JP2731613B2 - Cartridge for enzyme immunoassay, measuring method and measuring apparatus using the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial application field) The present invention utilizes an immunological reaction (antigen-antibody reaction) to produce a multi-component such as a biological sample (eg, blood, serum, plasma, urine, cerebrospinal fluid, etc.). The present invention relates to a cartridge for enzyme immunoassay suitable for measuring a specific substance contained in a trace amount in a system, a measuring method and a measuring apparatus using the same. The present invention will be described below the measurement of trace biological substances in clinical tests,
It can be applied to a wide range of fields such as pharmacy, biology, zoology, botany, agriculture, chemistry, and testing.

(Prior art) Substances involved in the biological activity of living organisms are generally trace amounts,
In addition, many play a very important role in living organisms. Therefore, quantitatively measuring such a trace amount of a physiologically active substance is important for biological fields such as medicine and biochemistry, and various measuring methods for the purpose have been devised and put into practical use. I have. Above all, an enzyme immunoassay using an enzyme as a label is widely used in the field of clinical testing.

In such a measurement method, first, a solid phase on which an antibody (or antigen) capable of specifically binding to an antigen (or antibody) to be measured is immobilized is a sample solution and a second antibody (or antigen) labeled with an enzyme. At the same time or successively to carry out an immune reaction, followed by washing, and then measuring the activity of the enzyme label remaining on the solid phase to determine the antigen ( Or antibody).

As a representative example of such a measurement method, a heterogeneous method EIA, so-called Enzyme Linked Immuno Sorbent Assay (ELISA) is known.

In ELISA, a test tube, a microplate, or the like is used as a solid phase on which an antibody (or antigen) that specifically binds to an antigen (or antibody) to be measured is captured in order to capture a substance to be measured in a sample solution. . In addition, when the measurement target is an antigen, an enzyme-labeled second antibody obtained by labeling an enzyme with a second antibody capable of binding to the antigen is used in the San Deutsch ELISA, and the same antigen as the measurement target antigen is used in the competition ELISA. An enzyme-labeled antigen labeled with an enzyme is used.

A substrate solution for the enzyme used as the label,
Then, if necessary, the coloring reagent is brought into contact with the solid phase.
Then, the optical properties of the substrate solution change with the decomposition reaction of the substrate solution, and the change is observed.

Several methods are conventionally known for observing changes in the optical properties of a substrate solution. Among them, a method using an instrument is to measure a change in the optical properties of a substrate solution with an absorptiometer, a fluorometer, a chemiluminescence photometer, etc. (for example, Ishikawa, Kawai, Miyai, enzyme immunoassay,
Medical Shoin (1982)).

As another method, there is a method in which the presence of an antigen (or antibody) is determined by comparing a substrate solution with a target substrate solution and visually observing a difference in color of the substrate solution with the naked eye (for example, Japanese Patent Application Laid-Open No.
-128369).

However, these optical measurement methods using equipment require a stable light source, a high-sensitivity photometer, a precision optical system amplifier circuit, and the like, and have to be expensive and large and complicated. In addition, since special techniques are required for measurement, a special engineer for handling has to be assigned.

On the other hand, the method of directly observing with the naked eye is a qualitative measurement method, and the variation in color change and the subjectivity of the observer are included. Furthermore, in the case of measuring a very small amount of a substance, the change in color is small and it is difficult to determine.

The present inventors have improved the drawbacks of the conventional measurement method, removed the ambiguity of the subjective judgment by the observer, and objectively measure the decomposition reaction of the substrate solution with high detection accuracy. As a method, a method of measuring a pH change of a substrate solution with a pH electrode was proposed in Japanese Patent Application Laid-Open No. 1-212347.

(Problems to be Solved by the Invention) In such a measuring method, a pH-sensitive field-effect transistor (pH-FET) is usually used as a pH electrode, an inner wall of a tip portion of a pipette-shaped small-diameter tube as a solid phase, urease as a labeling enzyme, Alternatively, urea is used as a substrate.

In the enzyme immunoassay using a pH electrode, (1) the structure of the pH electrode is extremely simple as compared with a conventional optical measurement system.
(2) Since the pipette tip-shaped thin tube is used as the solid phase, there is an advantage that all operations from sample dilution to washing can be performed by dispensing means.
Only a laboratory-scale enzyme immunoassay method and apparatus using a pH electrode has been proposed, and no practical measurement method and apparatus have been proposed yet.

Accordingly, an object of the present invention is to provide a practical method and apparatus for automatically performing an enzyme immunoassay using a pH electrode.

