JP3296078B2 - Enzyme immunoassay - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an enzyme immunoassay, and more particularly, to an enzyme immunoassay for easily detecting a trace amount of a target substance with high sensitivity.

[0002]

2. Description of the Related Art In order to identify and quantify antigens or antibodies with high sensitivity, high specificity of an antigen-antibody reaction is used, which is generally called an immunoassay (immunoassay). This immunoassay includes immunoturbidimetry (TIA), which optically detects precipitates and aggregates generated by an antigen-antibody reaction, and labeled immunoassay using an antibody or antigen labeled with a substance that can be easily separated and detected. Separated. In particular, in the latter, various systems are considered depending on the label, and typical examples include a radioimmunoassay (RIA) using a label containing a radioisotope, an enzyme immunoassay (EIA) using an enzyme as a label, There is a fluorescence immunoassay (FIA or FAT) using a fluorescent substance as a label. Looking at the tests currently applied, those using TIA include serum proteins such as transferrin and CRP, immunoglobulin IgG, IgA, and Ig.
M, rheumatoid factor in relation to autoimmunity, and hematological tests include quantification of plasminogen and antithrombin III, while RIA is a labeling immunoassay using an internal secretion test (eg, Erythropoietin, prostaglandin), tumor related tests (eg AFP,
CEIA), biochemical tests (for example, trypsin) and a wide range. EIA methods are hematological tests (for example, protein S, FPA, etc.), immunoserologic tests (for example, immunocomplexes, microglobulin, etc.), and FAT. It is used for virus antibody tests (eg, EBV, herpes, etc.) and immunoserologic tests (eg, antinuclear antibodies, etc.).

[0003]

[Problems to be Solved by the Invention] As described above, immunoassays are widely used for clinical examinations and the like, but RIA is generally used to detect a target substance with high sensitivity. is there. However, in this RIA method, there is a problem that radioisotope as a labeling substance cannot be stored, and handling is troublesome due to an influence on a human body.

The present invention has been made in view of such a problem, and has as its object to easily detect a target substance with high sensitivity.

[0005]

In the present invention, an antibody or an antigen is labeled by using ligase as an enzyme to form a detection reagent, and the detection reagent is reacted with an analyte to thereby form one or more target substances. After the separation of the unreacted detection reagent, two types of DNA strands having ends that can be joined to each other are reacted with the immune reaction complex as a substrate. By analyzing the DNA chain, the presence or absence and amount of the ligase, and thus the presence and absence and amount of the target substance were detected. If the target substance is estimated to be extremely small, a step of amplifying a partial chain containing a binding portion in the binding DNA chain is added, and the amplified partial chain is replaced with the original strand to shorten the time of the detection step. It was much shorter.

[0006]

According to the present invention, a ligase is attached to an antibody or an antigen to form a detection reagent, which is allowed to react with an analyte. This allows the antibody or antigen to bind with very high specificity if the antigen or antibody as the target substance is present in the subject. This is subjected to B / F separation to remove the unreacted detection reagent, whereby an immunoreactive complex is obtained. The condition at this time is that a small amount of the immune reaction complex has ligase. In order to detect this trace ligase, two types of DNA strands having ends that can be joined to each other are added and reacted. In this case, a large amount of long DNA chains bound by the action of ligase is generated. Therefore, if these are fractionated by, for example, gel electrophoresis, these DNA strands are localized at a specific position, and are easily detected by fluorescence observation by staining with ethidium bromide or the like. Alternatively, it is 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 in an amount sufficient for detection. At this time, two types of DNA strands used as substrates are used such that each strand has one unique sequence portion which is not present in the other strand, and each of the unique sequence portions becomes a primer sequence. . The two kinds of DNA strands are reacted with a very small amount of ligase, and the bound DNA strands produced by the reaction are amplified by a PCR method known in the art using primers in each of the strands. Is amplified, resulting in a large amount of DNA strand of a certain length. This can be analyzed and detected as before. Further, preferably the length of the strand resulting from the amplification is much shorter than that of each of the original strands.
If it is designed to be about 10 to 1/100, the electrophoresis time is shortened during analysis, and the result can be obtained in a shorter time.

