JPH055739A - Preparing method of specimen for laser magnetic immunoassay - Google Patents

Abstract

PURPOSE:To obtain a preparing method of a specimen which avoids cohesion between microbeads through the intermediary of an antigen and an antibody which causes a problem when the antigen and the antibody are caught, in a laser magnetic immunoassay. CONSTITUTION:A preparing method of a specimen comprises at least a first process wherein a liquid containing suspended microbeads with which a first specific antibody for an antigen to be detected is combined beforehand and the specimen are made to react with each other, a second process wherein the caught specimen and a second specific antibody for a specimen combined with biotin beforehand are made to react with each other, a third process wherein a liquid containing dispersely-suspended tagging material of magnetic particulates is added thereafter and the biotin in the specific antibody and avidin in the tagging material are made to react with each other, and a fourth process wherein the tagging maternal not having reacted yet is separated and removed. In another way, a process wherein a third specific antibody combined with the viotin beforehand is add to an anti-antibody for the second specific antibody and the second specific antibody and the third specific antibody are made to react with each other is provided behind the second process, and a part of the second specific antibody used in the second process is made to coexist in the first process.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to an immunoassay method utilizing an antigen-antibody reaction. More specifically, it relates to a sample preparation method used in the laser magnetic immunoassay method previously invented by the present inventors.

[0002]

2. Description of the Related Art Development of an immunoassay utilizing an antigen-antibody reaction is currently in progress worldwide as an early test method for new viral diseases such as acquired immunodeficiency syndrome, adult T-cell leukemia, and various cancers. Being promoted on a scale.

As a conventionally known microimmunoassay method, a radioimmunoassay (hereinafter referred to as RIA method),
Enzyme immunoassay (EIA), fluorescent immunoassay (FIA) method and the like have already been put to practical use. These methods are methods of detecting the presence or absence of an antibody or an antigen that specifically reacts with an antigen or an antibody to which an isotope, an enzyme or a fluorescent substance is added as a label, respectively.

The present inventors previously disclosed Japanese Patent Application No. 61-22456.
No. 7, No. 61-252427, No. 61-254164
No. 62-20262, No. 62-22603, No. 62-152791, No. 62-152792, No. 6
No. 2-184902, a patent application has been filed for an invention relating to a laser magnetic immunoassay method and measuring apparatus. These new immunoassays use magnetic particles as the labeling material,
For example, it is characterized in that the presence or absence of a magnetically labeled sample is detected from interference fringes, and it is possible to detect an ultratrace amount of a picogram or less without using an isotope. Based on the above-mentioned invention, the present inventors label the magnetic particles with an antigen or an antibody,
The virus was detected for the first time.

[0005]

An object of the present invention is to avoid aggregation between microbeads via an antigen (or antibody) which becomes a problem when capturing the antigen (or antibody) in the above-mentioned laser magnetic immunoassay. A method of preparing a sample is provided.

[0006]

The method generally used in the conventional ELISA method is to immobilize an antigen or an antibody on the bottom surface of a 96-well microplate, and to target it in the patient serum in the surface thereof. The antigen-antibody reaction is a solid-phase reaction because it captures an antibody or an antigen. On the other hand, in the case of the laser magnetic immunoassay method of the present invention, microbeads are used instead of the microplate as a method for improving the detection sensitivity. The reason for this is to increase the degree of freedom in the antigen-antibody reaction so that a minute amount of the antigen or antibody can be efficiently reacted with the antigen, and the unreacted magnetic fine particle labeling material is subjected to B / F separation (Bound / Fre).
e, that is, the magnetic fine particles bound to the sample are separated from the magnetic fine particles not bound. That is, if microbeads and magnetic fine particle labeling materials having diameters of 2 μm and 0.03 μm, respectively, are used, the microbeads and the magnetic fine particle labeling materials can be easily separated by centrifugal force because their sizes and masses are greatly different.

It is well known that when capturing an antigen on antibody-bound microbeads, agglomeration of the microbeads occurs via the antigen depending on the amount ratio of the amount of the antigen and the amount of the microbead. A similar phenomenon occurs when the antibody is captured by the antigen-bound microbeads. This phenomenon occurs because microbeads are bound via the antigen or antibody because the antigen or antibody to be detected is multivalent. By the way, the PA method (bead agglutination method) is a method for actively using this phenomenon for diagnosing an immune reaction.
Widely used for antigen or antibody tests. But,
The PA method has a detection sensitivity of about μg / ml and cannot detect a small amount of antigen.

