JPH0137694B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention provides a method and reagent for measuring the presence of a hapten, antigen, biotin, or avidin (target substance) in a liquid based on the affinity of the target substance for a specific (or specific) binding partner substance. It is related to. 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 synonymously. 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 means that
This is particularly the case 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 an unknown substance is detected by the appearance of a reaction product or the disappearance of a known reactant. In some cases, such analytical methods 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 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 has led to the creation of radioimmunoassay methods. According to this method, the amount of the radiolabeled substance to be detected is determined by the limited amount of antibodies that are unique to the unknown.
It is made to compete with the unknown. The amount of label that becomes bound to the antibody then varies inversely with the level of unknown material present. In radioimmunoassay technology,
Essentially, it is necessary to separate a labeled substance to be detected that becomes bound to an antibody 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. 3720760 and No. 3793445. However, all of these methods require at least one manual manipulation step such as filtration, centrifugation or washing to ensure efficient separation of bound-tagged from unbound-tagged bodies. need. If we could eliminate the need for separation operations, the analysis method would become much simpler.
It will be even more useful in clinical testing laboratories. The use of radioactive substances in immunoassays is
It has become possible to avoid this to some extent by using substances with enzymes instead of radioactive substances. As exemplified in U.S. Pat. The same requires a cumbersome separation step. Other disadvantages of using enzyme-conjugated materials include that each enzyme attached must be individually chemically modified in order to be used to form the attached conjugate. be. It has been proposed to use other attached substances, such as coenzymes or viruses (Nature, 219 , 186 (1968)), and it has also been proposed to use fluorescent labels (French Patent No. 2217350). 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. This method is therefore said to be homogeneous, as opposed to heterogeneous, where separation is required.
U.S. Pat. No. 3,817,837 describes a step in which a soluble complex consisting of an enzyme as a labeling substance that covalently binds to a substance (ligand) to be detected and a soluble receptor (usually an antibody) for the substance of interest is combined with a liquid to be analyzed. and a competitive binding analysis method comprising the steps of measuring the effect of a target substance on the enzyme activity in the 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 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 in order to block the enzymatically active sites due to the reaction between the complexed ligand and receptor. The size of the enzyme is
The molecular weight varies widely from about 10,000 to 1,000,000. Thus, the binding position of a receptor in the form of an antibody with a molecular weight between 150,000 and 300,000 is tightly controlled so that the active site of an average enzyme with a molecular weight of 500,000 or more can be physically blocked. There must be. 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 found in this homogeneous system. It may be considered that it can be used in analytical methods. Furthermore, in order to obtain quantitative test results, it is essential to tightly control the number of enzymes and the number of ligands for each enzyme-binding-ligand complex. Again, the complex peptide structure of the enzyme makes such control difficult. It is therefore likely that only a small number of enzymes have the appropriate molecular structure to ensure the necessary control of the ratio of ligand to enzyme. Prior art homogeneous analytical methods involve enzymatic amplification and are therefore said to be very sensitive. However, the applicability of this method is very limited since the labeling substance enzyme itself is a limiting factor determining the sensitivity of the prior art analytical methods. This sensitivity is clearly due to enzyme-
Limited by the catalytic activity of the particular enzyme in the 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. It is also limited by gender. 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-bound-ligand conjugate are present in significant amounts in the liquid sample being tested, thereby creating a background of uncontrollable activity that has a considerable impact on the accuracy of the analytical method. ). Therefore, in order to formulate analytical methods that can be used when testing human or animal biological fluids,
The need to select enzymes that are exogenous to such fluids and use them to form enzyme-binding-ligand conjugates further limits the applicability of this analytical method. will receive. Therefore, the object of the present invention is to provide a novel method for detecting haptens or antigens (target substances) in liquids that does not require a separation step and does not use inconvenient radioactive substances or partially modified enzymes as labeling substances. The purpose is to provide reagents. 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 a feature 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. This is one of the purposes. By using a wide variety of sensitive reaction systems, homogeneous characterization using conjugates containing labeled substances can more conveniently detect any change in enzyme activity than any change in enzyme activity in prior art methods. It is also an object of the present invention to provide a 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 hapten, antigen, biotin, or avidin in a liquid: (a) A liquid, (1) A labeling substance having predetermined characteristics and a hapten, antigen, or biotin. or conjugate with avidin, and (2) contact with a reagent consisting of an antibody to a hapten or antigen, an avidin to biotin, or a biotin to avidin, producing an antibody-bound phase and an antibody-free phase of the labeled conjugate. and (b) measuring the indicative properties of hapten, antigen, biotin or avidin in a liquid without separating bound and free phases, the method comprising: The labeling substance is an opposite substance of chemiluminescence reaction. Further, the homogeneous immunoassay reagent of the present invention is for analyzing hapten, antigen, biotin, or avidin in a liquid by a homogeneous immunoassay method: (1) A labeling substance having predetermined characteristics, a hapten, A conjugate of an antigen, biotin, or avidin; and (2) a reagent consisting of a hapten or an antibody to the antigen, avidin to biotin, or biotin to avidin, and the conjugate is labeled with the reagent and the hapten, antigen, biotin, or avidin. Antibodies - bound phase and antibodies -
forming a binding reaction system that produces a free phase, and the properties of either the bound phase or the free phase are a function of the amount of hapten, antigen, biotin, or avidin in the liquid, and the labeling substance in the conjugate is , is characterized by being a reactant in a chemiluminescent reaction. The present invention provides a very convenient, widely applicable, and sensitive homogeneous specific binding analysis method and system, in which a substance exhibiting reaction activity imparted as a component of a predetermined reaction is used as a labeling substance. It is provided on a usage-based basis. Such materials are referred to herein as "reactants."
