JPH07270419A - Enzyme immunity examining method - Google Patents

Abstract

PURPOSE:To determine whether limiting enzymes exist or not and, if exist, their quantities, and consequently to determine whether each aiming substance exists or not and, if exists, its quantity by using respectively different limiting enzymes as labels of antibodies and antigens which react with the aiming substances and adding the antibodies or the antigens labelled by the limiting enzymes as detection reagents to an object sample in the case where there are a plurality of aiming substances. CONSTITUTION:Detecting reagents produced by bonding limiting enzymes having respectively different cutting base equences with antibodies or antigens of a plurality of aiming substances are added to an object sample. Consequently, respective antibodies (or antigens) are bonded with respective antigens (or antibodies) with extremely high specificity. Immunologically reacted compounded bodies are obtained by removing not reacted detection reagents by B/F-separation of the reaction system. At that time, the respective immunologically reacted compounded bodies contain extremely slight amounts of the respectively different limiting enzymes. To detect them simultaneously, a DNA chain having base sequence which the enzymes cut is added as a substance to produce DNA chain fragments. If there are compounded bodies, due to the reaction of each enzyme and the DNA chain, a large quantity of DNA chain fragments are produced. These DNA chain fragments are located locally by electrophoresis and whether each aiming substance exists or not and, if exits, the quantity are determined.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an enzyme immunoassay method (enzyme immunoassay), and more particularly to an enzyme immunoassay method for detecting a small amount of a plurality of target substances with high sensitivity.

[0002]

2. Description of the Related Art The high specificity of an antigen-antibody reaction (immune reaction) is used to identify and quantify an antigen or an antibody with high sensitivity, and it is generally called an immunoassay method (immunoassay). ing. This immunoassay is largely classified into an immunoturbidimetric method (TIA) that optically detects precipitates and aggregates generated by an antigen-antibody reaction, and a labeled immunoassay method that uses an antibody or an antigen labeled with a substance that can be easily detected separately. Be separated.
In particular, various systems have been considered for the latter depending on the labeled substance, and typical examples thereof include a radioimmunoassay (RIA) that labels a substance containing a radioisotope, an enzyme immunoassay (EIA) that labels an enzyme, Fluorescent immunoassay (FIA or FA
T) etc.

Further, looking at the tests currently applied, those using the TIA method include transferrin and CRP, which are proteins in serum, and immunoglobulins IgG, Ig.
A, IgM, rheumatoid factor for autoimmunity, and quantification of plasmin-gen and antithrombin III for hematological tests. On the other hand, RIA as a labeled immunoassay is an endocrine test ( For example, from erythropoietin, prostaglandin, etc.) to virological tests (eg, HBs antibody, HCV antibody, etc.), tumor-related tests (eg, AFP, CEA, etc.), biochemical tests (eg, trypsin, etc.), and the EIA method is wide. Hematology tests (eg, Protein S, FPA, etc.), Immunoserologic tests (eg, immune complex, microglobulin, etc.), F
AT is used for a virus antibody test (for example, EBV, herpes, etc.) and an immunoserologic test (for example, antinuclear antibody, etc.).

[0004]

The immunoassay method is widely used in clinical tests and the like as described above, but the RIA method is often used to detect a target substance with high sensitivity. However, this RIA method has a problem that a plurality of target substances in the same subject cannot be detected at the same time. In addition, in other methods such as EIA and FIA, the problem that the use is limited in terms of sensitivity and when it is attempted to detect a plurality of target substances at the same time, the fractionation device of the enzyme reaction product and the spectral distribution of fluorescence and luminescence are There is a problem that an analyzer is required, the cost of the device is high, and the kinds of objects are considerably limited in terms of resolution.

The present invention has been made in view of the above problems, and an object thereof is to detect a plurality of target substances simultaneously and easily with high sensitivity.

