JPH0322587B2 - - Google Patents

Description

[Detailed description of the invention] The present invention relates to immunoassay methods.

The antigen-antibody reaction is a specific reaction that occurs between a specific antigen and an antibody, and the trace amount of antigen (antibody) in blood or body fluids determined using this reaction is
It is used for diagnosis and treatment. Analytical methods based on this antigen-antibody reaction include radioimmunoassay using radioisotopes, enzyme immunoassay using enzymes, and fluorescent substances. Among the flow immunoassays used, radioimmunoassay is known as a highly sensitive analysis method. In any of the analytical methods, when quantifying the antigen (antibody) to be measured, it is necessary to perform so-called B/f separation between the combined product of the antigen (antibody) and labeled antibody (antigen) and the advantageously labeled antibody (antigen). Then,
For example, as an immunoassay device using radioimmunoassay, B・
A once-through type device that performs f-separation is known. In this device, for example, when quantifying a predetermined antigen in a sample, a reaction chamber containing an immobilized antibody that reacts with the antigen to be measured is used, and the antibody chamber is first connected to the inlet of the reaction chamber (antibody chamber). A sample and an antigen labeled with a radioactive isotope are mixed in a tube as shown in FIG. 1A, and a reaction container containing an immobilized antibody corresponding to the antigen to be measured as shown in FIG. 1B is prepared. Here, as shown in Figure 1C, the antigen in the sample competes with the labeled antigen to bind to the immobilized antibody, and the free antigen that is not captured by the immobilized antibody is
The free labeled antigen is passed through the outlet of the reaction vessel to a scintillation counter and counted by its radioactivity. Next, the bound antigen captured by the immobilized antibody in the reaction vessel is eluted from the immobilized antibody with an elution buffer, and the eluted antigen is passed through the outlet of the reaction vessel to the scintillator as shown in Figure 1D. The labeled antigens are then sent to a radioactivity counter and counted according to their radioactivity. In this way, radioactivity characteristics as shown in Figure 2 were obtained, with both peak values, that is, the peak value of the count of free labeled antigen that was not captured by the immobilized antibody, and the peak value of the counted value of the free labeled antigen that was not captured by the immobilized antibody, and the captured and eluted label. The predetermined antigen in the sample is quantified based on a comparison of the antigen count to the PIC value.

However, in such analyzers, the sample and labeled antigen are mixed in a tube connected to the inlet of the reaction container, which requires a relatively long tube, which tends to cause contamination and mixing. The drawback is that it takes time to send the liquid to the reaction vessel, and the analytical throughput is low. Furthermore, since compounds labeled with radioactive isotopes are usually unstable, it is necessary to repeatedly adjust the labeling substance. Furthermore, radioactive isotopes are dangerous to humans;
It is difficult to dispose of it because of environmental pollution, and its handling requires expensive facilities that are certified under certain qualifications and standards, and the measuring equipment is also expensive.

In view of the above, recently enzyme immunoassays, in which radioactive isotopes are replaced with enzymes for labeling, and flow immunoassays, which use fluorescent substances, have been developed.
It has sensitivity and accuracy comparable to radioimmunoassay, and has been adopted as an excellent analytical method that can be performed safely.

