JPH0480657A - Immune measurement method and apparatus - Google Patents

Abstract

PURPOSE:To achieve quickness, handiness and higher sensitivity of a measurement method by solidifying an antigen/antibody reactant on a solid phase through a connecting medium. CONSTITUTION:Connecting medium 7 performs a function of connecting antigen/ antibody reactants in a liquid phase to a solid phase 6 and functions as a carrier of a plurality of specific reactive substances 4 and 5. By using the medium 7, all reactions are accomplished within the same container. The medium 7 herein used is preferably a substance which has two or more of one selected from the reactive substances 4 and 5 paired and is soluble in water. The reactive substance paired 4 and 5 employs combinations of (1) biotin and avidin, (2) antigen, an antibody thereof and the like. Then, a reagent and a coexisting substance yet to be reacted are washed to separate the reactive substance 5 from the solid phase 6 and composites 1-5 and 7 containing reactants 1-4 are separated. The reactants 1-4 isolated are shifted to a measuring device to inspect a concentration of a labeled substance 3 thereof thereby enabling measurement of the concentration of an antigen (antibody) 1 as substance to be measured.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to an immunoassay method suitable for clinical testing and biological sample measurement.

[Conventional technology]

Conventionally, as an immunoassay method in which the concentration of a labeled substance is measured by binding it to a solid phase after a liquid phase reaction and releasing the antigen-antibody reaction product into the liquid phase after B/F separation, the method described in Methods in Engineering, Vol. Volume 92 (1980), pages 345 to 3
Page 59 (Methods in Enzymol, 9
2 (1980) 345-359), an antibody is bound to a solid phase via an -S-8- (disulfide) bond, and the S-3 bond is cleaved with a reducing agent to generate an antigen-antibody reaction. Methods of liberating substances are known. In this method, antibodies are immobilized on a column packed with a carrier having SH groups, and antigen-antibody reaction products are captured on this column.

Furthermore, even if nonspecific adsorption of labeled antibodies occurs, ~5-
Since the 8-binding portion can separate specific reaction product complexes, it is possible to selectively measure down to low concentrations. Also, as described in JP-A-1-254868, is there any known method in which the antigen-antibody complex is directly captured on a carrier, dissociated after washing, and recaptured on another carrier? It is necessary to invent solid phase transition and the associated equipment.

[Problem to be solved by the invention]

The above conventional technology uses the reaction between the antibody on the solid phase and the reaction product to immobilize the antigen-antibody reaction product on the solid phase, so if a series of reactions are performed in the same reaction vessel, The antibody on the solid phase captures the labeled antibody or labeled antigen, and unless the reaction is allowed to proceed for a long time, the immobilization efficiency of the antigen-antibody reaction product will not increase. Furthermore, even if the reaction product is generated in the liquid phase, the rate of diffusion into the solid phase influences the immobilization efficiency. Therefore, in order to improve efficiency, it is necessary to perform the antigen-antibody reaction in the liquid phase and the immobilization on the solid phase in separate reaction vessels. There were problems such as deterioration.

Further, the method of transferring between two solid phases as disclosed in JP-A-1-254868 is complicated and difficult to implement, and therefore cannot achieve the speed and simplicity that are the objectives of the present invention.

The present invention aims to speed up and simplify such an immunoassay method as well as increase sensitivity.

[Means to solve the problem]

In order to achieve the above-mentioned object, the present invention combines one specific reactive substance of a linkage mediating substance that grows a pair of specific reactive substances and the corresponding specific reactive substance on a solid phase in immunoassay. and the binding of the other specifically reactive substance of the linking mediator to the corresponding antibody of the antigen-antibody reaction product or the specific reactive substance on the antigen, thereby linking the antigen-antibody reaction product to the mediating substance. The present invention aims to develop and provide a method for immobilization on a solid phase via .

Furthermore, the immunoassay method of the present invention includes a first step of producing an antigen-antibody reaction product in a liquid phase, and adding the linkage mediating substance into the liquid phase to connect the antigen-antibody reaction product in the liquid phase to the linker. The immunoassay method consists of a second step of immobilizing onto a solid phase via a mediator, and a third step of measuring the antigen-antibody reaction product.

