WO1985003356A1

WO1985003356A1 - Assay for immobilized reporter groups

Info

Publication number: WO1985003356A1
Application number: PCT/US1984/000138
Authority: WO
Inventors: Lyle J. Arnold, Jr.
Original assignee: Molecular Biosystems, Inc.
Priority date: 1984-01-27
Filing date: 1984-01-27
Publication date: 1985-08-01
Also published as: AU2577084A; NO853790L; DK436785D0; AU582341B2; JPS61501047A; DK436785A; EP0170652A4; EP0170652A1

Abstract

A sensitive and specific assay method for luminescent detection of support matrix-bound reporter groups smaller than about 10,000 daltons in size which comprises contacting such reporter groups with a detector complex which comprises a first component having a strong and specific affinity for such reporter group, and a second component capable of being readily coupled to a light emitting system, thereby to produce high affinity attachment of said detector complex to the immobilized reporter group. The amount of bound detector complex is determined with a luminescence coupled reaction. The light emitted is quantitated by means including a luminometer, light sensitive film, or light sensitive charge coupled device; and the amount of such light provides a measure of the reporter groups bound to the support matrix. In one application, the assay method provides means for monitoring the interactions by which the reporter groups are immobilized. Also disclosed are reagent means useful in practicing the method.

Description

DESCRIPTION

ASSAY FOR IMMOBILIZED REPORTER GROUPS

TECHNICAL FIELD

This invention relates to the field of immuno and diagnostic chemistry, and more particularly to the sensitive and specific luminescent detection of immobilized reporter groups smaller than about 10,000 daltons in size.

BACKGROUND ART

Over the last decade, a large nurnber of radiolabeled and enzyme-labeled assay systems have been developed. While radio- labeled reactions thus far developed exhibit high sensitivity by present day standards, there are several severe limitations inherent in radioactive detection. These include:

1) The radiolabel must be incorporated as a reporterc group by tedious chemical synthesis or by attaching it to appropriate functional groups;

2) The radiolabeled material must be purified under carefully controlled conditions to avoid dangerous exposures to radioactivity;

3) The radiolabeled material must also be handled and disposed of by special procedures;

4) The half-life of many radioactive substances is short, and as a result, the shelf-life of the corresponding radiolabeled reagent is short;

5) The instrumentation for detecting radioactive substances is expensive; and

6) The radioactivity decomposes the assay reagents, thereby adversely affecting the accuracy of the assay.

In contrast to the limitations associated with radioactive detection, assay methods which utilize luminescent reagents pose little environmental hazard, are stable for long periods of time, and require inexpensive instrumentation for detection. The enzyms-labeled reactions referred to earlier herein have the advantage that they do not utilize radioactivity, but they do not typically have the sensitivity of radioactive assays. Thus, there is a defined need for a nσnradioactive detection system capable of sensitivity comparable to that of radioactive detection systems.

The prior art enzyme-labeled systems typically utilize competitive binding interactions to detect the presence of soluble analytes, and they fall into two principal categories:

1) Heterogeneous - methods which require the separation of free from bound phases; and

2) Homogeneous - methods which do not require separation. U.S. Patents Nos. 3,850,752; 3,839,153; 3,654,090; 4,016,043; 4,228,237; and 4,318,980 all employ methods for detecting analytes in solution based upon a competition between bound and free forms. Some of these systems have been expanded to involve the use therein of bioluminescence or chemiluminescence. British Patent No. 1,578,275 describes a heterogeneous method for assaying insulin in solution using a competitive binding assay which employs an insulin-isoluminol conjugate. U.S. Patents Nos. 4,230,797 and 4,380,580 describe competitive binding assays for detecting ligands in a liquid medium and employ a conjugate of a ligand and an enzymatic reactant. U.S. Patent No. 4,383,031 discloses a homogeneous assay procedure using a similar type of conjugate to detect ligands in a liquid medium.

A good survey of the published literature in this area appears in a review by KRICKA, L.J. and CARTER, T.J.N. in "Clinical and Biochemical Luminescence", Vol. 12, pp. 153-178, edited by J.J. KRICKA and T.J.N CARTER, Marcel Dekker, New York, (1982). Since the preparation of this review article, several other papers have been published which report the use of chemiluminescence in the detecion of ligands in solution. Schroeder et al., used an isoluminol derivative-labeled antibody in a chemiluminescent system to measure hepatitis B surface antigen in human serum [Clin. Chem. 27, 1378-1384 (1981)3; Hinkkaneh et al., measured the quantities of immunoabsorbed proteins with a luminol system [Hoppe-Seyler's Z. Physiol Chem. 306, 407-411 (1983)]; and Pronovost and Baumgarten measured various proteins in solution using isoluminol [Experimentia 38, 304-306 (1982)]. Essentially all of these systems have been designed to detect analytes present in a liquid medium by competition with labeled analytes.

Historically, the detection of immobilized ligands has been limited principally to cytological and histological staining procedures employing fluorescent and colorimetric methods. For example, as reported in Virology, 126, 32-50 (1983) , Brigati et al detected DNA in paraffin-embedded tissue sections using a peroxidase complex and biotinylated DNA hybridization probes described in European Patent Publication No. 0063879,published 3 November 1982. These methods typically give qualitative and not quantitative measurements of the ligands present and are employed to test for specific sites already present within tissues or. cells.

A recently published abstract reported the detection of immobilized DNA using a microperoxidase covalently attached to DNA [Fed. Proc. 42, 1954, abs. 1149, (1983)].

This system employed luminol to detect the microperoxidase, and has little utility, since it is severly limited by a lack of sensitivity (detection limit ~10 ^-13 moles of target DNA).

The invention disclosed here is novel and differs markedly from the prior art. The definitive characteristics of the method of the invention are:

1) It detects immobilized reporter groups; 2) It is a direct method for assaying reporter groups; 3) It uses highly specific ligand-ligand interactions to visualize the reporter groups;

4) It has a detection limit in the range of about 10^-16 to about 10^-20 moles of reporter group. This places it close to the detection limits of radiolabels and is far greater than conventional nonisotopic detection systems

5) It uses nonhazardous chemiluminescent or bioluminescent reagents;

6) The reagents used are stable for a long period of time; 7) Only very small quantities of reagents are used making individual assays inexpensive;

8) Inexpensive instrumentation is used;

9) Only a few minutes are required for the assay to be run, in contrast to assays of radiolabeled reporter groups which may take days; and

10) The assay procedure affords high sensitivity because it involves detection of a single small signal, rather than a small difference between two large signals, as is done conventionally. This invention grew out of a need to detect very small quantities of support matrix-bound reporter groups by nonradioactive means. It is necessary in many instances to be able to quantitate the number of reactive sites or binding sites on the surface of a support matrix.

Moreover, it is imperative to be able to quantitate the efficiency of reactions by which substances are coupled to the surface of a support matrix. Finally, in many cases it is desirable to be able to quantitate the degree of interaction of a second molecule with the support matrix. For all these applications, a rapid and sensitive nonradioactive system is necessary. The above-noted characteristics of the invention fulfull this need. Because this invention correlates light emission with the presence of reporter groups of less than about 10,000 daltons in size, the very sensitive detection of such reporter groups is now possible. Current luminescent methods are capable of detecting as few as 100 photons/sec. If the quantum efficiency of the luminescent reaction is high,and light emission occurs within a few seconds, perhaps as few as 500 molecules can be detected. Theoretically, this makes luminescent detection as sensitive or more sensitive than radioactive detection. In cases where the detection of reporter groups can be amplified enzymatically prior to or coincident with the light emitting reaction, this system becomes even more sensitive.

Luminescent detection systems are preferable to radioactive detection systems for a number of reasons in addition to those previously mentioned. Radioactive particles slowly and continuously breakdown and emit radiation. Only a very small fraction of the radioactive isotopes present actually break down and emit radiation at any given time. For example, in the case of a substance labeled with ¹²⁵Iodine, 12.5 million of the ¹²⁵ Iodine atom labels must be present in order to produce 100 disintegrations per minute. Because of the slow breakdown of radioactive particles and the small number thereof which breakdown over any given time interval, radioactive detection systems necessarily require slow signal accumulation. In contrast, substantially all luminescent molecules are available for light emmission at any given time, and they can be made to emit photons rapidly when induced to do so. Hence, essentially all of the luminescent molecules present can, under user control, be made to emit photons simultaneously. This gives a benefit of sensitivity and short assay periods to luminescent systems when compared to systems employing radiolabels.

Another advantageous feature of this invention is that it provides a method which directly detects reporter groups immobilized on a support matrix. As noted earlier, the prior art enzymatic systems employing competitive binding assays for analytes in solution typically require that a small difference between two large signals be distinguished. The direct detection afforded by the present invention enhances its sensitivity over that of such prior systems, because it is far easier to detect a single small signal than it is to detect a small difference between two large signals.

Moreover, these prior art methods function to assay large size proteinaceous antibodies, antigens or analytes which tend to be unstable under certain reaction conditions. Reporter groups useful in determining the number of reactive groups on support matrices, determining the resultant efficiency of coupling reactions, or in quantitating the interaction of a second molecule with a support matrix,cannot be large proteins. In order to have a sensitive measure of matrix composition; the use of small size reporter groups is essential for accurate quantitative determinations, since such determinations cannot be made as easily with the use of large, sterically bulky proteinaceous reporter groups. In contrast to the unstable proteinaceous molecules assayed in the prior art methods, the reporter groups detected by the method of the present invention are comprised of substances which are stable under reasonable limits of pH, temperature, solvent, and salt concentrations.

