JPS6145776B2 - Specif combination analysis using prosthetic group as labelling component* reagent and composition for same* test kit* and ligand - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a homogeneous or heterogeneous specific binding analysis method for qualitatively or quantitatively measuring a ligand in a liquid medium, and reagents used in the method. More specifically,
The present invention relates to radioactive isotopes (radio isotopes)
It relates to specific binding assays using labels that are not labeled. The invention further relates to complexes of non-radioactive isotopic labels for use in the above-mentioned analytical methods. In conventional specific binding analysis methods, a detectable label component and a binding reaction system that produces two species or forms of label complex, a bound species and a free species, are combined with other components of the reagent (if The test sample is combined with reagents comprising various compositions comprising complexes with the binding moiety (if present) and the binding component involved in forming the test sample. The relative amounts or proportions of bound and free labeled complexes are a function of the presence (or amount) of the ligand to be detected in the test sample. As an example, a conventional competitive bond analysis method is described below. In traditional methods, the reagent contains (1) the ligand to be detected, which constitutes the binding component of the complex (e.g.
(2) a specific binding partner (counterpart) of the ligand (eg, an antibody); and (2) a specific binding partner (counterpart) of the ligand (eg, an antibody).
Binding of the test sample to the reagent causes the probed ligand and the bound component of the label complex to compete (competition) for non-covalent binding with the specific binding counterpart in a virtually indistinguishable manner. do. As a result, the amount of labeled complex that binds to the bound counterpart (which becomes the bound species) or the amount of labeled complex that remains free (which does not bind to the bound counterpart, which becomes the free species) can be measured as a function of the amount of competing ligand present. The amount of labeled complex that yields both species is the amount of labeled component in it,
For example, it can be measured by detecting (monitoring) or other methods. If the labeled complex in the bound species and the labeled complex in the free species are substantially indistinguishable depending on the means used to detect the labeled component, binding is performed to complete the analysis. Seeds and free species must be physically separated. This type of analysis is referred to in the art as "heterogeneous." If it is possible to distinguish between the bound and free forms of the labeled complex, a "homogeneous" approach is used and no separation step is necessary. The first highly sensitive specific binding assay to be discovered was the radioimmunoassay, which used a radioactive isotope as the labeling component. This method requires the use of a heterogeneous method since the detectable attributes of the label are qualitatively invariant between free and bound species.
Since it is inconvenient and difficult to handle radioactive substances, many efforts have been made to develop new analytical methods that use substances other than radioactive isotopes as labeling components. Examples of such substances include enzymes, fluorescent molecules, bacteriophage, metal or organometallic complexes, coenzymes, enzyme substrates, enzyme inhibitors, cycling reactants, and chemiluminescent reactants. . U.S. Patent Nos. 3654090, 3791932, 3839153,
Nos. 3850752 and 3879262, J.Immunol.Methods
1:247 (1972), and J.Immunol.109:129
(1972) describes various heterogeneous binding reaction systems using enzymes as labeling components. A heterogeneous binding assay using an inert precursor of a spectroscopically detectable substance as a label is described in US Pat. No. 3,880,934. For more background on heterogeneous analytical methods, see Principles of Competitive Protein—Binding.
Assays, ed. Odell and Daughaday (JB
Lippincott Co., Philadelphia 1972). US Pat. No. 3,817,834 describes a homogeneous enzyme-labeled immunoassay using a complex of a ligand and an enzyme. In this method, the enzyme activity of the complex in the bound species is less difficult to measure than the enzyme activity of the complex in the free species, so a homogeneous method can be used. Homogeneous and heterogeneous systems using particularly characteristic substances such as coenzymes, chemiluminescent molecules, cycling reactants, and cleavable fluorescent enzyme substrates as labels are described in German Open
Nos. 2618419 and 2618511 (assigned to the applicant)
U.S. Patent Application No. 667982 filed March 18, 1976;
Based on No. 667996). Although research into developing labels other than radioisotopes for use in binding assays has resulted in some practical solutions, there remains room for further improvements. Although the idea of using enzyme labels was initially the most promising, in practice various difficulties became apparent. The use of enzymes with high molecular weights and heterogeneous properties creates difficulties in the characterization, stabilization, and reproducibility of preparation of labeled conjugates. These problems can be overcome by replacing the enzyme label with a low molecular weight label whose reactive activity can be detected. By using these new labels, such as coenzymes, oxygen substrates, cycling reactants, and chemiluminescent reactants, highly sensitive and versatile analytical methods can be constructed. However, detecting these markers may require equipment that is not currently commonly used in hospital examination rooms. The primary object of the present invention is to provide excellent sensitivity, versatility, and ease of preparation and characterization of the reactant label, which is detectable with standard hospital laboratory equipment. The object of the present invention is to provide a method for binding a non-radioactive isotope system using a new label. According to the present invention, an organic prosthetic group can be used as a labeling component in a labeling complex.
It has been found that the use of the method provides an improved specific binding assay. Prosthetic groups are a group recognized in the art as enzyme cofactors. Enzyme cofactors are non-protein substances whose presence is necessary for the expression of the enzyme's catalytic activity, and which contributes to the catalytic reaction cycle of the enzyme involved (named the "parent enzyme"). Chemical changes occur during the process. A coenzyme is a type of enzyme cofactor that is chemically transformed during a catalyzed reaction by a parent enzyme. In order to regenerate the original form of the cofactor, it is necessary to engage the cofactor in another catalyzed reaction by an enzyme other than the parent enzyme. In contrast, a prosthetic group is one that is chemically transformed during the course of a catalytic reaction by a parent enzyme and returns to its original form, for example, by a second catalytic reaction by the same parent enzyme. It is an enzyme cofactor in which regeneration takes place. Organic prosthetic groups are usually characterized in that they are tightly bound relative to the protein moieties of the parent enzyme, known as apoenzymes, and are catalytic. The active parent enzyme is known as the holoenzyme. The equilibrium reaction regarding the interaction between the prosthetic group and the apoenzyme can be expressed as:

