JPS6052378B2 - Heterogeneous immunoassay method and reagent for analyzing haptens or antigens in liquids - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a method for measuring the presence of a hapten or antigen (target substance) in a liquid based on the affinity of the target substance for a partner substance of specific binding (specific binding). and reagents.

More specifically, the present invention relates to a method and a reagent used for analysis by specific binding without using radioactive substances or partially modified enzymes as labeling substances. In addition, in this specification, 1 specific cho and 1 specific cho are used interchangeably. It is clear that there is a need for a convenient, reliable, and non-hazardous method for detecting the presence of low concentrations of substances in liquids.

It is present in body fluids at concentrations as low as 10-11 molar. This is particularly true in the field of clinical chemistry, where components are of pathological significance. The difficulty of detecting such low concentrations of substances is increased in the field of clinical chemistry, where sample quantities are usually very limited. Previously, substances were detected in liquids based on reaction systems in which the substance being detected was necessarily a reactant. The presence of an unknown substance is detected by the appearance of a reaction product or the disappearance of a known reactant. In some cases,
Such analytical methods can also be quantitative by measuring the rate of appearance of products or disappearance of reactants or the total amount of product formed or reactants expended when equilibrium is reached. Obtained. The reaction system for each analysis is necessarily either limited to the detection of only a small group of substances or is non-specific. Research into analytical systems that are highly specific and can be applied to the detection of a wide range of substances has led to the creation of radioimmunoassay methods.

According to this method, the radiolabeled amount of the substance to be detected is made to compete with the unknown for a limited amount of antibodies that have specificity for the unknown. The amount of label that becomes bound to the antibody then varies inversely with the level of unknown material present. In radioimmunoassay technology, a labeled substance to be detected that becomes bound to an antibody (bound phase) is separated from a substance that does not cause such binding (free phase). Various means for accomplishing this required separation are described, for example, in U.S. Pat.
No. 6, No. 372,076 and No. 3,793,445. However, all of these methods
At least one manual step such as filtration, centrifugation, washing or column elution is required to ensure efficient separation of bound and free phases. Such separations accordingly consist of an insoluble part containing a bound phase and a liquid part containing a free phase, in which the amount of radioactive label in each part is a function of the magnitude of binding of the labeled substance and thus the assay. This is done by creating a system that is made to be a function of the amount of target substance in the sample. A heterogeneous system, as commonly used in this field of science and used herein, refers to specific binding assays that involve separation of free phases such as bound phases. There is. Such separation is necessary when performing specific binding assays in systems where the labeled substance in the bound phase is difficult to distinguish from the labeled substance in the free phase. Due to the dangers and difficulties of working with radioactive materials, there are specific methods that are as sensitive and rapid as radioimmunoassays, but easy to use and utilize non-radioactive properties as a means to monitor and detect binding reactions. Many attempts have been made to devise coupled analysis systems.

As described more fully herein below, labels that have been utilized in place of radioactive atoms or molecules include a variety of substances such as enzymes, fluorophores, and bacteriophages. It will be done. Examples of methods developed using enzymes used as labeling substances include U.S. Pat.
39153, and 3850752, and Journal of Immunological Methods, 1:247.
(1972) and Journal of Immunology,
109:129 (1972).

In each of the methods described, the enzyme is chemically bound to either the substance to be detected (ligand) or its binding partner, and after being placed with the sample, the insoluble or ligand portion is removed. An appropriate heterogeneous specific binding reaction scheme is constructed such that the amount of enzyme activity contained in either is a function of the amount of ligand (substance of interest) in the sample. Therefore, problems associated with the synthesis and characterization of enzyme conjugates represent a significant bottleneck to this solution. An interesting enzyme-added immunoassay is U.S. Patent No. 381.
No. 7837.

This method does not require the use of a separate (ie, insoluble part and liquid part) specific binding reaction system, and thus does not require a separate separation operation. This is because the target substance to which the enzyme is attached is made so that the enzyme activity is inhibited by the reaction of the target substance with the substance to which the target substance binds. The ratio of bound (bound) to free (free) phosphorylated substances is thus determined by measuring changes in enzyme activity. However, this method suffers from difficulties in preparing enzyme addition conjugates with excellent properties and in finding enzymes compatible with the basic scheme of this system. GB 1 392 403 and FR 2 2012 describe specific binding assays using inactive precursors of spectrophotometrically active substances as labeling substances.

After retaining the sample with the specific binding reaction system, the insoluble and liquid portions are separated and the amount of labeled substance present in the liquid portion is a function of the amount of analyte to be detected in the sample. It is determined by a reaction process consisting of converting the active labeling substance into a color-changing or fluorescently active substance, which is then measured by conventional means. Other specific binding analysis methods using different types of labeling substances are disclosed as follows. That is, US Pat. No. 3,850,578 discloses the use of electron spin resonance as a labeling means; US Pat. No. 3,901,654 discloses the use of stopping and increasing fluorescence emission as a labeling means. U.S. Department of Commerce National Technical Information Service (NTIS) Report No. PB-224875 (1973) describes an attempt (unsuccessful) to use hemin chloride as a labeling substance in a heterogeneous analytical system that monitors and detects by chemiluminescence reaction. Natya I, 219:186 (19
68) describes certain radioimmunoassay procedures in great detail and also discusses the possibility of using coenzymes and viruses instead of radioactive isotopes as labeling substances due to their highly active nature. It is mentioned incidentally. However, the authors do not teach how to perform the analysis using such alternative markers or whether, as a practical matter, such an analysis method is practicable. To further explain the background, 1
Principles of Competitive Protein Binding Analysis
sOfCompetitivePrOtein-Bin
dingAssaya) Odell and Doday, eds. (Shoo, Biichi, Litpencott Company, Philadelphia, 1972) describes various known analytical methods and the various substances and properties that have been used as labels for specific binding assays. have been extensively discussed. Although many new types of specific binding analysis methods have been suggested and investigated, radioimmunoassays and various enzyme-added immunoassays remain the most widely used and improved.

However, both types of systems have obvious drawbacks. That is, in radioimmunoassays, handling of radioactive substances is dangerous and requires caution, and in enzyme-addition immunoassays, it is difficult to prepare useful enzyme-added conjugates. Therefore, it is an object of the present invention to provide a new method and reagent for detecting a target substance (ligand) in a liquid without using inconvenient radioactive substances or partially modified enzymes as labeling substances.