This method consists, in part, in that a reaction between a hapten, antigen, biotin or avidin (target substance) and its specific binding partners (to one of which the reactant is bound) has previously been carried out. It is based on the fact that it changes the activity of the reactants in a given 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 operating schemes are direct binding techniques and competitive binding techniques. In the direct binding technique, the liquid presumed to contain the hapten, antigen, biotin or avidin to be detected is a conjugate containing a reactant bound to a specific binding partner of the target substance. and then analyzed for any changes in the activity of the reactants. 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 reactants is
Measurements are made by contacting the liquid with at least one reagent that together with the reactants forms a predetermined monitoring detection reaction. Qualitative determination of a target substance in a liquid is carried out by comparing the obtained reaction with a monitored detection reaction in a liquid that does not contain the target substance in terms of its characteristics (usually reaction rate). The difference between the two indicates a change in the activity of the reactant. 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 a chemiluminescent reactant. Typically, the monitoring detection reaction is
Selected 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. 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 presumed to contain a ligand or an antigen (substance of interest), a conjugate, and/or a partner of the specific binding of the analyte, has a significant value for the purpose of the analysis. 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 amount or 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 technology, the components of the specific binding reaction are a liquid presumed to contain the target substance;
A quantity of a conjugate containing a reactant that is bound to a specific binding partner of the target substance. 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. while,
The amount is greater than that which can bind all of the target substances that are thought to be present in the liquid. 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 the specific binding reaction are:
An amount of a conjugate comprising a reactant bound to the target substance (ligand) or a similar substance for a specific binding of the target substance, and an amount of a specific binding partner 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 will compete with the ligand in the conjugate or its specific binding analog to bind to the partner substance in the conjugate, so that the reactants of the conjugate when in contact with the liquid The activity of varies in direct proportion to the amount of binding between the target substance and the specific binding partner in the liquid. 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, the amount of a particular binding partner is chosen according to the criteria described above, based on the maximum expected amount of the target substance 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 of the target substance (ligand) or its analogous substance that is greater than the amount of the target substance (ligand) that can bind to the specific binding partner substance 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 conjugate that becomes bound to a specific binding partner substance in the first method;
This will be the amount of conjugate in the form of a complex in the second method, but this amount is usually determined by When the substance (or complex) and liquid are in contact, the amount is greater than that which can replace all of the partner substance in the liquid. 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 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 Additionally, the amount of target substance or analogue thereof in conjugate form is typically determined by the amount of unbound specific bonds remaining during contact of the conjugate with a liquid before evaluation of any changes in 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 anticipated that other addition orders and operating steps, including 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 Rukoto. Assessing any change in the activity of the conjugate reactant of the components of the predetermined monitored detection reaction comprises forming a reaction mixture of the specific binding reaction with at least one of the reactants of the conjugate forming the monitored detection reaction. This is conveniently accomplished by contacting a specific binding reaction with a substance 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. Suitable reaction components that together with the reactants in the conjugate form a monitoring detection reaction can be
Alternatively, simultaneously or subsequently, they are brought into contact with the reaction mixture of the specific binding reaction, either alone or in combined form. 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 incubated for a predetermined period of time before assessing any changes in the activity of the reactants in the conjugate. be done. 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. In situations where the substance of interest is not present in the liquid or is present in insignificant amounts, the predetermined monitoring detection response exhibits a relatively constant nature. When the substance of interest is present in the liquid, at least one property or property of the monitored detection reaction changes. In general, the activity of the reactants of the conjugate is
Defined as the amount or rate at which the reactant can participate in a monitored detection reaction. That is, the nature of the monitored detection reaction will vary depending on the presence of the substance of interest in the liquid, usually with respect to the overall reaction rate or the equilibrium amount of one or more 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. Decreased by 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 effect on the monitored 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 rate of appearance of reaction products. It is determined by measuring the Such measurements may be made by a wide variety of methods, including conventional chromatographic, gravimetric, potentiometric, spectrophotometric, fluorometric, turbidometric, volumetric, and other analytical techniques. Since this method is primarily intended for the detection of low concentrations of target substances, a highly sensitive reaction system has been developed for use in combination with the novel specific binding reaction system of the present invention. One preferred form of monitoring detection reaction is
Preferably, there is a luminescent reaction system catalyzed by an enzyme, 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 specific binding reaction is
Causes a change in the total amount, peak intensity, or nature of the light emitted. Examples of luminescent reaction systems include those listed in Table A, and the following abbreviations are used in this table. ATP: Adenosine triphosphate AMP: Adenosine monophosphate NAD: Nicotinamide adenine dinucleotide NADH: Reduced form of nicotinamide adenine dinucleotide
dinucleotide FMN: flavin mononucleotide FMNH ₂ : reduced flavin mononucleotide hν: electromagnetic radiation, usually in the infrared, visible or ultraviolet region

【table】

[Table] Further details and discussion regarding luminescent reaction systems that can be utilized in the method of the present invention can be found in the following references: Journal of Biological Chemistry, 236:48 (1961) Journal of the American Chemical Society, 89:3944 (1967) Cornier et al., Advances in Bioluminescence, Johnsson et al., eds., Princeton University Press. (New Jersey, 1966) pp. 363-84 Kries, B. Purification and Properties of Renilla Luciferase, Doctoral Dissertation, University of Georgia (1967) American Journal of Physiology, 41:454 (1916 Although not necessary in preferred embodiments of the present invention, the presence of the substance of interest in the liquid It may also be desirable to use heterogeneous analytical techniques in cases where the activity of the reactants of the conjugate is affected. Such situations arise where heterogeneous methods are particularly advantageous. Certain heterogeneous methods can increase the effective concentration of the analyte in the analytical system, thereby increasing sensitivity.
Examples of such heterogeneous methods include the specific binding partner of the conjugate or target substance of the invention, which is then transferred to a selected specific type of manipulation.
Some use column devices containing an insoluble matrix made of any of the following. All other heterogeneous analytical methods using radioactively labeled or enzymatically labeled 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 usually include peptides, proteins, carbohydrates, glycoproteins, steroids,
or other organic molecules for which the partner for specific binding exists within the biological system or from which the partner can be synthesized. In functional terms, this target substance is selected from the group consisting of antigens, their antibodies, haptens, their antibodies and vitamins, and their receptors and binding substances. Particular examples of target substances that can be detected using the present invention include:
Antigens and haptens such as ferritin, bradekinin, prostaglandin and tumor-specific antigens; pythione, vitamin B ₁₂ , folic acid, vitamin E
and vitamins such as ascorbic acid; antibodies such as microsomal antibodies, hepatitis antibodies, allergen antibodies; and specific binding receptors such as thyroxine-binding globulin, avidin, intrinsic factor and transcobalamin. 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, it is preferable to use a direct binding technique. Therefore, when the target substance to be detected is an antibody or a specific binding receptor, a conjugate containing a reactant bound to the antigen or a hapten bound to a low molecular weight counterpart of the specific binding of the antibody or receptor. Preferably, by direct bonding techniques in which If the molecular weight of the selected binding partner substance is 10 times or more larger than the molecular weight of the target substance to be detected, i.e. when the target substance is an antigen, hapten or vitamin, it is bound to a smaller target substance (ligand). It is particularly advantageous to utilize competitive binding techniques or sequential saturation techniques in which conjugates containing reactants 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. (either a similar substance for a specific bond or a partner substance for a specific bond of the target substance). The bond between the reactant and the specific binding substance is determined under analytical conditions in which the monitoring detection reaction using the active reactant is not designed to chemically break the bond, such as in the luminescence reaction system described above. , usually cannot be substantially changed. However, in some cases such bonds are designed to be broken or influenced by selected monitoring detection reactions as a means to assess changes in reaction activity. 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 the specific binding substance are connected by a bridging group containing from 1 to 50, preferably from 1 to 10 carbon atoms or heteroatoms such as nitrogen, oxygen, sulfur, phosphorous, etc. There is. Examples of bridging groups containing one atom include methylene groups (one carbon atom) and amino groups (one heteroatom). The bridging group usually has a molecular weight not exceeding 1000, preferably less than 200. Bridging groups contain chains of carbon atoms or heteroatoms, or combinations thereof, and usually include linking groups in the form of groups such as esters, amides, ethers, thioesters, thioethers, acetals, methylene or amino groups. the reactant and the specific binding substance or active derivative thereof. A reactant in a conjugate of the invention is a substance that has an endowed (eg, constant or known) reactive activity as a component of a predetermined monitoring detection reaction. More specifically, as disclosed herein, "reactant" and "reactive substance" are defined as defined and measurable substances that result in different - or several - products. chemical transformations (alterations), and the reactants and chemicals (e.g., other reactants, catalysts, other types of substances that participate in such chemical transformations or transformations). ) refers to a substance that produces electromagnetic radiation, thermal energy, or sonic energy upon interaction with a means for initiating a reaction, such as Accordingly, the group of substances defined 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 substance can function in different ways depending on its chemical environment, a single chemical substance may be classified into various different classes; It will be appreciated that the function of the disclosure is determined by the reactivity of the material with respect to the selected monitoring detection reactions described herein. 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. One or more components of the specific binding reaction and/or one or more components of the monitoring detection reaction may be included in the carrier. One way of thinking is that the carrier contains one or more of these components in its inner part, e.g. in liquid or loose solid form.
