Patent Application
20120309025
The present invention relates to a method of analyzing human sCD14-ST, which is known as a diagnostic marker of sepsis.
The term “analysis” as used herein includes a detection to judge the presence or absence of human sCD14-ST as a substance to be analyzed, and a measurement to quantitatively or semi-quantitatively determine the amount of the substance to be analyzed.
The CD14 molecule was named as a protein identified by a family of antibodies which recognize a glycoprotein expressed on the membrane surface of monocytes at the Third Leukocyte Typing Conference in 1986. In 1990, Wright et al. elucidated that the CD14 molecule was a receptor for an endotoxin, LPS (non-patent literature 1). The CD14 molecule is a glycoprotein having a molecular weight of 53 to 55 kDa, and analyses on cDNA revealed that 1.4 kb mRNA has a coding sequence of 356 amino acids (non-patent literature 2).
Human CD14 molecules include soluble CD14 as well as membrane-bound CD14, and it is known that a plurality of soluble CD14 subtypes having different molecular weights is present in blood. As these soluble CD14 subtypes, two soluble CD14 of about 55 kDa and 49 kDa (soluble high molecular weight cD14) and a soluble low molecular weight CD14 subtype of about 13 kDa (hereinafter referred to as human sCD14-ST) are known, and human sCD14-ST is clinically useful as a diagnostic marker of sepsis (patent literature 1).
Regarding human sCD14-ST, patent literature 1 discloses that when serum samples are repeatedly frozen and thawed, or allowed to stand at room temperature for a long time, it is assumed that human sCD14-ST in the serum samples is decomposed and cannot be measured in some cases, or it is assumed that high molecular weight CD14 in the serum samples is decomposed to human sCD14-ST or its similar structures and leads to incorrect measured values (paragraph [0151]). Patent literature 1 also discloses that human sCD14-ST is generated by digesting the full length of CD14 with proteases such as elastase, and that the generating sCD14-ST is stably present in the living body (in serum) at least in detectable quantities (paragraph [0126]).
As described above, the cases where incorrect measured values are obtained when serum samples are frozen and thawed, or allowed to stand at room temperature for a long time are assumed in patent literature 1, but it is general information about storage conditions of specimens, and does not caution any special handling. Patent literature 1 rather discloses that human sCD14-ST is stable in the living body and in serum.
[Non-patent literature 1] Science (the United States), 1990, vol. 249, p. 1431-1433
[Non-patent literature 2] Nucleic Acids Research (the United Kingdom), 1988, vol. 16, p. 4173
[Patent literature 1] Japanese Patent No. 4,040,666
Under the circumstances where it has been known that human sCD14-ST is relatively stably present in serum, the present inventors have conducted intensive studies to develop a more accurate method of analyzing human sCD14-ST contained in samples including plasma samples and whole blood samples, and as a result, unexpectedly found that the measured values of human sCD14-ST were affected when whole blood samples were used.
The present invention has been made based on this finding. An object of the present invention is to provide an analysis method capable of accurately measuring human sCD14-ST, even when a whole blood sample is used.
The problem may be solved by the present invention, that is, a method of analyzing human sCD14-ST, characterized by analyzing human sCD14-ST in a whole blood sample within 6 hours of the collection of the sample.
The term “human sCD14-ST” (also referred to as Presepsin (registered trademark)) as used herein means the “soluble CD14 antigen of the first aspect” disclosed in Japanese Patent No. 4,040,666, and more particularly, is a soluble CD14 antigen having the following characteristics:
1) Molecular weight of 13±2 kDa, as measured by SDS-PAGE under nonreducing conditions;
2) Having the amino acid sequence of SEQ ID NO: 1 at the N-terminal sequence; and
3) Binding specifically to an antibody prepared using a peptide consisting of 16 amino acid residues of SEQ ID NO: 2 as the antigen.
According to the present invention, human sCD14-ST, which is stable in serum or plasma samples, but measured values of which is fluctuating with the progress of time in whole blood samples, can be accurately analyzed using a whole blood sample, without preparing a serum or plasma sample from the whole blood sample.
The analysis method of the present invention may be carried out in accordance with known analysis methods for human sCD14-ST, except that a whole blood sample is used as the sample to be analyzed, and that its analysis is carried out within 6 hours of the collection of the sample.
Human sCD14-ST may be analyzed using various known analysis methods for proteins, for example, immunological analysis methods using one or more antibodies, or biochemical methods such as electrophoresis, and an automatic analyzer for clinical testing may be used.
