FI70726C - HOMOGENEAL IMMUNOANALYSFOERFARANDE MED FLAVINADENINNUCLOTID SOM MAERKESKONPONENT OCH DETEKTERING AV DET MED APOGLUCOSOXIDAS - Google Patents

Description

rBl f11. ANNOUNCEMENT r, n 0. «TilrlP B 11 UTLÄG G NIN G SSKRI FT 'J 1 6 C (45) Patent granted / &&& \ 3 j n, ·. ,,,; . · -Λ ίο Ί A. . V U U Ο. . - _ Ο >> -i- Ο J_ * / ν_; Ο (51) Kv.lk. * / Lnt.CI. * C 12 Q 1/00 FINLAND —FINLAND (21) Pitenttlhikemus - Patentansökning 791937 (22) Application date · -Ansökningjdag 1 8.06.79 (FI) (23) Starting date - Giltighctsdag 1 8.06.79 (41) Has become public - Blivit offentlig 23.12.79

National Board of Patents and Registration Date of publication and publication. -. the so-called

Patent and registration authorities '' Ansökan utlagd och utl.skriften publicerad 2d.0o.öo (86) Kv. application - Int. ansökan (32) (33) (31) Privilege claimed - Begärd priority 22.06.78 USA (US) 917961 (71) Miles Laboratories, Inc., P.O. Box 40, Elkhart, Indiana A6515, USA (72) William Edward Hornby, Bray, Berkshire, David Lindsay Morris, Stoke Poges, Buckinghamshire, UK-Storbritannien (GB) (71+) Berggren Oy Ab (5 ^ Homogeneous 1 mm immunoassay with flavin adenine nucleotide as a marker component and its determination using apoglucose oxidase - Homogeneous immunoassay for flavinate nucleotides and a heterogeneous specific type of binding, for the qualitative or quantitative determination of a ligand in a liquid medium, and more particularly, the invention relates to specific binding assays using non-radioisotopic labels, and to non-radioisotopically labeled conjugates thereof for use in such assays. istamiseksi.

In a conventional specific binding assay technique, a test sample is combined with reagents of different compositions containing a conjugate with a monitoring label component and a binding component that participates in other reagent components, if any, to form a bound reaction system that produces two species or forms. species. The relative amount or ratio of labeled conjugate to the bound species relative to the free species is a function of the presence (or amount) of ligand detected in the test sample.

2 n r. <7 o r i u / z 6

For illustrative purposes, conventional competitive binding assay techniques are presented. In such a technique, a reagent would consist of (1) a labeled conjugate in the form of a detectable ligand (e.g., an antigen or hapten, meaning a substance that can selectively react with antibodies of suitable specificity but stimulate the production of these antibodies in an animal only when associated with a carrier). , which ligand forms a binding component of the conjugate that is chemically linked to the labeling component, and (2) a specific binding partner for the ligand (e.g., an antibody) In combining the test sample and reagent, the detectable ligand and the binding component of the labeled conjugate would compete substantially non-selectively. As a result, either the amount of labeled conjugate that would be bound to the binding partner (i.e., that would be bound to the bound species) or the amount that would remain free (i.e., unbound to the binding partner and thus become free). species) can be measured as a function of the amount of competing ligand present. The amount of labeled conjugate that enters either species is determined by measurement, i.e., by monitoring the label component therein.

When the labeled conjugate in the bound species and the free species are substantially inseparable with the device used to monitor the label component, the bound species and the free species must be physically separated to complete the assay.

This type of assay is referred to in the art as "heterogeneous." When bound and free species of the labeled conjugate can be distinguished, a "homogeneous" assay format can be used and a separation step can be avoided.

The first type of highly sensitive specific binding assay invented is a radioimmunoassay using a radioactive isotope as a label component. Such an assay must necessarily follow a heterogeneous form, as the nature of the label to be monitored is qualitatively unchanged in the free and bound species. Due to the difficulty and difficulty of handling radioactive materials, many new assay systems have been discovered using non-radioisotopes as a label component, such as enzymes, fluorescent molecules, bacteriophages, metals and organometallic complexes, enzymes, enzymes, enzyme bases.

The following publication discloses several different heterogeneous binding reaction systems using the enzyme as a label component: U.S. Patent Nos. 3,654,090; 3,791,932; 3,839,153; 3,850,752 and 3,879,262; J. Immunol. Methods 1: 247 (1972); and J. Immunol. 109: 129 (1972). A heterogeneous binding assay using a non-active precursor of a spectrophotometrically detectable substance as a labeling agent is proposed in U.S. Patent No. 3,880,934. The Principles of Competitive Protein-Binding Assays, edited by Odell and Daughaday ( J. B: Lippincott Co., Philadelphia, 1972).

A homogeneous type of enzyme-labeled immunoassay is described in U.S. Patent No. 3,817,834, which uses a ligand-enzyme conjugate. The enzymatic activity of the conjugate in the bound species is measurably lower than in the free species, allowing the use of a homogeneous assay. The use of particularly unique materials as labeling agents, including coenzymes, chemiluminescent molecules, cyclic reagents and cleavable fluorescent enzyme agents in both homogeneous and heterogeneous assays is described in DE Patents Nos. 2,618,419 and 2,618,511.

FI C 54 034 and US 3 879 262 relate to an "ELISA" type (Enzyme Linked Immunosorbant Assay) immunoassay using enzyme markers, while the present invention uses a flavin adenine nucleotide as a marker, allowing a homogeneous assay without a separation step.

U.S. Patent No. 4,134,792 is directed to the use of modulators of intact enzymes (active enzymes) as a marker, while the present invention uses a non-proteinaceous prosthetic group of a conjugated enzyme as a marker. Such a marker form is detectable when it is reattached to the protein portion of its inactive apoenzyme to give a detectable active enzyme.

70726

Although the search for non-radioisotope labels in binding assays has yielded a number of useful solutions, there is still room for improvement. Enzyme-labeled approaches initially appeared to be the most promising; in practice, however, a number of difficulties have arisen. The high molecular weight and aberrant nature of the enzymes pose problems in the characterization, stabilization, and reproducible preparation of labeled conjugates. These problems were overcome by replacing enzyme labels with low molecular weight labels that can be monitored based on the activity of their reactant. These novel labels, including coenzymes, enzyme substrates, cyclic reagents, and chemiluminescent reagents, can be used to generate highly sensitive and useful assays. However, monitoring these labels often requires instrumentation, which is not currently common in a clinical laboratory.

It is a primary object of this invention to provide a non-radioisotope binding assay using novel labeling that can be monitored by standard clinical laboratory equipment while retaining the benefits of reagent labeling in terms of sensitivity, utility, and ease of synthesis and characterization of labeled conjugates.

It has now been found that an improved specific binding assay is achieved by using an organic residual prosthetic moiety as a labeling component in the labeled conjugate. Prosthetic groups are a class of enzyme cofactors known in the art. An enzyme cofactor is a non-proteinaceous substance whose presence is required for an enzyme to demonstrate its catalytic activity and which undergoes a chemical change during the catalytic cycle of that enzyme (referred to as the parent enzyme). A coenzyme is a type of cofactor of an enzyme that has been chemically modified during a reaction catalyzed by the parent enzyme. Regeneration of the original form of cofactor requires participation in a separate reaction catalyzed by an enzyme other than the parent enzyme. In contrast, a prosthetic group is a cofactor of an enzyme that is chemically modified during the reaction catalyzed by the parent enzyme and regenerated, i.e., converted back to its original form by another reaction catalyzed by the same parent enzyme.

It is usually characteristic of prosthetic groups that they are relatively tightly bound to a protein moiety of the parent enzyme, which protein moiety is known as an apoenzyme and which catalytically active parent enzyme is known as a holoenzyme. The equilibrium reaction to the interaction between the prosthetic group and the apoenzyme can be represented as follows:

Prosthetic group + apoenzyme i - - * conjugated or holoenzyme (Pr) (Apo) (Pr-Apo) As used herein, the term conjugated enzyme refers to a complex formed by the combination of a prosthetic group and an apoenzyme, whether or not this complex has catalytic activity and the statement holo catalytically active conjugated enzyme. For more information on substituent groups, their characteristics, and interactions with apoenzymes, refer to Dixon & Webb, Enzymes, 1st Edition, Longmans, Green & Co. (London 1958).

In the present invention, an organic prosthetic group is coupled to a binding component to form a labeled conjugate for use in a binding assay. A key feature of the invention is the resulting interaction between the conjugated prosthetic group and the apoenzyme, which is shown as follows: Q-Pr + Apo ((^) - Pr-Apo

Catalytically inactive Complex of active holoenzyme components

Although the above reaction is a normally observed interaction, it is expected that under certain conditions the conjugated enzyme complex will have little or no holoenzyme activity, such as when conjugation of a substituent group prevents normal holoenzyme-substrate interaction. In any case, labeled conjugates useful in the present invention may be formed if the binding component of the conjugated prosthetic group (represented by the symbol (^) - above) in the assay binding reaction, the bound conjugate enzyme complex, then inhibits holoenzyme activity, such as holoenzyme activity.

There are a number of possible ways to track a label, some of which are described below that follow any of the known homogeneous and heterogeneous binding methods. However, in all cases, the label component of the labeled conjugate involved in the binding reaction includes a residue of an organic prosthetic group, and the monitoring scheme comprises determining the label component in the bound species or free species, or both, as appropriate, based on holoenzyme activity measurement.

Residues of the prosthetic group that are preferred for use in this invention are residues of flavin adenine dinucleotide, flavin mononucleotide, and heme. Preferred pairs of prosthetic group residue and apoenzyme are flavin adenine dinucleotide / apoglucose oxidase residues and hemi / apoperoxidase residues. Corresponding holo enzymes formed from such pairs are easily monitored by a well-known hydrogen peroxide measurement technique, which includes colorimetric and fluorimetric methods. Due to the catalytic nature of the monitored holoenzyme, the presence of the label component is multiplied in the follow-up reaction, allowing for highly sensitive detection of the ligand to be determined (e.g. at 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 a component through which a label is attached to a binding component in a labeled association and the ability to achieve highly sensitive detection by spectrophotometric, especially colorimetric, measurement provides a unique advantageous specific binding.