The reporter groups detected by the method of the present invention are smaller than 10,000 daltons in size. The small size of such reporter groups affords several advantages, among which are that:

1) They can be easily attached to support matrices by methods which cannot be easily adapted for attachment of large or easily denatured reporter groups. 2) Such reporter groups can be attached to a given support matrix in much larger number than can larger size reporter groups. This higher density of reporter groups per unit surface area of support matrix facilitates the detection of the reporter groups within a given small area, because the higher density produces a more readily detected signal from such surface area; and 3) The use of small reporter groups affords a high degree of versatility which is not possible when large size reporter groups are used. The chemistry of attaching small reporter groups is easier to carry out, and the detection and quantitation of such groups is also easier. In addition, since a wide variety of substances may be chemically attached to such small reporter groups, a common detection system may be employed to monitor the immobilization of small reporter groups having such substances attached thereto. Such versatility is not possible with large reporter groups.

SUMMARY OF THE INVENTION

This invention relates to a method, complexes and reagent means for the sensitive detection of reporter groups bound to a support matrix, which permits monitoring of the interaction by which the reporter group is inmobilized on the matrix, as well as quantitation of the interaction of a second molecule with the support matrix. The invention also permits the monitoring of related interactions involving substances which may alter the ability of the reporter group complexes to become immobilized. In particular, this invention relates to the luminescent detection of immobilized reporter groups by the use of a novel detector complex which can be readily coupled to a light emitting system and which is capable of forming a high affinity interaction with a specific reporter group smaller than about 10,000 daltons in size. The reporter groups which are useful in the practice of the invention can be virtually any small molecule for which specific binding substances (i.e. ligands) are available. Such reporter groups include, but are not limited to, vitamins, such as biotin, iminobiotin, desthiobiotin, or pyridoxal phosphate, with which a highly specific interaction is formed with certain proteins; cofactors, such as porphyrins with which a highly specific interaction is formed with certain proteins, such as the cytochromes and the hydroperoxidases; antigens, such as dinitrophenol, biotin , iminobiotin , desthiobiotin , fluore scein and fluorescamine, or conjugates thereof, with which antibodies therefor form a highly specific interaction; and carbohydrates, such as mannose, galactose, and fucose, with which certain lectins form a highly specific interaction.

The presence of the immobilized reporter group is determined by first incubating it with an excess of detector complex free in solution. Due to the high affinity of the detector complex for the reporter groups, a portion of the detector complex binds to the reporter groups present. After the removal of unbound detector complex, the detector complex remaining bound is detected by contact thereof with a light emitting system capable of emitting light in the presence of the complex. Since the amount of bound detector complex is proportional to the amount of reporter group on the support matrix, the light emitted by the light emitting system on the support matrix is correlated with the number of reporter groups bound to the support matrix. The detector complex of the invention is comprised of a first component which has a high specific affinity for a reporter group having a size smaller than about 10,000 daltons, and a second component which can be readily coupled to a light emitting system. This second component can comprise either a chemical substance which can participate in light generation when additional components are added, or it can comprise an enzyme which plays an essential role in a light emitting cascade. Regardless of the exact nature of the second component of the detector complex, light is emitted by the light emitting system only when the detector complex is present. This light emission can be directly correlated with the presence of the immobilized detector complex. A large number of bioluminescent and chemiluminescent light emitting systems useful in practicing the method of the present invention have been elucidated in the prior art (c.f. Maulding, D.R. and Roberts, B.G., J. ORG. CHEM. 34, 1734 (1969); Tseng, S.S., et al., J. ORG. CHEM,, 44, 4113-4116 (1979); Gill, S.K., Aldrichimica Acta 16:59-61 (1983); Clinical and Biochemical Luminescence, supra; and Methods in Enzymology, Vol. 57, Academic Press, 1978). Since most of these require several components in order for the entire system to function, the luminescence coupling second component of the detector complex may be any of a wide variety of substances.

Similarly, since there are a wide variety of substances which have a high specific affinity for potential reporter groups having a size of less than about 10,000 daltons, the reporter group binding first component of the detector complex can be of a wide variety of substances. BEST MODE OF CARRYING OUT THE INVENTION

To facilitate an understanding of the present invention, the following definitions of certain terms used herein are provided: 1) Support Matrix - Any solid support composed of an insoluble polymer, such as nitrocellulose, agarose, etc., which may or may not have an organic coating which comprises a protein, a carbohydrate, a nucleic acid, or an analog thereof. 2) Reporter Group - Any chemical moiety which can be used to label specific chemical groups on a support matrix and which is reasonably stable to conditions of pH, temperature, solvent and salt. Such groups have a size of less than about 10,000 daltons, and are typically substituents which are nonproteinaceous. Reporter groups also include chemical moieties which are not normally present on the surfaces of molecules, but can be introduced as reporter groups through chemical processes.

3) Detector Complex - A complex having a first component comprising a reporter group binding substance and a second component comprising a coupling substance.

4) Light Emitting System - One or more reactions which, when coupled to a detector complex, culminate in the emission of light. The light emitting system can comprise a light emitting reaction, a bridging reaction and a substrate or other reactant which, when reacted with the detector complex, produces a product which is a constituent of the bridging reaction or the light emitting reaction. The basic method of the present invention employs the following steps:

1) The association of a detector complex with a reporter group affixed to a support matrix, and the complexation of the reporter group with the detector complex.

2) The removal of uncomplexed detector complex.

3) The coupling of a light emitting system to the bound detector complex which remains immobilized on the support matrix, causing light to be emitted.

4) The use of light sensing means such as a luminometer, photosensitive film or a light sensitive charge coupled device (CCD) and reference to standard curves to determine, from the amount of light emitted, the number of reporter groups present on the support matrix. In the light emitting reaction, additional steps and reagents may be required and will vary depending upon the nature of the detector complex. The second component of the detector complex is preferably of two principal types which respectively comprise: a) A substance, such as a luminescent compound, which is of itself an essential constituent of a light emitting reaction; or b) An enzyme, coenzyme, fluorescer, other factor or substrate which provides an essential or limiting substance in a bridging reaction or a light emitting reaction.

A wide variety of luminescent reactions can be used to carry out the method of the present invention. Discussions of light emitting systems useful in the practice, of the invention can be found in: Clinical and Biochemical Luminescence, supra, and Methods in Enzymology, supra. These references are representative but not inclusive of all of the light emitting reactions useful in the method of the present invention. The utility of these light emitting reactions can be expanded by the use of a variety of bridging reactions which can provide one of the limiting substances in the luminescent reaction. It should be pointed out that even though oxygen is a limiting substance, in many light emitting systems it is normally ubiquitous, and as a result, bridging reactions which produce oxygen are generally impractical.

A large number of detector complexes is possible. The coupling substance forming the second component of the detector complex may provide one of the necessary constituents of the luminescent reaction, or it may provide a constituent of a bridging system, the latter providing a constituent necessary for the luminescent reaction to function. In order to demonstrate the diversity of possible detector complexes, interaction of various forms thereof with a bioluminescense system derived from bacteria will now be discussed.

The second component can comprise a bacterial luciferase, and when such is the case, and FMNH, a long chain aldehyde and oxygen are contacted with the detector complex, light is emitted. The second component can also comprise a substance which is an essential component of a bridging reaction capable of providing an intermediate product which is essential or limiting in a luminescent reaction, such as a bacterial bioluminescence reaction. Typical substances useful as such second component are illustrated in the following light emitting systems which are grouped according to the type of bridging reaction employed. 1) FMNH producing bridging reactions a) Using a detector complex having a second component comprising FMN⁺ oxidoreductase and contacting the bound complex with NADH, aldehyde, oxygen, and bacterial luciferase, the NADH generates FMNH through the FMN⁺ oxidoreductase and, in the presence of the other components of the bacterial bioluminescent system, light is emitted; b) Using a detector complex having a second component comprising an NAD⁺ or NADP⁺ dependent dehydrogenase, and contacting the bound detector complex with NAD(P)⁺ and the appropriate reduced substrate, NAD(P)H is generated. Exemplary of such a dehydrogenase is glucose-6-phosphate dehydrogenase (G6PDH) from leuconostoc mesenteroides, which is capable of producing NADH or NADPH and is one of over 300 different NAD⁺ and NADP⁺ dependent dehydrogenases, many of which are useful in the detector complex.

Subsequent or simultaneous contact of the product NAD(P)H with FMN⁺, FMN⁺ oxidoreductase, aldehyde, oxygen and bacterial luciferase results in the formation of FMNH and in the emission of light. c) Using a detector complex having a second component comprising an NAD⁺ synthesizing enzyme such as ATP:NMN⁺ adenylate transferase, and contacting the bound detector complex with ATP and NMN⁺, NAD is generated.