Alternatively, it may be a liquid holding container such as a test tube or capsule contained within a coating on its internal surface. Alternatively, the carrier may be in the form of a matrix that is insoluble and porous, preferably absorbent, with respect to the liquid to be tested. Such matrices are, for example, absorbent papers; polymeric films, membranes, fluffs or masses; gels and other forms. In such a form, the device:
It 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 a hapten or antigen (target substance).
Usually, it is a liquid substance of an organism or a liquid obtained by diluting or otherwise processing it. Liquid substances from living organisms that can be analyzed by the method of the present invention include serum, plasma, urine, amniotic fluid, brain fluid, cough fluid, and the like. Other materials, such as solid materials such as cells or gaseous ones, can also be analyzed in liquid form by methods such as dissolving the solid or gas or extracting the solid. In contrast to prior art homogeneous analytical systems, the target substance in a biological fluid containing a substance with a reactive activity the same or similar to that of the labeled substance in the conjugate is isolated from the background. Analyzes can be performed without interference. Endogenous background reactant activity can be easily removed in a variety of ways. Biological fluids can be treated to selectively destroy endogenous reactant activity. Such a treatment includes, for example, a treatment in which a detergent that chemically destroys endogenous activity is applied, followed by a treatment to inactivate the destructive action of the detergent. The invention will be illustrated below by means of examples,
These do not limit the invention. Example 1 Production of biotin-isoluminol conjugate 6-(3-biotinoylamide-2-hydroxypropylamine)-2,3-dihydrofuratadine-1,4-dione A 4-(3-chloro-2 -Hydroxypropylamino)-N-methylphthalimide 25 g (0.142 mol) of 4-amino-N-methylphthalamide (obtained by the method described in Journal of the Chemical Society, 26: (1937)) and 20.7g (0.21mol)
1-chloro-2,3-epoxypropane
150 ml of 2,2,2-trifluoroethanol was added and the reaction mixture was refluxed with stirring for 48 hours. 70-80 ml of 2,2,2-trichloroethanol was removed by distillation and the remaining bath liquid was cooled to room temperature, forming a heavy yellow precipitate. The precipitate was triturated with ethyl acetate, collected by filtration, and dried to yield 29.5 g (77% yield) of the desired intermediate. Melting point 136-138.5℃ Calculated value (as C ₁₂ H ₁₃ ClN ₂ O ₃ ): C, 53.64; H,
4.88; N, 10.45. Analysis value: C, 53.87; H,
4.85; N, 10.81. B 4-[3-(N-phthalimido)-2-hydroxypropylamino]-N-methylphthalimide Intermediate obtained in A above (13.5 g, 0.05 mol)
and 15.7 g (0.085 mol) of phthalimide potassium salt were refluxed in 150 ml of dimethylformamide with stirring for 24 hours. Dimethylformamide was then removed and the residue was washed with water and filtered. The yellow mass remaining on the filter paper was recrystallized from a mixed solvent of acetic acid and water to obtain 12.8 g (67% yield) of the product. Melting point 247-248.5℃. Calculated value:
(as C ₂₀ H ₁₇ N ₃ O ₅ ): C, 63.32; H, 4.52;
N, 11.08. Analysis value: C, 63.16; H, 4.38; N,
10.93. C 6-[3-amino-2-hydroxypropylamino]-2,3-dihydrophthalazine-1,
4-dione Intermediate of B above (5.0 g, 13.2 mmol),
90 ml of absolute ethanol and 35 ml of 95% hydrazine were refluxed with stirring for 4 hours. Remove the solvent in vacuo and store the resulting solid at 120 °C in vacuo.
Let dry for 24 hours. This material was stirred with 70ml of 0.1N hydrochloric acid for 1 hour. Insoluble 2,3-dihydroxyphthalazine-1,4-dione was removed by filtration and the filtrate was adjusted to pH 6.5 with saturated sodium bicarbonate. The white precipitate formed was collected by filtration and dried to yield 2.2 g of product. Yield 67%. The compound recrystallized in water is
Decomposed at 273℃. Calculated value (as C ₁₁ H ₁₄ N ₂ O ₃ ): C, 52.79; H, 5.64; N, 22.39. Analyzed value: C, 52.73; H, 5.72; N, 22.54. D Biotin-isoluminol conjugate Biotin ( 0.29 g, 1.2 mmol) and 0.17 ml of triethylamine were dissolved in 20 ml of dry dimethylformamide under anhydrous conditions and cooled to -10<0>C. A solution of 0.141 ml of ethyl chloroformate in 2.86 ml of ether was slowly added and the mixture was stirred for 30 minutes. The formed precipitate was separated by filtration. 600 mg of the intermediate obtained in C above
(2.4 mmol), dry dimethylformamide 20
ml and 1 ml of dry pyridine were quickly added to the filtrate. The mixture was stirred at −10° C. for 30 minutes and then at room temperature overnight. During this time a solution was obtained. Dimethylformamide was removed by distillation at 60° C. under a pressure of 0.10 mmHg. 50ml of oily residue
The mixture was stirred for 1 hour with 0.1N hydrochloric acid. The white precipitate formed was filtered and washed with 0.1N hydrochloric acid and then with water. Drying in vacuo at room temperature overnight gave 0.55 g (97% yield) of product.
Melting point 170-3℃. Calculated value (as C ₂₁ H ₂₈ N ₆ O ₅ S): C, 52.92; H, 5.92; N, 17.64. Analytical value: C, 51.69; H, 5.90; N, 17.63. Example 2 Specific bond - chemiluminescence Analysis: Effect of avidin and biotin on the activity of biotin-isoluminol conjugate The chemiluminescent reaction system used in this example is based on the following reaction. Biotin-isoluminol + H ₂ O ₂ lactoperoxidase ----------→ Biotin-aminophthalate + N ₂ + hν Each with a total amount of 140μ, each with 0.1M Tris-(hydroxymethyl )-aminomethane hydrochloride buffer and 9 containing biotin, biotin-isoluminol conjugate (obtained in Example 1) and avidin (added last) at the concentrations shown in Table 1.
A variety of specific binding reaction mixtures were prepared. After holding for 5 minutes at 25°C, each reaction mixture was given 20
units/ml of lactoperoxidase (obtained from Sigma Chemical Company, St. Louis, Missouri, USA, "Methods of Enzymology" XVIIA,
(1970) p. 653 - 0.1 M tris-(hydroxymethyl)-aminomethane hydrochloride buffer at pH 7.4 (analyzed by the method described in Analysis 2).
Added 10μ. After holding at 25°C for an additional 2 hours,
A solution of 10μ of 0.95mM hydrogen peroxide in 10mM tris-(hydroxymethyl)-aminomethane hydrochloride buffer, pH 7.4 was injected into each reaction mixture, and the peak light intensity obtained for each was measured by DuPont. Model 760 Bioluminescence Spectrophotometer (E.I. Dupont, Wilmington, DE, USA)
de Nemours). The results are shown in Table 1.

