JPH051995A - New measurement method using metal porphin - Google Patents

Abstract

PURPOSE:To provide a highly sensitive measurement method for specific bonding, ensuring freedom from an adverse effect due to the use of an unstable reagent, and the saving of a measurement time, labor and a sample. CONSTITUTION:In a specific bonding measurement method in the title, a complex of a catalyst and an element bonded thereto is detected, and the existence of an analysis object in a sample is measured on the basis of one or more types of bonding reaction. A complex of metal porphin catalyst is caused to react with a chemical luminous substance at a high pH value in the presence of no oxidizing agent. Then, chemical luminosity in proportion to the catalyst complex is measured.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a novel quantitative method using a photosensitive substance such as metal porphine. More specifically, the present invention relates generally to the field of quantifying water-soluble analytes using metal porphine, and more particularly to chemiluminescent detection methods and catalysts used in diagnostics.

[0002]

2. Description of the Related Art Although a chemical method-based measurement method is very useful for detecting and quantifying various substances which appear in a liquid medium at extremely low concentrations and are important for medicine, environment and industry, Direct detection of low concentrations is difficult unless the substance to be analyzed itself exhibits strong fluorescence or chemiluminescence. Therefore, the presence of the analyte is typically measured indirectly at the detection stage using a signal-generating molecule (usually a catalyst) attached to the binding member. Prior to this detection step, another chemical reaction (generally involving an antibody or nucleic acid binding reaction) is performed. Binding members are characterized by having a high binding affinity for other molecules in the assay, such as antibodies or analytes. Enzymes are most often used as catalysts that are used to form conjugates for detection. The enzyme detection reaction is characterized by incubating the enzyme for a certain period of time under specific conditions (pH, salt concentration, temperature, substrate, buffer composition, etc.), and then measuring the enzyme reaction product. Since the enzyme is inherently unstable, the assay method incorporating the enzyme catalyst tends to have a short shelf life. Therefore, a more specific detection reaction that does not use the enzyme catalyst is demanded.

On the other hand, the sensitivity of the measuring method is very important,
The detection limit at the detection stage often limits the sensitivity of the assay, but chemiluminescent detection systems are the preferred method because they provide the highest sensitivity. In a chemiluminescent reaction, a chemiluminescent compound (a compound capable of generating light) is usually oxidized to generate photoelectrons, and the photoelectrons are counted by a very sensitive measuring instrument. The most common chemiluminescent system utilizes the oxidation reaction of luminol with hydrogen peroxide catalyzed by heme catalysts or heme-containing proteins. Studies of different oxidation systems with different catalysts have found that the system of microperoxidase with hydrogen peroxide has the highest detection sensitivity [Schroeder and Eger, Analytical Chemistry.
(Schroeder and Yeager, Anal. Chem.), 50
Vol., 1114-1120, 1978]. Horseradish peroxidase, a heme-containing protein, has found wide application in the oxidation reaction of luminol with hydrogen peroxide.

All of these methods require the addition of hydrogen peroxide or a strong oxidant such as its urea adduct or chlorite to the reaction mixture in high concentrations (0.1 mM-10 mM). However, these agents are unstable and inconvenient to store, use and treat. Furthermore, these methods have a high background resulting from the oxidation reactions of other substances (e.g. proteins) present in the reaction solution and the oxidation reactions of other substances such as iron which catalyze the oxidation reactions of chemiluminescent substances. There are problems that arise. Therefore, it is desired to develop a system that does not depend on adding a strong oxidant at a high concentration to the detection reaction solution.

