NO853790L - PROCEDURE FOR DISPOSAL OF IMMOBILIZED REPORT GROUPS - Google Patents

Description

The present invention relates to immunology

and diagnostic chemistry, specifically a sensitive and specific luminescence detection of immobilized reporter moieties smaller than approx. 10,000 daltons in size.

During the last decade, a large number of analytical methods with radioactive labeling and enzyme labeling have been developed. While reactions developed so far with radioactive labeling show high sensitivity by today's standards, there are several serious limitations associated with radioactive detection. These include: 1) The radioactive label must be incorporated as a reporter group by slow chemical synthesis or by binding it to appropriate functional groups.

2) The radioactively labeled material must be cleaned

under carefully controlled conditions to avoid dangerous exposure to radioactivity. 3) The radioactively labeled material must also be handled and disposed of using special procedures.

4) The half-life of many radioactive substances

is short and consequently the storage time for the corresponding radioactively labeled reagents is short. 5) The instrumentation for detecting radioactive substances is expensive.

6) The radioactivity decomposes the analysis reagents

and thereby adversely affects the accuracy of the analysis.

In contrast to the limitations associated

with radioactive detection, analysis methods that utilize luminescent reagents offer little environmental risk, are stable for long periods of time and require inexpensive instrumentation for the detection. The enzyme labeling reactions referred to above have the advantage that they do not use radioactivity, but they do not usually have the sensitivity of radioactive assays. Consequently, there is a clear need for a non-radioactive detection method with a sensitivity comparable to the sensitivity of the radioactive detection methods.

The previously known methods of enzyme labeling generally utilize competitive binding reactions to detect the presence of soluble analytes, and they fall into two main categories: 1) Heterogeneous. Processes requiring the separation of phases that are free from phases that are bound. 2) Homogeneous. Procedures that do not require separation.

According to US Patent Nos. 3,850,752, 3,839,153, 3,654,090, 4,016,043, 4,338,237 and 4,318,980, methods are used for the detection of analytes in solution based on a competition between bound and free forms. Some of these methods have been extended to include the use of bioluminescence or chemiluminescence. British patent document no. 1 578 275 describes a heterogeneous method for the analysis of insulin in solution where an analysis method with competitive binding is used which utilizes a conjugate of insulin and isoluminol. US Patent Nos. 4,230,797 and 4,380,580 describe analytical methods with competitive binding for the detection of ligands in a liquid medium and utilize a conjugate of a ligand and an enzyme reactant. US Patent No. 4,383,031 describes a homogeneous analysis method where a similar conjugate type is used to detect ligands in a liquid medium.

A good overview of the published literature in this area is published 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). After the preparation of this overview article, several other publications have been published which indicate the use of chemiluminescence in the detection of ligands in solution. Schroeder et al. used an antibody labeled with an isoluminol derivative in a chemiluminescence method to measure hepatitis B surface antigen in human serum [Clin. Chem. 27, 1378-1384 (1981)], Hinkkanen et al. measured the amounts of immunoabsorbed proteins by a luminol method [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)]. All these systems are mainly designed to detect analytes present in a liquid medium by means of competition with labeled analytes.

In the past, the detection of immobilized ligands has mainly been limited to cytological and histological procedures with staining where fluorescent and colorimetric methods have been used. As stated in Virology, 126, 32-50 (1983) demonstrated e.g. Brigati et al DNA in paraffin-embedded tissue sections using a peroxidase complex and biotinylated hybridization probes for DNA described in EP Patent Publication No. 0063879, issued November 3, 1982. These methods usually provide qualitative and not quantitative measurements of the ligands present and which are used to test for specific sites that are already present in the tissues or cells.

A recently published abstract discussed the detection of immobilized DNA using a microperoxidase covalently bound to DNA [Fed. Pro. 42, 1954, abs. 1149,

(1983)]. In this method, luminol was used to detect microperoxidase, but the method has limited applicability as it is severely limited due to a lack of sensitivity (detection limit ~10 -3 mol of target DNA).

The present invention is new and clearly differs from the prior art. The specific characteristics of the method according to the invention are:

1) It detects immobilized reporter groups.

2) It is a direct method for the analysis of reporter groups. 3) It uses highly specific ligand-ligand reactions to visualize the reporter groups. 4) It has a detection limit in the area from approx. 10 to approx. 10 mol reporter group. This places it close to the detection limits for radioactive markers and is far higher than conventional non-isotopic detection systems. 5) Harmless chemiluminescent or bioluminescent reagents are used.

6) The reagents used are stable for a long time.

7) Only very small amounts of reagents are used, something

which makes individual analyzes cheap.

8) Cheap instrumentation is used.

9) Only a few minutes are needed to perform the assay, in contrast to assays with radiolabeled reporter groups that can take several days. 10) The analysis procedure. has high sensitivity as it involves the detection of a single weak signal and not a small difference between two strong signals, as is done conventionally.

This invention grew out of a need for

to detect very small amounts of reporter groups bound to a carrier matrix by non-radioactive means. In many cases it is necessary to be able to quantify the number of reactive sites or binding sites on the surface of a carrier matrix. Furthermore, it is imperative to be able to quantify the effectiveness of reactions whereby substances are bound to the surface of a carrier matrix. Finally, in many cases it is desirable to be able

to quantify the degree of reaction between a second molecule and the carrier matrix. For all these applications, a fast and sensitive, non-radioactive method is needed. The above-mentioned characteristics of the invention fulfill these needs. As this invention correlates light emission with the presence of reporter groups with a size less than about 10,000 daltons, the highly sensitive detection of such reporter groups is now possible. Common luminescent methods are capable of detecting as few as 100 photons/second. If the quantum yield in the luminescent reaction is high and the 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 enhanced enzymatically before or simultaneously with the light-emitting reaction, this method becomes even more sensitive.

Luminescent detection methods are preferable to radioactive detection methods for a number of reasons in addition to those previously mentioned. Radioactive particles slowly and continuously break down and emit radiation. At any given time, only a very small proportion of the radioactive isotopes present break down and emit radiation. In the case of a substance labeled with 12 5iodine must

12.5 million of the 12 5iodine atom markers be present to

give 100 atomic fissions per minute. Because of the slow decay of radioactive particles and the small number that decay during any given time interval, radioactive detection methods necessarily require slow signal accumulation.

In contrast, virtually all luminescent molecules are available for light emission at any time and can be induced to emit photons rapidly when induced to do so. Consequently, practically all of the luminescent molecules present can be made to emit photons simultaneously under the control of the user. This gives luminescent methods an advantage in terms of sensitivity and short analysis time compared to methods where radioactive markers are used.

Another advantageous feature of the present invention is that it provides a method that directly detects reporter groups that are immobilized on a carrier substrate. As previously indicated, previously known enzymatic methods where analyzes with competitive binding are used on analytes in solution usually require that a small difference between two strong signals be distinguished. The direct detection provided by the present invention enhances its sensitivity compared to the sensitivity of such prior art methods as it is much easier to detect a single weak signal than it is to detect a small difference between two strong signals.

These previously known methods also work when analyzing large protein molecules in antibodies, antigens or analytes which tend to be unstable under certain reaction conditions. Reporter groups that are useful in determining the number of reactive groups on support masses, in determining the achieved efficiency of coupling reactions or in quantifying the reaction between a second molecule and a support mass, cannot be large proteins. To obtain a sensitive measure of mass composition, the use of small size reporter groups is essential for accurate quantitative determinations as such determinations cannot be made as easily using large, sterically bulky protein reporter groups. In contrast to the unstable protein molecules which are analyzed according to previously known methods, report tube groups detected by means of the method according to the present invention comprise substances which are stable within reasonable limits in terms of pH, temperature, solvent and salt concentrations.

The reporter groups detected using the method according to the present invention are less than 10,000 daltons in size. The small size of such reporter groups offers several advantages, including: 1) They can be easily bound to carrier masses using methods that cannot be easily adapted for binding large reporter groups or reporter groups that are easily denatured. 2) Such reporter groups can be tied to a specific carrier mass in much larger numbers than larger reporter groups can. This greater density of reporter groups per unit surface area of the carrier mass facilitates the detection of the reporter groups within a certain small area, as the greater density provides an easier detected signal from such a surface area. 3) The use of small reporting groups provides a higher degree of versatility that is not possible when using large reporting groups. The chemical method of binding small reporter groups is easier to perform and the detection and quantification of such groups is also easier. As a large number of different substances can be chemically bound to such small reporter groups, a common detection system can be used to monitor the immobilization of small reporter groups that have such substances bound to them. Such versatility is not possible with large reporting groups.