[0007]

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

[0008]

[Example 1] (1) Preparation of detection reagent (enzyme-labeled antibody) T4 DNA Ligase (ligase) was dissolved in 0.1 mol / 1 sodium phosphate buffer (pH 7.0) as an enzyme, and 500 to 100
An equivalent of N-succinimidyl-4- (N-maleimidomethyl) siloxane-hexan-1-carboxylate in N, N-dimethylformamide was added and reacted at 30 ° C. for 1 hour to precipitate the unreacted maleimide compound. After removal by centrifugation, the supernatant was equilibrated with 0.1 M sodium phosphate buffer (pH 6.0).
The target substance was purified using a Sephadex-G-25 column. This fraction was mixed with a sodium phosphate buffer containing 5 mmo1 / 1 EDTA in which an anti-CEA antibody was dissolved, and the mixture was incubated at 4 ° C for 20 hours.
After that, the target fraction was purified with a UItrogel AcA 44 column equilibrated with 0.1 M sodium phosphate buffer (pH 6.5), and the yield of anti-CEA antibody labeled with T4 DNA ligase was determined.
It was obtained at 85% and used as a detection reagent. (2) Preparation of substrate DNA strand for detection First, a DNA strand 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 their bases, and 3 ′ and 5 ′ indicate the ends of the strand. 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 not including this cleavage sequence, and both have a length of 196 bps. The DNA strand thus produced was cleaved with Not I and purified to obtain two detection substrate DNA strands as shown in FIG. The Not I cleavage site sequence is

[0009]

Embedded image

[0010] (3) Preparation of immobilized antibody A solution prepared by dissolving an anti-CEA antibody in a sodium phosphate buffer (pH 7.2) is added to a microtiter plate.
Incubated overnight. The solution was discarded and washed with a sodium phosphate buffer (pH 7.2) to carry out B / F separation to prepare an immobilized antibody on which an anti-CEA antibody was immobilized. (4) Preparation of Immobilized Antibody-Antigen-Labeled Antibody Complex (Labeled Immunoreactive Complex) A standard sample of CEA, which is a test sample, was added to a well and incubated at room temperature for 2 hours. After discarding the solution and washing with a sodium phosphate buffer (pH 7.2), the enzyme-labeled antibody, which is the detection reagent prepared above, is diluted with a sodium phosphate buffer and added to the wells, and the mixture is added at room temperature for 1 hour. Incubated. Thereafter, the solution is discarded and washed with a sodium phosphate buffer to remove the unreacted detection reagent, and the immobilized antibody-antigen (CE
A)-A labeled antibody complex (labeled immunoreactive complex) was formed. (5) Detection In a well, a buffer (50 mM Tris-HCl (pH 7.9), 10 mM MgCl ₂ , 20 mM DD) which is favorable for T4 DNA Ligase
T, 1.0 mM ATP) and the detection DNA strand of (2), and add
After incubating for 0 minutes, the solution in the well was subjected to electrophoresis on a polyacrylamide gel to obtain an electrophoretic pattern.
The gel was stained with ethidium bromide, the fluorescence was measured, and the original 202 bp
s, the amount of each DNA fragment of the bound band at 400 bps was calculated to quantify the antigen. The results are shown in FIG. 5 as an electrophoresis pattern. In the figure, 50 is a polyacrylamide gel plate, the direction of the arrow is the electrophoresis direction, 51 is 202 bp
s and 52 show the band of the 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 unused wells of plate
Then, the detection operation of (5) was performed under the same conditions as in the above-mentioned antigen-existing system, and the relationship between T4 DNA ligase and the amount of the bound DNA fragment produced was plotted and created. As a result, a calibration curve as shown in FIG. 7 was obtained.

[0011]

