CA1255213A - Assay for immobilized reporter groups - Google Patents

Abstract

ABSTRACT

A sensitive and specific assay method for luminescent detection of support matrix-bound reporter groups smaller than about 10,000 daltons in size is disclosed which comprises contacting such reporter groups with a detector complex which comprises a first component having a strong and specific affinity for such reporter groups, and a second component capable of being readily coupled to a bioluminescent or chemi-luminescent 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 inter-actions by which the reporter groups are immobilized.
Also disclosed are reagent means useful in practicing the method.

Description

ESCRIPTION

ASSAY FOR I ~ OBILIZED REPORTER GROUPS

TECHNICAL FIELD

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

BACKGROUND ART

Over the last decade, a large nu~ber of radiolabeled and enzy~.e-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 incor~orated as a reporter 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 nany 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 r~agents, thereby adversely affecting the accuracy of the assay.
In contrast to the limitations associated with radioactive detection, assay ~ethods which utilize luminescent reagents pose ~d~7 little environnental hazard, are stable for long periods of time, and require inexpensive iu~strumentation for detection. The enzyme-labeled reactions referred to earlier herein have the advantage that they do not utilize radioactivity, but they do not typically have ~he sensitivity of radioactive assays. Thus, there is a defined need for a nonradioactive 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 ~hich do not require separation.
U.S. Patents ~os. 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 systens have been expanded to involve the use therein of bioluminescence or chemiluminescence. British Patent No.
1,578,275 describes a heterogeneous nethod for assaying insulin in solution using a competitive binding assay which emplo~fs an insulin-isoluminol conjugate. U.S. Patents Nos. 4,230,i97 and 4,380,580 describe competitive binding assays for detecting ligands in a liquid medium and enpIoy a c~njugate 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, ~larcel Dekker, New York, (1982). Since the preparation of this review article, several other papers have been published which report the use of chemiluminesce~ce 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~];
Hinkkanen 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 ~een 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), Bri~ati 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, (lg83)].
This system employed luminol to detect the microperoxidase, and has little utility, since it is severly limited by a lack of sensltivi~y tdetection limit ~10 13 moles of tar~et DNA).
The invention disclosed here is novel and differs markedly from the prior art. The definitive character-istics 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 bio-luminescent 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 o a ~ingle s-r~lall sinnai, ratfner 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 -s-nonradioactive means. It is necessary in many instances to be able ~o quantitate the number of reactive sites or binding sites on the surface oE 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 o 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 ~o 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.i For example, in the case of a suhstance labeled wlth 125Iodine, 1~.5 million of the 125 Iodine atom labels must be present in order to produce 100 disintegrations per minute. Because of the slow break-down 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 ~mder certain reaction conditions. Reporter groups use~ul in determinin~
the number oE re~ctive groups on support matrices, de~ermining 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 determi-nations, since such determinations cannot be made as easily withthe use of large, sterically bulky proteinaceous reporter groups. In contrast to the unstable protein-aceous 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 ~e 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 n~mber than can larger size reporter groups. This higher density of reporter groups per unit surface area of support matrix facilitates ~he detection of the reporter groups within a given small area, because the higher density produces a more readily detected signal ~rom such surface area; and 3) The use of small reporter groups affords a high degree oE 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 ~o monitor the immobilization of small reporter groups having such sub-stances attached thereto. Such versatility is not possiblewith 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 immobilized 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.

~ I~e reporter groups whlch are useful ln ~he yractice of the invention can be virtually any small molecule for ~hich specific binding subs~ances (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 cer~ain proteins, such as the cytochromes and the hydroperoxidases; antigens, such as dinitrophenol, biotin, iminobiotin, desthiobiotin, fluorescein 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 group~, a portion of the detecto~ 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 matrqx is correlated with the number of reporter groups bound to the support matrix.

The detector co~plex of the in~ention is comprised of a first component which has a high speci~ic 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 ligh~ 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 sub-stances which have a high specifîc affinity for potential reporter groups having a size of less than about 10,000 daltons, the reportergroup binding first ccmponent of the detector complex can be of a wide variety of substances.