The present invention relates to a method, complexes and reagents for the sensitive detection of reporter groups bound to a carrier mass, which enables monitoring of the reaction whereby the reporter group is immobilized on the carrier mass, as well as quantification of the reaction between a second molecule and the carrier mass. The invention also enables the monitoring of related reactions which include substances which can alter the ability of the reporter group complexes to become immobilized. The present invention relates in particular to the luminescent detection of immobilized reporter groups using a new detector complex which can be easily connected to a light-emitting system and which is able to give a high affinity reaction with a specific reporter group which is less than approx. 10,000

daltons in size.

The reporter groups that can be used in the practice of the invention can be virtually any small molecule for which specific binding agents (ie, ligands) are available. Such reporter groups include, but are not limited to, vitamins such as biotin, iminobiotin, desthiobiotin or pyridoxal phosphate which react very specifically with certain proteins, cofactors such as porphyrins which react very specifically with certain proteins such as the cytochromes and hydroperoxidases, antigens , like for example. dinitrophenol, biotin, iminobiotin, desthiobiotin, fluorescein and fluorescamine or conjugates thereof with which antibodies react very specifically, and carbohydrates, such as e.g. mannose, galactose and fucose with which certain lectins react very specifically.

The presence of the immobilized reporter group is determined by first incubating it with an excess of free indicator complex in solution. Due to the strong affinity of the detector complex to the reporter groups, a part of the detector complex binds to the reporter groups present. After removal of unbound detector complex, the detector complex that is bound back is detected by contacting it with a light emitting system capable of emitting light in the presence of the complex. As the amount of bound detector complex is proportional to the amount of reporter group on the carrier mass, the light emitted by the light-emitting system on the carrier mass is correlated with the number of reporter groups bound to the carrier mass.

The detector complex according to the invention consists of a first component which has a high specific affinity to a reporter group with a size smaller than approx. 10,000 daltons, and a second component that can easily be connected to a light-emitting system. This second component can consist of either a chemical substance that can participate in light generation when additional components are added, or it can consist of an enzyme that plays an essential role in a multi-step light-emitting reaction. 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.

In the prior art, a large number of bioluminescent and chemiluminescent, light-emitting systems have come to light that can be used in the practice of the method of the present invention (cf. 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.L., Aldrichimia Acta 16, 59-61 (1983), Clinical and Biochemical Luminescence, supra; and Methods in Enzymology, Vol. 57, Academic Press, 1978) . As most of these require multiple components for the entire system to function, the luminescence-coupling second component in the detector complex can be any of a wide variety of substances.

As there is a wide range of substances that have one

high specific affinity to potential reporter groups with a size smaller than approx. 10,000 daltons, in the same way the reporter group that binds the first component in the detector complex can be a wide variety of different substances.

In order to facilitate the understanding of the present invention, the following definitions are given for certain terms used:

1) Carrier base material. Any fixed

carrier composed of an insoluble polymer, such as e.g. nitrocellulose, agarose, etc, which may, but need not, have a

organic coating comprising a protein, a carbohydrate, a nucleic acid or an analogue thereof.

2) Rapporteur group. Any chemical residue that can

used to label specific chemical groups on a carrier matrix, and which is reasonably stable to pH, temperature, solvent and salt conditions. Such groups have a size that is less than approx. 10,000 daltons, and are usually substituents that are not proteins. Reporter groups also include chemical residues which are not usually present on the molecular surfaces, but which can be introduced as reporter groups through chemical methods. 3) Demonstrator complex. A complex a first component consisting of a reporter group binding substance and a second component consisting of a binding substance. 4) Light emitting system. One or more reactions that, when coupled to a detector complex, culminate in the emission of light. The light-emitting system may consist of a light-emitting reaction, a transition reaction and a substance or other reactant which, when reacted with the detector complex, gives a product which is a component of the transition reaction or of the light-emitting reaction.

The basic method according to the present invention uses the following steps: 1) Binding of a detector complex to a reporter group which is bound to a carrier matrix, as well as the complex formation between the reporter group and the detector complex. 2) Removal of detector complex that has not formed a complex with the reporter group. 3) Coupling of a light emitting system with the detector complex which is bound and which remains immobilized on the carrier mass, causing light to be emitted. 4) Use of light-sensitive agents such as e.g. a luminometer, light-sensitive film or a light-sensitive charge-coupled device (CCD) and comparison with standard curves to determine from the amount of emitted light the number of reporter groups present on the carrier mass.

In the light-emitting reaction, additional steps and reagents may be required and will vary depending on the nature of the detector complex. The second component in the detector complex is preferably of two main types, which respectively comprise: a) A substance, such as e.g. a luminescent compound, which in itself is an essential component in a light-emitting reaction, or b) An enzyme, coenzyme, fluorescent compounds, another factor or substance which provides an essential

or limiting substance in a transition reaction or in a light-emitting reaction.

A wide variety of different luminescent reactions can be used to carry out the method according to the present invention. Discussion of light-emitting systems that can be used in the practice of the invention can be found in: Clinical and Biochemical Luminescence, supra, and Methods in Enzymology, supra. These literature references are representative, but do not include all light-emitting reactions that can be used in the method according to the present invention.

The applicability of these light-emitting reactions can be extended by the use of a wide variety of different transition reactions which can provide one of the limiting substances in the luminescent reaction. It should be emphasized that although oxygen is a limiting substance, in many light-emitting systems it is usually ubiquitous, and therefore transition reactions that produce oxygen are generally impractical.

It is possible to use a large number of detector complexes. The binding substance which forms the second component in the detector complex can form one of the necessary components in the luminescent reaction, or it can form a component in a transition system, as this provides a component that is necessary for the luminescent reaction to work. To demonstrate the diversity of possible detector complexes, reactions of different forms with a bioluminescent system derived from bacteria will be discussed below.

The second component may consist of a bacterial luciferase, and when this is the case, and FMNH, a long-chain alde hyd and oxygen are brought into contact with the detector complex, light is emitted. The second component can also consist of a substance that is an essential component in a transition reaction that gives an intermediate product that is essential or limiting in a luminescent reaction, such as e.g. a bacterial bioluminescence reaction. Typical substances that can be used as such a second component are illustrated in the light-emitting systems below, which are grouped according to the type of transition reaction used.

1) FMNH-producing transition reactions.

a) When a detector complex is used with a second component consisting of FMN<+>oxidoreductase and where the bound complex is brought into contact with NADH, aldehyde, oxygen and bacterial luciferase, NADH generates FMNH with the help of the FMN<+>oxidoreductase and , light is emitted in the presence of the other components of the bacterial bioluminescent system. b) When a detector complex is used with a second component consisting of an NAD<+->or NADP<+->dependent dehydrogenase, and where the detector complex is bound, it is brought into contact with NAD(P)<+>so that appropriately reduced substrate, NAD(P)H is generated. An example of such a dehydrogenase is glucose-6-phosphate dehydrogenase (G6PDH) from Leuconostoc mesenteroides: which is capable of producing NADH or NADPH and which is one of over 300 different NAD- and NADP<+->dependent dehydrogenases, many of which can be used in the detector complex. Contact between the NAD(P)H product and FMN<+>, FMN<+>oxidoreductase, aldehyde, oxygen and bacterial luciferase either sequentially or simultaneously results in the formation of FMNH and emission of light. c) When a detector complex is used with a second component consisting of an NAD<+->synthesizing enzyme which

e.g. ATP:NMN<+>adenylate transferase and the detector complex bound are brought into contact with ATP and FMN<+>, NAD is generated. By contacting the NAD<+->product with an NAD<+->dependent dehydrogenase and an appropriate reduced substrate for the dehydrogenase, either simultaneously or sequentially, NADH is generated. Contact between NADH and FMN<+>and FMN<+>oxidoreductase, aldehyde, oxygen and bacterial luciferase, either simultaneously or in series

follow, results in light emission.

d) When a detector complex with a second component consisting of active NAD<+> is used as a limiting agent,

and the bound detector complex is brought into contact with an appropriate reduced substrate and an appropriate hydrogenase, NADH is generated. Contact between NADH and FMN<+>, and FMN<+>oxidored-ductase, either simultaneously or sequentially, generates FMNH. Contact between FMNH and aldehyde, oxygen and bacterial luciferase produces light emission. Such a system requires that NAD<+>and NADH are catalytically active together with both dehydrogenase and FMN<+>oxidoreductase.

e) When a detector complex is used with a second component consisting of active FMN<+>and detector complex soy

is bound is brought into contact with NAD(P)H, FMN+ oxidoreductase, aldehyde, oxygen and bacterial luciferase, formation of FMNH and light emission are obtained.

