JPS5942453A - Homogeneous immunity analyzing 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 a hubten or antigen (target substance) in a liquid based on the affinity of the target substance for a specific (or specific) binding partner substance. method
It concerns reagents and reagents. In more detail: The present invention relates to a method and reagent for use in specific binding analysis that does not require a separation step and does not use a radioactive substance or a partially modified enzyme as a labeling substance. In addition, in this specification, "specific" and "specific" are used synonymously.

It is clear that there is a desire to detect 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 1,0-11 molar are of pathological significance. The difficulty of detecting such low concentrations of total IC increases in the field of clinical chemistry, where sample availability is usually very limited.

Previously, substances were detected in liquids based on reaction systems in which the substance to be detected was necessarily a reactant. The presence of unknown substances is detected by the appearance of reaction products or the disappearance of known reactants. In some cases, such analytical methods involve measuring the rate of appearance of products or disappearance of reactants or the afFr of products formed or reactants expended upon reaching equilibrium. Yes, I got a quantitative result as well. The reaction system for each analysis is necessarily either limited to the detection of only a small group of substances or is nonspecific.

Research into analytical systems that are highly individualized and can be applied to the detection of a wide range of substances led to the creation of radioimmunoassay.71.
Ta. According to this method, ■: of the substance to be detected with a radioactive label attached is an unknown substance with individuality.
It is made to compete with an unknown for a reduced amount of antibody. The amount of the compound labeled 1 that becomes bound to the antibody varies inversely with the level of the unknown substance present. In radioimmunoassay technology,
Essentially, it is necessary to separate the substance to be detected that is labeled with a label that can bind to antibodies from a substance that does not bind in such a way. Divine means for accomplishing this separation are described, for example, in U.S. Pat.
No. 505,019, No. 3,555,143, 3.646
, No. 346, No. 3,720,760 and No. 3,793,4
As shown in No. 45, the development formula J has been developed. However, all of these methods require at least one step of tying--unbinding of the labeled attachment agent--passage, centrifugation or washing A to ensure efficient separation from the labeled donor agent. It requires two manual operation steps. If separation operations could be eliminated, the analytical method would be much simpler9, and it would be even more useful in clinical laboratories.

Immunology/1) The use of radioactive substances in RU can now be avoided to some extent by using enzyme-containing substances instead of radioactive substances. US: Grant No. 3,654,090 and 3,791,
As exemplified in No. 932, the manual operation steps necessary to carry out complete fermentation and immunodistribution are essentially the same as those used in radioimmunoassay methods. , requiring cumbersome separation steps.

The one who got the enzyme? For use, each enzyme found must undergo individual chemical modification to be used in the formation of the conjugate found.
There are other disadvantages as well, such as not being able to do so. Other substances that can be detected, such as coenzymes or viruses (Nature, 219
, 186 (1968)), and furthermore, it is proposed to use fluorescent labels ('rfd'
a 2.217.350 'Pr') Mo proposal A i
(,-Cil.

Immunoassay methods using radioactive labels or detected enzymes may be improved in the future by expanding the range of detectable substances, or by reducing the risk of failure. , their essence and 1-, these methods (ryoyo-ru 掠 separation method) are definitely necessary.Recently, a different method that does not require the existence or necessity of The method is therefore said to be homogeneous, as opposed to heterogeneous, where separation is required.

U.S. Patent No. 3,817,837 describes the substance to be detected (
A step of combining a soluble complex consisting of an enzyme as a labeling substance (ligand) with a soluble complex of a receptor (usually an antibody) and a soluble complex for the target substance (usually an antibody) with the liquid to be analyzed; A competitive binding assay method involving T8 is disclosed that measures the effect of a target substance on enzyme activity in a complex.

This method is advantageous in that the reaction between the enzyme-bound-ligand complex and the receptor results in inhibition of the enzymatic activity of the enzyme in the complex, eliminating the need for a separation step. Nevertheless, this method does not meet the demands of extensive analysis due to severe limitations in its capabilities. For example, in the production of enzyme-conjugate-ligand complexes, the substance or ligand to be detected is carefully controlled so that its binding position is close to the enzymatically active site of the enzyme. It is clearly essential that the forces must be combined in a certain way.

This is necessary in order to block the enzymatically active sites due to the reaction between the complex and the Gant and the receptor. Enzymes vary widely in size, with molecular weights ranging from about 10,000 to 1,000,000. Thus, a receptor in the form of an antibody with a molecular weight between 1.50,000 and 300,000 can physically block the active site of an average enzyme with a molecular weight of 500,000 or more. The binding position of must be strictly controlled. Because the chemical structure of enzymes is complex, it is actually difficult to strictly control such chemical bonds, and even by screening a wide range of enzymes,
It would be expected that only a small number of enzymes could be used in this homogeneous analytical method.

Furthermore, in order to obtain quantitative test results, each enzyme-
Tight control of the number of enzymes and the number of ligands for binding-ligand complexes is essential. In this respect, Jk,
Furthermore, such control is difficult due to the enzyme's complex peptide structure. Therefore, it is thought that there are only a small number of enzymes that have such a unique molecular structure that the necessary control of the ratio of ligand 1 to the enzyme can be carried out reliably.

Prior art homogeneous analytical methods involve enzymatic amplification and are therefore said to be very sensitive. but,
The applicability of this method appears to be very limited since the enzyme itself is a limiting factor determining the sensitivity of prior art analytical methods. This sensitivity is clearly limited to the catalytic activity of the particular enzyme in the enzyme-bound-ligand complex. The applicability of the prior art method therefore lies not only in the requirement for conjugation for the formation of useful conjugates, but also in the dependence of the sensitivity of assays using such conjugates on the enzyme of the particular conjugate. It is also limited by gender.

Prior art homogeneous analytical methods have further disadvantages when applied to the testing of biological fluids such as urine and lymph.

Since the enzyme species contained in the enzyme-bound-ligand conjugate may be present in the liquid sample being tested, this control has a considerable impact on the effectiveness of the analytical method. The activity of the impossible begins to appear as a background. Therefore, in order to formulate analytical methods that can be used when testing human or animal biological fluids,
It is necessary to select an enzyme that is exogenous to such fluids and use it to convert the enzyme-linked conjugate to form J, and therefore this analysis The applicability of the method is further limited.

Therefore, the object of the present invention is to separate habten or antigen (target substance) in a liquid without requiring a separation process and without using inconvenient radioactive substances or partially modified enzymes as a labeling substance.
The purpose of the present invention is to provide a reliable method and trial for detecting.

It is a further object to provide a homogeneous immunoassay method and system that has a wider range of applications and is more convenient than the prior art methods.

fi of the prior art method for the target substance or its specific binding partner substance? It is also an object of the invention to provide a homogeneous immunoassay method and system using the 11λ substance so that the 11λ substance can be more conveniently bound to the 11λ substance.

It is also possible to provide a homogeneous immunoassay method using a conjugate of a marker substance, which has an activity that is more easily affected than the enzyme of the prior art method, by the ligation reaction of lPj W, and its threads. ! , is one of the objects of the present invention.

By using a wide variety of sensitive reaction systems, we have used conjugates containing labeled substances that allow us to detect any change in activity of the enzyme better than any change in the activity of the enzyme in prior art methods. It is one of the objects of the present invention to provide a homogeneous specific binding analysis method and its system.

It is further an object of the present invention to provide a homogeneous specific binding analysis method and system that can be more easily applied to the testing of biological fluids than the methods of the prior art.

The homogeneous immunoassay method of the present invention is for analyzing habten or antigen in a liquid: (a) one bottle per night; (1) binding of a labeling substance having predetermined characteristics to habten or antigen; (2) contacting the bound well and the free phase with a reagent consisting of an antibody to Habten or the antigen to produce an antibody-bound phase and an antibody-free phase of the labeled conjugate; A homogeneous immunoassay method consisting of the step of measuring the properties of habten or antigen as an indicator in a liquid without separation, characterized in that the labeled substance in the conjugate is a substrate in an enzyme-catalyzed reaction. That is.

Further, the uninvented homogeneous immunoassay reagent is for analyzing habuten or antigen in a liquid by a homogeneous immunoassay method: (1) Binding of habuten or antigen with a labeling substance having predetermined characteristics. and (2) a reagent consisting of an antibody against habten or antigen, which in turn forms a binding reaction system in which the reagent and conjugate 6 [body-bound phase and antibody-free phase are generated]. and the properties in either the bound or free phase are a function of the amount of habten or antigen in the liquid.c1 The labeling substance in the conjugate is a substrate in an enzyme-catalyzed reaction. It is.

