JPS63108264A - Magnetic enzyme measurement method and inspection reagent piece to be used for executing said method - Google Patents

Abstract

PURPOSE:To improve detection sensitivity and to provide the titled method suitable for automation of inspection by fixing an antigen to a base and bringing a ultrafine magnetic particle into antigen-antibody reaction with the immobilized. CONSTITUTION:The specimen is fixed by effecting the antigen-antibody reaction to the antigen or antibody fixed to the base 10 and a ultrafine magnetic particle labeling body 14 is brought into the antigen-antibody reaction with the fixed specimen. Since the ultrafine magnetic particle labeling body 14 is used as a label for the antigen-antibody reaction in the above-mentioned manner, the sepn. and removal of the labeling body 14 magnetically from the specimen is easy and the labeling body 14 making reaction with the specimen is eventually magnetically detected, by which the presence or absence and the degree of the antigen-antibody reaction of the specimen 22 are easily known. The detection accuracy is enhanced by making the magnetic detection after the magnetization direction of the labeling body 14 is matched by subjecting the labeling body 14 to magnetic writing.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to an immunoassay method using antigen-antibody reactions. More specifically, the present invention relates to an immunoassay method capable of detecting a specific antibody or antigen with high sensitivity and ease from a minute amount of a specimen, and a test reagent piece therefor.

BACKGROUND OF THE INVENTION The development of immunoassay methods that utilize antigen-antibody reactions is currently underway on a worldwide scale as an early detection method for new viral diseases such as AIDS and adult T-cell leukemia, as well as various cancers. This is a method that attempts to detect antibodies or antigens themselves by utilizing the property that antibodies formed when antigens such as viruses invade a living body specifically react with the antigen (antigen-antibody reaction). It is.

Conventional microimmunoassay methods for this purpose include radioimmunoassay (RIA) and enzyme immunoassay (EI).
A) Fluorescence immunoassay and the like have been put into practical use.

Problems to be Solved by the Invention RIA is currently the only method capable of measuring ultra-trace amounts on the order of picograms, but since it must handle radioactive materials, special equipment is required, and there are problems with half-life and disposal. There were restrictions on the timing and location of use due to material processing, etc.

On the other hand, although EIA has low detection sensitivity, it is suitable for testing automation and has recently become rapidly popular.

Furthermore, a simple immunoassay method that utilizes the aggregation of latex particles such as polystyrene is being developed, but its detection sensitivity is inferior to that of the EIA method.

As described above, the demand for automated testing has increased in recent years, and a simple immunoassay method with higher detection sensitivity has been required.

The applicant of the present invention proposed a 5QUID immunoassay method that uses magnetic ultrafine particles as a label to measure the magnetization of a specimen. However, although this 5QUID immunoassay method is a method with ultrahigh sensitivity equivalent to or higher than the RIA method, the measuring device is expensive because it is necessary to cool the 5QUID element to the temperature of liquid helium.

SUMMARY OF THE INVENTION Therefore, an object of the present invention is to provide an immunoassay method that has a detection sensitivity equal to or higher than that of EIA, is easy to automate, and is simple, and a test reagent piece therefor.

Means for solving the problem, that is, according to the present invention, is to prepare a test reagent consisting of a support on which an immobilized antigen or antibody is immobilized on a given region, and to A first step in which a specimen is dropped onto the region of the test reagent to cause an antigen-antibody reaction with the specimen; a magnetic ultrafine particle label is applied to the given region of the test reagent, and the first step a second step of causing an antigen-antibody reaction between the bound antigen or antibody and the antibody or antigen in the specimen; and magnetically removing the unreacted magnetic ultrafine particle label from the given region of the test reagent. a fourth step of fixing the reacted magnetic ultrafine particle label on the reagent; and magnetization of the magnetic ultrafine particle label within the given region of the test reagent. Provided is a magnetic immunoassay method characterized by comprising a fifth step of measuring with a magnetic detector.

In one embodiment of the method of the present invention, the unreacted magnetic ultrafine particle label is magnetically removed by bringing a magnet into contact with or in close proximity to the given region of the test reagent. Preferably, after the fourth step described above, magnetic writing is performed in the given region of the test reagent.