2) Aldehyde-producing transition reactions.

a) When a detector complex with a second component consisting of an appropriate alcohol dehydrogenase is used, and the bound detector complex is brought into contact with NAD(P)<+>and an appropriate alcohol, an aldehyde is produced. By bringing this aldehyde into contact with FMNH, oxygen and bacterial luciferase, either simultaneously or in sequence, light is emitted. b) when a detector complex is used with a second component consisting of active NAD(P)<+>which acts as a

limiting agent in the generation of aldehyde by alcohol dehydrogenase, and the bound detector complex is brought into contact with a suitable alcohol and a suitable dehydrogenase, production of an aldehyde and NAD(P)H is obtained. Simultaneous or consecutive contact between the product aldehyde and oxygen, FMNH and bacterial luciferase gives light.

A second dehydrogenase and its substrates added to this reaction system regenerate the NAD(P)<+->product from NAD(P)H.

From the above, it is obvious that the second component in the detector complex according to the present invention can vary widely, and it should be understood that those described are for illustrative purposes only, and are not intended to limit the scope of the invention. Although those skilled in the art will appreciate that hundreds of different detector complexes can be used with the various light emitting systems available, for practical reasons the preferred detector complexes are much fewer in number as many may include substances that are expensive, unstable, difficult to obtain , cumbersome to use or have little sensitivity.

The most preferred forms of the indicator complex

are those where the other component, when brought together with a light-emitting system, causes this to emit a large number of photons. Such detector complexes usually include in their other constituents an enzyme with a relatively high catalytic turnover, or a molecular substance that can go through several cycles during a short period of time,

with each cycle having a high probability of emitting a photon. A second component in such a complex can be connected directly to one of the luminescent reactions, or it can be connected directly to such a reaction and act as a limiting or essential substance through a transition reaction.

The chemical substances that the second component of the detector complex is preferably comprised of include such enzymes as the oxidoreductase, which e.g. FMrJ+ oxidoreductase, glucose-6-phosphate dehydrogenase (G6PDH), malate dehydrogenase, alcohol dehydrogenase, lactate dehydrogenase or triose phosphate dehydrogenase, alkaline phosphatase, adenyl transferase, 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 betagalactosidase, neuraminidase or fucosidase whose reaction products may be fluorescent substances, or enzymes whose reaction products may be other substances which may be linked directly or indirectly to light-emitting systems.

In addition, the second component in the detector complex can consist of luciferin or any enzymatically active coenzyme, such as e.g. 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 e.g. luminol, isoluminol, pyrogallol, lucigenin and lofin. The second component can also usefully consist of a fluorescent substance such as e.g. 7,10-diphenylanthracene, perylene, rubrene, bis[phenylethynyl]anthracene (PPEA) and umbellifero n- or dansyl derivatives, or substances that excite fluorescent compounds,

like for example. bis(2,4,6-trichlorophenyl)oxalate (TCPO) and bis(2-carbopentoxy-3,5,6-trichlorophenyl)oxalate (CPPO) and other oxalate analogs. For further discussion of fluorescent agents and exciting substances, such as oxalates, reference is made to Mauling, 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. Aldrichimia Acta, 16, supra. Further discussion of chemiluminescent and bioluminescent light-emitting systems can be found in Clinical and Biochemical Luminescence, 12, supra and Methods in Enzymology, 57, supra.

In the same way as the second component in the detector complex, the reporter-binding substance of which the first component in the detector complex consists can also be selected from a wide range of different substances. Reporter group-binding substances that can be used are those capable of giving high-specific affinity reactions with reporter groups that have a size smaller than approx. 10,000 daltons. It is preferred that the reaction between the reporter-binding substance and a reporter group exhibits an affinity greater than approx. 10 8. Examples of preferred first constituents in detector complexes are those consisting of proteins that bind certain vitamin reporter groups with high affinity, such as e.g. avidin or streptavidin that bind biotin reporter groups, proteins that bind risk factor reporter groups with high affinity, such as e.g. apomyoglobin that binds porphyrin reporter groups, antibodies that bind specific antigenic reporter groups with high affinity, such as e.g. antibodies for dinitrophenol, biotin, iminobiotin, desthiobiotin, fluorescein or fluorescamine reporter groups, lectins that bind carbohydrate reporter groups with high affinity, such as e.g. concanavalin A which binds the mannose reporter group, and chelating agents

which selectively binds metal reporter groups. As a first component, those consisting of chemical residues which give

highly specific reactions with specific parts of reporter groups. Recognition of a reporter group by means of the detector complex results in such cases in the formation of a covalent bond between the reporter group and the detector complex as a result of chemical residues on the reporter group and the first component of the detector complex forming a product that binds the two species together.

Formation of such a covalent bond between a reporter group and the first component of a detector complex

any of a number of different reactions can be carried out, such as those mentioned below where the reacting residues and resulting products are indicated: 1) Reaction of primary amines with active esters to amides. 2) Conversion of alcohols with active esters to esters. 3) Reaction of amines and alcohols with epoxides to respectively substituted amines and ethers. 4) Conversion of amines with isothiocyanates to thioureas. 5) Turnover of organic mercury salts

with olefins to substituted olefins. 6) Conversion of thiols with maleimides to thioethers. 7) Reaction of di-azonium salts with aromatic substances to diazo compounds.

Those skilled in the art will recognize that there are a number of additional chemical reactions that can be used to form the covalent bond. In all cases, however, the binding reaction used must be limited to the detector complex and reporter group involved.

As regards the synthesis of the detector complex, the binding of the first component to the second component can be carried out in several ways, including: 1) Direct covalent bonding between the components by the spontaneous covalent bonding between chemical residues

on the surfaces of the components so that a product is formed which connects the two species, such as e.g. an ether, ester, thiourea, amide, thioester, thioether, substituted olefin or diazo compound. Covalent bonding can also be produced by activating such chemical residues with a condensing agent, such as the reaction of amines, triols and alcohols with acids in the presence of a carbodiimide, thionyl chloride or other carboxylate activating agent.

2) Covalent bonding between the components through

a bifunctional coupling agent with peripheral functional groups that bind covalently to chemical residues on the respective constituents, and which has an internal part that provides a bond between the two species. Examples of such bifunctional coupling agents are those which bind together amines and/or thiols and/or alcohols. 3) Formation of a high-affinity, non-covalent reaction between the constituents.

In the preferred synthesis of the detector complex, a non-covalent reaction of the type referred to above is used for binding the detector complex to a reporter group, i.e. a reaction between vitamin and protein,

a reaction between cafactor and protein, a reaction between antigen and antibody, a reaction between carbohydrate and lectin or any other suitable high affinity non-covalent specific binding reaction.

An example of a detector complex where the components are bound together by means of a non-covalent reaction between protein and vitamin, e.g. avidin and biotin, is one in which the first component is avidin and the second component is bio-tinylated FMN<+>oxidoreductase. As 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.

If desired, the synthesis of the indicator complex can be carried out by a reaction which binds the second component to the first component after the latter is bound to a reporter group which is attached to a carrier mass, as in examples 1, 4 and 5 below. It is therefore to be understood that when reference is made to a reporter group being brought into contact with a detector complex, such contact includes the sequential contact between the constituents of the detector complex and the reporter group included in such synthesis, as well as contact between a reporter group and a detector complex synthesized before the contact with the rapporteur group. Regardless of which synthetic method is used, the requirement to preserve the chemical and/or biochemical activity of the respective components that are bound together is implied. If it turns out that a desired activity is lost during the complex formation, an alternative chemical method should be used in which the desired activity is preserved.

When choosing components for use in a detector complex and of the light-emitting system to be connected with the complex, several factors should be taken into account, including:

1) The individual components of an indicator complex

should work properly under conditions of pH, temperature, salt concentration, etc. which have an acceptable similarity.

2) The second component should, when linked to an enzyme, coenzyme or fluorescent compound in a product detectable by means of an appropriate light-emitting system. 3) The product referred to under 2) should be formed in a sufficiently large amount to provide the desired sensitivity with the particular light-emitting system. In this respect, it should be expected that the catalytic activity of a detector complex, when it is immobilized by binding to an immobilized reporter group, is lower than that of the catalytic component when it is not part of a complex. 4) If sensitive detection requires the accumulation of a reaction product over a certain time, it is necessary that such a product is stable for the required time. It would e.g. be ill-advised to use a light-emitting system that requires accumulation of FMNH for a long time as FMNH

is very unstable and has a half-life in the presence of oxygen of less than 1 second.