In recent years, studies of chemiluminescence have focused on chemical reactions that do not require the addition of high energy oxidants. One method is to use heat to generate a chemiluminescent detection signal [Hummelen et al., Pure Applied Chemistry.
Appl. Chemistry), 59, 639-644, 19
1987]. Another method is to develop a chemiluminescent enzyme substrate that already contains a high-energy but stable dioxetane [Bronstein et al., Journal of Bioluminescence and Chemiluminescence].
(Bronstein et al., J. Biolum. Chemilum.), 4
Vol., 99-111, 1989]. The stability and high sensitivity of the labels of these dioxetanes is a major improvement in the art, but the disadvantages of using enzyme labels remain.
The detection reaction usually takes a long time to obtain the highest sensitivity.
(10 minutes or more) is required. Most commercially available chemiluminescent systems use iron porphyrin protein complexes that interact with hydrogen peroxide to oxidize luminol, but compounds other than hydrogen peroxide act directly on the iron porphyrin protein and are required for catalysis. It has long been known that active oxidation intermediates can be produced [George, J. Biol. Chem., 201, 413-].
426, 1953). In addition, it has succeeded in luminol chemiluminescence without adding an oxidizing agent with divalent iron [Zeitz and Hercules, Analytical.
Chemistry (Seitz and Hercules, Anal, Che
m.), 44, 2143-2149, 1972]. However, this system has a poor detection limit (100p
M), the iron is, before oxygen and luminol react,
It must be stabilized so that it is not oxidized by oxygen. Moreover, this system cannot be applied to the measurement of antibody or DNA. This is because it is difficult to make a practical method of binding iron to a protein or nucleic acid so that iron can be used as a catalyst for chemiluminescence detection method.

Iron porphyrin (as microperoxidase or horseradish peroxidase) complexed with the above-mentioned protein is used as a label for an antibody and is detected by chemiluminescence. Hydrogen peroxide must be added. We succeeded in measuring hydrogen peroxide by substituting manganese for iron in porphine [Saito et al., Analytical Science (Saito et al., Anal. Sci.), 3
Vol. 171-174, 1987], the iron protein compound, peroxidase, is more active than the manganese porphine compound. Manganese porphine has also been shown to have catalase or peroxidase activity in systems using hydrogen peroxide, but does not show improved activity compared to natural iron protein compounds (e.g. horseradish peroxidase) [Saito et al. , Chemical and Pharmaceutical Bulletin (Saito et al., Chem. Pharm. Bul
l.), 34, 2885-2889, 1986, and 35, 869-872, 1987]. Therefore, although there have been some studies in the field of metal porphine catalyzed reactions, the produced compounds have less catalytic activity than natural iron porphyrin compounds, and in each case hydrogen peroxide is the substrate for the reaction. It is used.

[0007]

The present invention derives from research on photosensitive dyes in high pH luminol solutions. In this system, for example, a photosensitizing dye receives light to create singlet oxygen, which in turn produces chemiluminescence from luminol. The inventors have surprisingly found that some manganese porphines catalyze the chemiluminescent reaction of luminol very efficiently in the absence of light and in the absence of high energy oxidants. I found it. Further studies showed that metals were required for the porphine ring, manganese performed significantly better than iron, and that the electron-withdrawing groups on the phenyl ring of porphine chemiluminesce in the absence of hydrogen peroxide. It was found to further improve the reaction. As a result of further research, they have found that this activity is enhanced 100 times or more by an amphipathic substance such as a certain kind of surfactant or an organic acid as an additive. This is probably because these additives interact with the porphine ring to produce manganese,
It is believed to be by promoting the reaction with oxygen or luminol or both.

Thus, the main object of the present invention is to simplify the reactions used in the detection step in diagnostic methods.
This objective is achieved by eliminating the need for high energy compounds to generate chemiluminescence from luminol. For example, only the presence of sodium hydroxide and luminol in water is needed for chemiluminescent detection of metal porphines. Thus, the present invention is significantly simpler than prior art methods that require, for example, the mixing of hydrogen peroxide or other oxidants, or the heating or light irradiation to generate oxidants of energy. Can be converted. In a preferred embodiment, for example, an antibody labeled with manganese-mesotetra-4-carboxyphenylporphine is used in the assay system, and the manganeseporphine-labeled antibody bound as a result of the antigen-antibody reaction is incubated with an excess amount of luminol at pH 12.7 to obtain luminol. The generated light is integrated for 1 to 2 minutes after the reaction is started.

Another object of the invention is to improve the stability of the reagents required for immunoassays. This is accomplished in two ways, eliminating the need for unstable, high energy oxidants or oxidant precursors. In addition, the method of the present invention,
The commonly used enzymes can be replaced by the more stable metalloporphins. In a preferred embodiment, only one type of detection reaction solution can be prepared in advance and stored for a long period until the time of measurement. Still another object of the present invention is to reduce non-specific (incidental) binding of the labeled catalyst in a solid phase immunoassay system (eg, ELISA method). In many assays, sensitivity is limited by such nonspecific binding, and reducing nonspecific binding can directly improve sensitivity. To improve this sensitivity, a small porphine catalyst (molecular weight: 1000) that rarely binds to the solid phase is used instead of the enzyme catalyst (molecular weight: 40,000 to 200,000), which has a large molecular weight and easily binds to the solid phase. It will be possible.

