JP2005106694A - Early detection of blood poisoning and evaluation on seriousness - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for early detecting infectious disease and/or blood poisoning, and to provide an evaluation method on the seriousness of blood poisoning and on the seriousness of a multiple organ failure syndrome. <P>SOLUTION: This method for early detecting infectious disease and/or blood poisoning is characterized by measuring the amount of low molecular weight s-CD14 in blood of a tested person. This method for evaluation the seriousness of blood poisoning and the seriousness of a multiple organ failure syndrome is characterized by measuring the amount of low molecular weight s-CD14 in blood of a patient. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to an early detection method for infection and / or sepsis. The present invention also relates to a method for evaluating the severity of sepsis. Furthermore, the present invention relates to a method for evaluating the severity of multiple organ failure syndrome (MODS). Furthermore, the present invention relates to a low molecular weight s-CD14 measurement kit for use in a method for early detection of infection and / or sepsis, a method for evaluating the severity of sepsis, a method for evaluating the severity of MODS, or the like.

Sepsis is a systemic inflammatory response syndrome (SIRS) with infection, with infection, body temperature (<36 ° C. or> 38 ° C.), heart rate (> 90 times / min), breathing (respiration rate> 20 times / Min or PaCO ₂ <32 mmHg), white blood cell count (≧ 12,000 / mm ³ or ≦ 4,000 mm ³ , or appearance of juvenile spheres of 10% or more), two or more conditions It is defined as satisfying (see Non-Patent Document 1). In Non-Patent Document 1, the severity of sepsis is defined by the provisions of organ dysfunction, blood flow drop, blood pressure drop and tissue circulation disorder following sepsis, and a syndrome in which multiple important organs simultaneously fail As defined for multiple organ dysfunction syndrome (MODS).
Conventionally, diagnosis of infectious diseases and sepsis has been performed by confirming the presence of infectious bacteria by culturing the blood of a subject. However, this method has a fundamental drawback in that it takes a considerable amount of time for cultivation and the detection rate is low. Further, APACHE II score (see Non-Patent Document 2) has been proposed as a recent index reflecting the severity of sepsis, and SOFA score (see Non-Patent Document 3) has been proposed as an index of the severity of MODS. And is widely used clinically. However, these indices also require many parameters to be measured when evaluating the symptoms of sepsis and MODS, and are calculated by a specific arithmetic expression, which is complicated and requires time and effort for the measurement itself. C-reactive protein (CRP), whose value increases within a few days after infection, has also been measured clinically, but it is a factor that increases in patients with acute inflammation and tissue disruption. It is said that it is not a factor which increases (nonpatent literature 4, nonpatent literature 5).
Recently, as another blood marker reflecting SIRS and sepsis, a method using cytokines such as TNFα and IL-6 or endotoxin (ET) as an index has been studied, but the measurement method is complicated and the marker is unstable. However, due to reasons such as lack of sensitivity of measurement, established evaluation has not been obtained so far, and it is used only at the level of research reagents. In addition, a measurement method using procalcitonin as an index has been proposed (see Patent Document 1). However, the role of procalcitonin in sepsis and its usefulness in early diagnosis has not been established.

On the other hand, regarding the relationship between soluble CD14 molecules in blood and pathological conditions, Landmann et al. Conducted a Western blot analysis of soluble CD14 in septic patient serum, and about 55 kDa soluble CD14 was found to be a case of septic death or seizure nocturnal. It was high in hemoglobinuria (PNH) patients, and this molecule was not found in the serum of healthy volunteers. It was reported that 49 kDa soluble CD14 with a slightly lower molecular weight was detected in healthy volunteers (non-patented) Reference 6). Regarding the subtypes having different molecular weights, the difference in sugar chain is involved, and even if N- and O-linked sugar chains are removed, two types of soluble CD14 having different molecular weights are still present in the blood. This is reported by Stlter et al. (See Non-Patent Document 7).
A measurement system for soluble CD14 using an anti-CD14 antibody has been reported by Shutt et al. (See Patent Document 2), and soluble CD14 has been measured in many diseases including sepsis (Non-patent Document 8). Non-patent document 9).
However, even in diseases other than sepsis, the soluble CD14 containing about 55 kDa and 49 kDa as described above is associated with the degree of disease progression (the molecular weight differs depending on the report, so it is not limited to about 55 kDa and 49 kDa. The blood concentration of “high molecular weight CD14” (the same applies hereinafter) increased, and it was revealed that soluble CD14 is not a sepsis-specific marker (see Non-Patent Documents 10 to 12). Soluble CD14 was also expected as a marker for the severity of sepsis, but there was no correlation with septic shock (see Non-Patent Document 13), systemic inflammatory response syndrome (SIRS) and (See Non-Patent Document 14), it could not be a diagnostic agent for sepsis.

  In addition to the above-mentioned soluble CD14 of about 55 kDa and 49 kDa, etc. reported by Landmann et al., A soluble low molecular weight CD14 of about 36 kDa (hereinafter also referred to as “low molecular weight s-CD14”). A method for measuring low molecular weight s-CD14 (for example, low molecular weight s-CD14 protein by subtracting the amount of high molecular weight CD14 protein in body fluid from the total amount of soluble CD14 protein in body fluid) It has been proposed that the measurement is useful for the diagnosis of sepsis (see Patent Document 3).

International Publication No. 94/04927 Pamphlet West German Patent Application No. 286876 International Publication No. 01/22085 Pamphlet

“Critical Care Medicine”, Volume 20, USA, 1992, p. 864-874 “Critical Care Medicine”, Vol. 13, USA, 1985, p. 818-829 “Intensive Care Medicine”, Vol. 13, USA, 1996, p. 707-710 “American journal of surgery”, Vol. 175, USA, 1998, p. 325-327 Tadashi Kawai, Shinya Hashimoto, “ABC for Clinical Testing”, Japan Medical Association, February 15, 1999, p. 146-148 “The Journal of Infectious Disease”, Vol. 171, USA, 1995, p. 639-644 “European Journal of Biochemistry”, Vol. 236, Germany, 1996, p. 457-464 “Clinical Immunology And Immunopathology”, Vol. 80, USA, 1996 p. 307-310 “Clinical examination”, Vol. 38, 1994, p. 341-344 “Infection and Immunity”, Vol. 67, USA, 1999, p. 417-420 “Clinical and Experimental Immunology”, Volume 120, UK, 2000, p. 483-487 “Clinical and Experimental Immunology”, Vol. 96, UK, 1994, p. 15-19 “Pedatric allergy and immunology”, Vol. 8, Denmark, 1997, p. 194-199 “European Journal of Clinical Investigation”, Volume 28, UK, 1998, p. 672-678

  Under such circumstances, there is a desire in the clinical field to distinguish infection and / or sepsis at an early stage after suffering from the disease state. In addition, a method that can be more easily performed at the clinical site is desired as the discrimination method. Similarly, a simpler method is desired as a method for evaluating the severity of sepsis or MODS. Such a method for evaluating the severity is also a therapeutic effect such as surgery or drug therapy applied to infections, sepsis, or MODS (hereinafter also simply referred to as “therapeutic effect”). It can also be used to evaluate the need for clinical practice.

  As a result of intensive studies, the present inventors have rapidly increased the low molecular weight s-CD14 value in blood (usually expressed as “ng / mL”) after suffering from infection and / or sepsis. As a result, it has been proved that it is extremely useful to measure the low molecular weight s-CD14 value in blood as an early detection method for infection and / or sepsis. In addition, the low molecular weight s-CD14 value in the blood changes up and down according to the severity of sepsis in the patient, and the change is captured earlier than the conventional diagnostic parameters for sepsis such as APACHE II score. I found it. Furthermore, it has been found that the therapeutic effects of surgery, drugs and the like in infection, sepsis, MODS and the like can be evaluated by the low molecular weight s-CD14 value in blood. Based on the above findings, the present inventors have completed the present invention.

That is, the present invention provides the following early detection method for infectious diseases and / or sepsis.
(1) A method for early detection of infectious disease and / or sepsis, which comprises measuring the amount of low molecular weight s-CD14 in the blood of a subject.
(2) The method for early detection of infection and / or sepsis according to (1) above, wherein the amount of low molecular weight s-CD14 in the blood of a subject is measured and compared with a predetermined threshold value.
(3) The threshold value is a threshold value set between a previously measured average value of healthy persons + 0.5 SD to an average value of healthy persons + 12 SD, Early detection method.
(4) Early detection of the infectious disease according to any one of (1) to (3) above, wherein the infectious disease is a bacterial infection, a fungal infection, a viral infection or a parasitic infection Method.
(5) The method for early detection of sepsis according to any one of (1) to (3) above, wherein the sepsis is sepsis caused by bacterial infection, fungal infection, viral infection or parasitic infection.
(6) The infectious disease according to any one of (1) to (5) above, wherein the amount of low molecular weight s-CD14 in blood of a subject is measured over time and compared with a predetermined threshold value; / Or early detection method of sepsis.
Here, “measuring the amount of low molecular weight s-CD14 in blood over time” means measuring the amount of low molecular weight s-CD14 in each blood sampled over time.
(7) The method for early detection of an infectious disease and / or sepsis according to any one of (1) to (6), wherein the amount of low molecular weight s-CD14 in the blood of a subject is directly measured.