On the other hand, the antigen-antibody reaction step in the radioimmunoassay, enzyme immunoassay, and flow immunoassay requires a relatively long reaction time, and in order to shorten this reaction time, it is necessary to A reaction system for antigen-antibody reaction is required. In order to obtain such a homogeneous reaction system,
For example, in enzyme immunoassays, fine cellulose particles are used as solid carriers to
There is a method in which the interface between the reaction solution, which is the site of antibody reaction, and the surface of the solid phase carrier is increased, and the solid phase carrier particles are evenly suspended. This analysis method is carried out manually. For example, when quantifying α-fetoprotein antigen in a sample, first, as shown in Figure 3A, sample 1 and the cellulose particle surface are immobilized. Reagent 2 containing phased anti-α-fetoprotein antibody is dispensed into reagent tube 3 to perform an antigen-antibody reaction, and α-fetoprotein and anti-α-fetoprotein Combine with protein. Next, as shown in Fig. 3B, washing buffer 4 was added into the test tube 3, centrifuged at 3000 rpm for 30 seconds, the supernatant was removed by suction, and the cellulose carrier particles were transferred to the test tube. Leave it within 3. Thereafter, as shown in FIG. 3C, a reagent 5 containing conjugated anti-α-fetoprotein, which is an enzyme-labeled antibody, is added to the test tube 3 to perform an antigen-antibody reaction. Anti-α-
A fetoprotein-α-fetoprotein-conjugated anti-α-fetoprotein conjugate is formed.
Next, as shown in FIG. 3D, washing buffer 4 was added into the test tube 3, centrifuged at 3000 rpm for 30 seconds, the supernatant was removed by suction, and the cellulose carrier particles were transferred to the test tube. Leave it within 3. By repeating this washing step shown in FIG. 3D three times, the enzyme-labeled antibody that did not react in the antigen-antibody reaction shown in FIG. 3C is washed away and B/f separation is performed. after that,
As shown in FIG. 3E, an enzyme substrate solution 6 is added into the test tube 3 to cause an enzyme-substrate reaction with the labeled enzyme captured on the surface of the cellulose particles, thereby reducing the amount of the captured labeled enzyme. Produces proportional amounts of color-forming substances. After allowing this enzyme-substrate reaction to occur for a predetermined time, the test tube 3 is centrifuged at 3000 rpm for 30 seconds, and the supernatant containing the coloring substance is aspirated and discharged into the colorimetric cell 7 as shown in Figure 3F. Then, the absorbance was measured using a spectrophotometer 8, and based on this measured value, the α-fetoprotein antigen in sample 1 was determined from a calibration curve between the absorbance determined from the sample with a known concentration and the α-fetoprotein antigen. Quantify.

In the above-mentioned enzyme immunoassay, cellulose particles are used, which is the site of the antigen-antibody reaction, compared to assay systems that use beads or balls that are commonly used as solid phase carrier particles. A uniform reaction system is obtained by increasing the interface between the liquid and the solid support and by uniformly suspending the solid support particles. However, in this analysis method, the cellulose particles and their reaction solution are separated after the antigen-antibody reaction between sample 1 and reagent 2, which contains cellulose particles with immobilized antibodies. In order to separate the cellulose particles and their reaction solution after the antigen-antibody reaction with the reagent 5, and after the enzyme-substrate reaction between the cellulose particles and the enzyme substrate solution 6, the reaction solution (containing the coloring substance) is transferred to the colorimetric cell 7. Since centrifugation is required in order to separate and fractionate each, there is a drawback that the operation is troublesome. Furthermore, there is a technical difficulty in that cellulose particles are also aspirated when the supernatant is aspirated after centrifugation. Furthermore, in the above analysis method, it is difficult to remove the cellulose particles remaining in the test tube 3 after transferring the reaction solution containing the coloring substance to the colorimetric cell 7, so the test tube 3 is made of polystyrene. Although the test tube 3 is disposable, if the sample is a body fluid such as serum or plasma, and the reaction solution or other waste is to be sterilized after the measurement, both the reaction solution and the test tube can be used. The disadvantage is that the disposal process is extremely complicated, as it must be disinfected.

The object of the present invention is to eliminate the various drawbacks mentioned above,
The present invention aims to provide an immunoassay method that can easily and quickly quantify antigens or antibodies to be measured, and is especially suitable for automation.

In the immunoassay method of the present invention, an antigen-antibody reaction is performed by mixing a sample with a first reagent containing suspended particles immobilized with an antibody or antigen that specifically reacts with the antigen or antibody to be measured. After the reaction is carried out, the reaction solution is passed through a filter that does not allow the particles to pass through.
a step of separating the particles with a filter; and reacting the separated particles with a second reagent containing a labeling substance bound to an antibody or antigen that specifically reacts with the antigen or antibody to be measured; A step of passing the reaction solution through the filter and separating the particles with the filter, and reacting the separated particles with a third reagent capable of producing a specific substance in the presence of the labeling substance. , passing the reaction solution through the filter and colorimetrically measuring the specific substance contained in the liquid that has passed through the filter to quantify the antigen or antibody to be measured in the sample. This is a characteristic feature.

