CA1239580A - Hybridization assay employing labeled pairs of hybrid binding reagents - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

A nucleic acid hybridization assay wherein the hybrid formed with the probe has binding sites for two specific binding reagents, one of which comprises a first label and the other a second label, interaction of the first and second labels providing a detectable response which is measurably different when the two labeled reagents are both bound to the same hybrid compared to when the two labeled reagents are not so bound. The formation of the hybrid assay product brings the two labels within a proximate interaction distance of one another, e.g., as in the cases of sequential catalyst (enzyme) interaction and energy transfer.
Since the labels provide a response which is distinguishable when they are associated with a hybridized probe, no separation step is required.

Description

SUE

Hybridization ASSAY EMPLOYING
LABELED PAIRS OF HYBRID BINDING REAGENTS

FIELD OF THE INVENTION

This invention relates to nucleic acid hybridization assay methods and reagent systems for detecting specific polynucleotide sequences. The principle of nucleic acid hybridization assays was developed by workers in the recombinant DNA field as a means for determining and isolating particular polynucleotide base sequences of interest. It was found that single stranded nucleic acids, e.g., DNA
and RNA, such as obtained by denaturing their double stranded forms, will hybridize or recombine under appropriate conditions with complementary single stranded nucleic acids. By labeling such complementary probe nucleic acids with some readily detectable chemical group, it was then made possible to detect the presence of any polynucleotide sequence of interest in a test medium containing sample nucleic acids in single stranded form.
n addition to the recombinant DNA field, the analytical hybridization technique can be applied to the detection of polynucleotides of importance in the fields of human and veterinary medicine, agriculture, and food science, among others. In particular, the technique can be used to detect and Jo Lo it identify etiological agents such as bacteria and viruses, to screen bacteria for antibiotic resistance, to aid in the diagnosis of genetic disorders such as sickle cell anemia and thalassemia, and to detect cancerous cells. A
general review of the technique and its present and future significance is provided in biotechnology (August 1983), pp. 471-478.

INFORMATION DISCLOSURE

The following information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present invention. No admission is necessarily intended, nor should be construed, that an of the following information constitutes prior art against the present invention.
The state-of-the-art nucleic acid hybridization assay techniques generally involve a separation of hybridized and unhybridized labeled probe. This required separation step is usually facilitated by immobilizing either the sample - nucleic acids or the nucleic acid probe on a solid support. Commonly, hybridization between particular base sequences or genes of interest in the sample nucleic acids and a labeled form of the probe nucleic acid is detected by separating the solid support from the remaining reaction mixture which contains unhybridized probe, followed by detection of the label on the solid support.

pa There are continuing efforts to simplify the analytical procedure for performing nucleic acid ~L~395~3~

hybridization assays. A primary goal of these - efforts is to reduce the complexity of the procedure to the point that it can be conveniently and routinely performed in clinical laboratories.
The necessity of a separation step seriously impedes the progress of these efforts. The separation step requires considerable expertise in order to be accomplished in an analytically reproducible manner and is a physical manipulation not readily automated or suited to high volume testing.
Moreover, in conventional methods involving the immobilization of sample nucleic acids, two significant difficulties are encountered. Firstly, the procedures required to accomplish immobilization are generally time consuming and add a further step which is undesirable for routine use of the technique in a clinical laboratory.
Secondly, proteins and other materials in the heterogeneous sample, particularly in the case of clinical samples, can interfere with the immobilization process.
As alternatives to immobilizing sample nucleic acids and adding labeled probe, one can use an immobilized probe and label the sample nucleic acids in situ, or one can use a dual hybridization technique requiring two probes, one of which is immobilized and the other labeled. The former alternative, however, is even less desirable since 3Q the in situ labeling of the sample nucleic acids requires a high degree of technical skill which is not routinely found in clinical technicians and there are no simple, reliable methods for monitoring the labeling yield, which can be a - 4 ~23~580 significant problem if the labeling media contain variable amounts of inhibitors of the labeling reaction. The dual hybridization technique has the disadvantages of requiring an additional reagent and incubation step and the kinetics of the hybridization reaction can be slow and inefficient. The accuracy of the assay can also be variable if the complementary fly of the two probes with the sample sequence is variable.
Some of the problems discussed above are solved by employing an immobilized RNA probe and detecting resulting immobilized DNA-RNA or RNA-RNA hybrids with a labeled specific anti-hybrid antibody [Canadian Patent Application No. 481,294, filed May 10, 1985].
This technique still requires a separation step and thus has the disadvantages common to all hybridization techniques that require a separation step as discussed above.
U.S. Patent Nos. 3,996,345; 4,233,402; and 4,275,149 describe assays to detect antigens by immune-assay techniques involving enzyme pair and energy transfer interactions.
European Patent Application No. 70,685 proposes a hybridization assay technique that dispenses with the need to physically separate hybridized from us-hybridized probe. It is proposed to employ a pair of probes which hybridize to contiguous regions on a polynucleotide sequence of interest and to label one probe with a chemiluminescent catalyst such as the enzyme peroxides and the other with an absorber molecule for the chemiluminescent emission. The catalyst and absorber labels must be situated near the ~39S~30 contiguous terminal ends of the respective probes such that upon hybridization there is observed quenching of the chemiluminescent emission by energy transfer to the absorber molecule. III order to perform such an assay, one must be able to controllable synthesize two critical probe reagents such that the respective labels are brought into a quenching orientation upon hybridization to the sample nucleic acid and without affecting the affinity of the respectively labeled probe segments to actually undergo hybridization.

SUMMARY OF THE INVENTION

A nucleic acid hybridization assay has now been devised based on the use of proximal interacting labeled pairs which yield a detectable signal that is measurably different for the hybridized probe compared to unhybridized probe.
In this way there is no need to separate hybridized and unhybridized probe, greatly facilitating the performance and automation of the assay. In addition, the assay signal is nonradioisotopic in nature thereby meeting another criterion of assay convenience, the use of detection systems not involving radioactivity.
According to the present invention, specific polynucleotide sequences are detected in a test sample by forming a hybrid between the sequence to be detected and a nucleic acid probe having a complementary sequence such that the resulting 3Q hybrid possesses binding sites for two different specific binding reagents. One of such binding reagents comprises a first label and the other a Pi 35~0 second label where interaction between the two labels provides a detectable response which is measurably different, either in a positive or a negative sense, when the two labeled reagents are bound to the same hybrid compared to when not so bound. When bound to the same hybrid the two labels are brought to within a proximate interaction distance of one another, thereby substantially increasing signal affecting lo interactions between the two labels compared to the relatively less frequent interactions occurring in the bulk solution between the free diffusible labeled reagents.
A preferred interaction between the two labels is a sequential interaction wherein the first label participates in a first chemical reaction to produce a diffusible product that is a participant in a second chemical reaction with the second label to produce a detectable product. It is especially preferred that the first and second labels be catalysts, e.g., enzymes, for the first and second chemical reactions, respectively. For example, where the first label is glucose oxidize and the second is peroxides, hydrogen peroxide produced by action of glucose oxidize on glucose diffuses to the peroxides label to be detectable as an optical signal in the presence of suitable indicator dye compositions. Another proofer labeling pair is that involving energy transfer interaction such as 3Q between a fluoresce or luminescer and a quencher for the photo emission of the first label.
With regard to the nature of the specific binding agents involved, in principle at least one of the two must bind to a site which is unique to ~72395~

