JP7288428B2 - Use of anti-CD14 antibody useful for presepsin measurement - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for suppressing an increase in an apparent presepsin measurement value in a specimen due to physical stimulation of the specimen, in presepsin measurement in the specimen, and the like.

The CD14 molecule is a glycoprotein expressed on the membrane surface of mononuclear cells, and is known to function as a receptor for LPS (lipopolysaccharide). There are two types of CD14 molecules: membrane-bound CD14 (mCD14) expressed on the cell surface and soluble CD14 (sCD14). As sCD14, sCD14 with a molecular weight of about 55 kDa and about 49 kDa (hereinafter referred to as "high molecular weight soluble CD14" or "high molecular weight sCD14") is known, and is used for sepsis (SEPSIS), acquired immune deficiency syndrome (AIDS), It has been reported to show high levels in the blood of patients with many diseases such as acute respiratory distress syndrome (ARDS) and systemic lupus erythematosus (SLE). Therefore, these high-molecular-weight sCD14 are not considered to be disease-specific markers (Non-Patent Documents 1 and 2).

On the other hand, it has been reported that sCD14-ST (soluble CD14 antigen subtype, also called presepsin) exists as a new molecular species of sCD14 whose blood concentration is characteristically elevated in sepsis patients.

Among sCD14, sCD14-ST (preseptin) is characterized by having a molecular weight of 13±2 kDa in SDS-PAGE under non-reducing conditions, and retains the N-terminus of CD14. Compared to high-molecular-weight sCD14, it has an amino acid sequence with a large deletion on the C-terminal side, and unlike high-molecular-weight sCD14, it does not have LPS-binding ability. Also, since presepsin exhibits different immunogenicity than high molecular weight sCD14, antibodies can be used to distinguish between the two. Blood levels of presepsin are specifically elevated in sepsis patients (Patent Document 1). In addition, presepsin is a specific diagnostic marker for sepsis, as it has been reported that blood levels are higher in patients with sepsis than in patients with systemic inflammatory response (SIRS), which is difficult to distinguish from sepsis. (Non-Patent Document 3).

A rabbit-derived polyclonal antibody (S68 antibody) and a rat-derived monoclonal antibody (F1146-17-2) that specifically recognize presepsin have been disclosed (Patent Documents 1 and 2).

Currently, for the measurement of presepsin, a measurement system using a rabbit-derived polyclonal antibody as a specific antibody against presepsin has been put into practical use, and a measurement kit is available in Europe and Japan (PATHFAST (trade name) Presepsin, manufactured by LSI Co., Ltd.). Medience).

Further, a fully automatic measuring device for presepsin using a rabbit monoclonal antibody has been put into practical use (for example, STACIA (trade name) (LSI Medience Corporation), AIA (trade name) (Tosoh Corporation), etc.).

Regarding the presepsin measurement kit, if the sample to be used is subjected to physical stimulation such as vigorous stirring, the apparent presepsin measurement value in the sample may increase, and the original presepsin value in the sample may not be obtained. It is said.

International publication WO2005/108429 International publication WO2004/044005

Hayashi et al., Infection and Immunity, 67:417-420, 1999. Lawn et al., Clinical & Experimental Immunology, 120:483-487, 2000. Yaegashi et al., Journal of Infection and Chemotherapy, 11:234-238, 2005.

As mentioned above, with the presepsin measurement kit, if the sample to be used is subjected to physical stimulation such as vigorous agitation, the measured value of presepsin increases, and the original presepsin value in the sample cannot be obtained. are concerned. For this reason, it is said that preseptin assay kits should be handled with care so as not to subject them to vigorous agitation.

Under such circumstances, a method for obtaining the original measured value of presceptin in a sample without substantially increasing the measured value of presceptin even if a physical stimulus is applied to the sample to be used when measuring presceptin in the sample. was sought.

Alternatively, when measuring presceptin in a sample, even if a physical stimulus is applied to the sample, the apparent presceptin measurement value in the sample hardly increases, and a method that enables stable presceptin measurement regardless of how the sample is handled. was sought.

An object of the present invention is to provide a method and the like useful for presepsin measurement in a specimen, which can solve at least one of the conventional problems.

As a result of extensive studies, the present inventors have found that the phenomenon in which the measured value of preseptin increases when a physical stimulus such as vigorous stirring is applied to a specimen is caused by a product derived from sCD14 in the specimen caused by the physical stimulation. I found that Therefore, the present inventors believe that this sCD14-derived product can eliminate the influence of the product, for example, by contacting the specimen with an anti-CD14 antibody, or by separating it from the presepsin using chromatography. The present invention was completed based on the idea that the original presepsin level in a sample can be measured.

In the examples described later, normal human plasma (EDTA) was used to confirm the effect of shaking the sample on the measured value of the presepsin ELISA kit, and it was confirmed that shaking the sample increased the measured value of presepsin. .
In addition, in Examples, using human serum and CD14-absorbed serum from normal subjects, it was confirmed that the increase in presepsin level due to shaking was only in samples containing sCD14 in the blood, and that it was derived from sCD14 in the serum. .
In addition, in the Examples, using normal human plasma, presepsin was measured in the presence and absence of F1024-1-3 antibody (anti-CD14 antibody). -3 antibody was confirmed to suppress.
Moreover, in Examples, it was confirmed that an increase in the presepsin level due to shaking is related to sCD14 in the sample. Specifically, as a result of analyzing each fraction by gel filtration of the sCD14-derived product produced by shaking, the elution pattern of the control and the sample shaken after adding the anti-CD14 antibody was almost the same. It was confirmed that a higher molecular weight aggregate was generated from sCD14 by shaking (this high molecular weight aggregate is also referred to as "sCD14-derived product" or "pseudopresepsin"). It was also confirmed that the formation of high-molecular-weight aggregates can be effectively suppressed by coexisting an anti-CD14 antibody in the specimen. Furthermore, it was confirmed that the high-molecular-weight aggregates can be separated by chromatography.

From the above examples, it can be seen that the influence of sCD14-derived products can be eliminated by, for example, contacting the specimen with an anti-CD14 antibody, or separating the preseptin from the specimen using chromatography. It can be seen that it is possible to effectively prevent an increase in the measured value of presceptin due to shaking in measuring the presceptin in the sample, and that the original measured value of presceptin in the sample can be obtained.

