DE10247430A1 - Method for determining the level of endotoxins in liquids - Google Patents

Abstract

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.

Description

The invention relates to a method to determine the content of endotoxins in liquids Use in the method according to the invention and the use of certain separators in a process for Determination of the endotoxin content in liquids.

Liquids, for example Water, watery Liquids, biological liquids, for example sepsis blood, but also technical liquids, especially liquids, the one with the human body in touch can come or stand with cell activating components, e.g. Pyrogen, partly with significant ramifications for products made from it may be contaminated. These include e.g. Beverages, Infusion and injection solutions, Dialysis fluids, Disinfecting solutions e.g. For cleaning of medical materials, rinsing solution for commercial purposes and for use in medicine, e.g. for rinsing the Abdominal cavity, the cavities organs during surgery or the lungs to obtain bronchial lavage, Liquid additives in air conditioning Systems for air humidification or liquid mixtures, for cooling be called Materials during processing, e.g. in the steel industry.

• 1. den in vivo Kaninchen-Pyrogen-Test, der nach Injektion der Prüfsubstanz die Erhöhung der Körpertemperatur ermittelt, und
• 2. den in vitro Limulusamöbozytenlysattest (LAL), der die Koagulation eines aus dem Blut des Pfeilschwanzkrebses gewonnenen Lysates mißt, ausgelöst durch das Endotoxin, eines Zellwandbestandteiles gramnegativer Bakterien.

The absence of pyrogen in injectable drugs (parenterals) is of particular importance. These fever-inducing contaminants, which include endotoxins, pose a considerable risk for humans. In extreme cases, they mean death from shock. In contact with immunocompetent cells, pyrogens release messenger substances that act directly on the central thermoregulation center in the brain. The pharmacopoeia prescribes two test systems for pyrogen detection:

• 1. the in vivo rabbit pyrogen test, which determines the increase in body temperature after injection of the test substance, and
• 2. the in vitro limulus amebocyte lysate test (LAL), which measures the coagulation of a lysate obtained from the blood of horseshoe crab, triggered by the endotoxin, a cell wall component of gram-negative bacteria.

The one currently prescribed in the Pharmacopoeia Pyrogen test method is limited to detection in parenterals (Rabbit test: biological and pharmaceutical drugs; LAL: mainly pharmaceutical drugs). Testing medical materials is in the EU law for medical devices since 1995 regulated and writes the rabbit test in addition to the LAL test in front. The pyrogen detection should also include medical devices such as Implants, medical materials, dialysis products, cellular therapeutic agents and include specific therapeutics such as recombinant proteins.

To the rabbit pyrogen test, which of drug safety 50 Years (Flint, 1984, Alternatives to Animal Testing, New Ways in the Biomedical Science, Trends and Progress (27 - 43), Weinheim, VCH), a new pyrogen test was developed (Hartung and Wendel, 1995, ALTEX 12, 70-75; Fennrich et al., ALTEX 16, 164-149). In this test, the substance to be checked is incubated with a small amount of blood from a healthy donor. Regardless of their chemical nature, any pyrogenic activity induces the formation of interleukin-1β (IL-1β), the concentration of which can be determined by ELISA.

The known methods have some disadvantages on. The rabbit pyrogen test is very expensive due to animal consumption, it is also ethical establish desirable, to limit its use. Moreover is selectivity of the test unsatisfactory because it also responds to exotoxins. The LAL test is also not a satisfactory alternative since the Detection of endotoxins in sepsis blood or plasma by various confounders falsified becomes. For example, there is a clear vulnerability against disruptive factors like glucans from fungal infections. Furthermore, e.g. in the presence of Cellulose activation of the test was observed. efforts the influence of Disruptive factors too suppress, to lead usually to significant losses in sensitivity. Moreover selective detection of endotoxins is not possible. The LAL test also has the disadvantage that animal material is also is used. Also the well-known pyrogen tests based on incubation based on whole blood have some disadvantages. For one, it can interferences between sample matrix and whole blood come what lead to false positive results can. On the other hand, the sensitivity of the method is not always satisfactory. Moreover this method is also unable to distinguish between exo- and endotoxins to distinguish.