The assay in question is characterized by highly sensitive detection limits, easy applicability to automation, the ability to track the label with instrumentation commonly found in clinical laboratories, and the use of labeled conjugates that can be reproducibly and relatively simply prepared.

For the purposes of this specification, the following expressions are defined as follows: "ligand" means a substance or a category of similar substances whose presence or amount in a liquid medium must be determined; "specific ligand binding partner" means a substance or category of substances with specific binding affinity for ligand to exclude other substances. a "ligand specific binding analog" is any substance or group of substances that behaves substantially the same as a ligand with respect to the binding affinity of a specific binding partner for the ligand; "residue" is part of an organic molecule, for example a residue of a prosthetic group in a labeled conjugate is an organic part in such a in a conjugate derived from a prosthetic group and acting substantially as a prosthetic group without a hydrogen atom at the position through which the prosthetic group is attached to the remainder of the labeled conjugate (in the FAD ligand-labeled conjugate FAD represents a residue of a prosthetic flavin adenine nucleotide moiety); "reagent device" means a mixture, apparatus or set of test devices containing the reagents used to perform an assay method; "monitoring reaction" means a reaction in which the label component of a labeled conjugate is determined by measuring holoenzyme activity; a "lower alkyl group" is a straight-chain or branched alkyl group containing 1 to 6 carbon atoms, including both numbers, such as methyl-Li, ethyl, isopropyl and hexyl.

8 70726

The assay method in question can be applied to the detection of any ligand for which a specific binding partner exists. A ligand is usually a peptide, protein, carbohydrate, glycoprotein, steroid, or other organic molecule to which a specific binding partner exists in a biological system or that can be synthesized. The ligand is usually selected in a functional sense from the group consisting of antigens and antibodies thereto; haptens and their antibodies; and hormones, vitamins, metabolic and pharmacological agents and their receptors and binders. Typical examples of ligands that can be detected using this invention include hormones such as insulin, fetal gonadotropin, thyroid hormones, and estriol; antigens and hapten such as ferritin, bradykinin, prostaglandins and specific tumor antigens; vitamins such as biotin, vitamin B-, folic acid, vitamin E, vitamin A and ascorbic acid; metabolites such as 3 ', 51-adenosine monophosphate and 3', 5'-guanosine monophosphate; pharmacological agents or drugs such as antiemetics, bronchodilators, cardiovascular drugs, and drugs for abuse; antibodies such as microsomal antibody and antibodies to hepatitis and allergens; and specific binding receptors such as thyroxine-binding globulin, avidin, internal factor, and transcobalamin. This method of analysis is particularly useful for the detection of haptens and their analogues with a molecular weight between 100 and 1000, in particular for the detection of thyroid iodothyroid hormones, thyroxine and liothyronine.

The liquid medium to be tested may be a naturally occurring or artificially formed liquid suspected of containing a ligand, and is usually a biological liquid or a liquid obtained from its dilution or other treatment. Biological fluids that can be analyzed according to the method in question include serum, plasma, urine, saliva, and membrane and cerebrospinal fluids. Other materials such as a solid material, for example tissue, can be analyzed by converting it to a liquid form, such as by dissolving a solid in a liquid or by liquid extraction of a solid.

Labeled Conjugate The labeled conjugate of the present invention contains a residue of an organic prosthetic group in its label component and may be in one of two basic forms, shown schematically as follows: 70726

Schematic formula Stamp component

Pr-R-L Residual prosthetic group (Pr-)

Apo-Pr-R-L A holoenzyme or conjugated enzyme residue (Apo-Pr-) wherein Pr represents a residue of a prosthetic group and Apo an apoenzyme, R is a linking group and L is a binding component of a labeled conjugate, usually a ligand or binding analog thereof. Typically, the binding component and the remainder of the prosthetic moiety are joined together through a linking moiety at a site separate from its specific binding site (i.e., the binding component involved in the assay binding reaction, such as an immunochemical binding site where the binding component is an antigen, an oxygen, or an oxygen). and a site of the latter separate from its active apoenzyme binding site.

Assay Methods Various methods are available for monitoring the novel labeled conjugates of this invention. In any case, the residue of the prosthetic group is covalently attached to the binding component in the labeled component, and the monitoring reaction comprises measuring holoenzyme activity in the bound species or free species, or both, as the case may be.

For illustrative purposes only, the following are examples of several trace diagrams based on homogeneous and heterogeneous competitive binding techniques, while it is to be understood that other homogeneous and heterogeneous techniques may be practiced.

The following abbreviations are used in the presentation below, in accordance with the remaining description:

Saying Abbreviation

Residue of prosthetic group Pr apoenzyme Apo linking group r

ligands L

related party B

conjugated enzyme residue Apo-Pr holoenzyme residue (active) f Apo-Pr] 10 70726

Illustrative follow-up diagram No. 1

Homogeneous competitive binding method - apoenzyme coupled after initiation of binding reaction

L (sample) + Pr-R-L + B; » L-B + Pr-R-L-B

Λ + Apo (bound species)

V

Apo + [Apo-Pr-R-L

(free species; active holoenzyme) In this scheme, the residual prosthetic group as a label component is monitored by adding the apoenzyme substantially simultaneously with or after the initiation of the binding reaction, i.e. during or after the binding reaction equilibrium, and measuring the holoenzyme activity in the holoenzyme. . The assay is homogeneous by selecting conditions such that binding of the apoenzyme to the remainder of the prosthetic group is inhibited for the prosthetic group in the bound species. Alternatively, the conditions may be selected so that the apoenzyme can bind to the bound form of the labeled conjugate, but the resulting conjugated enzyme complex does not have enzymatic activity, such as that due to inhibition of enzyme-substrate interaction (this situation is not shown above). In either case, the total amount of enzyme activity measured in the system is the result of uninhibited binding of the apoenzyme to the free labeled conjugate and, consequently, is a direct function of the amount of ligand in the sample available for binding to the binding partner.

It might also happen (but not shown above) that, in contrast to the above schemes, the labeled conjugate (Pr-RL) is formed such that binding to the apoenzyme is inhibited, but that binding of the labeled conjugate to the binding partner facilitates this inhibition and the apoenzyme is able to bind to the conjugate. with a bound species to form an active holoenzyme complex. In such a case, the amount of holoenzyme activity entering the system is an inverse function of the amount of ligand present in the sample.

Illustrative follow-up diagram No. 2

Heterogeneous competitive binding method - apoenzyme coupled after initiation of binding reaction.

70726 11

L (sample) + Pr-R-L + B. > L-B + Pr-R-L-B

Λ + Apo

V

Apo + Apo-Pl-R-L + Apo-pj-R-L-B

(free species, (bound species, active active holoenzyme) holoenzyme) v separate This scheme uses the same reagents and addition order as in Scheme No. 1 described above, except that the conditions are such that the apoenzyme can bind to the labeled conjugate of the active species in the bound species. , which is qualitatively distinguishable from that formed by binding to the free species of the labeled conjugate. The bound species and the free species must be distinguished and the enzyme activity in one of them is a function of the amount of ligand in the sample.

Illustrative monitoring diagram No. 3

Homogeneous competitive binding method - apoenzyme coupled before initiation of binding reaction

Pr-R-L + Apo - * Apo-Pr-R-L

(active holoenzyme)

L (sample) + | Apo-Pr | -R-L + B ^ i L-B + Apo-Pr-R-L-B

(free species; (bound species; active inactive) holoenzyme) In this scheme, a residue of a prosthetic group is incorporated into a label component by attaching it to a ligand and combining it with an apoenzyme in the form of an active holoenzyme product. The residue of the prosthetic group is monitored by measuring the activity of the holoenzyme in the final reaction mixture. The assay is homogeneous by selecting conditions such that upon binding of the binding moiety to the active holoenzyme complex, the resulting bound form has such inhibited enzyme activity due to inhibition of the enzyme-substrate-12 7 0 7 2 6 interaction. The total amount of enzyme activity measured from the system is the result of the unbound active holoenzyme complex, i.e. the free form, and is therefore a direct function of the amount of ligand in the sample.

It might also happen (but not shown above) that, in contrast to the scheme above, the labeled conjugate has little or no enzymatic activity, but binding to a binding partner in a bound species actually has measurable or improved enzymatic activity. In such a case, the amount of holoenzyme activity obtained in the system is an inverse function of the amount of ligand present in the sample.

Illustrative follow-up diagram No. 4

Heterogeneous competitive binding method - apoenzyme attached before initiation of binding reaction

Pr-R-L + Apo -> Apo-Pr-R-L

(active holoenzyme)

L (sample) + Apo-Pr-R-L + B <....> L-B + Apo-Pr-R-L-B

(free species; (bound species; active active holoenzyme) holoenzyme) separate This scheme uses the same reagents and addition order as in Scheme No. 3 described above except that the conditions are such that both the bound and free forms are enzymatically active and qualitatively separable. . The bound species and the free species must be distinguished and the enzyme activity in one of them is a function of the amount of ligand present in the sample.

Schemes No. 3 and No. 4 are essentially enzyme-labeled assays except that, unlike in the prior art, the enzyme as such is attached to the binding component of the labeled conjugate via a residual of a prosthetic group. In this regard, the present invention then provides a novel method of preparing an enzyme-labeled conjugate comprising the steps of (a) covalently attaching a binding moiety (i.e., a detectable ligand or binding analog or partner thereof) to an organic prosthetic group capable of combining an apoenzyme. to form a conjugated enzyme, e.g., an active holoenzyme, (b) combining the resulting prosthetic group-labeled conjugate with an apoenzyme, and (c) isolating the resulting conjugated enzyme-labeled conjugate. It is noted that such a method is most valuable in the preparation of enzyme-labeled assemblies for use in assays due to the controlled manner in which a low molecular weight and well-characterized prosthetic group can be attached to a binding component.

It is apparent from the above description, that certain labeled conjugates and the ligand / binding partner pairs of one or more of the described homogeneous Schemes are suitable depending upon a number of factors, such as to bind the labeled konju-conjugate-bound form, and the effect that the bond binding partner is conjugated to the enzyme complex apoenzyme capacity. When the bound and free forms of the labeled conjugate have separable activity, a heterogeneous approach can be used to provide a useful assay. Thus, although the circumstances dictate which treatment or methods are most advantageous in each case, the assay method in question is generally applicable to any conventional homogeneous or heterogeneous technique.

