferably by freeze drying or, alternatively, by spray drying.
- Antibiotic-metal complexes isolated by the aforementioned procedures can, optionally, be further characterized by combustion analysis, NMR, and electronic spectroscopy. These procedures should also be accompanied by a bio-assay method to ensure preservation of bacterial binding activity, and/or anti-microbial activity.
- a useful bio-assay can be conducted using immobilized target residues, such as intact lipopolysaccharide, or lipid A, both of which are available from Sigma Chemical Co.
- an end-point determination for Minimum Inhibitory Concentration ( MIC ) of the antibiotic can be conducted according to standard microbiological procedures.
- the methods of the present invention are suitable for use in rapidly detecting gram negative pathogens in samples as diverse as drinking water, hamburger and blood.
- samples For drinking water and low protein solutions, samples require concentration with thin film type-membranes so that captured bacterial cells can be resuspended in a small volume for analysis. More concentrated samples such as biological fluids and foodstuffs lend themselves to processing with rapid isolation techniques such as immuno-magnetic micro- beads, or high density immuno-silica micro-beads.
- the core utility of the antibiotic-metal complexes of the present invention is the binding activity to both intact and bacterial residues.
- a preferred embodiment is a simple binding assay consisting of labeling gram positive cells in suspension, pelleting the cells by centrifugation or by immuno-sedimentation, washing unbound label, and detection with chemiluminescent reagents has been demonstrated.
- Bacterial cells are diluted from stock cultures containing in excess of 10 6 colony forming units (CFU) per mL in peptone water.
- the cell suspensions are labeled at room temperature with antibiotic-metal complex of the invention at concentrations from 0.01 to 0.05 mg/mL in peptone water for a minimum of ten minutes.
- the labeled cells can, optionally, be collected by centrifugation, filtration on micro-porous filters of the polycarbonate film type (Osmonics, Inc.) or rapid immuno- sedimentation using antibody-bound agarose or silica micro-beads.
- the labeled cells are then washed and resuspended in peptone water for assay with preferably, hydrogen peroxide/luminol or any number of oxidizable chemiluminescent, including lucigenin, penicillin and the like.
- Potential bacterial targets for the antibiotic-metal complex of the present invention include E.coli, Salmonella species, Campylobacter species, all other gram negative organisms within Enterobacteriace as well as all other aerobic and anaerobic gram negative organisms.
- Preferred separation methods for target pathogens include immuno-sedimentation using either magnetically accumulated micro-beads or gravity sedimentation.
- Several methods for isolation of pathogens from food and water have been published, e.g., Fratamico ( 1992) Food Microbiol. 9:105-1 13, and Pyle ( 1999) Appl. Environm. Microbiol
- Polymyxin B (0.1 mmole, 162 mg) was dissolved in 6 mL phosphate buffer, pH 8.0, and iminothiolane (15.1 mg, 0.11 mmole, Sigma Chemical Co.) in 0.2 mL methyl sulfoxide was added. The reaction mixture was incubated at room temperature for 18 hours.
- the thiolated antibiotic was purified by thiol exchange chromatography on thiopropyl sepharose. The sample was applied to a 2.0 x 20 column and washed with 0.1% acetic acid. The thiolated compound was eluted from the solid support by washing the column with the acetic acid containing 0.01 M mercaptoethanol. The fractions absorbing at 280 nm were pooled, desalted by flash chromatography on sephadex G-25 and freeze dried.
- Polymyxin B was glycosylated by reductive alkylation by treatment of the antibiotic with one equivalent of D-mannose in the presence of sodium cyanoborohydride, as depicted in scheme 3.
- a derivative of polymyxin B was prepared by coupling the terminal thiol group through thiol sepharose, which has a terminal blocked aldehyde group, by reductive alkylation, Scheme 4.
- MIC Minimum Inhibitory Concentrations
- the complex exhibited a blue fluorescent emission, when illuminated with 330 nm light, which co-eluted with the peptide from the column.