The present invention provides a very convenient, widely applicable, and sensitive homogeneous specific bond analysis method and its system to demonstrate the reaction activity imparted as a component of a predetermined reaction. It is provided based on the use of substances as labeling substances. Such substances are referred to herein as ``reactants''.

This method is based, in part, on a predetermined reaction between a compound or antigen (target material) and its specific binding partner, one of which is bound to a reactant. The method of the present invention is based on the fact that the activity of the reactants changes in the reaction.This reaction serves as a means for monitoring and detecting specific binding reactions. A variety of operating procedures are employed in the practice, including different test compositions and equipment; the preferred basic operating procedures are direct binding techniques and competitive binding techniques.

In direct binding techniques, the batten or liquid presumed to contain the antigen to be detected is brought into contact with a conjugate containing a reactant that is bound to a specific binding partner of the analyte. , any changes in the activity of the reactants are then analyzed. In competitive binding techniques, a liquid is brought into contact with the target substance for specific binding and contains a reactant that is bound to one or both of the target substance (ligand) or its analog for specific binding. The reactant is then contacted with the conjugate and then analyzed for any changes in the activity of the reactant. In both techniques, the activity of the reactant is measured by contacting the liquid with at least one reagent that together with the reactant forms a predetermined monitoring detection reaction.

Qualitative measurement of the target substance in a liquid is based on the obtained reaction and
Monitored detection reactions in liquids that do not contain the target substance are compared in terms of their characteristics (usually reaction rates). The difference between the two indicates the activity of the reactants. Quantitative measurement of the target substance in a liquid is performed by comparing the obtained reaction with a monitored detection reaction in a liquid containing a known amount of the target substance.

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. Preferred for 111 are circulating reaction systems, particularly preferred are reaction systems in which the reactants are circulating materials. 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, an enzyme substrate, and preferably has a molecular weight of less than 9,000 Jh.

Terms used in this specification are defined as follows. A "ligand" or "target substance" is a substance or a group of substances whose presence in a liquid or substance is to be measured. "A partner substance for a specific binding of a target substance (ligand)" is a substance or a group of substances, excluding other substances and only one target! I'7
It indicates a specific binding affinity for <<r> quality.

[A similar substance with respect to a specific bond to a target substance] is a substance or a group of substances that exhibits essentially the same left-right movement as the target substance in terms of the binding affinity of the substance in a specific bond to the target substance. It is.

In general, the proportion of the specific binding reaction, e.g., the liquid presumed to contain the ligand or antigen (substance of interest), the conjugate and/or the specific binding partner of the substance of interest, is sufficient for the purpose of the analysis. IIFn may be bound in any amount, manner, and order, provided that the activity of the reactants in the conjugate measurably changes when a significant amount or concentration of the substance of interest is present in the liquid. It is preferable that all components of the specific binding reaction are soluble in the liquid, so that it becomes a homogeneous analytical system.However, if the partner substance or conjugate of the specific binding of the target substance is insoluble Certain heterogeneous analytical systems may also be utilized if desired.

When using direct binding techniques, the components of the specific binding reaction are a liquid presumed to contain the target substance and a certain combination containing all of the reactants that are bound to the target substance and the partner substance of the specific binding. . The activity of a reactant in a conjugate when it comes into contact with a liquid varies inversely with the amount of binding between the target substance in the liquid and the partner substance of a particular binding in the conjugate. If the number of target substances increases, the activity of the reactant of the conjugate decreases. In order to obtain quantitative results, the amount of the partner substance of a particular binding in contact with the liquid must always be determined before the conjugate and the liquid are in contact. During this period, it is possible to combine with all of the target substances that are thought to be present in the liquid. In practice, the opening of a particular binding partner is chosen according to the criteria described above, based on the maximum expected amount of the target substance expected to be present in the liquid. Direct binding techniques are particularly useful for detecting high molecular weight target substances that have smaller specific binding partners1. When using competitive binding techniques, the components of the specific binding reaction are assumed to contain all of the target substance. a liquid containing a quantity of a conjugate containing a reactant bound to the target substance (ligand) or a similar substance for a specific binding of the target substance, and a partner substance for a specific binding of the target substance for a given week; . The specific binding partner is brought into contact with both the conjugate and the liquid substantially simultaneously. Any target substance (ligand) in the liquid
The activity of the reactants of the conjugate when in contact with the liquid is determined by the activity of the reactants of the conjugate upon contact with the liquid, since the conjugate also competes with Gant or its specific binding analog in the conjugate to bind to the target substance in the liquid. It changes depending on the amount of bond between the substance and the mating substance of the fixing table.

That is, as the amount of target substance in the liquid increases15, the activity of the reactant of the conjugate increases. In order to obtain consistent results, the reactants in the conjugate and the specific binding partner in contact with the liquid must be traced before any changes in the activity of the reactants in the conjugate are evaluated. During the contact between the specific binding partner substance, the bound body, and the liquid, all the target substances that are considered to be present in the liquid and the target substance in the form of an aggregate, or the substance similar to it. There is a smaller amount than the amount that can be combined with everything (i 7'1. In fact,
'! ? The amount of the constant binding partner is selected according to the criteria described above, based on the maximum expected amount of the target material expected to be present in the liquid. Generally, δ, the amount of target substance (ligand) or its analog in the form of a conjugate in contact with the liquid must not exceed the minimum amount of the target substance to be tested in the liquid. Competitive binding techniques are particularly useful for detecting target substances that have larger specific binding partners than themselves.

A variation of the competitive binding technique is the displacement binding technique, in which the conjugate is first brought into contact with the specific binding partner and then with a liquid. Competition for specific binding partners then occurs.

In such a method, the amount of the conjugate that comes into contact with the target substance for specific binding is the amount of the conjugate that comes into contact with the target substance for specific binding before the contact with the liquid estimated to contain all of the target substance. Target substance (ligand) of amount of specific binding present during
Or, it is a number that contains other similar substances. According to one method, the conjugate is brought into contact with a liquid that is estimated to contain all of the target substance (VC1 liquid). It is brought into contact with the partner substance for specific binding in the living environment. According to the second method, a liquid estimated to contain all of the target substance is brought into contact with a complex containing the conjugate and the specific binding partner substance. In this case, the 4':r constant binding substance and the specific binding partner substance in the conjugate are bound facing each other. In the first method, the left side of the binding substance that becomes bound to the partner substance of the specific binding, this! 1° will be the amount of conjugate in the form of a complex in the second method, but this amount is usually used to evaluate the apparent change in the activity of the reactants of the conjugate. The amount that can replace all of the target substance in the liquid when the liquid is in contact with the partner substance (or complex) of the previous specific binding is also increased.

Another variation of the competitive combination technique is the sequential saturation technique.

In this technique, the components of the specific binding reaction are the same as those used in the competitive binding technique, but the order of addition or the combination of each component and the relative ratio of each component is different from that in the competitive binding technique. According to the sequential binding technique, a specific binding partner of the target substance is brought into contact with a liquid presumed to contain the target substance during a period prior to contact of the liquid with the binder.

The amount of the specific bonding partner that comes into contact with the liquid is usually falsely assumed to be present in the liquid at the time the liquid comes into contact with the specific bonding partner before the liquid comes into contact with the conjugate. The amount is increased more than can be combined with all of the substances. Furthermore,
The amount of target substance or its analog in conjugate form is usually determined by the amount of unbound substance remaining during contact of the conjugate with a liquid before evaluation of apparent changes in the activity of the conjugate reactant is completed. The amount of the partner substance for specific binding and the amount that can be bound are also increased. In practice, the specific binding partner and the amount of the target substance or analog thereof in the conjugate are selected according to the criteria described above, based on the maximum expected amount that will be present in the liquid.

Operating steps involving other orders of addition and other relative proportions of the components of a particular binding reaction may also be devised to perform homogeneous specific binding assays without departing from the inventive concepts described herein. What will happen is to be expected.

The step of assessing any change in the activity of the reactants of the conjugate of the components of the predetermined monitored detection reaction comprises at least one step of forming a reaction mixture of the specified binding reaction with the reactants of the combined monitored detection reaction. This is conveniently accomplished by contacting one substance with another and then measuring the effect of a particular binding reaction on the properties of the reaction. The monitoring detection reaction consists of one or a series of multiple chemically transforming or transforming reactions. Unless otherwise specified, the term "reaction system" as used herein refers to all or part of a predetermined monitoring detection reaction.

When an enzyme-catalyzed reaction system is utilized, the thread comprises the conjugate reactants and also at least one enzyme;
The present invention also includes one or more enzyme reactants such as substrates.

