FI68324C - SPECIFIC BINDING MEASURES AND REAGENTS FOR THE PURPOSE - Google Patents

Description

Γ7-55—1 KU RELEASE PUBLICATION, Q -z 0 A

jST »8 11 utläggningsskrift 683 2 4 C (45) Patent granted 12 C8 1905,. Patent racddolat (51) Kv.lk. * / Lnt.CI.4 G Oi N 33/536 FINLAND —FINLAND (21) Patent application - Patentansökning 76111 * 8 (22) Application date - Ansöknlngsdag 2 6.0 4.76 (Fl) (23) Starting date - Giltighetsdag 26.04.76 (41) Has become public - Blivit offentlig 29.10.76

National Board of Patents and Registration Date of display and publication. - 30.04.85

Patents and Registration in 1 Ansökan utlagd och utl.skriften publicerad (32) (33) (31) Privilege claimed - Begärd priority 28.04.75 USA (US) 572008 (71) Miles Laboratories, Inc., Elkhart, Indiana 46514, USA (US) (72) Robert Charles Bogus 1 case, Elkhart, Indiana, Robert Joseph Carrico, Bremen. Indiana, James Edward Christner, Ann Arbor, Michigan, USA (74) Berggren Oy Ab (54) Specific tower analysis method and reagent used in it - Specific binding analysis method and method for the preparation of oils

The invention relates to a specific binding assay method for determining a ligand in a liquid medium and to a reagent used therein.

Due to the difficulty and hazard of handling radioactive materials, several attempts have been made to create suitable specific binding assay systems that are as sensitive and rapid as radioimmunoassays, but do not use radioactivity to monitor the binding reaction. As described in more detail below, the agents used as markers instead of radioactive atoms or molecules include a wide variety of agents such as enzymes, fluorescent molecules, and bacteriophages. First, specific binding assay methods of the "heterogeneous" type are performed, in which the separation of bound and free phase is performed. Such a separation is necessary to perform a specific binding assay when the label in the bound phase is indistinguishable from the label in the free phase.

Examples of heterogeneous methods developed using the enzyme as a marker are described in U.S. Patent Nos. 3,654,090, 3,791,932, 3,839,153, 3,850,752 and 3,879,262 and in Journal of Inmunological Methods 1: 247 (1972). and Journal 68324 of Immunology 109: 129 (1972). In all of the methods described, the enzyme is chemically bound to either the ligand of interest or its binding partner, and a suitable heterogeneous specific binding reaction scheme is constructed after incubation with the sample, the amount of enzymatic activity associated with either the insoluble portion or the liquid portion being: the amount of enzymatic activity in the sample. Problems with the synthesis and characterization of enzyme conjugates are serious drawbacks to this approach.

British Patent No. 1,392,403 and French Patent No. 2,201,299 describe a heterogeneous specific binding assay using a non-active precursor of a spectrophotometrically active substance as a tracer. After incubating the sample with a specific binding reaction system, the insoluble and liquid portions are separated and the amount of label present in the liquid portion as a function of the amount of ligand detected in the sample is determined by performing reaction steps to convert the inactive label to a color-generating or fluorometrically active agent.

Specific binding assay methods using other types of tracers are described in the following: U.S. Department of Commerce (1973) National Technical Information Service (NTIS) Report No. PB-224,875 Nature 219: 186 (1968) describes in detail certain methods of radioimmunoassay and is by nature a very common transient reference to the possible use of coenzymes and viruses as markers instead of radioisotopes. However, it does not shed any light on how the analysis should be performed using such alternative markers or whether such an analysis would in fact be possible. For further background, reference is made to the Principles of Competitive Protein-Binding Assays, Odell and Daughaday (J.B. Lippincott Co., Philadelphia, 1972), in which various known assay schemes are extensively described as well as various substances and features used as markers in specific binding assays.

The enzyme-linked immunoassay described in U.S. Patent No. 3,817,837 is of interest. This method is of the homogeneous type, as it does not require the use of a partitioned (i.e. insoluble part / liquid part) specific binding reaction system or the separation procedure required because the enzyme-labeled ligand is such that enzymatic activity is inhibited when it has reacted with the ligand. with a paired pair. Thus, the ratio of bound marker to free form can be determined by monitoring changes in enzymatic activity. Nevertheless, this method suffers from the difficulty of preparing very characteristic enzyme-labeled conjugates and finding enzymes that would fit the basic design of the system.

Although several new types of specific binding assays have been proposed and studied, radioimmunoassay and various enzyme-labeled immunoassays remain the most widely used and improved. However, both types of systems have obvious drawbacks, the use of a radioactive substance in radioimmunoassay that is hazardous and requires careful handling, and the difficulty of preparing enzyme-labeled conjugates useful in enzyme-linked immunoassays.

It is an object of the present invention to provide a well-suited, versatile and sensitive improved specific binding assay method and reagent wave based on the use of a substance as a tracer that exhibits reagent activity as a component of a predetermined monitoring reaction system.

The invention thus relates to a specific binding assay method for determining a ligand in a liquid medium, comprising the steps of: (a) contacting the liquid medium with a reagent arm containing a conjugate of a label and a specific binding agent, wherein the reagent and ligand form a binding reaction system; a bound phase of the labeled conjugate, wherein the specific binding agent is bound to it by a specific binding pair, and (ii) a free phase of the labeled conjugate, wherein the specific binding agent is not bound to it by a specific binding pair, j. 4,68324 (b) determining a label as a measure of a ligand in either a bound or free phase liquid medium, the method being characterized in that the label is a coenzyme and the method is of a homogeneous type, whereby the bound phase and the free phase of the coenzyme-labeled conjugate are not separated and wherein the activity of the coenzyme in the total liquid reaction mixture is measured and related to the amount of ligand in the test medium.

The invention also relates to a reagent for determining a ligand in a liquid medium, which reagent comprises a conjugate of a tracer and a specific binding agent, and which reagent and ligand form a binding reaction system that produces a bound phase of the labeled conjugate with a specific binding agent and a specific binding agent. wherein the specific binding agent is not bound to it by a specific binding pair, wherein the amount of label in either the bound or free phase is a function of the amount of ligand in the liquid medium characterized in that the label is a coenzyme and the coenzyme-labeled conjugate is measurable. different coenzyme activity in the bound phase compared to the free phase, wherein the assay method is of a homogeneous type, so that it is not necessary to physically separate the bound phase and the free phase of the conjugate.

The tracer according to the invention can be used in a new homogeneous analytical technique.

The monitoring reaction system is preferably enzyme catalyzed. Typically, a monitoring reaction system that is highly sensitive to the reagent in the conjugate is selected. Luminescent or fluorescent reaction systems are very useful in this regard. Particularly preferred are circulating reaction systems, especially those in which the reagent is a circulating substance. Of the preferred circulating reaction systems, enzyme-catalyzed are particularly preferred. According to the present invention, the reagent in the conjugate is a coenzyme having a molecular weight of preferably less than 9,000.

68324 In this context, the following attributes have the following meanings: ligand is the substance or group of substances whose presence or amount in a liquid medium is to be determined, a ligand-specific binding pair is any substance or group of substances with a specific tendency to bind to a ligand to the exclusion of other substances; an analog is any substance or group of substances that behaves in substantially the same manner as a ligand with respect to the binding tendency of a specific binding pair of a ligand.

The new homogeneous analytical technique is based in part on the fact that the reaction between the ligand and the specific binding pair from which the reagent is bound alters the activity of the reagent in a predetermined reaction, thus acting as an intermediary in monitoring the specific binding reaction. In view of this basic phenomenon, various manipulative designs, including various test assemblies and devices, may be applied in performing the method of the present invention. Popular basic processing plans are direct bonding technology and competitive bonding technology.

