JP3076699B2 - Prostate specific antigen measurement method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for measuring a prostate specific antigen suitable for examination of the prostate, and more particularly, to an immunological measurement after extracting blood retained on a carrier to improve the working efficiency. The present invention relates to a method for quantifying a prostate-specific antigen, which has improved expression.

[0002]

It is widely known that the measurement of tumor markers in blood is extremely useful not only in cancer diagnosis but also in patient follow-up.

As a tumor marker for prostate cancer, prosta
tic acid phosphatase (PAP), prostate specific antigen (P
A, also referred to as prostate specific antigen (PSA)) and γ-seminoprotein (γ-Sm) are clinically used, of which Wa in 1979
2. molecular weight found in human prostate tissue by ng et al.
"Prostate-specific antigen" which is 40,000 glycoproteins (hereinafter "PA")
Has a clinical advantage (see, e.g., Nissan Cancer, 26 , 632-643).

Conventionally, when performing the above-mentioned measurement of PA, at the screening site, at least 2 ml of whole blood is mainly collected from the median elbow vein using a blood sampling syringe, and after standing at room temperature for at least 1 hour, the serum is collected. Was separated. In this serum separation, in order to separate the serum portion and the clot portion, the mixture was centrifuged at 1500 G for 10 minutes in a cooling centrifuge, and the supernatant (serum) was further separated.
After aliquots were collected in separate test tubes, PA was quantified by various immunoassays using test samples stored frozen.

[0005]

In the above-described conventional PA measurement method using blood collection, the work efficiency is not good because the intervention time of the examiner is long, and the invasiveness of the blood collection given to the subject is low. It was big. In addition, to reduce the invasiveness of the subject, body fluids other than blood (urine, saliva, etc.)
When was used, the reproducibility of PA measurement and the detection sensitivity were reduced, and the method had to be a qualitative test method.

An object of the present invention is to provide a novel PA measuring method which has solved the above-mentioned drawbacks of the conventional PA measuring method.

Another object of the present invention is to provide a PA measurement method which further improves the inspection work efficiency.

It is still another object of the present invention to provide a method for measuring PA suitable for screening a large number of subjects.

It is still another object of the present invention to provide a method for measuring PA with reduced invasiveness to a subject while maintaining good quantitative performance.

[0010]

Means for Solving the Problems As a result of intensive studies, the present inventors have found that the component to be measured (PA) in a dried sample is stable after blood is adsorbed on a carrier such as filter paper. The present inventors have further studied and found that extracting the component to be measured from the carrier to which the blood component was adsorbed into the liquid phase and performing PA measurement on the extracted solution was extremely important for achieving the above object. Found to be effective.

The PA quantification method of the present invention is based on the above findings. More specifically, a predetermined portion of a carrier holding blood is punched out, and a test component containing prostate specific antigen (PA) is extracted from the predetermined portion. Is extracted, the prostate specific antigen in the extract is measured by an immunoassay using an antibody-antigen reaction, and a standard curve showing the relationship between the intensity and the prostate specific antigen concentration obtained in advance from the measured values. Calculating the prostate specific antigen concentration from

Hereinafter, the PA measurement method of the present invention will be described in detail.

(Blood) The place and method of collecting blood used in the PA measurement method of the present invention are not limited as long as it is human blood. The blood is preferably peripheral blood from the viewpoint of reducing the invasiveness of the subject. Among peripheral blood, it is preferable to use blood (earlobe blood) collected by incision or puncture from an ear or earlobe, since the invasiveness to a subject is particularly low.

According to the findings of the present inventors, the correlation between the analyte in the earlobe blood and the analyte in the serum (isolated from the blood collected from the median elbow vein) is very high. Are found. Therefore, according to the method of the present invention, a highly reliable measured value can be obtained even when earlobe blood is used as a test sample.

More specifically, as an earlobe blood collection method, more specifically, the earlobe of the subject is thoroughly massaged or warmed prior to puncturing before the puncture, and the punctured site is wiped dry using a disinfecting gauze, followed by disposable lancet or A method of puncturing the earlobe with a scalpel to obtain earlobe blood is preferably used. In this case, the wound is preferably as small as possible (about 3 mm or less) and deep (about 2 to 4 mm or more). It is preferable to wipe off the first blood droplet and adsorb or immobilize the next blood droplet on the carrier.

(Carrier) The material and shape of the carrier are not particularly limited as long as the blood component as a test sample can be adsorbed or retained by solidification. However, the carrier is easily retained by adsorption. Is preferably made of a porous material. Among the porous materials, it is particularly preferable to use filter paper as a carrier from the viewpoint that efficient storage and transfer of a large amount of samples are easy. When filter paper is used as a carrier, a predetermined part (all or part) of the filter paper to which blood components are adsorbed
Can be easily punched out and used for the next extraction operation.