Contacting the product NAD⁺ either simultaneously or subsequently with an NAD⁺ dependent dehydrogenase and an appropriate reduced substrate for the dehydrogenase generates NADH. Either simultaneous or subsequent contact of the NADH with FMN⁺ and FMN⁺ oxidoreductase, aldehyde, oxygen and bacterial luciferase results in the emission of light. d) Using a detector complex having a second component comprising active NAD⁺ as a limiting agent, and contacting the bound detector complex with an appropriate reduced substrate, and an appropriate dehydrogenase, NADH is generated. Either simultaneous or subsequent contact of the NADH with FMN⁺, and FMN⁺ oxidoreductase generates FMNH. Contact of the FMNH with aldehyde, oxygen, and bacterial luciferase, results in the emission of light. Such a system requires that the NAD⁺ and NADH be catalytically active with both the dehydrogenase and the FMN⁺ oxidoreductase. e) Using a detector complex having a second component comprising active FMN⁺ and contacting the bound detector complex with NAD(P)H, FMN⁺ oxidoreductase, aldehyde, oxygen, and bacterial luciferase, results in formation for FMNH and in the emission of light, 2) Aldehyde producing bridging reactions a) Using a detector complex having a second component comprising an appropriate alcohol dehydrogenase, and contacting the bound detector complex with NAD(P)⁺ and an appropriate alcohol, produces an aldehyde. By contacting this aldehyde either simultaneously or subsequently with FMNH, oxygen and bacterial luciferase, light is emitted. b) Using a detector complex having a second component comprising active NAD(P)⁺ which functions as a limiting agent in the generation of aldehyde by alcohol dehydrogenase, and contacting the bound detector complex with an appropriate alcohol and an appropriate dehydrogenase, results in the production of an aldehyde and NAD(P)H. Simultaneous or subsequent contact of the product aldehyde with oxygen, FMNH and bacterial luciferase produces light. A second dehydrogenase and its substrates added to this reaction system regenerates the product NAD(P)⁺ from NAD(P)H.

From the foregoing, it will be apparent that the second component of the detector complex of the present invention can vary widely, and it should be understood that those disclosed are for illustrative purposes only, and are not intended to limit the scope of the invention. While those skilled in the art will recognize that several hundred different detector complexes can be used with the various light emitting systems available, as a practical matter the preferred detector complexes are much smaller in number, since many may include substances which are expensive, unstable, difficult to obtain, cumbersome in use, or are low in sensitivity.

The most preferred forms of the detector complex are those whose second component, when coupled to a light emitting system, causes the latter to emit a large number of photons. Such detector complexes typically include in their second components an enzyme with a relatively high catalytic turnover, or a molecular substance which can undergo multiple cycles in a short period of time, with each cycle having a high probability of emitting a photon. The second component of such complex can be coupled directly into one of the luminescent reactions, or it can be indirectly coupled to such a reaction, functioning as a limiting or essential substance therefor through a bridging reaction.

The chemical substances of which the second component of the detector complex is preferably comprised include such enzymes as the oxidoreductases, such as FMN⁺ oxidoreductase, glucose-6-phosphate dehydrogenase (G6PDH), malate dehydrogenase, alcohol dehydrogenase, lactate dehydrogenase or triose phosphate dehydrogenase; alkaline phosphatase; adenyltransferase; NAD⁺ synthetase or ATP synthetase; pyruvate kinase, creatine kinase or adenylate kinase; glucose oxidase, xanthine oxidase, or monoamine oxidase; peroxidase; bacterial or firefly luciferase; pyranosidases such as beta galactosidase, neuraminidase or fucosidase whose reaction products can be fluorescers; or enzymes whose reaction products can be other substances which can be coupled directly or indirectly to light emitting systems.

In addition, the second component of the detector complex can comprise luciferin or any enzymatically active coenzyme, such as NAD⁺, NADP⁺, ATP, ADP, AMP, FMN⁺ or

FAD⁺; catalysts, such as iron-heme and various metals; and substances which are luminescent in the presence of a catalyst and oxygen or hydrogen peroxide, such as luminol, isoluminol, pyrogallol, lucigenin and lophine.

The second component can also usefully comprise a fluorescer such as, for example, 9, 10,diphenylathracene, perylene, rubrene, bis [phenylethynyl] anthracene (BPFA) and umbelliferone or dansyl derivatives; or fluorescer exciting substances such as bis (2, 4, 6-trichloroρhenyl) oxalate (TCPO) and bis (2-carbopentoxy-3, 5, 6-trichlorophenyl) oxalate (CPPO) and other suitable oxalate analogs.

For further discussion of fluorescers and exciting substances, such as oxalates useful therewith, see Maulding, D.R. and Roberts, B.G. J. Org . Chem . , 34 , supra ;

Tseng , S . S . , et al . , J . Org . Chem. , 44, supra; and Gill, S.K. Aldrichimica Acta, 16, supra. Additional discussion of chemiluminescent and bioluminescent light emitting systems can be found in Clinical and Biochemical Luminescence, 12, supra, and Methods in Enzymology, 57, supra.

Like the second component of the detector complex, the reporter group binding substance of which the first component thereof is comprised can also be selected from a variety of substances. Useful reporter group binding substances are those capable of forming high specific affinity interactions with reporter groups having a size smaller than about 10,000 daltons, It is preferable that the interaction of the reporter group binding substance with a reporter group exhibit an affinity greater than about 10⁸. Exemplary of preferred detector complex first components are those which comprise proteins which bind certain vitamin reporter groups with high affinity, such as avidin or streptavidin which bind biotin reporter groups; proteins which bind certain cofactor reporter groups with high affinity such as apomyoglobin which binds porphyrin reporter groups; antibodies which bind specific antigenic reporter groups with high affinity, such as antibodies for dinitrophenol, biotin, iminobiotin, desthiobiotin, fluorescein or fluorescamine reporter groups; lectins which bind carbohydrate reporter groups with high affinity, such as cocanavilan A which binds mannose reporter groups; and chelating agents which selectively bind metal reporter groups. Also useful as the first component are those comprising chemical moieties which give highly specific reactions with particular reporter group functionalities. In such cases, recognition of a reporter group by the detector complex results in the formation of a covalent bond between the reporter group and the detector complex as a result of chemical moieties on the reporter group and the first component of the detector complex forming a product coupling the two species.

Formation of such a covalent bond between a reporter group and a detector complex first component can be accomplished in any of a variety of chemical reactions, such as the following, wherein the reacting moieties and resultant products are indicated: 1) The reaction of primary amines with active esters to form amides; 2) The reaction of alcohols with active esters to form esters; 3) The reaction of amines and alcohols with epoxides to form substitued amines and ethers, respectively; 4) The reaction of amines with isothiocyanates to form thioureas; 5) The reaction of organic mercury salts with olefins to form substituted olefins; 6) The reaction of thiols with maleimides to form thioethers; and 7) The reaction of diazonium salts with aromatic substances to form diazo compounds.

Those skilled in the art will recognize the variety of additional chemical reactions useful for the formation of the covalent bond. In all cases, however, the binding reaction employed must be confined to the detector complex and the reporter group involved.

With respect to the synthesis of the detector complex attachment of the first and second components thereof to each other can be carried out in a number of ways which include the following: 1) The direct covalent attachment of the components to each other by the covalent spontaneous binding of chemical moieties on the surfaces thereof to form a product coupling the two species, such as an ether, ester, thiourea, amide, thioester, thioether, substituted olefin or diazo compound. Covalent attachment can also be produced by activation of such chemical moieties by a condensing agent, such as the reaction of amines, thiols, and alcohols together with acids in the presence of a carbodiimide, thionylchloride or other carboxylate activating agent;

2) The covalent attachment of the components to each other through a bifunctional coupling agent having peripheral functional groups which covalently bind to chemical moieties on the respective components and having an internal portion which provides a linkage between the two species. Exemplary of such bifunctional coupling agents are those which link amines and/or thiols and/or alcohols;

3) The formation of a high affinity noncovalent interaction between the components.

The preferred synthesis of the detector complex employs a noncovalent interaction of the type referred to earlier herein as useful for binding the detector complex to a reporter group, i.e. a vitamin-protein interaction; a cofactor-protein interaction; an antigen-antibody interaction; a carbohydrate-lectin interaction; or any other suitable noncovalent high affinity specific binding interaction.

An example of a detector complex in which the components thereof are bound by a noncovalent protein-vitamin, e.g., avidin-biotin, interaction is one in which the first component thereof is avidin, and the second component thereof is biotinylated FMN⁺ oxidoreductase. Since avidin has four biotin-binding sites, the detector complex has an excess of such binding sites, making it particularly useful for the detection of the presence of biotin as an immobilized reporter group. The FMN oxidoreductase component can be readily coupled to light emission using the bacterial bioluminescence system and NADH. The synthesis of the detector complex can, if desired, be carried out by an interaction binding the second component thereof to the first component thereof after binding of the latter to a reporter group fixed to a support matrix, as in Examples 1, 4, and 5 hereinafter. It is understood, therefore, that when reference is made herein to contact of a reporter group by a detector complex, such contact includes the sequential contact of the detector complex components with the reporter group inherent in such synthesis, as well as contact of a reporter group by a detector complex synthesized prior to contact thereof with such reporter group . Regardless of the synthetic procedure used, implicit therein is the requirement for retention of the chemical and/or biochemical activity of the respective components coupled thereby. If it is found that a desirable activity is lost during complexation, alternate chemistry in which the desired activity is retained should be employed.

The selection of components for use in a detector complex and of the light emitting system to be coupled therewith should take into consideration several factors, some of which are: 1) The individual components of a detector complex should function properly under acceptably similar conditions of pH, temperature, salt concentration, etc.

2) The second component, when coupled to an enzyme. coenzyme, or fluorescer, should yield a product which canbe detected by an appropriate light emitting system.

3) The product referred to in 2) should be formed in an amount sufficient to provide the desired sensitivity with the particular light emitting system. In this regard, it should be expected that the catalytic activity of a detector complex, when immobilized by attachment to an immobilized reporter group, is lower than that of its catalytic component when uncomplexed.

4) If sensitive detection requires the accumulation of a reaction product over a period of time, it is necessary that such product be stable over the required time period. For example, it would be ill advised to use a light emitting system requiring accumulation of FMNH over an extended period of time, since FMNH is very unstable, and in the presence of oxygen has a half-life of less than one second.