In the direct bonding technique, a liquid medium suspected of containing a detectable ligand is contacted with a conjugate in which the reagent is bound to a specific binding pair of the ligand and then analyzed for any change in reagent activity. In the competitive binding technique, a liquid medium is contacted with a specific binding pair of a ligand and a conjugate in which the reagent is bound to the ligand or its specific binding analog, or both, and then analyzed for any change in reagent activity. In both procedures, the activity of a reagent is determined by contacting a liquid medium with at least one reagent that forms a predetermined monitoring reaction with the reagent. Qualitative determination of a ligand in a liquid medium involves comparing the property of the reaction obtained, usually the rate with that of the observed reaction in the non-ligand liquid medium, with any difference between them indicating a change in reagent activity. Quantitative determination of a ligand in a liquid medium involves comparing the property of the reaction obtained to a corresponding property in a monitoring reaction in liquid media containing known amounts of ligand.

6,68324

In general, when following a homogeneous analytical scheme, the components of a specific binding reaction, i.e. combine the liquid medium suspected of containing the ligand, conjugate and / or ligand-specific binding pair in any amount, manner and order, provided that the activity of the reagent in the conjugate changes measurably when the liquid medium contains the ligand in an amount or concentration significant for analysis . All components of the specific binding reaction are preferably soluble in the liquid medium.

When using a homogeneous direct bonding technique, the components of the specific binding reaction are a liquid medium suspected of containing a ligand and a conjugate in which the reagent is bound to a specific binding pair of the ligand. The activity of the conjugated reagent in contact with the liquid medium is inversely proportional to the degree of binding between the ligand in the liquid medium and the specific binding pair in the conjugate. Thus, as the amount of ligand in the liquid medium increases, the activity of the conjugated reagent decreases.

Using a homogeneous competitive binding technique, the components of a specific binding reaction are a liquid medium suspected of containing a ligand, an amount of conjugate containing a reagent bound to a ligand or ligand-specific binding analog, and an amount of a specific ligand binding pair. The specific binding pair is contacted substantially simultaneously with both the conjugate and the liquid medium. Because any ligand in the liquid medium competes with the ligand or ligand in the conjugate. with its specific binding analog to bind to the specific binding pair, the activity of the conjugated reagent in contact with the liquid medium is directly proportional to the degree of binding between the ligand in the liquid medium and the specific binding pair. Thus, the activity of the conjugated reagent increases as the amount of ligand in the liquid medium increases.

A variation of the homogeneous competitive binding technique is a homogeneous displacement binding technique in which the conjugate is contacted with a specific binding pair of a ligand and then with a liquid medium. In this case, there is competition for a specific pair of binders. In such a method, the amount of conjugate contacted with the specific binding pair is usually that comprising the ligand or analog thereof in excess of the amount capable of binding the specific binding pair with the amount present while the conjugate and the specific binding pair are in contact. prior to contact with the liquid medium suspected of containing the ligand. This order of contact can be implemented in two suitable ways. In another method, the conjugate is contacted with a specific binding pair in a liquid environment prior to contact with a liquid medium suspected of containing a ligand. In another method, a liquid medium suspected of containing a ligand is contacted with a complex comprising a conjugate and a specific binding pair, wherein the specific binding agent in the conjugate and the specific binding pair are reversibly bound to each other. The amount of conjugate that binds to its specific agent in complex form in the second method is usually greater than the amount the ligand is capable of displacing in the liquid medium during the time the specific binding pair or complex and the medium are in contact before the conjugate reagent activity change analysis is completed.

Another variation of a homogeneous competitive bonding technique is a homogeneous serial impregnation technique in which the components of a specific binding reaction are the same as those used in the competitive binding technique, but the order or combination of additions of ingredients and their relative amounts used are different. According to the serial impregnation technique, a specific binding pair of a ligand is contacted with a liquid medium suspected of containing the ligand for a period of time before said liquid medium is contacted with the conjugate. The amount of specific binding pair contacted with the liquid medium is usually greater than the amount capable of binding to the total amount of ligand assumed to be present in the liquid medium during the time the specific binding pair and the liquid medium are in contact before the liquid medium comes into contact with the conjugate. The amount of ligand or ligand analog in conjugated form is usually greater than the amount capable of binding to the remaining unbound amount of the specific binding pair while the liquid medium and conjugate are in contact before analysis of any change in activity of the conjugated reagent is completed.

8 68324

Confirmation of the change in activity of the conjugated reagent as a component of a predetermined monitoring reaction is preferably accomplished by contacting the specific binding reaction mixture with at least one agent that forms a monitoring reaction with the conjugated reagent and determining the effect of the specific binding reaction on the nature of such reaction.

Below is a brief description of a few different binding reaction schemes.

Although the labeling property in a labeled conjugate, such as radioactivity or enzymatic activity in conventional heterogeneous specific binding assays, such as radioimmunoassays, and in heterogeneous enzyme-linked immunosorbent assays, . In this situation, the observation reaction is of a relatively constant nature when the ligand is not present in the liquid medium or when it is present in an insignificant amount. When the ligand is present in the liquid medium, the nature or nature of the monitoring reaction changes, as described above in connection with the homogeneous technique. The following explanations apply to the diagrams below: 68324 a

Symbol Definition L test ligand © ligand or its specific binding analog B ligand binding pair * marker, i.e. reagent | - insoluble phase -> incubation period followed by suitable separation (lim) limited; present in an amount less than that capable of binding to all available binding sites under the selected reaction conditions during the selected incubation period; i. component to which other components compete for binding (exc) excess, present in an amount greater than that capable of binding to all binding sites present under selected reaction conditions during the selected incubation period 1. Heterogeneous arrangements of competing bonds a) L + (Γ) * + B (lim) - > + insolubilizer for B or © * This is a classic approach to competitive binding. Examples of such insolubilizers are specific precipitating antibodies, specific insolubilized antibodies and, when B or (L) * is a proteinaceous substance, protein precipitants such as ammonium sulfate or when B or (L) * is a small molecule to be adsorbed, dextran coated with charcoal. A description of similar systems can be found in Biochem. J. 88: 137 (1963) and U.S. Patent No. 3,839,153 (FI-P 54034).

b) L + © * + | -B (lim) -> This approach is commonly referred to as the solid phase technique. Descriptions of similar radioimmunoassays. enzyme immunoassay techniques are disclosed in U.S. Patent Nos. 3,505,019, 3,555,143, 3,646,346 and 3,654,090.

c) L + B * + | - @ (iim) - »

Reference: U.S. Patent No. 3,650,090.

d) L + J "(L) + Bx (lim) ->

Reference: U.S. Patent No. 3,850,752 (FI-P 54033).

68324 10 2. Heterogeneous series impregnation arrangements (a) L + B (exc) -> + (D X (exc) -? + Insoluble matter for B or (E) *

In the batch impregnation technique, the tracer component binds to some or all of the binding sites in substance B that remain after the first incubation period.

b) L + J- B (exc) -} + © * (exc) - ^

Similar radioimmunoassay and enzyme immunoassay techniques are described in U.S. Patent No. 3,720,760 (FI-P 48785) and J. ImmunoL. 209: 129 (1972).

c) L + B * (exc) -> + | - (E) (exc) -> 3. Heterogeneous deposition arrangement L + | “B (exc) - ^ B * (exc) ->

In the deposition technique, the label component binds to some or all of the ligand molecules bound to the insolubilized bond pairs.

Reference: U.S. Patent No. 3,720,760 (FI-P 48785).

4. Heterogeneous Solid Phase Dilution Arrangement L + (E) x + | - (non-specific) —--> + B (lim) -y In this technique, a ligand and a label component are bound to their non-specific fences and then dissociated in relative amounts by binding pair binding. , which has a higher affinity for the ligand and the label component. In the most useful form of this technique, a non-specific binding column is used, as described in U.S. Patent No. 3,639,104. Such a technique is useful when the ligand is bound to endogenous binding agents in a sample that would interfere with a competitive binding reaction if not removed. . Once bound to a non-specific binder, endogenous binders can be removed by suitable washes.

Parameters related to conventional heterogeneous assay systems, such as assay arrangements and alternative separation methods, are described in more detail in Principles of Competitive Protein-Binding Assays, Odell and Daughaday (J.B. Lippincott Co., Philadelphia, 1972).

