FI69214C - SPECIFIC BINDNINGSANALYSMETOD OCH REAGENS FOER ANVAENDNING DAERVID - Google Patents

Description

1 69214

Specific binding assay method and reagent

By division separated from application 761148

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

British Patent 1,392,403 and French Patent 2,201,299 describe a heterogeneous specific binding assay, 2,69214 in which a non-active precursor of a spectrophotometrically active substance is used as a tracer. After incubation of the sample with a specific binding reaction system, the insoluble and liquid portions are separated and the amount of tracer 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 tracer to a colorant .

Specific binding assay methods using different types of tracers are described in the following: U.S. Department of Commerce (1973) National Technical Information Service (NTIS) Report No. PB-224 875 describes a failed attempt to use hemin chloride as a tracer in a chemiluminescent reagent. Nature 219: 186 (1968) describes in detail certain methods of radioimmunoassay and is by nature a very general 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 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 the 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 a partitioned (i.e. insoluble part / liquid part) I! 6921 4 the use of a specific binding reaction system and not the separation method required by it, because the enzyme-labeled ligand is such that the enzymatic activity is inhibited when it has reacted with the ligand binding 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 a radioimmunoassay that is hazardous and requires careful handling, and the difficulty of preparing enzyme-labeled conjugates for use 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 a reagent based on the use of a substance as a marker 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 containing a conjugate of a label and a specific binding agent, wherein the reagent and ligand form a binding reaction system; 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 by a specific binding pair, and (b) determining the label as a measure of ligand in either bound or free phase liquid medium; The process is characterized in that the tracer is a reaction component of a chemiluminescence reaction, such as luminescence or iso-luminescence or a derivative thereof.

The invention also relates to 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 and ligand form a binding reaction system that produces a bound phase of the labeled conjugate with a specific binding agent. a phase in which 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 a chemiluminescent reaction component such as luminescence or isolum.

The tracer of the invention can be used in a novel homogeneous assay technique or in any conventional heterogeneous assay technique.

The monitoring reaction system is enzyme catalyzed and a monitoring reaction system is selected that is highly sensitive to the reagent in the conjugate. Fluorescent reaction systems are very useful in this regard. According to the present invention, the reagent in the conjugate is a reaction component of the chemiluminescence reaction.

Il 5 69214 In this context, the following attributes have the following meaning: 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 a specific binding analogue is any substance or group of substances that behaves in substantially the same way as a ligand with respect to the specific binding tendency of the 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 concepts are direct bonding and competing bonding.

In the direct coupling 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 a possible difference between them indicating a change in the activity of the reagent. Quantitative determination of 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.

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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, the conjugate and / or the specific ligand of the ligand 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 relevant for analysis . All components of the specific binding reaction are preferably soluble in the liquid medium.

Using a homogeneous direct binding technique, the components of a 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. to the binding.stee. 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. Since any ligand in a liquid medium competes in the conjugate with a ligand or a specific binding analog thereof to bind to a 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 modification of a homogeneous competitive binding technique is a homogeneous displacement binding technique in which the conjugate is first 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 6921 4 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 and the specific binding pair in the conjugate 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 bonding 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 69214

The amplification of the change in activity of the conjugated reagent as a component of a predetermined monitoring reaction is preferably achieved 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.

The use of a reagent as a tracer can also be applied to conventional heterogeneous type analytical charts in which the bound and free phases of the tracer moiety are separated and in which the amount of tracer in either is determined. The reagent for performing such a heterogeneous analysis may be in various forms. Generally, such a spacer contains three basic components, which are (1) a ligand of interest, (2) a specific binding pair of the ligand, and (3) a label component, usually labeled with (a) a ligand, (b) a specific binding analog of the ligand, or (c) a specific binding pair. form. The components of the binding reaction are pooled simultaneously or in series and at appropriate incubation times or times, the label component binds to its respective competitive sicospares so that the degree of binding, i. the ratio of the amount of the labeled component to the unbound pair and the amount of unbound, not a function of the amount of ligand present. Below is a brief description of a few different binding reaction schemes that can be used in carrying out the process of the invention.

Although a label 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, the word as a tracer. 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:

Symbol Definition L ligand to be examined ligand or its specific binding analog B ligand binding pair x marker, i.e. reagent | "insoluble phase -> irububation and a period followed by a suitable separation (Iira) 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; the components compete (exc) in excess, present in an amount greater than that capable of binding to all binding sites present under the selected reaction conditions during the selected incubation period.

