WO1994002641A1

WO1994002641A1 - Analogues of reporter groups as background reducers in hybridization assays

Info

Publication number: WO1994002641A1
Application number: PCT/US1993/006702
Authority: WO
Inventors: Shy Chen Ju; Michael Lee Cubbage; Joel Bresser; Mark Blick
Original assignee: Aprogenex, Inc.
Priority date: 1992-07-17
Filing date: 1993-07-16
Publication date: 1994-02-03
Also published as: AU4681193A

Abstract

Assays for target molecules in and from cells and viruses whereby the use of a reporter group analogue is used to decrease non-specific background.

Description

ANALOGUES OF REPORTER GROUPS AS BACKGROUND REDUCERS IN HYBRIDIZATION ASSAYS

FIELD OF THE INVENTION

The invention pertains to the reduction of undesired background in hybridization assays for specific molecules in cells and viruses.

BACKGROUND OF THE INVENTION In an important type of in situ assay, a cell (or virus) is incubated with a target- specific probe carrying a reporter group. If the probe binds to the cell or virus, its presence can be detected by a process designed to detect the reporter group. Reporter groups include fluorescent moieties and moieties that participate in chemiluminescent reactions. Such assays are useful to detect viral nucleic acids, human genes of interest, specific cellular antigens, and other biologically important molecules.

A common limitation on the sensitivity that can be achieved with such assays is that the probes can bind to entities other than the target molecule of interest. Those entities will normally be in the target biological entity (cell or virus), but may also be on the solid support on which the biological entity or purified target molecules are immobilized. The present invention involves incubating the probes with an excess of an analogue of the reporter group, the analogue being sufficiently similar to the reporter group so that it will compete for the non-specific binding sites, but sufficiently different from the reporter group that it will not enter into the reaction used to detect the reporter group. In particular, the invention focuses on reporter groups that comprise an aromatic component.

The present invention involves the discovery of compounds useful as background reducers in in situ assays.

In addition to the general benefit of increased assay sensitivity that the background reducers provide, there is the additional advantage that, for a given level of sensitivity to be reached, cell treatment procedures such as cell fixation need not be as

SUBSTITUTE SHEET detrimental to cell integrity as would be the case without the use of the background reducers. As a result, the cells retain their structural integrity for a longer time, a consideration particularly important for flow cytometry.

BRIEF SUMMARY OF THE INVENTION

The invention is, in part, in situ assays in which free radical scavengers are added in order to reduce autofluorescence.

The solutions that contain the free radical scavengers are also inventions.

DETAILED DESCRIPTION

In a general aspect, the invention is a detection process or assay capable of detecting a target molecule in or on a biological entity (cell or virus). The process comprises the steps of:

(1) contacting the biological entity with a solution comprising a probe capable of binding to a target molecule in or on said biological entity, said contacting performed in a manner such that the probe binds to said target molecule so as to make that probe a biological entity-bound probe, said probe comprising a reporter group,

(2) contacting the biological entity with a solution comprising a structural analogue of the aromatic reporter group, (3) performing one or more steps that will detect the reporter group on the probe bound to the biological entity but that will not detect analogue bound to the biological entity, wherein step (1) takes place before step (2), after step (2), or during step (2).

Steps (1) and (2) are considered to take place simultaneously if the probe and the analogue are in the same solution.

Preferably steps (1) and (2) are performed simultaneously by including the probe and the analogue in the same solution.

In a subgeneric aspect of the invention, the reporter group is a cyclic compound. In a further subgeneric aspect of the invention, the cyclic group comprises an

SUBSTITUTE SHEET unsaturated bond. In a still narrower subgeneric aspect of the invention, the cyclic group is an aromatic compound (one or more benzene rings).

It is preferred that, on a molar basis, the analogue is in excess as regards the reporter group; it is highly preferred that there be at least 10 times as much analogue as reporter group.

It is hypothesized that the invention works because the analogue competes with the reporter group for nonspecific binding sites. In the case of ATA used in conjunction with a nucleic acid probe, an additional mechanism may involve ATA binding to the active site of proteins that would bind the reporter group. It is preferred that the analogue is selected so that it retains most or all of the structural features of the reporter group. The analogue may additionally have structural features not present in the probe.