The present invention lies thus the task based on the disadvantages of the known methods to overcome, a method for determining the level of endotoxin in liquids To be provided in vitro without the involvement of experimental animals carried out can be, moreover Interference caused by minimize the sample matrix and have a higher sensitivity should. Another task is to develop a selective method that is called unadulterated by exotoxins Providing determination of the endotoxin content in liquids.

This task is accomplished by the in the claims Chen characterized embodiments of the present invention solved.

• (b) Inkontaktbringen der Flüssigkeit mit einem oder mehreren Trennelement(en), die ein Trägermaterial umfassen, auf das ein Bindungsprotein für Endotoxine immobilisiert ist;
• (c) Trennung von Flüssigkeit und Trennelement(en); und
• (d) Bestimmung der Menge an Endotoxin, die in Schritt (b) an das Bindungsprotein gebunden worden ist.

In particular, a method for determining the level of endotoxin in liquids is provided, which comprises the following steps:

• (b) contacting the liquid with one or more separating element (s) comprising a carrier material on which a binding protein for endotoxins is immobilized;
• (c) separation of liquid and separation element (s); and
• (d) Determination of the amount of endotoxin bound to the binding protein in step (b).

To the examples liquids belong in the sense of the invention generally flowable media, especially low and medium viscosity, Newtonian liquids and brine. Water, aqueous solutions, emulsions, dispersions are preferred and suspensions. The liquids 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.

• (i) Körpertemperatur > 38 °C oder < 36 °C;
• (ii) Steigerung des Herzschlags (> 90 Schläge pro Minute);
• (iii) Tachypnoe (Rate >20/min) oder Hyperventilation (Pa_CO2 > 32 mm Hg);
• (iv) Leukocyten > 12.000/mm³ oder < 4.000/mm³ oder > 10 % unnreife Neutrophile.

The term "endotoxin" is not subject to any restriction and includes, within the scope of the invention, all compounds which have both a sugar and a lipophilic group and which can produce a phenotype in humans, which is called SIRS ("Systemic Inflammatory Response Syndrome") and which is characterized by at least two of the following symptoms:

• (i) body temperature> 38 ° C or <36 ° C;
• (ii) increased heartbeat (> 90 beats per minute);
• (iii) tachypnea (rate> 20 / min) or hyperventilation (Pa _CO2 > 32 mm Hg);
• (iv) leukocytes> 12,000 / mm ³ or <4,000 / mm ³ or> 10% immature neutrophils.

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 nucleus 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 alone without a carbohydrate nucleus and the polysaccharide O antigen and is a particularly preferred endotoxin within the scope of the invention.

Separators are inert, or in essential inert materials and devices capable of are from the. liquid possibly contained endotoxins in a sufficient for the detection To bind and withhold scope, so separate.

Within the scope of the invention, both a monolithic separator as well as a variety of separators (for example appropriately treated polymer beads) for use come.

The separating elements comprise a carrier material immobilized on the at least one binding protein for endotoxins is. It can also several different binding proteins on the carrier material be immobilized. The dissociation constant of the immobilized Binding protein can be much lower than in the non-immobilized Condition.

• - BPI (bactericidal/permeability increasing protein), (Evans, T.J. et al. J. Infect. Dis. 171, 153–160; Ganzzano-Satoro, H. et al. Infect. Immun. 63, 2201–2205; Elsbach P. et al.. Prog. Clin. Biol. Res. 388, 41–51)
• - LBP (lipopolysaccharide binding Protein), (Tobias P. S. et al. J. Biol. Chem. 263, 13479–13481)
• – CD14 Zellrezeptor von Makrophagen/Monocyten, (Kirikae T. et al. FEMS Immunol Med Microbiol 8, 13–26)
• – Faktor C (endotoxin neutralizing protein) aus dem Lysat von Limulus polyphemus. (Obayashi et al. Clina Chimca Acta 149, 55–56)
• – Serumalbumin
• – A1-Adenosinrezeptor (WO 97/44665)
• – EGFP (Enhanced green fluorescence protein) (Goh et al. Protein engineering 15 (2002) 493)
• – SAP (Serum amyloid P component) (WO 98/16556, WO 99/55731)