Homogeneous techniques

The homogeneous technique, ie. One that does not require physical separation of the bound species and the free species can be used in the reaction between the labeled conjugate binding component and the corresponding binding partner to cause a measurable change in either the positive or negative sense, the labeled conjugate labeled component in the ability to participate in the monitoring reaction, ie. Labeled conjugate's ability associate with an apoenzyme and / or have holoenzyme activity. In such a case, the distribution of the macrocomponent between the bound species and the free species can be determined without distinguishing between species. The holoenzyme activity in the reaction mixture is then determined by forming a reaction catalyzed by such a holoenzyme in at least a portion of it, e.g., adding a substrate and measuring the amount of product formed or the rate of reagent consumption by any conventional technique.

14 70726

Qualitative determination of a ligand in a liquid medium involves comparing the measured amount to the amount of monitoring reaction in a liquid medium without ligand, any difference between them being an indication of the presence of such a ligand in the liquid being tested. Quantitative determination of ligand in a liquid medium involves comparing the measured amount to the amount in the monitoring plot in liquid media containing different known amounts of ligand, e.g., comparing to a standard curve.

In general, following a homogeneous assay technique, the components of a specific binding reaction, i.e., a liquid medium suspected of containing a ligand, a labeled conjugate, and, in some systems (i.e., a competing binding system), a ligand-specific binding moiety, may be combined in any amount. the activity of the label component of the conjugate is measurably altered when the liquid medium contains a significant amount or concentration of ligand. Preferably, all components of the specific coupling reaction are soluble in the liquid medium.

Known modifications of the homogeneous methods briefly described above and further details regarding the specific technique described, including an alternative technique known as the direct binding technique, are readily found in the literature, e.g., German Patent No. 2,618,511.

Heterogeneous techniques

The use of the novel labels in question can also be applied to a conventional heterogeneous-type assay technique in which the bound and free species of the labeled conjugate are separated and the label component in either is determined. Reagent means for performing such a heterogeneous assay can take many different forms. In general, these devices comprise three basic components, which are (1) a detectable ligand, (2) a ligand-specific binding partner, and (3) a labeled conjugate. Binding of the reaction components are combined simultaneously or in a series of additions and labeled with an appropriate incubation period or periods the conjugate becomes bound to the corresponding binding partners such that the extent of binding, ie. The conjugate binding partner of the labeled bound (bound species) to volume ratio unbound (free species) amount is present in the function il 70726 of the amount of ligand. The bound and free species are physically separated and the amount of label in one of them is determined by measuring its holoenzyme activity and comparing it to a negative control or standard results, e.g., a standard curve.

Various means for performing the separation step and forming the coupling reaction systems are available in the art. The separation may involve conventional techniques such as those involving what is commonly known as a solid phase antibody or antigen, a second antibody or a solid phase second antibody, and the use of immune complex precipitants and adsorbents, etc. Binding reaction systems that may be followed are nk .competitive bonding techniques, sequential impregnation techniques, "sandwich" techniques, etc. Further details regarding various known heterogeneous systems can be readily found in the literature, e.g., German Patent No. 2,618,419.

It should be noted that processing techniques involving other addition sequences and other coupling reactions may be devised to perform homogeneous and heterogeneous specific binding assays without departing from the concept of the invention outlined herein.

Prosthetic group

As previously mentioned, prosthetic groups are a distinct class of enzyme cofactors recognized in the art, primarily characterized by the ability of the original apoenzyme to regenerate them into the active cofactor form. The present invention uses organic prosthetic groups that can be chemically attached to a binding component in a labeled conjugate. Generally, the binding constant for combining an apoenzyme and a prosthetic moiety whose residue is included in the labeled conjugate of this invention is greater than about 10® mol%, and preferably greater than about 10® mol. The actual binding affinity of the prosthetic moiety residue in the labeled conjugate can be reduced. by a few percent or even two orders of magnitude without affecting the usefulness of the residue of such a prosthetic group as a label component in the binding assay.

The following is a table listing several prosthetic groups useful in this invention, the conjugated enzyme formed by their association with the apoenzyme, and the binding constants for the association of the prosthetic group with the apoenzyme:

Prosthetic Binding Constituent Group Conjugated Enzyme (Molarity) Reference flavin adenyl glutathione reductase 2 x 10 ^ 1.3 nidinucleotide (human red blood cells) (FAD) 9 flavin monocytochrome reductase 10 2 nucleotide (yeast) (FMN) FMN NADPH: oxidized "old yellow enzyme") g FAD glucose oxidase> 10 4 (Aspergillus niger) g FAD lipoamide dehydrogenase 4 x 10 5 7 FMN pyridoxine phosphate 5 x 10 6 oxidase 9 hemi peroxidase (pepper-> 10 7 root) g hemi cytochrome C> 10 8 1) Scott et al., J. Biol. Chem. 238: 3928 (1963) 2) Haas et al., J. Biol. Chem. 143: 341 (1942) 3) Staal et al., Biochim. Biophys. Acta 185: 39 (1969) 4) Swoboda, Biochim. Biophys. Acta 175: 365 (1969) 5) Visser and Veeger, Biochim. Biophys. Acta 206: 224 (1970) 6) Arsenis and McCormick, J. Biol. Chem. 241: 330 (1966) 7) Theorell et al., Arch. Kemi. Min. 0. Geol. 148: 1 (1940) 8) Yonetani, J. Biol. Chem. 242: 5008 (1967).

The preferred pair of prosthetic group and apoenzyme is hemi and apo-peroxidase, and flavin adenine dinucleotide and apoglucose oxidase are particularly preferred. Such prosthetic groups provide an easy means of attachment to ligands, ligand analogs, and binding partners, and the resulting holoenzymes participate in hydrogen peroxide detection reactions that are well known and analytically used and can be selected to generate colorimetric reactions that are advantageous to the clinical laboratory technician.

Il 1 7 70726

It is to be understood that a particular cofactor may act as a coenzyme or prosthetic group depending on the enzyme system into which it is introduced (e.g., FAD); however, the present invention contemplates the use of such a cofactor only in the context of an enzyme system in which it functions as a prosthetic group as defined above.

Bringing group

It should be noted that there are many methods available for the labeled binding component of the conjugate, e.g. combining a detectable ligand, a binding analog thereof, or binding partner prosthetic group. The particular chemical nature of the linking group depends on the nature of the corresponding junctions available in the bonding component and the prosthetic group. selecting the important aspects of the connecting points are generally (1) connected to the bond component to the ability to store effectively participate in the selected sidontamäärityssysteemiin and (2) retention of residue prosthetic group associated with the ability to connect with an apoenzyme (or such a binding capacity inhibition, as this may alleviate the labeled binding the conjugate bond party tool -. see luminous tracking diagram of the in both cases, to the extent that a useful assay is obtained for the ligand to be determined and for the respective concentrations and amounts at which this ligand is to be detected. Usually the linking group consists of a chemical bond, usually a single but sometimes double bond or a chain containing 1-14, more generally 1-6 carbon atoms and 0-5, more generally 1-3 heteroatoms selected from nitrogen, oxygen and sulfur.

Both the prosthetic group and the binding component naturally offer a wide range of possibilities for the attachment of the group connecting the available functional sites. In general, the functional groups that can be expected to be available for a linking group are amino, usually primary amino, hydroxyl, halo, usually chlorine or bromo, carboxylic acid; aldehyde; field-; isothiocyanate; isocyanate group, etc. Thus, the chemical structure of the linking group itself varies widely with its end groups depending on the functional groups in the prosthetic group and the bonding component and its overall length being a matter of choice within the basic constraints of the essential nature of the resulting linker substitution group and bonding component. With respect to the length of the linking group in preparing the conjugate for use in a homogeneous assay, it is usually desirable to use as short a group as possible without significantly forming interfering activity with the prosthetic group of the conjugate. When the linking component has a low molecular weight (e.g., a hapten having a molecular weight between 100 and 1000), the linking group is preferably a chemical bond or a chain of 1 to 6 atoms, such as a lower alkyl, carbonyl, alkylcarbonyl, amido, alkylamide group, and the like. In other circumstances, such as when the binding component in the conjugate has a relatively high molecular weight, such as a polypeptide or protein (e.g., an antibody), a longer linking group is usually desirable to avoid an etheric barrier to the apo-enzyme binding site of the conjugate. In these cases, the linking group usually comprises 1 to 14 carbon atoms and 0 to 5 heteroatoms as described above. Chains of some significantly greater length sometimes result in conjugates in which the binding component tends to fold at the site linking the apoenzyme. In view of these considerations, examples of bridging groups are given in Table 1 below. Specific examples of bridging groups are seen below and further modifications are readily found to be in the art.

70726 19 TABLE I Connecting group "-R1-

X

1 "9 -Ri-C-R2-

X

-R1-C-X-R2

X

1 "2 -R ± -X-C-R-

X

1 "9

Prosthetic group -R-X-C-X-R * - - bonding component

X X

In the group -C-R1-C- -R1-X-R2- -X-R1- -R1-X- -X-R1-X-10, X is an imino, sulfur or, preferably, oxygen group and R and R are independently lower alkylene groups having 1 to 6 carbon atoms, such as methylene, ethylene, isopropylene, butylene or hexylene groups.

apoenzyme

The apoenzyme, or protein portion of a holoenzyme without a prosthetic group, is prepared by cleaving the conjugated enzyme and isolating the released apoenzyme protein. Cleavage of the conjugated enzyme can be accomplished in a number of known ways, such as by placing the enzyme in a highly acidic solution, e.g., having a pH below 2. Similarly, recovery of the apo enzyme can be accomplished by a number of known techniques, such as selective protein precipitation or low molecular weight released adsorbent prosthetic moiety.