Conventionally, when measuring presepsin in a specimen, it has been noted that the measured value may be affected if the specimen is agitated (including, for example, vigorous short-term agitation and gentle long-term agitation). However, sCD14 in the blood is the cause that affects this measurement value, and contacting the specimen with an anti-CD14 antibody (for example, F1024-1-3 antibody), or separating from presepsin using chromatography It was surprising that the influence of the product on presepsin measurements could be eliminated, such as by

The present invention can include the following aspects.
[1] A method for stabilizing the measured value of presepsin in a sample, comprising the step of contacting the sample with an anti-CD14 antibody immediately after collection of the sample.
[2] A method for suppressing pseudopreseptin production in a sample, comprising the step of contacting the sample with an anti-CD14 antibody immediately after collection of the sample.
[3] A method for inhibiting denaturation of sCD14 in a sample, comprising the step of contacting the sample with an anti-CD14 antibody immediately after collection of the sample.
[4] The method according to any one of the above aspects [1] to [3], which comprises adsorbing and removing sCD14 in the specimen by contacting the specimen with an anti-CD14 antibody.
[5] Any one of the aspects [1] to [3] above, wherein the step of contacting the specimen with the anti-CD14 antibody forms a bond between the sCD14 in the specimen and the antibody-CD14 antibody. described method.
[6] A method for quantifying the true amount of presepsin in a specimen, which comprises the step of contacting the specimen with an anti-CD14 antibody before presepsin measurement.
[7] The method according to [6] above, wherein the step of contacting the specimen with the anti-CD14 antibody adsorbs and removes the pseudo-resepsin using an anti-CD14 antibody that reacts with the pseudo-presceptin in the specimen. .
[8] A method for quantifying only true preceptsin, which comprises a step of separating true preseptin from pseudopresceptin before presepsin measurement.
[9] The method according to [8] above, wherein the separation of pseudopresceptin and true presepsin is chromatographic separation.
[10] The method according to [8] above, wherein the separation between pseudopreseptin and true preseptin is separation by a membrane.
[11] A method for quantifying only pseudopresceptin, which comprises a step of separating pseudopresceptin from true presepsin before presepsin measurement.
[12] A method of quantifying a true preceptsin level, comprising measuring the presepsin of a specimen to calculate a presepsin level, and subtracting the pseudo presepsin level obtained by the method according to the aspect [11] from the calculated presepsin level. .
[13] containing at least one selected from the group consisting of an anti-CD14 antibody, a chromatography column, and a membrane for use in the method according to any one of aspects [1] to [12]; Presepsin measurement kit.
[14] from the group consisting of a sample cartridge, a sample processing unit, a reagent cartridge unit, a preseptin measurement unit, a calculation unit, and a display unit for use in the method according to any one of the above aspects [1] to [12] A presepsin measurement system comprising at least one selected.

According to some embodiments, when measuring presceptin in a sample, even if a physical stimulus is applied to the sample to be used, the measured value of presceptin hardly increases, and the original value of presceptin in the sample is obtained. can be done. For example, the effect of sCD14-derived products can be eliminated by contacting the specimen with an anti-CD14 antibody, or by separating the presepsin using chromatography. It is possible to effectively prevent an increase in the measured value of presceptin due to a physical stimulus, and to obtain the original measured value of presceptin in the sample. In this case, even if a physical stimulus such as vigorous stirring is applied to the sample, that is, regardless of how the sample is handled, a correct presepsin measurement value can be obtained.

FIG. 4 shows the effect of addition of anti-CD14 antibody on presepsin concentration. FIG. 10 is a diagram showing the results of measuring each fraction of gel filtration in Example 4 with a preseptin ELISA kit and measuring the absorbance of the measurement solution at 450-650 nm. FIG. 10 is a diagram showing the results of measuring each fraction of gel filtration in Example 4 with a CD14 ELISA kit and measuring the absorbance of the measurement solution at 450-650 nm.

In some embodiments, when measuring presceptin in a sample, even if the sample to be used is subjected to a physical stimulus such as vigorous stirring, the measured value of presceptin in the sample hardly increases, and the original presceptin in the sample does not increase. It relates to a method useful for measuring presepsin in a sample that yields a value.

Further, some other embodiments relate to methods for removing presepsin-like reacting products (pseudopresceptins) produced by physical stimuli. Further, in some other aspects, in using the presepsin measurement kit, even if a physical stimulus such as vigorous stirring is applied to the specimen, the apparent presepsin measurement value in the specimen does not increase, regardless of how the specimen is handled. , to methods that allow stable presepsin measurements.

Here, the following aspects [1] to [14] are provided.
[1] A first aspect is a method for stabilizing the measured value of presepsin in a sample, comprising the step of contacting the sample with an anti-CD14 antibody immediately after collection of the sample.
[2] A second aspect is a method for suppressing pseudopreseptin production in a sample, comprising the step of contacting the sample with an anti-CD14 antibody immediately after collection of the sample.
[3] A third aspect is a method for inhibiting denaturation of sCD14 in a sample, comprising the step of contacting the sample with an anti-CD14 antibody immediately after collection of the sample.
[4] A fourth aspect, according to any one of the above aspects [1] to [3], comprising adsorbing and removing sCD14 in the specimen by the step of contacting the specimen with an anti-CD14 antibody. The method.
[5] A fifth embodiment is characterized in that the sCD14 in the specimen and the antibody-CD14 antibody form a conjugate by the step of contacting the specimen with the anti-CD14 antibody. The method according to any one aspect.
[6] A sixth aspect is a method for quantifying the true amount of presepsin in a sample, comprising the step of contacting the sample with an anti-CD14 antibody before presepsin measurement.
[7] A seventh aspect is characterized in that the anti-CD14 antibody that reacts with the pseudopresceptin in the sample is used to adsorb and remove the pseudopresceptin in the step of contacting the sample with the anti-CD14 antibody. 6].
[8] The eighth aspect is a method for quantifying only true presceptin, comprising the step of separating true presceptin from pseudopresceptin before measuring presceptin.
[9] A ninth aspect is the method according to the aspect [8], wherein the separation of pseudopresceptin and true presepsin is chromatographic separation.
[10] A tenth aspect is the method according to the aspect [8], wherein the separation of pseudopreseptin and true presepsin is separation by a membrane.
[11] The eleventh aspect is a method for quantifying only pseudopresceptin, which comprises a step of separating pseudopresceptin from true presepsin before measuring presceptin.
[12] A twelfth aspect comprises measuring the presepsin of the sample to calculate the presepsin value, and subtracting the pseudo presepsin value obtained by the method according to aspect [11] from the calculated presepsin value. A method for quantifying presepsin levels.
[13] A thirteenth aspect is selected from the group consisting of an anti-CD14 antibody, a chromatography column, and a membrane for use in the method according to any one of the above aspects [1] to [12]. A presepsin measurement kit comprising at least one.
[14] A fourteenth aspect is a sample cartridge, a sample processing section, a reagent cartridge section, a preceptin measurement section, a calculation section, and a and a display unit.

Terms used in this specification will be explained below.
“Immediately after specimen collection” means immediately after specimen collection from a patient (for example, immediately after blood collection) or at the same time as specimen collection (for example, at the same time as blood collection). Here, immediately after specimen collection, "pre-transport", i.e., before the specimen is collected from the patient (e.g., blood is drawn) and sent to a specialized center for measurement of various specimen parameters (e.g., blood parameter measurement). included. In particular, it means the period from immediately after the specimen is collected (for example, immediately after blood collection) until it is sent to the examination center.
"Before presepsin measurement" means before starting presepsin level measurement, for example, before starting measurement on the spot after collecting a sample, or before measuring presepsin level of a sample arriving at a testing center. .

