JP2002323499A - Method for determining tumor marker in blood - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for easily determining a tumor marker for cancer of a digestive system and a tumor marker for hepatoma. SOLUTION: In the method for determining the tumor markers in blood, a blood component is extracted from a blood-carried carrier, and the tumor marker for cancer of a digestive system and/or the tumor marker for hepatoma are determined from the extracted blood component. The tumor markers are preferably tumor markers for colorectal cancer, biliary cancer, pancreatic cancer, and/or hepatoma and further preferably a carcinoembryonic antigen(CEA), α-fetoprotein(AFP), and/or CA19-9. It is preferable that the blood-carried carrier is formed by making an absorber using filter paper carry blood, and it is further preferable that the blood-carried carrier is formed by making the absorber carry blood collected from a finger tip.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for quantifying a tumor marker in blood. More specifically, the present invention relates to a method for quantifying a tumor marker in blood used for diagnosis of digestive system cancer and liver cancer.

[0002]

2. Description of the Related Art Conventionally, as a method for quantifying tumor markers for gastrointestinal cancer and liver cancer, 2 mL or more of whole blood is mainly collected from a patient's median elbow vein using a blood sampling syringe and left at room temperature for 1 hour or more. After that, the mixture was centrifuged at 1500 G for 10 minutes in a cooling centrifuge to separate the serum portion and the clot portion, and the supernatant (serum)
A method is known in which a portion is collected in another test tube and quantified as a measurement sample (for example, Clinical Laboratory Manual, 1988, Bunkodo, pp. 311-316). Furthermore, in order to carry out the measurement efficiently, there is known a method of freezing or refrigerated storage of a measurement sample and quantifying a certain amount of the measurement sample at once (for example, immunoassay, 198).
4th year, JMC, pp. 173-174).

[0003]

In the conventional method, when blood is collected from a large number of subjects such as a health examination and a tumor marker in the blood is measured, an operation of separating the supernatant from the large number of bloods is necessary. In addition, there is a problem in that the separated serum needs to be transported and stored in a test tube by refrigeration or freezing, thereby increasing costs. That is, an object of the present invention is to provide a method for easily quantifying a tumor marker for gastrointestinal cancer and a tumor marker for liver cancer.

[0004]

Means for Solving the Problems As a result of intensive studies to achieve the above object, the present inventor has found that the use of a specific method makes it possible to easily quantify a tumor marker for gastrointestinal cancer and a tumor marker for liver cancer. And arrived at the present invention. That is, the present invention provides a method for quantifying a tumor marker in blood, comprising extracting a blood component from a blood-carrying carrier, and quantifying a tumor marker for gastrointestinal cancer or a tumor marker for liver cancer from the extracted blood component. Is the way.

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION The tumor marker (antigen) in the present invention is a tumor marker for diagnosing gastrointestinal cancer and / or liver cancer. Gastrointestinal cancer includes esophageal cancer, stomach cancer, duodenal cancer, small intestine cancer, large intestine cancer, rectal cancer, pancreatic cancer, gallbladder cancer and the like. Of these, preferred are duodenal cancer, small intestine cancer, large intestine cancer, rectal cancer, pancreatic cancer and gall bladder cancer, more preferably large intestine cancer, rectal cancer, pancreatic cancer and gall bladder cancer, particularly preferably large bowel cancer and pancreatic cancer. Cancer and gallbladder cancer.

[0006] Examples of tumor markers for gastrointestinal cancer include the following. For esophageal cancer, carcinoembryonic antigen (CEA), IAP, ferritin, polyamine, β2
-Microglobulin, POA and trypsin inhibitor and the like. For gastric cancer, α-fetoprotein (AF
P), CEA, CA19-9, KMO-1, DuPAN
-2, SPan-1, CA50, SLX, CA72-
4, IAP, TPA, polyamine, β2-microglobulin, ferritin, POA and trypsin inhibitor. In the case of duodenal cancer, small intestine cancer, large intestine cancer and rectal cancer, CEA, CA19-9, KMO-1, Span-
1, CA50, SLX, CA72-4, IAP, TP
A, β2-microglobulin, ferritin, POA and trypsin inhibitor.

[0007] In the case of gallbladder cancer, AFP, CEA, CA1
9-9, KMO-1, DuPAN-2, Span-1,
CA50, CA72-4, basic fetoprotein (B
FP), NCC-ST-439, IAP, TPA, β2
-Microglobulin, ferritin, PIVKA-II,
POA and trypsin inhibitor. For pancreatic cancer, CEA, CA19-9, KMO-1, DuPAN
-2, SPan-1, CA50, CA72-4, BF
P, IAP, TPA, β2-microglobulin, ferritin, POA, trypsin inhibitor and elastase 1.

[0008] As tumor markers for liver cancer, for example, A
FP, CEA, CA19-9, KMO-1, DuPAN
-2, SPan-1, CA50, SLX, basic fetoprotein (BFP), NCC-ST-439, alkaline phosphatase isozyme, γ-GTP isozyme, IAP, TPA, β2-microglobulin, ferritin, PIVKA-II, POA and Trypsin inhibitor and the like. Among these tumor markers, preferably AFP, CEA, CA19-9, KMO
-1, DuPAN-2, Span-1, SLX, CA5
0, CA72-4, BFP, IAP, TPA, β2-microglobulin, ferritin, POA, trypsin inhibitor, elastase 1 and PIVKA-II, more preferably AFP, CEA, CA19-9,
KMO-1, DuPAN-2, Span-1, SLX,
CA72-4, BFP, IAP, TPA, POA and P
IVKA-II, particularly preferably AFP, CEA
And CA19-9.

