JPS5942454A - 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 hapten or antigen (target substance) in a liquid based on the affinity of the target substance for a specific (or specific) binding partner substance. Concerning methods and reagents. More specifically, 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 for the same cormorant.

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, it could also be quantitative. The reaction system for each analysis is necessarily either limited to the detection of 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 has led to the creation of radioimmunoassay methods.

According to this method, the amount of radiolabeled substance to be detected is made to compete with the unknown for a limited stream of antibodies that are specific to the unknown. The amount of label that becomes bound to the antibody then varies inversely with the level of unknown material present. In radioimmunoassay techniques, it is essentially necessary to separate the substance to be detected that is labeled so that it becomes bound to antibodies from a substance that does not cause such binding. . Various means for accomplishing this required separation are described, for example, in U.S. Pat. No. 3.505.019.
No. 3.555.143, No. 3.646,346, 3
．． 720.760 and 3.793.445. However, all of these methods involve at least one manual step, such as filtration, centrifugation, or washing, to ensure efficient separation of the bound-labeled material from the unbound-labeled material. It requires a different operation process.

If separation operations could be eliminated, the analytical method would be much simpler and would be even more useful in clinical laboratories.

The use of radioactive substances in immunoassays can now be avoided to some extent by using substances with enzymes instead of radioactive substances. U.S. Patent No. 3.
654.090 and 3.791.932, the manual steps required to perform enzyme-based immunoassays are generally not required in radioimmunoassays. However, it requires a complicated separation process.

Other disadvantages of using enzyme-tagged materials include that each attached enzyme must be individually chemically modified to be used in forming its attached conjugate. Other substances attached, such as coenzymes or viruses (Nature, Ta, 186(1)
968)) and furthermore using fluorescent labels (French Patent No. 2.217.350)
has also been proposed.

Although immunoassay methods using radioactive labels or labeled enzymes may be improved in the future by broadening the range of detectable substances or simplifying procedures, their essence is that these methods A seed separation step is always required. Recently, another type of method has been published that does not require a separation step. 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 (
The process of combining a soluble complex consisting of an enzyme as a labeling substance (ligand) and a soluble receptor (usually an antibody) for the target substance with the liquid to be analyzed and the effect of the target substance on the enzyme activity in the complex. A competitive binding analysis method is disclosed that includes the step of measuring.

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 has severe limitations in its capabilities and cannot meet the demands of extensive analysis. For example, in the production of enzyme-binding-ligand complexes, the substance or ligand to be detected is attached to the enzyme.
It is clearly essential that the attachment site must be attached in a carefully controlled manner so that it is in close proximity to the enzymatically active site of the enzyme.

This is necessary for the purpose of blocking enzymatically active sites due to complexing or reaction between Gant and receptor. Enzymes vary widely in size, with molecular weights ranging from about io, ooo to 1.000.000. Thus, the binding of a receptor in the form of an antibody with a molecular weight between 150,000 and 300,000 so that the active site of an average enzyme with a molecular weight of 500,000 or more can be physically blocked. Position must be strictly controlled. Due to the complexity of the chemical structure of enzymes, it is actually difficult to strictly control such chemical bonds, and even by screening a wide range of enzymes, only a small number of enzymes can be analyzed using this homogeneous analysis method. It can be considered that it can be used in

Furthermore, in order to obtain quantitative test results, each enzyme-
It is essential to tightly control the number of enzymes and Gants for the binding-ligand complex. In this respect too,
The complex peptide structure of enzymes makes such control an added advantage. It is therefore likely that only a small number of enzymes have the appropriate molecular structure to ensure the necessary control of the ligand to enzyme ratio.

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

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

The enzyme species contained in the enzyme-conjugate-ligand conjugate are present in significant amounts in the liquid sample being tested, thereby creating a background of uncontrollable activity (batter background) that has a considerable impact on the accuracy of the analytical method. ). Therefore, in order to formulate an analytical method that can be used to test human or animal biological fluids, it is necessary to select enzymes that are exogenous to such fluids and use them to perform fermentation. ㅅ(-bond-ligand conjugate must be formed), which further limits the applicability of this analytical method.

