NO844846L - HYBRIDIZATION ANALYSIS USING A LABELED TEST AND ANTI-HYBRID - Google Patents

Description

The present invention relates to nucleic acid hybridization analysis methods and reagent systems for detecting specific polynucleotide sequences. It was found that single-stranded nucleic acids, e.g. DNA and RNA, pro-

made by denaturing the double-stranded forms, will hybridize or recombine under suitable conditions with complementary single-stranded nucleic acids. By

labeling such complementary nucleic acid samples with an easily detectable chemical group made it possible to detect the presence of any polynucleotide sequence of interest in an experimental medium containing nucleic acid samples in single-stranded form.

In addition to application in recombinant DNA technology,

analytical hybridization can be used to detect important polynucleotides in the fields of human and veterinary medicine, agriculture, food technology and the like.

In particular, the technique can be used to detect and identify etiological agents such as e.g. bacteria and viruses,

to examine bacteria with regard to antibiotic resistance, facilitate the diagnosis of genetic anaemias, such as sickle-

cell anemia, and thalassemia, and to detect cancer cells. A general overview of the technique and its current and future importance can be found in Biotechnology (August 1983), pp. 471-478.

The following information is included to make known background information with possible relevance to the present invention. The following information is not intended to constitute prior art to be weighed against the present invention.

The state of the art in terms of nucleic acid hybridization analysis techniques generally includes a separation of hybridized and unhybridized labeled probe. The required separation step is usually facilitated by immobilizing either the sample nucleic acids or the nucleic acid probe on a solid support. Typically, hybridization between particular base frequencies 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 containing unhybridized probe, followed by detection of label on the solid support.

Efforts are constantly being made to simplify the analysis procedure for carrying out nucleic acid hybridization analyses. The primary goal of these efforts is to reduce the complexity of the procedure to the point that dm can be easily and routinely performed in clinical laboratories. The necessity of a separation step complicates these endeavors. To perform the separation step in an analytically reproducible manner, considerable expertise is required, and the physical manipulation cannot be easily automated or made suitable for large test volumes.

Furthermore, with conventional methods of immobilizing sample nucleic acids, two significant problems arise. First, the procedures required to achieve immobilization are generally time-consuming and constitute an additional step that is undesirable for routine use of the technique in clinical laboratories. Second, proteins and other materials in the heterogeneous sample, especially in the case of clinical samples, can affect the immobilization process.

As alternatives to the immobilization of sample nucleic acids

and addition of labeled probe, one can use an immobilized probe and labeled sample nucleic acid in situ, or one can use a double hybridization technique that requires two probes, one of which is immobilized and the other labeled.

However, the first option is even less desirable since in situ labeling of sample nucleic acids requires a high degree of technical skill that is not found routinely

in clinical laboratory personnel and there are no simple, reliable methods for controlling the labeling yield, which can be a significant problem if the labeling medium contains variable amounts of inhibitors for the labeling reaction. The double hybridization technique has the disadvantage that it requires an additional reagent and an incubation step,

and the kinetics of the hybridization reaction may be slow and inefficient. The accuracy of the analysis can also

be variable if the complementarity of the two samples with the sample sequence is variable.

Some of the problems discussed above can be solved

by using an immobilized RNA probe and detecting the resulting immobilized DNA"RNA or RNA"RNA

hybrids with a labeled specific anti-hybrid antibody

(see U.S. Patent Application No. 616,132, filed June 1, 1984). This technique still requires a separation step

and therefore have the disadvantages common to all hybridization techniques that require a separation step as discussed above.

European Patent application no. 70,685 proposes a hybridization analysis technique where the necessity of physically separating hybridized from unhybridized probe is avoided.

It is suggested to use a pair of probes that hybridize

to adjacent regions on a polynucleotide sequence of interest, and labeling a probe with a chemiluminescent catalyst such as the enzyme peroxidase and the other met an absorbing molecule for the chemiluminescent emission. The catalyst and the absorbing labels must be located close to the adjacent terminal ends of the respective probes so that, upon hybridization, quenching of the chemiluminescent emission by energy transfer to the absorbing molecule is observed. To perform

such an analysis, one must be able to controllably synthesize two critical probe reagents so that the respective labels are brought into a quenching orientation by hybridization to the sample nucleic acid, without affecting-

the affinity of the respective labeled probe segments for hybridization.

One has now arrived at a nucleic acid hybridization assay based on modulation of the detectable response of hybridized labeled probe by binding an antibody-

reagent to the hybrid formed between the labeled probe and the particular polynucleotide sequence to be detected. That is, the label in the antibody-bound hybrid expresses

a response that is detectably different from the response expressed by the label in unhybridized labeled probe.

In this way, there is no need to separate hybridized and unhybridized probe, this greatly simplifies the execution and automation of the analysis. In addition, the analysis signal is non-radioisotope in nature and therefore satisfies another criterion for a usable analysis process, the use of detection systems that do not involve radioactivity.

According to the present invention; specific nucleotide sequences are detected in a test sample by forming a hybrid between any of the particular sequences to be detected and a labeled nucleic acid probe that includes a label and at least one single-stranded base sequence that is essentially complementary to the sequence to be detected. The hybrid is characterized by the fact that it has epitopes for an antibody reagent (anti-hybrid) which does not bind to a significant extent to single-chain nucleic acids. Anti-

hybrid is then added and will not significantly bind to the labeled probe unless it hybridizes to the sequence to be detected. The mark in the marked probe

provides a detectable response that is measurably different from, i.e. increased or decreased, when the labeled probe is included in a hybrid bound by the anti-hybrid compared to when it is not included in such a hybrid--Measurement of the resulting the detectable response will be a function of the presence of the sequence to be detected in the sample.

A preferred mechanism for modulation of the label by anti-hybrid binding involves steric hindrance. In such situations, the label interacts chemically with a reagent part of the label detection system, such as e.g. by reaction or binding;, and the presence of anti-hybrid bound to the hybrid provides steric hindrance of access to the label for such part of the detection system. Preferred labels used for such systems include enzyme reactions, i.e., label and the portion of the reagent with which the label interacts are selected from enzyme substrates, enzyme co-factors, enzyme inhibitors, and enzymes. Detectable responses that are colorimetric, fluorimetric, or luminometric can be obtained.

Another preferred mechanism for modulating the label

in the case of anti-hybrid is based on the use of the nearest interacting tag pair. The probe is labeled with one of a first label and a second label and the antibody reagent is labeled with the other, or contains in native form a chemical group that serves as the second label, where interaction between the two labels produces a detectable response that is measurably different, either in a positive or negative sense, when the labeled antibody reagent binds to a hybrid that includes the labeled sample compared to the cases where it is not bound in this way.

When associated with the same hybrid, the two tags are brought to a neighboring interaction distance apart, thereby greatly increasing the signal affecting the interaction between the two tags compared to the relatively less frequent interactions that take place in the bulk solution between the free , the fusing labeled reagents. A preferred interaction between the two labels is a stepwise interaction where the first label participates in a first chemical reaction and forms a defundable product which participates in a second chemical reaction with the second label to form a detectable product. It is particularly preferred that the first and/or the second label are catalysts for the first and the second chemical reaction, respectively. E.g.

the antibody reagent can be labeled with an enzyme and the probe with a catalyst that acts on a defundable product from the enzyme reaction so that a detectable product is formed, e.g. by an optical signal in the presence of a suitable indicator color composition. Another preferred tag pair is that which includes energy transfer interaction such as between a fluorescent or luminescent substance and a quencher, for photoemission from the first mark.

The present invention is characterized by a number of clear advantages. In addition to the main advantage that lies in the elimination of the separation steps, no immobilization of either sample or probe nucleic acids is required, which gives rise to non-specific binding and reproducibility problems as with the commonly used hybridization techniques. Furthermore, the kinetics of the hybridization is significantly faster in resolution compared to systems with entro of the hybridizable Dåre imTnobilized.

An additional advantage is that the analysis can be carried out without a washing step. The analysis reagents can be added stepwise to the hybridization medium without the need to wash insoluble carrier materials.

Another significant feature of the present invention

is that the detection system involved may be special

efficient since double-stranded duplexes can contain many labels and binding positions for the anti-hybrid reagent. This results in large amounts of the label and anti-rhybrid being bound in an interactive configuration per unit hybridized probe. Considering antibodies to RNA<*>DNA or RNA"RNA hybrids, a labeled antibody can bind approximately every 10 base pairs in the hybrid.

