DK158366B - SPECIFIC BINDING ANALYSIS PROCEDURE AND REAGENT USE IN THE PROCEDURE - Google Patents

Description

DK 158366 B

in

The present invention relates to a specific binding assay method for analyzing a liquid medium for a ligand.

Due to the risk and difficulty of handling radioactive materials, many attempts have been made to devise appropriate, specific binding assay methods that are as sensitive and rapid as radioimmunoassays, but employing features other than radioactivity as a means of controlling As will be discussed in more detail below, the materials that have been used as labeling material instead of radioactive atoms or molecules include various materials such as enzymes, fluorescent molecules and bacteriophages. The specific binding assay methods, which will be discussed first, are of the "heterogeneous" type by which separation of the 15 bound and free phases is carried out. Such separation is necessary to perform a specific bonding analysis when the labeled material in the bonded phase cannot be distinguished from the labeled. matter in the free phase.

From the specification to US Patent Nos. 3,654,090, 3,791,932, 3,839,153, 3,850,752 and 3,879,262, and from the Journal of Immunological Methods 1: 247 (1972) and the Journal of Immunology 109: 129 (1972) are known examples of heterogeneous methods which have been developed using an enzyme as a labeling material. In each of the described methods, an enzyme is chemically coupled to either the ligand to be determined or a binding partner thereof, and an appropriate specific binding reaction scheme is constructed, thereby increasing the amount of enzymatic activity associated with either the insoluble moiety. or the liquid portion, after incubation with a sample, is a function of the amount of ligand in the sample. The problems associated with the synthesis and characterization of the enzyme conjugates are serious disadvantages of this method.

From the specification to GB patent No. 1,392,403 and FR patent no.

2,201,299 discloses a heterogeneous, specific binding assay method using a non-active precursor for a spectrophotometrically active material as a labeling material. After incubating the sample with the specific binding reaction system, the insoluble and liquid parts and the amount of labeling material are separated.

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2, which is present in the liquid portion and which is a function of the amount of ligand to be detected in the sample, is determined by converting the inactive label to a chromogenic or fluorometric active material which is then measured in the usual manner.

Other heterogeneous specific binding assay methods using different types of labeling materials are described below: PB-224,875 of the National Technical Information Service 10 (NTIS) of the United States Department of Commerce (1973) describes a unsuccessful attempt to use hemin chloride as labeling material in a heterogeneous assay system followed by a chemiluminescence reaction. In Nature 219: 186 (1968), certain radioimmunoassay procedures are described in great detail, and in very general terms, mention is made of the possible use of coenzymes and viruses instead of radioisotopes as labeling materials. However, the article does not elucidate how an analysis using such alternative labeling materials is to be conducted and thus it is not clear whether such an analysis is feasible. Reference can also be made to Principles of Competitive Protein-Binding Assays, ed. Odell and Daughaday (J.B. Lippincott Co., Philadelphia, 1972), discussing the various known assay schemes and the various materials and agents used as labels in specific binding assays.

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An enzyme labeled immunoassay is described in the specification of US Patent No. 3,817,837. This process is of the "homogeneous" type in that it does not require the use of a partitioned reaction system (i.e., with an insoluble moiety and a liquid moiety) and separation procedure 30 for this. Namely, the enzyme-labeled ligand is of such a form that the enzymatic activity after reaction with the ligand's binding partner is inhibited. Thus, the relationship between bound, labeled material and free material can be determined by controlling changes in enzymatic activity. Nevertheless, this method 35 has the disadvantage that it is difficult to prepare well-characterized enzyme-labeled conjugates and to find appropriate enzymes.

Although many new types of specific binding assays have been proposed and investigated, radioimmunoassay and the various 3

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enzyme-labeled immunoassays most commonly used. However, both types of systems have obvious deficiencies, the radioimmunoassay due to its use of radioactive material which is hazardous and requires careful handling, and the enzyme-labeled immunoassays due to the difficulty of producing useful enzyme-labeled conjugates.

The present invention provides an extremely convenient, versatile and sensitive, improved specific binding assay method for determining a ligand in a liquid medium, comprising the steps of: (a) contacting the liquid medium with a reagent comprising a conjugate of a labeling substance; and a specific binding agent, wherein the reagent and ligand form a reaction binding system comprising (i) a bound form of the labeled conjugate wherein the specific binding agent is bound to a specific binding partner thereof, and (ii) a free form thereof labeled conjugate wherein the specific binding agent is bound to a specific binding partner thereof; (B) determining the labeling substance in either the bound or the free form thereof as a measure of the ligand present in the liquid medium.