It is a further object to provide methods and reagents for heterogeneous specific binding analysis that are more versatile and convenient than prior art methods. It is also an object of the present invention to provide a method and reagent for heterogeneous specific binding analysis using a labeling substance that is capable of combinatorial binding to a target substance or its specific binding partner substance more conveniently than the enzymes of the prior art methods. one of. By using a wide variety of sensitive reaction systems, heterogeneous systems using conjugates containing labeled substances can detect any change in the activity of the enzyme more conveniently than any change in the activity of the enzyme in prior art methods. It is also an object of the present invention to provide specific binding analysis methods and reagents.

The present invention provides a highly convenient, widely applicable, and sensitive heterogeneous specific binding analysis method and reagent for labeling a substance exhibiting a reactive activity imparted as a component of a predetermined reaction. It is provided based on the use as a.

Such materials are designated herein as 1 reactant. The labeling substance of the present invention can be used in any conventional heterogeneous specific binding analysis method.

The amounts of reactants present in each of the bound and free phases include at least one reagent and each reactant that together form a predetermined reaction system that serves as a means for monitoring and detecting specific binding constraints. Measured by bringing the phases into contact. Quantitative measurements are carried out by comparing the activity of the reactant measured in one phase with the value obtained by the same method in a liquid containing a known amount of the substance (ligand) to be measured. The improved method generally follows {
a), (b) and (c) engineering. It includes a degree.

That is, improved methods for analyzing haptens or antigens in liquids generally include the following (a), (b), and (c).
) process. That is, (a) a test liquid is mixed with a labeling substance having predetermined characteristics and a hapten or an antigen, or the like! A step of contacting a reagent consisting of an antibody against the antibody or a conjugate with a partner substance that specifically binds to the target substance (
(b) separating said bound phase and said free phase; and (C) said liquid in one of the separated layers. A heterogeneous immunoassay method consisting of a step of measuring properties as an indicator of a hapten or antigen in the conjugate, which is characterized in that the labeling substance in the conjugate is a nucleotide coenzyme for an enzyme-catalyzed reaction. be.

As discussed in more detail hereinafter, the present binding reaction system could take the form of any known conventional technique, such as those used in radioimmunoassay systems and heterogeneous enzyme-immunoassay systems. . Preferably, an enzyme catalyst is used in the monitoring detection reaction.

Typically, the monitoring detection reaction is chosen to be highly sensitive to the reactants in the conjugate. Luminescent or fluorescent reaction systems are very useful in this regard. Particularly preferred are circulating reaction systems, particularly those in which the reactant is a circulating material. Among the preferred cyclic reaction systems, systems utilizing enzymes as catalysts are particularly advantageous. The reactant in the conjugate of the present invention is an enzymatic reactant, ie, a nucleotide coenzyme for an enzyme-catalyzed reaction, and preferably has a molecular weight of less than 9,000. Terms used in this specification are defined as follows. A ligand, a target substance, or a target substance is a substance or a group of substances whose presence or amount in a liquid is to be measured.

A partner substance for specific binding of a target substance (ligand) is a substance or a group of substances that exhibits a specific binding affinity for the target substance to the exclusion of other substances.

A similar substance with respect to specific binding of a target substance is a substance or a group of substances that exhibit essentially the same behavior as the target substance in terms of binding affinity of a partner substance that specifically binds to the target substance.

The heterogeneous immunoassay reagent of the present invention is for analyzing a hapten or antigen in a liquid: a labeling substance having predetermined characteristics and a hapten or antigen, an antibody against the same, or a partner substance for specific binding thereto. and the reagent and the hapten or antigen form a binding reaction system to produce a bound phase or a free phase of the labeled conjugate, and the characteristics of either the bound phase or the free phase. is a reagent for a heterogeneous immunoassay, which is a function of the amount of hapten or antigen in the liquid, characterized in that the labeling substance in the conjugate is a nucleotide coenzyme for an enzyme-catalyzed reaction. be.

Specific binding reagents can take a variety of forms. Generally, such reagents contain three basic components. That is (1)
the substance to be detected (ligand), (2) the specific binding partner of the ligand, and (3) the substance (usually in labeled form).
a) a ligand; (b) a substance analogous to the specific binding of the ligand; or (c) a labeling component that is a partner substance for the specific binding. The components of this coupling reaction may be combined simultaneously or added sequentially. and proper retention (
During the period (storage), the labeled moiety is subject to changes in binding, such as the ratio of the amount of labeled moiety bound (bound) to the amount of unbound (unbound) labeled moiety to the binding partner. It is bound (bound) to the corresponding competitive binding partner substance such that the amount, size, etc. is a function of the amount of target substance present. Some of the various binding reactions used in carrying out the method of the present invention will be briefly described below.

In conventional heterogeneous specific binding assays such as radioimmunoassay and heterogeneous enzyme immunoassay, the labeling properties of the labeled conjugate, such as radioactivity and enzymatic activity, are
It is essentially the same for the conjugate in the bound (bound) and free (free) states.

On the other hand, according to the method of the present invention, the activity of the reactant as a labeling substance is influenced by the binding of the labeled binding complex in some cases. In such situations, the monitored detection reaction exhibits relatively constant properties when the substance of interest is not present in the liquid, or is present in insignificantly small amounts. When the target substance is present in the liquid, the characteristics of the monitoring detection reaction are activated. Change in gender or nature. Generally, the activity of the reactants in the conjugate will be the amount or rate at which the reactants can participate in the monitoring detection reaction. In this way, the nature of the monitored detection reaction is altered by the presence of the substance of interest in the liquid, usually in terms of its overall reaction rate or the equilibrium amount of reaction product or products produced. In this case, the active capacity of the reactant of the conjugate involved in the monitored detection reaction is generally determined by the reaction between the specific binding substance to which it binds and the reactant of the specific binding of the specific binding substance. decreases due to That is, the conjugate in its free state is more active in the monitored detection reaction than when it is in its bound state. The following symbols are used in the following schematic formulas.

Absent 5 filter oki? Flood? Formula L? Instep? -Jyo黶D-1+B or [F] Wood insolubilizer This is done by traditional competitive binding methods.

Examples of such insolubilizing agents include specific precipitating antibodies, specific insolubilizing antibodies, B or 5 protein precipitating agents such as ammonium sulfate when the water is a proteinaceous substance, B or [
F] When water is a small absorbable molecule, dextrin-coated charcoal may be mentioned.
Similar systems are described in Biochemical Journal 88:137 (1963) and U.S. Pat. No. 38391.
Seen in issue 53. 1υ1b! the law of nature! 1/KSIlυ
This method is usually called solid phase technology.

