JPWO2019167935A1 - Use of anti-CD14 antibody useful for preceptin measurement - Google Patents

Abstract

INDUSTRIAL APPLICABILITY The present invention provides a method for stabilizing a preceptin measurement value in a sample, which comprises a step of contacting the sample with an anti-CD14 antibody immediately after collecting the sample. This provides a method for obtaining the original preceptin value in the sample without increasing the measured value of preceptin even if the sample to be used is subjected to a physical stimulus such as vigorous stirring when measuring preceptin in the sample. Will be done.

Description

The present invention relates to a method for suppressing an increase in an apparent preceptin measurement value in a sample due to a physical stimulus of the sample in the measurement of preceptin in the sample.

The CD14 molecule is a glycoprotein expressed on the membrane surface of mononuclear cell cells, and is known to have a function as a receptor for LPS (lipopolysaccharide). There are two types of CD14 molecules, a membrane-bound CD14 (mCD14) expressed on the cell surface and a soluble CD14 (sCD14). As sCD14, sCD14 having 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 sepsis (SEPSIS), acquired immunodeficiency 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 erythematosus (SLE). Therefore, these high molecular weight sCD14s 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 referred to as preceptin) exists as a new molecular species of sCD14 whose blood concentration is characteristically increased in septic patients.

sCD14-ST (preceptin) is characterized in that it is electrophoresed to a molecular weight of 13 ± 2 kDa under non-reducing conditions under SDS-PAGE among sCD14, and retains the N-terminal portion of CD14. Compared with the high molecular weight sCD14, it has an amino acid sequence in which the C-terminal side is largely deleted, and unlike the high molecular weight sCD14, it does not have LPS binding ability. Moreover, since preceptin exhibits immunogenicity different from that of high molecular weight sCD14, it is possible to distinguish between the two by using an antibody. Preceptin specifically increases the blood concentration in septic patients (Patent Document 1). In addition, it has been reported that the blood level of sepsis patients is higher than that of patients with systemic inflammatory response syndrome (SIRS), which is difficult to distinguish from sepsis, and preceptin is a specific diagnostic marker for sepsis. (Non-Patent Document 3).

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

Currently, for the measurement of preceptin, a measurement system using a rabbit-derived polyclonal antibody as a specific antibody against preceptin has been put into practical use, and measurement kits have been put on the market in Europe and Japan (PATHFAST (trade name) Presepsin, LSI Co., Ltd.). Medience).

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

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

International release WO2005 / 108429 International release WO2004 / 044005

Hayashi et al., Infection and Immunoty, 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 described above, with regard to the preceptin measurement kit, if a physical stimulus such as vigorous stirring is applied to the sample to be used, the measured value of preceptin increases, and the original preceptin value in the sample may not be obtained. There is concern. For this reason, it is said that care should be taken when handling the preceptin measurement kit so as not to give vigorous agitation.

In such a situation, when measuring preceptin in a sample, even if a physical stimulus is applied to the sample to be used, the measured value of preceptin hardly increases, and a method for obtaining the original measured value of preceptin in the sample. Was required.

Alternatively, when measuring preceptin in a sample, even if a physical stimulus is given to the sample, the apparent preceptin measurement value in the sample hardly increases, and a method that enables stable preceptin measurement regardless of how the sample is handled. Was required.

An object of the present invention is to provide a method useful for measuring preceptin in a sample, which can solve at least one of the conventional problems.

As a result of diligent studies by the present inventor, the phenomenon that the measured value of preceptin increases when a physical stimulus such as vigorous stirring is applied to the sample is caused by a product derived from sCD14 in the sample generated by the physical stimulus. I found that. Therefore, the present inventor can eliminate the influence of the product by, for example, contacting the sample with the anti-CD14 antibody or separating it from preceptin by using chromatography. The present invention was completed with the idea that the original preceptin value 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 preceptin ELAISA kit, and it was confirmed that the measured value of preceptin increased by shaking the sample. ..
It was also confirmed that in the examples, the increase in preceptin level by shaking was increased only in the sample containing blood sCD14, and it was derived from sCD14 in the serum, using human serum and CD14-absorbed serum of normal humans. ..
Further, in the example, when preceptin was measured in the presence or absence of F1024-1-3 antibody (anti-CD14 antibody) using normal human plasma, the increase in preceptin level caused by shaking was measured in F1024-1. -3 It was confirmed that the antibody suppressed.
Moreover, in the example, it was confirmed that the increase in the preceptin level due to shaking was related to sCD14 in the sample. Specifically, as a result of analyzing each fraction of the sCD14-derived product produced by shaking by gel filtration, the elution pattern of the sample to which the control and the anti-CD14 antibody were added and shaken was almost the same, and the elution pattern of the sample was almost the same. It was confirmed that a higher molecular weight agglomerate was produced from sCD14 by shaking (this high molecular weight agglomerate is also referred to as "product derived from sCD14" or "pseudopreceptin"). It was also confirmed that the formation of the high molecular weight aggregate can be effectively suppressed by coexisting the anti-CD14 antibody in the sample. Furthermore, it was confirmed that the high molecular weight aggregates could be separated by chromatography.

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

Conventionally, when measuring preceptin in a sample, it has been noted that if the sample is agitated (this includes, for example, vigorous short-term agitation and gentle long-term agitation), the measured value may be affected. However, the cause affecting this measured value is sCD14 in the blood, and the sample is brought into contact with an anti-CD14 antibody (for example, F1024-1--3 antibody) or separated from preceptin by chromatography. It was surprising that the effect of the product on the measured value of preceptin could be eliminated by such means.

The present invention may include the following aspects.
[1] A method for stabilizing a preceptin measurement value in a sample, which comprises a step of contacting the sample with an anti-CD14 antibody immediately after collecting the sample.
[2] A method for suppressing the production of pseudopreceptin in a sample, which comprises a step of contacting the sample with an anti-CD14 antibody immediately after collecting the sample.
[3] A method for suppressing denaturation of sCD14 in a sample, which comprises a step of bringing the sample into contact with an anti-CD14 antibody immediately after collecting the sample.
[4] The method according to any one of the above aspects [1] to [3], which comprises adsorbing and removing sCD14 in the sample by a step of bringing the sample into contact with an anti-CD14 antibody.
[5] The embodiment according to any one of the above aspects [1] to [3], wherein the sCD14 in the sample and the antibody CD14 antibody form a conjugate by the step of contacting the sample with the anti-CD14 antibody. The method described.
[6] A method for quantifying the true amount of preceptin in a sample, which comprises a step of contacting the sample with an anti-CD14 antibody before measuring preceptin.
[7] The method according to the above aspect [6], wherein pseudoreceptin is adsorbed and removed using an anti-CD14 antibody that reacts with pseudopreceptin in the sample by a step of contacting the sample with the anti-CD14 antibody. ..
[8] A method for quantifying only true preceptin, which comprises a step of separating false preceptin and true preceptin before measurement of preceptin.
[9] The method according to the above aspect [8], wherein the fractionation of the pseudopreceptin and the true preceptin is a separation by chromatography.
[10] The method according to the above aspect [8], wherein the separation of pseudopreceptin and true preceptin is separation by a membrane.
[11] A method for quantifying only sham preceptin, which comprises a step of separating sham preceptin from true preceptin before measurement of preceptin.
[12] A method for quantifying a true preceptin value, which comprises measuring a sample of preceptin, calculating a preceptin value, and subtracting the pseudopreceptin value obtained by the method according to the above aspect [11] from the calculated preceptin value. ..
[13] Containing at least one selected from the group consisting of anti-CD14 antibody, chromatographic column, and membrane for use in the method according to any one of the embodiments [1] to [12]. Preceptin measurement kit.
[14] Consists of a group consisting of a sample cartridge, a sample processing unit, a reagent cartridge unit, a preceptin measuring 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 preceptin measurement system comprising at least one selected.