When the method according to the present invention is carried out using a light-emitting system which undergoes a first reaction where the production of the intermediate product is catalysed and a second reaction where light is emitted, the optimal conditions for catalyzing the production of the intermediate product may be different from the optimal conditions for the light emission. When this is the case, the catalytic activity of the second component of the detector complex can be maximized in several different ways, such as: 1) By performing the first reaction, which involves the catalytic activity of the detector complex, under conditions that are generally optimal for the formation of the intermediate, and then carrying out the second reaction under conditions that are generally optimal for light emission. 2) By solubilizing at least the catalytic component in the detector complex before it is used in the first reaction to generate the intermediate. This is done as soluble catalytic components, e.g. enzymes, usually have greater activity than immobilized constituents. Solubilization can be performed in several different ways, depending on the nature of the detector complex. These include: a) Dissociation of the detector complex from the reporter group to which it is bound, applying a change in pH,

temperature, salt concentration, etc. Heat separation can be carried out using a detector complex where the first component consists of a heat-unstable antibody and where the second component consists of a heat-stable substance. Separation of a detector complex from a reporter group as a result of a change in pH is facilitated by choosing a reporter group and a first component in the detector complex that forms a high-affinity bond that dissociates under weakly acidic or weakly basic conditions. E.g. is the bond between avidin as a reporter group and iminibiotin as the first component of the detector complex one that has a high affinity at neutral pH,

but which dissociates at the slightly acidic pH of 4.5.

b) The use of a detector complex where the component is bound together by means of a bond that can be cleaved

under mild conditions. Examples of such bonds are disulphides which are cleaved with the help of thiols, vicinal diols, which are cleaved with the help of periodates, and weakly active esters or carbamates which are hydrolysed with the help of weak bases.

c) The use of an enzymatically degradable carrier mass to which the reporter group is bound. An example of such a carrier mass is one formed from amylopectin which is degradable by amylase. d) The use of an enzymatically cleavable chemical bond for the immobilization of the reporter group.

As previously mentioned, the detector complexes most preferred for use in the method according to the present invention are those where the second component, when coupled to a light-emitting system, causes this to produce more photons for each detector complex molecule. Such emission of several photons is possible when the second component in the detector complex consists of or can be connected to an enzyme, a coenzyme or a fluorescent compound. The advantages of using enzymes consist in the fact that each enzyme molemil within 1 minute can convert a large number of molecules of a suitable substrate into a product. An effective catalytic rate of an enzyme is referred to as a high turnover rate, and it is advantageously at least 10/minutes and preferred

2 5

in the range from 10 to 10 /minutes or higher. Such preferred turnover figures can in principle give an increase of 2

to 5 orders of magnitude in terms of sensitivity, but there is usually at least some reduction in activity associated with complexation (See Sundaram, P.V. et al., Ca. J. Chem. 48, 1498-1504, (1970) and Carlsson, J, and Svenson, A., FEBS. Lett., 42, 183-186 (1974). However, enzymes usually retain reasonable activity both when complexed and when immobilized.

Improved sensitivity can also be achieved if the second component consists of a coenzyme or a cofactor.

This benefit does not arise as a result of more products being generated, but rather as a result of the catalytic action of the coenzyme or cofactor in activating the enzymatic reaction. Such an effect of the coenzyme NAD<+> is illustrated in the periodic reaction below:

In the illustrated reaction, enzyme 1 regenerates

NADH and enzyme 2 use NADH for a transition reaction that leads to light emission. This requires that enzymes 1 and 2

is able to use the complexed or conjugated coenzyme NAD<+>, or that an active coenzyme or an active cifactor can be released from the complex prior to analysis. Enzyme 1 can be any of a wide variety of dehydrogenases. Enzyme 2 can e.g. be FMN+ oxidoreductase, and the bacterial luminescent reaction can be used to produce light emission.

Conjugated and immobilized forms of the coenzymes NAD<+>and NADP<+> have been synthesized which retain catalytic activity. (See, e.g., Weibel, M.K. et al., in Enxyme 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, Mosback et al., Methods in enzymology, 44, 859-887 (1976).

The second component in the detector complex can also consist of ATP which can act 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 analogue of a long-chain aliphatic derivative in the N6 or N8 position of the purine ring, there is a good chance that its catalytic activity together with enzymes will be retained (Mosbach, K. et al., Methods in Enzymology, 44, supra One such use of ATP is illustrated in the reaction below.

Where ATP-m is ATP-modified and AMP-m is AMP-modified, and PRPP is phosphoribosyl pyrophosphate.

A detector complex can also be one that can catalyze the formation of a product that is a catalyst for a separate reaction. Systems where such a complex is applicable include the following:

When the second component of the detector complex consists of 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 enhancing effects of the two reactions. The enzymatic formation of NAD<+>, the enzymatic production of fluorescent molecules and the enzymatic activation of a second enzyme fall within this category. Common reactions for the generation of NAD<+> and fluorescent compound respectively, as well as respectively bioluminescent and chemiluminescent reactions that can be used together with these, can be illustrated in the following way:

A. Preparation reactions for NAD<+>

1. MNM<+>+ ATP adenyltransferase>^

2. NADP<+>alkaline phosphatase^NAQ<+>

B. Light-emitting reaction (bioluminescent)

a = an appropriate dehydrogenase

b = FMN<+>oxidoreductase

c = bacterial luciferase

:. Conversion reaction for precursor compound to fluorescent compound

3rofluorescent compound enzYm Fluorescent compound An example of this reaction is the conversion of the profluorescent compound 4-methylumbelliferyl-N-acetyl-beta-D-glucosamine to the fluorescent compound 4-methylumbelliferone in the presence of the enzyme beta-galactosidase.

D. Light-emitting reaction (chemiluminescence)

In this light-emitting reaction, an oxalate derivative, which e.g. bis(2,4,6-trichlorophenyl)oxalate (TCPO) or bis(2-carbopentoxy-3,5,6-trichlorophenyl)oxalate (CPPO) to a cyclodioxethane, and the energy released by the breakdown of the cyclodioxethane to CC>2 is absorbed by the fluorescent compound, which causes excitation of the fluorescent compound. Then the subsequent relaxation of the fluorescent compound to the ground state results in light emission.

As previously mentioned, the present invention has

a central purpose, i.e. detection of reporter groups bound to a carrier mass. As the presence of immobilized reporter groups is dependent on the reaction that binds them to the carrier mass, the method according to the invention can also be used to quantify reactions that affect the ability of the reporter group to be immobilized. The mutual influence by which a reporter group is bound to a carrier mass can be a chemical reaction, a bond between antibody and antigen, a bond between carbohydrate and lectin, a bond between vitamin and protein, a bond between cofactor and protein, a bond between nucleic acids, such such as a DNA-DNA, RNA-DNA or RNA-RNA hybridization bond, a metal chelating bond, or any suitable specific ligand-to-ligand bond or chemical reaction.

Such a mutual influence that can be monitored

by means of the method according to the invention is a bond between carbohydrate and lectin whereby a reporter group is immobilized on a carrier mass. E.g. a lectin can be adsorbed on a carrier mass and a reporter group can be covalently bound to a carbohydrate in an area on the latter which will not affect the lectin's recognition of the carbohydrate. When the complex between reporter group and carbohydrate is brought into contact with the lectin adsorbed on the carrier mass, the resulting bond between lectin and carbohydrate causes the reporter group to be immobilized on the carrier mass. The indicator complex can then be used in the method according to the invention to detect the presence of the specific, immobilized lectin involved in the binding.

In another application of this concept, the immobilization of a reporter group on a support mass through a DNA-DNA bond can be monitored. E.g. an organic polymer in the form of a DNA chain can be adsorbed on a carrier mass, and a reporter group can be covalently bound to a second DNA chain, such as a single-stranded oligonucleotide, which is complementary to the immobilized part of the DNA chain. Hybridization of the DNA chain binds the reporter group to the carrier mass. In a difficult case, the method according to the invention can be used to monitor the hybridization reaction. By detecting the immobilized reporter group, the method also detects the presence of the particular DNA chain adsorbed on the carrier mass.

In carrying out the method according to the present invention, various means can be used to record the light emitted by a light-emitting reaction, some of which are used in the examples below. Among such light-sensitive means are a luminometer, a light-sensitive film and a light-sensitive charge-coupled device or other suitable and desired light-sensitive means.

In each of the examples below, detection of immobilized reporter groups using the method according to the invention is demonstrated, as are various specific adaptations of the method.

Example 1

Detection of agarose-bound biotin reporter moieties using a detector complex of avidin and a biotin-rich aggregate of biotinylated G6PHD and avidin (BAC)

In this example, biotin is the reporter group and the detector complex has avidin as its first component and has as its second component a non-covalent biotin-rich aggregate consisting of several molecules of biotinylated glucose-6-phosphate dehydrogenase held together by avidin. The indicator complex is linked to the production of NADH from the oxidation of glucose-6-phosphate by an enzyme in the presence of NAD<+.>NADH produced by the action of the enzyme is brought into contact with the reagent Bactilight I (Beneckea harveyi bacterial luciferase and FMN<+>oxidoreductase , which is provided by the Analytical Luminescence Laboratory, San Diego, Ca. 92121, which produces 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

to restore the FMN<+> with the accompanying emission of a photon.