The "step of contacting the specimen with the anti-CD14 antibody" is, for example,
1) adding an anti-CD14 antibody to the specimen;
2) Specimens collected in a container such as a blood collection tube in which an anti-CD14 antibody is present in advance (for example, an anti-CD14 antibody is immobilized on beads or the like placed in a container such as a blood collection tube). housing the
3) A step of using a dedicated container such as a dedicated blood collection tube in which anti-CD14 has been immobilized in advance, and housing the sample collected in the container;
4) A step of adsorbing and removing sCD14 in the specimen using an anti-CD14 antibody (more specifically, for example, using an affinity chromatography column filled with a carrier in which an anti-CD14 antibody is bound to a resin or the like, the specimen is allowed to pass through the column),
any step selected from the group consisting of By going through these steps, sCD14 in the specimen acquires resistance to physical stimulation, or sCD14 is removed. It is possible to suppress the denaturation of

Adding an anti-CD14 antibody to a sample includes, for example, adding an anti-CD14 antibody to a sample collected from a patient.
In order to allow the anti-CD14 antibody to coexist with the specimen before transportation, the anti-CD14 antibody may be coexisted in the specimen by adding the anti-CD14 antibody to the specimen. It includes the coexistence of an anti-CD14 antibody with a specimen by putting blood into a blood collection tube.

The term "pseudopreseptin" refers to a product derived from sCD14, which undergoes aggregation, unfolding of the steric structure, etc., due to vigorous stirring, etc., and exhibits a presepsin-like reaction. Thus, the presepsin-like response of pseudoprespsin results in an apparent increase in presepsin measurements.
“Inhibiting the production of pseudopreseptin” or “suppressing the production of pseudopresceptin” means inhibiting the formation or production of pseudopresceptin derived from sCD14.
The term "denaturation of sCD14" means that sCD14 in a sample aggregates into, for example, two molecules and becomes higher in molecular weight due to vigorous stirring or the like. Due to this aggregation, a site that reacts to presepsin measurement appears on the surface of the molecule, resulting in a state in which it reacts with an antibody that measures presepsin.
“Suppression of denaturation of sCD14” means inhibition of sCD14-derived pseudopreseptin from responding to presepsin measurement.
"Physical stimulus" means vigorous stirring (including gentle long-term stirring) or the like when handling a specimen, and also means overturning stirring or shaking of a blood collection tube after a vote.
By "true presepsin value" is meant a true value indicative of the amount of presepsin present in the sample, eg, it is the measurement of presepsin in the sample prior to subjecting the sample to physical stimulation. There is a phenomenon in which sCD14 aggregates and responds to presepsin measurement, thereby increasing the apparent presceptin level. For example, by preventing sCD14 from aggregating, the presepsin level itself present in the specimen can be known, and a correct presepsin level can be stably obtained even under the influence of shaking or the like. However, it does not mean that the measured value of presepsin in the sample before the physical stimulation is completely matched.

“Stabilization of the presepsin measurement value” means to exhibit a true presepsin value even if the specimen receives a physical stimulus. That is, for example, by suppressing the aggregation of sCD14, a true presepsin level can be obtained even when subjected to physical stimulation.
“Resistant to destabilization by physical stimuli” means that sCD14 is inhibited from being destabilized by physical stimuli such as aggregation and unfolding of the three-dimensional structure. In addition to the value, it means blocking the increase in apparent presepsin levels.

The “true preseptin” is the “preseptin” described later, namely sCD14-st (soluble CD14 antigen subtype), which is a substance quantified by a conventional preseptin assay kit.
"Distinction between pseudopresepsin and true presepsin" means the following methods,
1) separation by chromatography (e.g. separation by gel filtration chromatography (e.g. Example 4)),
2) membrane separation;
3) Separation by an antibody that reacts with pseudopresepsin,
etc.

Quantifying the true preceptsin level by separating the sample before the preseptin measurement by chromatography (e.g., gel filtration chromatography) to obtain a fraction containing the true preseptin, and measuring the preseptin level. is possible. For quantification, the preceptsin measurement method described later is used. For example, in Example 4, by comparing the amount of preceptsin in fractions containing each preceptsin with a calibration curve prepared with a standard concentration of preceptsin, true preceptsin was obtained. value can be obtained. In addition, by constructing a system that performs the separation and quantification, it is also possible to construct a system that obtains a true presepsin value simply by directly applying a specimen.

To remove pseudoprespsin in a sample by a membrane, for example, the sample may be passed through a membrane (eg, a 30 kilodalton ultrafiltration membrane) that rejects substances with a higher molecular weight than presepsin. A true presepsin value can be obtained by measuring the presepsin level of the sample from which the pseudopresceptin has been removed by the membrane and the pseudopresceptin has been removed.

In addition, in order to separate sCD14-derived pseudopresceptin from a sample, the sample can be contacted with an antibody that reacts with sCD14 but does not react with true preceptsin but with pseudopresceptin to remove pseudopresceptin. included. In order to remove the pseudopresceptin with an antibody, the specimen and the antibody are brought into contact, for example, the specimen is allowed to pass through a column on which the antibody is immobilized, and the sCD14-derived pseudopresceptin is adsorbed and removed by the antibody. included.
“Quantifying only the true presepsin level” means separating the sample before presepsin measurement by, for example, gel filtration chromatography, and quantifying the presepsin level in the fraction containing the true presepsin. More specifically, according to Example 4 described later, the separated relevant fractions are quantified.
A "pseudopreseptin value" can also be obtained by performing a presepsin measurement on a chromatographically separated fraction containing a "pseudoprespsin". Once the pseudopreseptin level is obtained, the prepsin level of the specimen can be measured, and the measured value of pseudopresceptin can be obtained from the sum of the true presepsin level and the pseudopresceptin level (total presepsin amount).
The phrase "quantify only pseudopreseptin" means separating the sample before measurement of presceptin by, for example, gel filtration chromatography, and quantifying the pseudopresceptin level in the fraction containing pseudopresceptin. More specifically, according to Example 4 described later, the separated relevant fractions are quantified.
A "presepsin measurement system" means a device such as a fully automated device in which each element for performing presepsin measurement is incorporated into the system.

The present invention will be described in detail below. The following embodiments are examples for explaining the present invention, and are not meant to limit the present invention only to these embodiments. The present invention can be embodied in various forms without departing from the gist thereof.

1. Anti-CD14 Antibodies, Membranes and Chromatographic Columns 1.1. Anti-CD14 Antibodies In some embodiments, anti-CD14 antibodies are used. Anti-CD14 antibodies include all antibodies that bind to CD14, preferably human CD14. Preferably, the anti-CD14 antibody is at least one antibody selected from F1024-1-3 antibody, 3C10 antibody, and MEM-18 antibody, more preferably F1024-1-3 antibody, 3C10 antibody, or MEM -18 antibody.

As described in WO2001/72993 A1, the term "F1024-1-3 antibody" refers to the hybridoma F1024- obtained by fusing immune cells and myeloma cells obtained by immunizing rats with CD14 protein purified from human serum as an antigen. 1-3 is the F1024-1-3 antibody produced. More specifically, it can be obtained according to the known method of Example 1 "Preparation of anti-human CD14 antibody" of WO2001/72993 A1 by the method described in the publication.