Therefore, an object of the present invention is to provide a novel method and reagent for detecting haptens or antigens (target substances) in liquids that do not require a separation step and do not use inconvenient radioactive substances or partially modified enzymes as labeling substances. The goal is to provide the following.

It is a further object to provide a homogeneous immunoassay method and system that is more versatile and convenient than prior art methods.

It is also an object of the present invention to provide a homogeneous immunoassay method and system using a labeling substance that can bind to a target substance or its specific binding partner more favorably than the enzymes of the prior art methods. be.

It is also an object of the present invention to provide a homogeneous immunoassay method and system using a conjugate containing a labeled substance whose activity is more easily affected by specific binding reactions than the enzymes of the prior art methods. one of.

By using a wide variety of sensitive reaction systems, homogeneous systems using conjugates containing labeled substances can detect any change in activity of the enzyme more conveniently than any change in the activity of the enzyme in prior art methods. It is also an object of the present invention to provide a specific binding analysis method and 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 prior art methods.

The homogeneous immunoassay method of the present invention is for analyzing butene or antigen in a liquid: (a) For the liquid (1) A conjugate of a labeling substance having predetermined characteristics and a hapten or antigen , and (2) contacting the bound and free phases with a reagent consisting of an antibody to the hapten or antigen, producing an antibody-bound phase and an antibody-free phase of the labeled conjugate. A homogeneous immunoassay method comprising the step of measuring the properties of a hapten or antigen as an indicator in a liquid without separation, characterized in that the labeling substance in the conjugate is a reactant in a chemiluminescent reaction. That is.

Further, the homogeneous immunoassay reagent of the present invention is used to analyze hapten or antigen in a liquid by a homogeneous immunoassay method: (1) Binding of a labeling substance having predetermined characteristics with a hapten or antigen. and (2) a reagent consisting of an antibody against a hapten or an antigen, the antibody being a labeled conjugate of the reagent and the hapten or antigen.
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 reactant in a chemiluminescent reaction.

The present invention provides a very convenient, widely applicable, and sensitive homogeneous specific binding analysis method and system for labeling substances exhibiting an endowed reaction activity as a component of a predetermined reaction. It is provided on the basis of its use as a substance. Such materials are referred to herein as "reactants."

This method is based, in part, on a predetermined reaction between a hapten or antigen (target substance) and its specific binding partners (to which the reactant is bound). It is based on the fact that it changes the activity of the reactants in the reaction. This reaction serves as a means to monitor and detect specific binding reactions. Due to this basic phenomenon, different operating steps, including different test compositions and equipment, are employed when carrying out the method of the invention. The preferred basic operations are direct binding techniques and competitive binding techniques.

In direct binding techniques, a liquid suspected of containing the hapten or antigen to be detected is brought into contact with a conjugate containing a reactant that is bound to a specific binding partner of the substance of interest. , then any changes in the activity of the reactants are 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 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 forms a predetermined monitoring detection reaction with the reactant.

Qualitative measurement of the target substance in a liquid is based on the obtained reaction and
This is done by comparing the characteristics (usually reaction speed) of monitored detection reactions in a liquid that does not contain the target substance. The difference between the two indicates a change in the activity of the reactant. Constant determination of the substance of interest in a liquid is performed by comparing the obtained reaction with a monitored detection reaction in a liquid containing a known amount of the substance of interest.

Preferably, the monitoring detection reaction uses a chemiluminescent reactant. 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.

The 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 a specific bond 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 in a specific bond to the target substance.

