JP4224592B2 - Method for producing plasma - Google Patents

Description

  The present invention relates to a method for producing plasma. More specifically, the present invention relates to a method for producing plasma suitable for the quantification of annexin V associated with a disease.

  The relationship between Hasegawa's simplified intelligence assessment scale, which was the only measure of Alzheimer's disease so far, and Annexin V was studied, and it was reported that Annexin V was detected at high concentrations in all Alzheimer patients ( Non-patent document 1). And the method which can test | require annexin V simply is known using the bodily fluid (blood etc.) as a sample and using the ELISA method by a Western blot (patent document 1).

Hiroko Okawa et al., Journal of Geriatric Psychiatry, Vol. 14, No. 2, pp. 227-235, 2003, Journal of the Japan Geriatric Psychiatric Association Japanese Patent Laid-Open No. 2004-251794

  However, the conventional inspection method has a problem that the accuracy of determination as to whether the patient is an Alzheimer patient or a non-Alzheimer patient (healthy person) is not high. In particular, in order to effectively use an effective therapeutic agent (for example, donepezil hydrochloride) for the initial treatment of Alzheimer's disease, it has been desired to develop a method for determining whether or not the patient is highly accurate in the early stage of Alzheimer's disease. . That is, an object of the present invention is to provide a method capable of determining whether or not a disease (for example, Alzheimer's disease) has a high accuracy.

As a result of diligent research to solve the above problems, the present inventor has discovered that the measured value of annexin V fluctuates during preparation of a measurement sample and storage of the measurement sample, and by preventing this fluctuation, The inventors have found that the above object can be achieved and have reached the present invention.
That is, the method for producing plasma of the present invention is characterized in that after adding an anticoagulant to blood, a centrifugation method using a centrifugal force of 3000 to 6000 G. And removing the tangible body having a size of 0.1 μm or more from the blood to obtain plasma having a tangible body content of 0.5 × 10 ⁵ pieces / mL or less (1). The gist.
In addition, the annexin quantification method of the present invention is characterized by a centrifugal separation method using a centrifugal force of 3000 to 6000 G after adding an anticoagulant to blood. And removing the tangible body having a size of 0.1 μm or more from the blood to obtain plasma having a tangible body content of 0.5 × 10 ⁵ pieces / mL or less, The point which has the process (2) which immunoassays the annexin V contained is included.
Further, the plasma storage method of the present invention is characterized in that after adding an anticoagulant to blood, a centrifugal separation method using a centrifugal force of 3000 to 6000 G. And removing the tangible body having a size of 0.1 μm or more from the blood to obtain plasma having a tangible body content of 0.5 × 10 ⁵ / mL or less, The point is that it comprises the step (3) of storing the plasma obtained in 1) at −200 to 10 ° C.

  If the method for producing plasma, the method for quantifying annexin V or the method for storing plasma of the present invention is used, it can be determined whether or not the patient has a disease with high accuracy. In particular, it is highly effective in determining Alzheimer's disease. In addition, since it is possible to determine whether the patient is a patient with high accuracy in the early stage of Alzheimer's disease, it is possible to effectively use an effective therapeutic agent for the initial treatment of Alzheimer's disease (deterioration of disease progression or Can be stopped or cured).

<About step (1)>
The blood used in the present invention is not limited in the collection site and collection method, and the conventional collection site and collection method (for example, a method of collecting about 5 mL of whole blood mainly from the median cubital vein using a blood collection syringe). Etc. are used. According to Clinica Chimica Acta (Kaneko et al., Vol. 251, pp. 65-80, 1996), it is shown that hemolysis of collected blood has a positive error on the annexin V measurement value. As the blood used in the above, blood in which hemolysis is not observed is preferable.