If the probe e.g. is 500 bases long, 40-50 antisi substances can bind. The presence of multiple binding positions on the hybrids can be advantageously used when low levels of the hybrids are to be detected.

Figures 1 - 3 are schematic illustrations of preferred methods for carrying out the present invention. These procedures are described in detail below.

The application of nucleic acid hybridization as an analytical tool is essentially based on the double-stranded duplex structure of DNA. The hydrogen bonds between the purine and pyrimidine bases in the respective chains in the double-

stranded DNA can be broken reversibly. The two complementary single strands of DNA that result from this "melting"

or the "denaturation" of DNA will associate (also called reannealing or hybridization) so that the duplex structure is restored. It is now well known that if one brings into contact a first single-stranded nucleic acid, either DNA or RNA, which includes a base sequence sufficiently complementary to (ie "homologous to") another single-stranded nucleic acid under suitable conditions, DNA will be formed"DNA, RNA"DNA, or RNA"RNA hybrids, depending on the starting point.

The probe will include at least one single-stranded base sequence

which is substantially complementary to or homologous to the sequence to be detected. Such a base sequence

need not necessarily be a single continuous polynucleotide segment, but may include 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 regions of the probe may be flanked at the 3'- and 5<1->ends by non-homologous sequences, such as e.g. those which include the DNA or RNA of a vector into which the homologous sequence has been inserted for propagation. In any case, the probe, which is present as an analytical reagent, will show detectable hybridization at one or more points with the sample nucleic acids of interest. Linear or circular single-stranded polynucleotides can be used as probe elements, with the major part or a minor part duplexed with a complementary polynucleotide chain or chains, provided that the critical homologous segment or segments are in single-stranded form and available for hybridization with sample DNA or RNA . In general, it is preferred to use probes which are essentially in single-chain form. The preparation of a suitable probe for a particular analysis is a matter of routine engineering.

The brand can be chosen from a large number of materials.

As a label, one can use any material which when included in the labeled sample provides a detectable response which can be modulated by binding of anti-hybrid and which is sufficiently stable, under hybridization conditions, to provide a measurable response. Modulation or modification of the label response can be due to a number of different effects that take place when the anti-hybrid binds to the hybrid.

A particularly preferred mechanism for modulating the label response involves steric hindrance. Normally the label is chosen to provide the detectable assay response with important effect, e.g. by chemical reaction or binding with a part of a reagent detection system that includes one or more substances that participate with the label in providing the measured signal. Whether due to steric hindrance or some other phenomenon, the binding of the anti-hybrid will interrupt or in-

activate the mark from such signal generating participation. Such effects during the signal generation will show the presence

of hybridized labeled probe.

Preferably, the label, when included in a system based on steric hindrance, will be small, e.g. of molecular weight less than 10,000, usually less than 4,000, and preferably less than 2,000 daltons, and the interactive reagent in the preferred detection system will normally be significantly larger, e.g. more than three times larger, usually more than 10 times larger, and preferably 20 to 100 times larger than the label. Accordingly, in the most preferred systems with labels having masses on the order of 100 to 2,000 daltons, at least a portion of the detection system with which the label interacts to provide the detectable signal will be on the order of 10,000 to 200,000 daltons or greater. Such size ratios between the label and the part of the detection system increase the probability of a significant steric effect upon binding of the anti-hybrid to the labeled hybrid. Preferred brands are therefore participants in an enzyme-catalyzed reaction, such as e.g. enzyme substrates, co-enzymes, enzyme prosthetic groups, and enzyme inhibitors, since a large number of enzyme reactions are available from which the analysis components can be selected. Many small substrates, co-enzymes, and inhibitors are known for enzymes of sufficiently large molecular weight that the preferred size ratio between the label and its

interactive part of the detection system can be achieved.

This also applies to prosthetic groups and their corresponding apoenzymes. Non-proteinaceous and especially stable organic or inorganic compounds are preferred as labels because of the denaturing organic or ionic compounds are preferred as labels because of the denaturing conditions under which the hybridization is carried out.

Some particularly preferred brands and methods for

to prepare the labeled probe is discussed below.

In one system, the label is chosen so that the labeled probe

is a substrate for an enzyme, and the ability of the enzyme to act on the substrate-labeled probe is affected, either in a positive or negative sense, but usually by inhibition,

upon binding between the labeled probe and the anti-hybrid. The effect of the enzyme on the substrate-labeled probe

gives a product characterized by some feature, usually a chemical or physical feature such as chemical reactivity in an indicator reaction such as a photometric character, e.g. fluorescence or light absorption (colour).

Marks of this type are generally described in US Patent Application No. 894,836, filed April 10, 1978 (belonging to U.K.

Patent 1,552,607); and in Anal.Biochem. 48:1933(1 9761 ), Anal.Biochem. 75:55 (1977) and Clin.Chem. 23-1402(1977).

In such enzyme-substrate labeling techniques, the labeled probe will have the property that it can be affected by an enzyme,

by cleavage or modification, so as to form a product having a detectable property which distinguishes it from its conjugate compound. E.g. the conjugated compound may be non-fluorescent under the assay conditions, but upon reaction with enzyme a fluorescent product is formed.

A very useful class of substrate labels includes those that undergo simple hydrolysis reactions yielding fluorescent products. Such products can be detected at low concentrations. Examples of these types of substrates are phosphate esters, phosphordiesters, and glycosides of fluorescent dyes. Glycosides are particularly preferred since their rates of non-enzymatic hydrolysis are low, and 3-galactosides are particularly convenient because 3-galactosidase enzymes are readily available and very stable.

A special class of useful fluorogenic substrate-labeled probe conjugates are of the formula:

where G is a cleavable group such as phosphate, carboxylate, sulfate or glycone, D is a fluorogenic dye unit which, on removal of G, gives a fluorescent product,

e.g. may D be umbelliferone, fluorescein, rhodamine, or derivatives thereof, R is a linking group,

NA is the nucleic acid probe and n is in average

number of marks per molecule of the probe, e.g. mellon 1

and 50. Enzymatic cleavage (eg, by phosphatase, carboxylase, sulfatase, glycosidase, etc.) of the labeled conjugate compound is performed by binding the antihybrid to the labeled hybrid, see U.S. Pat. Patent No. 4,279,992.

A particularly preferred substrate-labeled assay method uses a labeled conjugate of the type:

where R, NA, and n are as defined above, and the ability of the enzyme B-galactosidase to cleave the conjugated compound to a product characterized by fluorescence is inhibited by binding between the labeled hybrid and the anti-hybrid.

Other useful substrate-labeled conjugated compounds are of the formula: where X is an enzyme-cleavable linking group, e.g. phosphate, carboxylate, and the like, NA and n are as defined above, and D is a fluorogenic dye unit which, upon cleavage of X, releases a fluorescent indicator. A labeled conjugate compound of this type has the formula:

where R is a bond or a chain connecting the labeled component NA to the cleavable phosphate group and R 2 is hydrogen or a substituent group such as e.g. lower alkyl, e.g. methyl and ethyl, N-alkylamido or N-(hydroxy-substituted lower alkyl)amido, e.g.

-CONH-(CH2)m-OH where m = 2-6 (see U.S. Patent no.

4,273,715). The umbelliferone residue may contain other or additional substituents (see Anal.ehem. 40:803

(1968)). Cleavage by phosphate esterase is effected by binding the anti-hybrid to the labeled hybrid. In this process some of the phosphorus diester bonds in the hybrid can also be cleaved but will not affect the analysis since the hybridization cycle has ended and the modulation of the cleavage of substrate the mark will be essentially the same for marks present in intact hybrids compared to degraded parts thereof.

Another preferred fluorescent dye is 6-carboxyfluorescin, which has fluorescence excitation and emission maxima at 490 and 520 nm, respectively, and can be synthesized by the method described by Ullman et al, (1976), J. Biol. Chem. 251:4172.

It can be transferred to di-3-galactoside by the method described by Rotman et al. Pro. Nat<1>1 Acad. Pollock. 50, 1 (1963). This product can be converted to N-hydroxysuccinic acid imide ester by the method described by Khanna and Ullman, Anal. Biochem. 108:156 (1980) for the preparation of the corresponding ester of 4<1>, 5<1>dimethoxy-5-carboxymethylphorescein.