For example, the following can be used as binding proteins in the context of the invention:

• - BPI (bactericidal / permeability increasing protein), (Evans, TJ et al. J. Infect. Dis. 171, 153-160; Ganzzano-Satoro, H. et al. Infect. Immun. 63, 2201-2205; Elsbach P et al. Prog. Clin. Biol. Res. 388, 41-51)
• - LBP (lipopolysaccharide binding protein), (Tobias PS et al. J. Biol. Chem. 263, 13479-13481)
• - CD14 cell receptor of macrophages / monocytes, (Kirikae T. et al. FEMS Immunol Med Microbiol 8, 13-26)
• - Factor C (endotoxin neutralizing protein) from Limulus polyphemus lysate. (Obayashi et al. Clina Chimca Acta 149, 55-56)
• - serum albumin
• - A1 adenosine receptor (WO 97/44665)
• - EGFP (Enhanced green fluorescence protein) (Goh et al. Protein engineering 15 (2002) 493)
• - SAP (Serum amyloid P component) (WO 98/16556, WO 99/55731)

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. In any case, 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 means a covalent or non-covalent interaction, which is preferably irreversible under the conditions of the method according to the invention. An example of a non-covalent immobilization would be immobilization by biotin / streptavidin. Covalent immobilization is preferred. This is preferably done via one or more carboxyl groups of the protein, as in EP-B1-0 858 831 described, for example using carbodiimide as a coupling reagent.

In a preferred embodiment of the method according to the invention will the liquid before Step (b) in step (a) incubated with a protease. This Step causes the proteolytic breakdown of endotoxin-binding proteins, e.g. the degradation of serum albumin, LBP, sCD14, and / or BPI, to the extent in the liquid available, which is especially true for biological fluids, especially for Serum is the case. It is now possible for the first time to mask endotoxins through in the liquid to avoid existing proteins and thus sensitivity to improve the method and, if necessary, simultaneously the linearity between Amount of endotoxin and measurement signal increase in step (d). Furthermore, it is preferred to use the protease after incubation to inactivate. This has the advantage that the protease does not contain the binding proteins, which are immobilized on the separating elements, attacks when in Step (b) the liquid is brought into contact with the separating elements. A particularly cheap choice represents proteinase K as a protease.

In a preferred embodiment of the method according to the invention is the binding protein serum albumin or a fragment thereof, the latter so chosen is that the binding capacity for endotoxins The dissociation constant is therefore retained by no more than 1.5 orders of magnitude, preferably by no more than an order of magnitude, especially not increases more than 50%. A fragment of the serum albumin can by proteolytic digestion of the intact protein arise or with Help from genetic engineering. The serum albumin preferably has the primary structure of human serum albumin.

Serum albumin has the beneficial one Ability to selectively bind endotoxins so that a selective Allows detection of endotoxins will, even if the sample is additional Contains exotoxins.

In a preferred embodiment the method is serum albumin via one or more carboxyl groups immobilized, the immobilization preferably via a covalent bond exists. about Carboxyl groups immobilized on polymer beads human serum albumin binds LPS from healthy plasma by a factor of 2.5 better than over Amino group immobilized human serum albumin (HSA). This increase in affinity for LPS is based et al on the shift of the isoelectric point as a result the immobilization of (human) serum albumin via carboxyl groups.

The preferred separating elements are polymer beads coated with serum albumin, preferably acyl resin beads, as described in the patent EP-B 0 858 831 described, to which full reference is made. The serum albumin is preferably covalently immobilized.

The separating element preferably has a capacity more than 1.5 ng LPS per ml separating element, preferably more than 2 ng / ml, especially of about 2.5 ng / ml.

In a preferred embodiment of the method according to the invention is the carrier material porous and / or dispersible. Suitable as a carrier material all substances on which proteins can be immobilized, such as Filter papers or filter fleeces, preferably containing organic fibers, or polymer beads. Preferred carrier materials are organic polymer particles, in particular polyacrylic beads, with a (mean) diameter of preferably 10 to 500 μm, particularly preferably from 100 to 300 μm, especially of about 200 microns that preferably porous are. With the carrier materials can they are also gels and membranes. The porosity is preferred chosen so that the Oberflächenvergrößererung due to the porosity at least is about 500, especially about 1000.

