JP2010243342A - Method for analyzing lipopolysaccharide or lipid a - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for analyzing lipopolysaccharide and/or lipid A, for obtaining exact values of the lipopolysaccharide and/or lipid A by using a simple method, even in a sample to be examined whose exact value is not obtained constantly until now. <P>SOLUTION: The analyzing method uses a carrier, to which a peptide bindable with the lipopolysaccharide and/or lipid A is immobilized, and includes: the step of bringing the carrier to contact with the sample to be examined possibly containing the lipopolysaccharide and/or lipid A; the step of separating the carrier from an unreacted component and washing the separated carrier by using washing liquid; and the step of analyzing the lipopolysaccharide and/or lipid A bound with the carrier, wherein (a) an organic solvent containing solution is used as the washing liquid, or (b) the contact of the carrier and the sample to be examined is carried out under the presence of a surface acting agent, a reducing agent or a chelating agent. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a method for analyzing lipopolysaccharide (LPS) and / or lipid A.

  LPS (endotoxin) is composed of a polysaccharide part and a phospholipid part (ie, lipid A), and the sugar chain part varies depending on the bacterial species or strain, whereas the lipid A part is almost identical to the bacterial species or strain. Have the same structure. The lipid A moiety also plays a central role in lipopolysaccharide toxicity.

  Known LPS measurement methods include, for example, a rabbit fever test, a quantification method using cell production markers, and an LAL (limulus amebocyte lysate) method. The rabbit fever test is expensive and has bioethical problems. Quantification methods using cell production markers have problems such as cell preparation and identity. Currently, the most widely used LPS measurement method is the LAL method, but accurate values cannot be obtained depending on the test sample (for example, antithrombin III-containing sample, blood, plasma, serum, etc.) Has been pointed out.

  On the other hand, the present inventor obtained a novel peptide having a high binding activity to lipopolysaccharide and / or lipid A, and further dissolved LPS in phosphate buffered saline (PBS). It has been reported that the amount of endotoxin can be adsorbed and recovered using a bead carrier on which the novel peptide is immobilized and quantified by the LAL method (Patent Document 1). Although not described in the above-mentioned Patent Document 1, the present inventor confirmed that even in the combination of the novel peptide-immobilized beads and the LAL method, an accurate value may not be obtained depending on the test sample. is doing.

  Examples of a pretreatment method performed before measuring a test sample such as blood by the LAL method include a simple dilution method, a dilution and heating method, and a method of heating by adding a surfactant or salt. As mentioned above, none of the methods had a significant improvement effect until satisfactory measurement results were obtained for all the test samples.

International Publication WO2007 / 060979 Pamphlet

  An object of the present invention is to provide a method for analyzing lipopolysaccharide and / or lipid A that can obtain an accurate value by a simple method even for a test sample in which an accurate value has not been obtained so far. It is to provide.

The present invention
[1] A method for analyzing lipopolysaccharide and / or lipid A using a carrier on which lipopolysaccharide and / or lipid A-binding peptide is immobilized,
A step of contacting a test sample that may contain lipopolysaccharide and / or lipid A with the carrier (hereinafter referred to as a contact step),
A step of separating the carrier and unreacted components, washing the separated carrier with a washing liquid (hereinafter referred to as a washing step), and a step of analyzing lipopolysaccharide and / or lipid A bound to the carrier (hereinafter referred to as analysis) Called process)
Including
(A) An organic solvent-containing aqueous solution is used as the cleaning solution, and / or (b) the contact between the test sample and the carrier is performed in the presence of a surfactant, a reducing agent, or a chelating agent. And the analysis method,
[2] The analysis method according to [1], wherein the cleaning liquid further contains a surfactant, a reducing agent, or a chelating agent,
[3] The analysis method according to [1] or [2], wherein the analysis step is performed by a LAL (limulus amebocyte lysate) method, an immunological analysis method, or an endotoxin measurement method using cells or tissues,
[4] The analysis method according to [1] to [3], wherein the test sample is a test sample that may contain antithrombin III.
[5] The analysis method according to [1] to [4], wherein the test sample is blood, plasma, serum, or a biological sample or a biological material-derived material, and an organic solvent-containing aqueous solution is used as the cleaning solution.
[6] The lipopolysaccharide and / or lipid A-binding peptide is
Comprising the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, or an amino acid sequence in which one or several amino acids have been deleted, substituted, and / or added in the amino acid sequence, and lipopolysaccharide and / or The analytical method of [1] to [5], which is a peptide showing lipid A binding activity, or a derivative thereof,
About.

