JPH08292A - Method for emission of acridinium derivative and detection of subject substance for inspection by the same method - Google Patents

Abstract

PURPOSE:To reproducibly detect a subject substance for inspection with high accuracy and identify various kinds of diseases by using ascorbic acid as a chemiluminescence inducer, allowing an acridinium derivative to efficiently emit light and improving the intensity of emission light. CONSTITUTION:An acridinium derivative such as 9-phenoxycarbonyl-10-methyl- acridinium fluorosulfonate of the formula [(n) is 0 to 8; R1 to R3 are each a substituent group; X<-> is a counter ion] is allowed to emit light by using ascorbic acid enzymatically produced from ascorbic acid phosphate and alkali phosphatase as a chemiluminescence inducer. In addition, a subject substance for inspection is detected preferably by using this emitted light as a signal from the target.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention also relates to a method for emitting light from an acridinium derivative and a method for detecting a substance to be inspected using the method.

[0002]

2. Description of the Related Art With the progress of diagnosis and medical technology, a method for detecting a specific substance contained in a trace amount in a biological sample such as serum is required for early detection of various diseases and confirmation of therapeutic effect. Methods have been developed. Among them, the chemiluminescence method is particularly attracting attention because of its high sensitivity, and has been expanding more and more in recent years. The chemiluminescent substance actually used in the chemiluminescence method is luminol, an acridinium derivative or an adamantane compound (PPD or AMPPD). Among them, the acridinium derivative is used by Woodhead etc. for immunoassay. Its high sensitivity has attracted particular attention (Clin. Chem., 29, 1474-1479, 1983).

In the conventional method of detecting a specific substance using an acridinium derivative, the acridinium derivative is used as a labeling substance. For example, in the case of an immunoassay called a sandwich method, first, when a sample is brought into contact with an antibody against a specific substance immobilized on an insoluble carrier such as polystyrene, the specific substance contained in the sample becomes insoluble due to its affinity with the antibody. It binds on the carrier. Next, the labeled antibody in which the acridinium derivative is chemically bound to the antibody against the specific substance is brought into contact with the insoluble carrier. By this operation, the labeled antibody binds to the insoluble carrier in an amount that correlates with the amount of the specific substance bound to the insoluble carrier. next,
Excess labeled antibody not bound to the insoluble carrier is separated by a washing operation or the like, and then chemiluminescence measurement is performed. The acridinium derivative emits intense light when reacted with hydrogen peroxide under strong alkaline conditions. Therefore, as a reagent for inducing chemiluminescence, an aqueous solution of sodium hydroxide and hydrogen peroxide is usually used. A reagent that induces such chemiluminescence is brought into contact with the labeled antibody previously bound on the insoluble carrier to cause the acridinium derivative to emit light by a chemical reaction. Since the emission intensity at this time is correlated with the concentration of the specific substance in the sample, the concentration of the specific substance in the sample can be obtained by measuring the emission intensity with a photon counter or the like.

Through such a series of operations, it has become possible to detect a specific substance in a sample with high sensitivity. The reason why high sensitivity has become possible is not only the appearance of high-performance antibodies such as monoclonal antibodies, but also the acridinium derivative, which has a high quantum yield.
It is clear that they have made a particular contribution. However, the method using such an acridinium derivative has some problems, and its improvement is demanded.

[0005]

The first problem is the stability of the acridinium derivative. The acridinium derivative can be stably stored for a long period of time under acidic conditions, but a pseudobase is formed when exposed to neutral or higher conditions, and the acridinium derivative used as the pseudobase is a chemiluminescence-inducing reagent. It is known that even if they come into contact with each other, they cannot emit light (I.Weeks, I.Beheshti, F.McCapra, AKCampbel
l, and JSWoodhead, Clin.Chem., 29,1474-1479,1983;
JSLittig and TANieman, J. Biolumin. Chemilumin.,
8,25-31,1993).