1. Hetercreenic arrangements of competing bonds a) L + (Τ '"+ B (lim) -> + insolubilizer for 3 or © * This is a classical approach to competitive binding. Examples of such insolubilizers are specific precipitating antibodies , specific insolubilized antibodies and, when E or (E) * is a proteinaceous substance, protein precipitants such as ammonium sulfate or when B or (L) x is a small adsorbable molecule, dextran-11a coated charcoal. J. 88: 137 (1963) and U.S. Patent No. 3,839,153 (FI-P 54,034).

b) The L + (E) * + J-E (lim) - ^ * ·· έ · \ * <ί approach is usually called the solid phase technique. Similar radioimmunoassay and e.n-enzyme immunoassay techniques are described in U.S. Patent Nos. 3,505,019, 3,555,143, 3,646,346 and 3,654,090.

c) L + BÄ + J- (T ^ (lim) - ^

Reference: U.S. Patent No. 3,654,090.

d) L + | - (Γ) + B * (lim) .- ^

Reference: U.S. Patent No. 3,850,752 (Fi-p 54,033).

10 6921 4 2. Heterogeneous si £ sar.i aky 1 las t y, ° '· i? · 1 -s a a) L + B (exc) _-> + © * (exc) ---? + insolubilizer for B or ©

In the series impregnation technique, Merih-ttinescsa binds to some or all of the binding sites in substance z that divide the first incubation period with the residue · b) L + J- E (exc) - + © “(ex n) -?

Descriptions of similar racic immunoassay techniques can be found in S-Patent No. 3,720,760 (FI-P 48,785) and in J · Itimunol. 209: 129 (1,972).

c) L + B * (exc) - ^ + [- © (exc) * 3 · Heterogeneous' deposited in the left - y L + | ~ 3 (exc) - E * (exc) * --- ve «V, cstustekn- · -5 kassa rne ^^ ikiamesosd sit ^ u ^ uu ^ cin - nxin tai x aix—« ·: ιm insoluble Ksi teqtyi: - wc ^ y ^ .------- o --- .u „un._n ligand molecules.

Reference: U.S. Patent No. 3-60 (FI-P 48,725).

. neterogemic solid phase group. In this technique, a ligand and an excipient component are bound to a non-specific binder and its thereafter, relative amounts of it are dissociated by binding pair binding, which has a higher affinity for the ligand and label component. In the most useful form of the ankle, a non-specific binding column is used, as described in U.S. Patent No. 3,959 Such a technique is useful when the 1S-ndi is bound to endogenous binding agents in a sample that would interfere with the competitive binding reaction if they were not removed. Once bound, it does not specify. endogenous binding agents can be removed by suitable washes.

Parameters related to conventional heterogeneous analytical systems, such as analytical arrangements and alternative separation methods,

B

11 6921 4 which are described in more detail in Principles of Competitive Protein-Binding Assays, Odell and Daughaday (J. B. Lippin-cott Co., Philadelphia, 1972).

Suitable reactants that together with the reagent in the conjugate form a monitoring reaction may be contacted with (i) a specific binding reaction mixture by a homogeneous technique or (ii) a separated bound or free phase by a heterogeneous technique, alone or in any combination either before, simultaneously or by a specific binding reaction. after starting. After initiating a specific binding reaction, the reaction mixture, which may contain some or all of the components necessary for the monitoring reaction, is usually incubated for a predetermined time before any change in reagent activity in the conjugate is confirmed (homogeneous technique) or reagents in separated phases are confirmed (heterogeneous technique). After the incubation period, all ingredients required for the monitoring reaction that are not yet present in sufficient amounts in the reaction mixture 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.

A preferred sensitive monitoring reaction involves a fluorescence phenomenon and is enzyme catalyzed. In such a reaction system, the reagent in the conjugate is a substrate in an enzymatic reaction that produces a product with fluorescent properties that differ from those of the conjugated substrate. Any change in the activity of the conjugated enzymatic reagent due to a specific binding reaction causes a change in the fluorescent properties of the reaction mixture. A general reaction scheme for such an enzyme-catalyzed reaction system is as follows: enzyme substrate - XZ),) a product wherein X is an enzyme-cleavable bond or a linking group such as an ester or amido group, and Z is a specific binding substrate, depending on the specific reactant used. a ligand-specific binding analog or a ligand-specific binding pair. Specific 12 6921 4 conjugates used in this type of reaction system include 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 include:

Derivative Formula R1 ___ o ^ R2 fluorescein

Cf \

Uy 0 R2. / x 0 umbellif eroni γΓ \ I, p 3 _ ^ R2 3-indole J | I *

A

a. .R2 R2 'r ^ V "3-pyridol o ^ vVy" 2

resorufin I

wherein H 1 is -OH or -X-z (as defined hereinabove), F 2 is -X-Z, and F 3 is -H or -CH-.