the hybrid formed in the assay compared to unhybridized nucleic acids since signal generation must be dependent upon the formation of such hybrid. Preferably then, such specific binding reagent will be an antibody selective for the hybrid over the single stranded nucleic acids present in the assay mixture. A variety of second binding agents can be used as is detailed below.
The present invention is characterized by a number of significant advantages. In addition to the principal advantage of elimination of the separation step, there is no requirement to immobilize either sample or probe nucleic acids which gives rise to nonspecific binding and reproducibility problems in commonly used hybridization techniques. Further, the hybridization kinetics are substantially faster in solution compared to systems with one strand of the hybridizable pair immobilized. An additional advantage is that the assay can be performed without wash steps. The assay reagents can be sequentially added to the hybridization medium without the need to wash insoluble support materials.
Another significant feature of the present invention is that the detection systems involved can be particularly efficient since the double stranded duplexes can contain many binding sites for the first and second labeled reagents. This I results in large amounts of the first and second labels becoming assembled into their interactive configuration per unit of hybridized probe. In considering antibodies to RNA-DNA or RNA-RNA
hybrids, one labeled antibody can bind for ~23~580 approximately each 10 base pairs of the hybrid. If the probe is for example 500 bases long, 40-50 antibodies could bird. Ligands recognized by labeled binding proteins can be introduced into probes at levels of one ligand per 5-20 bases. The presence of multiple binding sites on hybrids can be used advantageously when low levels of hybrid must be detected.

GRIEF DESCRIPTION OF THE DRAWINGS

Figs. 1-3 are schematic illustrations of preferred methods for performing the present invention. These methods are described in detail below.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The use of nucleic acid hybridization as an analytical tool is based fundamentally on the double-stranded, duplex structure of DNA. The hydrogen bonds between the Purina and pyrimidine bases of the respective strands in double-stranded I DNA can be reversibly broken. The two complementary single strands of DNA resulting from this "melting" or "denaturation" of DNA will associate (sometimes referred to as reannealing or hybridization to reform the duplexes structure.
As is now well known in the art, contact of a first single stranded nucleic acid, either DNA or RNA, which comprises a base sequence sufficiently complementary to (i.e., "homologous with") a second single stranded nucleic cold under appropriate pa conditions, will result in the formation Go ~L239~

DNA-DNA, RNA-DNA, or RNA-RNA hybrids, as the case may be.

The Probe The probe will comprise at least one single stranded base sequence substantially complementary to or homologous with the sequence to be detected however, such base sequence need not be a single continuous polynucleotide segment, but can be comprised of two or more individual segments interrupted by non homologous sequences. These non homologous sequences can be linear, or they can be self-complementary and form hairpin loops. In addition, the homologous region of the probe can be flanked at the 3'- and 5'-termini by non homologous sequences, such as those comprising the DNA or RNA
of a vector into which the homologous sequence had been inserted for propagation. In either instance, the probe as presented as an analytical reagent will exhibit detectable hybridization at one or more points with sample nucleic acids of interest.
Linear or circular single stranded polynucleotides can be used as the probe element, with major or minor portions being duplexes with a complementary polynucleotide strand or strands, provided that the critical homologous segment or segments are in s Nile stranded form and available for hybridization with sample DNA or RNA. It will generally be preferred to employ probes which are substantially in single stranded form. The pa preparation of a suitable probe for a particular assay is a matter of routine skill in the art.

~-1353 ~3C3~;~0 Lube King Pays According to the principle of the present invention, hybridization dependent upon the presence of the polynucleotide sequence of interest and binding of the labeled reagents to the hybrid results in the bringing together of the two labels within a certain distance such that the signal produced by their interaction is measurably different from that produced by their encounters lo during simple diffusion in the bulk assay medium.
Various interaction phenomena can be applied to the present invention. The interaction can be chemical, physical, or electrical, or combinations of these forces. The environment of the labels which is created upon hybridization and binding of the labeled reagents to the formed hybrid, herein referred to as the bound hybrid environment, must be distinctively different in at least one critical aspect from the bulk medium. Since hybridization determines the proportion of labels which result in the localized hybrid environment compared to the bulk phase, the resulting signal response is dependent upon the presence of the sequence to be determined in the assay medium.
A preferred interaction between the two labels involves two chemical reactions wherein one label participates in the first reaction to produce a diffusible mediator product which participates in the second reaction with the second label to yield a detectable product. The microenvironment of the bound hybrid will thus contain a higher localized concentration of the mediator product, so as to increase the rate of the signal producing second 123958~

label, reaction, than the bulk solution. The two labels, respectively, can participate in the no-actions as reactants or, as is particularly preferred, catalysts. Any involved catalysis can be either enzymatic or non enzymatic.
A variety of enzymes and catalysts can be applied to the present invention and their selection will be a matter of choice to one working in the field. Useful enzymes for the firs-t reaction include the oxidoreduc-teases, particularly those involving nucleotides such as nicotinamide adenine dinucleotide (NOD) or its reduced form (NASH), or adenosine triphosphate (AT) as co-factors or those producing hydrogen peroxide or other small diffusible products. A few examples are alcohol dehydrogenase, glycerol dehydrogenase, lactate dodder-jounce, palate dehydrogenase, glucose-6-phosphate de-hydrogenate, glucose oxidize, and unease. Other classes of enzymes such as hydrolyses, transferases, leases, and isomerases can also be employed.
It is particularly preferred that the second label also be an enzyme where a product of the first enzyme reaction is a substrate or cofactor for the second enzyme so as to result in rapid enzyme reaction in the environment of the bound hybrid.
For example, where the first enzyme is an oxidore-Dakotas producing hydrogen peroxide as a product, the second enzyme can be peroxides. A detailed listing and description of useful enzymes is pro-voided in U.S. Patent Nos. 4,233,402 and 4,275,149.
Other useful systems involve one enzyme as the first label which catalyses a reaction producing a prosthetic group for an apoenzyme or pro enzyme.

~3~35~0 Non enzymatic catalysts can also serve as labels as previously mentioned. By way of example, reference is made to U.S. Patent No. 4,160,645.
Another preferred interaction between the labels is that of energy transfer. The first label will be a photo emitting substance such as a fluoresce or luminescer, the former producing an emission upon irradiation and the second producing an emission upon chemical reaction. The lo photo emission is absorbable by the second label to either quench the emission or to provide a second emission such as where the absorbing label is a fluoresce itself. Pairings of compounds useful for this effect are described in detail in U.S.
Patent Nos. 4,275,149 and 4,318,981. Some preferred fluorescer/quencher pairs are naphthalene/anthracene, ~-naphthylamine/dansyl, tryptophan/dansyl, dansyl/fluorescein, fluorescein/rhodamine, tryptophan/fluorescein, N-[p-(2-benzoxazolyl)phenyl]maleimide (BPM)/thiochrome, BPM/8-anilino-1-naphthalenesulfonate (AWNS), thiochrome/N-(4-dimethylamino-3,5-dinitrophenyl) maleimide (DDPM), and ANS/DDPM. Some preferred luminescer/quencher pairs are luminol with fluoresce in, eosins or radiomen S.
If desired, various modifications of the labeled reagents can be employed without departing from the scope of the present invention. In one variation, one of the labeled reagents comprises a solid phase to which is linked the binding substance and separately the label. The solid phase can be the walls of the reaction container or a dispersed solid such as a polyacrylamide or Lo 3~5~0 agrees bead. One could also modify the binding substance with a bindable ligand such as a hasten or button and introduce the label by addition of a labeled anti~hapten antibody or labeled avid in before or after the hybridization reaction. Thus, it can be seen that the label can be linked to the binding substance directly or indirectly through intermediary components.