Plastics in particular come as a carrier material in question, namely Polyacrylates, polymethacrylates, polysulfones, polyethersulfones, which wear amines or amides as a functional group. Are also next to it as a carrier material Cellulose, cellulose esters, aggarose, polytetrafluoroethylene, polycarbonate, or glass fibers, ceramics or metal. On metal Immobilize proteins with thiol groups, similar immobilization methods exist for the other carrier materials mentioned.

As already mentioned, the carrier material itself With. the endotoxin interact and the function of a filter or molecular sieve. The Pore sizes one Filters are usually 0.2 to 20 μm. To hire the desired one conditions of filtrate speed and separability can be with pressure filtration be worked. Depending on the viscosity of the fluid and the type and Amount of the expected toxin is preferred with pore sizes of 1 to 10 μm, worked in particular from 3 to 8 μm. With liquids filters with larger pores have also proven their worth, whereby in any case also the particle size of the endotoxin to be expected to consider is. The filter effect of the carrier material supports in this case the endotoxin-retaining Properties of the on the substrate Binding protein.

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. In particular, in cases in which the carrier material is dispersible in the liquid, separation by the action of gravity is appropriate. This can be done by sedimentation or centrifugation. Otherwise, the contacting according to step (b) and separation according to step (c) can also take place in one process step gene, for example, by passing the liquid in the manner of affinity chromatography through the separating elements or through the separating element, the separating element or the separating elements representing the affinity column. In a preferred embodiment of the method according to the invention, 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 Na ⁺ -, K ⁺ -, Cal ⁺ -, Mg ^{2 +} -, Cl ^- - and HCO ^- ₃ ions (e.g. Na ⁺ 134; K ⁺ 4; Ca ^{2 +} 1.75; Mg ^{2 +} 0.5; Cl ^- 106.5; HCO ₃ ^- 36; concentration in mmol / l).

• (d1) Durchführung eines Vollblut-Inkubationsverfahren mit dem Trennelement oder den Trennelementen, wobei entsprechend der Menge des in Schritt (b) gebundenen Endotoxins Mediatoren gebildet werden;
• (d2) Bestimmung Menge eines oder mehrerer der in Schritt (c1) gebildeten

In 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 method in which step (d) comprises the following steps:

• (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);
• (d2) Determining the amount of one or more of those formed in step (c1)

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. Mediators include IL-1, especially IL-1β, IL-6, tumor necrosis factor (TNF), prostaglandin E ₂ , NO and neopterin.

Whole blood incubation methods are understood in the context of the present invention to be methods as described in EP-A-0 741 294 and or EP-A-0 851 231 and which are the content of this description by this express reference. For the rest, reference is made to the following passages: Fennrich (1999) ALTEX 16, 146-149; Hartung, T. and Wendel, A. (1995) ALTEX 12, 70-75; Hartung, T. and Wendel, A. (1996), In Vitro Toxicology 9, 953-359; Fennrich, S. et al. (1998) ALTEX 15, 123-128.

• – Bestimmung mit Hilfe des LAL-Tests, wobei durch die erfindungsgemäß durchgeführte selektive Abtrennung der Endotoxine die vorstehend genannten Störeinflüsse vermieden werden können.
• – Bestimmung mit Hilfe von Monocytenzellkulturen. Hierbei werden nach der erfindungsgemäß durchgeführten selektiven Abtrennung der Endotoxine Monocytenzellkulturen mit dem zur Abtrennung verwendeten Bindungsprotein, das zum Beispiel auf Beads immobilisiert sein kann, in Kontakt gebracht. Die Monocytenaktivierung wird mit Antikörpern, die gegen Oberflächenproteine der Zellen gerichtet sind, im Durchflußcytometer oder mit ELISA bestimmt.
• – Markierung der Endotoxine auf der Oberfläche der Trennelemente durch geeignete Bindungsproteine. Es können beispielsweise BPI, LBP, CD14, Faktor C, Serumalbumin, A1-Adensosinrezeptor, EGFP, SAP oder Antikörper markiert werden (z.B. mit einer fluoreszierenden oder radioaktiven Markierung) und mit dem zur Abtrennung verwendeten immobilisierten Bindungsprotein in Kontakt gebracht werden, wobei eine Markierung der Endotoxine auf der Oberfläche der Trennelemente erfolgt. Nach Entfernung der markierten Bindungsproteine, die nicht Endotoxin gebunden haben, kann die Endotoxinmenge mit Hilfe der Markierungsintensität bestimmt werden, was z.B. durch ein Cytometer oder durch ein Fluoreszenzmeßgerät erfolgen kann.

