JPH03181853A - Cartridge for enzyme immunoassay and measuring method and apparatus using the same - Google Patents

Abstract

PURPOSE:To automatically perform enzyme immunoassay using a pH electrode by providing a cartridge wherein a dilution cup in which a pipette chip-shaped dilution fine diameter tube is received and a reaction cup are integrally arranged. CONSTITUTION:A specimen and a diluent are injected in a dilution cup 3 to prepare a specimen solution diluted in predetermined dilution magnification. Next, the leading end part of the fine diameter tube 8 for a solid phase received in a reaction cup 4 is immersed in the specimen solution in the dilution cup 3 to perform primary immunoreaction. Subsequently, a dissolving solution is injected in the reaction cup 4 to dissolve the freeze-dried enzyme labelled reagent of the reaction cup 4 and, thereafter, the leading end part of the fine diameter tube 8 after primary immunoreaction is immersed in the enzyme labelled reagent solution to perform secondary immunoreaction. Next, the fine diameter tube 8 after the secondary immunoreaction is washed. The washed fine diameter tube 8 is allowed to cover the responsive part of the pH electrode 65 provided in a measuring cell filled with a substrate solution and the pH change accompanied by the decomposition reaction of the substrate solution between the fine diameter tube 8 and the responsive part of the pH electrode 65 is measured.

Description

Detailed Description of the Invention (Industrial Application Field) The present invention utilizes immune reactions (antigen-antibody reactions) to analyze multicomponent samples such as biological samples (e.g. blood, serum, plasma, urine, spinal fluid, etc.). The present invention relates to an enzyme immunoassay cartridge suitable for measuring a trace amount of a specific substance contained in a system, a measuring method using the same, and a measuring device. The present invention will be described below regarding the measurement of trace amounts of biological substances in clinical tests, but it can be applied to a wide range of fields including pharmacy, biology, zoology, botany, agriculture, chemistry, testing, etc.

(Prior Art) Substances that are involved in the physiological activities of living organisms are generally in trace amounts, and many of them play very important roles in living organisms. Therefore, quantitatively measuring such trace amounts of physiologically active substances is important in biology-related fields such as medicine and biochemistry, and various measurement methods for this purpose have been devised and put into practical use. Among them, enzyme immunoassay using an enzyme as a label is widely used in the field of clinical testing.

In this measurement method, first, a solid phase on which an antibody (or antigen) that can specifically bind to the antigen (or antibody) that is the substance to be measured is immobilized is mixed with a sample solution and a second antibody (or antigen) labeled with an enzyme.
The antigen in the sample solution is determined by contacting the solid phase simultaneously or sequentially with the solid phase to cause an immune reaction, washing, and then measuring the activity of the enzyme label remaining on the solid phase. (or antibodies).

A typical example of such a measurement method is a non-uniform EIA, so-called Enzyme Linked 1ml uno 5or
bent As5ay (ELISA) is known.

In ELISA, a test tube, microplate, etc. is used as a solid phase on which an antibody (or antigen) that specifically binds to the antigen (or antibody) to be measured is immobilized to capture the substance to be measured in the sample solution. . In addition, when the target to be measured is an antigen, an enzyme-labeled second antibody, which is an enzyme-labeled second antibody capable of binding to the antigen, is used in the Sand-Germany EL ISA, and an enzyme-labeled second antibody is used as the antigen to be measured in the competitive EL ISA. An enzyme-labeled antigen is used, which is the same antigen labeled with an enzyme.

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

Several methods are conventionally known for observing changes in the optical properties of substrate solutions. Instrumental methods include those that measure changes in the optical properties of substrate solutions using spectrophotometers, fluorometers, chemiluminescence photometers, etc. (for example, Ishikawa, Kaigo, Miyai, enzyme immunoassay; (See Igakushoin (1982)).

In addition, as another method, there is a method in which the presence of an antigen (or antibody) is determined by comparing a substrate solution and a control substrate solution and observing the difference in color of the substrate solution with the naked eye (for example, in
-1.28369).

However, these optical measurement methods using instruments require a stable light source, a highly sensitive photometer, a precise optical amplifier circuit, etc., and therefore have to be expensive, large, and complicated devices. Furthermore, since measurement requires special techniques, a specialist engineer must be assigned to handle the measurement.

On the other hand, the method of direct observation with the naked eye is a qualitative measurement method, and because it involves variations in color change and the subjectivity of the observer, individual differences in judgment are likely to occur. Furthermore, when measuring a very small amount of a substance, there is little change in color, making it difficult to judge.