In addition, the present invention provides the following methods for evaluating the severity or therapeutic effect of sepsis.
(8) A method for evaluating the severity of sepsis or the therapeutic effect, comprising measuring the amount of low molecular weight s-CD14 in the blood of a patient.
(9) The method for evaluating the severity or therapeutic effect of sepsis according to (8) above, wherein the amount of low molecular weight s-CD14 in the blood of a patient is measured and compared with a predetermined threshold value.
(10) The severity of sepsis according to (8) or (9) above, wherein the amount of low molecular weight s-CD14 in the blood of a patient is measured over time and evaluated by the degree of change in the measured value Method of evaluating degree or treatment effect.
Here, “measuring the amount of low molecular weight s-CD14 in blood over time” means measuring the amount of low molecular weight s-CD14 in each blood sampled over time.
(11) The degree of change in the measured value is calculated based on a change amount per day or a change amount with respect to a maximum value of the low molecular weight s-CD14 amount of the patient (10) The method for evaluating the severity of sepsis as described in 1. above.
(12) In the above (10), the degree of change in the measured value is expressed using a change rate per day or a change rate with the maximum value of the low molecular weight s-CD14 amount of the patient. The method for evaluating the severity of sepsis as described.
(13) The method for evaluating the severity or therapeutic effect of sepsis according to any one of (8) to (12), wherein the amount of low molecular weight s-CD14 in the blood of a patient is directly measured.

Furthermore, the present invention provides the following methods for evaluating the severity of MODS.
(14) A method for evaluating the severity of MODS, comprising measuring the amount of low molecular weight s-CD14 in the blood of a patient.
(15) The method for evaluating the severity of MODS as described in (14) above, wherein the amount of low molecular weight s-CD14 in the blood of a patient is measured and compared with a predetermined threshold value.
(16) The seriousness of MODS as described in (14) or (15) above, wherein the amount of low molecular weight s-CD14 in the blood of a patient is measured over time and evaluated by the degree of change in the measured value Degree evaluation method.
Here, “measuring the amount of low molecular weight s-CD14 in blood over time” means measuring the amount of low molecular weight s-CD14 in each blood sampled over time.
(17) The degree of change in the measured value is calculated based on a change amount per day or a change amount from the maximum value of the low molecular weight s-CD14 amount of the patient (16) The evaluation method of severity of MODS as described in 1.
(18) In the above (16), the degree of change in the measured value is expressed using a change rate per day or a change rate with the maximum value of the low molecular weight s-CD14 amount of the patient. The method for evaluating the severity of MODS as described.
(19) The method for evaluating the severity of MODS as described in (14) above, wherein the MODS is MODS following sepsis.
(20) The method for evaluating the severity of MODS according to any one of (14) to (19), wherein the amount of low molecular weight s-CD14 in the blood of a patient is directly measured.

Furthermore, this invention provides the use of the following low molecular weight s-CD14 measurement kits.
(21) Measure the amount of low molecular weight s-CD14 in blood used in the early detection method of infection and / or sepsis, the evaluation method of the severity or therapeutic effect of sepsis, or the evaluation method of the severity of MODS Possible low molecular weight s-CD14 measurement kit.
(22) The low molecular weight s-CD14 measurement kit according to (21), wherein the amount of low molecular weight s-CD14 in the blood of a subject is directly measured.
(23) The low molecular weight s-CD14 measurement kit according to (22), wherein low molecular weight s-CD14 is measured by sandwich immunization.
(24) The low molecular weight s-CD14 measurement kit according to (23) above, which comprises an antibody that binds to a peptide consisting of 16 amino acid residues described in SEQ ID NO: 1.
(25) Low molecular weight s-CD14, which is used for an early detection method for infection and / or sepsis, a method for evaluating the severity (or therapeutic effect) of sepsis, or a method for evaluating the severity of MODS. How to use the measurement kit.
(26) The method for using the low molecular weight s-CD14 measurement kit according to (25), wherein the amount of low molecular weight s-CD14 in the blood of a subject is directly measured.
(27) The method for using the low molecular weight s-CD14 measurement kit according to (26), wherein low molecular weight s-CD14 is measured by sandwich immunization.
(28) The method for using a low molecular weight s-CD14 measurement kit according to (27) above, comprising an antibody that binds to a peptide consisting of 16 amino acid residues described in SEQ ID NO: 1.

Furthermore, the present invention provides the following sepsis therapeutic agent.
(29) A therapeutic agent for sepsis, which is administered within 24 hours to a patient who is determined to be positive by the method for early detection of infectious disease and / or sepsis according to (1).
(30) A therapeutic agent for sepsis, which is administered under the monitoring of the severity of infection and / or sepsis or MODS by the low molecular weight s-CD14 measurement kit according to (21).

By measuring low molecular weight s-CD14 in the blood of septic patients, SIRS patients, or patients suspected of having septicemia, the presence or absence of infection or septic infection, evaluation of the severity of sepsis, and multiple organ failure An assessment of the syndrome can be made.
Moreover, by measuring the low molecular weight s-CD14 in the blood of a patient over time, the transition of the severity of sepsis and the transition of the severity of MODS can be determined from the degree of the change.

  In addition, since it is possible to determine whether or not the disease is septic at an early stage, it is possible to formulate treatment guidelines such as the start and change of surgery and medication, and to determine the therapeutic effect of surgery or medication from the degree of change in low molecular weight s-CD14 can do.

Hereinafter, the present invention will be described in more detail.
The “low molecular weight s-CD14” described in the present invention is a soluble protein present in human blood, and is a protein having a molecular weight of 35-45 kDa in gel filtration. Further, the low molecular weight s-CD14 binds to an antibody that binds to a peptide consisting of 16 amino acid residues described in SEQ ID NO: 1, and further binds to some antibodies of an anti-CD14 antibody that binds to human high molecular weight CD14. It is protein to do. The part of the anti-CD14 antibody is, for example, 3C10. As a specific example of the low molecular weight s-CD14, a human blood protein having a molecular weight of about 36 kDa described in WO 01/22085 pamphlet can be exemplified. “Low molecular weight s-CD14” is shown as described above, but is present in the blood as one protein or various protein groups.

The “low molecular weight s-CD14 amount (sometimes referred to as“ low molecular weight s-CD14 value ”)” described in the present invention is the content of low molecular weight s-CD14 contained in a specimen, particularly blood. Usually, when measuring “low molecular weight s-CD14 amount”, the content per fixed volume of the specimen is often measured. For example, “low molecular weight s-CD14 amount” is set as a concentration such as “ng / mL”. Often get. That is, in the present invention, “low molecular weight s-CD14 amount” includes the meaning of “low molecular weight s-CD14 concentration” in the specimen.
In addition, by the early detection method of the present invention, a non-infectious SIRS patient and an infectious SIRS patient, that is, a septic patient can also be distinguished, and the method of distinguishing is also included in the early detection method of the present invention. .

The first aspect of the present invention is a method for early detection of infection and / or sepsis characterized by measuring the amount of low molecular weight s-CD14 in the blood of a subject.
Preferably, the method is an early detection method for infectious diseases and / or sepsis, wherein the amount of low molecular weight s-CD14 in the blood of a subject is measured and compared with a predetermined threshold value. That is, the method for early detection of infectious diseases of the present invention is preferably a method characterized in that the amount of low molecular weight s-CD14 in the blood of a subject is measured and compared with a predetermined threshold value for infectious diseases. . In addition, the method for early detection of sepsis according to the present invention is preferably a method characterized in that the amount of low molecular weight s-CD14 in the blood of a subject is measured and compared with a predetermined threshold value for sepsis.
Also preferably, the method is an early detection method for infection and / or sepsis, characterized in that the amount of low molecular weight s-CD14 in the blood of a subject is measured over time and compared with a predetermined threshold value.

  Examples of the infectious disease targeted by the method for early detection of infectious diseases of the present invention include bacterial infections, fungal infections, viral infections, and parasitic infections. Mainly bacterial or fungal infections. Moreover, the sepsis which is the object of the method for early detection of sepsis of the present invention is exemplified by sepsis caused by bacterial infection, fungal infection, viral infection or parasitic infection. Mainly sepsis due to bacterial or fungal infection.