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 degradation or dissociation reaction with a single r+% elementary catalyst. In that system, the reactant of the conjugate is the enzyme substrate that undergoes degradation or dissociation, and the only component of the reaction system that is required to contact the reaction mixture of the double bond reaction is the Solution Ni? It is an enzyme that does not catalyze the reaction.A more complex enzyme-catalyzed reaction system may be a single enzyme reaction involving two or more reactants, or it may involve several reactions. The body may also be involved (although one of the reactions is enzyme-catalyzed).

In such systems, the reactant of the conjugate is one of the enzymatic reactants of the enzyme-catalyzed reaction, and the reaction mixture of the particular conjugation reaction is the enzyme catalyst selected separately from those in the conjugate. It is brought into contact with the appropriate enzymes and reaction components necessary to form the 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 specific microorganisms may be selected as reactants in the conjugate. Any change in the activity of the reactants will also result in a change in the growth characteristics of the microorganism when such a microorganism is placed in an environment where the only reactant nutrient source is the conjugate. It would be. That is, for example, a change in the growth rate of a microorganism upon contact with a reaction mixture of a specific binding reaction would indicate the presence of the substance of interest therein.

Suitable reaction components that form a monitoring detection reaction with the reactants in the conjugate may be added to the reaction mixture of the specific binding reaction, alone or in combination, prior to, concurrently with, or subsequent to the initiation of the determination binding reaction. be brought into contact. After the onset of the specific binding reaction, some of the essential components of the M5 visual detection reaction also occur at t+'. Incubate for a predetermined time before evaluating. After the incubation period, components needed for the monitoring detection reaction and not yet present in sufficient quantities in the reaction mixture are removed by 1. Add to. Then, any influence on the monitoring detection reaction is evaluated, and the presence of the target substance in the liquid is also used as an indicator of quantity.

In situations where the substance of interest is present in the liquid, either absent or in insignificant amounts, the predetermined monitoring detection response exhibits a relatively constant nature. If the target substance is present in the liquid, the monitoring detection reaction may be delayed.
・The characteristics or properties of one change. Generally, the activity of a reactant of a conjugate is defined as the rate or rate at which the reactant can contribute −1 to the monitored detection reaction. That is,
The nature of the monitoring detection reaction depends on the presence of the target substance in the liquid.
Usually the overall reaction rate or the equilibrium amount of several reaction products produced by the reaction will vary. In normal cases, the ability of a reactant in a conjugate to participate in a monitoring detection reaction is determined by the relationship between the specific binding substance to which the reactant is bound and the specific binding counterpart of that specific binding substance. It decreases due to reaction.

That is, the conjugate in its free state is more active for monitoring detection reactions than in its bound state.

The relative amounts of free I':iA conjugates and bound conjugates present after incubation of a specific binding reaction will be a function of the presence of the analyte in the liquid and will determine the overall effect on the monitoring detection reaction. .

When a change in the overall reaction rate of a monitored detection reaction is the characteristic used to determine the presence of a substance of interest (which is preferred), the rate is usually determined by the rate of disappearance of reactants or the appearance of reaction products. Determined by measuring speed. Such measurements include conventional chromatography, gravimetric analysis, potentiometric titration, spectrophotometry, fluorometry, turbidity analysis α111
This can be done by a wide variety of methods, including analytical techniques such as quantification and volumetric measurements. Since this method is considered as a closed method and is intended for the detection of low concentration target substances (71), a reaction system that is sensitive to non-71. It has been developed to be used together.

One preferred form of the monitoring and detection reaction is a light-emitting reaction system, preferably an enzyme-catalyzed reaction, such as Q'1, a reaction exhibiting the phenomenon of bioluminescence or chemiluminescence. The reactants in the conjugate may be reactants of a photogenerating reaction or a reaction that is a preparatory step for a light-emitting reaction with or without an enzyme. Any change in the reactants of the conjugate resulting from a particular binding reaction causes a change in the total amount, peak intensity, or nature of the light produced. 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 triphosphate NAD: nicotinamide adenine dinucleotide NADI (: reduced nicotinamide adenine dinucleotide F'MN: flavin mononucleotide FMNH2:
Flavin mononucleotide of the i archetype hv: Electromagnetic radiation, usually in the infrared, visible or ultraviolet range A table hv + FMN ten long chain acid + H20 MN NAD + FMNH.

2) FMNH, Shincho silver aldehyde + 0° luciferase hν 10 FMN 10 long-chain acid + H, 0 B. fiscieri D, 1) 3', 5'-adenosine triphosphate 10 reduced luciferin sulfate (salt) + oxidized luciferin peroxy Guze* Kawara Reduced luminol + H, 0, -1 → 5, + Acid level, O / - A + H, O Reduced par'-rubell ookintase 1 F, Reduced virocalol + H, 0, - → 59 ．． + Acid type. Ga. -A + H, O reduced type addition - Further details and discussion regarding the luminescent reaction system that can be used in the method of the present invention can be found in the following documents:

Journal on Biological Chemistry, 2
36:48 (1961) Journal on American Chemical Society, 89:3944 (1967) Cornier et al., Advances in Bioluminescence, edited by Jeong-Nun et al., Princeton Tai-Hyo Publishing Department, Hyu Shashi, 1966) 363 -page 84
Kr1e8 Purification and TLE quality of Renilla Luciferase, Georgia University doctoral dissertation (1967) American Journal on Physiology, 4
1:454 (1916) Biological Bulletin, 51:89 (19
26) Journal on Biological Chemistry, 243:4714 (1968) Other types of preferred sensitive monitoring detection reactions include those that involve fluorescence and that are obtained by enzyme catalysis. In such a reaction system, the reactant in the conjugate is a substrate in an enzymatic reaction, and the enzymatic reaction produces a product that has a different fluorescent property than the substrate in the conjugate. It is. Any change in the activity of the enzymatic reactants in the conjugate resulting from a specific binding reaction results in a change in the fluorescence of the reaction mixture. A general reaction formula for such an enzyme-catalyzed reaction system is as follows (substrate): In the above formula, X represents a bond or linking group that is cleaved by the enzyme, such as an ester group or an amide group. , 2 is a specific binding substance that becomes either a target substance (ligand), a similar substance related to a specific binding of the target substance, or a partner substance for specific binding of the target substance, depending on the determination binding reaction technique used. Certain conjugates that can be used in this type of reaction system include fluorescein, umbelliferone, 3-
Indole, β-naf)-ru, 3-L"J)"-ru,
Resorufin, Rhodamine B, and other various rust conductors that can be cleaved by enzymes. Examples of possible structural formulas for such visiting conductors are as follows.

In the above formula, ■71 is -OH or -x-z (x and 2
is the same as above), Rt is -xz, and R3 is -H or -CH.

Although not necessary in preferred embodiments of the invention,
The presence of the target substance in the liquid affects the activity of the reactant of the conjugate @
It may be desirable to use heterogeneous analytical techniques even in cases where the resultant force is significant. Such situations may arise where heterogeneous methods are particularly advantageous. Some heterogeneous methods can increase the effective temperature of the analyte in the analytical system, which can increase sensitivity.

An example of such a heterogeneous method is the conjugate method 2 of the present invention. < is the partner substance of the target substance that also has a constant bond (this is
Some use column equipment containing an insoluble matrix of either type (selected according to the particular type of operation chosen). All other heterogeneous analytical methods using radioactive labels or enzyme-linked substances as labeling substances are also similarly performed using the reactants of the present invention as a labeling substance.

The present invention can be applied to the detection of any habten or antigen (target substance) in which a partner substance for determination binding is present. Target substances are usually peptides, proteins, carbohydrates,
Glycoproteins, steroids, or organic molecules that have specific binding partners in biological systems or can synthesize partner substances. This target substance is used for functional purposes.
F is selected from the group consisting of antigens, antibodies thereof, conjugates, antibodies thereof, vitamins, and receptors and binding substances thereof. Particular examples of target substances that can be detected using the present invention include antigens such as ferritin, pradekinin, prostaglandin and tumor 1% specific antigen;
Vitamins such as folic acid, vitamin E and ascorbic acid; antibodies such as micronomic antibodies, hepatitis antibodies, allergen antibodies; and thyrogin-binding globulin, avidin,
Important binding receptors include intrinsic factor and transnucobalamin.