The Binding Reagents A critical aspect of the present invention is the formation of a hybrid between the probe and the polynucleotide sequence of interest which comprises binding sites for the two specific binding reagents. A principle of the assay is that hybridization of the probe with the desired sequence results in a bringing together of the respective binding sites for the two binding reagents such that the labels are brought within a proximate interaction distance of one another. Any design of the system can be used which results in a binding site for at least one of the two labeled reagents which is unique to the hybrid. It will thereby be assured that the desired localization of the labeling pair will occur only upon formation of the hybrid, The binding of the labeled reagents to the hybrid will normally involve a highly specific nonequivalent binding such as is characteric of a variety of biologically derived substances, pa particularly binding proteins such as immunoglobulins. A variety of binding substances can be used to provide at least one binding reagent o which has a unique binding affinity for the hybrid with inconsequential binding affinity for single stranded nucleic acids such as unhybridized probe and unbraided sample nucleic acids.
Particularly preferred binding substances are antibody reagents having anti-hybrid binding activity and can be whole antibodies or fragments thereof, or aggregates or conjugates thereof, of the conventional polyclonal or monoclonal variety.
Preferred antibody reagents will be those that are selective for binding (i) DNA-RNA or RNA-RNA
hybrids or (ii) intercalation complexes. It is currently known that antibodies can be stimulated which are selective for DNA RNA or RNA-RNA hybrids over the single stranded nucleic acids, however, it is presently considered infeasible to generate such selectivity in the case of DNA-DNA hybrids. To the extent that selective DNA-DNA antibodies are developed in the future, they will clearly be applicable to the present invention. Antibodies to DNA-RNA or RNA-RNA hybrids can be used where the probe is RNA and the sample nucleic acids are DNA
or RNA or where the probe is DNA and the sample RNA.
Further, it should be understood that in referring to an RNA probe used with an anti-DNA-RNA
or anti-RNA-RNA reagent, it is contemplated herein that not all nucleotides comprised in the probe be ribonucleotides, i.e., bearing a 2'-hydroxyl group.
The fundamental feature of an RNA probe as used herein is that it be sufficiently non-DNA in character to enable the stimulation of antibodies to DNA-RNA or RNA RNA hybrids comprising an RNA
probe which do not cross react to an analytically 1239~

significant degree with the individual single strands forming such hybrids. Therefore, one or more of the possessions on the nucleotides comprised in the probe can be in the Dixie form provided the antibody binding characteristics necessary for performance of the present assay are maintained to a substantial degree. Likewise, in addition or alternatively to such limited Dixie modification, an RNA probe can comprise nucleotides lo having other modifications, or in general any other modification along its rubs phosphate backbone provided there is not substantial interference with the specificity of the antibody to the double stranded hybridization product compared to its individual single strands.
Where such modifications exist in an RNA
probe, the immunogen used to raise the antibody reagent would preferably comprise one strand having substantially corresponding modifications and the 2Q other strand being substantially unmodified RNA or DNA, depending on whether sample RNA or DNA is intended to be detected. Preferably, the modified strand in the immunogen would be identical to the modified strand in an RNA probe. An example of an immunogen is the hybrid poly(2'-0-methyladenylic acid) poly(2'-deoxythymidylic acid). Another would be poly(2'-0-ethylinosinic acid)-poly(ribocytidylic acid). The following are further examples of modified nucleotides which could be comprised in an RNA probe: 2'-0-methylribonucleotide, 2-0 -ethylribonucleotide, 2'-azidodeoxyrlbonucleotide,

2'-chlorodeoxyribonucleotide, 2' -a -acetylribonucleotide, and the methyl phosphonates or phosphorothiolates of ribonucleotides or ~3~58~

deoxyribonucleotide. Modified nucleotides can appear in RNA probes as a result of introduction during enzymic synthesis of the probe from a template. For example, adenosine 5'-0-(1-thiotriphosphate) (AUTOPSY) and dATPS are substrates for DNA dependent RNA polymerizes and DNA polymerizes, respectively. Alliteratively, the chemical modification can be introduced after the probe has been prepared. For example, an RNA probe can be 2'-0-acetylated with acetic android under mild conditions in an aqueous solvent.
Immunogens for stimulating antibodies specific for RNA-DNA hybrids can comprise homopolymeric or heteropolymeric polynucleotide duplexes. Among the possible homopolymer duplexes, particularly preferred is poly(rA)-poly(dT) [Kitagawa and Stellar (1982) Mol. Immunol. 19:413J. however, in general, heteropolymer duplexes will be preferably used and can be prepared in a variety of ways, including transcription of ~X174 vision DNA with RNA polymers [Nakazato (1980) Become. 19:2835].
The selected RNA-DNA duplexes are adsorbed to a methylated protein, or otherwise linked to a conventional immunogenic carrier material, such as bovine serum albumin, and injected into the desired host animal [see also Stellar (1980) Moth. Enzymol.
70:70]. Antibodies to RNA-RNA duplexes can be raised against double stranded RNAs from viruses such as rovers or Fiji disease virus which infects sugar cane, among others. Also, homopolymer duplexes such as puller) poly(rC) or puller) puller), among others, can be used for immunization as above. Further information regarding antibodies to RNA-DNA and RNA-RNA hybrids - 17 - ~239580 is provided in Canadian Patent Application No. 481,294, filed May 10, 1985.
Antibodies to intercalation complexes can be prepared against an immunogen which will usually comprise an ionic complex between a cat ionic protein or protein derivative (e.g., methylated bovine serum albumin) and the anionic inter-calator-nueleic acid complex. Ideally, the interealator will be eovalently coupled to the double stranded nucleic acid. The intercalator-nucleic acid conjugate alternatively can be covalently coupled to a carrier protein.
The nucleic acid portion of the immunogen can comprise the specific paired sequences found in the assay hybrid or can comprise any other desirable sequences since the specificity of the antibody will generally not be dependent upon the particular base sequences involved. Further information regarding antibodies to intercalation complexes is provided in Canadian Application No. 469,908, filed December 12, 1984.
As stated above, the antibody reagent can consist of whole antibodies, antibody fragments, polyfunctional antibody aggregates, or in general any substance comprising one or more specific binding sites from an antibody. When in the form of whole antibody, it can belong to any of the classes and subclasses of known immunoglobulins, e.g., Gig, I'm, and so forth. any fragment of any such antibody which retains specific binding affinity for the hybridized probe can also be employed, for instance, the fragments of Gig conventionally known as Fob, Flab'), and Phoebe.

