JPS60179657A - Hybrid formation analyzing method using mark probe and anti-hybrid - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION &i Natsuki I The present invention relates to a nucleic acid hybridization analysis method and reagent system for detecting specific polynucleotide sequences.

The principles of nucleic acid hybridization analysis were developed by researchers in the field of recombinant DNA as a means to determine and isolate specific polynucleotide base sequences of interest.

- Heavy chain nucleic acids, e.g. NA and RNA as obtained by denaturing their double-stranded form, have been found to hybridize or recombine with complementary heavy chain nucleic acids under appropriate conditions. Ta. By labeling such complementary probe nucleic acids with some readily detectable chemical group,
It becomes possible to detect the presence of any polynucleotide sequence of interest in a test medium containing the sample nucleic acid in single-stranded form.

Besides the recombinant DNA field, this hybridization analysis technique can be applied to the detection of important polynucleotides in fields such as medicine and veterinary medicine, agriculture and food chemistry, among others. In particular, this technology is used to detect and identify pathogens such as bacteria and viruses, screen bacteria for antibiotic resistance, aid in the diagnosis of genetic disorders such as string cell anemia and Mediterranean anemia, and detect cancer cells. can do. A general review of this technology and its current and future importance can be found at Biotachnol
ogy (August 1883), pp. 471-478.

The information set forth below is provided for the purpose of publicizing information believed by the applicant to be potentially relevant to the present invention. However, it is not necessarily intended to, and should not be construed as, an admission that the following information constitutes prior art to the present invention.

Current nucleic acid/hybrid formation analysis techniques generally involve hybridization and separation of unhybridized labeled probes. This required separation step is usually facilitated by immobilizing either the sample nucleic acid or the nucleic acid probe on a solid support.

In general, there is a difference between a particular base sequence or gene of interest in a sample nucleic acid and a probe nucleic acid in labeled form.
Bond formation has been detected by separating the solid support from the remaining reaction mixture containing unhybridized probe, followed by detection of the label on the solid support.

Efforts continue to simplify analytical procedures for performing nucleic acid hybridization assays.

The primary goal of these efforts is to reduce the complexity of the operation to the point that it can be performed conveniently and routinely in the clinical laboratory. The need for separation steps significantly hinders the progress of these efforts. The analytical process is a physical operation that requires considerable skill to accomplish with analytical reproducibility and is not easily automated or amenable to high volume testing.

Furthermore, there are two significant difficulties with conventional methods involving immobilization of sample nucleic acids. First, the manipulations required to achieve immobilization are generally time consuming and add steps that are undesirable for routine technique use in clinical laboratories. Second, proteins and other substances in heterogeneous samples, especially in the case of clinical samples, may interfere with the immobilization procedure.

An alternative method to immobilizing a sample nucleic acid and adding a labeled probe is to use an immobilized probe,
Examples include a method of labeling the sample nucleic acid in situ, or a method of using a double hybridization technique that requires two probes, one of which is immobilized and the other labeled. However, the former alternative is even less desirable. This is because in-situ labeling of sample nucleic acids requires a high level of skill that clinical technicians do not normally have, and also poses serious problems when the labeling medium contains variable amounts of substances that inhibit the labeling reaction. This is because there is no simple and reliable way to monitor the labeling yield obtained. On the other hand, double hybridization techniques have the disadvantage of requiring additional reagents and incubation steps, and the kinetics of the hybridization reaction is slow and inefficiently usable. The accuracy of this assay may also vary if the complementarity of the two probes to the sample sequence is variable.

Some of the problems described above are solved by using immobilized RNA probes to detect the resulting immobilized DNA-RNA or RNA-RNA hybrids with labeled specific anti-hybrid antibodies. No. 818,132, 188
See application filed June 1, 4]. This technique still requires a separation step and therefore has a drawback common to all hybridization techniques that require a separation step as described above.

European Patent Application No. 70. [1/5] proposes a hybridization analysis technique that does not require physical separation of hybridized probes from unhybridized probes. Using a pair of probes that hybridize (to adjacent regions of a polynucleotide sequence of interest), one probe can be labeled with a chemiluminescent catalyst such as the enzyme peroxidase and the other with an absorber molecule for chemiluminescence. Proposed. These catalyst and sorbent labels must be located near adjacent ends of each probe so that upon hybridization, quenching of chemiluminescence is observed due to energy transfer to the sorbent molecule. To perform such an analysis, each label is brought into a position that quenches upon hybridization to the sample nucleic acid, and each labeled probe segment has an affinity for actually hybridizing to the sample nucleic acid. One must have the ability to tunably synthesize two important probe reagents without affecting the

11 Standing 1 Nucleic acid hybridization analysis method based on modulation of the detectable response of a hybridized labeled probe resulting from binding of an antibody reagent to a hybrid formed between the labeled probe and a specific detected polynucleotide sequence has been devised. That is, the label in the anti-binding hybrid exhibits a response that is detectably different from the response exhibited by the label in the unhybridized labeled probe. In this way, there is no need to separate hybridized and unhybridized probes, greatly facilitating analytical performance and automation.

Furthermore, since the analytical signal is non-radioactive in nature,
Another standard of analytical convenience is met in that the detection system does not involve the use of radioactivity.

According to the present invention, by forming a hybrid between any particular sequence to be detected and a labeled nucleic acid probe consisting of a label and at least one single base sequence substantially complementary to the sequence to be detected.

A specific nucleotide sequence is detected in the test sample. This hyperprint is characterized by having a binding tope for the antibody reagent (anti-hybrid) that does not substantially bind to the -heavy chain nucleic acid. The anti-hybrid is then added, but will not substantially bind to the labeled probe unless it is present hybridized to the detected sequence. The label in the labeled probe is measurably different when the labeled probe is included in a hybrid bound by an anti-hybrid as opposed to when it is not included in such a hybrid. ie, give an increased or decreased detectable response. The resulting measure of detectable response is a function of the presence of the detected sequence in the sample.

A preferred mechanism of modulation by anti-hybrid binding of the label is understood to involve steric hindrance. In such situations, the label chemically interacts with the reagents of the label detection system, such as by reaction or binding, and the presence of an anti-hybrid that binds to the hybrid reduces the accessibility of such detection system members to the label. steric hindrance occurs. Preferred labels that can be applied to such systems are those that involve an enzymatic reaction, i.e., the label and the reagent components with which it interacts are selected from enzyme substrates, enzyme cofactors, enzyme inhibitors, and enzymes. It will be done. A detectable response is obtained by colorimetric, fluorometric or luminometric methods.

Another preferred mechanism for modulation of anti-hybrid labels is based on the use of adjacent pairs of interacting labels. The probe is labeled with one of the first label and the second label, and the antibody reagent is labeled with the other or contains a chemical group that serves as the other label in its pure form, and between these two labels a detectable f Give a silly response. When meeting within the same hybrid,
These two labels are placed within adjacent interacting distance of each other such that there is no free, diffuse H in the total solution (as opposed to the relatively less frequent interactions that occur between active labeled reagents). , substantially increases the interaction that affects the signal. A preferred interaction between these two labels is such that the first label participates in the first chemical reaction to produce a diffusible product; It is a sequential interaction in which it becomes a participant in a second chemical reaction with a second label and produces a detectable product. For example, by labeling the antibody reagent with an enzyme and the probe with a catalyst that acts on the diffusible products of the enzymatic reaction, the optical reaction in the presence of a suitable indicator dye composition is particularly preferred. A detectable product can be provided by a signal, etc. Another preferred label pair is one that involves an energy transfer interaction, such as between a fluorescent or luminescent material and a quencher of the emission of the first label.

The invention is characterized by a number of important advantages. In addition to the main advantage of eliminating a separation step, there is no need to immobilize sample or probe nucleic acids, which creates non-specific binding and reproducibility problems in commonly used hybridization techniques. Furthermore, the formation kinetics of Vibrift formation are substantially faster in solution as compared to systems using single strands of immobilized hybridizable pairs. An additional advantage is that the analysis can be performed without a washing step. Analytical reagents can be added to the hybridization medium sequentially without the need to wash insoluble support material.

Another important feature of the invention is that the included detection system is highly efficient because the duplex molecule can contain many labels and binding sites for anti-hybrid reagents. This allows large amounts of label and anti-hybrid to be incorporated into their interaction configuration per unit of hybridizing probe.

Considering antibodies against RNA-DNA or RNA-RN'A hybrids, one labeled antibody can be used for approximately every lO
Hybrids can be attached to base pairs.

For example, if the probe is 500 bases long, 40 to 5
Can bind 0 antibodies. The presence of a large number of binding sites in the hybrid can be used advantageously when low concentrations of hyperprints have to be detected.

- Description of Preferred Embodiments The use of nucleic acid hybridization as an analytical tool is fundamentally based on the double-stranded structure of DNA. The hydrogen bonds between the purine and pyrimidine bases of each strand of double-stranded DNA can be reversibly broken. This DNA's
The two complementary single strands resulting from "melting" or "denaturation" associate (sometimes called reannealing or hybridization) to reform the double strand bond. As is well known, contacting a first single-stranded nucleic acid, either DNA or RNA, comprising a base sequence that is sufficiently complementary (i.e., "homologous") to a second single-stranded nucleic acid under appropriate conditions. As a result, DNA-DNA, RNA@
A DNA or RNA-RNA hybrid is formed.