The labeled probe in the system with enzyme co-factor labels is composed, in the label part, of a co-

enzyme-active functionality, and the ability of such a co-enzyme label to participate in an enzymatic reaction is affected by the binding between the labeled hybrid and the anti-hybrid. The rate of the resulting enzymatic reaction

is measurable by conventional detection systems, so that it gives a detectable signal. Marks of this type are described in U.S. Pat. Patent Application No. 894,836, filed April 10, 1978 (corresponding to U.K. Pat. Spee. 1,552,706);

and in Anal. Biochem. 72:271(1976), Anal. Biochem, 72:283

(1976) and Anal. Biochem. 76:95 (1976).

A useful co-enzyme for labeling is nicotamamide adenine dinucleotide (NAD). NAD can be linked to the probe by a bridge at the 6-nitrogen, or 8-carbon, of the adenine moiety. A method for introducing an aminoethyl bridge at the 6-nitrogen position is described by Carrico et al. Anal. Biochem. 72:271(1976). Lee and Kaplan, Arch. Biochem. Biophys^168:665 (1976) has described a synthesis for the introduction of a diaminohexane bridge at the 8-carbon. The NAD marks can be detected in a number of different ways, such as by enzymic ring closure with maleic acid dehydrogenase and alcohol dehydrogenase.

Another important co-enzyme trirosrate (ATP).. Trayer et al, Biochem. J. 139:609 (1974) has described the synthesis of ATP with a hexylamine bridge at the 6-nitrogen of adenine

the group. This can be detected by enzymatic cyclization with hexokinase and pyruvate kinase. ATP can also be attached to the probe by means of ribosylation. The ribose is oxidized with sodium periodate and then condensed with a dihydrazide (Wilchek and Lamed, Meth. in Enzymol., 34B:475(1974)). The resulting hydrazone is reduced with sodium borohydride. This mark can be detected sensitively with firefly

luciferase by bioluminescent detection.

A particularly useful class of cofactor labels is

prosthetic groups. In such systems, the ability of a catalytically inactive apoenzyme to combine with the prosthetic group label so that an active enzyme (holoenzyme) is formed will be affected by binding between the labeled hybrid and anti-hybrid. The resulting holoenzyme's activity is measurable by conventional detection systems,

so that a detectable signal is obtained. Marks of this type are described in U.S. Pat. Patent No. 4,238,565.

A particularly preferred esthetic group-labeled assay embodiment uses flavin adenine dinucleotide (FAD) as the label and apoglucose oxidase as the apoenzyme. Resulting glucose oxidase activity is measurable using a colorimetric detection system that includes glucose, peroxidase, and an indicator system that gives a color change in response to hydrogen peroxide. Fluorometric detection of hydrogen peroxide is also possible using a suitable fluorine substrate. FAD can be linked to the probe by a bridging group at the 6-nitrogen of the adenine moiety. Morris et al., Anal. Chem. 53:658(1981) describes a method for the synthesis of FAD with a hexylamine bridge at the 6-nitrogen, and Zappelli

et al., Eur. J. Biochem. 89:491 (1978) describes a method for introducing a 2-hydroxy-3-carboxypropyl-

group at the same position.

The labeled probe in the enzyme modulator label system

is composed, in the tag part, of an enzyme-modulating functionality such as e.g. an enzyme inhibitor or stimulator, and the ability of such a modulator label to modulate the activity of an enzyme is affected by the binding between the labeled hybrid and anti-hybrid. The rate of the resulting enzymatic reaction is measurable by conventional detection systems so that a detectable signal is obtained. Marks of this type are described in U.S. Pat. Patent Nos. 4,134,792 and 4,273,866. Particularly preferred is the use of methotrexate labeled with dihydrofolate reductase as the modulated enzyme.

If the brand is an enzyme inhibitor, it may interact

with the enzyme covalently or non-covalently, and can be a small molecule, e.g. methotrexate, or a large molecule, e.g. antibody to an enzyme (see U.S. Patent 4,273,866 and U.S. Patent Application No. 285,605, filed June 21, 1981).

In another system, the label is a catalyst and the activity of the catalyst label is affected by binding between the labeled hybrid and anti-hybrid. Resulting catalytic activity is measurable by conventional detection systems, and provides a detectable signal, e.g. absorption or fluorescence. Marks of this type are described in U.S. Pat. Patent No. 4,160,645.

In another system, the tag includes an epitope, i.e. a binding position for antibody, for another antibody, i.e. anti-tag or fragments thereof. The anti-label's ability to and bind to the label in the labeled hybrid is affected by binding to the anti-hybrid. Several control and detection designs can be used. In one example, the epitope tag can also be a fluorescent substance whose light emission changes, e.g. is reduced by binding to an anti-fluorescent substance. Anti-hybrid binding to the labeled hybrid limits the availability of the fluorescent label to quenching anti-fluorescent substance, (see U.S. Patent No. 3,998,943). In another method, an additional detector molecule is used which includes the epitope tag linked to an enzyme. Binding of the anti-tag to this epitope-enzyme conjugate results in inhibition of the enzyme activity.

The more the anti-brand is excluded from and bound to the brand

on the epitope-tagged hybrid at the anti-hybrid bond,

the more anti-label is available to and binds to and inhibits the enzyme activity in the epitope-enzyme reagent (see U.S.

Patent No. 3,935,074).

According to another preferred embodiment of the present invention, a pair of labels is used so that the hybridization, which is dependent on the presence of the polynucleotide sequence of interest and binding to a labeled antibody reagent to the labeled hybrid, results in the two labels being brought together at a certain distance such that the signal produced by interaction is measurably different from that produced when the marks meet by diffusion in the bulk of the analysis medium. Various interaction phenomena can be used in this embodiment. The interaction can be chemical, physical, or electrical,

or take place by combinations of these forces. The environment for the marks formed by hybridization

and binding of labeled antibody reagents to the formed labeled hybrids, henceforth the environment held bound hybrid, must have at least one critical feature that is distinctively different from the bulk medium. Since the hybridization determines the amount of labels formed in the surroundings of the localized hybrid compared to the bulk phase,

the resulting signal response will depend on the presence of the sequence to be determined in the assay medium.

A preferred interaction between two brands in-

involves two chemical reactions, where a label participates in the first reaction, which forms a definable intermediate that participates in the second reaction with the second label, yielding a detectable product. The micro-environment for the bound hybrid will therefore contain a higher localized concentration of the intermediate,

than the bulk resolution, so that the rate of the signal-producing second label reaction is increased. The two brands can participate in the reactions as reactants or, which is particularly preferred, as catalysts. Any catalysis can be either enzymatic or non-enzymatic.

A large number of enzymes and catalysts can be used

in this embodiment. Useful enzymes for labeling the antibody reagent include oxidoreductases,

especially those that include nucleotides such as nicotinamide adenine, dinucleotide (NAD) and its reduced form (NADH), or adenosine triphosphate (ATP) as co-factors or those that form hydrogen peroxide or other small definable products. A few examples are alcohol dehydrogenase, glycerol dehydrogenase, lactate dehydrogenase, malate dehydrogenase, glucose-6-

phosphate dehydrogenase, glucose oxidase, and uricase.

Other classes of enzymes such as hydrolases, transferases, lyases and isomerases can also be used. A detailed list and description of useful enzymes is provided in U.S. Pat. Patent Nos. 4,233,402 and 4,275,149. Other useful systems include an enzyme as the first label that catalyzes a reaction that forms a prosthetic group for an apoenzyme or proenzyme. Non-enzymatic catalysts can also serve as labels as described earlier. It can.e.g. appears to the U.S. Patent No. 4,160,645.

Another preferred interaction between label pairs is energy transfer. The first mark can be a photo-emitting substance such as e.g. a fluorescent or luminescent substance, the former giving emission by irradiation and the latter giving emission by chemical reaction. The photoemission is absorbable by the second label, like this

that it either extinguishes the emission or provides another emission if the absorbing label itself is a fluorescent substance. Pairs of compounds useful for this effect are described in detail in U.S. Pat. Patent Nos. 4,275,149 and 5,318,981. Some preferred fluorescent/quenching pairs are naphthalene/anthracene, α-naphthylamine/dansyl, tryptophan/dansyl, dansyl/fluorescein, fluorescein/rhodamine, tryptophan/fluorescein,' N-(p-(2-benzoxazolyl))phenyl)maleimide (BPM (/thiochrome, BPM/8-anilino-1-naphthalene sulfonate (ANS), thiochrome/N-(4-dimethylamino-3,5-dinitrophenyl)maleimide (DDPM),

and ANS/DDPM. Some preferred luminescent/quenching pairs are luminol with fluorescein, eosin or rhodamine S.

