NO892043L - PROCEDURE AND PREPARATION FOR DIAGNOSIS. - Google Patents

Description

Description

Homogeneous protection provision

This is a continuation-in-part of Arnold, L.J., Jr. and Nelson, N.C. App. Looking. No. 099,392, filed September 21, 1987.

Background

The present invention relates to diagnostic procedures and techniques for detecting and determining very small amounts of organic compounds.

More particularly, the present invention relates to homogeneous diagnostic assays wherein there is a difference in stability of the marker in its bound forms, as opposed to its unbound forms. The invention relates to the construction of conditions for diagnostic determination systems in which a marker is degraded differently in either its complexed bound form compared to its non-complexed or unbound form.

Background for the invention

Diagnostic determinations are common analytical techniques for the detection, localization or quantification of biological substances using labeled binding reagents. The presence of the labeled reagent can then be detected using a variety of known methods. The present invention can be used in all known diagnostic assay formats, including without limitation, direct binding and competition assays and sequence saturation. A special type of diagnostic test is the nucleic acid hybridization test. The hybridization assay system is based on the fact that single-stranded nucleic acids (DNA or RNA) will hybridize or recombine, under appropriate conditions, with complementary single-stranded nucleic acids. By

labeling the complementary probe nucleic acid with an easily detectable marker, it is possible to detect the presence of the target

the polynucleotide sequence of interest in a test sample containing single-stranded nucleic acid sequences.

Determination systems can be broadly characterized as heterogeneous or homogeneous. The term "heterogeneous" used for determination systems means the specific binding assays that require separation of the labeled substance from the unlabeled substance to be detected. Homogeneous determination systems, on the other hand, do not include any physical separation steps. Because homogeneous assay systems involve a minimal number of processing steps, they are generally more convenient and easier to use than heterogeneous assay systems.

A typical sandwich immunoassay may, for example, comprise incubation of an immobilized antibody with a test medium. Antigens, if present in the medium, will bind to the antibody. After incubation, unbound antigen is removed in a separation step. After a second or simultaneous incubation with a solution of labeled antibody, the bound antigen becomes "sand-wiched" between the immobilized antibody and the labeled antibody. After a second separation step, the amount of labeled antibody can be determined as a measure of the antigen in the medium. This system is time-consuming because it involves a series of incubation and separation steps.

On the basis of the previously known technique in terms of diagnostic determinations, efforts have now been made to develop homogeneous determinations and markers that can discriminate between minor differences in the amount of bound substances as opposed to unbound substances of interest. One of the purposes of the present invention is a determination system in which the marker itself undergoes a detectable change, which may for example be its ability to chemiluminesce, when it is in bound form as opposed to unbound form. Another object of the invention is a determination system in which the marker is degraded or destroyed to a significant extent in either its bound or unbound form, thereby immediately providing means for identification and quantification of the reaction of interest. An object of the present invention is the achievement of an improved method for sensitively detecting analytes using a homogeneous determination format. It is also an object of the present invention to provide improved methods for increasing the sensitivity of determinations involving separation, by combining the homogeneous method set forth herein with other separation methods to reduce non-specific background. The principle of the present invention is based on the different stability of a marker towards chemical or biochemical reagents. When special markers are conjugated to binding partners, it has been found that the stability of said markers changes or can change when said binding partner is bound to a binding substance of said binding partner. The present invention also includes a method by which said different marker stabilities can be used for a sensitive detection of a hearing aid using a homogeneous diagnostic determination system. A further object of the invention is the use of chemiluminescence-acridinium ester-labeled DNA probes in said homogeneous diagnostic assays for sensitive detection of the presence of complementary target polynucleotide sequences.

Description of known technique

There are a number of homogeneous provisions in the prior art that vary in complexity. In some systems, for example, the marker is a catalyst that can participate in a chemical reaction in the presence of other components, for example enzymes, coenzymes and cofactors. In other, albeit related, systems, the marker is a substrate for a chemical or biochemical reaction. In these systems, the formation of a specific easily detectable substance, eg glucose, lactate or alcohol, is used to examine and measure the target reaction. In other assay systems, the marker has unique physical properties that allow it to be detected directly. Examples of such types of markers include metals, hemoglobin and chlorophyll.

Still other homogeneous determination systems are based on enzyme-linked specific binding reactions, in which the analyte is a ligand for a specific binding reaction. When the analyte is present, it undergoes a specific binding reaction with a conjugate comprising a specific binding partner and a label substance. Either simultaneously or subsequently, other substituents are added that act in concert with the marker. The activity of the marker is different when the conjugate, of which the marker is a component, is in a complexed form as opposed to an uncomplexed form. Such systems have typically used enzymes as labeling reagents and substrates that form a colorimetric, fluorimetric or chemiluminescent endpoint. Examples of homogeneous enzyme immunoassays include US patent nos. 3,654,090, 3,817,837 and 4,190,496. Other examples of homogeneous determinations involving the use of chromophores that constitute fluorescent/quenching pairs are found in US Patent Nos. 4,199,559, 4,174,384 and 4,318,707. In some systems, however, the marker has been a substance that is not an enzyme, eg vitamins, NAD, FAD or biotin, which can nevertheless be linked to a "monitoring" reaction. An example of this type is US patent no. 4,383,031. In these systems, the surveillance reaction is based on a structural change that modifies the marker's activity.

Other homogeneous determination systems involve the technique of polarization fluorescence. Here, the analyte competes with a low molecular weight fluorescent conjugate for binding to a specific high molecular weight binding partner. As the polarization of the fluorescence changes when small molecules are displaced from the surface of the large molecule, it is possible to determine the amount of analyte in solution. An example of this type of determination system is US patent no. 4,668,640.

A further type of homogeneous determination system involves non-radiative energy transfer. In these systems, the light absorption from one molecule to another, when the two molecules come in close proximity to each other, is used as a monitoring reaction. These methods are generally described in two ways. In one method, the first molecule is chemiluminescent and upon excitation a part of the generated electromagnetic energy is transferred to another "absorbing" molecule which must be in close proximity. If the energy is absorbed by the other molecule and emitted at a different wavelength, part of the light will show a spectral shift that is proportional to the number of chemiluminescent molecules in the immediate vicinity of the absorption molecules.

In other types of non-radiative energy determination systems, the first molecule is fluorescent and serves as an "absorber" of external light. In the presence of another fluorescent molecule, part of the energy is transferred and light is emitted at a different wavelength. The emitted light shows a spectral shift that is proportional to the number of "absorbing" and "emitting" molecules in close proximity to each other.

Another type of dual probe determination systems is seen in US patent no. 4,670,379. The first probe is labeled with a catalyst; the other with an apoluminescent. Both probe "target" neighboring regions of the nucleic acid sequence. When both probes have hybridized together with the target, add the substrate. The catalyst converts the substrate into a transformation radical which in turn converts the apoluminescent on the other probe into a luminescent. This only happens if hybridization has taken place. By applying these principles, determinations have been developed based on two labeled substances that simultaneously bind a common analyte.