Probe A probe consists of at least one single-stranded base sequence that is substantially complementary or homologous to the sequence to be detected. However, such a base sequence need not be a single continuous polynucleotide segment, but may consist of two or more individual segments interrupted by non-homologous sequences.

These non-homologous sequences may be linear, or they may be self-complementary and form hairpin loops. Furthermore, the homologous regions of the probe may be located at the 3'- and 5'-ends by heterologous sequences, such as sequences consisting of the DNA or RNA of a vector into which the homologous sequences are inserted for propagation. In either case, the probe provided as an analytical reagent exhibits detectable /\iblint formation at one or more points with the sample nucleic acid of interest. As long as the key homologous segments are in single-stranded form and available for hybridization with the sample DNA or RNA, the line will remain intact even if the major or minor portion forms a backbone with a complementary polynucleotide strand. A single-stranded polynucleotide in the form of a shape or a circle can be used as a probe element. It is generally preferred to use probes that are in substantially single-stranded form. The preparation of appropriate probes for a particular analysis is no more than a routine matter for those skilled in the art.

Labels can be selected from a variety of substances. Any material that, when included in a labeled probe, gives a detectable response that is modifiable by anti-hybrid binding and is sufficiently stable under hybridization conditions to give a measurable response. You can essentially do that.

Modulation or modification of the label response may be the result of a variety of different effects resulting from the binding of anti-hybrid to the hybrid.

It is understood that particularly preferred mechanisms for modulating label response include steric hindrance. Typically, the label is selected to provide a detectable analytical response upon interaction, such as by chemical reaction or binding, with a member of a reagent detection system consisting of one or more substances that participate with the label to provide a measurement signal. Binding of the anti-hybrid, whether due to steric hindrance or other phenomena, prevents or inactivates the label from participating in such signal generation. Such an effect on signal generation reflects the presence of the hybridizing labeled probe.

Preferably, when included in a sterically hindered-based system,
The label is small, e.g. less than 10,000, more acceptably less than 4,000, preferably less than 2,000 daltons in molecular weight, and the interacting reagent in preferred detection systems is usually considerably larger, e.g. more than 3 times, more usually more than 10 times, preferably 20 to 100 times
That's more than double that. Thus, in the most preferred systems using labels with masses on the order of 100 to 2,000 Daltons, at least one member of the detection system with which the labels interact to give a detectable signal has a mass on the order of 10,000 to 2,000 Daltons.
It is of the order of 200,000 Daltons or more.

Such a size relationship between the label and the detection system members increases the likelihood of significant steric hindrance during binding of the anti-hybrid to the labeled hybrid. Therefore, preferred labels are substances that participate in enzyme-catalyzed reactions, such as enzyme substrates, coenzymes, enzyme prosthetic groups, and enzyme inhibitors, since a wide range of enzymatic reactions are available to select analytes. . For sufficiently large molecular weight enzymes, many small substrates, coenzymes, inhibitors, etc. have been shown to have favorable size relationships between the label and its interacting detection system members. This applies likewise to prosthetic groups and their corresponding apoenzymes. Depending on the denaturing conditions under which hybridization is carried out, non-proteins, especially stable organic or inorganic compounds, are also preferred as labels.

Certain particularly preferred labels and methods of preparing labeled probes are described below.

In this system, the labeled probe is the substrate for the enzyme, and the ability of the enzyme to act on the substrate-labeled probe is affected by the binding of the labeled probe by the anti-hybrid, either in a positive or negative sense. , but usually chosen to be influenced in a way that inhibits it. The action of an enzyme on a substrate-labeled probe produces a product that is usually distinguishable by chemical or physical characteristics, such as chemical reactivity in an indicator reaction or photometric properties, such as fluorescence or light absorption (color). generate. Marks of this type are generally assigned together with concurrently pending 5eri.
al No. 894,838 (April 1978 lO
(corresponding to British Patent No. 1.552.607) and Anal, Che+s, 48:1933 (
1!37B), Anal.

Bloche+*, 77:55 (1977) and Cl
1n, Chetx, 23:1402(19,77)
It is described in. In such enzyme substrate-labeling techniques, the labeled probe has the property that it is acted upon by the enzyme by cleavage or denaturation to produce a product with detectable properties that distinguish it from the complex, e.g. The complex is non-fluorescent under analytical conditions, but upon reaction with the enzyme a fluorescent product is produced.

A very useful group of substrate labels include those that perform simple hydrolysis reactions and yield fluorescent products.

Such products can be detected at low concentrations. Examples of these types of substrates include phosphate esters, phosphodiesters, and glucosides of fluorescent dyes. Glycosides are particularly preferred because of their low rate of non-enzymatic hydrolysis, and β-galactosides are particularly advantageous because β-galactosidase enzymes are readily available and they are extremely stable.

A particular group of useful fluorogenic substrate-labeled probe complexes are those represented by the following formula: [G-D-R1nN
A [where G is a cleavable group such as phosphate, carboxylate, sulfate, or glycone, and D is a fluorogenic dye moiety that generates a fluorescent product upon removal of G, e.g., D is umbelliferone. , fluorenscein, rhodamine and their derivatives, R is a binding group, NA is a nucleic acid probe, and n is the average number of labels per molecule of the probe, e.g. 1 to 50.
].

Enzymatic cleavage of the label complex (eg, by phosphatases, carboxylases, sulfatases, glycositases, etc.) is influenced by the binding of the anti-hybrid to the label hybrid. For example, U.S. Pat.
279', 992. Particularly preferred substrate-label analysis formats employ label complexes of the following type: where R, NA and n are as defined above.

The ability of the enzyme β-galactosidase to cleave this complex and yield a product distinguishable by its fluorescence is thereby inhibited by binding of the labeled hybrid to the anti-/hybrid.

Other useful substrate-label complexes are those of the formula: [D-X]nNA, where X is an enzyme-cleavable linking group, e.g., phosphate, carboxylate, etc., and NA and n is as defined above; D is a fluorogenic dye moiety that releases a fluorescent indicator upon cleavage of X, as above).

Label complexes of this type have the formula: where R1 is a bond or chain linking the labeling component NA to the cleavable phosphate group, and R2 is hydrogen or lower alkyl, such as methyl and ethyl. :N-alkylamide; or 1. ) is a N-(hydroxy-substituted lower alkyl)amide, e.g. -(ONH-(CH2), -OH(m =
[U.S. Patent No. 4,27
3?See specification No. 15]. This umbelliferone residue may have other f or additional substituents I, X[
Anal, Chell, 40; 803 (1988)
reference].

Cleavage by phosphogesterase is influenced by the binding of anti-/\ibrint to the labeled hybrid. In this procedure, some of the phosphodiester bonds in the hybrid are also cleaved, but hybridization is complete and a modulation of substrate label cleavage is present in the intact hybrid versus the digested fragment. It does not affect the analysis since it is woody identical to that for the marker.

Another preferred fluorescent dye is 480 and 52, respectively.
It has fluorescence excitation and emission maxima at 0 nm, and Ullman et al. [
Ullman et al. (197B), J.B.
iol, Cheap.

251:4172] can be synthesized by
It is carboxyfluorescein. That is, Rotman et at, Proc, Nat.
'l Acad, Sci.

50, 1 (1983)]. This raw rice export was carried out by Khanna and Ullman [Khanna and Ullman,
Anal.

Bioche Il, 108:158 (1980)] to the N-hydroxysuccinimide ester and used in the preparation of the corresponding ester of 4°,5'-dimethoxy-5-carboxymethylfluorescein. be able to.

The labeled probe in this system is composed of a coenzyme-active functional group in its labeled portion, and the ability of such a coenzyme label to participate in the enzymatic reaction is due to the ability of the labeled hybrid by the anti-hybrid. is influenced by the combination of The rate of the enzymatic reaction carried out can be measured by conventional detector systems and ultimately provides a detectable signal. This type of marking shall be as follows: - Co-pending 5erial No.
894.83 [No. 1 (filed on April 10, 1978,
GB 1,552,708) and Anal.

Biochem, 72:271 (197B), Anal
, Biochem, 78:283° (+978) and Anal, Biochem, 78:95 (111
7B).

A useful coenzyme for labeling is nutamamide adenine dinucleotide (NAD). NAD can be combed into a probe by a bridge at the 6-nitrogen or 8-carbon of the adenine moiety. A method for introducing an aminoethyl bridge at the 66-nitrogen position is described by Carrini et al.
tal, Anal, Biochem? 2:2?1
(187B)]. Lee and Japlan, Arch, Bi
ochem.

Biophys, 188:1385 (197B)] describes the introduction of a large San bridge to diamino at the 8-carbon. NAD labels can be detected by various methods, such as enzymatic cyclization using malate dehydrogenase and alcohol dehydrogenase.

Another useful coenzyme is adenosine triphosphate
(ATP). Trayer etc.
tal, Biache+s, J, 139:80
9 (1974)] describe the synthesis of ATP with a hexylamine bridge on the 6-nitrogen of the adenine group. This can be detected by enzymatic cyclization using hexokinase and pyruvate kinase. ATP can also be coupled to the probe via a repose ring. Repose is oxidized with sodium periodate,
It is then condensed with dihydrazide [Wilchek a
nd Lamed, Meth, in Enzymo
l.