The present inventors have improved the shortcomings of the conventional 1jll + constant method, removed the ambiguity of the judgment criteria based on the subjectivity of the observer, and measured the decomposition reaction of a substrate solution objectively and with high detection accuracy. One way to do this is to change the pH of the substrate solution.
A method of measuring with electrodes was proposed in JP-A No. 1-212347.

(Problem to be Solved by the Invention) In such a measurement method, the pH is usually set to 11.
A sensitive field effect transistor (pH-FET) is used, the inner wall of the tip of a pipette tip-shaped narrow tube is used as the solid phase, urease is used as the labeling enzyme, and urea is used as the substrate.

In the enzyme immunoassay method using a pH electrode, (1) the structure of the pH electrode is extremely simple compared to a conventional optical measurement system. (2) Since a small-diameter tube in the shape of a pipette tip is used as the solid phase, there is an advantage that all operations from diluting the sample to washing can be performed with a dispensing means.However, in the above proposal, a pH electrode was used. Only a laboratory-scale enzyme immunoassay method and device have been proposed, and no practical measurement method and device have been proposed yet.

Therefore, the object of the present invention is pH3 [! It is an object of the present invention to provide a practical method and apparatus for automatically performing enzyme immunoassay using electrodes.

Furthermore, by using the above 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 so on. Furthermore, in general, enzyme immunoassay uses many reagents such as a solid phase, an enzyme label, a substrate, a dilution buffer (hereinafter abbreviated as diluent), and a washing buffer (hereinafter abbreviated as washing solution). Since some of these reagents differ depending on the measurement item, these reagents have conventionally been sold in the form of a kit. In other words, each time the measurement item changes, the user takes out a dedicated kit and performs the measurement according to a predetermined procedure. In general, the size of a kit is for tens to hundreds of samples, and it is suitable for measuring one item of tens to hundreds of samples at once, but it is suitable for measuring any measurement item in any order. There was an inappropriate guess.

Therefore, another object of the present invention is to provide a cartridge for enzyme immunoassay that makes it possible to measure any measurement item in any order by packaging special reagents and other components for one measurement item into a cartridge. be.

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

The present invention also provides an enzyme immunoassay method using the enzyme immunoassay cartridge described above, comprising: (1) diluting the sample and diluent in the sample cup with a pipette tip-shaped thin dilution tube housed in the dilution cup; A step of preparing a sample liquid that is injected into a dilution cup and diluted to a predetermined ratio in the dilution cup.

(2) A step of performing a primary immune reaction by immersing the tip of a pipette tip-shaped small-diameter solid phase tube housed in a reaction cup into a sample liquid in a dilution cup.

(3) After injecting the dissolution solution into the reaction cup and dissolving the freeze-dried enzyme labeling reagent in the reaction cup, solids that have undergone the primary immune reaction are added to the dissolved enzyme label solution. A step of immersing the tip of a small-diameter phase tube to perform a secondary immune reaction.

(4) A step of washing the small-diameter tube for solid phase after the completion of the secondary immune reaction.

(5) Place the washed thin tube for solid phase over the sensitive part of the pH electrode installed in the measurement cell filled with the substrate solution, and remove the substrate solution between the thin tube and the sensitive part of the pH electrode. Step of measuring pH change accompanying decomposition reaction.

This is an enzyme immunoassay method consisting of:

Furthermore, the present invention provides an enzyme immunoassay apparatus using the above-mentioned enzyme immunoassay cartridge, comprising means for supplying the enzyme immunoassay cartridge to an operation station, and a diluent inlet and an overflow liquid provided at the operation station. A dilution station having a diluent cell having an outlet, a dilution station having a pump for supplying diluent to the diluent cell, a cleaning fluid cell having a cleaning fluid inlet and an overflow fluid outlet, and a pump for supplying cleaning fluid to the cleaning fluid cell. A measuring cell having a substrate solution inlet and an overflow liquid outlet and exposing a sensitive part of a pH electrode to a substrate solution flow path, and a pump for supplying the substrate solution to the measuring cell. A measurement station, and a pipette head that is inserted into the upper end opening of the pipette tip-shaped dilution tube and solid phase thin tube housed in the dilution cup and reaction cup of the cartridge supplied to the operation station. a dispensing means having one end and a syringe connected to the other end via a tube; a means for raising and lowering the pipette head; a means for reciprocating the raising and lowering means in a horizontal direction; This enzyme immunoassay device is equipped with a means for pushing down the upper end of the small diameter tube to detach the small diameter tube.