In the method of the present invention for quantifying a tumor marker in blood, only one tumor marker may be quantified.
Although two or more tumor markers may be quantified, it is preferable to quantify two or more tumor markers from the viewpoint of sensitivity and specificity of cancer diagnosis, and more preferably two to three tumor markers are quantified. That is. In order to quantify two or more tumor markers, the tumor markers can be appropriately selected from the above tumor markers. In the case of liver cancer, for example, AFP, P
From tumor markers consisting of IVKA-II and CEA,
In the case of colon cancer, for example, from the tumor marker consisting of CEA and CA19-9, in the case of pancreatic cancer, for example, CA19
-9 and CEA, tumor esophageal cancer, for example, carcinoembryonic antigen (CEA) and POA, and gastric cancer, for example, α-fetoprotein (AFP), CEA, CA19-9 In the case of duodenal cancer, small intestine cancer and rectal cancer, for example, in the case of gallbladder cancer, for example, AFP, CEA, C
It is preferable to select from tumor markers consisting of A19-9, and in any case of gastrointestinal cancer and / or liver cancer,
More preferably, it is selected from tumor markers consisting of AFP, CEA and CA19-9.

[0010] The blood in the blood carrier is not limited in its collection site and collection method, for example, the conventional collection site and collection method (for example, using a blood sampling syringe, mainly 2 mL or more of whole blood from the median elbow vein). It is of course possible to use a part of the blood obtained in the present invention in the present invention, but from the viewpoint of reducing the invasiveness to the subject, the blood is preferably collected from peripheral blood vessels, Among the collection from, from the viewpoint of reducing the invasiveness of the subject, blood collected by puncturing the earlobe or fingertips is more preferable, the point that the subject can collect blood himself, less painful puncture and Blood collected by puncturing a fingertip is particularly preferable from the viewpoint that blood is easily collected (dropped) as droplets.

The method for collecting peripheral blood is not particularly limited as long as blood can be collected. From the viewpoint that the test can be carried out by the subject himself, for example, the relevant site (eg, earlobe, fingertip, etc.) of the subject is used. ) Is well massaged and / or warmed before puncturing to congest the blood, and the punctured site is wiped dry with a disinfecting gauze, and the site is punctured with a disposable lancet or the like to obtain blood.

[0012] The carrier in the blood-carrying carrier is not particularly limited in the material and shape of the carrier, as long as the carrier can hold the blood. It is preferable that the material and the shape are easily eluted, and it is more preferable that the material is an absorber from the viewpoint of easy holding by adsorption. As the material of the carrier, known natural polymers and synthetic polymers can be used, for example, cotton, wool, cellulose, polystyrene, polyolefin, polyurethane, nitrocellulose, cellulose acetate, polyester, epoxy resin, phenol resin, silk, Examples include fibroin, lignin, hemicellulose, chitin, ebonite, rubber, glass, quartz and ceramics. Of these, natural polymers are preferred, and cellulose and cotton are more preferred, and cellulose is particularly preferred.

As the carrier, known carriers can be used, and examples thereof include an absorbent made of a filter paper, a nonwoven fabric, a woven fabric or a sheet-like foam made of the above-mentioned materials. The pore size of the absorber can be freely set and selected, but the range of the average pore size is 1
１００100 μm is preferable, and more preferably 2 to 80 μm
And particularly preferably in the range of 5 to 50 μm. As the filter paper, for example, JIS P3801 (1995)
Year) or TAPPI (Technical Assoc)
iation of the Pulp and Pa
per Industry) T205. As the nonwoven fabric, for example, polyolefin nonwoven fabric, nitrocellulose nonwoven fabric, celsole acetate nonwoven fabric, polyester nonwoven fabric, epoxy nonwoven fabric,
Glass nonwoven fabric and ceramic nonwoven fabric are exemplified.
As the woven cloth, for example, cotton cloth, wool cloth, cellulose cloth,
Examples include polyolefin cloth, nitrocellulose cloth, cellulose acetate cloth, epoxy cloth, glass cloth, and ceramic cloth.

Examples of the sheet-like foam include expanded polystyrene, expanded polyolefin, expanded polyurethane,
Foamed polyester, foamed epoxy resin, foamed glass, foamed ceramics and the like can be mentioned. Among them, filter paper, nonwoven fabric and sheet-like foam are preferable, and more preferably filter paper and nonwoven fabric, particularly from the viewpoint that the amount of blood absorbed per unit volume or unit area tends to be constant (improvement of quantitative accuracy). Preferred are filter paper, polyolefin nonwoven fabric, nitrocellulose nonwoven fabric, cersol acetate nonwoven fabric, polyester nonwoven fabric, epoxy nonwoven fabric, glass nonwoven fabric and ceramic nonwoven fabric, and most preferably filter paper.

The thickness of the filter paper, non-woven fabric, woven fabric, sheet-like foam or the like can be appropriately selected.
0 mm is preferable, and more preferably 0.2 to 1.0 m
m, particularly preferably 0.3 to 0.6 mm. filter paper,
Size (area) of non-woven fabric, woven fabric or sheet-like foam
The blood collection, can be freely set in consideration of operability, such as during storage and transport, preferably 1～200Cm ^2, more preferably 10～150Cm ^2, particularly preferably 25 to 1
00 cm ² .

The loading of the blood on the carrier is not particularly limited as long as the blood can be retained, and the blood can be carried on the carrier by bringing the carrier into contact with the blood. Examples of the method of bringing the carrier into contact with blood include a method of immersing the carrier in blood, a method of dropping blood on the carrier, and a method of pressing the carrier against the puncture part. Among these, from the viewpoint of simplicity, a method of dropping blood on the carrier and a method of pressing the carrier against the puncture portion are preferable, and a method of dropping blood on the carrier is more preferable.