~239580 In addition, aggregates, polymers, derivatives and conjugates of immunoglobulins or their fragments can be used where appropriate.
The immunoglobulin source for the antibody reagent can be obtained in any available manner such as conventional antiserum and monoclonal techniques. Antiserum can be obtained by well-established techniques involving immunization of an animal, such as a mouse, rabbit, guinea pig lo or goat, with an appropriate immunogen. The immunoglobulins can also be obtained by somatic cell hybridization techniques, such resulting in what are commonly referred to as monoclonal antibodies, also involving the use of an appropriate immunogen.
In those instances where an antibody reagent selective for intercalation complexes is employed as one of the binding reagents, a variety of intercalator compounds can be involved. In general I it can bye said that the intercalator compound preferably is a low molecular weight, planar, usually aromatic but sometimes polycyclic, molecule capable of binding with double stranded nucleic acids, e.g., DNA-DNA, DNA-RNA, or RNA-RNA duplexes, usually by insertion between base pairs. The primary binding mechanism will usually be nonequivalent, with covalent binding occurring as a second step where the intercalator has reactive or activatable chemical groups which will form covalent bonds with neighboring chemical groups on one or both of the intercalated duplex strands.
The result of intercalation is the spreading of adjacent base pairs to about twice their normal separation distance, leading to an increase in MS-`1353 - 19 _ ~2;~5~

molecular length of the duplex. Further, unwinding of the double helix of about 12 to 36 degrees must occur in order to accommodate the intercalator.
General reviews and further information can be obtained from Lehman, J. Mol. slot. 3:18(1961);
Bloom field et at, "Physical Chemistry of Nucleic Acids", Chapter 7, pp. 429-476, Harper and Rowe, NOAH); Waring, Nature 219:1320 (1968); Hart Mann et at, Anger. Chum., Engl. Ed. 7:693(1968);
Leopard, Acts. Chum. Rest 11:211(1978); Wilson, Intercalation Chemistry(1982), 445; and Berman et at, Ann. Rev. Buffs. Boeing. 10:87(1981); as well as from the above-referenced Canadian Application No. 469,908. Exemplary of intercalators are acridine dyes, e.g., acridine orange, the phenanthridines, e.g., ethidium, the fanciness, furocoumarins, phenothiazines, and queenliness.
The intercalation complexes are formed in the assay medium during hybridization by use of a probe which has been modified in its complementary, single stranded region to have the intercalator chemically linked thereto such that upon hybrid-ration the intercalation complexes are formed.
Essentially any convenient method can be used to accomplish such linkage. Usually, the linkage is formed by effecting intercalation with a reactive, preferably photo reactive intercalator, followed by the linking reaction. A particularly useful method involves the azidointercalators. Upon ox-posture to long wavelength ultraviolet or visible light, the reactive neutrons are readily generated.
The neutrons of arylazides prefer insertion reactions over their rearrangement products [see White et at, Methods in Enzymol.

I,,;

~Z39~30 46:644(1977)]. Representative azidointercalators are 3-azidoacridine, 9-azidoacridine, ethidium monoazides, ethidium dozed, ethidium diver aside [Mitchell et at, JAYS 104:4265(1982)], 4-azido-7-chloroquinoline, and 2-azidofluorene.
Other useful photoreactable intercalators are the furocoumarins which form [2+2] cycloadducts with pyrimidine residues. Alkylating agents can also be used such as bis-chloroethylamines and epoxies or assuredness, e.g., aflatoxins, polycyclic hydrocarbon epoxies, mitomycin, and norphillin A.
The intercalator-modified duplex is then denatured to yield the modified single stranded probe.
repeating what is stated previously, at least one of the binding reagents must be of the type discussed above, however, there is a greater latitude of choice possible for the other binding reagent since it need not discriminate between the hybrid and other nucleic acids, including the probe and sample materials, to accomplish the desired effect. Accordingly, the second binding reagent can be the same or a different binding material as used in the aforesaid reagent except that when the same, it is, of course, ultimately different as an assay reagent by being labeled with the second label. When employing an antibody reagent selective for binding intercalation complexes for both binding reagents, it will normally be desired to employ a probe which has been modified to have the intercalator chemically linked to the probe, as opposed to being added as a soluble compound.
Alternatively, the second binding reagent can be any material which binds the probe. thus, it can be an antibody reagent that binds to nucleic 3L;~395~3~

acids nonspecifically such as anti-DNA-DNA.
Another embodiment will employ a probe comprising a specific binding ligand moiety with the nonspecific binding reagent then being a binding partner for such ligand moiety. In such case, the ligand moiety/binding partner pair can be selected from such known materials as haptens/antibodies, antigens/antibodies, biotin/avidin, lectins/polysaccharides, hormones/receptor proteins, and the live. In this respect, reference is made to British Pat. 2,019,408; European Patent Apples. 63,879 and 97,373 and POT Published Apples.
83-1459 and 83-2286.
While it is generally preferred that the critical microenvironment of the bound hybrid be created in a portion or the entirety of the hybridized complementary region of the sample/probe duplex, in principle such microenvironment can also occur at the boundary of such complementary region and the flanking region. This can occur employing binding reagents as discussed above. Additionally, one can use as the nonspecific binding reagent a binding substance for a binding site on double stranded flanking regions of the probe. Examples of such binding site/binding partner pairs are promoter sequences (e.g., lac-promotor, trp-promoters, and promoters associated with bacteriophages)/polymerase; fag operator/lac repressor protein; and antigenic nucleotides and sequences (e.g., Z-DNA and rare nucleotides such as 5-halodeoxyuridines)/antibodies thereto.
With reference to the drawings and the examples which follow, a few specific embodiments of the present assay scheme can be described.

3~58~

The method depicted in Fig. 1 employs an unmodified,polynucleotide probe which is either RNA
or DNA when the sample sequence of interest is RNA
or when the sample sequence is DNA the probe is RNA. The binding reagents are the same or different antibody reagents (A) selective for RNA-DNA or RNA-RNA hybrids as the case may be. One antibody reagent is labeled with glucose oxidize (GOD) and the other with peroxides (POD). In the presence of glucose and a color indicator for hydrogen peroxide, the rate of color development will be dependent upon the extent of hybridization that takes place. In the bound hybrid, glucose oxidize and peroxides are brought within a distance a which results in an increased rate of color development due to rapid diffusion of hydrogen peroxide to peroxides. In the bulk medium, hydrogen peroxide must on average diffuse distance b in order to cause a color development event which reflects in a significantly different rate. Optionally, kettles can be included in the bulk solution at concentrations sufficient to effectively destroy all hydrogen peroxide that diffuses away from the microenvironment of the bound hybrid to reduce background color formation.
The use of a ligand-modified probe is illustrated in Fig. 2, with button (Boo) being the selected representative land In this case the GOD-labeled binding reagent is again an antibody to I RNA-DNA or RNA-RNA as the case may be, and the other binding reagent is POD-labeled avid in (A).
The color development readout will be understood as in the case of method A.

95~3 In the method shown in Fig. 3, the probe is doubly modified, with both button as in method B
and a chemically linked intercalator (I). An antibody to intercalation complexes involving I is labeled with a fluoresce (F) and avid in is labeled with a quencher (Q). The fluoresce and quencher labels are brought into energy transfer distance in the bound hybrid such that upon irradiation with light of a first wavelength (hv1), the emitted energy (hv2) is absorbed by the quencher and not detected. On the other hand, fluorescer-labeled antibody in the bulk solution remains on average distance b from the quencher, which is not close enough for efficient energy transfer, and the fluorescence emission is observed. The amount of hv2 light detected is inversely related to the amount of hybridization that occurs.