As an early detection method of the present invention, for example, by measuring the amount of low molecular weight s-CD14 in the blood of a patient, particularly a patient suspected of sepsis such as a burn, a patient with trauma, or a subject such as a SIRS patient. Preferably, when the measured low molecular weight s-CD14 amount (hereinafter sometimes simply referred to as “measured value”) is compared with a predetermined threshold value and the measured value is higher than the threshold value, A method for determining sepsis is mentioned.
In the method for early detection of infectious disease and / or sepsis according to the first aspect of the present invention, “measuring the amount of low molecular weight s-CD14 in blood over time” means that each of the samples collected multiple times over time is used. It means measuring the amount of low molecular weight s-CD14 in blood.
The frequency of the measurement over time depends on the health condition of the subject or the degree of stability of the medical condition. For example, the initial stage of observation, every few hours when the medical condition changes drastically, usually once or every two days, the medical condition When it is stable, measurement is performed once every few days.

In addition, when compared with the previous value or the average value of the low molecular weight s-CD14 amount of the subject up to that point, when the increase is 10 to 40 ng / mL or more, or when the increase is 2 to 10 times or more, the measurement frequency Is exemplified.
For example, when the amount of low molecular weight s-CD14 in the blood of a hospitalized subject has been measured once a day over time, a value much lower than the threshold, for example, 1/5 to 1 / When the value of about 10 was maintained, the value was lower than the threshold value at a certain point, but suddenly increased to a high value, for example, about 3 to 8 times the previous value. For example, by monitoring the amount of low molecular weight s-CD14 in the blood at a rate of once every 8 hours, infection and / or sepsis in the subject can be detected early.
The present inventors have found that when a patient is infected with bacteria or the like, the amount of low molecular weight s-CD14 in the blood of the patient immediately increases, and in the case of Example 6 described later, 24% than bacterial infection. It was confirmed that the low molecular weight s-CD14 in the blood increased within the time. Therefore, taking advantage of this knowledge, as an early detection method of the present invention, for example, by measuring the amount of low molecular weight s-CD14 in the blood of a patient suspected of being infected with bacteria or the like, a measurement value is preferably determined in advance. A method for early detection of whether or not the patient suffered from infection and / or sepsis by comparing with the threshold value.

The subject to which the early detection method of the present invention is applied is a patient who may have contacted or infected with bacteria, fungi, viruses or parasites causing bacterial infections, fungal infections, viral infections or parasitic infections. A patient with a possibility of sepsis due to a bacterial infection, a fungal infection, a viral infection or a parasitic infection, or a SIRS patient or a patient with a possibility of SIRS, but not limited to these patients, and healthy individuals It may be.
The blood of the subject for measuring the amount of low molecular weight s-CD14 may be whole blood, plasma, or serum, and a sample corresponding to the measurement system may be used. Plasma and serum are preferable. Further, the blood of the subject is not particularly limited by the method collected from the subject, and is preferably the blood of the subject collected by a commonly performed method.

The threshold for early detection of infection and / or sepsis used in the preferred method of the present invention and compared to the amount of low molecular weight s-CD14 in the blood of a subject It is preferable to set in advance according to the measurement system to be used.
As the threshold setting, the threshold is set based on the statistics of low molecular weight s-CD14 values in the blood of healthy persons and septic patients. Specifically, the threshold is set between an average value of healthy persons + 0.5 SD or more and an average value of healthy persons + 12 SD or less. For example, the average value of healthy persons + 0.5 SD or more and the average value of healthy persons + SD or less, the average value of healthy persons + SD or more and the average value of healthy persons + less than 2 SD, the average value of healthy persons + 2 SD or more, and healthy persons The threshold is set between the mean value of + 3SD or less, the mean value of healthy people + 3SD, the mean value of healthy people + 5SD or less, the mean value of healthy people + 5SD or more, and the mean value of healthy people + 12SD or less. The Preferably, the average value of healthy persons + SD and above the average value of healthy persons + less than 2SD, the average value of healthy persons + the average value of 2SD and above average value of healthy persons + 3 SD and below, the average value of healthy persons + average of healthy persons The threshold value is set between the value + 5SD or less. More preferably, the threshold value is set between the average value of healthy persons + SD and less than the average value of healthy persons + less than 2SD, and between the average value of healthy persons + 2SD and the average value of healthy persons + 3 SD or less. As a more specific example, the threshold value is set to an average value of healthy persons + SD, an average value of healthy persons + 2SD, or an average value of healthy persons + 3SD.

As described in Example 5 of the present invention to be described later, in the sandwich EIA system quantitative measurement using a standard product, the average value of healthy people + 0.5 SD to the average value of healthy people + 12 SD (“average value of healthy people + 0. It means “average value of healthy individuals at 5 SD or more + 12 SD or less” (described in the same manner below) was 35 to 200 ng / mL.
That is, the threshold value can be set to a value between 35 and 200 ng / mL in sandwich EIA-based quantitative measurement using a standard product. For example, a value of 35, 40, 50, 57, 60, 70, 100, 135, 170, 200 ng / mL or a value in the vicinity thereof can be set as the threshold value. Preferably, setting to a value between 40 and 100 ng / mL, and more preferable setting to a value between 40 and 70 ng / mL is exemplified. Standard products include human low molecular weight CD14 and human low molecular weight CD14 analogues. The human low molecular weight CD14 analog is, for example, a soluble polypeptide having an amino acid at positions 1 to 285 of human CD14 (hereinafter referred to as “s-CD14 (1-285)”) and N-terminal of human CD14. A recombinant polypeptide having the 1st to 307th amino acids and having the 286th serine substituted with cysteine (hereinafter referred to as “s-CD14 (1-307) S286C”) is exemplified. However, as long as it is measured in the same manner as human low molecular weight CD14 in the sandwich EIA system, it is not particularly limited. The preparation method of s-CD14 (1-285) and s-CD14 (1-307) S286C is described in WO 01/72993.

  In the preferable early detection method of the present invention, when the measured low molecular weight s-CD14 value in the blood of the subject exceeds a threshold value set in advance between 35 and 200 ng / mL, infection is caused. What is necessary is just to determine with being affected, and what is necessary is just to determine with sepsis, when this test subject is a SIRS patient.

The method for measuring the amount of low molecular weight s-CD14 is not particularly limited as long as the amount of low molecular weight s-CD14 in a patient's blood can be measured.
An immuno sandwich assay method using an antibody that can directly measure the amount of low molecular weight s-CD14 and binds to a peptide consisting of 16 amino acid residues described in SEQ ID NO: 1 is preferred, and the method will be described below. .

  The amino acid sequence of 16 amino acid residues described in SEQ ID NO: 1 corresponds to the 16 amino acid residues from 53 to 68 of human CD14 described in SEQ ID NO: 2, and this sequence is present in human proteins other than human CD14. Other proteins possessed are not known and are sequences specifically contained in human CD14.

An example of a method for preparing a kit for the measurement method is described below.
The antibody that binds to the peptide consisting of 16 amino acid residues described in SEQ ID NO: 1 may be a polyclonal antibody or a monoclonal antibody, and is a peptide comprising 8 or more consecutive amino acids of the amino acid residues described in SEQ ID NO: 1. It can be prepared by using a known technique as an antigen (see, for example, “Immune Experimental Manipulation Method”, edited by the Japanese Society for Immunology, published by the Japanese Society for Immunology).

Although the production method of the polyclonal antibody of this antibody is illustrated, the production method of this antibody is not limited to the following production methods.
A peptide containing 8 or more consecutive amino acid residues of the above-mentioned SEQ ID NO: 1 as an antigen has a small molecular weight and is not immunogenic, so it is bound to a carrier such as keyhole limpet hemocyanin (KLH). It can be used as an immunogen.
20 to 1000 μg of an immunogen obtained by preparing as described above is mixed with an adjuvant such as Freund's complete adjuvant and immunized to animals such as rabbits, rats and mice. As the immunization method, for example, subcutaneous administration is performed, and booster immunization is performed in the same manner or intravenously by administering an immunogen mixed with an adjuvant such as Freund's incomplete adjuvant at intervals of 1 to 4 weeks after the initial administration. The antiserum is prepared by collecting blood from an immunized animal by a normal blood collection method, for example, from the carotid artery, etc., and separating the serum by centrifugation or the like. The obtained antiserum is precipitated with a salting-out method using ammonium sulfate or the like, and a gamma globulin fraction is precipitated, dialyzed against an appropriate buffer, and then used with an affinity matrix that can specifically purify gamma globulin such as protein A. Prepare a purified polyclonal antibody of the IgG fraction against the peptide of interest. Further, specific purification can be performed by selecting an antibody that binds to the peptide used as the immunogen.
In addition, the preparation method of the peptide used as an immunogen can be prepared by a method using a generally used peptide synthesizer (peptide synthesizer 433A type, Perkin-Elmer Japan) or the like.