The synthesis of biotinylated glucose-6-phosphate dehydrogenase (G6PDH: E.C. 1.1.1.49) from Leuconostoc mesenteroids is carried out as follows. Half ml of G6PDH (2 mg per ml) was dialyzed against 1 liter of 0.1 M 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 µm N-hydroxysuccinimide biotin (2 mg per ml 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 enzyme activity had decreased to 50% of the original activity, 50 mg of ammonium sulfate was added to stop the reaction. The biotinylated enzyme was then dialyzed against 0.1M sodium bicarbonate for 20 hours and the resulting solution was stored at 4°C. Biotinylation of the enzyme was confirmed by showing that more than 90% of the enzyme 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 prepared by forming an aggregate with excess biotinylated hydrogenase over avidin (ie, the BAC component is biotin-rich). The BAC aggregate is formed by adding 4 µl of a solution

of Avidin-D (0.5 mg/ml PBS-Tween 20) to 1 ml PBS-Tween 20. Avidin-D is supplied by Vector Laboratories, Burlingame, CA, and PBS-Tween 20 is phosphate-buffered saline (0.9% NaCl ,

10 mM sodium phosphate, pH 7) containing 0.1% Tween 20.

To this diluted solution, 12 µl of biotinylated enzyme (0.4 mg enzyme/ml 0.1 m NaHCO 3 ) is added with stirring. The aggregate is allowed to form for at least 15 minutes and then stored at 4°C until use. Immediately before use, the BAC aggregate is centrifuged for 5 minutes in a tabletop centrifuge (approx. 3000 rpm) to remove overlying large aggregates that contribute to non-specific binding.

10^1 of a slurry of biotin-agarose beads

(Pierce Chemical Co., 1.5 x 10<-11>avidin binding capacity)

diluted in 1 ml PBS-Tween 20. This slurry is dispersed

so in 100 jul aliquots in 1.5 ml Eppendorf tubes. The contents of each tube are washed twice with 1 ml of PBS-Tween 20, then incubated for 30 minutes with 40 µl of avidin D (0.5 mg per ml,

0.3 nmol) to form complex with the biotin. The agarose-biotin: avidin aggregate is washed free of unbound avidin with 1 ml of PBS-Tween 20 and resuspended in 100 ul of the same.

10 µl 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 pmol with respect to biotinylated G6PDH) and the mixture is allowed to react for 15 min to form

a complex of agarose-biotin and avidin-BAC. Control beads were prepared by incubating agarose-biotin-avidin beads with excess avidin (40 nM) before the 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 0.015 M sodium citrate. Selected single beads of agarose were then removed from each tube under a microscope and analyzed for bioluminescence in a Monolight 4 01 luminometer (Analytical Luminescence Laboratories, San Diego, CA). A reagent solution was prepared from dodecylaldehyde (0.0005%), FMN<+>(3 x 10~<6>M), NAD<+>(3 mM), glucose-6-phosphate (5 mM), magnesium chloride (3 mM) 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 (prepared according to the manufacturer's instructions) and the progress of the reaction was monitored in the Monolight 4 01 luminometer at a sensitivity setting of 10x. In Table I below, typical results are given together with controls.

Table I

Bioluminescent detection of biotin reporter moieties bound to single agarose beads using an avidin-BAC detector complex

Degree of light emission Sample (arbitrary units) No sample (empty apparatus) 0.01

Empty test tube 0.02

FMN + aldehyde + buffer 0.03

FMN + aldehyde + buffer + Bactilight I

reagent (Complete reagent system) 0.15 Complete reagent system + agarose

biotin:avidin-BAC 2.43 Complete reagent system + agarose-biotin:avidin-biotin control 0.15

The avidin-binding capacity of the biotin-agarose was independently determined to be 1.5 picomoles per µl of the original slurry. A sphere with a diameter of 70 µm corresponds

-15

therefore to about 10 mol of biotin. Based on this data, it can be calculated that by using the method according to the present invention, the detection limit for agarose-bound avidin is approx. in 10 biotin.

Example 2

Detection of agarose-linked biotin reporter groups using an avidin-biotinylated G6PDH detection complex.

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

Based on these results, it is calculated that the detection limit for biotin bound to agarose using this detector complex is approx. 10 moles of biotin.

Example 3

Detection of agarose-linked biotin reporter moieties using an avidin-biotinylated G6PDH detection complex.

In this example, the detector complex was formed by

to add an excess of avidin to biotinylated glucose-6-phosphate dehydrogenase. This detector complex has the advantage that no intermediate treatment with avidin and subsequent washings 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 luminometer Monolight 4 01 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 per ml, Bethesda Research Laboratories, Bethesda, MD) with 3 µl of biotinylated glucose-6-phosphate dehydrogenase

(0.42 mg per ml) in 125 µl PBS-Tween 20.

300 µl of biotin-agarose suspension (Sigma Chemical. Company, St. Louis, MO) was washed twice with 1 ml of 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 shaking on

a cuff shaker for 1 hour at room temperature. The samples were then washed 3 times with 1 ml of PBS-Tween 20 containing 0.5 M NaCl. Analysis of the samples using a Monolight 4 01 luminometer following the procedures of Examples 1 and 2 gave results corresponding to those obtained in these Examples.

Example 4

Detection of biotin reporter moieties on a nitrocellulose-bound DNA using an avidin-BAC aggregate-detector complex

In this example, the reporter groups are bound to the carrier mass 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. DNA from lambda phage was biotinylated using nick translation in the presence of biotinylated dUTP where the biotin was covalently bound to the C-5 position of deoxyuridine via a chain of 11 atoms. The reagent system used to biotinylate phage SNA was the nick translation reagent system marketed by Enxo Biochemicals, New York, NY. The reaction yielded lambda DNA in which 31% of the thymidine residues were replaced with biotinylated deoxyuridine, as judged by the concomitant incorporation of tritium-labeled dCTP of known specific activity. DNA was purified from contaminating unincorporated dUTP using ethanol precipitation and Sephadex® G-20 chromatography.

Biotinylated lambda DNA was denatured by heating to 100°C for 5 min in distilled water followed by cooling on ice to prevent single strand rebinding. Different amounts of the denatured DNA were then bound to nitrocellulose filters (Schleicher and Schuell, Catalog no. BA 85) by direct application of DNA to the filters in the presence of 6 x SSC. The filters were allowed to air dry overnight and then heated for 1 hour at 60°C under vacuum to attach the biotinylated DNA to the filters. Amount of biotinylated DNA bound to each filter was quantified by liquid scintillation counting. The average binding efficiency of the filters was 56% of applied DNA. Control filters were prepared using unmodified calf thymus DNA (1 µg DNA per filter). The BAC aggregate yielded biotinylated G6PDH and avidin was prepared as in Example 1.

The procedure used to analyze the filter for the presence of immobilized biotinylated DNA was as follows. The unreacted sites on the filters were "covered" by incubation for 30 minutes at 37°C with 2% bovine serum albumin in PBS-Tween 20. The covered filters were then washed twice for 5 minutes at room temperature with 5 ml of PBS-Tween 20. At this point, the reactive groups in the filter-bound biotinylated DNA can be schematized 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), thereby forming a filter-DNA-biotin:avidin complex. Unbound streptavidin remained

then removed by two washings of 5 minutes duration with 5 ml of PBS-Tween 20 and the filters were dried to remove excess

of resolution.

The filter-DNA-biotin:avidin complex was then reacted with 15 µl of BAC aggregate in PBS-Tween 20 for 30 minutes at room temperature, whereby a filter-DNA-biotin:avidin-BAC complex was formed. The unbound detector complex was removed by washing the filters 6 times for 5 min with 5 ml of 6 x SSC and once for 5 min with 5 ml of Tris buffer. The center portion of each area on the filter carrying the filter-DNA-biotin-avidin-BAC complex was then punched out using a cork board. The punched portion was placed in a microtiter well containing 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 transferred to a 5 ml plastic cuvette containing 400 µl of 0.0005% dodecylaldehyde in 0.1 M potassium phosphate buffer, pH 7.

These samples were then placed in a luminometer Monolight 4 01 and analyzed for NADH content by adding 100 µl

Bactilight I reagent and measure the peak of light emission above

a 10 second interval according to the manufacturer's instructions. The amount of bound biotinylated DNA had a linear relationship with the amount of emitted light.