~ h~t ~

8EST MODE OF C~RRYING OUT T}IE INVENTION

To facili~ate an understanding o 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 speci~ic 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 ~roups also include chemical moieties which are not normally present on the surfaces of molecules, but can be intro-duced as reporter groups through chemical processes.
3) Detector Complex - ~ complex having a first component comprising a repor~er group binding substance and a second component comprising a coupling substance.
4) Light Emitting Sys~em - One or more reactions which, when coupled to a detector complex, culminate in the ~mission 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 ~ ~ ~ 5 complexation of the reporter group with the detector complex.
2) The removal o~ uncomplexed detector complex.
3) The coupling o~ 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 lumi-no~e$er, 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, coenæyme, 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 netho~ of the present invention.

t~

The utility of ~hese ligh~ emi~ing reactions can be expanded by the use of a variety of bricl~ing reac~ions which can provide one of the limiting substances in the luminescent reaction. It should be pointed out tha~
even though oxygen is a limiting substance, in many light emitting systems it is normally ubiquitous, and as a result, brid~ing 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 luci- -ferase,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 c ~ onent of a bridging reaction capable of providing an inter-mediate product which is essential or limiting in aluminescent 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~ Uslng 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+ dépendent 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 dehydro-genases, many of which are useful in the detector complex.
Subsequen~ or simultaneous contac~ of ~he 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 N~D+ dependent dehydrogenase and an appropriate reduced substrate for the dehydrogenase generates NADH. Either simultaneous or subsequent contact of the NA~H with FMN+ and FMN~ oxidoreductase, aldehyde, oxygen and bacterial luciferase results in the emission of light.

s~

d) Using a detector complex having a second component comprising active NAD~ as a limiting agent, and contacting th~ bound detector complex with an appropriate reduced substrate, and an appropriate dehydrogenase, NADH ls generated. Either simultaneous or subsequent contact of the NADH with FMN+, and FMN~
oxidoreductase generate;s FMNH. Contact of the FMNH
with aldehyde, oxygen, and b~cterial luciferase, resul~s in the emission of light. Such a system requires that the NADf 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 o 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 dehydro-genase, 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 9ubsequent contact oE the product aldehyde with oxygen, FMNH and bacterial luciferase produces light. ~ second dehydrogenase and its subs~rates added to this reaction system re~enerates the product N~D(P) ~rom 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 ar~ for illustrative purposes only, and are not intended to limit the scope of the invention. While those skilled in the art will reco~nize that several hundred di~ferent 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 sub-stances 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 largé 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 c~Jcles in a short period oE 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 ~he oxidoreductases, such as FMN~
oxidoreductase, glucose-6-phosphate dehydrogenase (G6PDH), malate dehydro~enase, alcohol dehydrogenase, lactate dehydro~enase or triose phosphate dehydrogenase;
alkaline phosphatase; adenyltransferase; NAD~ synthetase or ATP synthetase; pyruvate kinase, creatine kinase or adenylate ~inase; glucose oxidase, xanthine oxidase, or monoamine oxidasei peroxidase; bacterial or firefly luciferase; pyranosidases such as beta ~alactosidase, 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 N~D+, 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 usefulIy comprise a fluorescer such as, for example, 9,10,diphenylanthracene, perylene, rubrene, bis[phenylethynyl]anthracene (BP~) ~ndumbelliferone or dansyl derivatives; or fluorescer exciting substances such as bis(2,4,6-trichlorophenyl)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 use~ul therewith, see Maulding, D.R. and Roberts, B.G. J. Org. Chem.,34, supra;
Tseng, S.S., et al., J. ~ Chem., 44, supra;

and Gill, S.K. ~ldrichimica ~c~a, :L~,supra. ~dditional discussion of chemiluminescent and biolumi.nescent light emitting systems can bc Eound in Clinical and Biochemical Luminescence, 12, supra, and Methods in Enzymolo~y, 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 small~r than about 10,000 daltons. It is preferable that ~he interaction of the reporter group binding substance with a reporter group exhibit an affinity greater than about 10 . Exemplary of preferred detector complex ~irst 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 whi~h 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 cocanavalin A which binds mannose reporter groups; and chelating agents which selectively bind metal reporter groups. ~lso useful as the first component are those comprising chemical moieties which give highly specific reactions with particular reporter group functionalities.

~S~3 In such casas, recognition ol a reporter group by the detcctor complex results in ~he lormation oE 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 couplin~ 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, respect-ively; 4) The reaction of amines with isothiocyanatesto form ~hioureas; 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) T~le direct covalent attachment oE ~he components to each other by the covalent spontaneous binding o chemical moleties on the s~lrfaces thereof to Eorm 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 ac~ivating 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 ~pecies. Exemplary of such bifunctional coupling agents are those which link amines and/or thiols and/or ~ alcohols;
3) The formation of a high affinity noncova'lent interaction between the components.
The preferred synthesis of the detector complex employs a noncovalent interaction of the type referred to earlier herein as useful Eor 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 int'eraction; 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 irst componen~ thereof i9 avidin, and the second component thereo~ is biotinylated FMN~ oxido-reductas~. Since avidin has our 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~ oxidore~uctase component can be readily coupled to light emission using the bacterial biolumi-nescence 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. Re~ardless 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 - 25 is found that a desirable activity is lost during complex-ation, 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:

.