20 70726

As mentioned above, apoperoxidase and apoglucose oxidase are particularly preferred apoenzymes for use in this invention because their holoenzymes are involved in analytically preferred hydrogen peroxide reactions. It follows that other closely related conjugated enzymes, especially oxidoreuctases, which catalyze hydrogen peroxide-producing reactions and which can be cleaved to produce an inactive apoenzyme, find particular use in this invention. Published methods for isolating apoenzymes are available, especially for apoglucose oxidase, Biochim. Biophys. Acta 175: 365 (1969) and apoperoxidase, J. Biol. Chem. 206: 109 (1953) and Arkiv Kemi. Min. O. Geol. 148: 1 (1940).

Should the possibility of disruption of the assay arise due to the presence of an endogenous prosthetic moiety in a sample or reagent, or due to contamination of laboratory equipment, glassware, or plastic articles with a prosthetic moiety, such interfering prosthetic moiety can be readily removed by available inactivation techniques. For example, FAD disorder can be eliminated by sequential treatment with periodate and ethylene glycol solutions or by using other FAD inactivation techniques.

The present invention, which is precisely defined in the appended claims, will now be illustrated by the following examples.

Example 1

Homogeneous Binding Assays for N-2 ', 4'-Dinitrophenyl-6-Amino-Caproate A. Preparation of Labeled Conjugate - N ** - (6-Aminohexyl) -dinitrophenyl-Flavin Adenine Dinucleotide

Flavin N 1 -aminohexyladenine dinucleotide N6-trifluoroacetamidohexyl adenosine 5'-monophosphate was synthesized by the method of Trayer et al., Biochem.

J. 139: 609 (1974).

Fifty-six milligrams (mg) of N, N-trifluoroacetamidohexyl adenosine 5'-monophosphate (0.1 mmol) was dissolved in about 10 ml (ml) of water and 25 microliters (μl) of tri-n-butylamine (0.1 mmol). ) was added. The water was removed in vacuo and the residue was dissolved in 10 mL of dry dimethylformamide (DMF), which was then removed

II

70726 in a vacuum. The residue was evaporated from dry DMF three more times.

The final residue was dissolved in 10 measures of dry DMF. 80 mg of N, N'-carbonyldiimidazole (0.5 mmol) was added and allowed to react for 1.5 hours. Then 15 water was added and the solvent was removed in vacuo. The residue (N-trifluoroacetamidohexyladenosine-5'-monophosphate imidazolide) was dissolved in 10 volumes of DMF.

47 mg of riboflavin-5'-monophosphate (0.1 mmol) was dissolved in about 10 measures of water and 20 measures of acetone containing 43 μl of tri-n-octylamine (0.1 mmol) were added dropwise. A precipitate formed before the addition was complete. The solvent was removed on 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 evaporated to dryness. The residue was dissolved in 15-20 measures of dry DMF and evaporated to dryness (this process was repeated three times). The residue was dissolved in 5 measures of DMF and combined with the above solution of 10 measures of imidazolide in DMF.

The reaction mixture was allowed to stand at room temperature overnight and then the solvent was removed. The residue was dissolved in 50 mL of water and poured onto a 2.5 x 25 centimeter (cm) DEAE cellulose column in bicarbonate form (Whatman DE 23, Reeve Angel, Clifton, New Jersey, USA). The chromatogram was developed with a linear gradient obtained with two liters (1) of water and two liters of 0.3 molar (M) ammonium bicarbonate (23 ml tn fractions were collected). Thin layer chromatography on silica gel 60 F 254 (E. Merck, Darmstadt, Germany) using a mixture of ethanol and 1-M triethylammonium bicarbonate (pH 7.5) in a volume of 7t3 by volume (vtv) showed that fractions ntot 68-73 contained higher (Rf = 0, 75) and a smaller (Rf = 0.36) yellow compound. These fractions were pooled and had optical absorption spectra at maxima at 267, 373 and 450 nanometers (nm).

The solvent was removed from the combined material and the residue was dissolved in about 5 ml of water. This solution was adjusted to pH 11.0 with 5N sodium hydroxide and allowed to stand at room temperature for nine hours. Thin layer chromatography showed that the component with Rf = 0.75 disappeared, while a new yellow material with Rf = 0.37 emerged. The reaction mixture was adjusted to pH 8.0 with hydrochloric acid and poured onto a 2.5 x 20 cm DEAE cellulose column in the form of 707 2 6 bicarbonate. The chromatogram was developed with a linear gradient formed with one liter of water and one liter of 0.2 M ammonimbic carbonate. The yellow effluent from the column was combined and the solvent was removed. The residue was adsorbed onto 2 grams (g) of silica gel placed on top of a 50 g silica gel column equilibrated with a 9: 2 (v / v) mixture of ethanol and 1-M triethylammonium bicarbonate (pH 7.5). The column was eluted with an 8: 2 (v / v) mixture of ethanol and 1-M triethylammonium bicarbonate, 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%.

N6- (6-aminohexyl) -dinitrophenyl-flavin adenine dinucleotide The residue obtained from the above, which contained flavin-N6-aminohexyl-adenine dinucleotide, was purified by chromatography on Sephadex G-10 resin (Pharmacia Fine Chemicals, Uppsala, Sweden). To a 0.9 x 30 cm column equilibrated with 25 millimolar (mM) sodium bicarbonate (pH 7.5) at room temperature was poured 1 ml of a solution of about 10 mM N 2 -FAD derivative in water.

The first eluted peak of the material that absorbed at 450 nm was recovered and rechromatographed on Sephadex G-10 resin, and the first eluted peak was recovered again.

f.

0.5 ml (0.63 μmol) of purified N-FAD derivative in 25 mM sodium bicarbonate (pH 7.5) was mixed with 2 ml of ethanol and 10 μl of 3'H-dinitrofluorobenzene (6.3 mol, 50 (uCi) in ethanol was added (tritium-treated dinitrofluorobenzene was obtained from the Radiochemical Center, Amersham, England). The reaction mixture was shaken continuously overnight in the dark at room temperature and then poured onto a 0.9 x 30 cm Sephadex G-10 column equilibrated with 0.1 M phosphate buffer (pH 7.0) containing 0.1% sodium azide. . The only peak of the material that absorbs at 450 nm was recovered and found to contain 8% radiolabel.

B. Preparation of apoglucose oxidase

Purified low catalase glucose oxidase from Research Products Division 70726 23 of Miles Laboratories, Inc., Elkhart, Indiana, USA, was dialyzed twice for 12 hours at 0.5% w / v against mannitol (30 volumes each). Aliquots containing 100 milligrams (mg) of glucose oxidase each were dialyzed, 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 of activated carbon (RIA grade, manufactured by Schwarz-Mann, Orangeburg, New York, USA) and cooled. ° C. Lyophilized glucose oxidase (100 mg) was redissolved in 3.1 ml of water, and 3 ml was added to the stirred albumin activated carbon suspension, continuing to stir for 3 minutes. The suspension was then filtered through a 0.8 micron, 25 millimeter (mm) diameter Millipore filter (Millipore Corp., Redford, Massachusetts, USA) mounted on a Sweenex filter device (Millipore Corp.) on a 50 mL disposable plastic hand syringe. The filtrate was rapidly neutralized to pH 7.0 by the addition of 2 ml of 0.4 M phosphate buffer (pH 7.6) followed by 5N sodium hydroxide. Dry activated carbon (150 mg) was then added and stirred for 1 hour at 0 ° C. The resulting suspension was first filtered through a 0.8 micron Millipore filter and then through a 0.22 micron Millipore filter. Glycerol was added to the filtrate up 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 was 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 7.10) containing 0.1% bovine serum albumin to a concentration of 958 nM FAD binding site. The FAD binding site concentration of the apoenzyme preparation was determined experimentally by measuring the minimum amount of FAD required to obtain maximum glucose oxidase activity when incubated with the apoenzyme.

3. Antiserum - Antiserum against dinitrophenyl-bovine serum albumin pool was obtained from Miles-Yeda Ltd., Rehevot, Israel and diluted 31-fold in 0.1 M phosphate buffer (pH 7.0) containing 0.1% bovine serum albumin. .

70726 24 4. Standards - Standard solutions contained known concentrations of N-2 ', 4'-dinitrophenyl-6-aminocaproate, prepared in 0.05 M phosphate buffer (pH 7.0).

5. Monitoring Reagent - Glucose oxidase reagent was prepared by mixing 45 ml of 0.1 M phosphate buffer (pH 7.0) containing 15 mM ethylenediaminetetraacetic acid, 9 ml of 11.5 mM

3,5-Dichloro-2-hydroxybenzenesulfonate in water adjusted to pH 7 with sodium hydroxide, 9 ml of 11.5 mM 4-aminoantipyrine in water containing 1.25 mg / ml peroxidase (Miles Laboratories, Inc., Elkhart, Indiana, USA) ) and 6 ml of 1.0 M glucose in aqueous saturated benzoic acid solution.

D. Assay Procedures 1. Addition of Apoenzyme Prior to Initiation of the Binding Reaction Method No. 1 - The following were added sequentially to separate reaction cuvettes: 0.1 ml of labeled conjugate solution, 0.05 ml of selected standard solution, 0.1 ml of antiserum solution, 2 ml of monitoring reagent and 0.1 ml of apoenzyme solution. Each reaction mixture was incubated for 30 minutes at 30 ° C and the absorbance at 520 nm was measured.

Method No. 2 - The following were added sequentially to separate reaction cuvettes: 0.1 ml of labeled conjugate solution, 0.05 ml of selected standard solution, 0.1 ml of antiserum solution and 0.1 ml of apoenzyme solution. Each reaction mixture was incubated for 20 minutes at room temperature and then 2.0 ml of monitoring reagent was added to each. After an additional 15 minutes of incubation at 30 ° C, the absorbance at 520 nm was measured from each cuvette.

2. Addition of Apoenzyme After Injection of Binding Reaction Method No. 3 - The following were added sequentially to separate reaction cuvettes: 0.1 ml of labeled conjugate solution, 0.05 ml of selected standard solution and 0.1 ml of antiserum solution. Each reaction mixture was incubated for 20 minutes at room temperature, after which 2.0 ml of monitoring reagent and 0.1 ml of apoenzyme solution were added to each. After an additional 30 minutes of incubation at 30 ° C, the absorbance at 520 nm was measured from each cuvette.

