FI68915C - SPECIFIC BINDNINGSANALYSMETOD OCH REAGENS FOER ANVAENDNING DAERVID - Google Patents

Description

! 68915

Specific binding assay method and reagent

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

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

British Patent 1,392,403 and French Patent 2,201,299 describe a heterogeneous specific binding assay, 6891 5 2 in which a non-active precursor of a spectrophotometrically active substance is used as a tracer. After incubation of the sample with a specific binding reaction system, the insoluble and liquid portions are separated and the amount of tracer present in the liquid portion as a function of the amount of ligand detected in the sample is determined by performing reaction steps to convert the inactive tracer to a colorant .

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

The enzyme-linked immunoassay described in U.S. Patent No. 3,817,837 is of interest. This method is of a homogeneous type because it does not require the use of a partitioned (i.e., insoluble part / liquid part) 6891 5 specific binding reaction system or the separation method required because the enzyme-labeled ligand is such that enzymatic activity is inhibited when reacted with a ligand binding pair. Thus, the ratio of bound marker to free form can be determined by monitoring changes in enzymatic activity. Nevertheless, this method suffers from the difficulty of preparing very characteristic enzyme-labeled conjugates and finding enzymes that would fit the basic design of the system.

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

It is an object of the present invention to provide a well-suited, versatile and sensitive improved specific binding assay method, and a reagent based on the use of a substance as a marker that exhibits reagent activity as a component of a predetermined monitoring reaction system. The invention thus relates to a specific binding assay method for determining a ligand in a liquid medium, comprising the steps of: (a) contacting the liquid medium with a reagent containing a conjugate of a label and a specific binding agent, wherein the reagent and ligand form a binding reaction system; a bound phase in which the specific binding agent is bound to it by a specific binding pair, and (ii) a free phase of the labeled conjugate in which the specific binding agent is not bound by a specific binding pair, and (b) determining the label as a measure of ligand in either bound or free phase liquid medium; The process of 4,68915 is characterized in that the tracer is a reaction component of a chemiluminescence reaction, such as luminescence or iso-luminescence or a derivative thereof.

The invention also relates to a reagent for determining a ligand in a liquid medium, which reagent comprises a conjugate of a tracer and a specific binding agent, and which reagent and ligand form a binding reaction system that produces a labeled phase of the labeled conjugate. wherein the specific binding agent is not bound to it by a specific binding pair, wherein the amount of label in either the bound or free phase is a function of the amount of ligand in the liquid medium, characterized in that the label is a chemiluminescence reaction component such as luminescence or isolation.

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

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

5 68915 In this context, the following attributes have the following meanings: ligand is the substance or group of substances whose presence or amount in a liquid medium is to be determined, a ligand-specific binding pair is any substance or group of substances with a specific tendency to bind to a ligand to the exclusion of other substances; a binding analog is any substance or group of substances that behaves in substantially the same manner as a ligand with respect to the binding tendency of a specific binding pair of a ligand.

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

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

•, i 6 68915

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

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

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

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

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

8 68915

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

The use of a reagent as a tracer can also be applied to conventional analysis schemes of the heterogeneous type in which the bound and free phases of the tracer moiety are separated and in which the amount of tracer in either is determined. The reagent for performing such a heterogeneous analysis may be in various forms. In general, such a spacer contains three basic components, which are (1) the ligand to be tested, (2) a ligand-specific binding pair, and (3) a label component, which is usually labeled with (a) a ligand, (b) a ligand-specific binding analog, or (c) a specific binding pair. form. The components of the binding reaction are pooled simultaneously or in series and the marker component binds to its corresponding competing sicospairs at appropriate incubation times or times, so that the degree of binding, i.e. the ratio of the amount of the labeled component to the unbound pair and the amount of unbound is a function of the amount of ligand present. Below is a brief description of a few different binding reaction schemes that can be used in carrying out the process of the invention.

Although the labeling property in a labeled conjugate, such as radioactivity or enzymatic activity in conventional heterogeneous specific binding assays, such as radioimmunoassays, and in the case of heterogeneous enzyme-linked immunosorbent assays, . In this situation, the observation reaction is of a relatively constant nature when the ligand is not present in the liquid medium or when an insignificant amount is present internally. When the ligand is present in the liquid medium, the nature or character of the scavenging reaction changes, as described above in connection with the homogeneous technique. The following explanations apply to the diagrams below: 9 68915

Symbol Definition L Ligand to be studied © ligand or its specific binding analog 3 ligand binding pair «marker, i. reagent insoluble phase -> incubation period followed by appropriate separation (lim) limited; present in an amount less than that capable of binding to all available binding sites under the selected reaction conditions during the selected incubation period; i. an ingredient to which other ingredients compete for binding (exc) in excess, present in an amount greater than that capable of binding to all binding sites present under the selected reaction conditions during the selected incubation period.

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

b) L + © i * + j-E (lim) - ^. This approach is commonly referred to as the solid phase technique. Similar radioimmunoassay and enzyme immunoassay techniques are described in U.S. Patent Nos. 3,505,019, 3,555,143, 3,646,346 and 3,654,090.

c) L + B * + | - © (lim) - »

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

d) L +.] - © + B * (lim) .- »

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

10 6891 5 2. Heterogeneous serial impregnation arrangement.and a) L + B (exc) -> + © * (exc) -? + insolubilizer for S or © “

In a series of impregnations, the binder binds to some or all of the binding sites in substance 5 that remain after the first incubation period.

b) L + | - E (exc) - + © * (exc) - *

Similar radioimmunoassay and enzyme immunoassay techniques are described in U.S. Patent No. 3,720,760 (FI-P 48,785) and J. Immunol. 209: 129 (1972).

c) L + B * (exc) -> + [- © (exc) - * 3 · He t ercgeen in.en 'stratified by tel + L + j- 3 (exc) -? Ex (exc) ->

In the construction technique, the label moiety binds to some or all of the ligand molecules bound to the insoluble seedlings.