Furthermore, in a preferred embodiment of the present invention, a test reagent is prepared as the test reagent, which comprises a support in which a plurality of immobilized antigens or antibodies are immobilized on a plurality of regions at regular intervals for each type. , an anti-immunoglobulin or a specific antibody labeled with the magnetic ultrafine particles is added as the magnetic ultrafine particle label, and the antigen is transferred between the antibody in the sample bound to the immobilized antigen in the first step described above. Make antibody reaction.

Furthermore, the reacted magnetic ultrafine particle label can be fixed onto the reagent by placing a thin fixing film in close contact with the given area of the test reagent. Alternatively, the reacted magnetic ultrafine particle label is fixed on the test reagent by passing the test reagent through a fixing solution made of resin to form a coating of the resin on the given area and drying it. You can also do this.

In a more preferred embodiment, an immobilized antigen or antibody is immobilized on a given region of a continuous tape-shaped or strip-shaped support as the test reagent, and the immobilized antigen or antibody is A test reagent provided with a marker for identifying the type is prepared, and detection of the magnetization of the magnetic ultrafine particles in the given region of the test reagent piece is performed using the test reagent or a magnetic detector and the marker. By running one of the detectors, the magnetization of the magnetic ultrafine particles and the marker are detected.

Furthermore, according to the present invention, a support, an immobilized antigen or antibody immobilized on a given region of one side of the support, and a removable antigen or antibody on the one side of the support. A test reagent piece for a magnetic immunoassay method is provided, comprising a magnetized magnetic film affixed to the magnetized magnetic film and a fixing film releasably affixed onto the magnetized magnetic film.

Note that the support may be in the form of a continuous tape or strip, and it is preferable that the support is provided with a marker for each given area. Further, on the support, a plurality of types of immobilized antigens or antibodies are immobilized in a plurality of regions at regular intervals for each type.

Function As described above, the magnetic immunoassay method of the present invention involves immobilizing a specimen by causing an antigen-antibody reaction with an antigen or antibody immobilized on a support, and applying a magnetic ultrafine particle label to the immobilized specimen. Antigen-antibody reaction occurs. In order to use magnetic ultrafine particle labels as labels for antigen-antibody reactions,
It is easy to magnetically separate and remove unreacted magnetic ultrafine particle labels from the specimen, and as a result, by magnetically detecting the magnetic ultrafine particle labels that have reacted with the specimen,
The presence or absence and degree of antigen-antibody reaction in a specimen can be easily determined. Then, before magnetic detection, magnetic writing is performed on one magnetic ultrafine particle label to adjust the direction of magnetization of the magnetic ultrafine particle label before magnetically detecting it, thereby increasing detection accuracy. can.

As described above, the magnetic immunoassay method of the present invention does not have the risk of radioactivity unlike RIA, and does not have the problems caused by the half-life of isotopes. Since the measurement is carried out magnetically, the inspection can be easily automated and the measurement is simple.

Furthermore, a technology similar to the present invention is the 5QUID immunoassay method previously proposed by the applicant of the present invention, but the 5QUID immunoassay method directly measures the magnetization of labeled magnetic ultrafine particles individually. In contrast, in the method according to the invention,
If a test reagent consisting of a support in which multiple types of immobilized antigens or antibodies are immobilized on multiple regions at regular intervals for each type is used as the test reagent, multiple types of tests can be performed at once. Can be performed continuously. That is, like tape recorders and magnetic card leagues, multiple immobilized antigens (
Multiple tests can be performed in succession by writing with a known magnetic field onto a test reagent (or antibody) immobilized thereon and subsequently measuring the magnetization of a given region of the test reagent.

Although the magnetic immunoassay method according to the present invention has a drawback of lower magnetization detection sensitivity than the 5QUID immunoassay method, the EIA
It is possible to achieve a detection sensitivity equal to or higher than that of the conventional method, and has the advantage that patient diagnosis can be performed quickly with a simple device.

EXAMPLES Hereinafter, examples of the magnetic immunoassay method and test reagents therefor according to the present invention will be described with reference to the accompanying drawings.

Example 1' Fig. 1 is a diagram showing a first example of a test reagent for a magnetic immunoassay method according to the present invention, in which Fig. 1(a) is a plan view and Fig. 1(5) is a plan view. It is a side view in a partially peeled state.