The F1024-1-3 antibody is an anti-CD14 antibody that specifically recognizes an epitope comprising 8 or more amino acids in the region from 285th to 315th amino acids of human sCD14 shown in SEQ ID NO:3.

In addition, as a human anti-CD14 antibody that controls LPS signaling via human CD14, the 3C10 antibody that binds to positions 7-14 of human CD14 (Steinman: J. Exp. Med., 158: 126 (1983) and Juan TS Chem., 270:29, 17237 (1995)) and the MEM-18 antibody that binds positions 57-64 (Bazil: Eur. J. Immunol., 16:1583 (1986) and Juan TS: J. Chem., 270, 10, 5219 (1995)).
3C10 antibody and MEM-18 antibody are available from, for example, abcom.

The term "antibody" is used to mean "antibody or antigen-binding fragment thereof" unless otherwise specified. “Antigen-binding fragment” refers to a partial fragment of an antibody that has substantially the same antigen-binding ability as the original antibody. Antigen-binding fragments include, for example, Fab, Fab', F(ab') ₂ and the like.

1.2. Membranes In some embodiments, membranes are used that are capable of removing pseudopreseptins. Preferably, the membrane is a membrane capable of excluding substances with a higher molecular weight than the presepsin (eg, a 30 kilodalton membrane). Such membranes include, for example, ultrafiltration filters (eg, Amicon (trade name) Ultra (Merck Millipore), etc.).

1.3. Chromatographic Columns In some embodiments, a chromatographic column (eg, a gel filtration chromatography column) is used to separate true and false presepsins in a sample. Examples of such chromatographic columns include types such as Superdex (trade name), Sephacryl (trade name), Superose (trade name), and Sephadex (trade name).

2. Presepsin "Presepsin" is also referred to as sCD14-ST (soluble CD14 antigen subtype). CD14 includes membrane-bound CD14 (mCD14) and soluble CD14 (sCD14), and blood contains multiple soluble CD14s with different molecular weights. Presepsin is a soluble fragment of CD14 and refers to a substance having the following properties 1) to 3).

1) SDS-PAGE under non-reducing conditions has a molecular weight of 13 ± 2 kDa;
2) having the amino acid sequence of positions 1 to 11 of the amino acid sequence of SEQ ID NO: 3 (amino acid sequence of human full-length soluble CD14) in the N-terminal sequence, and
3) Specific binding to an antibody prepared using a peptide (S68 peptide) consisting of 16 amino acid residues set forth in SEQ ID NO: 2 (corresponding to the amino acid sequence from position 53 to position 68 in the amino acid sequence of SEQ ID NO: 3) as an antigen do.

Presepsin is human presepsin unless otherwise stated. The presepsin is, for example, a presepsin standard (rsCD14-ST as described in Example 16 of WO2005/108429). Alternatively, a substance obtained by modifying a part of presepsin, which has binding activity as presepsin, may be used.

3. sCD14
As sCD14, sCD14 with a molecular weight of about 55 kDa and about 49 kDa is known. sCD14 is also referred to herein as "high molecular weight soluble CD14" or "high molecular weight sCD14".
sCD14 may be produced, for example, by 3C10 antibody affinity column adsorption of normal human body fluids (see Example 23 of WO2005/108429).

4. Method for Measuring Presceptin “Presepsin measurement” is, for example, immunological measurement of presepsin. For immunological measurement of presepsin, for example, anti-presceptin antibody (e.g., specific antibody such as S68 polyclonal antibody, P03-recognizing monoclonal antibody, P03-specific polyclonal antibody) (hereinafter referred to as "antibody used for presepsin measurement ”) and a sample containing presepsin (eg, a blood sample). The term "measurement" can be used interchangeably with terms such as "detection,""quantitation," and "assay," and is used to mean both quantitative and qualitative determination. Measurement of presepsin is preferably performed in vitro.

A method for stabilizing the measured value of presceptin in a sample, a method for suppressing pseudopresceptin production derived from sCD14 in a sample, a method for suppressing denaturation of sCD14 in a sample, a method for quantifying the amount of true presceptin in a sample, and only true presceptin. can be used in presepsin measurement.

When measuring presceptin in a sample, even if the sample to be used is subjected to a physical stimulus, the measured value of presceptin hardly increases, and the original measured value of presceptin in the sample can be obtained. For example, the sCD14-derived product can eliminate the influence of the product by contacting the specimen with an anti-CD14 antibody, or by separating it from presepsin using chromatography, etc. It is possible to effectively prevent an increase in the measured value of presepsin due to stimulation, and the original measured value of presceptsin in the sample can be obtained. It is possible to suppress the increase in the apparent presepsin measurement value in .

The term "S68 antibody" refers to an antibody obtained by purifying a polyclonal antibody obtained from a non-human mammal immunized with the S68 peptide (SEQ ID NO: 2) as an immunogen using a column immobilized with the S68 peptide. S68 peptide polyclonal antibody. The "S68 peptide" is a peptide consisting of the amino acid sequence of SEQ ID NO: 2 (amino acid sequence from position 53 to position 68 of the amino acid sequence of SEQ ID NO: 3). A specific method for producing the S68 antibody is as described in Example 1 of WO2004/044005.

The "P03-recognizing monoclonal antibody" refers to the amino acid sequence represented by SEQ ID NO: 1 in presepsin (krvdadadpr: region corresponding to positions 52-61 of SEQ ID NO: 3 (human full-length soluble CD14): also called P03 sequence ), a monoclonal antibody that recognizes The P03-recognizing monoclonal antibody is, for example, the monoclonal antibody described in WO2015/129774.

The “P03-specific polyclonal antibody” is a conventional rabbit-derived anti-presepsin polyclonal antibody (S68 antibody), that is, a polyclonal antibody obtained by immunizing a rabbit with S68 peptide (SEQ ID NO: 2), and immobilizing the S68 peptide. It is produced by purification on an affinity column. By purifying the polyclonal antibody using an affinity column immobilized with the P03 peptide (SEQ ID NO: 1) instead of the column immobilized with the S68 peptide, an anti-preceptin polyclonal antibody highly reactive with preceptsin can be obtained. A specific method for producing a P03-specific polyclonal antibody is as described in WO2017/033281.

Since presepsin is known as a marker used to detect sepsis, the method for measuring presepsin is used in a method for detecting sepsis, which includes the step of contacting an antibody used for presepsin measurement with a sample containing presepsin. can do.

That is, the method for measuring presepsin is
(1) a step of measuring the presepsin concentration in a specimen of a subject using an antibody used for presepsin measurement;
(2) determining whether the presepsin concentration obtained in (1) above is higher than the cutoff value;
It can also be said to be a method for detecting sepsis.

The cut-off value when the sample is a blood sample is, for example, 314-600 pg/mL, preferably 400-580 pg/mL, more preferably 450-550 pg/mL, still more preferably 500 pg/mL.

"Detection of sepsis" may be read as "aid in detection of sepsis" or "aid in diagnosis of sepsis".