Preferably, the analogue should be able to permeate a cell or virus. In the case of analogues that are aurin derivatives (rosolic acid derivatives), it is preferred that the analogues have, in addition to ATA, a polar functional group such as a -C0₂, -NH₂, —

OH, or -S0₃ group, on an aromatic group; examples are chromoxane cyanine R and Chrome Azurol S. A subgroup of preferred analogues are those that block the NH₂ groups on lysines.

Fluorescent reporter groups are detected by allowing the reporter group to absorb a energy and then emit some of the absorbed energy; the emitted energy is then detected.

Chemiluminescent reporter groups are detected by allowing them to enter into a reaction, e.g., an enzymatic reaction, that results in energy in the form of light being emitted. Other reporter groups, e.g., biotin are detected because they can bind to groups such as streptavidin which are bound, directly or indirectly to enzymes, e.g. (alkaline phosphatase or horse radish peroxidase that can catalyze a detectable reaction.)

Fluorescent groups with which this invention can be used include fluorescein (or FITC), Texas Red, Coumarin, Rhodamine, Rhodamine derivatives, Phycoerythrin, and Perci-P.

SUBSTITUTE SHEET Chemiluminescent groups with which this invention can be used include isoluminol (or 4-aminophthalihydrazide; See catalogues of Aldrich Chemical Co. for 1990-91, or see Molecular Probes, Inc. catalogue)

In one preferred embodiment of the process, when the reporter group is fluorescein, step (4) comprises measuring light emitted at wavelengths between about

520 nm and 560 nm (especially at about 520 nm), most preferably where the absorption wavelengths of step (3) are less than 520 nm.

A preferred embodiment of the fluorimetric process further comprises a wash step between the steps numbered (2) and (3). A wash step can be performed by centrifuging the cell out of the solution in which it is suspended, then suspending it in a wash solution, and then centrifuging it out of the wash solution. A wash solution is generally a probe-free solution.

In a particular embodiment of the process, the solution that is used in step (2) comprises a probe (comprising a reporter group), an analogue of the reporter group, a and a fixative. Such a probe solution is itself an invention.

A fluorescent probe that binds to a target molecule is preferably one which binds to that target with high specificity. Such a probe may be a fluorescent dye unattached to a nucleic acid or antibody or other molecule. More preferably, a fluorescent probe is a fluorescent dye covalently attached to a nucleic acid molecule, antibody or other molecule capable of binding specifically to a target molecule. The fluorescent dye may be covalently attached directly to the molecule having target specificity or it may be covalently attached to a linker group which in turn is covalently attached to the molecule having target specificity. The probe nucleic acid molecule will be specific for a nucleic acid target molecule with a base sequence complementary to the probe nucleic acid molecule. The probe antibody will be specific for a target antigen.

Nucleic acid probes can be used against a variety of nucleic acid targets, viral, prokaryotic, and eukaryotic. The target may be a DNA target such as a gene (e.g., oncogene), control element (e.g., promoter, repressor, or enhancer), or sequence coding for ribosomal RNA, transfer RNA, or RNase P. The target may be RNA such

SUBSTITUTE SHEET as mRNA, ribosomal RNA, RNase P, tRNA, a viral genome or complementary copy thereof. Additionally, the target may be a "nucleic acid amplification product," i.e., a nucleic acid molecule, either DNA or RNA, which is the result of introducing an enzyme or enzymes into the cell so that such enzymes will make an nucleic acid molecule complementary to one already present in the cell. For example, O. Bagasra et al, The New England Journal of Medicine. 326. pp. 1385-1391 (1992), have disclosed the use of the polymerase chain reaction (PCR) with intact cells such that the introduction of polymerase molecules into a cell resulted in additional nucleic acid molecules being formed, each a copy of one previously existing in the cell, though not necessarily existing before the introduction of the enzymes.

A viral nucleic acid can be part of a virus, in which case the virus may or may not be inside a cell. Alternatively, a viral nucleic acid target may or may not be part of a virus, but may be inside a cell.

Many publications show how to attach a fluorescent dye to a nucleic acid probe or antibody probe.