Similar radioimmunoassay and enzyme immunoassay techniques are described in U.S. Pat.
No. 43, No. 3646346, and No. 365409 (G). Reference: US Patent No. 365409 (d) L+卜5+B水(Eim) → Reference: US Patent No. 385075? In the λ-sequential saturation assay+B or the Sukyu insolubilizer sequential saturation technique, some or all of the binding sites of B remaining after the initial retention period are bound to the labeled component.

Similar radioimmunoassay and enzyme immunoassay techniques are described in U.S. Pat. No. 37207(1) and Journal of Immunology 1, 209:129 (19
72).

3 Sand Deutsch analysis method BIlυ (しΔυノ 2LJ'to) (ν11ν In the Sand Deutsch analysis method, part or all of the ligand (target substance) molecule bound to the inactivated binding partner substance is bound to the labeled component. There is.

Reference: U.S. Patent No. 3,720,760S3±-Solid Phase Dilution Analysis In this technique, the ligand and the labeled moiety are bound to a non-specific binding agent, and a proportional amount of the ligand and the labeled moiety is then attached to a larger amount. Dissociates upon binding with a partner substance that exhibits affinity.

The most useful form of this technology is U.S. Pat.
There is a method using a column of non-specific binding agents as described in No. 104. Such techniques are useful when the ligand is bound to endogenous binding substances in the sample that will interfere with competitive binding reactions unless removed. After binding to the non-specific binding agent, endogenous binding material is removed by appropriate washing. For a description of the variables included in a typical heterogeneous analysis system, such as a more detailed description of analysis methods and alternative separation techniques, see 1. Principles of Competitive Protein-Binding Analysis.
J (supra) may be cited as a reference.

It is anticipated that other addition orders and other binding reaction configurations may also be devised to perform heterogeneous specific binding assays without departing from the inventive concepts described herein.

Evaluating the activity of the reactants of the conjugate of the components of the predetermined monitoring detection reaction in the bound or free phase comprises:
Monitoring detection is conveniently accomplished by contacting the reaction with at least one substance that forms its phase with the reactants of the conjugate and then measuring the characteristics of the reaction.

The monitoring detection reaction consists of one or a series of multiple chemically transforming or transforming reactions. If an enzyme-catalyzed reaction system is utilized, the system will include the conjugate reactants and at least one additional enzyme, and may also include one or more enzyme reactants such as substrates and coenzymes. good.

Such enzyme-catalyzed reaction systems may involve a single simple enzymatic reaction or a complex series of enzymatic and non-enzymatic reactions. For example, an enzymatic reaction system may consist of a single enzyme-catalyzed degradation or dissociation reaction. In such systems, the reactant of the conjugate is the enzyme substrate that undergoes degradation or dissociation, and the only component of the reaction system required to contact the selected bound or free phase is the enzyme substrate that undergoes degradation or dissociation. There are enzymes that catalyze reactions. More complex enzyme-catalyzed reaction systems may involve a single enzymatic reaction involving two or more reactants, or may involve several reactants (provided that only one of the (One is enzyme-catalyzed). In such systems, the reactant of the conjugate is one of the enzymatic reactants of the enzyme-catalyzed reaction, and the selected bound or free phase is selected separately from that in the conjugate. Contact is made with the appropriate enzymes and reaction components necessary to form the enzyme-catalyzed reaction system.
The enzyme-catalyzed reaction system is also intended to include complex biochemical systems such as the cellular ecosystem of living organisms such as microorganisms. For example, nutritional substances essential for the growth of a particular microorganism may be selected as reactants in the conjugate. When such a microorganism is placed in an environment where the only source of nutrients for the reactant is the conjugate, the activity of the reactant can be determined by monitoring and detecting characteristics of the microorganism, such as the growth rate of the microorganism. Suitable reaction components that together with the reactants in the conjugate form the monitoring detection reaction system can be selected and separated, alone or in combined form, prior to, concurrently with, or subsequent to the initiation of a particular binding reaction. phase (separated phase).

After initiation of the specific binding reaction, the reaction mixture containing some or all of the essential components of the monitoring detection reaction is typically incubated for a predetermined period of time before separation of the resulting bound and free phases. After separation, components required for the monitoring detection reaction and not already present in sufficient quantities in the selected separation phase are added thereto. The activity of the reactant therein is then evaluated and measured, and is used as an indicator of the presence or amount of the target substance in the liquid. When the kinetics of a monitored detection reaction is a property used to assess and measure the activity of a reactant in the selected bound or free phase (which is preferred), the rate is usually determined by the disappearance of the reactant. It is determined by measuring the rate or rate of appearance of reaction products.

Such measurements can be carried out using conventional chromatographic, gravimetric,
It is carried out by a wide variety of methods including analytical techniques such as potentiometric titration, spectrophotometry, fluorometry, turbidity, volumetric and the like. Since this method is primarily intended for the detection of low concentrations of target substances, a highly sensitive reaction system has been developed for use in combination with the novel specific binding reaction system of the present invention. One preferred form of monitoring detection reaction is a luminescent reaction system, preferably an enzyme-catalyzed reaction, such as a reaction exhibiting the phenomenon of bioluminescence or chemiluminescence.

The reactants in the conjugate may be reactants of a photogenerating reaction or a reaction preparatory to a light-emitting reaction with or without an enzyme. The activity of the reactants of the conjugate is determined by the rate of light production (rate),
It is evaluated based on the total amount, peak intensity, or nature of the light emitted. Examples of luminescent reaction systems include those listed in Table A, and the following abbreviations are used in this table. ATP: Adenosine triphosphate AMP: Adenosine monophosphate NAD: Nicotinamide ● Adenine dinucleotide NADH: Reduced nicotinamide ● Adenine ●
Dinucleotide FMN: flavin ● Mononucleotide FMNH2: Reduced flavin ● Mononucleotide hν
: Electromagnetic radiation, usually in the infrared, visible or ultraviolet range.Further details and discussion of luminescent reaction systems that can be utilized in the method of the present invention can be found in the following references.

Journal of Biological Chemistry 1, 2
36:48 (1961) Journal of American Chemical Society, 89:3944 (1967)
Comier et al., Advances in Bioluminescence, Johnsson et al., eds., Princeton University Press (New Jersey, 1966), pp. 363-84. Kr.
ies), Renilla Luciferase (RenillaL
uciferase) purification and properties, Georgia University doctoral thesis (1967) American Journal of Physiology 1, 41:454 (1916) Biological Journal, 51:89 (1926) Journal ●
Ob●Biological●Chemistry 1, 243:471
4 (1968).

Other types of preferred sensitive monitoring detection reactions include those that involve fluorescence and are enzymatically catalyzed.