As the anticoagulant added to the blood, normal anticoagulants can be used. Examples include anticoagulants that promote antithrombin action and anticoagulants that inhibit blood coagulation by chelating calcium ions. It is done. Examples of the anticoagulant that promotes the antithrombin action include heparin sodium and heparin lithium. Examples of the anticoagulant chelating calcium ions include disodium ethylenediaminetetraacetate (EDTA2Na), dipotassium ethylenediaminetetraacetate (EDTA2K), tripotassium ethylenediaminetetraacetate (EDTA3K), sodium fluoride and sodium citrate. It is done. Of these, anticoagulants that chelate calcium ions are preferable from the viewpoint of convenience during use, and more preferably, disodium ethylenediaminetetraacetate (EDTA2Na), dipotassium ethylenediaminetetraacetate (EDTA2K), and ethylenediaminetetraacetic acid triacetate. Potassium (EDTA3K), particularly preferably disodium ethylenediaminetetraacetate (EDTA2Na).
The addition concentration (mmol / liter: mM) of the anticoagulant may be a commonly used amount, for example, preferably 1 to 10, more preferably 2 to 7, particularly preferably 3 based on the blood volume. ~ 5.

Examples of the tangible body having a size of 0.1 μm or more include blood cell components and fibrin clots.
Examples of blood cell components include red blood cells, white blood cells (granulocytes, monocytes, lymphocytes and the like), platelets and the like. In addition, platelets include platelet mass formed when anticoagulants such as disodium ethylenediaminetetraacetate (EDTA2Na), dipotassium ethylenediaminetetraacetate (EDTA2K) and tripotassium ethylenediaminetetraacetate (EDTA3K) are used. It is.
Examples of the fibrin clot include a clot composed of fibrin formed by polymerizing a fibrin monomer produced by fibrinogen being decomposed by thrombin.

The content of tangible bodies having a size of 0.1 μm or more (pieces / mL) is preferably 0.7 × 10 ⁵ or less, more preferably 0.5 × 10 ⁵ or less, and particularly preferably 0.3 × 10 5. ⁵ or less. Within this range, it is possible to determine whether or not the patient has a disease with higher accuracy. In addition, the storage stability of plasma is further improved, and Annexin V involved in the disease can be more accurately quantified even after long-term storage.
For counting the content of a tangible body having a size of 0.1 μm or more, a counting method using a culture microscope having a phase difference function can be applied. The measurement sample is preferably used without pretreatment such as dilution, and it is not necessary to perform special treatment such as heat treatment. In addition, the magnification of the culture microscope at the time of counting is 800 times, and the counting is performed using a Burker-Turk calculation plate or Neubauer calculation plate.
The shape of the tangible body having a size of 0.1 μm or more to be counted is a sphere or a lump, and the longest diameter or the longest length is 0.1 μm or more.

In order to remove a tangible body having a size of 0.1 μm or more from blood, a centrifugal separation method, an adsorption removal method, a filtration method, or the like can be applied. Among these, from the viewpoint of simplicity (the operation can be performed quickly and easily), the centrifugation method is preferable.
The centrifuge and the centrifuge container used in the centrifuge method are ordinary ones (such as those used for blood separation), and a centrifuge with a cooling function or a vacuum blood collection tube used for blood collection is used. It is preferable.
The temperature (° C) during centrifugation is preferably 0 to 40, more preferably 0 to 37, particularly preferably 0 to 25, and most preferably 2 to 10.
The centrifugal force (G) at the time of centrifugation is preferably 2000 to 6000, more preferably 2500 to 5500, and particularly preferably 3000 to 5000G. Within this range, it is possible to determine whether or not the patient has a disease with higher accuracy. In addition, the storage stability of plasma is further improved, and Annexin V involved in the disease can be more accurately quantified even after long-term storage. That is, when the centrifugal force is less than 1800 G, measurement value fluctuations according to the blood cell component concentration of the subject tend to occur, and it is difficult to accurately quantify the annexin V involved in the disease. Since the centrifugal force for obtaining normal plasma is 800 to 1800 G, annexin V cannot be accurately quantified with plasma prepared by conventional centrifugal force. There is a problem in that the measured value of the rise is significantly increased. On the other hand, when the centrifugal force exceeds 6000 G, blood cell components and the like in the blood are easily destroyed, and as a result, annexin V not involved in the disease is detected, and the annexin V involved in the disease is accurately quantified. Things tend to be difficult.
The time (minutes) during which the centrifugal force (G) is applied is preferably 5 to 30, more preferably 6 to 25, particularly preferably 8 to 20, and most preferably 10 to 15.