~ ~5 ~ ~;3 l~ The individual components of a detector complex should function properly under acceptably similar conditions o pH, temperature, salt concentration, etc.
2~ The second component, when coupled to an enz~ne.
coenzyme, or fluorescer, should yield a product which can be 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, i~ 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 pxoduct 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 iess 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 inter-mediate product is catalyzed and a second reaction bywhich 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 ~23-in several ways, as fol~ows:
1) By carrying out the first reaction, which involv~s ~he catalytic ac~ivi~y of ~lle deLec~or complex, under condi~ions which are generally optimal for the formation of the intermediate reac~ion 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 groùp to which it is bound using a change in pH, temperature, salt concentration, 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 o~ a deLector 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 30 of-hi~h affinity at neutral pH, but it dissociates at ~he mildly acid pH of ~1. 5 .
b) The use of a detector complex whose components are coupled by ~ linkage which can be cleavecl 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 bne minute convert to product a lar~e 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, bu~ there are normally at least some decreases in activity associated with com-plexation (See Sundaram, P.V. et al., Can. J. Chem.

~t~

4~, 1498-1504, (1970) and Carlsson, J, and Svenson, A., FEBS Lett., 42, 183-186 (1974). In general, however, enzymes retain reasonabl~ 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 activa~ion of the enzymatic reaction. Such action of the coenzyme NAD is illustrated in the following cyclic reaction:

substrate ~ NAD+ ~ ~ product~ -a- light ~ nzyme ~ enzyme 2 product NADH ~ substrate 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 co~actor can be liberated from the complex prior to assay. Enzyme 1 can be any of a wide range of dehydrogenases. Enzyme 2 can, 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 retaln catalytic activity have been syn~hesized. (See, ~or 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, ll9 (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 ~8 position of the purine ring, there is a good chance that its catalytic activity with enzymes will be retained tMosbach, K. et al., Methods in Enzymology, 44, supra. One such use of ATP
is illustrated in the following reaction PRPP AMP-m Luciferin-Oxidized + Ligh~

YATP PRPP ~uciferase (Fire~ly) ATransferase ribose-5- ~ ~ ~ \
phosphate ATP-m Luciferin (Firefly) 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:

1. Procatalyst Detector complex ~_ Catalyst 2. Reactan~ Cata_y~t ~Produc~
Directly or 3. Product ~_ through lumines- ->_ Light cent reaction 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 reactions.
The enzymatic formation of NAD+, the enzymatic production of fluorescer molecules, and the enzymatic activation of a second enzyme fall into this category. Typical reactions for generation of NAD+ and fluorescer, respectively, as well as bioluminescent and chemiluminescent reactions respectively useful therewith can be illustrated as fol~lows:
+
A. NAD Formation Reactions 1. MNM+ + ATP adenvltransferase~_ NAD+

2. NADP+ alkaline phosphatase ~ NAD

~ 3 B. Light Emitting Reaction (Bioluminescent) Substrate ~ N~D X FMNH X aldehyde + oxygen Product NADH FMN acid + H20 ~ Light a = an appropriate dehydrogenase b = FMN+ oxidoreductase c = bac~erial luciferase C. Profluorescer to Fluorescer Conversion Reaction Profluorescer enzyme ~_ Fluorescer 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) O O
Oxalate 2 2 ~ Fluorescer . Light derivative l I ~ (ground stat cyclodioxetane ~ J
2 CO ~ ~ Fluorescer 2 (excited state) In this light emitti.ng reaction an oxalate derivative, such as bis(2,ll,6-trichlorophenyl)oxalate (TCPO~ or bis(2-carbopentoxy-3,5,6-tricllolorphenyl) oxalate (CPPO) is converted to a cyclodioxetane, and the energy liberated by the collapse the cyclodioxetane to CO2 is absorbed by the :Eluorescer, 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 immobili~ed 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 inter-action 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 int'er-action; 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 inter-action by which a reporter group is immobilized on a su~port 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 thc lcc~in. Whcn ~l~e rcl~or~cr ~roup-carl~olly(l~<l~
complex is brought into contact wlth 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 immobil-ization of a reporter group on a support matrix through aDNA-DNA interaction can be monitored. For example, an organic polymer in ~he 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 9 the method also de-tects 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. `~nong such light sensitive means are a lumin~neter, light sensitlvë fiLm, and a light sensitive charge coupled device or other suitable and desired li~ht 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 adap~ations of the method.