Generally, it is preferred that the conjugate comprises a reactant bound to a smaller σ target substance (ligand) and its selected specific binding partner. When detecting a target substance when the molecular weight of the selected specific binding partner substance is about [10]/lO or less than the molecular weight of the target substance, it is preferable to use surface binding techniques. Therefore, when the target substance to be detected is an antibody or a specific binding receptor, the reactant bound to the antigen or the conjugate containing Habten bound to the low molecular weight counterpart of the specific binding of the antibody or receptor is detected. Preference is given to the direct bonding technique used. When the molecular weight of the selected binding partner is 10 times or more larger than the molecular weight of the target substance to be detected, i.e. when the target is an antigen, habten or vitamin, the binding partner is bound to a more mysterious target substance (ligand). It is also particularly advantageous to utilize competitive binding techniques, in which conjugates containing reactants are used [2] or sequential saturation techniques.

In the conjugates of the present invention, the reactants are capable of binding to the specific binding substance (depending on the analytical method chosen, the target substance, the target substance, The target substance is either a similar substance for one-time binding or a partner substance for a specific binding of the target substance). The bond between the reactant and the specific binding substance is determined under analytical conditions in which the monitoring detection reaction using the active reactant is not designed to chemically break the force bond, as in the luminescence reaction system described above. , normally real T1 VC cannot be changed.

However, in some cases the VC is engineered such that such bonds are broken or influenced by monitoring detection reactions as a means of assessing changes in reaction activity. Such cases include the enzymatic fluorogenic substrate reaction systems described earlier in this specification.

The reactant may be 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 specific binding substances will contain from 1 to 50, preferably from 1 to 10 carbon atoms or nitrogen, oxygen, sulfur,
By bridging groups containing heteroatoms such as phosphorus and other! ll
I have been contacted.

An example of a bridging group containing one atom is a methylene group (
(1 carbon atom) and an amino group (1 heteroatom).

The bridging group usually has a molecular weight not exceeding 100 (+?), preferably less than 200. The bridging group contains a chain of carbon atoms or heteroatoms, or a combination thereof. The reactant and the internal binding substance (7 or its active derivatives) are usually linked by a linking group in the form of a Nisder, amide, needle, thioester, thioether, acetal, methylene or amine group. There is.

A reactant in the present conjugate is a substance with a (eg, constant or known) reactive activity that is placed as a component of a predetermined monitoring detection reaction. More specifically, as disclosed herein, "reactant" and "substance having reactive activity" refer to limited melting and measurable substances that result in different or several products. can undergo a chemical transformation (modification 1i-t), and furthermore, the reactant and the chemical (e.g., other reactants, catalysts, etc.) means a substance which, upon interaction with a means for initiating a reaction, such as other types of substances) produces electromagnetic radiation, thermal energy or sonic energy. Accordingly, the group of substances defined as "reactants" in this specification includes conventional inorganic and organic reagents and biochemical substances. However, those that are not suitable reactants for monitoring and detection reactions, such as catalysts (including enzymes) and radioactive isotopes, are excluded. Because sailing chemicals can function in different ways depending on their chemical environment, a single chemical may be classified into a variety of different classes; It will be appreciated that having such functionality is determined by the reactivity of the material with respect to the selected monitoring detection reactions described herein.

The reactant in the present invention is a reactant of an enzymatic reaction, such as an enzyme substrate or a partially modified substance or derivative thereof having an activity. An enzyme substrate is a compound or moiety that can be chemically transformed (Jt)'t with the enzyme as a catalyst. When the substrate is used as a reactant in the conjugate, its preferred molecular weight will be less than 9,000, more preferably less than 5,000. Substrates of that size are particularly convenient for use in making entire 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 contemplated for use in the present invention include fluorescent substances cleaved by the enzymes mentioned above, such as derivatives of fluorescein and umbelliferone, pH indicators 1 and indicator dyes for spectrophotometric measurements ( In particular, the colored type) is included.

In one form of the present invention, the components of the specific binding reaction combined with the liquid that is thought to contain the target substance may also be liquid (-< is in solid form. In a preferred homogeneous analysis system, The component is usually in solution or solid form that can be readily dissolved in the liquid. Since the liquid being tested is usually aqueous in nature, the component is generally in a water-soluble form. i.e., in the form of an aqueous solution or a water-soluble solid such as a powder or resin.
It can be carried out using constant concentration reaction components and components of the reaction system added thereto.

-Also(7〈(Components of several types of specific binding anti-LL, and/or components of - or several types of monitoring detection reactions!, :I, Kawakyu may include an inclusive form.One way of thinking As, the phase body is
A test tube or capsule containing one or more of these ingredients in its inner part, e.g. in liquid or loose solid form, or in a coating on its inner surface. Alternatively, the phase may be in the form of a matrix that is insoluble and porous with respect to the liquid being tested, and is absorbent. Textile paper; polymeric films, crotches, fluff, lumps; gels and other forms. In such a case, the device is
(provides a convenient means for contacting the liquid to be tested, carrying out specific binding reactions and/or monitoring detection reactions, and observing the resulting responses.

The liquid to be tested is naturally occurring or artificially produced, and is presumed to contain haftene or antigen (target substance).

Usually, it is a liquid of a biological body 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. Solid materials such as cells or other materials such as gaseous materials; can be analyzed.

In contrast to prior art homogeneous analytical systems, deposits of analytes in biological fluids containing substances that exhibit a reactive activity the same or similar to that of the labeled substance in the conjugate, You can analyze without being disturbed by the ground. Endogenous background reactant activity can be easily removed by several methods. The biological fluid can be treated to selectively destroy endogenous reactant activity.

Such processing and 1. An example of this is a treatment in which a detergent that chemically destroys endogenous activity is applied, and then a treatment is performed to inactivate the destructive action of the detergent.

The present invention will be explained below with reference to Examples, but these are not intended to limit the present invention.

Example 1 Production of nicotinamide 6-(2-amineethylamino-9purine dinucleotide) Nicotinamide adenine dinucleotide (NAD
) 217' ilo m, e (D was dissolved in water, then 0.6 m of ethyleneimine was added dropwise. At this time, 1 M
The pH is maintained below 7 by adding perchloric acid.

After dropping the ethyleneimine, adjust the pH to 45.
The reaction was then maintained at 20-25°C. Add 0.6 mJJ of ethyleneimine every 24 hours, pHi 4.
I revisited it on 5th and made 1 glaze. After 96 hours, the solution was poured into an aliquot at 10°C, and the resulting oil was collected and washed with ether, leaving about 50 ml in a flask. of water.

The resulting solution was oxidized to pH 7.0 with IJum.
-7.5, and 1 gram of sodium bicarbonate was added to the anus. The solution was flushed with nitrogen for 4-5 minutes and then 1 gram of sodium hydrosulfite was added.

The flask was sealed and left at room temperature for 45 minutes. Next, this solution was treated with oxygen for 15 minutes, the pH was adjusted to 11.3 with sulfur and lithium hydroxide, and the solution was heated at 75° C. for 1 hour.

The reaction mixture was cooled to room temperature and 0.6 grams of Tris-(
hydroxymethyl)-aminomethane, then 5N
The pH was adjusted to 7.5 using hydrochloric acid. To the resulting solution were added 1000 international units of alcohol dehydrogenase and 1-acetaltehyde. The decrease in optical density of the reaction mixture was monitored at 340 nm, and when the decrease could no longer be observed,
H was adjusted to 3.5. Dilute the solution 10 times the volume to 10°
The resulting oil was separated, washed with ether, and dissolved in 10-15 water.

The resulting solution was mixed with Sephadex G-equilibrated with water.
10 (Pharmazoa Ny, Uppsala, Sweden)
The mixture was introduced into a 2.5 x 90 m column (obtained from Bee).

Fractions of 12 rnl each were collected. The wavelength of maximum optical absorption in the ultraviolet region and the optical density at that wavelength were measured for each fraction. In addition, 3 of each fraction after reduction with alcohol dehydrogenase
The optical density at 40 nm was also measured. Fractions with a maximum optical absorption at 264 nm and a ratio of optical density at 340 nm to optical density at 264 nm greater than 05 were collected. The collected material was concentrated to 15-20rneK in a rotor 1.1-evaporator and equilibrated with water to DOWEX 1-X8 (IJ!1, obtained from Bio-Rad Laboratories, Richmond, California) It was passed through a 2.5xzs& column.

Add more water to the column to wash the collected material, 10-
I collected nine fractions of each. It has a maximum optical absorption at 264 planes and an optical density at 340 nm and 26
Fractions with a ratio of optical density at 4 nm greater than 01 were collected.