In the adsorption removal method, a tangible body can be removed by allowing blood to pass through a column filled with an adsorbent and adsorbing the tangible body on the adsorbent.
As the adsorbent, widely known ones can be used, and examples thereof include polyolefin fibers, nitrocellulose fibers, cellulose fibers, and polyethylene terephthalate fibers. Among these, polyethylene terephthalate fibers are preferable from the viewpoint of adsorption capacity. The liquid volume (mL) for allowing blood to pass is preferably 0.5 to 2, and more preferably 1 to 1.5, with respect to 0.1 g of the adsorbent.
In order to prevent the destruction of blood cell components such as red blood cells when blood is passed through the column, it is preferable that the blood falls naturally without being pressurized.

  In the filtration method, a tangible body can be removed by filtration using a filter medium (dialysis membrane, ultrafiltration membrane or the like). As the filter medium, those usually used can be used, and examples thereof include a cellulose membrane, a nitrocellulose membrane, and a glass filter. As an average hole diameter (micrometer) of a filter medium, 0.01-2 are preferable, More preferably, it is 0.02-1.6, Most preferably, it is 0.05-0.8.

If it has a process (1), the manufacturing method of the plasma of this invention can include the other normal process {The well-known process etc. which are applied in preparing plasma}.
In addition to the step (1), the method for producing plasma of the present invention may include a step of coexisting a protease inhibitor (protease inhibitor). In addition, the proteolytic enzyme inhibitor is added in order to prevent degradation of proteins in plasma. Examples of the protease inhibitor include inhibitors described in JP-A No. 2003-270238.

<About step (2)>
The plasma obtained as described above can be accurately quantified for annexin V involved in the disease by subjecting it to the step (2) of immunoassay (immunological assay) of annexin V.
Examples of immunoassay include radioimmunoassay (RIA method), enzyme immunoassay (EIA method), fluorescence immunoassay (FIA method), chemiluminescence immunoassay (CLIA method), and latex agglutination method. Applicable. Among these, radioimmunoassay (RIA method), enzyme immunoassay (EIA method), fluorescence immunoassay (FIA method) and chemiluminescence immunoassay (CLIA method) are preferable from the viewpoint of quantitative sensitivity.

As the anti-annexin V antibody used for immunoassay, both a polyclonal antibody and a monoclonal antibody can be used. In immunoassay, a sandwich assay method using a solid phase antibody in which an anti-annexin V antibody is immobilized on a solid phase and a labeled antibody labeled with a labeled compound is preferred.
As a method for immobilizing the anti-annexin V antibody on a solid phase, it can be carried out by a usual method (chemical bonding method and physical adsorption method) or the like.
As a chemical bonding method, a functional group (amino group, sulfo group, etc.) introduced on the solid surface and a functional group (amino group, sulfo group, etc.) of anti-annexin V antibody are combined with a binder (glutaraldehyde, succinaldehyde). , M-maleimidobenzoyl-N-hydrosuccinimide ester, o-phenylenebismaleimide, etc.) and the like (US Pat. Nos. 4,280,992 and 3,627,761, etc.).
As a physical adsorption method, when the solid phase is polystyrene, the polystyrene solid phase is immersed in a carbonate buffer solution (pH 9.0) containing 0.001 to 0.04% (W / V) of an annexin V antibody for an appropriate time. (BioSim Biophys Actor, Vol. 251, 427, 1971). This method can also be applied to substances whose solid phase is other than polystyrene, such as polypropylene, silicon, glass and cellulose.

As labeling compounds for labeling anti-annexin antibodies, ordinary compounds can be used, such as radioisotope atoms (such as ¹²⁵ I), fluorescent materials (such as europium complexes), luminescent materials (such as N-methylacridium esters) and Enzymes (horseradish peroxidase, alkaline phosphatase, β-galactosidase, etc.) are used. Of these, enzymes are preferred, and horseradish peroxidase and alkaline phosphatase are more preferred. As a labeling method, a normal method or the like can be applied {for example, “Sequence Biochemistry Experiment Course 5 Immunobiochemistry Experiment Method” (edited by the Japanese Biochemical Society, Tokyo Chemical Dojin, 1986, pages 102-112).