By using a linkage mediating substance in this way, the antigen-antibody reaction and the immobilization reaction can be performed in stages, making it possible to perform all reactions in the same container, so there is no need to exchange reaction containers as in conventional methods. It's no longer needed.

As the linkage mediating substance, it is preferable to use a substance that has two or more types of one of the pair of specific reactants and is soluble in water.

As the specific reactive substance pair, combinations such as (i) biotin and avidin or streptavidin, (ii) an antigen and its antibody, (ii) protein A or protein G and immunoglobulin G are used.

In addition to columns, various containers and carriers such as microtiter plates, test tubes, and petri dishes can be used as reaction containers.

In addition, in order to perform rapid measurements, an antibody or antigen conjugated with a label for use in detecting the reaction product and an antibody or antigen conjugated with a specific reactive substance capable of binding to a linkage mediator are simultaneously administered. Alternatively, a method is adopted in which the reaction is carried out continuously in a liquid phase.

Furthermore, after immobilizing the reaction product with a linkage mediator, the complex containing the reaction product is detached from the solid phase.
The reaction product can be specifically detected, and a decrease in sensitivity due to adsorption of antigen or antibody bound to a label can be prevented. As the desorption step, a method was adopted in which the linkage mediating substance was divided, the specifically reactive substance was desorbed from the solid phase, or the antibody or antigen on the solid phase bound to the substance to be measured was desorbed.

By incorporating one of a pair of two or more specific reactive substances into the linkage mediator, it has become possible to selectively bind the antigen-antibody reaction product and the solid phase. As a result, the antigen-antibody reaction can be carried out in the liquid phase and the reaction product can be immobilized on the solid phase quickly, and there is no need to transfer the reaction container.

Furthermore, by providing a plurality of different types of solid phases as the solid phase in the same reaction vessel, it is possible to simultaneously measure a plurality of different substances to be measured.

Furthermore, it becomes possible to use the same solid phase and linkage mediating substance regardless of the type of substance to be measured.

Furthermore, it is possible to detect a plurality of substances to be measured by appropriately selecting the type of antibody, the type of label, and the specific reactive substance on the linkage mediating substance.

The present invention also develops and provides a measuring device for detecting and measuring enzymes, fluorescent substances, luminescent substances, etc., as labeling substances for antibodies or antigens. The device consists of a container that functions as a solid phase, and a plurality of dispensing nozzles that can sequentially supply various substances necessary for performing an antigen-antibody reaction into the container. are designed to move relative to each other. Furthermore, in the device it is also possible to form a plurality of different solid phases within the container.

An overview of the present invention will be explained based on the accompanying drawings.

FIG. 1 is a diagram explaining the principle of the present invention.
Figure a) shows a state in which the analyte 1 in the sample forms a reaction product with the antibody (or antigen) 2 attached with the labeling substance 3 or the specific reactive substance 4 on the solid phase. In Figure 1 b), the linkage mediating substance 7 is reacted following the state in Figure 1 a),
This shows a state in which the reaction product is immobilized on the solid phase. Figure 1 C)
1b) shows a state in which unreacted reagents and coexisting substances are washed after the immobilization shown in FIG. 1b) is completed, and the immobilized reaction product is further cut and released. By transferring the released reaction product to a measuring device and assaying the concentration of the labeling substance 3,
The concentration of antigen (antibody) 1, which is the substance to be measured, can be measured.

[For production]

The linkage mediating substance functions to link the antigen-antibody reaction product in the liquid phase and the solid phase, and functions as a carrier for a plurality of specific reactive substances. If the linkage mediator is soluble in water, the reaction will be even more rapid.

The specific reactive substance pair is a combination of substances with high selectivity and strong binding, and is a substance that does not interfere with the label or antigen-antibody reaction, so that the reaction product can be immobilized quickly.

