WO2004036212A2 - Procedes pour determiner la teneur en endotoxines de liquides - Google Patents

Procedes pour determiner la teneur en endotoxines de liquides Download PDF

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Publication number
WO2004036212A2
WO2004036212A2 PCT/EP2003/011028 EP0311028W WO2004036212A2 WO 2004036212 A2 WO2004036212 A2 WO 2004036212A2 EP 0311028 W EP0311028 W EP 0311028W WO 2004036212 A2 WO2004036212 A2 WO 2004036212A2
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WO
WIPO (PCT)
Prior art keywords
carrier material
endotoxins
binding protein
endotoxin
liquid
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PCT/EP2003/011028
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German (de)
English (en)
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WO2004036212A3 (fr
Inventor
Veit Otto
Michael Zimmermann
Andrea SCHÖN
Frank Hacket
Thomas Hartung
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Fresenius Hemocare Gmbh
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Application filed by Fresenius Hemocare Gmbh filed Critical Fresenius Hemocare Gmbh
Priority to AU2003293601A priority Critical patent/AU2003293601A1/en
Publication of WO2004036212A2 publication Critical patent/WO2004036212A2/fr
Publication of WO2004036212A3 publication Critical patent/WO2004036212A3/fr

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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
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing

Definitions

  • the invention relates to a method for determining the content of endotoxins in liquids, a flow-through container for use in the method according to the invention and the use of certain separating elements in a method for determining the content of endotoxins in liquids.
  • Liquids e.g. water, aqueous liquids, biological liquids, e.g. sepsis blood, but also technical liquids, especially liquids that come into contact or are in contact with the human body, can contain cell-activating components, e.g. Pyrogen, sometimes with considerable consequences for products made from it.
  • cell-activating components e.g. Pyrogen
  • the pyrogen test method currently prescribed in the pharmacopoeia is limited to the detection in parenterals (rabbit test: biological and pharmaceutical drugs; LAL: mainly pharmaceutical drugs).
  • the testing of medical materials has been regulated in the EU law for medical devices since 1995 and prescribes the rabbit test in addition to the LAL test.
  • Pyrogen detection should also include medical devices such as implants, medical materials, dialysis products, cellular therapeutic agents and specific therapeutic agents such as recombinant proteins.
  • the known methods have some disadvantages.
  • the rabbit pyrogen test is very expensive due to the animal consumption, and it is also desirable for ethical reasons to limit its use.
  • the selectivity of the test is unsatisfactory because it also responds to exotoxins.
  • the LAL test is also not a satisfactory alternative because the detection of endotoxins in sepsis blood or plasma is falsified by various confounding factors. For example, there is a clear susceptibility to disruptive factors such as glucans from fungal infections.
  • activation of the test is observed, for example, in the presence of cellulose. Efforts to influence Suppressing the interference factors usually lead to significant losses in sensitivity.
  • selective detection of endotoxins is not possible.
  • the LAL test also has the disadvantage that animal material is also used.
  • the known pyrogen tests which are based on incubation with whole blood, also have some disadvantages. On the one hand, there can be interference between the sample matrix and whole blood, which can lead to false positive results. On the other hand, the sensitivity of the method is not always satisfactory. In addition, this method is also unable to differentiate between exo- and endotoxins.
  • the present invention is therefore based on the object of overcoming the disadvantages of the known methods, that is to provide a method for determining the content of endotoxin in liquids which can be carried out in vitro without the involvement of experimental animals, which furthermore minimizes interference from the sample matrix and a should have higher sensitivity.
  • a further object is to provide a method for the selective, that is to say, determination of the content of endotoxins in liquids that is not adulterated by exotoxins.
  • a method for determining the level of endotoxin in liquids comprises the steps of: (b) contacting the liquid with one or more separating element (s) comprising a carrier material on which a binding protein for endotoxins is immobilized;
  • step (d) Determination of the amount of endotoxin bound to the binding protein in step (b).
