JPS5951353A - Unhomogeneous system immunity analysis method and reagent for analyzing hapten or antigen in liquid - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a method for measuring the presence of hasithene or an antigen (target substance) in a liquid based on the affinity of the target substance for a partner substance of specific binding (specific binding). and il
l: It is related to medicine. More specifically, the present invention includes:
The present invention relates to methods and reagents used for analysis using specific binding (specific binding) without using radioactive substances or partially modified enzymes as labeling substances. Note that in this specification, "specific" and "specific" 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. This is particularly true in the field of clinical chemistry, where components present in body fluids at concentrations as low as 10-11 molar 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 unknown substances has been detected by the appearance of reaction products or the disappearance of known reactants. In some cases, such analytical methods involve determining the rate of appearance of products or disappearance of reactants or the total amount of product formed or reactants expended upon reaching equilibrium. Therefore, we obtained the constant 1′. The reaction system for each analysis necessarily detects only a small group of substances.
Either R is defined or there is no specificity.

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 i- of the substance to be detected is made to compete with the unknown for a limited amount of antibody that has 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, originally,
It is necessary to separate the labeled substance to be detected that becomes bound to the antibody (bound phase) from the substance that does not cause such binding (free phase). Various means for accomplishing this required separation are available, for example, in U.S. Pat.
, 646,346, 3,720.760, and 3,
No. 793,445. However, all of these methods require at least one manual step such as filtration, centrifugation, washing or column elution 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 test. This is done by creating a system that is made to be a function of the amount of target substance in the sample. As commonly used in this field of science and herein, "heterogeneous" refers to specific binding assays that involve separation of bound and free 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 m-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, those that have been utilized as labels for radioactive atoms or molecules include a variety of agents such as enzymes, fluorescent materials, and bacteriophages. .

Examples of methods developed using enzymes used as labeling substances include U.S. Pat.
, No. 791,932, No. 3.839,153, and No. 3,850,752, and Journal of Immunological ψMethods, 1:247 (1972).
and Journal of Immunology, 109:12.
9 (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 co-assembly with the sample, the insoluble portion or the ligand is removed. An appropriate heterogeneous specific binding reaction scheme is constructed such that the amount of enzyme activity contained in any of the moieties 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 in this direction.

An interesting enzyme-added immunoassay is U.S. Patent No. 3,
No. 817,837. This method takes minutes #(
There is no need to use a specific binding reaction system (that is, an insoluble part and a liquid part), and therefore no separation operation is required. This is because the target substance to which the enzyme has been added is made so that the p-enzyme activity is inhibited by the reaction of the target substance with the substance to which the target substance binds. In this way, the bound (bound) state of the enzyme-added substance and the free (free state)
The ratio between the two conditions is 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.

British Patent No. 1.392,403 and French Patent No. 2.201.299 describe specific binding assays using inactive precursors of spectrophotometrically active substances as labeling substances. has been done. After retaining the sample together with the specific binding reaction system, the insoluble part and the liquid part are separated, and the amount of labeled substance present in the liquid part is a function of the amount of target substance to be detected in the sample. It is determined by a reaction process consisting of converting an inert 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, U.S. Pat. No. 3,850,578 discloses the use of electron spin resonance as a labeling means; U.S. Pat. No. 3,90
No. 1,654 discloses the use of cessation and increase of fluorescence as a labeling means. U.S. Department of Commerce National Technical Information Service (NTIS) Report mPB-224,
875 (1973) describes an attempt (unsuccessful) to use hemin chloride as a labeling substance in a heterogeneous analytical system for monitoring and detection by chemiluminescence reaction. Nature.

219:186 (1968) describes in great detail certain radioimmunoassay procedures, and also discusses in great detail the possibility of using coenzymes and viruses in place of radioisotopes as labels. It is incidentally mentioned as a general property. However, the authors do not teach how to conduct an analysis using such a variety of labeling substances, or whether, as a practical matter, such an analysis method is possible.

