JP2002533679A - Diagnosis of sepsis using the LPS-binding portion of alkaline phosphatase - Google Patents

Abstract

  (57) [Summary] Diagnosis of signs of endotoxemia or sepsis due to Gram-negative bacterial infection, comprising monitoring the degree of AP occupancy of LPS binding sites on alkaline phosphatase in tissue or fluid samples from patients. As such, the degree of AP occupancy is related to the presence or absence of Gram-negative bacterial infection. Alkaline phosphatase LPS binding site binding ligand and instructions for performing the claimed assay, and optionally further components necessary for such an assay, e.g., detectable markers, buffers, vessels and comparisons. Kit comprising data relating to a sample or a data chart, such as a standard curve or relevant data for alkaline phosphatase values. A method of treating endotoxemia or sepsis, comprising administering the LPS binding site of alkaline phosphatase in a pharmaceutically effective amount in a systemically acceptable form, provided that the ligand is alkaline phosphatase. However, it is not a derivative of alkaline phosphatase having dephosphorylation activity. A method of removing LPS from a tissue or fluid, comprising contacting the LPS binding site of alkaline phosphatase with the tissue or fluid to be treated, and subsequently binding the LPS binding site to the LPS present in the fluid or tissue. Comprising separating the binding site and the tissue or fluid, except that the ligand is neither alkaline phosphatase nor a derivative of alkaline phosphatase having dephosphorylation activity. A method of purifying AP itself from tissues or body fluids or other biological production systems using a compound having an LPS binding site for alkaline phosphatase, such as LPS, lipid A or other ligands as claimed in the claims.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] Alkaline phosphatase (AP) is a glycosyl-phosphatidyl-inositol (GPI) -anchor on the plasma membrane of many types of cells including endothelial and epithelial cells, osteoblasts, fibroblasts and hepatocytes. Is an ecto-enzyme bound via This enzyme is also present in the special granula of neutrophils. As a result, many organs contain abundant alkaline phosphatase activity. In addition, enzyme activity is also present in plasma.

[0002] Elevated AP activity in the blood is found, for example, when embryogenesis and bone formation in childhood are evident (3). Liver diseases such as cholestasis are also characterized by high serum AP levels and enhanced expression of AP activity on the plasma membrane of hepatocytes. Therefore, this enzyme is generally accepted as a serum marker for the diagnosis of this disease (4).
Following activation of this type of cells, release from neutrophil granules may also contribute to the enhanced serum AP activity found in the inflammatory process. The physiological significance of this enzyme induction in liver disease and inflammatory processes is unknown. The role of AP in the process of bone formation has been demonstrated in vitro and in vivo.

[0003] Recent studies have demonstrated that AP can dephosphorylate endotoxin or lipopolysaccharide (LPS). LPS is a component of the cell wall of Gram-negative bacteria and is very toxic to humans and most animal species. This induces a severe inflammatory response, which may be life-threatening if it occurs in the bloodstream. This disease is
Sepsis "or" systemic inflammatory response syn
drome: SIRS), and about 35% of all cases die. LPS is associated with increased intestinal permeability during liver disease or, for example, during or after an ischemic event.
It can enter the bloodstream after a bacterial infection. Enterobacteriaceae Escherichia coli (E
coli), and high levels of LPS are produced in the gastrointestinal tract by production by many other bacterial species. Since LPS is hardly found in plasma, the gastrointestinal tract clearly has an effective barrier. This barrier is characterized by high AP expression, similar to the blood brain barrier where neuroendothelial cells express high AP activity.

[0004] Enhanced intestinal permeability to LPS has been shown in patients with gastrointestinal perforation, appendicitis or peritonitis, but with injurious signs such as burns, major gastrointestinal surgery and sepsis. Later it was shown in patients who overdose alcohol. Most of these latter cases are characterized by reperfusion injury to the intestinal wall after ischemia and side-by-side simultaneous release of inflammatory mediators that affect vascular permeability in the intestine, which is the intestinal tract of LPS. Causes leak into pulse. Under normal conditions, the liver plays an important role in removing LPS from the circulation, especially in this regard Kupffer cells.
However, in many liver diseases, this function is impaired, which can in turn lead to endotoxemia.