In addition, methods for measuring presepsin include, for example, discrimination between sepsis and systemic inflammatory response syndrome (SIRS); risk assessment of aggravation of sepsis; detection of postoperative infection; detection of disseminated intravascular coagulation (DIC); detection of infectious DIC; detection of cardiac disease; respiratory infection with bacterial infection, inflammatory bowel disease (Crohn's disease, ulcerative colitis), febrile neutropenia (FN), or detection of hemophagocytic syndrome (HPS) for the detection or evaluation of at least one disease can be used. That is, the method for measuring presepsin can also be said to be a method for detecting or evaluating the disease.

A postoperative infection is a general term for infections that develop after surgery, and means all infections caused by surgery and necessary adjuvant therapy. In addition, postoperative infections include all diseases diagnosed as postoperative infections based on the Guideline for prevention of surgical site infection, 1999 (CDC).
Heart diseases include, for example, acute coronary syndrome (ACS), acute heart failure, acute decompensated heart failure (ADHF), chronic heart failure, coronary artery disease, angina pectoris, myocardial infarction, ischemic stroke, hemorrhagic stroke and transient Cerebral ischemic attack is included.

Respiratory infections involving bacterial infections include lower respiratory tract infections or pneumonia. Lower respiratory tract infections include acute lower respiratory tract infections and chronic lower respiratory tract infections. Acute lower respiratory tract infections include acute tracheitis, acute bronchitis, and acute bronchiolitis. Infection continues. Antibiotics are indicated if signs of secondary bacterial infection are observed. Chronic lower respiratory tract infection is a pathological condition in which persistent bacterial infection is established in the lower respiratory tract, which has organic disorders such as bronchiectasis and chronic obstructive pulmonary disease, and there are persistent infection and acute exacerbation. Diseases that cause organic disorders of the lower respiratory tract include bronchiectasis, chronic obstructive pulmonary disease, chronic bronchitis, diffuse panbronchiolitis, old pulmonary tuberculosis, pneumoconiosis, nontuberculous mycobacterial disease, and allergies. Includes bronchopulmonary aspergillosis, pulmonary fibrosis, chronic bronchial asthma, and others. Antibiotic administration is indicated for both persistent infection and acute exacerbation. Pneumonia includes community-acquired pneumonia and hospital-acquired pneumonia. Community-acquired pneumonia is preferred.

Examples of methods for immunologically measuring presepsin using an antibody used for presepsin measurement include enzyme immunoassay ((hereinafter also referred to as EIA or ELISA), chemiluminescent enzyme immunoassay (CLEIA), chemiluminescent immunoassay Assay method (CLIA), fluorescent antibody assay (FAT), fluorescent enzyme immunoassay (FEIA), electrochemiluminescence immunoassay (ECLIA), radioimmunoassay (RIA), immunochromatography, agglutination method, competition method, etc. Examples include, but are not limited to, both direct and indirect methods, and a sensitizing method in which a biotin-avidin (streptavidin) complex is formed for detection.

EIA is one of immunoassay methods using an enzyme-labeled antibody, and includes a direct method, an indirect method, and the like. A preferred example is a sandwich ELISA (enzyme-linked immunosorbent assay).

Sandwich ELISA uses two or more types of antibodies with different antigen recognition sites, one of which is immobilized on a solid phase in advance, and the antigen to be detected is sandwiched between the two types of antibodies to form an antibody-antigen-antibody complex. This is a method of measuring by forming.

Chemiluminescent Enzyme Immunoassay (CLEIA) involves reacting an antigen in a specimen with an antibody immobilized on magnetic particles, beads, or the like, reacting with an enzyme-labeled antibody, and washing (B/F separation ), a chemiluminescent substrate is added, and the luminescence intensity is measured after enzymatic reaction.

For example, an antigen in a specimen and a biotin-conjugated antibody are reacted in a liquid phase, the antibody is trapped on streptavidin-conjugated magnetic particles, washed (B/F separation), and then reacted with an enzyme-labeled antibody, Measure the luminescence intensity.

When alkaline phosphatase (ALP) is used as the labeling enzyme, CDP-Star™, AMPPD™, and CSPD™ are preferably used as chemiluminescent substrates. When the labeling enzyme is HRP, luminol is preferably used as the chemiluminescent substrate.

Detection sensitivity is generally said to be higher in the order of chemiluminescence>fluorescence>absorption (coloration), and the measurement method can be selected according to the required sensitivity.

Chemiluminescence immunoassay (CLIA: Chemiluminescence immunoassay) is performed by reacting antigens in the sample with antibodies immobilized on magnetic particles or the like, reacting antibodies labeled with chemiluminescent substances, and washing (B/F separation). The second is a method of measuring the luminescence intensity. Acridinium or the like is used as the labeling substance.

Fluorescent Enzyme Immunoassay (FEIA) involves reacting an antigen in a sample with an immobilized antibody, reacting with an enzyme-labeled antibody, washing (B/F separation), and adding a fluorescent substrate. In this method, the fluorescence intensity is measured after the enzymatic reaction. HRP, ALP, or the like is used as the labeling enzyme. As the fluorescent substrate, when the labeling enzyme is HRP, AmplexTM Red or the like is preferably used, and when the labeling enzyme is ALP, 4-MUP (4-methylumbelliphenyl phosphate), AttoPhosTM or the like is preferably used.

Electrochemiluminescence immunoassay (ECLIA: Electro Chemiluminescence Immunoassay) involves reacting antigens in a sample with antibodies immobilized on magnetic particles and antibodies labeled with electrochemiluminescent substances, followed by washing (B/F separation). , is a method of measuring the luminescence intensity by electrical energy. Ruthenium or the like is used as the labeling substance. Ru(bpy)3 or the like is used as the labeling substance, and excitation luminescence is repeated by oxidation due to the charge on the electrode and reduction reaction with tripropylamine (TPA) or the like.

Radioimmunoassay (RIA: Radioimmunoassay) is a measurement method using a radioisotope-labeled body. ¹²⁵ I, etc.)-labeled antibody is allowed to react, and after washing (B/F separation), the radiation dose of ¹²⁵ I can be measured.

Immunochromatography is an immunoassay method that applies capillary action in which a test sample moves on a test strip while dissolving a reagent. In this method, the antigen in the sample forms an immune complex with two of the labeled antibody and the capture antibody on the test strip, and the color of the labeled substance is confirmed. Gold colloids, enzymes, fluorescent substances, and the like are used for antibody labeling. When using an enzyme-labeled antibody, the enzyme substrate is placed on the test strip to allow color development.

The flow-through method is a method in which an antibody-antigen-antibody complex is formed on a membrane, which is an insoluble carrier, with a solution in a sample and an antigen, which is a test substance. At this time, substances not immobilized on the membrane are usually removed vertically from the front to the back of the membrane.

The agglutination method is a method of reacting an antigen in a specimen with an antibody in a reagent and observing agglutination. Methods that do not use a solid phase, particle aggregation (PA) that uses artificially produced particles as a solid phase, latex aggregation (LA) that uses latex particles among PAs, and the like. .
In the competitive method, for example, an antibody is bound to a solid phase, a test sample and a certain amount of labeled antigen are simultaneously reacted, and the amount of antigen in the sample can be measured from the amount of bound label.
Antibodies used for presepsin measurement are preferably used in the above-described measurement method.