For example, Japanese Patent No. 4,040,666 discloses a method of measuring human sCD14-ST, more particular, a sandwich EIA system [Example 7-(1) of Japanese Patent No. 4,040,666] using the combination of a polyclonal antibody (S68 antibody) or a monoclonal antibody (F1146-17-2 antibody) prepared using a peptide (S68 peptide described in Japanese Patent No. 4,040,666) consisting of 16 amino acid residues of SEQ ID NO: 2 as the antigen, and an anti-CD14-antigen monoclonal antibody (for example, F1031-8-3 antibody or F1106-13-3 antibody), and it may be applied to the analysis method of the present invention.
In the analysis method used in the Examples described below, human sCD14-ST contained in each sample is bound to magnetic latex coated with an anti-sCD14-ST mouse monoclonal antibody (F1106-13-3 antibody) and an ALP (alkaline phosphatase)-labeled anti-sCD14-ST rabbit polyclonal antibody (S68 antibody) to form an immunocomplex by an antigen-antibody reaction; the excess ALP-labeled anti-sCD14-ST rabbit polyclonal antibody is removed from the immunocomplex; and the ALP enzyme activity of the immunocomplex is measured by a chemiluminescent enzyme immunoassay (CLEIA) using a luminescent substrate (CDP-Star; Tropix) to determine the concentration of human sCD14-ST contained in the sample.
In the analysis method of the present invention, a whole blood sample is used as the sample to be analysis. The expression “a whole blood sample is used” as used herein means that the sample used is a sample which has been stored in the form of whole blood for a period of time from the collection of whole blood to the beginning of the analysis of the sample (hereinafter referred to as the storage period after blood collection) or most of that period. The whole blood sample may be collected by a normal operation, i.e., using a blood collection tube containing an anticoagulant such as heparin, EDTA, or citric acid.
The whole blood sample collected using a blood collection tube may be stirred by inversion several times, and then, may be directly used for measurement or may be used after plasma separation. The expression “used after plasma separation” includes the case where whole blood is centrifuged to separate two layers of plasma and blood cells and the plasma component is used in that state, and the case where the plasma component is used after further dispensing the plasma component. In the analysis method of the present invention, the whole blood sample is used as the sample to be analyzed, and the case where whole blood after the storage period after blood collection is separated into plasma and the other components is used is included in embodiments of “a whole blood sample is used”. According to the findings of the present inventors including the Examples below, when human sCD14-ST contained in a whole blood sample is measured, the measured values may fluctuate with the progress of time in some cases, and excessive physical shock when preparing the sample or transporting the sample may be a factor which affects the measured values in some cases.
In the analysis method of the present invention, the analysis is carried out within 6 hours (preferably within 4 hours) of the collection of the sample. The expression “the analysis is carried out within 6 hours” as used herein means that the analysis begins within 6 hours.
More particularly, it is preferable that when the whole blood sample is collected using an EDTA blood collection tube, the analysis is carried out within 6 hours, and it is preferable that when the whole blood sample is collected using a heparin blood collection tube, the analysis is carried out within 4 hours.
As shown in the concrete experimental data described in the Examples below, an increase in the measured values of human sCD14-ST was observed in whole blood samples from healthy people, even when the measurement was after 6 to 8 hours from the blood collection, in which period no changes in measured values are observed in plasma samples or serum samples. It is known that the measured value of human sCD14-ST is different by 50-fold between healthy people and sepsis patients, and the concentration of human sCD14-ST contained in the blood of healthy people is extremely low. This enables sepsis to be diagnosed with high sensitivity and high specificity by setting an appropriate cut-off value (Example 12 of patent literature 1). However, where the measured values of human sCD14-ST in healthy people increase with the progression of time after collecting a whole blood sample, there is a possibility that the judgment of whether the measured value is lower or higher than the cut-off value is different depending on the measuring time. Under these circumstances, the present inventors found that the measured values of human sCD14-ST in a whole blood sample did not fluctuate within 6 hours (preferably within 4 hours) of the collection of a sample. Since the analysis method of the present invention is carried out within 6 hours of the collection of a whole blood sample, human sCD14-ST can be accurately measured without an increase in measured values.
The present invention now will be further illustrated by, but is by no means limited to, the following Examples.