(Example) An example of the enzyme immunoassay cartridge of the present invention will be explained with reference to the drawings. Figure 1 is a perspective view of the cartridge;
The figure is a sectional view. The cartridge (1) is composed of a sample cup (2), a dilution cup (3), and a reaction cup (4), and each of the dilution cup (3) and reaction cup (4) has a small pipette tip-shaped pipette tip for dilution. A tube (7) and a small diameter tube for solid phase (8) are accommodated. Further, a barcode label (9) for displaying measurement items is affixed to the side wall (5) of the cartridge. Furthermore, each of the above cups (2),
A sole piece (10) is attached to the upper end openings of (3) and (4) to airtightly close each cup. As shown in FIG. 2, a freeze-dried labeled antibody (or labeled antigen) (6) is stored at the bottom of the reaction cup (4). Further, an antibody (or antigen) is immobilized on the inner wall (It) of the tip end of the solid phase thin tube (8) housed in the reaction cup.

The cartridge of the present invention is preferably integrally molded,
Various plastics such as polypropylene, polystyrene, polyvinyl chloride, polytetrafluoroethylene, polymethyl methacrylate, polyethylene, polycarbonate, polyamide, and polyester may be used as the material. The top openings of the three cups are sealed with one seal piece (10
), the upper end openings of each cup are preferably flush. Since the seal piece (10) is especially used to preserve the small-diameter tube for solid phase and the freeze-dried enzyme label in a dry state, a material with low air permeability such as an aluminum laminate film is used. Although glass or the like may be used as the material for the thin tube for dilution (7) and the thin tube for solid phase (8), usually any of the resins listed above for cartridge molding is used.

The sample cup (2) is for charging the sample liquid into it. Since the required volume of sample liquid is usually 200 μQ or less, the internal volume of the sample cup should be 20 to 500 μQ.
μQ is preferred. The dilution cup (3) is used to dilute the sample liquid to a predetermined ratio. A thin dilution tube (7) housed within the dilution cup (3) is used for this dilution operation. The thin dilution tube (7) used for the dilution operation is immediately discarded. Small diameter tube for dilution (
By making 7) disposable in this way, it is possible to prevent so-called carryover that occurs when it is shared. The dilution cup (3) is also used to carry out the primary immunoreaction between the solid phase narrow tube (8) and the diluted sample liquid therein. The internal volume of the dilution cup (3) is usually 0.5 to 2.0 II (preferably. is stored, but a small diameter tube for solid phase (8
) is first used to carry out the primary immune reaction in the dilution cup (3) as described above. In addition, a lysis solution, such as a dilution solution of a common reagent or a washing solution, is aspirated and injected into the reaction cup, and the freeze-dried labeled substance (6) in the reaction cup (4) is aspirated.
) is also used to dissolve. After that, the narrow diameter tube (8
) is immersed in a dissolved label solution and subjected to a second immune reaction. The internal volume of the reaction cup (4) is preferably 0.5 to 2.0 m12 similarly to the dilution cup (3). The freeze-dried enzyme label (6) before dissolution is placed in the reaction cup (4).
) together with the small-diameter tube for solid phase (8), but at that time, the marker (6) and the small-diameter tube for solid phase (8) must never come into contact with each other.

For this purpose, the lyophilized label (6) is placed in the reaction cup (4).
The small-diameter solid phase tube (8) is housed in the upper part of the tube at a distance.

However, when the freeze-dried labeled substance (6) is redissolved in a small amount of a dissolution solution, such as a diluent, it is necessary to immerse the tip of the solid phase thin tube in the labeled solution, so freeze-drying at a distance is necessary. It is desirable that the distance between the top surface of the marker (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 label in such a position, it is preferable to provide a constriction part (12) near the bottom of the reaction cup (4) as shown in FIG. 2.

The cartridge for enzyme immunoassay of the present invention includes a thin tube for solid phase as a reagent specific to the measurement item, an enzyme label, a barcode for displaying the measurement item, and a disposable item necessary for measuring one sample. Equipped with sample cup, dilution cup, reaction cup, and small diameter tube for dilution. Therefore, the user simply puts a sample liquid into the sample cup of the cartridge and attaches it to the apparatus described later, and the measurement is automatically performed.

Next, an embodiment of an enzyme immunoassay apparatus using the above 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 the basic configuration of the device of the present invention.