By using the annexin V quantification method of the present invention, the disease can be determined more accurately.
Diseases include those related to annexin V contained in blood, and examples include heart disease and Alzheimer's disease.

In the determination method of the present invention, a reagent kit for immunoassay comprising a solid phase on which an anti-annexin V antibody is immobilized and a peroxidase-conjugated anti-annexin V antibody using peroxidase as a labeling enzyme is used as a constituent. The configuration of the immunoassay reagent kit is not limited, but preferably includes an immune reaction buffer, a B / F separation buffer, a control sample (a standard substance for preparing a calibration curve), and the like. As the buffer for immune reaction and the buffer for B / F separation, the buffer described in JP-A-2004-264294 can be used.
There is no limitation on the dosage form of each reagent contained in the reagent kit.

<About step (3)>
Step (3) is a step of storing the plasma obtained in step (1) at −200 to 10 ° C.
The plasma obtained in the step (1) can be stored using a normal storage device (freezer, refrigerator, etc.), and a storage container that is usually used can be used as a storage container.
The temperature (° C.) during storage is preferably −200 to 10, more preferably −100 to 4, particularly preferably −85 to −20.

EXAMPLES Hereinafter, although an Example demonstrates this invention further, this invention is not limited to this.
<Example 1>
(Selection of Alzheimer patients, etc.)
The Hasegawa-style simplified intelligence evaluation scale was used to examine intelligence scores. Ten people (5 men and 5 women) who were recognized as Alzheimer's disease and 10 healthy individuals (5 men and 5 women) who were not recognized as Alzheimer's disease. ) Was selected.

(Preparation of plasma specimen)
Using a vacuum blood collection tube (trade name “Benoject II” manufactured by Terumo Corporation) containing 5.4 mg of disodium ethylenediaminetetraacetate, 10 Alzheimer's disease patients (5 males and 5 females) and 10 healthy individuals ( After 5 mL of blood was collected from 5 men and 5 women), it was mixed uniformly to obtain blood with an anticoagulant. Thereafter, using a centrifuge H-3FR manufactured by KOKUSAN (4 ° C., 3500 G, 15 minutes), the anticoagulant-containing blood was centrifuged, and only the upper layer portion of the blood separated into the upper and lower layers was taken out. Plasma {plasma derived from Alzheimer patients (patients 1 to 10), plasma derived from healthy persons (healthy persons 1 to 10)} was obtained.

(Measurement of the number of tangible bodies with a size of 0.1 μm or more)
About the obtained plasma, the measurement of the tangible body with a magnitude | size of 0.1 micrometer or more was performed using the culture microscope CKX41 type | mold by OLYMPUS, and a BurKer-Turk type | mold count plate. The plasma used for measurement was used without pretreatment. Further, the magnification of the microscope was 800 times, and the measurement was performed three times for each plasma (the average value of three times was used as the measurement result).

(Storage of plasma samples)
As for plasma, 1 mL for immediately measuring annexin V was collected, and the remaining plasma was stored frozen (SANYO ultra-low temperature freezer, at −80 ° C. for 4 days).

(Preparation of immunoassay reagents)
・ Acquisition of anti-annexin V antibody Two mouse monoclonal antibodies (antibodies A and B) obtained using annexin V as an immunogen were obtained by the method described in the monoclonal antibody experiment manual (Toyama et al., Kodansha Scientific, 1987). Prepared.

Anti-Annexin V Antibody-Binding Polystyrene Beads Antibody A was uniformly mixed in a pH 9.0, 0.1 M carbonate buffer solution to a concentration of 20 μg / mL to obtain a mixed solution. Next, 2000 polystyrene beads (manufactured by Immunochemical Co., Ltd.) with a diameter of 3.2 mm were added to 50 mL of this mixed solution and allowed to stand at 4 ° C. for 48 hours, and then the mixed solution was removed by suction with an aspirator to bind anti-annexin V antibody. 2000 polystyrene beads were obtained.
Immediately, 2,000 of the anti-annexin V antibody-bound polystyrene beads were immersed in 50 mL of a phosphate buffer solution containing 0.1% by weight BSA, and stored refrigerated (2 to 10 ° C.).