In such a reaction system, the reactant in the conjugate is the substrate in an enzymatic reaction, and the enzymatic reaction produces a product with fluorescent properties that is distinguishable from the substrate in the conjugate. It is something to do. The general reaction equation for such an enzyme-catalyzed reaction system is as follows. In the above formula, X represents a bond or linking (bridging) group that is cleaved by an enzyme, such as an ester group or an amide group, and Z represents a target substance (ligand) or a target substance that can be identified by the specific binding reaction technology used. A specific binding substance that is either a similar substance for binding or a partner substance for specific binding of the target substance.

Specific conjugates that can be used in this type of reaction system are:
Fluorescein, umbelliferone, 3-indole,
β-naphthol, 3-pyridole, resorufin, rhodamine B1 and various other enzyme-cleavable derivatives. Examples of possible structural formulas for such derivatives are as follows. In the above formula, R1 is -0H or -X-Z
(X and Z are the same as above), R2 is -X-Z1 and R3 is -H or -CH3.

A particularly preferred reaction system used to assess the activity of the reactants of the conjugate in the selected separated phase is a cyclic or circulating reaction system.

Such a reaction system is one in which the product of the first reaction becomes the reactant of the second reaction, and the second reaction has as its product a substance that also becomes the reactant of the first reaction. One model of a cyclic reaction system is expressed by the following diagram: l In the cyclic reaction system of the above model, if sufficient amounts of reactants A and B exist, a small amount of circulating substances A larger amount of product A. (5B) can be produced.

Since the rate and amount of products produced by the reactions that make up the circulating reaction system are highly sensitive to the amount of circulating material present, said circulating material may be used as a reactant in the conjugate of the present invention. It is particularly preferred to use Examples of cyclic reaction systems intended to be used in combination with the novel specific binding reaction system of the present invention are shown in Tables B, C and D. It should be noted that the cyclic reaction systems listed in Tables B and C include all combinations of any one of the reactions listed in each table with any of the other reactions listed in the table. be. For example, reaction 1 in Table B can be combined with any one of reactions 2 through 8 to form a useful circulating reaction system. Therefore, Table B and C
The tables represent respectively 56 and 20 possible cyclic reaction systems for use in the present invention. In addition to the cyclic reaction systems represented in Tables B and C, there is also one of the cyclic reaction systems comprising an enzymatic or non-enzymatic modification of a spectrophotometric indicator (preferably a colorimetric one). contemplated by the present invention.

If a preferred colorimetric indicator is used, the change will be a color change. An example of a cyclic reaction system involving a change in indicator is one obtained by combining one of the reactions between reactant B and product B in Table B with a reaction involving an oxidation-reduction indicator and an electron transfer reagent. The system can be mentioned. Phenazine methosulfate can be used as the electron transfer reagent. Useful indicators include nitrotetrazolium, thiazole blue and the oxidized form of dichlorophenol indophenol. In the circulation reaction system described in Tables B, C, and D, each component is an ion,
Although shown in acid or salt form, in forming any of the reaction systems in those tables, each component can, of course, also be added in the form of a salt or acid that ionizes upon contact with the liquid. be.

Monitoring detection reaction systems are also intended to include exponential cycling reaction systems.

An example of an exponentially cyclic reaction system is as follows. Such a cyclic reaction is an autocatalytic reaction in the sense that the amount of circulating material doubles during each cycle.

The circulation rate therefore increases exponentially with time, resulting in high sensitivity. Further details and discussion of such cyclic reactions can be found in Journal of Biological Chemistry, 247; 3558-70 (1972). If a cyclic reaction system is used as a means of assessing any change in the activity of the reactants of the conjugate, the rate of disappearance of the reactants or the rate of appearance of the reaction products can be determined using one or more circulatory systems and then It can be determined by measuring the reaction rate using a conventional method.

The use of a cyclic reaction system combined with a heterogeneous specific binding reaction system provides high analytical sensitivity as well as high applicability.

Conjugates of specific binding substances in a single reactant are used in conjunction with multiple reactions to create a circulatory system that can vary in sensitivity over a wide range and yield a wide range of responses detectable by the sense organs or by artificial means. It's also good to let them do it. Such applicability is something lacking in the prior art. The invention can be applied to the detection of any hapten or antigen in which a specific binding partner is present.

These substances of interest are usually peptides, proteins, carbohydrates, glycoproteins, steroids, or other organic molecules for which specific binding partners exist in biological systems or for which partners can be synthesized. In functional terms, this target substance is selected from the group consisting of antigens, their antibodies, haptens (adherents), their antibodies and vitamins, and their receptors and binding substances. Particular examples of target substances that can be detected using the present invention include antigens and haptens such as ferritin, bradekinin, prostaglandin and tumor-specific antigens; biotin, vitamin Bl2,
Vitamins such as folic acid, vitamin E and ascorbic acid; antibodies such as microsomal antibodies, hepatitis antibodies, allergen antibodies; and thyroxane binding globulin, avidin,
Specific binding receptors such as intrinsic factor and transcobalamin are included. In the conjugate of the present invention, the reactant is
The specific binding substance (depending on the analytical method chosen, the target substance, an analogue for the specific binding of the target substance, or a partner substance).

The bond between the reactant and the specific binding substance is such that a predetermined monitoring detection reaction using an active reactant chemically breaks the bond, such as in the luminescent and cyclic reaction systems described above. Under conditions of analysis for which it was not designed, it usually cannot be substantially changed. However, in some cases such bonds are designed to be broken or influenced by selected monitoring detection reactions as a means to assess changes in reaction activity. Such cases include the enzymatic fluorogenic substrate reaction systems described herein above. The reactant may be combined and bound directly to the specific binding substance such that the molecular weight of the conjugate is equal to or less than the total molecular weight of the reactant and the specific binding substance.

However, usually the reactants and the specific binding substance are connected by a bridging group containing from 1 to 5 atoms, preferably from 1 to 10 carbon atoms or heteroatoms such as nitrogen, oxygen, sulfur, phosphorous, etc. There is. Examples of bridging groups containing one atom include methylene groups (one carbon atom) and amino groups (one heteroatom).

The bridging group usually has a molecular weight not exceeding 1000, preferably less than 200. Bridging groups contain chains of carbon atoms or heteroatoms, or combinations thereof, and usually include linking groups in the form of groups such as esters, amides, ethers, thioesters, thioethers, acetals, methylene or amino groups. the reactant and the specific binding substance or active derivative thereof. A reactant in a conjugate of the invention is a substance that has an endowed (eg, constant or known) reactive activity as a component of a predetermined monitoring detection reaction.