On the other hand, in order to bring a specific substance to be inspected in a sample into contact with an antibody or the like labeled with an acridinium derivative and efficiently capture the specific substance, the contact is carried out under a neutral condition. There is a need. If the substance labeled with the acridinium derivative is a protein such as an antibody, its storage condition is preferably neutral condition. However, when the acridinium derivative is used as the labeling substance, the neutral condition as described above is especially disadvantageous. As a measure against this, the pseudobase formation of the acridinium derivative reversibly changes depending on the pH of the solution, that is, even if the pseudobase is formed by exposure to neutral conditions, it can be returned to the original state by returning it to acidic. The property of returning to an acridinium derivative is used. Specifically, when detecting a specific substance using an acridinium derivative as a labeling substance,
After capturing a specific substance in a sample under neutral conditions with an antibody labeled with an acridinium derivative, acid such as hydrochloric acid or nitric acid is added to make it acidic, and the chemiluminescence operation is performed after holding for a certain period of time. It is becoming popular. However, the above method is not only complicated in operation, but also the pseudo base of the acridinium derivative is not completely returned to the original state by the acid, so that the original chemiluminescence amount cannot be obtained. Furthermore, in the process of storing a solution of an antibody or the like labeled with an acridinium derivative under neutral conditions, a part of the acridinium derivative is decomposed by a dark reaction (JP-A-63-101368, JP-A-1). -261461 and JP-A-6-9566). Therefore, it is difficult to always obtain stable results.

The second problem of the acridinium derivative is
The light emission time is very short. The luminescence peak is reached within 1 second after the addition of the chemiluminescence inducing agent, and then the luminescence intensity rapidly decreases and the reaction is completed within a few seconds. Because of such luminescence characteristics, it is necessary to measure the luminescence intensity at the same time as adding a certain amount of chemiluminescence inducing agent. Therefore, the device for measuring the emission intensity becomes complicated regardless of whether it is a fully automatic device or a simple semi-automatic device. The reason is that, in the measuring device, the part that performs the step of adding a constant amount of the chemiluminescence-inducing agent is for maintenance of the device,
This is because it is necessary to form an open structure, while it is necessary to solve the contradictory problem that the portion for performing the step of measuring the emission intensity must be completely sealed and darkened. If the luminescence time is long to some extent, a time and space margin is created between the step of adding the chemiluminescence-inducing agent and the step of measuring the luminescence intensity in the measuring device, which increases the degree of freedom in designing the measuring device, and It is possible to supply inexpensive and highly reliable equipment.

In view of such a situation, the present inventors have
As a result of earnest research on a method for detecting a specific substance using an acridinium derivative, when ascorbic acid was used,
It was found that an acridinium derivative can emit strong light under neutral to alkaline conditions, and further that this phenomenon can be used to detect a specific substance. The present invention is based on these findings.

[0009]

Therefore, the present invention relates to a method for emitting light from an acridinium derivative, which comprises using ascorbic acid as a chemiluminescence inducer. Further, the present invention is a method for detecting a test substance in a test sample by detecting a signal from a label, wherein the luminescence obtained by the above-mentioned luminescence method is a signal from the label. The present invention also relates to a method for detecting a substance to be inspected.

The present invention will be described in detail below. The acridinium derivative used in the present invention has a counter ion in which the ring nitrogen atom of the acridine ring is quaternized and has a substituent at the 9-position of the acridine ring via a -C (= O)-group. Then
And, a compound which may be optionally substituted by one or more substituents at 1 to 8 positions, that is, the following general formula (1):

Embedded image (In the formula, n is 0 or an integer of 1 to 8, and R ₁ , R ₂
And R ₃ are each independently a substituent, and X ⁻ is a counter ion).

The acridinium derivative represented by the general formula (1) is, for example, EP Publication No. 830629, JP-A-63-57572, JP-A-63-101368 and JP-A-63-112564. As described in the publication, they are already known as chemiluminescent substances, and these acridinium derivatives can also be used in the method of the present invention. Specifically, 9-phenoxycarbonyl-10
-Methylacridinium fluorosulfonate, 2 ',
6'-dimethylphenyl-10-methylacridinium-9-carboxylate fluorosulfonate, 2 ',
4 ', 6'-trichlorophenyl-10-methylacridinium-9-carboxylate fluorosulfonate, 9-chlorocarbonyl-10-methylacridinium chloride, or 10- (3-sulfopropyl) -N
-Tosyl-N- (3-sulfopropyl) -9-acridinium carboxamide and the like can be mentioned.