The cells containing the target molecules may be eukaryotic cells (e.g., human cells), prokaryotic cells (e.g., bacteria), plant cells, or any other type of cell. They can be simple eukaryotes such as yeast or be derived from the more complex eukaryotes such as humans. The target molecules can be in a non-enveloped virus or an enveloped virus

(having an enveloping membrane such as a lipid-protein membrane).

The hybridization assay may be done with fixed cells or (or fixed viruses). Useful precipitation fixatives include ethanol, acetic acid, methanol, acetone, and combinations thereof. Other useful fixatives will be obvious to one skilled in the art. Fixatives and hybridization of fixed cells, in general, are discussed in PCT international applications, WO 90/02173 and WO 90/02204 of Research Development Corp. Fixatives should provide good preservation of cellular morphology and preservation and accessibility of antigens, and high hybridization efficiency.

SUBSTITUTE SHEET The fixative may contain a compound which fixes the cellular components by cross-linking these materials together, for example, paraformaldehyde, glutaraldehyde or formaldehyde. Cross-linking agents, while preserving ultrastructure, often reduce hybridization efficiency; they form networks trapping nucleic acids and antigens and rendering them inaccessible to probes and antibodies. Some also covalently modify nucleic acids preventing later hybrid formation.

The hybridization solution may typically comprise a chaotropic denaturing agent, a buffer, a pore forming agent, a hybrid stabilizing agent.

The chaotropic denaturing agents (Robinson, D. W. and Grant, M. E. (1966) J. Biol. Chem. 241: 4030; Hamaguchi, K. and Geiduscheck, E. P. (1962) J. Am. Chem.

Soc. 84: 1329) include formamide, urea, thiocyanate, guanidine, trichloroacetate, tetramethylamine, perchlorate, and sodium iodide. Any buffer which maintains pH at least between 7.0 and 8.0 may be utilized.

The pore forming agent is for instance, a detergent such as Brij 35, Brij 58, sodium dodecyl sulfate, CHAPS™ Triton X-100. Depending on the location of the target biopolymer, the pore-forming agent is chosen to facilitate probe entry through plasma, or nuclear membranes or cellular compartmental structures. For instance, 0.05% Brij 35 or 0.1% Triton X-100 will permit probe entry through the plasma membrane but not the nuclear membrane. Alternatively, sodium desoxycholate will allow probes to traverse the nuclear membrane. Thus, in order to restrict hybridization to the cytoplasmic biopolymer targets, nuclear membrane pore-forming agents are avoided. Such selective subcellular localization contributes to the specificity and sensitivity of the assay by eliminating probe hybridization to complementary nuclear sequences when the target biopolymer is located in the cytoplasm. Agents other than detergents such as fixatives may serve this function.

Hybrid stabilizing agents such as salts of mono- and di-valent cations are included in the hybridization solution to promote formation of hydrogen bonds between complementary sequences of the probe and its target biopolymer. Preferably sodium chloride at a concentration from 0.15 M to 1 M is used. In order to prevent

SUBSTITUTE SHEET non-specific binding of nucleic acid probes, nucleic acids unrelated to the target biopolymers are added to the hybridization solution.

Many types of solid supports may be utilized to practice the invention. Supports which may be utilized include, but are not limited to, glass, Scotch tape (3M), nylon, Gene Screen Plus (New England Nuclear) and nitrocellulose. Most preferably glass microscope slides are used. The use of these supports and the procedures for depositing specimens thereon will be obvious to those of skill in the art. The choice of support material will depend upon the procedure for visualization of cells or viruses and the quantitation procedure used. Some filter materials are not uniformly thick and, thus, shrinking and swelling during in situ hybridization procedures is not uniform. In addition, some supports which autofluoresce will interfere with the determination of low level fluorescence. Glass microscope slides are most preferable as a solid support since they have high signal-to-noise ratios and can be treated to better retain tissue.

In one embodiment of the process, the target biological entity is immobilized on a solid surface (especially a glass slide where the entity is a cell) during steps (1) through (4). In another embodiment, the target cell or virus is suspended in liquid during the entire process and not immobilized on a solid surface.