If desired, various modifications of

the labeled probe is used within the scope of the present invention. In a variation, the labeled probe includes a solid phase to which the label is attached. The solid phase can be the walls of the reactor vessel or a dispersed solid such as e.g. polyacrylamide or agarose beads. One can also modify the probe with a bindable ligand such as a hapten or biotin and introduce the label by addition of a labeled anti-hapten antibody or labeled avidin before or after the hybridization reaction. Accordingly, it is seen that the label can be connected to the binding substance directly or indirectly by means of transition components.

A number of methods can be used to label polynucleotide samples. It is preferred to have the label distributed along the length of the polynucleotide rather than concentrated around one position. Several labeling methods are described below.

5-(3-amino)allyl deoxyuridine triphosphate (AA-dURP) can be synthesized by the method described by Langer et al, Proe. Night'1. Acad. Pollock. 78:6633(1981) and can be introduced into double-stranded DNA probes by nick-translation with DNA polymerase. The probe will have side chain amino groups that can react with the labels described above.

N-Hydroxysuccinimide esters of 6-carboxylfluorescein, 4<1>,,5<1->dimethoxy-6-carboxymethylfluorescein and their 3-galactosyl derivatives can be reacted directly with the nick-translated probe. The activated dyes are used in excess to maximize the incorporation of the label, and the uncoupled dyes are then separated from the labeled DNA by a gel filtration procedure.

The brands with terminal amines on the bridging groups can

is connected to the nick-translated DNA using bifunctional reagents such as dimethyl adipimidate, hexamethylene diisocyanate, and p,p'-difluoro-m,m'-dinitrophenyl sulfone. The labels can be reacted with an excess of the bifunctional reagent, followed by removal of unreacted reagent. The activated tag can be reacted with the nick-translated probe.

Certain planar aromatic compounds intercalate between the base pairs of double-stranded nucleic acids and form reversible complexes. Some intercalating agents can be covalently linked to the polynucleotides by photolysis of the intercalating complexes. Examples of photoactivatable intercalating agents are 8-azidothidium, 8-azidomethidium, and furocoumarins such as e.g. angelicin. These intercalators can be modified by bridge arms containing functional groups. 8-azidomethidium derivatives can be prepared as described by Hertzberg and Dervan, J. Am. Chem. Soc. 104:313(1982) and Mitchell and Dervan, J. Am. Chem. Soc. 104:4265(1972). The tags can be connected directly to the modified intercalators. E.g. N-hydroxysuccinic acid imide esters of 6-carboxyfluorescein or 4<1>,5<1->dimethoxy-6-carboxyfluorescein can be reacted with the intercalator derivatives so that amide bonds are formed. These intercalator-labeled conjugates can then be added to a polynucleotide probe and photolysed. Alternatively, the modified intercalators can be photolyzed with polynucleotide probe and then linked to the labeled primers. 8-azidomethidium can be photolytically coupled to single-stranded nucleic acids by a non-intercalating mechanism (Balton and Kerns (1978) Nucl. Acids Res. 5:4891). This method can be used to connect this intercalator derivative to single-stranded DNA and RNA.

A critical feature of the present invention is the formation of a hybrid between the probe and the polynucleotide sequence of interest that includes binding sites for the anti-hybrid antibody reagent. A principle of the assay is that the hybridization of the labeled probe with the desired sequence results in the formation of a hybrid that is bound by the anti-hybrid and produces a measurable effect on the labeling response. Any design of the system can be used that provides a binding position for the anti-hybrid reagent that is unique to the hybrid. It is thereby ensured that the desired effect of the binding of the anti-hybrid to the probe label will take place during the formation of the hybrid.

The binding of the anti-hybrid to the hybrid will normally involve a very specific non-covalent bond, which is characteristic of a number of biologically deposited substances, especially bonds in proteins, such as e.g. immunoglobulins. A variety of binding agents can be used to provide anti-hybrid which has a unique binding affinity for the hybrid and no binding affinity for single-stranded nucleic acids such as unhybridized probe and unhybridized nucleic acid samples.

Particularly preferred binding agents are antibody reagents

which have anti-hybrid binding activity and may be whole antibodies or parts thereof, or aggregates or conjugates thereof, of the conventional polyclonal or monoclonal type.. Preferred antibody reagents are those selective for binding (i) DNA<*>RNA or RNA 'RNA hybrids or (ii) intercalation complexes.

It is currently known that antibodies can be stimulated to be selective for DNA"RNA or RNA"RNA hybrids, compared to single-stranded nucleic acids, but it is currently considered impossible 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 able to be used in the present invention. Antibodies to DNA"RNA hybrids can be used where one of the probe and the sequence to be detected is DNA and the other is RNA and antibodies to RNA<*>RNA can be used when both the probe and the sequence to be detected are RNA.

Furthermore, it should be noted that when it is referred to here

an RNA probe used with anti-DNA"RNA or anti-RNA"RNA reagents, it is assumed that not all the nucleotides included in the probe are ribonucleotides, i.e. contain a 2<1->hydroxyl group. The fundamental feature of an RNA

probe used here is that it is sufficiently non-

DNA capable of enabling the stimulation of antibodies to DNA"RNA or RNA'RNA hybrids that include an RNA probe that does not cross-react to an analytically significant degree with the individual single chains constituting such hybrids. Therefore, one or more of the 2' positions may on the nucleotides included in the probe be in the deoxy form, provided that the antibody binding properties necessary for the performance of the present assay are substantially maintained. Similarly,

in addition to or alternatively to such limited 2'-deoxy modification, an RNA probe may include nucleotides having other 2'-modifications, or generally any modification along the ribose phosphate backbone provided that there is no significant interference with the specificity of the antibody to it the double-stranded hybridization product compared to its individual single-stranded counterparts.

Where such modifications exist in an RNA probe,

will the immunogen used to raise the antibody reagent preferably include a chain that has essentially corresponding modifications and the other chain is essentially unmodified RNA or DNA, depending on whether the sample to be detected is RNA or DNA. Preferably, the modified chain in the immunogen should

be identical to the modified chain in an RNA probe.

An example of an immunogen is the hybrid poly(2'-0-methyladenylic acid)*poly(21-deoxythymidyl acid). Another example is poly(2<1->O-ethylinosinic acid)"poly(ribocytidylic acid). The following compounds are further examples of modified nucleotides that can be included in an RNA probe: 2<1->O-methylribonucleotide, 2-0- ethylribonucleotide, 2'-azido-deoxyribonucleotide, 2<1->chlorodeoxyribonucleotide, 2-0-acetylribonucleotide, and methylphosphonates or phosphorothiolates of ribonucleotides or deoxyribonucleotides.Modified nucleotides may appear in RNA probes as a result of introduction during enzyme synthesis of the probe from a template. For example, adenosine 5<1->0-(1-thiotriphosphate) (ATPaS) and dATPaS are substrates for DNA-dependent RNA polymerases and DNA polymerases, respectively. Alternatively, the chemical modification can be introduced after the probe is For example, an RNA probe can be 2'-0-acetylated with acetic anhydride under mild conditions in an aqueous solvent.

Immunogens for stimulating antibodies specific for RNA"DNA hybrids may include homopolymeric or heteropolymeric polynucleotide duplexes. Among the possible homopolymeric duplexes, poly(rA)"poly(dT) is particularly preferred (Kitagawa and Stollar (1982) Mol. Immunol. 19 :413). In general, however, heteropolymeric duplexes are preferred and can be prepared in a variety of ways, including transcription of ØD174 virion DNA with RNA polymerase (Nakazato (1980) Biochem. 19:2835). The selected RNA"DNA duplexes are adsorbed onto a methylated protein, or otherwise bound to a conventional immunogenic carrier material, such as bovine serum albumin, and injected into the desired host animal (see also Stollar (1980) Meth. Enzymol 70 :70).Antibodies to RNA'RNA duplexes can be raised against double-stranded RNA from viruses such as reovirus or Fiji disease virus that infect sugarcane plants, e.g.

Also homopolymeric duplexes such as e.g. poly (ri) "poly(rC) or poly(rA)" poly(rU), among others, can be used for immunization as described above. Further information regarding antibodies to RNA-DNA and RNA-RNA hybrids is provided in U.S. Pat. Patent Application No. 616-132, filed June 1, 1984.

