IE48264B1 - Specific binding assay method with a prosthetic group as a label component - Google Patents

Abstract

A specific binding assay for determining a ligand in a liquid medium employing an organic prosthetic group residue, such as a residue of flavin adenine dinucleotide, flavin mononucleotide, or heme, as a label component in the labeled conjugate, Preferably, the label component is the prosthetic group residue alone or is a holoenzyme residue comprising such prosthetic group residue combined with an apoenzyme in the form of a holoenzyme complex. In the former case, the label component preferably is monitored in the assay by adding an apoenzyme after the binding reaction has been initiated and measuring the resultant holoenzyme activity. In the latter case, the label component is monitored simply by measuring holoenzyme activity. The assay method may follow conventional homogeneous and heterogeneous schemes. Preferred apoenzymes for use in the assay are apoglucose oxidase and apoperoxidase. The assay offers the advantages of colorimetric read-out and of being readily adaptable to automated techniques. Also disclosed are prosthetic group- labeled and enzyme-labeled conjugates for use in the assay, as well as a method for preparing the latter conjugates.

Description

This invention relates to assay methods, and reagent means for use therein, of the homogeneous or heterogeneous specific binding type for determining qualitatively or quantitatively a ligand in a liquid medium. In particular, the invention relate to specific binding assays employing non-radioisotopic labels.

Further, the invention relates to non-radioisotopic labeled conjugates for use in such assays anl to methods of their preparation.

In conventional specific binding assay techniques, the te. 10 sample is combined with reagent means of various compositions that include a conjugate having a monitorable label component and a binding component which participates with other constituents, if any, of the reagent means to form a binding reaction - system producing two species or forms of the labeled conjugate, 15 a bound-species and a free-species. The relative amount or proportion of the labeled conjugate that results in the bound-species compared to the free-species is a function of the presence (or amount) of the ligand to be detected in the test sample.

As an illustration a conventional competitive binding assay technique will now be described. In such a technique tlie reagent means would comprise (1) a labeled conjugate in the Conn of. the ligand to be detected (e.g., an antigen or hapten), such ligand constituting the binding component of the conjugate, chemically linked to a label component, and (2) a specific binding partner for the ligand (e.g., an antibody). Upon combination of the test sample and the reagent means, the ligand to he detected and the binding component of the labeled conjugate would compete in a substantially nondiscriminating manner for noncovalent binding to the specific binding partner. As a result, either the amount of labeled conjugate that would become bound to tlie binding partner (i.e., that results in the bound-species) or that amount which would remain free (i.e., unbound to the bind ing partner and thus that results in the free-species) can be measured as a function of tlie amount of competing ligand present. The amount of labeled conjugate resulting in either species is determined by measuring, i.e., monitoring, tlie label component therein.

Where the labeled conjugate in the bound-species and that in the free-species are essentially indistinguishable by the means used to monitor the label component, the bound-species and the free-species must be physically separated in order fo complete the assay. This type of assay is referred to in tlie art as heterogeneous”. Where the bound-species and free-species forms of the labeled conjugate can be distinguished, a homo geneous format may be followed and the separation step avoided.

The first discovered type of highly sensitive sped fitbinding assay was the radioimmunoassay which employs a radio active isotope as the label component. Such an assay necessarily must follow the heterogeneous format since the monitorable character of the label is qualitatively unchanged in the t'reeand bound-species. Because of the inconvenience and difficulty of handling radioactive materials, many new assay systems have been devised using materials other than radioisotopes as the label component, including enzymes, fluorescent molecules, ID bacteriophages, metals and organometallic complexes, coenzymes, enzyme substrates, enzyme inhibitors, cyclic reactants, and chemiluminescent reactants.

The following describe several different heterogeneous binding reaction systems in which an enzyme is employed as tlie label component: U.S. Patents Nos. 3,654,090; 3,791,932; 3,839,153; 3,850,752; and 3,879,262; J. Immunol. Metho,hi /:247 (1972); and J. Immunol. 109:129 (1972). Λ heterogeneous binding assay utilizing a non-active precursor of a spectrophotometrically detectable substance as the labeling substance is 2Q suggested in U.S. Patent No. 3,880,934. Of further background interest pertaining to heterogeneous assays is Principles of Competitive Protein-Binding Assays, ed. Odell and Daughaday (J.B.Lippincott Co., Philadelphia, 1972).

An enzyme-labeled immunoassay of the homogeneous type is described in U.S. Patent No. 3,817,834 wherein a ligand-enzyme conjugate is employed. The enzymatic activity of the conjugate in the bound-species is measurably less than that in the free-species, thereby allowing a homogeneous format to be used.

The use of particularly unique materials as labeling substance··, including coenzymes, chemiluminescent molecules, cyclic reac tants, and cleavable fluorescent enzyme substrates, in both homogeneous and heterogeneous formats, is described in German Offenlegungschriften Nos, 2,618,419 and 2,618,511 based on U.S. Patent Applications Serial Nos. 667,982 and 667,996, filed on March 18, 1976, and assigned to the present assignee.

While the search for non-radioisotopic labels in binding assays has produced a number of workable solutions, there remains room for improvement. The enzyme-labeled approaches appeared at first to be the most promising, however, in practice several difficulties have come to light. The high molecular weight and heterologous nature of enzymes creates problems in characterizing, stabilizing, and reproducibly preparing tbe labeled conjugates. These problems were overcome by replacing enzyme-labels with low molecular weight labels monitorable by their reactant activity. These novel labels, including coenzymes, enzyme substrates, cyclic reactants and chemiluminescent reactants, can be used to form assays of high sensitivity and versatility. However, monitoring of these labels often requires instrumentation which for the present is not commonly found in the clinical laboratory. it is a primary object of the present invention to provide a non-radioisotopic binding assay employing a novel label which is monitorable with standard clinical laboratory equipment while retaining the reactant-label advantages of sensitivity, versatility, and ease of synthesis and characterization of labeled conjugates.

Xt has now been found that an improved specific binding assay is provided by employing an organic prosthetic group residue as a label component in the labeled conjugate. Prosthetic groups are an art recognized class of enzyme cofactors.

An enzyme cofactor is a nonprotein substance whose presence is required for an enzyme to exhibit its catalytic activity and which undergoes a chemical change during the catalytic cycle of the enzyme involved (referred to as the parent enzyme). A coenzyme is a type of enzyme cofactor which is chemically 1° modified in the course of the reaction catalyzed by the parent enzyme. Regeneration of the original form of the cofactor requires its participation in a separate reaction that is catalyze; by an enzyme other than the parent enzyme. In contrast, a prosthetic group is an enzyme cofactor which is chemically 15 modified in the course of the reaction catalyzed by the parent enzyme and is regenerated, i.e., reconverted to its original form, by a second reaction catalyzed by the same parent enzyme.

Prosthetic groups are usually characterized by being firmly bound in relative terms to the protein portion of the parent enzyme, such protein portion being known as the apoenzyme and the catalytically active parent enzyme being known as the holoenzyme. The equilibrium reaction for the interaction between a prosthetic group and apoenzyme may be represented as follows: 2s prosthetic group + annonzymo j,1 · ^conjugated or holoenzyme (Pr) (Apo) (Pr-Apo) As used herein, the term conjugated enzyme refers to the complex formed by combination of the prosthetic group and προ enzyme whether or not such complex exhibits catalytic activity and the term holoenzyme refers only to a catalytically active conjugated enzyme. Further information concerning prosthetic groups, their characteristics, and their interactions with apoenzymes may be obtained by referring to Dixon and Webb, Enzymes, 1st ed., Longmans, Green § Co. (London 1958).

In the present invention an organic prosthetic group is 10 coupled to a binding component to form a labeled conjugate lotuse in binding assays. A central feature of the invention is the resulting interaction between the conjugated prosthetic group and apoenzyme illustrated as follows: σ Pr + Apo __________θ-Pr-Apo catalytically inactive active components holoenzyme complex While the above-represented reaction is the normally observed interaction it is anticipated that in certain circumstances the conjugated enzyme complex will exhibit little or no holoenzyme activity, such as where conjugation of the prosthetic group prevents normal holoenzyme-substrate interaction. Labeled conjugates of use in the present invention may nonetheless result if, upon binding of the binding component (represented by the symbol Q- above) of the conjugated prosthetic group in the assay binding reaction, the bound conjugated enzyme complex then does exhibit holoenzyme activity, such as where such bind ing relieves the inhibition of holoenzyme-substrate interaction 4826 4 There are numerous possible label monitoring schemes, some of which are illustrated hereinafter, following any of the various known homogeneous and heterogeneous binding formats, however, in all cases the label component of the labeled conju5 gate that participates in the binding reaction comprises an organic prosthetic group residue and the monitoring scheme comprises determining the label component in the bound-species or the free-species, or both, as the case may be, based on measurement of holoenzyme activity.

Prosthetic group residues which are preferably used in the present invention are residues of flavin adenine dinucleotide., flavin mononucleotide, and heme. Preferable prosthetic group residue/apoenzyme pairs are residues of flavin adenine dinucleotide/apoglucose oxidase and residues of heme/apo15 peroxidase. The respective holoenzymes formed from such pairs are readily monitored with the well-known hydrogen peroxide measurement techniques which include colorimetric and fluorometric methods. Due to the catalytic nature of the monitored holoenzyme, the presence of the label component is amplified manyfold in the monitoring reaction, permitting highly sensitive detection of the ligand under assay (e.g., at levels of ng/ml) using relatively insensitive but readily available and simple instrumentation, such as spectrophotometers. The combination of the ability to use a low molecular weight prosthetic group molecule as the component through which the label is coupled to the binding component in the labeled conjugate and the ability to attain highly sensitive detection with spectrophotometric, particularly colorimetric, read-out provides a uniquely advantageous specific binding assay.

The present assay is characterized by highly sensitive detection limits, by being readily adaptable to automation, by permitting the label to be monitored with instrumentation commonly found in clinical laboratories, and by utilizing labeled conjugates that can be reproducibly and relatively simply synthesized.

In the context of this disclosure, the following terms shall be defined as follows unless otherwise indicated: ligand is the substance, or class of related substances, whose presence or the amount thereof in a liquid med4um is to be determined; specific binding partner of the ligand is any substance, or class of substances, which has a specific binding affinity lor the ligand to the exclusion of other substances; specific binding analog of the ligand is any substance, or class of substances, which behaves essentially the same as the ligand with respect to the binding affinity of the specific binding partner for the ligand; residue is a moiety in an organic molecule, for example, a prosthetic group residue in a labeled conjugate is an organic moiety in such conjugate derived from and functioning essentially as a prosthetic group, such as the prosthetic group less a hydrogen atom at a position through which the prosthetic group is coupled to the remaining portion of the labeled conjugate (in an FAD-ligand labeled conjugate, FAD represents a residue of the prosthetic group flavin adenine dinucleotide); reagent means is a composition, device or test kit comprising the reagents used to perform an assay method; . 48264 monitoring reaction is the reaction in which the label component of the labeled conjugate is determined by measuring holoenzyme activity; lower alkyl is a straight chain or branched alkyl group comprising from 1 to 6 carbon atoms, inclusive, such as methyl, ethyl, isopropyl, and hexyl.

The present assay may be applied to the detection of any ligand for which there is a specific binding partner. The ligand usually is a peptide, protein, carbohydrate, glycoprotein steroid, or other organic molecule for which a specific binding partner exists in biological systems or can be synthesized.

The ligand, in functional terms, is usually selected from the group consisting of antigens and antibodies thereto; haptens and antibodies thereto; and hormones, vitamins, metabolites and pharmacological agents, and their receptors and binding substances. Specific examples of lig..nds which may be detected using the present invention are hormones such as insulin, chorionic gonadotropin, thyroid hormones, and estriol; antigens and haptens such as ferritin, bradykinin, prostaglandins, and tumor specific antigens; vitamins such as biotin, vitamin Bj?, folic acid, vitamin E, vitamin A, and ascorbic acid; metabolites such as 3', 5'-adenosine monophosphate and 3',51-guanosine monophosphate; pharmacological agents or drugs, such as anticonvulsants, bronchodialators, cardiovascular agents, and drugs of abuse; antibodies such as microsomal antibody and antibodies to hepatitis and allergens; and specific binding receptors such as thyroxine binding globulin, avidin, intrinsic factor, and transcobalamin. The present assay is particularly useful for the detection of haptens, and analogs thereof, of molecular weight between 100 and 1000, particularly the iodothyroninc thyroid hormones thyroxine and liothyronine. •18 2 6 4 The liquid medium to be tested may be a naturally occurring or artificially formed liquid suspected of containing the ligand, and usually is a biological fluid or a liquid resulting from a dilution or other treatment thereof. Biological fluids which may be assayed following the present method include serum, plasma, urine, saliva, and amniotic and cerebrospinal fluids. Other materials such as solid matter, for example tissue, may be assayed by reducing them to a liquid form, such as by dissolution of the solid in a liquid or by liquid extraction of θ the solid.

