IE42544B1 - Heterogenous specific binding assay method and test system for use therein - Google Patents

Abstract

A test process operating using a specific bond for the investigation of a liquid medium on a ligand is described. The test process comprises the following steps: (a) the liquid medium is brought into contact with a reagent which contains a conjugate of a labelling substance which has a predetermined characteristic and a specific binding substance, the reagent and the ligand forming a binding reaction system which (i) forms a bound phase of the labelling conjugate, in which the specific binding fraction is bound thereto by a specific binding component, and (ii) forms a free phase of the labelling conjugate, in which the specific binding fraction is not bound thereto by a specific binding component, (b) determination of the said characteristics either in the bound phase or in the free phase as a measure of the said ligands in the said liquid medium. The said characteristic of the labelling substance proves to be active in the following reaction systems: (1) as a substrate in an enzyme-catalysed reaction, (2) as a reactant in a cyclic reaction system, (3) as a reactant in a chemiluminescent reaction system, or (4) as a coenzyme in an enzyme-catalysed reaction.

Description

The present invention relates to a specific binding assay method and composition, compound, and device for use therein.

Because of the hazard and difficulty of handling radioactive materials, there have been many attempts to devise con5 venient specific binding assay systems which are as sensitive and rapid as radioimmunoassays but which utilize features other than radioactivity as the means for monitoring the binding reaction. As will be discussed more fully hereinafter, materials which have been utilized as the labeling substance in place of radioactive atoms or molecules include such diverse materials as enzymes, fluorescent molecules, and bacteriophages. The type of specific binding assay methods which Will be discussed first are those of the heterogenous type wherein a separation of the bound- and free-phases is 15 accomplished. Such a separation is necessary to carry out a specific binding assay where the labeled material in the bound-phase is indistinguishable from that in the free-phase.

Exemplary of heterogenous methods which have been developed using an enzyme as the labeling substance are those des2o cribed in U.S. Patents Nos. 3,654,090; 3,791,932; 3,839,153; 3,850,752; and 3,879,262 and in the Journal of Immunological Methode 1:247(1972) and the Journal of Immunology 109:129(1972) In each of the described methods an enzyme is chemically coupled to either the ligand to be detected or a bindihg partner thereof and an appropriate heterogenous specific binding reaction scheme is constructed whereby after incubation with a sample, the amount of enzymatic activity associated with either the insoluble portion or the liquid portion is a function of the amount of ligand in the sample. The problems associated with the synthesis and characterization of the enzyme-conjugates are serious short comings of this approach.

British Patent No. 1,392,403 and French Patent No. 2,201,299 describe a heterogenous specific binding assay which utilizes a non-active precursor of a spectrophotometrically-active substance as the labeling sub10 stance. After incubation of the sample with the specific binding reaction system, the insoluble and liquid portions are separated and the amount of labeling substance present in the liquid portion, which is a function of the amount of ligand to be detected in the sample, is determined by carry15 ing out reaction steps that transform the inactive labeling substance into a chromogen or fluorometrically-active material which is then measured by conventional means.

Other heterogenous specific binding assay methods employing different types of labeling substances are disclosed in the following: Report No. PB-224,875 of the National Technical Information Service (NTISJ of the United States Department of Commerce (1973) describes an unsuccessful attempt to use hemin chloride as a labeling substance in a heterogenous assay system monitored by a chemiluminescence reaction. Nature 279: 186(1968) describes in great detail certain radioimmunoassay procedures and makes a passing reference of a very general nature to the possible use of coenzymes and viruses in place of radioisotopes as labeling substances. However, the author provides no enlightenment as to how to carry out an assay using such alternative labeling substances, or in fact as to whether such an assay would be operable. For further back5 ground, reference may be had to Principles of Competitive Protein-Binding Assaye, ed. Odell and Oaughaday (J.B.Lippincott Co., Philadelphia, 1972) which discusses in breadth the various known assay schemes and the different materials and features that have been used as labels for specific binding assays.

Of interest is the enzyme-tagged immunoassay described in U.S. Patent No. 3,817,837. This method is of the homogenous type in that it does not require the use of a partitioned (i.e. insoluble portion/liquid portion) specific bind15 ing reaction system and the separation procedure necessitated thereby since the enzyme-tagged ligand is designed such that upon reaction with the binding partner of the ligand, enzymatic activity is inhibited. Thus, the ratio of bound tagged material to that in free form can be determined by monitoring changes in enzymatic activity. Nonetheless, this method suffers from the difficulty of preparing well-characterized enzyme-tagged conjugates and of finding enzymes that will fit the basic design of the system. liven though many new types of specific binding assays have been suggested and investigated, the radioimmunoassay and the various enzyme-tagged immunoassays remain the most widely used and improved. However, both types of systems have obvious shortcomings, the radioimmunoassay in its use of radioactive material which is hazardous and requires careful handling and the enzyme-tagged immunoassays in the difficulty of preparing useful enzyme-tagged conjugates.

The present invention provides a highly convenient, versatile, and sensitive improved specific binding assay method and means based on the use of, as labeling substance, a substance which exhibits reactant activity as a constituent of a predetermined monitoring reaction system, such substance being referred to herein as the reactant. The inventive labeling substance may be used’in a novel homogenous assay techi nique or in any of the conventional heterogenous assay schemes.

The invention provides a specific binding assay method for determining a ligand in a liquid medium, which method comprises the steps of: (a) contacting said liquid medium with reagent means comprising a labeled conjugate having a specific binding portion coupled to a labeling portion having a predetermined characteristic, said reagent means and the ligand forming a binding reaction system producing (1) a bound-phase of the labeled conjugate in which the specific binding portion is bound by a specific binding partner thereto, and (2) a free-phase of the labeled conjugate in which the specific binding portion is not bound by a specific binding partner thereto, (b) determining said characteristic in either the bound.. phase or the free-phase; characterized in that said predetermined characteristic of said labeling portion of the labeled conjugate is a predetermined activity as ι (1) a substrate in an enzyme-catalyzed reaction system; 425 14 (2) a reactant in a cyclic reaction system; (3) a reactant in a luminescent reaction system; or (4) a coenzyme in an enzyme-catalyzed reaction system.

The monitoring reaction system preferably is enzyme -catalyzed. Usually, a monitoring reaction system is selected which is highly sensitive to the reactant in the conjugate. Luminescent or fluorescent reaction systems are very useful in this regard. Particularly preferred are cyclic reaction systems, especially those in which the reactant is the cycled material. Of the preferred cyclic reaction systems, those which are enzyme-catalyzed are particularly advantageous. The reactant in the conjugate is usually an enzymatic reactant, such as an enzyme substrate or, as is particularly preferred, a coenzyme, and preferably has a molecular weight of less than 9000. 5a 42844 In the context of this disclosure, the following terms shall be defined as follows: ligand is the substance, or group of substances, whose presence or the amount thereof in a liquid medium is to be determined; specific binding partner of the ligand is any substance, or group of substances, which has a specific binding affinity for the ligand to the exclusion of other substances; and specific binding analog of the ligand is any substance, or group of substances, which behaves essentially the same as the ligand with respect to the binding affinity of the specific binding partner for the ligand.

The novel homogenous assay technique is based, in part, on the fact that the reaction between a ligand and a specific binding partner thereof to one of which the reactant is 15 coupled alters the activity of the reactant in the predetermined reaction, which reaction thus serves as means for monitoring the specific binding reaction. In view of this basic phenomenon, various manipulative schemes involving various test compositions and devices may be employed in performing the method of the present invention. The preferred fundamental manipulative schemes are the direct binding technique and the competitive binding technique.

In the direct binding technique, a liquid medium suspected of containing the ligand to be detected is contacted with a conjugate comprising the reactant coupled to a specific binding partner of the ligand, and thereafter any change in the activity of the reactant is assessed. In the competitive binding technique, the liquid medium is contacted with a specific binding partner of the ligand and with a conjugate S comprising the reactant coupled to one or both of the ligand or a specific binding analog thereof, and thereafter any change in the activity of the reactant is assessed. In both techniques, the activity of the reactant is determined by contacting the liquid medium with at least one reagent which forms, with the reactant, the predetermined monitoring reaction. Qualitative determination of the ligand in the liquid medium involves comparing a characteristic, usually the rate, of the resulting reaction to that of the monitoring reaction m a liquid medium devoid of the ligand, any dif15 ference therebetween being an indication of a change in activity of the reactant. Quantitative determination of the ligand in the liquid medium involves comparing a characteristic of the resulting reaction to that of the monitoring reaction in liquid media containing known amounts of the ligand.

In general, when following the homogenous assay scheme, the components of the specific binding reaction, i.e., the liquid medium suspected of containing the ligand, the conju gate, and/or a specific binding partner of the ligand, may be combined in any amount, manner, and sequence, provided that the activity of the reactant in the conjugate is measurably altered when the liquid medium contains the ligand in an amount or concentration of significance to the purposes of the assay. Preferably, all of the components of the specific binding reaction are soluble m the liquid medium.

Where a direct binding homogenous technique is used, the components of the specific binding reaction are the liquid medium suspected of containing the ligand and a quantity of a conjugate comprising the reactant coupled to a specific binding partner of the ligand. The activity of the conjugated reactant on contact with the liquid medium varies inversely with the extent of binding between the ligand in the liquid medium and the specific binding partner in the conjugate. Thus, as the amount of ligand in the liquid medium increases, the activity of the conjugated reactant decreases.

Where a competitive binding homogenous technique is used, the components of the specific binding reaction are the liquid medium suspected of containing the ligand, a quantity of a conjugate comprising the reactant coupled to the ligand or a specific binding analog of the ligand, and a quantity of a specific binding partner of the ligand. The specific binding partner is contacted substantially simultaneously with both the conjugate and the liquid medium. Since any ligand in the liquid medium competes with the ligand or specific binding analog thereof in the conjugate for binding with the specific binding partner, the activity of the conjugated reactant on contact with the liquid medium varies directly with the extent of binding between the ligand in the liquid medium and the specific binding partner. Thus, as the amount of the ligand in the liquid medium increases, the activity of the conjugated reactant increases.

A variation of the competitive binding homogenous 5 . technique is the displacement binding homogenous technique wherein the conjugate is contacted first with the specific binding partner of the ligand and thereafter with the liquid medium. Competition for the specific binding partner then occurs. In such a method, the amount of the conjugate con10 tacted with the specific binding partner is usually that which comprises the ligand or analog thereof in excess of that capable of binding with the amount of the specific binding partner present during the time that the conjugate and the specific binding partner are in contact prior to IS contact with the liquid medium suspected of containing the ligand. This order of contact may be accomplished in either of two convenient ways. In one method, the conjugate is contacted with the specific binding partner in a liquid environment prior to contact with the liquid medium suspected of containing the ligand. In the second method, the liquid medium suspected of containing the ligand is contacted with a complex comprising the conjugate and the specific binding partner, the specific binding substance in the conjugate, and the specific binding partner being reversibly bound to each other. The amount of the conjugate that becomes bound to the specific thereof which is in complexed form in the second method, is usually in excess of that capable of being displaced by all of the ligand in the liquid medium in the time 43544 that the specific binding partner, or complex, and the medium are in contact prior to the completion of the assessment of any change in the activity of the conjugated reactant.

Another variation of the competitive binding homogenous technique is the sequential saturation homogenous technique wherein the components of the specific binding reaction are the same as those used in the competitive binding technique, but the order of addition or combination of the components and the relative amounts thereof used arp different. Following a sequential saturation technique, the specific binding partner of tlie ligand js contacted with the liquid medium suspected of containing tlie ligand for a period of time prior to the contact of said liquid medium with the conjugate.