34B:475 (1974)]. The obtained hydrazone is reduced with sodium borohydride. This label can be sensitively detected using firefly luciferase in a bioluminescent readout device.

A particularly useful group of cofactor labels are prosthetic groups. In such systems, the ability of the catalytically inactive apoenzyme to combine with the prosthetic group label to form the active enzyme (holoenzyme) is influenced by the binding of the labeled hybrid by the anti-hybrid. . The activity of the holoenzyme obtained can be measured by conventional detection substance systems, ultimately resulting in a detectable signal. This type of label is described in co-owned US Pat. No. 4.238.5fi5. A particularly preferred prosthetic molecule-label analysis format uses flavin adenine dinucleotide (FAD) as the label and apoglucose oxidase as the apoenzyme. The resulting glucose oxidase activity can be measured by a colorimetric detection system consisting of glucose, peroxidase, and an indicator system that produces a color change in response to hydrogen peroxide. Fluorometric detection of hydrogen peroxide is also possible using appropriate fluorogenic substrates. FAD can be coupled to the probe via a bridging group at the 6-nitrogen of the adenine moiety.

Morris et at, Anal,
Chem, 53:858(+981)] describe a method for the synthesis of FAD with a hexylamine bridge on the 8-nitrogen, and Zappelli et al.
al, Eur, J, 'Biochem, 811
:494(197B)] describes a method for introducing a 2-hydroxy-3-carboxypropyl group at the same position.

Modulator - The labeled probe in this system is composed of an enzyme activity regulating function such as an enzyme inhibitor or activator in its labeled portion, and the ability of such a modulator label to regulate enzyme activity is achieved by the binding of the labeled hybrid by the anti-hybrid. affected. The rate of the resulting enzymatic reaction can be measured by conventional detection agent systems, ultimately resulting in a detectable signal. This type of label is disclosed in U.S. Patent No. 4,13
No. 4,792 and No. 4,273,888. Particularly preferred is the use of dihydrofolate reductase as the activity-regulated enzyme and methotrexate as the label. If the label is an enzyme inhibitor, it interacts covalently or non-covalently with the enzyme and may be a small molecule, such as methotrexate, or a large molecule, such as an antibody directed against the enzyme. [“U.S. Pat.
No. 273.8BE1 and co-pending application Serial No. 285. E105
(filed on July 2, 1981)].

In this system the label is a catalyst and the activity of the catalytic label is influenced by the binding of the anti-hybrid/hybrid. The resulting catalytic activity can be measured using a conventional detection agent system.

The final result is a detectable signal, eg absorption or fluorescence. This type of label is described in U.S. Pat. No. 4,180,84
It is described in the specification of No. 5.

In this system, the label is attached to an epitope, i.e. the second antibody (
i.e., the antibody binding site for the anti-label).

Or it consists of fragments thereof. The ability of the anti-label to bind to the label is influenced by the binding of the anti-label. Several monitoring or detection schemes are possible. - By way of example, an epitope label is also a fluorescent agent whose luminescence is altered, for example reduced by binding with an anti-fluorescent agent. Binding of the anti-hybrid to the labeled hybrid limits the accessibility of the fluorogen label to the quenching anti-fluorogen (
(See U.S. Pat. No. 3,9118,943). In another approach, an additional detection agent molecule consisting of an epitope label coupled to the enzyme is used. Binding of the anti-label to this epitope-enzyme complex results in inhibition of enzyme activity. By anti-hybrid binding,
The more anti-label is excluded from the bound label on the epitope-label hybrid, the more anti-label will be available to bind and inhibit the enzymatic activity of the epitope-enzyme reagent. (U.S. Patent No. 3,935,07
(See No. 4).

11 According to another preferred embodiment of the invention, hybridization dependent on the presence of the polynucleotide sequence of interest and binding of the labeled antibody reagent to the labeled hybrid results in the binding of these two labels to their interaction. A pair of labels is used such that the signal produced by the label comes at a distance that is measurably different from that produced by their encounter upon mere diffusion in the entire analytical medium. Ru. Various interaction phenomena may apply to this embodiment. This interaction may be chemical, physical, electrical, or a combination of these forces. The environment of the label formed upon hybridization and binding of the labeled antibody reagent to the formed labeled hybrid, referred to herein as the bound hybrid environment, is separated from the overall medium in at least one important aspect. It must be clearly different.

Since hybridization determines the proportion of label that results in a localized hybrid environment versus the global phase, the signal response obtained will depend on the presence of the sequence to be measured in the assay medium. .

A favorable interaction between two labels is such that one label participates in a first reaction to produce a diffusion-mediated product, which in turn participates in a second reaction with a second label, producing a detectable product. It involves two chemical reactions that lead to The microenvironment of the binding nullibrid is one that has a higher local concentration of 1/) mediator product than the total solution, thus increasing the rate of the second labeling reaction that generates the signal.

These two labels can each take part in the reaction as reactants or, particularly preferably, as catalysts. Any catalyst involved may be either enzymatic or non-enzymatic.

Various enzymes and catalysts can be applied to this embodiment, the selection of which is merely a matter of choice for the person skilled in the art. Enzymes useful for labeling antibody reagents include oxidoreductases, particularly nucleotides such as nicotinamide adenine dinucleotide (NAD), its reduced form (NADH), or adenosine triphosphate (ATP) as a cofactor. Contains/l/)t
± those that produce hydrogen peroxide or other small diffusible products. Specific examples of the enzymes include alcohol dehydrogenase, glycerol dehydrogenase, lactate dehydrogenase, malate dehydrogenase, glucose-6-phosphate dehydrogenase, glucose oxidase, and uricase.

Other classes of enzymes can also be used, such as hydrolases, transferases, lyases and isomerases. A detailed list and description of useful enzymes can be found in U.S. Pat.
No. 148, these patents apply mutatis mutandis to the present invention. Other useful systems include as the first label an enzyme that catalyzes a reaction that produces a prosthetic group for the apoenzyme or proenzyme. Non-enzymatic catalysts may also serve as labels, as described above.

See, eg, US Pat. No. 4,180,845.

Another preferred interaction between pairs of labels is through energy transfer. The first label is a luminescent substance such as a fluorescent substance or a chemiluminescent substance; the former produces luminescence when irradiated, and the latter produces luminescence upon chemical reaction.

The emitted light can be absorbed by a second label, which either quenches the emitted light or provides a second emitted light, as is the case if the absorbing label is a fluorescent material itself. A combination of compounds useful for this effect is disclosed in U.S. Pat. No. 4,275.
, No. 149 and No. 4,318,981. Preferred fluorescent emitter/quencher combinations include naphthalene/anthracene, α-naphthylamine/dansyl, tryptophan/dansyl, dansyl/fluorexein, fluorescein/rhodamine, tryptophan/fluorexein, N-[p-( 2
-benzoxazolyl) phenylcomaleimide (BPM)
/thiochrome, BPM/8-anilino-1-naphthalenesulfonate (ANS), thiochrome/N-(4-
Examples include dimethylamino-3,5-dinitrophenyl)maleimide (DDPM) and ANS/DDPM. Preferred chemiluminescent substance/quenching substance combinations include luminol and fluorescein, eosin or rhodamine S group.

Various modifications of labeled probes can be used if desired without departing from the scope of the invention. In one variation, the labeled probe includes a solid phase to which a label is attached. The solid phase may be the wall of the reaction vessel or may be a dispersed solid such as polyacrylaniide or agarose beads. It is also possible to modify the probe with a binding capable ligand such as hapten or biotin and introduce a label by adding labeled anti-/\butene antibody or labeled avidin before or after the hybridization reaction. In this way, the label can be attached to the binding agent directly or via an intermediate moiety.

Various methods can be used to label the polynucleotide probe. Preferably, the label is distributed over the length of the polynucleotide rather than concentrated in one location. Some means of marking are sketched below.

5-(3-amino)allyldeoxyuridine triphosphate (AA-dUTP) was prepared by the method of Langer et al.
ger et at, Proc, Nat'1. A
cad, sci.

78:f (833 (1981))] and can be introduced into a double-stranded DNA probe by nick translation using a DNA polymerase. It has a chain amino group.

The N-hydroxysuccinimide ester of 8-carpoxyfluorescein, 4′,5°-dimethoxy-6-carpoxymethylfluorescein, and their β-galactosyl derivatives can be directly reacted with nick-translated probes. I can do it. It is desirable to use an excess of the activating dye to maximize label introduction, and uncoupled dye is then separated from the labeled DNA by gel filtration.

Labels with terminal amines on the bridging group can be nick-translated using bifunctional reagents such as dimethyl adipimidate, hexamethylene diisocyanate, and p, p'-difluoro m, at'-dinitrophenyl sulfone. It can be coupled to DNA. These labels can be reacted with an excess amount of bifunctional reagent, and then unreacted bifunctional reagent can be removed. The activated label can be reacted with the nick-translated probe.

Certain planar aromatic compounds are intercalated between base pairs of double-stranded nucleic acids to form reversible complexes. Photolysis of this intercalation complex allows several intercalation agents to be covalently coupled to the polynucleotide. Photoactivatable intercalating agents are furocoumarins such as 8-azidoethidium, 8-azidomethidium and angelicin. These intercalating agents can be modified with bridging arms containing functional groups. 8-azidomethidium derivatives are described by Hertzberg and Dervan [Hertzberg and Dervan].
J, Am, Chell, Sac, 104=31
3 (1882)] and Mitchell and Delpin [Mit
chell and l1ervan.