- 2 4 _ ~X39 Rear t ion Mixture The test sample to be assayed can be any medium of interest, and will usually be a liquid sample of medical, veterinary, environmental, nutritional, or industrial significance. Human and animal specimens and body fluids particularly can be assayed by the present method, including urine, blood (serum or plasma), amniotic fluid, milk, cerebrospinal fluid, sputum, local matter, lung aspirates, throat swabs, genital swabs and exudates, rectal swabs, and nasopharnygal aspirates. Where the test sample obtained from the patient or other source to be tested contains principally double stranded nucleic acids, such as contained in cells, the sample will be treated to denature the nucleic acids, and if necessary first to release nucleic acids from cells. Denaturation of nucleic acids is preferably accomplished by heating in boiling water or alkali treatment (e.g., 0.1 N sodium hydroxide), which if desired, can simultaneously be used to lyre cells. Also, release of nucleic acids can, for example, be obtained by mechanical disruption (freeze/thaw, abrasion, sonication), physical/chemical disruption (detergents such as Briton, Tweet, sodium dodecylsulfate, alkali treatment, osmotic shock, or heat), or enzymatic louses (lysozyme, protons K, pepsin). The resulting test medium will contain nucleic acids in single stranded form which can 3Q then be assayed according to the present hybridization method. In those situations where RNA DNA hybrids are to be detected with labeled * Trade Clark ~3~35~3~

antibody reagents, Mona and Rena in the sample can be removed from participating in the binding reactions by conventional methods such as treatment with alkaline conditions, e.g., the same conditions used to denature the nucleic acids in the sample.
As is known in the art, various hybridization conditions can be employed in the assay.
Typically, hybridization will proceed at slightly elevated temperatures, e.g., between about 35 and lo 75C and usually around 65C, in a solution comprising buffer at pi between about 6 and 8 and with appropriate ionic strength (e.g., 5XSSC where l~SSC = 0.15M sodium chloride and 0.015M sodium citrate, pi 7.0). In cases where lower hybridization temperatures are desirable, hydrogen bonding reagents such as dim ethyl sulfoxide and formamide can be included. The degree of complementarity between the sample and probe strands required for hybridization to occur depends on the stringency of the conditions. Factors which determine stringency are known in the art.
Normally, the temperature conditions selected for hybridization will be incompatible with the binding of the anti-hybrid reagent to formed hybrids and detection of the label response.
Accordingly, the anti-hybrid binding step and label detection step will proceed after completion of the hybridization step The reaction mixture will usually be brought to a temperature in the range of pa from about 3C to about 40C and the binding and detection steps then performed. Dilution of the hybridization mixture prior to addition of the antibody reagent is desirable when the salt and/or formamide concentrations are high enough to interfere significantly with the antibody binding reaction.

Reagent System The present invention additionally provides a reagent system, i.e., reagent combination or means, comprising all of the essential elements required to conduct a desired assay method. The reagent system is presented in a commercially packaged form, as a composition or admixture where the computability of the reagents will allow, in a test device configuration, or more usually as a test kit, i.e., a packaged combination of one or more containers, devices, or the like holding the necessary reagents, and usually including written instructions for the performance of assays.
Reagent systems of the present invention include all configurations and compositions for performing the various hybridization formats described herein.
In all cases, the reagent system will comprise (1) a nucleic acid probe as described herein, and I the first and second labeled binding reagents.
A test kit form of the system can additionally include ancillary chemicals such as the components of the hybridization solution and denaturation agents capable of converting double stranded nucleic acids in a test sample into single stranded form. Preferably, there is included a chemical lying and denaturing agent, e.g., alkali, for treating the sample to release single stranded I nucleic acid therefrom.

39~

The present invention will now be illustrated, but is not intended to be limited, by the following examples.

Exam e Hybridization Assay for Bacterial RNA
Monitored with Labeled Antibodies to RNA DNA Hybrid A. Monoclonal antibody to RNA-DNA hybrid is raised by the method of Stuart et at (1981) PEAS
78, 3751. Antibody is isolated from Auschwitz fluid by affinity chromatography on protein A Suffers available from Pharmacia Fine Chemicals, Pussycat, NJ. About 1 milliliter (ml) of Auschwitz fluid per ml of protein A Suffers is applied to the column and then the column is washed with 50 millimolar (my) Bison buffer, pi 8.0, 0.15 molar (M) Nail until the absorbency of the effluent at 280 nanometers (no) is less than 0.05. The bound antibody is eluded with 0.1M Gleason buffer, pi 3.0 2Q and antibody is detected by measurement of absorbency at 280 no. The antibody pool is adjusted to pi 5.0 or above by addition of lo Bison buffer, pi 8.5. The pool is dialyzed into 0.1M borate buffer, pi 9.0 The antibody is labeled lightly with 5-(4,6-dichlorotriazin-2-yl)-aminofluorescein (DEAF) (Molecular Probes, Inch Junction City, Oregon) by the method of Blakeslee and Brines (1976) J. Immunol. Moth. 13, 305. A solution containing 1.44 micro moles (Molly) of DEAF in 361 micro liters (GIL) of borate buffer it added to 2.4 58~

ml of 0.014 my antibody and allowed to react for one hour at 25C. The reaction mixture is chromatographed on a 1 x 25 cm Bejewel P-6DG (Byrd Laboratories, Richmond, CA) column with Old sodium phosphate buffer, pi 7.0, Old Nikko as the fluent.
Absorbances at 280 no of 1 ml fractions are monitored and those comprising the first peak are pooled. The molar DTAF/protein ratio is determined by the method of The and Feltkamp (1970) Immunology 18, 865.
Free they'll groups are introduced into the antibody by reaction with N-succinimidyl 3-(2-pyridyldithio)propionate and subsequent mild reduction with dithiothreitol [Carson et at (1978) Become. J. 173, 723].

B. A glucose oxidize antibody conjugate is prepared as follows. Maleimide groups are introduced onto glucose oxidize (available from Miles Scientific, Naperville, IL) by reaction with the N-hydroxysuccinimide ester of N-(4-carboxycyclohexylmethyl)-maleimide (SMOCKS) as described by [Yoshitoake, et at, (1979) Eur. J.
Become. 101, 395 (1979)]. This glucose oxidize is immediately combined with a two-fold molar excess of the they'll derivatized antibody described above.
The reaction conditions and purification methods of Yoshitake et at are followed. Enzyme content of the purified conjugate is determined by measurement of enzyme activity and the antibody content is I determined by measurement of the DEAF content as described by The and Feltkamp, swooper.

~3~35f~0 C. Horse radish peroxides is reacted with SMOCKS and coupled to the they'll derivatized antibody by the method of Ishikawa et at (1983) J.
Immunoassay 4:209.

D. A 565 base pair fragment from a gene for the 16s ribosomal RNA from E. golf has been cloned between Hind III sites of a pBR322 vector [Bruises (1978) Pro. Natal. Aged. Sat. 75:4801]. The fragment is excised by digestion with Hind III
lo restriction endonuclease.