Reference: U.S. Patent No. 3,720,760 (FI-P 48,785).

4. Heterogeneous Solid Phase Delivery L + © x + (non-specific) - + 3 (lim) - ^ In this technique, a ligand and a label component are bound to a non-specific binder and then dissociated in relative amounts by a binding pair with a higher affinity for ligand and label component. In the most useful form of this technique, a non-specific binding column is used, as described in U.S. Patent No. 3,659,104. Such a technique is useful when the ligand is bound to endogenous binding agents in a sample that would interfere with the competitive binding reaction if not removed. Once bound to a non-specific binder, endogenous binders can be removed by suitable washes.

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

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

The monitoring reaction according to the invention comprises a luminoivar. a reaction system, which is preferably an enzyme-catalyzed reaction having a bioluminescent or chemiluminescent phenomenon. The reagent in the conjugate may be a reagent in either a light generating reaction or a reaction preceding an enzymatic or non-enzymatic luminescence reaction. Any change in the activity of the conjugated reagent due to a specific binding reaction causes a change in the rate of light development or in the total amount, peak intensity, or property of the light generated. Examples of luminescent reaction systems are given in Table A, where the following abbreviations are used: ATP adenosine triphosphate AMP adenosine monophosphate NAD nicotinamide adenine dinucleotide NADH reduced to the nucleotide region ».Hi o o

C -P CU Ό H C I C I

'Φ -¾ -H a; | .r-ι o · Η o M ^ ΛΉ c H J) E ίπ c l ω -H + J>, -H -rt · Η rH a rH a u x -h x s «un

- S O 4-3 3 S »>>, >> >> -H -H

P 0) C Ό pr, PP rH 4-3 4-> 4-3 4-3 i — i r — 4

-P ft'H ΉΗ <U Ή 4-3 4-3 43 4-3 O O

'X, V' O -p> »<u <u cu a> cc", ‘d ^ ^ W CÖ P P 43 43 -H 43 4-3 -H · Η · Η

öp cö cd C 43 i ¢ 43 to co i — i co to i — i ε E

3 43 Cm CMft) - rt CU vH »H · Ηθ · Η · Η O 3 3

'Z? Λ <H £ C 5 LH (m43C X rH J * JX rH, -H r-H

S P "<CO X * H ^ * CO CO Ή i — I i— (f— | rHrHrHOO

^ · 0 · Η · Η <D <U (ΰ (DOCtfcOCO

«<^ -1 (X, -f-s X ro <P Ph O, O, faO Ο. Ω. Bö -H -h

• H .H

C C · Η Ή -H i — l 3 · Η -H o

• P k ft. 3 I-I

+3 <D CL) 43 -H rH

<13 V-4 tn 43 rH CÖ OJ

P -H · Η CU O bO 2

Λ CO CO 43 3 C O

CÖ 3 3 Cl) 4-3 -H Ph + -C h I-H c, 43 E>,

CÖ CU 3 CL -H

+ I 3 X 43 H 43 • H 43 ft) 3 4-3 gö C 4-3 + 0.3-PCÖ S CM »-H (U CU 43 43 CÖ << rc 'H 433x :-p <uhcm

_ § ^ ft. Q, X <U 43 cö X

O + S · Η Ή ft) + Cö. + 43 φ 4_> J 'fc CO Ph Uh χ A cd Q, L-, +

«3 - CÖCU-H-H X> CL cö O

Co jC-h-h + cö X eo-P + · Η X! ci £ C’H

M «p C CO 3 43 CO X -H -P

ftO cö CU Q CU M i — I CÖ 3CÖ + E43 <^ cö x O <ChiCmO cö x cö o + s Cö EH -'ft.COCMXTHCM> j <ft. -a X p Cö

C CU Ή PC CO · X P (H .H -H 3X + rH

CULhLh A 3 CL. P »3 Λ CO CO X CÖ H C · Η + 'Ή r-" + 1) CO ϋ CÖ »-> p

co: cö CO · · Η 43 .H o O CÖ „^ X <M

: CO | CÖ O, 3 Oh O CO CM O CO CO ft-ι Jh T3 o -H O

E cö Ph πμ o, cö O o, ή u m cu · η · η co xC

-HPhW CLCÖ ft ä ci o Ω, CO CO CÖ a-h

CU CU CM CÖ C + CÖ i — I Ph ft-. CM X CÖCÖ -H E

43 Uh Ή OXCU XCUCUI O CM O CÖ C CO CÖ CO Ή rH bDl T-H 0, ^ 4 ► O Ph CCU CÖ <uco3 + c: oop: coin + cm cu CubO cö + • 73 3 P ft) ft. > s ft) + C I K oJ bO >> Ό ft <1 Γ— Ή C O £ C 3 Ή · γΗ -H> i CO -H - 3