More specifically, as disclosed herein, one reactant and one reactive substance are defined as defined and measurable substances that result in one or more products different from themselves. can undergo chemical transformation (alteration),
Furthermore, by interaction of the reactants with reaction initiation means such as chemicals (e.g., other reactants, catalysts, other types of substances to participate in such chemical transformations or alterations),
means a substance that provides electromagnetic radiation, thermal energy, or sonic energy. Accordingly, the group of substances defined herein as a reactant includes conventional inorganic and organic reagents and biochemical substances. However, catalysts (including enzymes) and radioisotopes that are not reactants in monitoring detection reactions are excluded. Because a particular chemical can function in different ways depending on its chemical environment, a single chemical may be classified into various different classes, and such substances are not included herein. It will be appreciated that the function of the disclosure is determined by the reactivity of the material with respect to the selected monitoring detection reactions described herein. The reactant of the present invention is a reactant of an enzymatic reaction, such as a nucleotide coenzyme or an active partially modified substance or derivative thereof.

Coenzymes are non-protein molecules that move from one enzyme protein to another when promoting the efficient catalytic function of enzymes. Known coenzymes have a molecular weight of less than 9000, and coenzymes with a molecular weight of less than 5000 are preferred. Examples of useful nucleotide coenzymes include adenosine phosphate (eg, mono-, di-, and triphosphate forms), nicotinamide adenine dinucleotide and its reduced form, and nicotinamide adenine dinucleotide phosphate and its Examples include those containing an adenine group such as a reduced type. Other useful nucleotide coenzymes include guanosine phosphate, flavin●mononucleotide and its reduced form, flavin●adenine●dinucleotide and its reduced form, coenzyme A and its thioesters (including succinyl-coenzyme A), 3 ″, 5″-adenosine diphosphate, and adenosine-3″-phosphate-5″. There is phosphosulfate. Useful coenzyme-active conjugates include direct binding of a specific binding substance (e.g., a ligand, an analog for a specific binding of a target substance, or a partner for a specific binding of a target substance) as described above or Includes nucleotide coenzymes with adenine groups linked by bridging groups.

Such coenzyme-active conjugates comprising adenosine phosphate, nicotinamide adenine dinucleotide or a reduced form thereof, or nicotinamide adenine dinucleotide phosphate or a reduced form thereof have the general formula: In the above formula, R1 or R3 represents or; R4 represents or; R5 represents -Y-Z (Y is a bond or a bridging group, Z is a target substance (ligand), and relates to a specific bond of the target substance) is a similar substance or a partner substance for specific binding of the target substance)
represents.

Although the above formula represents the ionized form of the coenzyme-active conjugate, the hydrogenated or salt forms are equally useful. The amount of hydrogenation depends on the PH of the environment. Salts of these conjugates may also be used where appropriate. Synthesis of such compounds is carried out by various methods.

It may be advantageous in producing useful compounds to follow the synthetic routes shown schematically below. In the synthetic scheme, the position of the adenine ring structure is shown as follows. It also indicates the following omissions: Rib indicates the ribose portion (see the figure below).

Rib'' indicates a phosphorylated ribose portion (lower figure).

Ph represents a phosphoric acid group. AP derivatives refer to derivatives of adenosine-5'' monophosphate, such as monophosphate (AMP), diphosphate (ADP), and triphosphate (ATP).

The N,AD derivative refers to nicotinamide*adenine*dinucleotide or its reduced derivative.

The non-NMW derivative refers to nicotinamide adenine dinucleotide phosphate or a reduced derivative thereof. P represents a specific binding substance or a partially modified substance thereof.

7X represents a dissociable group (usually halogen).

(1) Etsuchi●Gilford et al., Chemica●Scripter,
2:165 (1972) (2) Ai Pi, Treyer et al., Biochemistry Journal, 139:609 (1
974) 1-position derivative of NAP NAD derivative (3) 1-position derivative of NADP (3) 1-position derivative of NADP NADP derivatives (4) C. R. Rowe and K. Mosvatu 2 (P
Chemistry 1, 49:511 (1974) Ha, European ● Journal ● of ● Bio* N? Akebono ozuql?
Conductor (5) R.M. Acheson, Introduction to Heterocyclic Compound Chemistry, Interscience Publishing (New York, 196)
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0:2239 (1897) {7) Daboll et al., Journal of Chemical Society, 967 (1948)
(8) H. Gilford et al., previously mentioned (9) I*Pi* Treyer, mentioned above, 21-position derivative of NAD (10) N*A* Hughes et al., Journal of...
Chemical Society, 3733 (1957) NADP
2-position derivatives of AMP derivatives NADP derivatives (11) N.A. Hughes et al., 3-position derivatives of the aforementioned AP (12) A.H. Lister, Advances in Heterocyclic Chemistry, edited by Kablicki et al., Academic. ●Press (New York, 1966) p. 33 (13) N.G.A. Leonard and T.G. Fujii, Journal of American ●Chemical Society, 85:3719
(1963) (14) I I ● Fissia I, mentioned above (15) Daboll et al., mentioned above (16) Etsu Gilford et al. mentioned above, (17) I ● P I Treyer et al. mentioned above, NAD 3-
Positional derivatives (18) NA Hughes et al., the 3-position derivative of NADP (19) NA Hughes et al., the 6-position derivative of AP (20) H. Gilford et al., the 6-position derivative of the aforementioned NAD Derivatives (21) I.P. Treyer et al., mentioned above (22) H. Jiichi Nuw: I Morihagi Windmiller One and N.O.I.
Power Plan, Journal of Biological Chemistry 1 236:2716 (23) C1 R Roe and K Mosbach, 8-position derivative of AP (24) I P1 Treyer et al., NAD mentioned above 8-
Positional derivatives (25) Shi I ● Y ● Riichi et al., Archives ● of ● Biochemistry I ● and ● Biophysics, 163:5
61 (1974) 8-position derivatives of NADP (26) C. Y. Riichi et al., mentioned above (27) C. R. Roe and K. Mosva*
C. The above-mentioned 5Nτi = particle member derivative (28) N.G. Leonard and T.G.
Fujii, mentioned above (29) G.E.T.R. Star, mentioned (30) I. Fisher, mentioned above (31) Daboll et al., mentioned (32) Etsu Gilford et al., mentioned (33) I.P. Treyer. In addition, the aforementioned NAD 9-
Positional derivatives (34) NADP, et al.; 9-position derivatives of NADP (35) NADP, et al.; Useful enzymatically active conjugates also include adenosine phosphate.

Such compounds have the general formula: In the above formula, R1 represents or; R2 is -Y-Z (Y is a bond or bridging group, Z is a target substance (ligand), a specific bond of the target substance; a similar substance or a specific bond of the target substance) is the partner substance)
represents.