The target molecule to which the probe is bound need not be in or on a biological entity. It can be a purified target. A purified target is a molecule, such as a nucleic acid molecule that has been extracted from a cell or a virus, or has been synthesized in a cell-free system. Many procedures have been published for hybridizing a nucleic acid probe against a nucleic acid target that is either in solution or immobilized in single stranded form on a solid support such as a nitrocellulose filter or nylon. The hybridizations vary considerably, depending in part on the level of specificity desired. Some examples are the Southern Blot procedure ( J. Mol. Biol.. 98,

503-517 (1975)) for electrophoresed and immobilized DNA, the Northern Blot procedure (Seed, B., in Genetic Engineering: Principles and Methods. Setlow, J.K. and Hollaender, A., eds., 1982; P. S> Thomas, Proc. Natl. Acad. Sci. USA.. 77: 5201 (1980)) and the use of stringent conditions with short oligomer probes, S. V. Suggs et al, Proc. Natl. Acad. Sci. USA. 78, 6613-6617 (1981)).

SUBSTITUTE SHEET In another aspect, the invention is a kit which comprises a probe (comprising a reporter group) and an analogue of the reporter group. The probe and the analogue may be combined in a variety of ways. For example, the probe and analogue may (1) be present in the same solution, or (2) be present in separate solutions; alternatively, either the probe or the analogue or both of them may be present in solid form, so that they must be dissolved in a solvent or solution prior to use.

Abbreviations

Compound Abbreviation aurintricarboxylic acid ATCA fluorescein isothiocyanate FITC ethylene diamine tetraacetic acid EDTA dimethyl sulfoxide DMSO dithiothreitol DTT polyvinylpyrrolidone PVP polyethylene glycol (circa 4000 Mol. Wt.) PEG 4000

Napachrome green Nap Green

Targets in cells, tissue, and fluids

The hybridization assay can be done for targets in cells in liquid suspension, in cells on slides or other solid supports, and in tissue sections. Such hybridization procedures are well known in the art. They are, for example, described in more detail in PCT applications WO 90/02173 and WO 90/02204.

The target molecules can be in eukaryotic cells or prokaryotic cells. The cells can come from solid tissue (e.g., nerves, muscle, heart, skin, lungs, kidneys, pancreas, spleen, lymph nodes, testes, cervix, and brain) or cells present in membranes lining various tracts, conduits and cavities (such as the gastrointestinal tract, urinary tract, vas deferens, uterine cavity, uterine tube, vagina, respiratory tract, nasal cavity, oral cavity,

SUBSTITUTE SHEET pharynx, larynx, trachea, bronchi and lungs) or cells in an organism's fluids (e.g., urine, stomach fluid, sputum, blood and lymph fluid) or stool.

Flow cvtometrv A Coulter Profile II flow cytometer can be used.

For experiments with FITC as the probe dye, the dye is first excited with light of 488 nm and then the emitted light is measured. For the emitted light (for LFL1), a 540 bp (40) filter was used; i.e., only light with a wavelength between 520 nm and 560 nm is allowed to pass. The filter for LFL3 is a 635 long pass filter; i.e., it allows any light over 635 nm wavelength to pass.

Solutions

Fixation solution F has the following ingredients: 4 volumes ethanol, 5 volumes of 1 x PBS, 1 volume of glacial acetic acid. Hybridization cocktail HC has the following ingredients;

5 x SSC (0.75 M NaCl, 0.075 M sodium citrate); 30% Formamide (v/v); 3% Triton X- 100 (v/v); 0.4 M Guanidinium isothiocyanate; 0.16 M sodium phosphate buffer (pH 6); 15 x Ficoll/PVP (polysucrose 400,000 mol wt/polyvinylpyrrolidone); 1 mg/ml Sheared Salmon Sperm DNA; 10 mM EDTA; 25 mM DTT; 5% PEG 4000. In the foregoing, 500 x Ficoll/PVP is 5 g of Ficoll type 400 (polysucrose 400,00 mol wt) plus 5 g of PVP

(polyvinylpyrrolidone) dissolved in water to a total volume of 100 ml; 15 x Ficoll/PVP indicates that 500 x Ficoll/PVP has been diluted by a factor of 15/500 with water.

Ficoll (Pharmacia) is a nonionic synthetic polymer of sucrose.