  • liquids in the sense of the invention generally include flowable media, in particular low and medium viscosity, Newtonian Liquids and brine.
  • Water, aqueous solutions, emulsions, dispersions and suspensions are preferred.
  • the fluids can be of biological origin such as urine, blood, liquid blood components such as serum or plasma or other body fluids such as saliva, lymph, cerebrospinal fluid and the like.
  • endotoxin is not subject to any restrictions and, in the context of the invention, includes all compounds which are both a sugar and a lipophilic
  • SIRS Systemic Inflammatory Resp ⁇ nse Syndrome
  • tachypnea (rate> 20 / min) or hyperventilation (Paco2> 32 mm Hg);
  • the endotoxins are preferably components of the cell wall of gram-negative bacteria. At least the endotoxins are usually modeled on such components. Preferred endotoxins have a large molecular weight (between 20,000 and 100,000) and / or are heat stable. These endotoxins consist of lipid A (a glycosamine esterified with C 10 -C 20 fatty acids), a carbohydrate core and the polysaccharide-O-antigen (repeating sequences of linear or branched oligosaccharides, the chain length of which varies between the bacterial strains). The isolated lipid A can also be regarded as an endotoxin without carbohydrate core and the polysaccharide-O-antigen and represents a particularly preferred endotoxin within the scope of the invention.
  • Separating elements are inert, or essentially inert, materials and devices which are able to bind and retain, ie separate, endotoxins from the liquid to an extent sufficient for detection.
  • a monolithic separating element and a multiplicity of separating elements for example appropriately treated polymer beads
  • the separating elements comprise a carrier material on which at least one binding protein for endotoxins is immobilized.
  • binding proteins can also be immobilized on the carrier material.
  • the dissociation constant of the immobilized binding protein can be significantly lower than in the non-immobilized state.
  • binding proteins can be used as binding proteins in the context of the invention:
  • BPl bactericidal / permeability increasing protein
  • LBP lipopolysaccharide binding protein
  • the carrier material itself can interact with the microbiological toxins to be separated, for example by sorption, in particular by adsorption, by microfiltration or by molecular sieve effects.
  • the carrier material must be selected so that protein immobilization is possible.
  • the carrier material is preferably porous, the pore size being selected in such a way that the surface available for mass transfer is increased without an excessive back pressure being built up in the carrier material when the liquid flows through it.
  • Immobilization of the protein on the carrier material is understood to mean a covalent or non-covalent interaction, which is preferably irreversible under the conditions of the method according to the invention.
  • Non-covalent immobilization would be immobilization by biotin / streptavidin. Covalent immobilization is preferred. This is preferably carried out via one or more carboxyl groups of the protein, as described in EP-B1-0 858 831, for example using carbodiimide as a coupling reagent.
  • the liquid is incubated with a protease before step (b) in step (a).
  • This step brings about the proteolytic degradation of endotoxin-binding proteins, for example the degradation of serum albumin, LBP, sCD14, and / or BPI, if present in the liquid, which is particularly the case with biological liquids, especially with serum. It is now possible for the first time to avoid masking the endotoxins by proteins present in the liquid and in this way to improve the sensitivity of the method and, if necessary, to simultaneously increase the linearity between the amount of endotoxin and the measurement signal in step (d). It is further preferred to inactivate the protease after incubation.
  • the binding protein is serum albumin or a fragment thereof, the latter being chosen so that the binding capacity for endotoxins is retained, i.e. the dissociation constant is not more than 1.5 orders of magnitude, preferably not more than one order of magnitude, in particular does not increase by more than 50%.
  • a fragment of the serum albumin can result from proteolytic digestion of the intact protein or with the help of genetic engineering methods.
  • the serum albumin preferably has the primary structure of human serum albumin.
  • Serum albumin has the advantageous property of selectively binding endotoxins, so that a selective detection of endotoxins is made possible, even if the sample additionally contains exotoxins.
  • the serum albumin is immobilized via one or more carboxyl groups, the immobilization preferably consisting of a covalent bond.