[0005] The phosphate group in the lipid A portion of LPS determines some biological responses to this bacterial product. The lipid A moiety is a relatively constant moiety in other highly variable molecules. In addition to the various phosphate groups in the polysaccharide tail, generally two phosphate groups are present in the lipid A moiety. The phosphate group in the lipid A moiety is most important, while the phosphate group in the polysaccharide tail does not affect the inflammatory response to LPS. Mono-phosphoryl lipid A is a weak inducer of macrophage response and non-toxic in chickens and rabbits, whereas natural di-phosphoryl lipid A is a potent activator of macrophages and Lethal to.

[0006] Dephosphorylation of LPS by alkaline phosphatase therefore becomes important in vivo, and this may create new insights into the physiological role of this enzyme. This new insight can have many therapeutic implications. PCT / NL94 / 00189 describes the therapeutic application of alkaline phosphatase to treat all kinds of complications induced by LPS. DESCRIPTION OF THE INVENTION Briefly, in the examples, serum AP in patients with established Gram-negative sepsis
Although activity is reduced, serum AP activity is shown to be unaffected or slightly enhanced in patients with established Gram-positive sepsis. This new finding,
This can be explained by the binding of LPS to the AP enzyme as shown herein. This association is AP
, Which can either shorten the half-life in serum or directly affect enzyme activity. Based on all such considerations, and considering new insights into the role of AP in vivo as an LPS detoxifying enzyme (described in PCT / NL94 / 00189), AP
It has a binding site for S, and it can be concluded that binding of this ligand to the enzyme has important implications.

[0007] Early detection of endotoxemia in patients with liver failure, severe burns or other injuries (eg, after organ transplantation) is important but somewhat complex. Measure free LPS in serum,
And a reliable and sensitive method that also takes into account the endotoxin detoxification potential of serum is not yet available. Furthermore, the method of determining the amount of LPS in serum does not give any information on the situation described above. This is particularly relevant in terms of the short half-life of LPS in the bloodstream. Detection methods to determine that LPS binds to the AP itself are possible, and such methods can be used to distinguish between Gram-positive and Gram-negative sepsis or to assess disease severity. Can be applied for the purpose of diagnosis. The extent of binding site occupancy appears to be an early marker for this disease. That is, valuable new diagnostic tools are described herein. Furthermore, based on the consideration that AP has a specific binding domain for LPS, a novel LPS binding system that binds to this domain can be developed. Such domains can remove LPS from fluids or tissues. Ligands that interact with AP can also be used to purify AP from crude or partially purified biological sources (eg, milk, culture media, tissue samples). Can also be used for purpose.

[0008] Thus, the present invention describes the use of ligands for the LPS-binding site of alkaline phosphatase. Examples of such ligands include naturally occurring forms or Fa
Mono- or polyclonal antibodies against this ligand, either as b-fragments, single chains or other modified immunoglobulins. In addition, labeled LPS, lipid A, derivatives of both substances, or products mimicking the three-dimensional structure of lipid A can be used as ligands for the LPS binding site in the AP enzyme. They can be designed based on computer modeling. This product can be produced synthetically using chemical means. There are technical literatures detailing the production of lipid A-like structures using chemical means (1, 2). The use of recombinant DNA techniques to engineer the required structure is possible, as is the chemical modification of lipid A-like structures. Measurement of the concentration of free LPS-binding domain on alkaline phosphatase for diagnostic purposes can be performed using such a product. This concentration is then correlated with the total concentration of the AP enzyme or the total enzyme activity in the biological fluid, thereby providing further information regarding the presence of LPS in the biological fluid and the ability to detoxify LPS.