When practicing the method of the present invention by the use of a light emitting system which undergoes a first reaction in which the production of an intermediate product is catalyzed and a second reaction by which light is emitted, the optimal conditions for catalyzing the production of the intermediate product may be different from those for the emission of light. Where such is the case, the catalytic activity of the second component of the detector complex can be maximized in several ways, as follows:

1) By carrying out the first reaction, which involves the catalytic activity of the detector complex, under conditions which are generally optimal for the formation of the intermediate reaction product, and then carrying out the second reaction under conditions generally optimal for light emission.

2) By solubilizing at least the catalytic component of the detector complex before it is used in the first reaction to generate the intermediate reaction product. This is done because soluble catalytic components, e.g. enzymes, normally have greater activity than immobilized components. Solubilization can be achieved in several ways, depending upon the nature of the detector complex. These include: a) Dissociation of the detector complex from the reporter group to which it is bound using a change in pH, temperature, salt concentrations, etc. Thermal separation can be accomplished by the use of a detector complex whose first component comprises a thermally unstable antibody, and whose second component comprises a thermally stable substance. Separation of a detector complex from a reporter group in response to a change in pH is facilitated by selection of a reporter group and a detector complex first component which form a high affinity interaction which dissociates under mildly acid or mildly basic conditions. For example, the interaction between an avidin reporter group and an iminobiotin detector complex first component is one of high affinity at neutral pH, but it dissociates at the mildly acid pH of 4.5. b) The use of a detector complex whose components are coupled by a linkage which can be cleaved under mild conditions. Examples of such linkages are disulfides. which are cleaved by thiols; vicinal diols, which are cleaved by periodates; and mildly active esters or carbamates, which are hydrolyzed by mild bases. c) The use of an enzymatially digestible support matrix to which the reporter group is bound. An example of such a support matrix is one formed of amylopectin which is digestible by amylase. d) The use of an enzymatically digestible chemical bond by which the reporter group is immobilized.

As mentioned earlier herein, the detector complexes most preferred for use in the method of the present invention are those whose second component, when coupled to a light emitting system, causes the latter to yield multiple photons for each detector complex molecule. Such multiple photon emissions are possible when the second component of the detector complex comprises or can be coupled to an enzyme, a coenzyme, or a fluorescer. The benefits of using enzymes arise from the fact that each molecule of an enzyme can in one minute convert to product a large number of molecules of appropriate substrate. An efficient catalytic rate of an enzyme is referred to as a high turnover number, and it is advantageously at least 10/min., and preferably in the range of 10² to 10⁵/min. or higher. Such preferred turnover numbers can, in principle, give an increase of 2 to 5 orders of magnitude in sensitivity, but there are normally at least some decreases in activity associated with complexation (See Sundaram, P.V. et al., Can. J. Chem. 48, 1498-1504, (1970) and Carlsson, J, and Svenson, A., FEBS Lett., 42, 183-186 (1974). In general, however, enzymes retain reasonable activity when both complexed and immobilized.

Enhanced sensitivities may also be achieved if the second component comprises a coenzyme or cofactor. This benefit does not arise as a result of multiple products being generated, but rather as a result of the catalytic action of the coenzyme or cofactor in activation of the enzymatic reaction. Such action of the coenzyme NAD⁺ is illustrated in the following cyclic reaction:

In the illustrated reaction, enzyme 1 regenerates NADH and enzyme 2 utilizes NADH for a bridged light emitting reaction. This requires that enzymes 1 and 2 be able to use the complexed or conjugated coenzyme NAD⁺, or that an active coenzyme or cofactor can be liberated from the complex prior to assay. Enzyme 1 can be any of a wide range of dehydrogenases. Enzyme 2can, for example, be FMN oxidoreductase, and the bacterial luminescent reaction can be used therewith to provide light emission. Conjugated and immobilized forms of the coenzymes NAD⁺ and NADP⁺ which retain catalytic activity have been synthesized. (See, for example, Weibel, M.K. et al., in Enzyme Engineering (E.K. Pye and L.B. Wingard, eds.) Vol. 2, p. 203 - Plenum, New York, 1974; Larsson, P.O. and Mosback, K., FEBS Lett. 46, 119 (1974); Mosbach, et al., Methods in Enzymology, 44, 859-887 (1976)).

The second component of a detector complex may also comprise ATP which can function as a catalytic reagent. However, the use of ATP probably requires its release from the detector complex. As long as the released form of ATP is an analog with a long chain aliphatic derivative at the N6 or N8 position of the purine ring, there is a good chance that its catalytic activity with enzymes will be retained (Mosbach, K. et al., Methods in Enzymology, 44, supra. One such use of ATP is illustrated in the following reaction

Wherein ATP-m is ATP modified, and AMP-m is AMP modified, and PRPP is phosphoribosyl pyrophosphate. In addition, a detector complex may be one which can catalyze the formation of a product which is a catalyst for a separate reaction. Systems in which such a complex is useful include the following:

When the second component of the detector complex comprises a catalytic substance, such as an enzyme, which can cause the rapid formation of a catalyst product from a procatalyst, very sensitive detection can be expected due to the amplifying effects of the two reaction The enzymatic formation of NAD⁺ , the enzymatic productionof fluorescer molecules, and the enzymatic activation of a second enzyme fall into this category. Typical reaction for generation of NAD⁺ and fluorescer, respectively, as well as bioluminescent and chemiluminescent reactions respectively useful therewith can be illustrated as follows:

NAD Formation Reactions

B. Light Emitting Reaction (Bioluminescent)

a = an appropriate dehydrogenase b = FMN⁺ oxidoreductase c = bacterial luciferase

C. Profluorescer to Fluorescer Conversion Reaction

Exemplary of this reaction are the conversion of the profluorescer 4 methylumbelliferyl-N-acetyl-beta-D- glucosamine to the fluorescer 4 methylumbelliferone in the presence of the enzyme beta galactosidase.

D. Light Emitting Reaction (Chemiluminescent)

In this light emitting reaction an oxalate derivative, such as bis (2, 4, 6-trichlorophenyl) oxalate (TCPO) or bis(2-carbopentoxy-3,5,6-tricholorphenyl) oxalate (CPPO) is converted to a cyclodioxetane, and the energy liberated by the collapse the cyclodioxetane to CO₂ is absorbed by the fluorescer, causing exitation of the fluorescer. Subsequent relaxation of the fluorescer to the ground state results in the emission of light.

As mentioned earlier herein, the present invention has one central purpose, i.e. detection of reporter groups bound to a support matrix. Since the presence of immobilized reporter groups is dependent upon the interaction binding them to the support matrix, the method of the invention can also be used to quantitate interactions. which effect the ability of the reporter group to become immobilized. The interaction by which a reporter group is bound to a support matrix may be a chemical reaction; an antibody-antigen interaction; a carbohydrate-lectin interaction; a vitamin-protein interaction; a cofactor-protein interaction; a nucleic acid interaction, such as a DNA-DNA, RNA-DNA or RNA-RNA hybridization interaction; a metal-chelating interaction; or any other suitable specific ligand-ligand interaction or chemical reaction. One such interaction which can be monitored by the method of the invention is a carbohydrate-lectin interaction by which a reporter group is immobilized on a support matrix. For example, a lectin can be adsorbed to a support matrix, and a reporter group can be covalently attached to a carbohydrate in a region of the latter which will not interfere with its recognition by the lectin. When the reporter group-carbohydrate complex is brought into contact with the lectin adsorbed to the support matrix, the resulting lectin-carbohydrate interaction causes the reporter group to become immobilized on the support matrix. The detector complex can then be utilized in the method of the invention to detect the presence of the specific immobilized lectin involved in that interaction.

In another application of this concept , the immobilization of a reporter group on a support matrix through a DNA-DNA interaction can be monitored. For example, an organic polymer in the form of a DNA strand may be adsorbed on a support matrix, and a reporter group may be covalently coupled to a second DNA strand, such as a single strand oligonucleotide, which is complementary to the immobilized DNA strand. Hybridization of the DNA strands binds the reporter group. to the support matrix. In such a case, the method of the invention can be used to monitor the hybridization reaction. By detecting the immobilized reporter group, the method also detects the presence of the specific DNA strand adsorbed on the support matrix.

In practicing the method of the present invention, various means may be used to sense the light emitted by the light emitting reaction, some of which are utilized in the examples which follow. Among such light sensitive means are a lumincmeter, light sensitive film, and a light sensitive charge coupled device or other suitable and desired light sensitive means.

In each of the following examples detection of immobilized reporter groups by the method of the invention is demonstrated, as are various specific adaptations of the method. EXAMPLE 1

The Detection of Agarose-Bound Biotin Reporter Groups Using a Detector Complex of Avidin and a Biotin-Rich Aggregate of Biotinylated G6PDH and Avidin (BAC)

In this example, biotin is the reporter group, and the detector complex has avidin as its first componentand has as its second component a noncovalent biotinrich aggregate which consists of several molecules of biotinylated glucose-6-phosphate dehydrogenase held together with avidin. The detector complex is coupled to the generation of NADH from the oxidation of glucose-6- phosphate by the enzyme in the presence of NAD⁺. NADH generated by the action of the enzyme is contacted with Bactilight I reagent (Beneckea harveyi bacterial luciferase and FMN⁺ oxidoreductase, available from Analytical Luminescence Laboratory, San Diego, CA 92121) to produce light emission. In the light emission reaction, NADH is used to reduce FMN⁺ to FMNH, which is, in turn, oxidized in the presence of an aldehyde to reform FMN⁺ with the concomitant emission of a photon.