In the conventional light emitting method, as described above, the acridinium derivative is placed under a strong alkaline condition, and hydrogen peroxide as a chemiluminescence inducing agent acts thereon to cause strong light emission. On the other hand, the method of the present invention uses ascorbic acid as a chemiluminescence-inducing agent for an acridinium derivative, and the chemiluminescence-inducing agent can act under neutral to alkaline conditions. This is a new method that did not exist. Since the mechanism of the luminescence reaction according to the method of the present invention is not yet clear, the “chemiluminescence-inducing agent” used in the present specification is not limited to those having the same luminescence reaction mechanism as conventional hydrogen peroxide. It is sufficient that the chemiluminescence-inducing agent / ascorbic acid of the present invention is simultaneously present in the solution containing the acridinium derivative, and as a result, the acridinium derivative causes chemiluminescence. Therefore, it is not limited to the case where ascorbic acid directly causes a chemical reaction with an acridinium derivative to emit light, and acts on, for example, dissolved oxygen existing in a solution, and the luminescent reaction is caused via the dissolved oxygen. It is possible that it is progressing, and this case is not excluded.

In the method of the present invention, a chemiluminescence inducer, ascorbic acid, is allowed to act under neutral to alkaline conditions to cause the acridinium derivative to emit light. here,
The neutral to alkaline conditions usually mean pH 6 to 13, preferably pH 6.5 to 13, and more preferably pH 7 to 12. When the pH is lower than 6, the emission intensity is lowered and detection becomes practically difficult. On the other hand, if the pH exceeds 13, the rate of pseudobase formation of the acridinium derivative will be increased, resulting in shortened emission time and reduced emission intensity.
When an enzyme is used, it is preferably at a pH of 11 or lower at which the enzyme activity can be expressed.

In the method of the present invention, ascorbic acid or its salt (for example, an alkali metal salt or an alkaline earth metal salt) as a chemiluminescence inducer may be added as it is to the luminescence reaction system, but preferably it is an enzyme reaction. Is used to induce ascorbic acid or a salt thereof (for example, an alkali metal salt or an alkaline earth metal salt) in the reaction system. As the enzymatic reaction, any combination of an enzyme that induces ascorbic acid or a salt thereof and a substrate can be used. For example, when the enzyme alkaline phosphatase is reacted with ascorbic acid phosphate as a substrate, the enzymatic reaction Produces ascorbic acid.

The alkaline phosphatase which can be used in the present invention may be of any origin so long as it has an optimum pH from neutral to alkaline, but calf intestine-derived alkaline phosphatase is particularly preferable. Examples of the substrate of alkaline phosphatase that can be used in the present invention include ascorbic acid 2-phosphate ester or ascorbic acid-3-phosphate ester, or an alkali metal salt or alkaline earth metal salt thereof,
One or more of these compounds can be used in combination.

According to the method of the present invention, the acridinium derivative can be brought into contact with the acridinium derivative after the ascorbic acid is produced by the enzymatic reaction of the alkaline phosphatase and its substrate (eg, ascorbyl phosphate). It becomes possible to store as an acidic solution until just before the luminescence reaction.

By using the above-described luminescence method of the present invention, for example, in a method for detecting a specific trace amount of a component present in a biological sample, a signal from a label attached to a standard substance can be efficiently detected. In the method of detecting a specific substance using a label, various standard substances are used for the test substance present in the test sample, the standard substance is labeled, and various substances in which the test substance and the standard substance participate After the reaction of, the signal from the label is detected to detect the substance to be inspected.