According to some embodiments, when measuring preceptin in a sample, even if a physical stimulus is applied to the sample to be used, the original preceptin measurement value in the sample is obtained with almost no increase in the preceptin measurement value. Can be done. For example, the product derived from sCD14 can be eliminated from the influence of the product by contacting the sample with the anti-CD14 antibody or separating it from preceptin by using chromatography, and the physics in the measurement of preceptin in the sample. It is possible to effectively prevent an increase in the preceptin measurement value due to stimulus, and the original preceptin measurement value in the sample can be obtained. Further, according to some other aspects, the preceptin measurement value in the sample can be obtained. At that time, even if a physical stimulus such as vigorous stirring is given to the sample, that is, the correct preceptin measurement value can be obtained regardless of how the sample is handled.

It is a figure which shows the influence on the preceptin concentration of the addition of an anti-CD14 antibody. It is a figure which showed the result of having measured each fraction of the gel filtration in Example 4 with a preceptin ELAISA kit, and measured the absorbance of the measuring solution at 450-650 nm. It is a figure which showed the result of having measured each fraction of the gel filtration in Example 4 with a CD14ELAISA kit, and measured the absorbance of the measuring solution at 450-650 nm.

In some embodiments, when measuring preceptin in a sample, even if a physical stimulus such as vigorous stirring is applied to the sample to be used, the measured value of preceptin in the sample hardly increases, and the original preceptin in the sample is not increased. It relates to a method useful for measuring preceptin in a sample from which a value can be obtained.

In addition, some other aspects relate to a method of removing a preceptin-like product (pseudopreceptin) produced by a physical stimulus. In yet some other aspects, when using the preceptin measurement kit, even if a physical stimulus such as vigorous stirring is applied to the sample, the apparent preceptin measurement value in the sample does not increase, regardless of how the sample is handled. , Concerning a method that enables stable preceptin measurement.

Here, the following aspects [1] to [14] are provided.
[1] The first aspect is a method for stabilizing a preceptin measurement value in a sample, which comprises a step of contacting the sample with an anti-CD14 antibody immediately after collecting the sample.
[2] The second aspect is a method for suppressing the production of pseudopreceptin in a sample, which comprises a step of contacting the sample with an anti-CD14 antibody immediately after collecting the sample.
[3] A third aspect is a method for suppressing denaturation of sCD14 in a sample, which comprises a step of contacting the sample with an anti-CD14 antibody immediately after collecting the sample.
[4] The fourth aspect according to any one of the above aspects [1] to [3], which comprises adsorbing and removing sCD14 in the sample by a step of bringing the sample into contact with an anti-CD14 antibody. The method.
[5] The fifth aspect is characterized in that the sCD14 in the sample and the antibody CD14 antibody form a conjugate by the step of contacting the sample with the anti-CD14 antibody, the above-mentioned aspects [1] to [3]. It is the method according to any one aspect of.
[6] A sixth aspect is a method for quantifying the true amount of preceptin in a sample, which comprises a step of contacting the sample with an anti-CD14 antibody prior to measurement of preceptin.
[7] The seventh aspect is characterized in that pseudopreceptin is adsorbed and removed using an anti-CD14 antibody that reacts with pseudopreceptin in the sample by a step of contacting the sample with the anti-CD14 antibody [7]. 6] is the method described in.
[8] An eighth aspect is a method for quantifying only true preceptin, which comprises a step of separating false preceptin and true preceptin before measurement of preceptin.
[9] The ninth aspect is the method according to the above aspect [8], wherein the separation of the pseudopreceptin and the true preceptin is a separation by chromatography.
[10] The tenth aspect is the method according to the above aspect [8], wherein the separation of pseudopreceptin and true preceptin is separation by a membrane.
[11] An eleventh aspect is a method of quantifying only sham preceptin, which comprises a step of separating sham preceptin from true preceptin before measurement of preceptin.
[12] A twelfth aspect comprises measuring the sample preceptin, calculating the preceptin value, and subtracting the pseudopreceptin value obtained by the method according to the above aspect [11] from the calculated preceptin value. This is a method for quantifying the preceptin level.
[13] The thirteenth aspect is selected from the group consisting of an anti-CD14 antibody, a chromatographic column, and a membrane for use in the method according to any one of the above aspects [1] to [12]. A preceptin measurement kit containing at least one.
[14] A fourteenth aspect is a sample cartridge, a sample processing unit, a reagent cartridge unit, a preceptin measuring unit, a calculation unit, for use in the method according to any one of the above aspects [1] to [12]. A preceptin measurement system comprising at least one selected from the group consisting of and a display.

The terms and the like in the present specification will be described below.
"Immediately after collecting a sample" means immediately after collecting a sample from a patient (for example, immediately after collecting blood) or at the same time as collecting a sample (for example, at the same time as collecting blood). Here, immediately after the sample collection, "before transportation", that is, before the sample is collected from the patient (for example, blood is collected) and sent to a specialized center for various sample parameter measurement (for example, blood parameter measurement). included. In particular, it means the period from immediately after collecting a sample (for example, immediately after collecting blood) to sending it to a testing center.
"Before measurement of preceptin" means before starting measurement of preceptin value, and corresponds to, for example, before collecting a sample and starting measurement on the spot, or before measuring preceptin value of a sample arriving at a testing center. ..