The method of desorbing the antigen-antibody reaction product after immobilization with a linkage mediator can be performed without interaction with the binding of the labeled antibody to the solid phase by adsorption, increasing selectivity and expanding the range of application of subsequent measurement methods. be able to.

If the reaction container can also serve as a solid phase, measurement time can be shortened and operations can be simplified.

This purpose can be achieved by the invention of a method that allows immobilization without replacing the reaction container as in the present invention.

〔Example〕

Hereinafter, the present invention will be explained in detail with reference to Examples.

However, the present invention is not limited in any way by these Examples.

(Example 1) An example in which the present invention is applied to α-fetoprotein in serum will be described. First, 1 ml of a 1% solution of γ-aminopropyltriethoxysilane was added to a glass test tube, and the mixture was left at room temperature for 1 hour. After 1 hour, the test tube was washed with water and heated at 110"C for 1 hour to aminosilanize the glass surface. 1 ml (containing 6 mg) of a dimethylformamide solution of S-acetylmercaptosuccinic anhydride was added to the test tube. The aminosilane was then left at room temperature for 30 minutes to introduce an SH group into the aminosilane.Next, the crosslinking reagent N-succinimidyl-3-(2-pyridyldithio)-propionic acid (
After adding an ethanol solution (30 mM) of SPDP in an amount equivalent to 0.6% of the weight of the antibody and reacting for 30 minutes,
The 5PDP-bound antibody was separated from unreacted reagent using a Sephadex G-25 column. This antibody solution was added at a concentration of 0.2 mg/ml to form a -8-8 bond with the -SH group introduced at the bottom of the glass test tube, thereby preparing an anti-avidin antibody immobilized test tube. On the other hand, the anti-α-fetoprotein antibody was divided into two parts, and one part contained S, Avrame.
As and T, Ternyck; Immuno
chemistry, 8 (1971) p, 11
β-galactosidase (β-gal) was conjugated using gel taraldehyde in the method of 75, and biotinyl-N-hydroxysuccinimide ester (BNH
The dimethylformamide solution of 8) was reacted in a carbonate buffer of pH 8.5 at an antibody:biotin ratio of 10:l. A POD-labeled antibody and a biotinylated antibody were prepared by the above operations.

Immunoassay was performed using the reagents etc. prepared in this manner. 50 μl of human serum was added to the anti-avidin antibody immobilized test tube, and then 50 μl each of β-gal labeled anti-α-fetoprotein antibody solution and biotinylated anti-α-fetoprotein antibody solution were added and mixed well. After leaving it at room temperature for 1 hour, 100 μl of avidin solution was added, and the mixture was allowed to react at room temperature for 1 hour. Thereafter, the glass test tube was rinsed with a phosphate buffer (pH 7.0, sodium chloride O, 1 mol/l) to remove unreacted reagent coexisting substances. Next, add 300μI of dithiothreitol 10mM (pH 8) solution and
The light was on for 0 minutes.

50 μl of the reaction product solution released from the solid phase by reduction was placed in the well of a microtiter plate, and 0, OIM
Phosphate buffer (0.1M NaCl, 1mM MgCl
2, containing 0.1% bovine serum albumin) and diluted 1:1 with 50mM 2-nitrophenyl-β-, which is the substrate solution.
Add 50 μl of D-galactoside phosphate buffer solution and
React at 7°C for 130 min, then 0.5 M Na2C
The reaction was stopped by adding 150 μm of O3, and the absorbance at 420 nm was measured. A calibration curve for this example using a standard solution of α-fetoprotein is shown in FIG. In this example, avidin was used as a linkage mediator, and the antigen-antibody reaction product in the liquid phase was immobilized using its biotin and its specific binding reactivity with anti-avidin antibodies. Since it is an -8-8- bond, it was shown that elimination using a reducing agent is possible. By performing selective desorption by cleavage of the -8-8- bond as in this example, it was possible to prevent an increase in the blank value due to adsorption of the labeled antibody to the solid phase. Furthermore, the antigen-antibody reaction product could be immobilized in the same glass test tube, and there was no need to change the reaction container.