The synthesis of biotinylated glucose-6-phosphate dehydrogenase (G6PDH: E.C. 1.1.1.49) from Leuconostoc mesenteroides is accomplished as follows. One half ml G6PDH (2 mg/ml) was dialysed against one liter 0.1M sodium bicarbonate overnight. The dialysis was repeated for 2 hours and the dialysate removed from the dialysis bag. NADH and glucose-6-phosphate were added to a final concentration of 5 mM and 10 mM, respectively, in a volume of 1.4 ml. To this solution was added 50 μl of N-hydroxy- succinimide biotin ( 2 mg/ml in DMSO) , and the solution was incubated at room temperature. G6PDH activity was followed by removing small aliquots of the reaction mixture and assaying enzymatic activity by monitoring the production of NADH spectrophotometrically in 1 ml of 55 mM Tris (pH 7.8) containing 3.3 mM magnesium chloride, 1.7 mM NAD⁺ , and 2.8 mM glucose-6-phosphate. When the activity of the enzyme had decreased to 50% of its initial activity, 50 mg ammonium sulfate was added to stop the reaction. The biotinylated enzyme was then dialysed against 0.1 M sodium bicarbonate for 20 hours, and the resultant solution was stored at 4° C. Biotinylation of the enzyme was confirmed by showing that more than 90% of the enzyme activity was absorbed on an avidin- agarose column (Pierce Chemical Co., Rockford, IL), in the presence of 0.2 M NaCl. Control columns pretreated with an excess of biotin did not bind the biotinylated enzyme.

The BAC component of the detector complex is made by forming an aggregate with an excess of biotinylated dehydrogenase over avidin (i.e. the BAC component is biotin rich). The BAC aggregate is formed by adding four μl of a solution of Avidin-D (0.5 mg/ml PBS-Tween 20) to 1 ml of PBS-Tween 20. Avidin-D is available from Vector Laboratories, Burlingame, CA, and PBS-Tween 20 is phosphate buffered saline (0.9% NaCl, 10 mM sodium phosphate, pH7) containing 0.1% Tween 20. To this dilute solution 12 μl of biotinylated enzyme (0.4 mg enzyme/ml 0.1 M NaHCO₃) is added with mixing. The aggregate is allowed to form for at least fifteen minutes and is then stored at 4° C until used. Immediately before use, the BAC aggregate is centrifuged for 5 minutes in a tabletop centrifuge (ca 3000 rpm) in order to remove overly large aggregates which contribute to non-specific binding.

Ten microliters of a slurry of biotin-agarose beads (Pierce Chemical Co., 1.5 x 10^-11 avidin binding capacity) is diluted into 1 ml PBS-Tween 20. This slurry is then dispensed in 100 microliter aliquots into 1.5 ml Eppendorf tubes. The contents of each tube are washed twice with 1 ml PBS-Tween 20, then incubated for 30 minutes with 40 μl avidin D (0.5 mg/ml, 0.3 nmoles) in order to complex the biotin. The agarose-biotin:avidin aggregate is washed free of unbound avidin with 1 ml PBS-Tween 20 and resuspended in 100 μl of the same. Ten microliter portions of the suspension are then aliquoted into 1.5 ml Eppendorf tubes. To each tube is added 200 μl of the BAC complex (12 pmoles with respect to biotinylated G6PDH) and the mixture allowed to react for 15 minutes to form an agarose-biotin:avidin-BAC complex. Control beads were prepared by incubating the agarose-biotin-avidin beads with excess biotin (40 nM) prior to incubation with BAC. Unbound BAC was then removed by 6 washes with 2x standard sodium citrate buffer (SSC) consisting of 0.15 M NaCl, and 0.015M sodium citrate. Selected individual agarose beads were then removed from each tube under a microscope and assayed for bioluminescence in a Monolight 401 luminometer (Analytical Luminescence Laboratories, San Diego, CA). A reagent solution was prepared from dodecyl aldehyde (0.0005%), FMN⁺ (3 x 10^-6 M), NAD⁺ (3 mM), glucose-6-phosphate (5 mM), magnesium chloride (3mM) and tris buffer (30 mM,pH 7.8) in a final volume of 40 μl. After the addition of a single agarose bead to the reagent solution, the light emitting reaction was initiated by the addition of 10 μl Bactilight I reagent (made up as per the manufacturer's instructions), and the course of the reaction monitored in the Monolight 401 luminometer on a sensitivity setting of 10x. Typical results, along with the appropriate controls, are given in Table I.

The avidin binding capacity of the biotin-agarose was independently determined to be 1.5 picomoles per microliter of the original slurry. One bead 70 microns in diameter thus corresponds to about 10 moles of biotin. From these data it can be estimated that, using the method of the present invention, the detection limit for agarose bound biotin is about 10^-16 moles of biotin. EXAMPLE 2

The Detection of Agarose-Bound Biotin Reporter Groups Using an Avidin-Biotinylated G6PDH Detector Complex

This example is the same in all respects as Example 1, with the exception that the detector complex has as its first component avidin, and has as its second component biotinylated glucose-6-phosphate dehydrogenase (biotin- G6PDH). In this case, 200 μl of a biotin-G6PDH solution (~12 pmol) is added to each sample using the same procedure as in Example 1. The ultimate complex which is formed here is an agarose-biotin:avidin-biotin-G6PDH complex which is assayed for bioluminescence in the Monolight 401 luminometer as described in Example 1. The resulting data are given in Table II.

Based on these results, it is estimated that the detection limit for biotin bound to agarose using this detector complex is about 10^-16 moles of biotin.

EXAMPLE 3

In this example the detector complex was formed by adding an excess of avidin to biotinylated glucose-6- phosphate dehydrogenase. This detector complex has the advantage that no intermediate treatment with avidin and subsequent washes are necessary to detect biotin bound to the agarose beads as is the case in Examples 1 and 2. As in Examples 1 and 2, the reporter group is biotin, and a Monolight 401 luminometer is used for luminescence detection.

The complex of biotinylated glucose-6-phosphate dehydrogenase and avidin was formed by rapidly mixing 12 μl of streptavidin (0.5 mg/ml, Bethesda Research Laboratories, Bethesda, MD) with 3 μl of biotinylated glucose-6-phosphate dehydrogenase (0.42 mg/ml) in 125 μl PBS-Tween 20.

Three hundred μl of biotin-agarose suspension (Sigma Chemical Company, St. Louis, MO) was washed twice with 1 ml PBS-Tween 20 and suspended in 1 ml of the latter. Small aliquots of the biotin-agarose suspension were then reacted with 10 μl aliquots of the aggregate, followed by agitation on a wrist action shaker for 1 hour at room temperature. The samples were then washed 3 times with 1 ml PBS-Tween 20 containing 0.5 M NaCl. Assaying the samples using a Monolight 401 luminometer according to the procedures in Examples 1 and 2, produces results similar to those produced in such examples.

EXAMPLE 4

The Detection of Biotin Reporter Groups on a Nitrocellulose-Bound DNA Using an Avidin-BAC

Aggregate Detector Complex

In this example, the reporter group is bound to the support matrix through an immobilized ligand, and the detector complex has avidin as its first component, and has the biotin rich BAC aggregate described in Example 1 as its second component. Lambda phage DNA was biotinylated using nick translation in the presence of biotinylated dUTP in which the biotin is covalently attached to the C-5 position of deoxyuridine via an 11 atom linker arm. The reagent system used to biotinylate the phage DNA was the nick translation reagent system marketed by Enzo Biochemicals, New York, NY. The reaction yielded Lambda DNA in which 31 % of the thymidine residues had been replacedwith biotinylated deoxyuridine, as judged by the concomitant incorporation of tritium labeled dCTP of known specific activity. The DNA was purified away from contaminating unincorporated dUTP using ethanol precipitation and Sephadex G-25 chromatography.

Biotinylated lambda DNA was denatured by heating to 100° C for 5 minutes in distilled water followed by chilling on ice to prevent reannealing of the single strands. Various amounts of the denatured DNA were then bound to nitrocellulose filters (Schleicher and Schuell, Catalog #BA 85) by direct spotting of the DNA to the filters in the presence of 6x SSC. The filters were allowed to air dry overnight and were then baked for 1 hour at 60° C in vacuo in order to fix the biotinylated DNA thereto. The amount of biotinylated DNA bound to each filter was quantitated by liquid scintillation counting. The average binding efficiency of the filters was 56% of the input DNA. Control filters were prepared using unmodified calf thymus DNA (1 microgram of DNA per filter). BAC aggregates of biotinylated G-6-PDH and avidin were prepared as in Example I.

The procedure used to assay filters for the presence thereon of immobilized biotinylated DNA, was as follows. The unreacted sites on the filters were "capped" by incubation for 30 minutes at 37° C with 2% bovine serum albumin in PBS-Tween 20. The capped filters were then washed twice for 5 minutes at room temperature with 5 ml PBS-Tween 20. At this point the reactive grouping of the filter-bound biotinylated DNA can be diagrammed as filter-DNA-biotin. The filters were then treated for 30 minutes at room temperature with 20 μl streptavidin solution (prepared by diluting 15 μl streptavidin to 1.5 ml with PBS-Tween 20) to form a filter-DNA-biotin:avidin complex. Unbound streptavidin was then removed by two 5 minute washes with 5 ml PBS-Tween 20, and the filters were blotted dry to remove excess solution.