  A known technique can be used for the sandwich immunoassay using the purified polyclonal antibody. For the principle, application, and improvement of the measurement method, see, for example, Eiji Ishikawa, “Ultrasensitive Enzyme Immunoassay”, Academic Publishing Center (1993); “New use cases of immunoassay and diagnostic reagents and therapeutics” Application to Development ", Immunoassay Development Study Group, Management Education Publishing; Eiji Ishikawa et al.," Enzyme Immunoassay (3rd edition) ", Medical School (1987).

  Examples of the second binding substance used in the sandwich immunoassay include an antibody that binds to low molecular weight s-CD14. For example, a polyclonal antibody or a monoclonal antibody may be prepared using high molecular weight CD14, low molecular weight s-CD14, a mixture of high molecular weight CD14 and low molecular weight s-CD14 or recombinant CD14 as an antigen. An example of the production method of the second antibody using a mixture of high molecular weight CD14 and low molecular weight s-CD14 as an antigen is shown in Example 2- (1) described later.

In addition, before actually measuring the amount of low molecular weight s-CD14, an antibody that binds to a peptide consisting of 16 amino acid residues described in SEQ ID NO: 1 is preliminarily prepared in the same manner as in Example 2- (2) described later. It is preferable to construct a sandwich method system with the second antibody candidate antibody, confirm the measurement sensitivity, and select the second antibody.
Using an antibody that binds to a peptide consisting of 16 amino acid residues described in SEQ ID NO: 1 and a second antibody, for example, "an antibody that binds to a peptide consisting of 16 amino acid residues described in SEQ ID NO: 1"-"human The measurement may be performed by forming a complex of “low molecular weight s-CD14” — “second antibody that binds to human low molecular weight s-CD14”.

Although the measurement method which can measure low molecular weight s-CD14 used suitably for this invention directly below is illustrated, the measurement method used for this invention is not limited to the measurement method illustrated below.
The measurement method performs the following procedure.
That is, an immunoplate in which “an antibody that binds to a peptide consisting of 16 amino acid residues described in SEQ ID NO: 1” is immobilized by a thermal adsorption method or the like is prepared, and a sample is added to the immunoplate for reaction. Next, a “second antibody that binds to human low molecular weight s-CD14” labeled with peroxidase or the like by the periodate method or the like is added and reacted. Thereafter, 3,3 ′, 5,5′-tetrabenzidine or the like is added as a chromogenic substrate for reaction, and the reaction is stopped with a sulfuric acid solution or the like, and then the absorbance at 450 nm is measured with a spectrophotometer. And the amount of low molecular weight s-CD14 is measured.

  In the present invention, in addition to the above-mentioned method for directly measuring the low molecular weight s-CD14 amount, the total blood CD14 amount and the high molecular weight CD14 shown in Example 11 described in WO 01/22085 pamphlet are used. A method of measuring low molecular weight s-CD14 indirectly by measuring the amount and subtracting the high molecular weight CD14 amount from the total CD14 amount is also exemplified. Moreover, the method of detecting the low molecular weight s-CD14 shown in Example 10 described in WO 01/22085 pamphlet electrophoretically and quantifying the band is also exemplified.

According to a second aspect of the present invention, the severity of sepsis characterized by measuring the amount of low molecular weight s-CD14 in the blood of a patient (or the effect of surgery or drug treatment on sepsis) This is an evaluation method.
Hereinafter, differences from the early detection method according to the first aspect of the present invention will be described.
Preferably, it is a method for evaluating the severity of sepsis or the therapeutic effect, characterized in that the amount of low molecular weight s-CD14 in the blood of a patient is measured and compared with a predetermined threshold value. That is, the method for evaluating the severity of sepsis according to the present invention is preferably a method characterized by measuring the amount of low molecular weight s-CD14 in the blood of a patient and comparing it with a predetermined threshold. The method for evaluating the therapeutic effect of sepsis according to the present invention is preferably a method characterized by measuring the amount of low molecular weight s-CD14 in a patient's blood and comparing it with a predetermined threshold.

  More preferably, it is a method for evaluating the severity of sepsis or the therapeutic effect, characterized in that the amount of low molecular weight s-CD14 in the blood of a patient is measured over time and evaluated based on the degree of change in the measured value. That is, the method for evaluating the severity of sepsis according to the present invention is a method characterized by measuring the amount of low molecular weight s-CD14 in a patient's blood over time and evaluating the degree of change in the measured value. . In addition, the method for evaluating the therapeutic effect of sepsis according to the present invention is a method characterized by measuring the amount of low molecular weight s-CD14 in the blood of a patient over time and evaluating it according to the degree of change in the measured value.

  More preferably, the amount of low molecular weight s-CD14 in the patient's blood is measured over time and calculated based on the amount of change per day or the maximum amount of the patient's low molecular weight s-CD14 amount. It is a method characterized by evaluating the severity of sepsis according to the degree of change or evaluating the therapeutic effect of sepsis. In addition, the amount of low molecular weight s-CD14 in the patient's blood is measured over time, and the change represented by the rate of change per day or the maximum value of the amount of low molecular weight s-CD14 of the patient. It is a method for evaluating the severity of sepsis according to the degree of septicemia or evaluating the therapeutic effect of sepsis.

  In other words, the second aspect of the present invention is preferably evaluated by comparing the measured value with a predetermined threshold value. It is more preferable to compare the calculated or expressed degree of change with a predetermined change amount or change rate of the low molecular weight s-CD14 amount, and it is particularly preferable to use these in combination.

  In clinical practice, sepsis is usually diagnosed by measuring indices related to body temperature, heart rate, respiration, and white blood cells separately from confirmation of infection. For necessary patients, APACHE II score (Non-patent Document 2) is further calculated and used as an index of severity. The present inventors have found that the change in the amount of low molecular weight s-CD14 in the patient's blood meets the SIRS diagnostic criteria for body temperature, heart rate, respiration, and leukocytes, or APACHE II score early or At the same time, we found that it changed with the same trend. That is, in the case of Example 7 to be described later, the amount of low molecular weight s-CD14 in the blood decreases due to the improvement of the severity of sepsis, and in the case of Example 8 to be described later, the severity of sepsis It was confirmed that the amount of low molecular weight s-CD14 in the blood increased due to worsening of the blood, and the amount of low molecular weight s-CD14 in the blood decreased due to the improvement of the severity of sepsis. Based on these findings, the present inventors measure the amount of low molecular weight s-CD14 because the low molecular weight s-CD14 value changes in the septic patient according to the severity of the symptoms. Thus, the inventors have invented that it is possible to evaluate the severity of sepsis, preferably by measuring the amount of low molecular weight s-CD14 over time. For example, for example, by measuring the amount of low molecular weight s-CD14 in the blood of a septic patient over time and confirming the degree of change in the amount of low molecular weight s-CD14, the severity is evaluated. can do.

  In the severity evaluation method of the second aspect of the present invention, if the low molecular weight s-CD14 value measured over time decreases, it can be evaluated that the severity has improved. In the severity evaluation method of the second aspect of the invention, when the low molecular weight s-CD14 value measured over time is decreased and the measured value is lower than the above-described threshold, the symptoms are recovered. It is possible to evaluate.

  In the method for evaluating the severity of the second aspect of the present invention, “measuring the amount of low molecular weight s-CD14 in blood over time” refers to the low molecular weight of each blood sampled multiple times over time. It means measuring the amount of s-CD14.

Threshold for comparing the amount of low molecular weight s-CD14 in the patient's blood, degree of change (the amount of change per day or the maximum amount of low molecular weight s-CD14 in the patient, or one day It is preferable that the rate of change and the rate of change of the patient's low molecular weight s-CD14 amount with respect to the maximum value be set in advance according to the measurement system used.
The threshold and the degree of change are set according to, for example, statistics and experiences of low molecular weight s-CD14 values of healthy persons and septic patients. The threshold for recovery from sepsis is as described in the first aspect of the invention.

  The degree of change can also be set statistically and empirically as a diagnostic indicator. For example, the low molecular weight s-CD14 value measured last time is the previously measured low molecular weight s-CD14 value as a criterion for determining that the severity of sepsis has increased and / or the treatment guidelines need to be changed. In contrast, it can be increased by about 30 to 50 ng / mL / day or more. On the other hand, as a judgment criterion such as improvement of sepsis and / or recognition of therapeutic effect, for example, the measured low molecular weight s-CD14 value is about more than the maximum value of the low molecular weight s-CD14 value measured after hospitalization. Reduction of 100 to 400 ng / mL or more, or reduction of about 2/3 to 1/4 or less of the maximum value, or about 50 to 200 ng / mL / day with respect to the low molecular weight s-CD14 value measured last time This can be reduced.