, Dowex 5O-
A 5 x 45 column of X2 (obtained from Bio-Rad Laboratories, Richmond, California, USA)
passed through. More water was added to the column to wash the collected material and 20-fractions were collected. 264
The fractions with the maximum optical absorption at 340 nm and the ratio of the optical density at 264 nm of greater than 0.18 were collected. Collected 4-5
ml and purified by electrophoresis as described below.

The width perpendicular to the direction of liquid flow is L20.
Whatman 3 MM paper (obtained from Chris 7 Noribu Angel, New Chassis, USA). The paper was then moistened with 0.02M sodium phosphate at pH 6.0. Electrophoresis method is Science 121:829
(1955).
m) Performed according to the suspension assembly method at a potential gradient of 8.5 volts/camellia for 4-6 hours. The location of the desired pyridine dinucleotide visiting conductor appears after spraying on 0.5 M sodium anhydride test paper according to the method described in Journal of Biological Chemistry, 191:447 (1951). Determined by fluorescence. Cut out the area containing the desired derivative from the paper, 5t)
mJ! Extract 3 times with water. Nicotinamide 6-(2
The resulting extract i containing the -aminoetheramino) purine dinucleotide is collected and marked with a convex line at 3-4 tJ,
It was then stored at -20°C.

Example 2 Production of a conjugate of nicotinamide adenine dinucleotide and biotin In ln- water containing 22 mg of nicotinamide 6-(2-aminoethylamino)purine dinucleotide obtained in Example 1. 16m? of biotin was completely suspended. A few drops of 0. IN sodium hydroxide was added to help dissolve the biotin. 2404 of 1-cyclohexyl-3-(2-morpholinoenal)-carbodiimide metho-p-toluene sulfone l- was added to the resulting solution, and O, IN hydrochloric acid was added dropwise to form a solution. The reaction mixture was left at room temperature for 5 hours and then poured into 10-10°C acetone.

The produced oil was completely separated and 5-10-ether-C2
Washed twice and dissolved in 1-2 ml of water. The obtained product was purified by the frost pneumophoresis method using the paper described in Example 1. When sodium cyanide was sprayed, multiple fluorescent bands appeared, one moving towards the cathode and the others moving towards the anode. The latter zone contained the NAD-biotin conjugate, which was extracted with water and stored at -20°C.

Example 3 Nicotinamide adenine dinucleotide and 2.4-
Preparation of dinitrophenyl conjugate Nicotinamide, 6-(2-aminoethylamino), purine, obtained in Example 1
26 ml of bicarbonate (IJ) was dissolved in 1,5-water containing the dinucleotide 23rITg. To the resulting solution, 17 μl of ethanol containing 2,4-dinitrofluorobenzene was added, and the reaction mixture was incubated in the dark at room temperature for 5 minutes.
After stirring for an hour, 45ml of acetone at 10°C was added. The formed precipitate was separated, washed twice with 10 yd of acetone, and stirred with 5 ml of water. The separated yellow soluble substance was electrophoresed using the paper described in Example 1. Purification was carried out using 1 liter over 15 hours. The band that migrated to the anode contained a conjugate of NAD and 2,4-dinitrophenyl. This was extracted with water, concentrated to 3-5 ml, and then stored at -20°C.

Example 4 Effect of avidin and biotin on the enzyme circulation rate of NAD and NAD-biotin conjugates The cycling reaction system used in this example
H-ligand pyruvate (salt) (b) NAI)
H-ligand + thiacylinoa blue (oxidized form) in a total amount of 0.5- each, containing a buffer of 0.1 M N,N-bis-2-hydroxyethylglycine hydrochloride at pH 7,8, as shown in Table 1. NAD%NA with concentration and activity as shown in
Type 89 specific binding reaction compounds were prepared, each containing a D-biotin conjugate (Retamono prepared in Example 2), biotin and avidin (the latter having an affinity to bind to habiotin). One unit of avidin activity is the amount of avidin that binds 1 μg of biotin.

The reaction mixture was stored at room temperature for 2-3 hours. 1 of 0.14
M lithium lactate, 0.05-(1) 10 mM oxidized thiazolyl blue, and a sufficient amount of 081 at pH 7,8
2M N,N-bis-2-hydroxyethylglycine hydrochloride dilute solution (containing 0,3813:1 units of bovine heart lactate dehydrogenase and 15 international units of pig heart diaphorase). The reaction mixtures were contacted with the enzyme/substrate aqueous mixture by adding a 1-volume solution to each reaction mixture. The total change in optical density of each mixture at 570 nm was measured every 24 minutes within the first hour after addition of the enzyme/substrate mixture to determine the relative rate of production of reduced thiazolyl blue for each reaction mixture anastomosis. . The entire operation was repeated twice and the average results are shown in Table 1.

Reactions 1.4 and 8 are control experiments and show that in the absence of NAD and NAD-biotin conjugate, very little cycling occurs. Reactions 2 and 3 demonstrate that the NAD-biotin conjugate has a significant amount of enzymatic activity compared to native NAD. From the results of reactions 3 and 6,
The presence of avidin in the reaction mixture reduces the amount of NAD present.
is combined with biotin.

It can be seen that the production of reduced thiazolyl blue is inhibited when A comparison of the results of Reactions 6 and 7 shows that the presence of free biotin reduces the amount of inhibition of thiazolyl blue in proportion to the concentration of biotin in the reaction mixture.

Therefore, in this example, the activity related to the circulation reaction system of NAD in the conjugate of NAD and biotin is decreased by the presence of avidin, and the magnitude of such decrease in activity is greater than that in the presence of biotin. has been shown to decrease.

Example 5 Direct binding of avidin - Circulation analysis; Effect of variation of avidin amount on circulation rate The circulation reaction system used in this example is shown in Example 4 (-
It is the same as the previous one. Each total valve is 0.6-, each p
H7,8 in 0.12M N,N-bis-2-hydroxyethylglycine hydrochloride buffer and N purified in Example 2.
Seven types of specific binding reaction mixtures containing AD-biotin conjugate (250 nM) were prepared, and six of the reaction mixtures contained one avidin as shown in Table 2. do.

Leave the reaction mixture at room temperature for 2-3 hours. Shita. 01-IM milk 1゛lithium, 0.057nl of 10mM oxidized thiasiri A blue, and 100%), moon pH 7,
8012M N,N-bis-2-hydro-7ethyl phosphate buffer (contains 038 international units of bovine heart liver lactate dehydrogenase and 1.5 international units of porcine heart chamber diaphorase) The reaction mixture and enzyme/
Dissolved in contact with substrate aqueous mixture. The total change in optical density of each mixture at 570 nm was measured every 24 minutes within the first hour after addition of the yeast/substrate mixture to determine the relative rate of production of reduced thiazolyl blue throughout the reaction mixtures. did. The ratio (expressed as a percentage) of the change in optical density of each reaction mixture containing avidin to the change in optical density of the reaction mixture without avidin was calculated and the relative reactions are shown in Table 2 and the attached Figure 1. It is written as speed. The results are shown in Table 2 and also in Figure 1 of the accompanying drawings in the form of Cla 7.

Table 2 1 0.000 100 2 0.005 96 30.01093 4 0.045 84 5 0.090 68 6 0.120 51 7 0.180 8 As described above, in this example, the circulation reaction It has been shown that the relative circulation rate of the system, and thus the activity of NAD in the NAD-biotin conjugate, is an inverse function of the amount of avidin present in a particular binding reaction mixture. Accordingly, the present invention provides a normal phase reagent and method that utilizes a direct binding-circulation analysis technique for quantitatively determining the presence of the analyte avidin in a liquid.

Example 6 Competitive Binding of Biotin - Circulation Analysis; Effect of Variations in Navidin Opening on Circulation Rate The circulation reaction system used in this example is the same as that shown in Example 4. The total amount of each was 0.45 yd, and the NAD-biotin conjugate obtained in Example 2 (180 nM ) Seven types of specific binding reaction mixtures containing f were prepared.

Six of the reaction mixtures, numbered 1-6 in Table 3, contain an additional 0.11 units of avidin. In addition, five of the six reaction mixtures containing avidin, namely the third
Mixtures 2-6 in the table contain biotin at the concentrations shown in Table 3.