That is, in the case of using a test sample containing a test target substance and a standard substance having an affinity for the test target substance and capable of forming a complex with the test target substance,
By contacting the test sample with an excess amount of the standard substance, the test substance and the standard substance form a complex at a constant ratio. The amount of the complex formed here is in excess of the standard substance, and therefore correlates with the amount of the test substance contained in the test sample. Therefore, if the amount of the formed complex is known, the amount or concentration of the substance to be tested in the test sample can be determined. The method for determining the amount of the complex is such that the formed complex is separated by a known method as necessary, and the intensity of the signal generated by the label attached to the standard substance of the complex, that is, the chemiluminescence derived from the label. By measuring the emission intensity, the detection of the substance to be inspected is achieved.

When a standard substance that is chemically or immunologically the same as or similar to the test substance is used, there is affinity for both the test substance and the standard substance, and it is possible to form a complex. The reaction in which the substance participates is available. In this case, the test sample is brought into contact with a fixed amount of the standard substance and the third substance, respectively. By this step, the complex formation reaction of the test substance and the third substance and the standard substance and the third substance competes with each other, correlating with the amount of the test substance contained in the test sample, and The amount of complex formed with the third substance is reduced. Therefore, if the amount of the complex formed from the standard substance and the third substance is known, the amount or concentration of the test target substance in the test target substance can be obtained. The amount of complex may be determined by measuring the emission intensity of chemiluminescence derived from the label attached to the standard substance, as in the above method.

In the detection method according to the present invention, one of the compounds involved in the luminescence system is used as the label. For example, an acridinium derivative, ascorbic acid, an enzyme that reacts with a substrate to generate ascorbic acid, or a substrate that reacts with an enzyme to generate ascorbic acid can be used as a label. The selection of the labeling substance depends on the storage conditions of the reagent,
It can be appropriately performed in consideration of the conditions of the step of contacting the test sample with the labeled standard substance, the conditions of the luminescence reaction, and the like. When an enzyme is used as a label, an amplification effect by the enzyme is obtained,
This is advantageous in that it enables highly sensitive detection. Therefore, in the detection method according to the present invention, it is particularly preferable to use alkaline phosphatase as a labeling substance, induce ascorbic acid through an enzymatic reaction to cause the acridinium derivative to emit light, and detect the test substance.

The test sample containing the substance to be tested is not particularly limited, but in particular, a biological sample such as blood, serum, plasma, urine, saliva or spinal fluid, or a cell or tissue extract. Can be mentioned. The substance to be inspected is not limited, but for example, various proteins, polysaccharides,
It can be mentioned lipids or nucleic acids, and more specifically, fibrinogen, albumin, AFP, CRP, HB _S
Antibody, HB _C antibodies include HIV antibodies, HTLV antibodies, hormones and, antiepileptics and various drugs such as digoxin. In addition, in the nucleic acid of the substance to be tested,
A DNA fragment having a specific base sequence is also included. The labeled substance to which a label, for example, alkaline phosphatase is attached, is an antibody against the substance to be inspected, the substance to be inspected, complementary DNA, or the like.

The case of using alkaline phosphatase as a label and detecting a protein in a serum sample by an immunological method using the detection method of the present invention will be described below. In this case, an antibody that specifically recognizes the protein of the test substance is labeled with alkaline phosphatase by a known method, and an antigen-antibody reaction is carried out between the labeled antibody and the target protein in serum, which is necessary. Accordingly, the unreacted labeled antibody can be removed by B / F separation, and subsequently, the luminescence step can be carried out, for example, by the following method 2.

One method is to carry out the enzymatic reaction and the luminescent reaction in one step. Ascorbic acid phosphoric acid ester is added as a substrate for alkaline phosphatase, and an enzymatic reaction is performed at around pH 10, which is the optimum pH of alkaline phosphatase. At the same time, an acridinium derivative stored under acidic conditions is added to perform an enzymatic reaction. When it is brought into contact with ascorbic acid produced by the method, luminescence is generated. The luminescence generated by this is much longer than the luminescence of the acridinium derivative obtained by the conventional method, and therefore the luminescence intensity can be easily measured without using a specially prepared device such as the conventional one. You can

Another method is a two-step method in which the enzyme reaction and the luminescence reaction are carried out. A phosphoric acid ester of ascorbic acid is added, an enzyme reaction is carried out at around pH 10 for a certain period of time, and then a reagent for stopping the enzyme reaction, for example, p
The enzyme reaction is stopped by adding H7 phosphate buffer. Then, the acridinium derivative stored under acidic conditions is added to emit light under the condition of pH 7. The intensity of the light emission generated at this time is several thousandth of that of the one-step method, but the S / N ratio is equal to or higher than that of the method, and the light emission time is longer. Therefore, the target protein in serum can be detected with high sensitivity.