Antibodies to intercalation complexes can be raised against an immunogen that usually includes an ionic

complex between a cationic protein or protein-

derivative (eg methylated bovine serum albumin) and the anionic intercalation nucleic acid complex. Ideally, the intercalator should be covalently linked to the double-stranded nucleic acid. Alternatively, the intercalator-nucleic acid conjugate may be covalently linked to a carrier protein. The nucleic acid portion of the immunogen may include the specific paired sequence found in the assay hybrid or may include any other desired sequence since the specificity of the antibody will generally not depend on the particular base sequence involved. Further information regarding antibodies to intercalation complexes is provided in U.S. Pat. Patent Application No. 560,429, filed December 12, 1983.

As stated above, the antibody reagent can consist of whole antibodies, parts of antibodies, polyfunctional antibody aggregates, or generally any substance that includes one or more specific binding positions for an antibody. When present in whole antibody form, it may belong to any of the classes and subclasses of known immunoglobulins, e.g.,

IgG, IgM, etc. Any fragment of any such antibody, which has retained specific binding affinity for the hybridized probe, can also be used, e.g., fragments of IgG conventionally known as Fab, F(ab') , and F(ab<*>)2'1 addition, polymeric aggregates, derivatives and conjugates of immunoglobulins and fragments thereof, can be used where appropriate.

The immunoglobulin source for the antibody reagent can be obtained by any available technique, such as e.g. by conventional antiserum and monoclonal techniques. Antiserum can be obtained by well-known techniques which include immunization of animals, such as e.g. mouse, hare, guinea pig or goat, with a suitable immunogen. Immunoglobulins can also be obtained by the somatic cell hybridization technique, which results in monoclonal antibodies, which also involves the use of a suitable immunogen.

In cases where an antibody reagent is used which

is selective for intercalation complexes as one of the binding reagents, a large number of intercalator compounds can be used. In general, it can be said that the intercalator compound is preferably a planar, usually aromatic but sometimes polycyclic, low molecular weight 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 is usually non-covalent, but covalent binding takes place as a second step where the intercalator has reactive or activatable chemical groups that form covalent bonds with neighboring chemical groups on one or both of the intercalated duplex chains. The result of the intercalation is that when bt> base pairs. spread to approx. twice their normal separation distance, this leads to an increase in the molecular length of the duplex. Furthermore, the double spiral must be wound up at approx. 12 to 36° for the intercalator to find its place. General overviews and further information can be found in Lerman, J. Mol. Biol. 3:18(1961); Bloomfield et al, "Physical Chemistry of NucleidAGids", Chapter 7, pp. 429-476, Harper and Rowe, et al, NY(1974); Waring, Nature 219: 1320. (1968); Hartmann et al, Angew. Chem., Engl. Oath. 7:693.(1968); Lippard, Accts. Chem. Res. 11:211(1978); Wilson, Intercalation chemistry (1982), 445; and Berman

et al, Ann. Fox. Biophys. Bio meadow. 10:87(1981); as well as the aforementioned U.S. Patent application 560,429. Examples of intercalators are acridine dyes,

e.g. acridine orange, phenanthridines, e.g. ethidium, phenazines, furocoumarins, phenothiazines, and quinolines.

The intercalibration complexes are formed in the assay medium during hybridization using a probe that has been modified in its complementary, single-stranded region and has the intercalator bound to it so that the intercalation complexes are formed by hybridization. Any suitable method can be used to provide such binding. Typically, the bond is formed by intercalating with a reactive, preferably photoreactive, intercalator, followed by the bonding reaction. A particularly useful method involves the azido intercalators. Upon exposure to ultraviolet light of intermediate wavelengths or visible light, the reactive nitrenes are readily generated. The nitrenes of aryl azides preferentially undergo insertion reactions rather than the formation of conversion products (see White et al, Methods in Enzymol. 46:644(1977)). Examples of azidointercalators are 3-azidoacridine, 9-azidoacridine, ethidium monoazides, ethidium diazide, ethidium dimer azide

(Mitchell et al, JACS 104:4265.(1982) 1 , 4-azido-7-chloroquinoline, and 2-azidofluorene. Other useful photo-reactive intercalators are furocoumarins which form

(2. + 2) cycloaddition products with pyrimidine residues. Alkylating agents can also be used as e.g. bis-chloroethylamines and epoxides or azirides, e.g. aflatoxins, polycyclic hydrocarbon epoxides,

mitomycin j and norphyllin A. The intercalator-modified duplex is then denatured to yield the modified single-stranded probe.

With reference to the drawings and examples that follow,

a couple of specific embodiments of the present analysis method can be described.

The method illustrated in Figure 1 includes a FAD-labeled polynucleotide probe that is either RNA or DNA when the sample sequence of interest is RNA or is RNA when the sample sequence is DNA. An anti-hybrid antibody is chosen so that it is specific for RNA<*>RNA or RNA<*>DNA hybrids, depending on what is available. Upon formation of hybrids between the sequence of interest and the FAD-labeled probe, binding sites for the anti-hybrid are formed.

Binding of the anti-hybrid to the now FAD-labeled hybrid

rendering the FAD tag unable to recombine with apoglucose oxidase. The unhybridized or free probe, on the other hand, includes FAD that is available for recombina-

tion so that active glucose oxidase is formed which acts on glucose and releases hydrogen peroxide which can be detected using colorimetric or fluorescent in-

directions.

In the method shown in Figure 2, the probe is as in Figure 1, except that it is labeled with a fluorescent substance

(F) . Anti-hybrid and anti-fluorescent substances are added

the system. In the hybrid formed, the binding of

anti-hybrid binding of anti-fluorescent substance tri labeled which retains its ability to . to vf luor.ize light (hu2) by I irradiation (hv^). However, the label in the unhybridized probe is available for binding by anti-fluori-

curing substance which results in the extinguishing of fluorine-

the essence.

The method shown in Figure 3 uses a fluorescent (F)-labelled polynucleotide probe which is either RNA or DNA when the sample sequence of interest is RNA, or is RNA when the sample sequence is DNA. An antibody selective for RNA * RNA or RNA * DNA hybrids, whichever is present, is labeled with a quencher moiety (Q). The fluorescent substance and the quencher are brought to a distance from each other a which is such that energy transfer in the bound hybrid can take place, when irradiated with light of a first wavelength (hv^) the emitted energy (hv2) will thereby be absorbed by the quencher and.not detected. Fluorescent-labeled probe in the bulk solution, on the other hand, will be at an average distance b from the quencher, which is too large for efficient energy transfer to take place, and fluorescent emission is observed. The amount of h^ light detected is inversely related to the extent of hybridization that takes place.

The test samples to be analyzed can be any

any medium of interest, and will usually be a liquid sample of medical, veterinary, environmental, nutritional, or industrial importance. Samples from humans and animals, and especially body fluids,

can be analyzed by the present method, including urine, blood (serum or plasma), milk, amniotic fluid, cerebrospinal fluid, saliva, feces, lung aspirates, throat swabs, genital swabs and exudates, rectal swabs, and nasopharyngeal aspirates.

When the test samples obtained from the patient or other sources contain predominantly double-stranded nucleic acids,

like for example. in cells, the sample is treated so that the nucleic acids are denatured and, if necessary, first so that the nucleic acids are released from the cell. Denaturation of nucleic acids is preferably carried out by heating in boiling water or by alkali treatment (e.g. 0.1 N sodium hydroxide), as desired, these can also be used to lyse cells. Release of nucleic acids can also be achieved by e.g. mechanical disruption (freeze/thaw, abrasion, using sound waves), physical/chemical disruption (detergents such as Triton, Tween, sodium dodecyl sulfate, alkali treatment, osmotic shock or heat), or enzymatic lysis (lysozyme, proteinase K, pepsin). The resulting test medium will contain nucleic acids in single-chain form, which can then be analyzed according to the present hybridization method. In cases where RNA"DNA hybrids are to be detected with labeled antibody reagents, mRNA and

rRNA in the sample is prevented from participating in the binding reactions by conventional methods such as e.g. treatment under alkaline conditions, e.g. the same conditions used to denature the nucleic acids in the sample.

As is known, different hybridization conditions can be used in the analysis. The hybridization will typically: take place at moderately elevated temperatures, e.g. between approx. 35 and 75°C, and usually around 65°C, in a solution including buffer at pH between approx. 6 and 8, and with a suitable ion fragment (e.g. 5XSSC where 1XSSC = 0.15M sodium chloride and 0.015M sodium citrate, pH 7.0). In cases where lower hybridization temperatures are desired, hydrogen-binding reagents such as e.g. dimethylsulfoxide and formamide are included. The degree of complementarity between the sample and probe strands required for hybridization to occur depends on the stringency of the conditions. The factors that determine stringency are well known.