The specific binding assay method of the invention is characterized in that as an labeling substance an enzyme substrate is used and that the process is of a homogeneous type, i.e. that physical separation of the bound form and free form of the substrate-labeled conjugate is not performed without measuring and comparing the substrate activity throughout the liquid reaction mixture with the ligand present in the liquid medium tested.

The invention also relates to a reagent for use in the determination of a ligand in a liquid medium comprising a conjugate of

DK 158366B

4 is a labeling substance and a specific binding agent, which reagent together with the ligand forms a binding reaction system comprising a bound form of the labeled conjugate, wherein the specific binding substance is bound to a specific binding partner thereof, and a free form of the labeled conjugate wherein the specific binding agent is not bound to a specific binding partner thereof, whereby the amount of labeling substance in either the bound or the free form is a function of the amount of the ligand present in the liquid medium, which reagent is peculiar in that the labeling substance is an enzyme substrate and at For example, the substrate-labeled conjugate exhibits measurably different substrate activities in the bound form and in the free form, thus allowing homogeneous analysis.

The names given below in the specification will then be defined: ligand is the material or group of materials whose presence or quantity in a liquid medium is to be determined; specific binding partner for the ligand is any material or group of materials having a specific binding activity for the ligand with the exclusion of other materials; and specific binding analogue to the ligand is any material or group of materials which behaves in much the same manner as the ligand with respect to the binding activity of the specific binding partner for the ligand.

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The novel homogeneous assay technique of the invention is based in part on the reaction between a ligand and a specific binding partner, where the enzyme substrate is coupled to one, altering the activity of the enzyme substrate in the predetermined reaction, which reaction thus serves as a means of monitoring the specific binding reaction. In view of this basic phenomenon, various binding methods, including different assays and devices, can be used to practice the method of the invention. The preferred basic methods are the direct bonding technique and the competitive bonding technique.

By the direct binding technique, a liquid medium believed to contain the ligand to be detected is contacted with a conjugate comprising the enzyme substrate coupled to a specific

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binding partner for the ligand, and then a possible change in the activity of the enzyme substrate is measured. In the competitive binding technique, the liquid medium is contacted with a specific binding partner for the ligand and with a conjugate comprising the enzyme substrate coupled to the ligand and / or a specific binding analogue thereof, and then any change in the activity of the enzyme substrate is measured. In both techniques, the activity of the enzyme substrate is determined by contacting the liquid medium with at least one reagent which with the enzyme substrate constitutes the predetermined control reaction. Qualitative determination of the ligand in the liquid medium includes comparison of a property, usually the rate, of the resulting reaction with the same property of the control reaction in a liquid medium without the ligand, any difference being an indication of a change in the activity of the enzyme substrate. Quantitative determination of the ligand in the liquid medium takes place by comparing one property of the resulting reaction with the same property of the control reaction in liquid media containing known amounts of the ligand.

When the homogeneous assay scheme is followed, it is generally true that the components of the specific binding reaction, i.e. the liquid medium believed to contain the ligand, conjugate and / or a specific binding partner for the ligand can be combined in all conditions, in any manner and in any order, provided that the activity of the enzyme substrate in the conjugate changes measurably when liquid medium contains the ligand in an amount or concentration significant in relation to the assay method. Preferably, all of the components of the specific binding reaction are soluble in the liquid medium.

When a homogeneous direct binding technique is used, the components of the specific binding reaction are the liquid medium believed to contain the ligand and an amount of a conjugate comprising the enzyme substrate coupled to a specific binding partner for the ligand. The activity of the conjugated enzyme substrate upon contact with the liquid medium varies inversely with the degree of binding between the ligand in the liquid medium and the specific binding partner in the conjugate. When the amount of ligand in the liquid

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Thus, increasing the medium, the activity of the conjugated reactant decreases.

When a homogeneous competitive binding technique "is used, the components of the specific binding reaction are the liquid medium" which is believed to contain the ligand, an amount of a conjugate comprising the enzyme substrate coupled to the ligand or a specific binding analog for the ligand, and an amount of a specific binding partner for the ligand. . The specific binding partner is contacted substantially simultaneously with both the conjugate and the liquid medium. Since any 10 ligand in the liquid medium competes with the ligand or specific binding analog thereof in the conjugate for binding with the specific binding partner, the activity of the conjugated enzyme substrate upon contact with the liquid medium directly varies with the degree of binding between the ligand in the liquid 15. medium and the specific binding partner. Thus, as the amount of ligand in the liquid medium increases, the activity of the conjugated enzyme substrate increases.