[0006]

Therefore, in the present invention, when there are a plurality of target substances to be detected, different restriction enzymes are used as labels for the antibodies or antigens that react with them. By adding such antibodies or antigens labeled with different restriction enzymes to a test substance as a detection reagent, an antigen-antibody reaction occurs when the test substance contains a target substance, and one or more or more restrictions occur. An immune reaction complex with the enzyme is obtained. This is separated from the unreacted material by B / F. The obtained complex has different restriction enzymes depending on the target substance, and a DNA having a cleavage base sequence that is cleaved by each of these restriction enzymes.
DNA is obtained by reacting the complex with the chain as a substrate.
The chain is broken. In the present invention, cleaved DNA
By analyzing and measuring the chain fragment, the presence or absence of a restriction enzyme and its amount, and thus the presence or absence of each target substance and its amount are detected.

As the DNA chain used in the present invention, it is preferable to use a DNA chain in which a cleaved base sequence is arranged so that the length of the cleaved DNA chain fragment varies depending on each restriction enzyme. In particular, the cleavage base sequences are arranged in the same strand so that they can be cleaved by each restriction enzyme, and each DNA strand fragment has a different length even if the DNA strands are cleaved at multiple locations. It is possible to detect a plurality of target substances with one type of DNA chain by using the existing DNA chain.

Alternatively, a plurality of types of DNA having only a cleavage base sequence that is cleaved by each restriction enzyme
Use chains. The analysis of the cleaved DNA chain fragment includes
For example, it was decided to use the electrophoresis method or the chromatography method.

[0009]

According to the present invention, a detection reagent prepared by attaching a restriction enzyme having a different cleavage base sequence to an antibody or an antigen of each of a plurality of target substances is simultaneously added to a subject. This allows each of these antibodies (or antigens) to bind to each of the antigens (or antibodies) with very high specificity. An immune reaction complex can be obtained by subjecting this to B / F separation to separate the unreacted detection reagent. The state at this time is such that a very small amount of each immune reaction complex has a different restriction enzyme attached thereto. Therefore, in order to detect these different restriction enzymes at the same time, a DNA chain having a cleavage sequence of each restriction enzyme is added as a substrate and reacted to generate DNs having different lengths.
Generate an A chain fragment. When one kind of such DNA chain is used, it has a cleavage sequence of each restriction enzyme within it, and it is located at a position where a DNA chain fragment of the same length is not produced even by the combination of arbitrary cleavage points. It has a cleavage sequence. When a plurality of types of DNA strands are used, each cleavage sequence has one in each separate DNA strand, and the lengths of the DNA strand fragments after cleavage are different from each other. In this way, in the presence of each immunoreaction complex, a large amount of DNA chain fragments having different lengths are produced by the reaction of each restriction enzyme with the DNA chain. Therefore, in order to analyze these, for example, by gel electrophoresis, localizing different lengths at different locations, staining with ethidium bromide, etc., and observing fluorescence, multiple target substances can be easily detected simultaneously as gel patterns. it can. Furthermore, by measuring the fluorescence intensity, not only the presence or absence of a plurality of target substances but also the amount of the target substances can be measured.

Similarly, a plurality of target substances can be easily detected simultaneously by chromatography.

[0011]

Example Using the present invention, carcinoembryonic antigen (CEA) and α-fetoprotein (AFP), which are the target substances,
An example of simultaneously detecting with high sensitivity is shown. [Example 1] (1) Preparation of detection reagent (restriction enzyme-labeled antibody) EcoRI as a restriction enzyme was dissolved in 0.1 mol / l sodium phosphate buffer (pH 7.0), and 500-1000 equivalents of N- A solution of succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate in N, N-dimethylformamide was added and reacted at 30 ° C for 1 hour, and the precipitated unreacted maleimide compound was removed by centrifugation. , Supernatant to 0.
Equilibrated with 1 mol / l sodium phosphate buffer (pH 6.0)
The target product was purified on a Sephadex G-25 column. This fraction was mixed with 5 mmol / l EDTA-containing sodium phosphate buffer containing anti-CEA antibody, incubated at 4 ° C for 20 hours, and then 0.1 mol / l sodium phosphate buffer (pH 6.5).
The target fraction was purified on an Ultrogel AcA 44 column equilibrated with and the yield of anti-CEA antibody labeled with EcoR I was obtained.
% To obtain a detection reagent. In addition, Stu
An anti-AFP antibody labeled with I was prepared at a yield of 87% and used as a detection reagent. (2) Creation of substrate DNA strand for detection A total length of 400 bps, a DNA strand having a cleavage site of EcoR I only in its central part and a total length of 300 bps, only in its central part DNA with Stu I cleavage site
The strand was prepared by a DNA synthesizer and used as a substrate DNA strand for detection. FIG. 1 shows a schematic diagram of both substrate DNA strands.
In the figure, 5'and 3'indicate the ends of the DNA chain, each square box 11 indicates a nucleotide, and 200 bps and 150 bps indicate base sequence lengths, respectively. Moreover, A, G, T, and C represent nucleotide bases, and a, a ′, b, and b ′ are arbitrary sequences that do not include the cleavage sequences of EcoRI and StuI. The cleavage sequence of EcoR I is