When the carrier is immersed in blood, the amount of blood used is such that the carrier can be immersed and is determined by the size of the carrier, but is preferably 0.05 to 2 ml, more preferably 0.1 to 1 ml. Particularly preferably 0.15 to 0.5 ml
It is. When blood is dropped onto the carrier, the amount of blood used is determined by the size of the carrier, but is preferably 0.02 to 0.5 ml, more preferably 0.05 to 0.3 ml, and particularly preferably 0.1 to 0.5 ml. ０．２0.2 ml. When the carrier is pressed against the puncture part, the amount of blood used is determined by the size of the carrier, but is preferably 0.02 to 0.5 ml, more preferably 0.05 to 0.3 ml, and particularly preferably 0.1 to 0.3 ml. ~
0.2 ml.

When the blood-carrying portion of the blood-carrying carrier is cut into a predetermined size (described later), it is sufficient to drop more blood than the cut-off carrier can hold, and it is not necessary to precisely control the amount of blood dropped. . For example, when the filter paper is Whatman's BFC180 (0.49 mm thick) and the amount of dropped blood is 50 μL, the size of the portion holding the blood is about 12 mm in diameter. The volume of one drop is about 40-60μ
Since it is L, when the blood-carrying portion is punched out into a circular shape having a diameter of 6 mm, the required amount of blood is 1 to 2 drops.

Further, from the viewpoint of blood stability and reproducibility of quantification, it is preferable that the blood is dried after being held on the carrier, and the weight of the blood held on the carrier is 50% by weight.
It is more preferred to dry until it is below (preferably below 30% by weight). As the drying method, for example, vacuum drying, freeze vacuum drying, slight heating drying, simple drying (air drying) and the like can be applied, and among these, vacuum drying, freeze vacuum drying and simple drying are preferable, and more preferably vacuum drying and It is simple drying, particularly preferably simple drying.

The drying is preferably carried out under conditions that do not change the antigenicity of the tumor marker, more preferably at a temperature of 40 ° C. or lower, particularly preferably at 10 to 30 ° C. When reducing the pressure, 0.02 to 10 Pa is preferable,
More preferably, it is 0.05 to 2 Pa, particularly preferably 0.1 to 1 Pa. The humidity for simple drying is not particularly limited, but is preferably 10 to 90% RH, and more preferably 40 to 80% RH. The drying time can be appropriately set depending on the shape of the carrier and the amount of blood retained, but is about 20 minutes to 1 hour when the carrier is filter paper.

When storing the blood holding carrier, the humidity is 8
It is preferably 0% RH or less, more preferably 10 to 60%.
% RH, particularly preferably 20 to 40% RH, and the temperature is preferably 0 to 40 ° C, more preferably 2 to 30 ° C.
° C, particularly preferably 2 to 10 ° C. In addition, humidity 80
In a state where% RH or less can be maintained (eg, under a sealed condition or under a sealed condition in which a desiccant is present), the product can be transported by mail, courier service, or the like.

Before the extraction of the blood component, it is preferable to cut out the blood-carrying portion of the blood-carrying carrier into a certain size before carrying out the extraction. For example, it is preferable to use a method in which blood is dropped on a uniform absorber, excess blood is diffused and absorbed to the surroundings, dried, and then a cut out central part is used for extraction. As a method of cutting, a method of punching out with a punch having a constant diameter, a method of cutting out along a cutting line (perforation or the like) inserted in advance, or the like can be applied.

The extraction of blood components from the blood-carrying carrier can be performed without any particular limitation as long as the antigenicity of the tumor marker (antigen) in the blood does not change. For example, the blood-carrying carrier is immersed in an extraction solution, The supernatant can be used as an extract. As the extraction solution, a buffer having a neutral pH range can be used. For example, a good buffer having a pH of 6 to 8 and p
H6-8 phosphate buffer and the like are preferably used.

Further, a salt, a surfactant, a protein, an antigen stabilizer and the like can be added to the extraction solution. Examples of the salt include sodium chloride, potassium chloride, lithium bromide and the like. Examples of the surfactant include sorbitan lauric acid monoester ethylene oxide adducts (for example, Tween 20 and Tween 40 (I
CI America Corporation)) and the like. Examples of the protein include bovine serum albumin and casein. Examples of the antigen stabilizer include a chelating agent such as EDTA and a protease inhibitor.

From the viewpoint of quantitative reproducibility, it is necessary to keep extraction conditions such as the amount of the extraction solution used for the carrier and the extraction time constant. The amount of the extraction solution used is 0.05 to
5 ml is preferred, more preferably 0.1-1 ml,
Particularly preferably, the volume is 0.2 to 0.5 ml. The extraction time is preferably 0.5 to 480 minutes, more preferably 1 to 180 minutes, and particularly preferably 5 to 60 minutes. The stirring is preferably performed by shaking the container using a device such as a vortex mixer, and the number of times of shaking is 100 to
2000 rpm is preferred.

For example, a filter paper having a diameter of 6 mm (thickness: 0.49 mm) cut from a blood carrier (Whatman B
FC180), it can be extracted under the following conditions. Solution composition for extraction: 0.05 mol / L containing sodium chloride
Phosphate buffer (pH 7.2) (content of sodium chloride: 0.85 g per 100 mL of buffer, the same applies hereinafter) Amount of solution for extraction: 200 to 300 μL Extraction temperature: room temperature (15 to 25 ° C.) Extraction time : 20 minutes to 1 hour (leaving time after stirring) Extraction operation: The extraction solution is added to the carrier, stirred with a vortex mixer (500 rpm, 1 minute), and left at the above temperature for the above time. Stir again (500 with vortex mixer)
rpm, 5 seconds) and allowed to stand for 1 minute to sediment the dispersed filter paper fibers. The supernatant is collected and used as a sample (extract) for tumor marker measurement.