A variation of the homogeneous competitive binding technique is the homogeneous displacement binding technique in which the conjugate is first contacted with the specific binding partner for the ligand and then with the liquid medium. Then there is competition for the specific binding partner. In such a method, the amount of the conjugate contacted with the specific binding partner is usually so large that the ligand or analog thereof is in excess of the amount capable of binding with the amount of specific binding partner present during the time when the conjugate and specific binding partner are in contact with each other prior to contact with the liquid medium which is believed to contain the ligand. This contact order can be obtained in two convenient ways. In one method, the conjugate is contacted with the specific binding partner in a liquid environment prior to contact with the liquid medium which is believed to contain the ligand. In the second method, the liquid medium presumed to contain the ligand is contacted with a complex comprising the conjugate and the specific binding partner, the specific binding substance of the conjugate and specific binding partner being reversibly bonded to each other. The amount of conjugate that becomes 7

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bound to the specific binding partner, which in the second process is in complex form, is usually greater than the amount which can be displaced by the entire amount of the ligand in the liquid medium during the time when the specific binding partner or complex and the medium are present. contact with each other before completing the measurement of any activity change for the conjugated reactant.

Another variation of. the homogeneous competitive binding technique is the homogeneous sequential saturation technique in which the components of the specific binding reaction are the same as those used in the competitive binding technique, but the order of addition or combination of the components and the relative amounts used are different. By a sequential saturation technique, the specific binding partner of the ligand is contacted with the liquid medium which is believed to contain the ligand for a period of time before the liquid medium is contacted with the conjugate. The amount of the specific binding partner contacted with the liquid medium is usually greater than that capable of bonding with the entire amount of ligand believed to be present in the liquid medium during the periods of time in which the specific binding partner and the liquid medium are in contact with each other before the time when the liquid medium is contacted with the conjugate. Further, the amount of the ligand or ligand analog in conjugated form is usually greater than the amount capable of bonding with the remaining unbound amount of the specific binding partner during the period in which the liquid medium and the conjugate are in contact with the each other prior to completion of the measurement of the change of the activity of the conjugated enzyme substrate.

The measurement of any change in activity of the conjugated enzyme substrate, which enzyme substrate constitutes a component of the predetermined monitoring reaction, is conveniently achieved by contacting the specifically binding reaction mixture with at least one substance which, together with the conjugated enzyme substrate, provides the predetermined monitoring reaction and specific binding reaction to a property of this reaction.

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The reaction components which together with the enzyme substrate in the conjugate provide the predetermined monitoring reaction can be contacted with the specific binding reaction mixture by the homogeneous technique. The reaction components are added either individually or in any combination, either before, simultaneously with or after initiating the specific binding reaction. After initiating the specific binding reaction, the reaction mixture, which may include any or all of the necessary components of the monitoring reaction, is usually incubated for a predetermined period of time before any change in reactant activity in the conjugate is assessed. After the incubation period, the reaction mixture is added to all the components necessary for the monitoring reaction and which are not already present in sufficient quantities in the reaction mixture and the monitoring reaction is measured to indicate the presence or amount of ligand in the liquid medium.

A preferred sensitive monitoring reaction involves the phenomenon of fluorescence and is enzyme catalyzed. In such a reaction system, the reactant in the conjugate is a substrate in an enzymatic reaction which produces a product having fluorescent properties that differ from the conjugated substrate. Any change in the activity of the conjugated enzyme substrate resulting from the specific binding reaction causes a change in the fluorescence properties of the reaction mixture. A general reaction scheme for such an enzyme catalyzed reaction system is the following:

Enzymatic Reactant -XZ (Enzyme) Product 30 wherein X represents an enzyme cleavable bond or linker group, such as an ester or amido group, and Z a specific linker which, depending on the specific binding reaction technique used, is either the ligand, a specific linker analog to the ligand or a specific binding partner for the ligand. Specific conjugates that can be used in this type of reaction system are various enzyme cleavable derivatives of fluorescein, umbelliferone, 3-indole, β-naphthol, 3-pyridol, resorufin, and the like. Examples of possible structural formulas for such derivatives are the following:

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Derivative Formula. ". "-cc ^ y fV \ KÅ?

Y

10 0 rI ^ v. o 0

Nj V) /

umbelliferon | IN

R3 15 o

year

jT

H

R2

j8-naphthol [[^ lf X

25 ^ .2

3-pyridol in · J

\reach

Wherein R represents -OH or -X-Z (as defined above), R represents -1-1, and R3 represents -H or -CHg.

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10

One reaction system which may be particularly preferred for monitoring the novel specific binding reaction of the present invention is a cyclic or cyclization reaction system. Such a reaction system is a system in which a product of a first reaction 5 is a reactant in a second reaction, which second reaction as one of its products has a substance which is also reactant in the first reaction.