; CÖ Ό Ή> »CU» H * H Ph 43 C C + CM COii COX

‘-S-H>» 3 X Ό 3 43 43 43 · Η · Η O ϋΟ X

O c X Ό CU H ·! -} 43'H 10 · Η Ή · Η CM O O

· Η · Η Φ +3 Ö CÖ 43 CÖ43cÖPh Ph rH E CM Ph A

-P-H Ό ft) I ft) CÖ 43 U-h ft) <U ‘O O CU

ft. H ϋ KC ^ CmCOcOCmCPC + o CM a Cö ft) cö: cö Q cu xö co cö o · <η · η · η cm O + o <U <MX <CX O ft-i ft-ι CO CO E · Η B 43

fti * H ΡΡ P X -HP U-4I — II3 3 30 1 — I + · Η X

CO ft) · Η 3 · Η · Η 3-1-1 ι — I ι— (CM Ο 4 rH CÖ CÖ 3 e Λ. Ό Ο, Ό co ro Pep ι — I -Η Ο ι — i

> <Η · Η © 43 · Η I> j >>> s rH · Η rH r — I

• rH 3 + w CU + C · γΗ 434343-f CÖrH OrH CM

0 >> * r-5 * P * H C 43 43 43 bOOCCÖ O CM

C 43 -P X CCÖ X · Η 43 · Η ft) ft) ft) -H CÖrH-Η bO CMO

• H -P ft) S + ^ S CO 43 * rH 43 43 43 rH ΡηγΗ P Ο ΧΧ-Η

Eft) -XÖH 43 & hOCÖC0C0C0C0O> jCÖ3Ph CO

3 · ^: cö Ρ2 · η e cö Ο · Η · η · η C 0 <bO rH> j + + cö

Jeo ^ Η- ^ ρ, + ιο'Μχ ^^ ίϋ-Η o a co

• H P [X, OrHa) rHrHrHE> sPH >> · Η -H rH

-HP H-3 CÖ3T5D ft) <U3 p >> p> 5 1-1 ft £ + xicocoaacLrHpapp oop

ft) CM- CUCUPCCcO

Cfti + SCPCLPc PPCU-H'HPs;

. BS 'co co p e E

+ CM <S - · Η · Η CO 3 3 · Η _ 2 X CO OsC PsC »H rH rH CÖ

^ _ 1-1 rH OOP

φ ft) ft) CO CO

Cfe rH CM rH CM a 0.0.-HVHX

<«O q [ijfcaKHx 13 6891 5

Further details and description regarding luminescent reaction systems that can be used in the present method are given in the following references: J. Biol. Chem. 236: 48 (1961).

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

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

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

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

Biol. Bull. 51:89 (1926).

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

The present invention can be applied to the detection of any ligand having a specific binding pair. A ligand is usually a peptide, protein, carbohydrate, glycoprotein, steroid, or other organic molecule that has a specific binding pair in biological systems or that can be synthesized. In a functional sense, the ligand is usually selected from an antigen or an antibody thereof, a hapten or an antibody thereof, or a hormone, vitamin, metabolite or pharmacological agent, or a receptor or binding agent thereof. Specific examples of ligands detectable according to the invention include hormones such as insulin, chorionic gonadotropin, thyroxine, liothyronine and estriol, antigens and hapten such as ferritin, bradykinin, Prosta-gLandines and cancer-specific antibodies, vitamins such as biotin, vitamin , folinic acid, vitamin E, and ascorbic acid, metabolites such as 3T, 5'-adenosine monophosphate and 3T, 51-guanosine monophosphate, pharmacological agents such as dilantin, digoxin, morphine, digitoxin and barbiturates, antibodies such as microsomal antibodies antibodies to hepatitis and allergens, and specific binding receptors such as thyroxine and binding globulin, avidin, intrinsic factor, and transcobalamin.

i4 6891 5

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

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

In one aspect of the invention are the components of a specific binding reaction that must be combined with a liquid medium suspected of containing ligar, in liquid or solid form. In a preferred homogeneous system of analysis, the ingredients are usually in solution or solid form that is readily soluble in the liquid medium. Because the liquid medium to be tested is usually aqueous in nature, the ingredients are usually in water-soluble form, i. either in aqueous solution or in water-soluble is 6891 5 in solid form such as powder or resin. The assay method can be performed in a standard laboratory vessel such as a test tube, in which the components of the specific binding reaction and the components of the reaction system are added thereto in solid or liquid form.

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

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

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

ie 6891 5

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

Example 1

Preparation of nicotinamide adenine dinucleotide-biotin conjugate.

A. Mycinamide β- (2-aminoethylamino) purine dinucleotide.

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

The resulting solution was adjusted to a pK of 7.0-7.5 with 1 N sodium hydroxide, and 1 g of sodium bicarbonate was added. Nitrogen was bubbled through the solution for 4-5 min and 1 g of sodium hydrosulfite was added. The flask was sealed and allowed to stand at room temperature for 45 min. The solution was then oxidized for 15 min and adjusted to pH 11.3 with sodium hydroxide. The solution was heated at 75 ° C for 1 hour. The reaction mixture was then cooled to room temperature, and 0.6 g of (tris-hydroxymethyl) aminomethane was added, followed by the addition of 5 N hydrochloric acid to adjust the pH to 7> 5. To the resulting solution were added 1000 International Units of alcohol dehydrogenase and 1 ml of acetaldehyde. The decreasing optical density of the reaction mixture was monitored at 340 nm and when no further decrease was observed, the pH was adjusted to 3.5. The solution was poured into 10 volumes of acetone at -10 ° C. The resulting oil was separated and washed with ether, then dissolved in 10-15 ml of water.