Further, by using the above-described measuring method and apparatus, it is possible to measure a large number of antigens and antibodies of various infectious diseases, various hormones, various cancer markers, various drugs, and the like. In addition, generally, many reagents are used in the enzyme immunoassay, such as a solid phase, an enzyme-labeled substance, a substrate, a buffer for dilution (hereinafter abbreviated as a diluent), and a buffer for washing (hereinafter abbreviated as a wash). Since some of these reagents are different for each measurement item, conventionally, these reagents have been sold as a single kit. That is, the user takes out a dedicated kit every time the measurement item changes, and performs the measurement in a predetermined procedure. In general, the size of a kit is several tens to several hundreds of samples, and it is suitable for measuring several tens to several hundreds of specimens at once, but measuring arbitrary measurement items in an arbitrary order. Was inappropriate.

Therefore, another object of the present invention is to provide a cartridge for enzyme immunoassay in which an arbitrary measurement item can be measured in an arbitrary order by converting a dedicated reagent or the like for one measurement item into a cartridge. That is.

(Means for Solving the Problems) The present invention relates to an enzyme in which a sample cup for accommodating a sample to be measured, a dilution cup accommodating a pipette-shaped thin pipe for dilution and a reaction cup are integrally arranged. A cartridge for immunoassay, wherein a freeze-dried enzyme-labeled reagent is contained at the bottom of the reaction cup, and at least on the inner wall of the tip, an antibody (or antigen) that binds to an antigen (or antibody) as a substance to be measured. The pipette tip-shaped solid-phase small-diameter tube in which the tip was fixed was housed in the reaction cup with its tip separated from the enzyme labeling reagent, and the upper end opening of each of the cups was hermetically closed with a sealing piece. A cartridge for enzyme-linked immunosorbent assay, characterized in that:

The present invention also relates to an enzyme immunoassay method using the above-described cartridge for enzyme immunoassay, wherein (1) diluting the sample and the diluent in the sample cup with a pipette-shaped thin-diameter tube accommodated in the dilution cup. A step of injecting into a cup and preparing a sample liquid diluted to a predetermined magnification in the dilution cup.

(2) A step of immersing the distal end of the pipette-shaped solid-phase small-diameter tube accommodated in the reaction cup into the sample liquid in the dilution cup to perform a primary immune reaction.

(3) A lysis solution is injected into the reaction cup to dissolve the lyophilized enzyme labeling reagent in the reaction cup, and then the solid solution after the completion of the primary immunoreaction in the dissolved enzyme label solution. A step of immersing the tip of the phase-use small-diameter tube to perform a secondary immune reaction.

(4) a step of washing the small-diameter solid-phase tube after the completion of the secondary immune reaction;

(5) Put the washed small-diameter tube for solid phase over the sensitive part of the pH electrode provided in the measurement cell filled with the substrate solution, and transfer the substrate solution between the small-diameter tube and the sensitive part of the pH electrode. A step of measuring a pH change accompanying the decomposition reaction.

An enzyme immunoassay method comprising:

Further, the present invention relates to an enzyme immunoassay apparatus using the above-described cartridge for enzyme immunoassay, means for supplying the cartridge for enzyme immunoassay to an operation station, a diluent inlet and an overflow solution provided in the operation station. A diluent cell having an outlet, a diluting station including a pump for supplying diluent to the diluent cell, a cleaning liquid cell having a cleaning liquid inlet and an overflow liquid outlet, and a pump for supplying a cleaning liquid to the cleaning liquid cell Cleaning station and
A measurement cell having a substrate solution inlet and an overflow liquid outlet, and exposing a sensitive part of the pH electrode to a flow path of the substrate solution; a measurement station including a pump for supplying the substrate solution to the measurement cell; One end has a pipe head inserted into an upper end opening of a dilution tip of a cartridge supplied to the operation station and a pipette-shaped thin pipe for dilution and a thin pipe for solid phase accommodated in a reaction cup, and the other end. Dispensing means connected to a syringe via a tube, means for elevating and lowering the pipet head, means for reciprocating the elevating means in the horizontal direction, and pushing down the upper end of the small-diameter tube into which the pipet head is inserted. And a means for detaching a small-diameter tube.

(Example) An example of a cartridge for enzyme immunoassay of the present invention will be described with reference to the drawings. FIG. 1 is a perspective view of a cartridge, and FIG. 2 is a sectional view. The cartridge (1) comprises a sample cup (2), a dilution cup (2) and a reaction cup (4), and each of the dilution cup (3) and the reaction cup (4) has a pipette-shaped thin pipe for dilution. (7) and a small-diameter tube (8) for a solid phase are accommodated. A bar code label (9) for displaying the measurement item is attached to the side wall (5) of the cartridge. Further, a sealing piece (10) is provided at the upper end opening of each of the cups (2), (3) and (4).
Is adhered, and each cup is airtightly closed. As shown in FIG. 2, a lyophilized labeled antibody (or labeled antigen) (6) is stored at the bottom of the reaction cup (4). In addition, a small-diameter solid-phase tube (8) accommodated in a reaction cup
An antibody (or antigen) is immobilized on the inner wall (11) at the tip of the device.

