DK154670B - HOMOGEN, SPECIFIC BINDING ANALYSIS WITH A BRAND PROSTHETICAL GROUP AND REAGENT USE FOR ANALYSIS - Google Patents

Description

in

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The present invention relates to a homogeneous, specific binding assay for the determination of a ligand in a liquid medium, in which analysis provides a reaction mixture by combining the liquid medium with reagents comprising a labeled 5 conjugate having a labeling component comprising is coupled to a binding component which combines to form a binding reaction system in which a bound form and a free form of the labeled conjugate are formed, the ratio of the label component of the two forms being a function of the ligand in the liquid medium and the label component exhibiting a detectable response which is different in the two forms, after which the detectable response is measured in the reaction mixture.

The labeling component is preferably just the remainder of a prosthetic group or a holoenzyme residue containing the rest of the prosthetic group in combination with an apoenzyme in the form of a holoenzyme complex. In the first case, the labeling component is preferably determined by the addition of an apoenzyme after initiating the binding reaction and subsequently measuring the resulting holo-enzyme activity. In the latter case, the labeling component is simply determined by measuring the holoenzyme activity. The assay method according to the invention can be carried out according to conventional homogeneous or heterogeneous technique. The most preferred apoenzymes are apoglycose oxidase and apoperoxidase. The analysis method allows for 25 calorimetric determination and can be easily automated. The invention also relates to reagents, such as test compositions and test agents for determining a ligand in a liquid medium, as well as ligands labeled with a prosthetic group or a conjugated enzyme. The specific binding assay is performed without the use of 30 radioisotopes as labeling substance.

In the traditional specific binding assay methods, the assay samples are combined with reagents of various compositions comprising a conjugate having a detectable labeling component 35 and a binding component which, together with any other constituents of the reagents, participates in the formation of a binding reaction system which produces two species or forms. of the labeled conjugate, a bound form and a free form. The relative amount or part of the labeled conjugate which appears in the bound form as compared to 2

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the free form is a function of the presence (or amount) of the ligand to be detected in the assay sample.

By way of illustration, a traditional competitive bonding method will be described. In such a method, the reagent will comprise (1) a labeled conjugate in the form of the ligand to be detected (e.g., an antigen or hapten), which ligand constitutes the binding component of the conjugate and is chemically bound to a label component, and (2 ) a specific binding partner to the ligand (eg, an antibody). By combining the assay sample and reagent, the ligand to be detected and the binding component of the labeled conjugate compete in a substantially non-discriminatory manner for non-covalent binding to the specific binding partner. As a result, either the amount of labeled conjugate that would be bound to the binding partner (i.e., results in the bound form) or the amount of labeled conjugate that remains free (i.e., the free form which is not bound to the binding partner) can be determined. as a function of the amount of competing ligand present. The amount of resulting labeled conjugate in both forms is determined by measuring the labeling component thereof.

Where the labeled conjugate in the bound form and the free form is essentially indistinguishable from the means used for detecting the labeling component, the bound form and the free form must be physically separated to enable the assay to be performed. This type of analysis is referred to in the art as "heterogeneous analysis". Where the bound forms and free forms of the labeled conjugate can be distinguished analytically from one another, no separation step is required and such analysis is called "homogeneous analysis".

The first type of highly sensitive specific binding assays found was the radioimmunoassay utilizing a radioactive isotope as a labeling component. Such an analysis must necessarily follow the heterogeneous form, since the detectable property of the labeling substance is qualitatively unchanged in the free and bound form. Due to the difficulty and difficulty of handling radioactive materials, many new analytical systems have been devised which use as a labeling component materials other than radioisotopes, e.g. enzymes, fluorescent molecules, bacterial phages, metal and organometallic complexes, coenzymes, enzyme substrates, enzyme inhibitors,

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3 torics, cyclic reactants and chemistry luminescent reactants.

Several different heterogeneous binding reaction systems in which an enzyme is used as a labeling component are described in the specification of 5 U.S. Patents Nos. 3,654,090, 3,791,932, 3,839,153, 3,850,752 and 3,879,262, and in J. Immunol. Methods 1: 247 (1972) and J. Immunol. 109: 129 (1972). A heterogeneous binding assay utilizing a non-active precursor of a spectrophotometrically detectable substance as the labeling substance is proposed in the specification of U.S. Pat. 3880934. Heterogeneous assays are also described in Principles of Competitive Protein-Binding Assays, ed. Odell and Daughaday (J.B. Lippincott Co., Philadelphia, 1972).

An immunoassay of the homogeneous type is described by enzyme labeling 15 in U.S. Patent No. 3,817,834, which uses a ligand-enzyme conjugate. The enzyme activity of the conjugate in the bound form is measurably less than that in the free form, allowing a homogeneous assay form to be used. The use of special substances such as labeling substances, such as coenzymes, chemistry of the chemical seer-2o molecules, cyclic reactants and cleavable fluorescent enzyme substrates, in both homogeneous and heterogeneous assay forms, are described in the specification of German Publication Nos. 2,618,419 and 2,618,511 corresponding to Danish patent application no. 1891/76.

25

Although the search for non-radioactive labels for binding assays has provided a number of useful solutions, improvements are still needed. Methods of enzyme labeling initially appeared to be the most promising, but in practice, however, several difficulties have emerged. The high molecular weight and heterologous nature of the enzymes create problems in the characterization, stabilization and reproducible preparation of labeled conjugates. These problems were overcome by replacing the enzymes with low molecular weight labeling substances which can be detected by their reaction activity. These new labels, which include coenzymes, enzyme substrates, cyclic reactants and chemiluminescent reactants, can be used to perform high sensitivity and versatility assays. However, detection of these labels often requires instruments that are not currently found in the clinical laboratory.

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ride around.

It is a primary object of the present invention to provide a binding assay that does not use radioisotopes as a labeling substance, but which can be demonstrated by standard clinical laboratory equipment while providing a sensitive, versatile assay and the labels used. Conjugates are easy to synthesize and characterize.

This object is achieved by the method according to the invention, which is characterized in that the labeling component is flavin adenine dinucleotide, the reagents also comprise apoglucose oxidase which is capable of combining with flavin adenine dinucleotide to produce catalytically active glucose oxidase, and The ash activity is measured in the reaction mixture.

Prosthetic groups are a class of enzyme cofactors. An enzyme cofactor is a non-protein substance whose presence is required for the enzyme to exhibit catalytic activity, which enzyme factor 20 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 that is chemically modified during the parent-catalyzed reaction. Regeneration to the original form of the cofactor requires it to participate in a separate reaction that is catalyzed by a different enzyme than the parent enzyme. In contrast, a prosthetic group is an enzyme cofactor that is chemically modified during the reaction catalyzed by the parent enzyme and regenerated, i.e. genome is formed to its original form, by another reaction catalyzed by the same parent enzyme.