Example 5

Bioluminescence 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 the invention is the non-radioactive detection of DNA-DNA hybrids formed between an oligonucleotide labeled with a reporter group such as e.g. biotin, and immobilized complementary DNA bound to a matrix. 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. According to this example, a biotinylated oligonucleotide with 21 nucleotides and which was complementary to the gene for The HBV surface antigen, synthesized using the method described in US patent application no. 06/4 68 4 98 which is assigned to the applicant in the present application. In this oligonucleotide, two of the thymidine residues were substituted with biotinylated deoxyuridine. The biotin was covalently bound to the 5-position in deoxyoruridine by means of a chain of 12 atoms. The HBV-immobilized ligand was a recombinant DNA plasmid called pAM6 carrying the entire genome of HBV cloned into pBR322 (Moriarty et al., PNAS, 78, pages 2606-2610 (1981)).

HBV DNA contained in the plasmid pAM6 was adsorbed onto nitrocellulose filters essentially as described in Example 4 for lambda DNA. Control filters prepared at the same time contained only calf thymus or herring sperm DNA. The filters were then treated for 30 minutes in 6 x SSC (0.9M NaCl and 0.09M sodium citrate) containing 10 x Den-hardf's solution (0.02% bovine serum albumin, 0.2% polyvinylpyrrolidone and 0.2% Ficoll) to cover the unreacted sites on the nitrocellulose and prevent non-specific 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 mg/ml) overnight at 46°C in 6 x SSC containing 1 x Denhardt<1>'s solution to form a complex that can be schematized as filter-HBV : oligonucleotide-biotin. Unbound oligonucleotide was washed off the filter by three 15 minute washes with 6x SSC on ice followed by 1 minute washes in 6x SSC at 46°C. The filters were then covered again to prevent the non-specific 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 per ml in Buffer A) for 30 minutes on ice to form a filter-HBV:oligonucleotide-biotin-streptavidin complex. Unbound streptavidin was removed by three washes of 3 minutes each in Buffer A on ice. The filters were then treated for 30 minutes with the BAC aggregate essentially as described in Example 4 and unbound BAC aggregate was removed by three 3 minute washes with ice-cold Buffer A.

At this point, the entire complex bound consisted of filter-HBV:oligonucleotide-biotin:streptavidin-BAC.

To detect an amount of immobilized biotin reporter group and thereby amount of immobilized HBV DNA, as well as 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 produced NADH was detected in the luminometer Monolight 4 01 using the Bactilight I reagent in the following way. A 25 µl aliquot of each assay mixture was added to a 5 mL plastic reaction cuvette containing 400 µl of 0.005% dodecylaldehyde in 0.1 M potassium phosphate buffer (KPB), pH 7. This mixture was placed in the luminometer and the background emission of light was measured . The luminescence reaction was started by injecting 100 µl of Bactilight I reagent which contained

<+><+>+

FMN , NAD :FMN oxidoreductase and bacterial luciferase. The light pulse generated during 10 seconds was approximately linear in relation to the amount of bound biotin reporter group and the amount of bound hepatitis B virus DNA on the nitrocellulose filters.

Example 6

Bioluminescence detection of cellulose-bound anti-herpes antibody using a biotin reporter group-labeled herpes surface antigen and an avidin-biotinylated G6PDH detection complex.

Anti-herpes simplex virus (HSV) antibodies (obtained from Bethesda Research Laboratories, Bethesda, MD) were bound to cellulose plates using the metaperiodate method of Kricke, et al., (J. Clin. Chem. Clin. Biochem. 20:91- 94, (9182). HSV type 1 cell surface antigen (supplied by Flow Laboratories, McLean, VA) was biotinylated by adding 50 µl of N-hydroxysuccinimidobiotin (2 mg/ml in dimethylsulfoxide) to 1 mg of the antigen dissolved in 1 ml 0.1 M sodium bicarbonate, followed by incubation at room temperature for 1 hour. The reaction is then abruptly stopped by adding 50 mg of ammonium sulfate and the product is dialyzed twice against 0.1 M sodium bicarbonate. Alternatively, the surface antigens can be sent to a commercial laboratory for biotinylation to order.

To detect the presence of anti-riSV antibodies bound to the cellulose rafts, they were incubated for 30 min at 37°C with a 1 ng/ml solution of the biotinylated HSV antigen in PBS-Tween 20 containing 0.1% bovine serum albumin The antigens that were not bound were then removed by washing the plates with 3 washes of 5 ml in PBS-Tween 20-BSA. The detector complex used was the same as that used in Example 3, and the plates were then treated for 30 minutes with the detector complex essentially as described in Example 4. The unbound detector complex was then removed by three washes a 3 minutes with Buffer

A.

To detect the immobilized biotin reporter group and thereby the amount of bound anti-HSV antibody present on the plates, the plates were suspended in Bactilight I reagent as described in Example 1, and the degree of signal generation was measured as a function of time. These values can be compared to a standard curve prepared by following the same procedure and using different known amounts of anti-HSV antibody to determine the amount of anti-HSV antibody present. Using a similar method, anti-HSV antibodies can be quantified from human serum.

Example 7

Bioluminescence detection of nitrocellulose-bound HSV DNA

using a dinitrophenol reporter group-labeled oligonucleotide and an anti-dinitrophenyl antibody-G6PDH detection complex

Herpes simplex virus DNA (HSV DNA) that had been cloned into a plasmid (pHSVlOl supplied by Bethesda Research Laboratories, Bethesda, MD) was bound to nitrocellulose following the procedure described in Example 4. A herpes DNA oligonucleotide having dinitrophenyl reporter groups attached to the C-5 position of deoxyuridine via a chain of 11 atoms, was synthesized by the method of US Patent Application No. 06/468,498, which is assigned to the applicant in the present application.

Dinitrophenylated herpes oligonucleotide complementary to the immobilized HSV DNA was complexed with the latter at an oligonucleotide concentration of 10 ng/ml in 6 x SSC containing 10 x Denhardt's solution, overnight at 37°C in heat-sealed plastic bags. At the end of the incubation, unbound excess oligonucleotide was washed away with 10 washes of 5 ml using 6 x SSC on ice.

Then, filters carrying the DNA complex were incubated with 2% BSA in PBS-Tween 20 at 37°C for 30 minutes to cover any remaining protein binding sites on the filter. After three 5-min washes with PBS-Tween 20, the filters were incubated with anti-dinitrophenyl antibody-G6PDH detection complex (1 μmol) for 30 min at room temperature in 40 μl PBS-Tween 20.

The detector complex with antiDNP-Ab-G6PDH was prepared in a ratio of 1:3 according to the method of Carlsson et al., (Biochem. J. 173: 723-737, 1978) using -N-succinimidyl-3-(2- pyridyldithio)propionate. After washing the filter-bound complex 6 times with 6 x SSC/0.1% Tween 20, the emitted light is quantified as follows. Each individual filter is dried to remove excess moisture and transferred to an individual well on a microtiter plate. 100 ul of Bactilight I reagent is added to each well and the reaction now attains an equilibrium which is achieved after about 1 hour of incubation at room temperature. The microtiter plate is then placed 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 continue for a suitable length of time (usually from about 1 to about 15 hours).

At the end of the exposure time, the film was developed in an automatic developer and the amount of light emitted was quantified using a densitometer. The amount of light emitted was proportional to the amount of immobilized DNP reporter group and the amount of originally immobilized HSV DNA, as well as the amount of complex that had formed between the oligonucleotide and the immobilized DNA.

Example 8

Luminescence detection of agarose-bound antiHSV antibody by

using a biotin reporter group-labeled HSV surface antigen and an avidin-dansyl detection complex

Samples containing antiHSV antibody (obtained from Bethesda Research Laboratories, Bethesda, MD) were bound to CnBr-activated agarose beads using standard procedures. The beads were appropriately coated with non-specific goat IgG or bovine serum albumin (BSA). HSV types 1 or. 2 cell surface antigen of the type used in Example 6 can be biotinylated by any method or by commercial order biotinylation.

A streptavidin-dansyl detector complex was prepared by dansylation of streptavidin using dansyl chloride according to standard procedures (cf. Biochemical Journal 181: 251, 1979).

To detect the presence of anti-HSV antibodies bound to the agarose support material, the coated beads were incubated for 30 minutes at 37°C with a solution of 1 mg per ml of the biotinylated HSV antigens in PBS-Tween 20. The unbound antigens were then removed by washing the beads with three washes of 5 ml PBS-Tween 20. The filters were then treated for 30 minutes with the detector complex essentially as described in Example 4, and the detector complex that was not bound was removed by three 3 minute washes with Buffer A. At this point, the entire complex that was bound consisted of bead-antibody: antigen-biotin:streptavidin-dansyl.