  • Human serum albumin immobilized via carboxyl groups on polymer beads binds LPS from healthy plasma, for example, by a factor of 2.5 better than human serum albumin (HSA) immobilized via amino groups. This increase in affinity for LPS is based, among other things, on on the shift of the isoelectric point as a result of the immobilization of the (human) serum albumin via carboxyl groups.
  • the preferred separating elements are polymer beads coated with serum albumin, preferably acyl resin beads, as described in patent specification EP-B 0 858 831, to which reference is made in full.
  • serum albumin is preferably covalently immobilized.
  • the separating element preferably has a capacity of more than 1.5 ng LPS per ml separating element, preferably more than 2 ng / ml, in particular about 2.5 ng / ml.
  • the carrier material is porous and / or dispersible. All substances on which proteins can be immobilized, such as filter papers or filter nonwovens, preferably with a content of organic fibers or polymer beads, are suitable as carrier material.
  • Preferred carrier materials are organic polymer particles, in particular polyacrylic beads, with an (average) diameter of preferably 10 to 500 ⁇ m, particularly preferably 100 to 300 ⁇ m, especially approximately 200 ⁇ m, which are preferably porous.
  • the carrier materials can also be gels and membranes.
  • the porosity is preferably selected so that the surface enlargement due to the porosity is at least about 500, in particular about 1000.
  • the carrier material in particular is plastics, namely polyacrylates, polymethacrylates, polysulfones, polyethersulfones, which carry amines or amides as a functional group.
  • plastics namely polyacrylates, polymethacrylates, polysulfones, polyethersulfones, which carry amines or amides as a functional group.
  • cellulose, cellulose esters, aggarose, polytetrafluoroethylene, polycarbonate, or glass fibers, ceramics or metal are also conceivable as a carrier material. Proteins can be immobilized on metal by thiol groups, similar
  • the carrier material itself can interact with the endotoxin and act as a filter or molecular sieve.
  • the pore sizes of a filter are usually 0.2 to 20 ⁇ m. Pressure filtration can be used to set the desired ratios of filtrate speed and separability. Depending on the viscosity of the fluid and the type and amount of the expected toxin, pore sizes of 1 to 10 ⁇ m, in particular 3 to 8 ⁇ m, are preferably used. In the case of liquids, filters with larger pores have also proven successful in some cases, whereby the particle size of the endotoxin to be expected must also be taken into account in any case. In this case, the filter effect of the carrier material supports the endotoxin-retaining properties of the binding protein on the carrier material.
  • the separating elements After the liquid containing endotoxin has been brought into contact with the separating elements, the separating elements must be separated from the liquid again in step (b), the endotoxins being retained by the separating elements.
  • separation by the action of gravity is appropriate. This can be done by sedimentation or centrifugation.
  • the contacting according to step (b) and separation according to step (c) can also take place in one process step, for example by passing the liquid through the separating elements or through the separating element in the manner of affinity chromatography, the separating element or the separating elements represent the affinity column.
  • the separation in step (c) is completed by contacting the separating element or the separating elements with a suitable washing solution.
  • the washing solution is preferably an isotonic solution (for example 0.9% NaCl) and more preferably the so-called "priming" solution which contains Na + -, K + -, Ca 2+ -, Mg 2+ -, Cl ⁇ - and HCO 3 " ions (e.g. Na + 134; K + 4; Ca 2+ 1, 75; Mg 2+ 0.5; CI " 106.5; HCO 3 " 36; concentration data in mmol / l ).
  • step (d) the amount of endotoxin which has been bound to the binding protein in step (b) is determined.
  • the determination is not subject to any particular restriction and can generally be carried out using any method known in the art. For example, this is done by subjecting the separating element or elements to a whole blood incubation process and determining the mediators formed in the process.
  • a preferred embodiment of the invention relates to a
  • step (d) comprises the following steps:
  • step (d1) performing a whole blood incubation process with the separating element or the separating elements, mediators being formed in accordance with the amount of the endotoxin bound in step (b);
  • step (d2) Determining the amount of one or more of the mediators formed in step (d).