It can likewise be used in the form of hydrogen adducts, acids and, where appropriate, salts. Synthesis of such compounds can be accomplished by a variety of methods.

In preparing this useful compound, it may be advantageous to follow the synthetic route illustrated below. The abbreviations given above also apply to the diagrams below. ~? Evil? Humiliation (36) Ai Piichi Treya et al., Biochemical
Journal, 139:609 (1974) (37) I, P1, Treyer et al., As one form of the present invention described above, the component of the specific binding reaction that is combined with the liquid that is thought to contain the target substance is a liquid. Or in solid form.

The analytical method is carried out in standard laboratory vessels, such as test tubes, with solid or liquid components of the specific binding reaction and the components of the reaction system added thereto. One or more components of the specific binding reaction and/or one or more components of the monitoring detection reaction may be included in the carrier.

One way of thinking is that the carrier is a liquid-containing container, such as a test tube or capsule, containing one or more of these components in its internal part, for example in liquid or loose solid form, or in a coating on its internal surface. It may also be a container for use.
Alternatively, the carrier may be in the form of a matrix that is insoluble and porous, preferably absorbent, with respect to the liquid to be tested. Such matrices are, for example, absorbent papers; polymeric films, membranes, fluffs or masses; gels and other forms. In such a form, the device contacts the liquid to be tested and performs the specific binding reaction and/or
or provide a convenient means for performing a monitored detection reaction and performing the necessary separations and observing the resulting response. The liquid to be tested is naturally occurring or artificially produced, and is known or estimated to contain a hapten or antigen (target substance).

Usually, it is a liquid substance of an organism or a liquid obtained by diluting or otherwise processing it. Liquid substances from living organisms that can be analyzed by the method of the present invention include serum, plasma, urine, amniotic fluid, brain fluid, cough fluid, and the like. Other materials, such as solid materials such as cells or gaseous ones, can also be analyzed in liquid form by methods such as dissolving the solid or gas or extracting the solid. In contrast to prior art analytical systems, the target substance in the biological fluid containing a substance with a reactive activity the same or similar to that of the labeling substance in the conjugate is not disturbed in the background. can be analyzed without being

Endogenous background reactant activity can be easily removed in a variety of ways. Biological fluids can be treated to selectively destroy endogenous reactant activity. Such treatments include, for example, applying a detergent that chemically destroys endogenous activity and then inactivating the destructive action of the detergent. for example,
Correspondingly, there are enzymes present in the biological fluids that naturally degrade the reactants.

In particular, the reactants are NAD, . This is the case for coenzymes such as NADP or ATP. There are many inhibitors of enzymes with degraded coenzymes, such as chelating agents that act to deprive enzymes of essential metal ion active substances. As a particular example, NAD-degrading enzymes are found in normal serum, which has sufficient enzymatic activity to substantially remove all endogenous NAD activity from separated serum within a few hours. Such enzyme-degrading activity can be effectively inhibited by adding a chelating agent such as ethylenediaminetetraacetic acid. can be inhibited. Degrading activity can also be eliminated by adding specific enzyme inhibitors. For example, ATP degrading enzyme is βγ-methylene A.
It can be inhibited by adding TP or αβ-methylene ATP. The invention will be illustrated below by means of examples, but these are not intended to limit the invention.

Example 1 Production of nicotinamide/6-(2-aminoethylamino)/purine/dinucleotide 2g of nicotinamide/adenine/dinucleotide (NAD) in 10ml
of water, and then 0.6 mt of ethyleneimine was added dropwise.

At this time, the pH is maintained at 7 or less by adding 1M perchloric acid. After the dropping of ethyleneimine is completed, the pH
is adjusted to 4.5 and kept at 20-25°C for reaction. Add 0.6 mL of ethyleneimine every 2 casting intervals and adjust the pH.
Readjust to 4.5.

After % hours, the solution is poured into 1 volume of -10 DEG C. acetone, the oil formed is collected, washed with ether and dissolved in about 50 ml of water in a flask. The obtained solution is 1
pH7. with N sodium hydroxide. Adjust to O-7.5 and add 1 gram of sodium bicarbonate to it.

Pass nitrogen through the solution for 4-5 minutes, then add 1 gram of sodium hydrosulfite. Seal the flask and let stand at room temperature. Next, this solution was treated with one powder of oxygen, and the pH was adjusted to 11.3 with sodium hydroxide, and the pH was adjusted to 7.
Heat at 5°C for 1 hour. The reaction mixture was cooled to room temperature and 0
．． Add 6 grams of tris-(hydroxymethyl)-aminomethane and then adjust the pH to 7.5 using dihydrochloric acid. Add 100 pharyngeal units of alcohol dehydrogenase and 1 ml of acetaldehyde to the resulting solution. Monitor the reaction mixture for a decrease in optical density at 340 r1rn and adjust the pH to 3.5 when no decrease is observed. The solution is poured into 1 volume of −10° C. acetone and the oil formed is separated, washed with ether and dissolved in 10-15 wL of water. The resulting solution is introduced into a 2.5 x 90C7X column of Sephadex G-10 (obtained from Pharmacia E.B., Uppsala, Sweden) equilibrated with water.

Collect 12 ml of each fraction.

The wavelength of maximum optical absorption in the ultraviolet region and the optical density at that wavelength are measured for each fraction. The optical density at 340 nm of each fraction after reduction with alcohol dehydrogenase is also measured. 264r1
It has a maximum value of optical absorption at rn, and the ratio of the optical density at 340r1n1 and the optical density at 264nn1 is 0.5.
Collect larger fractions.

The collected material is evaporated by rotary evaporator to 15-
Dowex 1- concentrated to 20ml and equilibrated with water.
Pass through a 2.5 x 28 crn column of X8 (obtained from Bio-Rad Laboratories, Richmond, Calif., USA). Add more water to the column to wash the collected material and collect 10 ml fractions each. 2
It has the maximum value of optical absorption at 64r1rn, and 340n
The ratio of the optical density at n1 and the optical density at 264 nm is 0
．． Collect fractions greater than 1.

The collected material was washed with Dowex 50-X equilibrated with water.
2 (5 x 45) Cln (obtained from Bio-Rad Laboratories, Richmond, Calif., USA).

Add more water to the column to wash the collected material and add 20 m
Collect each fraction of t. It has a maximum optical absorption at 264 nm and an optical density at 340 nm of 2.
Collect the fraction with a ratio of optical density at 64 nm greater than 0.18.

The collected material is concentrated to 4-5 ml and purified by electrophoresis as described below. The concentrated material was poured into a thin strip of Watmann 3MM paper (1-2 cm wide perpendicular to the direction of liquid flow).
United States, New Jersey, Krypton's Leaf (obtained from Angel).