To provide further background, see ``Principles of Competitive Protein-Binding Analysis Methods J.
(Pr1nciples of Competitive
e Protein-Binding As5ays)
In O'Dell and Dody (eds., G.B. Rickton Company, Philadelphia, 1972),
The various known analytical methods and the various substances and properties that have been used as labels for specific binding assays are extensively discussed.

Although many new types of specific binding assays 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. In other words, in radioimmunoanalysis, handling of radioactive materials is dangerous and requires caution.
In enzyme-linked immunoassays, useful acetylated conjugates are difficult to prepare.

Therefore, an object of the present invention is to provide a novel method and reagent for detecting a target substance (ligand) in a liquid without using an inconvenient radioactive substance η or a partially modified enzyme as a labeling substance.

Furthermore, we have developed a method and reagent for heterogeneous specific binding (specific binding) analysis that has a broader range of applications and is more convenient than the prior art methods.
: One of the purposes is to provide.

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, any change in activity of the enzyme can be detected more conveniently than in prior art methods using conjugates containing labeled substances. It is also an object of the present invention to provide heterogeneous specific binding analysis methods and reagents.

The present invention provides highly convenient, widely applicable, and sensitive heterogeneous specific binding analysis methods and reagents for the use of substances exhibiting reactive activity as components of predetermined reactions. It is provided based on its use as a labeling substance. Such materials are referred to herein as "reactants."

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 that together with the reactants forms a predetermined reaction system that serves as a means for monitoring and detecting specific binding reactions. Measured by touching each phase.

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 target substance (ligand) to be measured. It will be done.

−”,’,’,’+’.

The improved method for analyzing bald tenes or antigens in a liquid generally includes the following steps (a), (b), and (e). That is, (
a) contacting the test liquid with a reagent consisting of a conjugate of a labeling substance with predetermined characteristics and a hapten or antigen, an antibody against it, or a partner substance for specific binding thereto; producing a bound phase or a free phase of the labeled conjugate; (b) separating the bound phase and the free phase; and (C) separating the bound phase and the free phase; A heterogeneous immunoassay method comprising the step of measuring the properties of a hapten or antigen in the liquid as an indicator in a layer, wherein the labeling substance in the conjugate is a substrate for an enzyme-catalyzed reaction. This is a special mission.

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 indeterminate enzyme immunoassay systems. .

Preferably, the monitoring detection reaction uses an enzyme catalyst.

Typically, the monitoring detection reaction is chosen to be highly sensitive to the reactants in the conjugate. In this regard, luminescent or fluorescent reaction systems are very useful. 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 enzyme-based reactant, ie, an enzyme substrate in an enzyme-catalyzed reaction.

Terms used in this specification are defined as follows. A "ligand", "target substance", or "target substance (ligand)" 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 to the target substance" is a substance or a group of substances that behave essentially the same as the target substance with respect to the binding affinity of a partner substance that specifically binds to the target substance.

The heterogeneous immunoassay reagent of the present invention is for analyzing hapten or antigen in a liquid: A labeling substance having predetermined characteristics and hapten or antigen, or an antibody against it 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 either the bound phase or the free phase A reagent for heterogeneous immunoassays whose properties are a function of the hapten or antigen in the liquid, characterized in that the labeling substance in the conjugate is a substrate for an enzyme-catalyzed reaction. .

Specific binding reagents can take a variety of forms. Generally, such reagents contain three basic components.

namely (]) the substance to be detected (ligand), the partner substance for the specific binding of the +21 ligand, and (3) the analogous substance for the specific binding of (a) the ligand, (b) the ligand, usually in labeled form. or (e) a labeling component that is a partner substance for specific binding. The components of this binding reaction may be combined simultaneously, or added sequentially. Then, during an appropriate retention (storage) period, the labeled component is stored, for example, at a ratio of the amount of the labeled component bound (bound) to the binding partner substance and the amount of the labeled component that is unbound (unbound). The amount, size, etc. of the binding is a function of the amount of the target substance present, so that it is bound (bound) to the partner substance of the corresponding competitive binding.