  • Mediators are endogenous pyrogens, i.e. inflammatory messengers that initiate the fever response in response to exogenous pyrogens.
  • the cells that the mediators release are mainly monocytes.
  • the mediators include IL-1, especially IL-1 ⁇ , IL-6, tumor necrosis factor (TNF), prostaglandin E 2 , NO and neopterin.
  • the amount of endotoxin which has been bound to the binding protein in step (b) can be determined in addition to the whole blood incubation method by one of the following examples:
  • Binding protein which can for example be immobilized on beads, brought into contact. Monocyte activation is determined with antibodies directed against surface proteins of the cells in a flow cytometer or with ELISA.
  • suitable binding proteins For example, BPI, LBP, CD14, factor C, serum albumin, A1 adensosin receptor, EGFP, SAP or antibodies can be labeled (for example with a fluorescent or radioactive labeling) and in contact with the immobilized binding protein used for the separation, the endotoxins being labeled on the surface of the separating elements. After removing the labeled binding proteins that have not bound endotoxin, the amount of endotoxin can be determined using the
  • Marking intensity can be determined, which e.g. can be done by a cytometer or by a fluorescence meter.
  • the determination of the amount of endotoxin can be preceded by a separation of endotoxin and the separating elements and, if appropriate, of the binding proteins, in which case the determination of the separated endotoxin then, for example, by cell activation (for example with the aid of the monocyte cultures mentioned above) or with labeled binding proteins (see the preceding section) or with antibodies or possibly recombinant antibody fragments competitively or in a sandwich arrangement.
  • the invention further relates to a flow-through container (1) comprising a supply and exit opening and, between the supply and exit opening in the flow-through region, one or more separating elements (3) which consist of a carrier material onto which a binding protein for endotoxins is immobilized.
  • the separating elements are detachably or non-detachably arranged in the flow-through container.
  • the separating elements are located as segments in a frame, for example made of polystyrene. Thanks to their interchangeability, the use of such segments has the advantage of being able to use a flow-through container more than once.
  • a detachable arrangement of separating elements with a carrier material has proven to be advantageous, since after the absorption capacity of the separating elements has been exhausted the flow device can be removed for incubation in an incubation vessel (and if necessary for regeneration).
  • the flow container itself is usually tubular, funnel-shaped or box-like made of inert material, such as plastic or metal with an oval, rectangular, circular, polygonal or any other cross-section, the size of the cross-sectional area in the area of the separating elements primarily from the viscosity of the test medium , the intended throughput, the predetermined throughput rate (filtration rate), the expected type and possible concentration of the endotoxins, whether in the main or secondary flow, and the like is determined.
  • inert material such as plastic or metal with an oval, rectangular, circular, polygonal or any other cross-section, the size of the cross-sectional area in the area of the separating elements primarily from the viscosity of the test medium , the intended throughput, the predetermined throughput rate (filtration rate), the expected type and possible concentration of the endotoxins, whether in the main or secondary flow, and the like is determined.
  • the filtration speed and quantity of the liquid flow can be based on the principle of pressure filtration by pumping or by suction and e.g. in the case of liquid, by using gravity through the flow-through container as a function of the cross-sectional area and the layer or filter cake thickness of the separating element. In this way, even with a flowable medium below the detection limit or extremely low endotoxin concentration, an endotoxin detection or a reliable detection is still possible by means of the enrichment.
  • the concentration of endotoxin in the liquid e.g. in blood or serum.
  • Flow-through containers in the present sense also mean, for example, two-chamber collecting containers, the two chambers of which are separated by a separating element. After loading a chamber, they can be used primarily for the examination of liquids, for example using gravity as a flow aid. Accordingly, the method also relates to a flow-through container with at least two openings, a feed opening and an outlet opening and with separating elements which are detachably or non-releasably connected thereto for separating endotoxins, the separating element in the flow-through region between the feed and the Outlet opening is arranged.