The enzyme immunoassay device of the present invention includes the above-mentioned cartridge (1).
to an operating station (A) in which a dilution station (B), a washing station (C) and a measuring station (D) are arranged in this order; Small diameter tube (7
) and a pipette head (28) inserted into the upper end opening of the solid phase narrow tube (8) to aspirate the limb into the narrow tube or discharge the liquid sucked into the narrow tube. E)
, an elevating means (F) for elevating the pipette head (28) to insert and remove the small diameter tube into each cup, a reciprocating means (G) for reciprocating the elevating means in the horizontal direction, and a reciprocating means (G) for reciprocating the elevating means in the horizontal direction; Means for detaching the inserted small diameter tube ()I)
It is equipped with

The cartridge supply means supplies the cartridge (1) to the operation station (A), and usually employs a turntable mechanism that moves the cartridge along the circumference, a conveyor mechanism that moves the cartridge linearly, or the like. 3 to 5 show examples using a conveyor mechanism (31). Also, during the movement of the cartridge within the operating station (^), an immunological reaction takes place in the reaction cup.

Although the immunoreaction may be performed at room temperature, it is usually performed at a constant temperature, for example 37° C., so it is preferable to maintain the cartridge at a constant temperature during cartridge transport. Therefore, as shown in Figure 4, the conveyor (31) is stretched over a heating block (29), or the conveyor mechanism (31) is covered with a sealing member to supply heated air into it. The surrounding area of the cartridge may be maintained at 37°C.

The conveyor mechanism is provided with, for example, cartridge gripping mechanisms (31) at regular intervals.1. Both ends of the cartridge are gripped by the gripping mechanism, and the cartridge is moved intermittently or continuously by the movement of the conveyor. At that point, the gripping mechanism (30) of the cartridge (1) whose measurement has been completed at the operation station (A) is released, and the cartridge (1) is placed in a cartridge waste container (
19).

At the entrance of the operation station of the conveyor mechanism (31), there is a barcode label (9) attached to the cartridge II + wall.
) is provided to read the barcode of the cartridge supplied to the operating station.

The operation station (8) includes a dilution station (B) and a cleaning station (C) in order from the population side of the station.
) and a measuring station (D) are arranged.

The dilution station (B) is provided with a diluent cell (36) having a diluent outlet opening at one end of a groove provided on the upper surface and an overflow outlet opening at the other end. The diluent is transferred from the container (44) to the diluent cell (3) by the pump (40).
6), overflows from the groove provided on the top surface of the cell, and is discharged from the overflow outlet to a waste liquid container (45) along with a cleaning liquid and a substrate solution, which will be described later, by a pump (41).

The cleaning station (C) is provided with a cleaning liquid cell (35) having the same shape as the diluting liquid cell (35).

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 top surface of the cell, and is transferred from the overflow liquid outlet to the waste liquid container (45) by the pump (41). is discharged.

When using the sample diluent and washing liquid at a predetermined temperature, for example, 37°C, the washing liquid cell (35) and the dilution liquid cell (3
6) needs to be kept at a constant temperature, in which case it is preferable to use these cells as a constant temperature heat block. Such cells (35) and (36) are usually made of metal such as aluminum.

The measurement station (D) is provided with a measurement cell (33) that has a substrate solution inlet and an overflow liquid outlet and houses a pH electrode (18) with a sensitive part exposed to the substrate solution flow path. ing. The substrate solution is pumped (38) from the washing liquid container (43) and the urea original container (42) to the washing liquid and urea source container, respectively.
) to the mixer (34), and the mixer (34)
prepared by mixing in the measuring cell (3
3). The overflowing liquid from the measurement cell is transferred to a waste liquid container (45) by a pump (41) from an overflowing liquid outlet.
is discharged.

As a urea substrate solution, there are two methods: diluting a concentrated raw wave and using an already diluted urea substrate solution.
38) will send only the urea substrate solution, and the mixer (
34) becomes unnecessary.

FIG. 6 is a detailed sectional view of the measuring cell, which consists of a metal block (60), a substrate solution inlet (61) in the lower side wall of the cell, a measuring chamber (62) communicating with the population;
Substrate solution overflow port (6) provided at the top of the measurement chamber (62)
3), and a substrate solution flow path comprising a substrate solution outlet (64) from the cell. A pH electrode (65) is housed in this measurement chamber (62) so that its sensitive part is exposed to the flow path of the substrate solution.

A measuring cell (60) consisting of a metal block contains a substrate solution (
Urea is usually used. ), titanium and nickel are usually preferably used. If aluminum or the like is used, the surface that will come into contact with the substrate solution must be coated with a corrosion-resistant resin. Also pi-
When measuring at a constant temperature, a metal block (6
An opening (90) for accommodating the heat transfer wire is bored in 0),
A heat transfer wire (91) is accommodated in this opening to heat the block to a constant temperature.