LABELED CONJUGATE The labeled conjugate of the present invention comprises an organic prosthetic group residue in its label component and may be in either of two basic forms schematically represented as follows: schematic formula label component Pr-R-L Apo-Pr-R-L prosthetic group residue (Pr-) holoenzyme or conjugated enzyme residue (Apo-Pr-) 1θ wherein Pr represents the prosthetic group residue and Apo the apoenzyme, R is a linking group, and I is the binding component of the labeled conjugate, usually the ligand or a binding analog thereof. Usually, the binding component and the prosthetic group residue are coupled through the linking group at a site on the former away from its specific binding locus (i.e., the location on the binding component which participates in the assay binding reaction, such as an immunochemical binding site where the binding component is an antigen, hapten or antibody thereto) and at a site on the latter away from its active binding site for the apoenzyme.

ASSAY METHODS There are various methods available for monitoring the novel labeled conjugates according to the present invention. In every case the prosthetic group residue is covalently coupled to the binding component in the labeled component and the uioni toring reaction comprises measuring holoenzyme activity in tlie bound-species or free-species, or both, as the case may be.

For purposes of illustration only, the following are examples of several monitoring schemes based on homogeneous and heterogeneous competitive binding techniques whereas it will be understood that other homogeneous and heterogeneous techniques can be followed in practice.

In the illustrations below, will be used consistent with the term prosthetic group residue apoenzyme linking group ligand binding partner conjugated enzyme residue holoenzyme residue (active) the following abbreviations remaining description herein abbreviation Pr Apo R L B Apo-Pr |Apo-Pr'| illustrative Monitoring Scheme #1 Homogeneous Competitive Binding Method Apoenzyme Introduced After Initiation of Binding Reaction L(sample) + Pr-R-L + B L-B + Pr-R-L-B 1 + Apo (boundspecies) Apo + |Apo-Pr}R-L (free-species; active holoenzyme) 48364 ϊη this scheme, the prosthetic group residue as label compnnenl is monitored by adding apoenzyme substantially simultaneous with or after initiation of the binding reaction, i.e., during the binding reaction or after the binding reaction has reached s equilibrium, and measuring the holoenzyme activity in the final reaction mixture. The assay is homogeneous by selection of conditions such that binding of apoenzyme with the prosthetic group residue is inhibited for the prosthetic group in the bound-species. Or conditions might be selected such that apoenzyme can bind to the bound-species form of the labeled conjugate but the resulting conjugated enzyme complex exhibits no enzymatic activity such as because of inhibition of enzyme-substrate interaction (this situation is not shown m the above schematic). In either case, tlie total amount of IS enzyme activity measured in the system is the result of uninhibited binding of apoenzyme to free labeled conjugate and accordingly is a direct function of the amount of ligand I rout the sample available for competition for binding to the binding partner.

It might also occur (but also is not shown above) that, in contrast to the above schemes, the labeled conjugate (Pr-R-I.) is formed such that binding with the apoenzyme i.s inhibited but that upon binding of the labeled conjugate by the binding partner such inhibition is relieved and apoenzyme is able in 2S bind with the bound-species form of the labeled conjugate to form an active holoenzyme complex. In such a case, the amount of holoenzyme activity resulting in the system is an inverse function of the amount of ligand present in the sample.

Illustrative Monitoring Scheme #S Heterogeneous Competitive Binding Method Apoenzyme Introduced After Initiation of Binding Reaction L(sample) + Pr-R-L + B L-B + Pr-R-L-B +Apo Apo + [Apo-Pr}-R-L + |Apo-Pr|-R-L-B (free-species, active holoenzyme) (bound-species, active holoenzyme) separate This scheme involves the same reagents and order of addition as above-described scheme #1 except that conditions are such that apoenzyme can bind to the labeled conjugate in the bound-species to form an active holoenzyme complex qualitatively indistin15 guishable from that formed by binding to the free-species form of the labeled conjugate. The bound-species and free-species must be separated and the enzyme activity in one thereof is a function of the amount of ligand present in the sample.

[LLuatrative Monitoring Scheme #3 Homogeneous Competitive Binding Method Apoenzyme Introduced Prior to Initiation of Binding Reaction Pr-R-L + Apo -»-[Apo-Prj-R-L (active holoenzyme) L(sample) » |Apo-Prf-R-L + B L-B + Apo-Pr-R-L-B (free species; active holoenzyme) (bound species; inactivated) In this scheme, the prosthetic group residue is comprised in the label component by being coupled co the ligand and combined with the apoenzyme in the form of an active holoenzyme product. The prosthetic group residue is monitored by measuring holoenzyme activity in the final reaction mixture; The assay is 35 homogeneous by selection of conditions such that upon binding of the binding partner to the active holoenzyme complex the bound-species form that results exhibits inhibited enzyme activity such as because of inhibition of enzyme-substrate interaction. The total amount of enzyme activity measured in 2() the system is the result of unbound active holoenzyme complex, i.e., the free-species form, and accordingly is a direct function of the amount of ligand present in the sample.

It might also occur (but is not shown above) that, in contrast to the above scheme, the labeled conjugate exhibits 23 little or no enzymatic activity but upon binding of the binding partner the bound-species form actually exhibits measurable or enhanced enzymatic activity. In such a case, the amount of holoenzyme activity resulting in the system is an inverse function of the amount of ligand present in the sample. tuu brative Monitoring Soheme tt4 Heterogeneous Competitive Binding Method Apoenzyme Introduced Prior to Initiation of Binding Reaction Pr-R-L + Apo ->-|Apo-Pr)-R-L (active holoenzyme) L(sample) + |Apo-Pr]-R-L + 63£L-J! + |Apo-Pr[-R-L B (free-species; active holoenzyme) (bound-spec it s; active holoenzyme) separate This scheme involves the same reagents and order of addition as above-described scheme #3 except that conditions are such that both the bound-species and free-species forms are enzymati cally active and are qualitatively indistinguishable. The bound-species and the free-species must be separated and the enzyme activity in one thereof s a function of tlie amount of ligand present in the sample.

Schemes #3 and #4 are essentially enzyme-labeled assays except that, in contrast to the prior art, the enzyme as such is coupled to the binding component of the labeled conjugate through a prosthetic group residue. In this respect then, the present invention provides a novel method of preparing an enzyme-labeled conjugate comprising the steps of (a) covalently coupling the binding component moiety (i.e., the ligand to be 5 detected or a binding analog or partner thereof) to an organic prosthetic group which is capable of combining with an apoenzyme to produce a conjugated enzyme, e.g., an active holoenzyme, (b) combining the resulting prosthetic group labeled conjugate with the apoenzyme, and (c) isolating the resulting conjugated enzyme-labeled conjugate. It is contemplated that such a method is most valuable in preparing enzyme-labeled conjugates for use in assays because of the controlled manner in which the low molecular weight and well characterized prosthetl· group can be coupled to the binding component. it is evident from the above discussion that for certain labeled conjugates and ligand/binding partner pairs one or more of the described homogeneous schemes will be appropriate depend ing upon several factors, including the ability of apoenzyme to bind to the bound-species form of the labeled conjugate and -° the effect of binding partner binding on the conjugated enzyme complex. Where the bound-species and free-species forms of the labeled conjugate exhibit indistinguishable activity, a heterogeneous format can be followed to provide a useful assay. Accordingly, while circumstances may dictate what particular “5 manipulative scheme or schemes will be most useful, the present assay method, in general, is adaptable to any conventional homogeneous or heterogeneous technique.

Homogeneous Techniques A homogeneous technique, i.e., one which does not require a physical separation of the bound-species and the free-species, is available where reaction between the binding component of the labeled conjugate and a corresponding binding partner causes a measurable change, either in a positive or a negative sense, in the ability of the label component o.f the labeled conjugate to participate in the monitoring reaction, e.g,, in the ability of the labeled conjugate to combine with apoenzyme and/or Id exhibit holoenzyme activity. Zn such a case, the distribution of the label component between the bound-species and the free-species can be determined without separation of the species The holoenzyme activity in the reaction mixture is then determined by forming in at least a portion thereof the chemical reaction catalyzed by such holoenzyme, e.g., by addition of substrate, and measuring by any conventional technique the rate or aggregate amount of product production or reactant consumption. Qualitative determination of the ligand in tlie liquid medium involves comparing the measured quantity to that of the monitoring reaction in a liquid medium devoid of the ligand, any difference therebetween being an indication of the presence of such ligand in the liquid tested. Quantitative determination of the ligand in the liquid medium involves comparing the measured quantity to that of the monitoring reaction in liquid media containing various known amounts of the ligand, e.g., a comparison to a standard curve.

In general, when following a homogeneous assay technique, the components of the specific binding reaction, i.e., the liquid medium suspected of containing the ligand, the labeled conjugate, and, in some systems (i.e., a competitive binding 5 system], a specific binding partner of the ligand, may be combined in any amount, manner, and sequence, provided that the activity of the label component of the labeled conjugate is measurably altered when the liquid medium contains the ligand in an amount or concentration of significance to the purposes of the assay. Preferably, all of the components of the specificbinding reaction are soluble in the liquid medium.

Known variations of the above briefly described homogeneous methods and further details concerning the specific techniques discussed, including an alternative technique known as the Is direct binding technique, are readily available in the literature, e.g., German OLS No. 2,618,511, corresponding to U.S. Patent Application Serial No. 667,996, filed March 18, 1976 and assigned to the present assignee.

Heterugeneous Techniques The use of the present novel labels can also be applied to the conventional heterogeneous type assay techniques wherein the bound- and free-species of the labeled conjugate are separated and the label component in one or the other is determined. The reagent means for performing such a heterogeneous assay may take many different forms. In general, such means comprises three basic constituents, which are (1) the ligand to be detected, (2) a specific binding partner of the ligand, and (3) the labeled conjugate. The binding reaction constituents are combined simultaneously or in a series of additions, and with an appropriate incubation period or periods, the labeled conjugate becomes bound to its corresponding binding partners such that the extent of binding, i.e., the ratio of the amount of labeled conjugate bound to a binding partner (the bound -species) to that unbound (the free-species), is a function of the amount of ligand present. The bound- and free-species are physically separated and the amount of label present in one thereof is determined by measuring the holoenzyme activity therein and comparing such to a negative control or standard results, e.g., a standard curve.

Various means of performing the separation step and of forming the binding reaction systems are available in the art. Separation may involve such conventional techniques as those involving what is commonly known as a solid-phase antibody or antigen, a second antibody, or a solid phase second antibody, as well as the use of immune complex precipitation agents and adsorbents, and so forth. Binding reaction systems that can be followed include the so-called competitive binding technique, the sequential saturation technique, the sandwich technique, and so forth. Further details concerning the various known heterogeneous systems are readily available in the literature, e.g., German OLS No. 2,618,419, corresponding to U.S. Patent Application Serial No. 667,982, filed March 18, 1976 and assigned to the present assignee.

It should be recognized that manipulative techniques involving other orders of addition and other binding react ion formats can be devised for carrying out homogeneous and heterogeneous specific binding assays without departing from the inventive concept embodied herein. 4-8 2 6 4 prosthetic group As previously stated, prosthetic groups are an art recognized distinct class of enzyme cofactors principally characterized by their ability to be regenerated to the active 5 cofactor form by the parent apoenzyme. The present invention employs organic prosthetic groups which can be chemically coupled to the binding component in the labeled conjugate. Usually, the binding constant for association of the apoenzyme and the prosthetic group whose residue is comprised in the labeled conjugate of the present invention is greater than ~ 1 8-1 about 10° molar , and preferably is greater than 10° molar The actual binding affinity of the prosthetic group residue appearing in the labeled conjugate may be decreased from that for the free prosthetic group by a few percent or as much as two orders of magnitude without affecting the utility of such prosthetic group residue as a label component in a binding assay.

Following is a table listing several prosthetic groups useful in the present invention, the conjugated enzyme produced by combination with apoenzyme, and binding constants for association of the prosthetic group and the apoenzyme: prosthetic group conjugated enzyme binding constant (molar '-*·) referen flavine adenine dinucleotide (FAD) glutathione reductase (human erythrocytes) 2 χ 106 1 , i 10 flavin mononucleotide (FMN) cytochrome reductase (yeast) 109 ) FMN NADPH: oxidoreductase (old yellow enzyme) 3 x 1()7 2 FAD glucose oxidase (Aspergillus nigerl >109 4 15 FAD lipoamide dehydrogenase 4 x 106 Γ .» FMN pyridoxine phosphate oxidase 5 χ 107 6 20 heme peroxidase (horse radish) >109 1 heme cytochrome C >109 8 iScott et al, J. Biol. Chem. 238:3928(1963). 3Haas et at, J. Biol, Chem. 143:341(1942). 3Staal et al, Biochim. Biophys. Acta 385:39(1969). ^Swoboda, Bioohim. Biophys. Acta 175:365(1969).

JVisser and Veeger, Bioohim. Biophys. Aota 200:224(1970). ^Arsenis and McCormick, J. Biol. Chem. 243:330(1966). 7Theorell et al, Arkiv. Kemi. Min. 0. Ceol. 348:1(1940). ^Vonetani, ./. liiol. Cham. 242:5008(1967).