The illustrated test reagent has a thickness of 0.2 [01! + Strip-shaped support 1 made of polyethylene terephthalate (PET)
Three areas 12 are provided on 0, and these three areas 1
2, three types of antigens immobilized on gelatin are immobilized. A magnetic marker 14 is provided in line with the area 12 for each area 12. Further, on the region 12 side of the support 10, there is a magnetized magnetic film 16 with a thickness of 0.1 mm in which fine permanent magnet particles such as fine Ba ferrite particles are dispersed in vinyl chloride, and a fixing film 18 made of PET with a thickness of 7 μm. are removably attached as shown in the figure.

Hereinafter, a piece consisting of a solid-phase antigen and a marker immobilized on a support will be referred to as a "test reagent piece."

Before use, the magnetized magnetic film 16 is inserted between the support 10 and the fixing film 18 and superimposed on the support.
Together with the fixing film 18, it has the role of protecting the immobilized antigen in the area 12. In use, the fixing film 18 is opened and the magnetized magnetic film 16 is peeled off once.

Further, a notch IOA indicating the direction of insertion into a magnetic detector, which will be described later, is inserted into the support.

The fixing film I8 is necessary to protect the marker and the magnetic head during magnetic detection, which will be described later. However, if the fixing film 18 is thick, it will have a negative effect on the magnetic detection sensitivity of the marker. In the case of detection using a magnetic head, it is preferable that the width be equal to or smaller than the head gap width.

Further, the magnetized magnetic film 16 is used to separate and remove unreacted magnetic ultrafine particle labels from the test reagent.

The magnetic ultrafine particle label that has reacted with the sample is left as is.
Since it is necessary to remove only unreacted substances with good reproducibility, in this example, the magnetized magnetic film is brought into close contact with the support.

FIG. 2 is a diagram illustrating a method for preparing the test reagent strip shown in FIG. 1, and FIG. Figure 2 (C) shows a step of immobilizing a labeled reagent consisting of a magnetic ultrafine particle label (for example, an anti-immunoglobulin or specific antibody labeled with magnetic ultrafine particles). In addition to the phased antigen region, the step of causing an antigen-antibody reaction between the antibody in the sample bound to the immobilized antigen in the above step is shown, and FIG. 2(C) shows the step of causing an antigen-antibody reaction between the unreacted magnetic ultrafine particles FIG. 2(d) shows the step of separating and removing the reacted magnetic ultrafine particles from the test reagent piece using a magnetized magnetic film, and the step of fixing the reacted magnetic ultrafine particles onto the test reagent piece using a fixing film.

In step (a) shown in FIG. 2(a), first, the fixing film 18 shown in FIG. 1 is opened and the magnetized magnetic film 16 is opened.
Once peeled off, the sample collected from the patient (for example, patient's blood a) is injected into three 50 μ
The sample is dispensed onto different types of immobilized antigen 12, and an antigen-antibody reaction is performed with the antibody 22 in the sample. FIG. 2(a) shows that among the three types of immobilized antigen regions 12, the reaction occurs with the antigen in the right region.

In the step shown in Fig. 2 et al.), after the step (a), anti-immunoglobulin 2
10μβ using a microsyringe 20
It is dispensed into three types of immobilized antigen regions 12, respectively, and an antigen-antibody reaction is performed with the antibody 22 in the specimen. Figure 2 (5) shows that the anti-immunoglobulin labeled with magnetic ultrafine particles reacts only with the antibody in the sample bound to the immobilized antigen in the right region of the three types of immobilized antigen regions. It shows.

In the step (C) shown in FIG. 2(C), the magnetized magnetic film 16 is brought into close contact with the support IO, and the unreacted labeling reagent (anti-immunoglobulin 24 labeled with magnetic ultrafine particles) is attached to the magnetized magnetic film 16. After attracting, magnetize magnetic film 1
Remove 6. If necessary, a step of washing and drying the test reagent piece can be added before or after this step (C).

In the step (d) shown in FIG. 2(d), the fixing film 18, which had been open until the previous step, is closed and brought into close contact with the support 10 to prevent the labeled magnetic material from being damaged by the magnetic detection head. do.