The "step of contacting the sample with the anti-CD14 antibody" is, for example,
1) Step of adding anti-CD14 antibody to the sample,
2) An anti-CD14 antibody is pre-existed in a container such as a blood collection tube (for example, the anti-CD14 antibody is immobilized on beads or the like placed in a container such as a blood collection tube), and a sample collected in the container is used. The process of accommodating,
3) A step of accommodating a sample collected in a dedicated container such as a dedicated blood collection tube on which anti-CD14 is immobilized in advance.
4) A step of adsorbing and removing sCD14 in a sample using an anti-CD14 antibody (more specifically, for example, using a column for affinity chromatography packed with a carrier in which an anti-CD14 antibody is bound to a resin or the like, the sample. The process of allowing the antibody to pass through the column),
It is any step selected from the group consisting of the above. By going through these steps, the sCD14 in the sample gains resistance to physical stimulation, or the sCD14 is removed, so that the preceptin measurement value in the sample is stabilized, the pseudopreceptin production in the sample is suppressed, or the sCD14 is suppressed. It is possible to suppress the degeneration of.

Examples of adding the anti-CD14 antibody to the sample include adding the anti-CD14 antibody to the sample collected from the patient.
Further, in order to coexist the anti-CD14 antibody in the sample before transportation, the anti-CD14 antibody may coexist in the sample by adding the anti-CD14 antibody to the sample, or the anti-CD14 antibody may be present in the blood collection tube in advance. This includes coexisting an anti-CD14 antibody with a sample by inserting blood into a blood collection tube.

The “pseudopreceptin” means a product or the like derived from sCD14, in which sCD14 aggregates, unfolds of a three-dimensional structure or the like occurs due to vigorous stirring or the like, and a preceptin-like reaction is exhibited. Therefore, the preceptin-like reaction of pseudopreceptin will apparently increase the preceptin measurement.
"Inhibiting the production of pseudopreceptin" or "suppressing the production of pseudopreceptin" means inhibiting the production or production of pseudopreceptin derived from sCD14.
The “denaturation of sCD14” means that the sCD14 in the sample is aggregated with, for example, two molecules to have a higher molecular weight due to vigorous stirring or the like. Due to the agglutination, a site that reacts with the preceptin measurement appears on the surface of the molecule, resulting in a state of reacting with the antibody that measures the preceptin.
"Suppression of denaturation of sCD14" means to inhibit the pseudopreceptin derived from sCD14 from reacting to the preceptin measurement.
"Physical stimulation" means vigorous agitation (including gentle long-term agitation) when handling a sample, and also means overturning agitation or shaking of a blood collection tube after voting.
The "true preceptin value" means the correct value indicating the amount of preceptin present in the sample, for example, this is the measured value of preceptin in the sample before the physical stimulus is applied to the sample. There is a phenomenon that the apparent preceptin value increases due to the aggregation of sCD14 and the reaction to the preceptin measurement. For example, by preventing sCD14 from aggregating, the preceptin value itself existing in the sample can be known, and a stable and correct preceptin value can be obtained even under the influence of shaking. However, it does not mean that it completely matches the measured value of preceptin in the sample before the physical stimulus is given to the sample.

"Stabilization of preceptin measurements" means ensuring that the specimen exhibits a true preceptin value even when subjected to physical stimulation. That is, for example, by suppressing the aggregation of sCD14, a true preceptin value can be obtained even when a physical stimulus is received.
"Resistance to instability due to physical stimulus" means that sCD14 aggregates due to physical stimulus and suppresses unfolding instability of the three-dimensional structure, thereby showing a preceptin-like reaction in preceptin measurement, and is a true preceptin. In addition to the value, it means blocking the increase in apparent preceptin level.

The "true preceptin" is "preceptin", which will be described later, that is, sCD14-st (soluble CD14 antigen subtype), which is a substance quantified by a usual preceptin measurement kit.
"Separation of false preceptin and true preceptin" is the following method,
1) Chromatographic separation (eg, gel filtration chromatography separation (eg, Example 4)),
2) Separation by membrane,
3) Separation with an antibody that reacts with pseudopreceptin,
And so on.

The true preceptin value is quantified by separating the sample before the preceptin measurement by chromatography (for example, gel filtration chromatography), obtaining a fraction containing the true preceptin, and measuring the preceptin value. Is possible. In the quantification, the preceptin measurement method described later is used. For example, in Example 4, the true preceptin is obtained by comparing the calibration curve prepared by the standard concentration of preceptin with the amount of preceptin in the fraction containing each preceptin. You can get the value. Further, by constructing a system for performing the separation and quantification, it is possible to construct a system for obtaining a true preceptin value only by directly applying the sample.

To remove pseudopreceptin in a sample by means of a membrane, for example, the sample may be passed through a membrane (eg, a 30 kilodalton ultrafiltration membrane) that can remove substances higher in molecular weight than preceptin. Pseudopreceptin is removed by the membrane, and the true preceptin value can be obtained by measuring the preceptin value of the sample from which the pseudopreceptin has been removed.

Further, in order to separate the pseudopreceptin derived from sCD14 from the sample, it is possible to remove the pseudopreceptin by contacting the sample with an antibody that reacts with the pseudopreceptin but reacts with the sCD14 but not with the true preceptin. included. To remove pseudopreceptin with an antibody, the sample and the antibody can be brought into contact with each other, for example, the sample can be allowed to pass through a column on which the antibody is immobilized, and the antibody can adsorb and remove pseudopreceptin derived from sCD14. included.
"Quantifying only the true preceptin value" means that the sample before the preceptin measurement is separated by, for example, gel filtration chromatography, and the preceptin value in the fraction containing the true preceptin is quantified. More specifically, the separated relevant fractions are quantified according to Example 4 described later.
In addition, a "pseudopreceptin value" can be obtained by performing preceptin measurement with a fraction containing "pseudopreceptin" separated by chromatography. Once the pseudopreceptin value is obtained, it is possible to measure the prepsin value of the sample and obtain the measured value of pseudopreceptin from the total value (total preceptin amount) of the true preceptin value and the pseudopreceptin value.
"Quantifying only pseudopreceptin" means that the sample before measurement of preceptin is separated by, for example, gel filtration chromatography, and the value of pseudopreceptin in the fraction containing pseudopreceptin is quantified. More specifically, the separated relevant fractions are quantified according to Example 4 described later.
The “preceptin measurement system” means a device such as a fully automatic device in which each element for carrying out a preceptin measurement is incorporated in the system.

Hereinafter, the present invention will be described in detail. The following embodiments are examples for explaining the present invention, and the present invention is not intended to be limited to this embodiment. The present invention can be implemented in various forms as long as it does not deviate from the gist thereof.

1. 1. Anti-CD14 antibody, membrane and chromatographic column 1.1. Anti-CD14 antibody In some embodiments, an anti-CD14 antibody is used. The anti-CD14 antibody includes 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, "F1024-1-3 antibody" is a hybridoma F1024- obtained by cell fusion of immune cells and myeloma cells immunized with CD14 protein purified from human serum as an antigen in rats. It is an F1024-1-3 antibody produced by 1-3. More specifically, it can be obtained by the method described in the publication according to the known method of Example 1 "Preparation of anti-human CD14 antibody" of WO2001 / 72993 A1.