J, Am, Chem, Soc, 104:42 [
15 (1972)].

These labels can be coupled directly to modified intercalating agents.

For example, 8-carpoxyfluorescein or 4″,
The N-hydroxysuccinimide ester of 5'-dimethoxy-6-carpoxyfluorescein can be reacted with a derivative of an intercalating agent to form an amide bond. These intercalating agent-label conjugates can then be added to polynucleotide probes and photolyzed. Alternatively, a modified intercalating agent can be photolyzed with a polynucleotide and then coupled to a labeling compound.

8-Azidomethidiram can be photolytically coupled to single-stranded nucleic acids by a non-intercalation mechanism [Balton and Kerns (1
978) Nucl, Ac1d, s Rea, 5:
4891], this means can be used to couple the intercalating chrysanthemum derivatives to single-stranded DNA and RNA.

One important aspect of the present invention is the formation of a hybrid between a probe and a polynucleotide sequence of interest that comprises a binding site for an anti-hybrid antibody reagent. The principle of this assay is that hybridization of the labeled probe with the desired sequence results in the formation of a hybrid bound by the anti-hybrid, which produces a measurable effect on the label response. Any system of design that can generate binding sites for anti-hybrid reagents that are unique to the hybrid can be used. This ensures that the desired effect of binding of the anti-hybrid to the labeled probe occurs only upon hybrid formation.

Binding of anti-hybrid to hybrid is typically 5
Various biologically derived substances, particularly those that involve highly specific non-covalent bonds such as those characteristic of binding proteins such as immunoglobulins. Use various binding substances that provide anti-hybrid properties that have unique binding activity to hybrids but have little binding activity to single-stranded nucleic acids such as unhybridized probes and unhybridized sample nucleic acids. be able to.

Particularly preferred binding substances are antibody reagents with anti-hybrid binding activity, which may be whole antibodies of conventional polyclonal or monoclonal variety, or fragments thereof, or aggregates or conjugates thereof. Preferred antibody reagents are (
It selectively binds to ilDNAIIRNA or RNA/RNA hybrid or (11) intercalation complex. Currently, DNA-RNA or R
NA-RNA hybrids are known to be able to activate antibodies that bind more selectively than -heavy chain nucleic acids; however, currently such selectivity cannot be achieved with DNA-DNA hybrids.
It is believed that it is not possible to generate this in the case of an A-hybrid. DNA-D with selectivity
Depending on the extent to which NA antibodies are developed in the future, they will clearly be applicable to the present invention. Antibodies against DNA@RNA hybrids can be used when one of the probe and the detected sequence is DNA and the other is RNA, and antibodies against RNA-RNA can be used when both the probe and the detected sequence are RNA. It can be used in case.

Furthermore, anti-DNA-RNA or anti-RNA/RN, A
Regarding RNA probes used with reagents in the present invention, it should be understood that not all nucleotides included in the probe are necessarily ribonucleotides, i.e., contain 2'-hydroxyl groups. A fundamental characteristic of the RNA probe used in the DNA consisting of probes
It is capable of activating antibodies against RNA or RNA@RNA. Thus, one or more 2°-positions on the nucleotides included in the probe may be in the deoxy form so long as the antibody binding properties necessary for carrying out the assays of the invention are retained to a substantial degree.

Similarly, in addition to, or instead of, such limited 2°-deoxy modification, RNA probes may
．． It is also possible to include nucleotides with other 2"-modifications, as long as they do not substantially interfere with the specificity for double-stranded hybridization products compared to the specificity for each single strand, or It is also possible to have any other modifications along the lipophosphate backbone.

If such modifications are present in the RNA probe,
The immunogen used to produce the antibody reagent preferably consists of a single chain with substantially corresponding modifications, the other chain depending on whether the sample RNA or DNA is intended to be detected. It is substantially unmodified RNA or DNA. Preferably, the modified chain in the immunogen is RN
Identical to the modified strand in the A probe. An example of an immunogen is hybrid poly(2°-0-methyladenylic acid).
-Poly(2°-deoxythymidylic acid) is mentioned. Another example is poly(2'-0-ethyrinosinic acid)
・Poly(lipocytidylic acid) is mentioned.

The following are further examples of modified nucleotides that can be included in RNA probes: 2'-0-methylribonucleotide, 2-0-ethylribonucleotide, 2'-azidodeoxyribonucleotide, 2'- Chlorobuokyliponucleotides, 2-0=niacetylribonucleotides and methylphosphonates or phosphorothiolates of ribonucleotides or deoxyribonucleotides.

Modified nucleotides are added to the template (te+
nplate) during enzymatic synthesis of the probe. For example, adenosine - 5° - 〇 = (
1-thiotriphosphate) (ATPαS) and d
ATPαS is a DNA-dependent RNA polymerase and D
It is a substrate for NA polymerase. Alternatively, chemical modifications can be introduced after the probe has been prepared. For example, the RNA probe can be 2°-0-acetylated with acetic anhydride under mild conditions in an aqueous solvent.

Immunogens that activate antibodies specific for RNA@DNA hybrids can consist of homopolymers or heteropolymers of polynucleotide duplexes. Among the possibilities are homopolymer duplexes, especially poly(r
A) Poly (dT) (Kitagawa and 5t
ollar (1882) Mo1. Immuno! .

+! II:413] is preferred. However, in general heteropolymeric duplexes are preferably used, and RNA
It can be prepared by various methods including transcription of φ×174 pillion DNA by polymerase [Nakaz
ato (111180) Biochem, 19:2
835].

The selected RNA-DNA duplex is absorbed into a methylated protein or coupled to a common immunogenic carrier material, e.g., calf serum albumin, and injected into the desired host animal [5tollar (1980) Met
h, see Enzymol 70 Near 0], RNA-R
Antibodies against NA duplexes can be raised against double-stranded RNA from viruses such as reovirus or Fiji disease virus, which infect sugarcane, among others. Homopolymer duplexes such as poly(rI)-poly(rC) or poly(rA)-poly(rU), among others, can also be used for immunization as described above. Furthermore, RNA
- Information regarding antibodies to DNA and RNA-RNA hybrids can be found in commonly assigned U.S. Patent Application No. 5eri.
al No. 816, 132 (June 1, 1984 M
) is given in

Antibodies against intercalation complexes typically consist of an ionic complex between a cationic protein or protein derivative (e.g., methylated calf serum albumin) and an anionic intercalator-nucleic acid complex. It is something. Ideally, the intercalator is covalently coupled to the double-stranded nucleic acid. Or again,
The intercalator-nucleic acid conjugate can also be covalently coupled to a carrier protein. The nucleic acid portion of the immunogen may consist of the specific paired sequences found in the assay hybrid, or any other sequence since the specificity of the antibody is generally independent of the particular base sequences involved. . Further information regarding antibodies to intercalation complexes is provided in commonly assigned US Patent Application Serial No. 580.429, filed December 12, 1983.

As mentioned above, the antibody reagent can consist of a whole antibody, an antibody fragment, a multifunctional antibody aggregate, or generally any material comprising one or more specific binding sites from an antibody. In the form of a whole antibody, it may belong to any of the known immunoglobulin classes and subclasses, such as IgG, IgM, etc.

Any fragment of any such antibody that retains specific affinity for the hybridization probe can also be used, for example, typically Fab, F(ab'),
Fragments of IgG known as F(ab')2 and F(ab')2 can be used. Furthermore, aggregates, polymers, derivatives and conjugates of immunoglobulins or fragments thereof can be used where appropriate.

Immunoglobulin sources for antibody reagents can be any available, including conventional antiserum methods and monoclonal methods. It can be obtained by this method. Antisera can be obtained by well-established techniques of immunizing animals, such as mice, rabbits, guinea pigs, or goats, with appropriate immunogens.

Immunoglobulins can also be obtained by somatic cell/\brid formation techniques that yield what are commonly referred to as monoclonal antibodies, which involve the use of appropriate immunogens.

When using an antibody reagent selective for intercalation complexes as one of the binding reagents, various intercalator compounds can be used. In general, the intercalator compound is preferably a low molecular weight, planar, usually aromatic but optionally polycyclic, double-stranded nucleic acid, such as a DNA@DNA, DNA-RNA or RNA-RNA duplex. Generally, it can be said that it is a molecule that can bind by insertion between base pairs. The primary bonding mechanism is usually non-covalent, but reactive or activatable in which the intercalator forms a covalent bond with an adjacent chemical group on the steel of one or both of the duplexes being intercalated. If the compound has a chemical group, covalent bonding also occurs as a second step. As a result of intercalation, adjacent base pairs are expanded to twice their normal separation, resulting in an increase in the molecular length of the duplex.

Additionally, approximately 12-3 to accommodate the intercalator
Eight degrees of double helix unwinding must occur.

General review and further information Rama? [Lerman,
J, Mo1. Biol, 3:18 (1981)
], Bloomfield et al.
tal, ”PhysicalChe+++1stry
of Nucleic Ac1ds''.

Chapter 7. pp, 429-47B], Harper and Rowe [) 1arper and Rowe,
NY (1974)], Waring [Woring,
Nature 219:1320 (1988) 1,
Hartmann et al, An
gew, Chew, Engl, Ed.