Below, some outlines of various binding reactions used in carrying out the entire method of the present invention will be briefly described.

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 essentially the same for the conjugate in the (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 may be influenced by the binding of the labeled conjugate. 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 substance of interest is present in the liquid, the characteristics or properties of the monitored detection reaction change. 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 specific binding counterpart of the specific binding substance. It gets worse. In other words, the conjugate in the free state is more active in the monitoring detection reaction than in the bound state.5 The following symbols are used in the schematic formula shown below. .

Symbol Definition L Target substance to be detected ■ Target substance or its analogous substance for specific binding B Partner substance for binding of target substance * Labeling substance, e.g., reactant Insoluble phase → Retention period (tim) following appropriate separation Limited amount; presence of less than the amount that can bind to all available binding sites under the selected reaction conditions during the selected retention period; e.g., a component that competes for binding with other components (exe ) Excess amount: greater than the amount that can bind to all possible binding sites under the selected reaction conditions during the selected retention period 1. Binding analysis method a) L 10 B (Aim) → 10B or [F]
Insolubilizing agent This is by traditional competitive binding methods. Examples of such insolubilizing agents include specific precipitating antibodies, specific insolubilizing antibodies, protein precipitating agents such as ammonium sulfate if B or In some cases, charcoal coated with dextrin is mentioned. Similar systems are described in Biochemical Journal, 88:
137 (1963) and U.S. Patent No. 3.839,153.
Seen in the issue.

b) L + (p” 33 (tim) →
This method is commonly referred to as solid phase technology. Similar radioimmunoassay and enzyme immunoassay techniques are described in U.S. Pat. No. 3,505,019;
No. 3,555,143, No. 3,646.346, and No. 3,654,090.

c) L0B*+I-■(tim)→ Reference: U.S. Patent No. 3,654,090 d) L0to■10B*(tim)→ Reference: U.S. Patent No. 3,850,752 a) L
10 B (exe) → 10 ■ * (exe) - 10 B or ■
In the insolubilizer sequential saturation technique of No. 8, some or all of the binding sites of B remaining after the initial retention period are bound to the labeled moiety.

b) Descriptions of similar radioimmunoassay and enzyme immunoassay techniques can be found in U.S. Pat. Nology, 209:129 (
(1972).

c) L 10B*(exe) → 10F(p-(exc)
→L ten B(exC) → B*(eXC)→
In the sandwich analysis method, part or all of a ligand (target substance) molecule bound to an inactivated binding partner substance is bound to a labeled component.

References: U.S. Pat. No. 3,720,7604,
Solid-Phase Dilution Analysis Method L + B(tim) → In this technique, the ligand and the labeled moiety are bound to a non-specific binding agent, and a proportionate amount of the ligand is then The labeling component is then dissociated upon binding with a binding partner substance that has greater affinity for the labeled component.

The most useful form of this technique is U.S. Pat.
There is a method using a column of non-specific binding agents as described in No. 9,104. 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 descriptions of variables included in normal heterogeneous analysis systems, such as more detailed descriptions of analytical methods and alternative separation techniques, please cite "Principles of Competitive Protein Binding Analysis Methods" (mentioned above) as a reference. I can do it.

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.

The step of evaluating the activity of the reactant of the conjugate of the components of the predetermined monitoring detection reaction in the bound phase or in the free phase comprises:
Monitoring detection is advantageously 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.

When an enzyme-catalyzed reaction system is utilized, the system must include the conjugate reactants and at least one enzyme;
It may also contain one or more enzyme reactants such as substrates and coenzymes. 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. It does not catalyze the dissociation reaction and is an enzyme. In short, a complex enzyme-catalyzed reaction system may be a single enzymatic reaction involving two or more phase reactants;
Alternatively, several reactants may be involved (provided that one of the reactions is enzyme-catalyzed).

In such systems, the reactant of the conjugate is one of the enzyme 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 an organism such as a microorganism. 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 characteristics of the microorganism, such as the growth rate of the microorganism. can.