E. A urine specimen containing greater than 105 bacteria per ml is diluted three-fold into an enriched culture broth and incubated at 35C until the cell population reached about 109 organisms per ml. Allocates of the culture are transferred to a series of tubes to give 105 to 109 bacteria per tube. The tubes are centrifuged at 10,000 x g for 10 minutes and the supernatants are discarded. The cells are lucid in 20 I per tube of 10 milligrams (my) egg white lysozyme (Sigma Chemical Co., St.
Louis, MO) in 10 my Tris-hydrochloride buffer, pi 8.0, Old Nail and 5 my ethylenediamine~etraacetic acid. Fifteen minutes later, 80 I ox a solution composed of 60% formamide and 40~ 0.16 M sodium phosphate buffer, pi 6.5, 1.44 M Nail, and 0.1%
(w/v) sodium dodecylsulfate.
The DNA probe described in section D above, in 0.2M sodium phosphate buffer, pi 6.5, is denatured in a boiling water bath for 5 minutes arid cooled on pa ice. Twenty micro liter allocates (80 no DNA) are added to each tube and incubated at 55C fox 18 hours. Then 500 I of peroxides labeled antibody ~L~3~35~

and glucose oxidize labeled antibody are added to each tube and allowed to stand at room temperature for 2 hours The concentrations of these enzyme conjugates are optimized in preliminary experiments to give optimum signal to background ratios.
The following substrate reagent is prepared and used within 3 hours: elm sodium phosphate, pi 6.5, 0.1% bovine serum albumin, 20 my 3,5-dichloro-2-hydroxybenzene sulfonate, elm glucose, 0.01 unit catalase/ml and 0.2 my

4-aminoantipyrine. One milliliter of substrate reagent is added to each tube and incubated at 37C
for 30 minutes. The the absorbances at 510 no are recorded. As the number of bacteria per tube increases the amount of ribosomal RNA'DNA hybrid will increase and as a result the absorbency will be increased.

I

Exam Z e II

Hybridization Assay for Detection of Bacterial Ribosomal RNA Using a Biotinylated DNA Probe A. Button duty and streptavidin-horse radish peroxides conjugate are available from Eons Become., Inc., New York, NY.

B. The 565 base pair probe for ribosomal RNA
is labeled with biotin-ll-dUTP by the method of Lunger et at (1981~ Pro. Natal. Aged. Sat., 78:663 3 .

C. Bacteria are processed as outlined in Example I, section E above. the lusts are combined with 80 I of a solution composed of 60%
formamide and 40% 0.16 M sodium phosphate buffer, pi 6.5, 1.44 M Nail and 0.1% (w/v) sodium dodecylsulfate. The biotinylated DNA probe is denatured in a boiling water bath for 5 minutes and then cooled on ice. The probe is in 0.2M sodium phosphate buffer pi 6.5 and 20 I allocates containing 80 no probe are added to each extract and incubated at 65C for 18 hours. Then 500 I of peroxides labeled streptavidin and glucose oxidize labeled antibody against RNA-DNA hybrid is added to each tube and allowed to stand at room temperature for 2 hours. The concentrations of these enzyme conjugates are optimized in preliminary experiments to give maximum signal to background ratios.

~3~58~3 The enzyme reactions are initiated by addition of 1 ml of the substrate reagent described in Example I, section D. These reactions are allowed to proceed at 37C for 30 minutes and then the absorbances at 510 no are recorded. The absorbances will increase as the number of bacteria added per tube increases.

Exam Zen III

Hybridization Assay for Bacterial Ribosomal RNA Using a Probe Intercalated with Ethidium Bromide A. Calf thymes DNA (Sigma Chemical Co.) is treated with prunes to digest protein contaminants and then precipitated in ethanol. The DNA is dissolved in 20 my tris-hydrochloride buffer, pi 8.0, 0.2M Nail.
A photo reactive derivative of ethidium bromide, 8-azidoethidium, is prepared by the method of Graves et at (1977) Become. Buffs. Act, 479:98. A solution is prepared containing about 1 my base pairs of the DNA and 0.5 my 8-azidoethidium. This solution is stirred in a glass reaction vessel immersed in a water bath maintained at 20-30C and is photolyzed 10 to 20 cm from a 15~ watt spotlight for 60 minutes.
Following the photolysis the nonequivalently bound ethidium derivatives are removed by extractions with water saturated n-butanol. The amount of ethidium coupled to the DNA is estimated using the 3¢ extinction coefficients of Eye = 4 x 103M cm for photolyzed ethidium aside, the relationship ~'~39~

between Aye and Aye for photolyzed ethidium bound 4 [1260 ; (Aye x 3.4)-0.011], and Eye = 1.32 x 10 M cm for the concentration of DNA base pairs of a given DNA being labeled.

B. Preparation of methylated thyroglobulin One hundred milligrams of bovine thyroglobulin (Sigma) is combined with 10 ml of an hydrous methanol and 400 I of 2.55 M Hal in methanol.
This mixture is stirred on a rotary mixer at room temperature for 5 days. The precipitate is collected by centrifugation and washed twice with methanol and twice with ethanol. Then it is dried under vacuum overnight. About 82 my of dry powder is obtained.

C. An immunogen is prepared by combining the DNA-ethidium complex with methylated thyroglobulin as outlined by Stroller (1980) Methods in En zymology 70:70. Twenty to fifty micrograms of the immunogen in Fronds adjutant are injected per mouse and booster injections are given weekly beginning two weeks after the original immunization. Antibody titers are measured by an LISA procedure and hybridomas are produced and screened for antibody by standard procedures [Galore and Millstone, (19813 Moth. Enzymol., 73:1; Pointer, et at (1982) Pro.
Natal. Aged. Sat., 79:6443~. Selection procedures were designed for antibodies which bind the DNA-ethidium complex with association constants of 101 or greater but do not bind to single or double pa stranded DNA or the ethidium residue alone.
Antibody is isolated from Auschwitz fluid as outlined in Example I, section A for antibody to ~2~9~
- 34 _ RNA DNA hybrid. This antibody is also labeled lightly with DEAF and coupled to glucose oxidize.

D. The 565 base pair fragment from the pKK115 plasm id is cloned into the Hind III site of M13 mp9 [Messing and Voyeur, (1982) Gene 19, 269;
commercially available from New England Bulbs, Beverly, MA]. The fragment inserts in two orientations and a clone with the vision DNA
sequence complementary to ribosomal RNA is selected for further work.
The modified M13 mp9 is propagated in E. golf JM103 [Luke, pro)thi, stray super end, sbcB15, hsdR4, F'tra~36, probe, lacIq, Z~M15] available from Bethesda Research Laboratories, Gaithersburg, MD. The phase is isolated from culture broth by precipitation with polyethylene glycol and the vision DNA is purified by phenol/chloroform extraction [Mounts et at (1982) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor].
An oligonucleotide primer ~CACAATTCCACACAAC;
available from New England Bulbs) is complementary to the M13 mp9 vector on the OWE
side of the inserted ribosomal RNA probe. This primer is used to initiate replication of the M 13 sequence by E. golf DNA polymers clown fragment) in the presence of limiting quantities of deoxynucleotides trinhosphates. The replication is limited to an extent that the inserted ribosomal RNA probe remains single stranded and available for pa hybridization [Hut and Messing (1982~ Gene 17:271].
This modified M 13 probe is mixed with 8-azidoethidium, photolyzed and purified as outlined in section A above.

~L2~958~

En Bacteria are prepared and lucid as described in Example I, part A. The lusts are mixed with 80 I of the formamide hybridization solution described in Example I, Part E. Twenty micro liters of the ethidium-probe complex (100 no DNA) (section D) is added to the extracts and incubated at 65C for 18 hours and then 500 us of peroxides labeled antibody to RNA-DNA and glucose oxidize labeled antibody to ethidium-DNA complex is added to each tube. The mixtures are allowed to stand at room temperature for 2 hours.
At the end of this incubation, 1.0 ml of the substrate reagent, Example I, part E, is added and incubated at 37C for 30 minutes. Then the absorbency at 510 no is recorded. As the number of bacteria per tube is increased, the absorbency will increase.