In the present invention, the method of adsorbing or immobilizing a blood component on the carrier is not particularly limited as long as the blood component can be retained on the carrier. From the viewpoint of the stability and reproducibility of the component to be measured including PA, it is preferable to air-dry (air-dry) after adsorbing or solidifying blood on the carrier. After sufficiently air-drying, the carrier is preferably stored in a refrigerator (2 to 8 ° C.) while avoiding moisture absorption.

(Extraction) (PA) supported on a solid phase carrier
Of the test component to the liquid phase substantially native (including
The extraction solvent or extraction method for performing this extraction is not particularly limited as long as tive) extraction is possible. From the viewpoint of performing the extraction as natively as possible, it is preferable to use a buffer (pH 6.8 to 7.6) such as a Tris buffer or a phosphate buffer as the extraction solvent.

From the viewpoint of extraction efficiency, the extraction solvent preferably contains a surfactant (preferably, a nonionic surfactant such as Tween-20). The concentration of this surfactant is preferably about 0.1 to 0.5%.

The extraction conditions are not particularly limited. For example, when a test component is held on a spot filter paper (thickness: about 1 mm) having a diameter of 3 mm, the following conditions are preferably used.

Extraction solvent: 0.1-0.4% Tween
-20 containing Tris buffer (0.05-0.2 mol /
L) or phosphate buffered saline Amount of solvent for extraction: 200-300 μL Extraction temperature: room temperature (about 25 ° C.) Extraction time: 1-4 hours (PA measurement method) PA in the above-mentioned extract can be measured. As long as the method of measuring PA is not particularly limited, from the viewpoint of reproducibility of the measured value, a biochemical method using a specific reaction such as an enzyme reaction or an antigen-antibody reaction is usually used. Preferably, it is used. From the viewpoint that accurate quantification of PA is easy, it is particularly preferable to use an immunoassay using an antigen-antibody reaction among biochemical methods.

As the above immunoassay, radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescence immunoassay (FIA) and the like can be used, but high sensitivity can be easily obtained. From the viewpoint, it is preferable to use a fluorescence immunoassay, particularly a time-resolved fluorescence immunoassay (TR-FIA).

In this time-resolved fluorescence immunoassay,
It is preferable to use a europium (Eu) -labeled antibody. In this embodiment, it is preferable to release Eu ³⁺ from the Eu-labeled antibody and to form an Eu-chelate in the solution during fluorescence measurement. This Eu-chelate has a long fluorescence wavelength (615 nm) and an excitation wavelength (340 nm).
nm) and the fluorescence lifetime is long (1000 μs
ec or more), and the fluorescent property that the fluorescence intensity is stronger than that of Eu ³⁺ alone makes it possible to perform measurement with high sensitivity and low background (based on excitation scattered light and coexisting proteins). Become.

In this embodiment, the above-mentioned Eu is further used.
-It is preferable to perform the measurement (time-resolved measurement) after the background due to coexisting proteins or the like has disappeared by utilizing the long fluorescence lifetime of the chelate. In this case, for example, it is preferable to perform time-resolved measurement of about 100 to 1000 times / sec for one sample and integrate the fluorescence intensities from the viewpoint of obtaining high sensitivity and a wide measurement range.

Hereinafter, the present invention will be described more specifically based on examples.

[0026]

Example 1 As a disposable lancet for collecting earlobe blood, a commercially available hemolet (manufactured by Midori Cross Co., Ltd.) was used. TYPE II (manufactured by Toyo Roshi Kaisha, Ltd.) was used as the filter paper for blood collection. For prostate specific antigen (PA) measurement,
DELFIA (trade name) PSA measurement reagent (Kabi P
Pharmacia) was used.

(Blood Collection Method) When collecting earlobe blood, the earlobe of the subject is thoroughly massaged or warmed prior to puncturing to congest the blood, and the site to be punctured is wiped dry using a disinfecting gauze, and the above-described disposable lancet or scalpel is used. Then, the earlobe was punctured to obtain earlobe blood. The first blood drop was wiped off, the next blood drop was absorbed by the filter paper for blood collection and air-dried sufficiently, and then stored in a refrigerator (4 ° C.) to avoid moisture absorption.