The specimen used for presepsin measurement is that obtained from a subject.
Subjects herein include, for example, humans, non-human mammals (eg, rabbits, goats, horses, sheep, pigs, rats, and mice). In some embodiments, the subject is human.
The specimen is not particularly limited, but is preferably an aqueous specimen, such as blood (whole blood, plasma, serum, etc.), urine, interstitial fluid, lymph, synovial fluid, milk, cerebrospinal fluid, pus, saliva, tears, mucus. , runny nose, phlegm, ascites, water, semen, etc., nasal cavity, bronchi, lungs, skin, peritoneal cavity, various organs, joints, bones, etc. Washing fluid after washing, cell culture supernatant, or column elution A liquid etc. are mentioned. These samples are used for measurement as they are, or diluted with various buffers or the like, or concentrated after being extracted. The sample used for presepsin measurement is preferably a blood sample, more preferably a human blood sample.

In addition, when using a whole blood sample as a sample to be used for presepsin measurement, within 72 hours, 48 hours, 24 hours, 12 hours, 6 hours, or 4 hours after collection of the whole blood sample Analysis may be performed. Whole blood specimens may also be collected with EDTA or heparinized blood collection tubes. Preferably, a whole blood sample is collected in an EDTA blood collection tube and analyzed within 6 hours, or a whole blood sample is collected in a heparin blood collection tube and analyzed within 4 hours.

4. Presepsin Measurement Kit Also provided herein is a presepsin measurement kit (also referred to as "measurement kit") containing at least one selected from the group consisting of an anti-CD14 antibody, a chromatography column, and a membrane.
The measurement kit preferably includes a method for stabilizing the presepsin level in a sample, a method for suppressing sCD14-derived pseudopresceptin production in a sample, a method for suppressing denaturation of sCD14 in a sample, and a quantification of the amount of true presceptin in a sample. A kit for measuring presceptin used for methods, methods for quantifying only true presepsin, methods for quantifying only pseudo presepsin levels, and the like. The assay kit preferably contains an anti-CD14 antibody, a chromatography column, or a membrane.
"Anti-CD14 antibody", "chromatographic column" or "membrane" are specifically as described above.

The measurement kit more preferably contains auxiliary reagents for presepsin measurement. Examples of auxiliary reagents include primary antibodies, secondary antibodies, labeling antibodies, labeling enzymes, labeling substances such as colloidal gold, chromogenic substrates, fluorescent substrates (Amplex™ Red, AttoPhos™, 4-MUP, etc.), chemiluminescent substrates (luminol, CDP-Star™, AMPPD™, CSPD™, etc.), specific binding substances such as biotin-streptavidin, insoluble carriers, blocking agents, diluents, washing solutions, standard substances and the like, but are not limited to these.

Auxiliary reagents for presepsin measurement are appropriately combined and used according to the method of presepsin measurement.

The primary antibody is preferably an antibody that binds to presepsin, more preferably an antibody that recognizes an epitope different from that of the antibody. Examples include F1106-13-3 antibody and F1031-8-3 antibody described in Example 3 of WO2004/044005. Reference can also be made to WO2005/108429.

Either the antibody used for preseptin measurement or the primary antibody may be the labeled antibody. If neither the antibody used for presepsin measurement nor the primary antibody are labeled, a labeled secondary antibody may be used.

Examples of insoluble carriers include magnetic particles, beads, glass, cellulose, nitrocellulose, porous synthetic polymers, glass fibers, polyacrylamide, nylon, polystyrene, polyvinyl chloride, polypropylene, plastic plates, latex particles, non-woven fabric, filter paper, and the like. mentioned.

Labels for antibodies used for presepsin measurement are preferably enzymes such as peroxidase (HRP), alkaline phosphatase (ALP), β-galactosidase, colloidal gold, etc., but are not limited to these.

For example, when HRP is used, chromogenic substrates include 3,3',5,5'-tetramethylbenzidine (TMB), o-phenylenediamine (OPD) and the like. When ALP is used, the chromogenic substrate includes p-nitrophenyl phosphate (pNPP) and the like. Examples of chromogenic substrates when using β-galactosidase include o-Nitrophenyl-β-D-Galactopyranoside (ONPD) and the like.

For example, a measurement kit for sandwich ELISA may contain antibodies and primary antibodies (either of the antibodies may be enzyme-labeled) used for presepsin measurement, chromogenic substrates, diluents, standard substances, and the like. If the antibody and primary antibody are unlabeled, it may contain a labeled secondary antibody.

A measurement kit for chemiluminescent enzyme immunoassay (CLEIA) may contain, for example, an antibody immobilized on magnetic particles or the like, an enzyme-labeled antibody, a chemiluminescent substrate, a diluent, a washing solution, and the like.

A measurement kit for fluorescence enzyme immunoassay (FEIA) can contain, for example, an antibody immobilized on magnetic particles or the like, an enzyme-labeled antibody, a fluorescent substrate, a diluent, a washing solution, and the like.

An electrochemiluminescence immunoassay (ECLIA) measurement kit can contain, for example, biotinylated antibody, Ru(bpy)3-labeled antibody, streptavidin-coated magnetic particles, tripropylamine, and the like.

A measurement kit for immunochromatography is a test strip provided with a sample application section, a reagent section, a detection section, and an absorption section so that the liquid specimen added to the test application section moves in the above order. For example, the reagent portion can be impregnated with a labeled second antibody, and an insoluble carrier to which the first antibody is bound can be placed in the detection portion.

As for the test strip, use of a porous carrier or the like is exemplified. Porous carriers include, for example, nitrocellulose, cellulose, cellulose derivatives, nylon, nylon fibers, glass fibers, porous synthetic polymers and the like. The absorbent part includes an absorbent polymer such as a sponge of a water-absorbent material, cellulose filter paper, filter paper, and the like.

Since it has been reported that the presepsin blood concentration is characteristically increased in patients with sepsis, the presepsin measurement kit is a kit for detecting sepsis or a kit for assisting in the detection or diagnosis of sepsis. good too.

In addition, the measurement kit can be used as a diagnostic agent for sepsis or as an auxiliary agent for sepsis diagnosis. For the purpose of detecting such sepsis, the kit for measuring presepsin is used to detect the possibility of sepsis in a subject when the presepsin concentration in the subject's specimen measured using an antibody is higher than the cutoff value. It can be determined to have potential and aid in detection or diagnosis. At this time, the cut-off value is 314-600 pg/mL, preferably 400-580 pg/mL, more preferably 450-550 pg/mL, still more preferably 500 pg/mL.

In some aspects, the kit for measuring preseptin is used, for example, to distinguish between sepsis and systemic inflammatory response syndrome (SIRS); to assess the risk of aggravation of sepsis; to predict the prognosis of sepsis (mortality detection of postoperative infection; detection of disseminated intravascular coagulation (DIC); detection of infectious DIC; detection of cardiac disease; inflammatory bowel disease (Crohn's disease, ulcerative colitis), febrile neutropenia (FN), or detection of hemophagocytic syndrome (HPS) It can be used for detection or evaluation. The kit for measuring presepsin may be a kit for detecting or evaluating at least one of the above diseases.