In general, the amount of endotoxin that 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 methods, which are listed as examples:

• - Determination with the aid of the LAL test, the above-mentioned interferences being able to be avoided by the selective separation of the endotoxins carried out according to the invention.
• - Determination using monocyte cell cultures. Here, after the selective separation of the endotoxins according to the invention, monocyte cell cultures are brought into contact with the binding protein used for the separation, which may be immobilized on beads, for example. Monocyte activation is determined with antibodies directed against surface proteins of the cells in a flow cytometer or with ELISA.
• - Labeling of the endotoxins on the surface of the separating elements by means of 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 label) and brought into contact with the immobilized binding protein used for the separation, a label the endotoxins occur on the surface of the separating elements. After removal of the labeled binding proteins that have not bound endotoxin, the amount of endotoxin can be determined with the aid of the labeling intensity, which can be done, for example, by a cytometer or by a fluorescence measuring device.

Determining the amount of endotoxin a separation of endotoxin and the separating elements and possibly preceded by the binding proteins, in which case the Determination of the separated endotoxin then, for example, by cell activation (for example using the monocyte cultures mentioned above) or with labeled binding proteins (see the previous section) or with antibodies or possibly recombinant antibody fragments can be done competitively or in a sandwich arrangement.

The invention further relates to a flow-through container ( 1 ), comprising feed and outlet opening and one or more separating elements arranged in the flow area between the feed and outlet opening ( 3 ), which consist of a carrier material on which a binding protein for endotoxins is immobilized.

The liquid flows through the flow-through container to separate the endotoxins, the separating elements and the endotoxin bound to them being retained. The separating elements are detachably or non-detachably arranged in the flow-through container. In the case of separating elements which are detachably connected to the flow-through container, it has proven useful if 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. in particular Especially when using liquid media such as blood or blood components, a detachable arrangement of separating elements with a carrier material (possibly based on an exchange resin) has proven to be advantageous, since after the absorption capacity of the separating elements has been exhausted, the flow-through device for incubation in an incubation vessel ( and if necessary for regeneration) can be removed. 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 mainly 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.

Filtration speed and - amount of liquid flow can be based on the principle of pressure filtration by pumping or by Sucking and e.g. with liquid by using gravity through the flow tank in Dependence on the cross-sectional area and the layer or filter cake thickness of the separating element is predetermined become. So even with a flowable medium below the Limit of detection of lying or extremely low endotoxin concentration on the way to enrichment at all still enables endotoxin detection or reliable detection.

By measuring the flow rate at quantitative separation of the endotoxins by binding to the binding protein (if necessary in connection with micro or ultrafiltration) Concentration of endotoxin in the liquid, e.g. in the blood or Serum to be determined.

Under flow tank in the present sense e.g. also understood two-chamber collection containers, whose two chambers are separated by a separating element. You can go after Feeding a chamber mainly for the examination of liquids are used, for example using gravity as a flow aid. Accordingly, the method also relates to a flow-through container with at least one two openings, a feed opening and an outlet opening as well as solvable with it or unsolvable connected separating elements for the separation of endotoxins, wherein the separator in the flow area is arranged between the feed and the outlet opening. Preferably the separating element is essentially vertical to the main flow direction and / or so arranged that a Forced flow of the separating element, e.g. by extending the Separator essentially over the entire cross-sectional area of the flow container in the area of the seat of the separating element. In particular, the flow container in the respective opening area (Feed opening, outlet opening) Sealing elements which have a toxin-impermeable closure, e.g. ensure with caps or other lid closures. To close the openings are the openings adapted, optionally provided with sealing elements cover. A particular advantage is that the endotoxins are separated and the whole blood incubation procedure is carried out in the same container can, so handling problems, those when changing containers could arise omitted. It is readily possible to start with the microbiological Separate toxins from the fluid and then through the flow container intended for this To close the lid and to be sent to microbiological laboratories for examination.