【table】

[Table] Reactions 1, 2, 5 and 7 are control experiments; in the absence of biotin-isoluminol conjugate,
This shows that only a slightly lower background amount of light is measured. 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. Comparing the results of reactions 4 and 8 with reaction 9 shows that the increase in photogeneration decreases inversely with the amount of free 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 resulting peak light intensity The chemiluminescence reaction system used in this example was the same as that described in Example 2. . 0.1 M tris-(hydroxymethyl)-aminomethane hydrochloride buffer, 84 nM biotin-luminol conjugate (obtained in Example 1), each with a total volume of 140 μ, pH 7.4, as shown in Table 2. Six specific binding reaction mixtures were prepared containing the indicated concentrations of biotin and 0.035 units/ml avidin (added last). After holding for 5 minutes at 25°C, add 10μ to each reaction mixture.
of lactoperoxidase (20 units/ml) was added. After holding for another 2 minutes, 10μ of 0.95mM
A 10 mM tris-(hydroxymethyl)-aminomethane hydrochloride buffer at pH 7.4 containing dissolved hydrogen peroxide was injected into each reaction mixture, and the peak light intensity obtained for each mixture was measured as in Example 2. .
The results are shown in Table 2 and in the form of a glass in Figure 1 of the accompanying drawings.

【table】

[Table] From the above, it can be seen that the magnitude of the peak light intensity obtained by the chemiluminescent reaction system is an inverse function of the amount of biotin present in the specific binding reaction mixture. Accordingly, the present invention provides test reagents and methods using competitive binding-chemiluminescence spectrometry techniques to determine the presence of the target 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 + KO ₂ → biotin-aminophthalate + N ₂ + hν Each in a total volume of 150μ, each in 0.1M tris-(hydroxymethyl)-aminomethane hydrochloride buffer at pH 8.0, 42nM biotin-luminol conjugate (conducted) Sixteen specific binding reaction mixtures were prepared containing biotin (obtained according to Example 1), biotin at the concentrations shown in Table 3, and 0.12 units/ml avidin (added last). After holding at 25°C for 5 minutes, in each reaction mixture:
0.15 M potassium peroxide (KO ₂ , obtained from Alpha Products, Beverly, WI, USA) and 0.10 M 1,4,7,10,13,16-hexaoxyacylcoocdecane (obtained from Alpha Products, Beverly, WI, USA). (obtained from Aldritch Chemical Company, Milwaukee) was injected with 10μ of dimethylformamide containing 100 μl of dimethylformamide. The peak light intensity obtained for each was measured 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] From the above, it can be seen that the magnitude of the peak light intensity obtained by the chemiluminescent reaction system is an inverse function of the amount of biotin present in the reaction mixture. According to the invention, therefore, there is provided a composition and method for testing the presence of the analyte biotin in a liquid using a competitive binding-chemiluminescent spectrometry technique that does not utilize an enzyme-catalyzed monitoring detection reaction. is provided. Example 5 Light Measurements The light intensities of the peaks resulting from the chemiluminescent reactions described herein were automatically recorded by a DuPont Model 760 Luminescence Biometer. Alternatively, a custom microprocessor connected to the biometer monitored the signal at 10-millisecond intervals and printed out the peak light intensity and total light produced in arbitrary units. The microprocessor was activated when the signal from the photomultiplier exceeded a threshold, ie, five times the electronic noise. Preparation of reagents Water for the preparation of reagents is filtered through activated carbon;
Distilled using a glass device. All glass containers except the 6 x 50 mm test tube for the chemiluminescence reaction were exposed to concentrated nitric acid overnight and thoroughly washed with distilled water. Reagents were measured using disposable glass pipettes or pneumatic pipettes with disposable plastic tips. Chemiluminescent compound: 1 mmol stock solution of isoluminol and its derivatives in 0.1 M Na ₂ CO ₃ ,
Although prepared in PH 10.5, these solutions are stable for several months at 4°C. Naphthyl hydrazides were dissolved in 0.1M _{Na2CO3-0.15M NaOH} , PH12.6. A few hours before use, further dilutions were prepared in water or an appropriate buffer. Heme catalyst: Microperoxidase (Sigma)
Chemical Co., St. Louis, Missouri;
USA) 1 milligram in 10mM Tris-HCl, PH
7.4 Dissolved in 2.5 ml. This 200 μM stock solution was stable for at least 1 month at 4°C.
A 2 μM solution was prepared in 75 mM sodium barbital buffer PH 8.6 within a few hours before use. 1mM of hematin in 0.1M _Na2CO3 , _PH10.5
Stock solutions were aged at 4° C. for at least one week to reduce variation in performance during testing. This solution was diluted to 1 μM just before use. The activity of this catalyst in chemiluminescent reactions gradually decreased, but the effect was compensated by increasing the amount of catalyst per reaction. This stock solution was thus usable for at least two months. Antibody to thyroxine: This antibody was raised in rabbits immunized with thyroxine-calf serum albumin complex. Antibodies were purified using known methods. Chemiluminescent reaction Reaction mixture containing chemiluminescent compound (150μ
) and a suitable catalyst in a 6 x 50 mm test tube (Kimble
Division of Owens-Illinois, No. 73500). Attach each test tube to a photometer,
10μ of the oxidizing agent was injected at once from a 25μ Hamilton syringe. Oxidation reaction system _H2O2 -microperoxidase: The reaction mixture ( _150μ ) contained 50mM NaOH, 0.27μM microperoxidase and chemiluminescent compound. In addition, these mixtures contained 5.9 mM sodium barbital, a buffer for diluting microperoxidase and chemiluminescent compounds. The final pH was approximately 12.6. The mixture was allowed to stand at room temperature for at least 10 minutes, and then the chemiluminescent reaction was terminated in 10 mM Tris.HCl, PH
Started by injecting 10μ of 90mM H ₂ O ₂ in 7.4. Alternatively, chemiluminescent reactions were performed in 75 mM sodium barbital, PH 8.6, containing 0.27 μM microperoxidase and various concentrations of chemiluminescent compounds. The mixture was left at room temperature for at least 10 minutes, then chemiluminescence was initiated by injection of 10 μ of 3mM H ₂ O ₂ in 10mM Tris.HCl, PH 17.4. _{H2O2 -} hematin: in addition to chemiluminescent compounds,
In this reaction mixture (150μ), 50mM
Contained NaOH and 0.07 μM hematin. Leave these at room temperature for at least 10 minutes, then
Light generation was initiated by injecting 10μ of mM H ₂ O ₂ . H ₂ O ₂ -Lactoperoxidase: This reaction system (150μ) uses 0.1M Tris・HCl (PH7.4 or 8.8)
Chemiluminescent compounds and lactoperoxidase in
It contained 10μg. After incubation at 25°C for 2 minutes, 10mM Tris-HCl, PH
10μ of 1mM H ₂ O ₂ in 7.4 was injected.

【table】

[Table] Synthesis of 6-N-(3-amino-2-hydroxypropyl)amino-2,3-dihydro-1,4-phthalazine-dione [See Table 4 ()] 4-Amino-N-methylphthalimide The synthesis was carried out by the method of Drew and Pearman [J.Chem.Soc., London
p.26 (1937)]. 211 g (1 mol) of 4-nitrophthalic acid was stirred with acetic anhydride under reflux. After complete dissolution, the mixture was kept at the same temperature for an additional 15 minutes. The solvent was removed under reduced pressure and the residue was recrystallized from toluene, giving a melting point (mp) of 119−
103 g of 4-nitrophthalic anhydride at 120°C (yield: 53
%)was gotten. 103 g (0.5 mol) of 4-nitrophthalic anhydride,
Stir with 200 ml of 20% methylamine for 30 minutes at 0°C. Adjust the solution to PH2.0 with concentrated hydrochloric acid,
A white precipitate was formed, which was collected, washed with water, and dried. This solid was then stirred with acetic anhydride at reflux temperature for 15 minutes after a complete solution was obtained. The product crystallized as the reaction mixture cooled. The crystals were collected and washed with acetic acid and water. When the crystals were dried, 61 g (yield 59%) of N-methyl-4-nitrophthalimide was obtained with a mp of 178°C. A solution of 281g (1.25mol) of SnCl ₂ 2H ₂ O was added to the water.