Suitable reactants which, together with the reagent in the conjugate, form a monitoring reaction can be contacted with a specific binding reaction step according to a homogeneous technique. After initiating a specific binding reaction, the reaction mixture, which may contain some or all of the ingredients necessary for the monitoring reaction, is usually incubated for a predetermined period of time before any change in the activity of the reagent in the conjugate is confirmed (homogeneous technique). After the incubation period, all ingredients required for the monitoring reaction that are not yet present in sufficient amounts in the reaction reaction are added to the reaction mixture, and the monitoring reaction is confirmed as an indication of the presence or amount of ligand in the liquid medium.

One preferred form of the monitoring reaction is a luminescent reaction system, preferably an enzyme-catalyzed reaction such as a bioluminescent or chemiluminescent phenomenon. The reagent in the conjugate may be a reagent in either a light generating reaction or a reaction preceding an enzymatic or non-enzymatic luminescence reaction. Any change in the activity of the conjugated reagent due to a specific binding reaction causes a change in the rate of light development or in the total amount, peak intensity, or property of the light generated. Examples of the luminescent reaction systems in Table A, which is used in the following abbreviations are used: ATP Adenosine triphosphate AMP adenosine diphosphate NAD: nicotinamide adenine dinucleotide NADH reduced nikotiiniamidiaueniinidinukleotidi PMN flavin mononucleotide reduced PMNHj flavin mononucleotide hp electromagnetic radiation, usually in the infrared range, the visible range ta'i ultraviolet region 68 324 12 'Ρ · Η · Η ω ι γη ρ Μ · Η ο Ο

C Ό β I β I

· Η · Η Ο · Η Ο M β H β g β · Η β> 3 β> 3 φ ή ι — ι ρ ρ ρ β 2 w ^ Ο ϊ> 3 ϊ> 3 1¾ Ρ Ή 3 Ρ β, Ρ Ρ ρ ρ ρ ρ

Ρ Φ 'Π Ρ Ρ Ρ p O O

• η · η ό -ρ α> α> φ φ cco CO Cti Ρ Ρ Ρ · Η ρ Ρ · Η · Η · Η 50 -Ρ I cd CO ΜΗ (Π WH gg 3 CM - Cd Ρ · Η O · Η · Η O Ρ 3 • -3 WK ιτ \ <β λ; j * ρ χ λ: ρ ρ ρ

β Ρ 2 Q *> CC ι — I ι — trHi — I ι — ji — IOO

O EH S <C -Ο Φ φ cd φ φ cd co co «c p 2 rocp a p 50 ρ p 50 ρ p

• H P

β β Ή

'*'! · Η i — I

β Ρ · Η O

p β β β Ρ Ρ φ φ ρ Ή (—j

φ * ρ <Μ Ρ rH cd CM

ρ · Η · Η φ Ο 50 2 Ρ co op β co «J β 3 φ Ρ Ρ β + X Η Ρ Q. Ρ g> 3 cd ω β ο, -η + ι Ρ χ ρ ρ ρ η Ρ φ β Ρ ρ £ ρ + ρ β ρ cd S CM λ. · Ι I φ cdppcd

<< ffi -H Ρ;> X Ρ Φ P CM

_ 2; ^ β Ρ X Φ -P cti 2

CP + S Ρ Ρ Φ + Cti. + Ρφ P

P P co β <H χ A φ ρ p + p? cdo-H-H IK p> p cd o 2 X · Η Ή + cd χ; CO Ρ + · Η X cd X β · Η

Ρ CO β β CO β P CO X · Η P

2> ^ cd φ Ρ φμ p cti? Cd A + gp <Λ cd χ o <PiP o cd x cd + cd cd

Hi ^ ^ cd fy 2l Ή oj> 3 Ρ Ό χ p. Cd

β Φ P EC CO · IX Ρ ι I. p -H;> X 4- ι-I

φρρ a. β p pa3 / vco co χ cd • H β .H + V r — I f— '+ Φ CO X cd a ^ 3 p

to: cd co · p Ρ P O o cd ^ χ P

: cd cd Ρ β p O CO CM O CO CO P £ Ό Λν. H O

g cd β pM Q Cd o ρ p cd co φ p -h co »β ρ β cover P cd ρ χ cd op co to β ^ ρ φ φ cm (d β + cd ρ β ρ cm χ cdcd -hg ρ Ρ ή ox; φ Χ3ΦΦΙ o cm o cd β co cd cOtHH M · γΙ Ρ P - O £ βφ β φΟβ + βΟΌβ COLO + C \ J Φ φ 50 Cti + n> 3 Ρ Φ β> 3 Φ + β IK p 50 > 3 Ό β 1 I 'Ρ β Ό X β cd · Η Ρ Ρ> 3 C0 Ρ ρ

; Cd Ό · Η> 3 Φ · Η · Η £ Ρ β β + CM ΟΛΙ COX

•> -3 Ή> 3ΧΧ Ό β Ρ Ρ ρ · Η · Η O XscJ Ο Ο β X Tj Φ Ρ · <~ 3 Ρ · Η Cd · Η · Η · Η CM OO a • η ή φρό cdpcdpcd ££ p X cm β 'Ρ · Η ΌΦΙ ΦCdPPφφO Οφ .ϋβ Η, ϋ S β Ä PcdcnP Ρ β + Ο CM Ρ edo cdxdQ φ ccd co cd o · η · η · η ρΟ + ο ΦΡ X <β ^ £ | OPPeO CO g g · ΗΧ Ρ β · Η βρχ · ΗΡ ΡΡΙβ β 30 Ρ + · Η X.

C0 Φ · γΗ β · Η · Ηβ · - (- ΙΡι — ICMO + Ρ Cd cd β 3P_ — iPT3corP Kp -HO Ρ

Ρ · Η Ρ Ρ I> 3> 3> 1 f — I · Η I — IP

• Η 3 + Φ + β -HPPp + ctiPOP CM

O> 3 "r-j! Χ ^ · Η --H β ρ ρ ρ 500 β cd Ο cm

β-ρ -PIS ad 2 Ρ Ρ · Η φ φ φ · Η cdP-H 50 cm O

Η ρ Φ2 + ΛΙ2 m ρήρ ρ ρ ρ βρ g ο χχ · η §Φ ΛΙΡ PpOcdcocococoo> 3Cd3 £ co p-Ρ '-Cd 2 · Η β cd Ο · Η Ή · Η β Ρ 50 ι — I> 3 + + cd

Po ^ ί + εΡ + ΦΡβ ^ Ι, ^ ΛΙ-Η Ο Ρ Cd

• Ρ Ρ Ρ OP <l) PPPg> jjH> 3 · Η · Η ι — I

γΜ -Η> + βββχ) ΦΦΦβρ> 3Ρ> 3 pped ρ ρ χ + Xicocdpppppppp οορ φ CM- φ φ ρ β β cd p + mmm ρρφ-Η-Η ^ ι 02 «cncopgg" ^ · CM S - Ή * H CO 3 β Ρ IX 2 Ρ ^ ^ χ ρ.-Η ρ cd Ρ 2 ΡΡ ^ ΙΟΟΡ Ε-ι S, ^ ^ ^ φ φ Φ co co <dpPCM ρ ρ ρ ρ Ρ Ρ ικ Ο ΡΡΟΧμρ 13 68324

For further details and description regarding luminescent reaction systems that can be used in the present process, see the following references: J. Biol. Chem. 236: 48 (1961).

J. Amer. Chem. Soc. 89: 3944 (1967).

Cornier et al., Bioluminescence in Progress, Johnson et al., Princeton University Press (New Jersey, 1966), pp. 363-84.

Kries, P. Purification and Properties of Renilla Luciferase, Ph.D., University of Georgia (1967).

Am. J. Physiol. 41: 454 (1916).

Biol. Bull. 51:89 (1926).

J. Biol. Chem. 243: 4714 (1968).

A reaction system that is particularly useful for monitoring a novel specific binding reaction of the present invention is a cyclic or rotational reaction system. Such a reaction system is one in which the product of the first reaction is a reagent in a second reaction, the product of which in the second reaction is a substance which is also a reagent in the first reaction.