A preferred prosthetic group/apoenzyme pair is heme and .ipoperoxida.se, and particularly preferred is flavin adenine dinucleotide and apoglucose oxidase. Such prosthetic groups provide ready means for attachment to ligands, ligand analogs and binding partners, and the resulting holoenzymes participate in hydrogen peroxide detection reactions that are well known and utilized analytically and that can be selected to generate colorimetric responses which are advantageous to the clinical laboratory technician. 1() It should be realized that a particular cofactor may function as a coenzyme or a prosthetic group depending on the enzyme system in which it is placed (e.g., FAD), however, the present invention contemplates the use of such a cofactor only in conjunction with an enzyme system in which it functions as •5 a prosthetic group as defined hereinbefore.

LINKING GROUP It will be recognized that there are many methods available for linking the binding component of the labeled conjugate, e.g., the ligand to be detected, a binding analog thereof, or a bind21' ing partner thereof, to the prosthetic group. The particular chemical character of the linking group will depend upon the nature of the respective available linking sites on the binding component and the prosthetic group. The important considerations in selecting the linking sites usually are (1) preservation of the ability of the linked binding component to participate effectively in the selected binding assay system and (2) preservation of the ability of the linked prosthetic group residue to com bine with apoenzyme (or inhibition of such binding ability where such can be relieved upon binding of the labeled conjugate hy a binding partner - see illustrative monitoring scheme SI), 8 3 6 4 in both cases, to the extent that a useful assay will result for the particular ligand under assay and for the particular concen trations or amounts in which such ligand is to be detected. Usually the linking group will comprise a chemical bond, usually a single, but sometimes a double bond, or a chain containing between 1 to 14, more commonly 1 to 6, carbon atoms and 0 to 5, more commonly 1 to 3, heteroatoms selected from nitrogen, oxygen, and sulfur.

Both the prosthetic group and the binding component, of 1,1 course, will offer a great diversity of available functionalities for attachment of the linking group. Commonly the Functionalities that can be expected to be available to the Jinking group are amino, usually primary amino; hydroxyl; halo, usually chloro or bromo; carboxylic acid; aldehyde; keto; isothiocyanate; isocyanate; and so forth. Accordingly, the chemical structure of the linking group itself will vary widely with its terminal groups depending on the functionalities available on the prosthetic group and the binding component and its overall length being a matter of choice within the basic constraints of maintaining the essential prosthetic group and binding component characters of the resulting conjugate.

With regard to the length of the linking group in preparing a conjugate for use in a homogeneous assay format, it is usually desirable to use as short a group as possible without causing the resulting binding component in the conjugate to intcrfer significantly with the prosthetic group activity of the conjugate. Where the binding component is of low molecular weight (e.g., a hapten of molecular weight between 100 and 1000), the linking group is preferably a chemical bond or a 1 to f) atom chain such as lower alkyl, carbonyl, alkyl carbonyl , amido, a 1kylamide, and the like. In other circumstances, such as 8 2 6 4 where the binding component in the conjugate is οΓ relatively high molecular weight, such as a polypeptide or protein (e.g., an antibody), a longer linking group is usually desirable to prevent steric hindrance of the apoenzyme combining site of the conjugate. In these cases, the linking group will comprise usually 1 to 14 carbon atoms and 0 to 5 heteroatoms as previously discussed. Chains of any significantly greater length sometimes result in conjugates in which the binding component will tend to fold back into the apoenzyme combining site. With these considerations in mind, examples of linking groups are shown in following Table 1. Particular examples of linking groups will be seen hereinafter and further variations will he readily recognized as being state-of-the-art.

TAP LB I linking group -R11 11 2 -R -C-R 1 11 2 -R -C-X-R 1 1 2 -R-X-C-R prosthetic group — -R1-X-C-X-R2binding component II τ II -C-R -C1 2 -R -X-R -X-R -R1-X15 -X-R -Xwherein X is imino, sulfur or, preferably, oxygen; and R1 and R are, independently, a lower alkylene having 1-ft carbon atoms, such as methylene, ethylene, isopropylene, butylene or hexylene. 8 2 6 4 ΑΡΟΕΗΖΪΜΕ The apoenzyme, or protein portion of the holoenzyme devoid of the prosthetic group, is prepared by splitting the conjugated enzyme and isolating the freed apoenzyme protein.

Splitting of the conjugated enzyme can be accomplished in a variety of known manners such as by placing the enzyme in a highly acid solution, e.g., pH less than 2. Likewise, recovery of the apoenzyme can be accomplished by a variety of known techniques, including selective precipitation of the protein or selective adsorption of the low molecular weight freed prosthetic group.

As indicated previously, apoperoxidase and apoglucose oxidase are particularly preferred apoenzymes for use in the present invention because their holoenzymes participate 1-5 in analytically advantageous hydrogen peroxide reactions. lt follows that other related conjugated enzymes, particularly the oxidoreductases, that catalyze reactions producing hydrogen peroxide and that can be split to yield an inactive apoenzyme will find particular application in the present invention. 1(1 Published methods of isolating apoenzymes are available, particularly for apoglucose oxidase, Biochim. Bioiihys. Aeba. 7/5:365(1969) and for apoperoxidase, J. Biol. Chem. ,10(7:109 (1953) and Arkiv. Kerni. Min. 0. Geo'l. 140:1(1940).

Should there arise the possibility of interference in the assay due to the presence of endogenous prosthetic group in the sample or in any of the reagents or due to prosthetic group contamination of laboratory equipment, glassware or plasticware, such interfering prosthetic group can be readily eliminated by available inactivation techniques. For example, FAD inter50 L'erence can be eliminated by sequential treatment witli periodate and ethylene glycol solutions, or by use of other FAD inactivation techniques. 48364 The present invention will now be illustrated, but is noi intended to be limited, by the following examples.

EXAMPLE 1 Homogeneous Binding Assays for N-2',4'-Dinitrophenyl-6-aminocaproate A. Preparation of the labeled conjugate - N6(6-aminohexyl) - dinitrophenyl-flavin adenine dinucleotide Flavin N^-aminohexyl-adenine dinucleotide.

N^-Trifluoroacetamidohexyl-adenosine-5'-monophosphate was ID synthesized by the method of Trayer et al, Bioohem. J. IJ.1?: 609(1974).

Fifty-six milligrams (mg) of N6-trifluoroacetamidohexyl -adenosine-5'-monophosphate (0.1 mmol) was dissolved in about 10 milliliters (ml) of water and 25 microliters (μI) of tri-n-butylamine (0.1 mmol) was added. The water was removed under vacuum and the residue was dissolved in 10 ml of dry dimethylformamide (DMF) which was then removed under vacuum.

The residue was evaporated from dry DMF three more times. The final residue was dissolved in 10 ml of dry DMF. Eighty milli2d grams of Ν,Ν'-carbonyldiimidazole (0.5 mmol) was added and allowed to react for 1.5 hours. Then 15 μΐ of water was added and the solvent was removed under vacuum. The residue (N&-trifluoroacetamidohexyl-adenosine-5'-monophosphate imidazolide) was dissolved in 10 ml of DMF.

Forty-seven milligrams of riboflavin-5'-monophosphate (0.1 mmol) was dissolved in about 10 ml of water and added dropwise to 20 ml of acetone containing 43 μΐ of tri-n-octylamine (0.1 mmol). A precipitate formed before the addition was 2 6 4 complete. The solvent was removed with a rotary evaporator until the riboflavin-5'-monophosphate dissolved. Then 5 ml of acetone and 5-10 ml of DMF were added and the mixture was taken to dryness. The residue was dissolved in 15-20 ml of dry DMF and taken to dryness (this process was repeated threo times). The residue was dissolved in 5 ml of DMF and combined with the above-mentioned 10 ml solution of the imidazolide in DMF.

The reaction mixture was allowed to stand at room temperature overnight and then the solvent was removed. The residue was taken up in 50 ml of water and applied to a 2.5x25 centimeter (cm) column of DEAE-cellulose in the bicarbonate form (Whatman DE23, Reeve Angel, Clifton, New Jersey USA). The chromatogram was developed with a linear gradient generated with two liters (1) of water and two liters of 0.3 molar (M) ammonium bicarbonate (23 ml fractions were collected). Thin -layer chromatography on silica gel 60 F254 (E. Merck, Darmstadt, West Germany) using a 7:3 volume:volume (v:v) mixture of ethanol - 1 M triethylammonium bicarbonate (pH 7.5) showed that fractions numbered 68 to 73 contained major (R^ = 0.75) and minor (R^ = 0.36) yellow compounds. These fractions were pooled and the optical absorption spectrum had maxima at 267, 373 and 450 nanometers (nm).

Tbe solvent was removed from the pooled material and the residue was dissolved in about 5 ml of water. This solution was adjusted to pH 11.0 with 5 N sodium hydroxide and allowed to stand at room temperature for nine hours. Thin-layer chroma tography showed that the component with = 0.75 disappeared while a new yellow material with = 0.37 appeared. The reaction mixture was adjusted to pH 8.0 with hydrochloric acid and applied to a 2.5x20 cm column of DEAE-cellulose in the bicarbonate form. The chromatogram was developed with a linear 8 2 6 4 gradient developed with one liter of water and one liter of (1.2 M ammonium bicarbonate. The yellow effluent from the column was pooled and the solvent was removed. The residue was adsorbed onto 2 grams (g) of silica gel which was placed •r> atop a 50 g column of silica gel equilibrated with a 9:2 (v:v) mixture of ethanol - 1 M triethylammonium bicarbonate (pH 7.5). The column was eluted with an 8:2 (v:v) mixture of ethanol - 1 to triethylammonium bicarbonate (pH 7.5), the yellow component with Rf = 0.37 was collected, and the solvent was removed. The yield based on absorbance at 450 nm was about 10’,. (6-aminohexyl)-dinitrophenyl-flavin adenine dinucleotide.

The residue from above containing flavin N1’-aminohexyl -adenine dinucleotide was purified by chromatography on Sephadex G-1O (Pharmacia Fine Chemicals, Uppsala, Sweden; Sephadex is a Registered Trade Mark). To a 0.9 x 30 cm column equilibrated with 25 millhnolar (nM) sodium bicarbonate (pH 7.5) at roan temperature was applied 1 ml of an approximately 10 mM solution of the Nb-FAD derivative in water. The first eluted peak of material absorbing at 450 nm was collected and rechromatographed on Sephadex G-10 and the first eluting peak collected again. 0.5 ml (0.63 pmol) of the purified N^-FAD derivative in mM sodium bicarbonate (pH 7.5) was mixed with 2 ml of ethanol and 10 μΐ of H-dinitrofluorobenzene (6.3 umol, 50 nCi) in ethanol were added (the tritiated dinitrofluorobenzenc was obtained from the Radiochemical Centre, Amersham UK). The reaction mixture was shaken continuously overnight in the dark at room temperature and then applied to a 0.9x30 cm column of Sephadex G-10 equilibrated with 0.1 M phosphate buffer (pH 7.0) containing 0.1% sodium azide. The single peak of material absorbing at 450 nm was collected and found to contain 8% of the radioactive label.

B. Preparation of apoglucose oxidase Purified glucose oxidase with low catalase activity obtained from the Research Products Division of Miles Laboratories, Inc., Elkhart, Indiana USA was dialyzed twice for 12 hours each against 0.5% weight:volume (w:v) mannitol (30 volumes each). Aliquots of the dialyzate containing 100 milligrams (mgi of glucose oxidase each were lyophilized and stored at -20°C.

Bovine serum albumin (200 mg) was dissolved in 12 ml of water adjusted to pH 1.6 with concentrated sulfuric acid, mixed with 150 mg charcoal (RIA grade from Schwarz-Mann, Orangeburg, New York USA), and cooled to 0°C. Lyophilized glucose oxidase (100 mg) was redissolved in 3.1 ml of water and 3 ml was added to the stirred albumin-charcoal suspension with continued stirring for 3 minutes. The suspension was then filtered through a 0.8 micron, 25 millimeter (mm) diameter Millipore filter (Millipore Corp., Bedford, Massachusetts USA) mounted in a Sweenex filter apparatus (Millipore Corp.) on a 50 ml disposable plastic syringe. The filtrate was quickly neutralized to pH 7.0 by addition of 2 ml of 0.4 M phosphate buffer (pH 7.6) and thereafter 5. N sodium hydroxide. Dry charcoal (150 mg) was then added and stirred for one hour at 0°C.

The resulting suspension was filtered first through a 0.8 micron Millipore filter and then through a 0.22 micron Millipore filter. To the filtrate was added glycerol to 25% (v:v) and the stabilized apoglucose oxidase preparation was stored at 4°C.