The cartridge of the present invention is preferably integrally molded, and its material is polypropylene, polystyrene,
Polyvinyl chloride, polytetrafluoroethylene, polymethyl methacrylate, polyethylene, polycarbonate,
Various plastics such as polyamide and polyester are used. Since it is desirable to close the upper end openings of the three cups with one sealing piece (10), it is preferable that the upper end openings of the respective cups be on the same plane. Seal piece (10)
In particular, a material having low air permeability, such as an aluminum laminate film, is used because the thin tube for solid phase and the freeze-dried enzyme label are stored in a dry state. Also, a small-diameter tube for dilution (7) and a small-diameter tube for solid phase (8)
May be used as the material, but usually, any of the resins listed above for cartridge molding is used.

The sample cup (2) is for charging a sample liquid therein. Since the required volume of the sample liquid is usually 200 μl or more, the internal volume of the sample cup is 20 to 500 μl.
Is preferred. The dilution cup (3) is for diluting the sample liquid therein to a predetermined magnification. In this dilution operation, a small-diameter pipe (7) for dilution housed in a dilution cup (3) is used. The small-diameter tube for dilution (7) used for the dilution operation is immediately discarded. Thin tube for dilution (7)
In this way, the so-called carry-over, which is caused when the device is shared, can be prevented. The dilution cup (3) is also used therein to carry out a primary immune reaction between the solid phase capillary (8) and the diluted sample liquid. Usually, the inner volume of the dilution cup (3) is preferably 0.5 to 2.0 ml. The reaction cup (4) contains a small-diameter tube for solid phase (8) and a freeze-dried enzyme-labeled body (6). Used in the dilution cup (3) to perform the primary immune response. Further, it is also used for dissolving the lyophilized label (6) in the reaction cup (4) by sucking a lysis solution, for example, a diluting solution or washing solution of the common reagent, and injecting the solution into the reaction cup. Thereafter, the distal end of the small-diameter tube (8) is immersed in a dissolved solution of the labeled substance and subjected to a secondary immune reaction. The inner volume of the reaction cup (4) is the dilution cup (3).
0.5 to 2.0 ml is preferable as in the above. The lyophilized enzyme label (6) before lysis is accommodated in the reaction cup (4) together with the solid-phase small-diameter tube (8). ) Must never touch. For this purpose, the freeze-dried label (6) is stored at the bottom of the reaction cup (4), and the small-diameter solid phase tube (8) is stored at the top of the reaction cup.

However, when the freeze-dried label (6) is redissolved in a small amount of a dissolving solution, for example, a diluent, it is necessary to immerse the tip of the small-diameter tube for solid phase in the label solution. It is desirable that the distance between the top surface of the label (6) and the tip of the small-diameter solid phase tube (8) be as short as possible. Usually, the distance is about 0.2 to 2 mm. In order to hold the freeze-dried marker at such a position, it is preferable to provide a squeezed portion (12) near the bottom of the reaction cup (4) as shown in FIG.

The cartridge for enzyme immunoassay of the present invention is a solid-phase small-diameter tube and an enzyme label as reagents specific to the measurement item, and a barcode indicating the measurement item and a disposable product necessary for performing the measurement of one sample. A sample cup, a dilution cup, a reaction cup, and a small-diameter tube for dilution are provided. Therefore, the user automatically puts the sample liquid into the sample cup of the cartridge and mounts it on a device described later, so that the measurement is automatically performed.

Next, an embodiment of the enzyme immunoassay apparatus using the cartridge for enzyme immunoassay will be described with reference to the drawings. FIG. 3 is a front view of the device of the present invention, FIG. 4 is a plan view, and FIG. 5 is a schematic diagram showing a basic configuration of the device of the present invention.

The enzyme immunoassay device of the present invention comprises: a means for supplying the cartridge (1) to the operation station (A) in which the dilution station (B), the washing station (C) and the measurement station (D) are arranged in this order; In the operation station, the liquid is sucked or thinned into the small-diameter tube via a pipet head (28) inserted into the upper end opening of the small-diameter tube for dilution (7) and the small-diameter tube for solid phase (8). Dispensing means (E) for discharging the liquid sucked into the diameter pipe, elevating means (F) for elevating and lowering the pipe head (28) to insert and remove the small diameter pipe from each cap, and moving the elevating means in the horizontal direction. Reciprocating means (G) for reciprocating the pipe, and means (H) for detaching the small-diameter tube inserted in the pipe head.