[0009] Thus, the method according to the invention comprises the diagnosis of signs of endotoxemia or sepsis due to Gram-negative bacterial infection, said method comprising LPS binding on alkaline phosphatase in a sample of tissue or fluid from a patient. Monitoring the extent of AP occupancy of the site, wherein the extent of AP occupancy is associated with the presence or absence of a Gram-negative bacterial infection.
Specifically, such a method has a lower AP occupancy of the LPS binding site on alkaline phosphatase in the sample than in an equivalent sample of individuals without Gram-negative infection. As an example, the extent of AP occupancy of the LPS binding site on alkaline phosphatase in a sample or tissue or fluid from a patient is monitored over a period of time, where a decrease in the extent of AP occupancy indicates a Gram-negative bacterial infection. In such cases, the degree of AP occupancy of the LPS binding site on alkaline phosphatase in the sample is determined, and when the onset of a decrease in the degree of AP occupancy is confirmed, this is indicative of a gram-negative bacterial infection. Also, depending on the measured value, the degree of AP occupancy of the LPS binding site on alkaline phosphatase can indicate a mixed infection of Gram-negative and Gram-positive bacteria. This test can show signs of sepsis, but is at risk of sepsis, such as at risk of sepsis, such as genetic deletion or rheumatoid arthritis, cerebral edema, Alheimer, ischemic heart disease Alternatively, patients with structurally low AP levels in tissues or blood due to chronic conditions such as inflammatory bowel disease can be identified. In addition, this test can be used to assess (potential) infection in pregnant women. Such a potential infection may induce HELLP syndrome or pre-eclampsia in the mother, or affect the condition of the child by causing fetal intrauterine growth retardation, or even induce preterm birth.
As a result, the tests described herein can be used to prevent neonatal illness. In the method according to the invention, the sample to be measured is subjected to binding to a ligand for the LPS binding site on alkaline phosphatase, and the degree of binding of the ligand can subsequently be determined. Any ligand with the required specificity can be used. The choice will depend on the type of sample being tested and the degree of sensitivity to be achieved. LPS, which can select appropriate ligands with very specificity
Lipid A, LPS binding site antibody to alkaline phosphatase, Fab fragment capable of binding LPS binding site on alkaline phosphatase, single chain fragment of immunoglobulin capable of binding LPS binding site on alkaline phosphatase. Alternatively, other ligands developed based on, for example, computer modeling of the binding system between alkaline phosphatase and LPS and lipid A can also be used. The ligand may be a naturally occurring molecule or a synthetic molecule. Synthetic molecules can be produced chemically or biochemically, ie, using recombinant DNA techniques. Preferably, the method according to the invention employs an LPS binding site binding ligand having at least an affinity for the LPS binding site of the alkaline phosphatase of LPS. The method according to the invention may also employ an LPS binding site binding ligand having at least an affinity for the LPS binding site of alkaline phosphatase of lipid A. The degree of specificity and affinity required can be confirmed using standard tests to compare the specificity and affinity of the compound for the substrate. In the method according to the invention,
The degree of AP occupancy of the LPS binding site on alkaline phosphatase can be determined by assessing the dephosphorylating ability of alkaline phosphatase in the sample. In such a method, the ratio of dephosphorylated alkaline phosphatase to non-phosphorylated alkaline phosphatase is determined. This ratio uses the value obtained by assessing total alkaline phosphatase activity using a biochemical method to measure dephosphorylation activity and by assessing the total amount of alkaline phosphatase using antibodies, and It can be determined by calculating the ratio of such values. Numerous methods are available to those skilled in the art to make such an assessment. We give references 5 and 6 as examples. The contents of which are incorporated herein by reference. It will be readily apparent that other alternative known methods can be applied to reach the required data.

[0010] The sample used in the method according to the invention may be a sample from a patient. Such a sample may be from blood or tissue. The sample must be treated so that any alkaline phosphatase present is not removed prior to the assay. Such a sample can be selected from the group consisting of blood and tissue. The blood sample can be, for example, serum. Tissue samples are non-bone because alkaline phosphatase levels are generally high in bone. Suitable tissues can be selected, for example, from liver and intestine. Biopsy tissue sample (biopt
) Can be collected in a manner known per se. Blood and serum samples may also be prepared in a customary manner.

[0011] The method according to the invention can be performed using a sample from a patient without cholestasis. The method according to the invention can also be carried out using samples from patients with cholestasis. The value determined for the degree of LPS binding can be compared to standard values for healthy and / or sick patients to ensure a correct diagnosis. Alternatively, the method according to the invention can comprise performing the above-described assay in any of the embodiments described, and furthermore in patients with other diseases associated with elevated alkaline phosphatase activity, such as cholestasis. It can also comprise an assay of the sample from which it is derived. This further assay should preferably employ a method that avoids measuring alkaline phosphatase levels.
Numerous documents describing alternative assays for such alkaline phosphatase-related diseases are available in the literature covering the disease, the contents of which are incorporated herein by reference. A suitable embodiment of the method according to the invention in which a further assay is performed is when the alkaline phosphatase assay according to the invention shows no reduction in AP occupancy of the LPS binding site of alkaline phosphatase. The method according to any of the aspects of the invention suitably comprises a method wherein the sample is obtained from an individual at risk of infection by Gram-negative bacteria. The sample can be taken, for example, from an individual before and / or after an injury. The sample can also be taken immediately after the injury. Very interesting injury cases relate to surgery, burns and ischemic injuries. Since hospitals are a well-known source of infection, suitably the method according to the invention may comprise the evaluation of samples taken from hospitalized individuals.