  According to the present invention, an accurate value of lipopolysaccharide and / or lipid A can be obtained by a simple method even for a test sample for which an accurate value has not been obtained so far.

It is a graph which shows the result of the endotoxin (LPS) measurement in the ATIII containing sample which performed the contact process in DTT presence. It is a graph which shows the result of the endotoxin (LPS) measurement in an ATIII containing sample using an acetonitrile containing washing | cleaning liquid. It is a graph which shows the result of the endotoxin (LPS) measurement in a plasma sample using an acetonitrile containing washing | cleaning liquid (this invention) or a NaCl containing washing liquid (comparative example). It is a graph which shows the LPS recovery rate computed from the result shown in FIG.

The analysis method of the present invention uses a carrier on which lipopolysaccharide and / or lipid A-binding peptide is immobilized, and the lipopolysaccharide and / or lipid A in the test sample is adsorbed and recovered by the carrier and bound to the carrier. By analyzing lipopolysaccharide and / or lipid A, the amount or concentration of lipopolysaccharide and / or lipid A in the test sample can be determined.
The analysis method of the present invention includes a contact process, a cleaning process, and an analysis process, and (a) an organic solvent-containing aqueous solution is used as a cleaning liquid used in the cleaning process, or (b) a test sample in the contacting process Contact between the carrier and the carrier is carried out in the presence of a surfactant, reducing agent, or chelating agent. In the present invention, only one of (a) and (b) can be performed, or both [that is, (a) and (b)] can be performed.

The test sample that can be analyzed by the analysis method of the present invention is not particularly limited as long as it may contain lipopolysaccharide and / or lipid A, and preferably contains antithrombin III. This is a sample that can be used. It is known that a sample containing antithrombin III cannot obtain an accurate value in the LAL method. According to the analysis method of the present invention, an accurate value can be obtained even for a sample containing antithrombin III. Can be obtained.
Samples that may contain lipopolysaccharide and / or lipid A include various biological samples (eg, blood, plasma, serum), or various samples of biological samples or biological materials (eg, plasmin preparations, antibiotics) Examples of samples that may contain antithrombin III include blood, plasma, and serum.

In the analysis method of the present invention, for example, when blood, plasma, or serum is used as the test sample, an organic solvent-containing aqueous solution is preferably used as the cleaning liquid used in the cleaning step. In addition, the test in the contact step More preferably, the contact between the sample and the carrier is carried out in the presence of a surfactant, a reducing agent, or a chelating agent.
Further, when a blood fraction preparation (human immunoglobulin preparation, etc.) is used as a test sample, contact between the test sample and the carrier in the contact step is carried out in the presence of a surfactant, a reducing agent, or a chelating agent. By carrying out in step (b), accurate measurement can be carried out without using the organic solvent-containing aqueous solution as a cleaning liquid. Even when such a sample is used, an organic solvent-containing aqueous solution can be used as a cleaning liquid regardless of whether a surfactant, a reducing agent, or a chelating agent is used.

  The lipopolysaccharide and / or lipid A-binding peptide used in the present invention is not particularly limited as long as it has a binding ability to lipopolysaccharide and / or lipid A. For example, WO2007 / 060979, WO2005 / 47313, Biochim. Various peptides described in Biophys. Acta Vol. 1611, p. 234-242 (2003), FASEB J. Vol. 14, p. 1801-1813 (2000) can be used, and can be used for lipopolysaccharide and / or lipid A. In view of having extremely excellent binding activity, it is preferable to use various peptides described in WO2007 / 060979.

  The term “peptide” in the present specification includes both oligopeptides and polypeptides, and is used in the sense of including peptide derivatives subjected to various modifications. Examples of the modification include D-formation of L-form amino acid (eg, D-formation of N-terminal amino acid, D-formation of C-terminal amino acid, D-formation of amino acids other than N-terminal and C-terminal), N-terminal amino acid Acetylation of the group, amidation of the C-terminal carboxyl group, substitution of natural amino acids with non-natural amino acids (similar in nature), or combinations thereof.