The diagram below shows a model of a cyclic reaction system: 10 products A. xyclized matter all reactants B \ 7 * (form 1)

REACTION A I [REACTION B

λ Λ

15 reactants A7 'cyclized material products B

. (form 2) In the above cyclic reaction system model, a small amount of the cycled material, if sufficient 20 quantities of reactants A and B are provided, will form large quantities of products A and B. Since the reaction rate and amount of product formed at While the reactions constituting the cyclic reaction system are highly sensitive to the amount of cycling present in the serous material, it is particularly preferred to use the cyclized material as the reactant in the conjugate of the present invention. Examples of cyclizing reaction systems devised for use in connection with the new specifically binding reaction system of the invention are seen in Tables B and C.

30 1

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TABLE B

product NAD * ^^. reactant B

5 enzyme enzyme k Λ

reactant K x NADH ** * product B

Reactant A Reactant B

10 or or

Reaction product B Enzyme product A

1 lactaldehyde al carbohydrate hydrogenase propanediol 15 2 alpha-ketoglutarate glutamic acid dehydro-glutamate + 3 genase 3 oxalacetate malic acid dehydrogenase mal 4 20 acetaldehyde all carbohydrate hydrogenase ethanol 5 phosphate dehydrogenase role phosphate 7 pyruvate lactic acid dehydrogenase lactate 30 8 1,3-diphosphoglyceraldehyde-3-phosglyceraldeglycerate phatdehydrogenase hyd-3-phosphate + phosphate 35 * nicotinamide adenine dinucleotide

** reduced NAD

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TABLE C

product NADP * ^^, reactant B

5 enzyme enzyme λ λ

reactant A NADPH ** Λ product B

Reactant A Reactant B

10 or or

Reaction product B Enzyme product A

1 6-phosphogluconate glucose-6-phosphate-glucose-6 dehydrogenase phosphate 15 2 oxidized glutathione glutathione reductase reduced thion glutathione 3β-benzoquinone quinone reductase hydroquinone 20 4 nitrate nitrate reductase nitrite 5 α-ketoglutarate giutamic acid + glutamic acid coti namidadeni ndi nueleotide phosphate

** reduced NADP

It should be noted that the cyclic reaction systems set forth in Tables B and C comprise the combination of any of the reactions set forth in the respective tables with any other reaction set forth therein. Eg. For example, reaction 1 of Table B can be paired with any of reactions 2-8 to form a useful cyclic reaction system. Tables B and C thus represent 35 and 56 possible 20 cyclic reaction systems, respectively.

In addition to the cyclic reaction systems represented in Tables B and C, it is conceivable that one of the reactions in the cyclic reaction system may include enzymatic or non-enzymatic 13 conversion of a spectrophotometric indicator, preferably colorimetric. In such a system, any change in reaction rate or cycle in the rate of reflection will be reflected by a change in the spectrophotometric properties of the indicator. When the preferred colorimetric indicators are used, such a change would be a color change. An example of a cyclic reaction system involving the conversion of an indicator is the system formed by combining one of the reactant B product B reactions from Table B with a reaction comprising an oxidation reduction indicator and an electron transfer agent. As an electron transfer agent, phenazine methosulfate can be used. Useful indicators include the oxidized forms of nitrotetrazolium, thiazoyl blue and dichlorophenolindophenol.

It is also conceivable that an exponential cyclic reaction system 15 may be included in the monitoring system. An example of an exponential cyclic reaction system is the following:

AMP + ATP my ° kinas ^ 2 ADP

20 ADP * PEP Pyiwtklnase „p + pyruvate

Such a cyclic reaction is autocatalytic in the sense that the amount of cycle in serous material is doubled over each cycle. Cycle in the rate of seeding therefore increases exponentially with time and 25 gives a high degree of sensitivity. Further details of and discussion of such cyclic reactions can be found in J. Biol. Chem. 247; 3558-70 (1972).

The present invention can be used to detect any ligand to which there is a specific binding partner.

The ligand is usually a peptide, protein, carbohydrate, glycoprotein, steroid or other organic molecule to which a specific binding partner exists in biological systems or to which a specific binding partner can be synthesized. The ligand 35 is usually selected from antigens and antibodies thereto, haptenes and antibodies thereto as well as hormones, vitamins, metabolites and pharmacological agents and their receptors and binding agents. Specific examples of ligands that can be detected by the method of the present invention are hormones such as

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14 insulin, chorionic gonadotropin, thyroxine, liothyronine and estriol; antigens and haptens such as ferritin, bradykinnin, prostaglandins and tumor-specific antigens; vitamins such as biotin, vitamin B 2 folic acid, vitamin E and ascorbic acid; metabolites such as 3 ', 5'-adeosine monophosphate and 3', 5'-guanosine monophosphate; pharmacological agents such as dilantin, digoxin, morphine, digitoxin and barbiturates; antibodies such as mi crosomal antibody and antibodies to hepatitis and allergens; as well as specific binding receptors such as thyroxine-binding globulin, avidin, and transcobalamin.