In the method according to the invention, the sample can be taken multiple times during the period. The data is compared to each other, which can reveal the level of AP occupancy during the period. However, this value can also be compared to a standard value for a healthy or sick individual. This value can be correlated with various characteristics of the patient, such as gender, age, weight, the type of tissue or fluid being tested. The method thus determines whether the degree of AP occupancy indicates endotoxemia or sepsis. The method according to the invention comprises taking a sample as long as the individual is at risk for infection, ie during hospitalization or after injury recovery.

The present invention also envisages a kit for performing the diagnostic method according to the present invention. Such a kit provides an alkaline phosphatase LP for performing an assay according to the above embodiments.
May comprise an S binding site binding ligand and instructions for use. The kit may further comprise additional components necessary for such an assay, such as a detectable marker, a buffer, a container. Also suitable for addition to the kit are comparative samples or data charts, such as data relating to standard curves or relevant data for alkaline phosphatase values. Alkaline phosphatase LPS binding site binding ligand for performing an assay according to the above embodiments, and the following detectable markers, buffers, vessels, comparative samples, data charts, e.g., standard curves or alkaline phosphatase values required for such assays A kit comprising further components selected from the group consisting of data relating to the relevant data is also part of the invention. Contemplated ligands are described in the method aspects and claims and examples disclosed above.

The invention further encompasses a method of removing LPS from a tissue or fluid, the method comprising contacting the LPS binding site of alkaline phosphatase with the tissue or fluid to be treated,
Subsequently, separating the LPS binding site and the tissue or fluid after the LPS binding site binds to the LPS present in the fluid or tissue, provided that the ligand is alkaline phosphatase or alkaline phosphatase having dephosphorylation activity. Not a derivative. Thus, LPS can be removed from tissue or fluid. Obviously, the binding must be performed under conditions that do not damage the fluid or tissue being purified.
Such conditions will be readily apparent to one skilled in the art of working with such tissues or fluids. By way of example, the LPS-binding domain of the AP itself can be conjugated to a non-soluble carrier to remove LPS from a tissue or fluid. Suitable fluids for processing include cell-culture media, fluids used for perfusion or storage of organs,
Blood, milk or other therapeutic products administered to others. Ligands that bind to the LPS binding site of the AP may be used to purify the AP itself from tissues or body fluids or other biological production systems.

[0015] The invention also encompasses a method of treating endotoxemia or sepsis, which comprises administering the LPS binding site of alkaline phosphatase in a pharmaceutically effective amount in a systemically acceptable form. Wherein the ligand is neither alkaline phosphatase nor a derivative of alkaline phosphatase having dephosphorylation activity. Any customary dosage form is sufficient. The dosage regimen to be followed will depend, for example, on the condition, sex, and weight of the patient, and the physician will be able to apply the best regimen. The state of the LPS-binding domain itself or a derivative of alkaline phosphatase, which does not have dephosphorylating activity, may be used to therapeutically treat patients with systemic inflammatory response syndrome.
Alternatively, it can be applied to prevent endotoxemia in patients at risk for such complications. Binding of circulating LPS to this specific domain may result in the interaction of LPS with other endogenous receptors in serum and on cell membranes of endothelial cells or macrophages, such as LPS-binding proteins, CD-14 and scavenger receptors. Can be prevented. As a result, the inflammatory response based on LPS is reduced.

The invention also relates to other therapeutic and diagnostic applications of the ligands described in the description of the invention, and to the purification of AP itself using LPS or any of the AP-binding derivatives thereof, ie the other ligands mentioned. It covers the law. Experimental data Experiment I These reports prompted us to investigate the serum levels of AP during endotoxemia. AP is generally accepted as a marker of liver injury, and LPS is known to induce direct or joint damage to this organ. Increased serum AP activity in patients with sepsis was often shown, and was also expected in endotoxemic mice. However, E. coli (055: B5 strain, Sigma Chemical Co.)
Co.), USA, 2.5 mg / kg, bw) LPS from Balb / c mice (male, 20-25 g)
) At t = 0, a sharp and sharp decrease in serum AP activity was observed as compared to mice receiving saline (FIG. 1). A similar decrease is seen in E. coli (
ATCC 25922 (2 × 109 CFU) after intraperitoneal administration to mice (FIG. 1).