5. Method for Differential Quantification of Pseudopresceptin and True Presceptin A method for differential quantification of pseudopresceptin and true presepsin in a specimen includes using a gel filtration method.
“Differential quantification of pseudopreseptin and true preseptin” specifically means separation of sCD14-derived pseudopresceptin and presceptin as in Example 4.
For example, according to the method according to Example 4, it can be performed using a gel filtration method, and the presepsin level in each fraction is measured. More specifically, the sample before presepsin measurement is separated by, for example, gel filtration chromatography to obtain a fraction containing true preseptin, and the preseptin level is measured to quantify the true presceptin level. It is possible to For quantification, the above-described method for measuring presceptin is used. For example, in Example 4, the amount of preseptin in fractions containing each presceptin is compared with a calibration curve prepared using a standard concentration of presceptin to determine true presceptin. value can be obtained.
Alternatively, the pseudopresepsin value can be obtained by obtaining a pseudopresceptin fraction instead of the true presepsin-containing fraction and measuring the pseudopresepsin value. If a false preseptin value is obtained, it is possible to measure the prepsin value of the specimen and obtain the true presepsin measurement value from the sum of the true preseptin value and the false preseptin value (total preseptin amount).

6. Presepsin measurement system Further, here, a method for stabilizing the presepsin level in a sample, a method for suppressing production of pseudoprespsin derived from sCD14 in a sample, a method for suppressing denaturation of sCD14 in a sample, and a method for measuring the amount of true presceptin in a sample. Provided is an apparatus such as a fully automated apparatus incorporating a quantification method, a method for quantifying only true presepsin, a method for quantifying only pseudo presepsin, etc. into the system.
Also, by constructing a system that performs the separation and quantification, it is possible to construct a system that obtains a true presepsin value simply by directly applying a specimen.
Each configuration of the system is, for example, as follows.
1) Specimen cartridge part: for example, a part that holds a microtube containing a collected specimen used for measurement;
2) Specimen processing unit: a part that performs specimen sampling, for example, a part that has a nozzle for direct sampling or a part that can be equipped with a disposable chip and collects a sample;
3) Reagent cartridge part: A part containing reagents and the like necessary for presepsin measurement.
4) Presepsin measurement part: A part that measures presceptin in a sample, and corresponds to the part that measures presceptin or the part that measures presceptin described in the item of the presepsin measurement kit;
5) Calculation part: corresponds to the part that calculates the presepsin value based on the measurement in the measurement part in 4);
6) display part: corresponds to the part that displays the presepsin value calculated in the calculation part of 5);
and the like, and a presepsin measurement system including at least one selected from the group consisting of 1) to 6) above. The above configurations 1) to 6) are basically processed in the order of 1) → 2) → 3) → 4) → 5) → 6) in the system. becomes.

All publications, patent publications, and other patent documents cited herein are hereby incorporated by reference.
The present invention will be described in more detail with reference to the following examples, but the present invention should not be understood as being limited to these examples.

Example 1: Effect of Specimen Shaking on Presepsin Measurement Value Two normal human plasma (EDTA) samples from TENNESSEE Blood Services (#R255292, #R255287) were used as specimens, and a presepsin ELISA kit (Mochida Pharmaceutical Co., Ltd., Lot: 2S015) was used to measure each presepsin level in each shaken specimen.
The presepsin values of the samples were measured before shaking (0 minutes) and 10 minutes and 60 minutes after shaking.
For shaking, 2 mL of the specimen was dispensed into a microtube, and a TAITEC Vortex Mixer was used for up to 60 seconds, followed by EYELA CUTE CM-1000 (1800 rpm) for up to 60 minutes. 50 µL of each sample was sampled from 2 mL of each sample for use.
Table 1 shows the measured values of each presepsin.

Table 1: Changes in presepsin concentration over time by shaking

From the above results, a phenomenon was observed in which the presepsin level in the sample increased over time due to vigorous shaking of the sample. This increased value was a cutoff value for diagnosing sepsis; a value greater than 500 pg/mL. It should be noted that the physical stimulation (shaking) model applied in this experiment is an extreme condition for examination, and unlike the normal handling or transport conditions of blood specimens, it is extremely abnormal and violent. Let me give a supplementary explanation.

Example 2: Investigation of cause of increase in presepsin measurement value 100 μL each was dispensed into each 1.5 mL microtube and shaken for 10 minutes (shaking conditions are the same as in Example 1). The CD14-absorbed human serum was prepared by binding the F1024-1-3 antibody (prepared by the method described in Examples 1 and 2 of WO2001/72993) to a chromatography resin (Sepharose 4FF). prepared using affinity chromatography. Normal human serum was passed through the affinity column, CD14 was adsorbed to the resin, and the non-adsorbed fraction (pass-through fraction) was used.
Presepsin concentration ( pg/mL) was measured. Also, the sCD14 concentration (ng/mL) in each sample was measured using a CD14 ELISA kit (R&D Systems).
Table 2 shows the presepsin concentration and sCD14 concentration in each sample.

Table 2: Presepsin concentration (pg/mL) and sCD14 concentration (ng/mL) in each sample

From the above results, normal human serum without special treatment showed a marked increase in presepsin concentration after 10 minutes of shaking. No significant increase was observed.
The sCD14 concentration decreased by more than half with 10 minutes of shaking in normal human serum, but remained almost unchanged in CD14-absorbed human serum.
It was confirmed that the sCD14 content in normal human serum could be reduced by two digits by CD14 absorption treatment by affinity chromatography. (Table 2)
These results suggested the possibility that the increase in presepsin level due to shaking was derived from sCD14 in serum.

Example 3: Examination of the effect of anti-CD14 antibody on changes in presepsin concentration About 30 μg of F1024-1-3 antibody (anti-CD14 antibody) was added to 1 mL of normal human plasma (EDTA), shaken for 10 minutes, and then The presepsin concentration of the sample was measured using a presepsin kit (PATHFAST (trade name)), and the effect of shaking the anti-CD14 antibody on changes in presepsin concentration was examined.
The results are shown in FIG. Using normal human plasma, presepsin was measured in the presence and absence of F1024-1-3 antibody (anti-CD14 antibody), and F1024-1-3 antibody suppressed the increase in presepsin level caused by shaking. was shown.

It was also confirmed that the addition of the anti-CD14 antibody suppressed the change in the presepsin concentration due to shaking even when the plasma of a septic patient was used.
On the other hand, when an antibody other than an anti-CD14 antibody, such as a non-anti-CD14 antibody such as an anti-arachidonic acid antibody or an anti-hCG antibody, was added to the plasma specimen, no effect of shaking on suppressing changes in presepsin concentration was observed.

From the above results, it was clarified that the addition of anti-CD14 antibody can suppress the increase in presepsin concentration due to shaking, ie, intense physical stimulation.