Furthermore, in the immunoassay method of the present invention, an antigen-antibody reaction is performed by mixing a sample with suspended particles immobilized with an antibody or antigen that specifically reacts with the antigen or antibody to be measured. After that, the reaction solution was
passing the particles through a reaction container having an injection port provided above and a filter placed at the bottom of the injection port that does not allow the particles to pass through, and separating the particles with a filter; and the antigen or antibody to be measured. A step of injecting a labeled enzyme bound to an antibody or antigen that specifically reacts with the labeled enzyme into the reaction container from the injection port to react with the particles, and then separating the particles with a filter; a step of injecting a substrate solution capable of producing a specific substance in its presence into the reaction container from the injection port to react with the labeled enzyme bound to the particles, and measuring a colored substance generated thereby; It is characterized by having.

The present invention will be described in detail below with reference to the drawings.

FIG. 4 is a sectional view showing the structure of an example of a reaction container used in the immunoassay method of the present invention. This reaction vessel 11 has an inlet 12 and an outlet 13 .
A part of the passage 14 connecting the inlet 12 and the outlet 13 is provided to block the passage 14 to prevent passage of particles immobilized with antibodies or antigens that specifically react with the antigen or antibody to be measured. A filter 15 made of glass, for example, is provided to allow the reaction liquid and the like to pass through. In addition, the discharge port 13 is located near the filter 15.
A stopper 16 made of, for example, a stainless wire mesh is provided on the side, and a plate valve 17 made of, for example, silicone rubber is arranged between the filter 15 and the stopper 16. The plate valve 17 is separated from the filter 15 as shown in FIG.
As shown in FIG. 2, the plate valve 17 is brought into close contact with the filter 15 to prevent reaction liquid and the like from passing through the filter 15. Note that it is preferable that the thickness of the filter 15 be made as thin as possible in terms of strength.

6A to 6I are diagrams for explaining the sequential steps of an example of the immunoassay method of the present invention using the reaction container 11 shown in FIG. 4, and the reaction container 11 is shown schematically. . In this example, the outlet 13 of the reaction vessel 11 is opened to the air via the two-way valve 21, and is also opened to the air via the two-way valve 22, the pump 23, and the two-way valve 24. First, in FIG. 6A, the two-way valve 21 and the two-way valve 22 are closed, and the two-way valve 24 is closed.
After opening the pump 23 and sucking air, the two-way valve 22 is opened and the two-way valve 24 is closed to discharge the pump 23 and the sucked air is drawn into the reaction vessel 11 from the discharge port 13. This causes the plate valve 17 to pass through the filter 15 as shown in FIG. 5B.
The sample 26 contained in the sample cup 25 is passed through the pump 27 and the sample nozzle 28 while the passage 14 in the reaction vessel 11 is closed (hereinafter this state is referred to as "closing the plate valve 17"). A first reagent 30 containing immobilized particles of an antibody (antigen) that specifically reacts with the antigen (antigen) to be measured stored in the reagent tank 29 and dispensed into the reaction container 11 from the injection port 12 is dispensed from the injection port 12 into the reaction container 11 via the pump 31 and the dispensing nozzle 32 to obtain a mixed solution 33, which contains the antigen (antigen) in the sample 26 and the immobilized antibody (antigen).
Allow the antigen-antibody reaction to occur at room temperature or in a thermostatic bath for a certain period of time.