The amount of the specific binding partner contacted with the liquid medium is usually in excess of that capable of binding with all of the ligand thought to be present in the liquid medium in the time that the specific binding partner and the liquid medium are in contact prior to the time that the liquid medium is contacted with the conjugate. Further, the amount of the ligand or ligand analog in conjugated form is usually in excess of that capable of binding with the remaining unbound amount of the specific binding partner during the time that the liquid medium and the conjugate are in contact prior to the completion of the assessment of any change in activity of the conjugated reactant.

The step of assessing any change in activity of the conjugated reactant as a constituent of the predetermined monitoring reaction is conveniently accomplished by contacting .. 435^4 the specific binding reaction mixture with at least one substance which forms with the conjugated reactant, the monitoring reaction, and determining the effect of the specific binding reaction on a characteristic of such reaction.

S HETEROGENOUS ASSAY SCHEMES The use of a reactant as labeling substance can also be applied to the conventional heterogenous type assay schemes wherein the bound- and free-phases of the labeled constituent are separated and the quantity of labeling substance in one or the other is determined. The reagent means for performing such a heterogenous assay may take on many different forms. In general, such means comprises three basic constituents, which are (1) the ligand to be detected, (2) a specific binding partner of tlie ligand, and (3) a labeled constituent which is normally a labeled Lorm of (a) the ligand, fb) a specific binding analog oi the ligand, or (c) the specific binding partner. The binding reaction constituents are combined simultaneously or in a series of additions, and with an appropriate incubation period or periods, the labeled con20 stituent becomes bound to its corresponding competing binding partners such that the extent of binding, i.e. the ratio of the amount of labeled constituent bound to a binding partner to that unbound, is a function of the amount of ligand present. To follow is a brief description of some of the different binding reaction schemes that may be used in carrying out the method of the present invention. - 11 42544 While in conventional heterogenous specific binding assay methods, such as radioimmunoassays and heterogenous enzyme immunoassays, the labeling characteristic in the labeled conjugate, such as radioactivity or enzymatic activity, is essentially the same for the bound- and free -forms of the conjugate, according to the present method, the activity of the reactant, as labeling substance, is in certain cases affected by binding of the labeled conjugate.

In such a situation the monitoring reaction exhibits a relatively constant character where the ligand is absent from the liquid medium, or is present in an insignificantly small amount. When the ligand is present in the liquid medium, a characteristic or property of the monitoring reaction would be altered as discussed previously with respect to the homogenous technique.

For tlie diagrams which are set out hereinafter, the following legend shall apply: Symbol Definition ligand to be detected ligand or specific binding analog thereof binding partner for the ligand labeling substance, i.e. reactant insoluble phase incubation period followed by appropriate separation (lim) limited; present in an amount less than that capable of being bound to the total available binding sites under the selected reaction conditions during the selected incubation period; i.e. the constituent for which the other constituents compete for binding (exc) excess, present in an amount greater than that capable of being bound by the total available binding sites under the selected reaction conditions during the selected incubation period 1. Competitive binding heterogenous formats a) L +(L? + B(lim) -+ insolubilizing agent for B or®* This is the classical competitive binding approach. Examples of such insolubilizing agents are specific precipitating antibodies, specific xnsolubilized antibodies, and, where B or®* is a proteinaceous material, protein precipitators such as ammonium sulfate, or where B or® is a small adsorbable molecule, dextran-coated charcoal. Description of parallel systems may be found in Biochem. J. 88:137(1963) and U.S. Patent No. 3,839,153.

This approach is commonly referred to as the solid-phase technique. Descriptions of parallel radioimmunoassay and enzyme immunoassay techniques may be found in U.S. Patents Nos. 3,505,019; 3,555,143; 3,646,346; and 3,654,090. lim) Reference: U.S. Patent No. 3,654,090.

L + Reference: U.S. Patent No. 3,850,752. 2. Sequential saturation heterogenous formats a) L + B(exc) + (L) (exc) -»-+ insolubilizing Λ agent for S or@ b) In the sequential saturation technique, some or all the binding sites on B remaining after the first incubation period are bound by the labeled constituent.

L + -B(exc) —► + © (exc) — Descriptions of parallel radioimmunoassay and enzyme immunoassay techniques may be found in U.S. Patent No. 3,720,760 and J. Immunol. 209:129(1972), c) L + B (exc) -O(exc) 3. Sandwich heterogenous format L + J-B(exc) -»- B- (exc) 15 In the sandwich technique, some or all of the ligand molecules bound to the insolubilized binding partners are bound by the labeled constituent.

Reference: U.S. Patent No. 3,720,760. 4.

Solid-phase dilution heterogenous format L +©* + l· (nonspecific) -i*- + B(lim) In this technique, the ligand and the labeled constituent are bound to a non-specific binder and thereafter proportional amounts are dissociated therefrom by binding with a binding partner having a greater affinity for the ligand and the IS labeled constituent. The most useful form of this technique employs a column of the non-specific binder as described in U.S. Patent No. 3,659,104. Such a technique is useful where the ligand is bound to endogenous binding substances in the sample which unless removed would interfer with the competitive binding reaction. Upon being bound to the non-specific binder, the endogenous binding substances may be removed by appropriate wa sties.

Por further discussion of the parameters involved in conventional heterogenous assay systems, such as more detailed descriptions of assay formats and alternative separation techniques, reference may be had to Principles of Competitive Protein-Binding Assays, ed. Odell and Daughaday (J.B.Lippincott Co., Philadelphia, 1972).

It is contemplated that manipulative schemes involving other orders of addition and other binding reaction formats may be devised for carrying out homogenous and heterogenous specific binding assays without departing from the inventive concept embodied herein.

MONITORING REACTION SYSTEMS The appropriate reaction constituents which form, together with the reactant in the conjugate, the monitoring reaction may be contacted with (i) the specific binding 42S44 reaction, mixture, following a homogenous technique, or (ii) with either the separated bound- or free-phase, following a heterogenous technique, singularly or in any combination either prior to, simultaneous with, or subsequent to initiation of the specific binding reaction. After initiation of the specific binding reaction, the reaction mixture, which may include any or all of the necessary components for the monitoring reaction is usually incubated for a predetermined period of time before assessing any change in the activity of the reactant in the conjugate (homogenous technique) or the amount of reactant activity in the separated phases (heterogenous technique). After the incubation period, any components which are necessary for the monitoring reaction and which are not already present in sufficient quantities in IS the reaction mixture are added thereto, and the monitoring reaction is assessed as an indication of the presence or amount of the ligand in the liquid medium.

One preferred form of the monitoring reaction includes a luminescent reaction system, preferably enzyme-catalyzed, such as a reaction exhibiting the phenomenon of bioluminescence or chemiluminescence. The reactant in the conjugate may be a reactant in either the light-producing reaction or a reaction which is preliminary to an enzymatic or non -enzymatic luminescent reaction. Any change in the activity of the conjugated reactant resulting from the specific binding reaction causes a change in the rate of light production 40844 or in the total amount, peak intensity, or character of the light produced. Examples of luminescent reaction systems are given in Table A in which the following abbreviations are used: 5 ATP adenosine triphosphate AMP adenosine monophosphate NAD nicotinamide adenine dinucleotide NADU reduced nicotinamide adenine dinucleotide FMN flavin mononucleotide 10 fmnii2 reduced flavin mononucleotide hv electromagnetic radiation, usually in the infrared, visible, or ultraviolet region (page 1 of 2 pages) fi •rl μ fi 4-1 P •fi fi fi υ •fi rt fi rt P r-t -fi υ υ rt 'fi 1 fi fi W) a U fi fi o X ’ti ’fi tfi X ο fi X P fi fi fi rt o fi fi M fi x o fi o 04 -fi •r~i X fi X fi X S xl Q o H r-t u < x rt X fi P nJ -fi X Vi o fi rfi-H CU H •ri a> •ti44 •ri fi 0 £ fi •rl r-4 o*fi fi fi fi u fi fi rt »fi ι fi - μ tn - fi tn o < w' .-1 cP H fi •H Cl fi m •ri a Of tn fi rt P fi rt fi 44 4-t rfi tn fi fi tn rt fi P 'fi fi N •r 4 •fi 4 «* Ol O + X e£ «**« i X X X fi Sh s U-\ 4 tn rt rS fi fi c H «0 + tn ta < fi 1 rt rC X 44 fi fi co •rl _C fi u * 44 O J ,, fi fti o •ri 04 fi H 04 ε fi . X cn X ο fi fti rt 04 P fi i-4 + fi C ) + tn tn X fi X rt r—i 04 fi bfi + fi cn fi 44 c > •rt o •ri fi u H fi 0 fi 44 fi 4 rt fi T > rt Q •ri fi X •r, u •rt fi fi -fi -r fi P rt 44 •fi •ri fi fi •ri u <-4 rt fi b rt rt j: -fi r— rfi fi fi fi υ X rt 0 fii •ri J 1 A bO fi r 4 bo •ii c P fi rt fi X fi fi fi υ 44 •«4 u fi ♦r4 o r4 rt rfi U o r4 tn fi rt 4- ) 4· fi i-4 -C 4 DO u X c X •rt •τ J 1 X c s £ fi DO Ch Λ r— X fi o fi X >P fi C ) 4- 4· + n: J r- t + fi fi 04 fi 4· -fi X £ -fi fi 1 2 4 Γ4 2 X 2 X Ph 2 E- H X s-\ <1 Ch I- t 04 & ω fi P rt r-i fi tn fi »ri fi fi •ri υ fi •fi fi υ fi fi V P rt -fi Λ <0 o rfi cu •ri fi fi fi •fi m o fi 0) 'fi rt -phosphate-5 1 -phosphosulfate + reduced luciferin. fi fi •fi in o fi a) •fi U fi fi (1) u fi fi Luminescent Reaction System Conjugated Reactant , 42844 Further details and discussion concerning luminescent reaction systems which may be used in the present method may be found in the following references: J. Biol. Chem. 236:48(1961). 3. Amer. Chem. Soc. 69:3944(1967).

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

Kries, P. Purification and Properties of Benilla 10 l.ucif erase, doctoral thesis University of Georgia (1967).

Am. J. Physiol. 42:454(1916).

Biol. Bull. 52:89(1926).

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

Another type of preferred, sensitive, monitoring reaction involves the phenomenon of fluorescence and is enzyme-catalyzed. In such a reaction system the reactant in the conjugate is a substrate in an enzymatic reaction which produces a product which has fluorescent properties that differ from those of the conjugated substrate. Any change in the activity of the conjugated enzymatic reactant resulting from the 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: enzymatic fenzVme1 reactant -X-Z product (substrate) wherein X is an enzyme-cleavable bond or linking group, such as an ester or amido group, and Z is a specific binding sub21 stance which, depending upon the specific binding reaction technique used, is the ligand, a specific binding analog of the ligand, or a specific binding partner of the ligand. Specific conjugates which may be used in this type of reac5 tion system are various enzyme-cleavable derivatives of fluorescein, umbelliferone, 3-indole, β-naphthol, 3-pyridol, resorufin, and so forth. Examples of possible structural formulas of such derivatives are as follows: Derivative Pormula β-naphthol 3-pyridol resorufin wherein R1 is -OH or -X-Z (as defined above in this paragraph) , RZ is -X-Z and R3 is -H or -CHr A reaction system which is particularly preferably for use in monitoring the novel specific binding reaction of the present invention is a cyclic or cycling reaction system. Such a reaction system is one in which a product of a first reaction is a reactant in a second reaction, which second reaction has as one of its products a substance that Is also a reactant in the first reaction.