[Table] Prosthetic group + apoenzyme complex or holoenzyme
←
(Pr) (Apo) (Pr〓Apo)
As used herein, the term "complex enzyme" refers to a complex formed by the combination of a prosthetic group and an apoenzyme, and whether or not the complex exhibits enzymatic activity is irrelevant. do not have. "Holoenzyme"
The term refers only to complex enzymes that exhibit enzymatic activity. Further information on prosthetic groups, their properties, and their interaction with apoenzymes can be found in Dixon and Webb, Enzymes, Ist ed.
Longmans, Green & Co. (London 1958). In the present invention, an organic prosthetic group is combined with a binding moiety to form a label complex for use in binding assays. The central element of the present invention is the interaction that occurs between the complex prosthetic group and the apoenzyme as shown below. Although the reactions shown above are the interactions that are normally observed, under certain conditions, such as when conjugation with a prosthetic group interferes with the normal holoenzyme-substrate interaction, In some cases, the complex enzyme exhibits no or very little holoenzyme activity.
Nevertheless, the labeled conjugate used in the present invention is the binding component of the complex prosthetic group (described above) in the binding reaction in the assay.

If the bound enzyme complex exhibits holoenzyme activity after the binding of the compound (denoted by the symbol [formula]) is completed, for example, if the binding removes the inhibition of the interaction between the holoenzyme and the substrate, , is what is generated. There are many marker detection methods that can be used according to various known homogeneous and heterogeneous combination schemes, some of which are listed below. In all cases, however, the labeling component of the labeling complex involved in the binding reaction contains residues of an organic prosthetic group, and the detection method involves detecting either the bound or the free species, or possibly both. It involves a measurement based on the measurement of the holoenzyme activity of the labeled component. The prosthetic group residue used in the present invention is flavin adenine dinucleotide, and the combination of prosthetic group residue and apoenzyme is flavin adenine dinucleotide residue and apoglucose dinucleotide. There is a combination of oxidases. Each of the holoenzymes formed from these combinations is
It can be easily detected by known hydrogen peroxide measurement techniques such as spectroscopy and fluorometry. Since the holoenzyme to be detected has catalytic activity, it is possible to greatly amplify the presence of the labeled component in the detection reaction, and it is possible to use relatively low-sensitivity and easily available methods such as spectrophotometers. By using a simple device, it is possible to detect the ligand to be analyzed with high sensitivity (eg, at the ng/ml level). The ability to use low molecular weight prosthetic group molecules as intervening moieties for label binding to binding components in label complexes and the ability to use spectroscopic measurements, particularly colorimetric readouts, for sensitive detection. This combined ability has created a uniquely superior specific binding assay. The present invention therefore provides a marker combination that can be easily automated, that allows the use of markers that can be detected with equipment normally found in hospital examination rooms, that is reproducible, and that is relatively easy to manufacture. It is unique in that it can be used with the human body and has highly sensitive detection limits. In this specification, the terms described below have the following meanings unless otherwise specified. "Ligand" means a substance or a series of related substances whose presence or abundance in a liquid medium is to be determined. "Specific binding partner (correspondent) of a ligand" means a substance or a series of substances that have specific binding affinity for the ligand to the exclusion of other substances. A "specific binding analog of a ligand" refers to a substance or a group of substances that exhibit substantially the same behavior with respect to the binding affinity of the specific binding counterpart for the ligand. A "residue" is a moiety in an organic molecule, for example, a residue of a prosthetic group in a labeling complex is a hydrogen atom that serves to bind the prosthetic group to the rest of the labeling complex. An organic molecule in a complex that is derived from a prosthetic group and substantially acts as a prosthetic group, such as a prosthetic group in which a is removed at one point. For example, in the FAD-ligand labeling complex, FAD represents the residue of the flavin adenine dinucleotide prosthetic group. "reagent"