In the conjugate of the present invention, the enzyme substrate is coupled or bound to a specific binding substance which is the ligand, a specific binding analogue of the ligand or a specific binding partner of the ligand according to the selected assay system so that a measurable amount of the reactant's activity is maintained. The bond between the enzyme substrate and the specific binding agent is usually largely irreversible under the assay conditions, such as when the monitoring reaction in which the enzyme substrate has activity is not destined to chemically destroy this binding as in the above-mentioned luminescence and cyclic reaction systems. However, in some cases, destruction or other effect of this binding occurs by the selected monitoring reaction as a means of measuring the change in the activity of the enzyme substrate. One such case is the enzymatic fluorescent substrate reaction system described herein.

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The enzyme substrate may be directly coupled to the specific binding material such that the molecular weight of the conjugate is less than or equal to the total molecular weight of the enzyme substrate and the specific binding substance. However, usually, the enzyme substrate and the specific binding substance are joined by a bridging group containing between 1 and 50, and preferably between 1 and 10 carbon atoms or heteroatoms such as nitrogen, oxygen, sulfur, phosphorus and so on. Examples of a bridging group comprising a single atom are a methylene group (one carbon atom) and an amino group (one heteroatom). The bridging group usually has a molecular weight not exceeding 1000 and is preferably less than 200. The bridging group comprises a chain of carbon atoms or heteroatoms or a combination thereof and is attached to the enzyme substrate and the specific binding agent or active derivative thereof.

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15 by a linking group, usually in the form of an ester, amido, ether, thioester, thioether, acetal, methylene or amino group.

An enzyme substrate is a compound or group capable of undergoing a chemical conversion catalyzed by an enzyme. The substrate has a preferred molecular weight of less than 9000 and preferably less than 5000. Substrates of this size are particularly suitable for use in the preparation of the conjugate due to their lack of molecular complexity. Furthermore, the activity of such substrates, when coupled to a specific binding agent, is easily affected by reaction between the conjugate and the specific binding agent. Examples of enzyme substrates which may be used for the present invention are the aforementioned enzyme cleavable fluorescent substrates such as fluorine rescein and umbel1 derivatives, pH indicators and spectrophotometric indicator dyes, especially chromogenic types.

In one embodiment of the invention, the components of the specific binding reaction to be combined with the liquid medium supposed to contain the ligand are in liquid or solid form. In the preferred homogeneous assay system, the components are usually in solution or in a solid form which is readily dissolved in the liquid medium. Since the liquid medium to be tested is usually of aqueous nature, the components are usually in water-soluble form, ie. either in aqueous solution or in a water-soluble solid such as a powder or resin. The assay method can be carried out in a conventional laboratory container such as a test tube, the components of the specific binding reaction and the components of the reaction system being added thereto in solid or liquid form.

Furthermore, one or more of the specific binding reaction components and / or one or more of the components of the monitoring reaction can be connected to a carrier. In one aspect, the carrier may be a container, such as a test tube or capsule, containing the component or components of an inner portion, e.g. in the form of a liquid or a loose solid or a coating on an inner surface of the container. In another aspect, the carrier may be in the form of a

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16 matrix which is insoluble and porous and preferably absorbent with respect to the liquid medium to be determined. The matrix may be in the form of moisture-absorbent paper, polymer films, membranes, veils or blocks, gels, etc. In this form, the matrix provides a convenient means for touching the liquid medium to be examined, for carrying out the specific bonding reaction and / or the monitoring reaction and for observing the resulting response.

The liquid medium to be examined may be a naturally occurring or artificially formed liquid which is believed to contain the ligand. The liquid medium is usually a biological liquid or a liquid derived from a solution thereof or another treatment thereof. Biological fluids which can be assayed according to the present method include serum, plasma, urine and amniotic fluid, cerebral and spinal fluid. Other materials such as solid material, e.g. Tissues, or gases, can be analyzed by bringing them into liquid form, such as by dissolving the solid or gas in a liquid or by extracting the solid.

Contrary to the known homogeneous methods, biological fluids containing substances having a reactant activity that is equivalent to or identical to the activity of the conjugated labeling substance can be analyzed for the ligand without background disturbance. Endogenous background reactant activity can be easily eliminated in a number of ways. The biological fluid can be treated so that the endogenous reactant activity is selectively destroyed. Such treatment may involve action with a clarifying agent that chemically destroys endogenous activity, followed by treatment to inactivate this agent used.

The invention will now be illustrated by the following examples, which are not to be construed as limiting: • 35

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17

Example 1

Preparation of 2,4-dinitrophenyl-fluorescein conjugate.