C. Assay Reagents 1. Labeled conjugate - N6-(6-aminohexyl)-DNP-FAD wa;· diluted in 0.05 M phosphate buffer (pH 7.0) to a concentration of 118 nM. 2. Apoenzyme - Apoglucose oxidase was diluted with 0.1 M phosphate buffer (pH 7.0) containing 0.1% bovine serum albumin to a concentration of 958 nM FAD binding sites. The FAD binding site concentration of the apoenzyme preparation was determined experimentally by measuring the minimum amount of FAD required to give maximum glucose oxidase activity when incubated with the apoenzyme. 3. Antiserum - Antiserum against a dinitrophenyl-bovine serum albumin conjugate was obtained from Miles-Ycda, 7-0 Ltd., Rehevot, Israel and was diluted 31 fold in 0.1 M phosphate buffer (pH 7.0) containing 0.1% bovine serum albumin. 4. Standards - The standard solutions contained known concentrations of N-2',4'-dinitrophenyl-ϋ -aminocaproate prepared in 0.05 M phosphate buffer (pH 7.0).

. Monitoring reagent - Λ glucose oxidase reagent was prepared by mixing 45 ml of 0.1 M phosphate hullei (pH 7.0) containing 15 mM ethylenediamine tctraacelii acid, 9 ml of 11.5 mM 3,5-dichIoro-2-hydroxybenzeiu’ sulfonate in water adjusted to pH 7 with sodium hydroxide, 9 ml of 11.5 mM 4-aminoantipyrine in water containing 1.25 mg/ml of peroxidase (Miles Laboratories, Inc., Elkhart, Indiana USA), and <> ml of 1.0 M glucose in aqueous saturated benzoic acid solution. 1). Assay Procedures 1. Addition of apoenzyme before initiation of hindi_ng reaction Method #1 - The following were added in sequence to separate reaction cuvettes: 0.1 ml of the labeled conjugate solution, 0.05 ml of a selected standard solution, 0.1 ml of the antiserum solution, 2.0 ml of the monitoring reagent, and 0.1 ml of the apoenzyme solution. Each reaction mixture was incubated for 30 minutes at 30°C and the absorbance at 520 nm measured.

Method H2 - The following were added in sequence to separate reaction cuvettes: 0.1 ml of the labeled conjugate solution, 0.05 ml of a selected standard solution, 0.1 ml of the antiserum solution, and (1.1 ml of the apoenzyme solution. Each reaction mixture was incubated for 20 minutes at room temperature and then 2.0 ml of the monitoring reagent was added to each. After further incubation for 15 minutes at 30°C the absorbance at 520 nm was measured in each cuvette. 2. Addition of apoenzyme after initiation of hinding reaction Method #3 - The following were added in sequence to separate reaction cuvettes: 0.3 ml of labeled conjugate solution, 0.05 ml of a selected standard solution, and 0.1 ml of the antiserum solution.

Each reaction mixture was incubated for 20 minutes at room temperature and then 2.0 ml of the monitoring reagent and 0.1 ml of the apoenzyme solution were added to each. After further incubation for minutes at 30°C the absorbance at 520 nm was measured in each cuvette.

Method #4 - The following were added in sequence to separate reaction cuvettes: O.i ml of the labeled conjugate solution, 0.05 ml of a selected standard solution, and 0.1 ml of the antiserum solution.

Each reaction mixture was incubated for 20 minutes at room temperature and then 0.1 ml of the apoenzyme solution was added to each. After further incubation for 20 minutes 2.0 ml of the monitoring reagent was added to each cuvette. Then each reaction mixture was incubated for 15 minutes at 30°C and tlie absor bance at 520 nm was measured in each cuvette. li. Results Following is Table 2 showing the results of the four assay procedures in measuring N-2',4'-dinitrophenyl-6-aminocaproate.

The concentrations of N-2',41-dinitrophenyl-6-aminocaproate are expressed as concentrations in the final 2.35 ml reaction mixture volumes. The absorbance results are expressed as the average of duplicate runs corrected for residual enzyme activity and background absorbance in the reagents. The correction factor is given for each assay method at the end of Table 2 and was deter10 mined by experimental runs substituting 0.1 M phosphate buffer (pH 7.0) for all of the labeled conjugate, standard, and antiserum solutions. r<4 co mi P fi O υ fi fi fi fi o w © < OJ oo fi fi 0) > < Ό 0) P υ o © CM © 00 r^· fi- ♦fi Γ» © Lfi © oo cn © ♦fi tn ♦fi ♦fi lfi lfi CM r-4 © © ♦fi ffi r-4 © r-- Lfi ffi CM CM CM rrt rrt i-4 r-l r-l © © o © © © © © O tfi r*. r·» ♦fi Lfi fi· ♦fi fM fi· © 1—4 © o © ¢0 Lfi ♦fi ♦fi CM o o O i—4 CM r-4 r-4 rd i—4 r-4 r-4 r-4 i—l r-l r-4 CM o © © © © © O © O © O © cn CM 00 © lfi Lfi © Lfi ♦fi r- r-4 o0 eo © © 00 fi- © CM © oo © o © 00 © ΙΛ ♦fi CM CM f—1 r-4 i-4 r-4 © © © O © © O © O O o fi· ♦fi lfi r-4 fi Lfi i-4 fi· CM ♦fi r- »—1 K. © ♦fi CM © © fi· ♦fi CM CM © fi- CM CM CM CM rrt rrt rrt rrt r-4 irt r-4 CM © © © O © O © © O O O o P fi o fi (X fi υ o fi Ο ι fi fi i O r-l •H >, P fi fi CM © r*4 in tn CM oO CM * * * * fi· CM © 00 <3· CM O fi lfi r-4 * * * * fi O P υ fi m fi o •rt P U Φ fi fi o u fi· CM ι <5 The results demonstrate that the present invention provides a specific binding assay method of the homogeneous type wherein apoenzyme is introduced either before or after initiation of the binding reaction.

EXAMPLE 2 Heterogeneous Binding Assay for Thyroxine A. Preparation of the labeled conjugate - N^-(2-aminoethyl) -thyroxine-flavin adenine dinucleotide 6-(2-Aminoethyl)-amino-9-(21,3'-O-isopropylidine-g-D 10 -ribofuranosyl) purine. 13.56 g (41.5 mmol) of 6-chloro-9-(2',3'-O-isopropylidene -g-D-ribofuranosyl) purine [Hampton et al, J. Am. Chem. Soe. H3: 150(1961)] was added with stirring over a 15 minute period to a cold excess of 1,2-diaminoethane (75 ml). The resulting solution was allowed to stand at room temperature for 24 hours. The solution was evaporated in vaauo and the resulting yellow oil was stirred with 50 ml of cold, saturated sodium bicarbonate.

The mixture was evaporated in vaauo and the resulting residue was further repeatedly evaporated in vaauo first from water (3 times from 50 ml) and then from 2-propanol (4 times from 50 ml) to obtain a yellow glass (15 g). A portion (3 g) of the glass was passed through a 25x55 centimeter (cm) Dowex 50W-X2 cation exchange column in the ammonium form (Bio-Rad Laboratories, Richmond, California USA). (Dowex is a Trade Mark). 8 2 6 4 The column was eluted with a linear gradient generated with 2 1 of water and 2 1 of 0.5 M ammonium bicarbonate. The elution was completed using a linear gradient generated with 2 1 each of 0.5 M and 1 M ammonium bicarbonate. The effluent from the column was collected in 19 ml fractions and monitored by elution on silica gel thin layer chromatography (TLC) plates (E. Merck, Darmstadt, West Germany) with a 9:1 (v:v) , mixture of ethanol and ammonium hydroxide. The developed TLC plates were examined under ultraviolet light, then sprayed with ninhydrin reagent [Randerath, Thin Layer Chromatography , Academic Press (1966)]. Fractions numbered 250 through 350 from the column chromatography were combined and evaporated in vacuo leaving the desired purine as a pale yellow amorphous glass (1.5 g).

Analysis: Calculated for 0, 51.42; H, 6.33; N, 23.99 Found: C, 50.92; H, 6.54; N, 23.01 NMR (60 MHz, CDClj): fi 1.37 (s,3H, isopropylidene), 1.63 (s,3H, isopropylidene), 5.92 (d, IH, l'-ribose), 7.90 (s, IH, purine), 8.26 (s, Hi, purine) Optical Rotation [<*]ρθ = -74.85° (c 1.0, CHjOH) The remaining crude product (12 g) was purified by chromatography on Dowex 50W-X2 as described above. The overall yield was 8 g (55i).

A 8 2 6 ·1 u-(N-Trifluoroacetyl)-amino-β-[3,5-diiodo-4-(3',5'-di iodo-4' -hydroxyphenoxy)-phenyl] propanoic acid.

This compound was prepared by the method of Blank, J. I'hcru, Sci. 53:1333(1964). To a cooled (0°C), stirred suspension of 5 5 g (6.4 mmol) of L-thyroxine (Sigma Chemical Co., St. Louis, Missouri USA) in 60 ml of dry ethyl acetate was added 11.5 ml of trifluoroacetic acid and 1.9 ml of trifluoroacetic anhydride. After 30 minutes the resulting clear solution was washed three times with 30 ml of water, once with 30 ml of 5» sodium bicar10 bonate, and twice with 50 ml of saturated sodium chloride. The combined aqueous washings were extracted twice with 20 ml of ethyl acetate. The ethyl acetate layers were combined and washed with 30 ml of water, then dried over magnesium sulfate. The dried ethyl acetate solution was evaporated in vacuo leaving a white solid. Recrystallization from a mixture of ethyl ether and petroleum ether gave a pinkish-white solid (3.95 g, 70.5Ί yield) melting at 228-230°C with decomposition.

Analysis: Calculated for ^H^F^I^NO^: C, 23.39; 11, 1.15; N, 1.60 Found: C, 23.00; H, 1.05; N, 1.65 NMR [60 MHz, DCON(CD3)2] δ 7.28 (s, 2H, aromatic), 8.03 (s, 2H, aromatic), 9.7 (m, 111, amido) IR (KCl): 1700 (>C=O) Optical Rotation [a]^ = -14.97° (c 1.0, dimethylsulfoxide) A second recrystallization produced a second precipitate (0.95 g) m.p. 224-228°C with decomposition. The overall yield was 87.5',',. Ν-{2-[N-(Trifluoroacetyl)-3,5',5,51-tetraiodothyronyl| aminoethyD-21,5'-0-isopropylidene adenosine.

A solution of 8.72 g (10.0 mmol) of α-(N-trifluoroacety1) amino-β-[3,5-diiodo-4-(3',S'-diiodo-4'-hydroxyphenyoxy) phony 1) propanoic acid and 3.86 g (11.0 mmol) of 6-(2-aminoethyl) amino-9-(2',3’-0-isopropylidene-f5-D-ribofuranosyl) purine in SO ml of dry dimethylacetamide was prepared under a dry argon atmosphere at -20°C. To this cold stirred solution was added a solution of 3.04 g (11.0 mmol) of diphenylphosphory1 azide (Aldrich Chemical Co., Milwaukee, Wisconsin USA) in 10 ml of dry dimethylacetamide followed by the addition of 1.6 ml (11.0 mmol) of dry triethylamine. The solution was left at room temperature for 22 hours. The solution was then added dropwise to 300 ml of cold (0°C) water with stirring. The resulting white IS precipitate was collected by filtration and dried i-n tuuuo (S6°C) to give 13.0 g of a light cream colored solid. The solid was dissolved in 500 ml of acetone and the solution was concentrated by boiling. The white solid which precipitated from the boiling acetone solution was collected by filtration while hot. Continued boiling of the filtrate produced two additional precipitates. The three precipitates were combined to give 8 g (66.6% yield) of a white solid, m.p. 198-200°C (ile composed). 4 Analysis: Calculated for ε32Η30ί-374^7θ8: C> H, 2.51; N, 8.14 Found: C, 31.95; H, 2.60; N, 7.86 NMR [220 MHz, (CD3)2SOJ δ 1.32 (s, 3H, isopropylidene), 1.55 (s, 3H, isopropylidene) , 6.14 (d, IH, 1'-ribose), 7.02 (s, 2H, thyroxine), 7.82 (s, 2H, thyroxine), 8.25 (s, IH, purine), 8.36 (s, IH, purine), 8.41 (t, 111, J=6, amido), 9.64 (d, IH, J=8, trifluoroacetamido) Optical Rotation [a]^3 = -11.82° (c 1.0, pyridine) N-{2 - [N-(Trifluoroacetyl)-3,3',5,51 - tetraiodothyronyl| aminoethyl}-21,31-0-isopropylidene-5'-adenylic acid monotriethylamine salt monohydrate. Λ solution of 1.2 g (1.0 mmol) of N-{2-[N-(trifluoroacetyI) 3,3',5,51-tetraiodothyronyl]-aminoethyl}-21,3'-0-isopropylidene adenosine in 10 ml of dry triethylphosphate was prepared under a dry argon atmosphere at 0°C. To the cold, stirred solution was added 0.45 ml (5 mmol) of phosphorous oxychloride. The resulting solution was kept for 24 hours at 0°C, then added dropwise with stirring to 1 1 of ice water. The resulting precipitate was collected by filtration and dried in vacuo to give 1.23 g of a white solid. The solid was dissolved in acetone and 0.32 ml (2.2 mmol) of triethylamine was added. A precipitate formed. The mixture was evaporated in vacuo and the resulting residue lixiviated with dry acetone, then re42 crystalized from a mixture of dry methy] alcohol and dry ethyl ether to give 390 mg (27.8% yield) of a white solid, m.p. 173-183°C (decomposed).