[0012]

[Chemical 1]

The cleavage sequence of Stu I is

[0014]

[Chemical 2]

[0015] (3) Preparation of immobilized antibody Anti-CEA antibody and anti-CEA were added to sodium phosphate buffer (pH7.2).
AFP antibody was added to a microtiter plate, incubated overnight at 4 ° C, discarded, washed with sodium phosphate buffer (pH7.2), and separated into B / F. A solid-phased antibody in which was immobilized was prepared. (4) Preparation of immobilized antibody-antigen-labeled antibody complex (labeled immunoreaction complex) Add standard samples of CEA and AFP, which are the analytes, to the wells,
Incubated for 2 hours at room temperature. After discarding the solution and washing with sodium phosphate buffer (pH 7.2), dilute the two types of restriction enzyme-labeled antibodies that were the detection reagents prepared above with sodium phosphate buffer and add them to the wells at room temperature. At 1
Incubated for hours. After that, the liquid is discarded and washed with sodium phosphate buffer to remove the unreacted detection reagent, and the immobilized antibody-antigen (CEA or AFP) -labeled antibody complex (labeled immune reaction complex) is removed. Formed. (5) Detection Well buffers (50mM sodium chloride, 7mM magnesium chloride, Tris) that are favorable conditions for EcoR I and Stu I
-HCl, pH 7.5) and (2) two DNA strands for detection were added, and the temperature was 37 ° C.
After incubating at 30 ° C. for 30 minutes, the solution in the well was electrophoresed on a polyacrylamide gel to obtain a migration pattern. This gel was stained with ethidium bromide, and its fluorescence was measured.
The amount of each DNA fragment in the band at ps, 150 bps, and 300 bps was calculated to quantify each antigen. The results are shown in FIG. 3 as a migration pattern. In the figure, 30 is a polyacrylamide gel plate, the direction of the arrow is the migration direction, and 31 is 15
0 bps, 32 shows 200 bps, 33 shows 300 bps, and 34 shows bands of DNA chain fragments at 400 bps. (6) Preparation of calibration curve To prepare the calibration curve, add each restriction enzyme of known concentration to an unused well of the same immunoassay titer plate used above, and perform the detection operation in (5) above in the antigen presence system. Was carried out under the same conditions as above, and the relationship between the amount of restriction enzyme and the amount of generated DNA chain fragment was plotted and created. As a result, a calibration curve as shown in FIG. 5 was obtained.

[Example 2] (1) Preparation of detection reagent The same as in Example 1. (2) Preparation of substrate DNA strand for detection As shown in FIG. 2, the total length is 450 bps, the EcoR I cleavage sequence is present only at a position 200 bps from one end, and the other is A DNA duplex having a StuI cleavage sequence only at a position of 150 bps from the end was prepared by a DNA synthesizer.