Normally, the temperature conditions chosen for the hybridization will be incompatible with the binding of the anti-hybrid reagent to hybrids formed and detection of the label response. Accordingly, the anti-hybrid binding step and the label detection step will follow after the hybridization step is completed. The reaction mixture will usually be brought to a temperature in the range from approx. 3°C to approx. 40°C, and then the binding step and the detection step are carried out. Dilution of the hybridization mixture before addition of the antibody reagent is desirable when the salt and/or formamide concentrations are high enough to significantly

to affect the antibody binding reaction.

The present invention also provides a reagent system, i.e. reagent combination or devices, which includes all the essential elements required to carry out a desired analysis method. The reagent system is present in a commercially packaged form, as a composition or addition where the compatibility of the reagents allows, in an experimental device embodiment, or usually as an experimental package, i.e. a packaged combination of one or more containers, devices or the like containing the necessary reagents, and which also usually include written instructions for carrying out the analyses. Reagent systems according to the present invention include all configurations and compositions for carrying out the different hybridization formats described here.

In all cases, the reagent system will include (1) a labeled nucleic acid probe as described, and (2) the antibody reagent. A form of trial package system can also contain auxiliary chemicals such as e.g. components of the hybridization solution and denaturants that can convert double-stranded nucleic acids in a test sample to single-stranded form. Preferably, a chemical lysing and denaturing agent is included, e.g. alkali,

for processing the sample so that single-stranded nucleic acid is released.

The present invention is illustrated, but not limited,

of the following examples.

Example 1.

Detection of ribosomal RNA from bacteria was used by a FAD-labeled DNA probe.

A. Flavin N -(6-aminohexyl)adenine dinucleotide ((aminohexyl)FAD) is prepared according to the method described by Morris et al, Anal. Chem. (1981)53:658. Apoglucose oxidase is prepared as described by Morris et al,

(1983) Meth. in Enzymol. 92:413. Antibody to RNA * DNA hybrid is prepared as described by Stuart et al,

(1981) Proe. Nat!l. Acad. Pollock. 78:3751. A 565 base pair fragment of the 16s ribosomal RNA sequence from E.coli has been cloned between the Hind III positions of a pBR322 vector (Brosius et al (1978) Proe. Nat<1>1. Acad. Sei. 75:4801). 5(3-amino)allyldeoxyuridine triphosphate is synthesized by the method described by Langer et al, (1981) Proe. Night<1>1. Acad. Pollock. 78:6633.

B. Preparation of labeled DNA probe. The pBR322 plasmid containing 565 baseor fragments of 16s ribosomal RNA is propagated in É. coli from HB101 Ree. The A and 16s RNA fragment is excised using Hind III restriction endonuclease. The fragment is nick-translated with 5-(3-amino) allyldeoxyuridine triphosphate using<3>H-dATP to control incorporation as described by Langer et al, supra. This method provides a double-stranded DNA with primary amino groups distributed along its length.

The complementary DNA chain is isolated by hybridization

with 16s RNA from E. coli available from Boehringer Mannheim Biochemicals, Indianapolis, IN. The nick-translated probe is hybridized with excess 16s RNA as described by Casey and Davidson, (1977) Nucl. Acids Ree. 4:1539. The hybridization mixture is fractionated at equilibrium closely

o

heat gradient centrifugation at 23 C for 48 hours at 33,000 rpm in a "SW39" rotor. The Cs2SO4 solution is initially set to a density of 1.50 g per cubic centimeter (g/cm ) and at the end of the experiment DNA has a density of 1.45 g/cm<3>, RNA"DNA has a density of 1.52 and the excess of RNA has a density of 1.6 g/cm . (Bassel, Hagaski and Spigelman, 52:796(1964)).

RNA * DNA is collected and the RNA chain hydrolyzed in 0.1 molar

(M) sodium hydroxide for six hours at room temperature. The hydrolysis mixture is then adjusted to pH 7.0 with dilute acetic acid and the DNA is precipitated with cold 80% ethanol.

DNA is dissolved in 1 M triethylammonium bicarbonate buffer,

pH 9.3, and this solution is made 3 millimolar (mM)

with diethyl adipimidate dihydrochloride and allowed to react for five minutes at room temperature. The excess of bifunctional binding reagent is removed by gel filtration on

"Sephadex G-50" of fine quality, which has been brought to equilibrium with 20 mM sodium carbonate buffer, pH 9.3 at 5°C. Chromatography was completed in less than 15 minutes and . x. the effluent containing DNA was immediately combined with an equal volume of 1 mM (aminohexyl) FAD in water.

This reaction mixture is allowed to stand at room temperature for

two and a half hours, and then the excess of (aminohexyl)FAD is removed by gel filtration in a column of "Sephadex G-25", of quality medium, in 0.1 M sodium phosphate buffer, pH 7.0. The reaction products separate into two yellow bands and the first to wash out is collected and used for the hybridization analysis. C. Hybridization Assay for Detection of 16s Ribosomal RNA. Aliquots of various sizes of a liquid culture of E. coli were dispensed into a series of centrifuge tubes such that each centrifuge tube contained 10 2 to 10 9 cells. The suspensions were centrifuged at 10,000 x g for 10 minutes and the supernatant was removed. The cells in each tube were suspended in 20 microliters (µl) of 10 milligrams per milliliters (mg/ml) of egg white lysozyme (Sigma Chemical Co., St. Louis, MO) in 10 mM Tris-hydrochloride buffer, pH 8.0, 0.1 M NaCl, and 5 mM ethylenediaminetetraacetic acid. The lysates were extracted with phenol/chloroform by the method of Maniatis et al, "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor (1982). Polynucleotides in the extracts are precipitated with ethanol.

The precipitate is dissolved in 20 ul of water and 80 ul of a

solution consisting of 60% formamide and 40% 0.16 M sodium phosphate buffer, pH 6.5, 1.44 M NaCl and 0.1% (w/v) sodium dodecyl sulfate is added. 20 ml of the labeled DNA probe, 5 ng DNA, in 0.1 M sodium phosphate buffer, pH 6.5, is added. The reaction tubes are sealed and incubated at 5°C for 18 hours. The tubes are then opened and 500 µl of antibody to the RNA * DNA hybrid is added and allowed to react for one hour at room temperature.

The following reagents are used for the analysis of glucose oxidase activity:

Composite reagent - 92 mM sodium phosphate,

pH 7.0, 0.1% bovine serum albumin, 2 mM 3,5-dichloro-2-hydroxybenzene sulfonate,

0.1 M glucose, 20 mg peroxidase per ml.

Apoglucose oxidase reagent - 4 uM apo-

glucose oxidase binding positions, 25%

glycerol, 4.0 mM 4-aminoantipyrine, and 0.01% (w/v) sodium azide.

After the hybridization reactions have been incubated with the antibody, add 1.9 ml of the composite reagent to each tube followed by 0.1 ml of the apoglucose oxidase reagent. The mixtures are incubated at 25°C for 30 minutes, and at the end of this period the absorbances at 510 nm are determined. As the amount of bacteria increases, the absorbances will decrease due to increasing amounts of ribosomal RNA

which is hybridized to the labeled probe.

Example II.

Hybridization analysis controlled by fluorescence quenching.

A. Antibody to fluorescein is cultured with a fluorescein bovine serum albumin conjugate (Ullman, (1976) U.S. Patent 3,998,943).

B. 6-carboxyfluorescein is synthesized by

the method described by Ullman et al, (1976) J. Biol. Chem. 251:41 72 . (The synthesis gives a mixture of isomers). N-hydroxysuccinic acid imide ester is prepared as described by Khanna and Ullman (1980) Anal. Biochem. 108-156 for the preparation of a corresponding ester of 4',5'-dimethoxy-6-carbomethylfluorescein.

C. The nick-translated probe containing the 4-(3-amino)allyl deoxyuridine monophosphate residue described in Example 1, Part B, is resolved from the ethanol precipitation

step in 100 mM sodium phosphate buffer, pH 8.0, and is done

2 mM with N-hydroxysuccinic acid imidester of 6-carboxymethyl-fluorescein. This reaction mixture is allowed to stand overnight and is then fractionated by gel filtration on "Sephadex G-25", quality medium, brought to equilibrium with 0.1

sodium phosphate buffer pH 8.0. The first dissolved peak for excitation of fluorescent materials, 4 90 nanometers (nm), emission 520 nm is the fluorescein labeled probe used for the hybridization assays.