  The above threshold value and change index are examples derived from the embodiments of the present invention, and differ depending on differences in measurement kits, measurement environments, and the like. It may also be corrected empirically. The index of diagnosis in the present invention is not limited to the above threshold value and the degree of change.

  In the severity evaluation method of the second aspect of the present invention, the determination is made by comparing with the degree of change of the measured value and the “threshold” used in the early detection method of the first aspect of the present invention. Is preferred.

The severity evaluation method of the second aspect of the present invention may be performed approximately 24 hours to 48 hours earlier than the conventional evaluation method using parameters, and its usefulness is great. .
In the severity evaluation method of the second aspect of the present invention, the frequency of measurement over time depends on the degree of stability of the disease state, for example, at the initial stage of observation, every few hours when the disease state is severely changed, usually The measurement is exemplified once every 1 to 2 days and once every few days when the medical condition is stable.

  In the second aspect of the present invention, the therapeutic effect can be evaluated in addition to the severity evaluation method. Evaluation of therapeutic effect means that, for example, if the low molecular weight s-CD14 value measured over time using the blood of a patient after surgery decreases, it can be confirmed that the surgery has been effective and the therapeutic effect can be evaluated. . In addition, when the low molecular weight s-CD14 value measured over time using the blood of a patient after medication is unchanged or increased, it can be confirmed that the medication is not very effective and the therapeutic effect can be evaluated.

In the second aspect of the present invention, it is also possible to evaluate the following treatment guidelines. For example, if the low molecular weight s-CD14 value measured over time using the patient's blood after treatment decreases, continue current treatment, decrease drug dosage, or change to a less effective drug Etc. can be set and evaluated. Furthermore, if the measured low molecular weight s-CD14 value falls below the threshold, it can be evaluated that the drug administration is terminated.
On the contrary, if the low molecular weight s-CD14 value measured over time using the blood of the patient increases, it can be evaluated that the severity has increased, that is, the disease state has deteriorated. At this time, for example, it is possible to set / evaluate a change or addition of the type of drug, an increase in drug dose, etc. from conservative therapy (drug administration) to surgical therapy (surgery). It is also possible to determine a guideline for the degree of condition monitoring. For example, determination of a guideline such as moving from an ordinary ward to an intensive care unit (ICU) can be mentioned.

  The explanation common to the first aspect of the present invention, such as the method for measuring low molecular weight s-CD14, the blood of the patient for measuring the amount of low molecular weight s-CD14, and the cause of sepsis, is the first aspect of the present invention. It is as described in. The same applies to the following embodiments.

A third aspect of the present invention is a method for evaluating the severity of MODS, which comprises measuring the amount of low molecular weight s-CD14 in the blood of a patient.
Preferably, it is a method for evaluating the severity of MODS, characterized in that the amount of low molecular weight s-CD14 in the blood of a patient is measured and compared with a predetermined threshold value.
More preferably, it is a method for evaluating the severity of MODS, characterized in that the amount of low molecular weight s-CD14 in the blood of a patient is measured over time and evaluated by the degree of change in the measured value.

  More preferably, the amount of low molecular weight s-CD14 in the patient's blood is measured over time and calculated based on the amount of change per day or the maximum amount of the patient's low molecular weight s-CD14 amount. This is a method for evaluating the severity of MODS, which is characterized by evaluating the degree of change. In addition, the amount of low molecular weight s-CD14 in the patient's blood is measured over time, and the change represented by the rate of change per day or the maximum value of the amount of low molecular weight s-CD14 of the patient. This is a method for evaluating the severity of MODS, characterized by evaluation based on the degree of the above.

  The MODS subject to the method for evaluating the severity of MODS according to the third aspect of the present invention does not need to be specified as long as there is a concern about the onset or involvement of infection or sepsis. Is done.

The relationship between the pathology of sepsis and the amount of low molecular weight s-CD14 in the blood of the patient is as described in the first and second aspects of the present invention. The first aspect and second aspect of the present invention also relate to the method of comparison with a predetermined threshold in the method of evaluating the severity of MODS of the present invention and the method of evaluation based on the degree of change in the measured value. This is the same as described in the embodiment.
Furthermore, the present inventors show that the change in the amount of low molecular weight s-CD14 in the patient's blood changes with the same tendency as that of the patient's SOFA score (Non-patent Document 3) either early or at the same time. I found out. That is, in the case of Example 8 described later, the amount of low molecular weight s-CD14 in the blood increases due to the worsening of the severity of MODS, and the low molecular weight s-CD14 in the blood increases due to the improvement of the severity of MODS. It was confirmed that the amount decreased. Based on this finding, the present inventors measure the amount of low molecular weight s-CD14 in a MODS patient because the amount of low molecular weight s-CD14 changes according to the severity of the symptoms. Thus, it was invented that the severity of MODS can be evaluated preferably by measuring the amount of low molecular weight s-CD14 over time. For example, by measuring the amount of low molecular weight s-CD14 in the blood of a MODS patient over time and confirming the degree of change in the amount of low molecular weight s-CD14, the severity is evaluated. can do. In particular, it is possible to evaluate the severity of MODS following sepsis and to make an early evaluation.
The measurement method of low molecular weight s-CD14 is as described in the first aspect of the present invention.

The fourth aspect of the present invention is a method for detecting an infectious disease or sepsis early, a method for evaluating the severity or therapeutic effect of sepsis, or a method for evaluating the severity of MODS. This is a low molecular weight s-CD14 measurement kit capable of measuring CD14.
When the low molecular weight s-CD14 measurement kit of the fourth aspect of the present invention is used, an early detection method for infection or sepsis, a method for evaluating the severity or therapeutic effect of sepsis, or a method for evaluating the severity of MODS Low molecular weight s-CD14 in blood as an index can be measured.

  The measurement principle of low molecular weight s-CD14 in the low molecular weight s-CD14 measurement kit of the present invention is that an early detection method for infection or sepsis, a method for evaluating the severity or therapeutic effect of sepsis, or an evaluation of the severity of MODS. If low molecular weight s-CD14 used as the parameter | index of a method can be measured, it will not specifically limit.

The low molecular weight s-CD14 measurement kit of the present invention is not particularly limited as long as it contains a necessary reagent according to the measurement principle and does not inhibit the measurement result based on the measurement principle.
The example of the measurement principle of the low molecular weight s-CD14 measurement method and the optional components according to the principle are as described in the description of the first aspect of the present invention.

The low molecular weight s-CD14 measurement kit of the present invention is preferably a method for early detection of infection or sepsis, severity of sepsis or therapeutic effect, characterized by measuring low molecular weight s-CD14 by sandwich immunization. It is a low molecular weight s-CD14 measurement kit used for the evaluation method of (2) or the evaluation method of the severity of MODS.
The low molecular weight s-CD14 measurement kit of the present invention preferably contains an antibody that binds to a peptide consisting of 16 amino acid residues described in SEQ ID NO: 1.

Examples of the kit for directly measuring low molecular weight s-CD14 include an antibody that binds to a peptide consisting of 16 amino acid residues described in SEQ ID NO: 1 and a second antibody that binds to low molecular weight s-CD14. The kit characterized by this. Here, the “second antibody” is an antibody which may bind to other blood soluble proteins such as high molecular weight CD14 as long as it binds to human low molecular weight s-CD14. One of the two antibodies may be contained as a solid phase immobilized on an insoluble carrier and the other as a labeled antibody.
Furthermore, optional constituents include a buffer solution of a specimen or a labeled antibody, a substrate and a coloring agent suitable for the enzyme when the enzyme is used for the labeled antibody, a blocking agent, a cleaning agent or a terminating agent.

Examples of the insoluble carrier used for the solid phase include immunoplates, microtiter plates, plastic beads, magnetic particles, and membranes.
Examples of the labeled antibody include an antibody labeled by enzyme labeling with peroxidase, oxidase, alkaline phosphatase, urokinase or β-galactosidase, chemiluminescence labeling with acridinium or aequorin, or fluorescence labeling with FITC or the like.
Examples of suitable substrates for the enzyme include tetramethylbenzidine and the like for peroxidase, and p-nitrophenyl phosphate and the like for alkaline phosphatase.

  A kit for measuring low molecular weight s-CD14 indirectly described in WO 01/22085 is also included as a kit of the present invention.

The fourth aspect of the present invention includes a method for using the low molecular weight s-CD14 measurement kit described above.
That is, the fourth aspect of the present invention is used for an early detection method for infection and / or sepsis, a method for evaluating the severity (or therapeutic effect) of sepsis, or a method for evaluating the severity of MODS. It is the usage method of the low molecular weight s-CD14 measuring kit characterized.
Preferably, it is a method of using a low molecular weight s-CD14 measurement kit characterized by measuring low molecular weight s-CD14 by sandwich immunization, and more preferably a peptide comprising 16 amino acid residues described in SEQ ID NO: 1 The low molecular weight s-CD14 measuring kit is a method for using the low molecular weight s-CD14 measuring kit, characterized in that it comprises an antibody that binds.
These low molecular weight s-CD14 measurement kits are used, for example, by the method described in the first aspect of the present invention.