(Extraction conditions) A portion of the dried earlobe blood filter paper obtained as described above was punched out with a 3 mm diameter punch to obtain a piece of filter paper (3 mm diameter spot filter paper). The components to be measured were extracted from the filter paper pieces. In this extraction, the following six extraction conditions were compared and examined (extraction temperature: 25 ° C.).

1) Type of extraction buffer: 0.1% Twe
0.1 mol / l Tris-HCl buffer (pH 7.4) containing en-20 and phosphate buffered saline (pH 7.2) 2) Extraction volume: 200 μl, 250 μl, 300 μl 3) Dispensing of extraction solution Volume: 150 μl, 125 μl 4) Number of 3 mmφ spot filter papers for extraction: 3, 4,
5) 5) Extraction time: 1 hour, 2 hours, 3 hours, 4 hours 6) Tween-2 in Tris-HCl buffer for extraction
0 concentration: 0.1%; 2%, 0.3%, 0.4
% Using the various extracts (extracts) obtained as described above as samples, the PA amount was measured using the DELFIA PSA measurement kit.

The measurement operation was performed as follows.

1) A strip (a plate having wells included in the kit) to be used is set on a frame (measurement table), and a washing machine (1296-024 DELFIA PLA) is set.
TEWASH, Wallac. The liquid in each well was removed by suction using LKB (manufactured by LKB).
Used within 30 minutes).

2) A specified amount (150 or 200 μl) of an extract (standard or sample) extracted from dried filter paper blood
l) was dispensed into each well of the strip.

3) Shaker (1296-002 PLAT)
E SHAKE, Wallac. The plate was incubated with shaking (scale: SLOW) for 1 hour at room temperature using LKB (manufactured by LKB).

4) The wells were washed twice with a washing solution using the above washing machine.

5) Using a shaker, the strip was incubated with the frame for 1 hour at room temperature with shaking (SLOW).

6) Tracer solution (Eu-labeled PSA antibody supplied with the kit, diluted with the buffer supplied with the kit)
200 μl was added to each well.

7) The wells were washed four times with a washing solution using a washing machine.

8) 200 μl of the enhancement reagent attached to the kit was added to each well.

9) Using a shaker, the strip was shaken (SLOW) together with the frame at room temperature for 5 minutes.

10) A dedicated time-resolved fluorescence measuring device (12
30 ARCUS Fluorometer, Wall
ac. (Manufactured by LKB), and time-resolved measurement was performed 1000 times / second, and the integrated fluorescence intensity was measured.

11) Self-adjusted standard PSA filter paper (6 types)
The data obtained based on the above measurements were plotted on a log-log graph paper to obtain a standard curve. Next, the PA concentration in each sample was determined using this standard curve.

(Results) The measurement was carried out under the above six conditions.
The obtained results are shown in the following (Table 1), (Table 2), (Table 3),
The results are shown in (Table 4), (Table 5) and (Table 6).

[0043]

[Table 1]

(In the above table, “RFU” means Relative-Flu
It means orescence-Unit. )

[0045]

[Table 2]

[0046]

[Table 3]

[0047]

[Table 4]

[0048]

[Table 5]

[0049]

[Table 6]

In the selection of the type of extraction buffer shown in Table 1 above, when the Tris-HCl buffer was used, the extraction ratio of the component to be measured was high.

In the comparison of the amount of the extract shown in the above (Table 2), the sensitivity was excellent and the shape of the calibration curve was good when measured at 200 μl.

In comparison of the dispensed amount of the extraction solution shown in the above (Table 3), 150 μl was better in terms of sensitivity.

In the comparison of the number of spot filter papers having a diameter of 3 mmφ shown in Table 4 above, the fluorescence intensity increased as the number of filter papers increased. However, from the relation of the amount of specimen, four sheets were appropriate for normal use. Was.

In the comparison of the extraction times shown in the above (Table 5), no significant difference was observed between 1 hour and 4 hours.

Tween-20 shown in the above (Table 6)
In the comparison of the concentrations, the extraction rate was improved when the concentration was 0.1%.

Based on the above results, P
The optimum conditions for A extraction were selected, and the measurement operation procedure was as shown below.

(Measurement procedure) 1) A piece of filter paper having a diameter of 3 mm (3 mmφ spot filter paper) was placed in a test tube from dried blood filter paper (standard product or sample to be measured).
Was punched out.

2) 0.1% T as an extraction solution in a test tube
0.1 mol / l Tris-HC containing ween-20
200 μl of 1 buffer was added.

3) Let stand at room temperature for 1 hour and remove PA from the filter paper.
Was extracted.