13 6921 4

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 a hormone such as insulin, chorionic gonadotropin, thyroxine, liothyronine and estriol, antigens and hapten such as ferritin, bradykinin, prostaglandins and cancer-specific antibodies, vitamins such as biotin, vitamin B, vitamin -E, and ascorbic acid, metabolites such as 31,5'-adenosine rosin phosphate and 3 ', 5'-guanosine monophosphate, pharmacological agents such as dilantin, digoxin, morphine, digitoxin and barbiturates, antibodies such as microsomal antibody and hepatitis antibodies to allergens, as well as specific binding receptors such as thyroxine and binding globulin, avidin, intrinsic factor, and transcobalamin.

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 spacers having 1 to 50 and preferably 1 to 10 carbon atoms or heteroatoms such as nitrogen, oxygen, sulfur, phosphorus, etc. Examples of the single atom spacer include a methylene group (one carbon atom) and an amino group. (one heteroatom).

The molecular weight of the spacer is usually at most 1000 and preferably less than 200. The spacer contains a chain of carbon atoms or heteroatoms or a combination thereof and is attached to the reagent and a specific binding agent or active derivative thereof by a linking group usually ester, amido, ether, thioester, in the form of a thioether, acetal, methylene or amino group.

The reagent enzyme substrate is a compound or group that can be chemically modified by enzyme catalysis. When the substrate is used as conjugated reagents, it preferably has a molecular weight of less than 9,000 and most preferably less than 5,000. Substrates of this size, due to their uncomplicated molecule, are very suitable for use in the preparation of a conjugate. In addition, the activity of such substrates, when bound to a specific binding agent, can be readily altered by reacting the conjugate with the specific binding agent. Examples of enzyme substrates useful in the present invention include the above-mentioned enzyme-cleavable fluorescent substrates such as fluorescein and umbelliferone derivatives; pH indicators; and spectrophotometric indicator dyes, especially dye-generating types.

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 analytical system, 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 a water-soluble form, i. either in aqueous solution or in water-soluble

II

15 6921 4 in solid form such as powder or resin. The assay method can be performed in a conventional laboratory vessel such as a test tube, in which the components of the specific binding reaction and the components of the reaction system are added thereto in solid or liquid form.

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 by 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 prior art 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.

16 6921 4

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

Example 1 Preparation of 2,4-dinitrophenyl fluorescein conjugate

Fluorescein-3 '/ £ - (2,4-dinitroanilino) heksanoaatti7.

The synthesis essentially involved the reaction of 6- (2,4-dinitroanilino hicanoic acid chloride) with the disodium salt of fluorescein.

6- (2,4-Dinitroanilino) hexanoic acid was prepared according to Biochem. J. 1 * 2-287-94 (1948).

A solution of 1.5 g (5 mmol) of 6- (2,4-dinitroanilino) hexanoic acid was converted to the acid chloride by reacting with 10 mL of warm thionyl chloride for 15 minutes, then cooled and diluted with 20 mL of hexane. The solid acid chloride formed was collected by filtration, and after thorough drying, 600 mg of the disodium salt of fluorescein in 10 ml of dry acetone was added thereto. After refluxing for 5 hours, the reaction was quenched 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 carbonate solution. The organic phase was separated and washed with 1% aqueous sulfuric acid, dried

II

6921 4 17 anhydrous magnesium sulfate and evaporated. The red oil was chromatographed on 60 g of silica gel 50 (E. Merck, Darmstadt, Germany) using 20% (w / v) acetone in carbon tetrachloride as eluent.

1.2 g of crude fcis ester was extracted into 60 g of tii gel using 10 μl (v / v) acetone in carbon tetrachloride. The appropriate fractions were combined and evaporated to give 180 mg of a boiled glassy solid.