In general, each component of a specific binding reaction, e.g., a liquid putatively containing a ligand or antigen (substance of interest), a conjugate, and/or a partner substance for specific binding of the conjugate and/or analyte, will contain a significant amount of gas or Any amount, method, and order of binding may be used so long as the activity of the reactants in the conjugate measurably changes when the concentration of the substance of interest is included in the liquid. All components of a specific binding reaction are preferably soluble in the liquid, resulting in a homogeneous analytical system. However, a heterogeneous analysis system in which the partner substance or conjugate for specific binding of the target substance is insoluble can also be used if desired.

When using direct binding techniques, the components of a specific binding reaction are a liquid presumed to contain the substance of interest and a quantity of conjugate containing a reactant that is bound to the specific binding partner of the substance of interest. . The activity of the reactant of the 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 specific binding partner in the conjugate. That is, as the amount of target substance in the liquid 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, the amount of the target substance that is considered to be present in the liquid is greater than that which can be combined with all of the target substance. In practice, the amount of a particular binding partner is selected according to the criteria described above, based on the maximum expected amount of the target material that is expected to be present in the liquid. Direct binding techniques are particularly useful for detecting high molecular weight analytes that have smaller specific binding partners.

When using competitive binding techniques, the components of a specific binding reaction are a liquid presumed to contain the analyte, a quantity containing the reactant bound to the analyte (ligand) or similar substance for specific binding of the analyte. and a specific binding partner substance of a certain amount of the target substance. 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 reactant of the conjugate of I3 in contact with the liquid is determined by the activity of the reactant of the I3 conjugate in contact with the liquid, since the conjugate competes with Gant or its specific binding analog in the conjugate to bind to the target substance in the liquid. and the specific binding partner substance.

That is, as the amount of target substance in the liquid increases, the activity of the reactant of the conjugate increases. To obtain quantitative results, the specific binding partner in contact with the reactant and liquid in the conjugate is typically During the contact between the partner substance, the conjugate, and the liquid, the amount that can bind to all of the target substance considered to be present in the liquid and all of the target substance or its similar substance in the form of the conjugate is also reduced. In practice, m of the specific binding partner is selected according to the criteria described above, based on the maximum expected amount of the target substance expected to be present in the liquid. Generally, the amount of analyte (ligand) or its analog in conjugate form that comes into contact with the liquid should not exceed the minimum amount of analyte 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 contacted with the specific binding partner and then with a liquid. Competition for specific binding partners then occurs.

In such methods, the amount of conjugate that contacts the specific binding partner is determined by the amount of conjugate that contacts the specific binding partner prior to contact with the liquid presumed to contain the target substance. This is the amount that contains the target substance (ligand) or its analogous substance in an amount that is greater than the amount that can bind to the nail of the target substance for specific binding that is present. This order of contact can be accomplished in any of two convenient ways. According to one method, the conjugate is brought into contact with a specific binding partner in a liquid environment prior to contact with a liquid suspected of containing the target substance. According to the second method, a liquid presumed to contain the target substance is brought into contact with a complex containing a conjugate and a specific binding partner substance. In this case, the specific binding substance and the specific binding partner substance in the conjugate are bonded facing each other. The amount of the conjugate that becomes bound to the partner substance of the specific binding in the first method, which becomes the amount of the conjugate in the form of a complex in the second method, is Usually, some change in the activity of the reactant of the conjugate is capable of displacing all of the target substance in the liquid when the liquid is in contact with the partner substance (or complex) of a particular binding before the completion of the titration. More than quantity.

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 1 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 secondary bonding technique, a partner substance for specific binding of a target substance is brought into contact with a liquid presumed to contain a substance in the target substance during a period prior to contact between the liquid and the binder.

The amount of the specific binding partner that comes into contact with the liquid is usually the target substance that is believed to be present in the liquid at the time the liquid comes into contact with the specific binding partner before the liquid contacts the conjugate. more than can be combined with all of the Furthermore,
In the case of target substances or similar substances in the form of conjugates, residual and unbound specific bonds are usually detected during contact of the conjugate with a liquid before evaluation of any changes in the activity of the reactants of the conjugate is completed. The amount of the partner substance is greater than the amount that can be combined. 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.