Yet another object of the present invention is to reduce the complexity and cost of the equipment used in the measurement method. This is accomplished by providing a chemical reaction that produces a long-lived chemiluminescent light signal without the need to mix two or more separate solutions prior to use. Many conventional methods require mixing two or more labile solutions just prior to the detection step. In some cases, this mixing must be done in the optical cell of the photoelectron detector. The present invention eliminates this need because it produces a light signal that lasts as long as 15 minutes or more. The present invention, which produces a long-lived light signal, allows light to be measured for various times after the initiation of the chemiluminescent reaction. Yet another object of the invention is to increase the amount of light produced by a chemiluminescent reaction. This is advantageous as it allows the photo-detecting device used to be simpler and reduces the time required to obtain a measurement. The increase in the amount of light is
This is accomplished by including certain additives in the chemiluminescent reaction solution to accelerate chemiluminescent generation of light by a factor of 100 or more.

Still another object of the present invention is to provide a method capable of conveniently performing two simultaneous measurements. This is achieved by merging the chemiluminescent reaction from the metal porphine and the chemiluminescent reaction from the photosensitive dye by photoexcitation. Chemiluminescence catalyzed by metal porphine can be measured in the presence of a photosensitive dye, which can be measured in the presence of metal porphine. From this, each can be detected separately using two separate labeled antibodies in the same assay step. Thus, the simultaneous assay method can be constructed to save assay time and effort and sample usage.

That is, the present invention provides (1) a specific binding assay method for detecting the presence of an analyte in a sample by detecting a complex of a catalyst and an element that binds to the catalyst and performing one or more binding reactions. In step a. Reacting a metal porphine catalyst with a chemiluminescent agent at high pH in the absence of an oxidant,
b. Specific binding assay, characterized by measuring chemiluminescence proportional to the concentration of the catalyst complex, and (2) detecting a complex of a catalyst and an element that binds to the catalyst to detect one or more species. In the specific binding measurement method for measuring the presence of an analyte in a sample by the binding reaction of
a. Reacting a metal porphine catalyst complex with a chemiluminescent substance at high pH in the absence of an oxidant, b. Promoting the reaction of the metal porphine catalyst with an amphipathic substance, c. A specific binding assay, characterized by performing chemiluminescence proportional to the concentration of the catalyst complex, (3) detecting two complexes of a catalyst and an element that binds to it, In a specific binding assay in which two analytes in a sample are measured by one or more binding reactions, the detection comprises: a. Reacting a photosensitive dye-catalyzed complex and a metal-porphine-catalyzed complex with a chemiluminescent substance at high pH in the same reaction mixture in the absence of an oxidant, b. Measuring the chemiluminescence emitted from the metal polyfin catalyst, which is proportional to the concentration of the metal porphine catalyst complex in the absence of light, c. Specific binding assay, characterized by measuring chemiluminescence from a photosensitive dye, which is proportional to the concentration of a photosensitive catalyst complex formed during or after light irradiation, (4) a. A first container containing a composite of a metal porphine and elements associated therewith; b. High pH
And a second container for accommodating a chemiluminescent substance to which an oxidizing agent is not added, and a kit for measuring specific binding, comprising: (5) a. A first container containing a composite of a metal porphine and elements associated therewith; b. A kit for measuring specific binding, comprising: a second container containing a chemiluminescent substance and an amphipathic substance at a high pH and containing no oxidizing agent.

The catalyst used in the present invention is, for example, phenylporphines having a metal atom in the central nucleus. As the metal atom, iron and manganese are preferable, and manganese is particularly preferable. That is, the catalyst made of manganese is more active than the catalyst made of iron. Other metals such as aluminium, zinc or lead are less preferred as the resulting catalyst has very weak or no activity. The phenyl ring in the phenylporphines is preferably one in which the para position is substituted with an electron-withdrawing residue such as a carboxyl group or a sulfone group. To bind to a binding member such as an antibody or DNA, it is preferable to use a metal porphine having a carboxyl group introduced. This is because there are many chemical methods by which this carboxyl residue can be linked to other residues in the protein or nucleic acid that are binding members. For attachment to proteins such as avidin or antibodies it is most desirable to make active derivatives such as succinimide, which can easily attach porphine catalysts to the amino groups of proteins.