Analysis: Calculated for C^gH^gF^I^NgO]C, 32.50; H, 3.45; N, 7.98 Found: C, 32.24; II, 3.08; N, 7.58 NMR [60 MHz, (CU3)2S0] 6 1.53 (s, 311, isopropylidene), 6.2 (d, 111, 1Ή-ribose), 7.1 (s, 2H, thyroxine aromatic), 7.87 (s, 2H, thyroxine aromatic), 8.27 (s, IH, purine), 8.52 (s, IH, purine) Optical Rotation [a]2® = -17.50° (c 1.0, CH^Oll) N-(2-[N-(Trifluoroacetyl)-3,3', S^'-tetraiodothyronyl1 aminoethyl}-5'-adenylic acid. 200 mg (0.14 mmol) of N-{2-[N-(trifluoroacetyl-3,3',5,5' tetraiodothyronyl]- aminoethyl}- 2',3'-O-isopropylidene- 51-adeny1i c acid monotriethylamine salt monohydrate was suspended in 1 ml of water (0°C) and trifluoroacetic acid (9 ml) was added dropwise with stirring. After 30 minutes a clear solution was obtained. The solution was kept cold (0°C) for an additional 15 hours, then evaporated in vacuo (30°C). The resulting residue was evaporated four times in vacuo (25°C) from 20 ml volumes of anhydrous ethyl alcohol and then dried in vaunt, (25°C) leaving a white solid. - 43 The solid was stirred for 30 minutes with 10 mJ ol cold methyl alcohol, then collected by filtration and dried in ujcua (25°C) to give a white solid (135 mg, 76% yield) whicli slowly melted with decomposition above 188°C.

Analysis: Calculated for ^gf^yF^I^NyOuP: C, 27.97; 11, 2.19; N, 7.87 Found: C, 28.11; H, 2.31; N, 7.65 NMR [220 MHz, (CD3)2S0] δ 5.95 (d, 111, 1'-ribose), 7.04 (s, 2H, thyroxine aromatic), 7.84 (s, 2H, thyroxine aromatic), 8.25 (s, IH, purine), 8.36 (s, IH, purine), 8.43 (m , III, amido), 9.66 (d, 111, trifluoroacetamido) Optical Rotation [a]2^ = -2.72° (c 1.0, pyridine) Flavin adenine dinucleotide - thyroxine conjugate. 498 mg (0.4 mmol) of N-{2-[N-(trifluoroacetyl)-3,3',5,5' tetraiodothyronyl]-aminoethyl)-5'-adenylic acid was dissolved in 10 ml of dry dimethylformamide and tri-n-butylamine (96 μΐ, 0.4 mmol) was added followed by the addition of 1,1'-carbonyldiimidazole (320 mg, 2.0 mmol). After stirring for 18 hours at room temperature in the absence of moisture, water (280 μΐ) was added and then the solvent evaporated in vacua.

The resulting oil was dried by repeated in vacuo evapora tion from dry dimethylforamide (4 times from 10 ml). The resulting phosphorimidazolidate was redissolved in 10 ml of dry dimethylformamide and added dropwise to a 0.4 mmol solution of the tri-n-octylamine salt of riboflavin-5'-monophosphate in ml of dry dimethylformamide. The salt was prepared by addim a solution of the ammonium salt of riboflavin-5'-monophosphate (192 mg, 0.4 mmol) in 10 ml of water to a stirred solution of tri-n-octylamine (176 pi, 0.4 mmol) in 100 ml of acetone. After minutes, the resulting mixture was evaporated in vacuo. The residue was dried by repeated evaporation in vacuo from dry dimethylformamide leaving the salt as an orange solid.

The above solution containing the phosphorimidazolidate and the riboflavin-5'-monophosphate salt was divided Into two equal aliquots after 24 hours and one aliquot was evaporated in vacuo. The resulting residue was chromatographed on a column (2.5x78 cm) prepared from 100 g of Sephadex 1,11-20 (Pharmacia Fine Chemicals, Uppsala, Sweden) which had been preswollen (18 hours) in a 19:1 (v:v) mixture of dimethylforma mide and triethylammonium bicarbonate (1 M, pH 7.5). The column was eluted with the above 19:1 (v:v) mixture and 10 ml fractions were collected. The effluent from the column was monitored by elution on silica gel 60 silanised RP-2 TLC plates (E. Merck, Darmstadt, West Germany). 2li The TLC plates were developed using a 40:40:25:1:1 (v:v) mixture of acetone, chloroform, methyl alcohol, water, and triethylamine. Fractions numbered 11 through 17 from the above -mentioned column chromatography were combined and evaporated in vacuo. The residue was chromatographed on a column (2.5x75 cm) prepared from 125 g of Sephadex LH-20 which had been preswollen (18 hours) in 0.3 M ammonium bicarbonate. The column was eluted with 0.3 M ammonium bicarbonate collecting 10 ml fractions. The effluent was monitored by absorption of ultraviolet light at 254 nm. The volume of the fractions was increased to 20 ml beginning with fraction number 150. The salt concentration of the eluent was decreased in a stepwise fashion as follows: 0.15 M ammonium bicarbonate at fraction number 295, 0.075 M ammonium bicarbonate at fraction number 5 376, and water at fraction number 430. A total of 480 fractions was collected. Fractions numbered 200 through 235 were combined and evaporated in vacuo leaving the labeled conjugate as a yellow-orange residue. An alkaline, aqueous solution of this residue exhibited ultraviolet absorption maxima at the following wavelengths: 266 nm, 350 nm, 373 nm, and 450 nm.

The yield, estimated from the absorption at 450, was about 5».

A phosphodiesterase preparation (Worthington Biochemical Corp., Freehold, New Jersey USA) isolated from snake venom (Crotalus Adamanteus} hydrolyzed the aoove product to riboflavin-5'-monophosphate and the thyroxine substituted '-adenylic acid wherein the trifluoacetyl blocking group had been removed.

Further description of the preparation of labeled conjugates of this type may be found in the U.S. patent application filed on even date herewith entitled Flavine Adenine Dinucleotide - Iodothyronine Conjugates (Docket No. 11795) and assigned to the present assignee. - 48264 B. Preparation of apoglucose oxidase Apoenzyme prepared according to part B of Example 1 was used.

C. Assay Reagents 1. Labeled conjugate - N^-(2-aminoethyl)-thyroxine-PAD was diluted in 0.1 M phosphate buffer (pH 7) to a concentration of 1 μΜ. 2. Apoenzyme - Apoglucose oxidase was diluted with 0.1 M phosphate buffer (pH 7) to a concentration of 0.6 μΜ PAD binding sites (defined as in part C-2 of Example 1). 3. Insolubilized antibody - A washed, moist cake of Sepharose 4B gel (Pharmacia Fine Chemicals, Uppsala, Sweden) activated by cyanogen bromide according to the method of March at al, Anal. Biochem. GO:119 (1974) was added to a solution of 85 mg of antibody (isolated from antiserum against a thyroxine-bovine serum albumin conjugate) in 20 ml of 0.1 M phosphate buffer (pH 7.0) and agitated slowly for 36 hours at 4°C. Upon completion of the coupling reaction, 1 ml of 1 M alanine was added and shaking continued for more hours to block unreacted sites. The resulting Sepharose-bound antibody was washed on a sintered funnel with 400 ml each of 50 mM sodium acetate 500 mM sodium chloride (pH 5), and 50 mM phosphate buffer - 500 mM sodium chloride (pH 7), and 800 ml of 100 mM phosphate buffer (pH 7).

The moist filter cake was then suspended in 100 mM phosphate buffer (pH 7) containing 0.01¾ sodium azide to give 22 ml of an about 50% suspension. 4. Standard - A 1.15 mM stock solution of thyroxine in mM sodium hydroxide was diluted to 2 pM in 0.1 M phosphate buffer (pH 7).

. Monitoring reagent - A glucose oxidase assay reagent was prepared to contain the following mixture per 130 pi: 25 pi of 1.2 mg/ml peroxidase (Sigma jo Chemical Co., St. Louis, Missouri USA) in 0.1 M phosphate buffer (pH 7), 5 pi of 10 mM 4-aminoantipyrine in water, 20 pi of 25 mM 3,S-dichloro-2-hydroxybenzene sulfonate in 0.1 M phosphate buffer (pH 7), 30 pi of 16.5% bovine serum albumin in 0.1 M phosphate buffer (pH 7), and 50 pi of 1 M glucose in aqueous saturated benzoic acid solution.

D. Assay Procedure Binding reaction mixtures were prepared by mixing 150 pi of the insolubilized antibody suspension, 80 pi of the labeled conjugate solution, various amounts of the standard thyroxine solution to give varying concentrations of thyroxine in the reaction mixtures, and a sufficient volume of 0.1 M phosphate buffer (pH 7) to make a total volume of 500 pi. The reaction mixtures were incubated with shaking for two hours at 25°C. Each reaction mixture was then vacuum filtered through a glass wool plugged, dry pasteur pipette previously treated 2 6 4 with periodate and ethylene glycol solutions to eliminate possible FAD contamination. To a 300 μΐ aliquot of end) fil trate was added 130 μΐ of the monitoring reagent and 50 |il of the apoenzyme solution. After one hour, the absorbance of each reaction mixture was measured nt 520 nm.

E. Results Following is Table 3 showing the results of the assay procedure in measuring thyroxine. The absorbance results are expressed as the average of duplicate runs corrected for residual enzyme activity in the apoenzyme solution (absorbance of 0.522) and for endogenous FAD in the antibody suspension (absorbance 0.142).

TABLE 3 Volume of Thyroxine Standard Added (ul) 250 Corrected Average Absorbance at 520 nm 0.223 0.221 0.281 0.286 The results demonstrate that the present invention provides a useful specific binding assay method of the heterogeneous type.

EXAMPLE 3 Homogeneous Binding Assay for Thyroxine A. Preparation of the labeled conjugate - N^-(6-aminohexyl) -thyroxine-flavin adenine dinucleotide (()-Aminohexyljamino-9-(2’,3'-O-isopropylidene-g-D -furanosyl) purine. 16.0 g (50 mmol) of 6-chloro-9-(2',3'-0-isopropylidencg-D-ribofuranosyl) purine [Hampton et al, J. Am. Chem. Son.

: I 501 (1 96 1 ) [ was added with stirring to a molten (70°C) sarnpl oi freshly distilled 1,6-diaminohexane (58 g, 500 mmol). The resulting mixture was stirred under argon at 40°C for 18 hours. The excess diamine was removed by distillation under reduced pressure (60°C, 0.91 mm Hg). The resulting pale yellow residue was adsorbed onto ISO g of silica gel 60 (E. Merck, Darmstadt, West Germany) and used to top a chromatographic column prepared from a slurry of silica gel 60 (2 kg) in a 9:1 (v:v) mixture of absolute ethyl alcohol and triethylammonium bicarbonate (pH 7.5, 1 M). The column was eluted with the above 9:1 (v:v) solvent mixture and 900 20 ml fractions were collected. The (i fractions were examined by thin layer chromatography (TLC) on silica gel 60 eluting with a 7:3 (v:v) mixture of absolute ethyl alcohol and triethylammonium bicarbonate (pH 7.5, IM). fractions numbered 391 through 900 from the column chromatography were combined and evaporated in vacuo leaving 15.0 g of 482C4 a glassy residue (.74« yield). A 1 g sample of tlie glass was A i ;·,.>« 1 veil ia a small volume of methyl a 1 x<.hot which was then applied lo the top of a column prepared 1 row 80 g of Sephadex 1.11-20 (Pharmacia lino Chemicals, Uppsala, Sweden) preswollen in methyl alcohol. The column was eluted with methyl alcohol.

A total of ninety 8 ml fractions were collected. The fractions wore examined by Tl.C on silica gel 60 eluting wirh a 7:3 (v;v) mixture of absolute ethyl alcohol and tri el hyianimoni um blear Donate (pH 7.5., IM). Fractions numbered 19 through 27 from the column chromatography were combined and evaporated in -no leaving 910 mg (91% recovery) of a white glass.

Analysis: Calculated for C-j: C, 56.14; 11, 7.44; N, 20.68.

Found: C, 53.91; H, 7.35, N, 19.18 NMR (60 MHz, CDClj): δ 1.40 (s, 3il, isopropylidene), 1.63 (s, 311, isopropylidene) 5.98 (d, IH, l'-ribose), 7.92 (s, 111, purine), 8.36 (s, 111, purine) Optical Rotation la]2p = -50.11“ (c 1.0, methyl alcohol) 1. ί ο -1 N- Tr i fluoroacetyl) - 3 ,]>' , 5,5' - tet ra iodo thyron/_l_£ amrnotiexvi )- 2 '_,_31 -O-isopropyi idone adeoosine.