means a composition, device, or test kit containing one or more reagents used to carry out an analytical method. "Detection reaction" means a reaction in which a labeled component of a labeled complex is measured by measuring holoenzyme activity. "Lower alkyl"
means a straight or branched alkyl group containing from 1 to 6 (inclusive) carbon atoms, such as, for example, methyl, ethyl, isopropyl and hexyl. The analytical method of the present invention can be used to detect any ligand for which a specific binding counterpart exists. Typically, the ligand is a peptide, protein, carbohydrate, glycoprotein, steroid, etc. for which a specific binding counterpart exists in biological systems or can be synthesized. In functional terms, ligands are usually selected from the group consisting of antigens and their antibodies, haptens and their antibodies, and hormones, vitamins, metabolites and pharmaceuticals, and their receptors and binding substances. Examples of ligands that can be detected using the present invention include, among others, hormones such as insulin, pilonidal gonadotropin, thyroid hormones and follicular hormones; ferritin, bradykinin, prostaglandins and tumor-specific antigens. Antigen or hapten:
Vitamins such as biotin, vitamin _B12 , folic acid, vitamin E, vitamin A and ascorbic acid;
Metabolites such as 3′,5′-adenosine-phosphate and 3′,5′-guanosine-phosphate: anticonvulsants, bronchial muscle relaxants (bronchodilators), inotropes (cardiovascular medicines or drugs such as drugs) and narcotics; antibodies such as microsomal antibodies and antibodies of hepatitis and allergic antigens; and specific binding receptors such as thyroxine-binding globulin, avidin, intrinsic factor and transcobalamin. I can do it. The analytical method of the present invention uses 100 to 1000
haptens and their analogs having a molecular weight between
It is particularly useful for detecting iodothyronine thyroid hormones, thyroxine and liothyronine. The liquid medium tested is generally a naturally occurring or artificially produced liquid that is assumed to contain the ligand, usually a biological fluid or a diluted or otherwise This is a liquid obtained by processing. Examples of biological fluids that can be analyzed by the method of the invention include serum, blood plasma, urine, saliva, amniotic fluid and brain cough fluid. Other materials, such as solid materials such as cells, can also be analyzed by bringing them into liquid form, such as by dissolving the solid in a liquid or extracting the solid with a liquid. Labeling complex The labeling complex of the present invention contains flavin adenine dinucleotide, which is an organic prosthetic group residue, in its labeling component, and has two basic types represented diagrammatically as shown below. It can be indicated by either.

[Table] In the above scheme, Pr represents the residue of the prosthetic group, Apo represents the apoenzyme, R represents the linking group, and L represents the binding component of the labeling complex (usually the ligand or its is a binding analog). The binding moiety and the prosthetic group residue are usually defined by their specific binding site (e.g., the immunochemical binding site when the binding moiety is an antigen, hapten, or an antibody thereof); The latter is bound via a linking group at a site remote from the site of the binding component involved in the binding reaction (the location of the binding component) and, for the latter, at a site remote from the active binding site with the apoenzyme. Analytical Methods There are many methods for detecting new labeled conjugates according to the present invention. In either case, the prosthetic group residues are covalently bound to the binding component in the labeling component, and the detection reaction involves the holoenzyme activity in the bound or free species, or optionally both. Includes measurements. Examples of various detection methods based on homogeneous and heterogeneous competitive combination schemes and techniques are presented below for illustrative purposes only. It will be appreciated that implementations may in fact be based on other homogeneous and heterogeneous schemes and techniques.