The diagram below shows a model of a cyclic reaction system:

Products A Recycled Material Reagents B

X (Form 1)

I REACTION B

Reagents A Recycled material Products B

(Form 2)

In the above cyclic model reaction system, a small amount of recycled material is generated if it is supplied with sufficient amounts of reagents A and B, large amounts of products A and B. Since the proportion and amount of product produced by the circulating reaction system is very sensitive to recycled material, it is preferable to use recycled material. as a reagent in the conjugate of the present invention. Tables B and C provide examples of cyclic reaction systems for use with the novel specific binding reaction system of the present invention.

68324 14

TABLE B

product A „- reagent B

enzyme enzyme reagent A product B

reagent A reagent B

or t ai reaction product B enzyme product A__ 1 lactaldehyde alcoholide ^ propanediol hydrogenase 2 α-ketoglutarate glutamide dehydrglutamate ti + NH-rogenase 3 oxaloacetate maleic dehydrate rogenase ^ acetaldehyde ti + CO2 dehydrogenase 6 dihydroxyaceto-α-glycerol phosphate La-glycerol phosphate phosphate dehydrogen phosphate genease 7 pyruvate lactic acid dactate hydrogenase 8 1,3-diphosphoglycerol glyceraldehyde glyceraldehyde 3-phosphate phosphate dehydrogenate

x nicotinamide adenine dinucleotide Kx reduced NAD

15 Table c 68324

product A ^^> NADFx reagent E

enzyme enzyme

reagent A NADPH product B

reagent A reagent B

or or

reaction product B enzyme product A

1 6-phosphoglucose-6-phosphate-glucose-6-cononate dehydrogenase phosphate 2 oxidized glutathione reductase reduced thathion glutathione 3 p-benzoquinone quinone reductase hydroquinone 4 nitrate nitrate reductase nitrite gututhin nitrite 3 α-ketoglutar

* nicotinamide adenine dinucleotide phosphate xx reduced NADP

It should be noted that the cyclic reaction systems shown in Tables B and C comprise any combination of the reactions listed in those tables with any of the other reactions listed therein. For example, reaction 1 in Table B can be combined with any of reactions 2 to 8 'to form a useful cyclic reaction system. Thus, Tables B and C represent 56 and 20 possible cyclic reaction systems useful in the present invention.

In addition to the cyclic reaction systems shown in Tables B and C, it is contemplated that one of the reactions in the cyclic reaction system may comprise a spectrophotometric indicator, preferably colorimetric, enzymatic or non-enzymatic conversion. In such a system, any change in reaction rate or rotational speed would be reflected in a change in the spectrophotometric properties of the indicator. Using popular colorimetric indicators, such a change would be a color change. An example of a cyclic reaction system comprising indicator conversion is system 16 68324, which is obtained by combining one of the reagent B products from the B reactions in Table B with a reaction containing an oxidation-reduction indicator and an electron transfer medium. Phenazine methosulfate can be used as the electron transfer medium. Useful indicators include oxidized forms of nitrotetrazolium, thiazoyl blue, and dichlorophenol-indophenol.

It is also conceivable to include a cyclic exponential reaction system in the observation reaction system. An example of an exponential rotational reaction system is as follows:

AMP * ATP rcyyjLina.as.i 2 ADP

ADP ♦ PEP Pyruvate ^ ij ^ asi .. ATP * pyruvate Such a circulating reaction is autocatalytic in the sense that the amount of recycled material doubles during each cycle. The rotational speed therefore increases exponentially with time and allows a high degree of sensitivity. Further details and a description of such cyclic reactions can be found in J. Biol. Chem. 247; 3558-70 (1972).

The present invention can be applied to the detection of any ligand having a specific binding pair. A ligand is usually a peptide, protein, carbohydrate, glycoprotein, steroid, or other organic molecule that has a specific binding pair in biological systems or that can be synthesized. In a functional sense, the ligand is usually selected from an antigen or an antibody thereof, a hapten or an antibody thereof, or a hormone, vitamin, metabolite or pharmacological agent, or a receptor or binding agent thereof. Specific examples of ligands detectable according to the invention include hormones such as insulin, chorionin-gonadotropin, thyroxine, liothyronine and estriol, antigens and hapten such as ferritin, bradykinin, prostaglandins and cancer-specific antibodies, vitamins such as biotin, vitamin Botin, vitamin , vitamin E, and ascorbic acid, metabolites such as 3 ', 5'-adenosine monophosphate and 3', 5'-guanosine monophosphate, pharmacological agents such as dilantin, digoxin, morphine, digitoxin and barbiturates, antibodies such as microsomal antibody and antibodies to hepatitis and allergens, as well as specific binding receptors such as thyroxine and binding globulin, avidin, intrinsic factor, and transco-balamine.

O

* 'VJL

17 68324

In the conjugate of the invention, the reagent is bound or coupled to a specific binding agent, which is a ligand, a specific binding analog of the ligand, or a specific binding pair of the ligand, depending on the chosen analytical scheme, so that a measurable amount of reagent activity is maintained. The bond between the reagent and the specific binding agent is usually substantially irreversible under the assay conditions, e.g., when the monitoring reaction in which the reagent has activity is not designed to chemically destroy such a bond, as in the luminescent and cyclic reaction systems mentioned above. However, in certain cases, such a bond is intentionally destroyed or otherwise affected by a selected monitoring reaction to analyze the change in activity of the reagent. Such is the case in the previously mentioned enzymatic fluorescent substrate reaction systems.

The reagent can be directly bound to a specific binding agent so that the molecular weight of the conjugate is less than or equal to the group molecular weight of the reagent and the specific binding agent.

However, the reagent and the specific binding agent are usually bonded with an intermediate group having 1 to 50 and preferably 1 to 10 carbon atoms or heteroatoms such as nitrogen, oxygen, sulfur, phosphorus, etc. Examples of the one-atom intermediate group include a methylene group (one carbon atom) and an amino group. (one heteroatoml). The molecular weight of the intermediate group is usually at most 1000 and preferably less than 200. The intermediate group contains a chain of carbon atoms or heteroatoms or a combination thereof and is attached to a reagent and a specific binding agent or an active derivative thereof by a linking group usually an ester, amido, ether, thioester -, in the form of a thioether, acetal, methylene or amino group.

Due to its versatility and suitability, coenzymes are used as reagents in the conjugate. A coenzyme is a non-protein molecule that moves from one enzyme protein to another, promoting the efficient catalytic activity of the enzyme. All known coenzymes have a molecular weight of less than 9,000, preferably less than about 5,000. Useful coenzymes include nucleotide coenzymes, especially those containing an adenine group, 68324 18 such as adenosine phosphates (i.e., mono-, di- and triphosphate forms), nicotinamide its reduced forms, nicotinamide adenine dinucleotide phosphates and its reduced forms. Other useful coenzymes include guanosine phosphates, flavin monomucleotide and reduced forms thereof, flavin adenine dinucleotide and reduced forms thereof, coenzyme A and its thioesters succinyl coenzyme phosphine A, including 3 ', 5'-adenosine adenosine diphosphate. including phosphosulphate.

Useful coenzyme-active conjugates include nucleotide coenzymes having an adenine group to which a specific binding agent, i. the ligand, the ligand-specific binding analog, or the ligand-specific binding pair is bound by a direct bond or an intermediate group, as described above. Coenzyme-active conjugates comprising adenosine phosphate, nicotinamide adenine dinucleotide, or a reduced form thereof, nicotine adenine dinucleotide phosphate, or a reduced form thereof, have the following general formula: succinyl coenzyme A, including 3 't5'-3idenosine diphosphate and adenosine 3'-phosphate-5'-phosphosulfate.

Useful coenzyme active conjugates include nucleotide coenzymes having an adenine group to which a specific binding agent, i. the ligand, the ligand-specific binding analog, or the ligand-specific binding pair is bound by a direct bond or an intermediate group, as described above. Coenzyme-active conjugates containing adenosine phosphate, nicotinamide adenine dinucleotide or a reduced form thereof, nicotinamide adenine dinucleotide phosphate or a reduced form thereof have the following general formula:, v f *; 3 'l.