D. The cell lysates described in Example I, part C,

combine with 80 µl of 60% formamide and 40% 0.16 M sodium phosphate buffer, pH 8.0, 1.44 M NaCl and 0.1% sodium dodecyl sulfate.

20 µl of fluorescein labeled probe (5 ng DNA) in 0.1 M sodium phosphate buffer, pH 8.0, is added to each extract tube. The tubes are closed tightly again and incubated at 55°C for 18 hours. Then 500 µl of antibody is added to the RNA * DNA hybrid

to each tube which is then allowed to stand for at least 1 hour at room temperature. The concentration of the antibodies is determined in initial experiments to be able to provide a large excess compared to the amount required to

bind all the RNA"DNA hybrids expected.

Finally, 400 μl of antibody are added to fluorine

escein and 10 minutes later the fluorescence is recorded with 495 nm for excitation and 519 nm for emission. As the number of bacteria increases, so will the amount of ribosomal RNA

increase, and the quenching of fluorescence by antibodies to fluorescein will decrease. Consequently, the fluorescence will increase as the number of bacteria increases.

Example III.

Hybridization assay for cytomegalovirus using fluorescence energy transfer

A. Preparation of a fluorescein-labeled probe for cytomegalovirus.

Cloned EcoRI restriction fragments of cytomegalovirus

DNA is prepared as described by Tamashiro, et al, (1982) Virology 42:547. 1500 base pair fragments designated

EcoRI e in the Tamashiro reference is used for production

of the probe. The fragment is removed from the plasmid with EcoRI restriction enzyme and cloned into the corresponding position on the M13 mp8 vector (New England Biolabs,

Beverly, MA). The virus is grown in E. coli K12JM101 and

the single-stranded virion DNA is isolated.

The viral DNA in 20 mM tris-hydrochloride buffer, pH 8.0,

containing 10 mM MgCl2 is recombined to a molar excess of a 17 base primer GTAAAACGACGGCCAGT (New England Biolabs) at 55°C for 45 minutes (Bankier and Barrell,

(1982) "Rechniques in Nucleic Acid Biochemistry",

Elsevier, Ireland). This primer is complementary to

a region of M13 mp8 DNA near the 31 —OH end of the EcoRI e — insert. The reaction mixture is then made 15 mM

in dATP, dCTP, dGTP, and 5(3-amino)allyl-dUTP.

(Langer et al, supra) and the Klenow fragment of DNA polymerase I is added. The reaction is incubated at 25°C

in a period of time that is determined by initial trials.

For these experiments, samples are taken from the reaction mixture at various times and electrophoresed in denaturing alkaline agarose gel (Maniatis et al, supra). The reaction time is optimized so that newly synthesized fragments are obtained that extend at least through the EcoRI e insert, and a certain extent beyond the insert into the M13 mp8 sequence is acceptable for the present application.

The residue is then linked to the DNA probe as intercalation complexes. 8-azidoethidium is prepared by the method of Graves et al, Biochim. Biophys. Acta 479:97(1977). The DNA probe prepared above is extracted with phenol/chloroform and precipitated with methanol. It is dissolved in 50 mM tris-hydrochloride buffer, pH 8.0, 0.2 M NaCl and made 0.5 mM with 8-azidoethidium. The mixture is prepared in a glass reaction vessel and immersed in a glass water bath maintained at 20 to 30°C. The photo lighting is carried out for one hour 10 to 20 cm from a 150 watt spotlight..

Non-covalently bound ethidium azide and photolysis by-products are removed from the reaction mixture by 10 subsequent extractions with n-butanol saturated with water. Residues of butanol are removed by precipitating the DNA with ethanol, and the DNA is dissolved in the tris buffer. Covalently bound ethidium residues are measured spectrophotometrically using the extinction coefficient 3-1-1

the sients E^q * 4 x 10 ; cm for photolysed ethidium azide, the relationship between & 260°^A490^or photolysed ethidium bound to DNA (A26Q = (A49Q x 3.4)-0.011) and E260x 10^ M cm for the DNA base pair concentration.

The 8-azidoethidium binds covalently to DNA mainly in the double-stranded region where intercalation complexes can form. The purpose is to incorporate an ethidium residue

per 20 to 50 base pairs. Incorporation can be reduced by reducing photolysis time or reducing 8-azddo-

the ethidium concentration. Increased incorporation can be achieved by repeating the photolysis with new 8-azidoethidium.

The primary amino groups in the 5(3-amino)allyl-dUMP residues

in the DNA probe is reacted with N-hydroxysuccinic acid imide ester of 6-carboxyfluorescein. This reaction is carried out as described in Example II, parts B and C above.

Finally, the fluorescein-labelled/ethidium is separated

modified the probe from the M13 mp8 template by electro-

foreset in alkaline agarose gel (Maniatis et al, supra).

Since the probe is shorter than vector DNA, it migrates

faster and can be recovered from the excised agarose piece by electroelution.

B. Preparation of monoclonal antibody to

ethidium modified DNA.

1. Preparation of covalent ethidium-DNA complexes.

About. 250 mg of salmon sperm DNA (Sigma Chemical Co., St. Louis, MO) is dissolved in 40 ml of 50 mM NaCl, and cut at five passes through a needle (23 gauge). The cut DNA is placed in a 250 ml bottle and diluted with 160 ml of buffer. 145 µl) of S^-nuclease, 200,000 per ml (Pharmacia P-L Biochemicals, Piscataway, NJ), is added and the mixture is incubated at 37°C for 50 minutes.

The extraction mixture is then extracted twice

with phenol/chloroform, once with chloroform and DNA ex-

washed twice with ethanol (Maniatis et al, (1982) "Molecular Cloning", Cold Spring Harbor Laboratory, NY).

The final precipitate is dissolved in 70 ml of 20 mM Tris hydrochloride buffer, pH 8.0.

This DNA is reacted with 8-azidoethidium under the following conditions. The reaction mixture is prepared with 33 ml of 2.7 mg DNA/ml, 13.5 ml of 4.95 mM 8-azidoethidium, 1-5.6 ml of 0.2 M tris-hydrochloride buffer, pH 8.0, 0.2 M NaCl, and 76 ml of water. The mixture is placed in a 250 ml beaker with a water jacket which is kept at 22°C. The mixture is stirred and illuminated for 60 minutes using a 150 watt spotlight at a distance of 10 cm. This photolysis is repeated with an identical reaction mixture.

The photolysed reaction mixtures are combined and extracted 10 times with an equal volume each time of n-butanol saturated with 20 mM tris-hydrochloride buffer,

pH 8.0, 0.2 M NaCl. The extracted DNA solution is combined with 23 ml of 4.95 mM 8-azidoethidium and 77 ml of 20 mM tris-hydrochloride buffer, pH 8.0, 0.2 M NaCl. This solution is photolyzed for 60 minutes as described above. The reaction products are extracted 10 times with buffer-saturated butane as described above, and the DNA is precipitated with ethanol. The precipitate is dissolved in 10 mM tris-hydrochloride buffer, pH 8.0, 1 mM EDTA and the absorbances at 260 and 590 nm are recorded,

2. Preparation of methylated thyroglobulin.

100 mg of bovine thyroglobulin (Sigma Chemical Co.) is combined with 10 ml of anhydrous methanol and 400 µl of 2.55 M HCl 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. It is then dried under vacuum overnight. It is achieved approx. 82 g of dry powder. 3. Preparation of ethidium DNA/methylated thyroglobulin complex.

Methylated thyroglobulin (5.5 mg) is dissolved in 1.0 ml of water and 1.1 ml of a 2.2 mg/ml ethidium-DNA solution is added.

A precipitate immediately forms and the suspension is diluted to 30 ml with 0.15 M NaCl. This suspension is emulsified with an equal volume of Freund's adjuvant. 4. Immunization of mice and production of monoclonal antibody.

BALB/c mice are immunized with 0.5 mL each of the emulsified immunogen. They are given a new injection every other week and blood samples are taken 5 to 7 days after each injection.