In addition to the so-called glass electrode, which has conventionally been most commonly used as the pH1 electrode (65) housed in the measurement chamber (62), a pH-sensitive field effect transistor (hereinafter referred to as pH-F
ET), surface oxidized metal wire type pH electrodes such as palladium oxide/palladium wires, and corchid wire type pH electrodes in which metal wires or carbon wires are coated with a pH-sensitive polymer film made of polyvinyl chloride containing proton acceptors. Various micro pH electrodes such as pH electrodes, etc. Poles can be used. However, when the diameter of the glass electrode type pH1K electrode is reduced, the induced noise tends to increase. surface oxidized metal linear p
The diameter of the H electrode can be easily reduced, but there are drawbacks such as a long underwater life. Corchid wire type pH electrodes are also easy to reduce in diameter, but have drawbacks such as a narrow linear response range to pH changes and a short underwater life. Therefore, when using these pH electrodes, it is necessary to solve the above problems in advance.

On the other hand, pH-PET (1) can be easily reduced in diameter; (2) Less induced noise when the diameter is reduced, (3) I
Since it is manufactured using C technology, variations in characteristics between electrodes can be reduced, and the pH-sensitive surface (gate portion) can be miniaturized. (4) Extremely fast response to pH changes;
and no hysteresis remains in the response curve; (5) wide linear response range to pH changes; (6) semi-permanent shelf life in water; and little change in characteristics such as pH sensitivity over time; (7) temperature detection. The measurement chamber (6
2) It is most suitable as a pH electrode housed in the interior. FIG. 6 shows an example using pH-PET. pH-F
E T (65) has pH-F'E at the tip of the outer cylinder (66).
The sensitive part of the T is made to protrude, the lead wire connected to the electrode is extended to the other end of the outer cylinder, a resin is sealed between the electrode part of the pH-FET and the inner wall of the outer cylinder, and the outer cylinder is closed. ing. Further, the outer tube (66) and the reference electrode (67) are inserted into a thicker outer tube (68), and the tip is closed with resin. This thick outer cylinder (68) is inserted into a housing (69) having a connector part (71) at the end, and a resin is sealed between the inner wall of the housing, the outer cylinder (68), and the reference electrode (67). There is. Connector II (71) houses pins for connecting lead wires, to which at least three lead wires, the source, drain, and reference electrode of pH-PET, are connected. pH-FET (65) is the outer cylinder (66
), the chip coupler (75) is inserted into the measurement chamber (62) of the cell (60) by the threaded part (72) provided on the outermost electrode cylinder, and then the O-ring (73) and the holding screw are inserted. (74), it is fixed at a predetermined position. During pH measurement, the small-diameter solid phase tube (8) is inserted into the measurement chamber from the top of the measurement cell, guided to the tip coupler (75), and then
It is placed on H-FET (65).

Chip Kabra (75) is a thin tube for solid phase (8) with pHF
It has a role as a guide for covering E T (65). The chip coupler is shown in FIGS.
As shown in the A-A arrow view in the figure, four cavities form a clover-shaped substrate solution flow path around a hollow part (80) that serves as a guide for the solid phase thin tube (8). (81) is provided.

The dispensing means (E) capable of sucking liquid into a small diameter tube or discharging the liquid inside the small diameter tube includes a pipette head (28) inserted into the upper end opening of the small diameter tube and a tube (28) attached to the end of the pipette head. It consists of a syringe (25) connected via a syringe (26). (27) is a tube opening/closing valve attached to a branch of the tube (26).

The pipette head (28) is connected to an elevating means (F), such as a cylinder, for inserting and removing a small diameter tube into and from the cup, and is raised and lowered in conjunction with the operation of the elevating means. The lifting means (F) further includes horizontal movement means (G), e.g.
It is connected to a shaft drive arm (22) and reciprocated in the X direction. Furthermore, if necessary, the X-axis drive arm can be connected to the Y-axis drive arm (21) for reciprocating movement in the Y direction.

Further, as shown in FIG. 9, the elevating means (F) includes a means for pushing downward the upper end of the small diameter tube inserted into the pipette head (28) to detach the small diameter tube from the pipette head (28). (I()) is provided. (37) is a container for discarding the thin dilution tube after the dilution operation has been completed.

FIG. 10 is a control circuit diagram of the apparatus of the present invention, and the operations of the above-mentioned parts are controlled by a computer.

Data processing for converting the pH3l polar output to the concentration of the substance to be measured is also performed by the computer, and the results are displayed on a display and/or printer.