Method No. 4 - The following were added sequentially to separate reaction cuvettes: 0.1 ml of labeled conjugate solution, 0.05 ml of selected standard solution and 0.1 ml of antiserum solution. Each reaction mixture was incubated for 20 minutes at room temperature and then 0.1 ml of apoenzyme solution was added to each. After an additional 20 minutes of incubation, 2.0 ml of monitoring reagent was added to each cuvette. The reaction mixture was then incubated for 15 minutes at 30 ° C and the absorbance at 520 nm was measured from each cuvette.

E. Results

The following is Table 2, which shows the results of these four assays for measuring N-2 ', 4'-dinitrophenyl-6-amine caproate. Concentrations of N-2 ', 4'-dinitrophenyl-6-aminocaproate are expressed as concentrations in final 2.35 ml reaction mixture volumes. Ab absorbance results are expressed as the mean of the two runs corrected for residual enzyme activity and background absorbance in the reagent. A correction factor is given for each assay method at the end of Table 2 and was determined experimentally by replacing all solutions of labeled conjugate, standard and antiserum with 0.1 M phosphate buffer (pH 7.0).

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The results show that the present invention provides a specific binding assay method of the homogeneous type in which the apoenzyme is incorporated either before or after the initiation of the binding reaction.

Example 2

Heterogeneous Binding Assay for Thyroxine A. Preparation of Labeled Conjugate - N®- (2-Aminoethyl) -Troxoxetlavine Adenine Dinucleotide 6- (2-Aminoethyl) -amino-9- (2 ', 3'-O-isopropylidine-BD-rifcofuranosyl) purine__ 13.56 g (41.5 mmol) of 6-chloro-9- (2,3'-O-isopropylidene-β-D-ribofuranosyl) purine / Hampton et al., J. Am. Chem. Soc. 83: 150 (1961 // was added with stirring over 15 minutes to cold excess 1,2-diaminoethane (75 ml). The resulting solution was allowed to stand at room temperature for 24 hours. The solution was evaporated in vacuo and the resulting yellow oil was stirred with 50 ml of cold The mixture was evaporated in vacuo and the resulting residue was further repeatedly evaporated in vacuo, first from water (3 times from 50 ml) and then from 2-propanol (4 times from 50 ml) to give a yellow glass (15 g). passed through a 25 x 55 cm Dowex 50W-X2 cation exchange column in ammonium form (Bio-Rad Laboratories, Richmond, California, USA).

The column was eluted with a linear gradient generated with 2 L of water and 2 L of 0.5 M ammonium bicarbonate. Elution was completed using a linear gradient generated with 2 L of both 0.5 M and 1 M ammonium bicarbonate. The effluent from the column was collected in 19 ml fractions and examined by eluting on silica gel thin layer chromatography (TLC) plates (E. Merck, Darmstadt, Germany) with a 9: 1 (v / v) mixture of ethanol and ammonium hydroxide. The developed TLC plates were examined under ultraviolet light and then injected with ninhydrin reagent / Randerath, Thin Layer Chromatography, Academic Press (1966/7) Fractions from column chromatography Nos. 250-350 were combined and evaporated in vacuo to leave the desired purine as a clear amine. g).

Analysis: Calculated from the formula ^ (^ 22 ^ 04: C 51.42; H 6.33; N 23.99 experimentally: C 50.92; H 6.54; N 23.01

O Q

70726 NMR (60 MHz, CDCl 3): δ 1.37 (s, 3H, isopropylidene), 1.63 (s, 3H, isopropylidene), 5.92 (d, 1H, 1'-ribose), 7.90 (s, 1H, purine), 8.26 (s, 1H, purine).

Optical rotation / * V7p ° = -74.85 ° (c 1.0, CH3OH).

The residual crude product (12 g) was purified by chromatography on Dowex 50 W-X2 resin as described above. The total yield was 8 g (55%).

α- (N-Trifluoroacetyl) -amino-β- [3 ', 5'-diiodo-4- (3', 5'-diiodo-4-hydroxyphenoxy) phenyl] propionic acid This compound was prepared by the method of Blank, J. Pharm. Sci. 53: 1333 (1964). To a cooled (0 ° C) stirred suspension of 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 clear solution was washed three times with 30 ml of water, once with 30 ml of 5% sodium bicarbonate 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 and then dried over magnesium sulfate. The dried ethyl acetate solution was evaporated in vacuo to leave a white solid. Recrystallization from a mixture of ethyl ether and petroleum ether gave a pinkish white solid (3.95 g, 70.5% yield) which melted at 228-230 ° C with decomposition.

Analysis: Calculated for C 17 H 10 F 3 J 4 NO 5: C, 23.39; H 1.15; N 1.60 experimentally: C 23.00; H 1.05; N 1.65 NMR / 60 MHz, DCON (CD 3) 2-7 67.28 (s, 2H, aromatic), 8.03 (s, 2H, aromatic), 9.7 (m, 1H, amido) IR (KCl) : 1700 (> C = 0)

Optical rotation [α] D = -14.97 ° (c 1.0 dimethyl sulfoxide).

The second recrystallization gave a second precipitate (0.95 g), m.p. 224-228 ° C with decomposition. The overall yield was 87.5%.

N- {2 - / [N- (trifluoroacetyl) -3,3 ', 5,5'-tetraiiodothyronyl-7-aminoethyl} -21,31-O-isopropylidene adenosine___ 70726 29

A solution of 8.72 g (10.0 mmol) of α- (N-trifluoroacetyl) amino-3- [3,5-diiodide-4- (3 ', 5'-diiodide-4'-hydroxyphenoxy) phenyl] propionic acid acid and 3.86 g (11.0 mmol) of 6- (2-aminoethyl) amino-9- (2 ', 3'-O-isopropylidene-BD-ribofuranosyl) purine in 50 ml of dry dimethylacetamide were prepared in dry under an argon atmosphere at -20 ° C.

To this cold stirred solution was added a solution of 3.04 g (11.0 mmol) of diphenylphosphoryl azide (Aldrich Chemical Co., Milwaukee, Wisconsin, USA) in 10 mL of dry dimethylacetamide, followed by the addition of 1.6 mL (11 mL) of .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 precipitate was collected by filtration and dried in vacuo (56 ° C) to give 13.0 g of a light cream 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 hot filtration. Continued cooking 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 (decomposed).

Analysis: Calculated for C 32 H 30 F 3 J 4 O 8: C 31.89; H 2.51; N 8.14 experimentally: C 31.95; H 2.60; N 7.86 NMR δ 220 MHz, (CD 3) 2 O 7 <$ 1.32 (s, 3H, isopropylidene), 1.55 (s, 3H, isopropylidene), 6.14 (d, 1H, 1'- ribose), 7.02 (s, 2H, thyroxine), 7.82 (s, 2H, thyroxine), 8.25 (s, 1H, purine), 8.36 (s, 1H, purine), 8 .41 (t, 1H, J = 6, amido), 9.64 (d, 1H, J = 8, trifluoroacetamide)

Optical rotation [α] D = -11.82 ° (c 1.0, pyridine).

N- {2- [N- (trifluoroacetyl) -3,3 ', 5,5'-tetraodithronylamino] ethyl} -2', 3'-O-isopropylidene-5'-adenylic acid monotriethylamine salt monohydrate__

Solution of 1.2 g (1.0 mmol) of N- {2- / N- (trifluoroacetyl) -3,3 ', 5,5'-tetraiiodothyronyl-7-aminoethyl} -2', 3'-O-isopropylidene -adenosine in 10 ml of dry triethyl phosphate, was prepared under a dry argon atmosphere at 0 ° C. To the cold, stirred solution was added 0.45 mL (5 mmol) of phosphorus oxychloride. The resulting solution was kept at 0 ° C for 24 hours and then added dropwise with stirring to 1 L of ice water. The resulting precipitate was collected by filtration t 70726 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 was extracted with dry acetone and then recrystallised from a mixture of dry methyl 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 38 H 48 F 3 J 4 N 8 O 12 P: C 32.50; H 3.45; N 7.98 experimentally: C 32.24; H 3.08; N 7.58 NMR / 60 MHz, (CD 3) 2 SO / 61.53 (s, 3H, isopropylidene), 6.2 (d, 1H, 1Ή-ribose), 7.1 (s, 2H, thyroxine aroma), 7.87 (s, 2H, thyroxine aroma), 8.27 (s, 1H, purine), 8.52 (s, 1H, purine). Optical rotation λmax = -17.50 ° (c 1.0, CH 2 OH).

N- {2- [N- (trifluoroacetyl) -3,3 ', 5,5'-tetraodithionyl] amino] -5'-adenylic acid __ 200 mg (0.14 mmol) of N- {2- (N - (trifluoroacetyl) -3,3 ', 5,5'-tetraiiodothyronylaminoethyl} -2', 3'-O-isopropylidene-5'-adenylic acid monotriethylamine salt monohydrate was suspended in 1 meter 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 another 15 hours and then evaporated in vacuo (30 ° C). The resulting residue was evaporated four times in vacuo (25 ° C) from a volume of 20 ml of anhydrous ethyl alcohol and then dried in vacuo (25 ° C) to leave a white solid.

The solid was stirred with 10 mL of cold methyl alcohol for 30 minutes, collected by filtration and dried in vacuo (25 ° C) to give a white solid (135 mg, 76% yield) which slowly melted with decomposition at 188 ° C: above.

Analysis: Calculated for C 29 H 27 F 3 J 4 N 7 O 11 P: C, 27.97; H 2.19; N 7.87 experimentally: C 28.11; H 2.31; N 7.65.

NMR / 220 MHz, (CD 3) 2 SO 7 δ 5.95 (d, 1H, 1'-ribose), 7.04 (s, 2H, thyroxine aroma), 7.84 (s, 2H, thyroxine aroma), δ, (S, 1H, purine), 8.36 (s, 1H, purine), 8.43 (m, 1H, amido), 9.66 (d, li, trifluoroacetamido)

Optical rotation / α _ / ^ = -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'-tetraiiodothyronyl-7-aminoethyl} -5'-adenylic acid were dissolved in 10 ml of dry dimethylformamide and tri-n-butylamine (96, 0.4 mmol) were added, followed by 1,1'-carbonyldiimidazole (320 mg, 2.0 mmol). After stirring for 18 hours at room temperature protected from moisture, water (280) was added and the solvent was then evaporated in vacuo.