30

Prosthetic groups are usually characterized by being relatively firmly bound to the protein portion of the parent enzyme. The protein moiety is called the apoenzyme, and the catalytically active parent enzyme is called the hol enzyme. The equilibrium reaction for the interaction between a prosthetic group and an apoenzyme can be reproduced as follows: prosthetic group + apoenzyme «HZ * conjugated or hollow enzyme (Pr) (Apo) (Pr-Apo)

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5

The term conjugated enzyme is herein to be understood as the complex formed by combination of the prosthetic group and an apoenzyme, whether or not this complex exhibits catalytic activity, and the term holoenzyme refers only to a catalytically active conjugated enzyme. Further information regarding prosthetic groups, their characteristics and their interactions with apoenzymes can be obtained from Dixon and Webb, Enzymes, first edition, Longmans, Green & Co. (London 1958).

According to the present invention, the organic prosthetic group of flavinadenine dinucleotide (FAD) is coupled to a binding component to form a labeled conjugate for use in binding assays. A central feature of the invention is the resultant interaction between the conjugated prosthetic group and the apoenzyme, which can be illustrated as follows: ØPr + Apn f '-' ~ ► ØPr-Apo

Catalytically inactive components active holoenzyme complex 20

While the above reaction is the normally observed interaction, the conjugated enzyme complex may in some circumstances exhibit little or no holoenzyme activity, such as where the conjugation of the prosthetic group prevents normal holoenzyme-substrate interaction. Nevertheless, labeled conjugates for use in the present invention may appear if the bound conjugated enzyme complex after binding of the conjugated prosthetic group's binding component (represented above by the symbol 0), in the assay binding reaction, exhibits the holoenzyme activity such as where such binding abolishes inhibition. of holoenzyme-substrate interaction.

There are many possible methods for detecting labeling substances, some of which are illustrated hereinafter and using 35 different known homogeneous methods. In all cases, however, the labeling component of the labeled conjugate which participates in the binding reaction comprises a FAD residue, and the assay includes a determination of the labeling component in the bound or free form, or both, the assay being based on the measurement of the holistic enzyme activity.

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e

According to the invention, flavinadenine dinucleotide is used in combination with apoglucose oxidase. The holoenzyme glucose oxidase formed by this pair is readily determined by the well-known hydrogen peroxide method which comprises colorimetric and fluorimetric methods. Due to the catalytic nature of the hol enzyme, the presence of the labeling component is enhanced many times in the detection reaction, which allows a very sensitive determination of the ligand under test (e.g., at levels of ng / ml), using relatively sensitive but readily available and simple instruments such as spectrophotometers or preferably colorimeters.

The assay method of the invention is characterized by very sensitive detection limits, by being easily automated, by labeling the substance can be detected with instruments commonly found in clinical laboratories, and by using labeled conjugates which can be reproducibly and relatively simply synthesized.

For the purposes of the present disclosure, the following terms are defined unless otherwise indicated as "ligand" is the substance or class of related substances whose presence or amount in a liquid medium is to be determined; "specific binding partner to the ligand" is any substance or class of 25 substances having a specific binding affinity for the ligand excluding other substances; "specific binding analogue to the ligand" is any substance or class of substances which behaves substantially in the same manner as the ligand with respect to the binding affinity of the specific binding partner to the ligand; "Residue" is part of an organic molecule, e.g. a prosthetic group residue in a labeled conjugate is an organic portion of such a conjugate derived from and substantially functioning as a prosthetic group such as the prosthetic group without a hydrogen atom in the position through which the prosthetic group is coupled to the other portion 35 of the labeled conjugate (in an FAD ligand labeled conjugate, FAD represents a residue of the prosthetic group of flavinadenine dinucleotide); "reagents" is a composition, device or sample kit comprising the reagents used to perform the assay; "detection reaction" is the reaction by which the labeling component

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The degree of labeled conjugate is determined by measuring the holoenzyme activity; "lower alkyl" is a straight or branched alkyl group comprising from one to six carbon atoms, including those such as methyl, ethyl, in sopropyl and hexyl.

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The assay method of the present invention can be used to detect any ligand for which a specific binding partner exists. The ligand is usually a peptide, protein, carbohydrate, glycoprotein, steroid or other organic molecule for which a specific binding partner exists in biological systems or which can be synthesized. The ligand is usually selected from antigens and antibodies thereto, haptenes and antibodies thereto and hormones, vitamins, metabolites and pharmacological agents and their receptors and binding substances. Specific examples of ligands that can be detected using the assay method of the present invention are hormones such as insulin, chorionic gonadotropin, thyroid hormones and estriol, antigens and haptenes such as ferritin, bradykinin, prostaglandins and tumor specific antigens, vitamins such as biotin, vitamin B »Folic acid, vitamin E, vitamin A and ascorbic acid, metabolites such as 3 ', 5'-adenosine monophosphate and 3', 5'-guanosine monophosphate, pharmacological agents or drugs such as anticonvulsants, bronchodilators, cardiovascular and abusive drugs, antibodies such as microsomal antibodies and antibodies to hepatitis and allergens, as well as specific binding receptors such as thyroxine binding globulin, avidin, intrinsic factor and transcobalamin. The assay method of the present invention is particularly useful for detecting haptens and analogs thereof having molecular weights of between 100 and 1000, in particular the iodothyronine thyroid hormones thyrosine and liothyronine.

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The liquid medium to be assayed may be a naturally occurring or artificially formed medium which is believed to contain the ligand, and usually it is a biological liquid or a liquid obtained by a dilution or other treatment thereof. Biological fluids which can be assayed by the assay method of the present invention include serum, plasma, urine, saliva, amniotic fluid and cerebrospinal fluid. Other topics such as solids, e.g. tissues can be analyzed after transferring them to liquid state, such as by dissolving the solid in

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8 the liquid or by liquid extraction of the solid.

The conjugate used according to the present invention can be represented by the formula Pr-R-L, where Pr represents the prosthetic group residue (ie, FAD), R represents a linking group and L represents the binding component of the labeled conjugate, usually the ligand or a binding analogue thereof. Usually, the binding component and prosthetic group residue are linked via the coupling group at a site of the binding component at some distance from the specific binding site (i.e., the site of the binding component participating in the binding reaction, such as an immunochemical binding site if the binding component is an antigen, hapten or antibody. thereof) and at a site on the prosthetic group at some distance from the active binding site of the apoenzyme.

15

Various assay methods are available for determining the labeled conjugates of the present invention. In each case, the prosthetic group residue is covalently coupled to the binding component of the labeled component and the detection reactions include measuring the holoenzyme activity in the bound or free form or both, as is now the case.