It is understood that other addition sequences and operating steps involving other relative ratios of components of specific binding reactions may also be devised to perform homogeneous specific binding assays without departing from the inventive concepts described herein. is to be pre-Δilled.

The step of determining any change in the activity of the reactant of the conjugate of the components of the predetermined monitoring detection reaction is performed by evaluating the reaction mixture of the specific binding reaction to the reactant of the conjugate of the components of the monitoring detection reaction. This is conveniently accomplished by contacting the binding reaction with at least one substance formed together and then measuring the influence of the specific 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 j herein refers to all or a portion of a predetermined monitoring detection reaction.

Suitable reaction components forming the monitoring detection reaction with the reactants in the conjugate may be added to the reaction mixture of the specific binding reaction, alone or in combined form, prior to, concurrently with, or subsequent to the initiation of the specific binding reaction. be brought into contact. After initiation of a specific binding reaction, the reaction mixture containing some or all of the essential components of the monitoring detection reaction is typically collected for a predetermined period of time before evaluating any changes in the activity of the reactants in the conjugate. Cultivated. After the incubation period, components required for the monitoring detection reaction and not already present in sufficient quantities in the reaction mixture are added thereto. Any influence on the monitoring detection reaction is then evaluated and used as an indicator of the presence or amount of the target substance in the liquid.

Target substance is not present in the liquid or fB is not significant
In situations in which a sharp cutter is present, the monitoring detection response determined by the cutting tool exhibits a relatively constant property. When the substance of interest is present in the liquid, at least one property or property of the monitored detection reaction changes. Generally, the activity of a reactant of a conjugate is defined as the rate at which that reactant can participate in a monitoring detection reaction. That is, the nature of the monitoring detection reaction is altered by the presence of the substance of interest in the liquid, usually with respect to the overall reaction rate or the equilibrium amount of the reaction--or several reaction products--produced by the reaction. 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. 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 and bound conjugates present after incubation of a particular binding reaction will be a function of the amount of analyte in the liquid and will determine the impact on the monitoring detection response.

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 rate of appearance of reaction products. It is determined by measuring the

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

A preferred form of monitoring and detection reaction is a luminescent reaction system, preferably an enzyme-catalyzed reaction, such as a reaction exhibiting the phenomenon of bioluminescence or chemiluminescence. The reactants in the conjugate may be reactants of a photogenerating reaction or a reaction 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.

ATPH adenosine triphosphate kMP: adenosine-phosphate NA, DH nicotinamide adenine dinucleotide NADHH reduced nicotinamide adenine dinucleotide 1” M N: flavin mononucleotide FMN■
-12: Reduced flavin mononucleotide hν: Electromagnetic radiation, usually in the infrared, visible or ultraviolet region 11 + AMP decaoxidized luciferin NAD + FMNH2 2) FMNH2 + long chain aldehyde + 0 P. fiscieri D, 1) 3', 5'-Adenosine triphosphate + reduced luciferin sulfate (salt) 2) Reduced luciferin + 02
-hν + oxidized luciferin 11ν deca oxidized pyrogallol 10 H20 quantifying enzyme H, reduced pyrogallol +02--→ reduced pyrogallol 11 ν deca oxidized pyrogallol h ci + aminophthalic acid (salt) 1 N2 J. Isolminol + KO2 Isolminol h + aminophthalic acid (salt) 1N2 Further details and discussion of the luminescent reaction system that can be utilized in the method of the present invention can be found in the following documents:

Journal of Biological Chemistry, 2
36:48 (1961) Journal of the American Chemical Society, 89: 3944 (1967) Cornier et al., Advances in Bioluminescence, edited by Ji Tianson et al., Princeton University Press. Pages 363-84 B.
Kries, Renilla luciferase (R
Purification and Properties of Enilla Luciferase 1 Doctoral Dissertation, University of Georgia (1967) American Journal of Physiology, 41
:454 (1916) Biological Bulletin, 51389 (1926)
Journal of: Biological Chemistry, 2
43:4714 (1968) Although not necessary in preferred embodiments of the invention,
It may also be desirable to use heterogeneous analytical techniques in cases where the presence of the substance of interest in the liquid affects the activity of the reactants of the conjugate. In such situations, heterogeneous methods are particularly advantageous.1. Hail in z case. Certain heterogeneous methods can increase the effective concentration of analyte in the analytical system, thereby increasing sensitivity. Examples of such heterogeneous methods include a specific binding partner substance of the conjugate or target substance of the present invention (this is
Some use column devices containing an insoluble matrix of either 100% or more (selected according to the type of pulp chamber operation chosen). All other heterogeneous analytical methods using radioactively labeled or gamma-conjugated substances as labeling substances are also similarly carried out using the reactants of the present invention as labeling substances.

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 within a biological system or can be synthesized. This target substance is used for functional purposes.
In other words, it is selected from the group consisting of antigens, antibodies thereof, haptens (adherents), antibodies thereof, vitamins, and their receptors and binding substances. Particular examples of target substances that can be detected using the present invention include antigens and haptens such as ferritin, bradekinin, prostaglandin, and tumor-specific antigens; biotin, vitamin B1,
Vitamins such as folic acid, vitamin E and ascorbic acid; antibodies such as microsomal antibodies, hepatitis antibodies, allergen antibodies; and thyroxine-binding globulin, avidin,
Specific binding receptors such as intrinsic factor and transcobalamin are included.

Generally, it is preferred that the conjugate comprises a reactant and its chosen specific binding partner bound to a smaller target substance (ligand). When detecting a target substance where the molecular weight of the selected specific binding partner is about 1/10 or less of the molecular weight of the target substance, direct binding techniques are preferably used. Therefore, when the target substance to be detected is an antibody or a specific binding receptor, the reactant bound to the antigen or the binding containing the hapten bound to the low molecular weight non-1 partner of the specific binding of the antibody or receptor. Preferably, by direct bonding techniques where the body is used. 1′
! When the molecular weight of the excellent binding partner substance is 10 times or more larger than the molecular weight of the target substance to be detected, that is, when the target substance is an antigen, hapten, or vitamin,
It is particularly advantageous to utilize competitive binding techniques or sequential saturation techniques in which conjugates containing reactants bound to smaller target substances are used.

In the conjugates of the present invention, the reactant is used in a manner that maintains a measurable amount of activity of the reactant. The target substance is either a similar substance for a specific bond or a partner substance for a specific bond of the target substance. Bonds between reactants and specific binding substances are monitored using active anti-15 f't' assays in which detection reactions are not designed to chemically break bonds, such as in the luminescence reaction systems described above. Under these conditions, the general pattern 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.

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 coexistence 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,
They are linked by bridging groups containing heteroatoms such as phosphorus and others.

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 1000, preferably less than 200. Bridging groups contain carbon atoms or heteroatoms of copper, or combinations thereof, and are typically esters, amides,
ether, thioester, thioether, acetal,
A linking group in the form of a methylene or amino group connects the reactant and the specific binding agent or active derivative thereof.

The reactant in the conjugate of the invention is a substance that has an endowed (eg, constant or known) reactive activity as a component of a defined monitoring detection reaction. More specifically, in the disclosure herein, "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
Refers to a substance that provides thermal energy or sound energy. Accordingly, the group of substances referred to herein as "reactants" includes conventional inorganic and organic reagents and biochemical substances. However, catalysts (including enzymes) and radioisotopes that are not reactants in monitoring detection reactions are excluded. Because a particular chemical can function in different ways depending on its chemical environment, a single chemical may be classified into a variety of different classes, and such materials are not covered by this disclosure. It will be appreciated that what function it has is determined by the reactivity of the material with respect to the selected monitoring detection reactions described herein.