(Example 2) The present invention was applied to the measurement of human chorionic gonadotropin (hCG) in serum. First, a chimeric antibody F(ab')2 fragment capable of reacting with two antigens was prepared. Anti-sushi serum 1gG
Antibody F (ab')2 fragment (immune prepared from rabbit)
and anti-DNP (dinitrophenyl) antibody F (ab'
) 2 fragments (immune prepared from rabbits) were prepared, a solution of equimolar concentration was prepared, and 0.2-mercaptoethanol was added to this solution.
Add 1M solution to reduce F(ab')2 fragments,
-3H group was generated. Put the reaction solution into a dialysis tube,
It was immersed in a phosphate buffer solution without 2-mercaptoethanol. When the two types of F(ab')2 fragments are left to stand after reduction, they are oxidized again, forming a -5-S= bond, or cross-recombining at this time, resulting in F(ab')2 fragments having anti-bovine IgG activity.
A chimeric antibody is generated between the ab' fragment and the Fab' fragment with anti-DNP activity. Next, DNP-bound albumin and bovine IgG were added to CNBr-activated 5epharos, respectively.
e 4B to prepare an affinity column. First, F (ab'
)2 recombination solution to F (ab'), which has anti-DNP activity.
) The two fragments were ligated. Next, anti-DNP activity F (ab'
) 2 fragments were eluted from the column with pH 2, 0, 0, 1M glycine-hydrochloric acid buffer, pH returned to neutral, dialyzed, concentrated and now injected onto a bovine IgG binding column. pH again
F(ab')2 fragments with anti-bovine IgG activity were eluted from the column with 2,0,0,1M glycine-hydrochloric acid buffer. After neutralizing this, it was dialyzed and concentrated, and chimeric antibody F (ab') which has both anti-DNP activity and anti-bovine IgG activity
) Two fragments were prepared.

Next, the bottom of the glass test tube was subjected to aminosilanization in the same manner as in Example 1, and bovine IgG was immobilized there. Also DN
A DNP-conjugated anti-hCG antibody was prepared by binding dinitrobenzene sulfonic acid, which is a type of P compound, to an hCG antibody.

hCG was measured using these reagents.

Dispense the serum sample into a glass test tube bound with bovine IgG, add β-D-galactosidase (βgal)-labeled anti-hCG antibody solution and DNP-conjugated anti-hCG antibody solution,
The reaction was carried out at 7°C for 2 hours. A chimeric antibody F(ab')2 fragment solution having anti-DNP activity and anti-bovine IgG activity was added to this test tube, and the mixture was reacted at 37°C for 2 hours with stirring. After the reaction, the test tube was washed five times with phosphate buffered saline at pH 7.2. Next, a 0.1M solution of dithiothreitol was added, and the mixture was left stirring for 30 minutes. After 30 minutes, a neutral solution of 0.1 M cystine was added to block dithiothreitol, and then the solution in the test tube was transferred to a measuring test tube. β-gal is added to the solution containing the desorbed antigen-antibody reaction product.
A 1 mM solution of the substrate 4-methylumbelliferyl β-D-galactoside is added to start the reaction. 3 at 30°C
After 0 minutes of reaction, 0.5M K containing 10mM EDTA
The reaction was stopped by adding 1O times the amount of H2PO4-NaOH buffer pH 10.5.

The fluorescence intensity of this solution was measured at an excitation wavelength of 360 nm and a fluorescence wavelength of 4.
Measured at 500m. hCGl, O using serum standard solution
A calibration curve could be obtained in the range from IU/ml to 2004 U/ml.