5 Fluorescein n-3 ', 6'-bi s- [6- (2,4-dihydroaniline) hexanate].

The synthesis included the reaction of the acid chloride of 6- (2,4-dinitro-aniline) hexanoic acid with the disodium salt of fluorescein. 6- (2,4-Dinitro-aniline) hexanoic acid was prepared by that of Biochem. J. 42: 287-94 (1948).

A solution of 1.5 g (5 minimol) of 6- (2,4-dinitroaniline) hexanoic acid was converted to the acid chloride by reaction with 10 ml of hot thionyl chloride for 15 minutes, followed by cooling and dilution with 20 ml of hexane. The solid acid chloride which was formed was collected by filtration and added after thorough drying to 600 mg of the di-sodium salt of fluorescein in 10 ml of dry acetone. After 5 hours of reflux, the reaction was stopped by the addition of 2 ml of water and 5 ml of acetone.

After 30 minutes at 25 ° C, the mixture was concentrated to dryness and the residue partitioned between ethyl acetate and aqueous sodium bicarbonate solution. The organic phase was separated and washed with 1% aqueous sulfuric acid, dried over anhydrous magnesium sulfate and evaporated.

The red oil was chromatographed on 60 g of silica gel 60, obtained from E. Merck, Darmstadt, Germany, using a mixture of 25 acetone and carbon tetrachloride as the eluent in a 2: 8 volume ratio. The 1.2 g of crude bisester thus obtained was rechromatographed on 60 g of silica gel using a mixture of acetone and carbon tetrachloride in a volume ratio of 1: 9. The fractions containing the desired product were combined and evaporated to 180 mg of a yellow glass-like solid.

Calculated for ¢ 44 ^ 3 ^ 0 ^: C: 59.33; H: 4.30; N: 9.43 Found: C: 60.92; H: 4.35; N: 6.65.

The infrared spectrum of the product exhibited the expected ester carbonyl stretch absorption at 1765 cm

35

Example 2

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Assays were performed by the homogeneous method for detecting derivatives of 2,4-dinitrophenyl and antibodies thereof using an enzyme substrate (modified fluorescein) as a label.

The specific binding reaction system used in the example is based on the reaction shown in Diagram 1 below.

10 15 20 25 30 35

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19 DIAGRAM 1

0 O

5 1 I

R "C" 0 \ ^ Y o Y o-c- «esterase rj ^ Y \ -> 10 H2 O, PR 7> °

ISLAND

2,4-Di ni trophenyl fluorescein conjugate 15

e ° G

2o \ + other products I ° o 25 (maximum fluorescence at 510 nm) 1 R = - (CH2) 5 - NH - ^ J ^ NO2

35 02N

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A. Homogeneous direct binding fluorescence assay for antibodies to 2,4-dinitrophenyl; effect of different antibody concentrations on the reaction rate.

Seven specifically binding reaction reactions were prepared and analyzed. For each reaction mixture, 20 µl of 1 μΜ 2,4-dinitrophenyl fluorescein conjugate (prepared according to Example 1) in dimethyl sulfoxide was combined with the volume of antiserum to 2,4-dinitrophenyl listed in Table 1 1 M 10 bis-hydroxyethylglycine hydrochloride buffer at pH 7.0 to a total volume of 2.0 ml. The background rate of hydrolysis of the ester bonds in the conjugate was determined over 3 minutes for each reaction binder by determining the rate of fluorescence intensity increase at 510 nm with the fluorometer set for excitation at 15 470 nm. A 10 µl volume of 0.1 M bishydroxyethylglycine hydrochloride buffer at pH 7.0 containing 0.54 units of Type I esterase (E.C.

No. 3.1.1.1, provided by Sigma Chemical Co., St. Louis, Missouri) was then added to each reaction mixture. The total reaction rate obtained was measured in the same manner as the background hydrolysis rate. The results are shown in Table 1.

TABLE 1

Background Reaction Amount of Anti-Hydrolysis Total Reactor Breathing Serum (µl) Speed Ten Rate 1 0 0.02 2.68 2 5 0.07 2.48 30 3 10 0.11 1.91 4 20 0.17 1.08 5 30 0.21 0.63 6 40 0.22 0.45 7 60 0.26 0.30 35

It can be seen from the above that the net reaction rate of the hydrolysis reaction was an inverse function of the amount of antibodies present in the specifically binding reaction mixture to the 2,4-dinitrophenyl present in the binding reaction mixture.

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Similarly, it is seen that the rate of reaction of the background hydrolysis reaction was a direct function of the amount of antibodies present in the specifically binding reaction mixture. The present invention therefore provides an assay and assay method for determining the presence of the ligand antibody to 2,4-dinitrophenyl in a liquid medium using a direct binding fluorescence assay technique.