3 ~ r The DetectioII of Agarose-Bound Biotin Repor~er Groups Using a Detector Complex of Avidin and a Biotin-Rich Aggre~ate of Biotinyla~ed G6PDH
~nd Avidin (BAC) In this example, bio~in is the reporter group, and the detec~or complex has avidin as its first compon-entand has as its second component a noncovalent ~iotin-rich 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 Lumi-nescence 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 con-comitant 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.lM 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 concen-tration of 5 mM and 10 mM, respectively, in a volume of 1.4 ml. To this solution was added 50 ~1 of N-hydroxy-succinimide biotin (2 mg/ml in DMSO), and the solution was incubated at room temperature. G6P~H acti~icy was followed by removing small aliquots of the reaction mixture and assaying enzymacic activicy by monicoring the production o NAD~ spectrophotometrically in 1 ml of 55 mM Tris (pH 7.8) containing 3O3 mM magnesium chloride, 1.7 mM NAD~, and 2.8 mM glucose-6-phosphate.
When the activity of the enzyme had decreased to 50Z 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. Biotiny-lation of the enzyme was confirmed by showing that more than 90Z of the enzyme activity was absorbed on an a~idin-agarose column ~Pierce Chemical Co., Roc~ford, 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 ~omponent of the detector complex is made by forming an aggregate with an excess of biotinylated dehydro genase over avidin (i.e. the BAC component is biotin rich).
The BAC aggregate is formed by adding four ~1 of a solution of A~idin-D (O.S mgJml PBS-Tween 20) to 1 ml of PBS-Tween ~0. A~idin-D is ~vailable from Vector Laboratories, Burlingame, CA, and PBS-Tween 20 is phosphate buffered saline (0.9Z NaCl, 10 mM sodium phosphate, pH7) ~ontaining 0.1~ ~ween 20. To this dilute solution 12 ~1 of biotiny-lated enzyme ~0.4 m~,en~m~/m~0.1 M NaHC03) is added with mixing. The aggregate is allowed to form for at least fifteen minutes and is then stored at 4 C until used.
Immedia~ely before use, the BAC aggregate is centri-fuged or 5 minutes in a tabletop centrifuge (ca 3000 rpmj * Trade Mark ~ 3 in order to remove overly lar~e aggregates whieh contribute to non-speciic binding.
Ten microliters oE a 91urry oE biotin-agarose beads (Pierce Chemical Co., 1.5 x 10 ll avidin binding capaci~y) is diluted into 1 ml PBS-Tween 20. This slurry is then dispensed in 100 microliter aliquo~s into 1.5 ml Eppen-dorf tubes. The contents of each tube are washed twice with 1 ml PBS-Tween 20, then incubated for 30 minutes with 40jul avidin D (0.5 mg/ml, 0.3 nmoles) in order to complex the biotin. The agarose-biotin:a~idin aggregate is washed free of unbound avidin with 1 ml PBS-Tween 20 and resuspended in 100 ~1 of the same. Ten microliter portions of the suspension are then aliquoted into 1.5 ml Eppendorf tubes. To each tube is added 200JUl 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 indivi-dual agarose beads were then removed from each tube under `a microscope and assayed for bioluminescence in a Mono-light 401 luminometer (Analytical l.uminescence 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 JUl.
After the addition of a single agarose bead to the reagent solution, the light emitting reaction was initiated by the r - ~ P ~; ~

additi.on of 10 ~1 Bac~ilight I reagent (made up as per the manufacturer's instructions), and the course of the reaction monitored in t~e Monoligh~ 401 luminometer on a sensitivity setting of lOx. Typical results, along with the appropriate controls, are given in Table I.

TABLE I
Bioluminescent Detection of Biotin Reporter Groups Bound to Single Agarose Beads Using an Avidin-BAC Detector Complex Rate of Light Output Sample (Arbitrary Un _ No sample (machine blank) 0.01 Empty sample tube 0.02 FMN + aldehyde + buffer 0.03 15 FMN + aldehyde ~ buffer + Bactilight I reagent 0.15 (Complete reagent system) Complete reagent system +
agarose-biotin:avidin-BAC 2.43 20 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 microliter of the original slurry. One bead 70 microns 25 in diameter thus corresponds to about 10 15 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.

EXAMPI,E 2 The Detection of Agarose-Bolmd Biotin Repor~er Groups Using an ~vidin-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 dehydro~enase (biotin-G6PDH). In this case 9 200 ~1 of a biotin-G6PDH solution (~ 12 pmol) is added to each sample using the same pro-cedure 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 Mono-light 401 luminometer as described in Example l. The resulting data are given in Table II.