Peroxidase-labeled anti-annexin V antibody Antibody B was prepared using a horseradish-derived peroxidase (manufactured by Toyobo Co., Ltd.), literature [S Yoshitake, M Imagawa, E Ishikawa, Etol; Biochem, Vol. 92 (1982) 1413-1424], a peroxidase-labeled anti-annexin V monoclonal antibody solution was prepared.
Next, the peroxidase-labeled anti-annexin V monoclonal antibody solution was diluted to a concentration of 1 μg / mL with a phosphate buffer solution containing 1% by weight of bovine serum albumin to prepare a labeled antibody solution {refrigerated until used for measurement (2 -10 ° C)}.

・ Chemiluminescence reagent (first liquid and second liquid)
Luminol sodium (manufactured by Wako Pure Chemical Industries, Ltd.) 2.80 g and 4- (cyanomethylthio) phenol 0.56 g are dissolved in 1 L of pH 8.0 phosphate buffer solution, and the chemiluminescent reagent (first solution) is dissolved. Obtained {refrigerated (2-10 ° C) until used for measurement}.
In addition, 200 μL of 35 wt% hydrogen peroxide solution was dissolved in 1 L of deionized water to obtain a chemiluminescent reagent (second liquid) (stored refrigerated (2-10 ° C.) until used for measurement}.

Preparation of Annexin V Standard Solution About 100 μg of human annexin V antigen (manufactured by ALEXIS) was dissolved in 100 μL of 1% by weight bovine serum albumin-containing phosphate buffer to obtain an annexin V solution. The concentration was determined by the BCA method. Next, this Annexin V solution was serially diluted with a phosphate buffer containing 1% by weight bovine serum albumin, and 20 ng / mL, 10 ng / mL, 7.5 ng / mL, 5.0 ng / mL, 2.5 ng / mL, 1.0 ng / mL of each solution was prepared and used as annexin V standard solutions (20, 10, 7.5, 5.0, 2.5, 1.0). 10 ° C.)}.

(Measurement of Annexin V concentration in sample)
Annexin V immunoassay was performed as follows for plasma immediately after preparation and plasma frozen at −80 ° C. for 4 days (thawed at 10 ° C. × 30 minutes).
20 μL of plasma, 300 μL of 1 wt% bovine serum albumin-containing phosphate buffer, and one anti-annexin V monoclonal antibody-bound polystyrene bead are placed in a test tube and reacted at 37 ° C. for 1 hour, and then the reaction solution is aspirator To obtain reaction beads. Subsequently, 5 mL of physiological saline was added to the reaction beads to wash the reaction beads, and the washing solution was removed with an aspirator to obtain reaction washed beads.
Next, after adding 300 μL of peroxidase-labeled anti-annexin V monoclonal antibody solution to the reaction washed beads and reacting at 37 ° C. for 1 hour, the reaction solution was removed with an aspirator, and 5 mL of physiological saline was added to wash the beads. The beads for measurement were obtained by removing with an aspirator.
A test tube (12 × 75 mm) containing measurement beads is set in a sample holder of a luminescence reader BLR-201 manufactured by Aloka, and 200 μL of chemiluminescent reagent (first liquid) and 200 μL of chemiluminescent reagent (second liquid) are added. 40 seconds after the addition, the integrated measurement of the chemiluminescence amount was started, and the chemiluminescence amount (integrated amount) for 10 seconds was obtained.
Then, the chemiluminescence amount of the annexin V standard solution is measured in the same manner, a calibration curve is created based on the luminescence amount of the obtained annexin V standard solution, and the annexin V concentration contained in the plasma is calculated from the calibration curve. Calculated.

<Example 2>
Plasma was prepared in the same manner as in Example 1 except that the centrifugation conditions (4 ° C., 3500 G, 15 minutes) were changed to (4 ° C., 2500 G, 15 minutes), and the number of tangible bodies was counted. The concentration of was calculated.

<Example 3>
Plasma was prepared in the same manner as in Example 1 except that the centrifugation conditions (4 ° C., 3500 G, 15 minutes) were changed to (4 ° C., 5500 G, 15 minutes), and the number of tangible bodies was counted. The concentration of was calculated.