The cartridge supply means supplies the cartridge (1) to the operation station (A), and usually employs a turntable mechanism for moving the cartridge along the circumference, a conveyor mechanism for linearly moving the cartridge, and the like. 3 to 5 show an example in which a conveyor mechanism (31) is used. In addition, while the cartridge moves in the operation station (A), an immune reaction is performed in the reaction cup. The immune reaction may be performed at room temperature,
Usually, the heating is performed at a constant temperature, for example, at 37 ° C., so it is preferable to keep the cartridge at a constant temperature during the movement of the cartridge. Therefore, as shown in FIG. 4, the conveyor (31) is stretched on the heating block (29),
Alternatively, the conveyor mechanism (31) may be covered with a sealing member, and heated air may be supplied therein to keep the periphery of the cartridge at 37 ° C.

The conveyor mechanism is provided with, for example, a cartridge gripping mechanism (31) at regular intervals. The gripping mechanism grips both ends of the cartridge and moves the cartridge intermittently or continuously by moving the conveyor. The cartridge (1) for which the measurement has been completed at the operation station (A) is released at that time from the gripping mechanism (30) and the conveyor mechanism (3).
Cartridge waste container at the end of 1) (19)
Is discharged.

A bar code reader (32) for reading a bar code label (9) attached to a side wall of the cartridge is provided at an operation station entrance of the conveyor mechanism (31), and reads a bar code of the cartridge supplied to the operation station.

In the operation station (A), a dilution station (B), a cleaning station (C), and a measurement station (D) are arranged in this order from the entrance side of the station.

The dilution station (B) is provided with a diluent cell (36) having a diluent inlet opening at one end of a groove provided on the upper surface and an overflow outlet opening at the other end. The diluent is supplied from the container (44) to the diluent cell (36) by the pump (40), overflows from a groove provided on the upper surface of the cell, and is pumped from the overflow outlet together with a washing solution and a substrate solution to be described later. 41) Drained into the waste liquid container (45).

The cleaning station (C) is provided with a cleaning liquid cell (35) having the same shape as the diluting liquid cell (36). The cleaning liquid is supplied from the cleaning liquid container (43) to the cleaning liquid cell (35) by the pump (39), overflows from the groove provided on the upper surface of the cell, and is discharged from the overflow liquid outlet by the pump (41). Discharged into container (45).

When using a sample diluent and a washing solution at a predetermined temperature, for example, at 37 ° C., it is necessary to make the washing solution cell (35) and the diluting solution cell (36) constant temperature. In such a case, these cells are used as a constant temperature heat block. Is preferred. A metal such as aluminum is usually used for the cells (35) and (36).

The measuring station (D) is provided with a measuring cell (33) having an inlet for a substrate solution and an overflow liquid outlet, and containing a pH electrode (18) having a sensitive part exposed in the flow path of the substrate solution. ing. The substrate solution is obtained by supplying the washing solution and the urea stock solution from the washing solution container (43) and the urea stock solution container (42) to the mixer (34) by the pump (38), and mixing by the mixer (34). It is prepared and then supplied to the measuring cell (33). The overflow liquid overflowing from the measuring cell is discharged from the overflow liquid outlet to a waste liquid container (45) by a pump (41).

As a urea substrate solution, there are a method of diluting a concentrated undiluted solution and a method of using a dilute urea substrate solution. When using a dilute substrate solution, the pump (38) uses only the urea substrate solution. This means that the mixer (34) becomes unnecessary.

FIG. 6 is a detailed cross-sectional view of the measuring cell, which is made of a metal block (60). The lower side wall of the cell has an inlet (61) for a substrate solution, a measuring chamber (62) communicating with the inlet, A substrate solution flow path having a substrate solution overflow port (63) provided above the measurement chamber (62) and a substrate solution outlet (64) from the cell is formed. The measuring chamber (62) has a pH electrode (6
5) is accommodated so that the sensitive part is exposed to the flow path of the substrate solution.

The measuring cell (60) made of a metal block has corrosion resistance to a substrate solution (usually urea is used), and usually titanium and nickel are preferably used. When aluminum or the like is used, it is necessary to coat the contact surface with the substrate solution with a corrosion-resistant resin. When the pH measurement is performed at a constant temperature, an opening (90) for accommodating the heat transfer wire is formed in the metal block (60), and the heat transfer wire (91) is accommodated in the opening. The block is warmed to a constant temperature.