[Table] Examples of detection methods - Part 1 Homogeneous competitive binding method: Introduction of apoenzyme after initiation of binding reaction

[Table] In the above scheme, the prosthetic group residue that is the labeling component can be added substantially simultaneously with the initiation of the binding reaction, or after the initiation of the binding reaction, e.g., during the binding reaction, or when the binding reaction reaches equilibrium. This can then be detected by adding apoenzyme and measuring the holoenzyme activity of the final reaction mixture. This analytical method is made homogeneous by selecting conditions such that the binding between the apoenzyme and the prosthetic group residue is inhibited for the prosthetic group in the binding species. Alternatively, the apoenzyme binds to the labeled complex in the form of a bound species;
Conditions can also be selected such that the resulting composite enzyme complex does not exhibit enzymatic activity due to inhibition due to interaction between enzyme and substrate. This situation is not shown in the diagram above. In both of the above cases, the total amount of enzymatic activity measured in the system is the result of the non-inhibitory binding of the apoenzyme, which liberates the labeled complex, and therefore the competition for binding with its binding counterpart. is directly proportional to the amount of ligand in the sample exposed to. Also not shown above, but in contrast to the scheme above, the labeled complex (Pr-R-L) is inhibited from binding to the apoenzyme, while binding to the bound counterpart of the labeled complex It is also possible that upon binding of this inhibition, the apoenzyme is able to bind to the labeled complex in the form of the bound species and thus form an active holoenzyme complex. It can happen. In such cases, the amount of holoenzyme activity obtained in the system will be inversely proportional to the amount of ligand present in the sample. Example of detection method - Part 2 Heterogeneous competitive binding method: Introduction of apoenzyme after initiation of binding reaction

[Table] ↑ ↑

Claims (1)

[Scope of Claims] 1. A specific binding analysis method for measuring a ligand in a liquid medium, comprising: (a) treating the liquid medium with a binding component and an organic prosthetic group bound to the binding component; contact with a labeling complex consisting of a labeling component containing a residue, and optionally,
The binding component is further contacted with a specific binding counterpart, and this contact generates bound and free species of the labeled complex, and the ratio of the two generated species of labeled component is determined in the liquid medium. (b) combining the liquid medium with the prosthetic group to form a holoenzyme; contacting the holoenzyme in the bound species, the free species, or both, with an apoenzyme that can be in the specific binding analysis method, the prosthetic group labeling component is flavin adenine dinucleotide, the apoenzyme is apoglucose oxidase, and the holoenzyme produced thereby is catalytically active. A specific binding analysis method characterized by glucose oxidase. 2. The analytical method according to claim 1, wherein after contacting the liquid medium with the labeled complex, the liquid medium is brought into contact with the apoenzyme. 3. The analytical method according to claim 1, wherein the liquid medium is brought into contact with the labeled complex and the apoenzyme substantially simultaneously. 4. Claim 3, in which the labeled complex and the apoenzyme are brought into contact with a liquid medium in separate forms.
Analytical method described in section. 5. A patent claim in which the labeling complex and the apoenzyme are brought into contact with a liquid medium by using, as a labeling component in the labeling complex, a residue of a composite enzyme formed of a prosthetic group bound to the apoenzyme. range 3rd
Analytical method described in section. 6. Any one of claims 1 to 5, wherein the labeling complex consists of a labeling component bound to a ligand or a binding analog thereof, and the liquid medium is further brought into contact with a specific binding counterpart of the ligand. or the analysis method described in item 1. 7. The ability of the flavin adenine dinucleotide label component to combine with apoglucose oxidase to produce catalytically active glucose oxidase is reduced when the label complex and the binding counterpart are combined, and the liquid medium is 7. The method of claim 6, which is homogeneous and measures glucose oxidase in the reaction mixture formed upon contact with the labeled complex, bound counterpart and apoenzyme as a function of the amount of ligand present. 8. Any of claims 1 to 6 of a heterogeneous system in which the bound and free species of the labeled complex are physically separated and glucose oxidase is measured in one or the other of them. or the analysis method described in item 1. 9. The analytical method according to any one of claims 1 to 8, wherein the activity of glucose oxidase is measured by a colorimetric method. 10. The analytical method according to any one of claims 1 to 9, wherein the ligand is an antigen or a hapten. 11 Formula: [Wherein, riboflavin-(phos) ₂ -ribose represents a riboflavin-pyrophosphate-ribose residue in flavin adenine dinucleotide; R represents a linking group; L represents a specifically binding ligand or a binding analog or counterpart thereof. ] A flavin-adenine-dinucleotide labeling complex.