The catalyst, when bound to another molecule which is thus a binding member, is referred to as a "conjugate". At the detection stage, this complex can be quantified by incubation in a buffer (aqueous solution) with Chemilumiphore used as chemiluminescent substance. The chemiluminophore is a molecule that produces light when it reacts with a catalyst under basic pH. Luminol is the most preferred chemiluminophore, but other chemiluminophores such as luminol derivatives (eg, isoluminol), lucigenin or benzopyrene and the like can also be used. It goes without saying that other chemiluminescent substances can also be used. Luminol is used at a concentration of at least 0.002 mM, preferably 0.01 mM to 2 mM. For maximum detection sensitivity, luminol is recrystallized from alkaline solution and stored in plastic containers. The composition of the buffer solution for maintaining the pH is not particularly important, but a simple aqueous sodium hydroxide solution is preferable. The reaction between the metal porphine catalyst and the chemiluminescent substance is carried out at a pH of 11 or more, preferably 12 to 13.5. The reaction temperature is also not particularly important, but a temperature of 15 to 35 ° C is preferable.

If high light output is desired, it is preferable to use enhancers obtained by diluting the amphiphile. For example, the following surfactants are preferred for addition to the detection solution to increase light output. That is, Tween 20, Tween 40, Tween 60, Tween 80, and Tween 85. Tween 20 is the most preferable because it has the highest promoting effect. Other surfactants such as sorbitan monolaurate, sorbitan monooleate and Triton X-100 are also advantageous, but their use has a relatively low light level enhancing effect. Utilizing the promoting effect of the surfactant may slightly change the optimum reaction conditions. In the presence of a surfactant, higher concentrations of luminol are preferred, concentrations of 0.2-2 mM being particularly preferred.

Other enhancers include, for example, organic acids. The organic acid preferably has about 10 or more carbon atoms, and unsaturated fatty acids are particularly advantageously used.
Among them, linoleic acid and linolenic acid are effective. The concentration is used in the range of about 0.01% to about 1%, preferably 0.05 to 0.2%. Suitable conditions for chemiluminescent reactions catalyzed by metal porphines are also suitable for chemiluminescent reactions catalyzed by photosensitive dyes. In the photosensitive dye system,
A dye such as methylene blue absorbs photoelectrons and is excited to generate singlet oxygen from dissolved oxygen molecules. Singlet oxygen reacts with luminol to emit light.
In addition, the excited dye directly reacts with luminol to emit light. In order to perform the simultaneous measurement of the metal porphine and the photosensitive dye, the same conditions as described above for the metal porphine in the absence of the enhancer and the light intensity of 5% or more of the maximum light intensity generated by the metal porphine under assay are used. It is preferable to use a photosensitive dye that forms. Chemiluminescence does not occur from the photosensitive dye in the absence of light, so metal porphine chemiluminescence detection methods are compatible even in the presence of high concentrations of the photosensitive dye. The measurement and quantification of chemiluminescence by the measuring method of the present invention can be performed by known methods.

According to the quantification method of the present invention, for example, various substances which are present in a liquid at an extremely low concentration and which are medically, environmentally and industrially important can be measured. In particular, substances that can be used for clinical diagnosis are mentioned, for example, human immunoglobulin G, human immunoglobulin M, human immunoglobulin A, human immunoglobulin E, human albumin, human fibrinogen (fibrin and their degradation products) contained in body fluids. , Α-fetoprotein,
Examples thereof include C-reactive protein, β ₂ -microglobulin, myoglobin, carcinoembryonic antigen, hepatitis virus antigen, human chorionic gonadotropin, human placental lactogen, insulin and other proteins, hormones, and administered drugs. The body fluids mentioned above include blood such as whole blood, serum, and plasma.
Examples include urine, spinal fluid and extracts from tissues. As a reagent for measurement, metal porphines, for example, hapten manganese porphine having a carboxyl group introduced,
Antigens or antibodies against them, as well as RNA and DN
A metal porphine-labeled product can be prepared by chemically bonding with A.