Examples of the peptide described in WO2007 / 060979 include an amino acid sequence represented by SEQ ID NO: 1 (= SEQ ID NO: 4 in WO2007 / 060979) or SEQ ID NO: 2 (= SEQ ID NO: 1 in WO2007 / 060979) shown below, or In the amino acid sequence, one or several amino acids (for example, 1 to 6, preferably 1 to 4, more preferably 1 to 3, more preferably 1 or 2, particularly preferably 1) are missing. Mention may be made of peptides or derivatives thereof which comprise a deleted, substituted and / or added amino acid sequence and which exhibit lipopolysaccharide and / or lipid A binding activity.
XYSSS ISSIX A (SEQ ID NO: 1)
[The first and tenth Xs in the amino acid sequence independently represent K, R, or H.]
XNYSS SISSI XA (SEQ ID NO: 2)
[The first and eleventh Xs independently represent K, R, or H]

Here, it is preferable that the amino acid substituted in order to maintain the function of a peptide is an amino acid which has the property similar to the amino acid before substitution. For example, amino acids belonging to each group as shown below are amino acids having properties similar to each other within the group. Substituting these amino acids with other amino acids in the group often does not compromise the essential function of the protein. Such amino acid substitution is called conservative substitution and is known as a technique for converting an amino acid sequence while retaining the function of a polypeptide.
Nonpolar amino acids: Ala, Val, Leu, Ile, Pro, Met, Phe, and Trp
Uncharged amino acids: Gly, Ser, Thr, Cys, Tyr, Asn, and Gln
Acidic amino acids: Asp and Glu
Basic amino acids: Lys, Arg, and His

  Further, whether or not a certain peptide has lipopolysaccharide and / or lipid A binding activity can be easily determined by a known method. For example, lipopolysaccharide or lipid A (preferably lipid A or a combination of lipopolysaccharide and lipid A) is immobilized on an appropriate carrier (for example, ELISA plate or bead carrier) and analyzed for the presence or absence of binding to the lipopolysaccharide. Or a method based on surface plasmon resonance (SPR) [for example, a method using a BIACORE system (for example, BIACORE 2000; BIACORE)], wherein a peptide is lipopolysaccharide and / or It can be easily determined whether or not it has lipid A binding activity.

  As the carrier used in the present invention, for example, silica beads, agarose beads, cellulose beads, magnetic beads, glass fibers, or the like can be used. The binding between the lipopolysaccharide and / or the lipid A-binding peptide and the carrier can be performed, for example, by a disulfide bond or a maleimide method using a maleimide group of the carrier.

  Examples of the surfactant that can be used in the contact step in the analysis method of the present invention include a nonionic surfactant, an anionic surfactant, a cationic surfactant, or an amphoteric surfactant. it can.

Specific examples of the nonionic surfactant include polyoxyethylene alkyl ether [eg, polyethylene glycol mono-para-iso-octylphenyl ester (Triton X-100)], polyoxyethylene alkyl allyl ether, polyoxy Ethylene derivatives, sorbitan fatty acid esters, polyoxyethylene sorbitan fatty acid esters [for example, polyoxyethylene monolaurate (Tween 20)], polyoxyethylene sorbitol fatty acid esters, glycerin fatty acid esters, polyoxyethylene alkylamines, alkyl alkanolamides, etc. Can be mentioned.
Specific examples of the anionic surfactant include fatty acid salts, alkyl sulfate esters, alkylbenzene sulfonates, alkyl naphthalene sulfonates, and alkyl sulfosuccinates.
Specific examples of the cationic surfactant include alkylamine salts and quaternary ammonium salts.
Specific examples of amphoteric surfactants include alkyl betaines, amine oxides, and dimethylammoniopropane sulfonic acid.

  The concentration of the surfactant in the contacting step can be appropriately determined depending on the type of surfactant to be used. For example, when using Triton X-100, it is usually carried out at 0.01 to 1%. Preferably 0.02 to 1%, more preferably 0.05 to 0.5%.

  Examples of the reducing agent that can be used in the contacting step in the analysis method of the present invention include dithiothreitol (DTT) and tris (2-carboxyethyl) phosphine hydrochloride (TCEP · HCl). )]and so on. As the concentration used, for example, when DTT is used, it can be usually carried out at 0.1 to 1000 mmol / L, preferably 1 to 1000 mmol / L, more preferably 10 to 500 mmol / L. Can do. When using TCEP, it can be carried out usually at 0.05 to 500 mmol / L, preferably 0.1 to 100 mmol / L, more preferably 0.5 to 50 mmol / L.