Table II
Bioluminescent Detection of Agarose-Bound Biotin Reporter Groups Using an Avidin-&6PDH Detector Complex Rate of Light Output 20 Sample (Arbitrary Units) No sample (machine blank) 0.04 Empty sample tube 0.04 FMN ~ aldehyde + buffer 0.03 FMN + aldehyde + buffer 25 + Bactilight I reagent (Complete reagent system)0.15 ~ ~ ~ S~ ~ 3 ~36-TABLE II (Cont'd) Rate o~ Light Output Sample (Arbitrary Units) Complete reagent system ~
agarose-biotin:avldin-biotinylated G6PDH 2.34 Complete reagent system +
agarose-biotin:avidin-biotin control 0.11 Based on these results, it is estimated that the detec~ion limit for biotin bound to agarose using this detector complex is about 10 16 moles of biotin.

The Detection of Agarose-Bound Biotin Reporter GroupsUsing an Avidin-Biotinylated G6PDH Detector Complex 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 ~ul of streptavidin (0.5 mg/ml, Bethesda Research Laboratories, Bethesda, MD) with 3 ~1 of biotinylated ~5~

glucose-6-phospha~e dehyclrogenase (0.42 m~/ml) in 125 ~1 PBS-Tween 20.
Tllree hundred ~1 of biotin-agarose suspension (Sigma Chemical Company, St~ Louis, MO) was washed twice wit~ 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 ~1 aliquo~s of the aggregate, followed by agitation on a wrist action shaker for 1 hour at room temperature. The samples were then washed 3 times wi~h 1 ml PBS-Tween 20 containing 0.5 M NaCl. Assaying the samples using a Monolight 401 luminometer accordin~ to the procedures in Examples 1 and 2, produces results similar to those produced in such examples.

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 ~he support matrix through an immobilized ligand, and the detector complex has avidin as its first component, and has the biotin rich BAC a~gregate 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 at~ached 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 Bio-chemicals, New York, NY. The reaction yielded Lambda DNA in which 31 % of the thymidine residues had been replacedwith biotinylated deoxyuridine, as judged by the ~f~

concomitant incorporation of tritium labeled dCTP of known speciic activity. The DNA was purif~ed away from c~ntaminating unincorporated dUTP using ethanol precipitation and Sephadex G-25 chromatography.
Biotinylated lambda DNA was denatured by heating to lOOD C for 5 minutes in distilled water follo~ed by chilling on ice to prevent reannealing of the single strands. Various amounts of ~he denatured DNA were then bound ~o 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 alr dry overni~ht and were then ~aked for 1 hour at 60~ C in vacuo in order to fix the biotinylated D~A thereto. The amount of biotinylated DNA bound to each filter was quantitated by liquid scintillation counting. The average bindin~ eficiency of the filters was 56Z of the input DNA. Control filters were prepared using unmodified calf thymus DNA (1 micro~ram of DNA per fil~er). BAC aggre~ates of biotinylated G-6-PDH and a~idin were prepared as in Example I.
The procedure used ~o assay filters for the presence thereon of immobilized biotinylated DNA, was as follows.
The unreacted sites on the filters were "capped" by incubation for 3D minutes a~ 37 C with 2X 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 reacti~e 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 ~1 streptavidin solution (prepared by diluting 15 ~1 streptavidin to 1.5 ml with PBS-Tween 20) to form a filter-DNA-biotin:avidin * Trade Mark f . , ., i 2~3 complex. Unbound strep~avidin was then removed by two 5 min~tte 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 ~1 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 o 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 ~1 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 ~ul 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 ~1 Bactilight I
reagent and measuring peak light output over a 10 second ` interval according to the manufacturer's instructions.
The amount of,,boun,d biotin~lated DN~ corresDonded linearly with the amou~ti,of li~ht emitted.

.

EXAMPLE` 5 The Bioluminescent Detection of a Nitrocellulose-Bound llepatitis B Virus DNA Using A Biotin Reporter Group-Labeled Oligonucleotide and an Avidin-BAC
Detector Complex One application of -this invention is the non-radioactive detection of DNA-DNA hybrids formed be-tween 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 nucleo-tides in length and complementary to the HBV surface antigen gene was synthesized. In this oligonucleo-tide two of the thymidine residues were substituted with biotinylated deoxyuridine. The biotin was co-valently 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 11981)).
The HBV DNA contained in the plasmid pAM6 was adsorbed to nitrocellulose filters essentially as described in Example 4 for lambda DNA. Control fil-ters made at the same time contained only calf thy-mus 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 orcler to cap the unreacted sltes on the nitrocellulose and prevent nonspecific binding of the probe to the filter material.
Ficoll i~ a sucrose polymer sold by Pharm~cia Fine Chcmicals, Piscataway, N.J. The filters were then treated with the biotinylated DNA probe (2 ng/ml) overnight at 46 C in 6x SSC containing lx Denhardt's solution in order to form a complex which may be diagrammed as filter-HBV:
oli~onucleotide-biotin. Th~ 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 o 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 ~g/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 cssentially 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 detec~ the ~mount 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 ~ 3 incubatecl for 10 minutes at room temperature in S0 ~1 of the NADH-generating solution described in Example 4.
The NADH genera~ed was detected in Monolight 401 lumino-meter using the ~actilight I reagent as follows. A 25 ~1 aliquot of each assay mix was added to a 5 ml plastic reaction cuvette containing 400 ~1 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 lumines-cence reaction was initiated by the injection of 100 ~1Bactilight I reagent containing FMN+, NADT:FMN+ oxido-reductase 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 nitro cellulose filters.