$ 'j - · *. fr Γ • ’.K; · 17 6 891 5

The resulting solution was applied to a 2.5 x 90 cm Sephadex G-10 column (Pharmacia AB, Uppsala, Sweden) equilibrated with water. Fractions with a volume of 12 ml were collected. The wavelength of maximum optical absorption in the ultraviolet range and the optical density at this wavelength were determined for each fraction. The optical density of each jade at 340 nm after reduction with alcohol dehydrogenase was also determined. Fractions with an optical absorption maximum at 264 nm and a ratio of optical densities at 340 nm to 26b nm greater than 0.05 were pooled. The collected material was concentrated to 15-20 mL on a rotary evaporator and passed through a 2.5 x 28 cm Dowex 1-X8 column equilibrated with water (Bic-Rad Laboratories, Richmond, CA). More water was added to wash the collected material through the column and 10 ml fractions were collected. Fractions with an optical absorption peak at 26 * 4 nm and an optical density ratio at 340 and 264 nm greater than 0.1 were collected.

The collected material was passed through a water equilibrated 5 x 45 cm Dowex 50-X2 column (Bio-Rad Laboratories, Richmond, CA). More water was added to wash the collected material through the column, and 20 ml fractions were collected. Fractions with an optical absorption peak at 264 nm and an optical density ratio at 340 and 264 nm greater than 0.18 were collected. The collected material was concentrated to 4 to 5 ml and purified by electrophoresis as follows.

The concentrate was applied to a Whatman 31--1 sheet of paper (Reeve Angel, Clifton, New Jersey) as a 1-2 cm wide strip in a direction transverse to the electric current. The paper was then moistened with 0.02 M sodium phosphate at pH 6.0. Electrophoresis was performed by the Durrum-dependent paper method as described in Science 121: 829 (1955) for 4-7 hours with a potential gradient of about 8.5 volts / cm. The location of the desired pyridine nucleotide derivative was determined by the fluorescence that developed after spraying the test strip taken from the paper with 0.5 M sodium cyanide in J. Biol. Chem. 191: 447 (1951). The area containing the desired derivative was cut from the paper and extracted with three 50 ml volumes of water. The resulting extracts containing nicotinamide-6- (2-aminoethylamino) purine dinuclectide were collected, concentrated to 3-4 ml and stored at -20 ° C.

ie 6891 5 B. Nicotinamide adenine dinucleotide-biotin conjugate.

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

Example 2

Homogeneous competitive siton bioluminescence analysis ·, the effect of different amounts of biotin on the generated “bundle light intensity.

The thioluminescence reaction system used in this example was based on the following reactions: (a) NAD ligand + ethanol debydrogenase> NADH ligand + acetaldehyde (b) NADH ligand + FMH * * H ® HeHyH ^ phenase T * NAD ligand + FMNH2 (c) FMNH2 + long aldehyde + O2> FMN + long chain acid + H2O + hp * flavin mononucleotide

The light generating solution used to carry out reactions (b) and Cc) was prepared as follows. A reagent mixture of 0.13 M phosphate buffer pH 7.0, 0.67 wt% bovine serum albumin, 15.7 flavin mononucleotide (FMN) and 13.3 mM sodium acetate was prepared and stored in the dark at -20 ° C. On the day on which the light generating solution was to be used, an emulsion containing 5 μl of dodecanal in 5 ml of water was prepared. Lyophilized luciferase extracted from Photobacterium fisheri (an enzyme manufactured by Worthington Eiochemical Ccrp., Freehold, New Jersey) was added to 0.013 M phosphate buffer at pH 7.3 to a concentration of 20 mg / ml. After 30 minutes, the resulting suspension was centrifuged at 1500 g for 10 min and the pellet was discarded. A light generating solution was then prepared within 5 minutes of use by combining 75 of the reagent mixture, 5 of the dodecanal emulsion and 20 of the luciferase solution.

To detect the light generated by the reaction (c), a light meter was constructed with a light detector and a 6 x 50 mm cuvette fitted to a light integrating circuit so that the light generated in the cuvette was reflected to the light electrodes. The electrical signal produced by the light detector was passed to the count cell. The peak light intensity, as it is called in this context, was measured from the instrument mark and given arbitrary units depending on the diagram strip divisions.

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

The reaction mixtures were incubated at room temperature for about 30 min. A volume of 10 μl of each reaction mixture was injected into a separate cuvette fitted to the light meter described above, in addition to 100 light generating solutions prepared as described above and pre-incubated at 28 ° C for 2-3 minutes. The whole process was run in duplicate and the average results are shown in Table 1.

20 6 8 9 1 5 TABLE 1 reaction mixture biotin concentration average peak _ _ (nM) __ light intensity_ 1 0 36 2 25 3 50 57 4 100 79 5 150 90 6 200 97 7 300 104 This example illustrated that the bioluminescence reaction system The magnitude of the peak light intensity developed by NAD and thus the activity of NAD in the NAD-biotin conjugate was directly proportional to the amount of biotin present in the specific binding reaction mixture. The present invention thus provides a test mixture and method for determining the presence of ligand antibiotin in a liquid medium using a competitive binding bioluminescence assay technique.

Example 3 Preparation of 2,4-dinitrophenyl-ATP conjugate (6-position bound derivative.

N- [2- (2,4-dinitrophenyl) aminoethyl] adenosine 5'-triphosphate.