The F1024-1-3 antibody is an anti-CD14 antibody that specifically recognizes an epitope containing 8 or more amino acids in the regions 285 to 315 of human sCD14 set forth in SEQ ID NO: 3.

In addition, as human anti-CD14 antibodies that control the signal transduction of LPS via human CD14, 3C10 antibody (Steinman: J. Exp. Med., 158: 126 (1983)) and Juan TS that bind to positions 7 to 14 of human CD14. : J. Biol. Chem., 270: 29, 17237 (1995)) and MEM-18 antibody (Bazil: Eur. J. Immunol., 16: 1583 (1986) and Juan TS: J. Biol. Chem., 270, 10, 5219 (1995)) is known.
The 3C10 antibody and the 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. The "antigen-binding fragment" refers to a fragment of an antibody that has substantially the same antigen-binding property as the original antibody. Examples of the antigen-binding fragment include Fab, Fab', F (ab') _{2, and the} like.

1.2. Membrane In some embodiments, a membrane capable of removing pseudopreceptin is used. Preferably, the membrane is a membrane (eg, a 30 kilodalton membrane) capable of removing substances having a higher molecular weight than preceptin. Examples of such a membrane include an ultrafiltration filter (for example, Amicon (trade name) Ultra (Merck Millipore) and the like).

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

2. Preceptin "Preceptin" is also referred to as sCD14-ST (soluble CD14 antigen subtype). CD14 includes soluble CD14 (sCD14) in addition to membrane-bound CD14 (mCD14), and a plurality of soluble CD14s having different molecular weights are present in blood. Preceptin is a soluble fragment of CD14 and refers to a substance having the following properties 1) to 3).

1) Under non-reducing conditions, SDS-PAGE has a molecular weight of 13 ± 2 kDa.
2) The N-terminal sequence has 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), and
3) Specific binding to an antibody prepared using a peptide (S68 peptide) consisting of the 16 amino acid residues shown in SEQ ID NO: 2 (corresponding to the amino acid sequences 53 to 68 of the amino acid sequence of SEQ ID NO: 3) as an antigen. To do.

Preceptin is human preceptin unless otherwise specified. The preceptin is, for example, a preceptin standard product (rsCD14-ST according to Example 16 of WO2005 / 108429). Alternatively, a substance obtained by modifying a part of preceptin, which has a binding activity as preceptin, may be used.

3. 3. sCD14
As sCD14, sCD14 having a molecular weight of about 55 kDa and about 49 kDa is known. The sCD14 is also referred to herein as a "high molecular weight soluble CD14" or a "high molecular weight sCD14".
The sCD14 may be prepared, for example, by adsorbing a 3C10 antibody affinity column in the body fluid of a normal person (see Example 23 of WO2005 / 108429).

4. Measurement method of preceptin “Preceptin measurement” is, for example, immunological measurement of preceptin. To immunologically measure preceptin, for example, an anti-preceptin antibody (for example, a specific antibody such as S68 polyclonal antibody, P03 recognition monoclonal antibody, P03 specific polyclonal antibody, etc.) (hereinafter, "antibody used for preceptin measurement" is used. ”) And a sample containing preceptin (for example, a blood sample) are brought into contact with each other. The term "measurement" can be used interchangeably with terms such as "detection,""quantitative," and "assay," and is used to include quantitative and qualitative decisions. The measurement of preceptin is preferably carried out in vitro.

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

When measuring preceptin in a sample, even if a physical stimulus is applied to the sample to be used, the original measured value of preceptin in the sample can be obtained with almost no increase in the measured value of preceptin. For example, the product derived from sCD14 can be separated from preceptin by contacting the sample with the anti-CD14 antibody or by using chromatography, etc., so that the influence of the product can be eliminated, and the physical effect in the measurement of preceptin in the sample can be eliminated. It is possible to effectively prevent the increase in the preceptin measurement value due to stimulation, and the original preceptin measurement value in the sample can be obtained. In addition, by using a membrane, pseudopreceptin in the sample can be removed and the preceptin measurement can be performed. It is possible to suppress an increase in the apparent preceptin measurement value in.

The "S68 antibody" is an anti-antibody obtained by purifying a polyclonal antibody obtained from a non-human mammal immunized using the S68 peptide (SEQ ID NO: 2) as an immunogen by using a column on which the S68 peptide is immobilized. S68 peptide polyclonal antibody. The "S68 peptide" is a peptide consisting of the amino acid sequence of SEQ ID NO: 2 (amino acid sequence at positions 53 to 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" is also referred to as a region corresponding to positions 52 to 61 of the amino acid sequence represented by SEQ ID NO: 1 (krvdadadpr: SEQ ID NO: 3 (human full-length soluble CD14): P03 sequence in preceptin. ) Is a monoclonal antibody. The P03 recognition monoclonal antibody is, for example, the monoclonal antibody described in WO2015 / 129774.

The "P03-specific polyclonal antibody" is a conventional rabbit-derived anti-preceptin polyclonal antibody (S68 antibody), that is, a polyclonal antibody obtained by immunizing a rabbit with an S68 peptide (SEQ ID NO: 2), and immobilizing the S68 peptide. It is prepared by purifying with an affinity column. By purifying the polyclonal antibody with an affinity column on which the P03 peptide (SEQ ID NO: 1) is immobilized instead of the S68 peptide-immobilized column, an anti-preceptin polyclonal antibody having high reactivity with preceptin can be obtained. A specific method for producing a P03-specific polyclonal antibody is as described in WO2017 / 033281.

Since preceptin is known as a marker used for detecting sepsis, the method for measuring preceptin is used for a method for detecting sepsis, which comprises contacting an antibody used for measuring preceptin with a sample containing preceptin. can do.

That is, the method for measuring preceptin is
(1) A step of measuring the preceptin concentration in a sample of a subject using an antibody used for preceptin measurement, and a step of measuring the preceptin concentration in the sample.
(2) Including a step of determining whether or not the preceptin concentration obtained in (1) above is higher than the cutoff value.
It can also be said to be a method for detecting sepsis.

When the sample is a blood sample, the cutoff value is, for example, 314 to 600 pg / mL, preferably 400 to 580 pg / mL, more preferably 450 to 550 pg / mL, and even more preferably 500 pg / mL.

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

In addition, the method for measuring preceptin is, for example, discrimination between sepsis and systemic inflammatory response syndrome (SIRS); risk assessment of aggravation of sepsis; prognosis prediction of sepsis (mortality prediction); severity of sepsis. Degree assessment; Detection of postoperative infections; Detection of disseminated intravascular coagulation (DIC); Detection of infectious DIC; Detection of heart disease; Respiratory infection with bacterial infection, inflammatory bowel disease For the detection or evaluation of at least one disease selected from the detection of (Clone's disease, ulcerative colitis), febrile neuropenia (FN), or hemophagocytic syndrome (HPS). Can be used. That is, the method for measuring preceptin can also be said to be a method for detecting or evaluating the disease.