A specific example of this type of energy transfer determination system is Elazar et al., EP patent application no. 85.105.130.0, publication number 0.159.719, published October 30, 1985, which includes the use of two single-stranded nucleic acid probes that are complementary to the same or against -set chains of the target genetic materials. Each probe is labeled with a residue that is only able to give a signal when the two markers are brought together. In contrast to the present invention, Elazar et al. formation and detection of double hybrids or multihybrids. Likewise, Heller et al., EPO patent application no. 82.303.699.1, publication number 0.070.685, dated January 26, 1983 and related Morrison et al., EPO patent application no. 82.303.700.7, publication number 0.070.686 of the same date, include homogeneous determination systems by application of two luminescent probes. At least one of the light markers is of the absorption/emission type so that when the two probes are hybridized to the target, the energy transfer takes place between the two markers. This causes the absorption/emission substance to re-emit at a different wavelength. The second emission shows hybridization. An antigen assay of this type is given in Morrison et al supra.

Most biological substances cannot be easily detected directly at a reasonable level of sensitivity and require a binding reaction of some kind. As can be seen from what has been mentioned above, the previously known technique is based on the detection of minor impairments between bound and unbound markers. The systems according to the prior art are not capable of significant discrimination between bound and unbound marker. Such provisions have been found to be applicable for the detection of analytes which are only present in high concentrations, for example in the monitoring of various drugs in blood and urine.

In clinical diagnostics, there is a need for a homogeneous assay for direct detection based on the ability of two binding partners to modify the stability of the marker, e.g. resulting in selective removal or destruction of the marker in either the bound or the unbound shape. Hirschfield, in Fluorescence Background Discrimination by Prebleaching, J. Histochemistry and Cytochemistry, 27/1, 96 - 101 (1979) describes a somewhat related electrophoresis technique involving photochemical decolorization which can destroy marker molecules, or at least their fluorescence. The invention disclosed herein does not teach or suggest the use of photochemical decolorization or any other technique for the selective removal or destruction of a marker in a diagnostic assay. The present invention fulfills a current need in diagnostic determinations as it is much more sensitive than prior art. The sensitivity range for the previously known technique is no better than in the range of 10<-1>^ mol, while the present invention is sensitive in the range of 10 mol.

Summary of the invention

The present invention relates to diagnostic determinations and methods. The method can be used to detect an analyte in a medium, as said analyte is part of a specific binding pair. When the medium believed to contain the analyte is combined with a binding partner, a marker substance attached to binding partners is capable of undergoing a detectable change in stability or differential degradation each time the analyte binds to the specific binding partner. In a specific embodiment, the single-stranded nucleic acid probe is modified to contain labels at virtually any desired position or location on the probe. The probe markers have different stability or are susceptible to differential degradation depending on whether the target nucleic acid sequence is hybridized to the probe.

The present invention particularly includes determination systems where the marker on the bound probe is stabilized in relation to the unbound probe. This stabilization can be remedied by intercalation. DNA intercalating compounds, including acridinium esters, are particularly, but not exclusively, suitable for use in the determination system according to the invention. DNA intercalators are known to bind non-covalently to duplex DNA and are characteristic flat molecules that can be inserted between the base pairs of the DNA double helix. There is evidence indicating that acridinium masters prefer to enter regions rich in adenine and thymidine base pairs.

It should be understood that while the present invention is hereinafter described with particular reference to acridium ester labeled probes, the present invention contemplates the use of equivalent labels and assay formats which are amenable to differential degradation or stability change when the conjugate of which the label is a component is bound. As will be explained in more detail herein, other suitable label compounds include substances that are destabilized by the amines present on the nucleic acids, particularly those near the label on unhybridized probes. In addition, marker substances that can act together with hydrophobic environments, e.g. by intercalation between base pairs, can also be used.

The present invention is generally applicable in any system involving substantially selective degradation of the marker when comparing its bound and unbound conjugated forms. Furthermore, due to the relative simplicity of the determination system, which does not involve any separation or washing steps, it is both faster and cheaper than conventional systems. The system may be more sensitive than conventional systems in cases where there are large differences in stability between the bound and unbound conjugated forms of the marker because all remaining or unbound marker conjugate is destroyed and therefore not detected. Furthermore, the system is very versatile and can either be used alone or in combination with other separation methods and produces very little background noise. The present invention can be used in probe diagnostics, including the detection of infection and genetic and viral diseases and for the diagnosis of cancer.

Brief description of the drawings

Fig. 1 is a graphical representation of HPLC purification of a

acridinium ester-labeled probe.

Fig. 2 is a graphical presentation of the results which are

given in Example 2.

Fig. 3 - 5 are graphical representations of the results given in

example 3.

Fig. 6 is a graphical representation of the results given in

example 4.

Fig. 7 is a graphical representation of the results given in example 5. Fig. 8 is a graphical representation of the results given in

example 6.

Best type and detailed description of preferred embodiments

The following definitions are used in the present description: 1. Acridinium ester: acridine derivative with a quaternary nitrogen center and which is derivatized in the 9-position to give a labile phenylester residue, especially 4,2-succinimidyloxycarbonylethyl)-phenyl-10-methylacridinium-9- carboxylate fluorosulfonate:

2. Acridinium master: residues of the following general type:

R-L = ALKYL, ALKENYL, ARYL, SUBSTITUTED AL CYL, SUBSTITUTED ALKENYL, SUBSTITUTED ARYL, ALKOKSY,

ARYLOXY OR IS ABSENT WHEN X = HALOGEN.

R2= H, ALKYL, ALKENYL, ARYL, SUBSTITUTED ALKYL,

SUBSTITUTED ALKENYL, SUBSTITUTED ARYL, ALKOXY,

ARYLOXY, IF AND ONLY IF X = N.

R3= H, AMINO, HYDROXY, THIOL, HALOGEN, NITRO, AMINO,

AMIDO, ACETYL, ALKYL, ALKENYL, ARYL, SUBSTITUTED ACETYL, SUBSTITUTED ALKYL, SUBSTITUTED ALKENYL,

SUBSTITUTED ARYL, ALKOXY, ARYLOXY.

R4= ALKYL, ALKENYL, ARYL, SUBSTITUTED ALKYL, SUBSTITUTED ALKENYL, SUBSTITUTED ARYL.

X = 0, N, S, HALOGEN, SUBSTITUTED PHOSPHORUS, SUBSTITUTED

SULFUR, SUBSTITUTED BORON OR SUBSTITUTED ARSENIC.

Y = 0, S OR NH.

Ri AND/OR R2 AND/OR R3 AND/OR R4 HAS A REACTIVE SITE

WHICH ALLOWS CONJUGATION.

3. Analyte - any substance capable of undergoing a binding reaction with one or more specific binding partners, including, without limitation, antigens and antibodies thereto, haptens and antibodies thereto; hormones, drugs, metabolites, vitamins, coenzymes and their binding partners, including receptors; polynucleotides, oligonucleotides and hybrids of polynucleotides or oligonucleotides and antibodies and binding substances thereto; polynucleotides or oligonucleotides and hybridizable polynucleotides or oligonucleotides

thereto; metals and chelating agents thereto.

4. Binding partner - any molecule or substance capable of undergoing a specific binding reaction with an analyte. 5. Duplex: double-stranded complex formed by base part matching of two complementary, single-stranded nucleic acid molecules. 6. Hybridization: the formation of stable duplexes between two complementary single-stranded DNA or RNA molecules or a DNA and complementary RNA molecule. Duplex formation is specific for complementary base pairs, as the genetic code for the hybridized specific gene

thereby reproduced.