The following diagram illustrates a model of a cyclic reaction system: IS products A REACTION A .cycled material, (form 1) reactants B REACTION B reactants A ^cycled material ’’•products B (form 2) 4254^ In 1 he above mode i ΤΛΒΓ.1· Β product Λ NAD* reactant B enzyme ΝΛΠΗ** ν' enzyme Ax'Vj reactant n-rnrhirt R s reaction reactant A or product B enzyme reactant B or product A 1 lactaldehyde alcohol dehydrogenase propanediol 10 2 a-ketoglutarate + NHj glutamic dehydrogenase glutamate 3 oxaloacetate malic dehydrogenase malate 15 4 acetaldehyde alcohol dehydrogenase ethanol 5 a-ketoglutaratc + C02 isocitric dehydrogen- ase isocitrate 20 6 dihydroxyacetone phosphate a-glycerol phosphate dehydrogen- ase L-a-glycerol phosphate 7 pyruvate lactic dehydrogenase lactate 25 8 1,3-diphos- phoglycerate glyceraldehyde-3-phosphate dehydrogenase glyceraldehyde -3-phosphate + phosphate 30 * nicotinamide adenine ** reduced NAD dinucleotide TABLE C product Λ NADP* reactant B enzyme reactant — enzyme ΝΑϋΡΗ**->^\^ product B reactant A or reaction product B enzyme reactant B or product A 1 6-phosphogluconate glucose-6 -phosphate dehydrogenase glucose-6 -phosphate 2 oxidized glutathione glutathione reductase reduced glutathione 3 p-benzoqui none quinone reductase hydroqui- none 4 ni trate nitrate reductase nitrite 5 a-ketoglutarate + NH^ glutamic dehydrogenase glutamate * nicotinamide adenine dinucleotide phosphate 20 ** reduced NADP 2544 It should be noted that the cyclic reaction systems illustrated in Tables B and C comprise the combination of any one of the reactions listed in the respective tables with any other reaction listed therein. For example, reac5 tion 1 in Table B may be paired with any one of reactions 28 to form a useful cyclic reaction system. Thus, Tables B and C represent respectively 56 and 20 possible cyclic reaction systems for use in the present invention.

In addition to the cyclic reaction systems represented 10 in Tables B and C, it is contemplated that one of the reactions in the cyclic reaction system may involve the enzymatic or non-enzymatic conversion of a spectrophotometric indicator, preferably colorimetric. In such a system, any change in the reaction or cycling rate would be reflected in a change IS in the spectrophotometric properties of the indicator.

Using the preferred colorimetric indicators such change would be a color change. An example of a cyclic reaction system involving a conversion of an indicator is the system produced by combining one of the reactant B - product B reactions from Table B with a reaction comprising an oxidation -reduction indicator and an electron transfer agent. As electron transfer agent, phenazinemethosulfate may be used. Useful indicators include the oxidized forms of nitrotetrazolium, thiazoyl blue, and dichlorophenoliridophenol.

It is also contemplated that an exponential cyclic reaction system may be included in the monitoring reaction system. An example of an exponential cyclic reaction system is as follows: AMI’ * ATP gX.°kjnase^. 2 ADP ADP + PEP P.H».yate_kina,S^ATP + pyruvate Such a cyclic reaction is autocatalytic in the sense that during each cycle the amount of cycled material is doubled. The cycling rate therefore increases exponentially with time and affords a high degree of sensitivity. Further details and discussion relating to such cyclic reactions may be found in The present invention may be applied to the detection ol any ligand lor wliich (here is a specific binding partner.

IS The ligand usually is a peptide, protein, carbohydrate, glycoprotein, steroid, or other organic molecule for which a specific binding partner exists in biological systems or can be synthesized. The ligand, in functional terms, is usually selected from the group consisting of antigens and antibodies thereto; haptens and antibodies thereto; and hormones, vitamins, metabolites and pharmacological agents, and their receptors and binding substances. Specific examples of ligands which may be detected using the present invention are hormones such as insulin, chorionic gonado25 tropin, thyroxine, liothyronine, and estriol; antigens and haptens such as ferritin, bradykinnin, prostaglandins, and 254 4 tumor specific antigens; vitamins such as biotin, vitamin Bj2> folic acid, vitamin H, and ascorbic acid; metabolites such as 3',5' adenosine monophosphate and 3',S’ guanosine monophosphate; pharmacological agents such as dilantin, digoxin, morphine, digitoxin, and barbiturates; antibodies such as microsomal antibody and antibodies to hepatitis and allergens; and specific binding receptors such as thyroxine binding globulin, avidin, intrinsic factor, and transcobalamin.

In the conjugate of the present invention, the reactant is coupled or bound to a specific binding substance, which is the ligand, a specific binding analog of the ligand, or a specific binding partner of the ligand depending upon the assay scheme selected, such that a measurable amount of activity of the reactant is retained. The bond between the reactant and the specific binding substance is usually substantially irreversible under the conditions of the assay such as where the monitoring reaction in which the reactant has activity is not designed to chemically destroy such bond as in the above-mentioned luminescent and cyclic reaction systems. However, in certain instances such bond is by design destroyed or otherwise affected by the selected monitoring reaction as a means for assessing the change in reactant activity. Such a case is the enzymatic fluorescent substrate reaction systems referred to previously herein.

The reactant may be directly coupled to the specific binding substance so that the molecular weight of the conjugate is less than or equal to the aggregate molecular weight of the reactant and the specific binding substance. Usually, however, the reactant and the specific binding substance are linked by a bridge group comprising between 1 and 50, and preferably between 1 and 10, carbon atoms or heteroatoms such as nitrogen, oxygen, sulfur, phosphorus and so forth. Examples of a bridge group comprising a single atom would be a methylene group (one carbon atom) and an amino group (one heteroatom). The bridge group usually has a molecular weight not exceeding 1000 and preferably less than 200. The bridge group comprises a chain of carbon atoms or heteroatoms, or a combination of both, and is joined to the reactant and the specific binding substance, or active derivative thereof, by a connecting group usually in the form of an ester, amido, ether, thioester, thioether, acetal, methylene, or amino group.

The reactant in the conjugate of the present invention may be any substance which has given (i.e. fixed or known) reactant activity as a constituent of a predetermined monitoring reaction. More particularly, for the purposes of this disclosure, the terms reactant and substance having reactant activity refer to any chemical substance which is capable of undergoing a finite measurable chemical transformation which yields one or more products different from itself and which results upon interaction of said reactant with reaction initiating means, such as a chemical substance (i.e. another reactant, a catalyst, or other type of material which participates in such chemical transformation), electro5 magnetic radiation, thermal energy, or sonic energy. The class of substances defined herein as reactants therefore includes conventional inorganic and organic reagents and various biochemical materials but excludes such materials as catalysts, including enzymes, and radioactive isotopes which arc not reactants in the monitoring reaction. It will be recognized that while a particular chemical substance may be classified in several different catagories because it is able to function in several ways depending on its chemical environment, it is the activity of such substance with respect to the selected monitoring reaction referred to herein which shall govern which functional identity such substance shall have in the context of this disclosure.

Preferably, the reactant is an enzymatic reactant such as an enzyme substrate, a coenzyme, or an active modification 2Q or derivative thereof. An enzyme substrate is a compound or inoeity capable of undergoing a chemical transformation that is catalyzed by an enzyme. Where a substrate is employed as the conjugated reactant, the preferred molecular weight thereof is less than 9000 and preferably less than 5000.

Substrates of such size, because of their lack of molecular complexity, are most convenient for use in the fabrication of the conjugate. Moreover, the activity of such substrates when coupled to a specific binding substance is readily affected by reaction of the conjugate with a specific binding substance. Examples of enzyme substrates which are contemplated for use in the present invention include the enzymecleavable fluorescent substrates referred to previously such as fluroescein and umbelliferone derivatives; pH indicators; and spectrophotometric indicator dyes, particularly chromo genic types.

For the above reasons and for reasons of versatility and adaptability, coenzymes are especially preferred for use as the reactant in the conjugate. A coenzyme is a non10 protein molecule which migrates from one enzyme protein to another in facilitating the efficient performance of the catalytic function of the enzyme. All known coenzymes have a molecular weight of less than 9000, the preferred coenzymes having a molecular weight of less than about 5000. Useful coenzymes include the nucleotide coenzymes, particularly those comprising adenine groups, such as the adenosine phosphates (i.e. the mono-, di-, and tri-phosphate forms), nicotinamide adenine dmucleotide and its reduced forms, and nicotinamide adenine dmucleotide phosphate and its reduced forms. Other useful coenzymes include the guanosine phosphates, flavin mononucleotide and its reduced forms, flavin adenine dinucleotide and its reduced forms, coenzyme A and its thioesters including succinyl-coenzyme A, 3',5' adenosine diphosphate, and adenosine-3'-phosphate-5'-phosphosulfate.

Useful coenzyme-active conjugates comprise nucleotide coenzymes having an adenine group to which the specific binding substance, i.e., a ligand, a specific binding analog of a ligand, or a specific binding partner of a ligand, is coupled through a direct bond or a bridge group as referred to hereinbefore. Such coenzyme-active conjugates which comprise an adenosine phosphate, nicotinamide adenine dinucleotide or its reduced form, or nicotinamide adenine dinucleotide phosphate or its reduced form, have the follow5 ing general formula: wherein R is -O-P-O0 ο® ο® o® o-p-o-i>O-P-O II ooo ,0 i/i OH R 0° 0° I I , -O-P-O-P-O J s Γ0 wherein R2 is -OH or -O-P-O®; 44 wherein R3 is -Y-Z; wherein Y is a bond or a bridge group; and wherein Z is a ligand, a specific binding analog of a ligand, or a specific binding partner of a ligand. The above formula represents the ionized forms of the coenzymeactive conjugate, however, the protonized or acid forms are equally useful. The extent of protonization depends on the pH of the environment. Also, the salts of such conjugates may also be used where appropriate.

In addition to the compounds mentioned above, useful coenzyme-active conjugates include the adenosine phosphates to which are coupled the specific binding substance through the phosphate grouping. Such compounds have the following general formula: wherein R1 is R1-CH. 0® 0H 0H οΘ 0® I 2 I I 2 -O-P-O-Rz -O-P-O-P-O-Rz ί Ί 11u >00 0® 0® 0® I I 2 'otoi°T'R or wherein R is -Y-Z; wherein Y is a bond or a bridge group; and wherein 2 is a ligand, a specific binding analog of a ligand, or a specific binding partner of a ligand. Also, the protonized or acid forms, as well as the salt forms where appropriate, may be used.

In one form of the present invention, the components of the specific binding reaction which are to be combined with the liquid medium suspected of containing the ligand are in a liquid or solid form. In the preferred homogenous assay system, the components are usually in solution or in a solid form capable of ready dissolution in the liquid medium.

Since the liquid medium to be tested is normally aqueous in character, the components are generally in a water soluble form, that is, either in aqueous solution or in a water soluble solid form such as a powder or resin. The assay method may be carried out in a standard laboratory vessel such as a test tube with the specific binding reaction components and the components of the reaction system being added thereto in solid or liquid form.

It is also contemplated that one or more of the specific binding reaction components and/or one or more of the components of the monitoring reaction may be incorporated with a carrier. In one aspect, the carrier may be a liquidholding vessel such as a test tube or capsule containing such component or components in an interior portion thereof, for instance, in tho form of a liquid or loose solid or a coating on an interior surface of the vessel. In another aspect, tiie carrier may be in the form of matrix which is insoluble and porous, and preferably absorbent, relative to the liquid medium to be tested. Such matrix may be in the form of bibulous papers; polymeric films, membranes, fleeces, or blocks; gels; and so forth. In such a form, the device would provide a convenient means for contacting the liquid medium to be tested, for carrying out the specific binding reaction and/or the monitoring reaction, and for observing the resulting response.