B. Homogeneous competitive binding fluorescence analysis for derivatives of 2,4-dinitrophenyl; effect of different levels of 2,4-dinitrophenyl - / J-alanine on the reaction rate.

Ten specific binding reaction mixtures were prepared, each with a total volume of 2.0 ml and each containing 0.1 M bis-hydroxyethylglycine hydrochloride buffer at pH 7.0 and 2,4-dinitrophenyl-β-alanine prepared according to that of J. Amer. . Chem. Soc. 76: 1328 (1954) with the concentrations described in Table 2 below. To the 2-10 numbered reaction mixtures in Table 2, a sufficient amount of antiserum was added to 2,4-dinitrophenyl to inhibit the rate of the esterase catalyzed reaction in the second mixture, i.e. No. 1, by 60%. After mixing, 20 µl of 1 μΜ 2,4-dinitrophenyl fluorescein conjugate (prepared as in Example 1) in di methyl sul foxide was added to each reaction mixture. A 10 µl volume 0.1 M bis-hydroxyethylglycine hydrochloride buffer at pH 7.0 containing 0.54 units of Type I esterase (EC No. 3.1.1.1, provided by Sigma Chemical Co., St. Louis, Missouri) was added. then to each reaction mixture. The reaction rate obtained was measured as in Part A of this example. The percent ratio of the rate of reactions 2 - 10 to the rate of reaction # 1 (no antibody present) was calculated. The results are listed in Table 2.

TABLE 2

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22

Concentration of Percent of 5 Reaction 2,4-Dinitrophenyl Reaction Rate of Mixture / i-Alanine (nM) Rate Reaction No. 1 1 0 2.78 2 0 1.04 37 10 3 5 1.01 36 4 10 1.04 37 5 20 1.24 45 6 30 1.51 54 7 50 1.54 56 15 8 75 1.80 65 9 100 1.85 67 10 150 2.33 84

It is evident from the above results that the rate of reaction of the hydrolysis reaction was a direct function of the amount of 2,4-dinitrophenyl-β-alanine in the reaction mixture. The present invention therefore provides an assay and assay method for determining the presence of ligands such as derivatives of 2,4-dinitrophenyl in a liquid medium using a competitive bond fluorescence assay technique.

C. Homogeneous competitive binding spectrophotometric analysis for derivatives of 2,4-dinitrophenyl; effect of different levels of 2,4-dinitrophenyl / J alanine on the reaction rate.

30

Eight bond reaction mixtures were prepared, each with a total volume of 1.0 ml and each containing 0.1 M tris- (hydroxymethyl) -aminomethane hydrochloride buffer at pH 7.0. The reaction mixtures further contained 2,4-dinitrophenyl-β-alanine at the concentrations listed in Table 3 below. To the reaction mixtures 2-8 of Table 3, a sufficient amount of antiserum was added to 2,4-dinitrophenyl to inhibit the rate of the esterase-catalyzed reaction in mixture # 1 by 82%. After mixing, 10 µm of 0.1 mM 2,4-dinitrophenyl fluorescein conjugate was added

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23 (prepared as in Example 1) in di methyl sul foxide for each reaction mixture. A 20 µl volume of 0.1 M tris (hydroxymethyl) aminomethane hydrochloride buffer at pH 7 containing 2.6 international units of Type I esterase (EC No. 3.1.1.1, supplied from 5 Sigma Chemical Co., St. Louis, Missouri ) was then added to each reaction mixture. The absorbance change for each reaction mixture at 489 nm per minute was recorded with a Gilford 2000 spectrophotometer. The results are listed in Table 3.

TABLE 3

Concentration of Absorbance

Reaction 2,4-dinitrophenyl change mixture β-alanine (µM) rate 15-1.0 0.0261 2 0. 0.0047 3 1.25 0.0118 4 2.5 0.0131 20 5 5.0 0 0185 6 7.5 0.0202 7 10.0 0.0192 8 12.5 0.0223 25 It is clear from the above results that the reaction rate was a direct function of the amount of 2,4-dinitrophenyl / J alanine in the reaction mixture. Therefore, the present invention provides an assay and assay method for determining the presence of ligands, such as 2,4-dinitrophenyl derivatives, in a liquid medium using a competitive bonding spectrophotometric assay technique.

D. Homogeneous competitive binding fluorescence analysis for derivatives of 2,4-dinitrophenyl; use of non-enzymatic monitoring reaction.

35

The specific binding assay system used in this part was the same as shown in Figure 1 except that esterase was not used to catalyze the hydrolysis of the ester bonds in the conjugate.