The application examples of the present invention described here are only some examples. For example, the pH at which the acridinium derivative and ascorbic acid contact to emit light is from pH 6 at which the emission intensity can be ensured to a minimum. The present invention can be carried out as long as the alkaline phosphatase has a pH range up to pH 11 at which the enzyme activity can be expressed. Further, according to the present invention, not only the immunological detection method using the above-mentioned antibody but also the nucleic acid can be detected by utilizing the affinity of the nucleic acid complementary to the nucleic acid. it can.

According to the method of the present invention, an acridinium derivative is not used as a labeling substance as in the conventional method, but alkaline phosphatase or a phosphoric acid ester of ascorbic acid as a substrate thereof is used as a labeling substance, and ascorbine is enzymatically reacted. Since the acridinium derivative can be contacted after the acid is generated, it is not necessary to allow the acridinium derivative to be present in the immune reaction system. Therefore, the acridinium derivative can be stored as an acidic solution until just before the luminescence reaction. Furthermore,
Since it is possible to emit light for a long time, various problems in the conventional method can be solved at once.

[0027]

The present invention will be described in detail below with reference to examples, but these do not limit the scope of the present invention. Example 1: Luminescent pattern of luminescent reaction by one-step method
Alkaline phosphatase solution 10μl from down calf intestine
(7 × 10 ⁻¹⁴ mol), 10 mM of magnesium salt of ascorbic acid-2-phosphate and about 1 of magnesium salt of ascorbic acid-3-phosphate prepared with 10 mM sodium carbonate buffer (pH 10) 90 μl of the mixture solution of 1: 1 and 50 μl of 25 μM 9-phenoxycarbonyl-10-methylacridinium fluorosulfonate (PMA) adjusted to pH 4 with hydrochloric acid were added, and the luminescence intensity with respect to the elapsed time was measured with a photon counter. It measured and obtained the light emission pattern. The results are shown in FIGS. 1 and 2.

In FIG. 1 and FIG. 2 (further shown in FIG. 3), the AP
(+) Indicates AP in the presence of alkaline phosphatase
(−) Indicates the result in the absence of alkaline phosphatase. As is clear from FIG. 1, the method of the present invention makes it possible to prolong the emission time and obtain a sufficient S / N ratio. FIG. 2 is an enlarged view of the initial portion of light emission in FIG. From this figure, it is understood that the emission intensity linearly increases in the initial stage of emission. Therefore, by obtaining the inclination of this straight line portion, highly accurate measurement can be performed.

Example 2: Luminescent reaction by two-step method
The same amount of alkaline phosphatase as in Example 1 was used for Example 1.
Add ascorbic acid phosphoric acid ester in the same manner as
The enzyme reaction was performed for a minute. Next, 100 μl of 0.5 M phosphate buffer (pH 7) was added to stop the enzyme reaction. To 100 μl of this reaction solution, 80 μl of the same acridinium derivative as in Example 1 was added, and the luminescence intensity was immediately measured to obtain a luminescence pattern. The result is shown in FIG. The unit of the vertical axis in FIG. 3 corresponds to 1/1000 of the unit of the vertical axis in FIG. As can be seen from FIG. 3, stable light emission was obtained for a longer period of time.

