JPH0518975A - Tumor marker measuring method and sensor therefor - Google Patents

Abstract

PURPOSE:To easily measure the presence/absence of a tumor marker in constituent parts of the blood. CONSTITUTION:An AT-cut crystal resonator 11 is coated with polystyrene. Then the polystyrene-coated crystal resonator 11 is dipped in a phosphoric acid buffer 13 containing constituent parts of the blood for one hour at the room temperature so as to make the resonator 11 to adsorb the constituent parts of the blood. While the resonator 11 is dipped in the buffer 13 after the resonator 11 adsorbs the constituent parts of the blood, a bicarbonate buffer 15 containing anti-CEA monoclonal antibodies is added to the buffer 13. The presence/absence of a tumor marker is measured from the frequency variation of the resonator 11 before and after the buffer 15 is added.

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 the presence or absence of a tumor marker in blood components and a sensor suitable for use in this method.

[0002]

2. Description of the Related Art As a method of discovering cancer, which is a type of tumor, a method of intravenously injecting a tracer into the body for the purpose of imaging to confirm the localization of cancer tissue, and an in vitro diagnosis using serum or cells as a sample A method can be considered. In the former, the tracer is in the development stage. The latter is widely used in cancer diagnosis. The latter is specifically performed by measuring various markers that appear in the blood of patients.

These various markers are called tumor markers or related antigens (hereinafter referred to as "tumor markers").

Blood levels of tumor markers are generally ng / m 2.
on the order of l. For this reason, a highly sensitive quantitative method was required for the measurement of tumor markers. Currently, the radiation immunity test method (RIE (RADIO IMMUNOASSAY) method) and the enzyme immunity test method (EIA (ENZYME IMMUNOASSAY) method) are used.

[0005]

However, all of these measuring methods have problems that they are complicated and that it takes several days to determine the presence or absence of a tumor marker.

The present invention has been made in view of the above points, and therefore an object of the present invention is to provide a method for easily measuring the presence or absence of a tumor marker in a blood component and a sensor suitable for use in the method. To do.

[0007]

In order to achieve this object, according to the method for measuring a tumor marker of the first invention of the present application, a crystal oscillator is used for measuring the presence or absence of a tumor marker in blood components. Blood component is attached to the blood component, and the crystal oscillator already attached to the blood component is brought into contact with an antibody that specifically binds to a tumor marker, and the change in the frequency of the crystal oscillator before and after the contact causes It is characterized by measuring the presence or absence of the tumor marker.

Here, the adhesion of the blood component attached to the crystal oscillator means that the blood component is detached from the crystal oscillator when the crystal oscillator is brought into contact with an antibody that specifically binds to a tumor marker. The form does not matter, provided that the adhesiveness is such that there is no such problem. It may be either physically attached or chemically attached. In Examples described later, the crystal oscillator is immersed in a buffer containing the blood component for a predetermined time, and the blood component is attached to the crystal oscillator by adsorption.

[0009] Further, the contacting of the crystal oscillator having blood components attached thereto with an antibody that specifically binds to the tumor marker means that the blood component attached to the crystal oscillator specifically binds to the tumor marker. Any means can be used as long as the antibody can be effectively contacted. In the examples described later, the above contact is performed by immersing the crystal oscillator to which the blood component is adhered in the antibody-containing buffer.

Further, in carrying out the first invention, it is preferable that the crystal unit is coated with polystyrene and the blood component is attached to the crystal unit coated with polystyrene. This is because polystyrene easily attaches proteins to the crystal oscillator, which facilitates attachment of blood components to the crystal oscillator.

Further, the tumor marker measuring sensor of the second invention of this application is characterized in that the crystal oscillator is coated with polystyrene. This sensor may have a structure (sensor kit) containing a buffer containing an oncofetal monoclonal antibody as an assay solution.

Incidentally, a so-called AT-cut crystal oscillator is suitable for use as the crystal oscillator for carrying out the first and second inventions. Since the frequency variation with respect to temperature change is small, measurement error due to temperature difference during tumor marker measurement is unlikely to occur, and the frequency variation with respect to the antibody specifically binding to the tumor marker due to thickness shear oscillation is remarkable,
This is because advantages such as being able to adsorb blood components on the main surface of the vibrator can be obtained. Its shape (round plate, etc.), size, oscillation frequency, etc. are not essential. AT cut quartz crystals of various shapes and frequencies can be used. Also,
Other cut orientation crystal units can also be used.