As an example of the measurement kit using the labeled metal porphine, a kit by the sandwich method will be described below. (1) Antibodies retained on a carrier (2) Metal porphine-second antibody complex (3) Standards of substances to be measured (4) For diluting reagents and test samples of (2) to (3) above Buffer to be used (any buffer may be used as long as it can be used for diluting the reagent and the test sample, and an example thereof is a phosphate buffer of pH 6 to 9 or a glycine buffer) ( 5) After incubation, a buffer solution used for washing the carrier (any buffer solution can be used for washing the carrier, and examples thereof include a phosphate buffer solution or a glycine buffer solution). 6) Reagents necessary for measuring metal porphine activity. An example thereof is luminol dissolved in an alkaline solvent. The above kit can be used, for example, by the following method. Approximately 10 to 2 of the standard sample or test solution
The reagent (4) is added to 00 microliters to dilute the solution, and a fixed amount of the reagent (1) and then 10 to 300 microliters of the reagent (2) are added, followed by reaction at about 0 to 40 ° C. About 1
After reacting for 0 minutes to 24 hours, it is washed with the reagent (5) and the catalytic activity of the metal porphine bound on the carrier is measured. That is, about 10 to 3000 microliters of the reagent (6) is added, and the chemiluminescence amount in the reaction solution is measured for a certain period of time with a chemiluminescence measuring device.

The measuring kit of the present invention is in the form of powder, granules, liquid or the like in which the above-mentioned complex and the chemiluminescent substance are contained in separate containers, and the chemiluminescent substance is optional. It may be mixed with a predetermined concentration of the amphipathic substance. Also, a buffer may be combined. The container is not particularly limited, and glass, plastic, and metal can be used, and the respective reagents can be manufactured by known methods such as dissolution, dispersion, mixing, drying, granulation, and freeze drying.

[0020]

EXAMPLES The present invention will be described in more detail with reference to examples below, but the invention is not limited thereto. Example 1 Manganese-mesotetra-4-sulfophenylporphine standard curve Manganese-mesotetra-4-sulfophenylporphine
[Porphyrin Brodacts, Logan, Utah, USA
(Porphyrin Products) was dissolved in 1 mM NaOH and diluted to various concentrations with a solution containing 0.01 mM luminol and 0.2 M NaOH. A 1 ml sample was taken in a test tube and placed in a photoelectron counting luminometer. It was inserted in a luminometer and the photoelectrons were counted for 50 to 60 seconds. The results are shown in Fig. 1. The limit of detection obtained from this experiment (minimum concentration with 95% probability of being distinguishable from 0 level) was 0.9 pg of manganese-mesotetra-4-sulfophenylporphine.

Example 2 Manganese-mesotetra-4-sulfophenylporphine Luminescence Enhancement by Tween 20 Manganese-mesotetra-4-sulfophenylporphine was dissolved in 1 mM NaOH. The resulting solution was applied to Tween 20 [Sigma chemical company, St. Louis, MO, USA]
%, 0.0025%, 0.005%, 0.01% and 0.0%.
A solution of 0.1 M NaOH, 1 mM luminol, containing 03%, is 100 pg / ml and 0 pg in 1 ml.
/ Diluted to milliliters. Take 1 ml of this sample in a test tube and insert each sample into the luminometer.
Photoelectrons were counted for ~ 62 seconds. The results are shown in Figure 2.

Example 3 Comparison of light emitting action of various porphyrin derivatives Manganese-mesotetra-4-sulfophenylporphine, iron-mesotetra-4-sulfophenylporphine,
Manganese-mesotetra-4-carboxyphenylporphine, mesotetra-4-carboxyphenylporphine, mesotetra-4-sulfophenylporphine, manganese-protoporphyrin, zinc-protoporphyrin,
Tin-protoporphyrin, aluminum-phthalocyanine tetrasulfonate and chlorin E6
E6) (Porphyrin Products, Inc., Logan, Utah, USA) and microperoxidase, copper chlorphyrin and hematine (St. Louis, MO, USA).
Sigma Chemical Company, Inc.) with 0.5 mM Luminol and 0.1 M NaOH and 0.01% Tween 20 respectively.
Was diluted to a concentration of 100 pg / ml and 1 ng / ml. The light was measured for 60 to 62 seconds after inserting each sample into the photoelectron counting chemiluminescence meter.
The results obtained from this experiment are summarized in Table 1. The result shows that the luminescence catalyzed by each compound is manganese-mesotetra-4-
It is shown as a ratio to the light emission catalyzed by sulfophenylporphine.