7. Bound: condition whereby a binding interaction is formed between a molecule and its specific binding partner. 8. Stable: resistant to chemical or biochemical degradation, reaction, cleavage, displacement or

modification.

9. Stability: resistance of a substance to chemical or biochemical degradation, reaction, cleavage, displacement or modification.

It is known that in solution the acridinium masters are in equilibrium with their corresponding bases. At high pH, base formation is favored; the quaternary nitrogen species are regenerated at low pH. It is also known that the chemiluminescence reaction can be affected by the addition of a base, in particular an aqueous solution of sodium hydroxide containing hydrogen peroxide. Chemiluminescence involves the attack of hydroperoxide ions on the acridinium compounds, which results in the formation of N-methylacridone with excited electrons. See generally Weeks et al., Acridinium Esters as High-Specific Activity Labels in Immunoassay, Clin. Chem. 2918, 1474-1479 (1983). The reaction is shown schematically below.

The present invention can be carried out as follows. First, binding partners comprising a binding substance and one or more binding partners are selected for the determination to be carried out. These pairs can be antigens and antibodies thereto; haptens and antibodies thereto; hormones, drugs, metabolites, vitamins, coenzymes and their binding partners, including receptors; polynucleotides, oligonucleotides, and hybrids of polynucleotides or oligonucleotides and antibodies and binding substances thereto; polynucleotides or oligonucleotides, and hybridizable polynucleotides or oligonucleotides thereto; metals and chelating agents therefore.

The determination type to be used is then selected. These can be selected from types including direct binding assays, competitive assays, sequential saturation methods and "sandwich" assays.

Third, a marker is selected for the determination to be performed. This can be a marker that can be detected directly or indirectly by colorimetric, fluorimetric, chemiluminescent or bioluminescent devices. The marker must further have the property that it can be broken down chemically or biochemically so that its ability to be detected is modified, said breakdown being possible under conditions that do not adversely affect the bond between the labeled binding partner and its binding substance and other binding partners and binding substances that participate in the reaction . Preferred markers are those which, after being exposed to acids, bases or selective oxidizing agents such as peroxide, or enzymes are affected with regard to their ability to be detected.

Fourthly, chemical methods known in the state of the art are used, the marker being attached to the binding substance in a binding site so that the marker's sensitivity to chemical and biochemical degradation is modified by interaction of the labeled binding partner with its specific binding substance(s). In some cases, several different sites can be tested for marker attachment and the site that gives the best differential degradation can be used.

Fifth, the degradation conditions, whether chemical or biochemical, are optimized to provide the best detection discrimination for the labeled binding partner in the presence and absence of its binding agent.

Finally, for the application of the selected types of determinations, the ability of the determination system to quantitatively or qualitatively detect the analyte is tested, generally applying the steps:

a. Incubation

b. Selective degradation

c. Demonstration or

a. Simultaneous incubation and selective degradation b. Detection.

In the application of the present invention, oligonucleotide probes labeled with chemiluminescent acridinium esters are particularly useful for the detection of sequence-specific polynucleotides through hybridization. Acridinium esters can be attached, through a variety of different binding sites, to DNA probes and/or mixed nucleotide/non-nucleotide polymers as described in US Patent Application Serial No. (not yet assigned), filed September 21, 1987 entitled "Non-Nucleotide Linking Reagents for Nucleotided Probes" by Arnold, et al. This includes the ability to label the nucleotide bases, the phosphate backbone, the 3'-terminus and the 5<1->terminus of oligonucleotides as well as the non-nucleotide monomer units of mixed nucleotide/non-nucleotide polymers.

Such acridinium ester-labeled probes can exhibit significantly different chemical stability when the probes, to which the esters are attached, are free in solution compared to when they are hybridized. This different stability depends on the position of the acridium ester in the probe, the nucleotide residue in the immediate vicinity of the acridium ester marker and the presence of other probe molecules that form stable hybrids with the target polynucleotide. A schematic representation is shown below.

In determining the factors contributing to the differential stability of the acridinium ester on DNA probe molecules, it was found that the free amines on the bases of non-hybridized probes (both nucleotide and mixed nucleotide/non-nucleotide) destabilized the acridinium ester, particularly in an alkaline environment . At the same time, if the acridinium ester is near the thermal end of the probe, it can also be alkali-destabilized with the help of amines coming from target sequences when hybridization occurs. When the probe hybridizes with a sequence-specific polynucleotide (binding substance), the probe amines participate in base pairing with complementary nucleotides and are limited in their ability to destabilize the acridinium ester, particularly in an alkaline environment. At the same time, it has been found that the acridinium ester was further stabilized with respect to degradation by intercalation into the hybrid duplex, particularly in regions of adenine/thymidine base partition. It has been found that a number of other deposit areas also give good differential hydrolysis, as explained in the following section. There are a number of ways in which the present up-

invention can be used in hybridization. These include without limitation: 1. Binding of the acridinium master to the central region of the probe and near a region of adenine/thymidine base pairs. A preferred method is to attach an acridinium ester to a non-nucleotide monomer unit as provided in US patent app.

application, serial number (not yet assigned) submitted September 21, 1987, entitled "Non-nucleotide Linking Reagents for Nucleotide Probes" by Arnold et al, which is bound as an insert to the nucleotide-monomer units complementary to immediately adjacent nucleotides in the target polynucleotide sequence. Such placement serves to confine the amines in the nucleotide bases on both sides of the acridinium ester and provide a site for intercalation.

2. Attaching the acridinium ester to either the 3' or 5' terminus of the probe and limiting the effects of nearby amines coming from the target polynucleotide with another probe that hybridizes adjacent to the first. This would also be desirable if the aforementioned second probe forms an A/T base pair-rich region during duplex formation. Although this double probe or sandwich system relies on the formation of two duplexes instead of just one, it may provide a method for sensitive detection of minor base pair changes that can be discerned by very short probes, i.e. probes with a

length of about 5-15 nucleotides.

3. Binding of the acridinium ester in or near the site of a mismatch with a polynucleotide sequence that is not the desired target polynucleotide. In this way, discrimination between polynucleotide sequences differing by only one nucleotide can be achieved, as the region of the duplex around a mismatch site is sufficiently destabilized to render the acridinium ester (if attached in or near this site) susceptible to degradation.

A preferred method for the above three types is to carry out the differential hydrolysis step at the same temperature as the hybridization step, typically from 50 - 70°C.

Another method is to carry out another differential hydrolysis step at room temperature. This allows the use of pH in the range from 10 - 11, which gives even greater differences in the degree of hydrolysis between hybridized and non-hybridized acridinium ester-labeled probe.

Under normal circumstances, such short probes have the ability to discriminate simple mismatches under appropriate hybridization conditions. However, for practical reasons they are not used because they are not sufficiently specific for single polynucleotide sites. This means that the short probe will hybridize a number of specific sites contained in a large number of present complex DNA or RNA sequences. With the added requirement that two different probes must hybridize immediately to adjacent polynucleotide regions, such regions can be specifically targeted and identified. Thus, the advantages of short-probe discrimination can be applied while maintaining the specificity of the region spanned by both probes.