Evaluation of the clinical data of patients with recurrent endotoxemia also revealed a rapid primary sharp drop in serum AP activity, followed by a strong rise the next day. The first sharp drop in AP is thought to be caused by LPS. This reduction allows the remaining LPS to trigger its inflammatory effects, thereby activating the destructive response seen in septic patients. This primary response to a clinical decrease in AP has not been previously considered. Therefore, to enable rapid detection of sepsis, rapid detection of a decrease in AP levels is of paramount importance. The methods described herein allow for the measurement of such rapid kinetic changes in AP levels. The basis for this lies in the fact that free and thus fully active plasma AP is rapidly reduced by binding to LPS. Secondary elevations in serum AP levels seen in many patients may reflect liver injury, or interleukin-6 or other inflammation that attenuates the expression of AP activity as noted in recent studies May be induced by mediators. Experiment II We subsequently surveyed 64 patients in an intensive care unit suffering from sepsis.
The pathogen causing sepsis syndrome was identified in all these cases. Patients with endotoxemia (n = 16) were treated with patients with Gram-positive bacterial infection lacking LPS in serum (n = 30).
). Patients had various potential illnesses, except for those with cholestasis associated with elevated AP excretion. Blood samples were routinely assayed for AP during hospitalization. Changes in serum AP levels were applied on day 0, the day of admission to the intensive care unit of the hospital. We calculated changes in serum AP activity for day 0 before and after the blood sample became positive for bacteria or bacterial products (LPS). In such experiments, a decrease in serum AP activity proved to be an early marker of sepsis: the decrease preceded bacteremia (FIG. 2). Further data indicate that patients with established Gram-negative bacterial infections show a decrease in serum AP activity (mean slope = -21.5), while patients with overt Gram-positive bacterial sepsis have a slight increase over day 0 ( Mean slope = 10.9), indicating that Gram-negative bacterial infections can be distinguished from Gram-positive bacterial infections.
Note that on day zero (the day of admission to the intensive care unit), the actual value of serum AP was already affected. Patients with mixed infections (gram-positive and gram-negative,
n = 18) showed an intermediate change in serum AP levels (mean slope = 0.6). Gram-
The rapid diagnosis of positive versus Gram-negative bacterial infections is particularly important in emergency situations, where it is essential that effective antimicrobial treatment be started immediately. To date, this diagnosis is only possible after successful culture of the pathogen, which usually takes two days. Experiment III We hypothesized that the decrease in serum AP levels was caused by increased turnover of the enzyme. We therefore investigated whether direct binding between LPS and the AP enzyme had occurred and whether this binding affected the enzyme activity. Placental AP (6.7 U / ml) isolated from fresh human placenta was purified from E. coli (055: B5 strain, Sigma Chemical Co .; 1 mg / kg) LPS. For 10 minutes at room temperature. Subsequently, the AP was subjected to FPLC analysis. The FPLC was equipped with an anion-exchange column (mono-Q HR 5/5, Pharmacia, Sweden) and a UV detector (280 nm). P1AP is a 0.2M Trial containing MgCl2
Injected in s / HCl buffer (pH 7.4) (with or without LPS). Elution was with Tris / HCl buffer (pH 7.4) at a flow rate of 0.5 ml / min and NaC in the range of 0.0 to 0.45 M
Performed on a gradient (30 minutes). The retention time was measured. The retention time of the p1AP sample without LPS was 20 minutes, while the retention time after incubation with LPS shifted to 18 minutes. The short retention time of the p1AP sample containing LPS reflects the reduced negative charge of the complex, indicating binding of LPS to the AP enzyme, ie, neutralization of the charge in the protein. EXPERIMENT IV To assess whether inhibition of the product, which accounts for the reduction in serum AP activity, occurred, we tested the effect of non-phosphorylated lipid A, the end product of the enzymatic reaction of AP activity. Formalin / macrodex fixed cryopreserved sections (4 um) of rat intestine, kidney and placenta are incubated in LPS, MgCl2 and lead nitrate (Pb2NO3) in 0.2 M Tris / maleic acid buffer (pH 7.4) Phosphatase activity was demonstrated according to standard procedures (K. Poelstra et al. Lab. Invest 76: 319,1997, Am J
.Pathol. 151: 1163, 1997).