Various examples of detection schemes based on homogeneous competitive binding are listed below, but other homogeneous methods can of course be used. The following abbreviations will be used in the present description:

Expression Abbreviation 30 Prosthetic group test Pr

Apoenzyme Apo

Coupling group R

Ligand L

Binding Partner B

35 Conjugated Enzyme Residue Apo-Pr

Holoenzyme residue (active) Apo-Pr 9

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Detection Scheme # 1

Homogeneous Competitive Binding Method - The apoenzyme is introduced after 5 initiation of the binding reaction

L (sample) + Pr-R-L + Bjprr + L-B + Pr-R-L-B

Al + Apo (bound form) jr_

Apo + lApo-PrfR-L

(free form; active hol enzyme)

In this method, the prosthetic group residue is detected as labeling s component by adding apoenzyme substantially simultaneously with or after initiation of the binding reaction, i.e. during the binding reaction or after the binding reaction has reached equilibrium and the holoenzyme activity in the final reaction mixture is measured. The assay becomes homogeneous by selecting such conditions that binding between the apoenzyme and the prosthetic group residue is inhibited for the prosthetic group in the bound form. The conditions can also be selected such that the apoenzyme can bind to the bound form of the 2Q labeled conjugate, with the result that the conjugated enzyme complex does not exhibit any enzymatic activity in this state due to inhibition of enzyme-substrate interaction (this situation is not shown on the chart above). In both cases, the measured total enzyme activity in the system is the result of non-inhibited binding between apoenzyme and freely labeled conjugate and is therefore a direct function of the amount of ligand in the sample available for competition for binding to the binding partner.

Detection Scheme # 2 30

Homogeneous competitive binding method - The apoenzyme is introduced prior to initiation of the binding reaction.

Pr-R-L + Apo- »| Apo-Pr] -R-L

(active hollow enzyme)

25 L (sample) + Apo-Pr-R-L + B «= ± L-B + Apo-Pr-R-L-B

(free form; (bound form; active hol oenzyme) inactivated) In this scheme, the prosthetic group residue in the labeling component is coupled to the ligand and combined with the apoenzyme to an active

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A holoenzyme product. The prosthetic group residue is detected by measuring the holoenzyme activity in the final reaction mixture. The assay becomes homogeneous by selecting the conditions in such a way that after binding between the binding partner and the active holoenzyme complex 5, the resulting bound form exhibits inhibited enzyme activity due to inhibition due to enzyme-substrate interaction. The total amount of enzyme activity measured in the system is the result of unbound active holoenzyme complex, i.e. the free form, and as a result, is a direct function of the amount of ligand present in the sample.

Detection Scheme # 2 essentially illustrates enzyme labeling assays in which, however, unlike the heretofore known, the enzyme is coupled as such to the binding component of the labeled conjugate via a residue of a prosthetic group.

By homogeneous analysis, the distribution of labeled component between the bound form and the free form can be determined without separating the two forms.

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The holoenzyme activity in the reaction mixture is then determined by providing, at least in part, the chemical reaction catalyzed by this hollow enzyme, e.g. by adding substrate, and by some conventional technique to measure the rate or accumulated amount of product formation or reactant consumption.

Qualitative determination of the ligand in the liquid medium involves comparing the measured quantity with that of the detection reactions measured in a liquid medium without the ligand, any difference therebetween being an indication of the presence of such ligand in the liquid under study. Quantitative determination of the ligand in the liquid medium involves comparing the measured quantity with that of the detection reaction in liquid media containing various known amounts of the ligand, e.g. when compared to a standard curve.

In the process of the invention, the components of the specific binding reaction, i.e. the liquid medium presumed to contain the ligand, the labeled conjugate and in some systems (i.e., a competitive binding system) a specific binding partner to the ligand is combined in any amount, in any

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π in any way and in any order, provided that the activity of the labeled conjugate labeling component changes measurably when the liquid medium contains the ligand in an amount or concentration that is of significance for the purpose of the assay. All of the components of the specific binding reaction are preferably soluble in the liquid medium.

Known variations of the above-described homogeneous methods and further details of the specific techniques discussed, including an alternative technique known as the direct binding technique, are readily available in the literature, e.g. in the specification to German Publication No. 2,618,511.

Prosthetic Group 15 As previously mentioned, prosthetic groups are a well-known, distinct class of enzyme cofactors, characterized mainly by their ability to be regenerated into an active cofactor form of the parent apoenzyme. According to the present invention, organic prosthetic groups are used which can be chemically coupled to the binding component of the labeled conjugate. Usually, the binding constant for the bond between the apoenzyme and the prosthetic group, the residue of which is included in the labeled conjugate of the present invention, is greater than about 8%. 10 moles and preferably greater than 10 moles. The current binding affinity for the prosthetic group residue included in the labeled conjugate may be reduced by a few percent over that of the free prosthetic group or decreased by as much as two orders of magnitude without affecting the utility of such a prosthetic group residue. as a labeling component in a binding assay.

30

The binding constant for binding between the prosthetic group FAD and g apoglycose oxidase is T> 10, see Swoboda, Biochim. Biophys. Acta 175: 365 (1969).

35 Using flavin adenine dinucleotide and apoglycose oxidase, systems to which ligands, ligand channels and binding partners can be attached, and the resulting glucose oxidase participate in assay reactions involving hydrogen peroxide formation.

Such reactions are well known and are used analytically to provide

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12 results of a colorimetric response that can be advantageously utilized in clinical laboratories.

It must be recognized that FAD may function as a coenzyme or a prosthetic group depending on the enzyme system in which it is placed.

However, the present invention relates only to the use of FAD in connection with an enzyme system in which it functions as a prosthetic group as defined above.

10 Couplin group

There are many possible methods for coupling the binding component of the labeled conjugate, e.g. the ligand to be detected, a binding analogue to or binding partner thereof, to the prosthetic group. The particular chemical nature of the coupling group will depend on the nature of the respective available coupling sites on the binding component and the prosthetic group. In selecting the coupling sites, the following conditions are usually important: (1) the ability of the coupled binding component to participate effectively in the selected binding assay system, and (2) the ability of the coupled prosthetic group residue to be reconciled with apoenzyme. This binding ability must be maintained to such an extent that a useful assay is obtained for the particular ligand under analysis and for the particular concentrations or amounts in which such a ligand is to be detected. Usually, the coupling group will comprise a chemical bond, usually a single bond, but sometimes a double bond or a chain containing between 1 and 14, preferably 1 and 6 carbon atoms and 0-5, preferably 1-3 heteroatoms selected from nitrogen, oxygen and sulfur.

Both the FAD prosthetic group and the binding component will, of course, offer many opportunities for functional groups to attach the coupling group. The functional groups which are generally expected to be available to the coupling group are amino groups, especially primary amino groups, hydroxyl groups, halogen groups, usually chlorine or bromine, carboxylic acid, aldehyde, keto, isothiocyanate, isocyanate groups, and the like. Accordingly, the chemical structure of the coupling group itself will vary greatly, as its end groups will depend on the functional groups available on the coupling group.

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13 the prosthetic group and the binding component, and the total length of the coupling group is chosen such that the essential properties of the prosthetic group and the binding component of the resulting conjugate are retained. In preparing a conjugate for use in a homogeneous assay, it is usually desirable to use as short a coupling group as possible without the resulting binding component of the conjugate significantly interfering with the activity of the prosthetic group in the conjugate. Where the bonding component has a low molecular weight (e.g., a hapten having a molecular weight of between 100 and 1000), the linker group is preferably a chemical bond or a chain of 1-6 atoms such as a lower alkyl group, carbonyl, al cool carbonyl, amide, alkyl, amide group and the like relieve other circumstances such as where the conjugate's binding component has a relatively high molecular weight, such as a polypeptide or protein (eg, an antibody), it is usually desirable to have a longer coupling group to prevent steric hindrance of the apoenzyme combination site in the conjugate. In these cases, the coupling group will usually comprise 1-14 carbon atoms and 0-5 heteroatoms as mentioned above. Significantly longer chains sometimes result in conjugates where the binding component is prone to refolding toward the apoenzyme's combining site. Taking these considerations into account, examples of linkage groups are given in Table 1. Specific examples of coupling groups are given in the embodiments, and other suitable coupling groups can be readily selected by one of ordinary skill in the art.