- Figure 4 depicts the overlayed spectra of the metal -polymyxin complexes at comparable concentrations.
- Lipopolysaccharide resin was prepared as follows. Five grams of epoxy activated sepharose 4B (Sigma) was washed with distilled water and subsequently dehydrated with water-ethanol 20:80 (v/v), water-ethanol 50:50 (v/v) and ethanol. Ten milligrams of purified lipopolysaccharide from Salmonella Enteridis (Sigma) was dissolved in 18 mL of dimethyl sulfoxide. The dehydrated epoxy resin was suspended in the LPS solution and 0.13 mL of tributylamine was added. The suspension was agitated eighteen hours at room temperature.
- Unbound epoxy groups were blocked by addition of 5mL of 0.2 M glucosamine, free base, water:DMSO and incubated a further 24 hours at room temperature. The resin was progressively washed with solvents to resuspend in 100% aqueous medium.
- Bacteria were diluted in sterile saline from cell concentrations of 10 8 CFU/mL to 10 CFU/mL.
- the cells were treated with the PMB-Co(II) complex @ 20 ⁇ g/mL for twenty minutes at room temperature.
- the cells were centrifuged, rinsed with 1.0 mL saline; centrifuged and re-suspended in 0.1 mL saline.
- Chemiluminescence was measured using 0.2mL of Luminol reagent purchased from NEN Life Sciences ( Boston, MA) in a Biotrace ® luminometer.
- Bacteria were diluted in sterile saline from cell concentrations of 10 8 CFU/mL to 10 CFU/mL.
- the cells were treated with the PMB-Co(II) complex @ 20 ⁇ g/mL for twenty minutes at room temperature.
- the cells were centrifuged, rinsed with 1.0 mL saline; centrifuged and re-suspended in 0.1 mL saline.
- Chemiluminescence was measured using 0.2mL of Luminol reagent purchased from NEN Life Sciences ( Boston, MA) in a Biotrace ® luminometer.
- Bacteria were diluted in sterile saline from cell concentrations of 10 8 CFU/mL to 10 CFU/mL. The cells were treated with the PMB-Co(II) complex @ 20 ⁇ g/mL for twenty minutes at room temperature. The cells were centrifuged, rinsed with 1.0 mL saline; centrifuged and re-suspended in 0.1 mL saline. Chemiluminescence was measured using 0.2mL of Luminol reagent purchased from NEN Life Sciences ( Boston, MA) in a Biotrace ® luminometer. The cells exhibited background levels of chemiluminescence at all cell concentrations tested.
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Abstract
La présente invention concerne des complexes d'antibiotiques et de métaux qui sont utilisés pour la détection de bactéries et d'autres analytes biologiques, notamment pour la détection de bactéries Gram négatif. Ces complexes sont de préférence des complexes chélatés dans lesquels l'antibiotique est une polymyxine, une colistine, un aminoglycoside ou un analogue ou fragment de ces derniers. Cette invention concerne également des procédés d'utilisation desdits complexes.