7:893 (11388)], Lipp
ard, Accts.

Chew, Res, 11:211 (1878)],
Wilson [Wilson.

Intercalationchea+1try (
1982), 455] and Berman et al.
net at, Ann, Rev, Biophy
s.

Bioeng.
1 etc. Specific examples of intercalators include acridine dyes such as acridine orange, phenanthricines such as ethidium, phenazines, furocoumarins, phenothiazines, and quinolines.

The intercalation complex is hybridized by using a modified probe to chemically bind the intercalator in its complementary-heavy region such that upon hybridization an intercalation complex is formed. formed in the analytical medium during

Essentially any convenient method can be used to achieve such conjugation. Typically, conjugation involves reactive,
Preferably, it is formed by performing intercalation with a photoreactive intercalator and then performing a binding reaction. A particularly useful method is one involving an azide intercalator.

Reactive nitrene is readily formed upon exposure to long wavelength ultraviolet or visible light. The nitrenes of the aryl azides preferentially undergo insertion reactions over their rearrangement products [White et at, Methods in
Enzymol, 46:844 (1977)].

Typical azide intercalators are 3-azidoacridine, 9-azidoacridine, ethidium monoazides, ethidium diazide, ethidium dimer azide [M
Itchel et al. JAC9104:428
5 (+982)], 4-azido-7-chloroquinoline, and 2-azidofluorene. Other useful photoreactive intercalators include furocoumarins that form [2+2] cycloadducts with pyrimidine residues. Alkylating agents such as bis-chloroethylamines and epoxides or aziridines such as aflatoxins, polycyclic hydrocarbon epoxides, midomycin, and norphyline A can also be used. The intercalator-modified duplex is then denatured to yield a modified-heavyweight probe.

In the following, some particular aspects of the analytical method of the invention can be explained with reference to the drawings and examples.

The method illustrated in FIG. 1 involves a FAD-labeled polynucleotide probe that is either RNA or DNA if the sample sequence of interest is RNA, or RNA if the sample sequence is DNA. anti-hybrid,
Depending on the case, the antibody may be an RNA in RNA or an RNA-D antibody.
One is selected that is specific for NA hyperprints. A binding site for the anti-hybrid is created upon formation of a hybrid between the sequence of interest and the FAD-labeled probe. Binding of the anti-hybrid to the now FAD-labeled hybrid makes it impossible for the FAD-label to recombine with apoglucose oxidase.

In contrast, an unhybridized or free probe contains FAD available for recombination and H1+L ffi ?
+w 4%? FRl-r LL-1h,! +=
h1. % +h,% base:)4- ss -F E
Forms active glucose oxidase which releases hydrogen peroxide which is detected by rb.

In the method shown in FIG. 2, the probe is identical to the method of FIG. 1 except that it is labeled with a fluorogen (F). Anti-hybrid and anti-fluorogenic substances are added to the system. In the hybrid formed, the binding of the anti-hybrid prevents the binding of the anti-fluorogenic substance to the label which retains the ability to fluoresce (hV2) upon illumination (hVl). However, the label on the unhybridized, doped probe is available for binding by anti-fluorogens, resulting in quenching of the fluorescence.

The method illustrated in FIG.
If the sample sequence is DNA, then the fluorogenic substance C is RNA or l±DNA, and if the sample sequence is DNA, it is RNA.
F) - uses a labeled polynucleotide probe. Depending on the case, RNA-RNA or RNA-D
An antibody selective for NA hybrids is labeled with a quenching moiety (Q). When irradiated with light of the first wavelength (hν1), the fluorescence generating substance and quenching substance have an emission energy (hν2).
is brought to the energy transfer hole #:a in the bound hybrid such that it is absorbed by the quencher and not detected. On the other hand, the fluorogen-labeled probe in the total solution remains at an average distance that is not close enough for efficient energy transfer, and therefore, fluorescence emission is observed. The amount of hv2 light detected is inversely proportional to the amount of high print formation that occurs.

The test sample to be analyzed can be any medium of interest, typically a liquid sample of medical, veterinary, environmental, nutritional or industrial importance. In particular, the method of the invention allows human and animal specimens and body fluids to be analyzed;
For example, urine, blood (serum or plasma), emulsion, amniotic fluid, spinal fluid,
Saliva, feces, liver aspirates, throat swabs, genital swabs and exudates, rectal swabs and nasopharyngeal aspirates can be analyzed. If the test sample to be tested, obtained from a patient or other source, primarily contains double-stranded nucleic acids, e.g. contained in cells, the sample may be treated to denature the nucleic acids and optionally , the nucleic acid is first released from the cell. Denaturation of the nucleic acids is preferably accomplished by heating in boiling water or by alkali treatment (eg, 0, IN sodium hydroxide), preferably used simultaneously, to lyse the cells. Nucleic acid release can also be achieved by, for example, mechanical disruption (freezing/thawing, abrasion, sonication), physical/chemical disruption (Tr
It can be obtained by detergents such as sodium chloride, Tween, F-decyl sulfate, alkaline treatment, osmotic shock or heat), or enzymatic lysis (lysozyme, proteinase K, pepsin).

The test medium obtained contains the nucleic acids in single-stranded form, which can then be analyzed by the hybridization method of the invention. In cases where RNA-DNA hybrids are to be detected with labeled antibody reagents, the mRNA and rRNA in the sample can be isolated by conventional treatment, such as treatment with alkaline conditions, e.g., the same conditions used to denature the nucleic acids in the sample. Depending on the method, it can be avoided to participate in the binding reaction.

As is known, various hybridization conditions can be used in the analysis. Typically, high print formation is performed under moderately high temperature conditions, e.g., about 35-75°C, usually 656°C.
approximately p) 16~ with appropriate ionic strength in the vicinity
Proceed in a solution consisting of a buffer of 1.8% (eg 5XSSC, pH 7, o where 1X5SC = 0.15M sodium chloride and 0.015M sodium citrate). Hydrogen bonding reagents such as dimethyl sulfoxide and formamide can be included if lower hybridization temperatures are desired. The degree of complementarity between sample and probe silver amounts required for hybridization to occur varies depending on the severity of the conditions. The factors that determine severity are well known.

Typically, the temperature conditions selected for hybridization are incompatible with binding of the anti-hybrid reagent to the formed hybrid and detection of the label response. Therefore, the anti-hybrid binding step and the label detection step are performed after the completion of the hyperprint formation step. The reaction solution is typically placed at a temperature in the range of about 40°C to about 40°C before the binding and detection steps are performed. If the salt and/or formamide concentration is high enough to significantly interfere with the antibody binding reaction, it may be desirable to dilute the hyprint-forming mixture prior to addition of the antibody reagent.

The invention further provides a reagent system, i.e., a reagent combination or means comprising all the essential elements needed to carry out a desired analytical method. The reagent system may be used in commercially packaged form as a composition or mixture when compatibility of the reagents permits, as a test device, or more commonly as a test kit, i.e., for holding one or more of the necessary reagents. It consists of a packaged combination of containers, equipment, etc., and usually includes written instructions for carrying out the analytical method. The reagent system of the present invention encompasses all devices and compositions for carrying out the various hybridization specifications described in the present invention.

In all cases, the reagent system will comprise (1) a labeled nucleic acid probe as described in the present invention, and (2) an antibody reagent. Test kit forms of this system can additionally contain the components of the hybridization solution and auxiliary chemicals such as denaturants capable of converting double-stranded nucleic acids in the test sample to single-stranded form. .

Preferably, chemical lysing and denaturing agents, such as alkalis, are included to process the sample and release the heavy chain nucleic acids therefrom.

Hereinafter, the present invention will be explained according to examples, but these are not intended to limit the present invention.

151' Detection of liposomal RNA from bacteria using FAD-labeled DNA probes: A, Flavin N6-(fl-aminohexyl) adenine dinucleotide [(aminohexyl)FAD
] using the method of Morris et al. [Morris et al.
nal, Chew.

(1981) 53:85B). Apoglucose oxidase was prepared by the method described by Morris et al.
3) Meth, 1nEnzy+mo1.92:41
3]. Antibodies against RNA-DNA hybrids were prepared according to the method described by Stuart et al.
rt et al. (1981) Proc.

Nat'l, Acad, Sci, 78:3751
]. E, 18s liposomal RNA from c'oli
A 565 base pair fragment of the sequence was inserted into the old n of pBR322 vector.
dII [cloned between the sites [Brosius e
tal (1878) Proc, Nat'1.
Acad.

Sci, 75:4801], 5-(3-amino)
Allyldeoxyuridine triphosphate was synthesized by the method of Langer et al.
(1981) Proc.

Nat'l Acad Sci, 78:8633].

B, Preparation of labeled DNA probe. 1[1s liposome R
The pBR322 plasmid containing the 565 base pair fragment of NA was transformed into E. coli strain HBIOI Rec,'
The 18s' RNA A fragment was grown in old ndm
It was excised by digestion using restriction enzymes. In order to track the introduction of this fragment by the method of Langer et al.
Nick translation was performed with 5-(3-amine)allyldeoxyuridine triphosphate using dATP. This procedure yields double-stranded DNA with primary amide 7 groups distributed along its length.