The filter-DNA-biotin:avidin complex was then reacted with 15 μl BAC aggregate in PBS-Tween 20 for 30 minutes at room temperature to form a filter-DNA-biotin : avidin- BAC complex . The unbound detector complex was removed by washing the filters 6 times for 5 minutes with 5 ml 6x SSC and one time for 5 minutes with 5 ml Tris buffer. The center portion of each area of the filter bearing the filter-DNA-biotin:avidin-BAC complex was then punched out using a cork borer. The punched-out portion was placed in a microtiter well which contained 50 μl of an NADH-generating solution composed of 55 mM Tris buffer, pH 7.8, 3.3 mM magnesium chloride, 2 mM NAD and 3.3 mM glucose-6-phosphate. After 1 hour of incubation at room temperature, the supernatant solution containing enzyme-generated NADH was transfered to a 5 ml plastic cuvette containing 400 μl 0.0005% dodecyl aldehyde in 0.1 M potassium phosphate buffer, pH 7. These samples were then placed in a Monolight 401 luminometer and assayed for NADH content by adding 100 μl Bactilight I reagent and measuring peak light output over a 10 second interval according to the manufacturer's instructions. The amount of bound biotinvlated DNA corresponded linearly with the amount of light emitted.

EXAMPLE 5

The Bioluminescent Detection of a Nitrocellulose-Bound Hepatitis B Virus DNA Using a Biotin Reporter Group Labeled Oligonucleotide and an Avidin-BAC Detector Complex

One application of this invention is the nonradio-active detection of DNA-DNA hybrids formed between an oligonucleotide labeled with a reporter group such as biotin, and immobilized matrix-bound complementary DNA. Such probes have recently found application in the detection of specific disease organisms such as hepatitis B virus (HBV) and herpes simplex virus (HSV) types 1 and 2. In this example a biotinylated oligonucleotide twenty-one nucleotides in length and complementary to the HBV surface antigen gene was synthesized by the method disclosed in U.S. Patent application Serial No. 06/468,498 which is assigned to the assignee of the present application. In this oligonucleotide two of the thymidine residues were substituted with biotinylated deoxyuridine. The biotin was covalently attached to the 5 position of deoxyuridine by a twelve atom linker arm. The HBV immobilized ligand was a recombinant DNA plasmid termed pAM6 which carries the entire HBV genome cloned into pBR322 (Moriarty et al., PNAS , 78, pages 2606-2610 (1981)). The HBV DNA contained in the plasmid pAM6 was adsorbed to nitrocellulose filters essentially as described in Example 4 for lambda DNA. Control filters made at the same time contained only calf thymus or herring sperm DNA.The filters were then treated for 30 minutes in 6x SSC (0.9M NaCl and 0.09M sodium citrate) containing 10x

Denhardt's solution (0.02% bovine serum albumin, 0.2% polyvinylpyrrolidone and 0.2% Ficoll) in order to cap the unreacted sites on the nitrocellulose and prevent nonspecific binding of the probe to the filter material. Ficoll is a sucrose polymer sold by Pharmacia Fine Chemicals Piscataway, N.J. The filters were then treated with the biotinylated DNA probe (2 ng/ml) overnight at 46° C in 6x SSC containing 1x Denhardt's solution in order to form a complex which may be diagrammed as filter-HBV: oligonucleotide-biotin. The unbound oligonucleotide was washed off the filter by three 15 minute washes with 6x SSC on ice, followed by a one minute wash in 6x SSC at 46° C. The filters were then capped once again to prevent the nonspecific binding of the Avidin-BAC detector complex. This was done by incubating the filters for 30 minutes on ice in 3% bovine serum albumin dissolved in Buffer A (0.5 M NaCl, 50 mM sodium phosphate buffer, pH 8, and 0.05% Tween 20). The capping solution was removed by three 3 minute washes with Buffer A on ice. The filters were then treated with streptavidin (20 μg/ml in buffer A) for 30 minutes on ice in order to form a filter-HBV: oligonucleotide-biotin-streptavidin complex. Unbound streptavidin was removed by three 3 minute washes in Buffer A on ice. The filters were then treated for 30 minutes with the BAC aggregate essentially as described in Example 4, and the unbound BAC aggregate was removed by three 3 minute washes with ice cold Buffer A. At this point the entire bound complex consisted of filter-HBV: oligonucleotide-biotin: streptavidin-BAC.

In order to detect the amount of immobilized biotin reporter group and thereby the amount of immobilized HBV DNA, as well as the amount of complex formed between the oligonucleotide and the immobilized DNA, the filters were incubated for 10 minutes at room temperature in 50 μl of the NADH-generating solution described in Example 4. The NADH generated was detected in Monolight 401 luminometer using the Bactilight I reagent as follows. A 25 μl aliquot of each assay mix was added to a 5 ml plastic reaction cuvette containing 400μl 0.0005% dodecyl aldehyde in 0.1 M potassium phosphate buffer (KPB) , pH 7. This mixture was placed into the luminometer and the backgound light emission was recorded. The luminescence reaction was initiated by the injection of 100 μl

Bactilight I reagent containing FMN +, NAD⁺:FMN⁺ oxidoreductase and bacterial luciferase. The pulse of light generated in 10 seconds was approximately linear with respect to the amount of bound biotin reporter group and the amount of bound hepatitis B virus DNA on the nitrocellulose filters.

EXAMPLE 6

The Bioluminescent Detection of Cellulose-Bound Anti-Herpes Antibody Using a Biotin Reporter Group-Labeled Herpes Surface Antigen and an Avidin-Biotinylated G6PDH Detector Complex

Anti-herpes simplex virus (HSV) antibodies (obtained from Bethesda Research Laboratories, Bethesda, MD) are bound to cellulose discs using the metaperiodate procedure of Kricke, et al. (J. Clin. Chem. Clin. Biochem. 20:91-94, (1982). HSV type 1 cell surface antigen (available from Flow Laboratories, McLean, VA) is biotinylated by adding 50 μl N-hydroxysuccinimidobiotin (2 mg/ml in dimethyl-sulfoxide) to 1 mg of the antigen dissolved in 1 ml of 0.1 M sodium bicarbonate, followed by incubation at room temperature for 1 hour. The reaction is then quenched by adding 50 mg of ammonium sulfate, and the product is dialysed twice against 0.1 M sodium bicarbonate. Alternatively, the surface antigens may be sent to a commercial laboratory for custom biotinylation.

In order to detect the presence of anti-HSV antibodies bound to the cellulose discs, they are incubated for 30 minutes at 37° C with a 1 ng/ml solution of the biotinylate HSV antigen in PBS-Tween 20 containing 0.1% bovine serum albumin. The unbound antigens are then removed by washing the discs with three 5 ml washes in PBS-Tween 20-BSA. The detector complex used is the same as that used in Example 3, and discs are then treated for 30 minutes with said detector complex essentially as described in Example 4. The unbound detector complex is then removed by three 3 minute washes with Buffer A.

In order to detect the immobilized biotin reporter group, and thereby the amount of bound anti-HSV antibody present on the discs, the discs are suspended in the Bactilight I reagent as described in Example 1, and the rate of signal generation is measured as a function of time These values can be compared to a standard curve prepared by following the same procedure and using various known amounts of anti-HSV antibody, in order to determine the amount of anti-HSV antibody present. Using a similar procedure, anti-HSV antibodies may be quantitated from human serum. EXAMPLE 7

The Bioluminescent Detection of Nitrocellulose-Bound HSV DNA Using a Dinitrophenol Reporter Group-Labeled Oligonucleotide and an Antidinitrophenyl Antibody- G6PDH Detector Complex

Herpes simplex virus DNA (HSV DNA) which has been cloned into a plasmid (pHSV101 available from Bethesda Research Laboratories , Bethesda, MD ) , is bound to nitrocellulose according to the procedure described in Example 4. A herpes DNA oligonucleotide which has dinitrophenyl reporter groups attached to the C 5 position of deoxyuridine via an 11 atom linker arm is synthesized by the method of U.S. patent application Serial No. 06/468,498, which is assigned to the assignee of the present application. The dinitrophenylated herpes oligonucleotide, which is complementary to the immobilized HSV DNA, is complexed to the latter at an oligonucleotide concentration of 10ng/ml in 6x SSC containing 10x Denhardt's solution overnight at 37° C in heat sealed plastic bags. At the end of the incubation, excess unbound oligonucleotide is washed away with ten 5 ml washes using 6x SSC on ice.

Filters bearing the DNA duplex are then incubated with 2 % BSA in PBS-Tween 20 at 37° C for 30 minutes to cap off any remaining protein binding sites on the filter. After three 5 minute washes with PBS-Tween 20, the filtersare incubated with antidinitrophenyl antibody-G6PDH detector complex (~1 pmol) for 30 minutes at room temper-ature in 40 μl PBS-Tween 20.

The antiDNP-Ab-G6PDH detector complex is formed in a ratio of 1:3 according to the procedure of Carlsson, et al. (Biochem. J. 173:723-737, 1978) using N-succinimdyl- 3-(2(pyridyldithio)propionate. After washing the filter bound complex 6 times with 6x SSC/0.1% Tween 20, the light emission is quantitated as follows. Each individual filter is blotted dry to remove excess moisture and is transfered to an individual well in a microtiter plate. To each well is added 100 μl of Bactilight 1 reagent, and the reaction is allowed to reach a steady state which is achieved after approximately 1 hour of incubation at room temperature. The microtiter plate is then set on top of an 8 x 10 sheet of Kodak®XOMAT ® XAR-5 X-ray film in the dark, and the exposure is allowed to proceed for an appropriate period of time (generally from about 1 to about 15 hours). At the end of the exposure time, the film is developed in an automatic processor, and the amount of light emitted is quantitated using a densitometer The amount of light emitted is proportional to the amount of immobilized DNP reporter group and the amount the initially immobilized HSV DNA, as well as the amount of complex formed between the oligonucleotide and the immobil- ized DNA.