Postoperative infection is a general term for infections that develop after surgery, and means all infections caused by surgery and the adjuvant therapy required for it. In addition, postoperative infections include all diseases diagnosed as postoperative infections based on 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, myocardial infarction, ischemic stroke, hemorrhagic stroke and transient. Examples include cerebral ischemic attacks.

Respiratory infections associated with 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, most of which are caused by viral infections of the upper respiratory tract that spread to the lower respiratory tract, but in some cases secondary to bacteria. Infection continues. If there are signs of secondary bacterial infection, antibiotics are indicated. Chronic lower respiratory tract infection is a condition in which persistent bacterial infection is established in the lower respiratory tract, which has organic disorders due to bronchiectasis or chronic obstructive pulmonary disease, and there are persistent infection and acute exacerbation. Diseases that cause organic damage to the lower respiratory tract include bronchiectasis, chronic obstructive pulmonary disease, chronic bronchitis, diffuse panbronchiolitis, old-fashioned pulmonary tuberculosis, pneumoconiosis, non-tuberculous mycobacteriosis, and allergies. Includes sexual bronchopulmonary aspergillosis, pulmonary fibrosis, chronic bronchial asthma, etc. Antibiotics are indicated for both persistent infection and acute exacerbation. Pneumonia includes community-acquired pneumonia and nosocomial pneumonia. Community-acquired pneumonia is preferred.

Examples of the method for immunologically measuring preceptin using the antibody used for preceptin measurement include enzyme immunoassay (hereinafter, also referred to as EIA or ELISA), chemoluminescent enzyme immunoassay (CLEIA), and chemoluminescent immunity. Measurement method (CLIA), fluorescent antibody method (FAT), fluorescent enzyme immunoassay (FEIA), electrochemical luminescence immunoassay (ECLIA), radioimmunoassay (RIA), immunochromatography, aggregation method, competitive method, etc. However, the method is not limited thereto. Either a direct method or an indirect method may be used. A sensitization method for detecting by forming a biotin-avidin (streptavidin) complex may be used.

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

In sandwich ELISA, two or more types of antibodies having different antigen recognition sites are used, one antibody is fixed to the 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. It is a method of measuring by forming.

In chemiluminescent enzyme-linked immunosorbent assay (CLEIA), an antigen in a sample is reacted with an antibody immobilized on magnetic particles or beads, and then the enzyme-labeled antibody is reacted for washing (B / F separation). ) After that, a chemiluminescent substrate is added, and after an enzymatic reaction, the luminescence intensity is measured.

For example, an antibody in which an antigen in a sample is bound to biotin is reacted in a liquid phase, the antibody is trapped in magnetic particles to which streptavidin is bound, washed (B / F separation), and then an enzyme-labeled antibody is reacted. Measure the emission intensity.

When alkaline phosphatase (ALP) is used as the labeling enzyme, CDP-StarTM, AMPPDTM, and CSPDTM are preferably used as chemiluminescent substrates. When the labeling enzyme is HRP, luminol is preferably used as the chemiluminescent substrate.

The detection sensitivity is generally said to be higher in the order of chemiluminescence> fluorescence> absorption (color development), and the measurement method can be selected according to the desired sensitivity.

In chemiluminescence immunoassay (CLIA), the antigen in the sample is reacted with an antibody immobilized on magnetic particles, and then the antibody labeled with the chemiluminescent substance is reacted and washed (B / F separation). After that, it is a method of measuring the luminescence intensity. Aclidinium or the like is used as the labeling substance.

In Fluorescent Enzyme Immunoassay (FEIA), an antibody immobilized with an antigen in a sample is reacted, then an enzyme-labeled antibody is reacted, and after washing (B / F separation), a fluorescent substrate is added. This is a method of measuring the fluorescence intensity after the enzymatic reaction. HRP, ALP, or the like is used as the labeling enzyme. When the labeling enzyme is HRP, AmplexTMRed or the like is used as the fluorescent substrate, and when the labeling enzyme is ALP, 4-MUP (4-Methyllumbelliphenyl phosphate), AttoPhosTM or the like is preferably used.

Electrochemiluminescence immunoassay (ECLIA) is performed by reacting an antigen in a sample with an antibody immobilized on magnetic particles and an antibody labeled with an electrochemical luminescent substance, and then washing (B / F separation). , A method of measuring chemiluminescence intensity due to 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 light emission is repeated by oxidation by charging the electrode and reduction reaction by tripropylamine (TPA) or the like.

Radioimmunoassay (RIA: Radioimmunoassay) is a measurement method using a labeled substance with a radioisotope. For example, after reacting an antigen in a sample with an antibody immobilized on beads or the like, the radioisotope (RIA: Radioimmunoassay) ^After reacting the antibody labeled with 125 I etc.) and washing (B / F separation), ^{the radioactivity amount of 125} I can be measured.

The immunochromatography method is an immunoassay method that applies a capillary phenomenon in which a subject moves on a test strip while dissolving a reagent. This is a method in which the antigen in the sample forms an immune complex with the labeled antibody and the captured 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, an enzyme substrate is placed on the test strip to develop color.

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

The agglutination method is a method of observing agglutination by reacting an antigen in a sample with an antibody in a reagent. Examples include a method that does not use a solid phase, a particle aggregation method (PA) that uses artificially produced particles as a solid phase, and a latex aggregation method (LA) that uses latex particles among PAs. ..
In the competitive method, for example, an antibody can be bound to a solid phase, a test sample and a certain amount of labeled antigen can be reacted at the same time, and the amount of antigen in the sample can be measured from the amount of bound labeled substance.
The antibody used for the measurement of preceptin is preferably used in the above-mentioned measurement method.

The sample used for preceptin measurement is taken from the subject.
Here, the subject includes, for example, humans, non-human mammals (eg, rabbits, goats, horses, sheep, pigs, rats, and mice). In some embodiments, the subject is a human.
The sample is not particularly limited, but an aqueous sample is preferable, and for example, blood (whole blood, plasma, serum, etc.), urine, tissue fluid, lymph, joint fluid, milk, cerebrospinal fluid, pus, saliva, tears, mucus. , Body fluids such as runny nose, sputum, ascites, irrigation water, and semen, and cleaning fluid after cleaning the nasal cavity, bronchi, lungs, skin, abdomen, various organs, joints, bones, etc., or cell culture supernatant, or column elution. Examples include liquids. These samples are used as they are, or diluted with various buffers or the like, extracted and concentrated, and used for measurement. The sample used for the preceptin measurement is preferably a blood sample, more preferably a human blood sample.