Experimental methods and materials

The following examples are given to illustrate the present invention and not to limit the invention. The examples are based on currently available data and on the most likely explanations of the phenomenon. Other factors and methods may emerge with further research. Although the invention is described in detail by means of illustrations and examples using acridinium ester markers, certain changes and modifications can be made within the scope of the invention and other marker systems can be used within the scope of the invention.

EXAMPLE 1

Construction of acridinium ester-labeled probes.

A. Synthesis and purification of amine linker-arm probes. Deoxyoligonucleotide probes were synthesized to contain an amine linker arm (ie, one ending with a primary amine for acridinium ester labeling) located either at the 5'-terminal end, at a preselected site along the polyphosphate chain, in the interior of the probe, or bound to one of the nucleotide bases.

(in)

To attach a 5'-amine linker arm to a probe, the following compound was used:

This compound will be referred to below as the terminal amine linker-arm reagent. This reagent was synthesized as follows: 6-aminohexanol was reacted with S-ethyl trifluorothioacetate in anhydrous ethyl acetate. The reaction product was precipitated in petroleum ether, treated with a mixture of 10% pyridine in water for 10 minutes to hydrolyze the O-trifluoroacetyl formed, and evaporated to dryness as a gum. This compound was then phosphitylated according to standard procedures in the literature to give the desired compound, namely terminal amine linker-arm reagent (1).

Probes containing a 5'-amine linker arm were synthesized as follows. Using an Applied Biosystems, Inc. Model 3 80A DNA synthesizer, probes with the desired nucleotide sequence were prepared using standard phosphoramidite chemistry, building the probes from the 3' end to the 5' end. After the desired sequence was complete, the amine linker arm was automatically linked to the 5<1->hydroxyl group in the probe using the terminal amine linker arm reagent in the same way that another phosphoramidite nucleoside would be linked. Using standard procedures, the probe was then cleaved from the solid support, deprotected and purified by polyacrylamide gel electrophoresis followed by Sephadex G-25 chromatography.

The following probe was synthesized and purified using this procedure:

5'~NH2-(CH2)6-GCTCGTTGCGGGACTTAACCCAACAT-3'

wherein NH2-(CH2)g represents the amine linker arm.

(ii)

To incorporate an amine linker arm into the internal portion of a probe, internal amine linker arm reagents, type 1 or type 2, were used as described in US patent application serial number 099,050 entitled "Non-Nucleotide Linking Reagents for Nucleotide Probes " submitted on September 21, 1987, by Arnold et al. Again, probes were synthesized using standard phosphoramidite chemistry and purified using polyacrylamide gel electrophoresis and Sephadex G25 chromatography.

The following probes were synthesized using this procedure: 1. ) A 30mer complementary to the 16S subunit of rRNA for E. coli, with an internal amine linker arm, type 1, replacing an adenine residue at position 18 in the sequence: Replaced by an internal amine linker arm, type 1

5'-CCA CTG CTG CCT CCC GT© GGA GTC TGG GCC-3'.

2. ) A 33mer complementary to the 16S subunit of rRNA from Chlamydia trachomatis, with an internal amine linker arm, type 1, replacing an adenine residue in the 21st position of the sequence,

Replaced with an internal amine linker arm, type 1

5"-CGT TAC TCG GAT GCC CAA AT@TTCG CCA CAT TCG-3' ,

or inserted between the 21 and 22 residues,

An internal amine linker arm, type 1 or type 2, inserted here

5'-CGT TAC TCG GAT GCC CAA ATA^TCG CCA CAT TCG-3'.

(iii)

Nucleotide bases containing the amine linker arm were incorporated into a probe as follows. Template and primer oligonucleotides with the following sequences were synthesized:

(The primer and extended primer sequences are complementary to the 16S subunit of C. trachomatis.)

Primer extension using the Klenow fragment was performed as described in Maniatis (ref.), with the exception that BSA was omitted from the 10X nick translation buffer, to incorporate amine linker arm modified base-amino (12) dUTP (Calbiochem , California).

The sequence of oligomer obtained is therefore:

Purification of the primer-extended complex was achieved using a NENSORB-20 column (DuPont) and according to the procedure provided by the supplier. (The primer extension reaction was diluted by adding 900 µl of NENSORB Reagent A prior to application to the column. The purified oligomers were diluted with 50% methanol which was then removed in a speed-vac.

B. Labeling of amine linker-arm probe with acridinium ester and subsequent purification.

A 25 mM stock solution of acridinium ester was prepared in distilled DMSO. The desired amount of probe (see a list of the various probes labeled in Part A above) was evaporated to dryness in a 1.5 mL conical polypropylene tube. The following liquid mixture was formed by adding the following component parts and in the order indicated:

3 µl H 2 O

1 µl 1 M HEPES (pH 8.0)

4 µl DMSO (distilled)

2 µl 25 mM acridinium ester in DMSO (distilled)

The mixture was vortexed, centrifuged in a microcentrifuge for 2 seconds (to bring contents to the bottom of the tube) and incubated at 37°C for 20 minutes. The following components were then added to the reaction liquid mixture in the order indicated: 3.0 µl 25 mM acridinium ester in DMSO (distilled) 1.5 µl H 2 O

0.5 µl 1 M HEPES (pH 8.0)

The liquid mixture was vortexed again, centrifuged and incubated for an additional 20 minutes at 37°C. Unreacted label was suppressed using a 5-fold excess of lysine by adding 5 µl of 0.125 M lysine in 0.1 M HEPES (pH 8.0), 50% DMSO, and incubating for 5 min at room temperature.

The acridinium ester-labeled oligomer was then purified using the following method. To 20 µl of the suppressed reaction mixture, 30 µl NaOAc (pH 5.0), 245 µl H 2 ° 0<3 5 µl glycogen was added as a carrier (glycogen was pretreated to remove any nuclease activity). The sample was then gently vortexed and 640 µl of absolute EtOH was added. The sample was vortexed gently and incubated on ice for 5-10 minutes, then centrifuged for 5 minutes at 15,000 rpm in a microcentrifuge. The supernatant was carefully removed and the pellet was redissolved in 20 µl of 0.1 M NaOAc (pH 5.0), 0.1% SDS. The samples were then purified by high pressure liquid chromatography (HPLC) as described below.

(in)

The AE-labeled probes containing 5' and internal amine linker arms were purified as follows: 20 µl of redissolved pellet was injected onto a Nucleogen-DEAE 60-7 ion exchange HPLC column mounted in an IBM® 9533 HPLC system. All buffers were prepared with HPLC grade water, acetonitrile (CH3CN) and sodium acetate (NaOAc) from Fisher Scientific, and glacial acetic acid (HoAc) and reagent grade LiCl. All buffers were filtered through nylon-66 filters with a pore size of 0.45 µm before use. The sample was eluted as described in fig. 1 in the drawing (in this case the probe was the 5'-amine linker arm containing the 26mer described in Part A above). Immediately after passage, 5 µl of 10% SDS was added to each tube followed by vortex mixing of each tube (this was done to ensure that the acridinium ester labeled probe did not stick to the tube wall). A 0.5 µl sample was removed from fractions 21 - 42 and added to 200 µl of water in a 12 x 75 mm tube (a separate pipette tip was used for each sample to avoid the carryover problem). The chemiluminescence of each sample was then determined in a Berthold Clinilumat using the following automatic injection and reading sequence: injection of 200 µl 0.25 N HN0 3 , 0.1% H 2 O 2 , a 1 second hold, injection of 200 µl 1 N NaOH; the chemiluminescent amount was read for 10 seconds.