EXAMPLE 8

The Luminescent Detection of Agarose-Bound AntiHSV Antibody Using a Biotin Reporter Group- Labeled HSV Surface Antigen and an Avidin-Dansyl Detector Complex

Samples containing anti-HSV antibody (obtaining from Bethesda Research Laboratories, Bethesda, MD) are bound to CNBr activated agarose beads by standard procedures. The beads are suitably capped with non- specific goat IgG or bovine serum albumin (BSA). HSV type 1 or 2 cell surface antigen of the type used in Example 6 may be biotinylated by any number of methods, or by commercial custom biotinylation. A streptavidin- dansyl detector complex is produced by dansylating streptavidin using dansyl chloride according to standard procedures (c.f. Biochemical Journal 181:251, 1979).

In order to detect the presence of anti-HSV antibodies bound to the agarose support matrix, the capped beads are incubated for 30 minutes at 37 C with a 1 mg/ml solution of the biotinylated HSV antigens in

PBS-Tween 20. The unbound antigens are then removed by washing the beads with three 5 ml washes in PBS-Tween 20. The filters are then treated for 30 minutes with the detector complex essentially as described in Example 4, and the unbound detector complex is removed by three 3 minute washes with Buffer A. At this point the entire bound complex consists of bead-antibody: antigen-biotin: streptavidin-dansyl.

In order to detect the biotin reporter groups and thereby the amount of bound antiHSV antibody present on the agarose beads, the beads are suspended in 10 μl of 0.1M Tris, pH 7.5, and transferred to a glass cuvette to which is added the exciting agent, 250 μl bis (2,4,6-tri-cholorphenyl) oxalate (TCPO) in ethyl acetate, and 100 μl hydrogen peroxide in acetone (diluted from 30% aqueous hydrogen peroxide), which cause the dansyl groups to emit light. The final concentrations of TCPO and hydrogen peroxide are 1.7 mM and 0.7 mM respectively, and the rate of light emission per unit of time is measured in a light sensitive CCD. The values obtained are compared to a standard curve prepared by following the same procedure and using various known amounts of anti-HSV antibody, in order to determine the amount of bound anti-HSV antibody present on the beads. EXAMPLE 9

The Bioluminescent Detection of Nitrocellulose-Bound HSV DNA Using a Dinitrophenol Reporter Group-Labeled Oligonucleotide and an Anti DNP Antibody-Pyruvate Kinase Detector Complex

This example is similar to Example 7, except that pyruvate kinase is used as the second component of the detector complex instead of the G6PDH. The pyruvate kinase is employed to generate ATP which causes the firefly bioluminescence reaction to emit light.

A herpes oligonucleotide complementary to the immobilized HSV DNA and labeled with dinitrophenol is prepared and is complexed to the HSV DNA as in Example 7. After washing, the DNA duplex is incubated with an excess of the antiDNP antibody-pyruvate kinase detector complex which is formed in a 1:3 ratio following the procedure of Yoshitake, et al, (Eur. J. Biochem. 101:395-399, 1979). The incubation is done for 30 minutes at room temperature in PBS-Tween 20. After washing six times in PBS/0.5M NaCl/0/1% Tween 20, the filters containing the bound detector complex are incubated in 0.5 ml 0.05 M imidazole buffer, pH 7.6, containing 1.5 mM ADP, 1.5 mM phosphoenol pyruvate and firefly luciferin and luciferase (Firelight, available from Analytical Luminescence Laboratories, San Diego, CA) according to the manufacturer's instructions The amount of immobilized HSV DNA, as well as the amount of complex formed between the oligonucleotide and said DNA, is determined by the amount of light emitted with time using a light sensitive charge coupled device.

Claims

What is Claimed as the Invention is:

1. The method of luminescent detection of a specific reporter group smaller than about 10,000 daltons in size which is bound to a support matrix, comprising the steps of bringing into contact such reporter group and a detector complex having as a first component a binding substance which exhibits high specific affinity for such reporter group, and having as a second component a substance capable of being readily coupled to a light emitting system, thereby to produce upon such contact high affinity attachment of said detector complex and such reporter group; and then contacting at least the second component of the detector complex with a light emitting system capable of emitting light in the presence of the second component of the detector complex.

2. The method of Claim 1 wherein contact of the bound reporter group and the detector complex is made by initially binding the reporter group and the first component of the detector complex to each other, and then binding the second component of the detector complex and the bound first component thereof to each other.

3. The method of Claim 1 wherein the high affinity attachment of the detector complex to the reporter group involves formation of a covalent bond; or a ligand-ligand type interaction.

4. The method of Claims 1 or 3 wherein the reporter group is a vitamin, a cofactor, an antigen or a carbohydrate.

5. The method of Claim 4 wherein said vitamin is biotin, iminobiotin, desthiobiotin or pyridoxal phosphate.

6. The method of Claim 4 wherein said cofactor is a porphyrin.

7. The method of Claim 4 wherein said antigen is dinitrophenol, biotin, iminobiotin, desthiobiotin, fluorescein or fluorescamine.

8. The method of Claim 4 wherein said carbohydrate in mannose, galactose or fucose.

9. The method of Claims 1 or 2 wherein the reporter group is bound to the support matrix through an organic polymner coating comprising a protein, a carbohydrate, a nucleic acid or an analog thereof.

10. The method of Claims 1 or 2 wherein the high affinity attachment of the detector complex and the reporter group involves a vitamin-protein interaction; a cofactor-protein interaction; an antigen-antibody interaction, a carbo-hydrate-lectin interaction or a covalent bond.

11. The method of Claims 1 or 2 wherein the reporter group is a vitamin, and the first component of the detector complex comprises a vitamin binding protein.

12. The method of Claim 11 wherein said vitamin is biotin, iminobiotin or desthiobiotin, and said protein is avidin, streptavidin or a complex of avidin or streptavidin.

13. The method of Claims 1 or 2 wherein the reporter group is a cofactor, and the first component of the detector complex comprises a cofactor binding protein.

14. The method of Claim 13 wherein said cofactor is a porphyrin, and said protein is apomyoglobin.

15. The method of Claims 1 or 2 wherein the reporter group is an antigen, and first component of the detector complex comprises an antibody to said antigen.

16. The method of Claim 15 wherein said antigen is dinitro-phenol , biotin , iminobiotin , desthiobiotin , fluorescein or fluorescamine.

17. The method of Claim 1 or 2 wherein the reporter group is a carbohydrate, and the first component of the detector complex comprises a lectin.

18. The method of Claim 17 wherein said carbohydrate is mannose, galactose or fucose.

19. The method of Claim 17 wherein said carbohydrate is mannose, and said lectin is concanavilan A.

20. The method of Claims 1 or 2 wherein the first component of the detector complex is conjugated with avidin or streptavidin.

21. The method of Claims 1 or 2 wherein the first component of the detector complex is conjugated with biotin, iminobiotin or desthiobiotin.

22. The method of Claims 15 or 16 wherein said antigen is ditnitrophenol, fluorescein or fluorescamine, and said antibody is conjugated with avidin or streptavidin.

23. The method of Claim 15 or 16 wherein said antigen is dinitrophenol, fluorescein or fluorescamine, and said antibody is conjugated with biotin, iminobiotin or desthiobiotin.

24. The method of Claims 1 or 2 wherein the first and second component of the detector complex are attached to each other by a vitamin-protein interaction; a cofactor-protein interaction; an antigen-antibody interaction, a carbohydrate-lectin interaction or a covalent bond.

25. The method of Claim 24 wherein said vitamin is biotin, iminobiotin, desthiobiotin or a conjugate thereof, and the protein of said vitamin-protein interaction is avidin, streptavidin or a conjugate thereof.

26. The method of Claim 1 or 2 wherein one of said detector complex first and second components is an avidin or is conjugated with an avidin, and the other is conjugated with a biotin.

27. The method of Claim 1 or 2 wherein the second component of the detector complex comprises an enzyme, enzymatically active coenzyme, catalyst, fluorescer, fluorescer exciter, luciferin, or a substance which is luminescent in the presence of a catalyst and oxygen or hydrogen peroxide.

28. The method of Claims 1 or 2 wherein said second component of the detector complex comprises a dehydrogenase.

29. The method of Claims 1 or 2 wherein said second component of the detector complex comprises glucose-6-phosphate dehydrogenase.

30. The method of Claims 1 or 2 wherein said second component of the detector complex comprises biotinylated glucose-6-phosphate dehydrogenase.

31. The method of Claims 1 or 2 wherein the second component of the detector complex comprises FMN⁺ oxidoreductase, glucose-6-phosphate dehydrogenase, malate dehydrogenase, alcohol dehydrogenase, lactate dehydrogenase, triose phosphate dehydrogenase, alkaline phosphatase, adenyltransferase, NAD⁺ synthetase, ATP synthetase, pyruvate kinase, creatine kinase, adenylate kinase, glucose oxidase, xanthine oxidase, monoamine oxidase, peroxidase, bacterial luciferase, firefly luciferase, beta galactosidase, neuraminidase, fucosidase, or another enzyme whose reaction product can be a substance which can be coupled directly or indirectly to a light emitting system.

32. The method of Claims 1 or 2 wherein the second component of the detector complex comprises enzymatically active NAD⁺, NADP⁺, ATP, ADP, AMP, FMN⁺ or FAD⁺.