4) The strip to be used is set on the frame, and the washer (1296-024 DELFIA PLA)
TE WASH, Wallac. The liquid in each well was removed by suction using LKB (manufactured by LKB) (the suctioned strip was used within 30 minutes).

5) 150 μl of the supernatant (standard or sample to be measured) as the extract extracted in 3) was dispensed to each well of the strip.

6) Shaker (1296-002 PLAT)
E SHAKE, Wallac. Using LKB), the strip was shaken with the frame at room temperature for 1 hour (S
LOW).

7) The wells were washed twice with a washing solution using a washing machine.

8) Using a shaker, the strip was incubated together with the frame at room temperature for 1 hour with shaking (SLOW).

9) 200 μl of the tracer solution was added to each well.

10) 4 wells were washed with a washing solution using a washing machine.
Washed twice.

11) 200 μl of enhancement reagent was added to each well.

12) Using a shaker, the strips were shaken (SLOW) together with the frame at room temperature for 5 minutes.

13) A dedicated time-resolved fluorescence measuring device (12
30 ARCUS Fluorometer, Wall
ac. (LKB) was used to measure the fluorescence intensity.

14) The PA concentration in each sample was read from the standard curve obtained based on the measured values of the standard PSA filter paper.

Example 2 Based on the measurement operation procedure set in Example 1, a basic evaluation of the earlobe blood filter paper collection PA measurement method of the present invention was carried out under the following seven conditions.

1) Stability of Standard Curve As the stability of the standard curve, the reproducibility within the measurement and the reproducibility between the measurements were confirmed using a standard PA filter paper and a sample filter paper. The intra-measurement reproducibility was evaluated by measuring 10 times of each sample at the same time, and the inter-measurement reproducibility was evaluated by performing each sample 6 times while changing the day by double measurement.

2) Dilution test Using five types of sample filter papers, the filter paper extract solution of each sample was diluted by a factor of several with a buffer solution, and the dilution linearity was confirmed.

3) Spike recovery test Blood of three different concentrations (different fold dilution with buffer)
Then, a 10% dilution series of a high-concentration standard substance was added thereto, adsorbed on filter paper, air-dried, and a recovery test was performed.

4) Detection limit and measurement range The average value of the 10-fold measurement values of the 0 concentration sample filter paper + 2 of the standard deviation
The reading from the standard curve, indicated by a RFU value of MEAN + 2SD, was taken as the detection limit.

The minimum measurement range is as follows: (MEAN + 4SD) RFU of 10-fold measured value of filter paper with 0 concentration.
The reading from the standard curve indicated by the value was determined.

The maximum measurement range is 500 ng / m
(MEAN-4S) of 10 replicates of 1 standard PA filter paper
The reading from the standard curve indicated by the RFU value of D) was determined.

5) Reproducibility As a confirmation of an accidental error, a variation was measured when sample filter papers of three kinds of concentrations were measured 10 times each. Further, the same sample filter paper as described above was subjected to double measurement on different measurement dates, and the fluctuation (systematic error) when the measurement was performed seven times was examined.

6) Storage stability Sample filter papers of three concentrations were stored at room temperature (25 ° C.) and refrigerated (4
C), and the stability of PA in the sample filter paper for one month was examined.

7) Correlation with PA value in serum The correlation between the PA value in the serum of 100 cases (serum separated from blood collected from the median elbow vein) and the PA value collected from earlobe blood filter paper was confirmed.

(Results) The measurement was performed under the above seven conditions.
The obtained results are shown in the following (Table 7), (Table 8), (Table 9),
The results are shown in (Table 10), (Table 11), (Table 12), (Table 13) and (Table 14).

[0082]

[Table 7]

[0083]

[Table 8]

[0084]

[Table 9]

[0085]

[Table 10]

[0086]

[Table 11]

[0087]

[Table 12]

[0088]

[Table 13]

[0089]

[Table 14]

In the stability of the standard curve shown in the above (Table 7), the coefficient of variation (CV; standard deviation divided by the average value) of the reproducibility in the measurement of each PA standard filter paper = 6.28 to 1
2.4%, and CV = 0.0 for inter-measurement reproducibility (Table 8).
A stable result of 00 to 8.35% was obtained. The obtained standard curve (reproducibility within the measurement) is shown in the graph of FIG. Since the shape of the standard curve showed a substantially straight line on the log-log graph, it was possible to read the value of the unknown concentration sample using the standard curve.

In the dilution test shown in the above (Table 9), as shown in the graph of FIG. 2, in all of the five samples to be measured, up to the measured value of 300 ng / ml converged to the origin. It showed good dilution linearity.