  Examples of the chelating agent that can be used in the contact step in the analysis method of the present invention include ethylenediaminetetraacetic acid (EDTA). The concentration used can be generally 0.5 to 500 mmol / L, preferably 1 to 100 mmol / L, and more preferably 10 to 50 mmol / L.

  In the contacting step, any one of these surfactants, reducing agents, or chelating agents, or a combination thereof can be used.

Examples of the organic solvent used in the washing liquid used in the washing step in the analysis method of the present invention include diethyl ether, tetrahydrofuran, dioxane, dimethoxyethane, acetonitrile, propionitrile, dichloromethane, chloroform, dichloroethane, N, N-dimethylformamide, Aprotic polar solvents such as N, N-dimethylacetamide, N-methylpyrrolidinone, dimethyl sulfoxide, or aprotic nonpolar solvents such as hexane, cyclohexane, benzene, toluene, xylene, chlorobenzene, or methanol, ethanol, propanol, Protic polar solvents such as butanol.
As the use concentration, for example, when acetonitrile is used, it can be carried out usually at 0.1 to 100%, preferably 1 to 80%, more preferably 10 to 50%. .
In addition, the organic solvent containing aqueous solution used at a washing | cleaning process can contain the said surfactant, a reducing agent, and / or a chelating agent further.

  In the analysis step of the analysis method of the present invention, the analysis can be performed by a known endotoxin measurement method capable of analyzing lipopolysaccharide and / or lipid A bound to a carrier. For example, LAL method, immunology using specific antibody Analysis method, analysis system using cells or tissues (for example, measurement of induced amount of TNFα using cell lines (West MA, Koons A et al., Endotoxin tolerance in sepsis: concentration-dependent augmentation or inhibition of LPS-stimulated macrophage TNF secretion by LPS pretreatment., J Trauma. 2008 Oct; 65 (4): 893-898; discussion 898-900.), induction of cytokines using peripheral blood), and surface plasmon resonance. In the present invention, the LAL method is preferably used. For example, the LAL method can be performed using a commercially available endotoxin measurement kit (for example, Endospecy ES-50M set; Seikagaku Corporation).

  EXAMPLES Hereinafter, the present invention will be specifically described by way of examples, but these do not limit the scope of the present invention.

Example 1: Endotoxin (LPS) measurement in an ATIII-containing sample using DTT
(1) Preparation of chip column As LPS and / or lipid A binding peptide, peptide Li5-025: kYSSSISSIRAc described in WO2007 / 060979
[In the amino acid sequence, amino acids (k, c) shown in lower case indicate D-form amino acids. That is, the amino acid sequence is a sequence in which the first K (L-form) is substituted with D-form lysine and the 12th C (L-form) is substituted with D-form cysteine in the sequence represented by SEQ ID NO: 3. is there]
It was used. In addition, as a comparative LPS conjugate, a commercially available poly ε-lysine immobilized bead (ET-Clean; Chisso) was used.

Peptide Li5-025 was immobilized on beads by adding a peptide solution to iodoacetamide cellulose particles (hereinafter referred to as beads) and using a thiol group of a C-terminal cysteine for immobilization by CS bonding.
Specifically, first, the peptide is dissolved with a minimum amount of distilled water, a buffer of 50 mmol / L Tris, 5 mmol / L EDTA · 2Na, pH 8.5 (hereinafter referred to as a coupling buffer) is added, and the peptide concentration Was adjusted to a final concentration of 0.25 to 0.5 μmol / mL. Meanwhile, the beads were washed with distilled water and equilibrated with a coupling buffer.
The washed beads and the peptide solution were mixed so that the peptide was 30 μmol per mL of beads (Peptide 30 μmol / mL Beads), and the mixture was allowed to react for 6 hours to overnight with gentle stirring at room temperature. The solution after the peptide immobilization was collected, and the amount of the peptide immobilized was calculated by measuring the concentration of the unbound peptide with an SH group quantification reagent (trade name: Ellman's Reagent, manufactured by PIERCE).