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 prior to 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. Then, the activity of the reactant therein is evaluated and measured and used as an index of the presence or amount of the target substance in the liquid.

When the kinetics of a monitoring detection reaction is a property used to assess and measure the activity of the reactants in the selected bound or free phase (which is preferred), the kinetics will typically be:
It is determined by measuring the rate of disappearance of reactants or the rate of appearance of reaction products. Such measurements may be made by a wide variety of methods including conventional chromatographic, gravimetric, potentiometric, spectrophotometric, fluorometric, turbidometric, volumetric, and other analytical techniques. 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.

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 fluorescent product that is distinguishable from the substrate in the conjugate. It is something to do. A general reaction equation for such an enzyme-catalyzed reaction system is as follows.

In the above formula, X is a bond or linkage that can be cleaved by an enzyme (
bridging) group, such as an ester group or an amide group, and 2 represents the target substance (ligand), an analogue for a specific bond of the target substance, or a specific binding partner of the target substance, depending on the specific binding reaction technique used. It is a specific binding substance that can be any of the substances. Specific conjugates that can be used in this type of reaction system include fluorescein, umbelliferone, 3-indole, β-naphthol, 3-pyridol, resorufin, rhodamine B, and various other enzymatically cleavable conjugates. It is a derivative. Examples of possible structural formulas for such derivatives are as follows.

In the above formula, RlH-OH or 1d -X-Z ()
1 and 2 are the same as above), R2 is -X-Z, and R3 is -H or -CHs.

The present invention can be applied to the detection of any hapten or antigen (target substance) for which a specific binding partner substance is present. Target substances are usually peptides, proteins, carbohydrates,
A glycoprotein, steroid, or other organic molecule for which a specific binding partner exists or can be synthesized within a biological system. In functional terms, this target substance is selected from the group consisting of antigens, their antibodies, haptens, 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 such as ferritin, pradekinin, prostaglandin and tumor-specific antigens;
, vitamins such as folic acid, vitamin E and ascorbic acid; antibodies such as microsomal antibodies, hepatitis antibodies, allergen antibodies; and specific binding receptors such as thyroxine-binding globulin, avidin, intrinsic factor and transcobalamin.

In the conjugates of the present invention, the reactant is used in a manner that maintains a measurable amount of activity of the reactant. (either a similar substance for a specific bond or a partner substance for a specific bond of the target substance). The bond between the reactant and the specific binding substance is determined by a predetermined monitoring detection reaction using an active reactant to chemically break the bond, such as in the luminescent and cyclic reaction systems described above. As such, under conditions of analysis for which it was not designed,
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 a case is the reaction system of a fluorogenic substrate by the enzyme of the present invention.

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 50, 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 are methylene groups (one carbon atom) and amine groups (one heteroatom). The bridging group usually has a molecular weight of not more than 1000, preferably less than 200. The bridging group is a chain of carbon atoms or heteroatoms,
or a combination thereof, usually through a linking group in the form of an ester, amide, ether, thioester, thioether, acetal, methylene or amine group, linking the reactant with a specific binding substance or and its active derivative.

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, in the present disclosure, "reactant" and [
A "reactive substance" is a substance that is capable of a limited and measurable chemical transformation (transformation) that results in a different product or products, and that also (e.g., other reactants, catalysts, other types of substances involved in such chemical transformations or alterations)
Electromagnetic radiation, by interaction with reaction initiation means such as
An object that provides thermal energy or sound energy
means. Therefore, the group of substances defined as "reactants" herein includes common inorganic and organic reagents and biochemical substances. Anything that is not a reactant in the detection reaction is excluded.Because a particular chemical can function in different ways depending on its chemical environment, a single chemical can be classified into many different classes. However, what function such a substance has in the disclosure of Unknown II is determined by its reactivity with respect to the selected monitoring detection reactions listed in Unknown Mi1 Layer 1. It will be recognized that

The reactant of the present invention is a reactant of an enzymatic reaction, such as an enzyme substrate or an active partially modified substance or derivative thereof. An enzyme substrate is a substance that undergoes chemical transformation (
It is a compound or moiety that can undergo a change in quality.