(Example 3) The present invention was applied to the measurement of β2-microglobulin (β2m) in urine. The surface of glass beads (diameter 6 mm) was subjected to aminosilanization in the same manner as in Example 1 using a γ-aminopropyltriethoxysilane solution. Next 0.
Glass beads were placed in a 1% gel taraldehyde solution and reacted for 1 hour at room temperature. After 1 hour, the glass beads were rinsed twice with ion-exchanged water, and then added to a protein A solution (containing 0.5 mg/ml,
Protein A-immobilized glass beads were prepared by contacting with 50 mM, pH 7, dissolved in diphosphate buffer). Unreacted aldehyde groups were blocked with 0.1% monoethanolamine solution. Next, prepare an anti-β2-m antibody obtained from a rabbit, put it into a beaker containing pepsin-immobilized Sepharose, and digest it.
b') 2 fragments and Sephadex G-150
It was purified by gel filtration using a column. This anti-β2-m
0, in which part of the antibody F (ab')2 fragment was replaced with nitrogen,
Diluted with 5 M TrisHC1 buffer pH 7,8,
2-Mercaptoethanol was added to this to a concentration of 10 mM, and a reduction reaction was performed at 37°C for 1 hour.
And so. Horseradish peroxidase (POD) reacted with a crosslinking agent m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) was bound to the -SH group of this Fab' fragment. MBS processing PO
D is MBS dissolved in dimethylformamide and pH 7
, 0 for 1 hour at room temperature. F ab'
The binding between POD and POD was carried out in 100mM phosphate buffer (EDTA containing 5mM gold) at pH 6.0, and unreacted Fab' and reagents were removed with a Sephadex G-150 column. An IgG antibody (one that selectively binds to the F(ab')2 portion of rabbit IgG) was prepared.

Measurements were performed using the above reagents. One protein A-conjugated glass bead was placed in a test tube, 500 μm of the diluted urine sample was added to it, and then rabbit-derived anti-β2-m Fab was added to the test tube.
'Add 100 μl of antibody solution (containing 0.1 mg/ml),
A mixed reaction was carried out at 30°C for 30 minutes. After 30 minutes, mouse anti-heron IgG antibody (F (ab') 2
specificity) solution (containing 0.1 mg/ml IgG) 20
0 μl was added and reacted at 37°C for 130 minutes. Mouse IgG antibody is rabbit anti-β=-m F (ab'
)! It was bound to the antibody and immobilized on the beads by protein A on the beads. Remove the solution inside this reaction test tube and add phosphate buffer (100mM, pH 7.0)
Washed 3 times with Next, add 5 m of non-specific mouse IgG.
500 μl of the solution was added at a concentration of g/ml, and the mixture was stirred at 37°C for 1 hour.

After 1 hour, the solution inside the test tube was sucked up and transferred to another test tube. Remove 100 μl of the solution from this test tube and
Add 1ml of 0mM phosphate buffer (pH 7.0), add 300μl of 20mM 3-(4-hydroxyphenyl)propionic acid and 200μl of 8mM hydrogen peroxide solution, react for 10 minutes, and after 10 minutes add 0.1M glycine. NaO
The reaction was stopped by adding 2 ml of H buffer (pH 10,3). The fluorescence intensity of this solution was measured at an excitation wavelength of 320 nm.
The concentration of β and -m was measured using a fluorescence wavelength of 405 nm.

When a standard solution was prepared and subjected to measurement, β22-m3n
As shown in FIG. 3, a calibration curve showing linearity between 200 ng/ml and 300 ng/ml was obtained. Actual samples could also be measured, and the concentration of βz-m could be determined from the calibration curve.

(Example 4) An immunization device using a linkage mediator was produced. Figure 4 shows a schematic diagram of the configuration. Test tubes containing biological samples such as serum or urine are placed in the sample tray 40, and reaction test tubes are placed in the test tube rack 41. As shown in the above example, the reaction test tube functions as a solid phase. In this embodiment, both the test tube rack 41 and the dispensing nozzle 42 are mounted so as to be able to slide on the moving rails L and L', and by moving either or both of them on the rails, the following can be achieved. The process can be performed continuously.

Using the dispensing nozzle 42, a certain amount of sample (
50-100 μl). The rack may be equipped with a temperature-regulating heater (not shown) to set the temperature appropriate for the reaction, or depending on the test environment, such heating equipment may not be necessary. Next, using the same dispensing nozzle 42, a solution A of labeled antibodies or antibodies bound to specific reactive substances is injected into the reaction test tubes on the test tube rack 41 to initiate an antigen-antibody reaction. In order to speed up the diffusion, rack 4 is used, although not particularly shown.
It is also possible to provide a means such as a magnetic stirrer in the test tube 1 to stir the solution in each test tube.