25 30 35

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Table 1

KoblinasaruDPe 5 Γ -r1 "

X

-R ^ C-R 2

X

-R ^ C-X-R2-10 in X 2

-R-X-C-R -X

1 2 prosthetic group - -R -X-C-X-R - -binding component

FAD X X

ις -C-R2-C- 15 1 2 -RXR - -X-R1- -R * -X- __ -X-RX-X- 12 where X is imino, sulfur or preferably oxygen, and R and R are independently of each other represents a lower alkylene group, having 1-6 carbon atoms such as methyl one, ethyl one, isopropyl one, butyl one or hexyl one.

25

Apoenzvm

The apoenzyme (apoglycose oxidase), i.e. The protein portion of the holoenzyme without the prosthetic group is prepared by cleavage of the 3Q conjugated enzyme and isolation of the released apoenzyme protein. Cleavage of the conjugated enzyme can be carried out by a number of known methods, such as by placing the enzyme in a highly acidic solution, e.g. with a pH of less than 2. Likewise, the recovery of the apoenzyme can be carried out by a number of known methods including selective precipitation of the enzyme or selective adsorption of the low molecular weight released prosthetic group.

Is there any risk of interference in the analysis due to the presence of endogenous prosthetic group in the sample or in any of the 15

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the reagents, or due to contamination of laboratory equipment, glassware or plastic products with prosthetic group, such interference of prosthetic group can be easily eliminated with known inactivation technique. FAD interference can e.g. is eliminated by sequential treatment with periodate and ethylene glycol solution or by using 5 other FAD inactivation methods.

The invention is further illustrated by the following examples.

EXAMPLE 1

Homogeneous bindinosalines for determination of N-27,4'-dinotroDhe-nyl-6-aminocaproate 1C A. Preparation of the labeled conjugate-N®- (6-aminohexyl) dinitro-lb phenyl-flavinadenine dinucleotide.

g

Flavin N-aminohexyl-adenine dinucleotide g 2Q N -Trifluoroacetamidohexyl-adenosine-5'-monophosphate was synthesized according to the method described by Trayer et al., Biochem. J. 139: 609 (1974).

56 mg of N-trifluoroacetamidohexyl-adenosine 5'-monophosphate (0.1 mmol) was dissolved in ca. 10 ml of water and 25 microliters (µl) of L b tri-n-butylamine (0.1 mmol) were added. The water was removed in vacuo and the residue was dissolved in 10 ml of dry dimethyl formamide (DMF) which was again removed under vacuum. The residue was evaporated from dry DMF three more times. The final residue was dissolved in 10 ml of dry DMF. 80 mg of N, N'-car-2Q bonyldiimidazole (0.5 mmol) was added and allowed to react for 1.5 hours. Then 15 µl of water was added and the solvent was removed in vacuo. The residue (N-trifluoroacetamidohexyl-adenosine-S'-mono-phosphatimidazolid) was dissolved in 10 ml of DMF.

47 mg of riboflavin 5'-monophosphate (0.1 mmol) was dissolved in ca. 10 ml of water and added dropwise to 20 ml of acetone containing 43 µl of tri-n-octylamine (0.1 mmol). A precipitate was formed before the addition was complete. The solvent was removed with a rotary evaporator until the riboflavin 5 'monophosphate was dissolved. Then 5 ml was added

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16 acetone and 5-10 ml of DMF and the mixture dried. The residue was dissolved in 15-20 ml of dry DMF and dried (this process was repeated three times). The residue was dissolved in 5 ml of DMF and combined with the above 10 ml solution of the imidazolid in DMF.

5

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 2.5 x 25 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 2 L of water and 2 L 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 vol umen: vol umen (v: v) mixture of ethanol-triethyl ammonium bicarbonate (pH 7.5) showed 15 contained yellow head (R f = 0.75) and minor (R f = 0.36) compounds. These fractions were combined and the optical absorption spectrum had maxima at 267, 373 and 450 nanometers (nm).

The solvent was removed from the joining material and the residue dissolved in ca. 5 ml of water. This solution was adjusted to a pH of 11.0 with 5 N sodium hydroxide and allowed to stand at room temperature for 9 hours. Thin layer chromatography showed that the component with R.p = 0.75 disappeared when a new yellow material with R3 = 0.37 appeared. The reaction mixture was adjusted to a pH of 8.0 with hydrochloric acid and applied to a 2.5 x 20 cm column of DEAE cellulose in the bicarbonate form. The chromatogram was developed with a linear gradient generated with 1 L of water and 1 L of 0.2 M ammonium bicarbonate. The yellow drain from the column was combined and the solvent removed.

The residue was adsorbed on 2 g of silica gel placed on top of a 50 g column of silica gel equilibrated with a 9: 2 (v: v) mixture of ethanol-1 M triethyl ammonium bicarbonate (pH 7.5). The column was eluted with an 8: 2 (v: v) mixture of ethanol-1 M triethylammonium bicarbonate (pH 7.5), the yellow component with R f = 0.37 was collected and the solvent was removed. The yield based on adsorbance at 450 nm was approx. 10%.

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N6- (6-Aminohexv1) flavin adenine dinucleotide--dinitroDhenvl

C

The residue obtained containing flavin N-aminohexyl-adenine dinucleotide was purified by chromatography on Sephadex® G-10 (Pharmacia Fine Cemicals, Uppsala, Sweden). To a 0.9 x 30 cm column equilibrated with 25 millimolar (mM) sodium bicarbonate (pH 7.5) at room temperature was applied 1 ml of a ca. 10 mM solution of the N6-FAD derivative in water. The first eluted peak of material 10 absorbed at 450 nm was collected and re-chromatographed on Sepahdex® G-10 and the first peak eluted again was collected.

C

0.5 ml (0.63 / tmol) of the purified N -FAD derivative in 25 mM sodium bicarbonate (pH 7.5) was mixed with 2 ml of ethanol and 10 μΐ-H-di

Nitrofluorobenzene (6.3 µol, 50 µCi) in ethanol was added (the tritiated dinitrofluorobenzene was obtained from the Radiochemical Center, Amersham, England). The reaction mixture was shaken continuously overnight in the dark at room temperature and then applied to 20 a 0.9 x 30 cm column of Sephadex® G-10 equilibrated with 0.1 M

phosphate buffer (pH 7.0) containing 0.1% sodium azide. The only 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 Di Miles Laboratories, Inc. Elkhart, Indiana, USA was dialyzed twice for 12 hours, each time against 0.5% 30 weight: v / v (w: v) mannitol (30 volumes each). Aliquots of the dialysate, each containing 100 mg of glucose oxidase, were lyophilized and stored at -20 ° C.