FIG. 3 is a diagram illustrating a magnetic detector' for magnetically measuring a test reagent piece prepared by the adjustment method shown in FIG. The illustrated magnetic detector 30 includes a write magnetization head 34 disposed along the wall surface of the slit 32 that guides the test reagent piece (support 10) shown in FIG. 1, and a sample detection head 36. , the sample detection head 36 includes:
Marker detection heads 38 are provided in parallel. The magnetizing head 34 is configured to magnetize a given area 1 of the support 10 of the test reagent strip.
The specimen detection head 36 has a width that magnetizes both the magnetic ultrafine particles in the test reagent strip and the marker 14, while the specimen detection head 36 has a width that allows it to detect only the magnetic ultrafine particles in a given region 12 of the support 10 of the test reagent piece. The marker detection head 38 has a width and a position to detect only the marker 14 on the support 10 of the test reagent piece. These magnetization heads and detection heads are
The ring-shaped magnetic head used in

The head gap is 10 μm.

Furthermore, when the adjusted test reagent piece described in FIG. 2 is inserted into the slit 32 of the magnetic detector 30 from the notch direction of the support, the magnetic detector 30 automatically performs the test like a bank cash card. The reagent piece is drawn into the magnetic detector, processed for detection, and then returned to the entrance. Inside the magnetic detector 30, the magnetic ultrafine particle labels are first magnetized by the magnetization head 34 using a known alternating current magnetic field, and then the magnetic ultrafine particle labels are quantified by the detection head 36.

FIG. 4 is an output waveform showing an example of magnetic immunoassay using the magnetic detector 30 shown in FIG. FIG. 4(a) is the output waveform of the sample detection radar 36, and FIG. 4(5)
is the output waveform of the marker detection head 38 that detects the marker 14.

As can be seen from FIG. 4, the marker indicates the position of the above-mentioned given region 12 of the test reagent piece, that is, the position of the sample, and the output of the detection head 36 when the marker detection head 38 detects the marker. Magnetic immunoassay can be performed by observing the waveform. The example in FIG. 4 shows that an output is seen in the sample of the third marker.

Therefore, it was found that the patient's serum reacted with the third immobilized antigen, and the disease name could be diagnosed.

This example was an attempt at early diagnosis of influenza, and it was found that while the conventional EIA method was undetectable unless there were 100 million or more antibody molecules, it was possible to detect as little as 100,000 molecules. Ta. In addition, in the magnetic detector, when the magnetic ultrafine particle labels were not magnetized in advance with the magnetic head, the detection sensitivity was comparable to EIΔ.

Note that in this example, a magnetic material was used as a marker, but
The marker is not limited to a magnetic material, and may of course be an optical one. In that case, an optical detection head is used for the marker detection head 38.

Example 2 Example 2 basically consists of the same steps as Example 1, but includes a step of separating and removing the unreacted magnetic ultrafine particles from the test reagent using a magnet, and a step of separating and removing the unreacted magnetic ultrafine particles from the test reagent. The process of fixing magnetic ultrafine particles to the test reagent is different. The details of those steps will be explained below.

FIG. 5 illustrates test reagents used in a second embodiment of the invention. The illustrated test reagent includes a set of four types of immobilized antigens 42 on a tape-shaped PET film 40.
They are continuously arranged in the longitudinal direction of the tape-like film 40. A magnetic strip 44 is provided in parallel to these immobilized antigens 42. Such a test reagent is hereinafter referred to as a "test reagent tape." If this test reagent tape is used for mass screening for cancer, etc., the preparation of test reagent pieces and the automation of magnetic measurement described in Example 1 can be easily performed.

Such test reagent tapes are shown in Figures 2 (a) and b).
In the same manner as in the method described above, a sample is dispensed onto the immobilized antigen 42, and an antigen-antibody reaction is caused to occur with the antibody in the sample. Next, the magnetic ultrafine particle label is dispensed onto the immobilized antigen 42 to cause an antigen-antibody reaction with the antibody in the sample.

Figure 6 shows that the unreacted magnetic ultrafine particles in the above-mentioned treatment are continuously removed from the test reagent tape using a magnetized magnetic film magnet tape with a thickness of 0.1+++m in which Ba ferrite fine particles are dispersed in vinyl chloride. It is a figure explaining the process of separation and removal.