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

Example 9

Bioluminescence detection of nitrocellulose-bound HSV DNA

using a dinitrophenol reporter group-labeled oligonucleotide and an anti-DNP antibody-pyruvate kinase detector complex

This example corresponds to example 7 with an exception

of pyruvate kinase being used as the second component in the display complex instead of G6PDH. The pyruvate kinase was used to produce the ATP that causes the firefly bioluminescence reaction to emit light.

A herpes oligonucleotide complementary to the immobilized HSV DNA and labeled with dinitrophenol was prepared, and a complex was formed between this and HSV DNA as in Example 7. After washing, the DNA duplex was incubated with an excess of the anti-DNP-antibody-pyruvate kinase detector complex which was prepared in a 1:3 ratio following the method of Yoshitake et al.,

(Eur. J. Biochem. 101: 395-399, 1979). The incubation was carried out for 30 minutes at room temperature in PBS-Tween 20. After washing six times in PBS/0.5 M NaCl/0/1% Tween 20, the filters containing the bound detector complex were incubated in 0.5 ml 0.05 M imidazole buffer, pH 7.6, containing 1.5 mM ATP, 1.5 mM phosphoenolpyruvate, and firefly luciferin and luciferase (Firelight, supplied by 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 oligonucelotide and DNA was determined by the amount of emitted light with time using a photosensitive charge-coupled device.

Claims (92)