Bovine serum albumin (200 mg) was dissolved in 12 ml of water adjusted to a pH of 1.6 with concentrated sulfuric acid, mixed with 150 mg of coal (RIA grade from Schwarz-Mann, Orangeburg, New York, USA) and cooled to 0 ° C. Freeze-dried glucose oxidase (100 mg) was redissolved in 3.1 ml of water and 3 ml was added to the stirred alumina solution by continuous stirring for 3 minutes. The solution then became

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18 filtered through a 0.8 micron, 25 mm diameter Milipore® filter (Mi 11 in pore Corp., Bedford, Massachusetts USA) mounted in a "Swee-nex" filtering apparatus (Milipore Corp.) on a 50 ml of disposable plastic spray.

5

The filtrate was rapidly neutralized to a pH of 7.5 by the addition of 2 ml of 0.4 M phosphate buffer (pH 7.6) and then 5 N sodium hydroxide. Then dry coal (150 mg) was added and stirred for 1 hour at 0 ° C. The resulting solution 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

C

1. Labeled conjugate - N - (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 phos phate 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 upon incubation with the apoenzyme.

3. Antiserum - Antiserum against a dinitrophenyl bovine serum albumin conjugate was obtained from Miles-Yeda, Ltd., Rehevot, Israel and diluted 31 times 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-6-aminocaproate prepared in 0.05 M phosphate buffer (pH 7.0).

35

5. Detection Reagent - A 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 11.5 mM

3,5-dichloro-2-hydroxybenzenesulfonate in water adjusted to one

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19 pH 7 with sodium hydroxide, 9 ml of 11.5 mM 4-amino antipyrin in water containing 1.25 mg / ml peroxidase (Miles Laboratories, Inc., Elkhart, Indiana USA) and 6 ml of 1.0 M glucose in an aqueous saturated benzoic acid solution.

5 D. Methods of analysis.

1. Addition of Anoenzyme Before Binding Reaction Initiation Method 1 - The following was added in succession to separate 10 reaction cuvettes: 0.1 ml of labeled conjugate solution, 0.5 ml of a selected standard solution, 0.1 ml of antibody solution, 2.0 ml of the detection reagent and 0.1 ml of the apoenzyme solution.

Each reaction mixture was incubated for 30 min at 30 ° C and the absorbance at 520 nm was measured.

15

Method 2 - The following was added sequentially to separate reaction cuvettes: 0.1 ml of labeled conjugate solution, 0.05 ml of a selected standard solution, 0.1 ml of the antibody solution and 0.1 ml of the apoenzyme solution. Each reaction mixture was incubated for 20 minutes at room temperature, and then 2.0 ml of the detection reagent was added to each. After a further 15 minutes of incubation at 30 ° C, the absorbance at 520 nm was measured in each cuvette.

2. Addition of apoenzyme after initiation of binding reaction

Method 3 - The following was added sequentially to separate reaction cuvettes: 0.1 ml of labeled conjugate solution, 0.05 ml of a selected standard solution and 0.1 ml of the antibody solution. Each reaction mixture was incubated for 20 minutes at room temperature and then 2.0 ml of the detection reagent and 0.1 ml of the apoenzyme solution were added to each. After a further 30 minutes of incubation at 30 ° C, the absorbance at 520 nm was measured in each cuvette.

Method 4 - The following was added in sequence to separate reaction cuvettes: 0.1 ml of the labeled conjugate solution, 0.05 of a selected standard solution and 0.1 ml of the antibody solution. Each reaction mixture was incubated for 20 minutes at room temperature and then 0.1 ml of the apoenzyme solution

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20 added to each. After a further 20 minutes of incubation, 2.0 ml of the detection reagent was added to each cuvette. Then, each reaction mixture was incubated for 15 minutes at 30 ° C and the absorbance at 520 nm was measured in each cuvette.

5 E. Results

Following is Table 2, which shows the results of the four assay methods for measuring N-2,4,4-dinitrophenyl-6-aminocaproate.

The concentrations of N-2 ', 4'-dinitrophenyl-6-aminocaproate are expressed as concentrations in the final 2.35 ml reaction mixture volumes. The resulting absorbance is expressed as the mean of double samples corrected for residual enzyme activity and background absorbance in the reagents. The correction factor is given for each assay method at the end of the table, and it was determined by experimental procedures in which all the labeled conjugate, standard and antibody solutions were replaced with 0.1 M phosphate buffer (pH 7.0).

20 25 30 35

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22

The results show that a specific homogeneous type binding assay of the present invention can introduce the apoenzyme either before or after initiating the binding reaction.

EXAMPLE 2

Homogeneous bindinosanalysis for determination of thyroxine

C

A. Preparation of the labeled conjugate - ff- (6-aminohexyl) -thyrox-10 inflavinadenine dinucleotide.

6- (6-Aminohexyl) amine no-9- (2 ', 3 / -O-isopropylene-1H-D-di-bofuranosvi) dure 15 16.0 g (50 mmol) 6-Chloro-9 (2 ', 3'-O-isopropylidene / JD-ribofuranosyl) purine (Hampton et al., J. Am. Chem. Soc. 83: 1501 (1961)) was added to a melted (70 ° C) with stirring. of 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 at reduced pressure (60 ° C, 0.91 mm Hg). The resulting pale yellow residue was adsorbed on 150 g of silica gel 60 (E.

Merck, Darmstadt, West Germany) and used to form a peak on a chromatigraphic column prepared from a slurry of silica gel 60 (2 kg) in a 9: 1 (v: v) mixture of absolute ethyl alcohol and triethyl ammonium bicarbonate (pH 7 , 5, 1 M). The column was eluted with the above mentioned 9: 1 (v: v) solvent mixture and 900 20 ml fractions were collected. The fractions were assayed by thin layer chromatography (TLC) on silica gel 60, eluting with a 7: 3 (v: v) mixture of absolute ethyl alcohol and triethylammonium bocatbonate (pH 7.5, 1M).

The fraction numbers 391-900 from the column chromatography were combined and evaporated in vacuo leaving 15.0 g of a glassy residue (74% yield). A glass sample of 1 g was dissolved in a small volume of methyl alcohol, which was then applied to the top of a column made from 80 g of Sephadex® LH-20 (Pharmacia Fine Chemicals, 35 Uppsala, Sweden) swollen in methyl alcohol.

The column was eluted with methyl alcohol. A total of 90 8 ml fractions were collected. The fractions were assayed by TLC on silica gel 60, eluting with a 7: 3 (v: v) mixture of absolute ethyl alcohol and triethyl ammonium bicarbonate (pH 7.5, 1 M). Fraction numbers 19-27 from the column chromatography were combined and

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23 evaporated in vacuo leaving 910 mg (91% recovery) of a white glassy substance.