In the recovery test shown in the above (Table 10), the average recovery rate of the three samples used in the test was 91.5 to
The result was as good as 98.2% (see the graph in FIG. 3). From these results and the results described above, it was found that the measurement method of the present invention was hardly affected by coexisting substances and the like and had sufficient quantitative properties.

The detection limit and minimum measurement sensitivity shown in the above (10) (Table 11) were 0.62 ng / ml, respectively.
It was 0.88 ng / ml, and it was confirmed that an extremely low concentration of the substance to be measured was detectable and that the measurement method was highly sensitive (see the graph in FIG. 4). Further, as the maximum read value of the substance to be measured, quantification up to 300 ng / ml was possible.

In examining the reproducibility using the sample filter paper shown in the above (11) (Table 12), the CV of the variation within the measurement was 4.0.
9 to 6.36%, CV of variation between measurements = 3.45 to 8.8
It was as good as 7%, and the substance to be measured could be accurately quantified.

In the examination of the storage stability of the earlobe blood filter paper-adsorbed PA shown in the above (12) (Table 13), almost the same tendency was observed in the three samples used. That is, when stored at room temperature, the measured value significantly decreased after the third day. On the other hand, in the refrigerated storage, it was found that the sample was sufficiently stable for the confirmed 28 days (about one month), and the batch processing (collective measurement) of the refrigerated storage samples was possible.

In the comparison between the measured value of PA according to the method of the present invention shown in the above (13) (Table 14) and the measured value of serum PA,
For 100 measured values (n = 100), linear regression equation Y = 0.902X + 1.02, correlation coefficient R = 0.889
And a very high correlation was observed between the two groups (see the graph in FIG. 5).

As described above, in the evaluation of the basic considerations regarding the measurement method of the present invention, sufficiently good results were obtained. Therefore, according to the method of the present invention, it was found that PA in a test sample consisting of a carrier (filter paper) to which blood was adsorbed could be accurately and stably quantified.

[0098]

As described above, according to the present invention, a test component containing a prostate specific antigen (PA) is extracted from at least a part of a carrier holding blood components, and a prostate specific antigen in the extract is extracted. There is provided a method for measuring a prostate-specific antigen, which is characterized by performing the measurement.

According to the PA measurement method of the present invention, the operation of separating serum from blood by centrifugation or the like becomes unnecessary, so that the work efficiency of the test can be greatly improved.

Furthermore, according to the present invention, storage, storage or transfer of a carrier holding blood as a test sample is extremely simple and easy (compared to serum as a test sample in a conventional method). A PA quantification method suitable for screening a large number of subjects is provided.

Furthermore, according to the present invention, since blood is used as a test sample, not only good quantification is maintained, but also a conventional method using a serum obtained by centrifugation or the like (a relatively large amount of blood). Is required), it is possible to reduce the invasiveness to the subject.

[Brief description of the drawings]

FIG. 1 is a graph showing the stability (within measurement) of a standard curve obtained in Example 2.

FIG. 2 is a graph showing the results of a dilution linearity test obtained in Example 2.

FIG. 3 is a graph showing the results of an addition recovery test obtained in Example 2.

FIG. 4 is a graph showing the results of a detection limit / minimum measurement range confirmation test obtained in Example 2.

FIG. 5 shows the results of measuring serum PA obtained in Example 2,
It is a graph which shows the correlation with the measurement result of PA in dried filter paper blood of this invention.

────────────────────────────────────────────────── ─── Continuation of the front page (56) References JP-A-64-63868 (JP, A) JP-A-2-497 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) G01N 33/53 G01N 21/64 G01N 33/574 BIOSIS (DIALOG) JICST file (JOIS)

Claims (5)

(57) [Claims] 1. A predetermined portion is punched from a carrier holding blood, a test component containing a prostate specific antigen (PA) is extracted from the predetermined portion, and a prostate specific antigen in the obtained extract is used as an antigen-antibody. Measured by an immunological assay using the reaction, from the measured values obtained by the measurement, the concentration of the prostate specific antigen based on a standard curve showing the relationship between the intensity and the prostate specific antigen concentration obtained in advance A method for quantifying a prostate-specific antigen, which is calculated. 2. The method according to claim 1, wherein the carrier is an absorbent containing filter paper. 3. The method for quantifying a prostate-specific antigen according to claim 1, wherein the blood is peripheral blood. 4. The method for quantifying a prostate-specific antigen according to claim 1, wherein the peripheral blood is blood collected from an earlobe. 5. The method for quantifying a prostate-specific antigen according to claim 1, wherein the immunological assay is a time-resolved fluorescence immunoassay (TR-FIA).