The liquid medium to be tested may be a naturally occurring or artificially formed liquid suspected of containing the ligand, and usually is a biological fluid Or a liquid resulting from a dilution or other treatment thereof. Biological fluids which may be assayed following the present method include serum, plasma, urine, and amniotic, cerebral, and spinal fluids. .Other materials such as solid matter, for example tissue, or gases may be assayed by reducing them to a liquid form such as by dissolution of the solid or gas in a liquid or by liquid extraction of the solid.

In contrast to the prior art homogenous assay system, biological fluids containing substances which have reactant activity similar or identical to that of the conjugated labeling substance may be assayed for the ligand without background interference. Endogenous background reactant activity can be readily eliminated in several manners. The biological fluid can be treated to selectively destroy the endogenous reactant activity. Such treatment may involve the action of a clearing agent which chemically destroys the endogenous activity followed by treatment to inactivate the destructive action of such clearing agent.

For instance, reactant-degrading enzymes often appear naturally in biological fluids, particularly if the reactant is a coenzyme such as NAD, NADP, or ATP. There are many inhibitors of such coenzyme-degrading enzymes, for example, chelating agents which operate to deprive the enzymes of essential metal ion activators. As a specific example, NADdegrading enzymes are found in normal serum and have suf5 ficient enzymatic activity to remove essentially all endogenous NAD activity from isolated serum within a few hours.

The degrading activity of such enzymes may be effectively inhibited by addition of a chelating agent such as ethylenediamine tetraacetic acid. Elimination of the degrading activity may also be accomplished by adding a specific enzyme inhibitor, for example, ΛΤΡ-degrading enzymes may be inhibited by addition of βγ methylene ATP or αβ methylene ATP.

The present invention will now be illustrated, but is not intended to be limited, by the following Examples. 2544 EXAMPLE 1 Preparation of nicotinamide adenine dinucleotide-biotin conjugate.

A. Nicotinamide 6-(2-aminoethylamino) purine dinucleotide. 5 Two (2) grams of nicotinamide adenine dinucleotide t (NAD) were dissolved in 10 ml of water and 0.6 ml of ethyleneimine was added dropwise, the pH being maintained below 7 by the addition of 1 M perchloric acid. When addition of ethyleneimine was complete, the pH was adjusted to 4.5 and the reaction was incubated at 20-25°C. At 24 hour intervals 0.6 ml of ethyleneimine was added and the pH readjusted to 4.5. After 96 hours, the solution was poured into 10 volumes of acetone at -10°C. The oil which formed 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.S with 1 N sodium hydroxide, and 1 gram of sodium bicarbonate was added. Nitrogen was bubbled through the solution for from 4 to 5 minutes and 1 gram of sodium hydrosulfite was added, The flask was sealed tightly and allowed to stand at room temperature for 45 minutes. The solution was then oxygenated for 15 minutes and adjusted to pH 11.3 with sodium hydroxide.

The solution was heated at 75°C for 1 hour. Then the reaction mixture was cooled to room temperature and 0.6 grams of tris-(hydroxymethyl)-aminomethane was added, followed by 5 N hydrochloric acid to adjust the pH to 7.5. To the resulting solution was added 1000 International units of alcohol dehydrogenase and I ml of acetaldehyde. The decreasing optical density of the reaction mixture was monitored at 340 nm. and when no further decrease was observed, the pH was 42S44 adjusted to 3.5. The solution was poured into 10 volumes of acetone at -10°C. The oil which formed was separated and washed with ether, after which it was dissolved in 10 to 15 ml of water.

The resulting solution was introduced into a 2.5x90 cm column of Sephadex GIO, available from Pharmacia AB, Uppsala, Sweden, equilabratcd with water. Fractions of 12 ml volume were collected. The wavelength of maximum optical absorption in the ultraviolet region and the optical density at such wavelength were determined for each fraction. Also, tlie optical density at 340 nm of each fraction after reduction with alcohol dehydrogenase was determined. The fractions which had an optical absorption maximum at 264 nm and had a ratio of optical density at 340 nm to that at 264 nm greater than 0.05 were pooled. The pooled material was concentrated to from 15 to 20 ml on a rotary evaporator and passed through a 2.5x28 cm column of Dowex 1-X8, available from Bio-Rad Laboratories, Richmond, California, equilabrated with water. Additional water was added to wash the pooled material through the column, and 10 ml fractions were collected. The fractions which had an optical absorption maximum at 264 nm and had a ratio of optical density at 340 nm to that at 264 nm greater than 0.1 were pooled.

The pooled material was passed through a 5x45 cm column of Dowex 50-X2, available from Bio-Rad Laboratories, Richmond, California, equilibrated with water. Additional water was added to wash the pooled material through the column and 20 ml fractions were collected. The fractions -. 42S44 which had an optical absorption maximum at 264 nm and had a ratio of optical density at 340 nm to that at 264 nm greater than 0.18 were pooled. The pooled material was concentrated 1 to from 4 to 5 ml and purified by electrophoresis as follows. 5 The concentrated material was applied to a sheet of Whatman 3MM paper, available from Reeve Angel, Clifton, New Jersey, in a 1 to 2 cm wide strip perpendicular to the direction of current flow. The paper was then wetted with 0.02 M sodium phosphate at pH 6.0. Electrophoresis was conducted according to the Durrum hanging paper method, as described in Soienae 122:829(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 developed after spraying a test strip of the paper with 0.5 M sodium cyanide according to the procedure described in J. Biol. Chem. 191:447(1951). The area containing the desired derivative was cut out of the paper and extracted with three (3) 50 ml volumes of water. The resulting extracts containing nicotinamide 6 (2 aminoethylamino) purine dinucleotide were pooled, concentrated to from 3 to 4 ml, and stored at -20°C.

B. Nicotinamide adenine dinucleotide-biotin conjugate.

A 16 mg quantity 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 was added to aid dissolution of the biotin A 240 mg quantity of 1-cyclohexyl-3-(2-morpho1inoethyl)-carbodiimide metho-p-tolulene sulfonate was added to the resulting solution and brought into solution by dropwise addition of 0.1 N hydrochloric acid. The reaction mixture was allowed to incubate at room temperature for 5 hours and was then poured into 10 ml of acetone at -10°C.

The oil which formed was separated, washed twice with from 5 to 10 ml of ether and dissolved in from 1 to 2 ml of water.

The resulting material was purified by electrophoresis on paper as in Example 1. Two fluorescent bands appeared after spraying with sodium cyanide, one having migrated toward the cathode and the other toward the anode. The latter band, which contained the NAD-blotin conjugate, was eluted with water and stored at 2()°(.'.

EXAMPLE 2 Homogenous specific binding assay; effect of avidin and biotin on the enzymatic cycling rate of NAD and NAD-biotin conjugates.

The cycling reaction system used in thi'; Example was based on the following reactions: (a) NAD-ligand + lactate aehy-^^^ase— NADH-ligand + pyruvate (b) NADH-ligand + thiazolyl blue (oxidized) diaphorase^ NAD-ligand + thiazolyl blue (reduced) Eight specific binding reaction mixtures were prepared, each having a total volume of 0.5 ml and containing 0.12 M N,N bis-2-hydroxyethylglycine hydrochloride buffer at pH 7.8 and respectively containing the concentrations and activities indicated in Table 1 of NAD, NAD-biotin conjugate prepared as in Example 1, biotin, and avidin, which latter has an affinity for binding with biotin. One (1) unit of avidin activity is that quantity of avidin capable of binding 1 pg of biotin. The reaction mixtures were incubated at room temperature for from 2 to 3 hours. Each reaction mixture was contacted with an aqueous enzyme/substrate mixture by the addition of 0.1 ml of 1 M lithium lactate, 0.05 ml of 10 mM thiazolyl blue in its oxidized form, and a sufficient quantity of 0.12 M N,N bis-2-hydroxyethylglycine hydrochloride buffer at pH 7.8 containing 0.38 International units Of bovine heart lactic dehydrogenase, and 1.5 International units of porcine heart diaphorase to give a total reaction volume of 1 ml. The relative rate of production of the reduced form of thiazolyl blue was then determined in each of the reaction mixtures by measuring the total change in the optical density in each thereof at 570 nm during a 24 minute period within the first hour after the addition of the enzyme/substrate mixture. The entire procedure was performed in duplicate and the averaged results appear in Table I.

TABLE β «Η >» (ϋ p 1/) ·γΗ fl (Λ Ο fl fl rt Ο fl υ ό α ο • γΙΗΝ rt ιη ο Ο ' οο·κ fl -ρ rt ft fl • Η Ρ Ο fl fl •rH τ) ΙΛ Ρ •Η fl ιΗ r* rt Ρ Ο fl Ο fl •rt Ρ rt fl ί-ι -w P P fl O O z~\ υ 5S fl fl'P fl rt Ρ Ρ O flOP Φ *rt fl υ β oc fl » Ο Ω Ο < Ρ ο fl Ο r-i •rt ΞΞ Ρ fl fl * rt Ρ Ω fl < Ο V3 υ β ο U υ fl ο rt ΓΗ Κ) Ο ο ο »—ι σ ο στ m r- ο ο ο Ο Ό tn tn m Ο ΓΗ rrt Ο στ τ—ί \Ο ο ο ο ο ο o β tn o \O ι tn ο ο Ό Ό ι tn tn ο ο ΓΗ I I ΓΗ ΓΗ ΓΗ γη tn rt· ιη t·- CO Reactions 1, 4, and 8 were controls and show that in the absence of NAD and the NAD-biotin conjugate essentially no cycling occurred. The results of reactions 2 and 3 demonstrate that the NAD-biotin conjugate has a significant amount of coenzyme activity relative to native 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 thiazolyl blue (reduced form) where the NAD present is conjugated with biotin. By comparing the results of reactions 6 and 7 it can be seen that the presence of free biotin reduces the amount of inhibition of thiazolyl blue (reduced form) formation in proportion to the concentration of biotin in the reaction mixture.

It was thus demonstrated in this Example that the activity of the NAD in the NAD-biotin conjugate relative to the cycling reaction system was decreased in the presence of avidin and that the magnitude of such decrease in activity was reduced by the additional presence of biotin. .42544 EXAMPLE 3 Homogenous competitive binding-bioluminescence assay; ci'('. o v,-nying levels ol biotin on the peak light intensity produced.

The bioluminescence reaction system used in this Example was based on the following reactions: (c) NAD-ligand + ethanol NADH-ligand + acetaldehyde (d) NADH-ligand + FMN* + abhySen^ NAD-ligand + FMNH2 (e) FMNIIZ + long-chain aldehyde + 02 luciferase^ FMN + long-chain acid + il20 + h? *flavin mononucleotide A light-generating solution for carrying out reactions 15 (d) and (e) was prepared as follows. A reagent mixture was prepared containing 0.13 M phosphate buffer at pH 7.0, 0.67 wt£ bovine serum albumin, 15.7 μΜ flavin mononucleotide (FMN), and 13.3 mM sodium acetate, and this mixture was stored in the dark at -20°C. An emulsion of 5 pi of dode20 canal in 5 ml of water was prepared the day the lightgenerating solution was to be used. Lypohilized luciferase extracted from rhobobaoterium J'ieheri (enzyme available from Worthington Biochemical Corp., Freehold, New Jersey) wa:. added to 0.013 M phosphate buffer at pH 7.3 to a concentra25 tion of 20 mg/ml. After 30 minutes the resulting suspension - 46 42544 was centrifuged at 1500xg for 10 minutes and the pellet was discarded. The light-generating solution was then prepared within 5 minutes of use by combining 75 μΐ of the reagent mixture, 5 μΐ of the dodecanal emulsion, and 20 μΐ of the luciferase solution.