Analysis: Calculated for C₂gH3oN604: c> 56 145 145 H> 7 44 44 N N> 20> 68 · 5

Found: C, 53.91; H, 7.33; N, 19.18 NMR: (60MHz, CDCl 3): δ 1.40 (s, 3H, isopropylidene), 1.63 (s, 3H, isopropylidene) 5.98 (d, 1H, Γ-ribose) 7.92 (s, 1H, purine), 8.36 (s, 1H, purine) pc n

Optical Rotation: (a) D = -50.11 (c 1.0, methyl alcohol) N-16-fN-Trifluoroacetyl-3.3, 5.5, -tetraiodthyronylaminohexyl-2/3 / -0-15 iso-propylidene adenosine

A solution of 4.36 g (5.0 mmol) of α- (N-trifluoroacetyl) amino- [f- (3,5-diiodo-4- (3 ', 5'-diiodo-4'-hydroxyphenoxy)) -phenyl propionic acid, the preparation described in Part A of Example 2 above, and 2.24 g (5.5 mmol) of 6- (6-aminohexyl) amino-9- (2 ', 3'-O-isopropylidene) (3-D-ribofuranosyl) purine in 100 ml of dry di methyl formamide 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) di phenyl phosphoryl azide (Aid-rich Chemical Co., Milwaukee, Wisconsin, USA) in 50 ml of dry dimethylformamide followed by the addition of 0.8 ml (5.5 mmol) of dry trimethylamine. The solution was left at room temperature for 1 h. The solution was then added dropwise with stirring to 600 ml of cold (0 ° C) water, the resulting white precipitate was collected by filtration and dried in vacuo (60 ° C) to give 4.90 g (78% yield) ) of a white solid. A sample of this substance was recrystallized from a mixture of acetone and water, to give a white solid, mp 205-207 ° C (decomposed).

Analysis: Calculated for 5 5 5¾¾¾¾¾: C ’34>> 285H, 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 COF

Optical rotation (a) p = -21.89® (c 1.0 pyridine)

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24 N- (6-N-trifluoroacetyl1 -3 ♦ 3 '. 5.5'-tetrahydronylaminohehexyl) -2'. 3'-O-iso-propylene-5'-adenyl acid monotriethylamine salt monohydrate

A solution of 1.89 g (1.5 mmol) of N- (6- (N- (trifluoroacetyl) -3,3 ', 5, -5 5'-tetraiodthyronyl) aminohexyl) -2', 3'-O- isopropylidene adenosine 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 maintained at -15 ° C for 18 hours, then added dropwise to 1.5 l of 10 ice water with stirring. 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, mp 151-154 ° C (dec.).

Analysis: Calculated for C42H56F3I4N8 ° 12P: C '34> 54; 3.86; 20 N, 7.67

Found: C, 35.24; H, 3.88; N, 7.75 Mass spectrum (20 ma) m / e: 1342 (MH +), 1244 (M + minus COCF

Optical rotation: (a) 25D = -17.20 ° (c 1.0, CH 2 OH) 25 N- (6- (N- (Trifluoroacetyl1-3.3'.5.5'-tetraiodthyronylaminohexyl1-5'-adenylacetic acid) 600 mg (0, 41 mmol) N- (6- (N- (Trifluoroacetyl) -3,3 ', 5,5'-tetraiodothyronyl) aminohexyl) -2', 3'-O-isopropylidene-5'-adenyl acid monotriethylamine salt monohydrate suspended in 0.6 ml of water (0 ° C) and trifluoroacetic acid (6 ml) were added dropwise with stirring, after 50 minutes a clear solution was obtained, the solution was kept cold (0 ° C) for an additional 15 hours, then evaporated to dryness. The resulting residue was evaporated in vacuo 5 times from 20 ml volumes of anhydrous ethyl alcohol, 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 white solid, mp 180-183 ° C (dec.).

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25

Analysis: Calculated for C33H35F3I4 NyO2jP: 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 + 5 minus COCFg) F1 avi nadeni ndi nuel eoti d-thyroxy nconiate 130.13 mg (0.1 mmol): N- (6- N- (Trifluoroacetyl) -3,3 ', 5,5'-tetraiod-thyronyl) aminohexyl) -5'-adenyl acid was placed in an argon atmosphere.

To this sample was added a solution of 14 beers (0.1 mmol) of triethylamine in 1 ml of dry dimethyl formamide, followed by the addition of a solution of 16.2 mg (0.1 mmol) of Ι, b-carbonyldimidazole in 1 ml. dry dimethyl formaid. After 24 hours, another equivalent of 1,1'-carbonyldi imidazole (16.2 mg) in 1 ml of dry dimethyl formamide was added. The above reaction was allowed to proceed for a total of 48 hours at room temperature only humidity. A sample of 47.3 mg (0.1 mmol) of riboflavin 5'-monophosphate ammonium salts was converted to the corresponding tri-n-octylamine salt as described in Part A of Example 2. This salt was divided into 3 ml of dry di methyl formamide and added to the above solution containing the phosphorimide azolate of the adenyl acid flour product.

The resulting solution was allowed to stand in the dark at room temperature only 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 (Pharmacia Fine Chemicals, Uppsala, Sweden), which was pre-swelled (18 hours) ) in a 19: 1 (v: v) -bl breath of dimethyl formamide and triethyl ammonium 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 drain from the column was detected by elution on the sieve in the cage! 60 silanized 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 35 acetone, chloroform, methylalcohol, water and triethylamine.

Fraction numbers 24-38 from the 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

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26 were swollen (18 hours) in 0.1 M ammonium bicarbonate. The column was eluted with a linear gradient generated from 2 L 0.1 M ammonium bicarbonate and 2 L water and 23 ml fractions were collected. The effluent was detected with ultraviolet absorption (254 nm). Fraction numbers 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, which was pre-swollen in 0.05 M ammonium bicarbonate. The column was eluted with a linear gradient generated from 2 1 0.05 M ammonium bicarbonate and 2 1 0.02 M ammonium bicarbonate. The effluent 10 was detected by ultraviolet absorption (254 nm). Elution was continued with 2 l 0.2 M ammonium bicarbonate, collecting 23 ml fractions. A total of 257 fractions were collected. Fraction numbers 70-110 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 maximum at the following wavelengths: 270 nm, 345 nm and 450 nm. The yield determined from the absorbance at 450 nm was approx. 5%.

A phosphorous diesterase preparation (Worthington Biochemical Corp., 20 Freehold, New Jersey, USA) isolated from snake venom (Crotalus Adaman teus) hydrolyzed the above product to riboflavin 5'-monophosphate and the thyroxine-substituted 5'-adenyl acid where the tri-fluoroacetyl bl growing group has been removed.

Further description of the preparation of labeled conjugates of this type can be found in the specification for U.S. Patent Application Serial No. 917,952, filed June 22, 1978.

B. Preparations of Apoglucose Oxidase 30

Glucose oxidase was dialyzed and lyophilized as in Part B of Example 1 above. Part of the freeze-dried glucose oxidase (80 mg) was dissolved in 20 ml of 30% (v: v) glycerol at 4 ° C and the solution was adjusted to a pH of 1.4 by the addition of concentrated sulfuric acid (HgSO4). 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 a pH value of 1.4 with concentrated HgSO4.