A solution of 4.36 g (5.0 mmol) of κ (N-trifluoroaeetyl) amino-0-(3,5 - Jiiodo-4-(3',5'-diiodo-4'-hydroxyphenoxy)-phenyl propanoic acid, prepared as described in part A of Example 2 above, and 2.24 g (5.5 mmol) of 6-ί 6-aiiiinohexyl)amino-9-( 2 1,31 O -isopropylidene-β-D-ribofuranosyl) purine in 100 ml of dry dimethylformamide was prepared under a dry argon atmosphere at -20°C. To this cold stirred solution was added a solution of 1.52 g (5.5 mmol) of diphenylphosphoryl azide (Aldrich Chemical Co., Milwaukee, Wisconsin USA) in 50 ml of dry dimethylformamide followed by the addition of 0.8 ml (5.5 mmol) of dry triethylamine. The solution was left at room temperature for 22 hours. The solution was then added dropwise to 600 ml of cold (0°C) water with stirring. The resulting white precipitate was collected by filtration and dried in vacuo (60°C) to give 4.90 g (78% yield) of white solid. A sample of this solid was recrystallized from a mixture of acetone and water giving a white solid, m.p. 205-207°C (decomposed).

Analysis: Calculated for D^gH^gF^I^N^Og: C, 34.28; H, 3.04; N, 7.77 Found: C, 34.22; H, 2.99; N, 7.41 Mass Spectrum (20 ma) m/e: 1262 [MH+], 1164 [M+ minus COCF^] Optical Rotation [a]3^ = -21.89° (c 1.0, pyridine) N-{6-[N-Trifluoroacetyl)-3,5',5,5'-tetraiodothyronyl] aminohexyl}-2',3'-0-isopropylidene-51-adenylic acid monotriethylamine salt monohydrate.

A solution of 1.89 g (1.5 mmol) of N-{6-[N-(trifluoroacetyl) -3,3',5,5'-tetraiodothyronyl]aminohexyl}-2',3'-O-isopropylidcne adenosine in 15 ml of dry triethylphosphate was prepared under a dry argon atmosphere at -10°C. To the cold stirred solution was added 0.68 ml (7.5 mmol) of phosphorous oxychloride. The resulting solution was kept for 18 hours at -15°C then added dropwise with stirring to 1.5 L of ice water. The resulting precipitate was collected by filtration and dried in vacuo to give 1.91 g (87% yield) of a white solid. The solid was dis5 solved in 10 ml methyl alcohol and 0.38 ml (2.6 mmol) of triethylamine was added. This solution was evaporated in vacuo and the resulting residue was recrystallized from a mixture of methyl alcohol and ethyl ether to give 720 mg (33% yield) of a white solid, m.p. 151-154°C (decomposed).

Analysis: Calculated for C, 34.54 ; H, 3.86; N, 7.67 Found: C, 35.24; H, 3.88; N, 7.75 Mass Spectrum (20 ma) m/e: 1342 [MH+J, 1244 [M+ minus COCFj] Optical Rotation [a]^ = -17.20° (c 1.0, CHjOIl) N-(6-[N-(Trifluoroacetyl)-3,3’,5,5’-tetraiodothyronyl] aminohexyl)-5'-adenylic acid. 600 mg (0.41 mmol) of N-(6-[N-(trifluoroacetyl)-3,3',5,5' -tetraiodothyronyl]aminohexyl}-2',3'-0-isopropylidene-5'adenylic acid monotriethylamine salt monohydrate was suspended in 0.6 ml of water (0°C) and trifluoroacetic acid (6 ml) was added dropwise with stirring. After 50 minutes a clear solution was obtained. The solution was kept cold (0°C) for an additional hours then evaporated in vaouo (30°C). The resulting residue was evaporated in vaouo five times from 20 ml volumes of anhydrous ethyl alcohol then triturated with 30 ml water and washed with a small volume of methyl alcohol. The resulting white solid (430 mg) was recrystallized from methyl alcohol to give 290 mg (54.6% yield) of white solid, m.p. 180-183°C (decomposed).

Analysis: Calculated for CggHjjFjI^NyO^^P: C, 30.46; H, 2.71; N, 7.54 Found: C, 30.77; H, 2.55; N, 7.29 Mass Spectrum (20 ma) m/e: 1302 [MH+], 1204 [M+ minus COCF^j Flavin adenine dinucleotide - thyroxine conjugate. 130.13 mg (0.1 mmol) of N-{6-[N-(trifluoroacetyl)-3,3',5,5' -tetraiodothyronyl]aminohexyl}-5'-adenylic acid was placed in an argon atmosphere. To this sample was added a solution of 14 pi (0.1 mmol) of triethylamine in 1 ml of dry dimethylformamide followed by the addition of a solution of 16.2 mg (0.1 mmol) of 1,1'-carbonyldiimidazole in 1 ml of dry dimethylformamide. After 24 hours, a second equivalent of 1,1' -carbonyldiimidazole (16.2 mg) in 1 ml of dry dimethylformamide was added. The above reaction was allowed to proceed a total of 48 hours at room temperature excluding moisture. A sample of 47.3 mg (0.1 mmol) of the ammonium salt of riboflavin - 5'-monophosphate was converted to the corresponding tri-n-octylamine salt as described in part A of Example 2.

This salt was dissolved in 3 ml of dry dimethylformamide and added to the above solution containing the phosphorimidazolidate of the adenylic acid intermediate.

The resulting solution was allowed to stand in the dark at room temperature excluding moisture for 24 hours. The solvent was evaporated in vaauo and the resulting residue was chromatographed on a column (2.5x78 cm) prepared from 100 g of Sephadex LH-20 (Pharmacia Fine Chemicals, Uppsala, Sweden) which had been preswollen (18 hours) in a 19:1 (v:v) mixture of dimethylformamide and triethylammonium bicarbonate (1 M, pH 7.5). The column was eluted with the above 19:1 (v:v) mixture and 5 ml fractions were collected. The effluent from the column was monitored by elution on silica gel 60 silanised RP-2 TLC plates (E. Merck, Darmstadt, West Germany). The TLC plates were developed using a 40:40:25:1:1 (v:v) mixture of acetone, chloroform, methyl alcohol, water, and triethylamine.

Fractions numbered 24 through 38 from the column chromatography were combined and evaporated in vaauo. The residue was chromatographed on a column (2.5x85 cm) prepared from 125 g of Sephadex LH-20 which had been preswollen (18 hours) in 0.1 M ammonium bicarbonate. The column was eluted with a linear gradient generated from 2 L of 0.1 M ammonium bicarbonate and 2 L of water and 23 ml fractions collected. The effluent was monitored by ultraviolet absorption (254 nm). Fractions numbered 170 through 182 were combined and evaporated in vaaur·. The residue was chromatographed on a column (2.5x55 cm) prepared from 80 g of Sephadex LH-20 which had been preswollen in 0.05 M ammonium bicarbonate. The column was eluted with a linear gradient generated from 2 L of 0.05 M ammonium bicarbonate and 2 L of 0.02 M ammonium bicarbonate. The effluent was monitored by ultraviolet absorption (254 nm). Elution was continued with 2 L of 0.2 M ammonium bicarbonate, collecting 23 ml fractions. 8 2 6 4 Λ total of 257 fractions was collected. Fractions numbered 70 through 110 were combined and evaporated in vacuo leaving the labeled conjugate as a yellow-orange residue. An alkaline, aqueous solution of this residue exhibited ultraviolet absorption maxima at the following wavelengths: 270 nm, 345 nm, and 450 nm. The yield, estimated from the absorption at 450 nm, was about 5%.

A phosphodiesterase preparation (Worthington Biochemical Corp. , Freehold, New Jersey USA) isolated from snake venom (Crotalus Adamanteus} hydrolyzed the above product to riboflavin-5 ' -monophosphate and the thyroxine substituted 5'-adenylic acid wherein the trifluoroacetyl blocking group had been removed.

Further description of the preparation of labeled conjugates of this type may be found in U.S. patent application Serial No. 917,962 filed June 22, 1978 and assigned to the present assignee.

B. Preparation of apoglucose oxidase Glucose oxidase was dialyzed and lyophilized as in part B of Example 1 above. A portion of the lyophilized glucose oxidase (80 mg) was dissolved in 20 ml of 30% (v:v) glycerol at 4°C and the solution adjusted to pH 1.4 by addition of concentrated sulfuric acid (H2SO^). The solution was incubated at 4°C for 2 hours and then passed through a column of Sephadex G-50 (Pharmacia Fine Chemicals, Uppsala, Sweden) at 4°C, equilibrated with 30% (v:v) glycerol adjusted to pH 1.4 with concentrated H-jSO^. The eluted protein peak was collected (27 ml containing 74% by weight of material added to the column) and 200 mg bovine serum albumin was dissolved in the pooled eluate. Charcoal (600 mg; RIA grade from Schwarz-Mann, Orangeburg, New York USA) was then added and the mixture neutra5 lized by addition of 4.0 ml of 0.4 M phosphate buffer (pH 8.0) and sufficient 2 N sodium hydroxide to adjust the pH to 7.0.

The mixture was stirred for 60 minutes at 4°C and filtered successively through 0.8 μ and 0.22 μ Millipore filters (Millipore Corp., Bedford, Massachusetts USA). Sodium azide (10¾ w:v) was added to give a final concentration in the mixture of 0.1¾.

C. Assay Reagents 1. Labeled conjugate - N6-(6-aminohexyl)-thyroxine-FAD was diluted in 0.1 M phosphate buffer containing 0.1% (w:v) bovine serum albumin (pH 7.0) to a concentration of 400 nM. 2. Apoenzyme - Apoglucose oxidase was diluted in 0.1 M phosphate buffer containing 0.1% (w:v) bovine serum albumin (pH 7.0) to a concentration of 4.0 micronormal (μΝ) FAD binding sites (see part C-2 of Example 1). 3. Antiserum - Saturated ammonium sulfate solution (7.5 ml) was pumped (0.1 ml/minute) at room temperature into ml of rabbit anti-thyroxine antiserum stirring on , 48264 an ice bath. The resulting suspension was allowed to stand for 2 hours at 4°C without stirring. The precipitate was collected by centrifugation, dissolved in 3 ml of ice cold 50 mM borate buffer (pH 8.6) and dialyzed overnight against 1 L of 50 mM borate buffer (pH 8.6). The immunoglobulin solution was then made up to the original antiserum volume by addition of SO mM borate buffer (pH 8.6). The immunoglobulin solution was further diluted in 0.1 M phosphate buffer containing 0.1¾ (w:v) bovine serum albumin (pH 7.0) for use in the assay procedures described below in the proportions indicated. 4. Standards - Thyroxine sodium salt (Sigma Chemical Co., St. Louis, Missouri USA) was used as a 1 mg/ml stock solution titrated into solution in distilled water with 2 N sodium hydroxide to a final pH of 10.2. Standards were prepared by dilution of this stock solution in 0.1 M phosphate buffer containing 0.1¾ (w:v) bovine serum albumin (pH 7.0).

. Monitoring reagent - A glucose oxidase assay reagent was prepared by mixing 11 parts 0.15 M phosphate buffer (pH 7.0) containing 1.8¾ (w:v) bovine serum albumin, 2 parts 2.0 mM 4-aminoantipyrine containing 0.6 mg/ml peroxidase, 2 parts 20 mM 3,5-dichloro-2 -hydroxybenzene sulfonate in 0.1 M phosphate buffer (pH 7.0); and 2 parts 1.0 M glucose in aqueous saturated benzoic acid solution.

I). Assay Procedures 1. Addition of all reagents without pre-incubation step The following were added in sequence to separate reaction cuvettes: 0.05 ml of the labeled conjugate solution, 0.05 ml of a selected standard solution, 0.1 ml of a 1:4 dilution of the antiserum solution in the phosphate buffer, 1.7 ml of the monitoring reagent, and 0.1 ml of the apoenzyme solution.

Each reaction mixture was incubated for 30 minutes at 20°C and the absorbance at 520 nm measured. 2. Pre-incubation with antiserum The following were added in sequence to separate reaction cuvettes: 0.05 ml of the labeled conjugate solution, 0.05 ml of a selected standard solution, and 0.1 ml of a 1:16 dilution of the antiserum solution in the phosphate buffer. Each reaction mixture was incubated for 20 minutes at 20°C and then 1.7 ml of the monitoring reagent and 0.1 ml of the apoenzyme solution were added to each in succession. After further incubation for 30 minutes at 20°C the absorbance at 520 nm was measured in each cuvette. 8 2 6 4 3. Pre-incubation with apoenzyme The following were added in sequence to separate reaction cuvettes: 0.05 ml of the labeled conjugate solution, 0.05 ml of a selected standard solution, and 0.1 ml of the apoenzyme solution. Each reaction mixture was incubated for 20 minutes at 20°C and then 0.1 ml of a 1:2 dilution of the antiserum solution in the phosphate buffer and 1.7 ml of the monitoring reagent were added to each in succession After further incubation for 30 minutes at 20°C the absorbance at 520 nm was measured in each cuvette. 4. Sequential pre-incubation with antiserum first and then apoenzyme The following were added in sequence to separate 15 reaction cuvettes: 0.05 ml of the labeled conjugate solution, 0.05 ml of a selected standard solution, and 0.1 ml of a 1:32 dilution of the antiserum solution in the phosphate buffer. Each reaction mixture was incubated for 20 minutes at 20 °C and then 0.1 ml of the apoenzyme solution was added to each. After further incubation for 20 minutes at 20°C, 1.7 ml of the monitoring reagent was added to each cuvette and the absorbance at 520 nm measured in each after a 30 minute incubation at 20°C.