The method for quantifying the tumor marker in the above extract is not particularly limited, but an immunological assay is preferred from the viewpoint of reproducibility of the quantitative value and measurement sensitivity. As the immunoassay, a conventionally known method can be used. However, since the concentration of the tumor marker in the extract is lower than that in the blood, a method with higher quantitative sensitivity is desirable. For example, radioimmunoassay (RI
A), enzyme immunoassay (EIA), fluorescence immunoassay (FI
A) and chemiluminescence immunoassay (CLIA) are preferred.

The radioimmunoassay (RIA) includes a two-site sandwich assay using a solid-phase antibody and an I125-labeled antibody, and many assay reagent kits are commercially available. Examples of the enzyme immunoassay (EIA) include a two-site sandwich assay using a solid-phase antibody and an enzyme-labeled antibody, and various assay reagent kits using peroxidase, alkaline phosphatase, glucose oxidase, etc. as enzymes are commercially available. Have been. Fluorescence immunoassay (FIA)
Using a solid-phase antibody and a europium-labeled antibody
Site sandwich measurement method and the like. Examples of the chemiluminescence immunoassay (CLIA) include a two-site sandwich assay using a solid phase antibody and an acridinium ester-labeled antibody, and various assay reagent kits are commercially available. Among these immunological assays, the enzyme immunoassay (EIA) is preferred, and the chemiluminescent enzyme immunoassay (CLEIA) is more preferred.

Various methods can be used to determine the concentration of the tumor marker in the blood from the concentration of the tumor marker in the extract. For example, (1) a blood-carrying carrier carrying blood with a known concentration of the tumor marker And (2) a method using a calibration curve prepared using an extract having a known concentration of a tumor marker and an extraction rate of a tumor marker.

In the method (2), the extraction rate is determined by preparing a blood-carrying carrier using blood containing a known concentration of a tumor marker, and quantifying the content of the tumor marker in an extract extracted from the carrier. The concentration of the tumor marker in the liquid is determined, and is calculated from the following equation. As for the extraction rate, it is preferable to perform the above operation at least twice (preferably at least three times) and use an average value thereof. Extraction rate = (concentration of tumor marker in extract) / (concentration of tumor marker in blood) The tumor marker concentration in the blood of the sample is obtained by dividing the tumor marker concentration in the extract prepared from the sample by the extraction rate. In this case, the conditions for preparing and extracting the blood-carrying carrier need to be the same as those for obtaining the extraction rate.

Since the tumor marker quantified in the present invention is present at a very low concentration in blood, the change in the extraction rate due to the change in the concentration is very small.
, The same extraction rate can be used in the range of 0.5 to 1,000 ng / mL. In the case of a high-concentration sample exceeding 1,000 ng / mL, the obtained extraction rate may differ from the actual extraction rate, but does not affect clinical judgment (ie, in the case of AFP, cancer Judgment boundary density is 10
ng / mL, and if it exceeds 1,000 ng / mL, it is positive in cancer judgment. ).

In the case of CEA, 0.5 to 200 ng
The same extraction rate can be used in the range of / mL. 200ng
In the case of a sample with a high concentration exceeding 1 mL / mL, the obtained extraction rate and the actual extraction rate may be different from each other, but this does not affect clinical judgment (ie, in the case of CEA, the boundary of cancer judgment). The concentration is 5 ng / mL, and if it exceeds 200 ng / mL, it is positive in cancer judgment.) The same applies to tumor markers other than AFP and CEA, and the concentration of the tumor marker does not affect clinical judgment.

In any of the methods (1) and (2),
It is preferable to use two or more types of blood or extract containing a known concentration of a tumor marker at different concentrations. The concentration of a blood or extract containing a known concentration of a tumor marker can be freely set depending on the type of the tumor marker to be measured. However, usually, the boundary between the range of a healthy person (normal range) and the range where the possibility of cancer is high is high. It is preferable to set the concentration (cutoff) at a concentration that can be clearly measured. For example, in the case of AFP, since the cutoff is usually around 10 ng / mL, in the case of the method (1), a tumor marker of a known concentration is used. Blood containing at least 10 ng / mL is sandwiched between two concentrations (eg, 5 ng / mL).
Concentration of 10 ng / mL and more than 10 ng / mL
It is preferable to set the concentration to be less than 50 ng / mL. Further, in the case of the method (2), since the concentration is the concentration of the tumor marker in the extract, two concentrations considering the extraction rate based on 10 ng / mL (for example, when the extraction rate is 0.02, A concentration of 10 ng / mL corresponds to a concentration of 0.2 ng / m in the extract.
0.1ng / mL or more and 0.2ng / m
(A concentration of less than L and a concentration of 0.2 ng / mL or more and less than 1 ng / mL).

In the case of CEA, the cutoff is usually 5
Since the concentration is around ng / mL, in the case of the method (1), blood containing a known concentration of a tumor marker is at least 5 ng / mL.
mL and 2 concentrations (for example, 2 ng / mL or more and 5 ng
/ Ml and a concentration of 5 ng / mL or more and less than 25 ng / mL). Further, in the case of the method (2), since the concentration is the concentration of the tumor marker in the extract, two concentrations considering the extraction rate based on 5 ng / mL (for example, when the extraction rate is 0.02, Concentration 5ng / mL
Is equivalent to 0.1 ng / mL in the extract,
A concentration of 5 ng / mL or more and less than 0.1 ng / mL and 0.1
(ng / mL or more and less than 0.5 ng / mL).

In the case of CA19-9, the cut-off is usually around 37 U / mL. Therefore, in the case of the method (1), the blood containing the tumor marker of a known concentration must be at least 2 concentrations across 37 U / mL. (For example, a concentration of 5 U / mL or more and less than 37 U / mL and a concentration of 37 U / mL or more and 100 U /
(concentration less than mL). Also,
In the case of the method (2), since the concentration is the concentration of the tumor marker in the extract, two concentrations considering the extraction rate based on 37 U / mL (for example, when the extraction rate is 0.02, the blood concentration 37
U / mL corresponds to a concentration of 0.74 U / mL in the extract, so it is set to a concentration of 0.1 U / mL or more and less than 0.74 U / mL and a concentration of 0.74 U / mL or more and less than 2.0 U / mL). Is preferred.