When a whole blood sample is used for the preceptin measurement, 72 hours, 48 hours or less, 24 hours or less, 12 hours or less, 6 hours or less, or 4 hours after the whole blood sample is collected. The analysis may be performed. In addition, a whole blood sample may be collected by an EDTA blood collection tube or a heparin blood collection tube. Preferably, a whole blood sample is collected from an EDTA blood collection tube and analyzed within 6 hours, or a whole blood sample is collected from a heparin blood collection tube and analyzed within 4 hours.

4. Preceptin Measurement Kit Also provided here is a preceptin measurement kit (also referred to as a "measurement kit") comprising at least one selected from the group consisting of anti-CD14 antibody, chromatographic column, and membrane.
The measurement kit preferably comprises a method for stabilizing the preceptin level in the sample, a method for suppressing the production of pseudopreceptin derived from sCD14 in the sample, a method for suppressing denaturation of sCD14 in the sample, and a quantification of the true amount of preceptin in the sample. It is a preceptin measurement kit used for a method, a method for quantifying only true preceptin, a method for quantifying only sham preceptin value, and the like. The measurement kit preferably comprises an anti-CD14 antibody, a chromatographic column, or a membrane.
The "anti-CD14 antibody", "chromatographic column", or "membrane" is specifically as described above.

The measurement kit more preferably contains an auxiliary reagent for the measurement of preceptin. Examples of auxiliary reagents include primary antibodies, secondary antibodies, labeled antibodies, labeling enzymes, labeling substances such as gold colloids, color-developing substrates, fluorescent substrates (AmplexTM Red, AttoPhosTM, 4-MUP, etc.), chemiluminescent substrates (luminol, etc.). CDP-StarTM, AMPPDTM, CSPDTM, etc.), specific binding substances such as biotin-streptavidin, insoluble carriers, blocking agents, diluents, cleaning solutions, standard substances, etc., but are not limited thereto.

Auxiliary reagents for preceptin measurement are used in appropriate combinations according to the method of preceptin measurement.

The primary antibody is preferably an antibody that binds to preceptin, and more preferably an antibody that recognizes an epitope different from the antibody. For example, the F1106-13-3 antibody and the F1031-8-3 antibody described in Example 3 of WO2004 / 044005 can be mentioned. WO 2005/108429 can also be referred to in the same manner.

Either the antibody used for the preceptin measurement or the primary antibody may be used as the labeled antibody. If neither the antibody used for the preceptin measurement nor the primary antibody is labeled, a labeled secondary antibody may be used.

Examples of the insoluble carrier include magnetic particles, beads, glass, cellulose, nitrocellulose, porous synthetic polymer, glass fiber, polyacrylamide, nylon, polystyrene, polyvinyl chloride, polypropylene, plastic plate, latex particles, non-woven fabric, filter paper and the like. Can be mentioned.

The labeling of the antibody used for the measurement of preceptin is preferably used, but is not limited to, such as peroxidase (HRP), alkaline phosphatase (ALP), enzymes such as β-galactosidase, and colloidal gold.

For example, when HRP is used, examples of the color-developing substrate include 3,3', 5,5'-tetramethylbenzidine (TMB), o-phenylenediamine (OPD) and the like. When ALP is used, p-nitrophenyl phosphate (pNPP) and the like can be mentioned as the color-developing substrate. Examples of the color-developing substrate when β-galactosidase is used include o-Nitrophenyl-β-D-Galactopyranoside (ONPD).

For example, a measurement kit for the sandwich ELISA method may contain an antibody and a primary antibody (any antibody may be enzyme-labeled) used for preceptin measurement, a chromogenic substrate, a diluent, a standard substance, and the like. If the antibody and primary antibody are unlabeled, labeled secondary antibody may be included.

The measurement kit for chemiluminescent enzyme-linked immunosorbent assay (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, or the like.

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

The electrochemiluminescent immunoassay (ECLIA) measurement kit may contain, for example, a biotinylated antibody, a Ru (bpy) 3-labeled antibody, streptavidin-coated magnetic particles, tripropylamine, and the like.

The measurement kit by the immunochromatography method is a test strip in which a sample addition section, a reagent section, a detection section, and an absorption section are provided so that the liquid sample added to the test addition section moves in the above order. For example, the reagent part can be impregnated with the labeled second antibody, and the insoluble carrier to which the first antibody is bound can be placed in the detection part.

For the test strip, it is exemplified that a porous carrier or the like is used. Examples of the porous carrier include nitrocellulose, cellulose, cellulose derivatives, nylon, nylon fibers, glass fibers, porous synthetic polymers and the like. Examples of the absorbing portion include an absorbing polymer such as a sponge, which is a water-absorbing material, a cellulose filter paper, and a filter paper.

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

In addition, the measurement kit can be used as a diagnostic agent for sepsis or as an adjunct to the diagnosis of sepsis. The preceptin measurement kit is capable of causing sepsis in a subject when the preceptin concentration in the subject's sample measured using an antibody is higher than the cutoff value for the purpose of detecting such sepsis. It can be determined to be sexual and can assist in detection or diagnosis. At this time, the cutoff value is 314 to 600 pg / mL, preferably 400 to 580 pg / mL, more preferably 450 to 550 pg / mL, and even more preferably 500 pg / mL.

In some embodiments, the preceptin measurement kit can be used, for example, to distinguish sepsis from systemic inflammatory response syndrome (SIRS); risk assessment of aggravation of sepsis; prognosis prediction of sepsis (mortality rate). Prediction); Severity assessment of sepsis; Detection of postoperative infections; Detection of disseminated intravascular coagulation (DIC); Detection of infectious DIC; Detection of heart disease; Respiratory infection with bacterial infection Of at least one disease selected from the detection of disease, inflammatory bowel disease (Clone's disease, ulcerative colitis), febrile neuropenia (FN), or hemophagocytic syndrome (HPS). It can be used for detection or evaluation. The preceptin measurement kit may be a kit for detecting or evaluating at least one of the above-mentioned diseases.

5. Fractional Quantification Method for Pseudopreceptin and True Preceptin The fractional quantification method for sham preceptin and true preceptin in a sample includes the use of a gel filtration method.
“Separate quantification of pseudopreceptin and true preceptin” specifically means separating pseudopreceptin and preceptin derived from sCD14 as in Example 4.
For example, according to the method according to Example 4, the gel filtration method can be used, and the preceptin value in each fraction is measured. More specifically, the true preceptin value is quantified by separating the sample before the preceptin measurement by, for example, gel filtration chromatography, obtaining a fraction containing the true preceptin, and measuring the preceptin value. It is possible to do. In the quantification, the above-mentioned preceptin measurement method is used. For example, in Example 4, the true preceptin is obtained by comparing the calibration curve prepared by the standard concentration of preceptin with the amount of preceptin in the fraction containing each preceptin. You can get the value.
Alternatively, a sham preceptin value can be obtained by obtaining a fraction containing sham preceptin instead of a fraction containing true preceptin and measuring the sham preceptin value. Once the sham preceptin value is obtained, it is also possible to measure the prepsin value of the sample and obtain the true preceptin measured value from the total value of the true preceptin value and the sham preceptin value (total preceptin amount).