Then the paper was heated to pH 6. Moisten with 0.02M sodium phosphate with O. Electrophoresis method is Science 121:829 (19
It is carried out for 4-6 hours with a potential gradient of about 8.5 volts/CTfl according to the Durrurn hanging paper method described in 55). The position of the desired pyridine dinucleotide derivative was determined by spraying 0.5 M sodium cyanide on the test strip according to the method described in Journal of Biological Chemistry, 191:447 (1951). Determined by the fluorescence that appears. Cut out an area containing the desired derivative from the paper, 50 m
Extract 3 times with 1 water. The resulting extract containing nicotinamide 6-(2-aminoethylamino)purine dinucleotide is collected, concentrated to 3-4 ml and then stored at -20°C. Example 2 Production of a conjugate of nicotinamide adenine dinucleotide and biotin Nicotinamide 6- obtained in Example 1
Suspend the biotin of 16Tn9 in 1 ml of water containing the (2-aminoethylamino)purine dinucleotide Nln9.

Add a few drops of 0.1N sodium hydroxide to help dissolve the biotin. Add 24'9 of 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide meth-P-toluene sulfonate to the resulting solution, and add 0.1N
Make a solution by adding hydrochloric acid dropwise. The reaction mixture is left at room temperature for 5 hours and then poured into 10 ml of -100C acetone. The oil formed is separated off, washed twice with 5-10 ml of ether and dissolved in 1-2 ml of water.
The obtained product is purified by the paper electrophoresis method described in Example 1. When sodium cyanide is sprayed, multiple fluorescent bands appear, one moving toward the cathode and the other toward the anode. The latter band contains the N75O-biotin conjugate, which is extracted with water and stored in -2CfC. Example 3 Production of biotin-umbelliferone conjugate (2-oxo-2-H-1-benzopyran-J■■So-1H-thieno-(3,4-d)-imidazole) butyric acid ester 300
A solution of wL9 (1.2 mmol) of anhydrous biotin in 20 mt dry dimethylformamide is stirred at -10°C under dry nitrogen, then 0.17 wLt (1.2 mmol) dry triethylamine is added.

Freshly distilled ethyl chloroformate (0.141 ml in 3 TfL1 dry ether) is added dropwise. 3. After maintaining the powder while stirring, the resulting precipitate was separated under dry nitrogen.
Cool immediately to -10°C.

A solution of 197Tn9 (1.2 mmol) of anhydrous 7-hydroxycoumarin dissolved in 3 ml of dry pyridine was added to the separated residue, followed by stirring at -10°C for 1 hour and then at 25°C for 2 hours. Stir. Solvent in high vacuum 4(s)
Evaporate with water. After cooling, the obtained solid is separated and recrystallized from methanol to obtain the desired product. Melting point 216-218°C Calculated value (as Cl9H2ON2P5S): Cl48.75; Hl4. l9:Nl7.2l.
Analysis value: C,. 58.4: Hl5. l2;Nl6.86
Example 4 Competitive Binding of Biotin - Bioluminescence Analysis: Effect of Varying the Amount of Biotin on the Obtained Peak Light Intensity The bioluminescence reaction used in this example is based on the following reaction.

(a) NAD-ligand + ethanol Alcohol-based hydrogen, NAD trigger, de-enzyme Ten acetaldehyde did.

PH7. A reagent mixture containing 0.13 M phosphate buffer of -2
Store in the dark at 0°C.

An emulsion consisting of 5 wLL of water containing 5 μ' of dodecanol is prepared on the day the photogenerating solution is used.

Luciferase extracted from Photobacterium fuicieri (enzyme obtained from Worthington Biochemical Corporation, Freehold, New Jersey, USA) and lyophilized was added to 0.013r at pH 7.3.
Add 1!4 phosphate buffer to a concentration of 20 mg/ml. After 3 cycles, the resulting suspension is centrifuged at 1500×g for 1 cycle, and the broken material is discarded.

The reagent mixer of 75 Pe, 5 μe of dodecanol emulsion, and 20 Pe of luciferase solution are then combined to prepare a photogenerating solution for use within 5 minutes. B. Production of insolubilized binding partner substance Avidin, which has a binding affinity for bition, is insolubilized by covalently bonding to water-insoluble polymer compound beads as follows.

Appropriate amounts of Sepharose 4B (obtained from Pharmacia E.I., Uppsala, Sweden) were conjugated to avidin using the method of March et al. (Analytical Biochemistry 1, 60:149 (1974)). Activate regarding.

Approximately 4m1. 8ml (7ml) of activated cepharose a
) Suspend in 0.1M tartrate buffer (PH7.O).
This suspension contains avidin 6m9 with an activity of 9.9 units/M9! Add t of water. One unit of avidin activity is the amount of avidin that can bind 1 μg of biotin. The resulting reaction mixture was heated to 7°C.
Stir for 6 hours. Next, separate the avidin-bound Sepharose A8 mixture, wash with 100 ml of 0.1 M sodium bicarbonate buffer (PH 9.0), and wash again with 240 ml of 0.1 M sodium bicarbonate buffer (PH 9.0).
Suspend in 1M Tris-(hydroxymethyl)-aminomethane hydrochloride buffer (PH 8.0). ) C control experiments, each with a total volume of 0.19 mL, were prepared using 0.1 M Tris-(hydroxymethyl)-aminomethane hydrochloride buffer (PH 8).
．． O), 0.6 M ethanol, 0.01 M semicarbazide hydrochloride, and NAD. , NAD-biotin conjugate, avidin-conjugated monosepharose a suspension (obtained in B of this example),
Then, a suspension of cephalose a (obtained by suspending solidified cephalose a in 60 ml (7) of 0.1 M tris-(hydroxymethyl)-aminomethane hydrochloride buffer (PH 8.0)) was added to each. Nine specific (specific) reaction mixtures are prepared containing:

The reaction mixture is gently shaken once at room temperature.

Then each reaction mixture was added with 0. Add n international units of alcohol dehydrogenase to initiate the reduction reaction. In reaction (a), semicarbazide combines with the acetaldehyde produced to form semicarbazone, thus moving reaction (a) in the desired direction. The reaction mixture is again shaken once at room temperature. A 10 μe volume of supernatant of each reaction mixture was added to 25
Separate cuvettes containing 100 μe of the previously prepared photogenerating solution that had been pre-stored at Inject it into the (installed in the ●I-du-Pont●de●Nemor). The results are shown in Table 1.