Prepared in 500 ml and concentrated hydrochloric acid 1.5. To this, N
-Methyl-4-nitrophthalimide 67g
(0.3 mol) was added with vigorous stirring. The reaction mixture became warm and a complete solution was obtained.
Next, this solution was allowed to stand overnight, and the pale yellow solid produced was collected. This product was washed with a large amount of water and then dried to obtain 36 g of 4-amino-N-methylphthalimide (yield: 67%) with a mp of 247°C. 25g of 4-amino-N-methylphthalimide
(0.142 mol) and 20.7 g (0.21 mol) of 1-chloro-2,3-epoxypropane were combined with 150 ml of 2,2,2-trifluoroethanol and stirred at reflux temperature for 48 hours. A portion of the solvent, 70-80 ml, was distilled off and the remaining solution was allowed to cool to room temperature. A yellow solid was formed, which was collected. Digesting it with ethyl acetate and taking it again yields 4-N-(3-chloro-2-hydroxypropyl) with a mp of 136-138°C.
Amino-N-methylphthalimide 29.5g (yield 77
%)was gotten. Potassium phthalimide 15.7g (0.085mol) and 4-N-(3-chloro-2-hydroxypropyl)
-Amino-N-methylphthalimide was stirred in 150 ml of dimethylformamide at reflux temperature for 24 hours. Dimethylformamide was removed under reduced pressure, and the residue was washed with water and collected. Recrystallization of the yellow solid from a glacial acetic acid-water mixed solvent yields N-methyl-4-N-[2-hydroxy-3-(N-phthalimido)propyl]-aminophthalimide with a mp of 247-248°C 12.8
g (yield 67%) was obtained. 35 ml of 95% hydrazine and N-methyl-4-N-
5 g (13.2 mol) of [2-hydroxy-3-(N-phthalimido)propyl]-aminophthalimide was combined with 90 ml of ethanol and stirred at reflux temperature for 4 hours. The solvent was removed under reduced pressure and the remaining solid was dried under vacuum at 120°C. 0.1N dry substance
After stirring with 70 ml of HCl for 1 hour, the insoluble by-products were removed. The solution was adjusted to pH 6.5 with a saturated solution of sodium bicarbonate. Collect the white precipitate and dry it to give 2.2g (yield 77%)
was gotten. Recrystallizing this from water gives 273
An analytically pure material was obtained that decomposed at °C. Synthesis of 6-N-[(3-bithionylamido)-2-hydroxypropyl]amino-2,3-dihydrophthalazine-1,4-dione [Table 4 ()] Biotin 0.29 g (1.2 mmol) and 0.17 ml of triethylamine was dissolved in 20 ml of dry dimethylformamide and cooled to -10°C under anhydrous conditions. A solution of 0.14 ml of ethyl chloroformate in 2.9 ml of diethyl ether was slowly added to the reaction mixture, which was then allowed to stand for 30 minutes. The triethylammonium chloride precipitate was removed. 600 mg (2.4 mmol) in 1 ml dry pyridine and 20 ml dry dimethylformamide
A suspension of was added to the solution. This mixture was heated to −10°C.
After stirring for 30 minutes, the mixture was left to stand overnight and the temperature was raised to room temperature. The solvent was removed under reduced pressure and the resulting oily residue was stirred with 50 ml of 0.1N HCl for 1 hour. The produced white solid was collected and washed with 0.1N HCl and water. The resulting product was dried under reduced pressure at room temperature overnight to give () 0.55 g (97% yield) of mp170-173°C.
was gotten. 6-[N-(3-amino-2-hydroxypropyl)-N-ethyl]amino-2,3-dihydro-1,4-phthalazine-dione [Table 4 ()]
Synthesis of diethyl sulfate 106g (0.7mol) and 4-amino-
61g (0.35mol) of N-methylphthalimide 2,
It was combined with 500 ml of 2,2-trifluoroethanol and heated at reflux temperature for 48 hours. The reaction mixture was then concentrated under reduced pressure and carefully neutralized with _a saturated solution of _Na2CO3 . The resulting precipitate was collected and dried to obtain 62 g of 4-(N-ethylamino)-N-methylphthalimide. Recrystallization from aqueous methanol gave 51 g (74% yield) of analytically pure pale yellow product with mp 149-151°C. A mixture containing 51 g (0.75 mol) of 4-(N-ethylamino)-N-methylphthalimide and 54 g (0.59 mol) of 1-chloro-2,3-epoxypropane was dissolved in 500 ml of 2,2,2-trifluoro-ethanol. Heated at reflux temperature for 48 hours. The resulting yellow solution was chromatographed using a 7×76 cm column of silica gel 60 (1000 g) (E. Merck, Darmschütt, West Germany) equilibrated with CCl ₄ /acetone (9:1). Elution was also performed using the same solvent (CCl ₄ /acetone), and the target product was eluted between eluents 4 and 6.9. This material was recrystallized several times from aqueous methanol, yielding 27 g of analytically pure 4-[N-(3-chloro-2-hydroxypropyl)-N-ethyl]amino-N-methylphthalimide at mp 122°C. 44%). Potassium phthalimide 23g (0.13mol) and 4
-[N-(3-chloro-2-hydroxypropyl)
-N-ethyl]amino-N-methylphthalimide
25g (0.08mol) of dry dimethylformamide
The mixture was stirred in 150 ml at reflux temperature for 36 hours. When the solvent was removed under reduced pressure, a brown residue remained, which was triturated with methanol to obtain 19 g of a yellow solid. When this solid is crystallized from an acetic acid aqueous solution,
4-{N-ethyl-N[2-hydroxy-3-(N
16 g (yield: 49%) of amino-N-methylphthalimide was obtained. Recrystallization from aqueous methanol gave an analytically pure compound with a mp of 158-160°C. 4-60ml of 95% hydrazine and dry alcohol
{N-ethyl-[N-2-hydroxy-3-(N-
phthalimide)propyl]} together with 15 g (0.037 mol) of amino-N-methylphthalimide,
Heated under reflux for 3 hours. The solvent was then removed under reduced pressure and the residue was dried at 110°C at 0.05 mmHg overnight.