[0013]

According to the tumor marker measuring method of the first invention of the present application, when a tumor marker is contained in the blood component attached to the crystal oscillator, the crystal oscillator is specifically targeted to the tumor marker. When brought into contact with an antibody to be bound, the antibody is attached to the oscillator due to this specific binding, so that the frequency of the oscillator is reduced. When the blood marker attached to the crystal oscillator does not contain a tumor marker, the above-mentioned frequency reduction does not occur. The presence or absence of a tumor marker in the blood component can be measured by utilizing such a difference in frequency change.

According to the pollen sensor of the second invention of this application, the first invention can be easily carried out.

[0015]

EXAMPLES Examples of a tumor marker measuring method of the present invention and sensors suitable for use in the method will be described below with reference to the drawings. It should be noted that the names of chemicals used, the amounts and concentrations of chemicals used, the temperature and time of each treatment, etc. described in the following description are only suitable examples within the scope of the present invention. Therefore, it should be understood that the present invention is not limited to the following conditions.

1. Preparation of Sensor for Measuring Tumor of Example First, an AT-cut crystal oscillator having an oscillation frequency of 9 MHz was immersed in acetone in which polystyrene was dissolved. Next, the crystal oscillator is pulled out from an acetone solution of polystyrene and then left to stand to volatilize acetone as a solvent. As a result, the tumor measuring sensor of the example in which the surface of the crystal unit is covered with polystyrene is obtained.

The polystyrene coating amount is such that the crystal unit can be covered and the characteristics of the crystal unit are not significantly deteriorated. The coating amount of polystyrene can be adjusted by the polystyrene concentration in an acetone solution of polystyrene, the speed of pulling up the crystal oscillator from this solution, and the like.

In this embodiment, the entire surface of the crystal unit is covered with polystyrene, but various modifications may be made such that only the electrodes of the crystal unit are covered with polystyrene if necessary.

2. Preparation of test solution Further, blood was collected from a mouse in which cancer cells were artificially generated in the digestive system and a normal mouse, and the sample solution was diluted 100-fold with phosphate buffer having a pH of 7.4. And

In this example, an anti-CEA monoclonal antibody is used as the antibody that specifically binds to the tumor marker. Therefore, carcinoembryonic antibody (CEA. In this example, Code No. 29109 manufactured by Seikagaku Corporation)
2. ) Is used to immunize a mouse, and an anticarcinoembryonic antibody monoclonal antibody (hereinafter referred to as "this anti-CEA monoclonal antibody") is collected from the mouse. This anti-CEA monoclonal antibody was added to a bicarbonate buffer of pH 9.6,
Mix at a ratio of 1 mg / ml.

3. Measurement of Presence or Absence of Tumor Marker Next, a polystyrene-coated crystal unit is immersed in a sample solution prepared as described above from mouse blood in which cancer cells have been artificially generated in the digestive system, at room temperature for 1 hour. .
As a result, the blood component containing the tumor marker is adsorbed to the polystyrene-coated crystal oscillator (sensor for measuring the tumor marker in the example). Next, this crystal oscillator taken out from the sample solution was washed three times with a phosphate buffer of pH 7.4, and then immersed in a phosphate buffer containing bovine serum albumin (BSA) adjusted to a temperature of 37 ° C for 15 minutes. To do.
Then, the crystal oscillator is washed three times with a phosphate buffer having a pH of 7.4.

The polystyrene-coated crystal unit taken out from the sample solution was washed three times with a phosphate buffer in order to remove blood components that were unstablely adsorbed to the crystal unit. Further, this crystal oscillator was treated with a phosphate buffer containing BSA because the region of the crystal oscillator where the blood component was not adsorbed (non-specific adsorption site) was
This is because it is covered with SA. This is because in order to minimize changes in the frequency of the crystal oscillator due to factors other than specific binding between the tumor marker and the anti-CEA monoclonal antibody.

Next, as shown in FIG. 1, the crystal unit 11 having the blood component adsorbed therein is placed in the phosphate buffer 13, and the frequency of the crystal unit 11 is measured in this state. In addition,
The crystal oscillator 11 is connected to an oscillation circuit (not shown), and this oscillation circuit is connected to a frequency counter (not shown). The frequency is measured by monitoring the value of this frequency counter. Further, it should be understood that in FIG. 1, the illustration of the crystal oscillator holder and the like is omitted.

Next, a crystal unit 1 having a blood component adsorbed thereon.
1 is immersed in the phosphate buffer 13 of FIG.
In this example, the ratio of the anti-CEA monoclonal antibody-containing bicarbonate buffer 15 (adjusted in the above-mentioned item 2) 15 to the anti-CEA monoclonal antibody-containing bicarbonate buffer is 1 ml in this example. To add. Then, the change in the frequency of the crystal unit after adding the buffer is measured.