After the antigen-antibody reaction is completed, the test tube rack 41 is moved to the right in FIG. 4 on the rail L'. Another dispensing nozzle 43 is located to the right of the dispensing nozzle 42, and the dispensing nozzle 43
The ligation mediator B of the present invention is added to the test tube. The reaction product is immobilized on the solid phase in the test tube by the ligation mediator.

The test tube rack 41 is further moved to the right and positioned under the next dispensing nozzle 44. A cleaning solution C such as a Tris-HCI buffer containing a surfactant (such as Tween 20) and bovine serum albumin (BSA) is added through the dispensing nozzle 44 to remove unreacted reagents.

After cleaning, the test tube rack 41 is moved to the fluorescence measuring section 45. A desorption agent is dispensed into the test tubes in the rack 41 by an appropriate means. After the desorption reaction, the desorbed liquid inside the test tube is transferred to a measurement test tube (not shown) in the measurement section.

In this example, since an enzyme label is used, enzyme substrate E is added to the measurement test tube by an appropriate means. After a certain period of time from the start of the reaction, stop solution F is added to stop the reaction,
A suitable measuring means, for example, a quartz optical fiber type fluorescence detection probe (a detector incorporating two excitation light input fibers and a fluorescence reception fiber) is introduced into the liquid in the test tube, and the fluorescence intensity is measured.

The operation of this device is performed using an appropriate control mechanism such as the rack 41,
It is also possible to automatically move the dispensing nozzle 40, add various test substances, etc. In the figure, 46 indicates a control panel for such operations, and 47 indicates a display window for displaying necessary data.

For measurement, the same operation is performed separately on a standard solution, a concentration curve is created from the fluorescence intensity, and the antigen or antibody in the sample is measured. In the case where Example 1 was applied to this measuring device, α-fetoprotein was measured, and a phosphate buffer containing dithiothreitol was used as the desorption solution. Since β-galactosidase was used as the enzyme label, a 1 mM solution of 4-methylumbelliferyl β-D-galactoside was used as the substrate, and the resulting fluorescent product was measured for fluorescence at an excitation wavelength of 360 nm and a measurement wavelength of 450 nm. . FIG. 5 shows a calibration curve for α-fetoprotein measured by this measuring device.

(Example 5) A method and apparatus for immobilizing two types of antigens on the surface of the same reaction container and measuring their respective concentrations will be described.

FIG. 6 shows a schematic diagram of the reaction inside the reaction vessel.

α-fetoprotein (AFP) 15 and carcinoembryonic antigen (
A method and apparatus for detecting CEA) 21 will be described. FIG. 5 shows a schematic cross-sectional view of the test tubes 8 in the test tube rack 23 set in the measuring device shown in FIG. 6. The test tube 8 is made of plastic (such as polycarbonate or polystyrene) or glass, and a sample and a reaction solution 9 are injected into the test tube 8 . Two porous glass rings 11, 12 were placed inside the test tube 8 and fixed with a sleeve 1O. 11.12 of this glass ring
Anti-avidin antibody 16 and bovine IgG are pre-coated on the inner surface of the
18 were immobilized, respectively. The ring was placed at the bottom of the test tube to reduce the amount of reaction liquid.

The measurements were carried out as follows. This will be explained using FIGS. 6 and 7. In FIG. 7, the measuring device 30 has a test tube rack 23 in which test tubes 8 are stored.

A long hole is opened parallel to the test tube rack 23, and the liquid nozzle arm 24 is provided so that it can move and rotate along the opening.