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. In preferred homogeneous analytical systems, the components are usually in solution or solid form that can be readily dissolved in a liquid. Since the target liquid is usually aqueous in nature, the components are generally in water-soluble form. ie in the form of an aqueous solution or a water-soluble solid such as a powder or resin. 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. Another consideration is that the carrier is insoluble and porous, preferably absorbent, with respect to the liquid to be tested.
It may also be in the form of a trix. Such matrices are, for example, absorbent papers; polymeric films, membranes, ribs or masses; gels and other forms. In such a form, the device 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 response.

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

Usually, it is a liquid substance of an organism or a liquid obtained by diluting or otherwise processing it. Liquid substances of living organisms that can be analyzed by the method of the present invention include serum, plasma,
Includes urine, amniotic fluid, brain fluid, cough fluid, etc. 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 homogeneous analysis systems, substances of interest in biological fluids containing substances with monoconjugate activity can be analyzed without interference from the background. Endogenous background reactant activity can be easily removed in a variety of ways. Biological fluids can be treated to selectively destroy endogenous anti-IiL activity.

Such treatments include, for example, applying a detergent that chemically destroys endogenous activity and then performing 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 Production of biotin-isoluminol conjugate 6-(3-bionanoylamide-2-hydroxypropylamine)-2
, 3-dihydrophthalane-1,4-cymene A, 4-(3-chloro-2-hydroxypropylamino)-N-methylphthalimide 25 (0,142 mol) of 4-amino-N-methylphthal amide (obtained by the method described in Journal of Chemical Society, 26: (1937)) and 20.77 (0.21 mol) of 1-chloro-2,
3-Eboxib four pans for 150m/! 2.2.2-
) and the reaction mixture was refluxed with stirring for 48 hours. 70-BOml 2.2.2-
) The refluoroethanol was removed by distillation and the remaining solution was cooled to room temperature. A heavy yellow precipitate formed. The precipitate was triturated with ethyl acetate, collected by washing, and dried to yield the desired intermediate 295y (77% yield). Melting point 136138.5℃ Calculated value (Ct, 2 Ht
3CL N 203): C, 53, 64; H
, 4, 88; N, 10.45. Analysis value: C, 53, 8
7; H, 4,85; Np 10.81°B, 4-(3-
(N-phthalimido)-2-hydroxypropylamino)-N-methyl 7-thalimide Intermediate obtained in A above (13.5 g, 0.005 mol) and 7-talimide potassium salt of 1572 (0.085 mol) 2 with stirring in dimethylpolamide at 150° C.
It was refluxed for 4 hours. The dimethylformamide was then removed, and the residual fragrance was washed with water and filtered. When the yellow mass remaining on the filter paper is recrystallized from a mixed solvent of acetic acid and water, it becomes 12.85'
(67% yield) of product was obtained. Melting point 247-2
48.5℃. Calculated value: (as C2DH17N305): C, 63, 32; H, 4° 52; Nv
11.08. Analysis value: C, 63, 16; I-(, 43
8; Nt 10.93°C, 6-(3-amino-2-hydroxypropylamide/
]-2,3-dihydrophthalazine-1,4-dione Intermediate of B above (5,0y, 13.2 mmol), 9
0 ml of absolute ethanol and 35 mJ of 95% hydrazine were refluxed with stirring for 4 hours. The solvent was removed in vacuo and the resulting solid was dried in vacuo at 120° C. for 24 hours. 70m of this material? The mixture was stirred for 1 hour with o1N hydrochloric acid. Insoluble 2,3-dihydroxyphthalazine
The 1,4-dione was removed by 1yt filtration) and the filtrate was adjusted to plJ 6.5 with saturated sodium bicarbonate. The white precipitate formed was collected by bowel movement and dried to yield product 222.