The reaction mixture was kept at room temperature for 2-3 hours. I of 0.14
10mM oxidized thiazolyl blue of M lactic acid
M N,N-bis-2-hydroxyethylglycine hydrochloride buffer (contains 0,388 international units of bovine heart lactate dehydrogenase and 1.5 international units of pig heart diaphorase)
The reaction mixtures were contacted with the enzyme/substrate aqueous mixture by adding a solution made to a total concentration of 1- to each reaction mixture. The total change in optical density of each mixture in the 570 fields was measured every 24 minutes within the first hour after addition of the enzyme/substrate mixture to determine the relative rate of production of reduced thiazolyl blue for each reaction mixture. The ratio of the change in optical density of each reaction mixture containing biotin to the change in optical density of the reaction mixture containing neither biotin nor avidin (
(expressed as a percentage) is calculated and recorded as the relative reaction rate in Table 3 and the accompanying Figure 2. The results are shown in Table 3 and also in graphical form in Figure 2 of the accompanying drawings.

Table 3 1 0
82 so
223 160
354 320
635 400
806 800
As mentioned above, in this example,
It has been shown that the relative reaction rate of the cycling reaction system, and thus the activity of the NAD of the 2'JAD-biotin conjugate IP, is a direct function of the amount of biotin present in the particular binding reaction mixture. Accordingly, the present invention provides test reagents and methods that utilize competitive binding-circulation analysis technology to consistently and consistently measure the presence of biotin in a target substance for liquid vaporization. Example 7 Direct binding of antibodies to 2.4-dinitrophenyl and its 1-conductor - Circulation analysis The t-circulation reaction system used in this example is shown in Example 4.
Same as ζ. 0.12M N,N-bis-2-hydroxyethylglycine hydrochloride buffer M at pH 7 and 8, and NAD and NAD in the amounts and concentrations shown in Table 4, respectively.
-2,4-dinitrophenyl conjugate (obtained in Example 3), 2,4-dinitrophenyl antiserum, and 8 types of 0.0000. A specific binding reaction mixture of fLm! was prepared.

The reaction mixture was stored at room temperature for 3-4 hours. 0.1 ml of 1 M lithium lactate, 0.05 me of 10 mM thiazolyl blue, and enough pT (7,8 o,
in 12M N, 1(-bis-2-hydroxyethylglycine hydrochloride field buffer containing 0.38 international units of bovine heart lactate dehydrogenase and 1.5 international units of pig heart diaphorase). The reaction mixtures were contacted with the enzyme/substrate aqueous mixture by adding a total solution of 1 to 1 to each reaction mixture. Within the first hour after addition of the enzyme/substrate mixture, The relative rate of formation of reduced thiazolyl blue was determined for each reaction mixture by measuring the total change in optical density of each mixture in 570 dishes over 24 hours.The entire procedure was repeated twice and the average of the results is shown in Table 4. Reaction J, shown in Figure 6, is a control experiment that shows that cycling is very unlikely to occur in the absence of NAD and the NAD-biotin conjugate. C shows that the dinitrophenyl conjugate is active in the enzyme cycling system. Reactions 3 and 5 show that the presence of 2,4-dinitrophenyl antibodies prevents NAD cycling.

Such inhibition is reversed by addition of NMN, as shown in the results of reaction 6 1f'. reactions 3 and 4
The results show that the presence of NMN increases the circulation rate by about 15% compared to its absence. This result is likely due to contamination with external NAD, since other measurements have shown that NMN does not affect circulation rate in the absence of antibody.

However, the antiserum has some activity with respect to NAD itself, and this activity is inhibited in the presence of NMN.

As described above, in this example, the activity related to the circulatory reaction system of NAD in the conjugate of NAD and 2,4-dinitrophenyl was decreased by the presence of the 2,4-dinitrophenyl antibody. I'm showing.

Accordingly, FIIJ provides test reagents and methods that utilize a direct bond-circulation analysis technique to determine the presence of the target substance 2,4-dinitrophenyl in a liquid.

Example 8 Competitive binding of 2.4-dinitropensen and its derivatives - Circulation analysis; Effect of varying the amount of N-(2,4-dinitrophenyl)-6-aminocaproate on circulation rate In this example The circulation reaction system used was the same as that shown in Example 4. 0.12 M N,N-bis-2-hydroxy-7ethylglycine hydrochloride buffer, 300 nM NAD at pH 7 and 8, respectively, in a total amount of 6 mtJ.
-dinitrophenyl conjugate (obtained in Example 3)
Seven specific binding reaction mixtures containing 50 μM and 50 μM nicotinamide mononucleotide were prepared. Six of the seven reaction mixtures, 1 through 6 of Table 5, also contained sufficient amounts of 2,4-dinitrophenyl antibodies to inhibit the circulation rate of the other reaction mixtures by 85%. tfl is there. N -(2,4-dinitrophenyl--1() ・f May ” Mitsu 7
・C-A゛-Tools, 42:287
2,4-dinitrobenzene derivative) prepared by the method described in '(194 FI) further contains a reaction mixture of 2,4-dinitrobenzene containing 6 Ci esters (I4. 5 scales, i.e. 2.- in Table 5,
Included exactly once as indicated in the table.

The anti-stone mixture was kept in all chambers for about 4 hours. 0.1 n1
1 lM milk lithium, 0.05ml 10mM fermented thiazolylbuno1. -1 and a sufficient amount. II 7.
8 ON2 F/+ N, N-bis-2-hydroquine ethyl glycine hydrochloride J4 buffer (0, 33 countries internationalized cow's heart) (1 lactic acid dehydration 1'1 accumulated and 1 , 5 International Single (
(contains pig heart diadrase) for a total amount of 1 r
The reaction mixture was brought into contact with the enzyme/substrate aqueous mixture ii*+ by adding the solution prepared in 1, 1 to each reaction mixture. For the first 1 hour, the entire emulsion of each mixture was measured at 570 nm every 24 minutes, and the reduced thiazolyl glue for each mixture was measured. The relative speed of was measured. N-(2,4-dinitrophenyl)-6
- Amitsukaprony) e Changes in optical density of each reaction mixture containing N-(2,4-dinitrophenyl)-
The ratio of the change in optical density of the reaction mixture containing neither 6-aminocaproate nor 2,4-dinitrophenyl antibody (
(expressed as a percentage) is calculated and recorded as the relative reaction rate in Table 5 and the attached Figure 3. The results are shown in Table 5 and also in graphical form in Figure 3 of the accompanying drawings.

Table 5 1 0 16 2 17 193
42 304 83
355 166
416 415 76 As described above, in this example, the relative circulation rate of the circulation reaction system, and therefore the activity of NAD in the NAD-dinitrophenyl conjugate, is determined by the amount of N-(2,
It has been shown to be a direct function of the amount of 4-dinitrophenyl)-6-aminocaproate. Therefore, according to the present invention, a competitive binding-circulation analysis technique is used to quantitatively determine the presence of the target substance N-(2,4-dinitrophenyl)-6-aminocaproate in a liquid. Reagents and methods for testing are provided.

Example 9 Production of conjugate of biotin and umbelliferone 100mf
of umbelliferone, 167mf of biotin and 141mf of
A reaction mixture was prepared by dissolving dicyclohexylcarbodiimide in d in 1 OnLe of dimethylformamide.

The reaction mixture was stored at 1-18°C for 4 hours, then 7
It was allowed to stand overnight at <RTIgt;C</RTI> and then at room temperature for 3-4 hours. 141 more
The dicyclohexylcarbodiimide of 4 was added and the reaction mixture was stirred at 7°C for 3-4 hours and left at room temperature overnight. The entire precipitate obtained was filtered off and discarded. 75 m of ice-cold water was added to the filtrate and the resulting mixture was stored at 0°C for 1 hour. The resulting precipitate was filtered off and discarded. The filtrate was evaporated to dryness, and the residue was dissolved in 3-4 m(l) of methylene chloride. 5 me of diethyl ether was added to the resulting solution. The resulting precipitate was the biotin-umbelliferone conjugate. , which was filtered off, dried and then stored at room temperature.

Example 10 Specific binding analysis of biotin and avidin using an enzyme substrate as a labeling substance The specific binding analysis system used in this example was (378
(fluorescence maximum at 448 nm) (fluorescence maximum at 448 nm) each in a total volume of 0.3-, each in 0.1M tris-(hydroxymethyl)-aminomethane hydrochloride buffer at pH 8.0 and each as shown in Table 6. umbelliferone-bionan conjugate (obtained in Example 9) at different or higher concentrations;
Ten specific binding reaction mixtures containing biotin and avidin were prepared.

The reaction mixture was stored at room temperature for 1-3 hours.