To detect the light produced by reaction (e) a photometer was constructed consisting of a photodetector and a 6 x 50 mm cuvette mounted within a light integrating sphere such that light generated in the cuvette was reflected onto the photodetector. The electronic signal produced by the photodetector was passed to a strip chart recorder. The peak light intensity, as the term is used herein, was measured from the recorder trace and assigned arbitrary units based on the chart paper divisions.

Seven specific binding reaction mixtures were prepared, each having a total volume of 0.2 ml and each containing 0.1 M tris-(hydrozymethyl)-aminomethane hydrochloride buffer at pH 8.0, 0.6 M ethanol, 0.01 M semicarbazide hydrochloride, 343 nM NAD-biotin conjugate prepared as in 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.e. nos. 2 through 7 in Table 2, in the concentrations indicated- in said Table.

The reaction mixtures were incubated at room tempera25 ture for about 30 minutes. A 10 μΐ volume of each reaction mixture was injected into a separate cuvette mounted in the photometer described above and containing 100 pi of a light-yielding solution prepared in the manner described above and pre-incubated at 28°C for from 2 to 3 minutes. The entire procedure was run in duplicate, and the averaged results appear in Table 2. TABLE 2 reaction mixture concentration of biotin (nM) average peak light intensity 1 0 36 2 25 44 3 50 57 4 100 79 5 150 90 fl 200 97 7 300 104 It was thus demonstrated in this Example that the magnitude of the peak light intensity produced by the bioluminescence reaction system, and thus the activity of the NAD in the NAD-biotin conjugate, was a direct function of the amount of biotin present in the specific binding reaction mixture. The present invention therefore provides a test composition and method' for quantitatively determining the presence of the ligand biotin in a liquid medium using a competitive binding-bioluminescence assay technique. 1-ΧΛΜΡΙ,Π -1 Preparation of 2,4-dinitroplienyl-fluorescein conjugate. Fluoresccin-3', -[6-(2,4-dinitroanilino)hexanoate].

The synthesis basically involved the reaction of the acid chloride of 6-(2,4-dinitroanilino)hexanoic acid with the disodium salt of fluorescein. 6-(2,4-dinitroanilino) hexanoic acid was prepared by the method described in Biochem, J. ^2:287-94(1948).

A solution of I.S g (5 mmol) of 6-(2,4-dinitroanilino) hexanoic acid was converted to the acid chloride by reaction with 10 ml of warm thionyl chloride foi- J 5 min followed by cooling and dilution With 20 ml of hexane. The solid acid chloride which formed was collected by filtration and after thorough drying was added to 600 mg of the disodium salt of fluorescein in 10 ml of dry acetone. After 5 hr at reflux, the reaction was quenched by the addition of 2 ml water and 5 ml acetone. After 30 min at 25°C, the mixture was concentrated to dryness and the residue partitioned between ethyl acetate and aqueous sodium bicarbonate solution. The organic phase was separated and washed with 1% aqueous sulfuric acid, dried over anhydrous magnesium sulfate and evaporated. The red oil. was chromatographed on 60 g of silica gel 60, available from E. Merck, Darmstadt, Germany, with 20% (v/v) acetone in carbon tetrachloride as eluant.

The 1.2 g of impure bis-ester was rechromatographed on 60 g of silica gel using 10% (v/v) acetone in carbon tetrachloride. Appropriate fractions were combined and evaporated to yield 180 mg of a yellow, glassy solid.

Calculated for C44I138N()O1 r_: C, 59.33; 11,4.30; N.9.43 Found: C,l>0.92; 11,4.35; N,6.65.

The infrared spectrum displayed the expected ester carbonyl stretching absorption at 1765 cm 5 EXAMPLE 5 Homogenous specific binding assays for derivatives of 2,4 dinitrophenyl and antibody thereto employing an enzyme substrate (modified fluorescein) as labeling substance.

The specific binding assay systems used in this Example was based on the reaction shown in Diagram 1.

A. Homogenous direct binding-fluorescent assay for antibody to 2,4-dinitrophcnyl; effect of various levels of antibody on the reaction rate.

Seven specific binding reaction mixtures were prepared 15 and analyzed. For each reaction mixture, 20 pi of 1 pM 2,4-dini t ropheny1·fluoresceιn conj ligate (prepared accord ing to Example 4) in dimethylsulfoxide was combined with a volume of antiserum to 2,4-dinitrophenyl as indicated in Table 3 and with a sufficient volume of 0.1 M bis-hydroxyethy1glycine hydrochloride buffer at pH 7.0 to make a total volume of 2.0 ml. The background rate of hydrolysis of the ester linkage in the conjugate was determined for three minutes for each reaction mixture by determining tlie rate of increase of fluorescence intensity at 510 nm DIAGRAM 1 Ο esterase HZO, pll 7.0 2,4-dinitrophenyl-fluorescein conjugate + other products (maximum fluorescence at 510 nm) =—(ch2)5 Nil NO, with the i'luorometer set for excitation at 470 nm. Λ 10 μΐ volume of 0.1 M bis-hydroxyethylglycine hydrochloride buffer at pH 7.0 containing 0.54 units of Type 1 esterase (1:.C. No. 3.1.1.1, obtained from Signa Chemical Co., St. Louis, Missouri) was then added to each reaction mixture. The resulting overall reaction rate was measured in the same manner as the background hydrolysis rate. The results appear in Table 3. 10 react ion TABLE 3 amount of ant ι serum background hydrolysis overall react ion ill ι x t ii re (nl) rate i a t e --------------- ------—----- 1 0 0.02 2.(.8 2 5 0.07 2.48 3 10 0.11 1.91 IS 4 20 0.17 1.08 5 30 0.21 0.63 6 40 0.22 0.45 7 60 0.26 0.30 It was demonstrated in this part of the Example that 20 the net reaction rate of the hydrolysis reaction was an inverse function of tho amount of antibody to the ligand, 2,4 -dinit roplicnyl , present in the specific bindi ng reaction mixture. It was likewise demonstrated that the reaction rate of the background hydrolysis reaction was a direct function of the amount of antibody present in the specific binding reaction mixture. The present invention therefore provides a test composition and method for determining the presence of the ligand antibody to 2,4-dinitrophenyl in a liquid medium using a direct binding-fluorescent assay technique.

B. Homogenous competitive binding-fluorescent assay for 5 derivatives of 2,4-dinitrophenyl; effect of various levels of 2,4-dinitrophenyl-B-alanine on the reaction rate.

Ten specific binding reaction mixtures were prepared, each having a total volume of 2.0 ml and each containing 0.1 M bis-hydroxyethylglycine hydrochloride buffer at pH 7.0 and 2,4-dinitrophenyl-B-alanine, prepared according to the method described in J. Amer. Chem. Soe. ?β:1328(1954), at the concentrations indicated in Table 4. To nine of the ten reaction mixtures, i.e. nos. 2 through 10 in Table 4, was added an amount of antiserum to 2,4-dinitrophenyl suf15 ficient to inhibit the rate of the esterase-catalyzed reaction in the other mixture, i.e. no. 1, by 60 percent. After mixing, 20 μΐ of 1 μΜ 2,4-dinitrophenyl-fluorescein conjugate (prepared as in Example 4) in dimethylsulfoxide Was added to each reaction mixture. A 10 μΐ volume of 0.1 M bis-hydroxyethylglycine hydrochloride buffer at pH 7.0 containing 0.54 units of Type I esterase (E.C. No. 3.1.1.1, obtained from Sigma Chemical Co., St. Louis, Missouri) was then added to each reaction mixture. The resulting reaction rate was measured as in Part A of this Example. The percen25 tage value of the rate of reactions nos. 2 through 10 to that of reaction no. 1 (no antibody present) was calculated. The results appear in Table 4. 43844 TABLE 4 concentration of percent of react ion 2,4 dinitropheny1 reaction rate of reac mixture -[{-alanine (nM) __rate __no. 1 1 0 2.78 2 0 1.04 37 3 5 1.01 3 b 4 10 1.04 37 5 20 1.24 45 6 30 1.51 54 7 50 1.54 50 8 75 1.80 65 9 100 1.85 6 7 10 150 2.33 84 It was demonstrated in this part of the Example that the reaction rate of tlie hydrolysis reaction was a direct function of the amount of 2,4-dinitrophenyl-B-alanine in the reaction mixture. The present invention therefore provi.de> a test composition and method for determining the presence of ligands such as derivatives of 2,4 dinitrophenyl in a liquid medium using a competitive binding-fluorescent assay technique.

C. Homogenous competitive binding-spectrophotometric assay for derivatives of 2,4-dinitrophenyl; effect of various levels of 2,4-dinitrophenyl-B-alanine on the reaction rate Eight specific binding reaction mixtures were prepared, each having a total volume of 1.0 ml and each containing 0.1 M tris-(hydroxymethyl)-aminomethane hydrochloride buffer at pH 7.0 and 2,4-dinitrophenyl-8-alanine at the concentrations indicated in Table 5. To seven of the eight reaction mixtures, i.e. nos 2 through 8 in Table 5, was added an amount of antiserum to 2,4-dinitrophenyl sufficient to inhibit the rate of the esterase-catalyzed reaction in the other mixture, i.e. no. 1, by 82 percent. After mixing, 10 pi of 0.1 mM 2,4-dinitrophenyl-fluorescein conjugate (prepared as in Example 4) in dimethylsulfoxide was added to each reaction mixture. A 20 pi volume of 0.1 M tris-(hydroxymethyl) -aminomethane hydrochloride buffer at pH 7.0 containing 2.16 International units of Type I esterase (E.C. No. 3.1.1.1, obtained from Sigma ι Chemical Co., St. Louis, Missouri) was then added to each reaction mixture. The change in absorbance 15 of each reaction mixture at 489 nm per minute was recorded with a Gilford 2000 spectrophotometer . The results appear in Table 5. TABLE 5 20 reaction mixture concentration of 2,4-dinitrophenyl -8-alanine (pM) rate of change of absorbance 1 0 0,0261 2 0 0.0047 3 1.25 0.0118 25 4 2.5 0.0131 S 5.0 0.0185 6 7.5 0.0202 7' 10.0 0.0192 8 12.5 0.0223 It was demonstrated in this part of the Example that tho reaction rate was a direct function of the amount of 2.4 d ini t ropheny 1 - fi-alanine in the reaction mixture. The present invention therefore provides a test composition and method for determining the presence of ligands sueli as derivatives of 2,4-dinitrophenyl in a liquid medium using a competitive binding-spectrophotometric assay technique.

IJ. Homogenous competitive binding-fluorescent assay for derivatives of 2,4-dinitrophenyl; use of non-enzymatic monitoring reaction.

The specific binding assay system used in this part was tlie same as shown in Diagram 1 except that no esterase was used to catalyze the hydrolysis of the ester linkage m the Conjugate.