The resulting oil was dried by repeated vacuum evaporations from dry dimethylformamide (4 times from 10 ml). The obtained phosphorimidazolate was redissolved in 10 ml of dry dimethylformamide, and a solution of 0.4 mmol of the tri-n-octylamine salt of riboflavin-5'-monophosphate in 10 ml of dry dimethylformamide was added dropwise.

The salt was prepared by adding 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 μL, 0.4 mmol) in 100 mL of acetone. . After 30 minutes, the resulting mixture was evaporated in vacuo. The residue was dried by repeated evaporation in vacuo from dry dimethylformamide to leave the salt as an orange solid.

The above solution containing phosphorus imidazolidate and riboflavin-5'-monophosphate salt was divided into two equal fractions after 24 hours and the second fraction was evaporated in vacuo. The resulting residue was chromatographed on a column (2.5 x 78 cm) prepared from 100 g of Sephadex LH-20 resin (Pharmacia Fine Chemicals, Uppsala, Sweden) pre-expanded (18 hours) with a mixture of dimethylformamide and triethylammonium bicarbonate relative to 19: 1 (v / v) (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 examined by eluting with silica gel 60 on silanized Rp-2 TLC plates (E. Merck, Darmstadt, Germany).

TLC plates were developed using a 40: 40: 25: 1: 1 (v / v) mixture of acetone, chloroform, methyl alcohol, water and triethylamine. Fractions 11-17 from 32 70726 obtained from the above column chromatography were combined and evaporated in vacuo. The residue was chromatographed on a column (2.5 x 75 cm) prepared from 125 g of Sephadex LH-20 resin pre-expanded (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 examined by absorption of ultraviolet light at 254 nm. The volume of the fractions was increased to 20 ml starting from fraction 150. The salt concentration of the eluent was gradually reduced as follows: 0.15 M ammonium bicarbonate from fraction 295, 0.075 M ammonium bicarbonate from fraction 376 and water from fraction 430. Total 480 the fraction was recovered. Fractions Nos. 200-235 were combined and evaporated in vacuo to leave a labeled association as a yellow-orange residue. The alkaline aqueous solution of this residue had ultraviolet absorption maxima at the following wavelengths: 266 nm, 350 nm, 373 nm and 450 nm. The yield estimated from the absorbance at 450 nm was about 5%.

Phosphodiesterase preparations (Worthington Biochemical Corp., Freehold,

New Jersey, USA) isolated from snake venom (Crotalus Adamanteus) hydrolyzed the above product to riboflavin 5'-monophosphate and thyroxine-substituted 51-adenylic acid from which the trifluoroacetyl protecting group had been removed.

B. Preparation of Apoglucose Oxidase An apoenzyme prepared according to Example 1 Part B was used.

C. Assay Reagents 1. Labeled conjugate - N®- (2-aminoethyl) thyroxine-FAD was diluted in 0.1 M phosphate buffer (pH 7) to a concentration of 1 μM.

2. Apoenzyme - Apoglucose oxidase was diluted with 0.1 M phosphate buffer (pH 7) to a concentration of 0.6 μM FAD binding site (determined as in Example 1, Part C-2).

3. Insolubilized antibody - Washed, wet cake of Sepharose 4B gel (Pharmacia Fine Chemicals, Uppsala, Sweden) activated with cyanogen bromide according to the method of March et al., Anal. Biochem. 60: 119 (1974), 70726 33 was added to a solution of 85 mg of antibody (isolated from anti-bovine anti-bovine serum albumin) in 20 ml of 0.1 M phosphate buffer (pH 7.0) and stirred slowly. hours at 4 ° C. At the end of the coupling reaction, 1 ml of 1-M alanine was added and shaking was continued for another 4 hours to protect the unreacted sites. The resulting Sepharose gel-bound antibody was washed in a sinter funnel using 400 ml of both 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 0.01% sodium azide to give 22 ml of about 50% suspension.

4. Standard - A 1.15 mM stock solution of thyroxine in 5 mM sodium hydroxide was diluted to 2 μM in 0.1 M phosphate buffer (pH 7).

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

D. Determination procedure

Binding reaction mixtures were prepared by mixing 150 μl of insoluble antibody suspension, 80 μl of labeled assembly solution, various amounts of standard thyroxine solution to give different thyroxine concentrations to the reaction mixtures, and a sufficient amount of 0.1 M phosphate buffer (pH 7) in a total volume of 500 μl. The reaction mixtures were incubated with shaking for two hours at 25 ° C. Each reaction mixture was then vacuum filtered through a glass wool-backed, dry Pasteur pipette pretreated with periodate and ethylene glycol solutions to eliminate any FAD impurity. To a 300 μl fraction of each filtrate was added 130 μl of monitoring reagent and 50 μl of apoenzyme solution. After one hour, the absorbance of each reaction mixture was measured at 520 nm.

34 E. Results 7072 6

The following is Table 3, which shows the results of the assay procedure for measuring thyroxine. Absorbance results are reported as the mean of the two runs corrected for residual enzyme activity in the apoenzyme solution (absorbance 0/522) and endogenous FAD in the antibody suspension (absorbance 0.142).

TABLE 3

Corrected mean absorbance volume (μl) of the added thyroxine standard at 520 nm 0 0.223 25 0.221 75 0.281 250 0.286

The results show that the present invention provides a useful heterogeneous type specific binding assay.

Example 3

Homogeneous Binding Assay for Thyroxine A. Preparation of Labeled Conjugate - N6- (6-Aminohexyl) Thyroxine-Flavin Adenine Dinucleotide 6- (6-Aminohexyl) -amino-9- (2 ', 3'-O-isopropylidene-BD-ribofuranosyl) purine ____ 16, 0 g (50 mmol) of 6-chloro-9- (2 ', 3'-O-isopropylidene-8-D-ribofuranosyl) purine / Hampton et al., J. Am. Chem. Soc. 83: 1501 (1961j_7) was added with stirring to a sample of freshly distilled 1,6-diaminohexane (58 g, 500 mmol) in molten (70 ° C) .The resulting mixture was stirred under argon at 40 ° C for 18 h. Excess diamine was removed by distillation. under reduced pressure (60 ° C, 0.91 mm Hg) The resulting pale yellow residue was adsorbed onto 150 g of silica gel 60 (E. Merck, Darmstadt, Germany) and used as the top on a chromatography column prepared from a slurry containing 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 and analyzed by thin layer chromatography (TLC) on silica.

II

70726 35 gel eluting with a mixture of absolute ethyl alcohol and triethylammonium bicarbonate (pH 7.5, 1-M) in a ratio of 7: 3 (v / v). Fractions 391-900 from column chromatography were combined and evaporated in vacuo to leave 15.0 g of a glassy residue (74% yield). A 1 g sample of glass was dissolved in a small volume of methyl alcohol, which was then poured onto the top of a column prepared from 80 g of Sephadex LH-20 resin (Pharmacia Fine Chemicals, Uppsala, Sweden) pre-expanded in methyl alcohol. The column was eluted with methyl alcohol. A total of 90 8 ml fractions were collected. Fractions were analyzed by TLC on silica gel 60 eluting with a 7: 3 (v / v) mixture of absolute ethyl alcohol and triethylammonium carbonate (pH 7.5, 1-M). Fractions 19-27 from column chromatography were combined and evaporated in vacuo to leave 910 mg (91% recovery) of white glass.

Analysis: Calculated for C 18 H 19 N 2 O 2: C, 56.14; H 7.44; N 20.68 experimentally: C 53.91; H 7.33; N 19.18.

NMR (60 MHz, CDCl 3): δ 1.40 (s, 3H, isopropylidene), 1.63 (s, 3H, isopropylidene), 5.98 (d, 1H, 1'-ribose), 7.92 (s , 1H, purine), 8.36 (s, 1H, purine).

Optical rotation / α_ / ε = -50.11 ° (c, 1.0, methyl alcohol).

N- {6- / N- (trifluoroacetyl) -3,3 ', 5,5'-tetraiiodothyronyl / aminohexyl} -2', 3'-O-isopropylidenedenosine_

A solution of 4.36 g (5.0 mmol) of α- (N-trifluoroacetyl) amino-g-1-3,5-diiodo-4- (3 ', 5'-diiodo-4'-hydroxyphenoxy) ) -phenylpropionic acid prepared as described in Example 2, Part A above and 2.24 g (5.5 mmol) of 6- (6-aminohexyl) -amino-9- (21,3'-O-isopropylidene) -gD-ribofuranosyl) purine in 100 ml of dry dimethylformamide, 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 36,70726 lis) of a white solid. A sample of this solid was recrystallized from a mixture of acetone and water to give a white solid, m.p. 205-207 ° C (decomposed).

Analysis: Calculated from the formula C36H38F3J4N7O8: C 34.28 ·, H $ 3.04 N 7.77 Experimentally: C 34.22) H 2.99) N 7.41

Mass spectrum (20 ma) m / e: 1262 (MH +), 1164 (M + minus COCF3)

Optical rotation / = = -21.89 ° (c 1.0, pyridine) N- {6- [N- (trifluoroacetyl) -3,3 ', 5,5'-tetraiiodothyronyl] aminohexyl} -2,3-O-O -isopropylidene-5'-adenylic acid monotriethylamine salt monohydrate 1 _______

A solution of 1.89 g (1.5 mmol) of N- {6- / N- (trifluoroacetyl) -3,3 ', 5,5'-tetraodionitronyl / aminohexyl} -2', 3'-O -isopropylidene-adenosine in 15 ml of dry triethyl phosphate, was prepared under a dry argon atmosphere at -10 ° C. To the cold, stirred solution was added 0.68 mL (7.5 mmol) of phosphorus oxychloride. The resulting solution was kept at -15 ° C for 18 hours and 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 dissolved in 10 mL of 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 42 H 56 F 3 J 4 O 12 F: C 34.54; H 3.86; N 7.67 experimentally: C 35.24; H 3.88; N 7.75

Mass spectrum (20 ma) m / e: 1342 (MH +), 1244 (M + minus COCl 3)

Optical rotation λmax = -17.20 (c 1.0, CH3OH).