In the results of reactions 1 and 9 of the control experiment, NAD and NAD
- It has been shown that in the absence of biotin conjugates, very little light is generated.

The results of reactions 2 and 3 indicate that a photogenerating reaction occurs when free NAD is added, and that such a reaction is avidin-binding-sepharose? It has been shown that there is almost no effect even in the presence of The results of reactions 4, 5, and 6 demonstrate that the NAD-biotin conjugate is active in the photogenerating reaction, and that the peak light intensity generated is dependent on the NA present.
This shows that the amount of D-biotin conjugate increases as the amount increases, and that the presence of avidin-conjugated monosepharose a inhibits photogeneration. A comparison of the results of Reactions 3 and 5 with Reactions 7 and 8 shows that the photogenerating reaction is not affected by the presence of intact cepharose a. D Analytical method A total volume of 0.19 mt was prepared using 0.1 M tris-(hydroxymethyl)-aminomethane hydrochloride buffer (PH8).
．． 0.6 M ethanol, 0.01 M semicarbazide, and N.O), 0.6 M ethanol, 0.01 M semicarbazide, and N. /1d)
-Biotin conjugate, free biotin and avidin monoconjugate-
Five additional specific binding reaction mixtures are prepared, each containing a Sepharose 4B suspension.

Each reaction mixture is processed in the same manner as the control reaction mixture in Example C.

The results are shown in Table 2. The results of reactions 11, 12, and 13 indicate that free biotin and N,AD-biotin conjugate effectively compete for the binding sites of insolubilized avidin.

This is understood because the peak light intensity generated is dependent on the amount of free biotin present. reaction 10
and 14 show that the peak light intensity generated in the absence of insolubilized avidin remains constant even when the concentration of free biotin is varied significantly. From the above, this example shows that the amount of NAD-biotin conjugate in the liquid phase has an inverse relationship to the amount of free biotin present, and therefore, the analytical method of the present invention The reagent is useful for the determination of a target substance (ligand) in an unknown liquid sample.

Example 5 Specific (specific) binding analysis of biotin and avidin using an enzyme substrate as a labeling substance The specific binding analysis system used in this example is based on the following reaction.

Biotin-Umbelliferone Conjugate＼]1V-&″l＼＼ｮｲｮｲｲｲｲｲｲｲｲｲｲｲｲｲｲｲｲｯｲｲｲｯｲｲｲｲｲｲｲｲｲｲｲｲｲｲｲｲｲｶｶｲｶｶｶｶｶｶｶｲｲｲｲｶｶｶｶｶｶｲｲｲｯAvidin is covalently bonded to water-insoluble polymer compound beads in the same manner as B in Example 4. This makes it insolubilized.

However, 100ml of 0.1M sodium bicarbonate buffer (
PH9. After washing with O), the avidin-conjugated-cepharose a was suspended in 12 ml of 0.1 M tris-(hydroxymethyl)-aminomethane hydrochloride buffer (PH 8.0) and 0.1 M bis- Dilute 1:1 with hydroxyethylglycine hydrochloride buffer (PH 7.0). Competitive binding analysis of B-biotin: Effect of varying the amount of biotin on the amount of umbelliferone released. Each in a total volume of 0.2 ml, each in 0.1 M bis-hydroxyethylglycine hydrochloride buffer (PH 7.0), 0.2 ml of 0.1 M bis-hydroxyethylglycine hydrochloride buffer (PH 7.0); 3 pM biotin-umbelliferon conjugate (obtained in Example 3), 15 μ' avidin-conjugated monocephalose heart suspension (obtained in A of this example), and the concentrations shown in Table 3. Prepare eight specific binding reaction mixtures containing biotin.

The reaction mixture is kept at room temperature for 20 minutes with gentle agitation. Each reaction mixture was centrifuged and the supernatant was
The μl volume is combined with 2 ml of 0.1 M bis-hydroxyethylglycine hydrochloride buffer (PH&2) containing 1.08 units of porcine esterase). 448I1n1 with excitation at 364 nm after 5 min at room temperature.
The intensity of the fluorescence generated in each reaction mixture is measured using an ArIlicO-BOwnman firefly spectrometer. The results are shown in Table 3. According to the above, in this example, the amount of NAD-biotin in the liquid phase is
It has been shown to be directly proportional to the amount of free biotin present.

Claims (1)

[Claims] 1. For analyzing haptens or antigens in liquid: (a
) A step in which a liquid is brought into contact with a reagent consisting of a conjugate of a labeling substance having predetermined characteristics and a hapten or antigen, or an antibody against it, or a partner substance for specific binding thereto; producing a bound phase or a free phase of a conjugate in which the target substance (ligand) that generates the reaction system is labeled; (b) separating the bound phase and the free phase; and (c) the separated layer. In one of the
A heterogeneous immunoassay method comprising the step of measuring the properties of a hapten or antigen in the liquid as an indicator, characterized in that the labeling substance in the conjugate is a nucleotide coenzyme for an enzyme-catalyzed reaction. Analysis method. 2. The analytical method according to claim 1, wherein the nucleotide coenzyme is adenosine phosphate, nicotinamide adenine dinucleotide or a reduced form thereof, or nicotinamide adenine dinucleotide phosphate or a reduced form thereof. 3. The analytical method according to claim 1, wherein the nucleotide coenzyme is adenosine triphosphate. 4. The analysis method according to claim 1, wherein the nucleotide coenzyme is flavin adenine dinucleotide. 5. For analyzing haptens or antigens in liquids: a reagent consisting of a conjugate of a labeling substance with predetermined characteristics and a hapten or antigen, or an antibody against it, or a partner substance for specific binding thereto; and the hapten or antigen form a binding reaction system to produce a bound phase or a free phase of the labeled conjugate, and the properties of either the bound phase or the free phase determine the amount of the hapten or antigen in the liquid. 1. A reagent for heterogeneous immunoassay in which the labeling substance in the conjugate is a nucleotide coenzyme for an enzyme-catalyzed reaction. 6. The reagent according to claim 5, which comprises (1) a conjugate in which a labeling substance is bound to a hapten or an antigen, and (2) an antibody against the hapten or antigen. 7. The reagent according to claim 5, which is incorporated in a carrier matrix. 8. The reagent according to claim 5, wherein the nucleotide coenzyme is adenosine phosphate, nicotinamide adenine dinucleotide or a reduced form thereof, or nicotinamide adenine dinucleotide phosphate or a reduced form thereof. 9. The reagent according to claim 5, wherein the nucleotide coenzyme is adenosine triphosphate. 10. The reagent according to claim 5, wherein the nucleotide coenzyme is flavin adenine dinucleotide.