Eight specific binding reaction mixtures were prepared, each having a total volume of 2 ml and each containing 0.1 M tris -(hydroxymethyl)-aminomethane hydrochloride buffer at pll 7.5 and 2,4-dinitrophenyl-0-alanine at the concentrations indicated in Table 0. To each reaction mixture was added 50 pi of antiserum to 2,4-dinitrophenyl. After mixing, 20 pi of 2 pM 2,4-dinitrophenyl-fluorescein conjugate (prepared as in Example 14) in dimethylsulfoxide was added to each reaction mixture and the resulting reaction rate was measured as in Part A of this Example. The results appear in Table 6. 4254 4 TABLE 6 concentration of reaction 2,4-dinitrophenyl reaction mixture -g-alanine (nM) rate IQ 0.96 2 12.5 0.94 3 31.2 0.84 4 62.5 0.78 5 94.0 0.70 6 125 0.59 7 187 0.57 8 250 0.53 It was demonstrated in this part of the Example that the background hydrolysis rate, in the absence of esterase, was an inverse function of the amount of 2,4-dinitrophenyl -g-alanine in the reaction mixture. The present invention therefore provides a test composition and method for determining the presence of ligands such as derivatives of 2,4-dinitrophenyl in a liquid medium using a competitive binding-fluorescent technique wherein the binding partner, upon becoming bound to the ligand in the conjugate, participates in the monitoring reaction.

EXAMPLE 6 Preparation of 2,4-dinitrophenyl-ATP conjugate (625 position derivative), N®-[2-(2,4-dinitrophenyl)aminoethyl] adenosine-5' -triphosphate. Α. Νΰ-(2-aminoethyl) adenosine-5'-monophosphate.

Two g (7 mmol) 6-chloropurine riboside (available from Sigma Chemical Co., St. Louis, Missouri) was stirred with 17 ml triethylphosphate and was reacted with phosphoryl chloride in the presence of water as described in Chem. Scrip. /9:105-70 (1072), After hydrolysis of the phosphodichloridate, 0.5 ml ethylenediamine (140 mmol) was added and allowed to react at room temperature for 3 hr. The reaction mixture was diluted to 4 liters with water and adjusted to pH 12 with sodium hydroxide. This solution was passed through a 5x30 cm column of Dowex 1x8 (available from Bio-Rad Laboratories, Richmond, California) in the acetate form. Then the column was washed with 3 liters 0.01 M ammonium chloride and th$ chromatogram was developed with a linear gradient generated with 3 liters water and 3 liters 1 M acetic acid. An isolated peak of uv absorbing material eluted between 1800 ml and 2050 ml of the gradient was concentrated to about 25 ml under vacuum. While this solution stood at 7°C overnight, white crystals formed and these were collected and dried to give 65 percent yield of the product. A sample was recrystallized from hot water for analysis. Calculated for C12 i11n6°71’-2II20: c>33.8; 11,5.45; N,19.7. Found: C,34.3; 11,5.22; N,19.7. Separate thin-layer chromatograms developed with two solvent systems, the first consisting of 4 parts 0.5 M ammonium acetate to 1 part ethanol and the second consisting of 3 parts isobutyric acid to 5 parts 1 M ammonium hydroxide, each showed one component which quenched fluorescence and reacted with ninhydrin. The compound in 0.1 N hydrochloric acid had an absorption maximum at 264 nm and the millimolar extinction coefficient was 17.7 which spectral properties are characteristic of N^-alkylated adenosine derivatives. Β. N6-[2-(Z,4-dinitrophenyl)aminoethyl] adenosine-5' -monophosphate.

Two hundred fifty mg N®-(2-aminoethyl)adenosine-5' -monophosphate (0.65 mmol) from Part A of this Example was dissolved in 20 ml water at pH 8. Then 168 mg sodium bicarbonate was added, followed by 0.2 ml l-fluoro-2,4-dinitrobenzene (1.58 mmol dissolved in 2 ml ethanol). The reaction mixture was stirred in the dark at room temperature for 4 hr and then an additional 0.1 ml l-fluoro-2,4-dinitrobenezene in ml ethanol was added. After the reaction mixture had stirred overnight, it was adjusted to pH 2.0 with hydrochloric acid and was poured into 200 ml ethanol. The precipitate which formed was dissolved in 200 ml water and this solution was adjusted to pH 8.0 with sodium hydroxide and chromatographed on a 2.5x30 cm column of DEAE-cellulose in the bicarbonate form (available from Reeve Angel, Clifton, New Jersey). The chromatogram was developed with a linear gradient generated with 1.5 liters water and 1.5 liters 0.7 M ammonium bicarbon25 ate. A major peak of yellow material which absorbed uv light was eluted between 1Z0O and 1500 ml of the gradient. Ammonium bicarbonate was removed by repeated evaporation to dryness to give 40 percent yield of the desired product. This product migrated as one yellow spot on thin-layer chromatograms developed with the same two solvents mentioned in Part Λ of this Example and on epichlorohydrintriethanolamine anionexchange paper developed with 0.25 M sodium acetate-acetic acid buffer, pH 5.0. In 0.02 H hydrochloric acid the product had optical absorption maxima of 264 nm and 363 nm with millimolar extinction coefficients of 21.8 and 14.2, respectively.

C. 2,4-dinitrophenyl-ATP conjugate. Νϋ-[2-(2,4-dinitrophenyl)aminoethylJ adenosine-5' monophosphate (from Part B of this Example) (0.3 mmole) was converted to the pyridinium salt by chromatography on a 1.5x20 cm column of Dowex 50x2 in the pyridinium form (available from Bio-Rad Laboratories, Richmond, California). The yellow effluent was concentrated to dryness and 15 ml dimethylformamide and 0.3 mmol tri-n-butylamine were added. This mixture was evaporated to dryness and tlie residue was dried further by repeated evaporation. The monophosphate intermediate was then converted to the triphosphate form using the method disclosed in J. Amer. Chem. Soc. 87:1785-8(1965).

The reaction products which were soluble in dimethylformamide were added to 250 ml water which was then adjusted to pH 8.0. This solution was passed into a 2.5x58 cm column of DEAE-cellulose in the bicarbonate form and the chromatogram was developed with a linear gradient generated with 3 liters water and 3 liters 0.5 M ammonium carbonate. The first eluted peak of yellow material was identified as the diphosphate derivative. A second peak of yellow material, which eluted between 4.15 and 4.4 liters of the gradient, was evaporated to dryness to give 20 percent yield of the desired conjugate which, by analysis, was found to contain-3.0 residues of phosphate per ribose residue.

EXAMPLE 7 Preparation of 2,4-dinitrophenyl-ATP conjugate (8positxon derivative). -[2 -(2,4-dinitrophenyl)aminoethyl]aminoadenosine-5' -triphosphate.

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

A reaction mixture consisting of 2,2 mmol 8-bromoadenosine -5*-monophosphate, prepared according to the method described in Arah. Bioahem. Biophye. 263:561-9(1974), 66 mmol ethylenediamine and 25 ml water was heated in an oil bath at 140° for 2 hr. The cooled mixture was adjusted to pH 11.5 with sodium hydroxide and passed into a 2.5x55 cm column of Dowex 1x8 (200-400 mesh, bicarbonate form). The column was washed with 300 ml water and then with a linear gradient generated with 3 liters water and 3 liters 0.5 M ammonium bicarbonate. The absorbance of the effluent at 254 nm was monitored and a major peak of absorbing material eluted between 4.6 and 5.8 liters of the gradient.

Ammonium bicarbonate was removed by repeated evaporation (five times, 20 to 30 ml water each time) to dryness under vacuum and the final residue was dissolved in 20 ml water by addition of 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. Crystals which formed were collected, dissolved at pH 8.0 and recrystallized at pll 5.0. The yield of the desired intermediate was 27 percent, tin examination by thin layer chromatography in a solvent con sisting of 4 parts O.S M ammonium acetate to 1 part ethanol, the product migrated as one ninhydrin positive spot which quenched fluorescence. The optical absorption maxiumum In 0.02 N hydrochloric acid was 275 nm and the millimolar extinction coefficient was 17.5, which spectral properties are characteristic of alkylated 8·aminoadenosine derivatives. Calculated for ΰ12Η2θΝ7Ο7Ρ·Η2Ο: C.34.0; H,5.33; N.23.2.

Found: C,34.1; 11,5.28; N.23.9, B. 8-[2 (2,4-dinitrophenyl)aminoethyi]aminoadenos ine -5'-monophosphate. 8-(2 ami noethyl1 aminoadenosine-5'-monophosphate from Part A of this Example (0.64 mmol) was dissolved in 20 ml water by addition of sodium hydroxide to pH 8.0. Then Ki8 mg sodium bicarbonate was added, followed by 0.2 ini i-fluoro-4-dinitrobenzene (1.58 mmol dissolved in 2 ml ethanol). Tlie reaction was stirred for 18 hr at room temperature and then 0.1 ml I-fluoro-2,4-dinitrobenzene in 1 ml ethanol was added. After stirring for an additional 4 hrs, the mixture was adjusted to pH 2.0 with hydrochloric acid and poured into 200 ml cold acetone (-10°C). The yellow precipitate which formed was collected by filtration, dissolved in 200 ml water and passed into a 2.5x45 cm column of DEAE-cellulose in the bicarbonate form. The chromatogram 43544 was developed with a linear salt gradient generated with 2 liters water and 2 liters 0.7 M ammonium carbonate. A peak of yellow material with an absorption maximum at 275 nm eluted between 2 and 3 liters of the gradient. Ammonium bi5 carbonate was removed from this material by evaporation under vacuum and the yield of the desired intermediate was 37 percent. Optical absorption maxima measured in 0.02 N hydrochloric acid occurred at 275 and 363 nm and the millimolar extinction coefficients were 21.8 and 15.5, respec10 tively. Further analyses showed 1.07 phosphate residue per ribose residue.

C. 2,4-dinitrophenyl-ATP conjugate.

The monophosphate intermediate (0.5 mmol) was converted to the tri-n-butylammonium salt by addition of 0,8 mmol tri15 n-butylamine. The mixture was dried by repeated evaporation from dry dimethylformamide (four times, 10-15 ml each). The final residue, dissolved in 1 ml dimethylformamide, was mixed with 2.0 mmol carbonyldiimidazole also in 1 ml dimethylformamide and was allowed to react at room temperature for 4 hr. The excess carbonyldiimidazole was destroyed by reaction with 15 μΐ methanol for 30 min. Finally, 3 mmol trin-butylammonium pyrophosphate in 4 ml dimethylformamide was added and allowed to react for 20 hr. The solid residue which formed was separated by centrifugation and washed twice with 5 ml portions of dimethylformamide. The combined supernatants were added to 200 ml water, which was then adjusted to pH 8 and chromatographed on a 2.5x25 cm column of DEAE-cellulose in the bicarbonate form. The chromatogram was developed with a linear gradient generated with 2 liters water and 2 liters 0.5 M ammonium bicarbonate. A peak of yellow material with an optical absorption maxima at 275 and 303 nm was eluted between 2.0 and 2.9 liters of the gradient. The ammonium bicarbonate was removed by evaporation to give a 22 percent yield of the desired conjugate. Results of analyses indicated that this product contained 3.2 residues of phosphate per residue of ribose.

EXAMPLE 8 Homogenous competitive binding-bioluminescent assay for derivatives of 2,4-dinitrohpenyl; use of ΛΤΡ as labeling substance.

The bioluminescence reaction system used in this Example was based on the following reaction: ATP-ligand + reduced lucifcrin —ferase AMP-ligand + oxidized luciferin + hv A. Assay using 6-position derivative of ATP.