If an analogue is added to the cocktail, its preferred concentration is from 0.01 to 0.5% w/v.

For hybridization cocktails used with a nucleic acid probe, the temperature for the hybridization reaction preferably between 30° C and 46° C; the time preferably is between 5 minutes and 16 hours.

SUBSTITUTE SHEET Wash solution #1 has the following composition: 0.4 M guanidinium isothiocyanate; 0.1% Triton X-100 (an alcohol derivative of polyoxyethylene ether, see Aldrich Chemical Co. catalogue for 1990-91; 0.1 % v/v); 0.1 x SSC; in deionized water. Wash solution #2 has the following composition: 0.1% Triton X-100 (v/v);

0.1 x SSC; in deionized water.

1 x SSC has the following composition: 0.15 M NaCl, 0.15 M sodium citrate, pH 7.0. 2 x SSC is composed so that upon a 1:1 dilution with water, SSC would be produced; 10 X SSC is composed so that upon a 1:10 dilution with water, SSC would be produced.

1 x PBS is phosphate-buffered saline and had the formula, 0.136 M NaCl, 0.003 M KC1, .008 M Na₂HP0₄.7H₂0, 0.001 M KH₂P0₄.

If a dye-labeled antibody is used as the probe, then the probe may be dissolved in PBS, possibly supplemented with bovine serum albumin (BSA) while it is allowed to react with target cells preferably at a temperature in the range 4 °C to 34 °C. The cells need not be fixed (e.g. when the antibody target is a cell-surface antigen), or may be fixed after the probe-target incubation is completed, or may be fixed prior or during the probe-target incubation. The analogue of the probe's reporter group is preferably present while the probe is being mixed with the target cells or virus.

Additional useful reagents and solutions

Useful reagents and solutions for executing the inventions described herein include 0.0025% Evans Blue and/or 10% dodecyl alcohol in the solution analyzed cytofluorimetrically; 5% Vitamin E in the hybridization cocktail used where the assay target is in a biological entity; about 8% DMSO (v/v) with about 5% or 10% squalane and about 5% or 10% pyrrolidinone in the hybridization cocktail when the target is in a biological entity; 5 μl of DTT and 5 μl of Proteinase K (1 mg/ml) solution are added to 100 μl of cocktail and the hybridization reaction is run, for example, at 42°C for 5

SUBSTITUTE SHEET min, then at 95°C for 5 min, and then at 42°C for 2 min, when the target is in a biological entity;

Where 30-mers are used, probes against both strands of a double-stranded target can be used, provided that the probes are "out-of phase" along the map of the target so that any probe is not complementary in base sequence to more than about 15 nucleotides of a probe to the other strand of the target. In that way, probes hybridize to the target and not to each other.

Cell lines used in the Examples H9 cells are a human-derived lymphoma cell line.

Reagents

Reagents can be purchased from any of a variety of sources including Aldrich Chemical Co., Milwaukee, Wisconsin, Sigma Chemical Co., St. Louis, Missouri, Molecular Probes, Inc., Eugene, Oregon, Clontech, Palo Alto, California, Kodak,

Rochester, NY, and Spectrum Chemical Manufacturing Corp., Gardenea, California.

EXAMPLES

Example 1

Preparation of Cells

The H9 cell line was used in the following experiment. Cultured cells were washed with nuclease-free Phosphate Buffered Saline (PBS) and placed in a single cell suspension at a concentration that resulted in clearly separated cells. The cells were spun down to a pellet and the supernatant drained off. The cells were resuspended in 40% ethanol, 50% PBS, and 10% glacial acetic acid and left for 12-16 hours at 4°C. After fixation, the cells were spun to remove the fixative and then washed once in IX PBS and resuspended in 2X SSC. (The cells should be used immediately.)

SUBSTITUTE SHEET H9- cells were uninfected with human immunodeficiency virus (HIV) H9+ cells had one copy of HIV strain BH102 integrated in DNA form.

Preparation of Probes

The HIV sequences used as probes were accessed via GenBank, version 69.0, and prepared (See Table 1). The negative probe, NR was derived from the nitrogen reductase gene found in bacteria and known not to hybridize to nucleic acid within eukaryotic cells. The cells were 39-bases in length.