Calculated for C 12 H 19 MgO 2: C 55.33, H 4.30, n 9.43 Found: C 60.92, H 4.35, N 5.65.

The expected ester-c & carbonyl adsorption at 1765 cm-1 was in the infrared spectrum.

Example 2

Homogeneous specific siton assays for 2, α-dinitrophenyl derivatives and their antibodies using an enzyme substrate (modified flucresine) as a marker.

The specific binding assay systems used in this example are based on the reaction shown in Scheme 1.

A. Homogeneous direct sitcmis-fluorescent. analysis as an antibody to 2,4-dinitrophenyl; the effect of different amounts of antibody on the reaction rate.

Seven specific binding reaction mixtures were prepared and analyzed. In each reaction mixture, 20 μl of 1 μl of 2,4-dinitrophenylfluorescein conjugate (prepared according to Example 1) in dimethyl sulfoxide was combined with the volume of 2,4-dinitrophenyl antiserum given in Table 5 and additionally containing sufficient 0.1 M bishydroxyethylglycine hydrochloride hydrochloride. at pH 7.0 to give a total volume of 2.0 ml. For each reaction mixture, the background hydrolysis rate of the ester bond in the conjugate over 3 minutes was determined by determining the increase in fluorescence intensity at 510 nm in the excitation state of the fluorimeter apparatus. at 470 nm.

18 6921 4 SCHEME 1 8 o p.-co n CJ - 1, ,, a ^ r; r_? Si_ ^ n ^ e, pH I> 0 2,4-dinitrophenyl-fluorescein conjugate © O C'vV ^ V \ ^

CuO '

Ci, / s (maximum fluoroisomer in 51C no.) R = - (CK2) 5 K02 o2n 10 .mu.l volume 0.1 M bis-hydroxyethylsilyl] hydrochloride buffer at pH 7.0 with 0, 5 units of type I esterase (C.C. lio. 3.1.1.1 »Signa Chemical Co., St. Louis, Missouri) were then added to each reaction mixture. The total reaction rate obtained was measured in the same way as the background hydrolysis rate. The results are shown in Table 1.

11 19 Table 1 6921 4 antiserum background hydro- Total reaction rate lysis- reaction mixture___ (, ui 1_ rate__noceus. 1 0 0.02 2.58. 2 5 0.07 2.48 3 10 0.11 1.91 4 20 G, 17 1.08 5 30 0.21 0.63 6 40 C, 22 0.4ρ 7 6.0 0.26 · C, 30

This part of the example illustrated that the hydrolysis reaction. the net reaction rate was inversely proportional to the amount of ligandir, 2,1-nitrophenyl antibody present in the specific binding reaction mixture. It was illustrated that the reaction rate of the inhibitory background hydrolysis reaction was directly proportional to the amount of antibody present in the specific reaction mixture. The present invention thus provides a test mixture and method for determining the presence of a ligand antibody in 2, t-tintrophenyl in a liquid medium using a direct binding-fluctuating assay technique.

B. Homogeneous competitive sit-fluorescent analysis of 2,4-dinitrophenyl derivatives; Effect of different amounts of 2, β-ditrophenyl-ε-alanine on the reaction rate.

Ten specific coupling reaction mixtures were prepared, each with a total volume of 2.0 ml and each containing 0.1 M bis-hydroxyethyl-glycine hydrochloride buffer at pH 7.0 and 2,4-dinitrophenyl-β-alanine, prepared in J. Amer. Chem. Soc. 76: 1328 (1954) at the concentrations shown in Table 2. To the nine reaction mixtures, i. to numbers 2-10 in Table 2, antiserum was added to 2,6-dinitrophenyl in an amount sufficient to inhibit the rate of esterase-catalyzed reaction in the second mixture, i. n: 1, oC%: Ha. After stirring, 20 μl of 1 μM 2, t-dinitrophenyl fluorescein conjugate (prepared as in Example 1) in dimethyl sulfoxide was added to each reaction mixture. A volume of 10 μl of 0.1 M bis-hydroxyethylglycine hydrochloride buffer at pH 7.0 with 0.54 units of type I esterase (EC No. 3 · 1 · 1 · 1 »Sigma) was then added to each reaction mixture. Chemical Co., St. Louis, Missouri). The obtained reaction rate was measured as in Part A of this Example 20 6921 4. The percentage value of reactions 2-10 compared to reaction No. 1 (without antibody) was calculated. · The results are shown in Table 2.

