JPH07270420A - Enzyme immunity examining method - Google Patents

Abstract

PURPOSE:To determine whether ligase exists or not and, if exists, its quantity and determine whether an aiming substance exists or not and, if exists, its quantity by producing a detection reagent by labelling an antibody or an antigen by using a ligase and reacting the reagent with an object sample. CONSTITUTION:Ligase is bonded with an antibody or an antigen to give a detection reagent. Consequently, the antibody or an antigen is bonded with, if exists, an aiming substance, which is an antigen (or an antibody), with extremely high specificity. An immunologically reacted compounded body is obtained by removing the not reacted detection reagent by B/F separation of the reaction system. At that time, ligase is bonded with a slight amount of immunologically reacted compounded body. To carry out detection, two types of DNA chains are added. Due to the reaction, a large quantity of DNA chain fragments are produced. The fragments are fractionized by gel electrophoresis to locate the DNA chain fragments in specific positions and fluorescence observation is made possible by dying the fragments and thus whether ligase exists or not and, if exists, the quantity can be determined easily.

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, and more particularly to an enzyme immunoassay method for easily detecting a trace amount of a target substance with high sensitivity.

[0002]

2. Description of the Related Art The high specificity of an antigen-antibody reaction is used to identify and quantify an antigen or an antibody with high sensitivity, and is generally called an immunoassay method (immunoassay). This immunoassay method is largely classified into an immunoturbidimetric method (TIA) that optically detects a precipitate or an aggregate generated by an antigen-antibody reaction, and a labeled immunomethod that uses an antibody or an antigen labeled with a substance that can be easily and separately detected. 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, There is a fluorescent immunoassay (FIA or FAT) using a fluorescent substance as a label. In addition, looking at the tests that are currently applied, those that use TIA are those that are proteins in serum such as transferrin and CRP, immunoglobulins IgG, IgA, and Ig.
M, rheumatoid factor as an autoimmune-related substance, and quantification of plasmin-gen and antithrombin III as a hematological test. On the other hand, the RIA method as a labeled immunoassay is an internal secretory test (for example, Erythropoietin, prostaglandin), tumor-related tests (eg AFP,
CEA, etc., biochemical tests (eg, trypsin, etc.), and EIA methods include hematology tests (eg, protein S, FPA, etc.), immunoserologic tests (eg, immune complex, microglobulin, etc.), and FAT. It is used for testing for viral antibodies (for example, EBV, herpes, etc.) and immunoserologic tests (for example, antinuclear antibody, etc.).

[0003]

Although the immunoassay method is widely used for clinical examinations as described above, the RIA method is generally used to detect a target substance with high sensitivity. is there. However, in this RIA method, there are problems that the radioisotope as a labeling substance cannot be preserved and the handling is troublesome due to the influence on the human body.

The present invention has been made in view of these problems, and an object thereof is to easily detect a target substance with high sensitivity.

[0005]

In the present invention, an antibody or an antigen is labeled with a ligase as an enzyme to form a detection reagent, and the detection reagent is reacted with an analyte to give one or more target substances. After the separation of the unreacted detection reagent, two types of DNA chains having ends that can be joined to each other are used as substrates to react with the immunoreaction complex, and a bond formed by the reaction is formed. It was decided to detect the presence or amount of ligase, and thus the presence or absence or amount of the target substance by analyzing the DNA chain. When the target substance is estimated to be in a very small amount, a step of amplifying a partial chain containing a binding part in the binding DNA chain is added, and the amplified partial chain is replaced with the original chain in order to shorten the detection step time. I made it much shorter.