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Eight bond reaction mixtures were prepared, each with a total volume of 2 ml, and each containing 0.1 M tri s-hydroxymethylamino methethane hydrochloric acid at pH 7.5. The mixtures also contained 2,4-dinitrophenyl-5-alanine at the 5 concentrations listed in Table 4. To each reaction mixture, 50 µl of antiserum was added to 2,4-dinitrophenyl. After mixing, 20 µl of 2 µM 2,4-di-nitrophenyl fluorescein conjugate (prepared as in Example 1) in dimethyl sulphoxide was added to each reaction mixture. The resulting reaction rate was measured as in Part A of this example. The results are listed in Table 4.

TABLE 4

Concentration of Reaction 2,4-Dinitrophenyl Reaction Mixture / f-alanine (nM) Rate 1 0 0.96 2 12.5 0.94 20 3 31.2 0.84 4 62.5 0.78 5 94, 0 0.70 6 125 0.59 7 187 0.57 25 8 250 0.53

It is clear from the above results that the rate of background hydrolysis in the absence of esterase was an inverse function of the amount of 2,4-dinitrophenyl-β-alanine in the reaction mixture. Therefore, the present invention provides an assay and assays for determining the presence of ligands, such as derivatives of 2,4-dinitrophenyl, in a liquid medium using a competitive binding fluorescence technique in which the binding partner after being bound to the ligand in the conjugate participate in the monitoring reaction.

Example 3

DK 158366B

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Preparation of biotin-umbelliferone conjugate.

5 (2-oxo-2-H-1-benzopyran-7-yl) -5- [cis-hexahydro-2-oxo-1H-thien- (3,4-d -) - in dazole] valeric acid ester.

A solution of 300 mg (1.2 millimoles) of anhydrous biotin in 20 ml of dry dimethyl formamide was stirred at -10 ° C under dry nitrogen gas and 0.17 ml (1.2 millimoles) of dry triethylamine was added. A solution of freshly distilled ethyl chloroformate (0.141 ml in 3 ml dry ether) was added dropwise. After incubation for 30 minutes with stirring, the resulting precipitate was filtered under a dry nitrogen atmosphere and immediately cooled to -10 ° C. To the filtered residue was added a solution of 197 mg (1.2 millimoles) of anhydrous 7-hydroxycoumarin in 3 ml of dry pyridine and stirred for 1 hour at -10 ° C and then for 20 hours at 25 ° C. The solvents were evaporated under high vacuum at 40 ° C.

After cooling, the resulting solid was filtered and the recrystallized from methanol. The desired product was thereby obtained (m.p.

20 = 216 - 218 ° C).

Calcd. H: 4.19; N: 7.21

Found: C: 58.4; H: 5.12; N: 6.86

Claims (7)

A specific binding assay method for analyzing a liquid medium for a ligand, comprising the steps of: (a) contacting the liquid medium with a reagent comprising a conjugate of a label and a specific binding agent, wherein the reagent and the ligand forms a reaction binding system comprising (i) a bound form of the labeled conjugate wherein the specific binding agent is bound to a specific binding partner thereof, and (ii) a free form of the labeled conjugate in which the specific binding agent is bound to a specific binding partner thereof; (B) the labeling substance is determined in either the bound or the free form thereof as a target for the ligand present in the liquid medium, characterized in that as an labeling substance an enzyme substrate is used and the method is of a homogeneous type, i.e. that physical separation of the bound form and free form of the substrate-labeled conjugate is not performed without measuring and comparing the substrate activity throughout the liquid reaction mixture to the ligand present in the liquid medium tested. 30 A method according to claim 1, characterized in that the substrate-labeled conjugate is a substrate for an enzyme capable of acting on the conjugate and, by enzymatic cleavage, releasing a product having a detectable property different from that of the conjugate. . 3. A method according to claim 2, characterized in that the released product is fluorescent. ί 27 DK 158366B A reagent for use in the method of claim 1, comprising a conjugate of a label and a specific binding agent, and which reagent together with the ligand forms a binding reaction system comprising a bound form of the labeled conjugate wherein the specific binding agent is bound to a specific binding partner thereof, and a free form of the labeled conjugate wherein the specific binding substance is not bound to a specific binding partner thereof, whereby the amount of labeling substance in either the bound or the free form is a function of the amount of that in the liquid. medium-containing ligand, characterized in that the labeling substance is an enzyme substrate and in that the substrate-labeled conjugate exhibits measurably different substrate activities in the bound form and in the free form, thus allowing homogeneous analysis. 15 Reagent according to claim 4, characterized in that it comprises: 1. a labeled conjugate comprising the labeling substance bound to the ligand or a specific binding analog thereto, and 20 2) a binding partner to the ligand. Reagent according to claim 4, characterized in that the substrate-labeled conjugate is a substrate for an enzyme capable of acting on the conjugate by enzymatic cleavage to release a product having a detectable property different from conjugate. Reagent according to claim 6, characterized in that the released product is fluorescent. 30 35