In addition, when using a pH-FET as a pH electrode, the output of the pH-FET can be read using JP-A-60-4851 and JP-A-60-4851.
A source follower type measurement circuit described in eg No. 0-225056 is used.

Next, regarding the enzyme immunoassay method using the cartridge of the present invention, a two-step German assay will be described. In the following explanation, an antibody (or antigen) for capturing the antigen (or antibody) to be measured is immobilized on the solid phase thin tube. Furthermore, a labeled antibody (or labeled antigen) for sandwiching the substance to be measured is used as the freeze-dried label. The user takes out the cartridge for the desired measurement item, peels off the seal piece (10) affixed to the top of the cartridge, and inserts the sample cup (2).
When about 20 μQ of sample liquid is placed in the cartridge and set on the cartridge supply conveyor mechanism (31), the first
The measurement circuit shown in FIG. 0 automatically performs all of the following operations.

■When the cartridge (L) is supplied to the operation station (A) by the above conveyor, the barcode reader (3
2) reads the measurement items etc. from the barcode (9) and selects the operating program and calibration curve accordingly.

■Next, when the cartridge is sent to the dilution station (B), the elevating means is activated and the pipette head (28) attaches the thin dilution tube (7) in the dilution cup (3) and moves it to the sample cup (2). Aspirate a predetermined amount (for example, 10μ) of the sample solution in the sample solution, immediately move it to the diluent cell (36), and aspirate an additional predetermined amount of diluent (for example, 90μ).Then, the pipetter head returns to the dilution cup (3), A mixed solution of the sample and diluent (e.g., 100μ0) is discharged into the sample, and the solution is then repeatedly re-suctioned and re-discharged to mix the sample and diluent, thereby diluting the sample to a predetermined ratio (e.g., 10 times). Prepare the sample solution.The small diameter tube for dilution is the container (37
) will be discarded.

■Next, the pipetter head (28) attaches the solid phase thin tube (8) in the reaction cup (4), and connects it to the dilution cup (3).
A predetermined M (for example, lOμQ)
) After aspirating the diluted sample solution, the thin tube (8) is removed from the dilution cup (3) and allowed to stand still, and in this state the next immune reaction is carried out for a predetermined period of time (for example, 10 minutes).

(2) After completing the next immune reaction, move the cartridge to the washing station (B). Then, at this station, the pipettor head reinstalls the small-diameter tube for the solid phase, moves it to the diluent cell (36), and aspirates the diluent overflowing the groove provided on the top of the cell several times. Discharge to clean the small diameter tube.

Then, aspirate a predetermined amount (for example, 20μQ) of the diluted liquid,
The pipettor head is moved to the reaction cup (4) of the cartridge and the aspirated diluent is expelled into it to dissolve the lyophilized label (6). In order to dissolve the label more completely, the dissolved label solution in the reaction cup is aspirated and poured out several times to completely dissolve it, and then the solid phase small diameter tube (8)
is removed from the reaction cup (4) and allowed to stand still in this state for a predetermined period of time (for example, 5 minutes), after which the secondary immune reaction is carried out, and then the cartridge is moved to the measurement station (C).

■The small-diameter solid phase tube (8) that has undergone the second immune reaction is reattached to the pipettor head, moved to the washing liquid cell (35), and overflowed through the groove provided on the top surface of the cell. The small diameter tube is cleaned by aspirating and discharging the liquid several times. After that, the thin tube is inserted into the measurement chamber provided in the measurement cell (33), and after suctioning the urea substrate solution flowing through the measurement cell, it is placed over the pH-FET, and the thin tube and pH- FE
Measure p) [changes in the substrate solution encapsulated during T.

During pH change measurement, when the temperature of the tube (26) changes, the volume of air inside the tube changes, which causes pH-F
This may induce flow of the substrate solution sealed in the gap formed by the ET and the small diameter tube, leading to measurement errors. To do this, open the tube on-off valve (27) during pH change measurement.
Preferably, the pressure within the tube is released to atmospheric pressure.

The pH change is read as a change in the source potential of the pH-FET. Then, the rate of change in the source potential during a period of several tens of seconds after the small-diameter tube is placed over the pH-FET is calculated, and the rate of change is calculated using a conversion formula calculated in advance for the amount of the substance to be measured. Converted to concentration and displayed.

The cartridge of the present invention, a measuring method using the same, and an apparatus can be used for enzyme immunoassay using the Sand-Deutsch method and the competitive method. Furthermore, although the present invention describes the so-called 2-step Sander-German search method and the 2-step competitive method, it is also possible in principle to apply to the I-step Sanderutsch method and the 1-step competitive method. These 1. It is possible to use a step method. When employing these I-step methods, it is necessary to set assay conditions that prevent the so-called prozone phenomenon, in which the detection output decreases when the concentration of the substance to be measured becomes extremely high.