Example 3: AFP (α-fetoprotein)
1) Anti-AFP antibody dilution 1 on a 96-well microplate
An anti-AFP antibody was adsorbed and immobilized on a microplate by dispensing 00 μl and leaving it to stand at 4 ° C. for 17 hours.
Next, place the microplate on TBSBT (0.05% -B
Tris hydrochloric acid buffer solution containing SA, 0, 5 ml / liter Tween 20, and 8.77 g / liter NaCl) was washed 3 times to remove unadsorbed anti-AFP antibody. To this microplate, 200 μl of a 1% BSA solution prepared with Tris-HCl buffer was dispensed, and allowed to stand at room temperature for 2 hours for blocking. It was washed three times with TBSBT to remove unadsorbed BSA. The standard solution of AFP diluted to each concentration was dispensed into a microplate, and an antigen-antibody reaction was performed at room temperature for 2 hours. TBSB
After washing three times with T, 100 μl of an alkaline phosphatase-labeled anti-AFP antibody solution was dispensed, and the mixture was allowed to stand at room temperature for 3 times.
The reaction was allowed for 0 minutes. It was washed 5 times with TBSBT to remove unreacted labeled antibody. Next, 100 μl of the phosphoric acid ester solution of ascorbic acid used in Examples 1 and 2 was dispensed as a substrate for the enzyme, and the enzyme reaction was performed for 15 minutes. Next, 100 μl of 0.5 M phosphate buffer (pH 7) was dispensed to stop the enzyme reaction. 100 μl of this reaction solution was placed in a luminescence measuring container, 80 μl of the solution of the acridinium derivative used in Examples 1 and 2 was injected, and the luminescence intensity after 90 seconds was measured. The detection curve of AFP obtained by this series of operations is shown in FIG. In Fig. 4, a solid line and ・ (-
・-) Is the detection curve of AFP, and the broken line (---) is A
The average value of the luminescence intensity at FP0 g / ml + 3SD is shown. In this example, AF at a concentration of 5 picogram / ml was used.
It was possible to detect up to P.

[0031]

According to the luminescence method of the present invention, by using ascorbic acid as a chemiluminescence inducer, an acridinium derivative can emit light under neutral to alkaline conditions, and all the problems of the conventional method can be solved. Can be resolved. At the same time, the emission intensity is increased and the emission time can be extended, so that the substance to be inspected can be detected with high accuracy. By applying this luminescence method to detect the substance to be inspected, for example, alkaline phosphatase is used as a labeling substance, and an acridinium derivative which is unstable under neutral to alkaline conditions is kept under acidic conditions until just before the luminescence reaction. It became possible to save. Moreover, since the light emission time is significantly longer than that of the conventional method, not only the degree of freedom in the configuration of the light emission measuring device is increased, but also a more accurate and highly accurate detection method can be provided. According to the present invention, a method for detecting a substance to be tested in a sample by using a chemiluminescence analysis method, for example, an enzyme such as CLEIA (chemiluminescent enzyme immunoassay) as a labeling substance and measuring the enzyme activity by the chemiluminescence method. In the above, a very effective means is provided for a method of detecting an inspection target substance using an acridinium derivative as a chemiluminescent substance.

[Brief description of drawings]

FIG. 1 is a graph showing a luminescence pattern when the enzymatic reaction of alkaline phosphatase and the luminescence reaction of an acridinium derivative are carried out in one step.

FIG. 2 is an enlarged view of the initial stage of FIG.

FIG. 3 is a graph showing a luminescence pattern when the enzymatic reaction of alkaline phosphatase and the luminescence reaction of an acridinium derivative are carried out in two steps.

FIG. 4 Anti-AFP labeled with alkaline phosphatase
It is a calibration curve when AFP is detected using an antibody.

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Claims (6)

[Claims] 1. A method for emitting light from an acridinium derivative, which comprises using ascorbic acid as a chemiluminescence inducer. 2. The method according to claim 1, wherein ascorbic acid produced by an enzymatic reaction is used. 3. The method according to claim 2, wherein ascorbic acid produced by an enzymatic reaction of ascorbic acid phosphoric acid ester and alkaline phosphatase is used. 4. A method for detecting a substance to be inspected in a test sample by detecting a signal from a label, wherein the luminescence obtained by the luminescence method according to any one of claims 1 to 3 is used. Characterized by being a signal from a sign,
Method of detecting substances to be inspected. 5. The method according to claim 4, wherein the method of detecting the test substance is an immunological reaction. 6. The method according to claim 4, wherein an enzyme that reacts with a substrate to produce ascorbic acid is used as a label.