<Comparative Example 1>
Plasma was prepared in the same manner as in Example 1 except that the centrifugation conditions (4 ° C., 3500 G, 15 minutes) were changed to (4 ° C., 1000 G, 15 minutes), and the number of tangible bodies was counted. Immunoassays were performed.

<Comparative example 2>
Plasma was prepared in the same manner as in Example 1 except that the centrifugation conditions (4 ° C., 3500 G, 15 minutes) were changed to (4 ° C., 7000 G, 15 minutes), and the number of tangible bodies was counted. The concentration of was calculated.

  Table 1 (Example measurement result), Table 2 (Comparative example measurement result) and FIG. 1 show the immunoassay results of Annexin V in Examples 1 to 3 and Comparative Examples 1 to 2 and the intelligence score on the Hasegawa-type simplified intelligence evaluation scale. -5.

About the result of Examples 1-3, the relationship between annexin V measured value and a Hasegawa type | formula simple intelligence evaluation scale was shown to FIGS.
On the other hand, the relationship between the measured value of annexin V and the Hasegawa simple intelligence evaluation scale is shown in FIGS.

From these results, it is clear that the methods of Examples 1 to 3 have a remarkably high correlation with the Hasegawa simple intelligence evaluation scale as compared with the methods of Comparative Examples 1 and 2. In particular, in the vicinity of 20 points on the Hasegawa simplified intelligence evaluation scale in the early stage of Alzheimer's disease, the methods of Examples 1 to 3 were significantly more accurate than the methods of Comparative Examples 1 and 2.
In addition, the method of Examples 1 to 3 showed extremely little fluctuation in the measured value immediately after plasma preparation and the measured value when stored at −80 ° C. for 4 days, and the plasma could be stored extremely remarkably stably.
From this, it is presumed that annexin V contained in a tangible body does not originate from a disease (Alzheimer's disease or the like), and only annexin V not contained in a tangible body originates from a disease (Alzheimer's disease). When the centrifugal force is weak, it is considered that the tangible body is destroyed little by little and the annexin V measurement value changes little by little (particularly the destruction of the tangible substance occurs during the melting treatment after freezing, It is thought that the annexin V measurement value fluctuated greatly compared to the value before storage.) On the other hand, when the centrifugal force is increased, it is considered that the destruction of the tangible component during centrifugation further progresses, causing the annexin V measurement value immediately after plasma preparation to fluctuate.

It is the graph showing the relationship between the annexin V measured value in Example 1, and the Hasegawa simple intelligence evaluation scale. It is the graph showing the relationship between the annexin V measured value in Example 2, and Hasegawa type | formula simple intelligence evaluation scale. It is the graph showing the relationship between the annexin V measured value in Example 3, and a Hasegawa type | formula simple intelligence evaluation scale. It is a graph showing the relationship between the annexin V measured value in Comparative Example 1 and the Hasegawa simple intelligence evaluation scale. It is a graph showing the relationship between the annexin V measured value and the Hasegawa simple intelligence evaluation scale in Comparative Example 2.

Claims (5)

After adding an anticoagulant to blood, centrifuge by centrifugal force of 3000-6000G And removing a tangible body having a size of 0.1 μm or more from the blood to obtain plasma having a tangible body content of 0.5 × 10 ⁵ pieces / mL or less (1). A method for producing plasma. After adding an anticoagulant to blood, centrifuge by centrifugal force of 3000-6000G And removing the tangible body having a size of 0.1 μm or more from the blood to obtain plasma having a tangible body content of 0.5 × 10 ⁵ pieces / mL or less, And a step (2) of immunoassay for the annexin V contained therein. The quantification method according to claim 2, wherein annexin V associated with heart disease or Alzheimer's disease is quantified . The quantification method according to claim 2, wherein annexin V associated with Alzheimer's disease is quantified. After adding an anticoagulant to blood, centrifuge by centrifugal force of 3000-6000G And removing the tangible body having a size of 0.1 μm or more from the blood to obtain plasma having a tangible body content of 0.5 × 10 ⁵ / mL or less, A step (3) of storing the plasma obtained in 1) at −200 to 10 ° C.

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