[0023]Table 1   Comparison of chemiluminescence catalysis of various porphyrin derivatives     Test compound relative activity   Manganese-mesotetra-4-sulfophenylporphine 1   Iron-mesotetra-4-sulfophenylporphine 0.53   Manganese-mesotetra-4-carboxyphenylporphine 0.77   Mesotetra-4-carboxyphenylporphine 0   Mesotetra-4-sulfophenylporphine 0   Manganese-protoporphyrin 0.23   Zinc-protoporphyrin 0   Tin-protoporphyrin 0   Aluminum-phthalocyanine tetrasulfonate 0   Chlorine E60   Microperoxidase (containing heme) 0   Copper chlorphyrin 0   Hematin (containing heme) 0.06

Example 4 Use of manganese-mesotetra-4-carboxyphenylporphine for immunoassay determination of Endothelin White plastic microtiter plates [DYNATECH, Chantilly, VA, USA]
MicroFLUOR, Dynatech Laboratories
Incorporated (Dynatech Laboratories In
c.)], 0.1 ml of 0.1 M carbonate buffer (pH 9.5) containing 50 μg / ml of monoclonal anti-endothelin antibody was added to each well and treated at room temperature for 3 hours. The method for producing this antibody is described in Suzuki et al., Journal of Immunological Methods (Suzuki et al.,
J. Immun. Methods), 118, 245-250, 1
989. The wells were rinsed three times with phosphate buffer solution, and then 0.3 ml of phosphate buffer solution containing 25% of Block Ace [Snow Brand Milk Products Co., Ltd.] was added to each well. The plates thus prepared were stored at 6 ° C until use.

Endothelin-1 (Peptide Institute)
Dilute with% BSA and finally assay buffer (10
% Block Ace, 0.4 M NaCl, 1 mM disodium ethylenediaminetetraacetate, 20 mM sodium phosphate p
It was diluted 100 times with H7.0). Next, 0.2 ml of endothelin-containing assay buffer (0 pg /
(3 ml, 20 pg / ml, 50 pg / ml, 100 pg / ml and 200 pg / ml, all in triplicate) were added to each well of the prepared microtiter plate and incubated for 16 hours. Each well was washed 3 times with phosphate buffer solution. Complex incubation buffer containing a 1: 25-fold dilution of polyclonal anti-endothelin antibody-manganese-mesotetra-4-carboxyphenylporphine complex [10% Block Ace, 0.5% BSA, 0.4
20 mM sodium phosphate buffer (pH 7.0) containing M NaCl, 1 mM disodium ethylenediaminetetraacetate]
100 microliters was added to each well.

The antibody complex was produced as follows.
100 microliters dry dimethylformamide containing 5 micromoles N-hydroxysuccinimide
100 parts of (DMF) containing 7.5 micromoles of dicyclohexylcarbodiimide and 5 micromoles of manganese-mesotetra-4-carboxyphenylporphine.
Mix with microliter dry DMF and in the dark overnight at 4 ℃
It was made to react with. Contains 5 mg of anti-endothelin antibody 1
A milliliter of 0.05M phosphate buffer pH8 was mixed with 40 microliters of the succinimide ester solution prepared above and allowed to react overnight at 4 ° C in the dark. 0.1M
Column of Ultragel ACA34 resin (1.5 cm x 4) equilibrated with sodium phosphate buffer pH 6.5.
5 cm) to separate the complex. The titration plate was incubated at 4 ° C. for 16 hours. After the reaction, the microtiter plate was washed three times with a cold phosphate buffer solution, and the activity of the antibody complex reacted with endothelin was measured. In the step of detecting the antibody complex, 100 microliter of a solution containing 1.0 mM luminol (purified from alkaline solution), 0.07 M NaOH, 0.03 M glycine and 0.1% Tween 20 was added to each well of the microtiter plate. Was added. Luminescence from each well was measured with a ML1000 type microtiter plate chemiluminescence meter (Dynatech, Chantilly, Virginia, USA). FIG. 3 shows an endothelin standard curve prepared by the above method. Increasing the concentration of endothelin linearly increased the light generated in the detection step.

Example 5 Use of Mn-mesotetra-4-carboxyphenylporphine in the measurement of α-fetoprotein (AFP) by the ELISA method In the same manner as in Example 4, the anti-α-fetoprotein antibody [A-44 type, Tokyo standard] was used. Serum (Tokyo)] Sensitized white microtiter plate wells were prepared. α-fetoprotein [Scrrips Laboratories, TorreyPines, California, USA (Scr
ipps Laboratories)] in PBS containing 25% Block Ace and immediately before use, incubation buffer (10% Block Ace, 0.5% BSA, 0.35).
100% final dilution with 100% sodium alginate in PBS). 0, 5, 12.5, 3 in each well of the plate
195 microliters of incubation buffer containing 7.5 or 125 ng / ml α-fetoprotein was added. Then 5 microliters of 2 μg / ml manganese-mesotetra-4-carboxyphenylporphine antibody conjugate was added to each well. The complex was prepared as follows. Manganese
Mesotetra-4-carboxyphenylporphine 3 mg
Was dissolved in 2.1 ml of dimethylformamide. Add 25 mM phosphate buffer (pH 6.0) to this solution.
1.0 mg of N-hydroxysuccinimide 4 mg and 25 mM phosphate buffer (pH 6.0) 1.
A solution prepared by dissolving 6.3 mg of water-soluble carbodiimide was added to 05 ml. After incubation at room temperature for 1 hour, 1.5 mg of anti-α-fetoprotein [A2
95, Tokyo standard serum (Tokyo)], and 4.2 ml of 25 mM phosphate buffer (pH 6.0) was added. This was incubated overnight at 6 ° C. The derivatized complex is then subjected to gel filtration using Sephadex G-50 in the presence of 0.1M phosphate buffer (pH 6.8).
Separated from low molecular weight compounds. 45 with shaking at 6 ° C
After incubating for minutes, the plate was washed twice with PBS. A solution containing 0.1 M NaOH, 1 mM luminol and 0.1% linolenic acid was added (10 per well).
(0 microliter), and chemiluminescence was measured with a chemimetric meter for microtiter plates in the same manner as in Example 4. FIG. 4 shows a standard curve of α-fetoprotein prepared by this method. The limit of detection (the amount of analyte that gives a signal equal to twice the average background value) is 1.
It was calculated to be 5 ng / ml. With this method, 1
Plasma concentrations up to 5% can be used and it has been found that typical adult plasma AFP concentrations are below 10 ng / ml.

Example 6 Simultaneous Measurement by Chemiluminescence Method Using Two Catalysts This example shows that two different chemiluminescence catalyst systems coexist in one assay volume and separate measurements are performed. One catalyst, methylene blue, reacts with luminol to emit chemiluminescence. The second catalyst is manganese-mesotetra-4-carboxyphenylporphine.
Both catalysts were dissolved in a solution of 20% ethanol and 80% water containing 1 mM luminol and 50 mM NaOH. Methylene blue in the presence of 10 ng / ml manganese-mesotetra-4-carboxyphenylporphine, 1 ng / ml, 0.5 ng / ml,
Diluted to 0.25 ng / ml, 0.1 ng / ml and 0 ng / ml. 5mW 670nm laser diode to excite the dye catalyst, 6
43 to prevent 70nm light from entering the photomultiplier tube
The above chemiluminescence was measured with a photoelectron counting chemiluminescence meter equipped with a filter for cutting off light having a wavelength longer than 5 nm. 1 ml of each solution was placed in a 12 mm test tube, and after inserting each test tube into a chemiluminescence meter, photoelectrons were counted for 30 to 60 seconds. The count value in the background dark area was subtracted from the measured value. The obtained results are shown in FIG. In the next experiment, manganese-mesotetra-4-carboxyphenylporphine was added to the same buffer containing 1 ng / ml methylene blue at 10 ng / ml, 5 ng / ml, 2.5 ng / ml,
1ng / ml, 0.5ng / ml and 0n
Diluted to a concentration of g / ml. Chemiluminescence was measured by the same method as above except the laser light was turned off and no background was subtracted. The results are shown in FIG. Figures 5 and 6 show that both catalysts can be quantified independently from the same reaction mixture.

Example 7 Chemiluminescence enhancing action of amphiphile In this example, it was shown that chemiluminescence is enhanced by adding an amphiphile to a solution of luminol and manganeseporphine under alkaline conditions. It is a thing. 1 ml of 0.5 mM luminol dissolved in a 0.1 N NaOH aqueous solution was dispensed into a test tube. Next, 10 microliters of a 10% aqueous solution of chemiluminescence enhancing test compound was added and mixed, and the chemiluminescence amount was measured for 1 minute (B). Further, 100 ng / min of manganese-mesotetra-4-sulfophenylporphine dissolved in 10 mM NaOH aqueous solution
10 microliters of a milliliter solution was added and mixed, and the amount of chemiluminescence for 1 minute was measured (S). As a control, distilled water was used instead of the test compound aqueous solution. The intensity of the chemiluminescence enhancing action of the test compound was calculated by the following formula.

[Equation 1] The results of this experiment are shown in Table 2.

[0030]Table 2   Comparison of chemiluminescence enhancing effect

[0031]

EFFECTS OF THE INVENTION According to the present invention, a specific reaction can be measured with high sensitivity by a simplified reaction, the inconvenience caused by the use of an unstable reagent can be eliminated, and the assay time, labor and sample usage can be saved. Measurements can be made.

[Brief description of drawings]

FIG. 1 is a graph showing the effect of increasing concentration of manganese-mesotetra-4-sulfophenylporphine on light output.

FIG. 2 is a graph showing the enhancement of chemiluminescence catalysis of manganese-porphyrin by Tween 20.

FIG. 3: Standard curve of endothelin in an immunoassay obtained with a complex of anti-endothelin antibody and manganese-mesotetra-4-carboxyphenylporphine.

FIG. 4 is a standard curve of α-fetoprotein in an immunoassay obtained using a complex of anti-α-fetoprotein antibody and manganese-mesotetra-4-carboxyphenylporphine.

FIG. 5: Standard curve of methylene blue dye catalyst in the presence of manganese-mesotetra-4-carboxyphenylporphine.

FIG. 6: Standard curve of manganese-mesotetra-4-carboxyphenylporphine in the presence of methylene blue dye catalyst.

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Claims (5)

[Claims] 1. A specific binding assay in which a complex of a catalyst and an element that binds to it is detected to determine the presence of an analyte in a sample by one or more binding reactions, the detection comprising: a. Reacting a metal porphine catalyst complex with a chemiluminescent substance at high pH in the absence of an oxidant, b. A method for measuring specific binding, which comprises performing chemiluminescence proportional to the concentration of the catalyst complex. 2. A specific binding assay method for detecting the presence of an analyte in a sample by detecting a complex between a catalyst and an element that binds to the catalyst, the detection comprising the steps of: a. Reacting a metal porphine catalyst complex with a chemiluminescent substance at high pH in the absence of an oxidant, b. Promoting the reaction of the metal porphine catalyst with an amphipathic substance, c. A method for measuring specific binding, which comprises performing chemiluminescence proportional to the concentration of the catalyst complex. 3. Detecting two kinds of complexes of a catalyst and an element that binds to the catalyst, and detecting two kinds of complexes in a sample by one or more kinds of binding reactions.
In a specific binding assay that measures two analytes, the detection is performed by a. Reacting a photosensitive dye-catalyzed complex and a metal-porphine-catalyzed complex with a chemiluminescent substance at high pH in the same reaction mixture in the absence of an oxidant, b. Measuring chemiluminescence emitted from the metal porphine catalyst, which is proportional to the concentration of the metal porphine catalyst complex in the absence of light, c. A specific binding assay method, which is performed by measuring chemiluminescence from a photosensitive dye, which is proportional to the concentration of a photosensitive catalyst complex formed during or after light irradiation. 4. A. A first container containing a composite of a metal porphine and elements associated therewith; b. A kit for measuring specific binding, comprising: a second container having a high pH and containing a chemiluminescent substance to which an oxidizing agent is not added. 5. A. A first container containing a composite of a metal porphine and elements associated therewith; b. A kit for measuring specific binding, comprising: a second container containing a chemiluminescent substance and an amphipathic substance at high pH and containing no oxidizing agent.