Complementary DNA strands are Boehringer Mannhe
E, c, commercially available from ia+ Bio-chemicals (Indianapolis, Indiana).
It was isolated by hybridization with 18sRNA from Oli. This nick-translated probe was prepared by Kasey and Davidson [Ca5ey
andDavidson (+977) Nucl,
Aclds Rec, 4:1539] with excess 1flsRNA. The hybridization mixture was fractionated by mean gradient centrifugation in a 5W390-tar (Beckman Instruments) at 33.00 Orpm for 48 hours at 23°C. Cs2SO4 solution at first 1
．． Adjust the density to 50 g/cm3 and remove the DNA at the end of the experiment.
performed band formation at a density of 1.45 g/cm3, and RN
A. DNA was run at 1.52, excess RNA was run at 1.52. [1g
7cm, 3 [Ba5sel, Hagaski
and Spiegelman.

52:? 9B (11af14)].

The RNA/DNA was collected and the RNA strands were hydrolyzed in 0.1 molar (M) sodium hydroxide at room temperature for 6 hours. The hydrolysis mixture was then adjusted to pH 7.0 with dilute acetic acid and the DNA was precipitated with cold 80% ethanol. This DNA was added to 1 liter of triethylammonium bicarbonate buffer (
This solution was made up to 3 mmol (mM) with dimethyladipimidate dihydrochloride and allowed to react at room temperature for 5 minutes. Excess bifunctional ligation reagent was added to fine Sephadex G-50 (Pharmacia) equilibrated with 20mM sodium carbonate buffer pH 9.3 at 5°C.
was removed by gel filtration over Fine Chemicals, Biscadoway, New Chassis). Chromatography was completed within less than 15 minutes, and the eluate containing the DNA was immediately diluted with lmM (aminohexyl)FAD in an equal volume of water! l: United. The reaction was left at room temperature for 2.5 hours, then excess (aminohexyl)FAD was removed in 0.1 M sodium phosphate buffer pH 7.
was removed by gel filtration in a column of medium 5 ephadex G-25 in . The reaction product was separated into two yellow bands, and the first eluted portion was collected and used for hybrid analysis.

C, Hyprint formation analysis for 1fls liposomal RNA detection. Aliquots of E. coli liquid cultures of various sities were weighed into a series of centrifuge tubes.

105-109 cells were obtained per tube. These suspensions were centrifuged at io, ooox g for 10 minutes and the supernatants were discarded. Cells in each tube were suspended in 20 g of 10 mg/Jj egg white lysozyme (Sigma Chemica 1, St. Louis, MO) in 10 mM Tris-HCl buffer pH 8.0, 0.1 M NaCl, and 511 M ethylenediaminetetraacetic acid. It got cloudy. Cell lysates were collected by Maniatis et al.
Molecular Cloning.

A Laboratory Manual, Gold
Spring Harbor (IH2')] was used for extraction with phenol/chloroform. Polynucleotides in the extract were precipitated with ethanol.

The precipitate was mixed with 20 ml of water and 60% formamide and 4 ml of water.
0% 0.18M sodium phosphate buffer pHf1.5.
It was dissolved in 80 μl of a solution made up of 1.44 M Mail and 0.1% (w) sodium dodecyl sulfate was added. 20#L9. labeled DNA probe, 0. IM
5 ng DNA in IJ sodium disodium buffer pH 8
was added. The reaction tube was sealed and incubated at 5°C for 18 hours. Then open the tube and add 500 ml of antibody against RNA/DNA. was added and allowed to react at room temperature for 1 hour.

The following reagents were used for the analysis of glucose oxidase activity: Complex reagent - 9'2mM sodium phosphate P per ml
H7.0, 0.1% baby plum serum albumin, 2mM3.
5-dichloro-2-hydroxybenzenesulfonate,
0.1M glucose, 20mg0mg peroxidase apoglucose oxidase reagent-0] apoglucose oxidase binding site, 25% glycerol, 4.0m
M 4-7 minoantipyrine, and 0.01% (Kaha)
sodium azide.

After incubating the hybridization reaction with antibody 1.
8mM of complex reagent was added to the tube followed by 0.1mM apoglucose oxidase reagent. The mixture was incubated at 25°C for 30 minutes and the absorbance at 510 nm was recorded at the end of this period.

As the amount of bacteria increases, the absorbance decreases due to the increasing amount of liposomal RNA hybridized to the labeled probe.

Example 1 I\brid formation assay tracked by quenching of fluorescence: A. Antibodies against fluorescein produced from fluorescein calf serum albumin conjugate [UII]
man, (197B) U.S. Patent No. 3,998,943
No. Specification].

B,6-carpoxyfluorescein was synthesized by the method of Ullman et al. [Ullman et al. (19
711) J. Biol, Cheap, 251:4
172], (this synthesis yields a mixture of isomers), the hydroxysuccinimide ester was synthesized by kana and kana for the preparation of the corresponding ester of 4′,5°-dimethoxy-6-carpomethylfluorescein. [Kha
nna and Ullman (1980) Anal
, Biochem, 108:1581° C1 Example Engineering, The translated probe containing the 4-(3-amino)allyldeoxyuridine monophosphate residue described in Section B was precipitated with 100 mM sodium phosphate from an ethanol precipitation step. Dissolved in buffer p) 18.0 and made to 2mM with N-hydroxysuccinimide ester of 6-carpoxymethylfluorenscein. After leaving the reaction solution overnight, a medium 5ephade solution equilibrated with 0.1 M sodium phosphate buffer pH 8.0 was added.
Fractionation was performed by gel filtration on x G-25. The first elution peak of the fluorophore with excitation at 490 nm and emission at 520 nm was the fluorescein-labeled probe and was used for hybridization analysis.

D. Example work, the cell lysate described in section 0,
60% formamide and 40% O, 18M sodium phosphate buffer pH 8, 0, 1, 44M NaCl and 0.
A solution of 1% sodium dodecyl sulfate was combined with 80 wells. To each extract tube, 20 pl of fluorescein-labeled probe (5 ng pNA) in 0.1 M sodium phosphate buffer pH 8.0 was added. The tubes were capped and incubated at 55°C for 18 hours. Next, add RNA-DNA/Iburi 1. to the container. Add 500 ml of antibody against the virus and incubate for at least 1 hour at room temperature.
I left it for a while. The concentration of antibody was determined in a preliminary experiment, and total RNAφ
A large excess was given over the amount expected to be needed to bind the DNA hybrid.

Finally, 400 μL of antibody against fluorescein was added and after 10 minutes fluorescence was recorded at 495 nm for excitation and 519 nm for emission. As the number of bacteria increases, the amount of ribosomal RNA increases,
Fluorescence quenching by antibodies against sulorescein is reduced. Therefore, fluorescence increases with increasing number of bacteria.

Hybridization analysis of cytomegalovirus using fluorescence energy transfer: A. Preparation of fluorescein-labeled probe for cytomegalovirus Cloning of cytomegalovirus DNA The EcoRI restriction fragment was prepared by Tamashiro et al.
.

(+982) Virology 42:547]. A 1500 base pair fragment called EcoRI e in the Tamashiro literature was used for probe preparation. This fragment was removed from the plasmid using the EcoRI restriction enzyme and cloned into the corresponding sites of the A13 mp8 vector (New England Biolabs, He/Heley, MA). This virus is E.coli K12JM101
The heavy virion DNA was isolated.

20mM Tris- containing 10mM MgCl2
Viral DNA in hydrochloride buffer, p) 18.0
Annealed for 45 min at
Base primer GTAAAAGGACGGCCAGT (
New England Biolabs) [B
Ankier and Barrell, (1982
) Techniquesin Nucleic Ac
1d Biochemistry, Elsev'ie
r.

Ireland], this primer is EcoRI
e A13 mp8 near the 3°-OH end of the insert
Complementary to a region of DNA. Then, the reaction solution was
ATP, dcTP, dGTP and 5-(3-amino)allyl-dUTP
er etc.).

Klenow fragment of DNA polymerase was added.

Reactions were incubated at 25° C. for the time specified in preliminary experiments. For these experiments, samples were removed from the reaction mixture at any time and electrophoresed in a denaturing alkaline agarose gel (Maniatis et al., supra). Reaction times were optimized to yield new synthetic fragments extended by at least the EcoRI e insert and, for the present application, A13 m beyond the insert.
Some extensions to the p8 sequence are also acceptable.

The ethidium residue was then coupled to the DNA probe as an intercalation complex. 8-Acid ethidium was prepared by Graves et al.
t, Biochem, Biophys, Acta
, 47! ]:97(+977)]. The above prepared DNA probe was fused with phenol/
Extracted with chloroform and precipitated with ethanol. Add it to 50mM Tris-HCl buffer p) 18.0,0
．． Dissolved in 2M Na11 and made to 0.5mM with ethidium 8-azido.

This mixture was prepared in a glass reaction vessel and 20-30°
The sample was immersed in a glass water bath maintained at C. The photolysis reaction was carried out for 1 hour at a distance of 10-20 cm from a 150 Watt spotlight.

Non-covalently bound ethidium azide I. and photolysis by-products were removed from the reaction by 10 sequential extractions with water-saturated n-butanol. Residual butanol was removed by precipitating the DNA with ethanol, and D
N and A were dissolved in Tris-H test solution. For photolysis of covalently bound ethidium residue E 490 = 4 x 103M −' c
For the extinction coefficient of m-' and photodecomposed ethidium bound to DNA, the relationship between A280 and A490 [A280= (
A490X 3.4) -0,011] and E2[(021-3X 104M-
Measured spectrophotometrically using 'cm-'.