The cutoffs of tumor markers other than AFP, CEA and CA19-9 are listed, for example, IAP: 5
01 μg / mL, ferritin: 200 ng / mL, polyamine: 45 μmol / g creatinine, POA: 20
U / mL, KMO-1: 600 U / mL, DuPAN-
2: 400 U / mL, CA50: 36 U / mL, CA7
2-4: 4U / mL, TPA: 130U / L, PIVK
A-II: 0.1 AU / mL.

In the method (1), there is a point that it is necessary to perform an extraction operation to prepare a calibration curve, and since blood for preparing a calibration curve cannot be stored for a long period of time, there is troublesome preparation in the measurement. There is an advantage that accurate measurement can be performed even if the value fluctuates. On the other hand, in the method (2), a large amount of a sample can be accurately and simply measured if the extraction rate is determined in advance and the conditions such as the extraction operation are fixed. (1) and (2)
Of the methods, the method (2) is preferable from the viewpoint of simplicity.

[0038] The quantification method of the present invention comprises the steps of:
It can be held, dried and transported on a carrier (eg, bring, mail, courier, etc.) and is ideal for collecting and quantifying a large number of samples from a wide area, such as a health checkup for gastrointestinal cancer and liver cancer. It is. In addition to cancer screening, the present invention can also be applied to follow-up of cancer treatment for a patient in a remote place.

[0039]

The present invention will be further described below with reference to examples, but the present invention is not limited to these examples.

<Example 1> This example shows that the tumor marker (AFP) can be accurately quantified even if the blood carrier is stored for a long period of time. 1. Collection of blood and preparation of blood-carrying carrier (blood-dried filter paper) The fingertips of six volunteers (subjects A to F) are thoroughly massaged and warmed to congest the blood. -Puncture with a lancet (trade name "Hemolet", manufactured by Midori Cross Co., Ltd.), wipe off the first blood drop, and apply the next 2 drops to 10 blood sampling filter papers (part number BFC180, Whatman) (Manufactured by Co., Ltd.). Then, indoors (25 ± 1 ° C, 65 ± 5% R
H) and air dry (air drying time 5, 20, 40, 60 or 12)
0 min) to obtain a blood-carrying carrier (blood-dried filter paper). The weight of the blood-carrying carrier (blood-dried filter paper) was measured every air-drying time, and the degree of dryness was determined as the ratio to the initial blood weight (the value excluding the filter paper weight) (average value of two blood-carrying carriers). The degree of drying is shown in Table 1. Next, the blood-carrying carrier (blood-dried filter paper) was stored under the storage conditions shown in Table 1 (storage days 0,
3, 7, 14, 28).

[0041]

[Table 1]

2. Extraction Operation The following operation was performed in a room at a temperature of 20 to 25 ° C. Table 1
The center of the blood-carrying portion of each blood-carrying carrier (blood-dried filter paper) stored under the conditions described in (1) was punched out with a 6-mm-diameter 1-hole punch (commercially available for business use) to obtain a piece of filter paper.
Filter paper pieces punched into test tubes and sodium chloride containing
250 μL of a 05 mol / L phosphate buffer (pH 7.2) (solution for extraction) was added, the mixture was stirred for 30 seconds (500 rpm with a vortex mixer), and then left for 30 minutes. Thereafter, the mixture was similarly stirred for 30 seconds and allowed to stand for 1 minute, and the supernatant was separated to prepare an extract, which was used for quantification of a tumor marker.

3. Quantification of tumor marker (AFP) in the extract As a reagent for quantification, "Sphereite AFP" sold by Wako Pure Chemical Industries, Ltd. as a clinical test agent
Was used. As a standard solution for determining the amount of the tumor marker in the extract, "Sphereite AFP Control Set" sold by Wako Pure Chemical Industries, Ltd. was used. As the quantitative device, SphereLight 180 manufactured by Olympus Optical Co., Ltd. was used. By using this reagent and the device, AF
P can be quantified. The quantification reagent used is a clinical test agent for measuring the tumor marker concentration in serum. The amount of a sample used for measurement is usually set to 10 μL, and the reaction time is about 15 minutes in total. In the case of quantification of the tumor marker in the extract, 100 μL of the extract was used because the concentration was lower than that in the serum.

4. Quantitative results Tables 2 to 11 show the quantitative results and the stability determined by the following formula. (Stability) = (quantitative value after predetermined storage period) × 100 /
(Initial Quantitative Value) The initial quantitative value is a value obtained by immediately putting the blood-carrying carrier into the extraction solution after preparing the blood-carrying carrier, starting the extraction operation, and quantifying the value. . The tumor marker (antigen: AFP) in the extract obtained from the blood-carrying carrier (blood-dried filter paper) prepared and stored under each condition was stored for 28 days at a storage temperature of 4 ° C., except when the drying time was 5 minutes.
At 5 ° C., the stability showed a value of 90% or more for 7 days. Therefore, it was found that when the drying time was 20 minutes or more, the tumor marker (antigen: AFP) in the blood-carrying carrier (blood-dried filter paper) was stable.

[0045]

[Table 2]

[0046]

[Table 3]

[0047]

[Table 4]

[0048]

[Table 5]

[0049]

[Table 6]

[0050]

[Table 7]

[0051]

[Table 8]

[0052]

[Table 9]

[0053]

[Table 10]

[0054]

[Table 11]

<Example 2> In this example, it was shown that the tumor marker (CEA) can be accurately quantified even when the blood carrier is stored for a long period of time. 1. Blood collection and preparation of blood-carrying carrier (blood-dried filter paper) Six volunteers (subjects A to
F) a blood-carrying carrier (blood-dried filter paper) was prepared and stored under the storage conditions shown in Table 1 (storage days 0, 3, 7, 14,
28th). 2. Extraction operation An extract was prepared in the same manner as in Example 1 and used for quantification of a tumor marker.