The fifth aspect of the present invention provides a therapeutic agent for sepsis.
That is, it is a therapeutic agent for sepsis characterized in that it is administered within 24 hours to a patient judged positive by the method for early detection of infection and / or sepsis according to the first aspect of the present invention. In addition, the present invention is a therapeutic agent for sepsis characterized by being administered under the monitoring of the severity of infection and / or sepsis or MODS by the low molecular weight s-CD14 measurement kit according to the fourth aspect of the present invention. .

  The therapeutic agent for sepsis characterized by being administered within 24 hours to a patient judged positive by the method for early detection of infection and / or sepsis according to the first aspect of the present invention is mainly an antibiotic. It is. For example, penicillin and cephalosporin β-lactam antibiotics, aminoglycoside antibiotics, glycopeptide antibiotics, macrolide antibiotics, tetracycline antibiotics, chloramphenicol antibiotics, cephem antibiotics Oxacephem antibiotics, carbapanem antibiotics, and the like.

What is also a therapeutic agent for sepsis, characterized by being administered under the monitoring of the severity of infection and / or sepsis or MODS by the low molecular weight s-CD14 measurement kit according to the fourth aspect of the present invention, In addition to the above antibiotics, immune supplements such as immunoglobulins, plasma preparations and fibronectin, immunopotentiators such as picibanil, levamisole and krestin, aprotinin, gabexate mesylate, jafamostat mesylate, urinastatin, anti-PAF antibody, Agents for mediators such as anti-TNF antibody, α-tocophenol, allopurinol and G-CFS, activated protein C preparation, anti-CD14 antibody and the like can be mentioned as therapeutic agents for sepsis.
In addition to these, circulatory improvement agents such as dopamine, epinephrine and antiarrhythmic agents, arachidone metabolism inhibitors, respiratory improvement agents such as steroids, blood anticoagulants such as heparin and ATIII, etc. are also treated for sepsis as drugs for organ damage. Included as a medicine.

The therapeutic agent for sepsis according to the fifth aspect of the present invention is administered within 24 hours to a patient determined to be positive by the method for early detection of infection and / or sepsis according to the first aspect of the present invention. As long as it is characterized, it is administered under the monitoring of the severity of infection and / or sepsis or MODS by the low molecular weight s-CD14 measurement kit according to the fourth aspect of the present invention. As long as all the therapeutic agents for sepsis are included, they are not limited to the drugs exemplified above. For example, Toru Yamaguchi et al., “Today's Treatment Guidelines 2003 Edition”, Medical School; Noboru Toda et al., “Mechanism of Cardiovascular Treatment”, Nanedo (1998), etc. can be used as a reference for treatment of sepsis.
The sepsis therapeutic agent of the fifth aspect of the present invention is appropriately selected and used as a single agent or a concomitant agent.

  EXAMPLES The present invention will be described more specifically with reference to the following examples. However, these are shown as examples, and the present invention is not limited to these examples. In addition, the abbreviations used in the following description are based on the abbreviations commonly used in the field. In addition, the amino acid number of the amino acid sequence is the number described in SEQ ID NO: 1 in the sequence listing.

(Example 1) Preparation of S68 antibody 1- (1) Preparation of peptide as immunogen Peptide having the sequence described in SEQ ID NO: 1 (corresponding to the 53rd to 68th sequences described in SEQ ID NO: 2) , Described as S68 peptide) was synthesized by inserting a cysteine at the N-terminus in order to bind to the carrier protein via the SH group at the N-terminus. That is, using a peptide synthesizer ABI433A (Applied), amino acid columns were arranged according to the amino acid sequence, and an amino acid column for cysteine was installed at the N-terminal for automatic synthesis. The synthesized peptide was cut out from the resin by a conventional method, precipitated with ether, recovered, dissolved again in distilled water, and lyophilized. The obtained crude purified peptide was dissolved, and then eluted with a linear gradient of 5-70% acetonitrile concentration using C18 reverse phase HPLC (CAPCELL-PAK, Shiseido), and a fraction containing the target peptide was collected. The collected fraction was lyophilized to obtain 2 to 3 mg of purified peptide.

1- (2) Preparation of peptide carrier antigen using synthetic peptide Each peptide prepared in 1- (1) was dissolved in distilled water to 10 mg / mL, and 10 mg / mL maleimidated keyhole limpet hemocyanin (Image Maleimed). Equal volume mixed with activated keyhole limpet hemocyanin (KLH) (PIERCE), reacted for 2 hours at room temperature, desalted with NAP-10 column (Amersham Bioscience) equilibrated with physiological saline, and S68 peptide carrier antigen (hereinafter referred to as “S68 peptide carrier antigen”) 1 mg of S68 peptide-KLH) The protein concentration described in the following examples was obtained by dividing the amount of KLH used by the amount of liquid.

1- (3) Preparation of polyclonal antibody using synthetic peptide as immunogen In order to prepare a polyclonal antibody against S68 peptide-KLH prepared in 1- (2), rabbits were immunized using S68 peptide-KLH. That is, 100 μg each of S68 peptide-KLH was diluted in 500 μL of physiological saline, mixed with 500 μL of Freund's complete adjuvant (DIFCO), and then subcutaneously in the back of a New Zealand white rabbit (Kitayama Labes) female 2.1-2.2 kg. Administered. Two weeks later, 100 μg each of S68 peptide-KLH was diluted in 500 μL of physiological saline, mixed with 500 μL of Freund's incomplete adjuvant (DIFCO) and administered subcutaneously to the back. Two weeks later, 100 μg of S68 peptide-KLH was diluted in 1 mL of physiological saline and administered into the ear vein.

  One week after the completion of administration, blood was collected from the ear vein, antiserum was separated according to a conventional method, and the antibody was purified. First, ammonium sulfate was added to the antiserum so as to have a final saturation concentration of 33%, followed by stirring at 4 ° C. for 1 hour, and then the deposited precipitate was centrifuged. Next, the precipitate was dissolved in 76 mM phosphate buffer (hereinafter referred to as PBS (pH 6.4)) and dialyzed overnight. After filtering the dialysate, it was applied to a protein A column (Prosep A, Millipore), and the bound IgG fraction was eluted with 0.1 M glycine hydrochloride buffer (pH 3.0) to obtain a purified antibody. After dialysis with PBS (pH 6.4), the protein concentration was calculated from the absorbance at 280 nm (absorption coefficient: 0.533 mg / mL). Hereinafter, the obtained antibody is referred to as S68 peptide polyclonal antibody.

1- (4) Preparation of specific purified polyclonal antibody In order to purify only the antibody against S68 peptide from S68 peptide polyclonal antibody, specific purification was performed by the following method. First, in order to bind S68 peptide having cysteine inserted (hereinafter referred to as C-S68 peptide) to a carrier via SH group, 200 μg of C-S68 peptide was mixed and reacted per 1 mL of SulfoLink Coupling Gel (PIERCE) according to the manual. . After the reaction, the remaining active groups were blocked to prepare an S68 peptide binding affinity column. Next, 7.92 mg of the purified IgG fraction described in 1- (3) was applied, and columned with a phosphate buffer (pH 7.4) (Dulbecco, hereinafter referred to as D-PBS (pH 7.4)). Then, the bound anti-S68 peptide antibody was eluted with 0.1 M glycine hydrochloride buffer (pH 3.0). After elution, the pH was neutralized, dialyzed with PBS, and the protein concentration was calculated from the absorbance at 280 nm (absorption coefficient: 0.533 mg / mL). As a result, 0.52 mg of anti-S68 peptide antibody (hereinafter referred to as S68 antibody). )was gotten.

(Example 2) Examination of measurement system of low molecular weight s-CD14 Using the antibody described in Example 1, a measurement system of human low molecular weight s-CD14 by sandwich EIA method was examined.