After immobilization of peptide Li5-025, in order to wash away unreacted peptides, the beads were washed with distilled water and further equilibrated with a coupling buffer. On the other hand, 2-mercaptoethanol (manufactured by Kanto Chemical Co., Inc.) was dissolved in a coupling buffer so as to be 100 mmol / L to prepare a blocking buffer. It was added to the equilibrated beads, mixed, and allowed to stand at room temperature for 6 hours to overnight with gentle stirring to perform blocking to prevent nonspecific adsorption.
After blocking, the plate was washed well with distilled water to remove unreacted mercaptoethanol. Finally, distilled water was removed cleanly, 0.2N NaOH (95% ethanol) solution was added, mixed, reacted for 30 minutes with gentle stirring, and then washed well with distilled water until the beads became neutral. This operation was repeated about 3 times to perform alkali washing for removing the mixed endotoxin.
Then, it was replaced with 20% ethanol or phosphate buffered saline (PBS) and stored at 4 ° C. until use.

The obtained Li5-025-immobilized beads, control beads (only the above-described blocking treatment), and commercially available poly-ε-lysine-immobilized beads were each filled in 200 μL filter chips (Fukae Kasei Co., Ltd.) in 15 μL, and the supernatant was After the removal, it was washed once with distilled water for injection (Otsuka Pharmaceutical).
(2) Endotoxin measurement As a sample for measurement, commercially available ATIII solution [Product name: Neuart, biological preparation standard, dry concentrated human antithrombin III, Mitsubishi Pharma Corporation] was used as an endotoxin standard [E.coli. UKT-B, Japan Public Standards Association] was added to a predetermined concentration (0, 0.025, 0.05, 0.1 EU / mL) and incubated at room temperature for 1 hour.
DTT was added to each sample solution so as to have a final concentration of 100 mmol / L, and the mixture was incubated at room temperature for 10 minutes, and then 200 μL was passed through each of the tip columns prepared previously. After passing through, it was washed 5 times with 200 μL of 20 mmol / L NaCl aqueous solution.

Cut the filter part of the chip column, and add 50 μL of endotoxin-free pure water to each well of the 96-well endotoxin-free plate (product name: Toxipet Plate LP, manufactured by Biochemical Biobusiness). Extruded.
After adding 50 μL of a commercially available endotoxin assay reagent (Endospecy, manufactured by Biochemical Biobusiness) to each well and incubating at 37 ° C. for 30 minutes, a reaction terminator (Product name: Toxicolor DIA-MP set, Biochemical Biobusiness) The product was converted to an azo dye by further adding a diazotizing reagent, and measured with an absorptiometer [545 nm (target wavelength: 630 nm)].

The results are shown in FIG.
Regarding Li5-025 immobilized beads, in addition to ATIII and endotoxin-containing sample solutions, the same applies to sample solutions in which endotoxin is dissolved in water (endotoxin aqueous solution) and sample solutions in which endotoxin is dissolved in PBS (endotoxin-containing PBS). The operation of was carried out.
As shown in FIG. 1, when ATIII was included in the sample, polyε-lysine, which is a known LPS conjugate, almost lost the LPS binding ability. On the other hand, the peptide Li5-025 retained the same LPS binding ability as when the sample contained ATIII but did not contain ATIII (endotoxin aqueous solution, endotoxin-containing PBS).

<< Example 2: Endotoxin measurement in ATIII-containing sample using acetonitrile for washing >>
As a tip column, a tip column in which a filter tip was packed with Li5-025 immobilized beads was prepared in the same procedure as in Example 1 (1).
Moreover, each sample solution (DTT additive-free) prepared in the same procedure as Example 1 (2) was prepared as a measurement sample.

200 μL of each sample solution (no DTT added) was passed through the chip column, followed by washing 5 times with 5 mmol / L Tris (pH 7.4) buffer containing 25% acetonitrile.
Cut the filter part of the chip column, and add 50 μL of endotoxin-free pure water to each well of the 96-well endotoxin-free plate (product name: Toxipet Plate LP, manufactured by Biochemical Biobusiness). Extruded.
After adding 50 μL of a commercially available endotoxin assay reagent (Endospecy, manufactured by Biochemical Biobusiness) to each well and incubating at 37 ° C. for 30 minutes, a reaction terminator (Product name: Toxicolor DIA-MP set, Biochemical Biobusiness) The product was converted to an azo dye by further adding a diazotizing reagent, and measured with an absorptiometer [545 nm (target wavelength: 630 nm)].

The results are shown in FIG.
The Li5-025 immobilized beads retained the LPS binding ability unchanged even when ATIII was included in the sample.