3. Quantitation of Tumor Marker (CEA) in Extract The measurement reagent used was "Sphereite CEA" sold by Wako Pure Chemical Industries, Ltd. as a clinical test reagent. As a standard solution for determining the amount of the tumor marker in the extract, "Sphereite CEA Control Set" sold by Wako Pure Chemical Industries, Ltd. was used. Olympus Optical Industries, Ltd. Sp
HereLight180 was used. CEA can be quantified by a chemiluminescent enzyme immunoassay using this reagent and the apparatus. The quantification reagent used is a clinical test agent for measuring the tumor marker concentration in serum. The amount of a sample used for measurement is usually set to 40 μL, and the reaction time is about 15 minutes in total. In the case of quantification of the tumor marker in the extract, the extract is 10
0 μL was used. Also, the reaction time was extended to 29 minutes.

4. Table 1 shows the quantitative results and the stability obtained in the same manner as in Example 1.
2 to 21. Incidentally, the initial quantitative value, immediately after the blood carrier is prepared, immediately put the blood carrier into the extraction solution,
It was quantified after the start of the extraction operation, and was shown as a storage period of 0 days. The tumor marker (antigen: CEA) in the extract obtained from the blood-carrying carrier (blood-dried filter paper) prepared and stored under each condition was stored at a storage temperature of 4 ° C. for 28 days and 25 days, except when the drying time was 5 minutes. At 7 ° C., the stability was 90% or more for 7 days. Therefore, it was found that when the drying time was 20 minutes or more, the tumor marker (antigen: CEA) in the blood-carrying carrier (blood-dried filter paper) was stable.

[0058]

[Table 12]

[0059]

[Table 13]

[0060]

[Table 14]

[0061]

[Table 15]

[0062]

[Table 16]

[0063]

[Table 17]

[0064]

[Table 18]

[0065]

[Table 19]

[0066]

[Table 20]

[0067]

[Table 21]

<Example 3> In this example, the tumor marker (CA19-9) was accurately obtained even when the blood carrier was stored for a long period of time.
Is shown in some cases. 1. Blood collection and preparation of blood-carrying carrier (blood-dried filter paper) Six volunteers (subjects A to
F) a blood-carrying carrier (blood-dried filter paper) was prepared and stored under the storage conditions shown in Table 1 (storage days 0, 3, 7, 14,
28th). 2. Extraction operation An extract was prepared in the same manner as in Example 1 and used for quantification of a tumor marker.

3. The tumor marker (CA19-
9) Quantitative measurement The reagent used as a clinical test agent is “Sphereite CA19-9” sold by Wako Pure Chemical Industries, Ltd.
Was used. The standard solution for determining the amount of the tumor marker in the extract is “Sphereite C19-9A Control Set” sold by Wako Pure Chemical Industries, Ltd.
Was used. As the quantitative device, SphereLight 180 manufactured by Olympus Optical Co., Ltd. was used. CA19-9 can be quantified by a chemiluminescent enzyme immunoassay using this reagent and apparatus.

The quantification reagent used is a clinical test agent for measuring the tumor marker concentration in serum. The amount of a sample used for measurement is usually set to 10 μL, and the reaction time is about 15 minutes in total. In the case of quantification of the tumor marker in the extract, the extract is 10
0 μL was used. Also, the reaction time was extended to 29 minutes.

4. Table 2 shows the quantitative results and the stability obtained in the same manner as in Example 1.
2 to 31. Incidentally, the initial quantitative value, immediately after the blood carrier is prepared, immediately put the blood carrier into the extraction solution,
It was quantified after the start of the extraction operation, and was shown as a storage period of 0 days. The tumor marker (antigen: CA19-9) in the extract obtained from the blood-carrying carrier (blood-dried filter paper) created and stored under each condition, except when the drying time is 5 minutes,
28 days at 4 ° C, 7 days at 25 ° C, stability 9
It showed a value of 0% or more. Therefore, it was found that when the drying time was 20 minutes or more, the tumor marker (antigen: CA19-9) in the blood-carrying carrier (blood-dried filter paper) was stable.

[0072]

[Table 22]

[0073]

[Table 23]

[0074]

[Table 24]

[0075]

[Table 25]

[0076]

[Table 26]

[0077]

[Table 27]

[0078]

[Table 28]

[0079]

[Table 29]

[0080]

[Table 30]

[0081]

[Table 31]

<Embodiment 4> This embodiment shows that there is a good correlation between the AFP concentration obtained by the method of the present invention and the AFP concentration obtained by the conventional method. 1. Preparation of standard AFP-added blood-carrying carrier 1.8 mL of blood from 5 volunteers (subjects G to K)
Was collected and divided into 0.9 mL × 2 tubes, and 100 μL of a standard AFP solution (400 ng / mL) of “Sphereite AFP Control Set” was added to one of them,
Standard AFP-supplemented blood was prepared. Filter paper (BFC180,
Whatman Co., Ltd.)
μL was dropped and dried at room temperature (25 ± 2 ° C.) for 1 hour to prepare a standard AFP-added blood carrier. Similarly, 0.9 ml of the other blood was dropped on filter paper and dried to prepare a blood-carrying carrier.

2. Extraction and Measurement In the same manner as in the extraction method described in Example 1, extracts were prepared from the standard AFP-added blood carrier and the blood carrier, respectively, and the AFP concentration in the extract was measured. The results are shown in Table 32.