33. The method of Claims 1 or 2 wherein the second component of the detector complex comprises iron-heme or a metal.

34. The method of Claims 1 or 2 wherein the second component of the detector complex comprises 9,10, diphenylanthracene, perylene, rubrene, BPEA or an umbelliferone derivative.

35. The method of Claims 1 or 2 wherein the second component of the detector complex comprises TCPO or CCPO.

36. The method of Claims 1 or 2 wherein the second component of the detector complex comprises luminol, isoluminol, pyrogallol, lucigenin or lophine.

37. The method of Claims 1 or 2 wherein the second component of the detector complex comprises an enzyme which in the presence of a substrate therefor, has a turnover number of at least 10/minute.

38. The method of Claims 1 or 2 wherein the detector complex second component comprises a molecular substance which, when coupled to the light emitting system, undergoes multiple cycles which result in the production of light.

39. The method of Claims 1 or 2 wherein the light emitting system comprises components of a light emitting reaction and components of a bridging reaction therefor, and the second component of the detector complex, when coupled to said system, provides an essential or limiting constituent of the bridging reaction.

40. The method of Claims 1 or 2 wherein contact, of the light emitting system, and the detector complex causes a first reaction in which an intermediate product is produced, and a second reaction in which the light is emitted, the first reaction being carried out under conditions generally optimal for the production of the imtermediate product, and the second reaction being carried out under conditions generally optimal for light emission.

41. The method of Claims 1 or 2 wherein the light emitting system is bioluminescent.

42. The method of Claims 1 or 2 wherein the light emitting system is chemiluminescent.

43. The method of Claims 1 or 2 wherein, prior to said contact with the light emitting system,at least the second component of the detector complex is solubilized.

44. The method of Claim 43 wherein the second component if the detector complex is solubilized by chemical separation thereof from the bound first component of said complex.

45. The method of Claim 43 wherein the detector complex is solubilized by chemical separation thereof from the reporter group.

46. The method of Claim 43 wherein the reporter group and the detector complex bound thereto are solubilized by dissolution of the support matrix.

47. The method of any of the preceding claims which comprises the additional step of quantitating the light emitted by the light emitting system as a measure of the reporter group bound to the support matrix upon contact therewith.

48. The method of Claim 47 wherein the light emitted by the light emitting system is quantitated by the use of means including a luminometer or light sensitive charge coupled device.

49. The method of Claim 47 wherein the light emitted by the light emitting system is quantitated by the use of means including light sensitive film.

50. The method of any preceding claim wherein the reporter group is contacted with an excess of the detector complex, and the unbound detector complex is separated from the bound detector complex prior to contact of the second component thereof with the light emitting system.

51. A detector complex useful for the detection of low levels of an immobilized reporter group smaller than about 10,000 daltons in size, comprising a first component exhibiting a high affinity for a specific reporter group smaller than about 10,000 daltons in size; and a second component capable of being readily coupled to a light emitting system.

52. The detector complex of Claim 51 wherein the first and second components thereof have a high. affinity attachment to each other which involves covalent linking of said components through a bifunctional coupling agent; a covalent bond formed between chemical moieties on said components as a result of activation of such moieties by a condensing agent; a noncovalent ligand-ligand type interaction; or a covalent bond spontaneously formed between chemical moieties on said components.

53. The detector complex of Claim 51 wherein the components thereof are noncovalently attached to each other by a vitamin-protein interaction, a cofactor- protein interaction, a carbohydrate-lectin interaction or an antigen-antibody interaction,

54. The detector complex of Claim 51 wherein the first and second components thereof are attached to each other by a biotin-avidin type interaction, said first component comprising an avidin, a complex of an avidin or a complex of a biotin, and said second component comprising a complex of an avidin or a complex of a biotin.

55. The detector complex of Claim 51 wherein said first component comprises a vitamin binding protein and said second component comprises a vitamin.

56. The detector complex of Claim 55 wherein said protein is avidin or streptavidin, and said vitamin is biotin, iminobiotin or desthiobiotin.

57. The detector complex of Claim 51 wherein said second component comprises an antigen and said first component comprises an antibody to said antigen.

58 . The detector complex of Claim 57 wherein said antigen is dinitrophenol, biotin, iminobiotin, desthiobiotin, fluorescein or fluorescamine.

59. The detector complex of Claim 51 wherein said components are attached to each other by a bifunctional coupling agent linking amine, thiol or alcohol moieties on said components.

60. The detector complex of Claim 51 wherein said components are covalently attached to each other as a result of the reaction of amine, thiol or alcohol moieties on said components with a carboxylate which has been activated by a condensing agent.

61. The detector complex of Claim 60 wherein said condensing agent is a carbodiimide or thionychloride.

62. The detector complex of Claim 51 wherein the second component thereof, when coupled to a light emitting system, directly or indirectly provides a necessary constituent of a luminescent reaction.

63. The detector complex of Claim 51 wherein the second component comprises an enzyme, enzymatically active coenzyme, catalyst, fluorescer, fluorescer exciter, luciferin, or a substance which is luminescent in the presence of a catalyst and oxygen or hydrogen peroxide.

64. The detector complex of Claim 51 wherein the second component comprises a substance capable of converting a procatalyst to a catalyst.

65. The detector complex of Claim 51 wherein the second component comprises a substance capable of converting a profluorescer to a fluorescer.

66. The detector complex of Claim 51 wherein said second component comprises a dehydrogenase.

67. The detector complex of Claim 52 wherein said second component comprises glucose-6-phosphate dehydrogenase.

68. The detector complex of Claim 51 wherein said second component comprises biotinylated glucose-6-phosphate dehydrogenase.

69. The detector complex of Claim 51 wherein the second component comprises FMN⁺ oxidoreductase, glucose-6-phosphate dehydrogenase, malate dehydrogenase, alcohol dehydrogenase, lactate dehydrogenase, triose phosphate dehydrogenase, alkaline phosphatase, adenyltransferase,

NAD⁺ synthetase, ATP synthetase, pyruvate kinase, creatine kinase, adenylate kinase, glucose oxidase, xanthine oxidase, monoamine oxidase, peroxidase, bacterial luciferase, firefly luciferase, beta galactosidase, neuraminidase, fucosidase, or another enzyme whose reaction product can be a substance which, can be coupled directly or indirectly to a light emitting system.

70. The detector complex of Claim 51 wherein the second component comprises enzymatically active NAD⁺ ,

NADP⁺, ATP, ADP, AMP, FMN⁺ or FAD⁺.

71. The detector complex of Claim 51 wherein the second component comprises iron-heme or a metal.

72. The detector complex of Claim 51 wherein the second component comprises 9, 10, diphenylanthracene, perylene, rubrene, BPEA, or an umbelliferone or dansyl derivative.

73. The detector complex of Claim 51 wherein the second component comprises TCPO or CPPO.

74. The detector complex of Claim 51 wherien the second component comprises luminol, isoluminol, pyrogallol, lucigenin or lophine.

75. The detector complex of Claim 51 wherein the second component comprises an enzyme which, in the presence of a substrate therefor, has a turnover number of at least 10/minute.

76. The detector complex of Claim 51 wherein the second component comprises a molecular substance which, when coupled to the light emitting system, undergoes multiple cycles which result in the production of light.

77. The detector complex of Claim 51 wherein the first component thereof comprises a chemical moiety capable of forming a covalent bond with a reporter group, or a ligand capable of forming a noncovalent ligand-ligand interaction with a reporter group.

78. The detector complex of Claim 51 wherein the first component comprises a protein capable of forming a vitamin-protein interaction with a vitamin reporter group, a protein capable of forming a cofactor-protein interaction with a cofactor reporter group, an antibody capable of forming an antigen-antobody interaction with an antigenic reporter group, or a lectin capable of forming a carbohydrate-lectin interaction with a carbohydrate reporter group.

79. The detector complex of Claim 51 wherein the first component comprises avidin or streptavidin.

80. The detector complex of Claim 51 wherein the first component comprises apomyoglobin .

81. The detector complex of Claim 51 wherein the first component comprises an antibody to dinitrophenol, biotin, iminobiotin, desthiobiotin, fluorescein or fluroescamine.

82. The detector complex of Claim 51 wherein the first component comprises a lectin.

83. The detector complex of Claim 51 wherein the first component comprises concanavilan A.

84. The detector complex of Claim 51 wherein the first component is conjugated with avidin or streptavidin.

85. The detector complex of Claim 51 wherein the first component is conjugated with biotin, iminobiotin or desthiobiotin.

86. Reagent means comprising the detector complex of any of Claims 51 to 61 and 77 to 85, and a light emitting system to which the second component of said complex can be readily coupled , and which is capable of emitting light responsive to such coupling.

87. The reagent means of Claim 86 wherein the light emitting system comprises the constituents of a bioluminescent reaction.

88. The reagent means of Claim 86 wherein the light emitting system comprises the constituents of a chemiluminescent reaction.

89. The reagent means of Claims 86 in which the light emitting system comprises constituents of a light emitting reaction, as well as constituents of a bridging reaction, and wherein the second component of the detector complex provides a limiting or essential constituent of the bridging reaction.

90. The reagent means of Claim 89 wherein the bridging reaction is one which, when in contact with the second component of the detector complex, is capable of generating ATP, NADH, NADPH, FADH, FMNH, aldehyde, hydrogen peroxide or a fluorescer.

91. The reagent means of Claim 90 wherein the light emitting reaction is capable of emitting light in the presence of the product generated by the bridging reaction.

92. Reagent means comprising the detector complex of any of Claims 62 to 76, and a light emitting system to which the second component of said complex can be readily coupled, and which is capable of emitting light responsive to such coupling.