As indicated earlier, cryopreserved sections of rat intestine and kidney expressed abundant LPS phosphatase activity. LPS-dephosphorylation activity in rat placenta was abundant but low. The localization of this enzyme activity was similar to the distribution of AP activity in all three organs. However, such cryopreserved sections and lipid A (Salmonella Minnesota)
ella minnesota) R595, List Biological Laboratories (List Biolo)
gical Laboratories Inc.), a monophosphoryl lipid A from Campbell, USA; 1 mg / ml) strongly attenuated intestinal and renal phosphatase activity when LPS was later added to the substrate. . No phosphatase activity was detectable in placenta pre-incubated with lipid A. Such data revealed that upon binding to LPS, AP was not temporarily utilized for further LPS dephosphorylation. This can be explained by the relatively high affinity of LPS for the AP binding site and vice versa. References: LPS Chemical Derivatives of Lipid A Qureshi N; Javis B; Takayama K; Sattar N; Hofman J; Stutz P; natural and synthetic LPS and lipid homologs or partial structures that antagonize or induce tolerance to LPS (Natural and synthetic LPS and lipid a analogs) or partial structu
res that antagonize or induce tolerance to LPS.). Prog Clin Biol Res 199
8; 397: 289-300 2. Brandenburg K; Kusumoto S; Seydel U; Conformational studies of synthetic lipid A homologues and partial structures by infrared spectroscopy
pid A analogues and partial structures by infrared spectroscopy.) Bioch
im Biophys Acta. 1997: 1329; 183-201 Conditions for increasing serum AP levels. Ross PD; Knowlton W: Rapid bone loss is associated with increased levels of biochemica
l markers.). J Bone Miner Res. 1998: 13; 297-302 4. Dourakis SP; Mayroyannis C; Alexopoulou A; Hadziyannis SJ; Prolonged cholestatic jaundice aft after endoscopic retrograde cholangiography
er endoscopic retrograde cholangiography.). Hepatogastroenterology 1997:
44; 667-80 Method for measuring serum AP Peake MJ; Pejakovic M; White GH; Quantitative method for measuring alkaline phosphatase isozyme activity in serum: prediction of intestinal constituents (Quantitative method for de
rermining serum alkaline phosohatase isoenzyme activity: estimation of in
testinal component.). J. Clin pathol 1988: 41; 202-206 6. McComb RB; Bowers GN; Posen S: Alkaline phosphatase, Plenum Publishing (P
lenum Press), New York and London, 1979: Chapter 7, pages 289-372 (Measurement of alkaline phosohatase activ).
ity.).

[Brief description of the drawings]

FIG. 1. Alkaline phosphatase levels in mouse serum after administration of saline (iv), LPS (iv) or E. coli (ip) at t = 0 (n = 4 per group)
. Diamonds represent saline, squares represent E. coli, and triangles represent LPS. t is set in time along the x-axis, and AP activity is expressed in U / L on the y-axis.

FIG. 2. Before and after a patient's blood sample is positive for bacteria or bacterial products (LPS).
Changes in serum AP levels for days admitted to the intensive care unit. Patients were divided into two groups: patients with established Gram-negative bacterial infection (n = 16) and patients with established Gram-positive bacterial infection (n = 30). Patients with mixed bacterial infection (n = 18) or SIRS without bacteremia identification are not shown here. Dark symbols indicate gram negative. Light symbols indicate gram positive.

[Procedural Amendment] Submission of translation of Article 34 Amendment

[Submission date] January 19, 2001 (2001.1.19)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Contents of correction]

[Claims]

25. A method of removing LPS from a tissue or fluid, comprising contacting a compound having a specific binding domain that blocks the LPS of alkaline phosphatase with the tissue or fluid to be treated, followed by contacting the compound with the fluid or tissue. LPS inside
Separating the compound and tissue or fluid after binding to the compound , provided that the compound is not an alkaline phosphatase or a derivative of alkaline phosphatase having dephosphorylating activity.

26. A method of treating endotoxemia or sepsis, said method administered in a form of LPS binding site of the alkaline phosphatase may be systemically acceptable in a pharmaceutically effective amount as activating compounds Wherein the active compound is neither an alkaline phosphatase nor a derivative of alkaline phosphatase having dephosphorylation activity.