As the pH electrode (65) housed in the measurement chamber (62), in addition to the so-called glass electrode, which has been most frequently used,
A pH-sensitive field-effect transistor (hereinafter referred to as pH-FET), a pH electrode of a metal oxide surface type such as palladium oxide / palladium wire, and a pH-sensitive polymer membrane made of polyvinyl chloride containing a proton acceptor. Various kinds of micro pH electrodes, such as a coated wire type pH electrode coated on a carbon wire and the like, can be used. However, when the diameter of the glass electrode type pH electrode is reduced, induction noise tends to increase. Although the surface oxidized metal wire type pH electrode can be easily reduced in diameter, it has a drawback in long-term underwater life and the like. The coated wire type pH electrode can also be easily reduced in diameter, but has disadvantages such as a narrow linear response range to a pH change and a short underwater life.
Therefore, when these pH electrodes are used, it is necessary to solve the above problems in advance.

In contrast, pH-FETs (1) are easy to reduce in diameter.
(2) Induction noise when the diameter is reduced is small. (3) Since it is manufactured by IC technology, variation in characteristics between electrodes can be reduced, and the pH-sensitive surface (gate portion) can be miniaturized. (4) The response to pH change is extremely fast and no hysteresis remains in the response curve. (5) The linear response range to pH change is wide. (6) The shelf life in water is semi-permanent, and the characteristics such as pH sensitivity (7) It is most suitable as a pH electrode housed in the measurement chamber (62) because it has excellent features such as a small change with time and (7) a diode for temperature detection can be attached to the substrate. FIG. 6 shows an example using a pH-FET. The pH-FET (65) protrudes the sensitive part of the pH-FET at the tip of the outer cylinder (66), extends the lead wire connected to the electrode to the other end of the outer cylinder, and connects with the electrode part of the pH-FET. Resin is sealed between the inner walls of the outer cylinder, and the outer cylinder is closed.
Further, the outer cylinder (66) and the reference electrode (67) are inserted into the thicker outer cylinder (68), and the tip is closed with a resin. This thick outer cylinder (68) is inserted into a housing (69) provided with a connector portion (71) at the end, and the inner wall of the housing and the outer cylinder (6) are inserted.
A resin is sealed between 8) and the reference electrode (67). The connector part (71) contains pins for connecting lead wires, to which at least three lead wires of a source, a drain and a reference electrode of the pH-FET are connected. pH
-After inserting the tip coupler (75) into the outer cylinder (66), the FET (65) is inserted into the measuring chamber (62) of the cell (60) by the screw (72) provided on the outermost cylinder of the electrode. It is inserted and fixed in place by an O-ring (73) and a retaining screw (74). At the time of pH measurement, the small-diameter solid phase tube (8) is inserted into the measurement chamber from above the measurement cell, guided to the chip coupler (75), and put on the pH-FET (65).

The tip coupler (75) uses a small-diameter solid-phase tube (8) for pH-FET.
It has a role as a guide to cover (65). The chip coupler is shown in FIGS. 7 and 8 (A-
As shown in Fig. A), four cavities (81) are provided around a hollow portion (80) as a guide for the small-diameter tube for solid phase (8) to form a cloverleaf substrate solution flow path. ing.

Dispensing means (E) for sucking liquid into or discharging liquid from the small-diameter tube comprises a pipe head (28) inserted into the upper end opening of the small-diameter tube and a tube (26) at the end of the pipe head. It is composed of a syringe (25) connected via a. (27) is a tube opening / closing valve attached to a branch of the tube (26).

The pipe head (28) is connected to elevating means (F) comprising a cylinder or the like for inserting and removing a small-diameter tube into and from the cup, and moves up and down in conjunction with the operation of the elevating means. The elevating means (F) is further provided with a horizontal moving means (G), for example, X
It is connected to the shaft drive arm (22) and reciprocates in the X direction. If necessary, the X-axis drive arm can be reciprocated in the Y-direction by connecting it to the Y-axis drive arm (21).

As shown in FIG. 9, the lifting means (F) presses the upper end of the small-diameter tube inserted into the pipe head (28) downward to detach the small-diameter tube from the pipe head (28). ) Is provided. (37) is a container for discarding the diluted small-diameter tube after the dilution operation.

FIG. 10 is a control circuit diagram of the device of the present invention, and the operation of each of the above components is controlled by a computer. Data processing for converting the output of the pH electrode into the concentration of the substance to be measured is also performed by the computer, and the result is displayed on a display and / or a printer.

When a pH-FET is used as the pH electrode, a source follower type measuring circuit described in JP-A-60-4851 and JP-A-60-225056 is used to read the output.