The sample solution was prepared from the blood components of normal mice, and the blood components were adsorbed to the crystal unit in the same manner as described above.
The frequency change before and after the addition of the monoclonal antibody is measured.

FIG. 2 shows a sensor in which a blood component of a mouse in which cancer cells are artificially generated in the digestive system (ie, a blood component containing a tumor marker) is adsorbed and a sensor in which a blood component of a normal mouse is adsorbed. It is a characteristic view which shows the frequency change of each sensor at the time of making anti-CEA monoclonal antibody contact each. In FIG. 2, I is the characteristic of the sensor adsorbing the blood component containing the tumor marker, and II is the characteristic of the sensor adsorbing the normal blood component. In FIG. 2, the horizontal axis indicates the elapsed time (unit: × 1), which is indicated by the time when the carbonate buffer containing the anti-CEA monoclonal antibody is added to the phosphate buffer in which the sensor is immersed.
0 is ³ seconds) and the vertical axis represents the amount of frequency variation (unit: is Hz).

As is clear from FIG. 2, it is understood that in the sensor in which the blood component containing the tumor marker is adsorbed, there is a decrease in frequency which is not seen in the sensor in which the normal blood component is adsorbed. This is because in the crystal oscillator in which the blood component of the mouse having cancer cells in the digestive system is adsorbed, the anti-CEA monoclonal antibody specifically binds to the tumor marker present in the blood component and causes mass increase. . Further, as is clear from FIG. 2, it can be seen that the above frequency change occurs immediately after adding the carbonate buffer containing the anti-CEA monoclonal antibody to the phosphate buffer in which the sensor is immersed. Therefore, according to the method of the present invention, it can be seen that the presence or absence of the tumor marker in the blood component can be measured more easily than before.

[0028]

As is clear from the above description, according to the method for measuring a tumor marker of the first invention of this application,
The presence or absence of a tumor marker in the blood component can be measured more easily than before by utilizing the frequency change of the crystal oscillator.
Therefore, for example, in vitro diagnosis of cancer can be easily and quickly performed.

According to the tumor marker measuring sensor of the second invention of this application, the tumor marker measuring method of the first invention can be easily carried out.

[Brief description of drawings]

FIG. 1 is a diagram for explaining an example of a tumor marker measurement experiment and schematically shows a state in which an antibody that specifically binds to a tumor marker is brought into contact with a crystal oscillator to which blood components have been attached. Is.

FIG. 2 shows a case where an antibody that specifically binds to a tumor marker is brought into contact with each of the crystal oscillator to which a blood component containing a tumor marker is attached and the crystal oscillator to which a blood component not containing a tumor marker is attached. FIG. 6 is a characteristic diagram showing a change in frequency of each crystal oscillator.

[Explanation of symbols]

11: Crystal oscillator to which blood components are attached 13: Phosphate buffer 15: Bicarbonate buffer containing an antibody that specifically binds to a tumor marker

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Masaaki Kaifu             1-7-12 Toranomon, Minato-ku, Tokyo Oki Electric             Industry Co., Ltd. (72) Inventor Masakazu Kato             1-7-12 Toranomon, Minato-ku, Tokyo Oki Electric             Industry Co., Ltd.

Claims (5)

[Claims] 1. When measuring the presence or absence of a tumor marker in a blood component, a blood component is attached to a crystal oscillator, and the crystal component-attached crystal oscillator is contacted with an antibody that specifically binds to the tumor marker. A method for measuring a tumor marker, which comprises measuring the presence or absence of a tumor marker in the blood component from the change in the frequency of the crystal oscillator before and after the contact. 2. The method for measuring a tumor marker according to claim 1, wherein the crystal oscillator having blood components attached thereto is brought into contact with an antibody that specifically binds to the tumor marker, the crystal oscillator is treated with the antibody. A method for measuring a tumor marker, which is performed by immersing the tumor marker in a containing buffer. 3. The method for measuring a tumor marker according to claim 1 or 2, wherein the crystal unit is coated with polystyrene, and the blood component is attached to the polystyrene-coated crystal unit. Tumor marker measurement method. 4. A sensor for measuring a tumor marker, characterized in that a quartz oscillator is covered with polystyrene. 5. The tumor marker-measuring sensor according to claim 4, wherein the polystyrene-coated crystal oscillator is used.
A tumor marker measuring sensor comprising a buffer containing an anti-carcinoembryonic monoclonal antibody as an assay solution.