[0084]

[Table 32]

3. Setting of extraction rate The extraction rate was calculated from the measured values shown in Table 32 by the following formula.
The results are shown in Table 32. The average value of the extraction rates of the blood of the five patients was set as the extraction rate (0.0199) when the sample was created and extracted under the above conditions. Extraction ratio = (BA) / CA A: AFP concentration in extract prepared from blood-carrying carrier B: AFP concentration in extract prepared from standard AFP (400 ng / mL) added blood-carrying carrier C: 40 ng / mL (AFP amount added)

[0086] 4. Blood collection Blood was collected from 20 patients including liver cancer patients. Blood was collected by the method described in Example 1 (blood collection from the fingertip) and the conventional method from the median elbow vein (2 m of whole blood from the median elbow vein using a blood sampling syringe).
L or more). Whole blood collected from the median elbow vein was allowed to stand at room temperature for at least one hour,
The mixture was centrifuged at 0 G for 10 minutes under cooling, and the supernatant (serum) was further collected.

5. Preparation of blood-carrying carrier (blood-dried filter paper) In the same manner as in Example 1, a blood-carrying carrier (blood-dried filter paper) was prepared. The drying time was one hour, which was the same as in the case of setting the extraction rate.

6. Extraction and Quantification The blood carrier was subjected to extraction and AFP concentration measurement in the same manner as in the case of setting the extraction rate. On the other hand, the serum collected and separated by the conventional method was measured using “Sphereite AFP” described in Example 1 under the conditions according to the instructions attached to the measurement reagent (sample volume: 10 μL).

7. Determination of AFP concentration in blood The AFP concentration in the extract prepared from the blood-carrying carrier was divided by the extraction rate (0.0199) to determine the AFP concentration in the blood. Table 33 shows the AFP concentration in serum obtained by the conventional method and the AFP concentration in blood obtained by the method of the present invention.
FIG. 1 shows a correlation diagram between the AFP concentration according to the method of the present invention and the AFP concentration according to the conventional method using these values. As can be seen from FIG. 1, there was a good correlation between the serum AFP concentration according to the conventional method and the blood AFP concentration according to the method of the present invention. In FIG. 1, the equation is a correlation equation (X: A in the conventional method).
FP concentration, Y: AFP concentration in the method of the present invention), and R indicates a correlation coefficient.

[0090]

[Table 33]

<Embodiment 5> This embodiment shows that the CEA concentration obtained by the method of the present invention and the CEA concentration obtained by the conventional method have a good correlation. 1. Preparation of standard CEA-supplemented blood-carrying carrier 1.8 mL of blood from 5 volunteers (subjects G to K)
Was collected and divided into 0.9 mL × 2 tubes, and one of the standard CEA solutions (0 or 150 ng / mL) of “Sphereite CEA Control Set” was added to each of the 10 tubes.
Add 0 μL of standard CEA-supplemented blood (0 ng / mL, 1
50 ng / mL). Standard CEA-supplemented blood (150 n
g / mL) and dried at room temperature (25 ± 2 ° C.) for 1 hour to prepare a standard CEA-added blood-carrying carrier. Similarly, the other blood (0 ng / mL) 100
μL was dropped on filter paper and dried to prepare a blood-carrying carrier.

[0092] 2. Extraction and Measurement In the same manner as in the extraction method described in Example 2, extracts were prepared from the standard CEA-added blood carrier and the blood carrier, respectively, and the CEA concentration in the extract was measured. The results are shown in Table 34.

[0093]

[Table 34]

3. Setting of extraction ratio The extraction ratio was calculated from the measured values shown in Table 34 by the following formula.
The results are shown in Table 34. The average value of the extraction rates of the blood of the five patients was set as the extraction rate (0.0199) when the sample was created and extracted under the above conditions. Extraction ratio = (BA) / CA A: CEA concentration in extract prepared from blood carrier B: CEA concentration in extract prepared from standard CEA (150 ng / mL) added blood carrier C: 15 ng / mL (CEA amount added)

4. Blood collection Blood was collected from 20 patients including colorectal cancer patients. Blood was collected by the method described in Example 1 (blood collection from fingertips) and the conventional method from the median elbow vein (whole blood 2 from the median elbow vein using a syringe for blood collection).
mL or more). Whole blood collected from the median elbow vein was allowed to stand at room temperature for at least 1 hour, and then
After centrifugation at 00G for 10 minutes, the supernatant (serum) was further fractionated.

5. Preparation of blood-carrying carrier (blood-dried filter paper) In the same manner as in Example 1, a blood-carrying carrier (blood-dried filter paper) was prepared. The drying time was one hour, which was the same as in the case of setting the extraction rate.

6. Extraction and Quantification The blood carrier was subjected to extraction and CEA concentration measurement in the same manner as in the case of setting the extraction rate. On the other hand, the serum collected and separated by the conventional method was subjected to measurement using “Sphereite CEA” described in Example 2 under the conditions according to the instructions attached to the measurement reagent (sample volume: 40 μL).

7. Determination of CEA Concentration in Blood The CEA concentration in the extract prepared from the blood carrier was divided by the extraction rate (0.0199) to obtain the CEA concentration in the blood. Table 35 shows the serum CEA concentration determined by the conventional method and the blood CEA concentration determined by the method of the present invention.
FIG. 2 shows a correlation diagram between the CEA concentration according to the method of the present invention and the CEA concentration according to the conventional method using these values. As can be seen from FIG. 2, there was a good correlation between the serum CEA concentration by the conventional method and the blood CEA concentration by the method of the present invention. In FIG. 2, the equation is a correlation equation (X: C in the conventional method).
EA concentration, Y: CEA concentration in the method of the present invention), and R represents a correlation coefficient.