Fractions 29 - 33 were then precipitated with EtOH as follows: to each fraction add 5 µl glycogen, vortex, add 1 ml EtOH, vortex, incubate 5-10 minutes on ice and centrifuge 5 minutes at 15,000 rpm in a microcentrifuge. Each supernatant was carefully removed, the pellet was redissolved in 20 µl of 0.1 M NaOAc, pH 5, 0.1% SDS and the fractions were pooled.

In this way, very pure acridinium ester-labeled probes were obtained. The specific activity for such probes was typically 5-10 • 10^ chemiluminescent light pulse number (Berthold Clinilumat) per picomole oligomer.

(ii)

The AE-labeled probe containing the amine linker arm-modified base (amino-12-U) was generally purified as described above, with the following exceptions: a Vydac C4 reverse-phase column was used; buffer A was 0.1 M triethylammonium acetate (Applied Biosystems, Inc., Foster City, Calif.) and buffer B was CH 3 CN; the labeled probe was eluted as a hybrid using a linear gradient of 10-15% solvent B for 25 minutes at a flow rate of 1 ml/min. The largest chemiluminescent peak was then identified and

induced as described above.

The foregoing discussion generally describes how to prepare and label probes with acridinium ester. In the specific example herein, probes were labeled on the end and inside the probes, as well as labeled in a nucleotide base.

EXAMPLE 2

Stability at pH 6 of hybridized probe compared to non-hybridized probe that was internally labeled with acridinium ester

An internally labeled 33mer probe specific for Chlamydia trachomatis was prepared as described above (adenine replacement, type 1 linker arm). The probe was hybridized with its target rRNA (in this case Chlamydia trachomatis) according to the following procedure:

The hybridization mixture

2 µl AE probe (0.5 pmol)

0.3 µl 4% (w/v) SDS

5.8 µl 1 M PB, pH 5

4.4 µl C. trachomatis rRNA (2 pg), or 4.4 µl H2O for control

The hybridization and control mixtures were incubated for 40 minutes at 60°C, after which each mixture was analyzed for percent hybridization using hydroxyapatite (HAP) as follows. A 0.1 µl sample of the hybrid or control mixture was added to 200 µl of 0.14 M PB, pH 6.8, containing 2% HAP. Each mixture obtained was vortexed for five seconds, incubated for five minutes at 60°C, vortexed for

20 seconds, and then centrifuged for 30 seconds in a microcentrifuge at 15,000 rpm. The supernatant was removed and saved, 200 µl of 0.14 M PB, pH 6.8, was added to the HAP pellet, the mixture was vortexed for ten seconds and then centrifuged for 30 seconds in a microcentrifuge at 15,000 rpm. The supernatant was removed and saved and the pellet was resuspended in 200 µl of 0.14 M PB, pH 6.8. A 50 µl sample of the resuspended HAP and each of the two supernatants was analyzed for chemiluminescence as described above, and percent hybridized probe, i.e., percent chemiluminescent signals associated with HAP, was calculated.

The stability of the hybridized probe compared to the non-hybridized probe (ie, control) was tested at pH 6 according to the following procedure:

Stability test mixture

12.3 pl hybrid or control from above

50 µl 1 M PB, pH 6

2.5 µl 4% SDS

65 pl H20

These mixtures were gently vortexed, and 5 µl of sample was removed immediately (thawed) and analyzed for chemiluminescence as described below. The remainder of the mixture was incubated at 60°C, and 5 µl of sample was removed at various time points (see below) and analyzed immediately for chemiluminescence.

The chemiluminescence of each sample was measured by adding a sample amount to 200 pl H2O in a 12 • 25 mm tube and chemiluminescence is measured in a clinilumat (automatic injection of 200 pl 0.25 HNO3, 0.1% H2O2 and after a second dwell, autoinjection of 200 µl 2 M potassium PB, pH 13.2, after which chemiluminescence was read for ten seconds).

RESULTS

1. Percent Hybridization (HAP Analysis)

Hybrid - 96%

Control - 1.3% (non-specific binding)

2. The course of stability time

See fig. 2 in the drawing.

These results show that the hybridized probe is well protected against degradation and subsequent loss of chemiluminescence (half-life of chemiluminescence loss equal to 3,370 minutes), while unhybridized sample is highly susceptible to degradation and loss of chemiluminescence (half-life equal to 29.2 minutes; discrimination between hybridized and unhybridized probe is therefore equal to 115 - times). These data show the ability of the homogeneous determination described herein to locate target DNA sequences by imparting differential stability between hybridized and unhybridized probe and measuring the same.

EXAMPLE 3

Stability at pH 9. for hybridized probe compared to unhybridized probe labeled internally with acridinium ester. Internally labeled probe (all three internally labeled 3 3mer probes described in Example 1 were tested) were hybridized with its target rRNA (in this case Chlamydia trachomatis) and analyzed for percent hybridization as described in Example 2.

The stability of hybridized probe compared to unhybridized probe (ie control) was tested at pH 9 according to the following procedure:

Stability test mixture

5 yl hybrid or control from above

4 5 yl 0.2 M sodium borate, pH 9

The mixtures were then incubated, and samples were removed and analyzed for chemiluminescence as described in Example 2.

RESULTS

1. Percent Hybridization (HAP Analysis)

a. Adenine replacement, type 1 linker arm

Hybrid - 95%

Control - 0.5% (non-specific binding)

b. Insertion, type 1 linker arm

Hybrid - 98%

Control -0.3%

c. Insertion, type 2 linker arm

Hybrid - 98%

Control -0.2%

2. Course of stability time

See fig. 3 - 5 in the drawing. Fig. 3 is a curve for adenine substitution, type 1 linker arm; fig. 4 is a curve for insertion, type 1 linker arm; fig. 5 is a curve for insertion, type 2 linker arm.

As in Example 2, hybridized probe is protected from degradation while unhybridized probe is not. This is the case for all three types of internally labeled probes. In this example, it has been shown that sodium borate at elevated pH accelerates the process (compared to example 2) while the differential degradation properties are still maintained between hybridized and unhybridized probes.

EXAMPLE 4

Stability at pH 6 for probe end-labeled with acridinium ester as hybrid with nearby probe compared to non-hybrid.

This example involves the use of a probe of sequence 5'-CCG GAC CGC TGG CAA CAA AGG ATA AGG GTT GC-3' (prepared using standard forforamidite chemistry) that hybridizes to E. coli rRNA immediately adjacent to 5<1-> end of the AE-labeled probe used in this example (see below), thus leading to capping off of all the amines in the target rRNA in the vicinity of the acridinium ester marker.

Probe end-labeled with acridinium ester (preparation described in Example 1) (hereafter referred to as AE probe) and the adjacent probe described above were hybridized according to the following procedure:

The hybridization and control mixtures were incubated for 40 minutes at 60°C, and then analyzed for percent hybridization using hydroxyapatite as described in Example 2.