Example IV

Hybridization Assays for Cylomegalovirus Monitored with a Fluorescer-Quencher Pair A. A 1500 base pair fragment of the cytomegalovirus gnome is cloned into the M13mp8 vector. A complementary cop of the inserted fragment is synthesized in vitro with DNA
polymers in the presence of a biotinylated nucleated triphosphate to provide a biotinylated probe. This probe is modified further by covalent incorporation of ethidium residues as intercalation complexes.
pa An EcoRI digest of cytomegalovirus DNA is prepared and the fragments are cloned into the ~;~3~5~

plasm id pesky [Timeshare et at (1982) J.
Virology 42:547]. The plasmids are propagated in E. golf strain HB101 Roe A and the EcoRI e fragment described in the Timeshare reference is used for preparation of the probe. The fragment is removed from the plasm id by digestion with EcoRI
restriction enzyme and cloned into the EcoRI site of the M13mp8 vector (New England Bulbs) which is propagated in the E. golf K12JM101 host. The virus lo is isolated from the culture fluid by precipitation with polyethyleneglycol and the DNA is purified by pheno~/chloroform extraction.
The DNA is annealed with a 17 base primer with the sequence GTAAAACGACGGCCAGT (New England Bulbs) which is complementary to a region of the M13mp8 sequence near the OWE end of the EcoRI e insert. The DNA is 20 my Tris-hydrochloride buffer, pi 8.0, containing 10 my McCauley is combined with a molar excess of the primer and incubated at 55C for 45 minutes [Banker and Barrett (1983) Techniques in Nucleic Acid Biochemistry, Elsevier, Ireland]. Then the reaction mixture is made 15 my in date, dCTP, dGTP and ~io-11-dUTP (available from Eons Become) and finally the Clown fragment of DNA polymers I is added. This reaction is incubated at 25C for a period determined in preliminary experiments. In these experiments samples are taken from the reaction mixture at various times and electrophoresed in alkaline agrees gel [Mounts et at, swooper]. The objective is to find reaction conditions which give button labeled probes that extend through the EcoRI e insert. This procedure will provide button labeled DNA with some variation in length; however, 95~3 extension of the labeled DNA beyond the EcoRI e insert into the M13 sequence is acceptable for the present purpose.
The next step is the covalent coupling of ethidium residues to the button labeled DNA as intercalation complexes. This is accomplished by photolysis of the DNA with 8-azidoethidium prepared by the method of Graves et at, swooper. The button labeled DNA is extracted with phenol/chloroform and precipitated with ethanol. It is dissolved in 20 my Tris-hydrochloride buffer, pi 8.0, 0.2 M Nail, to give about 50 go DNA/ml and made 0.5 my with 8-azidoethidium. The mixture is photolyzed for 1 hour 10 to 20 cm from a 150 watt spotlight. The mixture is stirred during this period in a glass reaction vessel immersed in a glass water bath.
The bath maintains a reaction temperature of 20 to 30C.
Then the nonequivalently bound 8-azidoethidium and photolysis by-products are removed by the successive extractions with water saturated n-butanol. The DNA is precipitated with ethanol and dissolved in the Tris-buffer. The covalently bound ethidium is measured spectrophotometrically as described in Example III, section A.
8-Azidoethidium binds covalently to the DNA
almost exclusively in the double stranded region where intercalation complexes can form. The objective is to covalently couple one ethidium I residue per 10 to 50 base pairs. Since the photolytic reaction goes nearly to completion in one hour, the incorporation can be decreased by reducing the reaction time or by reducing the 8-azidoethidium concentration. The incorporation - ~395~

can be increased by repeating the photolysis with fresh 8-azidoethidium.
The final step is separation of the button labeled/ethidium modified probe from the M13mp8 template. The labeled probe is substantially smaller than the M13mp8 template and can be isolated by alkaline agrees gel electrophoresis [Mounts et at, swooper]. The gel containing the probe is cut from the agrees gel slab and recovered by electroelution as described in the Mounts reference.

B. Fluoresce in labeled antibody to ethidium-DNA intercalation complex.

Monoclonal antibody to ethidium-DNA complex described in Example III, part B above is dialyzed into 0.1 M sodium borate buffer, pi 9.0, and 0.5 ml containing 5 my antibody is combined with 0.5 ml of 10 my 5(4,6-dichlorotriazin-2-yl)-aminofluorescein (Molecular Probes, Inc., Junction City, OR) in the borate buffer. The mixture is allowed to stand for 7 hours at room temperature and then it is chromotographed on a 1 x 26 cm column of Bejewel P6DG resin Byrd Laboratories) in 0.1 M
Tris-hydrochloride buffer, pi 8Ø The absorbances at 280 no of 1.0 my fractions are measured and fractions from the first eluded peak are pooled.
The fluorescein/protein ratio is measured spectrophotometrically by the method of The and Feltkamp (1970) Immunology 18:865.

Lo C. Preparation of streptavidin labeled with 4,5-dimethoxy-6-carboxyfluorescein.

The N-hydroxysuccinimide ester of 4',5'-dimethoxy-6-carboxyfluorescein is synthesized by the method of Cowan and Pullman (1980) Anal.
Become. 108:156. (The synthesis actually gives a mixture of the 5 and 6- carboxyfluorescein isomers). Streptavidin isolated from Streptomyces avidini [Hoffman et at (1980) Pro. Natal. Aged.
Sat. 77:4666] is labeled with the N-hydroxysuccinimide ester of 4',5'-dimethoxy-6-carboxyfluorescein by the method described by Cowan and Pullman for labeling immunoglobulins.

D. Hybridization assay for cytomegalovirus in urine.

The procedure uses centrifugation to isolate the virus from urine and the viral DNA is hybridized in solution with the button labeled/ethidium modified probe. Then the labeled avid in and antibody to ethidium-DNA intercalation complex are allowed to bind to the hybrids formed and fluorescence is used to estimate the amount of hybrid present.
Cellular and particulate materials are removed from urine by centrifugation in a Servile GLC-3 centrifuge at 3000 rum for five minutes. The supernatant is placed in a polyallomer ultra centrifuge tube and run at 25,000 rum in a pa Beckman Tao rotor for 75 minutes. The pellets are * Trade Mask I

dissolved in 0.1 M Noah and incubated at 37C for 30 minutes.
One hundred fifty micro liters of I M sodium phosphate buffer, pi 6.0, containing 1.8 M Nail, 0.1% sodium dodecylsulfate (w/v) and 1 my ETA is added. Then 20 AL of the button labeled/ethidium modified probe (50 my) is added and the hybridization mixture is incubated at 65C for 10 hours. After the hybridization reaction, 650 AL of 0.1 M Tris-hydrochloride buffer, pi 8.2, containing the labeled avid in and antibody to ethidium-DNA
intercalation complex is added. The concentrations of the labeled proteins in this reagent are optimized in preliminary experiments to maximize the fluorescence quenching to background ratio.
The protein binding reactions are allowed to proceed at room temperature for 1 hour.
Then the fluorescence of the reaction mixture is recorded using 495 no light for excitation and 519 no for emission. A reference assay is run in parallel with a urine known to be free of cytomegalovirus and the fluorescence signal obtained with this reference assay will be higher than that for samples containing the virus, thus the fluorescence signal will increase as the virus level decreases.

The present invention has been particularly described and exemplified above. Obviously many other variations and modifications of the invention pa may be made without departing from the spirit and scope thereof.