68324 20 κ1'Ί \ Γ ° ^ 15 OH l2 where R1 is 0 ° f f--O-P-O0,, -O-P-O-Ij-O®

0 CO

οΘ οΘ 00 o0 f 4 -O-P-O-i-O-P-O®, or -O-P-O-P-O-CH-O R; 6 J to 4 ft \

OH OH

Wherein R 2 is -OH or -O-P-O 0; In part j, r3 is

NH2 R5 NH2 NHRS

HY; <Vj «<-" rS

χνΛν ^ T2 R5 NH2 YH2

</ ΝΊΐ ni® 1N / Y ^ N

I I * I | 0

Dii n5

where R is K

-®Q> - Q

. * * conh2 conh.

V L

21 68324 wherein R 5 is -Y-Z, wherein Y is a bond or an intermediate group, and wherein Z is a ligand, a ligand-specific binding analog, or a ligand-specific binding pair. The above formula represents ionized forms of a coenzyme-activated conjugate, however, protonated or acidic forms are equally useful. The extent of protonation depends on the pH of the environment. Salts of such conjugates may also be used where appropriate.

In addition to the above-mentioned compounds, useful coenzyme-active conjugates are adenosine phosphates to which a specific binding agent is attached via a phosphate group. Such compounds have the following general formula: nh2 / Y ^ n

R1-ch2 0 \ -KkJ

o

OH OH

β β β • 1 I p | I 9 wherein Fr is -O-P-O-Fr, -O-P-O-P-O-Fr or 0 0 8 β β β -O - ^ - O - ^ - O-P-O-R2 8 8 8 wherein R2 is -Y-Z; Y is a bond or spacer and Z is a ligand, a ligand-specific binding analog, or a ligand-specific binding pair. In suitable cases, protonated or acidic forms as well as salt forms may also be used.

In one aspect of the invention are the components of a specific binding reaction that must be combined with a liquid medium suspected of containing a ligand in liquid or solid form. In a preferred homogeneous system of analysis, the ingredients are usually in solution or solid form that is readily soluble in the liquid medium. Because the liquid medium to be tested is usually aqueous in nature, the ingredients are usually in water-soluble form, i. either in aqueous solution or in water-soluble 22 68324 solid form, such as powder or resin. The assay method can be performed in a standard laboratory vessel such as a test tube, with the components of the specific binding reaction and the components of the reaction system being added in solid or liquid form of the hedgehogs.

It is also contemplated that one or more of the components of the specific binding reaction and / or one or more of the components of the monitoring reaction will be added with the carrier. In one embodiment, the carrier may be a liquid-containing container, such as a test tube or capsule, containing such ingredient or ingredients in its interior, e.g., in liquid or loose solid form, or in the form of a layer on the interior surface of the container. In another embodiment, the carrier may be in the form of a matrix that is insoluble and porous and preferably absorbs the liquid medium to be tested. Such a matrix may be blotting paper, polymeric films, films, lint or pieces, gels, etc. In a conventional form, the device would form a suitable spacer for contacting the liquid medium to be tested, performing a specific binding reaction and / or monitoring reaction, and detecting the result.

The liquid test medium may be a naturally occurring or artificially formed liquid suspected of containing a ligand and is usually a biological fluid or a liquid obtained as a result of this dilution or other treatment. The biological fluids to be analyzed by the method of the invention include serum, plasma, urine and amniotic fluid, as well as cerebrospinal fluid. Other substances such as a solid, e.g. tissue, or gases can be analyzed by liquefying them, such as by dissolving a solid or gas in a liquid or by liquid extraction of a solid.

In contrast to the previously known homogeneous assay system, biological fluids containing substances with reagent activity similar or identical to the conjugated label can be analyzed for ligand without background interference. Endogenous background reaction activity can be eliminated in several ways. The biological fluid can be treated to selectively destroy endogenous reagent activity. Such treatment may involve the use of a clearing agent that chemically destroys endogenous activity, followed by treatment to inactivate the destructive effect of such a clearing agent.

23 68324

Reagent-degrading enzymes, e.g., often occur naturally in biological fluids, especially if the reagent is a coenzyme such as NAD, NADP, or ATP. For such coenzyme degrading enzymes, there are several inhibitors, e.g., chelating agents, that rob essential metal ion activators of the enzymes. NAD-degrading enzymes are found in normal serum and have sufficient enzymatic activity to eliminate substantially all endogenous NAD activity from isolated serum within a few hours. The degrading effect of such enzymes can be effectively prevented by the addition of a chelating agent such as ethylenediaminetetraacetic acid. The detrimental effect can also be eliminated by adding a specific enzyme inhibitor. For example, ATP-degrading enzymes can be inhibited by the addition of βγ-methylene ATP or αβ-methylene ATP.

The invention is described in more detail below by means of examples.

Example 1

Preparation of nicotinamide adenine dinucleotide-biotin conjugate.

A. Nicotinamide-6- (2-aminoethylamino) purine dinucleotide.

Two grams of nicotinamide adenine dinucleotide (NAD) was dissolved in 10 ml of water and 0.6 ml of ethyleneimine was added dropwise, keeping the pH below 7 by the addition of 1 M perchloric acid. When the addition of ethyleneimine was complete, the pH was adjusted to 4.5 and the reaction was incubated at 20-25 ° C. Every 24 hours, 0.6 ml of ethyleneimine was added and the pH was readjusted to 4.5. After 96 hours, the solution was poured into 10 volumes of acetone at -10 ° C. The resulting oil was collected, washed with ether and dissolved in approximately 50 ml of water in a flask.

The resulting solution was adjusted to pH 7.0-7.5 with 1 N sodium hydroxide, and 1 g of sodium bicarbonate was added. Nitrogen was bubbled through the solution for 4-5 min and 1 g of sodium hydrosulfite was added. The flask was sealed and allowed to stand at room temperature for 45 min. The solution was then oxidized for 1 min and adjusted to pH 11.3 with sodium hydroxide. The solution was heated at 75 ° C for one hour. The reaction mixture was then cooled to room temperature, and 0.6 g of tris- (hydroxymethyl) aminomethane was added, followed by 5N hydrochloric acid to adjust the pH to 7.5. To the resulting solution were added 1000 International Units of alcohol dehydrogenase and 1 ml of 24,68324 acetaldehyde. The decreasing optical density of the reaction mixture was monitored at 340 nm and when no further reduction was observed, the pH was adjusted to 3.5. The solution was poured into 10 volumes of acetone at -10 ° C. The resulting oil was separated and washed with ether, then dissolved in 10-15 ml of water.

The resulting solution was applied to a 2.5 x 90 cm Sephadex G-10 column (Pharmacia AB, Uppsala, Sweden) equilibrated with water. Fractions with a volume of 12 ml were collected. The wavelength of maximum optical absorption in the ultraviolet range and the optical density at this wavelength were determined for each fraction. The optical density of each jade at 340 nm after reduction with alcohol dehydrogenase was also determined. Fractions with an optical absorption maximum at 264 nm and a ratio of optical densities at 3 nm to 264 nm greater than 0.05 were pooled. The collected material was concentrated to 15-20 mL on a rotary evaporator and passed through a 2.5 x 28 cm Dowex 1-X8 column equilibrated with water (Bio-Rad Laboratories, Richmond, CA). Additional water was added to wash the collected material through the column and 10 ml fractions were collected. Fractions with an optical absorption peak at 264 nm and an optical density ratio at 340 and 264 nm greater than 0.1 were collected.

The collected material was passed through a water equilibrated 5 x 45 cm Dowex 50-X2 column (Bio-Rad Laboratories, Richmond, CA). More water was added to wash the collected material through the column, and 20 ml fractions were collected. Fractions with an optical absorption peak at 264 nm and a ratio of optical densities at 340 and 264 nm with a higher dip were collected. 0,1.8. The collected material was concentrated to 4 to 5 ml and purified by electrophoresis as follows.