[0006]

According to the present invention, ligase is attached to an antibody or an antigen to form a detection reagent, which is reacted with an analyte. As a result, the antibody or antigen binds with extremely high specificity if the antigen or antibody, which is the target substance, is present in the subject. An immune reaction complex can be obtained by subjecting this to B / F separation and removing the unreacted detection reagent. At this time, a small amount of the immune reaction complex is attached with ligase. In order to detect this trace amount of ligase, two types of DNA chains having ends that can be joined to each other are added and reacted. In this way, a large amount of long DNA chains bound by the action of ligase is produced. Therefore, when these are fractionated by, for example, gel electrophoresis, these DNA chains are localized at a specific position, and can be easily detected by fluorescence observation by staining with ethidium bromide. Alternatively, it can be easily detected by fractionation and staining by chromatography. However, when the amount of the target substance is extremely small, it may be difficult to form a binding chain sufficient for detection. At this time, as the two types of DNA chains used as substrates, one having each unique sequence part which is not present in the other strand, and each unique sequence part becomes a primer sequence is used. . The two kinds of DNA chains thus formed are reacted with an extremely small amount of ligase, and the bond D generated by the reaction is reacted.
When the NA chain is amplified by the known PCR method using the primers in each chain, the partial chain including the binding portion of the binding DNA chain is amplified, and a large amount of DNA chain of a certain length is produced. become. This can be analyzed and detected as before. Furthermore, the length of the strands resulting from the amplification is then much shorter than each of the original strands, preferably 1/1.
If the design is made from 0 to 1/100, the migration time will be shortened during analysis, and the results will be obtained in a shorter time.

[0007]

Example A method for easily detecting carcinoembryonic antigen (CEA) using the present invention with high sensitivity will be described below. All materials used are easily available from the market.

[0008]

[Example 1] (1) Preparation of detection reagent (enzyme-labeled antibody) T4 DNA Ligase (ligase) as an enzyme was dissolved in 0.1 mol / 1 sodium phosphate buffer (pH 7.0), and 500 to 10
A solution of 00 equivalents of N-succinimidyl-4- (N-maleimidomethyl) siloxane-1-carboxylate in N, N dimethylformamide was added and reacted at 30 ° C for 1 hour to precipitate the unreacted maleimide compound. After removing by centrifugation, the supernatant was equilibrated with 0.1 M sodium phosphate buffer (pH 6.0).
The target substance was purified on a Sephadex-G-25 column. This fraction was mixed with 5 mM o1 / 1 EDTA-containing sodium phosphate buffer in which anti-CEA antibody was dissolved and incubated at 4 ° C for 20 hours.
After purification, the target fraction was purified by UItrogel AcA 44 column equilibrated with 0.1M sodium phosphate buffer (pH 6.5) to obtain the enzyme-labeled anti-CEA antibody with T4 DNA ligase.
It was obtained at 85% and used as a detection reagent. (2) Preparation of detection substrate DNA chain First, a DNA chain having a total length of 400 bps as shown in FIG.
Prepared by NA synthesizer. In the figure, each box 11 indicates a nucleotide, G and C indicate its base, and 3'and 5'indicate the end of the chain. In addition, the portion indicated by N has a length of 8 bps and is a cleavage sequence of the restriction enzyme Not I.
R'is an arbitrary sequence that does not include this truncated sequence, and both have a length of 196 bps. The DNA chain thus prepared was cleaved with Not I and purified to obtain two substrate DNA chains for detection as shown in FIG. The cleavage site sequence of Not I is

[0009]

[Chemical 1]