N- [6- (N- (trifluoroacetyl) -3,3 ', 5,5'-tetraiiodothyronyl] -aminohexyl] -5-adenylic acid 600 mg (0.41 mmol) of N- {6- [N- (trifluoroacetyl) - 3,3 ', 5,5'-Tetraiodothyronyl-7-aminohexyl} -2', 3'-O-isopropylidene-5'-adenylic acid monotriethylamine salt monohydrate was suspended in 0.6 ml of 37,70726 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 another 15 hours and then evaporated in vacuo (30 ° C). The residue obtained was evaporated in vacuo five times from 20 ml of anhydrous ethyl alcohol and then triturated with 30 ml of 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 a white solid, m.p. 180-183 ° C (decomposed).

Analysis: Calculated from the formula C33H35F3J4N7 ° i] _p '· C 30.46; H 2.71; N 7.54 experimentally: C 30.77; H 2.55; N 7.29.

Mass spectrum (20 ma) m / e: 1302 (MH +), 1204 (M + minus COCF3).

Flavin adenine dinucleotide-thyroxine conjugate 130.13 mg (0.1 mmol) of N- {6- / N- (trifluoroacetyl) -3,31,5,51-tetraiodothyronyl-7-aminohexyl} -5 ', adenylic acid was placed under an argon atmosphere. To this sample was added a solution of 14 (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 1. in ml of dry dimethylformamide. After 24 hours, another equivalent of 1,1'-carbonyldiimidazole (16.2 mg) in 1 ml of dry dimethylformamide was added. The above reaction was allowed to proceed for a total of 48 hours at room temperature protected from moisture. A 47.3 mg (0.1 mmol) sample of the ammonium salt of riboflavin-5'-raonophosphate was converted to the corresponding tri-n-octylamine salt as described in Example 2, Part A. This salt was dissolved in 3 ml of dry dimethylformamide and added to the above solution containing the adenyl acid intermediate phosphorimidazolidate.

The resulting solution was allowed to stand in the dark at room temperature protected from moisture for 24 hours. The solvent was evaporated in vacuo and the resulting residue was chromatographed on a column (2.5 x 78 cm) prepared from 100 g of Sephadex LH-20 resin (Pharmacia Fine Chemicals, Uppsala, Sweden) pre-expanded (18 hours) with dimethylformamide. and a mixture of triethylammonium bicarbonate (1-M, pH 7.5) in a ratio of 19: 1 (v / v). The column was eluted with the above 19: 1 (v / v) mixture and 5 ml fractions 70726 38 were collected. The effluent from the column was examined by eluting with silica gel 60 on silanized RP-2 TLC plates (E. Merck, Darmstadt, Germany). TLC plates were developed using a 40:40: 25: 1: 1 (v / v) mixture of acetone, chloroform, methyl alcohol, water and triethylamine.

Fractions 24-38 from column chromatography were combined and evaporated in vacuo. The residue was chromatographed on a column (2.5 x 85 cm) prepared from 125 g of Sephadex LH-20 resin pre-expanded (18 hours) in 0.1 M ammonium bicarbonate. The column was eluted with a linear gradient of 2 L of 0.1 M ammonium bicarbonate and 2 L of water, and 23 mL fractions were collected. The effluent was examined by ultraviolet absorption (254 nm). Fractions 170-182 were combined and evaporated in vacuo. The residue was chromatographed on a column (2.5 x 55 cm) prepared from 80 g of Sephadex LH-20 resin pre-expanded in 0.05 M ammonium bicarbonate. The column was eluted with a linear gradient formed from 2 L of 0.05 M ammonium bicarbonate and 2 L of 0.02 M ammonium bicarbonate. The removal current was checked by ultraviolet absorption (254 nm). Elution was continued with 2 L of 0.2 M ammonium bicarbonate, collecting 23 mL fractions. A total of 257 fractions were collected. Fractions Nos. 70-110 were combined and evaporated in vacuo to leave a labeled association as a yellow-orange residue. The alkaline aqueous solution of this residue had absorption maxima at the following wavelengths: 270 nm, 345 nm and 450 nm. The yield, estimated from the absorbance at 450 nm, was about 5%.

A phosphodiesterase preparation (Worthington Biochemical Corp., Freehold, New Jersey, USA) isolated from snake venom (Crotalus Adamanteus) hydrolyzes the above product to riboflavin-5'-monophosphate and thyroxine-substituted 5'-adenylic acid deprotected with trifluoroacetyl.

B. Preparation of apoglucosidase

Glucose oxidase was dialyzed and lyophilized as in Example 1, Part B above. A portion of the lyophilized glucose oxidase (80 mg) was dissolved in 20 ml of 30% (v / v) glycerol at 4 ° C 70726 39 and the solution was adjusted to pH 1.4 by the addition of concentrated sulfuric acid (H 2 SO 4). The solution was incubated at 4 ° C for 2 hours and then passed through a Sephadex-G-50 column (Pharmacia Fine Chemicals, Uppsala, Sweden) at 4 ° C equilibrated with 30% (v / v) glycerol, which was adjusted to pH 1.4 with concentrated I 2 SO 4. The eluted protein peak was collected (27 ml containing 74% by weight of material added to the column) and 200 mg of bovine serum albumin was dissolved in the combined eluate. Activated carbon (600 mg; RIA grade, manufactured by Schwarz-Mann, Orangeburg, New York, USA) was then added and the mixture was neutralized by the addition of 4.0 mL of 0.4 M phosphate buffer (pH 8.0) and sufficient 2N sodium hydroxide To adjust the pH to 7.0. The mixture was stirred for 60 minutes at 4 ° C and filtered successively through a 0.8 μm and 0.22 μm Millipore filter (Millipore Corp., Bedford, Massaschusetts, USA). Sodium azide (10% w / v) was added to give a final concentration of 0.1%.

C. Assay Reagents 1. Labeled Coalition - N®- (6-aminohexyl) -thyroxine-FAD was diluted in 0.1 M phosphate buffer containing 0Γ1% (w / v) bovine serum albumin (pH 7.0) at 400 nM. a concentration.

2. Apoenzyme - Apoglucose oxidase was diluted in 0.1 M phosphate buffer containing 0.1% (w / v) bovine serum albumin (pH 7.0) to 4.0 micronormal concentration (μN) FAD binding sites (see Part C-2 of Example 1).

3. Antiserum - Saturated ammonium sulfate solution (7.5 ml) was pumped (0.1 ml / min) at room temperature into 15 ml of rabbit anti-thyroxine antiserum with stirring in 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 volume of antiserum by the addition of 50 mM borate buffer (pH 8.6). The immunoglobulin solution was further diluted in 0.1 M phosphate buffer containing 0.1% bovine serum albumin (pH 7.0) for use in the ratios indicated in the assays described below.

_ - τ 40 70726 4. Standards - The sodium salt of thyroxine (Sigma Chemical Co., St. Louis, Missouri, USA) was used as a stock solution of 1 mg / ml as a titrated solution in distilled water with 2N sodium hydroxide to a final pH of 10, 2. Standards were prepared by diluting this stock solution in 0.1 M phosphate buffer containing 0.1% bovine serum albumin (pH 7.0).

5. Monitoring Reagent - Glucose oxidase assay reagent was prepared by mixing 11 parts of 0.15 M phosphate buffer (pH 7.0) containing 1.8% (w / v) bovine serum albumin, 2 parts of 2.0 mM 4-aminoantipyrine, containing 0.6 mg / ml peroxidase, 2 parts of 20 mM 3,5-dichloro-2-hydroxybenzenesulfonate in 0.1 M phosphate buffer (pH 7.0) and 2 parts of 1.0 M glucose in saturated aqueous benzoic acid .

D. Assay Procedures 1. Addition of All Reagents Without Pre-d-nknh ^ fluorol Step The following were added sequentially to separate reaction cuvettes: 0.05 mL of labeled conjugate solution, 0.05 mL of selected standard solution, 0.1 mL of a 1: 4 dilution of antiserum in phosphate buffer, 1 , 7 ml of monitoring reagent and 0.1 ml of apoenzyme solution. Each reaction mixture was incubated for 30 minutes at 20 ° C and the absorbance at 520 nm was measured.

2. Pre-incubation with antiserum

The following were added sequentially to separate reaction cuvettes: 0.05 ml of labeled conjugate solution, 0.05 ml of selected standard solution, and 0.1 ml of a 1:16 dilution of antiserum in phosphate buffer. Each reaction mixture was incubated for 20 minutes at 20 ° C, after which 1.7 ml of monitoring reagent and 0.1 ml of apoenzyme solution were added to each successively. After an additional 30 minutes of incubation at 20 ° C, the absorbance at 520 nm was measured in each cuvette.

3. Pre-inkpbOittti with apoentswmin

The following were added sequentially to separate reaction cuvettes: 0.05 ml of labeled conjugate solution, 0.05 ml of selected standard solution, and 0.1 ml of apoenzyme solution. Each reaction mixture was incubated for one minute at 20 ° C and then 0.1 ml of a 1: 2 dilution of antiserum in phosphate buffer and 1.7 ml of monitoring reagent 41 70726 were added sequentially. After an additional 30 minutes of incubation at 20 ° C, the absorbance at 520 nm was measured in each cuvette.

4. Successive pre-incubation first with antiserum and then with apoenzyme_

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

5. Successive pre-incubation first with apoenzyme and then with antiserum__________

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

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

E. Results Assays were performed in duplicate with appropriate blank experiments. It was found that in Methods 1-3, the concentration of thyroxine in 70726 42 standard solutions was directly proportional to glucose oxidase activity, while in Methods 4-6, the concentration of thyroxine was inversely proportional to enzyme activity. In methods 1-3 without thyroxine, enzyme activity was inhibited, while in methods 4-6, enzyme activity improved. Methods 4-6 were also found to be somewhat more sensitive to thyroxine than other methods. The reason or reasons why enzyme activation variably inhibited or improved depending on the reaction sequence is not fully understood; however, the results showed that the present invention provides a homogeneous specific assay method for the determination of thyroxine using a number of different reaction sequences.