The test reagent tape 40 is moved from roller 46A to roller 46B.
sent to. Similar to the test reagent tape 40, the roller 4
A magnetized magnetic tape 50 sent from 8A to roller 48B is guided to the test reagent tape by a guide roller 52, and is pressed onto the test reagent tape 40 by a pair of contact rollers 54. As a result, unreacted magnetic ultrafine particles are transferred to the magnetized magnetic tape and separated and removed from the test reagent tape.

FIG. 7 is a diagram illustrating the process of fixing reacted magnetic ultrafine particles on the test reagent tape from which unreacted magnetic ultrafine particles have been removed. The test reagent tape 40 being sent from the roller 56A to the roller 56B is immersed in a fixer bath 62 made of ester resin by a guide roller 58 and a fixing roller 60 on the way, and is coated with the ester resin. An infrared heater 6 is installed on the take-up roll side of the fixer bath 62.
4 is provided to dry the ester resin. Through this treatment, a fixing film with a thickness of 2 μm was formed on the test reagent tape, and the magnetic ultrafine particles were fixed to the detection reagent tape with the fixing film.

In this example, a tape recorder type magnetic detector was used for the detection reagent tape 40, but its basic configuration, output method, and detection sensitivity are the same as in Example 1.

The detection head in the above embodiment is a ring-shaped magnetic head used in VTRs, etc., but it is not limited to ring-shaped magnetic heads, and can also be used with semiconductors such as magnetoresistive elements and magnetic diodes (Hall elements). A magnetic sensing element or the like can also be used as the sensing head.

In addition, instead of the method of closely adhering the magnetized magnetic tape of this example, a rare earth magnet with a large energy product is attached directly above the running test reagent tape, and unreacted magnetic ultrafine particles can be separated from the test reagent tape without contact. Of course, it is also possible to remove it.

In the above two embodiments, a known antigen (virus antigen) is immobilized on the support of the test reagent, and the antigen and the specimen (virus antibody in the patient's blood) are subjected to an antigen-antibody reaction, and further, The antibody is reacted with magnetically labeled anti-immunoglobulin labeled with magnetic ultrafine particles that specifically react with the antigen-antibody, and then, after magnetically separating and removing unreacted magnetically labeled anti-immunoglobulin, magnetic measurement is performed. are doing.

However, instead of immobilizing antigens on the support of the test reagent, known virus antibodies are immobilized on the support of the test reagent, and the immobilized virus antibodies are applied to the patient's gargle fluid. An unknown viral antigen is injected to cause an antigen-antibody reaction. Next, a magnetically labeled virus antibody is reacted with the virus antigen. After that, the unreacted magnetically labeled virus antibody is magnetically separated and removed, and then magnetic measurement is performed. By doing so,
Immunoassays can also be performed.

As described in detail, the magnetic immunoassay method according to the present invention includes:
It has higher detection sensitivity than the conventional EIA method and is most suitable for test automation. Furthermore, the test reagent strip for magnetic immunoassay according to the present invention has a very simple structure, and therefore can be provided at low cost. Therefore, the magnetic immunoassay method and the test reagent strip for the magnetic immunoassay method according to the present invention are particularly effective when used in screening tests for various viruses, cancers, etc. required in mass medical examinations. The method of the present invention can be useful for early diagnosis and treatment of patients, and will be of great benefit to the medical community.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing a first embodiment of a test reagent for a magnetic immunoassay method according to the present invention, in which (a) is a plan view and FIG. 2(a) to 2(d) are diagrams explaining the method for preparing the test reagent piece shown in FIG. 1. FIG. 3 shows the test reagent prepared by the preparation method shown in FIG. 4 is a diagram illustrating a magnetic detector of the present invention. FIG. 4 is an output waveform showing an example of magnetic immunoassay using the magnetic detector 30 shown in FIG. 3. FIG. FIG. 6 is a diagram showing a second example of a test reagent for a magnetic immunoassay method using a magnetic tape. FIG. FIG. 7 is a diagram illustrating the step of removing the reacted magnetic ultrafine particles on the test reagent tape. (Main reference numbers) 10. Support, 12.・Region where immobilized antigen is immobilized, 14. Magnetic marker, 16. Magnetized magnetic film, 18. Fixing film, 20. Microsyringe, 22. Antibody in specimen, 24. Magnetic super Anti-immunoglobulin labeled microparticles, 3
0... Magnetic detector, 32... Slit, 34... Magnetization head, 36... Sample detection head, 38... Marker detection head 40... Test reagent tape, 42... Solid-phase antigen, 46... Magnetic strip, 50...Magnetized magnetic tape, 54...Tight roller, 60...Fixing roller, 62...Fixer bath, 64...Infrared heater