After capturing the antigen or antibody using microbeads, the magnetic fine particle labeling material is bound in the form of sandwiching the antigen or antibody, and the amount of the antigen or antibody is measured from the existing amount of the magnetic fine particles. In the case of laser magnetic immunoassay, when agglutination of microbeads occurs, the antigen or antibody will be buried in the agglutination mass of microbeads, and it will not be able to react with the second biotinylated specific antibody. Cannot be combined further.

According to the first method of the present invention, it is possible to prevent the aggregation of microbeads via the antigen at the time of capturing the antigen. That is, the first specific antibody against the antigen to be detected is pre-bound microbead suspension, the sample is reacted, the first step of capturing the sample on the surface of the microbeads, the captured sample, Further reacting with a second specific antibody against the sample previously bound with biotin,
A second step of sandwiching the sample with a first and a second specific antibody, and after the second step, a dispersion suspension of magnetic fine particle labeling material is added, and biotin in the second specific antibody,
A third step of reacting with avidin in the magnetic fine particle labeling material to magnetically label only the sample captured by the microbeads, and separating / removing the magnetic fine particle labeling material that has not reacted in the third step In the method for preparing a specimen, which comprises at least the fourth step, there is provided a method for preparing a specimen, characterized in that, during the first step, a part of the second specific antibody used in the second step is allowed to coexist.

Further, as a versatile sample preparation method, it can be applied to the secondary antibody method. That is, a first step of reacting a microbead suspension previously bound with a first specific antibody against an antigen to be detected and a sample, and capturing the sample on the surface of the microbead, the captured sample, and A second step of further reacting with a second specific antibody against the sample, sandwiching the sample with the first and second specific antibodies, and after the second step, an anti-antibody against the second specific antibody is previously prepared. Add a third specific antibody bound to biotin,
A third step of reacting the second specific antibody with the third specific antibody, and a dispersion suspension of the magnetic fine particle labeling material is added after the third step, and biotin in the third specific antibody and the magnetic A fourth step of reacting with avidin in the fine particle labeling material to magnetically label only the sample captured by the microbeads, and a step of separating and removing the magnetic fine particle labeling material that has not reacted in the fourth step In a sample preparation method comprising at least five steps, there is provided a sample preparation method characterized by allowing a part of the second specific antibody used in the second step to coexist during the first step.

Although the method for testing an antigen such as a virus as a sample has been described above, the sample preparation method of the present invention can be applied to an antibody test as well as an antigen. That is, in this case, as the microbeads for capturing the antibody, those obtained by previously binding the antigen that specifically reacts with the target antibody to the microbeads are used. In the above-mentioned first sample preparation method, an antibody is captured on the microbeads, and then a biotinylated anti-antibody for the target antibody is reacted, and then the magnetic fine particle labeling material of the present invention is added, and the sample is magnetically added. Just label it. Also in the case of the above-mentioned second sample preparation method (secondary antibody method), similarly, after capturing the antibody on the microbeads, an anti-antibody against the antibody is reacted, and a biotinylated secondary antibody against the anti-antibody is further reacted. After the reaction with the above, the magnetic fine particle labeling material of the present invention may be added to magnetically label the sample.

The sample preparation method of the present invention can also be applied to the detection of trace amounts of proteins such as hormones and physiologically active substances in addition to the above-mentioned antigens and antibodies.

[0013]

EXAMPLES The sample preparation method of the present invention will be described in detail below based on examples. (Example 1) Polymer beads labeled with antibody (bead concentration: 0.2 wt%) 30 μl suspension, α-fetoprotein dilution series as antigen (protein concentration 10 ⁻¹⁵ to 10 −10)
Each step of 10-fold dilution in a range of ^-8 g / ml 8 steps 3
0 μl, and 5 μl of anti-α-fetoprotein antibody (antibody concentration 10 ⁻² mg / ml) previously bound with biotin
Were mixed and reacted at 37 ° C. for 2 hours with stirring.

Next, anti-α-in which each reaction solution was bound with biotin
Fetoprotein antibody (antibody concentration 0.3 mg / ml)
Was added and reacted at 37 ° C. for 1 hour, and then unreacted biotin-conjugated antibody was separated.