Example 4 Gel Filtration Analysis of Shaking-Processed Human Plasma Normal human plasma (#STL130145) was dispensed in 0.5 mL aliquots into 3 tubes, and one tube was allowed to stand as a control, followed by gel filtration analysis. used for The remaining two
After shaking treatment for 10 minutes, gel filtration analysis was performed respectively. At this time, about 200 μg of F1024-1-3 antibody was added to one tube, which was allowed to stand at room temperature for 10 minutes and then shaken.
・Column Superdex 75 10/300 GL (1cmφ×30cm)
・Fraction 0.5 mL/tube ・Amount of specimen added to column 100 μL

(1) Each fraction of gel filtration was measured with a preseptin ELISA kit, and the absorbance of the measured solution at 450-650 nm is shown in FIG.

(2) Each gel filtration fraction was measured with a CD14 ELISA kit (R&D Systems), and the absorbance of the measured solution at 450-650 nm is shown in FIG.

As shown in FIG. 2, normal plasma as a control was eluted as a single peak near fraction 19. This elution position is the elution position of true presepsin with a molecular weight of 13 KD.
On the other hand, two peaks near fractions 13 and 19 were observed in the gel filtration analysis results of the shake-treated human plasma sample. Fraction 13 corresponds to the position of the void in the gel filtration column and is the high molecular weight fraction.
The sample to which the anti-CD14 antibody was added and shaken showed an elution pattern similar to that of the control.
Based on this fact, shaking the sample generates a high-molecular-weight presepsin-like substance (a substance that reacts with anti-presceptin antibodies, although it is different from true presceptin; hereinafter sometimes referred to as "pseudopresceptin"). It was found that an increase in the measured value of presepsin (increase in apparent concentration) occurred with the production. In addition, it was revealed that the production of pseudopresepsin was effectively suppressed by the coexistence of anti-CD14 antibody.

In addition, the chromatographic elution pattern of the substance detected by the CD14 measurement kit shown in FIG. changed to a peak with shoulders. This suggested that sCD14 with a higher molecular weight was produced by shaking. In addition, since this shoulder portion coincided with the elution position where pseudopreseptin was detected, it was suggested that pseudopresceptin was the result of sCD14 molecules being denatured and aggregated to have a high molecular weight.

Consideration will be made based on the above experimental results.
sCD14-ST (preseptin) was characterized by having a molecular weight of 13±2 kDa in SDS-PAGE under non-reducing conditions, and was eluted on the low molecular weight side in gel filtration of plasma specimens (FIG. 2). On the other hand, CD14 present in the blood is also called soluble CD14 (sCD14), and molecular species with a molecular weight of about 55 kDa or about 49 kDa are known, and this is observed in the chromatographic pattern in control normal human plasma in FIG. It is considered that It was also suggested that the higher-molecular-weight substances produced by shaking reacted with the presepsin assay kit and caused the abnormal values of the presepsin assay as false presepsin. This is because the presepsin measurement kit is an analytical quantification method that immunologically recognizes the specific sequence (steric structure) of the sCD14 molecule, so even denatured high-molecular-weight sCD14 molecules react with the presepsin measurement kit. There are some things that must be assumed, and the results of this experiment suggest that such a situation is occurring.

Based on the above, the following invention is born. In order to suppress the production of pseudopresepsin due to physical stimulation, etc., the sCD14 molecule in the sample should be stabilized, and this can be achieved, for example, by contacting the sample with an anti-CD14 antibody immediately after collection of the sample. can be done. That is, it is possible to prevent occurrence of an abnormal presepsin measurement value due to shaking or the like in the presepsin value measurement. Also, if the sCD14 molecules are removed by absorption or the like immediately after sample collection, pseudopresepsin will not occur.

Furthermore, even in the measurement of a sample that has already produced pseudopresceptin, it is possible to quantify true presceptin by distinguishing true presceptin from pseudopresceptin. For example, when presepsin is measured, (i) pseudopresceptin is removed by passing through a 30 kDa ultrafiltration membrane, (ii) an antibody that reacts with pseudopresceptin but does not react with true presceptin is used. Pretreatment such as adsorption removal/separation of pseudo-presepsin enables correct presceptin measurement.

Furthermore, as in Example 4, it is possible to separate and analyze pseudopresceptins and true presceptins on a chromatographic graph. Presepsin levels in fractions containing true presepsin can be quantified. Also, if the pseudopreseptin can be quantified by any means, the true presceptin level can be obtained by calculation. For example, a "pseudopreseptin value" can be obtained by performing a presepsin measurement on a chromatographically separated fraction containing the "pseudoprespsin". If a pseudopreseptin value is obtained, it is also possible to measure the prepsin value of the sample and obtain the measured value of the pseudopresceptin from the total value of the true presceptin value and the pseudopresceptin value (total presepsin amount).
Furthermore, by screening a measurement system that responds only to true preseptin and not to pseudopreseptin, it is possible to quantify only the true presceptin level without requiring any special treatment.

SEQ ID NO: 1: Amino acid sequence of P03 peptide.
SEQ ID NO: 2: amino acid sequence of S68 peptide.
SEQ ID NO: 3: Amino acid sequence of human full-length soluble CD14.

Claims (14)

A method for stabilizing the measured value of presepsin in a sample, comprising the step of contacting the sample with an anti-CD14 antibody immediately after sample collection and before presepsin measurement . A method for suppressing pseudopreseptin production in a sample, comprising the step of contacting the sample with an anti-CD14 antibody immediately after collection of the sample. A method for inhibiting denaturation of sCD14 in a specimen, comprising the step of contacting the specimen with an anti-CD14 antibody immediately after collection of the specimen. 4. The method according to any one of claims 1 to 3, comprising adsorbing and removing sCD14 in the specimen by the step of contacting the specimen with the anti-CD14 antibody. 4. The method according to any one of claims 1 to 3, wherein the step of contacting the specimen with the anti-CD14 antibody forms a conjugate between sCD14 in the specimen and the antibody-CD14 antibody. A method for quantifying the true amount of presepsin in a sample, comprising the step of contacting the sample with an anti-CD14 antibody before presepsin measurement. 7. The method according to claim 6, wherein the step of contacting the specimen with the anti-CD14 antibody adsorbs and removes the pseudopresceptin using an anti-CD14 antibody that reacts with the pseudopresceptin in the specimen. A method for quantifying only true preceptsin, comprising the step of separating true presceptin from pseudopresceptin before presepsin measurement. 9. A method according to claim 8, characterized in that the differentiation between pseudopresceptins and true presepsins is a chromatographic separation. 9. A method according to claim 8, characterized in that the differentiation between pseudopreseptins and true preseptins is membrane separation. A method for quantifying only pseudopresceptin, comprising a step of separating pseudopresceptin from true presepsin before presepsin measurement. A method for quantifying a true presepsin level, comprising measuring the presepsin of a specimen to calculate a presepsin level, and subtracting the pseudo presepsin value obtained by the method of claim 11 from the calculated presepsin level. 13. A presepsin measurement kit for use in the method according to any one of claims 1 to 12, comprising at least one selected from the group consisting of an anti-CD14 antibody, a chromatography column, and a membrane. selected from the group consisting of a sample cartridge unit, a sample processing unit, a reagent cartridge unit, a preseptin measurement unit, a calculation unit, and a display unit for use in the method according to any one of claims 1 to 12 Presepsin measurement system comprising at least one.

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