Example 4

Homogeneous binding assay for theophylline A. Preparation of labeled conjugate - theophylline-FAD.

To a solution of 2.4 μmol of flavin-N 2 -aminohexyladenine dinucleotide prepared as described in Example 1A above in 200 μl of dimethyl sulfoxide under argon was added 0.9 mg of 1,3-dimethyl-1,6, 7,8-Tetrahydropyrido [1,2-e] purine-2,4,9 (3H) -trione (3.62 μmol) prepared according to the method of Cook et al., Res. Comm. in Chem. Pathol, and Pharm. 13: 497 (1976), to which an additional 1.8 mg (7.3 μmol) of the same substance was added after 4 hours. After stirring overnight, the solvent was evaporated in vacuo (0.1 mm Hg) and the residue was chromatographed on a 2.5 x 90 cm Sephadex LH-20 column (Pharmacia Fine Chemicals, Uppsala, Sweden) and eluted with 0.3 g. -M with triethylammonium bicarbonate buffer (pH 7.8). The crude product, eluting with an effluent in the range of 210-246 ml, was collected, placed on a 20 x 20 cm x 1000 silica gel plate and eluted with an 8: 2 mixture of ethanol and 1-M triethylammonium bicarbonate buffer (pH 7.8). The zone containing the desired product (Rf = 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 Sephadex LH-20 resin eluting with 0.3 M buffer afforded 1.26 μmol of labeled association as determined by absorbance measurement at 450 nmr, representing 53% yield.

B. Antibody Binding Reactions

The antibody was grown in rabbits against 8- (3-carboxypropyl) -1,3-43,70726 dimethylxanthine BSA immunogen as described by Cook et al., Res. Comm. in Chem. Pathol. Pharm. 13: 497 (1976).

Antibody binding reactions were performed at room temperature in 0.1 M sodium phosphate buffer (pH 7.0) and measurements of glucose oxidase activity were performed 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-FAD labeled conjugate or 160-nM N- (6-aminohexyl) FAD (FAD derivative) C theophylline antiserum (diluted 10-fold). ti reagent (A) D apoglucose oxidase (50 nM FAD binding site / ml) Monitoring reagent E: 200 ug peroxidase / ml, 0.71 mM mM 4-aminoantipyrine? 7.1 mM 3.5-dichloro-2-hydroxybenzenesulfonate, 353 mM glucose and 35 mg BSA / ml.

Reagents A, B and C were combined in separate reaction cuvettes in the ratios indicated in Table 4 below. Reagent D (100 μl) and Reagent E (283 μl) were then added rapidly and sequentially to each reaction mixture, followed by incubation for 30 minutes at 20 ° C. The absorbance at 520 nm was then measured for each cuvette. The results shown in Table 4 are averages of the double runs.

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70726 45

The results of reactions 2-6 showed that the antiserum did not affect the activity of the FAD derivative (i.e., was not associated with theophylline). Reactions 6-11 showed that increasing amounts of theophylline antiserum decreased the activity of the labeled conjugate relative to its ability to bind to the apoenzyme.

C. Competitive Binding Assays These reactions were performed as outlined in Part B above, except that the indicated amounts of theophylline were combined with Reagents A and B before Reagent C was added and also that a 100-fold dilution of antiserum was used for Reagent C. The results are shown in Table 5 below.

Reactions 1-3 were comparative and showed that the theophylline antibody impairs the ability of the labeled association to bind to the apoenzyme. Reactions 4-9 show that the FAD activity decreases in proportion to the amount of theophylline in the reaction mixture.

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Example 5

Homogeneous Binding Assay for Human IgG A. Preparation of Labeled Conjugate - IgG-FAD To 4.24 mg of flavin-N-aminohexyladenine dinucleotide prepared as described in Example 1, Part A above, 2.5 mg of dimethyl adipimidate dihydrochloride (Pierce Chemical Co., Rockford, Illinois, USA) in 1 ml of water and 5 μl of triethylamine. The reaction was stirred at room temperature for 10 minutes and 40 mg of human immunoglobulin (IgG) in 1 ml of 0.1 M sodium pyrophosphate buffer (pH 8.5) was then added. After stirring for an additional 3 hours at room temperature, the reaction mixture was poured onto a 2.5 x 50 cm Sephadex G-25 column (Pharmacia Fine Chemicals, Uppsala, Sweden) equilibrated and eluted with 0.1 M sodium phosphate buffer (pH 7.0). . Fractions from the first eluted peak with an absorbance at 450 nm were collected and dialyzed sequentially against 4 L of 0.1 M sodium phosphate buffer (pH 7.0) for 16 hours, against 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 a concentration of 0.1% (w / v). The reaction material was filtered through a 0.22 μm lipo filter and stored.

B. Antibody Binding Reagents The reagents used in the assay were as follows:

Reagent Composition A 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-hydroxybenzenesulfonate in reagent A,

E 1.2 mg / ml horseradish peroxidase in reagent A

F 30% (w / v) bovine serum albumin (Research Products Division, Miles Laboratories,

Inc., Elkhart, Indiana, USA) G IgG-FAD labeled association in solution stored above H apoglucose oxidase I rabbit antiserum against human IgG (obtained from Behring Diagnostics, Somerville, NJ) J standards - human IgG in Reagent A predetermined amounts 70726 48

Reagent mixtures were prepared as follows:

Mixture No. 1 - 180 ul of reagent A, 20 ul of reagent B, 100 ul of reagent D and 5 ul of reagent G (5.4 ul).

Mixture No. A total volume of 2-0.3 ml in various proportions, including the amounts of Reagent I shown in Table 6 below, with the final volume consisting of Reagent A.

Mixture No. 3-80 of reagent D, 50 of reagent E, 33 of reagent F, 137 of reagent A and 1.6 of reagent H (4,2-YUN FAD binding site).

To 300 of mixture No. 1 was added 300 of mixture No. 2 and 300 of reagent A in separate reaction cuvettes. After incubation for at least 10 minutes at room temperature, 300 of mixture # 3 was added to each reaction. After an additional 30 minutes of incubation at 20 ° C, the absorbance at 520 nm was measured from each cuvette.

The results were as follows: TABLE 6

To prepare mixture No. 2 Absorbance volume of antiserum added (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 show that as the amount of antibody increases, the glucose oxidase activity generated by FAD-labeled IgG decreases.

C. Competitive Binding Assays These reactions were performed using the reagents described in Part B above. The indicated amounts of human IgG in 300 μl volumes of Reagent A were combined with Mixture No. 1 in separate reaction cuvettes. A mixture of 12 reagent I and 288 reagent A was then added to each reaction. After 10 minutes, 300 μl

II

49 No. 70726 mixture No. 3 was added and the reaction mixtures were incubated at 20 ° C for 30 minutes. After this time, the absorbance at 520 nm was measured in each cuvette. The results were as follows: TABLE 7

Increased Absorbance Amounts of Human IgG (μg) (520 nm) 0 0.579 4 0.653 3 0.751 12 0.844 16 0.986 24 1.06 Accordingly, it was shown that the present invention provides a specific binding assay for the determination of a high molecular weight ligand, human IgG, in liquid media.

Reagenssiväliaineet

As will be apparent to those skilled in the art from the above description and example, the reagent means used to carry out this method can take a number of different forms. In particular, such reagent means may take the form of a uniform test composition, such as a suitable solid or liquid form. As an example of a determination of the test Coalition form of a ligand in accordance with the present invention comprises (a) a labeled consortium consisting of an organic substitute group, which is linked to the ligand or its sidosanalogiin, (b) an apoenzyme ligand specific bonding partner and (c), which is able to mate with the replacement group producing a holoenzyme. Such a test composition may also contain reagents for measuring holoenzyme activity and, if desired, and where appropriate, the labeled association and apo enzyme may be combined in the form of their conjugated enzyme complex. Of course, conventional diluents, buffering materials, stabilizers, etc. may also be included in the test composition.

The reagent means may also take the form of a test kit, i.e. a packaged combination of containers containing the necessary reagent parts. As another example, one embodiment of a test kit for determining a ligand in accordance with the present invention comprises one or more containers having (a) a labeled moiety consisting of an organic substituent attached to the ligand or binding analog thereof, (b) a specific ligand binding moiety, and ( c) an apoenzyme capable of joining a substituent group to form a holoenzyme. Such a set of test media may also provide reagents for measuring holoenzyme activity in one or more of the same or different containers, and if desired, and when appropriate, the labeled association and apoenzyme may be combined in the form of a conjugated enzyme complex thereof. In one embodiment, the test medium kit comprises at least two separate containers, one with a labeled association and optionally reagents for determining holoenzyme activity, and the other with a binding partner and an apoenzyme. Of course, the test kit may contain other reagents known in the art and which may be commercially desirable, such as buffers, diluents, standards, etc.

Il

Claims (5)

51 70726 A quantitative homogeneous immunoassay method for determining a hapten and an antigen in a liquid medium, the method comprising forming a reaction mixture by combining the liquid medium with an antibody to the hapten or antigen and a labeled conjugate having a label component attached to the hapten or antigen. is different when the labeled conjugate is bound to the antibody than if it is not bound and the detectable reaction in the reaction mixture is measured, characterized in that the label component is a flavin-adenine dinucleotide and said reagents also contain apoglucose oxidase capable of binding to a flavin-adenine dinucleotide glucose oxidase, and that glucose oxidase activity is measured in the reaction mixture. Method according to Claim 1, characterized in that the glucose oxidase activity is measured by a colorimetric method. Process according to Claim 1, characterized in that the hapten or antigen is theophylline, thyroxine, IgG or N-2 ', 4'-dinitrophenyl-6-aminocaproate. Reagent system for the determination of a hapten or antigen by the method of claim 1, characterized in that it comprises 1. a labeled conjugate in which the label component is a flavin-adenine dinucleotide linked to a hapten or antigen, 2. a hapten or antigen antibody, and 3. an apoglucose oxidase. Labeled conjugate for use in the method of claim 1, characterized in that the label component is a flavin adenine dinucleotide.