A plurality of nozzles 25 are formed at the tip of the liquid nozzle arm 24 for supplying liquid to the test tube. Further, a plurality of reagent bottles 26 are located at contrasting positions across the test tube rack 23 and the long hole. Further, a metered liquid inlet 28 is provided at a position within the movement range of the liquid nozzle arm 24. This device is also designed to be controllable by conventionally known control means, and has an operation panel 29 and a display window 27 formed on the front side of the device.

In the measurement, a sample was first injected into the test tube 8 of the test tube rack 23, and two types of antibody solutions were injected into the test tube 8 using the liquid nozzle 25. After 30 minutes, the connection mediator solution was injected from the liquid nozzle 25. After one hour, the cleaning liquid is sucked up from the reagent bottle 26, cleaning is performed, and the liquid nozzle 2 is again
It was discharged at 5. As the antibody against AFPI5, a mixture of alkaline phosphatase-conjugated anti-AFP antibody 14 and biotinylated anti-AFP antibody 13 was used, and as the antibody against CEA21, β-D-galactosidase-conjugated anti-CEA was used.
Antibody F(ab')2 fragment 22, DNP-conjugated antibody, and αF
(ab')! A mixed solution with Fragment 19 was used. As shown in FIG. 6, AFP15 binds to two types of antibodies in the liquid phase, and is immobilized on the ring 11 by adding the linkage mediator avidin 17. Similarly, CEA21 binds two types of antibodies, and when an antibody F(ab')z fragment having anti-DNP and anti-DNP IgG activities is added as a linkage mediator, it becomes immobilized on ring 12. . After AFP15 and CEA21 are immobilized, a washing solution is injected to remove unnecessary reagents and components. As in Example 1, an anti-avidin antibody was bound to the inside of the ring 11 via a -5-8- bond. Therefore, both AFP 15 and CEA 21 are -5-
3- Bonded to the glass ring surface via a bond;
When dithiothreitol is added to this test tube, -5-S
-The bond is broken and the antigen-antibody complex is released.

This free liquid was introduced into the reaction container through the measurement liquid introduction part 28 in FIG. 7, and an enzyme reaction was carried out. Substrates that generate fluorescence were used for both alkaline phosphatase and β-D-galactosidase. For alkaline phosphatase, a substrate solution containing 0.5mM of 4-methylumbelliferyl phosphate was used, and for β-D-galactosidase, a substrate solution containing 0.5mM of 4-methylumbelliferyl-D-galactoside was used. The products were measured at the same fluorescence wavelength of 450 nm.

AFP.

We have created a calibration curve for CEA and were able to measure the concentrations of both in the same serum. The number of measurement items is not limited to two types, and three or more types of antigens can be measured by providing a large number of solid phase rings. In addition, by placing the rings in separate reaction vessels and measuring enzyme activity,
Even if the same labeling substance is used, it is possible to measure individual components. In addition to enzymes, fluorescent substances, chemiluminescent substances, and bioluminescent substances could also be used as labeling substances.

(Example 6) An example in which a plurality of measurement items were measured using the same solid phase will be described. In the same manner as in Example 1, test tubes with immobilized anti-avidin antibodies were prepared. Prepare two test tubes, add 50 μl of serum to each tube, and then add β-gal labeled anti-AF to each tube.
A mixture of P antibody and biotinylated anti-AFP antibody was added to one tube, and a mixture of β-gal labeled anti-CEA antibody and piotinylated anti-CEA antibody was added to the other tube. After leaving it at room temperature for 1 hour, add 100 μl of avidin solution to the test tube, and add 100 μl of avidin solution to the test tube.
Coupling was allowed to take place at room temperature for an hour. After 1 hour, add phosphate buffer (
0.1M, pH7.0, NaC10.1M) 30
Washed 4 times with C1czl. The anti-avidin antibody immobilized on the test tube was 5PDP-8-3 as shown in Example 1.
- Since it is immobilized in the test tube through a bond, -5-
It can be liberated with S-bond reducing agents. In this example, a 10 mM (pH 8) solution of dithiothreitol was added to a test tube and reduced at room temperature for 50 minutes with stirring.

Antigen-antibody reaction products could be released in both test tubes to which anti-AFP antibody and anti-CEA antibody were added.