The concentrated material was applied to a Whatman 3MM sheet of paper (Reeve Angel, Clifton, New Jersey) as a 1-2 cm wide strip in a direction transverse to the electric current. The paper was then moistened with 0.02 M sodium phosphate at pH 6.0. Electrophoresis was performed by the Durrum-dependent paper method as described in Science 121: 829 (1955) for 4-7 hours with a potential gradient of about 8.5 volts / cm. The location of the desired pyridine nucleotide derivative was determined by fluorescence, which after development of a test strip taken from paper 25,68324 was sprayed with 0.5 μM sodium cyanide in J. Biol. Chem. 191: 447 (1951). The area containing the desired derivative was cut from the paper and extracted with three 50 ml volumes of water. The resulting extracts containing nicotinamide-6- (2-aminoethylamino) purine dinucleotide were collected, concentrated to 3-4 ml and stored at -20 ° C.

B. Nicotinamide adenine dinucleotide-biotin conjugate.

16 mg of biotin was suspended in 1 ml of water containing 22 mg of nicotinamide-6- (2-aminoethylamino) purine dinucleotide prepared as in Part A above. A few drops of 0.1 N sodium hydroxide were added to facilitate dissolution of biotin. To the resulting solution was added 240 mg of 1-cyclohexyl-3- (2-morpholinoethyl) carbodiimide metho-p-toluenesulfonate, and the solution was added dropwise with 0.1 N hydrochloric acid. The reaction mixture was allowed to incubate at room temperature for 5 hours and then poured into 10 ml of acetone at -10 ° C. The resulting oil was separated, washed twice with 5-10 ml of ether and dissolved in 1-2 ml of water. The obtained material was purified by paper electrophoresis as in Example 1. Fluorescent strips appeared after sodium cyanide spraying, one moved towards the cathode and the other towards the anode. The latter band containing the NAD-biotin conjugate was eluted with water and stored at -20 ° C.

Example 2

Homogeneous specific binding assay> Effect of avidin and biotin on the enzymatic turnover of NAD and NAD-biotin conjugates.

The circulating reaction system used in this example was based on the following reactions: (a) NAD ligand + lactate — Mj-iL9j? APP, ° ψ dehydrogenase NADH ligand + pyruvate (b) NADH ligand + thiazolyl blue (oxidized) —SA_ »NAD ligand + thiazolyl blue (reduced)

Eight specific binding reaction mixtures were prepared, each with a total volume of 0.5 ml and containing 0.12 MN, N-bis-2-hydroxyethylglycine hydrochloride buffer at pH 7.8 and containing 68324 26 concentrations and activities of NADria, respectively. , the NAD-biotin conjugate prepared in Example 1, biotin and avidin, which tend to bind to biotin. One unit of avidin activity is the amount of avidin capable of binding one microgram of biotin. The reaction mixture was incubated at room temperature for 2-3 hours. Each reaction mixture was contacted with an aqueous enzyme / substrate mixture by adding 0.1 ml of 1 M lithium lactate, 0.05 ml of 10 mM thiazolyl blue in its oxidized form, and sufficient 0.12 MN, N-bis-2-hydroxyethylglycine hydrochloride buffer at pH 7. , 8 with 0.38 International Units bovine cardiac lactic dehydrogenase and 1.5 International Units porcine cardiac diaphorase to a total reaction volume of 1 ml. The relative rate of formation of the reduced form of thiazolyl blue was then determined in each reaction mixture by measuring the total change in optical density at 570 nm in 2 * 1 minutes within the first hour after the addition of the enzyme / substrate mixture. The whole procedure was performed in duplicate and the average results are shown in Table 1.

68324 27

B

C rH

φ ό e> j ω ^ φ c Ξ (- »ej_j ^

• H: cö CM Γ ^ Λ CJ \ ΚΛ rft LP \ CTi i — I

p ^ o ooc - oocm'Po: c0 f- οΗοοΉοοο £: tÖ LPi · * ·> n n n n n n

Φ H - ‘OOOOOOOO

co -h • h ft co -P M> 3 ft Φ: <0 o ft ra

•B

-P

X

CO: c0 i i — i i — ii — ii — i

• H · Ρ: θ 1 — I rH i — li — I

CP ϋ I I I I ·> *> · »«

• ri P ft O O O O

• H <11 Ή O Φ ffl Ή P X ί> · Η cO> -

•B

O

rH P

B

O ft M C ^ o o

ft -H S I I I VO I I VO I

ft G C ^ ft -H '-r ft Ή ft p w 0 es • H 3 p co

, r ~) CO

G 3 o zs

ft CO Ή * H

c o

• H P

• H · Η ^ O O O

P ft S I I MD I I VO VO 0 C K '* ΙΌ ΙΌ • H C -

ft -H

1 p O cti <cO S bO

•B

O

P ^

• H S O O

ftp I CV I I CM I I I

- CM C \ J

G

·· CO., LTD

O 3 <3 S to

O

•B

^ HOjr ^ ftTLTNVOC ^ -CO

CD

<D

in 68324

Reactions 1, 4 and 8 were comparative reactions and show that without NAD and NAD-biotin conjugate no substantial circulation occurred ·. The results of reactions 2 and 3 show that the NAD-biotin conjugate had a significant amount of coenzyme activity compared to pure NAD. It can be seen from the results of reactions 3 and 6 that the presence of avidin in the reaction mixture inhibits the formation of thioazolyl blue (reduced form) when the present NAD is conjugated to biotin. Comparing the results of reactions 6 and 7, it is seen that the presence of free biotin reduces the inhibition of the formation of thiacyllyl blue (reduced form) relative to the concentration of biotin in the reaction mixture.

Thus, it was illustrated in this example that the activity of NAD in the NAD-biotin conjugate with respect to the circulatory system decreased in the presence of avidin and that the magnitude of such a decrease in activity decreased in the presence of additional biotin.

Example 3

Homogeneous competitive binding bioluminescence assay; the effect of different amounts of biotin on the peak luminosity generated.

The bioluminescence reaction system used in this example was based on the following reactions: (c) NAD ligand + ethanol - * -> & dehydrogenase NADH ligand + acetaldehyde (d) NADH ligand + FMN * + F. ® -r-> ° dehydrogenase NAD ligand + FMNH2 (e) FMNH2 + long chain aldehyde + 02 —u ---— -> FMN + long chain acid + H2O + hv * flavin mononucleotide

The light generating solution used to carry out reactions (d) and (e) was prepared as follows. A reagent mixture was prepared with 0.13 M phosphate buffer at pH 7> 0, 0.67 wt% bovine serum albumin, 15.7 μM flavin mononucleotide (FMN) and 13 mM sodium acetate and stored in the dark at -20 °. C. On the day on which the light generating solution was to be used, an emulsion containing 5 μl of dodecanal in 5 ml of water was prepared. Lyophilized luciferase extracted from Photobacterium fi.sheri (an enzyme manufactured by Worthington Biochemical Corp., Freehold, New Jersey) was added to 0.013 M phosphate buffer at pH 7.3 to a concentration of 20 mg / ml. After 30 minutes, the resulting suspension was centrifuged at 1500 g for 10 min and the pellet was discarded. A light generating solution was then prepared within 5 minutes of use by combining 75 of the reagent mixture, 5 of the dodecanal emulsion and 20 of the luciferase solution.

To detect the light generated by the reaction (e), a photometer was constructed with a light detector and a 6 x 50 mm cuvette fitted to a light integrating circuit so that the light generated in the cuvette was reflected by a light electrode. The electrical signal produced by the light detector was passed to a calculator. The peak light intensity, as it is referred to herein, was measured from the instrument sign and was given arbitrary units depending on the diagram strip divisions.

Seven specific binding reaction mixtures were prepared, each with a total volume of 0.2 ml and each containing 0.1 M tris- (hydroxymethyl) aminomethane hydrochloride buffer at pH 8.0, 0.6 M ethanol, 0.01 M semicarbazide hydrochloride, 3 ^ 3 nM NAD-biotin conjugate prepared according to Example 1, 0.025 International units of alcohol dehydrogenase and 0.055 units of avidin. Biotin was added to six of the seven reaction mixtures, i. numbers 2-7 in Table 2, at the concentrations shown in said table.