Yield 67%. The compound recrystallized from water decomposed at 273°C. Calculated value (as C11H14N203): C, 52
,79;H,5,64;N,22.39°Analysis value:C,
52,73; Ht 5.72; N, 2254°D+ Biotin-isoluminol conjugate biotin (“0
．． 291ii', 1.2 mmol) and 0.17 ml of triethylamine were dissolved in 20 ml of dry dimethylbormamide under anhydrous conditions and cooled to -10<0>C. 2.8
A solution of 0.141 mJ of ethyl chloroformate in 6 rugs of ether was slowly added and the mixture was stirred for 30 minutes. The formed precipitate was separated by filtration. 600 mp (2,4 mmol) of the intermediate obtained in C above,
20ml dry dimethylformamide and 11ml dry pyridine
The punishment was quickly added to the filtrate. This mixture was heated to -10℃ for 30 minutes.
Stirred for a minute and then at room temperature overnight. During this time a solution was obtained. 0.10 in. of dimethylformamide
It was removed by distillation at 60° C. under pressure of Hf. The oily residue was stirred for 1 hour with 50++ l of 0, IN hydrochloric acid. The white precipitate formed was filtered and washed with 01N hydrochloric acid and then with water. 0.55 f when dried overnight in vacuum at room temperature
(97% yield) of product was obtained. Melting point 1.70-3
°C o subtraction value (as C21H2gC21H2): C95
2,92;) Ding, 5.92;N, 17.64° Analysis value 20.

51.69; I■+ 5.90; Np 17.63
Example 2 Specific binding - chemiluminescence analysis: Effects of avidin and biotin on the activity of biotin-isoluminol-1 complex and the chemiluminescence reaction system used in this example is based on the following reaction.

Each with a total amount of 140 μt, each with a pl-I of 7.4 0.
11VI tris-(bitroxymethyl)-aminomethane hydrochloride buffer and biotin at the concentrations shown in Table 1, biotin-inluminol conjugate (obtained in Example 1)
Nine specific binding reaction mixtures containing avidin and avidin (added last) were prepared.

After holding for 5 minutes at 25°C, each reaction mixture was charged with 20 units/yrte of lactoperoxidase (obtained from Sigma Chemical Company, St. Louis, MO, USA, "Enzymology Methods JXVIIA").

(1970) page 653 - Analysis 2) in 0.1 M monoamino/methane hydrochloride buffer at pH 7.4 was injected into each reaction mixture; The peak light intensity obtained for each was measured using a DuPont 760 bioluminescence meter (Plaue State, USA).
Measurements were made using E.I. DuPont de Nemours, Wilmington.

The results are shown in Table 1.

Reactions 1.2.5 and 7 are control experiments and show that only a slightly lower amount of background light is measured in the absence of the biotin-isoluminol conjugate. The results of reactions 3 and 6 show that the biotin-isoluminol conjugate is active in the chemiluminescent reaction and that the presence of free biotin does not significantly affect its activity. The results of Reaction 4 show that the activity of the biotin-isoluminol conjugate increases when avidin, which is a substance that binds to biotin, is present. This result is surprising. This is because it is believed that the binding of avidin to the conjugate should limit the efficacy of the isoluminol moiety to the chemiluminescent reaction. This observed increase in photoproduction is difficult to understand. reaction 4
Comparing the results of reaction 9 and 8 with reaction 9, it can be seen that the increase in photogeneration decreases inversely with the amount of free N# biotin present.

In this example, the target substances, avidin and biotin, can be measured by using a specific binding-chemiluminescence analysis technique, and according to the present invention, the labeling substance in the conjugate and the corresponding binding partner It has been shown that the effect of binding with the substance is not inhibition of the activity of the labeled substance, but rather an increase thereof.

Example 3 Competitive binding of biotin - chemiluminescence analysis; Effect of varying the amount of biotin on the peak light intensity obtained The chemiluminescence reaction system used in this example was the same as that described in Example 2. .