Next, the enzyme immunoassay using the cartridge of the present invention will be described for the case of two-step sans serbia. In the following description, an antibody (or antigen) for capturing the antigen (or antibody) to be measured is immobilized on the small-diameter tube for solid phase. As the lyophilized label, a labeled antibody (or labeled antigen) for sanitizing the substance to be measured is used. The user takes out the cartridge for the desired measurement item, peels off the sealing piece (10) attached to the upper surface of the cartridge, puts about 20 μl of the sample liquid into the sample cup (2), and puts this into the cartridge supply. When the conveyor mechanism (31) is set,
The following operations are all performed automatically by the measurement circuit shown in FIG.

When the cartridge (1) is supplied to the operation station (A) by the conveyor, first, the bar code reader (3) is supplied.
2) reads the measurement items and the like from the barcode (9), and selects an operation program and a calibration curve according to the measurement items.

Next, when the cartridge is sent to the dilution station (B), the lifting / lowering means is operated, and the pipet head (28) attaches the small-diameter pipe (7) for dilution in the dilution cup (3) to the inside of the sample cup (2). A predetermined amount (for example, 10 μl) of the sample solution is aspirated, immediately moved to the diluent cell (36), and a predetermined amount of the diluent (for example, 90 μl) is additionally aspirated. Then, the pipette head returns to the dilution cup (3), and discharges a mixed solution (for example, 100 μl) of the sample and the diluent into the diluent cap, and repeats re-suction-redischarge of the liquid to mix the sample and the diluent. To prepare a sample solution diluted to a predetermined magnification (for example, 10 times). The thin tube for dilution is discarded in a container (37).

Next, the pipe head (28) is equipped with a small-diameter tube (8) for the solid phase in the reaction cup (4), and this is inserted into the diluted sample solution in the dilution cup (3) to a predetermined amount (for example, 10 μl). ), The small-diameter tube (8) is removed from the dilution cup (3) and allowed to stand for a predetermined period of time (for example,
Perform primary immune reaction for 10 minutes).

When the primary immune reaction is completed, the cartridge is moved to the washing station (B). Then, the pipette head reattaches the small-diameter tube for the solid phase in the station, moves it to the diluent cell (36), and aspirates the diluent overflowing the groove provided on the upper surface of the cell several times. Discharge to wash the small diameter tube. Thereafter, a predetermined amount (for example, 20 μl) of the diluent is aspirated, the pipethead is moved to the reaction cup (4) of the cartridge, and the aspirated diluent is discharged therein to dissolve the lyophilized label (6). Let it. In order to completely dissolve the labeled substance, the labeled substance solution dissolved in the reaction cup is suction-poured several times to completely dissolve, and then the solid-phase small-diameter tube (8) is placed in the reaction cup (4). Then, after a secondary immune reaction is performed for a predetermined time (for example, 5 minutes) in this state, the cartridge is moved to the measurement station (C).

After the secondary immune reaction, the solid-diameter small-diameter tube (8) is reattached to the pipethead, moved to the washing cell (35), and the liquid overflowing the groove provided on the upper surface of the cell is washed. Is suctioned and discharged several times to wash the small-diameter tube. Thereafter, the small-diameter tube is inserted into a measurement chamber provided in the measurement cell (33), and after the urea substrate solution flowing in the measurement cell is sucked, the small-diameter tube is covered with a pH-FET. The change in pH of the substrate solution enclosed between the FETs is measured.

If the temperature of the tube (26) changes during the pH change measurement, the volume of air in the tube changes, and this is the pH-FET
In this case, the flow of the substrate solution sealed in the gap formed by the small-diameter tube may be induced to cause a measurement error. PH
It is preferable to open the tube opening / closing valve (27) during the change measurement to release the pressure in the tube to the atmospheric pressure.

The pH change is read as a change in the source potential of the pH-FET. Then, a change measurement of the source potential is usually calculated in any time period of several tens of seconds after the thin tube is covered with the pH-FET, and the change speed is measured by a conversion formula obtained in advance. It is displayed in terms of the concentration of the substance.

The cartridge of the present invention, the measuring method and the apparatus using the cartridge, can be used for enzyme immunoassay by the San Germanti method and the competitive method. Although the present invention has been described with respect to the so-called two-step Sanche-Marche method and the two-step competitive method, the present invention can be applied in principle to the one-step San-Germanche method and the one-step competitive method. It is possible to use these one-step methods. When these one-step methods are employed, it is necessary to set assay conditions that prevent the so-called prozone phenomenon, in which the detection output decreases rather when the concentration of the substance to be measured becomes extremely high. .