8-Azidoethidium binds covalently to DNA primarily in double-stranded regions where interaction complexes can occur. The aim is to introduce one ethidium residue every 20 to 50 base pairs.

The amount introduced can be decreased by decreasing the photolysis time or decreasing the concentration of 8-azidoethidium. An increase in the amount introduced can be achieved by repeating photolysis using new 8-azidoethidium.

5-(3-amino)allyl-dUM in DNA probe
The primary amine 7 group of the P residue reacts with the tohydroxysuccinimide ester of 6-carpoxyfluorenscein.

This reaction is described in Example! This is done in the same manner as described in Sections B and 0 of I.

Finally, the fluorexein-labeled/ethidium-modified probe was electrophoresed in an alkaline acarose gel.
Separate M13 from the mp8 template (aniat
is etc.). Since this probe is shorter than the vector DNA, it migrates faster and can therefore be recovered from the excised 7 callose slabs by electroelution.

B. Preparation of monoclonal antibodies against ethidium-denatured DNA: 1. Preparative 250 covalent ethidium-DNA complexes
mg of salmon sperm DNA (Sigma Chem
ica1, St. Louis, Missouri) at 40 mJ.
1 in 50mM NaCl2 and a 23 gauge button.
By passing it through, shear deformation is caused. This shear-deformed DNA was put into a 25 (NaM) flask, and then 180
M! ; Diluted with L buffer. 145 le sentence 81-
Nuclease, 200,000 units per mfL (Ph
armacia P-LBiochemicals, Inc.
Bispower, New Jersey) was added and the mixture was incubated at 37°C for 50 minutes.

The reaction solution was then extracted twice with phenol:chloroform and once with chloroform, and the DNA was precipitated twice with ethanol [Maniatis at (1982)
Mo1ecular Cloning'', Co1d
Spring Harbor Laboratory,
Gold Spring Harbor, NYI,
The final precipitate was dissolved in 70II ml of 20mM Tris-HCl buffer pH 8.0.

This DNA was reacted with 8-azidoethidium under the following conditions. The reaction solution was 2.7mgDN of 33Pengbun.
A /mJlj, 4.95n+M of 13.5mJ1
8-Azidoethidium, 13.6 + 0.2 of nJl
M Tris-hydrochloride buffer p) 18.0.0.2M
It was prepared using NaCl and 78m water. The mixture was heated to 250I with a water bath maintained at 22°C.
119. I put it in a beaker. Stir the mixture, +50
60 from a distance of 10 cm by the spotlight of the wand.
Irradiated for minutes. This photolysis was repeated using the same reaction solution.

Combine the photolyzed reaction solutions and add an equal volume of 20mM each time.
Tris-11X liquor salt pH8,0,0,2A Na1ll
: Extracted 10 times with n-butanol saturated with l.

The extracted DNA solution was diluted with 23 mM of 4.85 mM 8-azidoethidium and 20 mM Tris-77II.
Combined with hydrochloride buffer pH 8,0, 0,2M NaCl. This solution was photolyzed for 60 minutes in the same manner as described above. The reaction product was diluted with the same buffer saturated butanol as above.
After 0 extractions, the DNA was precipitated with ethanol. This precipitate was dissolved in 10mM Trrs-hydrochloride buffer pua,o,
Dissolved in 11M EDTA, 260 and 590nm
The absorbance was recorded.

2. Preparation of methylated thyroglobulin Bovine thyroglobulin (Sigma Chemica1
(Company) was combined with 10ml of anhydrous methanol and 2.55M MCI 400 in methanol. This mixture was stirred on a rotary mixer at room temperature for 5 days. The precipitate W was collected by centrifugation and washed twice with methanol and twice with ethanol. It was then dried under vacuum overnight. Approximately 82 mg of dry powder was obtained.

3゜Preparation of Ethidium-DNA/Methylated Thyroglobulin Complex Methylated thyroglobulin (5.5 mg) was dissolved in 1.0 mM water and 1.1 mfL of 2.2 mg/mJ1 x
Thidium-DNA solution was added. A precipitate formed immediately and the suspension was diluted to 3 oIII with 0.15M Mail.

This suspension was emulsified with an equal volume of Freund's adjuvant.

4. Immunization of mice and monoclonal antibody (7) Preparation BALB/c mice were each immunized with 0.5 mfL of emulsified immunogen. For them, 2N! Booster injections were given at intervals, and test blood was collected 5 to 7 days after the booster injection.

Antibody titers were analyzed by standard enzyme-labeled immunosorbent procedures. -Heavy-stranded DNA, double-stranded DNA, or covalently bound ethidium-DNA intercalation by placing Immulonll (Dynateck, Alexantria, VA) microtiter wells with 50 μl of 5p-g1m solution in each well. coated with a cation complex. These polynucleotides are 0.1
5M sodium citrate buffer pH 6,8, 0,15M
It was dissolved in Na11. After these solutions were left in the wells for 2 hours at room temperature, the wells were treated with 5 mg calf serum albumin/ml and 0.5% ty) Tw.
een 2 (0,02 containing l detergent (v/V)
Washing was carried out with M sodium phosphate buffer pH 7.4. Antiserum at an appropriate dilution was added to the wells to allow binding of the antibody to the immobilized polynucleotide. The peach antiserum was washed away, and bound antibodies were detected with enzyme-labeled anti-mouse IgG using well-known procedures.

Mice with high titers for covalently bound ethidium-DNA complexes and extremely low titers for double-stranded and -heavy-stranded DNA were selected and further treated with immobilized non-covalently bound ethidium-DNA complexes. Screening was conducted. This consisted of coating the wells with double-stranded DNA and including 0.1 mM ethidium bromide in all reagents and washing solutions except for the antibody binding solution and the reagent for measuring enzyme activity. there were. Spleen cells from mice with high titers for non-covalent ethidium-DNA complexes were fused with myeloma cells to generate hybridomas [Po1rer et al.

Proc, Nat'l Acad, Sci, 7
9:E1443 (19B2); Ga1fre and
MiLstein, Meth, in Enzym
ol, 73:1 (19B+)].

The cloned hybridoma was grown intraperitoneally in mice and produced large amounts of antibodies.

Albumin was extracted from ascites in 10 mM Tris-HCl buffer pH 8.0, O, 15 M NaCl equilibrated with A
ffigel-blue resin (Bio-Rad Labo
Ratories, Inc., Lynchmond, Calif.) was removed by chromatography. The antibody was passed directly through the column using a linear gradient ramping from lomM Tris-HCl buffer, pH 8.0, to this buffer containing 0.2M NaCl.
D E A, E-” Sepharose (Par
macia Fine Che+oicals, New Jersey.

Chromatographed on a bistorage column.

The main peak of eluted protein contains antibody substantially free of other proteins.

C. Labeling of monoclonal antibodies against ethidium-DNA with 4°,5-dimethoxy-6-carpoxyfluorenscein: 4-5′-dimethoxy-6-carpoxyfluorescein (4°,5°-dimethoxy- (Synthesized as a mixture with 5-carpoxyfluorenscein isomers) was converted to the N-hydroxysuccinimide ester by the method of Riki-Anna and Ullman described above. This dye was coupled to an antibody against ethidium-DNA intercalation complex by the method described in ref.

D. Cytomegalovirus hybridization analysis: Urine samples were incubated at 300 °C in a 5orvall FLC-3 instrument.
Cells and particulate matter were removed by centrifugation in Orpm for 5 minutes. The supernatant was run in a polyaromer-ultracentrifuge tube for 75 minutes at 25.00 Orpm in a Beckman Ti 50 rotor. The resulting pellet was dissolved in 0.1M NaOH and incubated at 37°C for 30 minutes.

100ml of 0.2M sodium phosphate buffer containing 1.8M NaCl, 0.1% dodecyl sulfate and 1mM EDTA Iflt, 0
was added. 20p-1 fluorescein-labeled/ethidium-denatured probe (50 ng) was then added and the mixture was incubated at 65°C for 10 hours. The reaction solution was cooled to room temperature, and 850ILfL containing a labeled antibody against ethidium-DNA intercalation complex was added.
0.1 M Tris-HCl buffer pH 8.2 was added. The concentration of labeled antibody in this reagent was optimized in preliminary experiments to give maximum fluorescence quenching relative to background. The reaction solution was left at room temperature for 1 hour.

The fluorescence intensity of the reaction solution was then recorded with 495n+i light for excitation and 519nm light for emission. Urine without cytomecalovirus infection was also run in parallel, and the fluorescence obtained with this control was higher than that of the samples with virus. The fluorescence signal increases with decreasing proportion of virus.

The present invention has been described and illustrated in detail above. Obviously, many changes and modifications can be made without departing from the spirit and scope of the invention.