The Bioluminescent Detection of Cellulose-Bound 'Anti-Herpes Antibody Using a Biotin Reporter' Group-Labeled Herpes'Surface Antigen'and'an Avidin-BiotinyIated G~PDH 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,ul 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 ~ 5 temperature for 1 hour. The reaction is then quenched by adding 50 mg of ammonium sulate, and the product is dialysed twice against 0.1 M sodium bicarbonate. Alter-natively, ~he 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 biotinylated HSV antigen in PBS-Tween ~0 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 detec~or 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 de~ect 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, an~ the rate of signal generation is measured as a function of time.
These values can be compared ~o a standard curve prepared - by following the same procedure and using various known amoun~s 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.

l~S~
-4~-EXA~PL~ 7 The Bioluminescent De-tec-tion of Nitrocellulose-Bound HSV DNA Using a Dinitrophenol Reporter Group-Labelecl 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 oligonucleo-tide which has dinitrophenyl reporter groups at-tached to the C 5 position of deoxyuridine via an

11 atom linker arm is synthesized.
The dinitrophenylated herpes oligonucleotide, which is complementary to the immobilized HSV DNA, is complexed to the latter at an oligonucleotide concentration of 10 ng/ml in 6x SSC containing lOx Denhardt's solution overnight at 37C 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 incu-bated with 2% BSA in PBS-Tween 20 at 37C for 30 minutes to cap off any remaining protein binding sites on the filter. After three 5 minute washes with PBS-Tween 20, the filters are incubated with antidinitrophenyl antibody-G6PDH detector complex (- 1 pmol) for 30 minutes at room temperature in 40 ~1 PBS-Tween 20.
- 30 The antiDNP-Ab-G6PDH detector complex is formed in a ratio of 1:3 according to the procedure of Carl-sson, et al. (Biochem. J. 173:723-737, 1978) using N-succinimdyl-~ .

~s~

3-(2-pyridyldithio)propionate. After washing the filter bound complex 6 times with 6x SSC/0.1% Tween 20, the light emission is quantltated as follows. ~ach individual filter is blotted dry to remove excess moisture and is transfered to an individual we1l in a microtiter plate.
To each well is added lO0 ~l of Bac~ilight l reagent, and the reaction is allowed to reach a steady state which is achieved after approximately l hour of incubation at room temperature. The microtiter plate is then set on top of an 8 x lO sheet of Kodak ~ XOMAT ~ XAR-5 X-ray film in ~he dark, and the exposure is allowed to proceed for an appropriate period of time (generally from about l 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.

. .
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 anti-bodies bound to the agarose support matrix, the capped beads are incubated ~or 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 repor-ter groups and thereby the amount of bound antiHSV antibody present on the agarose beads, the beads are suspended in lO ~1 of O.lM Tris, pH 7.5, and transferred to a glass cuvette to which is added the exciting agent, 250 JUl bis(2,4,6-tri-cholorphenyl)oxalate (TCPO) in ethyl acetate, and 100 ~1 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 ligh~ 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 pro-cedure and using various known amounts of anti-HSV anti-body, in order to determine the amount of bound anti-HSV antibody present on the beads.

~t~5~ ~ 3 -~17-The Biolumin~scent Detection of Nitrocellulose-Bound HSV DNA Vsing 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 ~mployed to generate ATP which causes the firefly bioluminescence reaction to emi~ 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 w~ich 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 Analytica~ 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 (27)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A heterogeneous 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 an excess of 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 bioluminescent or chemiluminescent light emitting system, thereby to produce upon such contact high affinity attachment of said detector complex and such reporter group; separating the unbound excess detector complex from the detector complex bound to the reporter group; and then contacting at least the second component of the bound detector complex with a bioluminescent or chemiluminescent 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 which is preferably a vitamin-protein interaction, a cofactor-protein interaction, an antigen-antibody interaction, or a carbohydrate-lectin interaction.