[0099]

[Table 35]

<Example 6> In this example, the concentration of CA19-9 obtained by the method of the present invention was compared with that of CA19-9 by the conventional method.
This shows that there is a good correlation with the 19-9 concentration. 1. Preparation of Standard CA19-9 Added Blood Carrier 1.8 mL of blood from 5 volunteers (subjects G to K)
Was collected and divided into 0.9 mL × 2 pieces, and one of the pieces was referred to as “Sphereite CA19-9 Control Set”
Of standard CA19-9 solution (0 or 200 U / mL) was added thereto to prepare blood with standard CA19-9 added thereto. 100 μL of standard CA19-9 added blood (200 U / mL) was dropped on filter paper (BFC180, manufactured by Whatman Co., Ltd.) and dried at room temperature (25 ± 2 ° C.) for 1 hour to prepare a standard CA19-9 added blood-carrying carrier. .
Similarly, 100 μL of the other blood (0 U / mL) was dropped on a filter paper and dried to prepare a blood-carrying carrier.

2. Extraction and Measurement In the same manner as in the extraction method described in Example 3, standard CA19-
Extracts were respectively prepared from the 9-added blood carrier and the blood carrier, and the CA19-9 concentration in the extract was measured. The results are shown in Table 36.

[0102]

[Table 36]

3. Setting of extraction ratio The extraction ratio was calculated from the measured values shown in Table 36 by the following formula.
The results are shown in Table 36. The average value of the extraction rates for the five blood samples was set as the extraction rate (0.0204) when the sample was created and extracted under these conditions. Extraction ratio = (BA) / CA: CA19- in the extract prepared from the blood-carrying carrier
9 Concentration B: CA19-9 concentration in an extract prepared from a blood carrier with standard CA19-9 (200 U / mL) added C: 20 U / mL (Amount of added CA19-9)

4. Blood collection Blood was collected from 20 patients including pancreatic cancer patients. Blood was collected by the method described in Example 1 (blood collection from fingertips) and the conventional method from the median elbow vein (whole blood 2 from the median elbow vein using a syringe for blood collection).
mL or more). Whole blood collected from the median elbow vein was allowed to stand at room temperature for at least 1 hour, and then
After centrifugation at 00G for 10 minutes, the supernatant (serum) was further fractionated.

5. Preparation of blood-carrying carrier (blood-dried filter paper) In the same manner as in Example 1, a blood-carrying carrier (blood-dried filter paper) was prepared. The drying time was one hour, which was the same as in the case of setting the extraction rate.

6. Extraction and Quantification The blood carrier was subjected to extraction and CA19-9 concentration measurement in the same manner as in the case of setting the extraction rate. On the other hand, the serum collected and separated by the conventional method was measured using “Sphereite CA19-9” described in Example 3 under the conditions according to the instructions attached to the measurement reagent (sample volume).
10 μL).

7. Determination of CA19-9 concentration in blood The CA19-9 concentration in an extract prepared from a blood-carrying carrier was divided by the extraction rate (0.0199) to obtain a CA19-9 concentration in blood.
Nine concentrations were determined. CA19 in serum determined by conventional methods
-9 and the concentration of CA19- in blood determined by the method of the present invention.
The nine concentrations are shown in Table 37. Using these values, the CA19-9 concentration according to the method of the present invention and the CA19-9 according to the conventional method are used.
The correlation diagram with the 19-9 concentration is shown in FIG. As can be seen from FIG. 3, the CA19-9 concentration in serum by the conventional method and the CA19-9 concentration in blood by the method of the present invention showed a good correlation. In FIG. 2, the equation is a correlation equation (X: CA1 in the conventional method).
9-9 concentration, Y: CA19-9 concentration in the method of the present invention), R indicates a correlation coefficient.

[0108]

[Table 37]

[0109]

According to the method for quantifying tumor markers in blood of the present invention, tumor markers for gastrointestinal cancer and tumor markers for liver cancer can be quantified extremely easily. Furthermore, since the subject can collect the blood by himself, there is no need for an expert or the like for collecting the blood, and the subject does not have to go to a hospital or the like for collecting the blood, which is also convenient.
In addition, since the blood-carrying carrier can be easily transported, a large number of specimens can be collected and measured at one place, and the cost of transport and measurement can be reduced. Therefore, the quantification method of the present invention is most suitable for collecting and measuring a large number of samples from a wide area, such as a health checkup for digestive system cancer and liver cancer.

[Brief description of the drawings]

FIG. 1 is a graph showing a correlation between an AFP concentration in serum by a conventional method and an AFP concentration in blood by a method of the present invention.

FIG. 2 is a graph showing a correlation between a serum CEA concentration according to a conventional method and a blood CEA concentration according to the method of the present invention.

FIG. 3 is a graph showing the correlation between the concentration of CA19-9 in serum by the conventional method and the concentration of CA19-9 in blood by the method of the present invention.

Claims (6)

[Claims] 1. A blood tumor marker characterized by extracting a blood component from a blood carrier and quantifying a tumor marker for gastrointestinal cancer and / or a tumor marker for liver cancer in the blood from the extracted blood component. Quantitation method. 2. The method for quantifying a tumor marker in blood according to claim 1, wherein the tumor marker is a tumor marker for colorectal cancer, gallbladder cancer, pancreatic cancer and / or liver cancer. 3. The method according to claim 1, wherein the tumor marker is carcinoembryonic antigen (CE).
A), α-fetoprotein (AFP) and / or CA
The method for quantifying a tumor marker in blood according to claim 1 or 2, which is 19-9. 4. The method for quantifying a tumor marker in blood according to any one of claims 1 to 3, wherein the blood-carrying carrier carries blood on an absorbent body using filter paper. 5. The method for quantifying a tumor marker in blood according to claim 1, wherein the blood-carrying carrier carries blood collected from a fingertip. 6. The method for quantifying a tumor marker in blood according to claim 1, wherein the concentration of the tumor marker in blood is determined from the concentration of the tumor marker in the extract and the extraction rate of blood components.