This was stirred in dilute HCl and then filtered to remove insoluble by-products. When the liquid was adjusted to pH 7.0 using a saturated solution of sodium carbonate and the generated precipitate was collected, 4.6 g (46% yield) was obtained.
was gotten. When recrystallized from water, mp208-211℃
An analytically pure sample was obtained. 6-{N-ethyl-N-[2-hydroxy-3-
(Thyroxynylamide)propyl]}amino-
Synthesis of 2,3-dihydro-1,4-phthalazine-dione [Table 4 ()] 20 g (25.6 mmol) of L-thyroxine was stirred with 240 ml of anhydrous ethyl acetate at 0°C;
46ml of trifluoroacetic acid and trifluoroacetic anhydride
Added 7.6ml. The reaction mixture becomes clear after stirring for 1 hour, and is then poured with cold water (0°C) at 200 °C.
ml was added and the mixture was saturated with NaCl. Undissolved NaCl was removed and the organic and aqueous layers were decomposed. The organic layer was extracted twice with saturated NaCl (100 ml each) and then dried over MgSO ₄ . The solvent was removed under reduced pressure at 35°C, and the resulting white residue was washed twice with water and dried at 60°C at 0.05 mmHg to obtain 21 g of N-trifluoroacetylthyroxine (95% yield). Recrystallization from ether-petroleum ether gave an analytically pure material with a mp of 233-235°C (decomposition). 0.17 ml of triethylamine and 1.05 g (1.2 mmol) of N-trifluoroacetylthyroxine were dissolved in 20 ml of dimethylformamide at -10°C under anhydrous conditions. Add a solution consisting of 0.14 ml (1.2 mmol) of ethyl chloroformate and 2.9 ml of anhydrous diethyl ether,
The reaction was allowed to proceed for 30 minutes. The precipitate of triethylammonium chloride was removed, and the N-trifluoroacetylthyroxynyl ethyl carbonic anhydride (ethyl carbonic anhydride) in the solution was used in the next synthesis step without being isolated. Compound () (670 mg: 2.4 mmol) was suspended under anhydrous conditions in 20 ml of dimethylformamide and 1 ml of pyridine at -10<0>C and mixed with the above-mentioned anhydride. The reaction mixture was left at room temperature for 2 days. Then, the solvent was removed under reduced pressure and the residue was dissolved in 10% HCl.
Washed with. The solid material was removed and dried. The trifluoroacetyl protecting group was removed by treatment with alkali. 0.1M solid
Stir with 40 ml of Na ₂ CO ₃ , PH 10.5 and add a small amount of dimethylformamide to obtain a clear solution. After 24 hours, the solvent was distilled off under reduced pressure. The residue was dissolved in 30 ml of water and the resulting solution was adjusted to pH 7.2 using dilute HCl. When the white precipitate formed is taken and dried at 0.05mmHg and 60℃, mp＞240℃
(decomposition) () 600 mg (yield 50%) was obtained. 6-[N-(4-aminobutyl)]amino-2,
3-dihydro-1,4-phthalazine-1,4-
Synthesis of dione [Table 4 ()] 42 g of N-(4-bromobutyl)phthalimide
(0.15 mol) and 51.5 g (0.29 mol) of 4-amino-N-methylphthalimide were stirred with 300 ml of dimethylformamide at reflux temperature for 24 hours.
The yellow precipitate that forms when cooled to room temperature is collected and dried to give N-methyl-4-N-[4-(N-
Phthalimide) Butyl] Aminophthalimide 38.5
g (yield 68%) was obtained. Recrystallization from aqueous acetic acid gave an analytically pure product with a mp of 217-218°C. 15 ml of 95% hydrazine in 75 ml of dry ethanol were mixed with 4 g of N-methyl-4-N-[4-(N-phthalimido)butyl]aminophthalimide and heated at reflux temperature for 3 hours. The solvent was removed under reduced pressure and the residue was dried at 110°C at 0.1 mmHg. 10 of this stuff
% HCl and filtration to remove insoluble by-products. The pH of the solution was adjusted to 7 using sodium bicarbonate, and the resulting precipitate was collected to obtain 400 mg (yield: 15%). Samples recrystallized from water decomposed at 335°C. According to chemical ionization mass spectrometry, 249 P
A peak of +1 was shown and a peak of P+2 was shown at 250.
Calculated molecular weight is 248. 6-[N-(4-aminobutyl)-N-ethyl]
Synthesis of amino-2,3-dihydro-1,4-phthalazine-1,4-dione [Table 4 ()] 100 ml (0.77 mol) of diethyl sulfate and N-methyl-4-N-[4-(N- Phthalimido)butyl]-
Aminophthalimide was heated in an oil bath at 160°C under anhydrous conditions.
heated for 45 minutes. The mixture was cooled to room temperature and poured into ice water. Take the generated yellow precipitate,
Dry. When recrystallized from acetic acid aqueous solution, mp164~
Analytically pure 4-[N-ethyl-N-
4-(N-phthalimido)butyl]amino-N-
29 g (yield 72%) of methylphthalimide was obtained. 80ml of 95% hydrazine in 300ml of dry ethanol
N-[N-ethyl-N-(4-N-phthalimido)butyl]amino-N-methylphthalimide 29
g (0.07 mol) and heated at reflux temperature for 3 hours. Then, the solvent was removed under reduced pressure and the residue was reduced to 0.05 mm
Hg, dried at 110°C. The dry product was stirred with dilute HCl and filtered to remove insoluble by-products. When the liquid was adjusted to pH 9.0 using KOH, a precipitate was formed. When this solid is recrystallized from 50% dimethylformamide aqueous solution, mp255~257
°C analytically pure () 6.5 g (33% yield)
was gotten. 6-[N-ethyl-N-(4-thyroxynylamido)butylamino-2,3-dihydro-1,4
Synthesis of -phthalazine-dione [Table 4 ()] 0.8 g (5.0 mmol) of carbonyldiimidazole
was added to a solution of 4.4 g (5.0 mmol) of N-trifluoroacetylthyroxine in 50 ml of dry tetrahydrofuran and the mixture was heated at reflux temperature for 10 minutes. Solvent was removed under reduced pressure. A suspension of 1.4 g (5 mmol) of ( ) in dry dimethylformamide was added and stirred at room temperature for 48 hours. The solvent was removed under reduced pressure and the residue was stirred with 80 ml of 10% HCl. The insoluble material was removed and dried at 0.1 mmHg and room temperature. A portion of this, 2.1 g, was dissolved in 35 ml of 0.5 NaOH and reacted for 1 hour at room temperature to remove the trifluoroacetyl protecting group. The solution was adjusted to PH5.0 with concentrated hydrochloric acid, the generated precipitate was collected, washed with water, and then reduced to 0.1
mmHg, dried at room temperature. The resulting material was chromatographed on 200 g of silica gel 60 using ethanol/triethylammonium bicarbonate (7:3), PH 7.5 as solvent. The product eluted between 490 and 650 ml of eluate. The solvent was then removed under reduced pressure to yield 680 mg of a cream colored solid. This stuff, 50% dimethylformamide 50
ml and filtered, and water was added to the liquid to precipitate the product. 20000G white precipitate
Collected by centrifugation for 25 min at room temperature and dried at mp200 °C (digested) analytically pure ()
200mg was obtained. Synthesis of 7-[N-aminobutyl)-N-ethyl]aminonaphthalene-1,2-dicarboxylic acid hydrazide [Table 4 ()] The first step in the synthesis of () was performed by Gundermann's method [KDGundermann,
W. Horstmann and G. Bergmann, Justus
Liebigs Ann. Chem. 684 , 127 (1965)].
Tetrachloroethane 1, nitrobenzene 250ml
and 108 g (1 mol) of anisole and succinic anhydride
111 g (1.1 mol) was stirred at room temperature for 30 minutes and then cooled to 0°C. Next, 280 g (2.1 mol) of AlCl ₃
was added and the reaction mixture was stirred at 0°C for 3 days. The reaction was stopped by adding ice until no more HCl gas was evolved. 200 ml of 10% hydrochloric acid was added and the organic layer was removed by steam distillation. When cooled, a light brown solid precipitates from the aqueous layer, but
When this is crystallized from aqueous ethanol, mp149~
188 g (yield 91%) of 4-(4-methoxyphenyl)-4-oxobutyric acid at 150°C was obtained. Zinc amalgam was mixed with 374 g (5.7 gram atoms) of mossy zinc, 37.4 g (0.18 mol) of mercuric chloride, and water.