A. N- (2-aminoethyl) adenosine-51-monophosphate.

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

St. Louis, Missouri) was mixed with 17 mL of triethyl phosphate and reacted with phosphoryl chloride in the presence of water as described in Chem. Scrip. 19: 165-70 (1972). After hydrolysis of the phospho-dichloridate, 9.5 ml of ethylenediamine (140 mmol) were added and allowed to react at room temperature for 3 hours. The reaction mixture was diluted with 4 liters of water and the pH was adjusted to 12 with sodium hydroxide. The solution was passed through a 2i 6,891 5 5 x 30 cm Dowex 1 x 8 column (Bio-Rad Laboratories, Richmond, CA) in acetate form. The column was then washed with 3 liters of 0.01 H ammonium chloride and the chromatogram was developed with a linear gradient of 3 liters of water and 3 liters of 1 M acetic acid. An isolated gradient of UV absorber between 1800 ml and 2050 ml of the eluted peak was concentrated to about 25 ml in vacuo. Upon standing at 7 ° C overnight, this solution formed white crystals which were collected and dried to give a 65% yield of product. A sample of hot water was recrystallized for analysis.

Calculated for c 12 H 19 N 6 O 7 P · 2H 2 O: C 33.8, H 5.45, N 19.7 Found: C 34.3, H 5.22, N 19.7.

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

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

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

168 mg of sodium bicarbonate were then added, followed by 0.2 ml of 1-fluoro-2,4-dinitrobenzene (1.58 mmol dissolved in 2 ml of ethanol). The reaction mixture was stirred in the dark at room temperature for 4 hours and then an additional 0.1 ml of 1-fluoro-2,4-dinitrobenzene in 1 ml of ethanol was added. After stirring overnight, the reaction mixture was adjusted to pH 2.0 with hydrochloric acid and poured into 200 ml of ethanol. The precipitate formed was dissolved in 200 mL of water and this solution was adjusted to pH 8.0 with sodium hydroxide and chromatographed on a 2.5 x 30 cm column of DEAE cellulose (Reeve Angel, Clifton, New Jersey) in bicarbonate form. The chromatogram was developed with a linear gradient of 1.5 liters of water and 1.5 liters of 0.7 M ammonium bicarbonate. A gradient between 1200 and 1500 ml eluted the largest peak of the ultraviolet light absorbing yellow substance. Ammonium bicarbonate was removed by repeated evaporation to dryness to give a 40 ° yield of the desired product. This product 22 6 8 9 1 5 passed as a single yellow spot on thin layer chlorate grams developed with the two solvents mentioned in Part A of this example and epichlorohydrin triethanolamine on anicin exchange paper developed with 0.25 M sodium acetate-acetic acid buffer. In 0.02 M hydrochloric acid, the product had an optical absorption peak at 26¾ nm and 365 nm with millirolar extinction coefficients of 21.8 and 14.2.

C. 2, ¾-dinitrophenyl-ATP conjugate.

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

Example 4 Preparation of 2,4-dinitrophenyl-β-conjugate (8-substituted substituent).

8- / 2- (2,4-dinitrophenyl) aninoetyyli7aminoaaenosiini-5'-triphosphate.

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

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

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

Calculated for C 20 H 20 N 2 O 2 • H 2 O: C 34.0, H 5.33, N 23.2.

Found: C 34.1, H 5.28, N 23.9.

B. 8- [2- (2,4-Dinitrophenyl) aminoethyl] aminoadenosine 5'-monophosphate.

8- (2-Aminoethyl) aminoadenosine-5'-monophosphate from Part A of this example (0.64 mmol) was dissolved in 20 mL of water with the addition of sodium hydroxide to pH 8.0. 168 mg of sodium bicarbonate were then added, followed by 0.2 ml of 1-fluoro-4-dinitrobenzene (1.58 mmol dissolved in 2 ml of ethanol). The reaction mixture was stirred for 18 hours at room temperature, and then 0.1 ml of 1-fluoro-2,4-dinitrobenzene in 1 ml of ethanol was added. After stirring for an additional 4 hours, the mixture was adjusted to pH 2.0 with hydrochloric acid and poured into 200 mL of cold acetone (-10 ° C). The yellow precipitate formed was collected by filtration, dissolved in 200 ml of water and applied to a 2.5 x 45 24 68915 cm column of DEAE cellulose in bicarbonate form. Krcmatcgranmi was developed with a linear salt gradient developed from 2 liters of water and 2 liters of 0.7 M ammonium carbonate. Between a gradient of 2 and 3 liters, a yellow substance with an absorption maximum of 275 nm eluted. Ammonium bicarbonate was removed from the material by evaporation in vacuo and the yield of the desired intermediate was; The optical absorption peak measured in C2 V. hydrochloric acid occurred at 275 and 363 nm and the millinolar extinction coefficient was 21.5 and 15.5. Further analysis showed that there were 1.07 phosphate residues per ribose residue.

C. 2, ^ -dinit rcf er.yy li-AT? - conjugate.

The monophosphate intermediate (0.5 mmol) was reacted to give the tri-n-butylammonium salt by the addition of 0.3 mmol of tri-n-butylamine. The mixture was dried by repeated evaporation from dry dimethylformamide (10-15 ml each), the final residue dissolved in 1 ml of dimethylformamyc was mixed with 2.0 mmol of carbonyldiimicazil in 1 ml of dimethylformamide and allowed to react at room temperature for b hours. Excess carbonyldiimidazole was destroyed by reacting it with 15 methanol for 30 min. Finally, 3 mmol of tri-n-butylammonium pyrophosphate in 1 ml of dimethylformamide were added and allowed to stand for 20 hours. The solid residue formed was separated by centrifugation and washed twice with 5 ml portions of dimethylformamide. The combined supernatants were added to 200 ml of water, which was then adjusted to pH 8 and chromatographed on 2.5 x 25 cm DEAE-celluloco in bicarbonate. column. The chromatogram was developed with a linear gradient of 2 liters of water and 2 liters of 0.5 M ammonium bicarbonate. Between a gradient of 2.0 and 2.9 liters, a yellow substance eluted with an optical absorption maximum at 275 and 363 nm. Ammonium bicarbonate was removed by evaporation to give the desired conjugate in 22 "yield. Analysis showed that this product contained 3.2 phosphate residues for each ribose titre.