[0010] (3) Preparation of immobilized antibody Sodium phosphate buffer solution (pH 7.2) with anti-CEA antibody dissolved was added to a microtiter plate and the mixture was allowed to stand at 4 ° C for 1 hour.
Incubated overnight. The liquid was discarded and washed with a sodium phosphate buffer (pH 7.2) for B / F separation to prepare a solid-phased antibody having an anti-CEA antibody immobilized thereon. (4) Preparation of immobilized antibody-antigen-labeled antibody complex (labeled immune reaction complex) A standard sample of CEA as a test sample was added to the wells and incubated at room temperature for 2 hours. After discarding the solution and washing with sodium phosphate buffer (pH 7.2), the enzyme-labeled antibody, which is the detection reagent prepared above, is diluted with sodium phosphate buffer and added to the wells, and then at room temperature for 1 hour. Incubated. After that, the liquid is discarded and washed with a sodium phosphate buffer solution to remove the unreacted detection reagent, and the immobilized antibody-antigen (CE
A) -labeled antibody complex (labeled immune reaction complex) was formed. (5) Detection A well buffer (50 mM Tris-HCl (pH 7.9), 10 mM MgCl ₂ , 20 mM DD) is used in wells for T4 DNA Ligase.
(T, 1.0 mM ATP) 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 was stained with ethidium bromide, and its fluorescence was measured.
s, the amount of each DNA fragment in the bound band at the 400 bps position was calculated to quantify the antigen. The results are shown in FIG. 5 as a migration pattern. In the figure, 50 is a polyacrylamide gel plate, the direction of the arrow is the migration direction, and 51 is 202 bp.
s and 52 are bands of a DNA fragment of 400 bps. (6) Preparation of calibration curve The calibration curve was prepared using the same immunoassay titer used above.
T4 DNA ligase of known concentration in the unused wells of the plate
Was added, and the detection operation of (5) was performed under the same conditions as in the above-mentioned antigen-presenting system to plot the relationship between T4 DNA ligase and the amount of bound DNA fragments produced. As a result, a calibration curve as shown in FIG. 7 was obtained.

[0011]

Example 2 (1) Preparation of detection reagent The same as in Example 1. (2) Preparation of substrate DNA strand for detection The sequence of λgt10 primer is used as a sequence unique to each of the primer portions in the two types of substrate DNA strands. λgt
The sequence for the 10 primer forward direction is 5 '(GCTGGGTAGTCCCCACC
TTT) 3'and the reverse sequence is 5 '(CTTATGAGTATTTCTTCCA
GGGTA) 3 ′, and these are referred to as λgt10F and λgt10R here. First, as shown in FIG. 3, a DNA strand having a total length of 2000 bps, a Not I cleavage sequence only in the central portion, and a λgt10F sequence and a λgt10R sequence on each side of the DNA chain was synthesized by a DNA synthesizer. It was prepared in. Here, the description of FIG. 3 will be given. 117 and 118 are 8 bps sequences showing Not I cleavage sequences, the same sequences as N in FIG. 1, 121 is a λgt10F sequence, 122 is a sequence complementary thereto, 114 is a λgt10R sequence, 1
13 shows the complementary sequence, 5'end of 114 and 3'of 122
The length between the ends is 200 bps. 111 and 112,115 and 116,11
9 and 120, 123 and 124 are complementary to each other and Not I, λgt10F,
It is an arbitrary sequence that does not include any sequence of λgt10R.
In addition, 3'and 5'are conventional notations indicating the ends of DNA chains.
The DNA chain thus created is cut with Not I,
Purification yielded two substrate DNA strands for detection as shown in FIG.
Reference numerals 113, 114, 117, 118, 121 and 122 in FIG. 4 are the same as those in FIG. (3) Preparation of immobilized antibody Pyrex glass micro test tubes (size that fits in the heater part of a commercial PCR amplification device) are dried at 500 ° C for 5 hours and then at 45 ° C.
It was immersed in a 2% acetone solution of 3-aminopropyltriethoxysilane for 24 hours, washed, dried and then immersed in a 1% glutaraldehyde aqueous solution for 1 hour. A solution of the CEA antigen antibody dissolved in sodium phosphate buffer (pH7.2) was added to this and incubated overnight at 4 ° C. Then, the solution was discarded and the sodium phosphate buffer (pH7.2) was added. Washed. As a result, a solid-phased antibody immobilized on a test tube was obtained. (4) Preparation of immobilized antibody-antigen-labeled antibody complex (immune reaction complex) The test tube containing the immobilized antibody treated in (3) is added with the CEA antigen standard sample, which is the analyte, Was incubated at room temperature for 2 hours. After discarding the solution and washing with sodium phosphate buffer (pH 7.2), the T4 DNA ligase-labeled antibody that is the detection reagent prepared above is diluted with sodium phosphate buffer and added to the test tube at room temperature. At 1 hour incubator
I got it. (5) Detection of label After discarding the solution in the test tube, washing with buffer and B / F separation, a buffer solution (50 mM Tris-HCl (pH 7. 9), 10mM MgCl ₂ , 20mM DD
T, 1.0 mM ATP, 16 ° C) and (2) DNA strand for detection were added,
Incubated for 30 minutes. In this solution, Taq-Polymerase as DNA polymerase and λgt10F, λg as primer
t10R, adding dNTPs dissolved in a buffer as a material for amplification, and further adding KCl, TritonX-100, the final composition of 10mM Tris-HCl, 50mM KCl, 1.5mM MgCl ₂ , 0.1% TritonX
-100, and PCR was performed by a known method.
Then, the liquid in the test tube was electrophoresed on a polyacrylamide gel to obtain an electrophoretic pattern. This gel was stained with ethidium bromide, its fluorescence was measured, and the amount of each DNA fragment in the original 200 bps and bound band at 2000 bps was calculated from the calibration curve prepared at the same time to quantify the antigen. It was The results are shown in FIG. 6 as a migration pattern. In the figure
60 is a gel plate of polyacrylamide, the direction of the arrow is the migration direction, 61 is the band of 200 bps, and 62 is the band of the DNA fragment of 2000 bps. (6) Preparation of calibration curve The calibration curve of Example 1 was applied.