It was prepared by mixing 560 ml and 19 ml of concentrated hydrochloric acid. This mixture was stirred vigorously for 5 minutes. Remove the liquid by decantation and add 280 ml of water.
ml, concentrated hydrochloric acid 655 ml, toluene 375 ml, glacial acetic acid 20 ml and 4-(4-methoxyphenyl)-4-oxobutyric acid.
Added 188g. heating the reaction mixture at reflux temperature;
200 ml of concentrated hydrochloric acid was added every 6 hours for the next 24 hours.
After cooling, separate the aqueous layer and add ether three times (500ml each)
I washed it with These extracts were then combined with the organic layer obtained by the reaction. The combined organic extracts were mixed with 500 ml of 2N NaOH and the organic solvent was removed by steam distillation. Then dimethyl sulfate 88
g (0.7 mol) was added and the temperature was maintained at 80-90°C. When the reaction mixture becomes neutral, add another 2N
NaOH (approximately 300ml) was added. After 5 hours, the reaction mixture was cooled, acidified with concentrated hydrochloric acid, and diluted with ether for 3 hours.
Extracted twice (500 ml each). These extracts were combined, dried, and the solvent was removed. The residue was recrystallized from aqueous ethanol to give 90 g (52% yield) of 4-(4-methoxyphenyl)-butyric acid as white crystals with a mp of 59-61°C. 4-(4-methoxyphenyl)butyric acid 90g
(0.47 mol), boron trifluoride-methanol complex 124 g (0.94 mol) and methanol 400
ml of the mixture was refluxed for 4 hours. After cooling, the reaction mixture was extracted with ether three times (400 ml each) and the solvent was removed from the extract to give 96 g (98% yield) of methyl 4-(4-methoxyphenyl)butyrate as a yellow oil. . In a dry three-neck round-bottomed flask equipped with a mechanical stirrer, reflux condenser, and argon inlet, add 18.2 g (0.48 g atom) of potassium, 800 ml of dry ether (distilled from LiAlH ₄ ), and anhydrous ethanol.
Added 28.3ml (0.48ml). When the reaction mixture is refluxed for 5 hours, potassium reacts and a white suspension is obtained, to which 94 ml of dimethyl oxalate is added.
(0.69 mol) was added dropwise over 15 minutes to obtain a clear brown solution. Next, 95.6 g (0.46 mol) of methyl 4-(4-methoxyphenyl)butyrate was added,
While the reaction mixture was refluxed overnight, a pale brown solid formed was collected and washed with ether. When dried,
106 g of ethyl potassium valerate [2-hydroxy-3-methoxycarbonyl-5-(4-methoxyphenyl)] potassium enolate at 120-122°C (yield 67
%)was gotten. Add this product (0.31 mol) little by little to 5 to 15
Added in 326 ml of 60% H ₂ SO ₄ at °C. After addition,
Heat the reaction mixture on a steam bath for 15 min, then 20 min.
Cooled to ℃. An additional 652 ml of 80% H ₂ SO ₄ was added and heating was repeated for 15 minutes. During this time a yellow precipitate formed and the mixture was poured into cold water 1. If you take this product, mp163~165℃7
-methoxy-3,4-dihydronaphthalene-1,
24.9 g (yield 35%) of 2-dicarboxylic anhydride was obtained as yellow crystals. A mixture of 19.9 g (0.087 mol) of this 7-methoxy-3,4-dihydronaphthalene-1,2-dicarboxylic acid and 2.8 g (0.087 gram atom) of sulfur was heated at 250 DEG C. for 3 hours under an argon atmosphere. When cooled,
The brown solution solidifies, and when this is crystallized from benzene, 15 g of 7-methoxynaphthalene-1,2-dicarboxylic anhydride (yield 80
%) were obtained as yellow crystals. 15 g (0.07 mol) of 7-methoxynaphthalene-1,2-dicarboxylic anhydride, 240 ml of glacial acetic acid and 48%
The mixture of 240 ml of HBr was refluxed for 2 hours. During this time, a dense yellow precipitate formed. After cooling and removing the precipitate, 7-hydroxynaphthalene-1,2-dicarboxylic anhydride with a mp of 285°C was obtained.
g (yield 75%) was obtained. 240ml of 20% ammonium hydroxide aqueous solution at 0℃
A solution was prepared by dissolving 180 g of SO ₂ in . Add 20% ammonium hydroxide solution to this solution.
200ml and 7-hydroxynaphthalene-1,2-
25 g (0.12 mol) of dicarboxylic anhydride was added.
The reaction mixture was placed in a stainless steel autoclave and heated at 170°C for 8 hours. After cooling, the reaction mixture was concentrated under reduced pressure to remove _NH3 . Neutralization with acetic acid gave 7-aminonaphthalene-1,2-dicarboxylic acid as a red solid. This solid was heated on a steam bath for 2 hours in 70 ml of dry methanol and 40 ml of concentrated sulfuric acid. The reaction mixture was then cooled and poured into crushed ice containing excess Na ₂ CO ₃ . This neutralized mixture was treated with ether three times (each
250 ml) and the combined extracts were dried over anhydrous _MgSO4 . When the solvent was removed, 20 g of dimethyl 7-aminonaphthalene-1,2-dicarboxylate (yield
66%) was obtained as a red oil. 25.9 g (0.1 mol) of dimethyl 7-aminonaphthalene-1,2-dicarboxylate and 14.1 g (0.05 mol) of N-(4-bromobutyl)phthalimide were added to 2,
The mixture was refluxed in 100 ml of 2,2-trifluoroethanol under an argon atmosphere for 16 hours. The solvent was removed under reduced pressure and the residue was partitioned between 400ml ether and 300ml water. The ether layer was dried over anhydrous _MgSO4 ,
It was made clear by passing through it. Next, the solvent was distilled off under reduced pressure. Benzene the resulting dark red residue
Dissolve in 100ml benzene/methanol (9:1)
Chromatography was performed on 1200 g of silica gel 60 equilibrated with. When developed with this same solvent, the target product was eluted between eluates 5.1 and 5.9. The solvent was removed under reduced pressure to obtain 9 g (40% yield) of dimethyl 7-N-(4-phthalimidobutyl)aminonaphthalene-1,2-dicarboxylate as a red oil. 7-N-(4-phthalimidobutyl)aminonaphthalene-1,2- dissolved in 20 ml of diethyl sulfate.
9 g (0.02 mol) of dimethyl dicarboxylate was heated at 130° C. for 2 hours under an argon atmosphere. The reaction mixture was then poured into 200 ml of saturated NaHCO ₃ containing crushed ice. The resulting aqueous solution was extracted with 250 ml of ether,
The extraction operation was repeated twice. The combined ether extracts were dried and the solvent removed to give a red oil contaminated with diethyl sulfate. This oil was used as the elution solvent with benzene/methanol (19:
1) was used for chromatography through a 600 g column of silica gel 60. The yellow fractions were collected and concentrated under reduced pressure to give an oil containing some diethyl sulfate. This residual impurity is 0.01
When sublimated at mmHg and 50℃, 7-[(N-ethyl)
4.0 g (yield 42%) of dimethyl -4-(N-phthalimidobutyl)]aminonaphthalene-1,2-dicarboxylate was obtained as a red oil. 15 ml of 85% hydrazine, 7-[N-ethyl)-4-
(N-phthalimidobutyl)]aminonaphthalene-
Dimethyl 1,2-dicarboxylate 4g (8mmol)
and 50 ml of dry methanol were refluxed for 3 hours. next,
The solvent was removed under reduced pressure leaving a crystalline residue of 0.01 mm
Hg and dried overnight at 80°C. The obtained substance was mixed with ethanol/1M as a solvent.