(Effects of the Invention) As described above, the cartridge for enzyme-linked immunosorbent assay of the present invention, and the measuring method and apparatus using the same, are as follows: (1) Since a desired item can be measured in an arbitrary order, a small number of items can be measured. In addition to the features that can be applied to small and medium-sized hospitals without loss of reagents even for specimens, (2) full automation can be performed with simple hardware, and (3) thin pipe tip shape Since the immune reaction and enzyme reaction are performed in a small area called the small diameter part at the tip of the diameter tube,
Each reaction is very rapid, the reaction time is short, and the amount of sample liquid used is small.

(4) In the enzyme immunoassay, it is generally important to wash a small-diameter tube for a solid phase, which is called separation of a conjugate / free body. However, in the present invention, this is performed by a dispensing means. Does not require a special drive unit.

It has an excellent effect.

Experimental Example 1 The cartridge for enzyme-linked immunosorbent assay of the present invention was applied to an assay device shown in FIG.
P) measurements were made. In this case, anti-AFP
The antibody was immobilized, and a freeze-dried urease-labeled anti-AFP antibody was stored at the bottom of the reaction cup.

The following were used as common reagents regardless of the measurement items.

Diluent: 0.0915 M disodium hydrogen phosphate + 0.0085 M sodium phosphate (pH 7.8) Washing liquid: 0.1 M ammonium chloride + 0.154 M sodium chloride Substrate: 0.1 M ammonium chloride + 0.154 M sodium chloride + 0.1 M urea AFP measurement After taking out the cartridge for use and opening the sealing piece, 20 μl of the serum sample was put into the sample cup. Next, the cartridge was mounted on a measuring device, and the measurement was performed in the following order.

a) A dispenser attaches a small-diameter tube for dilution housed in a dilution cup, aspirates 10 μl of the sample in the sample cup and 90 μl of the diluent in the diluent cell, and aspirates this in the dilution cup. To prepare a 10-fold diluted sample solution. The dilution tube was then discarded in a waste bottle.

b) The primary immunoreaction was started by attaching a small-diameter tube for solid phase housed in the reaction cup by the dispensing means and detaching it into the 10-fold diluted sample solution. .
Thereafter, in this state, the small-diameter tube in the dilution cup was incubated at 37 ° C. for 10 minutes. Then, the dispensing means again attached the small-diameter tube, moved it to the diluent cell, and aspirated and discharged the diluent to perform washing.

c) While the dispensing means is equipped with the small-diameter tube for solid phase, aspirate 30 μl of the diluent, discharge it into the reaction cup, and aspirate.
The lyophilized marker at the bottom of the cup was dissolved by repeating the ejection. A second immune response was then initiated by removing the chip into the cup. In this state, the chips in the cup were incubated at 37 ° C. for 5 minutes.

d) The dispensing means reattaches the small-diameter tube for the solid phase, moves the small-diameter tube to the washing liquid cell, and aspirates and discharges the washing liquid to wash the small-diameter tube and discharge the washing liquid. Thereafter, the substrate solution (urea and ammonium chloride solution) was sucked into the small-diameter tube while inserting the small-diameter tube into the pH measurement cell.
Next, the small-diameter tube was housed in the pH measurement cell, and the tip of the small-diameter tube was covered with the pH-FET.

e) Read the output (source potential) of pH-FET for 20 seconds,
The average value of the output change rate for 10 seconds from 10 seconds to 20 seconds after the start of measurement was determined.

f) The substrate solution was pumped into the pH measurement cell for 10 seconds to replace the substrate solution in the cell with fresh one and prepared for the next measurement.

Table 1 shows the relationship between the change rate of the output of the pH-FET and the concentration of AFP measured in this manner.

Experimental Example 2 In the same manner as in Experimental Example 1, measurement of huerytin was performed. In this case, an anti-huetin antibody was immobilized in the small-diameter tube for solid phase, and a freeze-dried urease-labeled anti-huetin antibody antibody was stored at the bottom of the reaction cup.

Table 2 shows the results of the measurement performed in exactly the same operation procedure as in Experimental Example 1.

Experimental Example 3 As in Experimental Example 1, the measurement of carcinoembryonic antigen (CEA) was performed. In this case, immobilize the anti-CEA antibody on the small-diameter tube for solid phase,
A freeze-dried urease-labeled anti-CEA antibody was stored in the reaction cup, and the measurement was performed in the following order.

a) The dispensing means is equipped with a small-diameter tube for dilution housed in the dilution cup, and the serum sample 10 μl and the diluent 20 μl in the sample cup are attached.
1 was sucked and discharged into a dilution cup to prepare a sample solution diluted three times. Thereafter, the thin tube for dilution was discarded.

The results of performing the same operations as in b) to e) of Experimental Example 1 are shown in Table 3 below.