2- (1) Preparation of anti-CD14 monoclonal antibody F1031-8-3 The F1031-8-3 antibody was prepared by the method described in Example 7 of WO 01/22085 pamphlet. Briefly, this antibody is an antibody prepared by immunizing mice with CD14 protein in human blood, and the subtype determined using IsoStripe Mouse Monoclonal Isotyping Kit (Roche) was IgG2b / κ. .
To confirm the specificity of the F1031-8-3 antibody, s-CD14 (1-356) prepared with COS cells using the method described in Example 8 and Example 9 of WO 01/72993 The binding activity was measured using s-CD14 (1-307) S286C. First, s-CD14 (1-356), s-CD14 (1-307) S286C or BSA was immobilized on Hybond-C extra (Amersham Bioscience) at 250 ng / spot each, dried, and 0.05 g / Blocking was performed with 0.05% Tween 20, PBS (pH 6.4) containing mL skim milk (Meiji Dairies). After standing at room temperature for 1 hour, 0.05% Tween 20 containing 0.5% BSA, F1031-8-3 antibody diluted to 3 μg / mL with PBS (pH 6.4) was added, and reacted at room temperature for 1 hour. Washed with 0.05% Tween 20, PBS (pH 6.4).

Next, a peroxidase-labeled anti-mouse immunoglobulin antibody (DAKO) diluted 500-fold with 0.05% Tween 20 containing 10% rabbit serum and PBS (pH 6.4) was added, reacted at 37 ° C. for 30 minutes, and the like. And the binding activity was confirmed with an ECL kit (Amersham Bioscience). As a result, as shown in Table 1, the F1031-8-3 antibody bound to s-CD14 (1-307) S286C and s-CD14 (1-356), but not to BSA. It was revealed that the CD14 protein was specifically recognized.
“+” In Table 1 indicates a case where a spot is detected on the film by ECL, and “−” indicates a case where a spot is not detected.

2- (2) Examination of measurement system of low molecular weight s-CD14 In order to prepare a system capable of specifically detecting low molecular weight s-CD14, a sandwich using the antibodies described in Examples 1 and 2- (1) An EIA system was prepared.
[1] Preparation of peroxidase-labeled antibody Peroxidase-labeled antibody was prepared by dissolving 4 mg of peroxidase (Toyobo) in distilled water according to the method of Nakane et al. (J. Histochem. Cytochem., 22, p. 1084, 1974). Periodic acid was added and reacted at 25 ° C. for 20 minutes. After completion of the reaction, 1.5% ethylene glycol was added and reacted at 25 ° C. for 10 minutes and dialyzed against 1 mM acetate buffer (pH 4.4). Purified F1106-13-3 antibody was dialyzed against 10 mM carbonate buffer (pH 9.5), and 4 μg of peroxidase activated by adding 70 μL of 0.2 M carbonate buffer (pH 9.5) to 4 mg of antibody And the mixture was reacted at 25 ° C. for 2 hours. Next, 4 mg / mL sodium borohydride was added and reacted at 4 ° C. for another 2 hours. The reaction solution was dialyzed against PBS to obtain a peroxidase-labeled F1106-13-3 antibody (hereinafter sometimes referred to as F1106-13-3-HRP). The liquid concentration was measured, and the antibody concentration was calculated from the amount of antibody used.

[2] Preparation of Sandwich EIA System A sandwich EIA system using the S68 antibody prepared in Example 1 as a solid phase antibody and the antibody prepared in Example 2- (1) as a labeled antibody was prepared. That is, S68 antibody was diluted to 10 μg / mL with D-PBS (pH 7.4), and 50 μL was added to each well of an immunoplate (Maxisorb, NUNC). After overnight reaction at 4 ° C., the plate was washed 5 times with ion-exchanged water, and 100 μL of D-PBS containing 0.1% StabilGuard (SurModics, Inc) and 0.1% Tween 20 was added to each well for blocking. Next, 1% healthy human serum (serum from which soluble CD14 was removed using 3C10, hereinafter referred to as CD14-absorbed serum), PBS containing 0.1% BSA (pH 7.4) was used as a diluent. Diluted samples were prepared by diluting normal human serum and human sepsis patient serum 20-fold. The diluted specimen was added at 50 μL per well and reacted at 37 ° C. for 2 hours. After completion of the reaction, it was washed 3 times with physiological saline containing 0.05% Tween 20, and 0.6 μg / mL with 76 mM PBS (pH 8.0) containing 5% rat serum, 1% mouse serum and 0.1% Tween 20 F1106-13-3-HRP diluted to 50 μL was added to each well. After 2 hours of reaction at 37 ° C., the plate was washed 5 times in the same manner, and a tetramethylbenzidine solution (TMB, BioFix) was added to each well. After 20 minutes of reaction at room temperature, the reaction was stopped with a 0.5 M sulfuric acid solution, and the absorbance at 450 nm was measured with a plate spectrophotometer (NJ-2100, Nippon Intermed). As a result, in the healthy human serum, the absorbance was the same value as that of the diluted solution, and the sepsis patient activity showed an absorbance 4 times or more that of the diluted solution. From this, it was possible to measure a soluble protein in blood that can be confirmed specifically in septic patients, that is, low molecular weight s-CD14 defined in the present invention.

(Example 3) Human low molecular weight s-CD14 measurement kit 3- (1) Example of sandwich EIA system measurement kit Example 2- (2) shows a high value in the measurement of septic patients, and a low value in the measurement of healthy persons An example of the construction of a soluble protein kit using a combination of a solid phase antibody and a labeled antibody is shown.
(1) Solid phase antibody: Plate on which S68 antibody is immobilized
(2) Labeled antibody: peroxidase-labeled F1031-8-3 antibody
(3) Substrate solution (tetramethylbenzidine solution)
Other accessory plate system configuration examples
(4) Plate washing solution (0.9% NaCl, 0.05% Tween 20 solution)
(5) Sample dilution (PBS solution containing 0.1% BSA)
(6) Reaction stop solution (0.5 MH ₂ SO ₄ solution)
(7) Standard product (s-CD14 (1-307) S286C)
Measuring instrument when using the above measurement kit <Reference example>
(8) Plate spectrophotometer (for example, E-Max (Molecular Devices))

3- (2) Standard curve of sandwich EIA measurement kit The measurement was performed in the same manner as in Example 2- (2) using the measurement kit of (1). That is, S68 antibody was diluted to 10 μg / mL with D-PBS (pH 7.4), and 50 μL was added to each well of an immunoplate (Maxisorb, NUNC). After overnight reaction at 4 ° C., the plate was washed 5 times with ion-exchanged water, and 100 μL of D-PBS containing 0.1% StabilGuard (SurModics, Inc) and 0.1% Tween 20 was added to each well for blocking. Next, 0, 3, 25, 60, 100, 150 ng / mL of s-CD14 (1-307) S286C protein using 76 mM PBS (pH 7.4) containing 1% CD14-absorbed serum and 0.1% BSA as a diluent. A standard dilution series was prepared. 50 μL of a standard dilution series was added per well and reacted at 37 ° C. for 2 hours. After completion of the reaction, it was washed 3 times with physiological saline containing 0.05% Tween 20, and 5% rat serum, 1% mouse serum, peroxidase-labeled F1031-8-3 antibody was added to 76 mM PBS (pH 8) containing 0.1% Tween 20. 0.0), 50 μL of diluted labeled antibody diluted to 0.6 μg / mL was added to each well. After 2 hours of reaction at 37 ° C., the plate was washed 5 times in the same manner, and a tetramethylbenzidine solution (TMB, BioFix) was added to each well. After 20 minutes of reaction at room temperature, the reaction was stopped with a 0.5 M sulfuric acid solution, and the absorbance at 450 nm was measured with a plate spectrophotometer (NJ-2100, Nippon Intermed). The standard curve created is shown in FIG. A highly sensitive and simple measurement system with a measurement sensitivity of 0.6 ng / mL (blank + 3SD) was realized.

3- (3) Specificity of sandwich EIA system In order to examine the effect of high molecular weight CD14 present in human serum on the prepared measurement system, soluble human serum-derived soluble CD14 having a concentration of 0 to 4 μg / mL Was added to the standard product of s-CD14 (1-307) S286C, and the measurement was performed in the same manner as in (2). As a result, as shown in FIG. 2, the healthy human serum-derived soluble CD14 did not affect the measured value even at 4 μg / mL. From this result, it was found that the cross-reactivity with high molecular weight CD14 was 0.3% or less in this sandwich EIA system. That is, this system did not detect human serum high molecular weight CD14, and it was confirmed that it is specific for a soluble protein showing a high value in the serum of a septic patient.

3- (4) Evaluation of sandwich EIA measurement kit The reproducibility of the kit of (1) was evaluated. As in (2), the coefficient of variation (CV) for simultaneous reproducibility using three types of specimens is 5.8, 3.6, 3.5%, and the reproducibility between measurements is 6.2, 5.2, 5 The result was as good as 1%. Further, the recovery rate of the addition recovery test was as good as 88 to 109%, and the influence of anticoagulants (heparin, citric acid, EDTA) was not recognized. From the above results, it was shown that this kit has sufficient performance for measuring human low molecular weight s-CD14.