4. The method of Claim 1 wherein the reporter group is a) a vitamin, preferably biotin, iminobiotin, desthiobiotin or pyridoxal phosphate, or b) a cofactor, preferably a porphyrin, or c) an antigen, preferably dinitrophenol, biotin, iminobiotin, desthiobiotin, fluorescein or fluorescamine, or d) a carbohydrate, preferably mannose, galactose or fucose.

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

6. The method of Claim 1 wherein the reporter group is a) a vitamin, preferably biotin, iminobiotin or desthiobiotin, and the first component of the detector complex comprises a vitamin binding protein, preferably avidin, streptavidin or a complex of avidin or strep-tavidin, or b) a cofactor, preferably a porphyrin, and the first component of the detector complex comprises a cofactor binding protein, preferably apomyoglobin, or c) an antigen, preferably dinitrophenol, biotin, iminobiotin, desthiobiotin, fluorescein or fluorescamine, and the first component of the detector complex comprises an antibody to said antigen.

7. The method of Claim 1 wherein the first component of the detector complex is a) conjugated with avidin or streptavidin, or b) conjugated with biotin, iminobiotin or desthiobiotin, or c) an antibody to dinitrophenol, fluorescein or fluorescamine, and said antibody is conjugated with avidin, streptavidin, biotin, iminobiotin or desthio-biotin.

8. The method of Claim 1 wherein the first and second components of the detector complex are attached to each other by a covalent bond; a cofactor-protein interaction; an antigen-antibody interaction; a carbohydrate-lectin interaction; or a vitamin-protein interaction wherein a) 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, or b) the vitamin is biotin, iminobiotin, desthio-biotin or a conjugate thereof, and the protein is avidin, streptavidin or a conjugate thereof.

9. The method of Claim 1 wherein the second component of the detector complex comprises a) an enzyme, preferably glucose-6-phosphate dehydrogenase, biotinylated glucose-6-phosphate dehydrogenase, malate dehydrogenase, alcohol dehydro-genase, lactate dehydrogenase, triose phosphate dehydrogenase, FMN+ oxidoreductase, alkaline phospha-tase, adenyltransferase, NAD+ synthetase, ATP synthe-tase, pyruvate kinase, creatine kinase, adenylate kinase, glucose oxidase, zanthine 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 bioluminescent or chemiluminescent light emitting system, or b) an enzymatically active coenzyme, preferably enzymatically active NAD+, NADP+, ATP, ADP, AMP, FMN+
or FAD+, or c) a substance capable of converting a procatalyst to a catalyst, or d) a substance capable of converting a profluor-escer to a fluorescer, or e) a catalyst, preferably iron-heme or a metal, or f) a fluorescer, preferably 9,10, diphenylanthra-cene, perylene, rebrene, BPEA or an umbelliferone or dansyl derivative, or g) a fluorescer exciter, preferably TCPO or CCPO, or h) luciferin, or i) a substance which is luminescent in the presence of a catalyst and oxygen or hydrogen peroxide, preferably luminol, isoluminol, pyrogallol, lucigenin or lophine, or j) an enzyme which, in the presence of a substrate therefor, has a turnover number of at least 10/minute, or k) a molecular substance which, when coupled to the light emitting system, undergoes multiple cycles which result in the production of light.

10. The method of Claim 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.

11. The method of Claim 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 intermediate product, and the second reaction being carried out under conditions generally optimal for light emission.

12. The method of Claim 1 or 2 wherein, after the separa-tion step and prior to said contact with the light emitting system a) at least the second component of the detector com-plex is solubilized, or b) the second component of the detector complex is solubilized by chemical separation thereof from the bound first component of said complex, or c) the detector complex is solubilized by chemical separation thereof from the reporter group, or d) the reporter group and the detector complex bound thereto are solubilized by dissolution of the support ma-trix.

13. The method of any of Claims 1, 2 or 3 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 when said reporter group and detector complex are brought into contact, such quan-titation being preferably accomplished by the use of means including a luminometer, a light sensitive charge coupled device, or a light sensitive film.

14. The method of Claim 8 comprising 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 when said reporter group and detector com-plex are brought into contact, such quantitation being preferably accomplished by the use of means including a luminometer, a light sensitive charge coupled device, or a light sensitive film.

15. 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 ex-hibiting 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 biolumi-nescent or chemiluminescent light emitting system.