6. Preceptin measurement system Here, a method for stabilizing the preceptin level in the sample, a method for suppressing the production of pseudopreceptin derived from sCD14 in the sample, a method for suppressing denaturation of sCD14 in the sample, and a method for suppressing the true amount of preceptin in the sample. An apparatus such as a fully automatic apparatus incorporating a quantification method, a method of quantifying only true preceptin, a method of quantifying only sham preceptin value, and the like in the system is provided.
In addition, by constructing a system for performing the separation and quantification, it is possible to construct a system for obtaining a true preceptin value simply by directly applying the sample.
Each configuration of the system is, for example, as follows.
1) Specimen cartridge part: For example, a part in which a sample collected for measurement is placed in a microtube and the microtube is held is applicable;
2) Specimen processing unit: A part for sampling a sample, for example, a part having a nozzle for direct sampling or a part to which a disposable chip can be attached and collecting a sample;
3) Reagent cartridge part: A part containing reagents and the like necessary for preceptin measurement, and corresponds to, for example, the reagents and the like required for the preceptin measurement described in the above-mentioned preceptin measurement or the item of the preceptin measurement kit;
4) Preceptin measurement unit: A part that performs preceptin measurement in a sample, and corresponds to the part that performs preceptin measurement or the part that performs preceptin measurement described in the item of the preceptin measurement kit;
5) Calculation unit: The part that calculates the preceptin value based on the measurement in the measurement unit of 4) corresponds to;
6) Display unit: The part that displays the preceptin value calculated by the calculation unit in 5) corresponds to;
Etc., and examples thereof include a preceptin measurement system containing at least one selected from the group consisting of 1) to 6) above. The above configurations 1) to 6) basically proceed in the order of 1) → 2) → 3) → 4) → 5) → 6) in the system. It becomes.

All documents cited in this specification, as well as published publications, patent publications, and other patent documents are incorporated herein 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 only in these examples.

Example 1: Effect of sample shaking on preceptin measurement values Preceptin ELAISA kit (Mochida Pharmaceutical Co., Ltd., Lot: Using 2S015), each preceptin value in each shaken sample was measured.
The preceptin level of the sample was measured before shaking (0 minutes) and 10 minutes and 60 minutes after shaking.
For shaking, 2 mL of the sample was dispensed into a microtube, and EYELA CUTE CM-1000 (1800 rpm) was used with a Vortex Mixer manufactured by TAITEC for up to 60 seconds and thereafter for up to 60 minutes. Each measurement sample was used by sampling 50 μL from 2 mL of each sample.
The measured values of each preceptin are shown in Table 1.

Table 1: Changes in preceptin concentration over time due to shaking

From the above results, it was confirmed that the preceptin level in the sample increased with time due to the vigorous shaking of the sample. This increased value was larger than the cut-off value for sepsis diagnosis; 500 pg / mL. The physical stimulation (shaking) model added in this experiment is an extreme condition for examination, and it is extremely abnormal and intense, unlike the normal handling or transportation state of blood samples. A supplementary explanation will be given.

Example 2: Examination of the cause of the increase in the preceptin measurement value Using normal human serum (# STL130145) manufactured by Access Biologicals and human serum pretreated with anti-CD14 antibody (hereinafter referred to as CD14-absorbed human serum), each sample was used. 100 μL of each was dispensed per 1.5 mL microtube and shaken for 10 minutes (shaking conditions were the same as in Example 1). To prepare the CD14-absorbed human serum, an F1024-1-3 antibody (prepared by the method described in Examples 1 and 2 of WO2001 / 72993) was bound to a chromatographic resin (Sephalose 4FF). Prepared using affinity chromatography. Normal human serum was passed through the affinity column, CD14 was adsorbed on the resin, and the non-adsorbed fraction (pass-through fraction) was used.
Preceptin concentrations in the samples of normal human serum and CD14-absorbed human serum before and after shaking for 10 minutes using PathFirst Presupsin, PATHFAST (trade name) (LSI Medience Corporation). pg / mL) was measured. In addition, the sCD14 concentration (ng / mL) in each sample was measured using a CD14ELISA kit (R & D systems).
Table 2 shows the preceptin concentration and the sCD14 concentration in each sample.

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

From the above results, a significant increase in preceptin concentration was observed in normal human serum that had not been specially treated after 10 minutes of shaking, but in CD14-absorbed human serum, the measured value of preceptin was observed even after shaking. No significant increase was observed.
The sCD14 concentration decreased by less than half in normal human serum with shaking for 10 minutes, but hardly changed in CD14-absorbed human serum.
It was confirmed that the sCD14 content in normal human serum could be reduced by double digits by the CD14 absorption treatment by affinity chromatography. (Table 2)
These results suggest that the increase in preceptin level due to shaking may be derived from sCD14 in serum.

Example 3: Examination of the effect of anti-CD14 antibody on changes in preceptin concentration After adding F1024-1-3 antibody (anti-CD14 antibody) to about 30 μg of 1 mL of normal human plasma (EDTA), shaking for 10 minutes, and then each. The preceptin concentration of the sample was measured using a preceptin kit (PassFirst Presepsin, PTTHAST (trade name)), and the effect of shaking the anti-CD14 antibody on the preceptin concentration change was examined.
The results are shown in FIG. When preceptin was measured in the presence or absence of F1024-1-3 antibody (anti-CD14 antibody) using normal human plasma, the F1024-1--3 antibody suppressed the increase in preceptin level caused by shaking. Was shown.

It was also confirmed that the addition of the anti-CD14 antibody suppressed the change in the preceptin concentration due to shaking even when the plasma of a septic patient was used.
On the other hand, when an antibody other than the anti-CD14 antibody, for example, a non-anti-CD14 antibody such as an anti-arachidonic acid antibody or an anti-hCG antibody was added to the plasma sample, the effect of suppressing the change in preceptin concentration by shaking was not observed.