Antibody types are analyzed by standard enzyme-labeled immunoabsorbent methods. "Immulon II" microtype wells are coated with single-stranded DNA, double-stranded DNA, or the covalent ethidium-DNA intercalation complex by placing 50 µL of a 5 µg/mL solution in each well. The polynucleotides are in 0.15 M sodium citrate buffer, pH 6.8, 0.15 M NaCl. After the solutions have stood in the well at room temperature for 2 hours, the wells are washed with 0.02 M sodium phosphate buffer, pH 7.4, containing 5 mg bovine serum albumin/mL and 0.5%

Tween 20 cleaning agent (v/v). Suitable dilutions of antiserum are added to the wells to bind the antibodies to the immobilized polynucleotides. The diluted antiserum is washed away and the bound antibodies are detected with enzyme labeled antimouse IgG by well-known methods.

Mice with high titers to the covalent ethidium-DNA complex and very low titers to double- and single-stranded DNA are selected for further sorting with immobilized non-covalent ethidium DNA complex. This includes coating the wells with double-stranded DNA, and includes 0.1 mM ethidium bromide in the antibody binding solution and all subsequent reagents and wash solutions except the reagent for measuring the enzyme activity. Spleen cells from mice with high titers to non-covalent ethidium-DNA complexes are fused with myeloma cells to form hybridomas (Poirer, et al, Proe.

Night'1. Acad. Sci., 79:6443 (1982); Galfre and Milstein, Meth. in Enzymol. 73:1 (1981)).

Cloned hybridomas are grown intraperitoneally in mice so that

large amounts of antibody are grown. Albumin is removed from the ascites fluid by chromatography on "Affigel-

blue resin" in equilibrium with 10 mM tris-hydrochloride buffer, pH 8.0, 0.15 M NaCl. The antibody passes directly through the column and is chromatographed on "DEAE-Sepharose"

using a linear gradient of 10 mM Tris-hydrochloride buffer, pH 8.0, to this buffer containing 0.2 M NaCl. The main peak for the eluted protein contains the antibody essentially free of other proteins.

C. Labeling of monoclonal antibody to ethidium DNA with 4', 5<1->dimethoxy-6

carboxyfluorescein.

4<1>,5<1->dimethoxy-6-carboxyfluorescein (synthesized as a mixture with 4 1 ,5 1-dimethoxy-5-carboxyl fluorescein isomer" ) is transferred to the N-hydroxysuccinic acid ester as described by Khanna and Ullman, supra. This dye was conjugated to the antibody to the ethidium-DNA intercalation complex by the method described in the reference.

D. Hybridization assay for cytomegalovirus.

Urine samples are centrifuged at 3000 rpm for 5 minutes in a "Sorvall FLC-3" instrument to remove cellular and particulate material. The supernatant is run in a poly-allomer ultracentrifuge tube at 25,000 rpm in a "Beckman Ti50" rotor for 75 minutes.

The pellet is dissolved in 0.1 M NaOH and incubated at 37°C

for 30 minutes.

150 µl of 0.2 M sodium phosphate buffer, pH 6.0, containing 1.8 M NaCl, 0.1% sodium dodecyl sulfate (w/v) and 1 mM EDTA is added. Then 20 µl of the fluorescein-labelled/ethidium modified probe (50 ng) is added and the mixture is incubated at 65°C for 10 hours. The reaction mixture is cooled to room temperature and 650 µL of 0.1 M tris-hydrochloride buffer, pH 8.2, containing the labeled antibody to the entidium-DNA intercalation complex is added. The concentration of the labeled antibody in this reagent has been optimized in initial experiments so that a maximum ratio between fluorescence quenching and background is achieved. The reaction mixture is allowed to stand at room temperature for 1 hour.

The fluorescence of the reaction mixture is then recorded using 495 nm light for excitation and 519 nm for emission. A urine not infected with cytomegalovirus is run in parallel and the fluorescence obtained with this control is higher than the fluorescence of the sample with virus. The fluorescence content will increase when the virus level

sinks.

Claims (13)

1. Method for detecting a particular polynucleotide sequence in a test sample comprising single-stranded nucleic acids, which includes bringing the test sample into contact with a labeled nucleic acid probe which comprises at least one single-stranded base sequence which is essentially complementary to the base sequence to be is detected, and determination of the resulting hybrids, characterized in that the hybrids are formed so that they have hybrid epitopes for an antibody reagent that do not substantially bind to single-stranded nucleic acids, wherein any hybrid that is formed is brought into contact with the antibody reagent, the label in the labeled probe provides a detectable response that is measurably different when the labeled probe is included in a hybrid bound by the antibody reagent compared to when it is not included in such a hybrid, and the detectable response is measured as a function of the presence of the sequence to be detected in the sample. 2. Method according to claim 1, characterized in that the antibody reagent is (i) selective binding to DNA"RNA hybrids where either the probe or the sequence to be detected is DNA and the other is RNA, (ii) selective binding to RNA'RNA hybrids where both the probe and the sequence to be detected are RNA, or (iii) selective binding to intercalation complexes where the duplexes formed by the assay include a nucleic acid intercalator bound to it so that intercalation complexes are formed, preferably the labeled probe includes the intercalator chemically bound to the probe as the single-stranded complementary region of the probe whereby the intercalation complexes are formed i ■ the resulting hybrid by hybridization with the sequence to be detected. 3. Method according to claim 1, characterized in that the label interacts with a reagent component in the label detection system so that the detectable response is provided and the detectable response is measurably different when the labeled probe is included in a hybrid which is bound to the antibody reagent which is similar to the in the event that it is not included in such a hybrid due to steric hindrance between the component of the detection system and the label, the label is preferably a substrate, cofactor, or inhibitor of an enzyme that is a component of the label detection system with which the label interacts so as to provide a detectable response. 4. Method according to claim 1, characterized in that the probe is labeled with one of a first label and another label and the antibody reagent is labeled with the second, the interaction between the first and the second label provides a detectable response that is measurably different when the labeled the probe and the labeled anti-substance f reagent are both bound to the same hybrid compared to the case where they are not so bound. 5. Method according to claim 4, characterized in that the first label participates in a first chemical reaction which forms a definable product which is a participant in another chemical reaction with the second label so that a product is formed which provides the detectable response, the first and the second mark is preferably a catalyst for the first and the second chemical reaction, respectively. 6. Method according to claim 4, characterized in that the first and second marks participate in an interaction with energy transfer. 7. Method according to claim 1, characterized in that the test sample includes a biological sample that has been exposed to conditions that release and denature the nucleic acids present in the sample. 8. Reagent system for detecting a particular polynucleotide sequence in a test sample, which comprises a labeled nucleic acid probe that includes a label and at least one single-stranded base sequence that is essentially complementary to the sequence to be detected, characterized in that it also includes an antibody reagent capable of binding to hybrids formed between any of the particular polynucleotide sequences to be detected in the sample and the labeled probe, but incapable of substantial binding to single-stranded nucleic acids, the label provides a measurable response that is measurably different when the labeled probe is included in a hybrid bound to the antibody reagent compared to the case that it is not included in such a system. 9. Reagent system according to claim 8, characterized in that the antibody reagent is (i) selective for binding to DNA"RNA hybrids where either the probe or the sequence to be detected is DNA and the other is RNA, (ii) selective binding to RNA'RNA hybrids where both the probe and the sequence to be detected are RNA, or (iii) selective binding to intercalation complexes where the duplexes formed by the analysis include a nucleic acid intercalator bound to it so that intercalation complexes are formed, preferably the labeled probe includes the intercalator chemically bound to the probe in the single-stranded complementary regions of the probe, whereby the intercalation complexes are formed in the resulting hybrid, by hybridization with the sequence to be detected. 10. Reagent system according to claim 8, characterized in that the label interacts with a reagent component in the label detection system so that the detectable response is provided and the detectable response is measurably different when the labeled probe is included in a hybrid that is bound to the antibody reagent compared to that case that it is not included in such a hybrid due to steric hindrance between the component of the detection system and the label, the label is preferably a substrate, cofactor or inhibitor of an enzyme which is a component of the label detection system. 11. Reagent system according to claim 8, characterized in that the probe is labeled with one of a first label and a second label and the antibody reagent is labeled with the second, interaction between the first and the second label provides a detectable response that is measurably different when the labeled the probe and the labeled antibody reagent are both bound to the same hybrid compared to the case that they .. are not bound in this way. 12. Reagent system according to claim 11, characterized in that the first label participates in a first chemical reaction which forms a definable product which is a participant in another chemical reaction with the second label so that a product is formed which provides the detectable response, the first and the second label are preferably catalysts for the first and second chemical reactions, respectively. 13. Reagent system according to claim 11, characterized in that the first and the second label participate in an interaction with energy transfer.