(Effects of the Invention) As described above, the Cartlitz for enzyme immunoassay of the present invention, the measurement method and apparatus using the same, (1) can measure any measurement item in any order; In addition to the following features: (2) it can be fully automated with simple hardware without any loss of reagents even with a small number of samples, and (3) it has the shape of a pipette tip. Because immunoreactions and enzymatic reactions are carried out in a minute area called the narrow end of a narrow tube,
Each reaction is extremely rapid, the reaction time is short, and the amount of sample liquid used is small. Although this is called body/educt separation, in the present invention, this is carried out by a dispensing means, so there is no need for a special drive unit for cleaning.

It has this excellent effect.

Experimental Example 1 The cartridge for enzyme immunoassay of the present invention was applied to the measuring device shown in FIG.
P) was measured. In this case, anti-A
The FP antibody was immobilized, and a lyophilized urease-labeled anti-AFP antibody was stored at the bottom of the reaction cup.

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

Diluent: 0.0915M disodium hydrogen phosphate +0
, ON5M sodium phosphate (pH 7,8) washing
: 0.1M ammonium chloride + 0.154M sodium chloride substrate solution: 0.1M ammonium chloride + 0.154M sodium chloride + 0.1M urea After taking out the cartridge for AFP measurement and opening the seal piece, put 20 μg into the sample cup. A serum sample was added. Next, the cartridge was attached to a measuring device, and measurements were performed in the following order.

a) The dispenser attaches the thin dilution tube housed in the dilution cup, sucks up 10 u (l) of the sample in the sample cup and 90 μe of the diluent in the diluent cell, and dilutes this. A sample solution diluted 10 times was prepared by discharging it into a cup.

The dilution tube was then discarded into a waste bottle.

b) The first immune reaction was started by attaching the solid phase thin tube housed in the reaction cup using the dispensing means and removing it into the 10-fold diluted sample solution. . After that, in this state, the small diameter tube inside the dilution cup was heated to 1
Incubated for 0 minutes. Next, the dispensing means reattached the small-diameter tube, moved it to the diluent cell, and sucked and discharged the diluent to perform cleaning.

C) With the dispensing means attached to the small-diameter tube for solid phase, 30 μe of the diluted liquid is sucked, and it is discharged into the reaction cup, and by repeating the suction and discharge, the lyophilized label at the bottom of the cup is removed. was dissolved. A second immune reaction was then initiated by withdrawing the squeeze tip into the cup. In this state, the chip in the cup was placed at 37° C. for 5 minutes.

d) The dispensing means reinstalled the small diameter tube for solid phase, moved the small diameter tube to the washing liquid cell, cleaned the small diameter tube by sucking and discharging the washing liquid there, and discharged the washing liquid. After that, while inserting the small diameter tube into the pH measuring cell, add the substrate solution (l
ammonium chloride solution) was aspirated into a small diameter tube. Next, the thin tube was placed in the pH measurement cell, and the tip of the thin tube was placed over the pH-FET.

e) Set the output (source potential) of pH-FET to 2
Read for 0 seconds, 10 for 10 to 20 seconds after starting measurement
The average value of the rate of change in output per second was determined.

f) Substrate solution was pumped into the pH measurement cell for 10 seconds to replace the substrate solution in the cell with fresh one, in preparation for the next measurement.

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

AFPlkK pH-PET Output change rate 3.9 7.9 15.6 31.3 62.5 L25. Q 50 0 500.0 1000.0 0.024 0.080 0.149 0.245 0.445 0 , 768 1.404 2.568 4.065 6.676 Experimental Example 2 Ferritin was determined in the same manner as Experimental Example I. Measurements were made. In this case, an anti-ferritin antibody was immobilized on the solid phase narrow tube, and a lyophilized urease-labeled anti-ferritin antibody was stored at the bottom of the reaction cup.

Table 2 shows the results of measurements performed using the same operating procedure as in Experimental Example 1.

Output change rate 3.9 9 15.6 31.3 62.5 125.0 250.0 500.0 0.021 0.031 o, iog 175 0.488 0.879 1.737 3.331 Experimental example 3 Carcinoembryonic antigen (CEA) was measured in the same manner as in Experimental Example 1. In this case, an anti-CEA antibody was immobilized on the solid phase narrow tube, a lyophilized product of the urease-labeled anti-CEA antibody was stored in the reaction cup, and measurements were performed in the following order.

a) The dispensing means is equipped with a thin dilution tube housed in a dilution cup, and the serum sample 10 μQ and diluent 2 in the sample cup are
0 μQ was aspirated and discharged into a dilution cup to prepare a 3-fold diluted sample solution. The dilution tube was then discarded.