Example 5 Homogeneous Competitive Binding Bioluminescence Assay for 2,4-Dinitrophenyl Derivatives; Use of ATP as a marker.

The bioluminescence reaction system used in this example was based on the following reaction: ATP ligand + reduced luciferin - ^ S1? Sr $, as ± y AMP ligand + oxidized luciferin + hv A. Analysis using a 6-substituted A? P: n derivative.

Three specific binding reaction mixtures were prepared, each with a total volume of 100 μl and each containing 10 mM tris- (hydroxymethyl) aminomethane hydrochloride buffer at pH 7.4, 10 mM ethylenediaminetetraacetic acid, 20 μl antiserum 2,4-dinitrophene for 512 nM 2,4-dinitrophenyl-AT? conjugate (6-substituted derivative prepared according to Example 3) and 2,4-di-nitrophenyl-S-alanine at the concentrations shown in Table 2. After incubation at 25 ° C for 2 hours, 10 μl aliquots of each reaction mixture were analyzed by injecting them into a volume of 0.1 ml of the above light-generating solution, which had previously been incubated at 25 ° C for at least 2 minutes, and which was in a test tube fitted to a Bioluminescence light meter on a Dupont Model 760 (EI duPont de Nemours, Willmington, Delaware). The peak light intensity was read on a light meter. The average peak light intensity for each reaction mixture was calculated, as was the relative intensity (100% times the ratio of the average peak intensity of the sample to the absence of antiserum). The results are shown in Table TABLE 2 Concentration of 2,4-dinitrophenyl-8-reaction alanine Mean peak work_ _ (, μM) _______ light intensity__ 1 0 7 2 25 14 3 2500 185 B. Analysis using an 8-substituted derivative of ATP.

Four specific binding reaction mixtures were prepared, each with a total volume of 100 μl and each containing 20 ml of tris- (hydroxymethyl) aminomethane hydrochloride buffer at pH 7.4, 10 mM ethylenediaminetetraacetic acid, 20 μl antiserum, 2,4-dinitrofe -nyl, 594 nM 2,4-dinitrophenyl-ATP conjugate (8-substituted derivative, prepared according to Example 4) and 2,4-26891 5 dinitro-S-alanine at the concentrations shown in Table 3. After incubation at 25 ° C for 2 hours, 10 μl aliquots of each reaction mixture were analyzed as in Part A above and the average peak luminosity was calculated for each. The results are shown in Table 3.

TABLE 3 2,4-Dinitrophenyl-6-reaction-alanine concentration Mean peak mixture _ (μM) __ Luminous intensity 10 18 2 25 51 3 250 191 4 2500 190 This example shows that the tracer, A ?? E, can be substituted at various positions around its structure to prepare a conjugate useful in the specific binding assay method of the present invention.

Esimerkki.6

Preparation of bicinine-isoium conjugate.

6- (3-bictinoyyliamido-2-hyarok3ipropyyliamiini) -2,3 'dihydroftalat-toxin-1, ^ - dione.

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

25 e (0.142 mol) of 4-amino-H-methylphthalamide, prepared according to J. Chem. Scc. 26: (1937), and 20.7 g (0.21 mol) of 1-chloro-2,3-epoxypropane was added to 150 ml of 2,2,2-trifluoroethanol, and the reaction mixture was heated under reflux with stirring for 4 hours. 70-80 ml of 2,2,2-trifluoroethanol was removed by distillation and a heavy yellow precipitate formed when the remaining solution was cooled to room temperature. This precipitate was triturated with ethyl acetate, collected by filtration, dried to give 29.5 g (77 S yield ') of the desired intermediate, m.p. 136-138.5 ° C Calculated for C 11 H 24 Cl 2 Cy C 53.64, H 4.88, N 10.45 Found: C 53.87, H 4.85, N 10.8l.

B. 4- [N- (N-phthalimido) -2-hydroxypropylamino] -N-methylphthalimide.

27 6 8 9 1 5

The intermediate prepared in Part A (13 x 5 g, 0.05 mol) and 15.7 g (0.085 mol) of potassium phthalimide were heated under reflux and stirred in 150 ml of dimethylformamide for 24 hours. 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 to 248.5 ° C.

Calculated for C 20 H 17 N 3 O 5: c 63.32, H 4.52, N 11.08.

Found: C 63.16, H 4.38, N 10.93-C. 6- (3-amino-2-hydroxypropylamino) -2,3-dihydrophthalazine-1,4-dione.

The intermediate from Part B (5.0 g, 13.2 mmol), 90 mL of absolute ethanol, and 35 mL of 95% hydrazine were refluxed with stirring for 4 hours. The solvent was removed in vacuo and the resulting solid was dried under vacuum at 120 ° C for 24 hours. This material was stirred for 1 hour with 70 ml of 0.1 N hydrochloric acid. Insoluble 2,3-dihydroxyphthalazine-1,4-dione was removed by filtration and the filtrate was adjusted to pH 6.5 with saturated sodium bicarbonate. The white precipitate formed was collected by filtration and dried to give 2.2 g of product (67% yield). After recrystallization from water, the compound decomposed at 273 ° C.

Calculated for C 52.79, H 5.64, N 22.39.