Three specific binding reaction mixtures were prepared, each having a total volume of 100 pi and each containing 10 mM tris-(hydroxymethyl)-aminomethane hydrochloride buffer at pH 7.4, 10 mM ethylenediaminetetraacetic acid, 20 pi antiserum to 2,4-dinitrophenyl, 512 nM 2,4-dinitrophenyl-ATP conjugate (6-position derivative, prepared according to Example 6), and 2,4-dinitrophenyl-B-alanine at the concentrations indicated in Table 7. After incubation at 25°C for 2 hours, duplicate 10 pi aliquots of each reaction mixture were assayed by injection into a 0.1 ml volume of the above -described light generating solution previously incubated at 25°C for at least 2 minutes and contained in a test tube mounted in a Dupont Model 760 Bioluminescence Photometer (E.I. duPont de Nemours, Willmington, Delaware). The peak light intensity was read from the photometer. The average peak light intensity for each reaction mixture was calculated as well as the relative intensity (100¾ times the ratio of average peak intensity for the sample to that in the absence of antiserum). The results appear in Table 7.

TABLE 7 reaction mixture concentration of 2,4-dinitrophenyl ^β-alanine (pM) average peak light intensity 1 0 7 2 25 14 3 2500 185 B. Assay using 8-position derivative of ATP.

Four specific binding reaction mixtures were prepared, each having a total volume of 100 pi and each containing 20 mM tris-(hydroxymethyl)-aminomethane hydrochloride buffer at pH 7.4, 10 mM ethylenediaminetetraacetic acid, 20 pi antiserum to 2, 4-dinitropheny1, 594 nM 2,4-dinitrophenyl ATP conjugate (8-position derivative, prepared according to Example 7), and 2,4-dinitrophenyl-β-alanine at the concentrations indicated in Table 8. After incubation at 25°C for 2 hours, duplicate 10 pi aliquots of each reaction mixture were assayed as in Part A above and the average peak light intensity was calcu- lated for each. The results appear in Table 8. 10 TABLE 8 react ion mixture concentration of 2,4-dinitrophenyl -β-alanine (pM) average peak light intensity 1 0 18 15 2 25 51 3 250 191 4 2500 190 The results of this Example demonstrate that the labeling substance, ATP, may be derivatized at various positions about its structure in the preparation of a useful conjugate for use in the specific binding assay method of the present invention. 6t> EXAMPLE 9 Preparation of biotin-isoluminol conjugate. 6-(3-Biotinoylamido-2-hydroxypropyiamine)-2,3 -dihydrophthalazine-1,4-dione.

A. 4-(3-chloro-2-hydroxypropylamino)-N-methylphthalimide.

Twenty-five grams (0.142 mole) 4-amino-N-methylphthalamide, prepared according to the method described in J. Chem. Soe. g(>: (1937), and 20.7 g (0.21 mole) l-chloro-2,3-epoxypropane were added to ISO ml 2,2,2-trifluoroethanol and the reaction mix10 ture was heated to reflux with stirring for 48 hrs. Seventy to eighty ml of 2,2,2-trifluoroethanol was removed by distilla tion and a heavy yellow precipitate formed when the remaining solution cooled to room temperature. This precipitate was triturated with ethyl acetate, collected by filtration and dried to give 29.5 g (77% yield) of the desired intermediate m.p. 136-138.5°C. Calculated for C12H13C1N2°3: C.53.64; 11,4.88; N, 10.45. Found: C,53.87; H.4.85; N,10.81.

B. 4-[3-(N-Phthalinido)-2-hydroxypropylamino]-N 20 methyIphthalimide.

The intermediate prepared in Part A (13.5 g, O, 05 mole) and 15.7 g (0.085 mole) potassium phthalimido were heated to reflux with stirring in 150 ml dimethylformamide for 24 hrs. The dimethylformamide was removed and the residue was washed with water and filtered. The yellow filter cake was recrystallized from acetic acid-water to give 12.8 g (67% yield) of product, m.p. 247-248.5°C. Calculated for : C.63.32; H,4.52; N,11.08.

Found: C,63.16; H,4.38; N,10.93.

C. 6-[3-Amino-2-hyilroxypropylamino)-2,3 -di hydrophalaz ine-l,4-dione.

The intermediate from Part B (5.0 g, 13.2 mmole), 1)0 ml absolute ethanol and 35 ml 95¾ hydrazine were refluxed with stirring Cor 4 hrs. The solvent was removed under a vacuum and the resulting solid was dried for 24 hrs under vacuum at 120°C. This material was stirred i'or 1 hr with 70 ml of 0.1 N hydrochloric acid. The insoluble 2,3-dihydroxyphthalazine-1,4-dione was removed by filtration and the filtrate was adjusted to pH 0.5 with saturated sodium bicarbonate. The white precipitate which formed was collected by filtration and dried to give 2.2 g of the product (07¾ yield). After re crystallization from water, the compound decomposed at 273°C. Calculated for C.52.79; 11,5.04; N,22.39. Pound: C.52.73; 11,5.72; N.22.54.

D. Biot in - isoluminol conjugate.

Biotin (0.29 g, 1.2 mmole) and 0.17 ml triethylamine were dissolved in 20 ml dry dimethylformamide under anhydrous conditions and cooled to 10°C. A solution of 0.141 ml ethylchloroformate in 2.80 ml ether was added slowly and the reaction was stirred for 30 min. A precipitate which formed was separated by filtration, A suspension consisting of 000 mg (2.4 mmole) of the intermediate from Part C, 20 ml dry dimethylformamide and 1 ml dry pyridine was added to the filtrate quickly. This mixture was stirred at -10°C for 30 min and then at room temperature overnight. During this period a solution was obtained. The dimethylformamide was removed by distillation at 60°C and 0.10 mm Hg pressure. The oily residue was stirred with 50 ml 0.1 N hydrochloric acid for 1 hr. A white solid which formed was filtered and washed with 0.1 N hydrochloric acid and then water. After drying under a vacuum at room temperature overnight, 0.55 g (97¾ yield) of the product was obtained, m.p. 170-3°C. Calculated for C21ll2gNi)O5S: C,52.92; 11,5.92; N.17.64. Found: C,51.69; H,5.90; N,17.63.

EXAMPLE 10 Homogenous competitive binding-chemiluminescent assay for biotin.

The chemiluminescent reaction system used in this Example was based on the following reaction: biotin-isoluminol + K02 15 biotin-aminophthalate + N2 + hv Sixteen specific binding reaction mixtures were prepared, each having a total volume of 150 pi and each containing 0.1 M tris-(hydroxymethyl)-aminomethane hydrochloride at pH 0.0, nM biotin-luminol conjugate (prepared as in Example 9), biotin at the concentrations indicated in Table 9 and 0.12 units/ml avidin (added last). After incubation at 25°C for 5 minutes, 10 pi of dimethylformamide containing 0.15 M potassium superoxide (K02) (available from Alpha Products, Beverly, Massachusetts) and 0.10 M 1,4,7,10,13,16 -hexaoxyacylcooctadecane (available from Aldrich Chemical Co., Milwaukee, Wisconsin) were injected into each reaction mixture. - 42544 After incubation at 25°C for 2 additional minutes, 10 pi 0.95 mM hydrogen peroxide in 10 mM tris-(hydroxymethyl) -aminoinethane hydrochloride buffer at pH 7.4 was injected into eacli reaction mixture and the peak light intensity produced in each was measured using a Dupont Model 7(>0 Bioluminescence Photometer (li.i. dul’ont de Nemours, Willmington, Delaware).

The results appear in Table 9.

TABLE 9 reaction mixture concentration of biotin (nM) peak light intensity 1 0 38.5 2 13 38.5 3 27 34.3 4 40 36.1 15 5 S3 35.2 6 67 36.2 7 101 34.0 8 133 31.7 9 166 29.1 20 10 200 24.2 11 267 22.8 12 333 20.5 13 400 13.4 14 534 8.6 2S 15 667 8.3 16 800 7.0 It was demonstrated that the magnitude of the peak light intensity produced by the chemiluminescent reaction system was an inverse function of tho amount ol biotin present in the specific binding reaction mixture, 'ihe present invention therefore provides a test composition and method for determining the presence of tlie ligand biotin in a liquid jnedium using a competitive binding-chemiluminescent assay technique which does not employ an enzyme-catalyzed monitoring reaction.

EXAMPLE 11 Preparation of biotin-umbelliferone conjugate. (2-0xo-2-H-l-benzopyran-7-yl)-5-[cis-hexahydro -Z-oxo-lH-thieno-(3,4-d-)-imidazolejvaleric acid ester.

A solution of 300 mg (1.2 mmol) anhydrous biotin in ml dry dimethylformamide was stirred at -10°C under dry nitrogen gas and 0.17 ml (1.2 mmol)dry triethylamine was added. A solution of freshly distilled ethyl chloroformate (0.141 ml in 3 ml of dry ether) was added dropwise. After incubation for 30 min with stirring, the resulting precipi20 tate was filtered under a dry nitrogen atmosphere and cooled Immediately to -10°C. To the filtered residue was added a solution of 197 mg (1.2 mmol) anhydrous 7-hydroxycoumarin in 3 ml dry pyridine and stirred for 1 hour at -10°C fol lowed by 20 hours at 25°C. The solvents were evaporated under high vacuum at 40°C. After cooling, the resulting solid was filtered and recrystallized from methanol to yield the 435 14 desired product (melting point = 216-218°C). Calculated for C19112[)N21’sS: C,48.75; H.4.19; N,7.21.

Found: C,58.4; H,5.12; N,6,86, EXAMPLE 12 Heterogenous competitive binding - Xioluminescence assay for biotin; effect of varying levels of biotin on the peak light intensity produced.

The bioluminescence reaction system used in this Example was based on the reactions shown in Example 3.

A. Preparation of light-generating solution A light-generating solution was prepared as in Example 3.

B. Preparation of insolubilized binding partner.

Avidin, which has a binding affinity for biotin, was insolubilized by being covalently bound to a water insoluble polymer bead as follows. A quantity of Sepharose 4B (available from Pharmacia AB, Uppsala, Sweden) was activated for bonding to avidin using the method of March et al, Analytical Pioahemistry 80:149(1974). Approximately 4 ml of the activated Sepharose 4B was suspended in 8 ml of 0.1 M citrate buffer at pH 7,0. To the suspension was added 6 mg of avidin having an activity of 9.9 units/mg in 3 ml of water. One unit of avidin activity is that quantity of avidin capable of binding 1 ng of biotin. The resulting reaction mixture was stirred for 6 hours at 7QC.

The avidin-bound-Sepharose 4B was then filtered, washed with 100 ml ol' 0.1 M sodium bicarbonate buffer at pll 9.0, 425 44 anil resuspending in 240 mi of 0.1 M tris· (hydroxymethyl) -aminomethane hydrochloride buffer at pll 8.0.

C. Control experiments Nine specific binding ι-eaction mixtures were prepared, each having a total volume of 0.19 ml and each containing 0.1 M tris-(hydroxymethyl)-aminomethane hydrochloride buffer at pll 8.0, 0.6 M ethanol, 0.01 M semicarbazide hydrochloride, and respectively the amounts or concentrations indicated in, Table 10 of NAD, NAD-biotin conjugate, avidin-bound Sepharose 4B suspension (prepared according to Part B of this Example), and Sepharose 4B suspension (formed by .suspending 1 ml of packed Sepharose 41! in 61) ml of 0.1 M tris-(hydroxymethyl)-aminometbane hydrochloride buffer at pll 8.0). The reaction mixtures were shaken gently for 15 minutes at room temperature. Then, 0.22 International units of alcohol dehydrogenase was added to each reaction mixture to initiate the reduction reaction. Semicarbazide combines with the acetaldehyde produced in reaction (c) to form a semicarbazone and thus to drive reaction (c) in tlie desired direction.

Tlie reaction mixtures were shaken again for IS minutes at room temperature. A 10 pi aliquot of the supernatant from eacii reaction mixture was then injected into a separate cuvette mounted in a DuPont Model 760 Bioluminescence Photometer (E.I. duPont de Nemours, Willmington, Delaware) containing 100 pi of the previously prepared light-generating solution which had been pre-incubated for from 2 to i minutes at 25°C. The results appear in Table 10.