When the substrate is used as a reactant in the conjugate, its preferred molecular weight is less than 9000, more preferably less than 50
It will be smaller than 00. Substrates of that size are particularly convenient for use in making conjugates because of their low molecular complexity. Furthermore, when bound to a specific binding substance, the activity of the substrate to that extent is easily influenced by the reaction of the conjugate with the specific binding counterpart of that specific binding substance. Examples of enzyme substrates that are all contemplated for use in the present invention include fluorescent substances that are cleaved by the enzymes mentioned above, such as derivatives of fluorescein and umbelliferone; pH indicators; and indicator dyes for spectrophotometric measurements (
fF includes color-forming type).

In one form of the invention, the components of the specific binding reaction that are combined with the liquid believed to contain the substance of interest are in liquid or solid form. The analytical method is carried out in standard laboratory containers, such as test tubes, with solid or liquid components of the specific binding reaction and the components of the reaction system added thereto.

-or several components of the specific binding reaction and/or 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 nuclear device is convenient for contacting the liquid to be tested, performing specific binding reactions and/or monitoring detection reactions, performing the necessary separations, and observing the resulting responses. provide the means.

The liquid to be tested is naturally occurring or artificially produced, and contains nozotene or antigen (target substance).
are known or presumed to contain. 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 substances with the same or similar reaction activity to that of the labeling substance in the conjugate is not disturbed by 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 a treatment includes, for example, a treatment in which a detergent that chemically destroys endogenous activity is applied, followed by a treatment to inactivate the destructive action of the detergent.

The invention will be illustrated below by means of examples, but these are not intended to limit the invention.

Example 1 Preparation of biotin-umbelliferone conjugate (2-oxo-2-H-1-benzobilan-7-yl)-5-4cis-hexahydro-2-oxo-IH-cheno-(3,4
-d)-imidazole]butyric acid ester 300 WMi (1,2 mmol) of anhydrous biotin
A solution of dried seaweed in dimethylformamide
℃ under dry nitrogen, then 0.17 tnl (1,
2 mmol) of dry triethylamine is added. Freshly distilled ethyl chloroformate (1 in 3 ml dry ether)
141 ml) was added dropwise. After being kept under stirring for 30 minutes, the resulting precipitate is separated under dry nitrogen and immediately cooled to -10°C. 1,971 ng of leftovers were collected from door to door.
(1,2 mmol) of anhydrous 7-hydroxycoumarin'f
After adding a solution of t3ml in dry pyridine, -
Stir at 10°C for 1 hour, then at 25°C for 20 hours. The solvent is evaporated in high vacuum at 40°C. After cooling, the obtained solid is separated and recrystallized with methanol to obtain the desired product. Melting point 216-218°C calculated value (C
+sI (as +oNzPsS): C, 48,75
;H, 4,19:N, 7.21o Analysis value: C
, 58,4:H, 5,12:N, 6.86゜Example 2 Specific (specific) binding analysis of biotin and avidin using an enzyme substrate as a labeling substance Specific binding used in this example The analytical system is based on the following reaction.

Biotin-umbelliferone conjugate (448 μml C fluorescence maximum) A. Preparation of insolubilized binding partner substance Avidin, which has binding affinity for biotin, is insolubilized by covalently bonding to water-insoluble polymer compound beads as follows.

Appropriate 1 Sepharose (5 epharose) 4
B (obtained from Pharmacia N.B., Uppsala, Sweden) is activated for binding to avidin using the method of March et al. (Analytical Biochemistry, 60:149 (1974)).