Found: C 52.73, H 5.72, N 22.54.

D. Biotin-isoluminol conjugate.

• 1

Biotin (0.29 g, 1.2 mmol) and 0.17 mL of triethylamine were dissolved in 20 mL of dry dimethylformamide under anhydrous conditions and cooled to -10 ° C. A solution of 0.141 ml of ethyl chloroformate in 2.86 ml of ether was added slowly and the reaction mixture was stirred for 30 min. The precipitate formed was filtered off. A suspension of 600 mg (2.4 mmol) of Part C intermediate, 20 ml of dry dimethylformamide and 1 ml of dry pyridine was added rapidly to the filtrate. This mixture was stirred at -10 ° C for 30 min and then at room temperature overnight. A solution was obtained during this period. Dimethylformamide was removed by distillation at 60 ° C and 0.10 mm Hg, the oily residue was stirred with 50 ml of 0.1 N hydrochloric acid for 1 hour. The resulting white solid was filtered and washed with 0.1 N hydrochloric acid followed by water. After drying at room temperature overnight, 0.55 g (97 "yield) of product was obtained, mp 170-173 ° C.

Calculated for C 21 H 28 N 9 O 5 S: C 52.92, H 5.92, N 17.64.

Found ": C 51.59, H 5.90, N 17.65-

Example 7

Homogeneous competitive binding-chemiluminescence assay for biotin.

The chemiluminescence reaction system used in this example was washed for the following reaction: biotin isoluminol + KO2 --—> biotin aminophthalate + + hp

Sixteen specific binding reaction mixtures were prepared, each with a total volume of 150 μl and each containing 0.1 M tris- (hydroxymethyl) aminomethane hydrochloride at pH 8.0, 42 nM biotin-luminol conjugate (prepared according to Example 6), biotin in the table 9 and 0.12 units per ml avidin (last added). After incubation at 25 ° C for 5 min, 10 μl of dimethylformamide with 0.15 M potassium superoxide (CO 2) (Alpha Products Beverly, Massashusetts) and 0.10 M 1.4.7 were injected into each reaction mixture. 10,13,16-hexoxycyclooctadecane (Aldrich Chemical Co., Milwaukee, Wisconsin).

After an additional two minutes of incubation at 25 ° C, 10 μl of 0.95 mM hydrogen peroxide in 10 mM tris- (hydroxymethyl) aminomethane hydrochloride buffer at pH 7.4 was injected into each reaction mixture, and the luminosity intensity formed in each case was measured using a Dupont model. 760 bioluminescence photometers (EI duPont de Nemours, Willmington, Delaware). The results are shown in Table 4.

29 68915 TABLE 4 reaction biotin concentration peak light mixture _ (nx ~) _ intensity 1 G 38.5 2 13 38.5 3 27 3 * 2.3 4 240 36.1 5 53 35.2 6 67 36.2 7 101 3 ^, 0 8 133 31.7 9 166 29.1 10 200 224.2 11 267 22.8 12 333 20.5 13 40C 13.4 14 53 ^ 8.6 15 667 8.3 16 8CO 7 , 0

It was found that the magnitude of the peak light intensity produced by the kenilumir sensing reaction system was inversely proportional to the amount of biotin present in the specific binding reaction mixture. The present invention therefore provides a test mixture and method for determining the presence of ligand-dibiotin in a liquid medium using a competitive binding-chemiluminescence assay technique that does not utilize an enzyme-catalyzed monitoring reaction.

Example 3

Heterogeneous competitive binding-bioluminescence assay for biotin; the effect of different amounts of biotin on the resulting peak light intensity.

The bioluminescence reaction system used in this example was based on the reactions shown in Example 2.

A. ‘Preparation of the light-emitting solution

A light generating solution was prepared as in Example 2.! B. Preparation of insolubilized bonding pair 68915

Avidin, which tends to bind to biotin, was rendered insoluble by covalently binding it to a water-insoluble polymer bead as follows. An aliquot of Sepharose 4B (Pharmacia AB, Uppsala, Sweden) was activated for binding to avidin using the method described by March et al. In Analytical Eiochemistry 60: 149 (1974). Approximately 4 ml of activated Sepharose 4B was suspended in 8 ml of 0.1 M citrate buffer at pK 7.0. To the suspension was added 6 mg of avidin having an activity of 9.9 units per mg in 3 ml of water. One unit of avidin activity is the amount of avidin capable of binding one microgram of biotin. The resulting reaction mixture was stirred for 6 hours at 7 ° C. Avidin-bound Sepharose 4E was then filtered, washed with 100 ml of 0.1 M sodium bicarbonate buffer at pK 9.0, and resuspended in 240 ml of 0.1 M tis- (hydroxymethyl) aminomethane hydrochloride buffer at pH 8.0.

C. Verification tests

9 specific binding reaction mixtures were prepared, each with a total 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 semicarbazide hydrochloride, and Table 10, respectively. amounts or concentrations of NAD, NAD-biotin conjugate, avidin-bound Sepharose 4E suspension (prepared according to Part B 'of this example) and Sepharose 4B suspension (formed by suspending 1 ml of sealed Sepharose 4B in 60 ml of 0.1 M tris- (hydroxymethyl) aminomethane hydrochloride buffer at pH 8.0). The reaction mixtures were gently shaken for 15 min at room temperature. Then, 0.22 International Units of alcoholide hydrogenase was added to each reaction mixture to initiate the reduction reaction. The semicarbazide combines with the acetaldehyde formed in reaction C to form semi-carbazone and thus drives reaction C in the desired direction.