The reaction mixtures were incubated at room temperature for about 30 min. A volume of 10 of each reaction mixture was injected into a separate cuvette fitted to the light meter described above, in addition to 100 of the light generating solution prepared as described above and pre-incubated at 28 ° C for 2-3 minutes. The whole process was run in duplicate and the average results are shown in Table 2.

30 68324 TABLE 2

Reaction mixture Biotin content Mean peak _ _ (nM) __ luminance i__ 10 36

2 25 M

3 50 57 4 100 79 5 150 90 6 200 97 7 300 104 This example thus illustrated that the magnitude of the peak luminosity generated by the bioluminescence reaction system and thus the activity of NAD in the NAD biotin conjugate was directly proportional to the amount of biotin present in the specific binding reaction mixture. thus, a test mixture and method for determining the presence of ligand-biotin in a liquid medium using a competitive bind-bioluminescence assay technique.

Example 4 Preparation of 2,4-dinitrophenyl-ATP conjugate (6-position bound derivative) N, N-2- (2,4-dinitrophenyl) aminoethyl] adenosine 5'-triphosphate.

g A. N- (2-aminoethyl) adenosine 5'-monophosphate.

Two g (7 mmol) of 6-chloropurine riboside (Sigma Chemical Co.,

St. Louis, Missouri) was mixed with 17 mL of triethyl phosphate and reacted with phosphoryl chloride in the presence of water as described in Chem. Scrip. 19: 165-70 (1972). After hydrolysis of the phosphodichloridate, 9.5 ml of ethylenediamine (140 mmol) was added and allowed to react at room temperature for 3 hours. The reaction mixture was diluted with 4 liters of water and the pH was adjusted to 12 with sodium hydroxide. The solution was passed through a 31 68324 5 x 30 cm Dowex 1 x 8 column (Bio-Rad Laboratories, Richmond, CA) in acetate form. The column was then washed with 3 liters of 0.01 M ammonium chloride and the chromatogram was developed with a linear gradient of 3 liters of water and 3 liters of 1 M acetic acid. An isolated gradient of UV absorber between 1800 ml and 2050 ml of the eluted peak was concentrated to about 25 ml in vacuo. Upon standing at 7 ° C overnight, white crystals formed and were collected and dried to give 65% yield of product. A sample of hot water was recrystallized for analysis.

Calculated for C 11 H 19 N 5 O 7 • · 2 O: C 33.8, H 5.45, N 19.7 Found: C 34.3, H 5.22, N 19.7.

Separate thin layer chromatograms developed with two solvent systems, the first containing four parts of 0.5 M ammonium acetate per 1 part of ethanol and the second 3 parts of isobutyric acid per 5 parts of 1 M ammonium hydroxide, each contained 1 component which quenched the fluorination and reacted with. The 0.1 N hydrochloric acid compound had an absorption peak at 264 nmr and a millimolar extinction coefficient of 17.7, a spectrum characteristic of N 2 -alkylated adenosine derivatives.

B. N- (2,4-dinitrophenyl) aminoethyl] adenosine 5'-monophosphate.

250 mg of N- (2-aminoethyl) adenosine-5'-monophosphate (0.65 mmol) from Part A of this example was dissolved in 20 ml of water at pH 8.

168 mg of sodium bicarbonate were then added, followed by 0.2 ml of 1-fluoro-2,4-dinitrobenzene (1.58 mmol dissolved in 2 ml of ethanol). The reaction mixture was stirred in the dark at room temperature for 4 hours and then an additional 0.1 ml of 1-fluoro-2,4-dinitrobenzene in 1 ml of ethanol was added. After stirring overnight, the reaction mixture was adjusted to pH 2.0 with hydrochloric acid and poured into 200 ml of ethanol. The precipitate formed was dissolved in 200 mL of water and this solution was adjusted to pH 8.0 with sodium hydroxide and chromatographed on a 2.5 x 30 cm column of DEAE cellulose (Reeve Angel, Clifton, New Jersey) in bicarbonate form. The chromatogram was developed with a linear gradient of 1.5 liters of water and 1.5 liters of 0.7 M ammonium bicarbonate. A gradient between 1200 and 1500 ml eluted the largest peak of the ultraviolet light absorbing yellow substance. Ammonium bicarbonate was removed by repeated evaporation to dryness to give 40% yield of the desired product. This product 32 68324 passed as a single yellow spot on thin layer chromatograms developed with the same two solvents mentioned in Part A of this example and epichlorohydrin triethanolamine on anion exchange paper developed with 0.25 M sodium acetate-acetic acid buffer, pH 5.0. In 0.02 N hydrochloric acid, the product had an optical absorption peak at 264 nm and 363 nm mi Himo lari 11a with extinction coefficients 21.8 and 14.2.

C. 2,4-Dinitrophenyl ATP Conjugate.

C N- [2- (2,4-dinitrophenyl) aminoethyl] adenosine 5'-monophosphate (from Part B of this example, 0.3 mmol) was converted to the pyridinium salt by chromatography on a 1.5 x 20 cm Dowex 50 x 2- column (Bio-Rad Laboratories, Richmond, CA). The yellow effluent was concentrated to dryness and 15 mL of dimethylformamide and 0.3 mmol of tri-n-butylamine were added. This mixture was evaporated to dryness and the residue was dried repeatedly by evaporation. The monophosphate intermediate was then reacted to the triphosphate form using the method described in J. Amer. Chem. Soc. 87: 1785-8 (1965). Dimethylformamide-soluble reaction products were added to 250 ml of water, which was then adjusted to pH 8.0. This solution was applied to a 2.5 x 58 cm column of DEAE cellulose in bicarbonate form and the chromatogram was developed with a linear gradient of 3 liters of water and 3 liters of 0.5 M ammonium carbonate. The first eluted peak of the yellow substance was identified as a diphosphate derivative. The second peak of yellow material, eluting with a gradient between 4.15 and 4.4 liters, was evaporated to dryness to give the desired conjugate in 20% yield, which contained 3.0 phosphate residues per ribose residue in the analysis.

Example 5 Preparation of 2,4-dinitrophenyl-ATP conjugate (8-substituted derivative).

8- / 2- (2,4-dinitrophenyl) aminoetyy1x7aminoadenosiini-5'-triphosphate.

A. 8- (2-aminoethyl) aminoadenosine-5'-monophosphate.

2.2 mmol of 8-bromoadenosine-5'-monophosphate prepared according to Arch. Biochem. Biophys. 163: 561-9 (1974), the reaction mixture containing 66 mmol of ethylenediamine and 25 ml of water was heated in an oil bath at 140 ° C for 2 hours. The cooled mixture was adjusted to pH 11.5 with sodium hydroxide and applied to a 2.5 x 55 cm Dowex 1 x 8 column (200-400 mesh, bicarbonate form). The column was washed with 300 ml of water and then with a linear gradient of 3 liters of water and 3 liters of 0.5 M ammonium bicarbonate. The absorbance of the effluent at 254 nm was monitored and the largest peak of absorbent eluted with a gradient between 4.6 and 5.8 liters.

The ammonium bicarbonate was repeatedly removed by evaporation (five times, 20-30 ml of water each time) to dryness in vacuo and the final residue was dissolved in 20 ml of water by adding ammonium hydroxide to pH 8.0. The solution was filtered, adjusted to pH 5.0 with formic acid and allowed to stand at 5 ° C for one day. The formed crystals were collected, dissolved at pH 8.0 and recrystallized at pH 5.0. The yield of the desired intermediate was 27%. · When monitored by thin layer chromatography in a solvent of 4 parts of 0.5 M ammonium acetate per 1 part of ethanol, the product migrated as a single ninhydrin-positive spot which turned off the fluorination. The optical absorption peak in 0.02 N hydrochloric acid was 275 nm and the millimolar extinction coefficient was 17.5, which spectrum characteristics are characteristic of alkylated 8-aminoadenosine derivatives.

Calculated for C 12 H 20 N 7 O 7 P · H 2 O: C H 5.33, N 23.2.