[Brief description of the drawings]

FIG. 1 is a perspective view of a cartridge for enzyme immunoassay of the present invention, and FIG. 2 is a cross-sectional view of the cartridge. Third
FIG. 4 is a front view of the measuring device of the present invention, FIG. 4 is a plan view, FIG. 5 is a schematic diagram showing a basic configuration of the device of the present invention, and FIG. 6 shows a detailed structure of the measuring cell. FIG. 7 is a sectional view showing the structure of the chip coupler.
FIG. 8 is a view taken in the direction of arrows AA in FIG. 7, FIG. 9 is a front view showing the lifting / lowering means, and FIG. 10 is a control circuit diagram of the device of the present invention. A: Operation station B: Dilution station C: Cleaning station D: Measurement station E: Dispensing means, F: Elevating means G: Reciprocal moving means, H: Detachment means 1: Cartridge 2 ... Sample cup 3 ... Dilution cup 4 ... Reaction cup 6 ... Lyophilized marker, 7 ... Thin tube for dilution, 8 ... Thin tube for solid phase 10 ... Seal piece, 33 ... … Measurement cell 35… wash cell, 36… dilution cell 65… pH electrode.

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Takuichiro Watanabe 1-2-1, Kaigandori, Okayama City, Okayama Prefecture Inside Kuraray Co., Ltd. (72) Inventor Masuo Makino 1621 Sakurazu, Kurashiki City, Okayama Prefecture Kuraray Co., Ltd. (72) Inventor Michihiro Nakamura 2045 Sakurazu, Kurashiki-shi, Okayama Prefecture Kuraray Co., Ltd.Examiner, Hiroyuki Kameda

Claims (3)

(57) [Claims] 1. A cartridge for enzyme immunoassay in which a sample cup for accommodating a sample to be measured, a dilution cup accommodating a pipette-shaped thin tube for dilution and a reaction cup are integrally arranged. A freeze-dried enzyme-labeled reagent is contained in the bottom of the reaction cup, and at least an antigen (or antibody) as a substance to be measured is placed on the inner wall of the tip.
A pipette-shaped small-diameter tube for solid phase to which an antibody (or antigen) that binds to is fixed is accommodated in the reaction cup with its tip separated from the enzyme labeling reagent, and the upper end opening of each of the cups is closed. A cartridge for enzyme-linked immunosorbent assay, wherein the cartridge is hermetically closed with a sealing piece. 2. An enzyme immunoassay method using the cartridge for enzyme immunoassay according to claim 1, wherein (1) a sample in a sample cup with a thin pipe for dilution in the form of a pipette chip accommodated in the dilution cup. And injecting the diluent into the diluting cup to prepare a sample liquid diluted to a predetermined magnification in the diluting cup. (2) A step of immersing the distal end of the pipette-shaped solid-phase small-diameter tube accommodated in the reaction cup into the sample liquid in the dilution cup to perform a primary immune reaction. (3) A lysis solution is injected into the reaction cup to dissolve the lyophilized enzyme labeling reagent in the reaction cup, and then the solid solution after the completion of the primary immunoreaction in the dissolved enzyme label solution. A step of immersing the tip of the phase-use small-diameter tube to perform a secondary immune reaction. (4) a step of washing the small-diameter solid-phase tube after the completion of the secondary immune reaction; (5) Put the washed small-diameter tube for solid phase over the sensitive part of the pH electrode provided in the measurement cell filled with the substrate solution, and transfer the substrate solution between the small-diameter tube and the sensitive part of the pH electrode. A step of measuring a pH change accompanying the decomposition reaction. An enzyme immunoassay method comprising: 3. An enzyme immunoassay device using the cartridge for enzyme immunoassay according to claim 1, comprising: means for supplying the cartridge for enzyme immunoassay to an operation station; and a diluent provided on the operation station. A diluent cell having an inlet and an overflow liquid outlet, a dilution station having an on-board for supplying diluent to the diluent cell, a cleaning liquid cell having a cleaning liquid inlet and an overflow liquid outlet, and a cleaning liquid flowing into the cleaning liquid cell. A washing station equipped with a supply pump, a measurement cell having a substrate solution inlet and an overflow solution outlet, and exposing the sensitive part of the pH electrode to the substrate solution flow path, and supplying the substrate solution to the measurement cell A measuring station provided with a pump for performing the operation, a dilution cup of a cartridge supplied to the operation station and a small diameter for dilution of a pipette chip contained in a reaction cup. Dispensing means having at one end a pipe head inserted into the upper end opening of the small-diameter tube for solid phase, and a syringe connected to the other end via a tube; means for raising and lowering the pipe head; An enzyme immunoassay device comprising: means for reciprocating the pipe in the horizontal direction; and means for pushing down the upper end of the small-diameter pipe in which the pipe head is inserted to detach the small-diameter pipe.