(Example 4) Detection of human low molecular weight s-CD14 In order to analyze substances in sepsis patient serum detected by the measurement kit described in Example 3- (1), the sepsis patient serum was subjected to gel filtration chromatography column Superdex 200PC3. 2/30 (Amersham Bioscience), and each fraction was measured using the measurement kit described in Example 3- (1) and a commercially available CD14-EIA kit (IBL-Hamburg). The molecular weight is calculated by calibrating the column using aldolase (158 kDa), BSA (67 kDa), ovalbumin (43 kDa), chymotrypsin (25 kDa) in the LMW calibration kit and the HMW calibration kit (Amersham Bioscience). went.

As a result, as shown in FIG. 3, the commercially available CD14-EIA kit detected soluble CD14 having a molecular weight of about 57 kDa, and was determined to be a conventionally reported 49 to 55 kDa high molecular weight soluble CD14 protein. On the other hand, in the kit described in Example 3- (1), a peak derived from human low molecular weight s-CD14 detected in a sepsis patient was detected in the vicinity of a molecular weight of 35 to 45 kDa, and no peak was detected in the vicinity of 57 kDa. From this, it was confirmed that the kit described in Example 3- (1) specifically detected only the soluble protein present in the blood.
From these facts, low molecular weight s-CD14 binds to an antibody specific for a peptide having a sequence detected only in human CD14, and binds to some anti-CD14 antibodies. Moreover, it was found by gel filtration that the molecular weight was 35 to 45 kDa, which was lower than the high molecular weight CD14 (conventional native CD14) present in the blood of healthy people.

(Example 5) Measurement of low molecular weight s-CD14 in septic patients 55 low-molecular-weight s-CD14 in plasma of 55 patients with septicemia and 71 healthy persons as a control, using the kit of Example 3- (1) It measured by the method similar to Example 2- (2). As a result, the healthy group was 27.4 ± 14.4 ng / mL (mean ± SD). The average value of the sepsis group was 248.2 ng / mL, and when the average value of the healthy group + 2SD, 57 ng / mL, was used as the sepsis threshold, the positive rate was 95.5%. Moreover, the average value of the non-infectious SIRS patient group was 41.4 ng / mL, and the positive rate was 16.3%.
From this result, it took several days to determine sepsis based on confirmation of infection such as blood culture in a conventional patient. However, by measuring low molecular weight s-CD14, the measurement can be easily performed in a short time. I understood that I can judge. Furthermore, it has been found that the determination of sepsis serves as an indicator of the start or increase in dosage of antibiotics to patients. Moreover, it turned out that it can use also for determination of a non-infectious SIRS patient group and a sepsis group.

(Example 6) Time-lapse measurement of low molecular weight s-CD14 (1)
Low molecular weight s-CD14 in the plasma of the following patients was measured over time. The patient was hospitalized for multi-organ damage due to a traffic accident. Surgery for liver injury was performed immediately after admission. Thereafter, it was found by ascites culture that the animal was infected on the 6th day. Symptoms were alleviated by drainage with antibiotics and drainage.
The time course of low molecular weight s-CD14 and CRP is shown in FIG. CRP was measured by the method described in Journal of Infectious Diseases, Vol. 73, 197 (1999). The left vertical axis in FIG. 4 indicates the low molecular weight s-CD14 value, and the right vertical axis indicates the CRP value.
The low molecular weight s-CD14 value was 90 ng / mL on day 6 and exceeded the threshold of 57 ng / mL used in Example 5. The CRP measured at the same time was confirmed to rise after 3 days. From this, it was found that the low molecular weight s-CD14 value is a parameter capable of detecting sepsis earlier than in the prior art. For this reason, it is understood that low molecular weight s-CD14 measurement is useful for determining treatment guidelines.

(Example 7) Time-lapse measurement of low molecular weight s-CD14 (2)
Low molecular weight s-CD14 in the plasma of the following patients was measured over time. The patient had peritonitis due to perforation of the large intestine. After the transportation, emergency operation for excision of the large intestine and construction of the colostomy was performed, and it was stored in the ICU. Moved to the general ward as the medical condition improved.
The time course of low molecular weight s-CD14, APACHE II score, and CRP is shown in FIG. The left vertical axis in FIG. 5 represents the low molecular weight s-CD14 value, and the right vertical axis represents the APACHE II score and CRP value (× 1/2).
The low molecular weight s-CD14 value before surgery was as high as 600 ng / mL. After the operation, the low molecular weight s-CD14 value decreased and became lower than the threshold used in Example 5 on the fifth day. The state of change of this value was the same tendency as APACHE II score. CRP had the same tendency, but changed later than APACHE II score.
From the above, it was confirmed that the low molecular weight s-CD14 value was useful for determining the severity of sepsis and its therapeutic effect.

(Example 8) Time-lapse measurement of low molecular weight s-CD14 (3)
Low molecular weight s-CD14 in the plasma of the following patients was measured over time. The patient was hospitalized with peritonitis due to perforation of duodenal ulcer. Initially, conservative therapy was given and the condition was stable. However, on the 8th day, it was judged that peritonitis was exacerbated due to the spread of abdominal pain and an increase in SOFA score, and surgery for perforation and peritoneal lavage was performed. The postoperative course was good.
FIG. 6 shows the number of compatible items in the diagnostic criteria for low molecular weight s-CD14, SIRS, SOFA score, and CRP over time. The left vertical axis in FIG. 6 indicates the low molecular weight s-CD14 value, and the right vertical axis indicates the number of items conforming to the SIRS diagnostic criteria, SOFA score (× 1/2), and CRP value (× 1/4).
At the time of hospitalization, the low molecular weight s-CD14 value was as high as 350 ng / mL. Thereafter, although the low molecular weight s-CD14 value decreased, the value exceeded the threshold value used in Example 5 was maintained, and at the time of exacerbation of peritonitis, the value was further increased to 480 ng / mL. As the disease recovered, the low molecular weight s-CD14 value decreased. These circumstances were the same for the number of items conforming to the SIRS diagnostic criteria and SOFA score. In this case, CRP did not show a change reflected in the pathology.

  From the above, it was confirmed that the low molecular weight s-CD14 value is useful for determining the severity of sepsis and its therapeutic effect, and for determining the severity of MODS.

  From Example 5 to Example 8 described above, low molecular weight s-CD14 was specifically increased in septic patients, and a useful threshold for determining sepsis could be derived. It was also found that infection or sepsis can be detected early by measuring low molecular weight s-CD14 in patients with sepsis, SIRS, or patients suspected of having sepsis. Furthermore, by measuring the low molecular weight s-CD14 of the patient over time, the severity of the reduction or worsening of sepsis from the degree of the change, and the severity of improvement or worsening of the symptoms of MODS that develop from sepsis It was found that the transition of can be judged. Since sepsis was detected at an early stage and the transition of severity was judged, it was found that treatment guidelines such as surgery and start / change of medication can be formulated. Furthermore, it was found that the therapeutic effect by surgery or medication can be determined from the degree of change in low molecular weight s-CD14.

It is the figure which showed the standard curve of the low molecular weight s-CD14-EIA kit of this invention using s-CD14 (1-307) S286C protein. It is a figure showing that healthy human serum-derived soluble CD14 protein does not affect the measurement value of the low molecular weight s-CD14-EIA kit of the present invention using s-CD14 (1-307) S286C protein. It is the figure which showed the result of having analyzed low molecular weight s-CD14 and high molecular weight CD14 in the blood of a sepsis by a low molecular weight s-CD14-EIA kit and a commercially available CD14-EIA kit (IBL-Hamburg) by gel filtration chromatography. . It is the figure which showed the time-dependent change of the low molecular weight s-CD14 value and CRP of the clinical case whose blood low molecular weight s-CD14 value increased immediately after the infection. It is the figure which showed the time-dependent change of the low molecular weight s-CD14 value, APACHEII value, and CRP value of the clinical case which shows that the blood low molecular weight s-CD14 value represents the severity of sepsis. Of low molecular weight s-CD14 value in clinical cases showing that the low molecular weight s-CD14 value in blood represents the severity of sepsis and the severity of MODS, the number of conforming items of SIRS diagnostic criteria, SOFA value and CRP value It is the figure which showed a time-dependent change.

Claims (4)

  A method for early detection of infection and / or sepsis, comprising measuring the amount of low molecular weight s-CD14 in the blood of a subject.   A method for evaluating the severity of sepsis, which comprises measuring the amount of low molecular weight s-CD14 in the blood of a patient.   A method for evaluating the severity of multiple organ failure syndrome, comprising measuring the amount of low molecular weight s-CD14 in the blood of a patient.   It is possible to measure the amount of low molecular weight s-CD14 in blood used in an early detection method for infection and / or sepsis, a method for evaluating the severity of sepsis, or a method for evaluating the severity of multiple organ failure syndrome Low molecular weight s-CD14 measurement kit.

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