Claims (12)

[Claims] (1) Prepare a test reagent consisting of a support on which immobilized antigens or antibodies are immobilized on a given region, drop a specimen onto the given region of the test reagent, and mix between the test reagent and the specimen. A first step of causing an antigen-antibody reaction, applying a magnetic ultrafine particle label to the given region of the test reagent to cause the antibody in the specimen to bind to the immobilized antigen or antibody in the first step. or a second step of causing an antigen-antibody reaction with the antigen; a third step of magnetically removing the unreacted magnetic ultrafine particle label from the given region of the test reagent; a fourth step of fixing a magnetic ultrafine particle label on the reagent; and a fifth step of measuring magnetization of the magnetic ultrafine particle label within the given region of the test reagent with a magnetic detector. A magnetic immunoassay method comprising: (2) The magnetic immunoassay method according to claim (1), characterized in that, after the fourth step, magnetic writing is performed in the given area of the test reagent. (3) A patent claim characterized in that, as the test reagent, a test reagent consisting of a support in which a plurality of types of immobilized antigens or antibodies are immobilized in a plurality of regions at regular intervals for each type is prepared. The magnetic immunoassay method according to item (1) or item (2). (4) The unreacted magnetic ultrafine particle label is magnetically removed by bringing a magnet into contact with or in close proximity to the given region of the test reagent. ) to (3). (5) As the test reagent, a test reagent consisting of a support having a solid-phase antigen immobilized on a given region is prepared, and as the magnetic ultrafine particle label, an anti-immunoglobulin labeled with magnetic ultrafine particles is prepared. Alternatively, a specific antibody is provided to cause an antigen-antibody reaction between the antibodies in the specimen bound to the immobilized antigen in the first step. 4
) The magnetic immunoassay method according to any one of the preceding items. (6) The reacted magnetic ultrafine particle label is fixed onto the reagent piece by bringing a thin fixing film into close contact with the given area of the test reagent. The magnetic immunoassay method according to any one of items 1) to (5). (7) Fixing the reacted magnetic ultrafine particle label onto the reagent by passing the reagent through a fixing solution made of resin to form a resin coating on the test reagent and drying it. Claim No. 1 (1) characterized by
) to (5). (8) As the test reagent, an immobilized antigen or antibody is immobilized on a given area of a continuous tape-shaped or strip-shaped support, and the type of the immobilized antigen or antibody is identified. A test reagent is provided with a marker for detecting the magnetization of the magnetic ultrafine particle label in the given region of the test reagent, and detection of the magnetization of the magnetic ultrafine particle label in the given region of the test reagent is performed using the test reagent or a magnetic detector and a marker detector. Claims (1) to (7) are characterized in that the detection is performed by detecting the magnetization and marker of the magnetic ultrafine particles by running one of them.
) The magnetic immunoassay method according to any one of the preceding items. (9) a support; an immobilized antigen or antibody fixed to a given area on one side of the support; and a releasably affixed to the one side of the support. A test reagent piece for a magnetic immunoassay method, comprising a magnetized magnetic film and a fixing film releasably attached to the magnetized magnetic film. (10) The test reagent piece for a magnetic immunoassay method according to claim (9), wherein the support is in the form of a continuous tape or strip. (11) The support body is provided with a marker for each given area.
A test reagent piece for magnetic immunoassay method according to item ) or item (10). (12) Claim (9) characterized in that, on the support, a plurality of types of immobilized antigens or antibodies are immobilized in a plurality of regions at regular intervals for each type. A test reagent piece for a magnetic immunoassay method according to any one of items 1 to 11.