Reaction mixture numbers 2-10 of Table 6 also contain 0.26 international units of bovine liver carboxylic acid hydrolase (esterase). The relative reaction rate for each reaction mixture was then set to 364 nm excitation f? :, 1
11-inch Turner Fluorometer (Palo, California, USA)
Aldo, Pulgas Stogy) 2524 G.K.
The fluorescence emitted from each was monitored and measured at 448 nm using a fluorochrome analyzer (obtained from Turner Associaton). The fc electric signal generated from the fluorescent side is transmitted to a long and thin chart recorder.The amount of fluorescence generated per minute is measured from the shape of the recorder, and is expressed in arbitrary units based on the sections of the chart paper. did. The results are shown in Table 6.

Reaction 1 is a control experiment and shows that no reaction occurs in the absence of esterase. The results of Reactions 2 and 3 showed that the umbelliferone-biotin conjugate was active during the enzymatic reaction, and comparing the results of Reactions 4-8, the presence of avidin was proportional to the amount of avidin in the reaction mixture. It can be seen that the reaction rate is inhibited. Reactions 8.9 and 10
A comparison of the results shows that the amount of inhibition of reaction rate by avidin is a direct function of the amount of biotin present in the reaction mixture.

As described above, in this example, the rate of fluorescence generated by the esterase reaction, that is, the activity of the substrate of the umbelliferone-biotin conjugate, was decreased by the presence of avidin, and the decrease in activity was The expression has been shown to be reduced by the presence of biotin. Therefore, according to the present invention,
Test reagents and methods are provided that utilize specific binding analysis techniques using enzyme substrates as labels to determine the presence of target substances, biotin and avidin, in a liquid.

Example 11 2. Preparation of conjugate of 4-dinitrophenyl and fluorescein Fluorescein-3',6'-bis-(6-(2,4-
dinitroanilino)hexanoate] This synthesis basically consists of the acid chloride of 6-(2,4-dinitroanilino)hexanoic acid and the acid chloride of fluorescein)
Includes reaction with IJium salt. 6-(2,4-dinitroanilino)hexanoic acid is prepared by the method described in Biochemical Journal, 42:287-94 (1948).

A solution of 1.5 g (5 mmol) of 6-(2,4-dinitroanilino)hexyl acid was reacted with warm 10-thionyl chloride for 15 minutes, cooled, and then 20 g of hexane was distilled off. It was converted to acid chloride by doing this. The resulting solid acid chloride was collected by filtration, dried, and added to a solution of floml of the disodium salt of fluorescein, 60077f, in dry acetone. After refluxing for 5 hours, 27 nl of water and 51 nl of acetone were added to stop the reaction. After heating at 25° C. for 30 min, the mixture was concentrated to dryness (7, and the residue was partitioned between ethyl acetate and aqueous sodium bicarbonate solution. The organic phase was separated and 1
% aqueous sulfuric acid and dried over anhydrous magnesium sulfate.

It was then allowed to evaporate. The resulting red oil was dissolved in silica gel 60 (obtained from Nie Merck & Co., Darm 7 Uttat, West Germany) as a dissolving liquid by dissolving acetone in carbon tetrachloride (20 volume ratio). It was chromatographed using 1.2y impure bisester, 60
Acetone was dissolved in carbon tetrachloride and chromatographed again using silica gel 60 (10% B volume ratio). Combine the appropriate fractions,
Evaporation awn I80 m,'? A yellow glassy solid was obtained.

Word 1 calculation value (as C44H8, 1N60□): C, 5
9,33; Ti.

4.30; N, 9.43. Analysis value: C, 60
, 92i IT, /1.35; N, 6.65 In the infrared spectrum, the expected carbonyl stretch of varnish-ful was observed at 1765c+s-' i%.

Example 12 Identification result analysis of 2,4-pseudonitrophenyl derivatives and their antibodies using enzyme substrate (partially eclipsed, shaped fluorescein) as a labeling substance The specific binding analysis system used in this example was as follows: figure
Formula 1 2,4-dinitrophenyl-fluorescein
(Maximum fluorescence at 510 nm) Conjugate 1 -11□〜□〜μm□−−−−□−
1□−□−Hanrin, ! -i-

□-1. . Ht)8NH7,-\),. 2\==Rio2〈A, Direct binding of 2.4-dinitrophenyl antibody-
Fluorescence analysis; Effect of varying the amount of antibody on the reaction rate Seven specific binding reaction mixtures were prepared and analyzed. In each reaction mixture, 1 μ4 of 2,4-dini 1-0
Phenyl-fluoreneptin conjugate (obtained from run f!Hx) in dimethyl sulfoxide (20 μl) #
2,4-dinitrophenyl antiserum in the volumes shown in Table 7 and a sufficient volume of 0.1 M bishydroxyethylglycine hydrochloride buffer, pH 7.0, to bring the total volume to 2.0 ml. did. The background rate due to hydrolysis of the ester bond in the conjugate was determined by the rate of increase in fluorescence intensity at 510 nm using the general technique described in Example IO using a fluorometer with excitation set at 470 nm. )
was measured for 3 minutes for each reaction mixture. Then, 0.54 units of the
Obtained from Michal Company, E, C, A 3.1
．． 1.1) containing 0.1 M bishydroquine ethylglycine hydrochloride buffer, pH 7.0, was added to each reaction mixture. The resulting total reaction rate was determined in a similar manner as for the background hydrolysis rate. The results are shown in Table 7. The background hydrolysis rate was subtracted from the total reaction rate, thus giving the net reaction rate due to the entelase catalyzed reaction. The relationship between the net enzyme-catalyzed reaction rate and the background alkaline hydrolysis reaction rate and the amount of antiserum present in the reaction mixture is shown in graphical form in Figure 4 of the accompanying drawings.

Table 7 1 0 0.02 2.682
5 0.07 2.483
In O,111,914200,171,0
8 5300,210,63 6400,220,45 7600,260,30 In this part of this example, the net reaction rate of the hydrolysis reaction is It has been shown that J f-1 is an inverse function of the antibody for 2,4-dinitrophenyl, which is the target substance.
It has been shown that the kinetics of the background hydrolysis reaction is a direct function of the amount of antibody present in a particular binding reaction mixture. Therefore, according to the invention, objects in a liquid are 1 and 2. A assay chamber and method are provided for determining the presence of antibodies to /I-dinitrophenyl using direct binding fluorescence analysis.

B. Competitive and competitive binding of derivatives of 2,4-dinitrophenyl - Fluorescence analysis; influence of variation of 2,4-dinitrophenyl-β-alanine on the reaction rate, each total 552.0
ml of 01 M bishydrogyethyl chloride hydrochloride (preoperatively and 2,4-dini) r-phenyl-β-alanine (Journal Op.・American Chemical Company, 76
1328 (1954) K.A. 1328 (1954). Nine of the ten reaction mixtures, i.e., 2 to 10 in Table 8, contain sufficient amount of 2,
4-dinitrophenyl antiserum was added.

After combining 1 and 1 ml of a solution of 2,4-dinitrophenyl-fluorescein conjugate (obtained in Example 11) in dimethyl sulfoxide (20μ), add 1/IN to each reaction mixture. Then, 0.54 international units of type 1 esterase (Sigma, St. Louis, Missouri, USA) was used.
Obtained from Chemical Company, E.G3. Garden 3.1.
1.1) f: Contains 0 μm volume of gold containing 0.1 M bibis droxyethylglycine hydrochloride before bombardment, pH 7.0;
added to each reaction mixture. The reaction rate obtained was measured in the same manner as in Example A. The percentage ratio of the reaction rate of each reaction 2-10 to the reaction rate of reaction 1 (absence of antibody) was calculated. The results are shown in Table 8 and in graph form in Figure 5 of the accompanying drawings.

Table 8 1 0 2
．． 78 -2 0 1.04 3
7 3 5 1.01 36 4 10 1.04 37 5 20 1
．． 24 456 30 1.51 5
4 7 50 1.54 56 8 75 1.80 65 9 100 1.85 67 1o i50
2.33 84 In this part of the example, the reaction rate of the hydrolysis reaction is
It has been shown to be a direct function of the amount of dinitrophenyl-β-alanine. According to the invention, therefore, there are provided test reagents and methods for determining the presence of a substance of interest, such as a derivative of 2,4-dinitrophenyl, in a liquid using a competitive binding-fluorometric technique. Ru.

c, competitive binding of derivatives of 2,4-dinitrophenyl - spectroscopic analysis; influence of varying the amount of 2,4-dinitrophenyl-β-alanine on the reaction rate, each total -3t1.0
ynl, each containing 0.1 M tris-(hydroquinemethyl)-aminomethane hydrochloride buffer at pH 7.0 and 2,4-dinitrophenyl-β-alanine at the concentrations shown in Table 9. '1 binding reaction mixture was prepared.