. Sequential pre-incubation with apoenzyme first and then antiserum The following were added in sequence to separate reaction cuvettes: 0.05 ml of the labeled conjugate solution, 0.05 ml of a selected standard solution, and 0.1 ml of the apoenzyme solution. Bach reaction mixture was incubated for 20 minutes at 20°C and then 0.1 ml of a 1:32 dilution of the antiserum solution in the phosphate buffer was added to each.

After further incubation for 20 minutes at 20°C, 1.7 ml of the monitoring reagent was added to each cuvette and the absorbance at 520 nm measured in each after a 30 minute incubation at 20°C. 6. Pre-incubation with both antiserum and apoenzyme The following were added in sequence to separate reaction cuvettes: 0.05 ml of the labeled conjugate solution, 0.05 ml of a selected standard solution, 0.1 ml of the apoenzyme solution, and 0.1 ml of a 1:32 dilution of the antiserum solution in the phosphate buffer. Each reaction mixture was incubated for 20 minutes at 20 °C and then 1.7 ml of the monitoring reagent was added to each. After a further incubation of 30 minutes at 20°C, the absorbance at 520 nm was measured in each cuvette.

B. Results The assays were run in duplicate with appropriate blanks.

It was found that for methods 1-3 the concentration of thyroxine in the standard solution was directly related to glucose oxidase activity whereas for methods 4-6 the thyroxine concentration was inversely related to enzyme activity. For methods 1-3 in the absence of thyroxine, enzyme activation was inhibited whereas for methods 4-6 enzyme activation was enhanced. Methods 4-6 were also found to be somewhat more sensitive for thyroxine than the other methods. The reason or reasons why enzyme activation was variously inhibited or enhanced depending on the reaction sequence is not fully understood, however, the results demonstrated that the present invention provides a homogeneous specific binding assay method for determining thyroxine using a wide variety of reaction sequences.

EXAMPLE 4 Homogeneous Binding Assay For Theophylline A. Preparation of the labeled conjugate - theophylline-FAD To a solution of 2.4 pmol flavin N^-aminohexyl-adenine dinucleotide, prepared as described in part A of Example 1 above, in 200 μΐ dimethylsulfoxide under argon gas was added 0.9 mg 1,3-dimethyl-l,6,7,8-tetrahydropyrido[l,2-e]purine -2,4,9(3H)-trione (3.62 pmol), prepared according to the method of Cook et al, Res. Comm, in Chem. Pathol, and Pharm. 13:497 (1976), followed after 4 hours by addition of a further 1.8 mg (7.3 pmol) of the same. After stirring overnight, the solvent was evaporated under vacuum (0.1 mm Hg) and the residue chromatographed on a 2.5x90 cm LH-20 Sephadex (Pharmacia Fine Chemicals, Uppsala, Sweden) column and eluted with 0.3 M triethylammonium bicarbonate buffer (pH 7.8). The crude product eluted between 210 and 246 ml of the effluent, was collected, applied to a 20x20cmxl000p silica gel plate and eluted with an 8:2 ethanol: 1 M triethylammonium bicarbonate buffer (pH 7.8) mixture. The band containing the desired product (R.£=0.77) was scraped from the plate, extracted with 1 M triethylammonium bicarbonate buffer (pH 7.8), filtered and concentrated. Final purification by rechromatography on LH-20 Sephadex eluted with the buffer at 0.3 M gave 1.26 pmoles of the labeled conjugate, ns determined by absorbance measurement at 450 nm, which was a yield of 53%.

B. Antibody binding reactions Antibody was raised in rabbits against the immunogen 8-(3-carboxypropyl)-1,3-dimethylxanthine-BSA as described by Cook et al, Res. Comm, in Chem. Pathol. Pharm. 33:497(1976).

Antibody binding reactions were conducted at room temperature in 0.1 M sodium phosphate buffer (pH 7.0) and measurements of glucose oxidase activity were carried out in the same buffer at 20°C.

The reagents used in the assay were as follows: Reagent Composition A 0.1 M sodium phosphate buffer (pH 7.0) B 565 nM theophylline-FADglabeled conjugate or 160 nM N°-(6-aminohexyl) FAD (the FAD derivative) C antiserum to theophylline (diluted -fold in Reagent A) D apoglucose oxidase (50 nM FAD binding sites per ml) E monitoring reagent: 200 pg peroxidase/ml; 0.71 mM 4 -aminoantipyrine; 7.1 mM 3,5-dichloro-2-hydroxybenzene sulfonate; 353 mM glucose; and 35 mg BSA/ml.

Reagents A, B, and C were combined in separate reaction cuvettes in the proportions indicated in Table 4 below.

Reagent D (100 μΐ) and Reagent E (283 pi) were then rapidly and successively added to each reaction mixture followed by incubation for 30 minutes at 20°C. The absorbance at 520 mm was then measured for each cuvette. The results shown in Table 4 are the averages of duplicate runs. 8 2θ4 <υ υ β £ (ϋ £ -Ο ρ ο Ο CM ΙΛ LO Χ> < Zt*£ U P O p if) q «π 4) P ω q rt rt T3 P 4) rt r-t OO 4) □ /—'rO ·Ι“Ί r-t rt q 3.rH O v—(J P q kj 4) 4) > ro rt ·η <3: © p «·« Cd Ω to < > Ci -rt P TJ < P 4) p M-t q 'p © q rd rt' 4) Cd q o •r-t P P © υ .ω rt £ © q cd z \Ο «Μ* t—t cn CO «3- m CM O tZ) \Q Ό Ό O O O O O ο o © rH ^t· Ό rH r-. r- rrH rH O O LT) LD r- cr- lo T3- tn cm tn *3* LO O CD rH \O HD O O \Q *3 i-i CM CM CM O © CM rH CM CM O O O © © CM ^3 CO O O O O O CM CM CM CM CM c- e'en oo tn Γ-. CM r- tn LO LO 517 C-. CO © o rH O CM The results of reactions 2-5 show that the antiserum did not influence the activity of the FAD derivative (i.e., not coupled to theophylline). Reactions 6-11 demonstrated that increasing levels of theophylline antiserum decreased the activity of the labeled conjugate relative to its ability to combine with the apoenzyme.

C. Competitive binding assays These reactions were carried out as outlined in part B above except that designated levels of theophylline were combined with Reagents A and B before addition thereto of Reagent C and also that for Reagent C a 100-fold dilution of antiserum was used. The results are shown in Table 5 below.

Reactions 1-3 were controls showing that antibody to theophylline decreases the ability of the labeled conjugate to combine with apoenzyme. Reactions 4-9 demonstrate that FAD activity increases in proportion to the theophylline level in the reaction mixture. φ /—> υ 6 c c rt X» ο p (Μ Ο ·Λ (Λ '—' Α < cm tn σι tO sO Ο to ΓΟΊ Γρ- ο \Ο ΙΛ «Ρ 00 ΟΟ «Ρ Ρ- Ρ ΓΡ*Κ) g □ Ο Ρ φ ρ ω β ·Η 0) Ρ 00 β rt rt Φ Ρί β Ο •Ρ Ρ rt Ρ Ο Ρ /—s β C2 •Ρ Φ β«ρ υ ιΡ β ο Α ο Ρη ο φ /-¾ ,β «-* Η Φ 3. S β •Ρ Ό Ο Φ > π3 Ό /—> rt «Ρ oo o zt β ’·—'·-» U4 β •P CQ o CQ υ P P e-. βτί φ Φ OOlP rt Φ Φ A Pi rt < P Φ P M-4 β φ β 00,0 rt Φ Pi β O P >P φ PrO u 6 rt β φ Z Pi ο CM γ·-- r*. «Ρ ΟΊ vO m CM ο ο rp σ ο ο ο ο ο r-H rP «Ρ Ο Ο Ο Ο rp rP Ο Ο γΡ ο ο ο ΙΛ Ο Ο Ο ο tn σ ιΡ «Ρ ο ο ο ο ο CM CM CM CM CM r* r-σι στ ρ- tn r-> rρ· σι ρ* tn tn ρ m sO O r- t*» ρ σι ρ tn tM «Ρ ο O CM cm ρ ρoo cn EXAMPLE 5 Homogeneous Binding Assay for Human IgG A. Preparation of the labeled conjugate - IgG-FAD To 4.24 mg flavin N6-aminohexyl-adenine dinucleotide, prepared as described in part A of Example 1 above, was added 2.5 mg dimethyladipimidate dihydrochloride (Pierce Chemical Co., Rockford, Illinois, U.S.A.) in 1 ml of water and 5 pi of triethylamine. The reaction was stirred at room temperature for 10 minutes and 40 mg human immunoglobulin (IgG) in 1 ml of 0.1 M sodium pyrophosphate buffer (pH 8.5) was then added.

After further stirring at room temperature for 3 hours, the reaction mixture was applied to a 2.5x50 cm G-25 Sephadex (Pharmacia Fine Chemicals, Uppsala, Sweden) column equilibrated and eluted with 0.1 M sodium phosphate buffer (pH 7.0). Frac15 tions from the first eluting peak having absorbance at 450 nm were collected and dialyzed successively against 4 L of 0.1 M sodium phosphate buffer (pH 7.0) for 16 hours, 4 L of 0.1 M sodium phosphate buffer (pH 7.0) containing 1 M sodium chloride for 24 hours, and 0.01 M sodium phosphate buffer (pH 7.0) for 48 hours. Sodium azide was then added to 0.1% (w:v). The reaction material was filtered through a 0.22μ Millipore filter and stored.

B. Antibody binding reactions The reagents used in the assay were as follows Reagent Composition A 5 0.1 M sodium phosphate buffer (pH 7.0) B 10 mM 4-aminoantipyrine C 1.0 M glucose D 25 mM 3,5-dichloro-4-hydroxybenzene sulfonate in Reagent A 10 e 1.2 mg/ml horseradish peroxidase in Reagent A F 30% (w:v) bovine serum albumin (Research Products Division, Miles Laboratories, Inc., Elkhart, IN U.S.A.) 15 G IgG-FAD labeled conjugate in solution stored from above H apoglucose oxidase I rabbit antiserum against human IgG (obtained from Behring Diagnostics, 20 Somerville, New Jersey, U.S.A.) J standards - human IgG in Reagent A at predetermined levels Reagent mixes were prepared as follows Mix #1 25 - 180 pi Reagent A, 20 μΐ Reagent B, 100 pi Reagent D and 5 pi Reagent G (5,4 pM) Mix #2 · - 0.3 ml total volume of various proportions containing the volume of Reagent I indicated in Table 6 below with the remaining volume being made up of Reagent A 30 Mix #3 - - 80 pi Reagent D, 50 pi Reagent E, 33 pi Reagent F, 137 pi Reagent A and 1.6 pi Reagent H (4.2 pN FAD-binding sites) To 300 μΐ of Mix #1 was added 300 μΐ of Mix #2 and 300 μΐ of Reagent A in separate reaction cuvettes. After at least 10 minutes incubation at room temperature, 300 μΐ of Mix S3 was added to each reaction. After further incubation for 30 minutes at 20°C, the absorbance at 520 nm was measured in each cuvette. The results were as follows: TABLE β Volume of antiserum added to prepare Mix #2 Absorbance (520 nm) 0 0.859 2 0. 750 4 0.602 6 0.494 8 0.443 10 0.415 12 0.408 14 0.392 16 0.375 The results demonstrate that as antibody level increases, the glucose oxidase activity generated by the FAD label conjugated to IgG decreases.

C. Competitive binding assays These reactions were carried out using the reagents described in part B above. Designated levels of human IgG in 300 μΐ volumes of Reagent A were combined with Mix <1 in separate reaction cuvettes. Then a mixture of 12 pi of Reagent I and 288 pi of Reagent A was added to each reaction. After 10 minutes 300 pi of Mix #3 was added and the reaction mixtures incubated at 20°C for 30 minutes. At the end of thi period, the absorbance at 520 nm was measured in each cuvette The results were as follows: TABLE 7 Amounts of Human IgG Added (pg) 16 Absorbance (520 nm) 0.579 0.653 0.751 0.844 0.986 1.06 Thus, it was demonstrated that the present invention provides a specific binding assay for determining the high molecular weight ligand, human IgG, in liquid media.