Table 3 shows the results of the same operations as b) to e) of Experimental Example 1.

Below margin output change rate 0.0 0.0071.25
0.12g2.5
0.2555.0 0.51
010.0 0.99520,Q
1.91640.0
3 430go, o 5
．． 918160.0 9 375

[Brief explanation of drawings]

FIG. 1 is a perspective view of a cartridge for enzyme immunoassay of the present invention, and FIG. 2 is a sectional view of the cartridge. Third
The figure 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 the basic configuration of the device of the present invention, and FIG. 6 is a detailed structure of the measuring cell. 7 is a sectional view showing the structure of the chip coverr, FIG. 8 is a view taken along the line A-A in FIG. 7, and FIG. 9 is a front view showing the elevating means; FIG. 10 is a control circuit diagram of the device of the present invention. A...Operating station B...Dilution station C...Pre-cleaning station D...Measurement station E...Dispensing means, F...Elevating means G...Reciprocating means, H...・Detachment means I・
... Cartridge, 2... Sample cup 3... Dilution cup, 4... Reaction cup 6... Lyophilized label, 7... Thin tube for dilution, 8... Thin tube for solid phase. Diameter pipe 10...
・Seal piece, 33...Measuring cell 35...Cleaning cell,
36... Dilution cell 65... pH electrode.

Claims (1)

[Claims] 1. For enzyme immunoassay, in which a sample cup for accommodating a sample to be measured, a dilution cup accommodating a thin tube for dilution in the shape of a pipette tip, and a reaction cup are integrally arranged. A cartridge containing a freeze-dried enzyme-labeled reagent at the bottom of the reaction cup, and at least an antibody (or antigen) that binds to the antigen (or antibody) that is the substance to be measured is immobilized on the inner wall of the tip. A small-diameter pipette tip-shaped solid phase tube was housed in the reaction cup with its tip separated from the enzyme labeling reagent, and the upper end opening of each cup was airtightly closed with a seal piece. Characteristic enzyme immunoassay cartridge. 2. An enzyme immunoassay method using the enzyme immunoassay cartridge according to claim 1, which comprises: (1) a pipette tip-shaped thin dilution tube housed in the dilution cup; A step of injecting a liquid into a dilution cup and preparing a sample liquid diluted to a predetermined ratio in the dilution cup. (2) A step of performing a primary immune reaction by immersing the tip of a pipette tip-shaped small-diameter solid phase tube housed in a reaction cup into a sample liquid in a dilution cup. (3) After injecting the dissolution solution into the reaction cup and dissolving the freeze-dried enzyme labeling reagent in the reaction cup, solids that have undergone the primary immune reaction are added to the dissolved enzyme label solution. A step of immersing the tip of a small-diameter phase tube to perform a secondary immune reaction. (4) A step of washing the small-diameter tube for solid phase after the completion of the secondary immune reaction. (5) Place the cleaned solid phase thin tube over the sensitive part of the pH electrode installed in the measurement cell filled with the substrate solution, and the substrate solution between the thin tube and the sensitive part of the pH electrode is Step of measuring pH change accompanying decomposition reaction. An enzyme immunoassay method consisting of 3. An enzyme immunoassay device using the enzyme immunoassay cartridge according to claim 1, comprising means for supplying the enzyme immunoassay cartridge to an operating station, and a diluent inlet and overflow provided at the operating station. A dilution station having a diluent cell having a flow outlet, a dilution station having a pump for supplying diluent to the diluent cell, a cleaning fluid cell having a cleaning fluid inlet and an overflow fluid outlet, and a pump for supplying cleaning fluid to the cleaning fluid cell. a measuring cell having a substrate solution inlet and an overflow liquid outlet and exposing the sensitive part of the pH electrode to the substrate solution flow path, and a pump for supplying the substrate solution to the measuring cell. A measuring station equipped with the same, and a pipette inserted into the upper end opening of the pipette tip-shaped dilution tube and the solid phase thin tube housed in the dilution cup and reaction cup of the cartridge supplied to the operation station. a dispensing means having a head at one end and a syringe connected to the other end via a tube; means for raising and lowering the pipette head; means for reciprocating the raising and lowering means in a horizontal direction; An enzyme immunoassay device comprising a means for pushing down the upper end of the inserted thin tube to remove the thin tube.