Further, 20 μl of avidin-bonded magnetic fine particles (magnetite concentration 0.1 mg / ml) was added to each reaction solution from which unreacted biotin-conjugated antibody was separated, and 37
After reacting at 1 ° C. for 1 hour, unreacted magnetic fine particles were separated and then diluted with 1% BSA (bovine albumin) -HEPES buffer to a total volume of 200 μl to prepare a measurement sample. 30 μl of 200 μl of each measurement sample was sampled and the interference intensity was measured by a laser magnetic immunoassay device to obtain a calibration curve. The result is shown in FIG.

(Comparative Control Example) In order to compare Example 1 described above, a sample in which a biotin-bound antibody does not coexist was prepared in the step of capturing an antigen on a microbead as in the usual immunoassay method. First, 30 μl of a suspension of polymer beads (bead concentration: 0.2 wt%) labeled with an antibody, a dilution series of α-fetoprotein as an antigen (protein concentration: 10
^-10 to 10-fold dilution in the range of ^-15 to 10 ^-8 g / ml 8 steps)
Each step (30 μl) was mixed and reacted at 37 ° C. for 2 hours with stirring. Then, the same treatment as in Example 1 was performed to prepare a total of 200 μl of measurement sample. 30 μl of 200 μl of each measurement sample was sampled and the interference intensity was measured by a laser magnetic immunoassay device to obtain a calibration curve. The measurement result is shown in FIG.

The horizontal axis of FIG. 1 represents the detected concentration of α-fetoprotein and a control containing no antigen,
The vertical axis represents the interference light intensity measured by the laser magnetic immunoassay method. The polygonal line (a) in the figure is a calibration curve when a biotinylated antibody was made to coexist during antigen capture of the present invention, and the polygonal line (b) is a calibration curve when a biotinylated antibody, which is a comparative control example, was not made to coexist. is there.

As is clear from the measurement results shown in FIG. 1, the coexistence of the biotin-bound antibody when capturing the antigen on the microbeads greatly improves the detection sensitivity in the entire antigen region. It can be seen that the effect of coexisting a biotinylated antibody at the time of antigen capture is a significant improvement in sensitivity.

Regarding the mechanism of this effect, in the reaction of capturing an antigen by the antibody on the microbead, the antigen captured by the microbead binds to another microbead and the microbead aggregates via the antigen. become. Such aggregates are less likely to react with the biotinylated antibody to be reacted next due to steric hindrance. On the other hand, when the biotinylated antibody is allowed to coexist as in the sample preparation method of the present invention, the biotinylated antibody first reacts with the antigen and then with the antibody on the microbeads, so that they are captured by the microbeads. The biotinylated antibody is bound to the antigen. For this reason, the antigen is less likely to react with other microbeads, less likely to be aggregated, and the biotinylated antibody is surely reacted.
Therefore, it has sensitivity to the extremely low concentration of the antigen region,
In addition, even in the high concentration region, the aggregation of many microbeads is suppressed, and the effect of significantly suppressing the decrease in sensitivity can be obtained.

In this example, the case where α-fetoprotein was an antigen was described, but the effect obtained as apparent from the above mechanism does not depend on the antigen. Further, the amount of biotinylated antibody coexisted depends on the range in which the antigen is to be detected.

Next, an example of the secondary antibody method will be shown as an example of detecting hepatitis B surface antigens (HBs). (Example 2) Polymer beads labeled with antibody (bead concentration: 0.2 wt%) 30 μl of a suspension, a dilution series of hepatitis B surface antigens (HBs) as an antigen (protein concentration 10
^-15 to 10 ^-8 g / ml 10-fold dilution 8 steps)
Was mixed with 20 μl of mouse anti-HBs monoclonal antibody (antibody concentration 10 ⁻⁴ mg / ml), and the mixture was reacted at 37 ° C. for 1 hour with stirring.

Next, 10 μl of mouse anti-HBs monoclonal antibody (antibody concentration 0.25 mg / ml) was added to each reaction solution.
After adding and reacting at 37 ° C. for 1 hour, unreacted antibody was separated. Furthermore, a goat anti-mouse antibody (0.1 mg / m 2) previously biotinylated as a secondary antibody was added to each of the obtained reaction solutions.
1) was added and reacted at 37 ° C. for 1 hour. After the reaction, unreacted secondary antibody was separated.

Magnetic particles (magnetite concentration: 0.1 mg) in which avidin was previously bound to each of the finally obtained reaction solutions
/ Ml) and 10 minutes of reaction at 37 ° C., unreacted magnetic fine particles are separated. The total amount of the obtained reaction liquid is 80
A measurement sample was prepared by diluting with a buffer solution to give μl. The total amount of each measurement sample was measured with a laser magnetic immunoassay device, and a calibration curve was obtained from the interference light intensity. The results are shown in Table 1.
It was shown to.