In this example, the first reagent 30 is a solid phase carrier particle of an antibody (antigen) with an average diameter of 100 μm and a specific gravity of 1.03 to 1.05.
Using spherical nylon particles, the particles were hydrolyzed in 1NHCl solution at 60°C for 3 hours, washed with pure water, reacted in 1% glutaraldehyde at 24°C for 1 hour, and then treated with 0.25M sodium phosphate buffer. Liquid (PH7.5)
After washing at 4°C in a 0.25M sodium phosphate buffer containing an antibody (antigen) that specifically reacts with the antigen (antibody) to be measured at a rate of 1.0mg/ml.
The antibody (antigen) was immobilized by reaction for 24 hours, and then the reaction solution was removed and washed with _0.25M sodium phosphate buffer.
% bovine serum albumin, suspended in 0.01 M sodium phosphate buffer containing 0.1% _NaN3 .
The first reagent 30 made of suspended particles is stored at 4°C.

In this way, as solid phase carrier particles, the average diameter
By using suspended spherical nylon particles with a diameter of 100 μm and a specific gravity of 1.03 to 1.05, it is possible to create a large interface between the mixed liquid, which is the site of the antigen-antibody reaction, and the surface of the solid support. can be suspended uniformly in the water, and therefore a homogeneous reaction system can be obtained.

Next, in FIG. 6B, the pump 23 is operated for suction with the two-way valve 22 open and the two-way valve 24 closed, thereby opening the plate valve 17 and draining the mixed liquid 33 in the reaction vessel 11. After separating it from the nylon particles and sucking it through the filter 15, the two-way valve 21 is opened and the pump 23 is operated to discharge the sucked liquid mixture.

Thereafter, in FIG. 6C, the two-way valves 21, 22, 24 and the pump 2 are closed so that the plate valve 17 is closed.
3 is activated, the cleaning liquid 35 contained in the cleaning liquid tank 34 is dispensed from the injection port 12 into the reaction vessel 11 via the pump 36 and the dispensing nozzle 37, and then the process is explained with reference to FIG. 6B. As described above, the two-way valves 21, 22, 24 and the pump 23 are operated to open the plate valve 17 to discharge the dispensed cleaning liquid, thereby cleaning the filter 15 and the nylon particles. This washing step is performed once or multiple times.

Next, in FIG. 6D, the plate valve 17 is opened and the second reagent 39 containing an enzyme label in the resistance (antigen) against the antigen (antibody) to be measured stored in the reagent tank 38 is poured into the pump 40 and the dispensing nozzle. 41 into the reaction vessel 11 from the injection port 12,
The antigen-antibody reaction is carried out for a certain period of time at room temperature or in a thermostatic bath, thereby binding the enzyme-labeled antibody (antigen), the antigen to be measured (antibody), and the immobilized antibody (antigen). Examples of the labeling enzyme for this second reagent 39 include malate dehydrogenase, glucose 6.
Phosphate dehydrogenase, glucose oxidase, horseradish peroxidase, acetylcholinesterase, alkaline phosphatase, peroxidase, colicoamylase, lysozyme, β
An enzyme having high activity such as -D.galactosidase, whose substrate is relatively inexpensive, and is stable can be used.

After the above antigen-antibody reaction, Fig. 6B and C
The filter 15 and the nylon particles are washed with a cleaning liquid by performing the operation described in the sixth section.
As shown in FIG. The enzyme-substrate reaction is carried out for a certain period of time at room temperature or in a constant temperature bath, thereby producing a coloring substance. This third reagent 43 substrate solution is the second reagent 3
When the labeling enzyme in 9 is alkaline phosphatase, it is phenyl phosphate 2-sodium and 4-aminoantipyrine, and when it is peroxidase, it is 0.
-phenylenediamine, 0-nitrophenol/β-D-galactoside for β-D-galactosidase, β-D- for glucose oxidase
Glucose etc. can be used.

After carrying out the enzyme-substrate reaction for a certain period of time,
As shown in FIG. 6F, the reaction stop solution 4 is stored in the reaction stop solution tank 46 with the plate valve 17 closed.
7 is dispensed into the reaction vessel 11 from the injection port 12 via the pump 48 and the dispensing nozzle 49, thereby obtaining a reaction liquid 50 in which the enzyme-substrate reaction is stopped and the increase in the coloring substance is stopped. Enzyme-substrate reaction
Since the reaction can be easily stopped by changing the pH, the reaction stop solution 47 is
HCl, H ₂ SO ₄ solutions, etc. can be used.