TABLE 2 Reactivity of 2, -dinitrophenyl-β-alanine pitci reaction No. 1 reaction percentage mixture ___ (ca. i) rate rate 1 0 2.78 2 0 1.014 37 3 5 1.01 36 ^ '12 1.014 '37 5 20 1,214 145 6 30 1.51 5¾ 7 50 1.514 56 8 75 1.30 65 9 100 1.85 67

10,150 2.33 SH

In this part of the example, it was illustrated that the reaction rate of the hydrolysis reaction was directly proportional to the amount of 2,4,4-dinitrophenyl-S-alanine in the reaction mixture. The present invention thus provides a test mixture and method for determining ligands such as 2,4-dinitrophenyl derivatives in a liquid medium using a competitive binding-fluorescent assay technique.

C. Homogeneous competitive binding spectrophotometric analysis of 2,14-dinitrophenyl derivatives; Effect of different amounts of 2,1-dinitrophenyl-β-alanine on reaction rate.

Eight specific binding 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 pK of 7.0 and 2,14-cinitrophenyl-6-alanine at the concentrations shown in Table 3. . Seven of the eight reaction mixtures, i. to numbers 2-8 in Table 3, a sufficient amount of 2, β-dinitrophenyl antiserum was added to inhibit the rate of the esterase-catalyzed reaction by 82 5 in the second mixture, i. No. 1. After stirring, 10 μl of 0.1 mM 2-N-dinitrophenyl-fluorescein conjugate (prepared as in Example 1) was added to each reaction mixture. To each reaction mixture 21 6921 4 was added 20 μl volume of 0.1 μl. M tris- (hydroxymethyl) aminomethane hydrochloride buffer at pH 7.0 with 2.16 international units of type I esterase (EC No.

3.1.1.1, Sigma Chemical Co., St. Louis, Missouri). The change in absorbance of each reaction mixture at 489 nm per min was measured on a Gilford 2000 spectrophotometer. The results are shown in Table 3.

Table 3 Mixture of reaction-2,4-dinitrophenyl absorption rate 8-alanine concentration change _ _ (/ UM) _ _ 1 0 0.0261 2 0 0.0047 3 1.25 0.0118 4 2.5 0.0131 5 5.0 0.0185 6 7.5 0.0202 7 10.0 0.0192 8 12.5 0.0223 This part of the example illustrates that the reaction rate was directly proportional to the amount of 2,4-dinitrophenyl-β-alanine in the reaction mixture. The present invention thus provides a test mixture and method for determining the presence of ligands such as a competitive binding spectrophotometric analysis technique in the liquid medium of 2,4-dinitrophenyl derivatives.

Example 3

Preparation of biotin-umbelliferohic conjugate (2-oxo-2-H-1-benzopyran-7-yl) -5- / β-hexahydro-2-oxo-1H-thieno [3,4-d] imidazole-7-valeric acid ester.

A solution of 300 mg (1.2 mmol) of anhydrous biotin in 20 mL of dry dimethylformamide was stirred at -10 ° C under a dry nitrogen atmosphere and 0.17 mL (1.2 mmol) of dry triethylamine was added. A solution of freshly prepared 22 6 9 2 1 4 ethyl chloroformate (0.14 ml in 3 ml of dry ether) was added dropwise.

After boiling and stirring for 30 minutes, the resulting precipitate was filtered under a dry nitrogen atmosphere and immediately cooled to -10 ° C. The filtered residue was added to a solution of 197 mg (1.2 mmol) of anhydrous 7-hydroxycoumarin in 3 ml of dry pyridine and stirred for one hour at -10 ° C and then for 20 hours at 25 ° C. The dissolved solvents were evaporated under reduced pressure at 40 ° C. After cooling, the resulting solid was filtered and recrystallized from methanol to give the desired product (m.p. 216-218 ° C).

Calculated for C 18 H 19 N 2 O 2 S 2: C 48.75, H 4.19, N 7.21.

Found: C 58.4 H 5.12, N 6.86.

Example 4

Heterogeneous specific binding assays for amidine and biotin using an enzyme substrate as a marker.

The specific binding assay systems used in this example were based on the following reaction:

II

/ \ is__! S / esterase β 2 H 2 O, pH 8.0 biotin-umbelliferone conjugate 0 n ΠΘ j 1 H + + biotin (maximum fluorination at 448 nm) A. Preparation of insoluble coupling pair.