Triethylammonium bicarbonate (7:3), PH7.5
Chromatography was performed on a 200 g column of silica gel 60 equilibrated with . The target is the eluate
It eluted between 0.5 and 1.5. Removal of the solvent gave a yellow solid. When recrystallized twice from pyridine, 7-[N-(4-aminobutyl) with a mp of 246-247°C
1.1 g (42% yield) of -N-ethyl]aminonaphthalene-1,2-dicarboxylic acid phthalazide () was obtained as yellow fine crystals. Synthesis of 7-[N-ethyl-N-(4-thyroxynylamido)butyl]aminonaphthalene-1,2-dicarboxylic acid hydrazide (Table 4 ()) 1.7 g of N-trifluoroacetylthyroxine in 20 ml of dry pyridine A solution of (2 mmol) was stirred at -10°C under an argon atmosphere, and 450 mg (2.2 mmol) of dicyclohexylcarbodiimide was added thereto. After 45 minutes, 980 mg of ( ) was added and reacted for 3 hours. Next, the reaction mixture The temperature was allowed to rise to room temperature overnight. To this was added 100 ml of pyridine and 10 g of silica gel 60, and the pyridine was removed under reduced pressure. The silica gel thus impregnated was mixed with ethanol/1M triethylammonium bicarbonate (7:
3) When placed on top of a 200 g column of silica gel 60 equilibrated at pH 7.5 and developed with this same solvent, the target product eluted between 0.78 and 1.0.
Removal of the solvent gives a yellow crystalline residue, which is dissolved in 1M triethylammonium bicarbonate, pH 7.5.
The solution was dissolved in 300 ml and refluxed for 3 hours to remove the trifluoroacetyl protecting group. The hot reaction mixture was filtered to remove the oil. Next, silica gel 60
and the slurry was dried on a rotary evaporator. Place the impregnated silica gel on top of a 200 g column of silica gel 60 and add ethanol/
1M triethylammonium bicarbonate (7:3), PH
Deployed in 7.5. The target product was eluted between 0.6 and 1.0. Removal of the solvent gave 0.9 g of a yellow solid. Sephadex LH (Sephadex LH) equilibrated with methanol was added to 110 mg of this solid.
When chromatography was performed on a 3.2 x 45 cm column, the target product was eluted between 450 ml and 350 ml of eluate, and 60 mg (22% yield) of mp218℃ (decomposition) was obtained as a yellow solid. . Heterogeneous Competitive Binding Test Method Heterogeneous test method is a test method that requires separation of protein bound and free forms of labeled ligand. For example, antibody-bound thyroxine complex () and free thyroxine complex () are
Can be separated using a small G25 column. The free complex is bound to Sephadex and the antibody conjugate is washed directly from the column and used for chemiluminescence measurements. Titration of () with antibodies: Binding reaction mixtures (250 μ) containing 19.3 nM () and various concentrations of rabbit antibody against thyroxine were prepared in 75 mM sodium barbital, PH 8.6 and titrated at room temperature for 1
Incubated for hours. each binding reaction mixture
Sephadex G25 equilibrated with 200μ aliquots in 75mM sodium barbital buffer, pH 8.6.
was applied to a separate 2 ml column. These security columns were fitted with Vyon disks (ESB Inc., Merztown, USA) at the top and bottom.
(Sherwood Medical, St. Louis, Mo., USA) with 8 x 65 mm polyethylene barrels (Sherwood Medical, St. Louis, Missouri, USA).
The columns were washed with 2 ml of the same buffer and the eluate was collected. A portion (95μ) of each eluate was tested in the _H2O2 - _hematin system. Typical results are shown in Fig.3. In this example, the 5.6μ antibody binds 50% of the thyroxine complex. Similar results were obtained for (). Chemiluminescent immunoassay for thyroxine:
Competitive binding reaction mixtures containing 19.3 nM (), various concentrations of thyroxine as shown in Figure 2, and 8.3μ of antibody were prepared in 75mM barbital buffer, pH 8.6 (antibody was added last). After incubating the reaction mixtures at room temperature for 1 hour, 200μ aliquots of each mixture were applied to separate 2 ml columns of Sephadex G25 for measuring the antibody titers described above. 95 μ of the eluate was added three times to H ₂ O ₂ −
Analyzed using hematin system. The results shown in FIG. 2 show that according to this test method, thyroxine can be quantified in a clinically important range, ie, 10 to 60 nM. The specificity of this competitive binding assay can be demonstrated by replacing the thyroxine complex with isoluminol. Isoluminol 3.4nM
bound to Cephadex G25, but was not eluted by this buffer or by antibodies to thyroxine. Thyroxine derivatives with other chemiluminescent labels [Table 4 () and ()] can also be used in the above immunoassays and gave similar results.

[Brief explanation of drawings]

Figure 1 shows the titer measurement of the thyroxine-isoluminol complex () using antibodies () 19.3 nM and 75 m
The two reaction mixtures containing various concentrations of antibodies in M sodium barbital buffer, PH 8.6, were left at room temperature for 1 hour. 200μ of each reaction mixture was applied to a Sephadex G25 column, and () bound to the antibody was eluted with 2ml of barbital buffer. Three samples (95μ each) were taken from this eluate.
), the antibody-bound () was analyzed using the H ₂ O ₂ -hematin system. Results are expressed as the percentage of () bound to the antibody. Figure 2 is a standard curve for a competitive protein binding test for thyroxine () 19.3 nM, an antibody against thyroxine.
Two binding reaction mixtures (250μ) containing 8.3μ and the indicated concentrations of thyroxine were left at room temperature for 1 hour. The antibody-bound () was then isolated and tested as outlined in FIG.

Claims (1)

[Claims] 1. For analyzing a hapten, antigen, biotin or avidin in a liquid: (a) A liquid, (1) A labeling substance having predetermined characteristics and a hapten, antigen, biotin or avidin. (2) contacting the antibody-bound phase and the antibody-free phase of the labeled conjugate with a reagent consisting of an antibody to a hapten or antigen, avidin to biotin, or biotin to avidin; (b) A homogeneous immunoassay method comprising the step of measuring the indicator properties of a hapten, antigen, biotin or avidin in a liquid without separating the bound and free phases, the labeling substance in the conjugate; is a reactant of 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 or isoluminol or a derivative thereof. 4. For analyzing haptens, antibodies, biotin, or avidin in liquids by homogeneous immunoassay methods: (1) A conjugate of a labeling substance with predetermined characteristics and a hapten, antigen, biotin, or avidin, and (2) A reagent consisting of an antibody to a hapten or antigen, avidin to biotin, or biotin to avidin, which is a labeled conjugate of the reagent and hapten, antigen, biotin, or avidin - bound phase and antibody -
forming a binding reaction system that produces a free phase, and the properties of either the bound phase or the free phase are a function of the amount of hapten, antigen, biotin, or avidin in the liquid, and the labeling substance in the conjugate is , a reagent characterized by being 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.