1. Procedure for luminescent detection of a specific reporter group that is smaller than approx. 10,000 daltons in size and which is bound to a carrier mass, characterized by the steps of bringing the reporter group into contact with a detector complex which has, as a first component, a binding substance which exhibits high specific affinity for the reporter group, and which has, as a second constituent, a substance capable of being readily coupled to a light-emitting system, thereby, after such contact, providing high-affinity binding between the detector complex and the reporter group, and then bringing at least the other constituent of the detector complex into contact with a light-emitting system which is capable of emitting light in the presence of the other component of the detector complex. 2. Method according to claim 1, characterized in that contact between the bound reporter group and the detector complex is obtained by first binding the reporter group and the first component in the detector complex to each other, and then binding the second component in the detector complex and the bound first component to each other. 3. Method according to claim 1, characterized in that the high-affinity binding of the detector complex to the reporter group comprises the formation of a covalent bond, or a reaction of the ligand-ligand type. 4. Method according to claim 1 or 3, characterized in that the reporter group is a vitamin, a cofactor, an antigen or a carbohydrate. 5. Method according to claim 4, characterized in that the vitamin is biotin, iminobiotin, desthiobiotin or pyridoxal phosphate. 6. Method according to claim 4, characterized in that the cofactor is a por-phyr in . 7. Method according to claim 4, characterized in that the antigen is dinitrophenol, biotin, iminobiotin, desthiobiotin, fluorescein or fluorescamine. 8. Method according to claim 4, characterized in that the carbohydrate is mannose, galactose or fucose. 9. Method according to claim 1 or 2, characterized in that the reporter group is bound to the carrier mass through an organic polymer coating consisting of a protein, a carbohydrate, a nucleic acid or an analogue thereof. 10. Method according to claim 1 or 2, characterized in that the high-affinity binding of the detector complex to the reporter group comprises a reaction between vitamin and protein, a reaction between cofactor and protein, a reaction between antigen and antibody, a reaction between carbohydrate and lectin, or a covalent binding. 11. Method according to claim 1 or 2, characterized in that the reporter group is a vitamin and that the first component in the detector complex consists of a vitamin-binding protein. 12. Method according to claim 11, characterized in that the vitamin is biotin, iminobiotin or desthiobiotin, and that the protein is avidin, streptavidin or a complex of avidin or streptavidin. 13. Method according to claim 1 or 2, characterized in that the reporter group is a cofactor, and that the first component in the detector complex consists of a cofactor binding protein. 14. Method according to claim 13, characterized in that the cofactor is a porphyrin, and that the protein is apomyoglobin. 15. Method according to claim 1 or 2, characterized in that the reporter group is an antigen, and that the first component in the detector complex consists of an antibody for the antigen. 16. Method according to claim 15, characterized in that the antigen is dinitrophenol, biotin, iminobiotin, desthiobiotin, fluorescein or fluorescamin. 17. Method according to claim 1 or 2, characterized in that the reporter group is a carbohydrate, and that the first component in the detector complex consists of a lectin. 18. Method according to claim 17, characterized in that the carbohydrate is mannose, galactose or fucose. 19. Method according to claim 17, characterized in that the carbohydrate is mannose, and that the lectin is concanavalin A. 20. Method according to claim 1 or 2, characterized in that the first component in the detector complex is conjugated with avidin or streptavidin. 21. Method according to claim 1 or 2, characterized in that the first component in the detector complex is conjugated with biotin, iminobiotin or desthiobiotin. 22. Method according to claim 15 or 16, characterized in that the antigen is dinitrophenol, fluorescein or fluorescamine, and that the antibody is conjugated with avidin or streptavidin. 23. Method according to claim 15 or 16, characterized in that the antigen is dinitrophenol, fluorescein or fluorescamine, and that the antibody is conjugated with biotin, iminobiotin or desthiobiotin. 24. Method according to claim 1 or 2, characterized in that the first and second components of the detector complex are bound to each other by means of a reaction between vitamin and protein, a reaction between cofactor and protein, a reaction between antigen and antibody, a reaction between carbohydrate and lectin or a covalent bond. 25. Method according to claim 24, characterized in that the vitamin is biotin, iminobiotin, desthiobiotin or a conjugate thereof, and that the protein in the reaction between vitamin and protein is avidin, streptavidin or a conjugate thereof. 26. Method according to claim 1 or 2, characterized in that one of the first and second components of the detector complex is an avidin or is conjugated with an avidin, and that the second component is conjugated with a biotin. 27. Method according to claim 1 or 2, characterized in that the second component in the detector complex consists of an enzyme, an enzymatically active coenzyme, catalyst, fluorescent compound, exciting compound for fluorescent compound, luciferin or a substance that is luminescent in the presence of a catalyst and oxygen or hydrogen peroxide. 28. Method according to claim 1 or 2, characterized in that the second component in the detector complex consists of a dehydrogenase. 29. Method according to claim 1 or 2, characterized in that the second component in the detector complex consists of glucose-6-phosphate dehydrogenase. 30. Method according to claim 1 or 2, characterized in that the second component in the detector complex consists of biotinylated glucose-6-phosphate dehydrogenase. 31. Method according to claim 1 or 2, characterized in that the second component in the detector complex consists of FMN <+> oxidoreductase, glucose-6-phosphate dehydrogenase, malate dehydrogenase, alcohol dehydrogenase, lactate dehydrogenase, triose phosphate dehydrogenase, alkaline phosphatase, adenyl transferase, 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 that can be linked directly or indirectly to a light emitting system. 32. Method according to claim 1 or 2, characterized in that the second component in the detector complex consists of enzymatically active NAD <+> , NADP <+> , ATP, ADP, AMP, FMN+ or FAD <+> . 33. Method according to claim 1 or 2, characterized in that the second component in the detector complex consists of iron-heme or a metal. 34. Method according to claim 1 or 2, characterized in that the second component in the detector complex consists of 9,10-diphenylanthracene, perylene, rubrene, BPEA or an umbelliferone derivative. 35. Method according to claim 1 or 2, characterized in that the second component in the detector complex consists of TCPO or CPPO. 36. Method according to claim 1 or 2, characterized in that the second component in the detector complex consists of luminol, isoluminol, pyrogallol, lucigenin or lofin. 37. Method according to claim 1 or 2, characterized in that the second component in the detector complex consists of an enzyme which, in the presence of a substrate for the enzyme, has a turnover rate of at least 10/minute. 38. Method according to claim 1 or 2, characterized in that the second component of the detector complex consists of a molecular substance which, when connected to the light-emitting system, undergoes several reaction steps that result in the production of light. 39. Method according to claim 1 or 2, characterized in that the light-emitting system consists of components in a light-emitting reaction and components in a transition reaction for this, and that the other component in the detector complex, when connected to the system, provides a significant or limiting component in the transition reaction. 40. Method according to claim 1 or 2, characterized in that contact between the light-emitting system and the detector complex causes a first reaction where an intermediate product is produced, and a second reaction where the light is emitted, the first reaction being carried out under conditions which are generally optimal for the production of the intermediate, and the second reaction is carried out under conditions which are generally optimal for light emission. 41. Method according to claim 1 or 2, characterized in that the light-emitting system is bioluminescent. 42. Method according to claim 1 or 2, characterized in that the light-emitting system is chemiluminescent. 43. Method according to claim 1 or 2, characterized in that, before the contact with the light-emitting system, at least the second component in the detector complex is made soluble. 44. Method according to claim 43, characterized in that the second component in the detector complex is made soluble by chemical separation thereof from the bound first component in the complex. 45. Method according to claim 43, characterized in that the indicator complex is made soluble by chemical separation thereof from the reporter group] 46. Method according to claim 43, characterized in that the reporter group and the detector complex bound to it are made soluble by dissolution of the carrier mass. 47. Method according to any one of the above claims, characterized in that it comprises the further step of quantifying the light emitted by means of the light-emitting system as a measure for the reporter group which is bound to the carrier mass after contact with it. 48. Method according to claim 47, characterized in that the light emitted by the light-emitting system is quantified using means comprising a luminometer or a light-sensitive charge-coupled device. 49. Method according to claim 47, characterized in that the light emitted by means of the light-emitting system is quantified using means comprising light-sensitive film. 50. Method according to any one of the above claims, characterized in that the reporter group is brought into contact with an excess of the detector complex, and that the detector complex that is not bound is separated from the detector complex that is bound before the other component of the complex is brought into contact with the light-emitting system. 51. Detector complex for use in detecting small amounts of an immobilized reporter group that is less than approx. 10,000 daltons in size, characterized by a first component that exhibits a high affinity to a specific reporter group that is less than approx. 10,000 daltons in size, and a second component capable of being easily coupled to a light-emitting system. 52. Detector complex according to claim 51, characterized in that the first and second constituents thereof have a high affinity when binding to each other, which comprises covalent binding of the constituents via a bi-functional linking compound, a covalent bond formed between chemical residues on the constituents as a result of activation of the residues using a condensing agent, a non-covalent bond of the ligand-ligand type or a covalent bond that forms spontaneously between chemical residues on the constituents. 53. Detector complex according to claim 51, characterized in that the components are non-covalently bound to each other by a bond between vitamin and protein, a bond between cofactor and protein, a bond between carbohydrate and lectin or a bond between antigen and antibody. 54. Detector complex according to claim 51, characterized in that the first and second components thereof are bound to each other by means of a bond of the biotin-avidin type, the first component consisting of an avidin, a complex of an avidin or a complex of a biotin, and the second component consists of a complex of an avidin or a complex of a biotin. 55. Detector complex according to claim 51, characterized in that the first component consists of a vitamin-binding protein and the second component consists of a vitamin. 56. Detection complex according to claim 55, characterized in that the protein is avidin or streptavidin and that the vitamin is biotin, iminobiotin or -, desthiobiotin. 57. Detector complex according to claim 51, characterized in that the second component consists of an antigen and the first component consists of an antibody for the antigen. 58. Detector complex according to claim 57, characterized in that the antigen is dinitrophenol, biotin, iminobiotin, desthiobiotin, fluorescein or fluorescamine. 59. Detector complex according to claim 51, characterized in that the components are bound to each other by means of a bifunctional coupling agent which is binding amine, thiol or alcohol residues on the components. 60. Detector complex according to claim 51, characterized in that the components are covalently bound to each other as a result of the reaction between the amine, thiol or alcohol residues on the components and a carboxylate that has been activated by means of a condensation forge 1. 61. Detector complex according to claim 60, characterized in that the condensing agent is a carbodiimide or thionyl chloride. 62. Detector complex according to claim 51, characterized in that the second component, when connected to a light-emitting system, directly or indirectly provides a necessary component in a luminescence reaction. 63. Detector complex according to claim 51, characterized in that the second component consists of an enzyme, enzymatically active coenzyme, catalyst, fluorescent compound, excitation compound for fluorescent compounds, luciferin or a substance that is luminescent in the presence of a catalyst of oxygen or hydrogen peroxide. 64. Detector complex according to claim 51, characterized in that the second component consists of a substance capable of converting a procatalyst into a catalyst. 65. Indicator complex according to claim 51, characterized in that the second component consists of a substance capable of converting a precursor compound for a fluorescent compound into a fluorescent compound. 66. Detector complex according to claim 51, characterized in that the second component consists of dehydrogenase. 67. Detector complex according to claim 52, characterized in that the second component consists of glucose-6-phosphate dehydrogenase. 68. Detector complex according to claim 51, characterized in that the second component consists of biotinylated glucose-6-phosphate dehydrogenase. 69. Detector complex according to claim 51, characterized in that the second component consists of FMN <+> oxidoreductase, glucose-6-phosphate dehydrogenase, malate dehydrogenase, alcohol dehydrogenase, lactate dehydrogenase, triose phosphate dehydrogenase, alkaline phosphatase, adenyl transferase, NAD <+> synthetase, ATP synthetase , pyruvate kinase, creatine kinase, adenylate kinase, glucose oxidase, xanthine oxidase, monoamine oxidase, peroxidase, bacterial luciferase, firefly luciferase, beta-galactoxidase, neuraminidase, glucosidase or fucosidase or form an enzyme whose reaction product can be a substance that can be directly or indirectly coupled to a light-emitting system. 70. Indicator complex according to claim 51, characterized in that the second component consists of enzymatically active NAD <+> , NADP <+> , ATP, ADP, AMP, FMN <+> 'or FAD <+.> 71. Detector complex according to claim 51, characterized in that the second component consists of iron-heme or a metal. 72. Detector complex according to claim 51, characterized in that the second component consists of 9,10-diphenylanthracene, perylene, rubrene, BPEA or an umbelliferone or dansyl derivative. 73. Detector complex according to claim 51, characterized in that the second component consists of TCPO or CPPO. 74. Detector complex according to claim 51, characterized in that the second component consists of luminol, isoluminol, pyrogallol, lucigenin or lofin. 75. Detector complex according to claim 51, characterized in that the second component consists of an enzyme which, in the presence of a substrate for the enzyme, has a turnover rate of at least 10/minute. 76. Detector complex according to claim 51, characterized in that the second component consists of a molecular substance which, when connected to the light-emitting system, undergoes several reaction cycles that result in the production of light. 77. Detector complex according to claim 51, characterized in that the first component consists of a chemical residue capable of forming a covalent bond with a reporter group, or a ligand capable of forming a non-covalent ligand-ligand- binding with a reporting group. 78. Detector complex according to claim 51, characterized in that the first component consists of a protein capable of forming a vitamin-protein bond with a vitamin-reporter group, a protein capable of forming a cofactor-protein- binding with a cofactor reporter group, an antibody capable of forming an antigen-antibody bond with an antigen-reporter group, or a lectin capable of forming a carbohydrate-lectin bond with a carbohydrate reporter- group. 79. Detector complex according to claim 51, characterized in that the first component consists of avidin or streptavidin. 80. Detector complex according to claim 51, characterized in that the first component consists of apomyoglobin. 81. Detector complex according to claim 51, characterized in that the first component consists of an antibody for dinitrophenol, biotin, iminobiotin, desthiobiotin, fluorescein or fluorescamin. 82. Detector complex according to claim 51, characterized in that the first component consists of a lectin. 83. Detector complex according to claim 51, characterized in that the first component consists of concanavalin A. 84. Detector complex according to claim 51, characterized in that the first component is conjugated with avidin or streptavidin. 85. Detector complex according to claim 51, characterized in that the first component is conjugated with biotin, iminobition or desthiobiotin. 86. Reagent set, characterized in that it consists of the detector complex according to any one of claims 51 to 61 and 77 to 85, and a light-emitting system to which the second component of the complex can be easily coupled, and which is capable of emitting light as a result of such coupling . 87. Reagent set according to claim 68, characterized in that the light-emitting system comprises the components of a bioluminescent reaction. 88. Reagent set according to claim 86, characterized in that the light-emitting system comprises the components of a chemiluminescent reaction. 89. Reagent set according to claim 86, characterized in that the light-emitting system comprises the components of a light-emitting reaction, as well as the components of a transition reaction, and where the second component in the detector complex forms a limiting or essential component of the transition reaction. 90. Reagent kit according to claim 89, characterized in that the transition reaction is one which, when in contact with the second component of the detector complex, is capable of producing ATP, NADH, NAD PH, FADH, FMNH, aldehyde, hydrogen peroxide or a fluorescent connection. 91. Reagent set according to claim 90, characterized in that the light-emitting reaction is capable of emitting light in the presence of the product produced by the transition reaction. 92. Reagent set, characterized in that it comprises the detector complex according to any one of claims 62 to 76 and a light-emitting system to which the second component of the complex can be easily connected, and which is capable of emitting light as a result of such connection.