TABLE 10 β -ρ tdj-M •η tn •Η fl Φ β fl fl φ ·γΗ Ρ Φ fl tn o O • rt μ fl fl β Φ Π» P« Φ tn cn fl tn rrt Ό fl. fl CQ s—’ flrt O c β Φ o σ» • rH fl O M • rt μ fl T3 rt Φ •rt β P ϊ> CU tn fl Φ fl cn tn Mrt O fl 55 O fl fl • rt •rt rt rt rt o Φ μ •rl rt rt β rt fl M Φ Ω fl υ •1"1 fl X fl o o υ υ ΓΗ στ m Γ· rt Οτ ΙΛ tn rt Ο 00 rt rrt Cl «η rrt rH O « · O CM CM © rrt rrt rrt CM CM \Ο rt rrt «rt ο O I rrt rrt O CM M-i O fl O •rt z—s >: rt fl μ +J Q t rrt rrt fl < f ’ Φ X υ fl o u rrt I I Cl fl o υ co φ μ ch tn rt kO c^. co στ The results of control reactions 1 and 9 show that in the absence of NAD and NAD-biotin conjugate very little light was produced. Reactions 2 and 3 yielded results ί indicating that the light producing reaction occurred when 5 free NAD was added and that such reaction was substantially unaffected by the presence of avidin-bound-Sephprose 4B.

The results of reactions 4,5, and 6 show that the NAD-biotin conjugate was active in the light producing reaction, that the peak light intensity produced increased as more NAD-biotin conjugate was present, and that the presence of avidin-bound-Sepharose 4B inhibited light production. Com parison of the results of reactions 3 and 5 with those of 7 and 8 shows that the light producing reaction was not affected by the presence of plain Sepharose 4B.

IS D. Assay method Five additional specific binding reaction mixtures were prepared, each having a volume of 0.19 ml and each containing 0.1 M tris -(hydroxymethyl)-aminomethane hydrochloride buffer at pH 8.0, 0.6 M ethanol, 0.01 M semicar20 bozide, and respectively the amounts or concentrations indicated in Table 11 of NAD-biotin conjugate, free biotin, and avidin-bound-Sepharose 4B suspension, Each reaction mixture was treated in the same manner as the control reaction mixtures in Part C of this Example, The results appear in Table 11. 4-* >. λ: ff -Ί Ifl CD ty. ff «< ff Φ H u« Tl ff) Ί c Tt 'w—‘ 3 o Φ ff X) tn O O •ff c ff in •ff rt C .ff •ff cu 0- t* 4> tn rt CQ 3 in σ> t σι cn in i ooo CM (M r»j m u « ty H M-t o ff —' o η α +.* nl ff ff H ff +-» Φ O rj .ff ff X) O Iff .

O /. ff ff ff O ff -7 ty ff ff Ο Φ rt -ff ff ff .η rt ff · ofl ff Q 3 Oj <1 ·ι-ι U ty ff ff o o υ u CM cm CM CM CM ff O •rd u rt o ff γμ tn *dt—j r-d rd The results of reactions 11,12, and 13 show that free biotin and NAD-biotin conjugate compete effectively for the binding sites on the insolubilized avidin since the peak light intensity produced was dependent upon the amount of free biotin present. Reactions 10 and 14 gave results indicating that in the absence of insolubilized avidin, the peak light intensity produced was constant for vastly different concentrations of free biotin.

It was thus demonstrated in this Example that the 10 amount of NAD-biotin conjugate in the liquid phase was inversely related to the amount of free biotin present and thus the assay method and means of the present invention are useful in the determination of a ligand in an unknown liquid sample.

EXAMPLE 13 Heterogenous specific binding assays for avidin and biotin employing an enzyme substrate as labeling substance.

The specific binding assay systems used in this Example were based on the following reaction: Λ w:-(CH2 ; esterase . _ H?O, pH 8.0^ biotin-umbelliferone conjugate (max. fluorescence at 448 nm) + + biotin · Λ. Preparation of insolubilized binding partner.

Avidin was insolubilized by being covalently bound to a water insoluble polymer bead as in Part B of Example 12 except that after washing with 100 ml of 0.1 M sodium bicarbonate buffer at pll 9.0, tlie avidin-bound-Sepharose 4B was suspended 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-hydroxyethylgiycine hydrochloride buffer at pll 7.0.

B. Competitive binding assay for biotin; effect of various levels of biotin on the amount of umbel1 iferone liberated.

Eight specific binding reaction mixtures were prepared, each having a total volume of 0.2 ml and each containing 0.1 M bis-hydroxyethylglycine hydrochloride buffer at pH 7.0, 0.3 μΜ biotin-umbelliferone conjugate (prepared as in Example 11), 15 μΐ of the avidin-bound-Sepharose 4B suspension prepared as in Part A of this Example, and biotin in the concentrations indicated in Table 12. The reaction mixtures were allowed to incubate at room temperature with gentle shaking for 20 minutes. Each reaction mixture was centrifuged and a 100 μΐ aliquot of the supernatant was combined with 2 mi of 0.1 M bis-hydroxyethylglycine hydrochloride buffer at pH 8.2 containing 1.08 units of porcine esterase. After a 5 minute incubation at room temperature, the fluorescence intensity produced in each reaction mixture at 448 nm with excitation at 364 nm was measured using an Amico-Bowman spectrophotofluometer. The results appear in Table 12.

TABLE 12 reaction mixture concentration of biotin (uM) fluorescence 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 1.00 0.688 It was thus demonstrated in this Example 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 means of the present method are useful in the determination of a ligand in an unknown liquid sample.

Claims (28)

1. CLAIMS:1. A specific binding assay method for determining a ligand in a liquid medium, which method comprises the steps of: (a) contacting said liquid medium with reagent means comprising 5 a labeled conjugate having a specific binding portion coupled to a labeling portion having a predetermined characteristic, said reagent means and the ligand forming a binding reaction system producing (1) a bound-phase of the labeled conjugate in which the specific binding portion is bound by a specific binding partner thereto and (2) a free-phase of the labeled conjugate in which the specific binding portion is not bound by a specific binding partner thereto, 15 (b) determining said characteristic in either the bound-phase or the free-phase; characterized in that said predetermined characteristic of said labeling portion of the labeled conjugate is a predetermined activity as (1) a substrate in an enzyme-catalyzed reaction 20 system;

2. (2) a reactant in a cyclic reaction system;

3. (3) a reactant in a luminescent reaction system; or (4) a coenzyme in an enzyme-catalyzed reaction system?2. A homogeneous method as in Claim 1 wherein said predetermined 25 characteristic of the labeling portion of the labeled conjugate in said bound-phase is measurably different from that in said free-phase and the amount thereof is a function of the amount of the ligand present in the liquid medium under assay.

4. A method as in Claim 2 wherein said reagent means comprises (1) said labeled conjugate, said specific binding portion thereof being a specific binding analog of said ligand or a moiety which binds specifically with said ligand, and (2) if said specific binding portion 5 is said analog, a specific binding partner of said ligand. • 4. A heterogeneous method as in Claim 1 wherein said predetermined characteristic of the labeling portion of the labeled conjugate in said bound-phase is substantially the same as in said free-phase and the amount thereof is a function of the amount of the ligand present in 10 the liquid medium under assay; and which comprises the additional step between steps (a) and (b) of separating said bound-phase and said free-phase.

5. A method as in Claim 1 wherein said labeling portion has the activity of a nucleotide coenzyme. 15

6. A method as in Claim 1 wherein said labeling portion has the activity of a substance selected from the group consisting of the adenosine phosphates, nicotinamide adenine dinucleotide and reduced forms thereof, and nicotinamide adenine dinucleotide phosphate and reduced forms thereof. 20

7. A method as in Claim 1 wherein said labeling portion has the activity of a substrate in an enzyme-catalyzed reaction and said reaction produces, by enzymatic cleavage of said labeled conjugate, a product which has a property which distinguishes it from said conjugate.

8. A method as in Claim 7 wherein said distinguishing property is 25 a fluorescent property.

9. A method as in Claim 8 wherein said fluorescent product is umbelliferone or a derivative thereof. lfl.

10. A method as in Claim 8 wherein said fluorescent product is fluorescein or a derivative thereof. 42S44

11. A method as activity of luminol, in Claim 1 wherein said labeling portion has the isoluminol, or a derivative thereof.

12. A method as in Claim 1 wherein said labeling portion exhibits a predetermined activity as a reactant in a cyclic chemical reaction and 5 wherein said cyclic chemical reaction is autocatalytic.

13. A method as in Claim 1 wherein said labeling portion exhibits a predetermined activity as a reactant in a cyclic chemical reaction and wherein said labeling portion has the activity of a material which is cycled in said cyclic reaction. io

14. Reagent means for use in determining a ligand in a liquid medium, which means comprises a labeled conjugate having a specific binding portion coupled to a labeling portion having a predetermined characteristic, and which means and the ligand form a binding reaction system producing a bound-phase and a free-phase of the labeled conjugate, characterized in that said predetermined characteristic of said labeling portion of the labeled conjugate is a predetermined activity as (1) a substrate in an enzyme-catalyzed reaction system, (2) a reactant in a cyclic reaction system, (3) a reactant in a luminescent reaction system, or (4) a coenzyme in an enzyme-catalyzed reaction system.

15. Homogeneous reagent means as in Claim 14 wherein said predetermined characteristic of the labeling portion of the labeled conjugate in said bound-phase is measurably different from that in said free-phase and the amount thereof is a function of the amount of the ligand present in the 25 liquid medium under assay.

16. Means as in Claim 15 which comprises (1) said labeled conjugate, said specific binding portion thereof being a specific binding analog of said ligand, and (2) a specific binding partner of said ligand.

17. Heterogeneous reagent means as in Claim 14 wherein said predetermined characteristic of the labeling portion of the labeled conjugate in said bound-phase is substantially the same as in said free-phase and the amount thereof is a function of the amount of the 5 ligand present in the liquid medium under assay.

18. Means as in Claim 14 wherein said labeling portion has the activity of a nucleotide coenzyme.

19. Means as in Claim 14 wherein labeling portion has the activity of a substance selected from the group consisting of the adenosine 10 phosphates, nicotinamide adenine dinucleotide and reduced forms thereof, and nicotinamide adenine dinucleotide phosphate and reduced forms thereof.

20. Means as in Claim 14 wherein said labeling portion has the activity of a substrate in an enzyme-catalyzed reaction and said chemical 15 reaction produces, by enzymatic cleavage of said labeled conjugate, a product which has a property which distinguishes it from said conjugate.

21. Means as in Claim.20 wherein said distinguishing property is a fluorescent property.

22. Means as in Claim 21 wherein said fluorescent product is 20 umbelliferone or a derivative thereof.

23. Means as in Claim 21 wherein said fluorescent product is fluorescein or a derivative thereof,

24. Means as in Claim 14 wherein said labeling portion has the activity of luminol, isoluminol, or a derivative thereof. ST3

25. Means as in Claim 14 wherein said labeling portion exhibits a predetermined activity as a reactant in a cyclic chemical reaction and wherein said cyclic chemical reaction is autocatalytic. '

26. Means as in Claim 14 wherein said labeling portion exhibits a 5 predetermined activity as a reactant in a cyclic chemical reaction and wherein said labeling portion has the activity of a material which is cycled in said cyclic reaction.

27. Means as in Claim 14 incorporated with a carrier matrix.

28. Means as in Claim 27 wherein said matrix is absorbent relative 10 to said liquid medium.