Table 1

HIV HUMHB102 fluorescein

Probe Synthesis & Labeling

The above mentioned sequences are divided into several 25-base oligonucleotides and synthesized using DNA synthesizers (Applied Biosystem DNA

Synthesizer, Model 380B) and using the recommended ABI reagents. The oligonucleotides were then coupled to at their 5' end to FTIC, a fluorescent dye (Molecular Probes, FITC) and purified by column chromatography and HPLC. Coupling was through an aminohexyl moiety, aminolink, purchased from Applied Biosystems, inc.

SUBSTITUTE SHEET Hybridization

For the hybridization procedure, to pelleted cells were added 50 μl of a hybridization cocktail consisting of 30% formamide, 5X SSC, 0.16M sodium phosphate buffer, pH 7.4, 1 μg/μl sheared DNA, 3% (v/v) Triton X-100, 5% PEG 4000, 25mM DTT, 0.4M guanidinium isothiocyanate, 15X Ficol/PVP, and the probe was added at a concentration of 2.5 μg/ml. Hybridizations were carried out at 42°C for 30 minutes.

Additionally, as specified below, either 0.05% ATCA, 0.1% ATCA. 0.05% Nap Green or 0.1% Nap Green was added to the cocktail.

Washing

Proper washing after the hybridization reaction is essential to minimize background due to non-specific binding of probe. Post-hybridization the cells were placed in a 15 ml conical tube to which was added 10 ml of a wash solution, consisting of .IX SSC, .4M guanidinium isothiocyanate, and .1% Triton at a temperature of 42°C. The solution was agitated until the cells were a single cell suspension and then spun at

250 X g for 5 minutes. The supernatant was removed and to the pellet was added 10 ml of a wash solution, consisting of .IX SSC, .1% Triton at a temperature of 42°C. The solution was agitated until the cells were a single cell suspension. The cells were spun at 250 X g for 5 minutes. The supernatant was removed and the cell pellet resuspended in 0.2 ml counterstain solution consisting of .0025% Evans Blue in IX

PBS.

Flow Cytometer Use and Interpretation

The cells were analyzed on a Profile II™ made by Coiulter Instruments. The instrument uses a 488 nm argon laser, a 525nm band pass filter for FL1 and a 635nm band pass filter for the counterstain. For each sample analyzed the sample containing the negative probe is analyzed first and the quad-stats are set so that less than 0.01% of the cells fall in the upper-right quadrant or lower-right quandrant. Next the sample analyzed with the HIV probes is analyzed under the exact same parameters as the sample analyzed with the negative probe. Since the quad-stats are set correctly and the

SUBSTITUTE SHEET two samples have been handled identically, any number of cells (above 0.01%) that are recorded in the upper right quadrant are scored as positive.

Results

Tables B and C show results obtained with an FITC-labeled DNA probe specific for HIV sequences in H9- (uninfected) and H9+ (HIV infected) cells. In Table 4, the results were done with and without Evans Blue in the solution containing the cells during flow cytometry.

Table B

Table C

SUBSTITUTE SHEET The results show ATCA was effective at concenctrations 0.05% and 0.1% for reducing background. The results also show increased signal when napachrome green and ATCA were used with Evans Blue present during flow cytometry.

End of Example 1

The foregoing description of the preferred embodiments of the invention has been presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and its practical application to thereby enable others skilled in the art to best utilize the invention in various embodiments and with various modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims appended hereto.

SUBSTITUTE SHEET

Claims

CLAIMSHaving thus described the invention, what is desired to protect by Letters Patent and hereby claim is:

1. A detection process which process comprises the steps of:

(1) contacting a biological entity with a solution comprising a probe capable of binding to a target molecule in or on said biological entity, said contacting performed in a manner such that the probe binds to said target molecule so as to make that probe a biological entity-bound probe, said probe comprising an a reporter group, (2) contacting the biological entity with a solution comprising a structural analogue of the reporter group,

(3) performing one or more steps that will detect the reporter group on the probe bound to the biological entity but that will not detect analogue bound to the biological entity, wherein step (1) takes place before step (2), after step (2), or during step (2), wherein said biological entity is a cell or a virus.