Example 3: Endotoxin measurement in plasma using acetonitrile-containing washing solution
As a filter chip, a chip column in which Li5-025 immobilized beads were packed in a filter chip was prepared in the same procedure as in Example 1 (1).
In addition, as a measurement sample, endotoxin standard was added to human plasma at 0, 0.1, 0.5, 1 EU / mL, incubated for 30 minutes in ice, and then Triton X-100 was added at a final concentration of 0. Further added to 1% and mixed well by vortexing.

20 μL of each sample solution was passed through the chip column. Subsequently, the chip column was washed 3 times with 200 μL of 5 mmol / L Tris (pH 7.5) buffer containing 25% acetonitrile and then once with distilled water. At the end, the water was completely removed.
As a comparative example, instead of 5 mmol / L Tris (pH 7.5) buffer containing 25% acetonitrile, a cleaning solution containing 0.5 mol / L NaCl, 50 mmol / L Tris, 20 mol / L EDTA was used. Was also evaluated at the same time.

The filter portion of the chip column was cut, and the beads were each extruded into each well of a 96-well endotoxin-free plate (product name: Toxipet Plate LP, manufactured by Seikagaku Biobusiness) with 50 μL of distilled water.
To each well, 50 μL of a commercially available reagent for measuring endotoxin (LAL reagent) was added, and a diazotizing reagent was further added to convert it into an azo dye, followed by measurement with an absorptiometer [545 nm (target wavelength: 630 nm)].

FIG. 3 shows the relationship between the added LPS and the measured LPS, and FIG. 4 shows the LPS recovery rate calculated from FIG. 3 and 4, the black diamonds indicate the results of the acetonitrile-containing cleaning solution (the present invention), and the open squares indicate the results of the NaCl-containing cleaning solution (comparative example).
Although it could not be measured at all when washed with a 0.5M NaCl-containing solution, the LPS recovery rate was about 100% by washing with an organic solvent-containing solution, and low-concentration LPS was efficiently measured. It was shown that it can be done.
Moreover, it was shown that LPS in plasma can be quantified by adding Triton X-100 to plasma.
It is considered that the measurement was possible because the LAL reaction-inhibiting substance such as lipids non-specifically bound to the beads could be washed with acetonitrile cleanly.

  The analysis method of the present invention can be applied to endotoxin measurement.

Each amino acid sequence represented by the sequence numbered 1 to 3 in the sequence listing is a lipopolysaccharide and / or a lipid A binding peptide.
Each of the first and tenth amino acids “Xaa” in each amino acid sequence represented by the sequence of SEQ ID NO: 1 in the Sequence Listing independently means lysine, arginine, or histidine.
The first and eleventh amino acids “Xaa” in the respective amino acid sequences represented by the sequence of SEQ ID NO: 2 in the sequence listing independently mean lysine, arginine, or histidine.

Claims (6)

A method for analyzing lipopolysaccharide and / or lipid A using a carrier on which a lipopolysaccharide and / or lipid A-binding peptide is immobilized,
Contacting a test sample that may contain lipopolysaccharide and / or lipid A with the carrier;
Separating the carrier and unreacted components, washing the separated carrier with a washing solution, and analyzing the lipopolysaccharide and / or lipid A bound to the carrier,
(A) An organic solvent-containing aqueous solution is used as the cleaning solution, and / or (b) the contact between the test sample and the carrier is performed in the presence of a surfactant, a reducing agent, or a chelating agent. The analysis method.   The analysis method according to claim 1, wherein the cleaning liquid further contains a surfactant, a reducing agent, or a chelating agent.   The analysis method according to claim 1 or 2, wherein the analysis step is performed by a LAL (limulus amebocyte lysate) method, an immunological analysis method, or an endotoxin measurement method using cells or tissues.   The analysis method according to any one of claims 1 to 3, wherein the test sample is a test sample that may contain antithrombin III.   The analysis method according to any one of claims 1 to 4, wherein the test sample is blood, plasma, serum, or a biological sample or a biological material-derived material, and an organic solvent-containing aqueous solution is used as the washing solution. The lipopolysaccharide and / or lipid A-binding peptide is
Comprising the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, or an amino acid sequence in which one or several amino acids have been deleted, substituted, and / or added in the amino acid sequence, and lipopolysaccharide and / or The analysis method according to any one of claims 1 to 5, which is a peptide exhibiting lipid A binding activity or a derivative thereof.

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