The reaction mixtures were shaken again for 15 min at room temperature. A 10 ul aliquot of the liquid on top of each reaction mixture was then injected into a separate cuvette fitted to a DuPont Model 760 bioluminescence light meter (du.Ι. duPont de Nemours, V.'illmington, Delaware) containing 100 ul of the light prepared above. generating solution pre-brewed for 2-3 min at 25 ° C. The results are shown in Table 5.

3i 6 8 91 5

• H I

I 40

O <D

rH d) Λ -P C \ J (Τ 'tn \ Ω t> * ρΗ η r λ η

3 CO pH O. t ~ - LT "'O CO - f-H

CC 3 LO -C -40 r- · C \ J LO p— · CL Φ r — i p — i r — i i — t

• rH 4-3 O £ -CC -pH I

CQ rH

= r 3

OF

c -Φ o

CO · Η I I I I I I O O I

0 CQ (- (pH

i-4 3 Π3 o CL Cp j Φ 3

C, Q CO

C to 3 3 4-3 01 o Ό CC C ή H - ~ • pH Cl

• pH Cp - · C ~ C

CO I t C \ i t I Γ, 'I I (Λ;

I -rl C, C

LO1 -r-1 ra —i O Ή CC 3 CC> O o cc <to CC | .

D-C

CC I

<3 C- <Ό C to

O 3 c *: 3 CO

• H Ή c o • H 4-3 • rH * H LL I I I O pH i — I I «H |

• P CC C h t \ i (M OJ

0 ^ • pH C Χ3 · Η 1 4-3

q ra <c ra 2 fcO

to 3 3 to

•B

o

4LP

• H ^

CCH1 I I — I pH I | 1 I — i | I

C <M raj ra- C - ·

•B

Q

<

O

• pH

-P

Ci ΗίνίΛΐιΛνΟΝΧιΟΝ ra Φ

Cp 32 6 8 9 1 5

The results of control reactions 1 and 9 show that when MAD and the NAD-biotin conjugate were not present, very little light was generated. Reactions 2 and 3 gave results showing that a light-generating reaction occurred when free MAD was added and that the presence of avidin-bound Sepharose 9E did not substantially convince this reaction. The results of reactions M, 5 and 6 show that the NAD-biotin conjugate was active in the light-generating reaction, that the generated peak light intensity increased when more NAD-biotin conjugate was present and that the presence of avidide-bound Sepharose 9E inhibited light generation. A comparison of the results of Reactions 3 and 5 with the results of Reactions 7 and c shows that the presence of Sepharose® E alone did not convince the light-generating reaction.

D. Method of analysis

Five more specific binding reaction mixtures were prepared, each with a volume of 0.19 ml and each containing 0.1 M tris- (hydroxymethylD-aminomethane hydrochloride buffer at pH 8.0, 0.6 M ethanol, 0.01 M semicarbazide, and the like. the amounts or concentrations of NAD-biotin conjugate, free biotin and avidin-bound Sepharose 9B suspension shown in Table 6. Each reaction mixture was treated in the same manner as the control reaction mixtures in Part C of this example.

33 6 8 9 1 5 f—

•B

0 »H r-4 4-> C3 J-> * -! -ΞΓ r-H CT \ C ^ -> O * λ * s t \ rs

3 ^ N “Λ O

C- Ο- r-i * C "Lf \ n · - .S'3

d 3 I

23 j

C CO 3 3 J-> CO

o Ό 3 • H ..

^ tncs

“O I O O O I

3 3 CM CM (M

• HO) · '* • rt tO —r

»M C

i — i * u o <a l! * Χ>. £ 3 tX 3 • rt * rt

- O

<0 -p E .'- 'm I | te cc 3 3 t »· tr. tr.

• ^ rH (—i 3 • rt • rt J-5

O

•B

jQ

cd 3 tO'- '3 ε •• -3 3 3 »-

O

λ; to • rt 3

(Rt rH (rt rH rH

• rt tO CM CM CU CM CM

• rt * rt 4-> O

0 4-> • rt * rt 3i a a e

<-rt S -P

O

• rt • P O rH CM Κ'ι ~ .iC rH r-i r — l r-l

, CO., LTD

<U Sh 34 6891 5

The results of reactions 11, 12 and 13 show that free biotin and the NAD-biotin conjugate compete effectively for the insolubilized avidin binding sites, as the resulting peak luminosity depended on the amount of free biotin present. The results of reactions 10 and 14 show that in the absence of insolubilized avidin, peak light intensity was produced for very different concentrations of constant free biotin.

Thus, in this example, it was illustrated that the amount of NAD-biotin conjugate in the liquid phase was inversely proportional to the amount of free biotin present, and thus the assay method and reagent of the present invention are useful in determining the ligand in an unknown liquid sample.

Claims (6)

A specific binding assay method for determining a ligand in a liquid medium, the method comprising the steps of: (a) contacting the liquid medium with a reagent containing a conjugate of a label and a specific binding agent, wherein the reagent and the ligand forms a binding reaction system which forms (i) a bound phase of the labeled conjugate, wherein the specific binding medium is bound thereto with a specific binding pair and (ii) a free phase of the labeled conjugate, where the specific binding medium is not bound thereby, with a specific binding pair, and (b) determining the labeling agent either in the bonded or free phase as a center for the ligand in the liquid medium, characterized in that the labeling agent is a reaction component of a chemiluminescent reaction, such as luminol or isoluminol or a derivatives thereof. Method according to claim 1, characterized in that the activity of the chemiluminescent reaction component used as a labeling agent is different in the bound phase than in the free phase.