27. A method of removing LPS from tissue or fluid, the method comprising contacting the tissue or fluid for treating a compound having a specific binding domain related LPS alkaline phosphatase, followed by compound fluid or tissue Separating the compound and the tissue or fluid after binding to the LPS present therein, provided that the compound is not alkaline phosphatase or a derivative of alkaline phosphatase having dephosphorylating activity.

28. A method for purifying AP from tissue or body fluids or other biological systems using a ligand that binds to the LPS binding site of AP.

29. ligand LPS, a lipid A or a ligand that binds to the LPS binding site of AP according to any of the preceding claims, The method of claim 28.

30. A therapeutic or preparation of a medicament for the diagnosis, as the active compound, LPS, lipid A or alkaline phosphatase, such as a ligand binding to the AP of the LPS binding site according to any preceding claim Use of compounds with LPS binding sites.

31. A endotoxemia or preparation of a medicament for the treatment or diagnosis of sepsis, as active compounds, LPS, lipid A or a ligand that binds to the AP LPS binding site according to any preceding claim LPS for alkaline phosphatase such as
Use of compounds with binding sites.

Claims32】 OrganizationsampleOr fluidsampleHow to remove LPS from the
Wherein the method comprises alkaline phosphataseSpecific binding domain for LPS
Compound havingTissue to treatsampleOr fluidsampleAnd then contact Compound After binding to LPS present in fluid or tissue samplesCompound
And organizationsampleOr fluidsampleThe method comprising separating

33. A method of removing LPS from a tissue sample or fluid sample, the method comprising contacting a tissue sample or fluid sample treating a compound having a specific binding domain related LPS alkaline phosphatase, followed by compound Although but comprising separating the compound and tissue sample or fluid sample after binding to LPS present in the fluid sample or tissue sample, provided that the compound is a derivative of alkaline phosphatase-transfer agent over peptidase having dephosphorylation activity at alkaline phosphatase Not the above method.

34. A method for purifying AP from a tissue sample, a body fluid sample, or a sample of another biological system using a ligand that binds to the LPS binding site of AP.

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE , KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW (72) Inventor Manintverd, Ali Jacob N.N. B Zuterdam Lahnbanypenhof 93 F-term (reference) 4C084 AA02 AA17 BA34 BA44 BA50 CA25 CA38 NA14 ZA51 ZB35 4C086 AA01 AA02 MA01 MA04 NA14 ZA51 ZB35

Claims (30)