The stability of the neighboring probe hybridized probe compared to unhybridized probe (ie, control) was tested at pH 6 exactly as described in Example 2.

RESULTS:

1. Percent Hybridization (HAP Analysis)

Hybrid - 93.5%

Control - 2.7% (non-specific binding)

2. Course of stability time

See fig. 6 in the drawing.

As in Example 2, hybridized AE probe, in this case with a neighboring probe that is also hybridized, was protected from degradation while unhybridized probe was not. The protection was as good as in example 2, because it depends on two hybridizations instead of one.

EXAMPLE 5

Stability at pH 9 of probe end-labeled with acridinium ester as hybrid with "nearby" probe compared to non-hybrid.

Hybridization was carried out exactly as described in Example 4. Stability of hybridized probe with neighboring probe compared to unhybridized probe (ie, control) was tested at pH 9 exactly as described in Example 2, except for the composition of the stability test mixture, which was as follows:

5 ul hybrid or control

50 µl 0.2 M sodium borate, pH 9.0

Hybridized AE probe with a nearby probe (also hybridized) was again protected from degradation while unhybridized probe was not protected from degradation. As in Example 3, sodium borate at elevated pH accelerated the process while still maintaining the differential degradation properties between hybridized and unhybridized probe. For a graphical presentation of these results, see fig. 7.

EXAMPLE 6

Stability at pH 6 of probe end-labeled with acridinium ester as hybrid compared to non-hybrid.

The probe was hybridized to its target rRNA (in this case E. coli) according to the following procedure:

The hybridization and control mixtures were incubated for 40 minutes at 60°C, and then analyzed for percent hybridization using hydroxyapatite as described in Example 2.

The stability of the hybridized probe compared to unhybridized probe (ie, control) was tested at pH 6, exactly as described in Example 2.

RESULTS:

1. Percent hybridization

Hybrid - 94%

Control - 2.7% (non-specific binding)

2. Course of stability time

See fig. 8 of the drawings.

Unhybridized probe was again preferentially degraded compared to hybridized probe, although the difference in degradation is not as great as in the previous examples. The reason for this is that only part of the amines were "capped off" in the vicinity of the acridinium ester marker. This further demonstrates the ability to discriminate between hybridized end-labeled probe in the presence and absence of hybridized neighboring probe.

EXAMPLE 7

Stability at pH 7.6 of hybridized probe compared to unhybridized probe labeled on a nucleotide base with acridinium ester

The probe labeled on a nucleotide base (amino-12-U) with the acridinium ester described in Example 1 was hybridized with its target rRNA (in this case C. trachomatis) in accordance with the following procedure:

Hybridization mixing

0.1 M lithium succinate, pH 5.4

10% lithium lauryl sulfate

2 µg 1.3 pmol AE probe

Total volume - 30 pl

The hybridization and control mixture was incubated for five minutes at 80°C, and then 60 minutes at 60°C. The resulting solutions were each diluted to 300 µl with 0.1 M lithium succinate, pH 5.4, 10% lithium lauryl sulfate and analyzed for percent hybridization using hydroxyapatite as described in Example 2.

The stability of hybridized probe compared to unhybridized probe (ie, control) was tested at pH 7.6 with a series of identically prepared samples according to the following procedure:

Stability test mixture

15 pl hybrid or control from above

100 µl 0.2% sodium tetraborate, pH 7.6, 5% Triton X-100

These mixtures were then incubated at 60°C, and samples were removed at various time points and chemiluminescence was determined using a gene-probe Leader™ I Luminometer using automatic injection of 200 µl 0.1 M H2O2, then holding in one second, followed by automatic injection of 200 µl 1 N NaOH, and chemiluminescence was read for five seconds. From these data, the degree of hydrolysis was determined using regression analyses.

RESULTS

1. Percent Hybridization (HAP Analysis)

Hybrid - 53.8%

Control - 0.5%

2. Stability

Half-life of ester- Ratio of hydrolysis (min) half-life

As in the previous examples, these data show that hybridized probe is protected from degradation while unhybridized probe is not, in this case with the label bound to a base in the probe. This demonstrates the principle that multiple sites of AE binding are acceptable for use in the homogeneous protection provision described herein.

EXAMPLE 8

Stability at pH 7.6 of hybridized probe compared to unhybridized probe internally labeled with acridinium ester in a range of sequence dimer sites using both rRNA and DNA targets.

Probes containing the internal linker arm, type "X", inserted at a number of sequence dimer sites (see below) were synthesized, labeled with AE, and purified as described in Example 1. The sequence and linker arm locations of these probes is as follows:

These probes were hybridized as described in Example 7, with the exception that the incubation step at 80°C was omitted, and the amounts and types of target nucleic acids used were as follows:

The stability of hybridized probe compared to unhybridized probe was tested at pH 7.6 as described in Example 7.

RESULTS

Stability

Relationship between half-lives

These data show that the linker arm (and therefore AE) can be inserted at a number of sequence dimer sites and still be substantially protected from ester hydrolysis in the hybridized form. Furthermore, this example shows that DNA targets provide good protection of hybridized AE probes when used with these homogeneous determination types. This example also shows that the long (9 atoms) linker arm used herein provides different hydrolysis conditions that are substantially equivalent to the other linker arms set forth earlier in the present patent application, and establishes that a variety of lengths of linker arms can are used in the HPA format described herein.

EXAMPLE 9

Stability at pH 7.6 of internally labeled AE probe hybridized with perfectly matched target compared to target with a single mismatch.

A 24mer probe complementary to the 16S subunit of rRNA from

E. coli, with an internal amine linker arm, type "X", inserted between residues 6 and 7, was synthesized. The probe was then labeled with acridinium ester and purified as described in Example 1. The sequence is as follows (# = linker arm position):

5<*->CAA GCT# TGC CAG TAT CAG ATG CAG-3'

This probe has a single T-G mismatch at position 6 (numbered from the 5' end of the probe strand) with the 16S subunit of C. diversius.

The probe was hybridized with its target rRNA (in this case either E. coli or C. diversius) in accordance with the procedure described in Example 8. The stability of the hybridized probes obtained, as well as a sample of unhybridized probe, was tested by pH = 7.6 as described in example 7.

Stability

Relationship between half-lives

<*>The low half-life of ester hydrolysis is not due to a small degree of hybridization, as HAP analysis (as described in Example 2) showed 73% hybridization.

These data show that in the case of hybrid with perfectly matched target, the AE probe is protected from ester hydrolysis

(as described in the previous examples), while in the hybrid in which the target contains a single base mismatch, the AE probe is poorly protected. This evokes a 17-fold

difference in degree of hydrolysis for the AE probe between the perfect target and a target with a single site mismatch. Therefore, the method described herein is easily capable of distinguishing between nucleic acid targets containing simple base changes.

EXAMPLE 10

Stability at room temperature under different conditions of hybridized probe compared to unhybridized probe internally labeled with acridinium ester.

Internally labeled probe (insert, type 1; see Example 1) was hybridized with 1 µg of C. trachomatis rRNA as described in Example 8. The stability of the hybridized probe compared to the unhybridized probe was measured at room temperature under a range of different reagent and pH -conditions (see "Results") in accordance with the procedure described in Example 7.