[Brief explanation of the drawing]

1-3 are schematic illustrations of a preferred method of carrying out the invention. Engraving of the drawings (no changes to the contents) lG1 2, FAD-1m, O.°” Bakreo 111-ra-
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, company property 1jFIG, 3 3, nsha 尤＠＠ -#--＾ζa い+ Procedural amendment (method) April 1985, Commissioner of the Japan Patent Office Manabu Shiga 1, Indication of the case 1988 patent Application No. 260100 2, Title of the invention 3, Relationship with the amended case Patent applicant name Miles Laboratories 9 Incorporated 4, Agent

Claims (1)

[Claims] 1. - A method for detecting a specific polynucleotide sequence in a test sample comprising a heavy chain nucleic acid, the method comprising: (a) detecting any specific polynucleotide to be detected in the sample; A hybrid is formed between the sequence and a labeled nucleic acid probe consisting of a label and at least one single-stranded base sequence substantially complementary to the detected sequence, and the hybrid is substantially complementary to the single-stranded nucleic acid. (b) contacting any hybrids formed with an antibody reagent, the label in the labeled probe being present in the hybrid to which the labeled probe is bound by the antibody reagent; (C) providing a detectable response that is measurably different than when it is not contained in such a hybrid; A method characterized by: measuring as a function of . 2. Whether the antibody reagent (1) selectively binds to a DNA-RNA hyperprint in which one of the probe and the detected sequence is DNA and the other is RNA, or (11) the probe and the detected sequence are (iii+) consisting of a nucleic acid intercalator to which the duplex formed during the analysis is bound in the form of an intercalation complex. 2. The method of claim 1, wherein the label selectively binds to the intercalation complex. 3. The label interacts with a reagent member of the label detection system to provide a detectable response; If the detectable response is due to steric hindrance of the access of such detection system members to the label,
If the labeled probe is included in a hybrid bound by an antibody reagent, it is measurably different than if it were not included in such a hybrid. The method described in Section 1. 4. The label according to claim 3, wherein the label is a substrate, cofactor or inhibitor of an enzyme that is a member of the label detection system, and the label interacts with the detection system to give a detectable response. Method. 5. The method according to claim 4, wherein the label is a substrate that generates a colorimetric, fluorescent, or chemiluminescent signal under the action of an enzyme. 6. The label is a prosthetic group of the enzyme, the apoenzyme of such an enzyme is a member of the label detection system, and the detection system and the label interact to produce a catalytically active holoenzyme. The method according to claim 4. 7. The method according to claim 6, wherein the prosthetic group is FAD and the apoenzyme is apo(glucose oxidase). 8. The label is a ligand capable of specifically binding to a binding substance that is a member of a label detection system, and the label interacts with the detection system to give a detectable response. The method described in Scope No. 3. 9. The method according to claim 8, wherein the label is a hapten and the binding substance is a second antibody reagent. 10. The method of claim 9, wherein the label is a fluorogenic substance and binding of the second antibody reagent to it results in quenching of the fluorescence. 11. Claim 9, wherein the detection system further comprises a complex of a hapten label or a binding analog thereof with an enzyme, and the enzyme activity of the complex is changed by binding to that of a second antibody reagent. Method described. 12. The probe is labeled with one of the first label and the second label, and the antibody reagent is labeled with the other, and the interaction between the first and second labels causes the labeled probe and the labeled antibody reagent to 2. The method of claim 1, wherein the method of claim 1 provides a measurably different detectable response when both are bonded to the same hyperprint as compared to when they are not so bonded. 13. The first label participates in a first chemical reaction to produce a diffusion product, and the diffusion product becomes a participant in a second chemical reaction with a second label and is detectable. 13. A method according to claim 12, which produces a product that gives a specific response. 14. The method of claim 13, wherein the first and second labels are catalysts for the first and second chemical reactions, respectively. 15. The method according to claim 14, wherein the antibody reagent is labeled with an enzyme. 16. The method according to claim 15, wherein the probe is labeled with a non-enzymatic catalyst. 1? 13. The method of claim 12, wherein the first and second labels participate in an energy transfer interaction. 18. Claim 1, wherein the first label is a fluorescent substance or chemiluminescent substance, and the second label is a quencher.
The method described in Section 7. 18. If the probe is labeled with a fluorogenic or chemiluminescent substance, energy transfer interactions between the label and the native groups on the antibody reagent can be induced in the hybrid in which the labeled probe is bound to the antibody reagent. 2. The method of claim 1, wherein Al11, if present, provides a detectable response that is quantifiably different than if it were not included in such a high print. 20, the antibody reagent selectively binds to the intercalation complex, and the labeled probe also consists of a nucleic acid intercalator chemically linked to the probe in the single-stranded complementary region of the probe, thus The intercalation complex upon hybridization with
A method according to claim 1 formed in the resulting hybrid. 21. The method of claim 1, wherein the label contained in the labeled probe is chemically bonded to the probe in its single-stranded complementary region. 22. The method of claim 1, wherein the label contained in the labeled probe is chemically bonded to the probe in a region other than its complementary-heavy region. 23. The method of claim 1, wherein the test sample comprises a biological sample exposed to conditions that release and denature the nucleic acids contained therein. 24. A reagent system for detecting a specific polynucleotide sequence in a test sample, comprising a labeled nucleic acid probe comprising an m4m marker and at least one single-stranded base substantially complementary to the detected sequence;
and (2) an antibody reagent capable of binding to the hybrid formed between any particular polynucleotide sequence to be detected in the sample and the labeled probe, but substantially incapable of binding to single-stranded nucleic acids. and a measurably different detectable response when the label is included in a hybrid in which the labeled probe is bound to an antibody reagent as compared to when it is not included in such hybrid. A reagent system characterized by giving. 25. Whether the antibody reagent (1) selectively binds to a DNA/RNA hybrid in which one of the probe and the detected sequence is DNA and the other is RNA, or (11) the probe and the detected sequence are A nucleic acid intercalator which selectively binds to an RNA-DNA hybrid, both of which are RNA, or [iii+ a nucleic acid intercalator to which the duplex formed during the analysis is bound in the form of an intercalation complex. 25. The reagent system according to claim 24, which selectively binds to an intercalation complex consisting of: 28, a label interacts with a reagent member of a label detection system to give a detectable response, and the detectable response is caused by a label due to steric hindrance of access of such detection system members to the label. Claim 24 wherein the probe is measurably different when contained in a hybrid bound by an antibody reagent as compared to when it is not contained in such a hybrid. Reagent system as described. 27. The label according to claim 26, wherein the label is a substrate, cofactor or inhibitor of an enzyme that is a member of a label detection system, and the label interacts with the detection system to give a detectable response. 28. The reagent system according to claim 27, wherein the label is a substrate that generates a signal for a colorimetric method, a fluorescence method, or a chemiluminescence method when subjected to the action of an enzyme. 29. The label is a prosthetic group of an enzyme, and the apoenzyme of such an enzyme is a member of a label detection system, and the detection system and the label interact to produce a catalytically active holoenzyme. The reagent system according to claim 27. 30. The reagent system according to claim 29, wherein the prosthetic group is FAD and the apoenzyme is apo(glucose oxidase). 31. A patent claim in which the label is a ligand capable of specifically binding to a binding substance that is a member of a label detection system, and the label interacts with the detection system to give a detectable response. The reagent system according to scope item 26. 32. The reagent system according to claim 31, wherein the label is a hapten and the binding substance is a second antibody reagent. 33. The reagent system of claim 32, wherein the label is a fluorogenic substance and binding of the second antibody reagent to it results in quenching of the fluorescence. 34. The detection system further comprises a complex of a hapten label or a binding analog thereof with an enzyme, and the enzyme activity of the complex is changed by binding of a second antibody reagent thereto. reagent system. 35, the probe is labeled with one of the first label and the second label, and the antibody reagent is labeled with the other, and the phase 1 interaction between the first and second labels is such that the labeled probe and the labeled antibody 25. The reagent system of claim 24, wherein when the reagents are identically hybridized together, they provide a measurably different detectable response as compared to when they are not so bound. 36. The first label participates in a first chemical reaction to produce a diffusion product, which diffusion product becomes a participant in a second chemical reaction with a second label and is detectable. 25. A reagent system according to claim 24, which produces a product that gives a specific response. 37. The reagent system of claim 36, wherein the first and second labels are catalysts for the first and second chemical reactions, respectively. 38. The reagent system according to claim 37, wherein the antibody reagent is labeled with an enzyme. 39. The reagent system according to claim 38, wherein the probe is labeled with a non-enzymatic catalyst. 40. The reagent system of claim 35, wherein the first and second labels participate in energy transfer interactions. 41. Claim 4, wherein the first label is a fluorescent substance or chemiluminescent substance, and the second label is a quencher.
Reagent system described in item 0. 42. The probe is labeled with a fluorogenic or chemical generator, and energy transfer interactions between this label and the native groups on the antibody reagent can occur during hyperprinting, where the labeled probe is attached to the antibody reagent. 25. The reagent system of claim 24, which provides a measurably different detectable response when present in such a hybrid as compared to when not included in such hybrid. 43, the antibody reagent selectively binds to the interforce Resigon complex, and the labeled probe also consists of a nucleic acid intercalator chemically linked to the probe in the single-stranded complementary region of the probe, thus The intercalation complex upon hybridization with
A reagent system according to claim 24 formed in the resulting hybrid. 44. The reagent system of claim 24, wherein the label contained in the labeled probe is chemically bonded to the probe in its single-stranded complementary region. 45. The reagent system of claim 24, wherein the label contained in the labeled probe is chemically bonded to the probe in a region other than its complementary heavy chain region. 46. The reagent system of claim 24, further comprising a denaturing agent capable of converting double-stranded nucleic acids in the test sample to single-stranded form.