To test for endotoxins, 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. Finally, 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-1β , 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. The use of mammalian blood is preferred, especially that of human whole blood. The leukocytes are thus in their natural composition and environment. This applies in particular to the monocytes, which mainly release the mediators. At the same time, 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. A dilution of the whole blood preparation, for example with isotonic solutions or with a cell culture medium, for example with RMPI 1640 or with a physiological saline solution (20% dilution) is advantageous. As a precaution, 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 a period of about 2 to 24 hours. When carrying out the whole blood incubation, care must be taken to avoid disruptive contamination of the device and reagents.

Preparations from gram-negative and gram-positive bacteria, such as endotoxin and lipoteichoic acid Negative control e.g. pyrogen-free physiological saline is used become.

After the whole blood incubation procedure becomes the body's own primary reaction the formation of mediators for the administration of endotoxin was used for the investigation. There are all blood components that may be needed for an interaction the toxins with leukocytes are necessary, e.g. LPS binding protein LBP, bactericidal permeability-increasing protein BPI, soluble CD14, Definsine, etc. The whole blood incubation procedure can also with the whole blood of those affected with a liquid that comes from the individual Environment originated, carried out become. So when applying a defined amount of endotoxin the individual sensitivity determined and a hypersensitive Patient to be identified.

For serial tests or comparative tests over certain periods, the use of frozen blood or of collective frozen blood in the form of standardized blood unit doses has proven itself for reasons of standardizability ( EP-A 0 951 231 ).

To carry out the method according to the invention, 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.

The figures show:

1 shows a comparison of the IL-1 measured values which were achieved with untreated sepsis plasma and with a protease-incubated sepsis plasma. 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-1. The error bars refer to the standard deviations for triple determinations.

2 shows a comparison of the IL-6 measured values, which were obtained 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 the 1 , The error bars refer to the standard deviations for triple determinations.

3 shows an example of a flow container according to the invention. The through-flow container shown, which is about 5 to 10 times larger than preferred containers 1 has a substantially cylindrical shell 2 , a separating element 3 between the feed opening located below and the outlet opening visible above, and as a means for the toxin-impermeable closure, the lids 4 and 5 on. The direction of flow for the fluid is essentially axially parallel to the cylinder axis. The separating element, which can be in one or more pieces, is in 3 made in one piece, which facilitates the removal of the separating element.

The present invention will now with respect to examples explains which in no way limits the subject matter of the invention represent.

Examples

Preparation of the Matisse beads

5 ml of Matisse beads (Fresenius Hemocare) transferred to 50 ml Falcon tubes and overlaid with 0.9% NaCl to a total volume of 50 ml. Occasionally The beads are swirled for about 10 minutes.

By centrifugation (2 minutes 3000 rpm Heraeaus varifuge, ~ 2100 g) the beads are sedimented and the supernatant completely removed.

The beads are now covered with a priming solution (Fresenius Hemocare) and with occasional swiveling at least 10 Minutes equilibrated. After centrifugation again (2 minutes 3000 rpm Heraeus varifuge, ~ 2100 g), is obtained by removing a part of the supernatant the total volume reduced to 10 ml. Then 2% DMSO (Sigma, "cell culture tested") are added and mixed.

Contacting the Matisse beads with plasma samples containing different amounts of endotoxin

100 μl suspension of the prepared beads are mixed with 100 μl plasma samples in 2 ml increments village vessel mixed. The beads are either aliquoted with a cut pipette tip (sterile & pyrogen-free) or with an Eppendorf Multipette (also sterile and pyrogen-free with a cut tip).