Each sample to measure human sCD14-ST was measured using reagents for measuring human sCD14-ST. These reagents included a solution of magnetic latex coated with an anti-sCD14-ST mouse monoclonal antibody (F1106-13-3 antibody; Japanese Patent No. 4,040,666) as the first antibody solution, a solution of an ALP (alkaline phosphatase)-labeled anti-sCD14-ST rabbit polyclonal antibody (S68 antibody) as the second antibody solution, a washing solution for B/F separation, a substrate solution, and the like, and were packaged in a cartridge applicable to an automatic luminescent immunoanalyzer described below.
An automatic luminescent immunoanalyzer (PATHFAST; Mitsubishi chemical Medience Corporation), which is similar to that disclosed in Japanese Patent No. 3,115,501 and which can automatically perform immunoassay using magnetic particles, was used for the measurement. This apparatus is capable of efficiently performing B/F separation by magnetic force in a tip arranged as a unit of liquid suction and discharge, and exhibits high efficiency in washing. The measurement steps of the apparatus are as follows.
A chemiluminescent substrate “CDP-Star” (Tropix) was used as the substrate, and emission counts detected by a photomultiplier tube (PMT) were regarded as the measurement results.
Each cartridge for an automatic measurement was filled with each sample, a sample diluent solution, the solution of magnetic particles (coated with the first antibody), the washing liquid for B/F separation, the solution of the second antibody, the substrate solution, and the like, and loaded to the automated analyzer. The following steps were carried out in accordance with the normal procedure:
Whole blood samples, serum samples, and plasma samples were allowed to stand at room temperature for predetermined periods of time, and then used to measure human sCD14-ST in accordance with the measuring method described in Example 1.
More particularly, whole blood samples, serum samples, and plasma samples, which had been collected from three healthy people using blood collection tubes containing an anticoagulant (heparin or EDTA), were allowed to stand at room temperature for 0, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, or 10 hours (only for the whole blood samples and the plasma samples), and each measurement was immediately carried out at each time point to confirm the time course of the measured values for each sample. As the serum samples, whole blood samples were collected using plain blood collection tubes (Terumo) and centrifuged and separated into sera, and these serum samples were used. EDTA plasma samples were prepared using EDTA blood collection tubes (Terumo), and heparin plasma samples were prepared using heparin blood collection tubes (Terumo). The whole blood samples were collected using the above blood collection tubes, and directly used without plasma separation.
The results of the whole blood samples and the plasma samples prepared using heparin blood collection tubes are shown in
In addition, when excessive physical shock, such as stirring, was applied to the whole blood samples, a remarkable increase in the measured values was observed (data not shown).
The results of the serum samples are shown in
Further, it was confirmed that the measured values were sufficiently stable in the serum samples and the plasma samples, even when they had been allowed to stand at room temperature for at least 24 hours (data not shown).
The cause of the instability of the measured values of the whole blood samples was examined.
Condition 1: Whole blood sample
Condition 2: Plasma sample prepared by allowing the whole blood sample to stand at room temperature for predetermined periods of time and performing plasma separation before the measurement
Condition 3: Plasma sample
The preparation and the measurement of the samples were carried out, using heparin blood collection tubes, in accordance with Example 2. The results are shown in
It was suggested from these results that the increase in the measured values was caused by factor(s) not from the plasma component but from the blood cell component. It was necessary to measure a whole blood sample directly within 4 hours of the blood collection, and further, unless plasma separation was carried out within 4 hours of the blood collection of a whole blood sample, an increase in the measured values after the plasma separation was observed, and therefore, it was found that the plasma separation within 4 hours of the blood collection was necessary to obtain accurate measured values.
Whole blood samples were collected from three healthy people using blood collection tubes containing an anticoagulant (heparin or EDTA), and were allowed to stand at room temperature for 0, 2 hours, 4 hours, and 8 hours, and each measurement was immediately carried out at each time point, using a commercially available CRP measuring reagent (PATHFAST hs CRP measuring reagent; Mitsubishi chemical Medience Corporation) to confirm the time course of the measured values for each sample. The preparation and the measurement of the samples were carried out in accordance with Example 2, except that the CRP measuring reagent was used.
The results are shown in
The analysis method of the present invention may be used in, for example, the diagnosis of sepsis.
Although the present invention has been described with reference to specific embodiments, various changes and modifications obvious to those skilled in the art are possible without departing from the scope of the appended claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2010-018340 | Jan 2010 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP2011/051778 | 1/28/2011 | WO | 00 | 7/26/2012 |