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

Example 4: Gel filtration analysis of shake-treated human plasma Normal human plasma (# STL130145) was dispensed into three tubes of 0.5 mL each, and one tube was allowed to stand as a control and then gel filtration analysis. Used for. The remaining two are
After shaking for 10 minutes, each gel filtration analysis was performed. At this time, about 200 μg of F1024-1-3 antibody was added to one of them, and the mixture was allowed to stand at room temperature for 10 minutes and then shaken.
-Column Superdex 75 10/300 GL (1 cmφ x 30 cm)
・ Fraction 0.5 mL / tube ・ Sample addition amount to column 100 μL

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

(2) Each fraction of gel filtration was measured with a CD14ELISA kit (R & D systems), and the absorbance of the measurement solution at 450-650 nm is shown in FIG.

As shown in FIG. 2, the controlled normal plasma was eluted with a single peak near fraction 19. This elution position is the elution position of true preceptin having a molecular weight of 13 KD.
On the other hand, as a result of gel filtration analysis of the human plasma sample subjected to the shaking treatment, two peaks near fractions 13 and 19 were observed. Fraction 13 corresponds to the position of the void in the gel filtration column and is a high molecular weight fraction.
The sample treated with the addition of the anti-CD14 antibody and shaken showed the same elution pattern as the control.
From this, a high molecular weight preceptin-like substance (a substance that reacts with an anti-preceptin antibody, which is different from true preceptin: sometimes referred to as "pseudopreceptin") is generated by shaking the sample, and the pseudopreceptin is produced. It was found that the preceptin measurement value increased (apparent increase in concentration) with the production. It was also clarified that the production of pseudopreceptin was effectively suppressed by the coexistence of an anti-CD14 antibody.

In addition, the chromatographic elution pattern of the substance detected by the CD14 measurement kit shown in FIG. 3 is obtained by shaking the plasma from the pattern in which the control sample showed a single peak near the fraction 17 to the vicinity of the void position. It changed to a peak with shoulders. From this, it was suggested that sCD14 having a high molecular weight was produced by shaking. In addition, since this shoulder portion coincides with the elution position where pseudopreceptin was detected, it was suggested that pseudopreceptin is due to the denaturation and aggregation of the sCD14 molecule and the increase in molecular weight.

Consider based on the above experimental results.
sCD14-ST (preceptin) was characterized by being electrophoresed on SDS-PAGE under non-reducing conditions to a molecular weight of 13 ± 2 kDa, and was eluted on the low molecular weight side by gel filtration of plasma samples (FIG. 2). On the other hand, CD14 present in blood is also called soluble CD14 (sCD14), and a molecular species having a molecular weight of about 55 kDa or about 49 kDa is known, which is observed in the chromatography pattern in controlled normal human plasma in FIG. It is probable that it was done. Then, it was suggested that a higher molecular weight substance generated by shaking reacts with the preceptin measurement kit and causes an abnormal value of the preceptin measurement value as pseudopreceptin. This is an analytical quantitative method in which the preceptin measurement kit recognizes the specific sequence (three-dimensional structure) of the sCD14 molecule immunoscientifically, so even a denatured high molecular weight sCD14 molecule reacts with the preceptin 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 inventions are born. In order to suppress the generation of pseudopreceptin due to physical stimulation or the like, the sCD14 molecule in the sample may be stabilized. For example, this can be achieved by contacting the sample with the anti-CD14 antibody immediately after collecting the sample. Can be done. That is, it is possible to prevent the occurrence of an abnormal value of the preceptin measured value due to shaking or the like in the preceptin value measurement. Further, if the sCD14 molecule is removed by absorption or the like immediately after the sample is collected, pseudopreceptin is not generated.

Furthermore, even in the measurement of a sample that has already produced pseudopreceptin, it is possible to quantify the true preceptin by distinguishing the false preceptin from the true preceptin. For example, in the measurement of preceptin, (i) pseudopreceptin is removed by passing through an ultrafiltration membrane of 30 kDa, and (ii) by utilizing an antibody that reacts with pseudopreceptin without reacting with true preceptin. By performing pretreatment such as adsorption removal / separation of pseudopreceptin, correct preceptin measurement becomes possible.

Furthermore, as in Example 4, pseudopreceptin and true preceptin can be separated and analyzed by a chromatographic graph. The preceptin value in the fraction containing the true preceptin may be quantified. In addition, if pseudopreceptin can be quantified by any means, the true preceptin value can be obtained by calculation. For example, a "pseudopreceptin value" can be obtained by performing a preceptin measurement on a fraction containing "pseudopreceptin" separated by chromatography. Once the pseudopreceptin value is obtained, it is also possible to measure the prepsin value of the sample and obtain the measured value of pseudopreceptin from the total value (total preceptin amount) of the true preceptin value and the pseudopreceptin value.
Furthermore, by screening a measurement system that does not react with pseudopreceptin but only with true preceptin, it is possible to quantify only the true preceptin value without requiring any special treatment.

SEQ ID NO: 1: The amino acid sequence of the 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 a preceptin measurement value in a sample, which comprises a step of contacting the sample with an anti-CD14 antibody immediately after collecting the sample. A method for suppressing the production of pseudopreceptin in a sample, which comprises a step of contacting the sample with an anti-CD14 antibody immediately after collecting the sample. A method for suppressing denaturation of sCD14 in a sample, which comprises a step of bringing the sample into contact with an anti-CD14 antibody immediately after collecting the sample. The method according to any one of claims 1 to 3, which comprises adsorbing and removing sCD14 in the sample by a step of bringing the sample into contact with an anti-CD14 antibody. The method according to any one of claims 1 to 3, wherein the sCD14 in the sample and the antibody CD14 antibody form a conjugate by the step of contacting the sample with the anti-CD14 antibody. A method for quantifying the true amount of preceptin in a sample, comprising contacting the sample with an anti-CD14 antibody prior to preceptin measurement. The method according to claim 6, wherein the pseudopreceptin is adsorbed and removed using the anti-CD14 antibody that reacts with the pseudopreceptin in the sample by the step of bringing the sample into contact with the anti-CD14 antibody. A method of quantifying only true preceptin, which comprises the step of separating false preceptin from true preceptin prior to preceptin measurement. The method according to claim 8, wherein the separation of the pseudopreceptin from the true preceptin is a separation by chromatography. 8. The method of claim 8, wherein the separation of pseudopreceptin from true preceptin is by membrane separation. A method of quantifying only sham preceptin, which comprises the step of separating sham preceptin from true preceptin prior to preceptin measurement. A method for quantifying a true preceptin value, which comprises measuring a sample of preceptin, calculating the preceptin value, and subtracting the pseudopreceptin value obtained by the method according to claim 11 from the calculated preceptin value. A preceptin measurement kit comprising at least one selected from the group consisting of an anti-CD14 antibody, a chromatographic column, and a membrane for use in the method according to any one of claims 1 to 12. It is selected from the group consisting of a sample cartridge unit, a sample processing unit, a reagent cartridge unit, a preceptin measurement unit, a calculation unit, and a display unit for use in the method according to any one of claims 1 to 12. A preceptin measurement system, including at least one.