16. The detector complex of Claim 15 wherein the first and second component thereof have a high affinity attachment to each other which involves a) covalent linking of said components, preferably through a bifunctional coupling agent which links amine, thiol or alcohol moieties on said components, or b) a covalent bond formed between chemical moieties on said components, preferably 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 ag-ent, preferably a carbodiimide or thionychloride, or c) a noncovalent ligand-ligand type interaction, pre-ferably a vitamin-protein interaction a cofactor-protein interaction, a carbohydrate-lectin interaction, or an antigen-antibody interaction, or d) a covalent bond spontaneously formed between chem-ical moieties on said components.

17. The detector complex of Claim 15 wherein the first and second components thereof are attached to each other by a biotin-avidin type interaction, said first component com-prising 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.

18. The detector complex of claim 15 wherein said first component comprises a vitamin binding protein, preferably avidin or streptavidin, and said second component comprises a vitamin, preferably biotin, iminobiotin, or desthiobiotin.

19. The detector complex of Claim 15 wherein said second component comprises an antigen, preferably dinitrophenol, biotin, iminobiotin, desthiobiotin, fluorescein or fluoresc-amine, and said first component comprises an antibody to said antigen.

20. The detector complex of Claim 15 wherein the second component thereof, when coupled to a bioluminescent or chemiluminescent light emitting system, directly or indi-rectly provides a necessary constituent of a luminescent reaction.

21. The detector complex of Claim 15 wherein the second component comprises a) an enzyme, preferably glucose-6-phosphate dehydro-genase, biotinylated glucose-6-phosphate dehydrogenase, ma-late dehydrogenase, alcohol dehydrogenase, lactate dehydro-genase, triose phosphate dehydrogenase, FMN+ oxidoreductase, alkaline phosphatase, adenyltransferase, NAD+ synthetase, ATP synthetase, pyruvate kinase, creatine kinase, adenylate kinase, glucose oxidase, xanthine oxidase, monoamine oxi-dase, 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 bioluminescent or chemiluminescent light emitting system, or b) an enzymatically active coenzyme, preferably enzy-matically active NAD+, NADP+, ATP, ADP, AMP, FMN+, or FAD+, or c) a substance capable of converting a procatalyst to a catalyst, or d) a substance capable of converting a profluorescer to a fluorescer, or e) a catalyst, preferably, iron-heme or a metal, or f) a fluorescer, preferably 9,10 diphenylanthracene, perylene, rubrene, BPEA, or an umbelliferone or dansyl de-rivative, or g) a fluorescer exciter, preferably TCPO or CCPO, or h) luciferin, or i) a substance which is luminescent in the presence of a catalyst and oxygen or hydrogen peroxide, preferably luminol, isoluminol, pyrogallol, lucigenin or lophine, or j) an enzyme which, in the presence of a substrate therefor, has a turnover number of at least 10/minute, or k) a molecular substance which, when coupled to the light emitting system, undergoes multiple cycles which re-sult in the production of light.

22. The detector complex of Claim 15 wherein the first component thereof comprises a chemical moiety capable of forming a covalent bond with a reporter group smaller than about 10,000 daltons in size, or a ligand capable of form-ing a noncovalent ligand-ligand interaction with such a reporter group.

23. The detector complex of Claim 15 wherein the first com-ponent a) comprises a protein capable of forming a vitamin-protein interaction with a vitamin reporter group, prefer-ably avidin or streptavidin, or b) a protein capable of forming a cofactor-protein interaction with a cofactor reporter group, preferably apomyoglobin, or c) an antibody capable of forming an antigen-antibody interaction with an antigenic reporter group, preferably an antibody to dinitrophenol, biotin, iminobiotin, desthiobio-tin, fluorescein or fluorescamine, or d) a lectin capable of forming a carbohydrate-lectin interaction with a carbohydrate reporter group, preferably concanavalin A, or e) a conjugate with avidin or streptavidin, or f) a conjugate with biotin, iminobiotin or desthio-biotin.

24. Reagent means comprising the detector complex of Claim 15 and a bioluminescent or chemiluminescent light emitting system to which the second component of said complex can be readily coupled, and which is capable of emitting light re-sponsive to such coupling.

25. The reagent means of Claim 24 in which the light emit-ting system comprises, in addition to constituents of a bio-luminescent or chemiluminescent light emitting reaction, constituents of a bridging reaction as to which the second component of the detector complex functions as a limiting or essential constituent.

26. The reagent means of Claim 25 wherein the bridging re-action is one which, when in contact with the second com-ponent of the detector complex, is capable of generating ATP, NADH, NADPH, FADH, FMNH, aldehyde, hydrogen peroxide or a fluorescer.

27. The reagent means of Claim 26 wherein the bioluminescent or chemiluminescent light emitting reaction is capable of emitting light in the presence of the product generated by the bridging reaction upon contact of the latter with the second component of the detector complex.