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US7034113B2 (en) | 2002-02-22 | 2006-04-25 | Paradigm Diagnostics, Llc | Bacteriocin-metal complexes in the detection of pathogens and other biological analytes |
WO2006123343A2 (fr) * | 2005-05-18 | 2006-11-23 | Ramot At Tel Aviv University Ltd. | Proteines revetues de metal biologiquement actives |
WO2010085927A1 (fr) * | 2009-01-31 | 2010-08-05 | Moessmer Raimund | Composition et système de détection d'oxygène et de ligands biologiques |
US7951913B2 (en) | 2006-06-02 | 2011-05-31 | Biotika A.S. | Method of polymyxin B recovery from fermentation broth |
US8119371B2 (en) | 2006-06-15 | 2012-02-21 | Biotika A.S. | Process for the preparation of polymyxin B employing (PAENI) Bacillus polymyxa |
US9057088B2 (en) | 2005-01-26 | 2015-06-16 | Ramot At Tel-Aviv University Ltd. | Biologically active silver-coated proteins |
US20160016979A1 (en) * | 2013-03-13 | 2016-01-21 | Lantheus Medical Imaging, Inc. | Process for manufacture of gadofosveset trisodium monohydrate |
FR3088652A1 (fr) * | 2018-11-21 | 2020-05-22 | Nanotracks Diagnostics | Détection in vitro de la présence d’au moins un agent pathogène microbien dans un échantillon biologique |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112088023B (zh) * | 2018-01-05 | 2024-05-14 | 帕斯艾克斯公司 | 用于捕获和去除流体中的疾病物质的装置 |
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US5714121A (en) * | 1995-09-28 | 1998-02-03 | Optical Sensors Incorporated | Optical carbon dioxide sensor, and associated methods of manufacture |
US5750357A (en) * | 1994-05-18 | 1998-05-12 | Microquest Diagnostics, Inc. | Method of rapid analyte detection |
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- 2000-10-13 WO PCT/US2000/028358 patent/WO2001026673A1/fr active Application Filing
- 2000-10-13 AU AU12067/01A patent/AU1206701A/en not_active Abandoned
- 2000-10-13 WO PCT/US2000/028577 patent/WO2001027628A1/fr active Application Filing
- 2000-10-13 AU AU10835/01A patent/AU1083501A/en not_active Abandoned
Patent Citations (2)
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US5750357A (en) * | 1994-05-18 | 1998-05-12 | Microquest Diagnostics, Inc. | Method of rapid analyte detection |
US5714121A (en) * | 1995-09-28 | 1998-02-03 | Optical Sensors Incorporated | Optical carbon dioxide sensor, and associated methods of manufacture |
Cited By (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1335932B1 (fr) * | 2001-10-19 | 2005-11-30 | Natimmune A/S | Isolation des lectines |
US7034113B2 (en) | 2002-02-22 | 2006-04-25 | Paradigm Diagnostics, Llc | Bacteriocin-metal complexes in the detection of pathogens and other biological analytes |
US9057088B2 (en) | 2005-01-26 | 2015-06-16 | Ramot At Tel-Aviv University Ltd. | Biologically active silver-coated proteins |
WO2006123343A2 (fr) * | 2005-05-18 | 2006-11-23 | Ramot At Tel Aviv University Ltd. | Proteines revetues de metal biologiquement actives |
WO2006123343A3 (fr) * | 2005-05-18 | 2007-06-28 | Univ Ramot | Proteines revetues de metal biologiquement actives |
US7951913B2 (en) | 2006-06-02 | 2011-05-31 | Biotika A.S. | Method of polymyxin B recovery from fermentation broth |
US8119371B2 (en) | 2006-06-15 | 2012-02-21 | Biotika A.S. | Process for the preparation of polymyxin B employing (PAENI) Bacillus polymyxa |
WO2010085927A1 (fr) * | 2009-01-31 | 2010-08-05 | Moessmer Raimund | Composition et système de détection d'oxygène et de ligands biologiques |
US20160016979A1 (en) * | 2013-03-13 | 2016-01-21 | Lantheus Medical Imaging, Inc. | Process for manufacture of gadofosveset trisodium monohydrate |
US10106562B2 (en) * | 2013-03-13 | 2018-10-23 | Lantheus Medical Imaging, Inc. | Process for manufacture of gadofosveset trisodium monohydrate |
FR3088652A1 (fr) * | 2018-11-21 | 2020-05-22 | Nanotracks Diagnostics | Détection in vitro de la présence d’au moins un agent pathogène microbien dans un échantillon biologique |
Also Published As
Publication number | Publication date |
---|---|
WO2001026673A1 (fr) | 2001-04-19 |
WO2001026673A9 (fr) | 2002-08-01 |
AU1083501A (en) | 2001-04-23 |
AU1206701A (en) | 2001-04-23 |
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