After that, in FIG. 6G, the two-way valve 22 is opened,
The two-way valve 24 is closed, the pump 23 is operated for suction, the plate valve 17 is opened, and the reaction liquid 50 in the reaction vessel 11 is separated from the nylon particles through the filter 15 and sucked, and then the two-way valve 21 is opened. As a result, the pump 23 is discharged and the reaction liquid 50 is sucked.
is housed in the colorimetric cell 51.

Next, as shown in FIG. 6H, the absorbance of the reaction solution 50 contained in the colorimetric cell 51 is measured using a spectrophotometer
2, and based on this measured value, the antigen to be measured (antibody) in the sample 22 is quantified from a calibration curve between the absorbance determined from the sample of known concentration and the antigen to be measured (antibody). After the reaction vessel 11 is cleaned with a cleaning liquid by performing the operations explained in FIGS. 6B and 6C, the cleaning liquid and nylon particles remaining in the reaction vessel 11 are removed by suction nozzle 53 and as shown in FIG. 6I. It is evacuated via suction pump 54 to prepare for the next sample analysis.

The above-mentioned suction nozzle 53 has a notch 55 formed in the end face of the nozzle tip through which nylon particles can pass, as shown in the front and bottom views of FIGS. 7A and 7B, respectively. If such a suction nozzle 53 is used, the nylon particles and the remaining cleaning liquid can be effectively removed by suction by bringing the tip of the suction nozzle into contact with the filter 15 for suction.

FIG. 8 is a sectional view showing the structure of another example of a reaction container used in the immunoassay method of the present invention. This reaction vessel 61 differs from the one shown in FIG. 4 only in that the surface of the filter 15 on the injection port 12 side is processed into a concave shape. If this reaction vessel 61 is used, the nylon particles will gather at the center of the concave surface of the filter 15 in the suction step shown in FIG. 6I, and therefore can be easily and reliably removed by suction.

Note that the present invention is not limited to the above-mentioned example, and can be modified or changed in many ways. For example, although the plate valve 17 and the stopper 16 are provided in both the reaction vessels 11 and 61 described above, if the hole diameter of the filter 15, the inner diameter of the reaction vessel, the amount of reaction (mixed) liquid, etc. are appropriately set, these can be used. The reaction (mixture) liquid, etc. can be effectively held in the reaction vessel by air pressure from the outlet 13 side without using the plate valve 17 and the stopper 16. In addition, in the above example, nylon particles were used as solid phase carrier particles for antibodies (antigens) that specifically react with the antigen (antigen) to be measured, but cellulose, ABS,
It is also possible to use particles of polystyrene, styrene-butadiene resin, or the like, or microcapsules whose specific gravity is controlled by coating the surface of a core material with a high specific gravity such as iron powder with a film with a low specific gravity. Furthermore, in the analysis method described above, Fig. 6F
In the above, the enzyme-substrate reaction was stopped by dispensing the reaction stop solution 47, but if the reaction solution 50 is discharged into the colorimetric cell 51 through the filter 15 after the enzyme-substrate reaction for a certain period of time, the nylon particles The labeled enzyme captured by the filter 15 is separated into the nylon particles and the substrate liquid, and the substrate is no longer subjected to enzyme action and the reaction is stopped, so the step in Figure 6F can be omitted. can. Furthermore, in the above example, the reaction solution 50 is transferred to the colorimetric cell 51.
Although photometry was carried out by transferring the light to the reaction vessel 11, direct photometry can also be carried out through the reaction vessel 11.

According to the above embodiments, the solid phase carrier particles that immobilize the antibody (antigen) that specifically reacts with the antigen (antibody) to be measured have a large interface between the liquid mixture and the surface of the solid phase carrier, and Since the specific gravity, shape, and size of the particles were controlled so that they could float uniformly, a uniform reaction system could be obtained.