REAGENT MEANS As will be evident to those working in the relevant field from the foregoing description and examples, the reagent means employed for carrying out the present method may assume a wide variety of forms. In particular, such reagent means may take the form of a unitary test composition, such as an appropriate solid or liquid form. As an illustration, one form of a test composition for determining a ligand according to the present invention comprises (a) a labeled conjugate comprising an organic prosthetic group coupled to the ligand or a binding analog thereof, (b) a specific binding partner of the ligand, and (c) an apoenzyme capable of combining with the prosthetic group to produce a holoenzyme. Such a test composition can also comprise reagents for measuring the activity of the holoenzyme and, where desired and appropriate, the labeled conjugate and the apoenzyme can be combined in the form of their conjugated enzyme complex. Of course, conventional diluents, buffering materials, stabilizers, and so forth can also be comprised in the test composition.

Also, the reagent means can take the form of a test kit, i.e., a packaged combination of containers holding the necessary reagent elements. Again as an illustration, one form of a test kit for determining a ligand according to the present invention comprises one or more containers holding (a) a labeled conjugate comprising an organic prosthetic group coupled to the ligand or a binding analog thereof, (b) a specific binding partner of the ligand, and (c) an apoenzyme capable of combining with the prosthetic group to produce a holoenzyme. Such a test kit can also provide, in one or more of the same or different containers, reagents for measuring the activity of the holoenzyme and, where desired and appropriate, the labeled conjugate and the apoenzyme can be combined in the form of their conjugated enzyme complex. In one embodiment, the test kit comprises at least two separate containers, one holding the labeled conjugate, and optionally, reagents for measuring holoenzyme activity, and the other hold ing the binding partner and the apoenzyme. Of course, the test kit can include other reagents as are known in the art and which may be desirable from a commercial and user standpoint, such as buffers, diluents, standards, and so forth.

Claims (5)

CLAIMS 1. (1) a labelled conjugate, (2) a specific binding partner, and (3) an apoenzyme, all as defined in any of claims 32 to 38. 40. The test kit of claim 39 comprising at least two separate containers, one holding said labelled 20 conjugate and the other holding said binding partner and said apoenzyme. 41. A prosthetic-group-labeled ligand of the formula: Pr-R-L wherein Pr is an organic prosthetic group, R is a linking group, and L is a specifically bindable ligand or a binding 5 analog thereof. 42. The labeled ligand of Claim 41 wherein said ligand, or analog thereof, and said prosthetic group are coupled at a site on the ligand or analog away from its specific binding locus and at a site on the prosthetic group away from its 10 active binding locus for an apoenzyme. 43. A conjugated enzyme-labeled ligand of the formula; Apo-Pr-R-L wherein Pr represents an organic prosthetic group, Apo is an apoenzyme combined with said prosthetic group forming a con15 jugated enzyme complex, R is a linking group, and L is a specifically bindable ligand or a binding analog thereof. 44. The labeled ligand of Claim 43 wherein and said ligand, or analog thereof, and said prosthetic group are coupled through said linking group at a site on the ligand 20 or analog away from its specific binding site and at a site on the prosthetic group away from its active binding site for the apoenzyme. 4826 4 45. The labeled ligand of Claim 43 or 44 wherein said conjugated enzyme complex exhibits holoenzyme activity. 45. The labeled ligand of any one of Claims 43 to 45 wherein the binding constant for association of said 5 apoenzyme and said prosthetic group is greater than about 10 6 molar -1 . 47. The labeled ligand of any one of Claims 41 to 45 wherein the said prosthetic group is flavin adenine dinucleotide, flavin mononucleotide or heme. 1q 48. The labeled ligand of Claim 47 wherein the said prosthetic group is flavin adenine dinucleotide or heme, and said apoenzyme is apoglucose oxidase or apoperoxidase, respectively. 49. The labeled ligand of any of Claims 41 to 48 15 wherein the said linking group It is a chain containing 1 to 14 carbon atoms and 0 to 5 heteroatoms selected from nitrogen, oxygen and sulfur. 50 The labeled ligand of any of Claims 41 to 49 wherein the said ligand is an antigen or an antibody 20 thereto; a hapten or an antibody thereto;’ a hormone, vitamin, metabolite or drug, or a receptor or binding substance therefor. 51. The labeled ligand of Claim 50 wherein said ligand is a hapten of molecular weight between 100 and 1000. 52. The labeled ligand of Claim 51 wherein da id ligand ia an iodothyronine thyroid hormone. 53, The labeled ligand of Claim 52 wherein said thyroid hormone is thyroxine.

1. A specific binding assay method for determining .·> ligand in a liquid medium, which method comprises: contacting said liquid medium with (a) reagent means including a labeled conjugate having a label component comprising 3 residue of an organic prosthetic group and also having a binding component, such contact producing a binding reaction system in which a bound-spccies and a free-species of said labeled conjugate are formed, the proportion of label component in said two formed species being a function of the presence of said ligand in said liquid medium, and (b) an apoenzyme capable of combining with said prosthetic group to produce a holoenzyme, and determining the proportion of label component in said two formed species by measuring holoenzyme activity in said bound-species or said free-species, or both.

2. The method of Claim 1 wherein said liquid medium is contacted with said apoenzyme after contact with said reagent means.

3. The method of Claim 1 wherein said liquid medium is contacted with said reagent means and said apoenzyme substantially simultaneously.

4. The method of claim 3 wherein said reagent means and said apoenzyme are contacted with said liquid medium as separate entities. 5. The method of claim 3 wherein said reagent means and said apoenzyme are contacted with said liquid medium by employing as said label component in the labelled conjugate a residue of a conjugated enzyme formed of said prosthetic group combined with said apoenzyme. 6. The method of any one of claims 1 to 5 wherein the binding constant for the association of said apoenzyme and the prosthetic group whose residue is comprised in the labelled conjugate is greater than about 10θ molar \ 7. The method of claim 6 wherein the said 8 —1 binding constant is greater than 10 molar . 8. The method of any one of claims 1 to 7 wherein the said ligand to be determined is an antigen or an antibody thereto; a hapten or an antibody thereto; or a hormone, vitamin, metabolite or drug, or a receptor or binding substance therefor. 9. A specific binding assay method for determining a ligand in a liquid medium, which method comprises: contacting said liquid medium with reagent means including (1) a labelled conjugate comprising an organic prosthetic group coupled to said ligand or a binding analog thereof and (2) a specific binding partner of said ligand, such contact producing a binding reaction in which are formed a bound-species of said labelled conjugate in which said coupled ligand or analog in said conjugate is bound to said binding partner and a free-species of said labeled conjugate in 5 which said coupled ligand or analog in said conjugate is not bound to said binding partner, the proportion of said prosthetic group in said two formed species being a function of the presence of said ligand in said liquid medium; 10 contacting the formed bound-species or freespecies, or both, with an apoenzyme capable of combining with said prosthetic group to produce a holoenzyme; and determining the proportion of said prosthetic group in said two formed species by measuring holoenzyme 15 activity in the species with which said apoenzyme was contacted. 10. The method of claim 9 of the heterogeneous type wherein said bound-species and said free-species are physically separated, said apoenzyme is contacted 20 with one thereof, and said holoenzyme activity is measured therein. 11. A specific binding assay method for determining a ligand in a liquid medium, which method comprises: contacting said liquid medium with reagent means 25 including (1) a labelled conjugate comprising an organic prosthetic group coupled to said ligand or a binding analog thereof, (2) a specific binding partner of said ligand, and (3) an apoenzyme capable of combining with said prosthetic group to produce a holoenzyme, such 30 contact producing a binding reaction in which are formed a bound-species of said labelled conjugate in which said coupled ligand or analog in said conjugate is bound to said binding partner and a free-species of said labelled conjugate in 5 which said coupled ligand or analog in said conjugate is not bound to said binding partner, the proportion of said prosthetic group in said two formed species being a function of the presence of said ligand in said liquid medium; and 10 determining the proportion of said prosthetic group in said two formed species by measuring holoenzyme activity in said bound-species or said free-species, or both. 12. The method of claim 11 of the heterogeneous 15 type wherein said bound-species and said free-species are physically separated and said holoenzyme activity is measured in one thereof. 13. The method of any one of claims 9 to 12 wherein the ability of said coupled prosthetic group in 20 the labelled conjugate to combine with said apoenzyme to produce a product having holoenzyme activity is different when said labelled conjugate is in said bound-species than when in said free-species. 14. The method of claim 13 of the homogeneous 25 type wherein said holoenzyme activity is measured without separation of said bound-species and said free-species. 15. A specific binding assay method for determining a ligand in a liquid medium, whioh method comprises: contacting said liquid medium with reagent means including (1) a labeled conjugate comprising (aj a complex of an organic prosthetic group and an apoenzyme which combine to form a holoenzyme and (b) said ligand or a binding analog thereof coupled to said complex through said prosthetic group, and (2) a specific binding partner of said ligand, such contact producing a binding reaction in which are formed a bound-species of said labeled conjugate in which said coupled ligand or analog in said conjugate is bound to said binding partner and a free-species of said labeled conjugate in which said coupled ligand or analog in said conjugate is not bound to said binding partner, the proportion of said complex in said two formed species being a function of the presence of said ligand in said liquid medium, and determining said proportion by measuring holoenzyme activity in said bound-species or free-species, or both. 16. The method of Claim 15 wherein the holoenzyme activity of said complex in the labeled conjugate is different when said labeled conjugate is in said bound-species than when in said free-species. 17. The method of Claim 16 of the homogeneous type wherein said holoenzyme activity is measured in said bound-species and said free-species combined. 18. The method of claim 15 of the heterogeneous type wherein said complex has holoenzyme activity and wherein said bound-species and said free-species are physically separated and said holoenzyme activity is 5 measured in one thereof. 19. The method of any one of claims 9 to 18 wherein said ligand to be determined is an antigen or hapten and said binding partner is an antibody thereto. 20. The method of any one of claims 1 to 19 10 wherein said ligand is a hapten of molecular weight between 100 and 1000. 21. The method of any one of claims 9 to 18 wherein said ligand to be determined is an iodothyronine thyroid hormone. 15 22. The method of any one of claims 1 to 21 wherein said prosthetic group is heme and said apoenzyme with which it combines is apoperoxidase. 23. The method of any one of claims 1 to 21 wherein said prosthetic group is flavin adenine dinucleo2o tide and said apoenzyme with which it combines is apoglucose oxidase. 24. A reagent means for determining a ligand in a liquid medium, which comprises a conjugate having a label component and a binding component, the said label 25 component being an organic prosthetic group residue, and additionally an apoenzyme with which the said prosthetic group residue combines to produce a conjugated enzyme complex. 25. A reagent means for determining a ligand in a liquid medium, which comprises a conjugate having a label component and a binding component, the said label component being a residue of a conjugated enzyme 5 formed of an organic prosthetic group residue combined with an apoenzyme, and the said binding component being coupled to the said conjugated enzyme residue through the said prosthetic group. 26. The reagent means of claim 24 or 25 wherein 10 said conjugated enzyme complex exhibits holoenzyme activity. 27. The reagent means of claim 24 or 25 wherein said binding component in said labelled conjugate is said ligand or a binding analog thereof. 15 28 . The reagent means of claim 27 wherein said ligand, or analog thereof, and said prosthetic group are coupled at a site on the ligand or analog away from its specific binding locus and at a site on the prosthetic group away from its active binding locus 20 for the apoenzyme. 29. The reagent means of any of claims 24 to 28 wherein said ligand to be determined is an iodothyronine thyroid hormone. 30. The reagent means of any of claims 24 to 25 28 wherein said ligand to be determined is an antigen or an antibody thereto; a hapten or an antibody thereto; or a hormone, vitamin, metabolite or drug, or a receptor or birding substance therefor. 31. The reagent means of claim 30 wherein said ligand is a hapten of molecular weight between 100 and 1000. 32. A test composition for determining a ligand 5 in a liquid medium, comprising (a) a labelled conjugate comprising an organic prosthetic group coupled to said ligand or a binding analog thereof, (b) a specific binding partner of said ligand, and (c) an apoenzyme capable of combining with said prosthetic group to produce a j-θ holoenzyme. 33. The test composition of claim 32 which additionally comprises reagents for measuring the activity of said holoenzyme. 34. The test composition of claim 32 or 33 15 wherein said labelled conjugate and said apoenzyme are combined in the form of their conjugated enzyme complex. 35. The test composition of any of claims 32 to 34 wherein said ligand is an antigen or a hapten and said binding partner is an antibody thereto. 2q 36. The reagent means of any one of claims 24 to 31 or the test composition of any one of claims 32 to 35 wherein the binding constant for the association of said apoenzyme and the prosthetic group whose residue is comprised in the labelled conjugate is greater than 25 about 10^ molar 37. The reagent means of any one of claims 24 to 31 or 36 or the test composition of any one of claims 32 to 36 wherein said prosthetic group residue is a residue of flavin adenine dinucleotide, flavin mono· 5 nucleotide, or heme. 38. The reagent means or test composition of daim 37 wherein said prosthetic group residue is a residue of flavin adenine dinucleotide or heme, and said apoenzyme with which it combines is apoglucose 10 oxidase or apoperoxidase, respectively. 39. A test kit for determining a ligand in a liquid medium, comprising one container or, in a packaged combination, more than one container holding:

5. 54. A specific binding assay method for determining a ligand in a liquid medium substantially as described in any one of the foregoing Examples.