Claims (39)

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

1. A method for detecting a particular polynucleotide sequence in a test sample comprising single stranded nucleic acids, comprising the steps of:
(a) forming a hybrid between any of the particular polynucleotide sequence to be detected in the sample and a nucleic acid probe comprising at least one single stranded base sequence that is substantially complementary to the sequence to be detected, the hybrid having binding sites for two specific binding reagents, (b) contacting any hybrid that is formed with the two specific binding reagents, one of which comprises a first label and the other comprising a second label, interaction between the first and second labels providing a detectable response which is measurably different when the two labeled reagents are both bound to the same hybrid, compared to when the two labeled reagents are not so bound, and (c) measuring the detectable response as a function of the presence of the sequence to be detected in the sample.

2. The method of Claim 1 wherein the first label participates in a first chemical reaction that produces a diffusible product which is a participant in a second chemical reaction with the second label to produce a product that provides the detectable response.

3. The method of Claim 2 wherein the first and second labels are catalysts for the first and second chemical reactions, respectively.

4. The method of Claim 3 wherein the first and second labels are enzymes.

5. The method of Claim 4 wherein the first label is glucose oxidize and the second label is peroxides.

6. The method of Claim 1 wherein the first and second labels participate in an energy transfer interaction.

7. The method of Claim 6 wherein the first label is a fluoresce or luminescer and the second label is a quencher.

8. The method of Claim 1 wherein at least one of said binding sites on the hybrid is present substantially only in the hybridized region of such hybrid.

9. The method of Claim 1 wherein one of the specific binding reagents is an antibody reagent.

10. The method of Claim 9 wherein the antibody reagent is one that is selective for binding DNA?RNA hybrids wherein one of the probe and the sequence to be detected is DNA and the other is RNA, or is one that is selective for binding RNA?RNA hybrids wherein both the probe and the sequence to be detected are RNA.

11. The method of Claim 10 wherein the other specific binding reagent comprises the same antibody reagent.

12. The method of Claim 10 wherein the nucleic acid probe comprises a specific binding ligand moiety and wherein the other specific binding reagent is a binding partner for such ligand moiety.

13. The method of Claim 10 wherein the nucleic acid probe comprises a double stranded portion having a binding site for a protein and wherein the other specific binding reagent is such protein.

14. The method of Claim 13 wherein the double stranded probe portion comprises intercalation complexes and wherein the other specific binding reagent is an antibody selective therefor.

15. The method of Claim 9 wherein the antibody reagent is one that is selective for binding intercalation complxes wherein the duplexes formed in the assay comprise a nucleic acid intercalator bound thereto in the form of intercalation complexes.

16. The method of Claim 15 wherein the probe comprises a nucleic acid intercalator chemically linked thereto in its single stranded complementary region whereby upon hybridization with the sequence to be detected said intercalation complexes are formed in the resulting hybrid.

17. The method of Claim 16 wherein the other specific binding reagent comprises the same antibody reagent.

18. The method of Claim 16 wherein the nucleic acid probe comprises a specific binding ligand moiety and wherein the other specific binding reagent is a binding partner for such ligand moiety.

19. The method of Claim 16 wherein the nucleic acid probe comprises a double stranded portion having a binding site for a protein and wherein the other specific binding reagent is such protein.

20. The method of Claim 1 wherein the test sample comprises a biological sample which has been subject to conditions to release and denature nucleic acids present therein.

21. A reagent system for detecting a particular polynucleotide sequence in a test sample, comprising:
(1) a nucleic acid probe comprising at least one single stranded base sequence that is substantially complementary to the sequences to be detected, and (2) first and second specific binding reagents capable of binding to hybrids formed between the sequence to be detected in the sample and the probe, such binding reagents comprising first and second labels, respectively, which interact with each other to provide a detectable response which is measurably different when the two labeled reagents are both bound to the same hybrid compared to when the two labeled reagents are not so bound.

22. The reagent system of Claim 21 wherein the first label participates in a first chemical reaction that produces a diffusible product which is a participant in a second chemical reaction with the second label to produce a product that provides the detectable response.

23. The reagent system of Claim 22 wherein the first and second labels are catalysts for the first and second chemical reactions, respectively.

24. The reagent system of Claim 23 wherein the first and second labels are enzymes.

25. The reagent system of Claim 24 wherein the first label is glucose oxidize and the second label is peroxides.

26. The reagent system of Claim 21 wherein the first and second labels participate in an energy transfer interaction.

27. The reagent system of Claim 26 wherein the first label is a fluoresce or luminescer and the second label is a quencher.

28. The reagent system of Claim 21 which additionally comprises a denaturation agent capable of converting double stranded nucleic acids in a test sample into single stranded form.

29. A test kit for detecting a particular polynucleotide sequence in a test sample, comprising:
(1) a nucleic acid probe comprising at least one single stranded base sequence that is substantially complementary to the sequence to be detected, (2) a first specific binding reagent which is an antibody reagent that is either selective for binding DNA?RNA hybrids wherein one of the probe and the se-quence to be detected is DNA and the other is RNA, or selective for binding RNA?RNA hybrids wherein both the probe and the sequence to be detected are RNA, and (3) a second specific binding reagent capable of binding to hybrids formed between the probe and the sequence to be detected, such binding reagents comprising first and second labels, respectively, which interact with each other to provide a detectable response which is measurably different when the two labeled reagents are both bound to the same hybrid, whereby the labels are situated within a proximate interaction distance of one another, compared to when the two labeled reagents are not so bound.

30. The test kit of Claim 29 wherein the second specific binding reagent comprises the same antibody reagent as the first specific binding reagent.

31. The test kit of Claim 29 wherein the nucleic acid probe comprises a specific binding ligand moiety and wherein the second specific binding reagent is a binding partner for such ligand moiety.

32. The test kit of Claim 29 wherein the nucleic acid probe comprises a double stranded portion having a binding site for a protein and wherein the second specific binding reagent is such protein.

33. The test kit of Claim 32 wherein the double stranded probe portion comprises intercalation complexes and wherein the second specific binding reagent is an antibody selective therefore

34. The test kit of Claim 29 which additionally comprises a denaturation agent capable of converting double stranded nucleic acids in a test sample into single stranded form.

35. A test kit for detecting a particular polynucleotide sequence in a test sample, comprising:
(1) a nucleic acid probe comprising at least one single stranded base sequence that is substantially complementary to the sequence to be detected and comprising a nucleic acid intercalator chemically linked thereto in its single stranded complementary region whereby upon hybridization with the sequence to be detected intercalation complexes are formed in the resulting hybrid, (2) a first specific binding reagent which is an antibody that is selective for binding said intercalation complexes, and (3) a second specific binding reagent capable of binding to hybrids formed between the probe and the sequence to be detected, such binding reagents comprising first and second labels, respectively, which interact with each other to provide a detectable response which is measurably different when the two labeled reagents are both bound to the same hybrid, whereby the labels are situated within a proximate interaction distance of one another, compared to when the two labeled reagents are not so bound.

36. The test kit of Claim 35 wherein the other specific binding reagent comprises the same antibody reagent.

37. The test kit of Claim 35 wherein the nucleic acid probe comprises a specific binding ligand moiety and wherein the other specific binding reagent is a binding partner for such ligand moiety.

38. The test kit of Claim 35 wherein the nucleic acid probe comprises a double stranded portion having a binding site for a protein and wherein the other specific binding reagent is such protein.

39. The test kit of Claim 35 which additionally comprises a denaturation agent capable of converting double stranded nucleic acids in a test sample into single stranded form.