The eluted protein peak was collected (27 ml containing 74 wt.

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27 cents of material added to the column) and 200 mg of bovine serum albumin were dissolved in the united el uate. Charcoal (600 mg; RIA grade from 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 5 buffer (pH 8.0) and sufficient 2 N sodium hydroxide to set the pH to 7.0. The mixture was stirred for 60 minutes at 4 ° C and filtered successively through 0.8 β and 0.22 β Millipore® filters (Millipore Corp, Bedford, Massachusetts USA). Sodium azide (10% w: v) was added to give a final concentration in the mixture 10 of 0.1%.

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 ml of crown normal (μΜ) FAD binding sites (see Part C 2 in 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-hydroxynean antiserum stirred 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 1 50 mM borate buffer (pH 8.6). The immunoglobulin solution was then made up to the original antiserum volume 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% (w: v) bovine serum albumin (pH 7.0) for use in the assay procedures described below in the ratios given.

4. Standards - Thyroxine sodium salt (Sigma Chemical Co., St. Louis,

Missouri, USA) was used as a 1 mg / ml stock solution titrated for 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% (w: v)

DK 154670B

28 bovine serum albumin (pH 7.0).

5. Detection Reagent - - A 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-aminoane-tipyrin containing 0.6 mg / ml peroxidase, 2 parts 20 mM 3,5-dichloro-2-hydroxybenzenesulfonate in 0.1 M phosphate buffer (pH 7.0) and 2 parts 1.0 M glucose in aqueous saturated benzoic acid solution.

D. Procedure 1. Addition of all reagents without pre-incubation step The following were sequentially added 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 diluting the antiserum solution in the phosphate buffer, 1.7 ml of the detection reagent and 0.1 ml of the apoenzyme solution. Each reaction mixture was incubated for 30 minutes at 20 ° C and the absorbance measured at 520 nm.

2. Pre-incubation with antiserum The following were sequentially added to separate reaction cuvettes: 0.05 ml of the labeled conjugate solution, 0.05 ml of a selected standard 25 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 detection reagent and 0.1 ml of the apoenzyme solution were added one after the other. After further incubation for 30 minutes at 20 ° C, the absorbance at 30 520 nm was measured in each cuvette.

3. Pre-incubation with apoenzyme The following were sequentially added 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 detection reagent was added to one cuvette after another.

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29

After further incubation for 30 minutes at 20 ° C, the absorbance at 520 nm was measured in each cuvette.

4. Sequential pre-incubation with first antiserum οα then with 5 apoenzyme The following were sequentially added 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 1:32 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 cuvette. After further incubation for 20 minutes at 20 ° C, 1.7 ml of the detection reagent was added to each cuvette and the absorbance at 520 nm was measured in each cuvette after: a 30 ml nut incubation at 20 ° C.

5. Sequential pre-incubation with first apoenzyme and then with antiserum 20 The following were sequentially added 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 1:32 dilution of the antiserum solution in the phosphate buffer was added to 25 each cuvette. After further incubation for 20 minutes at 20 ° C, 1.7 ml of the detection reagent was added to each cuvette and the absorbance at 520 nm was measured in each cuvette after a 30 minute incubation at 20 ° C.

6. Pre-incubation with both antiserum οα apoenzyme The following were sequentially added 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 detection reagent was added to each cuvette. After further incubation for 30 minutes at 20 ° C, the absorbance at 520 nm was measured in each cuvette.

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E. Results

Duplicate tests were performed with the appropriate blank values. 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 activity was inhibited, whereas for methods 4-6, enzyme activity 10 was increased. Methods 4-6 were also found to be somewhat more sensitive to thyroxine than the other methods. The reason or reasons why the enzyme activity was severely inhibited or increased depending on the reaction sequence are not fully understood, but the results show that the present invention provides a homogeneous specific binding assay method useful for the determination of thyroxine using several different reaction sequences.

EXAMPLE 3 20

Homogeneous binding assay to determine theophylline A. Preparation of the labeled conjugate theophylline FAD

C

To a solution of 2.4 µmol of flavin N-aminohexyl-adenine dinucleotide prepared as described in Part A of Example 1 above, in 200 µl of dimethyl sul foxide under argon gas 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 described by Cook et al, Res.

Comm, in Chem. Pathol. and Pharm. 13: 497 (1976), 4 hours later followed by addition of an additional 1.8 mg (7.3 µmol) of the same.

After stirring overnight, the solvent was evaporated under vacuum (0.1 mm Hg) and the residue chromatographed on a 2.5 x 90 cm LH-20 Sephadex® (Pharmacia Fine Chemicals Uppsala, Sweden) column and eluted with 0.3 M triethylammomium bicarbonate buffer. (pH 7.8). The crude product eluted between 210 and 246 mn of the drain was collected, loaded on a 20 x 20 cm x 1000 µ silica gel plate and eluted with a mixture of 8: 2 ethanol: 1 M triethyl ammonium bicarbonate buffer (pH 7.8). The band containing the desired product (R 2 = 0.77) was

DK 154670B

31 scraped off the plate, extracted with 1 M triethylammonium bicarbonate buffer (pH 7.8), filtered and concentrated. Final purification by rechromatography at LH-20 Sephadex® eluted with the buffer at 0.3 M yielded 1.26 µmol of the labeled conjugate, determined by the absorbance measured at 5 at 450 nm, which corresponds to a yield of 53%.

B. Antibody binding reactions

Antibody was produced in rabbits with the immunogen 8- (3-carboxypropyl) -1,3-dimethylxanthine BSA 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 glucose oxidase activity measurements were performed in the same buffer at 20 ° C.

The reagents used in the assay were as follows:

Composition 20 A 0.1 M Sodium phosphate buffer (pH 7.0) B 565 nM Theophylline-FAD labeled conjugate or 160 nM N 2 - (6-aminohexyl) FAD (FAD derivative) C Antiserum against theophylline (diluted 10 25 times in reagent A) D Apoglucose oxidase (50 nM FAD binding sites per ml.) E Detection reagent: 200 μg peroxidase / ml;

0.71 mM 4-aminoantipyrine; 7.1 mM

3,5-dichloro-2-hydroxybenzenesulfonate; 353 mM

giucose and 35 mg BSA / ml.

Reagents A, B and C were combined in separate reaction cuvettes in the ratios given in Table 4 below. Reagent D (100 µl) and reagent 35 E (283 / hr) were then rapidly and successively added to each reaction mixture followed by incubation for 30 minutes at 20 ° C. The absorbance at 520 nm was measured for each cuvette. The results listed in Table 4 are averages of duplicate determinations.

32

DK 154670 B

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The results for 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 increased levels of theophylline antiserum decreased the activity of the labeled conjugate 5 relative to its ability to combine with the apoenzyme.

C. Competitive Binding Assays.

These reactions were carried out as indicated in Part B above with the exception that the indicated levels of theophylline were combined with Reagents A and B prior to the addition of Reagent C and also that a Reagent C was used 100 times dilution of antiserum. The results are shown in Table 5 below.

Reactions 1-3 are 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 relative to theophylline level in the reaction mixture.