As described above, in the present embodiment, electrophoresis was used for detection, but chromatography can be easily performed.

[0013]

As described above, when ligase is used as a label, the presence or absence of an antigen (or antibody) is replaced by different DNA lengths, amplified, and detected, so that detection can be performed easily with high sensitivity.

[Brief description of drawings]

FIG. 1 is a structural diagram of a substrate DNA chain.

FIG. 2 is a structural diagram of a substrate DNA chain.

FIG. 3 is a structural diagram of a substrate DNA chain.

FIG. 4 is a schematic diagram of a substrate DNA chain.

5 is a migration pattern of a detection substrate DNA chain in Example 1. FIG.

6 is a migration pattern of a detection substrate DNA chain in Example 2. FIG.

FIG. 7 is a diagram showing a calibration curve obtained from the relationship between ligase and the amount of bound DNA fragments produced.

[Explanation of symbols]

 111-124 DNA strand base sequence 51 202 bps DNA fragment band 52 400 bps DNA fragment band 61 200 bps DNA fragment band 62 2000 bps DNA fragment band

Claims (3)

[Claims] 1. An enzyme for detecting an objective substance by using an enzyme of the complex after reacting an antigen or antibody which is the objective substance with an antibody or antigen labeled with an enzyme to obtain an immune reaction complex. In an immunoassay, a ligase is used as a label for an antibody or an antigen that reacts with a target substance, an immune reaction complex having one or more ligases is obtained by the reaction and separation, and the ligase of the complex joins each other. Two kinds of DNA chains having ends are used as substrates to react with the immune reaction complex, and the bound DN generated by the reaction
An enzyme immunoassay method characterized by detecting a target substance by analyzing the A chain. 2. The enzyme immunoassay method according to claim 1, wherein the two kinds of DNA chains have ends capable of binding to each other by ligase, and each chain is not present in the other chain. An enzyme immunoassay characterized by including a step of amplifying a partial chain including a binding portion of a binding DNA strand, each step having one unique sequence portion and these two unique sequence portions serving as primer sequences. Law. 3. The enzyme immunoassay method according to claim 2, wherein the length of the amplified partial strand in the binding DNA strand is 1/10 to 1/100 of the original length of each DNA strand. The enzyme immunoassay method is characterized in that