As detailed above, according to the first invention, the reaction using suspended particles and the separation of the particles by a filter are repeated three times, and a specific substance contained in the liquid that has passed through the filter is measured colorimetrically. As a result, the presence of particles does not have an adverse effect on the colorimetric measurement, and the antigen or antibody to be measured in the sample can be accurately quantified.

Further, according to the second invention, analysis is performed using a reaction vessel having an injection port provided above and a filter disposed at the bottom of the injection port that does not allow suspended particles to pass through. Therefore, the particles can be separated without centrifugation, thus simplifying the analytical operation and facilitating automation.

[Brief explanation of the drawing]

Figures 1 A to D are diagrams for explaining the sequential steps by radioimmunoassay, Figure 2 is a diagram showing an example of counts by radioimmunoassay shown in Figure 1, and Figure 3A. -F are diagrams for explaining the conventional sequential steps of enzyme immunoassay, FIG. 4 is a sectional view showing the configuration of an example of a reaction vessel used in the immunoassay of the present invention, and FIGS. B is a diagram for explaining the operation of the plate valve of the reaction vessel shown in FIG. 4, FIGS. 7A and 7B are a front view and a bottom view showing the configuration of an example of the suction nozzle shown in FIG.
The figure is a sectional view showing the structure of another example of the reaction container used in the immunoassay method of the present invention. 11, 61...Reaction container, 12...Inlet, 1
3...Discharge port, 14...Passway, 15...Filter, 16...Stopper, 17...Plate valve, 21,2
2, 24...Two-way valve, 23, 27, 31, 36,
40, 44, 48, 54... pump, 25... sample cup, 26... sample, 28... sample nozzle, 29, 38, 42... reagent tank,
30...First reagent, 32, 37, 41, 45, 4
9...Dispensing nozzle, 33...Mixed liquid, 34...Washing liquid tank, 35...Washing liquid, 39...Second reagent, 43...Third reagent, 46...Reaction stop liquid tank, 47...Reaction Stop solution, 50...Reaction solution, 51
... Colorimetric cell, 52 ... Spectrophotometer, 53 ... Suction nozzle, 55 ... Notch.

Claims (1)

[Claims] 1. Performing an antigen-antibody reaction by mixing a sample with a first reagent containing suspended particles immobilized with an antibody or antigen that specifically reacts with the antigen or antibody to be measured. After that, the reaction solution is passed through a filter that does not allow the particles to pass through, and the particles are separated by the filter; a step of reacting with a second reagent containing a labeling substance bound to an antigen, passing the reaction solution through the filter to separate the particles with the filter; and the presence of the separated particles and the labeling substance. After reacting with a third reagent capable of producing a specific substance, the reaction solution is passed through the filter, and the specific substance contained in the liquid that has passed through the filter is measured colorimetrically to obtain the sample. An immunoassay method characterized by comprising the step of quantifying an antigen or antibody to be measured in the sample. 2. The immunoassay method according to claim 1, wherein a labeled enzyme is used as the labeling substance of the second reagent, and a substrate solution for the labeled enzyme is used as the third reagent. 3. After the sample is mixed with the sample and suspended particles immobilized with an antibody or antigen that specifically reacts with the antigen or antibody to be measured, an antigen-antibody reaction is performed, and then the reaction solution is poured upward. an injection port provided;
separating the particles with a filter by passing the particles through a reaction container having a filter placed at the bottom of the injection port that does not allow the particles to pass; and an antibody or antigen that specifically reacts with the antigen or antibody to be measured. A step of injecting a labeled enzyme bound to the labeled enzyme into the reaction container from the injection port to react with the particles, and then separating the particles with a filter, and producing a specific substance in the presence of the labeled enzyme. An immunoassay method comprising the step of injecting the obtained substrate solution into the reaction container from the injection port to react with the labeled enzyme bound to the particles, and measuring the colored substance generated thereby. .