[Claims] 1. A method of diagnosing signs of endotoxemia or sepsis due to Gram-negative bacteria, wherein the degree of AP occupancy of the LPS binding site on alkaline phosphatase in a sample of tissue or fluid from the patient is monitored. The AP
Such a method, wherein the degree of occupancy is associated with the presence or absence of a Gram-negative bacterial infection. 2. The AP of the LPS binding site on alkaline phosphatase in a sample
2. The method of claim 1, wherein the degree of occupancy is lower than an equivalent type of sample of an individual not infected with Gram-negative bacteria. 3. The degree of AP occupancy of the LPS binding site on alkaline phosphatase in a sample or tissue or fluid from the patient is monitored over a period of time, wherein a decrease in the degree of AP occupancy indicates a gram-negative bacterial infection. The method according to claim 1. 4. The AP of the LPS binding site on alkaline phosphatase in a sample
The method of any of the preceding claims, wherein the degree of occupancy is measured, and the onset of a decrease in the degree of AP occupancy is indicative of a gram-negative bacterial infection. 5. The degree of AP occupancy of the LPS binding site on alkaline phosphatase can also indicate a mixed or single infection of Gram-negative and Gram-positive bacteria.
A method according to any of the preceding claims. 6. The method according to any of the preceding claims, wherein the sample is subjected to binding of the LPS binding site on alkaline phosphatase with the ligand, and subsequently the extent of binding of the ligand is determined. 7. The ligand for an LPS binding site on alkaline phosphatase, wherein the ligand is a naturally occurring ligand, a chemically modified or genetically modified derivative of a natural LPS binding site binding substance, A method according to any preceding claim, wherein the method is selected from the group consisting of: 8. A sample comprising LPS, lipid A, an LPS-binding site antibody against alkaline phosphatase, and Fa having an LPS-binding site-binding ability on alkaline phosphatase.
b fragment, LPS on alkaline phosphatase selected from single chain fragments of immunoglobulin capable of binding to LPS binding site on alkaline phosphatase
A method according to any of the preceding claims, which is subjected to binding with a ligand for a binding site. 9. The method according to any of the preceding claims, wherein the LPS binding site binding ligand has at least an affinity for the LPS binding site of alkaline phosphatase of LPS. 10. The method according to any of the preceding claims, wherein the LPS binding site binding ligand has at least an affinity for the LPS binding site of alkaline phosphatase of lipid A. 11. The degree of AP occupancy of the LPS binding site on alkaline phosphatase is determined by assessing the dephosphorylating ability of alkaline phosphatase in the sample.
The method according to claim 1. 12. The method of claim 11, wherein the ratio of dephosphorylated alkaline phosphatase to non-phosphorylated alkaline phosphatase is determined. 13. The ratio is determined by assessing total alkaline phosphatase activity using a biochemical method for measuring dephosphorylation activity, and assessing the total amount of alkaline phosphatase using, for example, antibodies or other identifying entities. 13. The method according to claim 12, wherein the value obtained by is used and the ratio of such values is calculated and determined. 14. The method according to any of the preceding claims, wherein the sample is from a patient without cholestasis. 15. The method further comprises the further assay of a sample from a patient with another disease associated with increased alkaline phosphatase activity, wherein the further assay employs a method that avoids measuring alkaline phosphatase levels. Item 14. The method according to any one of Items 1 to 13. 16. The method of claim 15, wherein a further assay is performed according to the method of any of claims 1 to 14, when no decrease in AP occupancy of the LPS binding site of alkaline phosphatase is detected. 17. The method according to any of the preceding claims, wherein the sample is taken from an individual at risk of infection by Gram-negative bacteria. 18. The method of claim 17, wherein the sample is taken from an individual either before and after the injury, or immediately after the injury, wherein the injury relates particularly to surgery, burns or ischemic injury. 19. The method according to any of the preceding claims, wherein the sample is taken from a hospitalized individual. 20. The method according to any of the preceding claims, wherein the sample is taken multiple times during a period and the data is compared, thereby revealing the level of AP occupancy during the period. 21. The method according to any of the preceding claims, wherein said period of time is as long as the individual is at risk of infection, ie during hospitalization or after injury recovery. 22. Any of the preceding claims, wherein the results of the assay are compared to a standard value to determine whether the degree of AP occupancy is indicative of endotoxemia or sepsis or a risk thereof. The method described in Crab. 23. The sample is a sample selected from the group consisting of blood and tissue, wherein said blood sample is, for example, serum, said tissue sample is other than bone, and said tissue sample is, for example, from liver and intestine. A method according to any of the preceding claims, which is selected. 24. Alkaline phosphatase LPS binding site binding ligand and instructions for performing an assay according to any of the preceding claims, and optionally further components necessary for such an assay, such as a detectable marker. , A buffer, a container and a sample for comparison or a data chart, for example a standard curve or data relating to data corresponding to alkaline phosphatase values. 25. An alkaline phosphatase LPS binding site binding ligand for performing an assay according to any of claims 1 to 23 and the following detectable markers, buffers, vessels, comparative samples, data charts, eg standards A kit comprising the additional components necessary for such an assay selected from the group consisting of curves or data relating to data corresponding to alkaline phosphatase values. 26. A method of treating endotoxemia or sepsis, comprising administering an LPS binding site of alkaline phosphatase in a pharmaceutically effective amount in a systemically acceptable form. Wherein said ligand is neither alkaline phosphatase nor a derivative of alkaline phosphatase having dephosphorylation activity. 27. A method of removing LPS from a tissue or fluid, comprising contacting the LPS binding site of alkaline phosphatase with the tissue or fluid to be treated, wherein the LPS binding site is subsequently contacted with LPS present in the fluid or tissue. The above method comprising separating the LPS binding site and the tissue or fluid after binding, provided that the ligand is not alkaline phosphatase or a derivative of alkaline phosphatase having dephosphorylating activity. 28. A method for purifying AP itself from tissue or body fluids or other biological production systems using a compound having an LPS binding site for alkaline phosphatase, such as LPS, lipid A or other ligands of the preceding claims. . 29. LPS as an active compound in a therapeutic or diagnostic agent
Use of a compound having an LPS binding site for alkaline phosphatase, such as, lipid A or the other ligands of the preceding claims. 30. As active compound in therapy or diagnosis, LP of alkaline phosphatase such as LPS, lipid A or other ligands according to the preceding claims.
Use of compounds with S binding sites.