Avidin, which tends to bind to biotin, was rendered insoluble by covalently binding it to a water-insoluble polymer sphere as follows. An aliquot of Sepharose 4b (Pharmacia AB, Uppsala, Sweden) was activated for binding to avidin using the method described by March et al. In Analytical Biochemistry 60: 149 (1974). Approximately 4 ml of activated Sepharose 4B was suspended in 8 ml of 0.1 M citrate buffer at pH 7.0.

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To the suspension was added 6 mg of avidin having an activity of 9.9 units per mg in 3 ml of water. One unit of avidin activity is the amount of avidin capable of binding one microgram of biotin. The resulting reaction mixture was stirred for 6 hours at 7 ° C. Avidin-bound Sepharose 4B was then filtered, washed with 100 ml of 0.1 M sodium bicarbonate buffer at pH 9.0 and resuspended in 12 ml of 0.1 M tris- (hydroxymethyl) aminomethane hydrochloride buffer at pH 8. 0 and diluted 1: 1 with 0.1 M bis-hydroxy-ethylglycine hydrochloride buffer at pH 7.0.

B. Competitive Binding Assay for Biotin; the effect of varying amounts of biotin on the amount of umbelliferone released.

Eight specific binding reaction mixtures were prepared, each with a total volume of 0.2 ml and each containing 0.1 M bis-hydroxyethylglycine hydrochloride buffer at pH 7.0, 0.3 μM biotinium belliferone conjugate (prepared as in Example 3), 15 μl of avidin-bound A suspension of Sepharose 4B prepared as in Part A of this example and biotin at the concentrations shown in Table 4. The reaction mixtures were allowed to incubate at room temperature with gentle shaking for 20 min. Each reaction mixture was centrifuged, and a 100 μl aliquot of onset nephete was combined with 2 ml of 0.1 M bis-hydroxyethylglycine hydrochloride buffer at pH 8.2 with 1.08 units of porcine esterase. After incubation for 5 minutes at room temperature, the fluorination intensity developed in each reaction mixture was measured at 448 nm with 364 nm excitation using an Amico-Bowman spectrophotometer. The results are shown in Table 4.

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Table 4 reaction mixture biotin content fluorination (μM) intensity 1 0.00 0.355 2 0.10 0.495 3 0.20 0.469 4 0.30 0.503 5 0.40 0.547 6 0.50 0.502 7 0.75 0.580 8 1, This example shows that the amount of NAD biotin in the liquid phase was directly proportional to the amount of free biotin present, and thus the assay method and reagent of the present invention can be used to determine the ligand in an unknown liquid sample.

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Claims (2)

25 6921 4 1. A specific binding assay method for the preparation of a ligand and a medium, the method comprising the following methods: (a) a compound for the preparation of a compound with a reagent medium and a conjugate of a label and a specific binding medium, colors; reagents containing a ligand or a binding system, such as (i) a compound having a specific conjugate, a color specific to a compound having a specific binding and (ii) a mixture having a specific conjugate, the color being specific (b) the best use of a label binder, and (b) the best label of the label having a specific bonding agent, and (b) the best label of the label having a specific binding agent; image of the best product, including fluorescent product. 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 a labeled conjugate (i) , wherein the specific binding agent is bound to it by a specific binding pair and (ii) the free phase of the labeled conjugate, wherein 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, characterized by that the label is an enzyme substrate that is catalytically affected by the enzyme to form a detectable product, such as a fluorescent product. Process according to Claim 1, characterized in that the ability of the enzyme to act catalytically on the substrate marker is different in the bound phase than in the free phase. Homogeneous type method according to Claim 2, characterized in that the activity of the enzyme substrate in the liquid reaction mixture is measured without distinguishing between the bound phase and the free phase of the labeled conjugate. The heterogeneous type method according to claim 1, characterized in that the bound phase and the free phase of the obtained labeled conjugate are separated and the activity of the enzyme substrate is measured in one of them. 5. 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 26 6921 4 ligand form a binding reaction system that produces a bound phase of the labeled conjugate, wherein the specific binding agent is bound to it. wherein 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 an enzyme substrate catalytically affected by the enzyme to form a detectable product such as a fluorescent product. Reagent according to claim 5, 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. 2. A method according to claim 1, which comprises the enzymatic formulation of a catalytic pA substrate-labeling medium which can be applied to the bundle and to the bacon. Il