  • the separating element / the separating elements is preferably arranged essentially perpendicular to the main flow direction and / or in such a way that a forced flow through the separating element is achieved, for example by extending the separating element essentially over the entire cross-sectional area of the flow container in the region of the seat of the separating element.
  • the flow-through container has sealing elements in the respective opening area (feed opening, outlet opening), which ensure a toxin-impermeable closure, for example with caps or other lid closures.
  • openings which are adapted to the openings and optionally have sealing elements are provided.
  • a particular advantage is that the separation of the endotoxins and the whole blood incubation process can be carried out in the same container, so that handling problems which could arise when changing the container are eliminated. It is easily possible to first separate the microbiological toxins from the fluid and then to close the flow-through container by means of the cover provided and to send it to specialist microbiological laboratories for examination.
  • the separating elements or the separating element are first brought into contact with the liquid containing endotoxin in step (b), which preferably had previously been incubated with a protease.
  • the liquid is then separated from the separating elements or the separating element in step (c), which may also include a washing step.
  • the separating elements or the separating element in step (c) are incubated with preparations containing whole blood and, in a manner known per se, for example with the aid of the ELISA or RIA, for the formation of mediators, in particular on IL-1, especially on IL-1 ß, also examined for IL-6, TNF, prostaglandin E 2 , NO and / or neopterin.
  • Whole animal or human blood such as freshly obtained, possibly diluted, blood from healthy human donors can be used without separation of individual components.
  • Mammalian blood is preferred, especially that of whole human blood.
  • the leukocytes are thus in their natural composition and environment. This applies in particular to the monocytes, which mainly release the mediators.
  • the serum components are present that could influence the effect of a toxin.
  • Whole blood generally contains anticoagulant or anticoagulant components, such as citrate, for example in a final concentration of 0.38%, or heparin, such as Na-heparin or heparin fractions, it being important to recognize that the anticoagulant or anticoagulant components also do not impair or even falsify the incubation reaction.
  • Dilution of the whole blood preparation for example with isotonic solutions or with a cell culture medium, for example with RMPI 1640 or for example with a physiological saline solution (20% dilution) is advantageous.
  • antibiotics such as penicillin or streptomycin can be added without disturbing the reaction.
  • the incubation is carried out at elevated temperatures, preferably in the range from 35 to 38 ° C. over a period of about 2 to 24 hours.
  • preparations of gram-negative and gram-positive bacteria such as endotoxin and lipoteichoic acid, for negative control e.g. pyrogen-free physiological saline can be used.
  • the body's primary reaction of mediator formation to endotoxin is used for the investigation.
  • All blood components are available which may be necessary for an interaction of the toxins with leukocytes, e.g. LPS-binding protein LBP, bactericidal pemneability-increasing protein BPI, soluble CD14, definsins, etc.
  • the whole blood incubation method can also be used with whole blood be carried out by those affected with a liquid that comes from their individual environment.
  • the individual sensitivity can be determined and a hypersensitive patient identified.
  • the use of deep-frozen blood or of collective deep-frozen blood in the form of standardized blood unit doses has proven useful for reasons of standardizability (EP-A 0 951 231).
  • whole blood preparation can be used, for example, from 8 ml of physiological saline of clinical quality and 2 ml of heparenized whole blood (blood collection with 7.5 ml of heparinized monovettes, Sarstedt) from healthy donors.
  • the flow-through containers equipped with whole blood preparation are incubated in the incubation cabinet, for example at 37 ° C., 5% CO 2 overnight (18-24 hours).
  • the incubated solution can be mixed well with a pipette, transferred to sterile flacon tubes and centrifuged for two minutes at 4000g and room temperature. The supernatants formed are stored temporarily in the cold if necessary.
  • the amount of mediator formed is then determined, for example, by ELISA (enzyme linked immunosorbent assay) or by RIA (radioimmunoassay) or by similar methods using monoclonal or polyclonal, optionally recombinant antibodies.