RESULTS:

This data shows that there are a number of conditions under which the homogeneous protection provision described herein will work, which means that it can be adapted to a number of conditions of provision. Furthermore, systems other than borate are acceptable, e.g. phytic acid systems at room temperature where very high half-life ratios are achieved.

EXAMPLE 11

Detection of a dilution series of Chlamydia rRNA in buffer using probe internally labeled with acridinium ester. Internally labeled probe (as in Ex. 2) was hybridized to decreasing amounts of its target rRNA (in this case Chlamydia trachomatis) according to the following procedure:

Hybridization Mixture

0.5 µl probe (12.5 fmol)

1 µl RNA (10-<2>,10~<3>, 10-<4>or 10"<5>ug)

2 µl 20% SDS

1.7 pl H20

4.8 µl 1 M PB, pH 6.8

The control mixture was the same as the hybridization mixture except that it contained water instead of rRNA, and the reagent blank mixture was the same as the control mixture except that it contained water instead of probe. The mixtures were incubated for 20 minutes at 60°C.

After hybridization, 90 µl of 0.2 M borate, pH 9 was added to each sample, followed by incubation at 60°C for 14 minutes. Each sample was then read for chemiluminescence as described in example 2, with the exception that injection 1 was only 0.1% H2O2, injection 2 had a pH of 13.8 instead of 13.2 and the reading time was 7 seconds instead of 10 seconds .

RESULTS:

Reagent blank - 116 rlu

Control - 124 rlu

Reagent blank and control represented the mean of three samples; all others represented the mean of two samples.

These results demonstrate that the invention described herein was capable of detecting a linear dilution series of target rRNA to a sensitivity limit of approximately 10<-4>ug in a clean buffer system.

EXAMPLE 12

Detection of a dilution series of Chlamydia rRNA in clinical medium using probe internally labeled with acridinium ester.

Internally labeled probe (as in Example 2) was hybridized in a clinical sample (throat secretion) to decreasing amounts of its target rRNA (in this case Chlamydia trachomatis) in accordance with the following procedure:

Hvbridiserina mixture

50 ul throat secretions

6 µl 4.8 PB, pH 4.7

2 pl rRNA (3 x 10~<4>, 3 x 10~<3>, 3 x 10~<2>or 3 x 10"<1>ug)

2 µl probe (1 pmol)

The control mixture was the same as the hybridization mixture except that it contained water instead of rRNA, and the reagent blank mixture was the same as the control mixture except that it contained water instead of probe. The mixture was incubated for 60 minutes at 60°C.

After hybridization, one-third of each mixture (20 UD) was added to 50 µl of 0.2 M borate, final pH = 9 (after addition of the hybridization mixtures), followed by incubation at 60°C for 40 min. Each sample was then read for • chemiluminescence as described in example 11.

RESULTS:

Reagent blank - 163 rlu

Control - 6560 rlu

The data represent the average of two values.

These data show that the invention as described herein is capable of detecting a linear dilution series of target rRNA to a sensitivity limit of approximately 10<-3>ug in a system containing a clinical medium.

EXAMPLE 13

Detection of a dilution series of bacterial rRNA in urine using probe internally labeled with acridinium ester

An internally labeled 30mer probe specific for E. coli was prepared as described previously (adenine replacement, type 1, linker arm). The probe was hybridized in urine to decreasing amounts of its target rRNA (in this case E. coli) according to the following procedure:

Hvbridiserina mixture

79.6 yl urine

0.4 µl 0.25 M EDTA, 0.25 M EGTA

10 µl 4.8 M PB, pH 6.8

5 µl 20% SDS

5 µl probe (0.125 pmol)

1 µl RNA (10~<3>, 10-<2>or 10_<1>yg)

The control mixture was the same as the hybridization mixture except that it contained water instead of rRNA, and the reagent blank mixture was the same as the control mixture except that it contained water instead of probe. The mixtures were incubated for 30 minutes at 60°C.

After hybridization, 300 µl of 0.2 M borate, pH 9, was added to each sample, after which the samples were incubated at 60°C for 16 minutes. Each sample was then read for chemiluminescence as described in Example 11.

RESULTS:

Reagent blank test - 684 5 rlu

Control - 9250 rlu

The results represent the average of two values.

These data demonstrate that the invention described herein is capable of detecting a linear dilution series of target rRNA to a sensitivity limit of approximately 5 x 10<-3>ug rRNA in a system containing urine.

EXAMPLE 14

Selective degradation applied in combination with a separation step for detection of a dilution series of Chlamydia rRNA in clinical medium using probe internally labeled with acridinium ester

Internally labeled probe (as in Example 2) was hybridized in a clinical sample (throat secretion) as described in Example 8, including control and blank mixtures. After hybridization, one-third of each mixture was removed and subjected to selective degradation exactly as described in Example 8, while one-third was simply removed and allowed to stand at room temperature (ie, no selective degradation). Both sample sets, with and without selective degradation, were then subjected to separation of hybridized probe from unhybridized probe using HAP as described in example 2 with the following exceptions: a. 1 ml of HAP solution was used (instead of 200 ul) b. 1 ml wash solution was used (instead of 200 µl), c. 3 washes were carried out (instead of 1) d. the final HAP pellets were resuspended in 100 µl wash solution, all of which were measured for chemiluminescence (as described in Example 2).

RESULTS:

Selective degradation

The results represent the average of two values with the reagent blank already subtracted. S:B = ratio between signal and background, i.e. chemiluminescence at a particular rRNA concentration divided by chemiluminescence of control.

These data show that selective degradation prior to separation results in less background which leads to a higher ratio of signal to background, and therefore improved sensitivity.

EXAMPLE 15

Improved stringency using differential hydrolysis The following probe was constructed to contain an internal linker arm, type "X" (position for insertion indicated by # below), labeled with acridinium ester, and purified as described in Example 1:

This probe is exactly complementary to the 16S subunit of Neisseria gonorrhoeae. However, it is also closely related to N. meningitidis, and a cross-reaction is typically observed under hybridization stringency conditions given in Examples 2 and 8. This probe was hybridized with 0.5 µg of N. meningitidis as described in Example 8, (except in a 200 ul format) and incubated for 10 min at 60°C in 1 ml of 0.2 M sodium tetraborate, pH 7.6, 5% Triton X-100 to effect differential hydrolysis (+ D.H.), or it was not subjected to these differential hydrolysis conditions (-D.H.). The obtained hybrids were then separated on magnetic microspheres and measured for chemiluminescence as described below. 1 ml of 0.4 M PB, pH 6.0, containing 150 µg of magnetic amine microspheres is added (BioMag M4100, Advanced Magnetics, Inc, Cambridge, Mass.). Vortex mix for 10 seconds. Incubate at 60°C for five minutes. Vortex mix for 10 seconds. Magnetically separate the beads from the solution using the Pace-Mate™ Magnetic Separation Rack (gen-probe, Inc, San Diego, CA). Remove the liquid. Wash the beads by adding 1 ml of 0.3 M PB, pH 6.0, votex mix for 10 seconds, separate magnetically and remove the liquid. Repeat for a total of three washes. Elute the hybrid by adding 300 µl 0.2 M PB, pH 6.0, 50% formamide, vortex for 10 seconds, incubate at 60°C for five minutes, vortex for 10 seconds, and magnetically separate beads from the solution. Transfer the solution to a Clinilumat tube and measure chemiluminescence as described in Example 7.