Found: C 34.1, H 5.28, N 23.9- B. 8 - [2- (2,4-dinitrophenyl) aminoethyl] aminoadenosine 5'-monophosphate.

8- (2-Aminoethyl) aminoadenosine-5'-monophosphate from Part A of this example (0.64 mmol) was dissolved in 20 mL of water with the addition of sodium hydroxide to pH 8.0. 168 mg of sodium bicarbonate were then added, followed by 0.2 ml of 1-fluoro-4-dinitrobenzene (1.58 mmol dissolved in 2 ml of ethanol). The reaction mixture was stirred for 18 hours at room temperature, and then 0.1 ml of 1-fluoro-2,4-dinitrobenzene in 1 ml of ethanol was added. After stirring for an additional 4 hours, the mixture was adjusted to pH 2.0 with hydrochloric acid and poured into 200 mL of cold acetone (-10 ° C). The yellow precipitate formed was collected by filtration, dissolved in 200 ml of water and applied to a 2.5 x 45 34 68324 cm column of DEAE cellulose in bicarbonate form. The chromatogram was developed with a linear salt gradient developed from 2 liters of water and 2 liters of 0.7 M ammonium carbonate. Between a gradient of 2 and 3 liters, a yellow substance peak with an absorption maximum of 275 nm eluted. Ammonium bicarbonate was removed from this material by evaporation in vacuo and the yield of the desired intermediate was 37% · The optical absorption peaks measured in 0.02 N hydrochloric acid occurred at 275 and 363 nm and the millimolar extinction coefficients were 21.8 and 15.5. Further analysis showed that there were 1.07 phosphate residues per ribose residue.

C. 2,4-Dinitrophenyl-ATP Conjugate

The monophosphate intermediate (0.5 mol) was reacted to give the tri-n-butylammonium salt by the addition of 0.8 mmol of tri-n-butylamine. The mixture was dried by repeated evaporation from dry dimethylformamide (A times, 10-15 ml each). The final residue dissolved in 1 ml of dimethylformamide was mixed with 2.0 mmol of carbonyldiimidazole in 1 ml of dimethylformamide and allowed to react at room temperature for 4 hours. Excess carbonyldiimidazole was destroyed by reacting with 15 methanol for 30 min. Finally, 3 mmol of tri-n-butylammonium pyrophosphate in ml of dimethylformamide were added and allowed to stand for 20 hours. The solid residue formed was separated by centrifugation and washed twice with 5 ml portions of dimethylformamide. The combined supernatants were added to 200 ml of water, river, then adjusted to pH 8 and chromatographed on 2.5 x 25 cm bicarbonate. On a DEAE-cellulose column. The chromatogram was developed with a linear gradient of 2 liters of water and 2 liters of 0.5 M ammonium bicarbonate. Between a gradient of 2.0 and 2.9 liters, a yellow substance eluted with an optical absorption maximum at 275 and 363 nm. The ammonium bicarbonate was removed by evaporation to give the desired conjugate in 22% yield. The results of the analysis showed that this product contained 3.2 phosphate residues for each ribose residue.

Example 6 Home Competitive Binding Bioluminescence Assay of 2β-Dinitrophenyl Derivatives; Use of ATP as a Marker.

The bioluminescence reaction system used in this example was based on the following reaction: 68324 ATP ligand + reduced luciferin AMP ligand + oxidized luciferin + hv A. Analysis using a 6-substituted ATP derivative.

Three specific binding reaction mixtures were prepared, each with a total volume of 100 μl and each containing 10 mM tris- (hydroxymethyl) aminomethane hydrochloride buffer at pH 7.4, 10 mM ethylenediaminetetraacetic acid, 20 μl of antiserum 2,4-dinitrofen. , 512 nM 2,4-> dinitrophenyl-ATP conjugate (a 6-substituted derivative, prepared as an example) and 2,4-di-nitrophenyl-S-alanine at the concentrations shown in Table 7. After incubation at 25 ° C for 2 hours, 10 μl aliquots of each reaction mixture were analyzed by injecting them into a volume of 0.1 ml of the above light-generating solution, which had previously been incubated at 25 ° C for at least 2 minutes and was in a test tube cl.i fitted to a Bioluminescence light meter on a Dupont Model 760 (EI duPont de Nemours, Willmington, Delaware). Peak light intensity was read on a light meter. The average peak light intensity for each reaction mixture was calculated, as was the relative intensity (100% times the ratio of the average peak intensity of the sample to the absence of antiserum). The results are shown in Table 7 · TABLE 7 Concentration of 2,4-dinitrophenyl-6-reaction alanine Mean peak work___ (μM) ___ light intensity__ 10 7 2 25 14 3 2500 185 B. Analysis using an 8-substituted derivative of ATP.

Four specific binding reaction mixtures were prepared, each with a total volume of 100 μl and each containing 20 mM tris- (hydroxymethyl) -aminomethane hydrochloride buffer at pH 7.4, 10 mM ethylenediaminetetraacetic acid, 20 μl antiserum, 2.4 μl for dinitrophenyl, 594 nM 2,4-dinitrophenyl-ATP conjugate (8-substituted derivative, prepared according to Example 5) and 2,4-36,68324 dinitro-3-alanine at the concentrations shown in Table 8. After 2 hours of incubation at 25 ° C, 10 μl aliquots of each reaction mixture were analyzed as in Part A above and the average peak luminosity was calculated for each. The results are shown in Table 8.

TABLE 8

Reaction 2,4-dinitrophenyl-β- Mean peak mixture alanine content luminance intensity 1 nM) 10 18 2 25 51 3 250 191 k 2500 190 This example shows that the tracer, ATP, can be substituted at various positions around its structure in the preparation of the present compound. a conjugate useful in the specific binding assay method of the invention.

Claims (11)

37 68324 A 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 label and a specific binding agent, wherein the reagent and ligand form a binding reaction system that produces (i) a label a bound phase in which the specific binding agent is bound to it by a specific binding pair, and (ii) a free phase of the labeled conjugate in which the specific binding agent is not bound to it by a specific binding pair, and (b) determining the label as a measure of ligand in either bound or free phase liquid medium; that the tracer is a coenzyme and that the method is of a homogeneous type, wherein the bound phase and the free phase of the coenzyme-labeled conjugate are not physically separated and the activity of the coenzyme in the total liquid reaction mixture is m germinated and proportional to the amount of ligand in the medium tested. A method according to claim 1, characterized in that the coenzyme is a nucleotide coenzyme. Process according to Claim 1, characterized in that the coenzyme is adenosine phosphate, nicotinamide adenine dinucleotide or a reduced form thereof, or nicotinamide adenine dinucleotide phosphate or a reduced form thereof. li. Process according to Claim 1, characterized in that the coenzyme is adenosine triphosphate. The method of claim 1, characterized in that the coenzyme is a flavin adenine dinucleotide. A reagent for determining a ligand in a liquid medium, which reagent comprises a conjugate of a label and a specific binding agent, and which reagent 68324 38 and the ligand form a binding reaction system that produces a bound phase of the labeled conjugate, wherein the specific binding agent is bound to a specific binding agent. the specific binding agent is not bound to it by a specific binding pair, wherein the amount of label in either bound or free phase is a function of the amount of ligand in the liquid medium, characterized in that the label is coenzyme and the coenzyme-labeled conjugate has measurably different coenzyme activity the assay method is of a homogeneous type, so that it is not necessary to physically separate the bound phase and the free phase of the conjugate. Reagent according to claim 6, characterized in that it comprises (1) a labeled conjugate comprising a label bound to a ligand or a specific binding analogue thereof, and (2) a ligand binding pair. Reagent according to Claim 6, characterized in that the coenzyme is a nucleotide 'coenzyme'. Reagent according to Claim 6, characterized in that the coenzyme is adenosine phosphate, nicotinamide adenine dinucleotide or a reduced form thereof or nicotinamide adenine dinucleotide diphosphate or a reduced form thereof. Reagent according to Claim 6, characterized in that the coenzyme is adenosine nitrous phosphate. Reagent according to Claim 6, characterized in that the coenzyme is flavin adenine dinucleotide. 39 68324