Spiked with endotoxin plasma samples

Plasma from 5 donors (A-E) will by centrifugation (10 '~ 2100 g) from Li-heparin blood (Sarstedt, "Monovette"). A part of these are stored in aliquots at -80 ° C and used as zero controls.

The rest will be in equal parts mixed and mixed with LPS from E. coli 0-113 (0, 12.5, 25, 50, 100 and 200 pg / ml) incubated for 2 hours at room temperature. In this Time should be solved LPS of binding proteins can be bound as this in physiological Way also happens. The aliquots are stored at -80 ° C.

Probenvorbreitung

The plasma samples are on the laboratory bench thawed.

Components of the complement system inactivated by incubation at 56 ° C for 30 minutes. Then will Vortexed for 1 minute and centrifuged (2 minutes 6000 rpm, Heraeus pico or fresco, corresponds to ~ 2100 g). 100 μl of supernatant are transferred to a 2 ml Eppendorf tube.

If 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 in the 1 and 2 shown. 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.

Claims (22)

Procedure for determining the level of endotoxin in liquids, comprehensive following steps (b) contacting the liquid with one or more separating element (s) comprising a carrier material, on that a binding protein for Endotoxins are immobilized; (c) separation of liquid and separator (s); and (d) determining the amount of endotoxin, which was bound to the binding protein in step (b). The method of claim 1, wherein the liquid incubated with a protease in step (a) before step (b) becomes. The method of claim 2, wherein in step (a) the Protease is inactivated after incubation. The method of claim 2 or 3, wherein it is the protease is Proteinase K. Method according to one of claims 1 to 4, wherein the separating elements step (c) with an isotonic solution or with a solution containing Na ⁺ -, K ⁺ -, Ca ^{2 +} -, Mg ^{2 +} -, Cl ^- - and HCO ₃ ^- -Ions to be washed. Method according to one of claims 1 to 5, wherein the liquid a biological fluid is. The method of claim 6, wherein the biological liquid Blood is. The method of claim 6, wherein the biological liquid Is sepsis plasma. Method according to one of claims 1 to 6, wherein step (d) includes the following steps (D1) execution a whole blood incubation process with the separating element or Separating elements, according to the amount of bound in step (b) Endotoxin mediators are formed; (d2) Determination of quantity one or more of the mediators formed in step (c1). A method according to any one of claims 1 to 9, wherein the binding protein Serum albumin or a fragment thereof. The method of claim 10, wherein the serum albumin the primary structure of human serum albumin. A method according to any one of claims 1 to 9, wherein the protein an antibody or is a fragment of it. A method according to any one of claims 1 to 12, wherein the binding protein to the carrier material is covalently bound. Method according to one of claims 1 to 13, wherein the binding protein via a or more of its carboxyl groups is bound to the carrier material. Method according to one of claims 1 to 14, wherein the carrier material is porous. Method according to one of claims 1 to 15, wherein the carrier material dispersible is. Method according to one of claims 1 to 16, wherein the carrier material Filter papers or filter fleeces, preferably with an organic content Fibers, or polymer beads, foams, Includes gels or membranes. Method according to one of claims 1 to 17, wherein the carrier material Polyacrylates, polymethacrylates, polysulfones, polyethersulfones, cellulose, cellulose esters, Agarose, polytetrafluoroethylene, polycarbonate, polyacrylates, glass fibers or ceramic. Method according to one of claims 9 to 18, wherein the mediators quantified in step (d2) are interleukin (IL) IL-1, in particular IL-1β, or IL-6, tumor necrosis factor (TNF) and / or prostaglandin E ₂ . A method according to any one of claims 9 to 19, wherein the mediator or the mediators in step (c2) are determined using an ELISA or RIA become. Flow tank ( 1 ) for a method according to one of claims 1 to 16, comprising feed and outlet opening and one or more separating elements arranged between the feed and outlet opening in the throughflow area ( 3 ), which comprise a carrier material on which a binding protein for endotoxins is immobilized. Use of separating elements that are a carrier material on which a binding protein for endotoxins is immobilized is in a method for determining the content of endotoxins in liquids.