RESULTS:

<*>Hybrid signal - control signal (ie no rRNA), given in relative light units (rlu). Signal from the exact target (ie N. gonorrhoeae) in this format is typically 7-10 X 10 rlu.

In conclusion, it is seen that application of the differential hydrolysis technique described herein as an additional stringency discrimination step greatly reduces signal from unwanted cross-reacting sequences. In this example, cross-reaction of an N. gonorrhoeae probe with N. meningitidis was reduced more than 200-fold. The differential hydrolysis step increases sensitivity to unstable hybrids, which are in equilibrium with unhybridized probe, by constantly reducing the chemiluminescence (via hydrolysis) of the probe when it is in the unhybridized form, thus reducing chemiluminescence due to cross-reaction.

EXAMPLE 16

Detection of a chimeric target sequence associated with chronic myelogenous leukemia using a probe internally labeled with acridinium ester

A 24mer probe (sequence given below) with an internal linker arm, type "X", inserted as indicated was synthesized, labeled with AE and purified as described in Example 1. This probe is complementary to the chimeric mRNA transcript (regular cut). associated with chronic myelogenous leukemia (CML) and is called the bcr/- abl probe. This chimeric mRNA is a product of the chimeric gene formed by the translocation of a region of the abl gene into a region of another chromosome containing the bcr gene.

Synthetic DNA 60mer targets representing the cutpoint junction region of the bcr/abl chimeric target, as well as the normal ber and abl targets in the same region, were synthesized as described in Example 1 (sequences given below). An mRNA target was also prepared by transcription of a pGEM clone containing a 450 nucleotide segment of the chimeric bcr/abl gene centered around the cut point. An mRNA transcript of the same 450 nucleotide segment in probe context was also prepared as a negative control.

BCR/ABL PROBE SEQUENCE

The asterisk inset above denotes a linker arm insertion site for acridinium ester binding and the underline indicates abl sequences.

The bcr/abl probe was hybridized with approximately 1 pmol of each of the targets given above as described in Example 8, and the stability of these hybrids and the unhybridized probe was tested at pH 7.6 as described in Example 7.

RESULTS:

These data demonstrate that an AE-labeled probe designed to span the cutpoint junction region of the chimeric bcr/abl mRNA transcript associated with CML was able to discriminate between chimeric target and normal sequences (as well as unhybridized probe) using HPA - the technology described herein. This is a demonstration showing that the method according to the invention can be used to specifically detect chimeric targets (typically associated with genetic disorders) in the presence of normal, non-chimeric component nucleic acids.

This example further demonstrates that targets other than rRNA, in this case both mRNA and DNA, provide protection against AE hydrolysis when hybridized to an AE-labeled probe.

Claims (8)

1. Method for determining the presence or quantity of an analyte in a medium which combines with a binding partner to form a binding pair, characterized by the steps: a) one combines, with said medium or a part thereof, (1) a residue comprising the binding partner , the analyte or an analog of the analyte forming a binding pair with the binding partner, said residue being conjugated to a substance whose stability is different when the residue is a member of a binding pair compared to its stability in an unbound form; and (2) if said residue is said analyte or the analogue, combining said binding partner in the medium; b) selective degradation of the less stable form of the substance; and c) relating the amount of intact substance that remains after said substance degradation to the presence or amount of analyte in the medium. 2. Method for determining, in a medium, the presence or quantity of an analyte that combines with a binding partner to form a binding pair, characterized by the steps: a) one combines, with said medium or a part thereof, said binding partner that is conjugated to a substance whose stability is different when said binding partner is a member of a binding pair, compared to its stability in an unbound form; b) selective degradation of the less stable form of the substance; and c) relating the amount of intact substance that remains after said substance degradation to the presence or amount of analyte in the medium. 3. Method for determining, in a medium, the presence or quantity of an analyte that combines with a binding partner to form a binding pair, characterized by the steps: a) one combines, with said medium or a part thereof, (1) a residue comprising the analyte or an analog of the analyte that forms a binding pair with the binding partner, said residue being conjugated to a substance whose stability is different when the residue is a member of a binding pair compared to its stability in an unbound form; and (2) said bonding partner; b) selective degradation of the less stable form of the substance; and c) relating the amount of intact substance remaining after said substance degradation to the presence or amount of analyte in the medium. 4. Method for determining, in a medium, the presence or quantity of an analyte that combines with a binding partner to form a binding pair, characterized by the steps: a) one combines, with said medium or a part thereof, (1) a first binding partner of said analyte, said first binding partner being conjugated to a substance whose stability is different when said first binding partner is a member of a binding pair compared to its stability in an unbound form; and (2) a second binding partner of said analyte that is different from said first binding partner; b) selective degradation of the intermediate stable form of the substance; and c) relating the amount of intact substance remaining after said substance degradation to the presence or amount of analyte in the medium. 5. Method for detecting a nucleotide sequence in a medium, characterized by the steps: a) one combines, with said medium or a part thereof, said nucleotide sequence and a probe which is essentially complementary thereto, said probe being conjugated to a substance whose stability is different when said probe is hybridized to said nucleotide sequence compared with its stability in an unhybridized form; b) selective degradation of the less stable form of the substance; and c) relating the amount of intact substance remaining after said substance degradation to the presence or amount of nucleotide sequence in a medium. 6. Method for determining, in a medium, the presence or amount of an analyte that combines with a binding partner to form a binding pair, characterized by the steps: a) one combines, with said medium or a part thereof, (1) a residue comprising a first binding partner of said analyte, or an analogue of said first binding partner which forms a binding pair participating in a binding reaction, said residue being conjugated to a substance whose stability is different when the residue is a member of a binding pair compared to its stability in an unbound form; and (2) another binding partner of said analyte that is different from said first binding partner in said binding reaction; b) selective degradation of the less stable form of the substance; and c) relating the amount of intact substance remaining after said substance degradation to the presence or amount of analyte in the medium. 7. Method as stated in claims 1-6, characterized in that the substance is an acridinium ester. 8. Procedure as specified in claims 1 - 6, characterized in that the substance is a member selected from the group consisting of: wherein X is selected from the group consisting of O, N, S, halogen, substituted phosphorus, substituted sulfur, substituted boron or substituted arsenic; Y is selected from the group consisting of 0, S or NH; R 1 is selected from the group consisting of alkyl, alkenyl, aryl, substituted alkyl, substituted alkenyl, substituted aryl, alkoxy, aryloxy, or is absent when X is a halogen; R 2 is selected from the group consisting of H, alkyl, alkenyl, aryl, substituted alkenyl, substituted, aryl, alkoxy or aryloxy if, and only if X is N; R 3 is selected from the group consisting of H, amino, hydroxy, thiol, halogen, nitro, amido, acetyl, alkyl, alkenyl, aryl substituted acetyl, substituted alkyl, substituted alkenyl, substituted aryl, alkoxy or aryloxy; R 4 is selected from the group consisting of alkyl, alkenyl, aryl, substituted alkyl, substituted alkenyl or substituted aryl; and at least one of R 1 , R 2 , R 3 or R 4 contains a reactive site allowing chemical conjugation.