Each total RL is 140 μt, each pi (0.1 of 7,4
M tris-(hydroxymethyl)-aminomethane hydrochloride buffer, 84 nM biotin-luminol conjugate (obtained in Example 1), biotin at the concentrations shown in Table 2 and 0.035 units/m , l of avidin (added last).
, JIIA Shita.

After a 5 minute hold at 25°C, 10 μt of lactoperoxidase (20 units, 7 rytl) was added to each reaction mixture. After a further 2 min hold, 10 μt of 0.95 mM
10mM of hydrogen dioxide dissolved at pH 7.4)
Su(hydroxymethyl)-aminomethane hydrochloride buffer was injected into each reaction mixture, and the peak light intensity obtained for each mixture was determined in the same 4J period as in Example 2. The results are shown in Table 2 and in Figure 1 of the attached Figure mj in the form of a glass.

Table 2 1o23.5 2 67 21.1 3 134 15.5 4 200 12.6 5 268 12.3 6 400 8.1 As described above, the light beam obtained by the chemiluminescent reaction system.

- We know that the magnitude of light intensity is an inverse function of the amount of biotin present in a particular binding reaction mixture. Accordingly, in accordance with the present invention, there is provided a test sample and method using a competitive binding-chemiluminescence spectrometry technique to determine the presence of the chemical substance biotin in a liquid.

Example 4 Competitive binding of biotin - chemiluminescence analysis The chemiluminescence reaction system used in this example is based on the following reaction.

Biotin-isoluminol + KO2- → biotin-aminophthalate + N2 + hν each with an extra 150 μt, each with pI-18, 01M of O
Tris-(hydroxymethyl)-aminomethane hydrochloride buffer, 42 nM biotin-luminol&V conjugate (obtained according to Example 1), biotin at the concentrations shown in Table 3 and avidin at 012 units/me (finally Add)
A specific binding reaction mixture of 16 keyaki species containing the following was prepared.

After holding at 25°C for 5 minutes, 015
Potassium peroxide (KO2, obtained from Alpha Products, Beverly, MA, USA) and 010
M 1.4.7.10.13.16-hexaoxyacylcoocdecane (obtained from Aldrich Chemical Company, Milwaukee, WI, USA)
10 μt of dimethylformamide containing The peak light intensity obtained in each case was determined in the same manner as in Example 2. The results are shown in Table 3 and in graph form in Figure 2 of the accompanying drawings.

Table 3 0 38.5 2 13 38.5 3 27 34.3 4 40 36.1 5 53 35.2 6 '67 36.2 7 101 34.0 8 133 31.7 9 166 29.1 10 200
24.211 267
22.812
333 20.513
400 13.
414 534
8.615 667
8.316
8007,0 and above shows that the magnitude of the peak light intensity obtained by a chemiluminescent reaction system is an inverse function of the amount of biotin present in the reaction mixture. According to the invention, therefore, there are compositions and methods for testing the presence of biotin, a substance of interest, in a liquid, using a competitive binding-chemiluminescent spectrometry technique that does not utilize a fermentation-catalyzed monitoring detection reaction. is provided.

[Brief explanation of the drawing]

Figures 1 and 2 each graphically illustrate the effect of varying the amount of target substance on the peak light intensity obtained in two different competitive binding-chemiluminescence analysis techniques.

Claims (1)

[Claims] 1. For analyzing a hapten or antigen in a liquid: (a) A liquid containing (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 reactant in a chemiluminescent reaction. 2. The analytical method according to claim 1, wherein the characteristic is measured by measuring the total amount of emitted light or the peak intensity of emitted light. 3. The analytical method according to claim 1, wherein the labeling substance is luminol, isoluminol, or a derivative thereof. 4. For analyzing hebutene 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 hapten or antigen A reagent consisting of an antibody against a conjugate of the reagent and a labeled hapten or antigen.
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 reactant in a chemiluminescent reaction. 5. The reagent according to claim 4, which is incorporated in a carrier matrix. 6. The reagent according to claim 4, wherein the labeling substance is luminol or isoluminol or a derivative thereof.