Approximately 4-activated Sepharose 4 B f 8 m
l! 0.0, IM tartrate buffer (pH 7.0). To this suspension is added 3 wt of water containing 6 kg of avidin with an activity of 9.9 units/mq. One unit of avidin activity is the amount of avidin that can bind 1 μ2 of biotin. The resulting reaction mixture′ft
Stir at 7°C for 6 hours. Avidin-conjugated Sepharose 4B was then separated and washed with 100 ml of 0.1 M sodium bicarbonate buffer (pH 9,0), followed by 0.1 M of avidin-conjugated Sepharose 4B'k12-.
) suspended in lis-(hydroxymethyl)-aminomethane hydrochloride buffer (pH 8,0) and suspended in IM bis-hydroxyethylglycine hydrochloride buffer (pH 7,0).
) and dilute 1:1.

B. Competitive binding analysis of biotin: Effect of variation in the amount of biotin on the 1− of leaving umbelliferone, each with a total amount of 0.2−, each in 0.1 M bis-hydroxyethylglycine hydrochloride buffer (pH 7). , 0), 0.3 μM biotin-umbelliferone conjugate (obtained in Example 1)
, 15 μt of avidin-conjugated-Sepharose 4B suspension (obtained in A of this example), and eight specific binding reaction mixtures containing biotin at the concentrations shown in Table 1.

The reaction mixture is kept at room temperature for 20 minutes with gentle shaking. Each reaction mixture was centrifuged and the supernatant was
The μ volume is pressurized with two volumes of IM bis-hydroxyethylglycine hydrochloride buffer (pH 8.2) containing 1.08 units of porcine esterase. After a 5 min hold at room temperature, the fluorescence intensity generated in each reaction mixture at 448 nm with excitation at 364 nm was measured using Amico Bowman (
Measured using an Amico-Bowman) fluorescence spectrometer. The results are shown in Table 1.

Table 1 0.00 0.355 2 0.10 0.495 3 0.20 0.469 4 0.30 0.503 5 0.40 , 0.547 6 0.50 0.502 7 0.75 0.580 8 1.00 0. fi88 Thus, in this example it is shown that the amount of NAD-biotin in the liquid phase is directly proportional to the amount of free biotin present. Therefore, the analytical method and reagent of the present invention are useful for measuring target substances (ligands) in unknown liquid samples.

275

Claims (1)

[Claims] 1. For analyzing hapten or antigen in a liquid: (a) A liquid is treated with a labeling substance having predetermined characteristics and hapten or an antigen, or an antibody against the same or a specific binding thereof. A step of contacting with a reagent consisting of a conjugate with a partner substance, in which a target substance (ligand) forming a binding reaction system with the reagent produces a bound phase or a free phase of a labeled conjugate; (b ) a step of completely separating the bound phase and the free phase; and (e) a step of measuring the characteristics of the hapten or antigen in the liquid as an indicator in - of the separated layer. An analysis method characterized in that the labeling substance in the conjugate is a substrate for an enzyme-catalyzed reaction. 2. The analytical method according to claim 1, wherein the labeling substance is a substrate for an enzyme-catalyzed reaction that produces a product with detectable properties distinguishable from that of the conjugate. 3. Claim 1 in which the detectable property is fluorescence
Analytical method described in section. 4. For analyzing haptens or antigens in liquids; consisting of a conjugate of a labeling substance with predetermined characteristics and nobutene or antigen, or an antibody against it, or a partner substance for specific binding thereto; , and the reagent and the hasithene or antigen form a binding response 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 are such that the liquid A reagent for heterogeneous immunoassays in which the amount of hasitene or antigen in the conjugate is a function of the amount of the antigen in the conjugate, the reagent being characterized in that the labeled substance in the conjugate is a substrate for an enzyme-catalyzed reaction. 5. 5. The reagent according to claim 4, which comprises (]) the conjugate in which the labeling substance is bound to hapten or antigen, and (2) an antibody against the hapten or antigen. 6. The reagent according to claim 4, which is incorporated in a carrier matrix. 7. The labeled conjugate is a substrate for an enzyme capable of acting on the conjugate to release, by enzymatic cleavage, a product having detectable properties distinguishable from that of the conjugate. A reagent according to claim 4. 8. The reagent according to claim 4, wherein the detectable property is fluorescence.