Example 2 (1) Preparation of detection reagent The same procedure as in Example 1 was used. (2) Preparation of substrate DNA chain for detection The sequence of λgt10 primer is used as a unique sequence for each of the primer portions in the two types of substrate DNA chains. λgt
The sequence for the forward direction of 10 primers is 5 ′ (GCTGGGTAGTCCCCACC
TTT) 3 'and the sequence for the reverse direction is 5' (CTTATGAGTATTTCTTCCA
GGGTA) 3 ', which are referred to herein as λgt10F and λgt10R. 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 thereof, and a λgt10F and λgt10R sequence on each side thereof, one each, was synthesized using a DNA synthesizer. Was prepared. Here, FIG. 3 will be described. 117 and 118 are 8 bps sequences showing Not I cleavage sequence, the same sequence as N in FIG. 1, 121 is a sequence of λgt10F, 122 is a sequence complementary thereto, 114 is a sequence of λgt10R, 1
13 shows the sequence complementary thereto, 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,
Any sequence not including any sequence of λgt10R.
Incidentally, 3 'and 5' are conventional notations indicating the ends of the DNA strand.
The DNA strand thus produced is cut with Not I,
Purification yielded two detection substrate DNA chains as shown in FIG.
4, 113, 114, 117, 118, 121 and 122 are the same as those described in FIG. (3) Preparation of immobilized antibody Pyrex glass micro test tube (size that fits into the heater part of a commercially available PCR amplifying device) is dried at 500 ° C for 5 hours, and then dried at 45 ° C.
For 24 hours in a 2% acetone solution of 3-aminopropyltriethoxysilane, then washed and dried, and then immersed in a 1% aqueous glutaraldehyde solution for 1 hour. A solution prepared by dissolving the CEA antigen antibody in sodium phosphate buffer (pH 7.2) was added thereto, and the mixture was incubated at 4 ° C. overnight. Then, the solution was discarded and the solution was added to sodium phosphate buffer (pH 7.2). And washed. Thus, an immobilized antibody immobilized on the test tube was obtained. (4) Preparation of immobilized antibody-antigen-labeled antibody complex (immune reaction complex) A test sample containing the immobilized antibody treated in (3) was added with the CEA antigen standard sample as a test sample, 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, which is the detection reagent prepared above, is diluted with sodium phosphate buffer and added to a test tube. 1 hour incubation at
I did it. (5) Detection of label The solution in the test tube is discarded, washed with a buffer, and subjected to B / F separation. After that, a buffer (50 mM Tris-HCl (pH7. 9), 10mM MgCl ₂ , 20mM DD
T, 1.0 mM ATP, 16 ° C) and the detection DNA strand of (2), and add
For 30 minutes. This solution contains Taq-polymerase as DNA polymerase and λgt10F and λg as primers.
T10R, a solution obtained by dissolving dNTP in buffer was added as an amplification materials, further KCl, added TritonX-100, the composition of the final liquid is 10mM Tris-HCl, 50mM KCl, 1.5mMMgCl 2, 0.1% TritonX
-100, and PCR was performed by a known method.
Thereafter, the liquid in the test tube was electrophoresed on a polyacrylamide gel to obtain an electrophoresis pattern. The gel was stained with ethidium bromide, its fluorescence was measured, and the amount of each DNA fragment of the original band at the position of 200 bps and the bound 2000 bps was calculated from the calibration curve created at the same time to quantify the antigen. Was. The results are shown in FIG. 6 as migration patterns. In the figure
Reference numeral 60 denotes a polyacrylamide gel plate, the direction of the arrow indicates the direction of electrophoresis, 61 indicates a band of a DNA fragment of 200 bps, and 62 indicates a band of a DNA fragment of 2000 bps. (6) Preparation of calibration curve The one in Example 1 was applied.

As described above, in the present embodiment, electrophoresis is used for detection, but it can be easily performed by means of chromatography or the like.

[0013]

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

[Brief description of the drawings]

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

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

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

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

FIG. 5 is an electrophoresis pattern of a substrate DNA strand for detection in Example 1.

FIG. 6 is an electrophoresis pattern of a substrate DNA strand for detection in Example 2.

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 to 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

 ──────────────────────────────────────────────────続 き Continuation of front page (56) References JP-A-3-167474 (JP, A) JP-A-5-149949 (JP, A) JP-A-6-43135 (JP, A) JP-A-7-43 265076 (JP, A) JP-A-7-270418 (JP, A) JP-A-7-270419 (JP, A)

Claims (3)

(57) [Claims] An immunoreactive complex is obtained by reacting an antigen or antibody as a target substance with an antibody or antigen labeled with an enzyme, and the complex is separated from unreacted substances by B / F separation. In an enzyme immunoassay for detecting a target substance using an enzyme of a complex, a ligase is used as a label of an antibody or an antigen that reacts with the target substance, and the immunoreaction complex having one or more ligases by the above reaction and separation Body and two types of DNs having ends joined together by the ligase of the complex
An enzyme immunoassay comprising reacting an A-chain as a substrate with an immunoreactive complex and analyzing a bound DNA chain produced by the reaction to detect a target substance. 2. The enzyme immunoassay according to claim 1, wherein the two types of DNA strands have ends that can be linked to each other by ligase, and each strand is not on the other strand. An enzyme immunoassay comprising a step of amplifying a partial chain containing a binding portion of a binding DNA strand, wherein each of the two unique sequence portions serves as a primer sequence. Law. 3. The enzyme immunoassay according to claim 2, wherein the length of the amplified partial strand in the bound DNA strand is 1/10 to 1/100 of the length of each original DNA strand. An enzyme-linked immunosorbent assay, characterized in that: