JPH035539B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to sensitized luminescence or luminometric assay methods, particularly immunoassay methods, as well as diagnostic kits designed to facilitate this assay method. Immunoassay is one of the most widely used analytical techniques in clinical laboratories. Currently, most immunoassays use radioactive isotopes, particularly ¹¹²⁵ , as labels. However, radioisotopes have some major drawbacks. First, this labeling method uses highly radioactive and therefore potentially dangerous agents. Second, the shelf life of radiolabeled materials is short, not only because the radioactive isotope essentially constantly decays, but also because radiolabeled proteins are often unstable. Third, it is often difficult to label proteins sufficiently to obtain sensitive and rapidly detectable reagents. Fourth, it is inconvenient to dispose of radioactively labeled substances. These shortcomings have led to an active search for viable alternatives to radioactive labels. In order for a substance to be suitable as a label, it must meet at least the following three requirements. (a) When bound to a ligand, for example an antigen or an antibody, it must be detectable in very small quantities and rapidly. (b) It must be possible to bind it to a ligand such as an antigen or an antibody without affecting its measurement. (c) Once bound, the properties of the ligand should not change significantly. One of the most promising alternative labels are substances that can themselves participate in the reaction that causes luminescence, or that, upon appropriate treatment, yield compounds that can participate in the luminescence reaction. be. Luminescence reactions (ie, chemical reactions that produce luminescent emissions) generally have a duration sufficient to detect and measure the emitted light and thereby quantify the labeled material. On the other hand, measuring luminescence is a quick operation;
Measurement of radioactivity can be completed in seconds, whereas it normally takes several minutes. Luminescence has been used in three major luminescence immunoassay systems: (a) organic luminescence immunoassays; In this test,
A chemiluminescent or bioluminescent compound that directly participates in a luminescence reaction (i.e., is converted to an excited state and then returns to an unexcited state by emitting a photon) may be a chemical compound that directly participates in a luminescence reaction (i.e., converts to an excited state and then releases a photon to return to an unexcited state). It has been used to label steroids, nucleic acids, intermediate metabolites, antigens and/or antibodies. Examples of suitable compounds include luminol and isoluminol. (b) Luminescence catalyzed or cofactor immunoassay.
Catalysts or cofactors of the luminescence reaction have been used as labels in this assay. Examples of suitable catalysts include peroxidases. (c) Enzyme-linked immunoassay. In this assay, a luminescence reaction has been used to measure the product produced by the action of an enzyme label on a suitable substrate. Examples of this type of immunoassay include enzyme/
By a method of reacting an antibody reactant with glucose to produce hydrogen peroxide, then adding luminol to this under controlled conditions to initiate a luminescence reaction, and measuring the amount of hydrogen peroxide produced. , quantification of antibody-bound glucose oxidase. The sensitivity of the above immunoassays is determined in part by the lower limit of detection of the label or product from the label.
In luminescent immunoassays, the sensitivity of the assay system is determined in part by the amount of light emitted per unit of labeled material in the luminescent reaction. One of the objects of the present invention is the luminescence with increased sensitivity achieved by quantifying products from labeled materials or labels by the improved luminescence reaction of the present invention. An object of the present invention is to provide an immunoassay method. The improved luminescence reactions of the present invention are particularly useful in measuring immunoassay labels or products thereof, but are not limited to this use. A further object of the present invention is to provide a luminescence assay method (immunoassay method, etc.) with increased sensitivity, which is achieved by incorporating the improved luminescence reaction of the present invention into the assay procedure. It is to provide. Examples of assays that, although not immunoassays, may incorporate the luminescence reactions of the present invention include: (a) Elastase assay based on peroxidase release from insoluble peroxidase-elastin samples. (b) Glucose assay based on co-immobilized glucose oxidase and peroxidase. (c) Peroxidase, 2,3-dihydro-1,
Assay for 4-phthalazinedione compounds, or oxidizing agents such as hydrogen peroxide (if not the label or the product from the label). Accordingly, in accordance with the broadest aspect of the invention, a peroxidase, an oxidizing agent, and a chemiluminescent 2,3
The chemiluminescence reaction between -dihydro-1,4-phthalazinedione compound is expressed by the formula [In the formula, (i) A and B are hydrogen atoms, and R is a halogen atom, a phenyl group,

[Formula] (where Y is -CH ₂ - or -O-, and V is a hydrogen atom, or Y is -O-, -S- or -
S-S- and V is a hydroxyl group),

[Formula] (where W is a hydrogen atom or a carboxyl group),

[Formula] Group -CH=CH-Z (where Z is carboxyl group or 2,4-dinitrophenyl group), group -
( _{ii) A is a hydrogen atom, B is a halogen atom or a C1-C6 alkyl group , and R is} a _halogen atom; (iii) A is a halogen atom, B is a hydrogen atom, and R is a halogen atom or a phenyl group, or (iv) A is a hydrogen atom or a halogen atom, and R
and B are together the expression

[Formula] (In the formula, X is a hydrogen atom or a halogen atom) Represents a naphthalene core constituent chain represented by In each case of (i) to (iv) above, the halogen atom means a chlorine atom, a bromine atom, or an iodine atom. A luminescence or luminometric assay method is provided. The assay method is preferably an immunoassay method. The present invention shows that the addition of certain readily available phenols or naphthols to the known 2,3-dihydro-1,4-phthalazinedione compound/oxidant/peroxidase system significantly increases the sensitivity of the luminescence reaction. This is based on the surprising discovery that As used herein, the term "sensitized" means that the total light emission of the luminescence reaction of the invention and/or the signal/background ratio of the luminescence reaction of the invention is −
This means that it is larger than that obtained with the 1,4-phthalazinedione compound/oxidant/peroxidase system. Only assay methods that incorporate such "sensitized" luminescence reactions are within the scope of the present invention. It is a particular advantage of the system of the invention that the sensitization by addition of these phenols or naphthols is specific for reactions using peroxidases. The chemiluminescent 2,3-dihydro-1,4-phthalazinedione (DPD) compounds of the present invention include:
Any DPD compound that can be converted to an excited state in a chemiluminescence reaction and then return to an unexcited state by emitting light. chemiluminescent 2,3-dihydro-1,4
-The general formula for phthalazinedione compounds is (wherein R ¹ is an unsubstituted or substituted amino group, R ² , R ³ and R ⁴ are each hydrogen atom, unsubstituted or substituted C ₁ -C ₆ alkyl group, unsubstituted or substituted C ₁ -C ₆ alkenyl group,
is a hydroxyl group, a _C1 - _C6 alkoxy group, a carboxyl group or an unsubstituted or substituted amino group, or ^R2 is an unsubstituted or substituted amino group, and R ¹ , R ³ and R ⁴ are each hydrogen atom, unsubstituted or substituted C ₁ -C ₆ alkyl group, unsubstituted or substituted
_C1 - _C6 alkenyl group, hydroxyl group, _C1 - _C6
is an alkoxy group, a carboxyl group or an unsubstituted or substituted amino group, or R ¹ and R ² are jointly an unsubstituted or substituted amino derivative of a benzo group; where R ³ and R ⁴ are each hydrogen atom, unsubstituted or substituted C ₁ -C ₆ alkyl group, unsubstituted or substituted C ₁ -C ₆ alkenyl group, hydroxyl group , C ₁ −
C ₆ alkoxy, carboxyl or unsubstituted or substituted amino groups are preferred. Particularly preferred phthalazinedione compounds for use in the present invention are luminol and isoluminol. As used herein, substituted amino groups include amide groups. The form of the chemiluminescent DPD compound used in the luminescence assay method of the present invention varies depending on the type of assay method in question. In the case of assay methods using phthalazinedione compounds as labels, such as organoluminescent immunoassay methods, chemiluminescent DPD compounds are used in assays in which the amino group is linked to a ligand such as a protein, hormone, hapten, steroid, nucleic acid, intermediate metabolic Substituted amino derivatives of 2,3-dihydro-1,4-phthalazinedione conjugated to substances, antigens or antibodies. The amino group may be bonded directly to the ligand or via a bridging limb. Suitable bridging limbs are described, for example, in GB 2008247A and US 4104029.
This is well known to those skilled in the art, as is clear from the discussion in the specification. Preferred bridging limbs include those derived from, for example, hemisuccinate, hemiglutarate, hemimaleate, carboxymethyl, glucuronide, mercaptoacetate or carboxymethyl derivatives. The amino group can be attached to the ligand by any known method, some of which are also described in British Patent No.
No. 2008247A and US Pat. No. 4,104,029. Examples of preferred attachment methods include the use of mixed anhydrides, carbodiimides, and/or active esters. Chemiluminescent compounds suitable for these assays using phthalazinedione compounds as labels.
Although the DPD compound may be any substituted amino derivative of 2,3-dihydro-1,4-phthalazinedione in which the amino group is attached to the ligand, preferred materials are 5-amino-2 , 3-dihydro-1,4-phthalazinedione (luminol) and 6-amino-2,3-dihydro-1,4-phthalazinedione (isoluminol), in which case the amino group is particularly It is bound to an antibody. For assays other than those using phthalazinedione compounds as labels, chemiluminescent
DPD compounds are not bound to ligands, 2,3
-dihydro-1,4-phthalazinedione compound,
Particularly of the preferred types mentioned above. In this case the chemiluminescent DPD compound can be free in solution or immobilized on the matrix. Particularly preferred materials are luminol and isoluminol. Any phenol or naphthol of general formula (1) that enhances the luminescence reaction between the chemiluminescent DPD compound, the oxidizing agent, and the peroxidase can be used in this assay. However, it has been found that the following sensitizers exhibit a particularly high degree of sensitization, especially when the chemiluminescent DPD compound is luminol or isoluminol. Sensitizers include 4-chlorophenol, 4-bromophenol, 4-iodophenol, 4-bromo-2-chlorophenol, 2,4
-dichlorophenol, 3,4-dichlorophenol, 4-methylphenol, 4-tert-butylphenol, ethyl 3-(4-hydroxyphenyl)propionate, 4-benzylphenol, 4-
(2',4'-dinitrilostyryl)phenol, 4
-Hydroxycinnamic acid, 4-phenylphenol, 2-chloro-4-phenylphenol, 4-
(4'-hydroxyphenyl)benzophenone, 4
-(phenylazo)phenol, 4-(2'-carboxyphenylazo)phenol, 4-phenoxyphenol, 4-(4'-hydroxyphenoxy)
Phenol, 4-hydroxyphenyl sulfide, 4-hydroxyphenyl disulfide, 2-
naphthol, 1-bromo-2-naphthol, 6-
Bromo-2-naphthol and 1,6-dibromo-2-naphthol. Among these sensitizers,
4-iodophenol, 4-phenylphenol and 2-chloro-4-phenylphenol are particularly effective, with 4-iodophenol being the most effective. Any peroxidase (defined as donor) that catalyzes the luminescence reaction of 2,3-dihydro-1,4-phthalazinedione compounds, especially luminol. EC No. 1.11.1.7) can be used in the luminescence assay method of the invention. Examples include plant perosidase. However, a preferred enzyme is horseradish peroxidase (EC No. 1.11.1.7). The form of peroxidase used in sense assay methods differs depending on the type of assay method being used.In assay methods that use peroxidase as a label, especially immunoassay methods, peroxidase can be used as a ligand such as protein, hormone, hapten, or steroid. , nucleic acids, intermediate metabolites,
Combined with antigen or antibody. Generally, peroxidases are attached to a ligand via a bridging limb. Suitable bridging limbs and conjugation operations are chemiluminescent
Same as described above for DPD compounds. In assay methods other than those using peroxidase as a label, the enzyme is in a free form, unbound to ligands, both in solution and when immobilized on a matrix. 2,3-dihydro-
Any oxidizing agent that reacts with the 1,4-phthalazinedione compound, particularly luminol or isoluminol, to excite the DPD compound to emit light can be used in the luminescence reaction of the present invention. Particularly preferred oxidizing agents are perborate ion and hydrogen peroxide. In assay methods, particularly immunoassay methods, in which 2,3-dihydro-1,4-phthalazinedione compounds or peroxidases are used as a label for the ligand, a known amount of oxidizing agent is usually added to the reaction mixture from its source. do. However, in certain other assay methods, the amount of oxidizing agent (usually hydrogen peroxide) present is unknown. In this second type of assay method, the label is a substance involved in the conversion of the substrate to the oxidizing agent, often an enzyme such as glucose oxidase. Thus, in this case the luminescence reaction of the invention is used to quantify the labeled ligand by measuring the oxidant concentration in the luminescence reaction mixture. The emission of light from the luminescence assay method of the invention depends primarily on the choice of peroxidase, oxidant, sensitizer and chemiluminescent DPD compound, but also on secondary factors such as temperature, PH, reaction It is also determined by agent concentration, mixing speed and photometric method. To maximize the sensitivity of the system, these secondary factors can be adjusted to obtain maximum luminescence at the highest possible signal-to-background ratio using a reproducible and easy-to-measure method. You must do so. Selection of conditions is generally a compromise between the catalytic activity of the enzyme or peroxidase, the kinetics of the reaction, the equipment used, the signal-to-background ratio and the required sensitivity, etc. We believe that for optimal results the luminescence reactions of the present invention should be carried out under mild conditions in the temperature range 10-50°C and PH range 6-10, most commonly 7-9. I discovered something. Suitable buffering substances for the process of the invention are phosphates, tris(hydroxymethyl)aminomethane, 2-amino-2-methyl-1,3-propanediol, acetates, carbonates and borates. Generally, the concentration of reactants in the luminescent reaction mixture is kept constant except for the material to be measured. Variables include, for example, the product from the label, the oxidizing agent or unbound peroxidase, and the concentration of labeled ligand. The following reactant concentrations are particularly suitable for use in the luminescence reactions of the present invention: Peroxidase 0.1 μg-5000 mg/liter Oxidizing agent 10 μmol-300 mmol/liter chemiluminescent DPD compound
0.5 μmol-200 mmol/liter Sensitizer 1 μmol-100 mmol/liter To carry out the luminescence reaction of the present invention, some of the four main reactants (but not at least one) are placed in a sample tube. The missing primary reactant is then added to the tube to initiate the luminescence reaction. The emitted light is quantified by standard measurement equipment, such as a photomultiplier, and the signal therefrom is sent to a recorder, oscilloscope or scalar for display or recording. In other cases, the light is observed visually or recorded on a photographic plate. Preferably, however, the light is determined by a luminometer of the type described in GB 2025609A. The luminescence assay method of the present invention can be used in three major types of immunoassay methods, the distinguishing feature of each of which is the type of label attached to the ligand. The label is: (a) an amino or substituted amino derivative of a 2,3-dihydro-1,4-naphthalidinedione compound in which the amino group is attached to a ligand; (b) a peroxidase enzyme, and (c) a substance other than (a) and (b) above, which participates in the conversion of the substrate into a material (generally hydrogen peroxide or peroxidase) that can be quantified by the luminescence reaction, generally an enzyme, e.g. glucose oxidase. In the above-mentioned immunoassay method, it is possible to label the substance to be assayed or to label an antibody for such a substance. Depending on the type of label used, the assay method may be heterogeneous or homogeneous. In the former case, analysis of complex fluids such as serum is possible, while in the latter case preliminary extraction or purification steps may be necessary. A summary of typical heterogeneous or homogeneous luminescence immunoassay methods is provided below: 1. Heterogeneous luminescence immunoassay In this type of immunoassay, the substance to be assayed is reacted with its antibody. Next, free antibodies and bound antibodies are separated. The reaction is quantified by labeling the antibody, the substance to be assayed, or another molecule that can react with the free or bound moiety after separation. 2 Competitive Heterogeneous Luminescence Immunoassay In this case, an unknown amount of the substance to be assayed is mixed with a known amount of the substance to which a label and a known, limited amount of its antibody are bound. A competitive reaction against the antibody occurs between the labeled and unlabeled substances. Complexes of the antibody and unlabeled material and of the antibody and labeled material are separated from free labeled and unlabeled material. The amount of labeled material bound to the antibody is related to the amount of unlabeled material in the solution to be assayed. These amounts can be quantified by measuring the amount of label bound to the antibody or by measuring the amount of free labeled material remaining. An example of this type of assay method, in which peroxidase is the label and the antibody is bound to a solid phase, ie the wall of a glass test tube, is described in GB 2044927A. 3. "Two-Position" Heterogeneous Luminescence Immunoassay In this type of immunoassay method, the substance to be assayed is first bound to its unlabeled antibody and then the antibody is bound to a solid support, such as plastic. The complex (of antibody and substance) is then treated with a labeled antibody. Analysis of the labeled antibody in the resulting solid complex is performed by separating the solid complex from solution and then determining the amount of label present in the separated solid complex or any remaining dissolved in solution. This is done by measuring the amount of label present in the labeled antibody. In another embodiment of this type of immunoassay method,
The substance to be assayed is bound sequentially to a labeled antibody and to an unlabeled, solid-supported antibody, or to both labeled and unlabeled antibodies in one step. 4 Homogeneous luminescence immunoassay This method uses a method in which the label is 2,3-dihydro-1,4
- Can be applied to immunoassay methods that are amino or substituted amino derivatives of phthalazinedione compounds. This means that the intensity or wavelength of the light emitted from the free labeled substance of interest (or its antibody) is different from the light emitted from the bound labeled substance of interest (or its antibody). It is based on As an example, it has been found that the intensity of light emitted by the reaction of the (progesterone-isoluminol-derived) complex, heme catalyst and hydrogen peroxide is lower than the intensity when the same reaction is carried out in the presence of anti-progesterone IgG. In other words, in this assay method, an unknown progesterone sample is first treated with a known amount of antiprogesterone.
Incubated with IgG. After reaching equilibrium, a known amount of progesterone-isoylminol-derived complex was added, followed by known amounts of heme and hydrogen peroxide. The emitted light was measured and from this the amount of progesterone present in the unknown sample was determined from the standard curve. (The greater the amount of progesterone in the unknown sample, the less free IgG remains in equilibrium and the lower the light yield of the luminescence reaction.) In this way, quantification of progesterone can be achieved without the need for a separation step. In all of the above immunoassay methods, the luminescence reaction of the present invention can be applied to the quantification, detection and exploration steps. Antibodies used in the above immunoassay method can be purchased from the market or prepared by known immunological techniques. The antibody may be in the form of a complex mixture of antibodies or one or more monoclonal antibodies. Generally, the volume of antibody required is small, and the antibody is maintained at appropriate PH, ionic strength, and temperature conditions for its activity. The following substances may be advantageously used in the immunoassay method of the present invention using luminescence reactions, including but not limited to: proteins such as insulin, alphafuetoprotein and ferritin, hormones such as growth hormone. ,
Parathyroid hormones, follicle-stimulating hormone, luteinizing hormone, thyrotropin-stimulating hormone, adrenocorticotropic hormones, glucagon, prolactin, and calcitonin, haptens/steroids such as estriol, progesterone and cortisol, pharmaceuticals such as digoxin, antigens such as cell surface Antigens and carcinogenic antigens, and antibodies such as mumps virus antigen, human immunoglobulin G (IgG), rabbit
IgG, sheep IgG, guinea pig IgG, donkey IgG and human immunoglobulins E and M. The luminescence assay method of the present invention can also be used for assay methods other than the above-mentioned immunoassay methods. As examples, the following may be mentioned: 1. Elastase assay based on peroxidase release from insoluble peroxidase elastin samples. In this assay method, solid elastin-
The peroxidase complex is incubated with various amounts of elastase. After a predetermined period of time, unreacted complexes are removed by centrifugation, and the supernatant is assayed for unbound peroxidase. The amount of unbound peroxidase present in the supernatant is related to the activity of elastase in the test sample. 2 Proteinase assay based on isoluminol release from synthetic peptide substrates. In this assay method, an immobilized synthetic polypeptide substrate, Affigel 10, is used.
-Ala-Ala-Ala-Phe-isoluminol is treated with various amounts of proteinase. After a predetermined period of time, unreacted substrate is removed by centrifugation, and the supernatant is assayed for isoluminol. The amount of isoluminol present in the supernatant is related to proteinase activity in the test sample. 3 Glucose assay based on co-immobilized glucose oxidase and peroxidase. In this assay method, glucose oxidase and peroxidase are co-immobilized on a support such as Sepharose® or a plastic tube. To this is added a solution of luminol and sensitizer. Finally add a solution of glucose and record the light emission. Light emission is directly related to the amount of glucose in solution. The primary use of the assay method of the present invention is in clinical laboratories or surgery. It is common for such laboratories and/or surgeries to obtain the materials to be used in a given assay procedure in the form of assay kits. The invention therefore also provides (a) a peroxidase, (b) a sensitizer of the general formula (1), in which R, A and B are as defined above, and (c) a chemiluminescent agent. 2,3-dihydro-
An assay kit for enhanced luminescence or luminometric assays of the present invention is provided comprising a 1,4-phthalazinedione compound. Assay kits may also include an oxidizing agent, although in many cases the oxidizing agent is provided separately or is the substance to be assayed. Preferably, the peroxidase, oxidizing agent, sensitizer, and chemiluminescent DPD compound are each of the substances listed above as preferred for use in the assays of the invention. In one particularly preferred embodiment of the assay kit, at least one of the peroxidase and the chemiluminescent DPD compound is bound to the antibody of the substance to be assayed. Optionally, the assay kit may also include one or more standard solutions and/or one or more preferred buffers, each containing a known amount of the substance to be assayed. The assay kit also conveniently includes a reaction vessel suitable for sharing with the equipment used to measure emitted light in performing the assay. A mixing device for use in thoroughly mixing the reactants can also be included in the assay kit. The assay method and assay kit of the present invention will be described below, but this is merely an example and the present invention is not limited thereto. Materials and Methods Reactant Horseradish peroxidase [HRP: Purity Index (Reinheit Zahl) (ratio of optical density at 250 nm to optical density at 403 nm, RZ) approximately 1.0]
Ultrogel AcA 34, 2.6 cm x 4.0 cm, obtained from Hughes and Hughes (Romford, Essex)
Column (LKB Instruments, South
Purified by gel filtration using a gel filtrate (Croydon, Surrey). Column contains 0.15mol/NaCl
Elution was performed using 0.015 mol/phosphate buffer (PH7.2). of the obtained purified peroxidase.
RZ was approximately 3.0. alphafuet protein (AFP), rabbit anti-human AFP (code 100−008) and rabbit anti-human
AFP/HRP complex is Mercia Brocades Dako
Products (Brocades House, Pyrford, West
Byfleet, Weybridge, Surrey). Thyroxine ( _T4 ), rabbit anti- _T4 coated tubes and
The T ₄ /HRP complex was manufactured by Boehringer (Lewes,
Obtained from Sussex, UK). Rubella virus (human) coated beads and anti-human
The IgG (goat)/HRP complex was obtained from the Affott Laboratories, Diagnostics Division, Brighton Hill Parade, Basingstoke.
obtained from Hampshire). Anti-IgE (goat) coated tubes and anti-IgE (rabbit) peroxidase conjugate were obtained from Behringwerke (Marburg). Luminol (5-amino-2,3-dihydro-
1,4-phthalazinedione) and isoluminol (6-amino-2,3-dihydro-1,4-phthalazinedione) from Sigma Chemical (Fancy
Road, Poole, Dorset, UK).
The monosodium salt of luminol was prepared by a previously reported method (Ham et al., Anal. Lett., 1979, 12;
535). 7-dimethylaminonaphthalene-1,2-dicarboxylic acid hydrazide (in formula (2), R ¹ and R ² jointly represent a benzo group substituted with dimethylamino, and R ³ = R ⁴ = H) compound) is
Obtained from Boehringer (Mannheim). N-(6-aminohexyl)-N-ethylisoluminol was obtained from LKB, Finland. Phenols and naphthols include 2-chlorophenol and 2,4,6-trichlorophenol [Fluka, Chemische Fabrik (CH9470,
Buchs, Switzerland)], 2-naphthol [BDH Chemicals, (Atherstone, Switzerland)], 2-naphthol [BDH Chemicals, (Atherstone,
All were obtained from Aldrich Chemical with the exception of 4-hydroxyphenyl sulfide (obtained from Fluorochem, Glossop, Derbyshire). Elastin, elastase, and glucose oxidase were obtained from Sigma Chemical (Dorset). Analyzer Chemiluminescence reaction is 10mm x 10mm with a volume of 4ml
Plastic disposable cuvette (W.
Sarstedt, Leicester LE31UQ, UK). Synchrotron radiation can be applied to a previously reported luminometer (see Carter et al., UK Patent No. 2025609A), which allows several cuvettes to be positioned sequentially in front of the photocathode of a photomultiplier tube with precision and reproducibility. Quantification was performed with modifications. The results were displayed on a high-speed potentiometric chart recorder [Model PM8202, Philipo (Eindhoven, The Netherlands), total scale deviation time 0.25 seconds or less]. Example 1 Peroxidase luminescence assay using luminol and 4-iodophenol Sodium luminol (50 mg) and hydrogen peroxide (62 μ, 30% w/v) are added to 200 ml of Tris buffer (0.1 molar, PH 8.5). . The solution was prepared several hours before use and used to start the luminescence reaction. Place 10μ of 4-iodophenol in dimethyl sulfoxide (4.54 mmol/liter) in one corner of the cuvette and add rabbit anti-AFP.
- 10μ of HRP complex (1000 times diluted) was placed in another corner. The reaction was started by injecting Luminol/H ₂ O ₂ reagent (0.9 ml). Table 1 shows the results of measuring the emitted light. The improvement in signal/background ratio was also measured and the results are shown in Table 2. Example 2-3 The amount of 4-iodophenol solution added is
The procedure was as in Example 1 except that the thickness was increased to 20μ and 90μ. Table 1 shows the results of measuring the emitted light. We also measured improvements in signal/background ratio,
The results are shown in Table 2. Example 4-8 The procedure was as in Example 1, except that the following sensitizers were used instead of 4-iodophenol: 4-bromophenol 4-chlorophenol 4-bromo-2-chlorophenol 2,4-dichloro Phenol 3,4-dichlorophenol The results are shown in Tables 1 and 2. Example 9-13 The procedure of Example 4-8 was repeated except that the amount of sensitizer solution added was increased to 90μ. The results are shown in Tables 1 and 2. Examples 14-16 The procedure was as in Example 1 except that the following sensitizers were used in place of 4-iodophenol: 4-hydroxycinnamic acid 2-naphthol 6-bromo-2-naphthol The results are shown in Table 1 and Examples shown in Table 2 17-20 The procedure was as in Example 1, except that 5μ of the following sensitizers were used instead of 10μ of 4-iodophenol: 4-phenylphenol 1,6-dibromo-2-naphthol 1-Bromo-2-naphthol 2-chloro-4-phenylphenol The results are shown in Tables 1 and 2. Examples 21-25 The procedure was as in Example 1, except that 5μ of the following sensitizers were used instead of 10μ of 4-iodophenol: 4-Hydroxyphenyl sulfide 4-Hydroxyphenyl disulfide 4-(2 ',4'-dinitrostyryl)phenol 4-(4'-hydroxyphenyl)benzophenone 4-(4'-hydroxyphenoxy)phenol The improvement in signal/background ratio was measured and the results are shown in Table 3. . Examples 26-29 The procedure was as in Example 1, except that 20μ of the following sensitizers were used instead of 10μ of 4-iodophenol: 4-(phenylazo)phenol 4-(2′-carboxyphenylazo)phenol 4 -Benzylphenol Ethyl 3-(4-hydroxyphenyl)propionate Table 3 shows the results of measuring the improvement in signal/background ratio. Comparative Example The procedure was as in Example 1, except that no sensitizer was added to the luminescence reaction mixture. The results are shown in Tables 1 and 2.

[Table] Fixed

【table】

[Table] Enol

[Table] Propionate
Chill Examples 30-33 The procedure was as in Example 1, except that 4-iodophenol was replaced by various other phenols and naphthols (10 μ, 1 mg/ml solution in DMSO). The amount of emitted light was compared with that of the luminescence reaction without sensitizer. The luminescence reaction was considered enhanced if the amount of emitted light was greater in the presence of phenols or naphthols than in their absence. The results are shown in Table 4.

[Table] Example 34 N-(6-aminohexyl) instead of luminol
The procedure was as in Example 1 except that -N-ethylisoluminol was used. Example 35 The procedure was as in Example 1 except that isoluminol was used instead of luminol. Example 36 Using 7-dimethylaminonaphthalene-1,2-dicarboxylic acid hydrazide (9-dimethylamino-2,3-dihydrobenzo[f]phthalidine-1,4-dione) in place of luminol, the phthalazinedione compound The procedure was as in Example 1, except that the /H ₂ O ₂ was diluted 1:10 before use. Example 37 The procedure was as in Example 1 except that sodium perborate was used instead of hydrogen peroxide. Example 38 Assay for alphafuetoprotein (AFP) (a) Coating polystyrene beads with anti-AFP (1) Glycine/NaOH in a 500 ml wide-mouth glass bottle
125ml of buffer solution (0.1mol/, PH8.8) and
0.25g of BSA was added. The solution was then mixed for 2 hours on a roller mixer at 20°C. After completion, add glycine buffer (50ml x 3 times) to the bottle (and lid).
The solution was rinsed to remove unbound albumin, and finally drained. (2) 1000 polystyrene beads were washed with Lipsol (registered trademark, 1%, 500 ml) for 30 minutes. After washing with distilled water, the beads were placed in a Buchner flask with 200 ml of glycine buffer and degassed. (3) Transfer 400 ml of glycine buffer to a pre-coated glass bottle and add 1.5 ml of rabbit anti-human AFP.
After the solution was mixed well, the degassed beads were added. Place the whole thing on a roller mixer for 6 hours, 20
Mixed at ℃. (4) After 6 hours, decant the glycine buffer-antibody solution and add the beads to 400 ml of PBS (phosphate
0.015 mol/, NaCl 0.15 mol/, pH 7.2) twice. After decanting and removing the second PBS washing solution, a solution of BSA in PBS (400 ml) (0.5
%) was added. The whole was mixed again on a roller mixer at 20°C. After 30 minutes, take the PBS-albumin solution and add it to an acetate buffer (0.1 mol/, PH4.2,
400ml) was added. After 10 minutes of mixing, the acetate buffer was added to PBS containing 0.05% Tween.
It was replaced with buffer solution (400ml x 2). Finally PBS
was decanted and the beads were dried on paper for 30 minutes at 20°C. Place the beads in a screw-top bottle 4
Stored at °C. (b) (1) Sufficient antibody-coated beads in PBS [described above]
Step (a)] was degassed for 15-30 minutes. (2) PBS/BSA/50μ sample containing AFP
Tween (phosphate 0.015mol/,
NaCl0.15mol/, BSA0.2%, Tween20 0.05
%, PH7.2) to the cuvette along with 0.95 ml.
The solution was warmed to 37°C and incubated with beads for 1 hour. (3) Next, decant the solution and remove the beads in PBS/
Washed with Tween (200ml for 15 seconds, then 200ml
ml for 5 minutes). (4) The beads were then incubated with 0.9 ml of a 1:1000 dilution of rabbit anti-human AFP/HRP complex for 1 hour. (The complex was diluted in PBS/SBA/Tween and warmed to 37°C before addition to beads). (c) (1) The solution was again decanted and the beads were washed twice with deionized distilled water. After removing the water by decanting, the beads were carefully transferred to cuvettes (one per cuvette). (2) At the corner of each cuvette there is also a 10μ 4-
Iodophenol in Tris buffer (0.1mol/
, PH8.0) solution (1 mg/ml) was added. Inject 0.9 ml of Luminol/H ₂ O ₂ solution [sodium luminol (50 mg), H ₂ O ₂ (62 μ, 30% w/v), Tris (0.1 mol/, PH 8.0, 200 ml)] and perform the luminescence reaction. started. The light emission after 30 seconds was measured and compared with a pre-prepared standard curve to determine the AFP concentration. Example 39 Assay of T ₄ T ₄ was assayed using a competitive enzyme-linked immunosorbent assay. A sample mixture containing unlabeled T ₄ and T ₄ /HRP complex was treated with a limited amount of antibody.
Incubate with tubes coated with T ₄ (rabbit). After reaching equilibrium, unbound material is washed from the tube,
A solution of 10μ of 4-iodophenol in Tris buffer (0.1 mol/, PH 8.0) (1 mg/ml) was placed in each tube. Luminol/H ₂ O ₂ solution [sodium luminol (50 mg), H ₂ O ₂ (62 μ, 30% w/v),
A luminescence reaction was started by injecting 0.9 ml of Tris (0.1 mol/, PH 8.0, 200 ml). The light emission after 30 seconds was measured and compared with a pre-prepared standard curve to determine the _T4 concentration. Example 40 Assay for Anti-Rubella IgG Test samples containing anti-Rubella IgG were incubated with Rubella virus (human) coated beads in Tris buffer. After washing to remove unreacted components,
The beads were incubated with anti-human IgG (goat)/HRP complex, again in Tris buffer. The solution was decanted and the beads were washed with deionized distilled water. The washed beads were separated from the water and then transferred to a cuvette (one per cuvette). Also in the corner of each cuvette was 10μ of iodophenol in Tris buffer (0.1mol/, PH8.0).
solution (1 mg/ml) was added. Luminol/H ₂ O ₂
Solution [Sodium Luminol (50mg), H ₂ O ₂ (62μ
, 30%w/v), Tris (0.1mol/, PH8.0,
200 ml)] was injected to start the luminescence reaction. The light emission after 30 seconds was measured and compared with a standard curve prepared in advance to determine the anti-rubella IgG concentration. Example 41 Assay for IgE Samples containing human IgE were added to anti-IgE (goat) coated tubes and incubated at 20°C for 2 hours. After completion, wash the tube to remove unreacted components and add a known amount of anti-reactant.
IgE (rabbit) peroxidase conjugate was added to the tubes and incubated for an additional 2 hours at 20°C. Excess enzyme and antibody complexed with the antibody were then washed away, and the bound enzyme activity was measured as follows: In the corner of each tube, add 10μ of 4-iodophenol in Tris buffer (0.1mol/, PH8.0) ( 1mg/ml)
I put it in. Luminor/H ₂ O ₂ [Luminor (50
The luminescence reaction was initiated by injecting 0.9 ml of H ₂ O ₂ (62 μ, 30% w/v), Tris (0.1 mol/, PH 8.0, 200 ml). The light emission after 30 seconds was measured and compared with a standard curve prepared in advance to determine the IgE concentration. Example 42 Elastase Assay GCSunders horseradish peroxidase
(Anal.Biochem.1982, 126 , 122). Wash 1 ml of elastin peroxidase suspension and add CaCl ₂
(2 mmol/) in 2 ml of 0.01 mol/Tris buffer (PH9). The complex was centrifuged and the supernatant was discarded. Various amounts (0-100ng/
ml) of elastase in Tris-CaCl ₂ buffer was added and incubated with elastin-HRP at 37°C. Unreacted elastin after 60 minutes
HRP was removed by centrifugation and an aliquot of the supernatant was transferred and recentrifuged. Take 200μ of the supernatant liquid, put it in a cube, and add Luminol/H ₂ O ₂ /
4-iodophenol solution [sodium luminol (50 mg), H ₂ O ₂ (62 μ, 30% w/v), 4-iodophenol (2 ml, 1 mg/ml in DMSO), Tris buffer (0.1 mol/, PH 8. 0) 0.9 in 200ml
ml was added to start light emission. Light emission was measured after 30 seconds. Example 43 Glucose assay using co-immobilized glucose oxidase and peroxidase Glucose oxidase (5 mg, Sigma) and horseradish peroxidase (5 mg, Sigma)
Ford and Deluca's method (Anal.
Biochem., 1981, 110 , 43), and co-immobilized on Sepharose activated with cyanogen bromide. The immobilized enzyme was dissolved in phosphate buffer (0.1 mol/,
PH7.0). Immobilized enzyme suspension (100
mg/, 50 μ) was added to 1 ml of an aqueous luminol solution (25 mg/100 ml) and 50 μ of 4-iodophenol (1 mg/ml in DMSO) placed in a cuvette. A 10μ sample of the glucose-containing aqueous solution was then added, the cuvette contents mixed, and the light emission recorded. The peak light emission is glucose 50−
It was linear within 500 mmol. Alternatively, glucose oxidase and peroxidase can be immobilized on a plastic support (e.g. Leon et al., Clin.Chem., 1977, 23 ;
1556).

Claims (1)

[Scope of Claims] 1. The chemiluminescence reaction between peroxidase, oxidizing agent, and chemiluminescent 2,3-dihydro-1,4-phthalazinedione compound is expressed by the formula [wherein (i) A and B are hydrogen atoms, and R is a halogen atom, a phenyl group,
[Formula] (where Y is -CH ₂ - or -O-, and V is a hydrogen atom, or Y is -O-, -S- or -
S-S-, and V is a hydroxyl group), [Formula] (where W is a hydrogen atom or a carboxyl group),
[Formula] Group -CH=CH-Z (where Z is carboxyl group or 2,4-dinitrophenyl group), group -
( _{ii) A is a hydrogen atom, B is a halogen atom or a C1-C6 alkyl group , and R is} a _halogen atom; (iii) A is a halogen atom, B is a hydrogen atom, and R is a halogen atom or a phenyl group, or (iv) A is a hydrogen atom or a halogen atom, and R
and B together represent a naphthalene core constituent chain represented by the formula [formula] (wherein, and in each case of (i) to (iv) above, halogen atom means chlorine atom, bromine atom or iodine atom]. luminescence or luminometric assay method. 2 chemiluminescent 2,3-dihydro-1,
General formula as a 4-phthalazinedione compound (wherein R ¹ is an unsubstituted or substituted amino group, R ² , R ³ and R ⁴ are each hydrogen atom, unsubstituted or substituted C ₁ -C ₆ alkyl group, unsubstituted or substituted C ₁ -C ₆ alkenyl group,
is a hydroxyl group, a _C1 - _C6 alkoxy group, a carboxyl group or an unsubstituted or substituted amino group, or ^R2 is an unsubstituted or substituted amino group, and R ¹ , R ³ and R ⁴ are each hydrogen atom, unsubstituted or substituted C ₁ -C ₆ alkyl group, unsubstituted or substituted
_C1 - _C6 alkenyl group, hydroxyl group, _C1 - _C6
is an alkoxy group, a carboxyl group or an unsubstituted or substituted amino group, or R ¹ and R ² are jointly an unsubstituted or substituted amino derivative of a benzo group; where R ³ and R ⁴ are each hydrogen atom, unsubstituted or substituted C ₁ -C ₆ alkyl group, unsubstituted or substituted C ₁ -C ₆ alkenyl group, hydroxyl group , C ₁ −
The assay method according to item 1 above, which uses a C ₆ alkoxy group, a carboxyl group, or an unsubstituted or substituted amino group. 3 chemiluminescent 2,3-dihydro-1,
The assay method according to item 2 above, wherein luminol or isoluminol is used as the 4-phthalazinedione compound. 4 Using horseradish peroxidase as peroxidase Previous section 1~
3. The assay method described in any one of 3. 5. The assay method according to any one of items 1 to 4 above, which uses hydrogen peroxide or perborate ions as the oxidizing agent. 6. The assay method according to any one of items 1 to 5 above, wherein peroxidase exists in a free form or in a form bound to a ligand, and the presence or amount of peroxidase is estimated from chemiluminescence detection or measurement, respectively. 7. The assay method according to any one of items 1 to 6 above, wherein 4-iodophenol, 4-phenylphenol, or 2-chloro-4-phenylphenol is used as the phenolic compound. 8 Using luminol or isoluminol as the 2,3-dihydro-1,4-phthalazinedione compound and horseradish peroxidase present in a ligand-bound form as peroxidase, the presence or amount of horseradish peroxidase was determined chemically. The assay method according to the preceding item 1, which is estimated from detection or measurement of luminescence, uses hydrogen peroxide as the oxidizing agent, and uses 4-iodophenol as the phenolic compound. 9 chemiluminescent 2,3-dihydro-1,
4-phthalazinedione compounds, peroxidases, and formulas [wherein (i) A and B are hydrogen atoms, and R is a halogen atom, a phenyl group,
[Formula] (where Y is -CH ₂ - or -O-, and V is a hydrogen atom, or Y is -O-, -S- or -
S-S-, and V is a hydroxyl group), [Formula] (where W is a hydrogen atom or a carboxyl group),
[Formula] Group -CH=CH-Z (where Z is carboxyl group or 2,4-dinitrophenyl group), group -
( _{ii) A is a hydrogen atom, B is a halogen atom or a C1-C6 alkyl group , and R is} a _halogen atom; (iii) A is a halogen atom, B is a hydrogen atom, and R is a halogen atom or a phenyl group, or (iv) A is a hydrogen atom or a halogen atom, and R
and B together represent a naphthalene core constituent chain represented by the formula [formula] (wherein, and in each case of (i) to (iv) above, the halogen atom means a chlorine atom, a bromine atom, or an iodine atom]. A kit for use in luminescence or luminometric assays, comprising: 10 Chemiluminescent 2,3-dihydro-
General formula as a 1,4-phthalazinedione compound (wherein R ¹ is an unsubstituted or substituted amino group, R ² , R ³ and R ⁴ are each hydrogen atom, unsubstituted or substituted C ₁ -C ₆ alkyl group, unsubstituted or substituted C ₁ -C ₆ alkenyl group,
is a hydroxyl group, a _C1 - _C6 alkoxy group, a carboxyl group or an unsubstituted or substituted amino group, or ^R2 is an unsubstituted or substituted amino group, and R ¹ , R ³ and R ⁴ are each hydrogen atom, unsubstituted or substituted C ₁ -C ₆ alkyl group, unsubstituted or substituted
_C1 - _C6 alkenyl group, hydroxyl group, _C1 - _C6
is an alkoxy group, a carboxyl group or an unsubstituted or substituted amino group, or R ¹ and R ² are jointly an unsubstituted or substituted amino derivative of a benzo group; where R ³ and R ⁴ are each hydrogen atom, unsubstituted or substituted C ₁ -C ₆ alkyl group, unsubstituted or substituted C ₁ -C ₆ alkenyl group, hydroxyl group , C ₁ −
9. The kit according to item 9 above, which uses a C ₆ alkoxy group, a carboxyl group, or an unsubstituted or substituted amino group. 11 Chemiluminescent 2,3-dihydro-
11. The kit according to item 10 above, wherein luminol or isoluminol is used as the 1,4-phthalazinedione compound. 12. The kit according to any one of items 9 to 11 above, which uses horseradish peroxidase as the peroxidase. 13. The kit according to any one of items 9 to 12, wherein the peroxidase is present in a free form or in a form bound to a ligand. 14 Items 9 to 13 above using 4-iodophenol, 4-phenylphenol or 2-chloro-4-phenylphenol as the phenolic compound
Kits listed in any of the above. 15 Using luminol or isoluminol as the 2,3-dihydro-1,4-phthalazinedione compound, horseradish peroxidase present in a ligand-bound form as the peroxidase, and 4-iodophenol as the phenolic compound. Use the kit described in the previous section 9.