  • ELISA enzyme linked immunosorbent assay
  • RIA radioimmunoassay
  • Figure 1 shows a comparison of the IL-1 readings incubated with untreated sepsis plasma and with a protease
  • Sepsis plasma were obtained. The values obtained with untreated sepsis plasma are marked with filled squares. The values obtained with sepsis plasma, which was previously incubated with a protease, are marked with filled triangles. Abscissa: pg / ml LPS; Ordinate: pg / ml IL-
  • the error bars refer to the standard deviations for triple determinations.
  • 2 shows a comparison of the IL-6 measured values which were achieved with untreated sepsis plasma and with a protease-incubated sepsis plasma. Abscissa: pg / ml LPS; Ordinate: pg / ml IL-6. Otherwise the legend corresponds to figure 1.
  • the error bars refer to the standard deviations for triple determinations.
  • Fig. 3 shows an example of an inventive
  • Flow-through container 1 enlarged 5 to 10 times, has an essentially cylindrical jacket 2, a separating element 3 between the feed opening located below and the outlet opening recognizable above, and covers 4 and 5 as a means for the toxin-impermeable closure.
  • Fluid runs essentially axially parallel to the cylinder axis.
  • the separating element which can be in one or more pieces, is made in one piece in Fig. 3, whereby the removal of the separating element is facilitated.
  • Matisse beads (Fresenius Hemocare) are transferred to 50 ml Falcon tubes and covered with 0.9% NaCl to a total volume of 50 ml. The beads are washed for about 10 minutes with occasional swirling.
  • the beads are sedimented by centrifugation (2 minutes 3000 rpm Heraeaus varifuge, ⁇ 2100 g) and the supernatant is completely removed.
  • the beads are now covered with a priming solution (Fresenius Hemocare) and equilibrated for at least 10 minutes with occasional swirling.
  • a priming solution Fresenius Hemocare
  • 2% DMSO Sigma, "cell culture tested" are added and mixed.
  • Plasma from 5 donors is obtained by centrifugation (10 ' ⁇ 2100 g) from Li-heparin-BIut (Sarstedt, "Monovette”). Part of this is stored in aliquots at -80 ° C and used as zero controls.
  • the plasma samples are thawed on the laboratory bench.
  • Components of the complement system are inactivated by incubation at 56 ° C. for 30 minutes. Then vortexing for 1 minute and centrifuging (2 minutes 6000 rpm, Heraeus pico or fresco, corresponds to ⁇ 2100 g). 100 ⁇ l of supernatant are transferred to a 2 ml Eppendorf tube.
  • a protease incubation is to take place, the samples are incubated with 0.8 mg / ml Proteinase-K (16h at 37 ° C), then inactivated 15 'at 95 ° C. The results are shown in Figs. 1 and 2. It can be seen that the protease incubation increases the sensitivity of the measuring system to endotoxins. Pooled plasma from various sepsis patients is measured. This pool plasma is also used to measure aliquots that are enriched with LPS spikes of different concentrations. The spike concentrations correspond to the values given on the abscissa.

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Abstract

L'invention concerne un procédé pour déterminer la teneur en endotoxines de liquides. Elle concerne également un contenant traversé par lesdits liquides, utilisé lors de la mise en oeuvre dudit procédé, ainsi que l'utilisation de certains éléments séparateurs lors de la mise en oeuvre d'un procédé servant à déterminer la teneur en endotoxines de liquides.
PCT/EP2003/011028 2002-10-11 2003-10-06 Procedes pour determiner la teneur en endotoxines de liquides WO2004036212A2 (fr)

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AU2003293601A AU2003293601A1 (en) 2002-10-11 2003-10-06 Method for the determination of the endotoxin content in liquids

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DE10247430.3 2002-10-11
DE2002147430 DE10247430A1 (de) 2002-10-11 2002-10-11 Verfahren zur Bestimmung des Gehalts an Endotoxinen in Flüssigkeiten

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