2-8K in Table 9, an amount of 2,4-dinitrophenyl sufficient to inhibit the enuterase-catalyzed reaction rate of the other mixture, reaction mixture 1. Serum was added.

After mixing, 0.1 mM 2,4-dinitrophenyl-
A solution of the fluorescein conjugate (obtained in Example 11) in dimethyl sulfoxide (10 μl) was added to each mixture. Next, 2,16 international units of type 1 esterase (
Sigma Chemical Co., Ltd., St. Louis, Missouri, USA
Obtained from Company, E, C.

pu containing A 3.1.1.1) 7. 0 of O
．． A 20 μl volume of 1M) lis-(hydroxymethyl)-aminomethane hydrochloride buffer was added to each reaction mixture. The change in absorbance of each reaction mixture at 489 nm per minute was recorded on a Guilford 2000 spectrophotometer. The results are shown in Table 9 and in graphical form in Figure 6 of the accompanying drawings.

Table 9 0 0.0261 2 0 0.0047 3 1.25 0.0118 4 2.5 (1,0131 55,00,0185 67,50,0202 710,00,0192 812,50,0223 This implementation In this part of the example, it is shown that the reaction rate is a direct function of the amount of 2,4-dinitrophenyl-β-alanine in the reaction mixture.

According to the invention, therefore, there are provided test reagents and methods for determining the presence of a substance of interest, such as a derivative of 2,4-dinitrophenyl, in a liquid using competitive binding-spectroscopy techniques. Ru.

D. Competitive Binding of Derivatives of 2.4-Dinitrophenyl - Fluorescence Analysis: Utilization of Non-Enzymatic Monitoring and Detection Reactions The specific binding assay system used here is an esterase that catalyzes the hydrolysis of the ester bond in the conjugate. Other than that, it is the same as shown in Scheme 1.

Two shrines in full view, each with 0.1M Tris at pH 7.5.
(Hydroxymethyl)-aminomethane salt dispersion buffer and 2,4-strophenyl- at the concentrations shown in Table 10.
Eight specific binding reaction mixtures containing β-alanine were prepared. 50'pl of 2,4-dinitrophenyl antiserum was added to each reaction mixture.

After mixing, 2 μM of 2,4-dinitrophenino L-fluorescein conjugate (obtained in Example 11''C)
A dimethyl Nurhokind solution (20 μl) was added to each reaction mixture, and the resulting reaction rates were measured in the same manner as in Example A. The results are shown in Table 10.

1st O Table 1 0 0.962
12.5 0.943
31.2 0844
62.5 0.785
94.0 0.706 1
25 0.597 187
0.578 250
0.53 In this part of the example, the background hydrolysis rate is
In one case, 2,4-dinitrophenyl-
It has been shown that 1 is an inverse function of 4 for β-alanine.

Therefore, according to the present invention, the presence of a target substance, such as a derivative of 2,4-dinitrophenyl, in a liquid is monitored after the binding partner has bound with the target substance (ligand) in the conjugate. Test reagents and methods for measuring anti-(J+) using a binding-fluorescence technique involving the involvement of anti-(J+).

Example 13 Preparation of cortisol-umbelliferone conjugate Cortisol-21-hemisuccinate-umbelliferone A, drying of cortisol-21-hemisuccinate anhydrous nuccin branched (0,5y) in 10 ml containing 0.51 cortisol (hydrocortisone) It was added to the pyridine solution and stirred at room temperature overnight. After adding 100ml of water, the mixture was extracted with 100ml of ethyl acetate. The organic phase was washed once with water and extracted with 100-saturated sodium bicarbonate solution. The aqueous phase is separated and brought to pH 4 with JO dihydrochloric acid. The formed precipitate was collected by filtration, dried, and then diluted with hexane.
The desired intermediate was obtained by recrystallization with an acetone mixed solvent.

Melting point: 171-2°C B, cortisol-umbelliferon axis combination 50 m2 of carbodiimide was added to a solution of the intermediate obtained in step 8 of this example in 1 portion of dry dimethylformamide, and 30 min. , 71 stirred.

50 ynf of 7-hydrocyclocoumarin in dimethylpolamide (2-) was added and the reaction mixture was stirred for one stream at room temperature. Separate the generated precipitate and discard it.

Water was added to the filtrate and the mixture was extracted with ethyl acetate.

The organic phase was washed once with water, separated, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness in a potted plant. When the residue was crystallized with a mixed solvent of acetone and hexane, the desired conjugate was obtained at 21η.

Melting point: 126°C Example 14 Specific thread occupancy analysis of cortisol using enzyme substrate (B1; edible form of umbelliferone) as a labeling substance.
The qI constant binding analysis used in 1 was based on a first-order reaction.

Eight categories of specific binding reaction mixtures were prepared, each in a total volume of 2 ml, each containing 0.1 M bishydroquine ethylglycine hydrochloride buffer at pH 8:5 and cortisol at the concentrations shown in Table 6. . 7 glazes of the 8 reaction mixtures, i.e. 2-8 of Table 11, and the other reaction mixture i.e. reaction mixture 1.
A sufficient amount of cortisol anti-inhibitor was added to inhibit the rate of the esterase-catalyzed reaction by 70 degrees.

After mixing, each reaction mixture was mixed with 20 μe of 0.1 M pishydroxyethylgrin hydrochloride buffer vi′ff at pH 8.5 in which 2 μM of the cortisol-umbelliferone conjugate (obtained in Example 13) was dissolved. added to.

After further mixing, add 15 μl of porcine esterase (0,
81 units/ml) was added to each reaction mixture. The resulting reaction rates were determined for each reaction mixture in a manner similar to that described in Example 10. The results obtained are shown in Table 11.

Table 11 Reaction mixture Cortisol concentration (nM)
Reaction rate 1 0
0.08382 0
0.02563 2, 5
0.02964 10
0.03065 2
0 0.03816
30 0.04087
50' 0.06548
100 0.0603
This example shows that the reaction rate is a direct function of the amount of cortisol in the reaction mixture. Accordingly, in accordance with the present invention, there are provided test reagents and methods that utilize competitive binding-fluorometric techniques to determine the presence of cortisol in a fluid.

[Brief explanation of drawings]

FIG. 1 graphically illustrates the effect of varying the amount of analyte (licand) on the overall reaction rate in a direct binding-circulation analysis technique. Figures 2 and 3 each graphically illustrate the effects of varying the magnitude of two different target substances on the overall reaction rate in a competitive binding-circulation analysis technique. Figure 4 shows the effect of varying the amount of analyte on the net reaction rate of two different reactions in the direct binding-fluorometry technique, one with enzyme catalysis and the other without. The impact is shown graphically. Figures 5 and 6 respectively show that the reaction rate of (rj) is 7. (This is a graph that shows the effect of varying the purity of the target substance. Figure 3, Figure 4, 2.4-"; 50
too 1502.4-dini) Rofueni Ire β-alanine (nanomole) Figure 6

Claims (1)

[Claims] 1. For analyzing a hapten or antigen in a liquid: (a) a liquid, (1) a conjugate of a labeling substance having predetermined characteristics and a hapten or antigen, and (2) ) contacting the labeled conjugate with a reagent consisting of an antibody to the hapten or antigen, producing an antibody-bound phase and an antibody-free phase; and (b) contacting the labeled conjugate with a reagent consisting of an antibody to the hapten or antigen, without separating the bound and free phases; 1. A homogeneous immunoassay method comprising the step of measuring the properties of a hapten or antigen in the conjugate as an indicator, the method being characterized in that the labeling substance in the conjugate is a substrate in an enzyme-catalyzed reaction. 2. The analytical method according to item 1, wherein the labeling substance is a substrate for an enzyme-catalyzed reaction that produces a product with detectable properties that can be distinguished from that of the conjugate; . 3. Claim 1 in which the detectable property is fluorescence
Analytical method described in section. 4. For analyzing a hapten or antigen in a liquid by a homogeneous immunoassay method: (1) a conjugate of a labeling substance with predetermined characteristics and a hapten or antigen, and (2) a conjugate for the hapten or antigen. A reagent consisting of an antibody, which is a conjugate of the reagent and a hapten or antigen labeled.
forming a binding reaction system that produces a bound phase and an antibody-free phase, and the properties of either the bound phase or the free phase are a function of the amount of hapten or antigen in the liquid, and the labeling substance in the conjugate is a substrate in an enzyme-catalyzed reaction. 5. The reagent according to claim 4, which is incorporated in a carrier matrix. 6. A claim in which 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. The reagent according to item 4. 7. Claim 4, wherein the detectable property is fluorescence
Reagents listed in section.