[Table 1]

The detection limit is the antigen dilution series (10 ^-15 to 10
^The interference light intensity was measured with a laser magnetic immunoassay device at 10-fold dilution in the range of ^-8 g / ml (8 steps), and the interference intensity of the control sample containing no antigen was shown as an antigen concentration showing a significant difference.
It can be seen that the detection sensitivity is improved 10 ³ times or more in Example 2 as compared with the conventional EIA method.

In this Example, the detection of HBs antigen was described as an example, but the effects of the secondary antibody method and antibody coexistence do not depend on the type of antigen, as can be inferred from the principle of this measuring method.

[0026]

The laser magnetic immunoassay method of the present invention is not limited to the AFP antigen detection described in the examples,
Of course, it can also be applied to antibody tests that are currently widely practiced. When applied to an antibody test, it can be applied to an allergy diagnosis because, for example, IgE antibody having a low antibody amount in blood can be detected in a short time by utilizing the feature of high detection sensitivity.

Further, the method of the present invention is versatile because the same magnetic fine particle labeling material can be used even if the target sample is changed. That is, in the case of the conventional method of directly binding an antibody to dextran-coated magnetite, it was necessary to prepare a magnetic fine particle labeling material for each virus. In the case of the present invention, it is only necessary to prepare a biotinylated antibody for each virus. This difference is extremely remarkable when it is put to practical use as a diagnostic kit.

Further, according to the present invention, not only the antigen-antibody reaction but also the measurement of various hormones such as peptide hormones to which the RIA method has been conventionally applied or various enzymes, vitamins, drugs and the like can be applied. Is possible. Therefore, it is possible to perform accurate measurement in a general environment, which was previously impossible without the RIA method in a limited facility.
It becomes possible to carry out quickly. That is, in a general situation such as mass screening, if accurate measurement such as screening tests for various viruses and cancers can be widely carried out, early diagnosis of cancer or viral diseases becomes possible and effective early treatment. Can be carried out accurately. As described above, the effect of the present invention in the fields of medicine and medical treatment is immeasurable.

[Brief description of drawings]

FIG. 1 is a graph showing an experimental result of AFP antigen detection based on Example 1 of the present invention, where (a) is a case where the sample preparation method of the present invention is carried out, and (b) is a comparative control example.

 ─────────────────────────────────────────────────── ─── Continuation of front page (72) Inventor Shuichi Shibata 1-6, Uchisaiwaicho, Chiyoda-ku, Tokyo Nihon Telegraph and Telephone Corporation (72) Hiroko Mizutani 6-11, Udagawacho, Shibuya-ku, Tokyo

Claims (1)

Claim: What is claimed is: 1. A first step of reacting a microbead suspension in which a first specific antibody against an antigen to be detected is bound in advance with a sample, and capturing the sample on the surface of the microbead. A second step of further reacting the captured sample with a second specific antibody against the sample bound with biotin in advance, and sandwiching the sample with the first and second specific antibodies; After the step, a dispersion suspension of the magnetic fine particle labeling material is added, and biotin in the second specific antibody is reacted with avidin in the magnetic fine particle labeling material, and only the sample captured by the microbeads is magnetized. In a sample preparation method comprising at least a third step of labeling and a fourth step of separating / removing the magnetic fine particle labeling material which has not reacted in the third step, the second step is carried out at the time of the first step. In laser magnetic immunoassay for sample preparation method characterized by the coexistence of a portion of the second specific antibody used. 2. A first step of reacting a microbead suspension preliminarily bound with a first specific antibody against an antigen to be detected with a sample to capture the sample on the surface of the microbead, and the captured sample And a second step of further reacting the second specific antibody against the sample, sandwiching the sample with the first and second specific antibodies, and after the second step, an anti-antibody against the second specific antibody. A third specific antibody in which biotin is bound to the antibody in advance, and a third step of reacting the second specific antibody with the third specific antibody; and a dispersion suspension of the magnetic fine particle labeling material after the third step. And reacting biotin in the third antibody with avidin in the magnetic fine particle labeling material to magnetically label only the sample captured by the microbeads, and the reaction in the fourth step. Did not do the magnetic fine In a sample preparation method comprising at least a fifth step of separating / removing a particle labeling material, at the time of the first step, a part of the second specific antibody used in the second step is made to coexist. Sample preparation method for magnetic immunoassay.