2. A process of Claim 1 wherin the reporter group is a cyclic compound. In a

3. A process of Claim 2 wherein the cyclic moiety of the cyclic compound comprises an unsaturated bond.

4. A process of Claim 3 wherein the cyclic compound is an aromatic compound.

5. A process of Claim 1 wherein said probe comprises either a nucleic acid moiety or an antibody moiety.

6. A process of Claim 1 wherein steps (1) and (2) are performed simultaneously by including the probe and the analogue in the same solution.

SUBSTITUTE SHEET

7. A process of Claim 1 wherein the reporter group is a fluorescent moiety and step (3) comprises detecting said reporter group by a fluorimetric detection process.

8. A process of Claim 1 wherein the reporter group may participate in a chemiluminescent reaction and step (3) comprises detecting said reporter group by a chemiluminescent detection process.

9. A process of Claim 1 which further comprises a wash step between the steps numbered (2) and (3), said wash step comprising washing the biological entity in a probe-free solution.

10. A process of Claim 1 wherein prior to step (1) the biological entity has been treated with a fixative.

11. A process of Claim 1 wherein the probe comprises a nucleic acid moiety.

12. A process of Claim 8 wherein the fluorescent moiety is fluorescein.

13. A process of Claim 1 wherein the biological entity is suspended in liquid and not immobilized on a solid surface.

14. A process of Claim 1 wherein the biological entity is immobilized on a solid support.

15. A process of Claim 14 wherein the biological entity is not part of a tissue section.

16. A process of Claim 1 wherein the biological entity is present in a tissue section.

SUBSTITUTE SHEET

17. A process of Claim 1 wherein the biological entity is a cell.

18. A process of Claim 17 wherein the cell is a eukaryotic cell.

19. A process of Claim 18 wherein the eukaryotic cell is a human cell.

20. A process of Claim 19 wherein the probe is specific for a viral nucleic acid.

21. A process of Claim 17 wherein the cell is a prokaryotic cell.

22. A process of Claim 17 wherein the cell is a plant cell.

23. A process of Claim 1 wherein the biological entity is a virus.

24. A method of Claim 11 wherein the nucleic acid probe reacts with a specific

DNA sequence in the biological entity.

25. A method of Claim 11 where the nucleic acid probe reacts with a specific RNA sequence in the biological entity.

26. A method of Claim 1 where the probe comprises an antibody moiety.

27. A method of Claim 11 where the target is viral DNA.

28. A method of Claim 11 where the target is viral RNA.

29. A method of Claim 11 wherein the target is human immunodeficiency virus RNA or DNA.

SUBSTITUTE SHEET

30. A process of Claim 1 wherein the analogue comprises most or all of the structural components of the reporter group.

31. A process of Claim 7 wherein the reporter moiety is selected from the group, fluorescein, Texas Red, Coumarin, Rhodamine or Rhodamine derivative, Phycoerythrin, and Perci-P.

32. A process of Claim 31 wherein the reporter group is fluorescein or a rhodamine derivative and the analogue is Aurin or a derivative thereof.

33. A process of Claim 32 wherein the reporter group is fluorescein and the analogue is Aurin or a derivative thereof.

34. A process of Claim 33 wherein the analogue is aurintricarboxylic acid.

35. A process of Claim 5 wherein the reporter moiety is isoluminol.

36. A detection process which process comprises the steps of:

(1) contacting a target molecule with a solution comprising a probe capable of binding to said target molecule, said contacting performed in a manner such that the probe binds to said target molecule, said probe comprising an a reporter group,

(2) contacting the target molecule with a solution comprising a structural analogue of the reporter group,

(3) performing one or more steps that will detect the reporter group on the probe bound to the target molecule but that will not detect analogue bound to the biological entity, wherein step (1) takes place before step (2), after step (2), or during step (2).

37. A solution that comprises the following:

SUBSTITUTE SHEET 1) a probe that comprises a reporter group and further comprises an antibody or a nucleic moiety, and

2) a structural analogue of said reporter group.

38. A kit that comprises the following:

1) a probe that comprises a reporter group and further comprises an antibody or a nucleic moiety, and

2) a structural analogue of said reporter group.

SUBSTITUTE SHEET