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DK 154670 B

EXAMPLE 4

Homogeneous binding assay for human IaG determination

5 A. Preparation of the labeled conjugate IgG-FAD

To 4.24 mg of flavin N® aminohexyl adenine dinucleotide prepared as described in Part A of Example 1 above was added 2.5 mg of dimethyl adipimidate dihydrochloride (Pierce Chemical Co., Rockford, Illinois, USA) in 1 ml of water and 5 µl of tri ethyl amine. 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 further stirring at room temperature for 3 hours, the reaction mixture was charged with a 2.5 x 50 cm G25 Sephadex® (Pharmacia Fine 15 Chemicals, Uppsala, Sweden), column equilibrated and eluted with 0.1 M sodium phosphate buffer (pH 7.0). Fractions from the first elution peak having an 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, 41 0.1 M sodium phosphate buffer (pH 7, 0) containing 1 M 20 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 0.22 μ Millipore® filter and stored.

B. Antibody Binding Reactions

The reagents used in the assay were as follows:

Reagent Composition 30 A 0.1 M sodium phosphate buffer (pH 7.0) B 10 mM 4-aminoantipyrin C 1.0 M glucose D 25 mM 3,5-dichloro-4-hydroxybenzenesulfonate

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, IN, USA) G IgG-FAD labeled conjugate in solution

DK 154670 B

36 stored from above H Apoglucose oxidase I rabbit antiserum against human IgG (obtained from

Behring Diagnostics, Somerville, New Jersey, USA) J standards - human IgG reagent A in predetermined amounts.

Reagent mixtures were prepared as follows: 10

Mixture 1: 180 µl reagent A, 20 µl reagent B, 100 µl reagent D

and 5 µl of reagent G (5.4 μΜ)

Mixture 2: 0.3 ml total volume of various amounts containing the volume of Reagent I listed in Table 6 15 below, making up to total volume with

reagent A

Mixture 3: 80 µl of reagent D, 50 µl of reagent E, 33 µl of reagent F, 137 µl of reagent A and 1.6 µl of reagent H (4.2 μΝ FAD binding sites).

20

To 300 ml of mixture 1, 300 μ 300 of mixture 2 and 300 μΐ of reagent A were added in separate reaction cuvettes: After at least 10 minutes of incubation at room temperature, 300 μΐ 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 in each cuvette. The results were as follows: TABLE 6

Added volume of antiserum to prepare mixture 2 Absorbance (520 nm) 30 0 0.859 2 0.750 4 0.602 6 0.494 35 8 0.443 10 0.415 12 0.408 14 0.392 16 0.375

DK 154670 B

37

The results demonstrate that as the antibody level increases, the glucose oxidase activity produced by the FAD labeling conjugated to IgG decreases.

5 C. Competitive Binding Analysis

These reactions were performed using the reagents described in Part B above. Stated levels of human IgG in 300 µL volumes of reagent A were combined with mixture 1 in separate reaction cuvettes. Then a mixture of 12 µl of Reagent I and 288 µl of Reagent A was added to each reaction. After 10 minutes, 300 µl of mixture 3 was added and the reaction mixtures were incubated at 20 ° C for 30 minutes. At the end of this period, the absorbance at 520 nm was measured in each cuvette. The results were as follows: TABLE 7

Amounts added of human IgG (w) Absorbance (520 nm) 0 0.579 4 0.653 20 8 0.751 12 0.844 16 0.986 24 1.06 25 It is thus apparent that the present invention provides a specific binding assay for the determination of the high molecular weight ligand, human IgG, in liquid media.

REAGENTS

30

From the foregoing description and examples, it will be apparent to those skilled in the art that the reagents used to carry out the assay method of the invention can take many different forms. In particular, the reagents may take the form of a unit sample composition, such as a suitable solid or liquid form. By way of illustration, a test composition for determining a ligand of the present invention may comprise (a) a labeled conjugate comprising FAD coupled to a ligand or a binding analog thereto, (b) a specific binding partner to the ligand, and (c)

DK 154670 B

38 cose oxidase capable of combining with FAD to form glucose oxidase. Such a test composition may also comprise reagents for determining the activity of glucose oxidase, and where desired and appropriate, the labeled conjugate and apoenzyme may be combined in the form of conjugated enzyme complex. Of course, the sample composition may also contain traditional thinners, buffers, stabilizers and the like.

The reagents may also take the form of a test set, i.e. a packet-10 combination of containers containing the necessary reagent elements. Again, by way of illustration, a test kit for determining a ligand of the present invention may comprise one or more containers containing (a) a labeled conjugate comprising FAD coupled to the ligand or a binding analog thereto, (b) a specific binding partner to the ligand and ( c) apoglucose oxidase capable of combining with FAD to form glucose oxidase. Such a test kit may also contain reagents for determining the glucose oxide activity in one or more of the same or different containers, and where desired and appropriate, the labeled 20 conjugate and apoenzyme may be combined in the form of conjugated enzyme complex. In one embodiment, the test kit comprises at least two separate containers, one containing the labeled conjugate and optional reagent for measuring glucose oxidase activity, and the other containing the binding partner and apoglucose oxidase. Of course, the test set may also comprise other reagents well known in the art and which may be desirable from a commercial and utility standpoint such as buffers, thinners, standards and the like.

30 35

Claims (7)

A homogeneous, specific binding assay for determining a ligand in a liquid medium, wherein assay provides a reaction mixture by combining the liquid medium with reagents comprising a labeled conjugate having a labeling component coupled to a binding component. which combination forms a bonding reaction system in which a bound form and a free form of the labeled conjugate are formed, the relationship between the labeling component of the two forms being a function of the ligand in the liquid medium and the labeling component exhibiting a detectable response which is different in the two forms after which the detectable response is measured in the reaction mixture, characterized in that the labeling component is flavinadenine dinucleotide, the reagents also comprise apoglucose oxidase capable of combining with flavinadenine dinucleotide to produce glucose catalyticase , and that glucose oxidase activity is measured in rea do not mix. Analysis according to claim 1, characterized in that the reagents comprise (1) a labeled conjugate of flavinadenine dinucleotide linked to the ligand or a binding analogue thereof, (2) a specific binding partner to the ligand, and (3) apoglucose oxidase. Analysis according to claim 1, characterized in that the xylucose oxide ash activity is measured by a colorimetric method. Analysis according to claim 1, characterized in that the ligand is an antigen or a hapten. 30 Analysis according to claim 2, characterized in that the ligand to be determined is an antigen or a hapten and that the binding partner is an antibody thereto. The reagent system for use in the assay of claim 1, characterized in that it comprises (1) a labeled conjugate which has as a labeling component flavinadenine dinucleotide and (2) apoglucose oxidase which combines with the flavinadenine dinucleotide labeling component to generate glucose. DK 154670 B The reagent system of claim 6, characterized in that it comprises (1) a labeled conjugate comprising as a labeling component flavinadenine dinucleotide coupled to the ligand or a binding analog thereof, (2) a specific binding partner to the ligand and (3) apo-5 glucose oxidase. 10 15 20 25 30 35