In the figure, 20 is the cleavage base sequence of Stu I and 21 is EcoR
The cleavage base sequence of I is shown, and 22 is an arbitrary sequence that does not contain both of them. (3) Preparation of immobilized antibody The same as in Example 1. (4) Formation of immobilized antibody-antigen-labeled antibody complex (labeled immune reaction complex) The same procedure as in Example 1 was performed. (5) Detection Well buffers (50mM sodium chloride, 7mM magnesium chloride, Tris) that are favorable conditions for EcoR I and Stu I
-HCl, pH 7.5) and (2) DNA strand for detection were added, and
After incubating for 0 minutes, the solution in the well was electrophoresed on a polyacrylamide gel to obtain a migration pattern.
This gel is stained with ethidium bromide, its fluorescence is measured, and 100bps, 150bps, 200bps, 250bps, 300bp from the calibration curve prepared under the same conditions from the calibration curve prepared at the same time.
The number of DNA fragments in the band at the s and 450 bps positions was calculated to determine the amount of antigen. The migration pattern is shown in FIG.
In the figure, 40 is a polyacrylic acid plate, migration is performed in the direction of the arrow, 41, 42, 43, 44, 45, 46, 100bps, 150bps, respectively.
The band positions of DNA chain fragments of 200 bps, 250 bps, 300 bps, and 450 bps are shown. (6) Preparation of calibration curve The same as in Example 1.

In the above examples, examples for two types of antigens have been shown, but it is possible to easily use even more antigens by making the lengths of the DNA chain fragments different. Although the electrophoretic method was used in the two examples, it can be detected by chromatography in exactly the same manner.

[0019]

INDUSTRIAL APPLICABILITY As described above, in the enzyme immunoassay method of the present invention, by using a plurality of restriction enzymes as enzymes for detecting a plurality of target substances, a plurality of restriction enzymes can be easily detected with high sensitivity. The target substance can be detected at the same time. Moreover, the detected object data can be rechecked anytime later. Furthermore, there is an advantage that mutual comparison of samples can be easily performed with a single electrophoretic pattern.

[0020]

[Brief description of drawings]

FIG. 1 is a structural example of a DNA chain used as a substrate.

FIG. 2 is a structural example of a DNA chain as a substrate used.

FIG. 3 is a migration pattern of a DNA chain fragment in Example 1.

FIG. 4 is a migration pattern of a DNA chain fragment in Example 2.

FIG. 5 is a calibration curve, in which the horizontal axis represents the amount of restriction enzyme and the vertical axis represents the band concentration of the DNA chain fragment after electrophoresis.

[Explanation of symbols]

 11 nucleotides

Claims (6)

[Claims] 1. An immunoreactive complex is obtained by reacting an antigen or antibody as a target substance with an enzyme-labeled antibody or antigen, and then the complex and an unreacted product are separated to obtain a reaction product. In an enzyme immunoassay method for detecting a target substance using an enzyme, there are a plurality of target substances, and each antibody or antigen that reacts with them is labeled with a different restriction enzyme, and 1 or 2 or more depending on the reaction and separation. Each immunoreaction complex having a restriction enzyme is obtained, each DNA chain having a cleavage base sequence that is cleaved by the restriction enzyme of each complex is cleaved by the restriction enzyme of the complex, and each cleaved DNA An enzyme immunoassay method characterized by detecting a plurality of target substances by analyzing and measuring chain fragments. 2. The enzyme immunoassay method according to claim 1, wherein the DNA chain having a cleavage base sequence that is cleaved by the restriction enzyme of the reaction product has a length of each cleaved DNA chain fragment. An enzyme immunoassay method characterized by using a DNA chain in which a cleavage base sequence is arranged so as to differ depending on the type. 3. The enzyme immunoassay method according to claim 2, wherein the DNA strand having a cleavage base sequence that is cleaved by the restriction enzyme of the reaction product has the same cleavage base sequence that is cleaved by each restriction enzyme. An enzyme-linked immunosorbent assay characterized in that the cleaved nucleotide sequences are arranged such that each DNA strand fragment has a different length even if the DNA strands are cleaved inside and the DNA strands are cleaved at multiple positions. 4. The enzyme immunoassay method according to claim 2, wherein the DNA strand having a cleavage base sequence that is cleaved by the restriction enzyme of the reaction product is a cleavage base that is cleaved by each restriction enzyme. DN cleaved by each restriction enzyme using plural kinds of DNA chains having only sequences
An enzyme immunoassay method, wherein each A chain fragment has a different length. 5. The enzyme immunoassay according to claim 1, wherein a plurality of target substances are detected by analyzing and measuring the cleaved DNA chain fragment by an electrophoresis method. Inspection method. 6. The enzyme immunoassay according to claim 1, wherein a plurality of target substances are detected by analyzing and measuring the cleaved DNA chain fragment using chromatography. Inspection method.