Suction up the solution containing free substances with a 100 μm dispensing nozzle,
The mixture was injected into a measuring test tube, and a solution of enzyme substrate 2-nitrophenyl-β-D-galactoside was added thereto to react, and after color development, the absorbance was measured. The reaction conditions were set in the same manner as in Example 1. Perform the same procedure for standard solutions of AFP and CEA to create a calibration curve, and calculate the amount of AFP and CEA in serum.
could be measured using the same anti-avidin antibody-immobilized test tube.

〔Effect of the invention〕

According to the present invention, it is possible to perform everything from antigen-antibody reaction to immobilization of reaction products and separation of coexisting unnecessary substances in one reaction vessel, so that selective reactions that require movement between reaction vessels as in the past can be performed. Compared to conventional methods, measurements can be made quickly and operations are simpler.

Furthermore, since a common pair of specific reactants can be used, the same type of reaction vessel can be used regardless of the object to be measured.

[Brief explanation of drawings]

FIG. 1 is a diagram showing the principle of the present invention, FIG. 2 shows a calibration curve of Example 1, and FIG. 3 shows a calibration curve of βx-m in Example 3. FIG. 4 is a diagram showing the configuration of the immunoassay device of Example 4. FIG. 5 shows a calibration curve for α-fetoprotein using the immunoassay device of Example 4. FIG. 6 shows a schematic reaction diagram of Example 5, and FIG. 7 shows an external appearance of an example of the apparatus of Example 5. 1...Measurement object, 2...Antibody or antigen, 3...
- Labeling substance, 4... Specific reactive substance, 5... Second specific reactive substance, 6... Solid phase, 7... Connection mediating substance.

Claims (1)

[Claims] 1. Binding of one specific reactive substance of a pair of coupling mediating substances having specific reactive substances to the corresponding specific reactive substance on a solid phase, and the bonding of the coupling mediating substance. The antigen-antibody reaction product is immobilized on the solid phase via the linkage mediator by binding the other specific reactive substance to the corresponding antibody of the antigen-antibody reaction product or the specific reactive substance on the antigen. An immunoassay method characterized by: 2. In the immunoassay method according to claim 1, the first step is to generate an antigen-antibody reaction product in a liquid phase, and the step of adding the linkage mediating substance to the liquid phase to generate an antigen-antibody reaction in the liquid phase. An immunoassay method comprising: a second step of immobilizing a substance on a solid phase via the linking mediator; and a third step of measuring the antigen-antibody reaction product. 3. The immunoassay method according to claim 1 or 2, wherein the generation of the antigen-antibody reaction product and its immobilization on a solid phase are performed in the same reaction vessel. 4. The immunoassay method according to claim 2, wherein the third step further includes a step of detaching the antigen-antibody reaction product immobilized on the solid phase. . 5. The immunoassay method according to claim 1, wherein the specific reactive substance possessed by the linkage mediating substance is one specific reactive substance of each of two or more pairs of specific reactive substances. immunoassay method. 6. In the immunoassay method according to claim 1, the specific reactive substance pair is (i) biotin and avidin or streptavidin, (ii) an antigen and its antibody, (iii) protein A or protein G and immunoglobulin G. An immunoassay method characterized by being any of the following. 7. The immunoassay method according to claim 1, wherein the linkage mediating substance is soluble in water. 8. The immunoassay method according to claim 1, wherein a plurality of different types of solid phases are present in the same reaction container, so that a plurality of different substances to be measured can be measured simultaneously. immunoassay method. 9. The immunoassay method according to claim 3, wherein the same solid phase and linkage mediating substance are used regardless of the type of substance to be measured. 10. It consists of a container that functions as a solid phase, and a plurality of dispensing nozzles that can sequentially supply various substances necessary for performing the antigen-antibody reaction and various substances necessary for the desorption process into the container, An immunoassay device, wherein the container and the dispensing nozzle are configured to move relative to each other. 11. The immunoassay device according to claim 10, wherein a plurality of different types of solid phases are formed within the container.