# US 5,707,801 A · Manual in situ hybridization assay

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- Number: US 5,707,801 A
- Document ID: US5707801A
- Status: Granted
- Kind: A
- Application number: 08/421,705
- Filed: 1995-04-13
- Granted: 1998-01-13
- Earliest claimed: 1988-08-31
- Assignees: Aprogenex, Inc.
- Inventors: Joel Bresser; Mary Jean Evinger-Hodges
- Web page: https://patentgrep.com/patent/US5707801A

## CPC

- C12Q 1/703: {Viruses associated with AIDS}
- C12Q 1/68: involving nucleic acids
- C12Q 1/6841: In situ hybridisation
- C12Q 1/6886: for cancer (immunoassay for cancer G01N33/575)
- C12Q 1/70: involving virus or bacteriophage {(immunoassay for viruses G01N33/56983)}
- C12Q 1/705: {for herpetoviridae, e.g. herpes simplex, varicella zoster}
- C12Q 2600/112: Disease subtyping, staging or classification
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

A rapid, sensitive in situ hybridization assay is provided which will detect as few as 1-5 copies of target biopolymer per cell and may be accomplished in 2-4 hours. There is provided a quantitative assay which may be used to diagnose and monitor treatment of diseases.

## Claims

### Claim 1

A method for assaying target polynucleotides in a cellular specimen, said method comprising the steps of:
contacting a cellular specimen with a fixation medium to yield a fixed specimen having therein cells with substantially intact membranes, said fixation medium comprising at least one agent that is a precipitating agent or a cross-linking agent, said agent being an agent that allows double-stranded cellular polynucleotides to shift under hybridizing conditions to about 70% single-stranded cellular polynueleotides while maintaining cellular spatial relationships;
incubating said fixed specimen with a hybridization solution comprising a denaturing agent, a hybrid stabilizing agent, a buffering agent, a membrane pore-forming agent and at least one probe having a nucleotide sequence at least substantially complementary to a specific target nucleotide sequence to be detected, said incubating being under hybridizing conditions so that double-stranded cellular polynucleotides shift to about 70% single-stranded cellular polynucleotides, and
detecting duplexes formed between said probe and said specific target nucleotide sequence in said cells with substantially intact membranes;
wherein the time for performing said steps of contacting, incubating, and detecting does not exceed a total of about 4 hours;
whereby target polynucleotides are assayed in a cellular specimen;
wherein said shift under hybridizing conditions to about 70% single-stranded cellular polynucleotides while maintaining celluar spatial relationships is determinable by a process comprising the following four steps:
(a) staining cells with a stain able to stain polynucleotides within said cells to yield a different emission spectnun upon ultraviolet excitation of stained double-stranded and stained single-stranded polynucleotides;
(b) determining the total mount of emitted fluorescence of a cell sample not treated under hybridizing conditions;
(c) determining the total amount of emitted fluorescence of cell samples treated by various fixation and hybridization procedures and times;
(d) assessing when the nmount of fluorescence corresponding to the amount of double-stranded polynucleotide has decreased about 70%.

### Claim 2 (depends on 1)

The method of claim 1, wherein:
probes having nucleotide sequences at least substantially complementary to specific target nucleotide sequences to be detected are used at a concentration of at least 1.0 microgram per milliliter hybridization solution, and said method is capable of detecting as few as five copies of said target nucleotide sequence per cell.

### Claim 3 (depends on 2)

The method of claim 2, comprising an additional step of:
quantitating said detection of duplexes formed between said probe and said target nucleotide sequence.

### Claim 4 (depends on 3)

The method of claim 3, wherein:
said quantitating comprises:
determining the quantity of said duplexes formed between said probe and said target nucleotide sequence detected in one or more cells of said cellular specimen, or determining the quantity of said cells of said cellular specimen, wherein said duplexes between said probe and said target nucleotide sequence are detected.

### Claim 5 (depends on 1)

The method of claim 1, wherein said step of detecting duplexes is accomplished by means of a detectable label attached to said probe.

### Claim 6 (depends on 1)

The method of claim 1, wherein said step of detecting duplexes is accomplished by means of a detectable label which is added after formation of said duplexes is complete.

### Claim 7 (depends on 1)

The method of claim 1, wherein said detectable label is selected from the group consisting of: radioactive labels, fluorescers, chemiluminescers, and enzyme labels.

### Claim 8 (depends on 1)

The method of claim 1, wherein said detectable label is selected from avidin or streptavidin.

### Claim 9 (depends on 1)

The method of claim 1, wherein said precipitating agent is selected from the group consisting of: ethanol, methanol, acetone, combinations thereof, and combinations of any of the foregoing with formaldehyde.

### Claim 10 (depends on 1)

The method of claim 1, wherein said cross-linking agent is selected from the group consisting of: paraformaldehyde, formaldehyde, dimethylsuberimidate and ethyl-dimethylamino-propylcarbodiimide.

### Claim 11 (depends on 1)

The method of claim 1, wherein said denaturing agent is selected from the group consisting of: formamide, urea, sodium iodide, thiocyanate, guanidine, perchlorate, trichloroacetate and tetramethylamine.

### Claim 12 (depends on 11)

The method of claim 11, wherein said denaturing agent is formamide.

### Claim 13 (depends on 12)

The method of claim 12, wherein said denaturing agent is formamide at a concentration of 20% to 80%.

### Claim 14 (depends on 1)

The method of claim 1, wherein said hybrid stabilizing agent is selected from the group consisting of: sodium chloride, lithium chloride, magnesium chloride and ferric sulfate.

### Claim 15 (depends on 1)

The method of claim 1, wherein said pore-forming agent is a selective pore-forming agent.

### Claim 16 (depends on 1)

The method of claim 1, wherein said pore-forming agent is selected from the group consisting of: polyoxyethylene(23) lauryl ether, polyoxyethylene(20) cetyl ether, 3-((3-cholamidopropyl)-dimethylammonio)-1-propanesulphonate, octylphenoxypolyethoxyethanol, sodium deoxycholate and sodium dodecyl sulfate.

### Claim 17 (depends on 1)

The method of claim 1, wherein said hybridization is-carried out at a temperature from 15.degree. C. to 80.degree. C.

### Claim 18 (depends on 17)

The method of claim 17, wherein said temperature is from 50.degree. C. to 55.degree. C.

### Claim 19 (depends on 1)

The method of claim 1, wherein the formamide concentration is 50% and the NaCl concentration is 0.75M.

### Claim 20 (depends on 1)

The method of claim 1, wherein the formamide concentration is 50%, the NaCl concentration is 0.75M, and the sodium citrate concentration is 0.075M.

### Claim 21 (depends on 1)

The method of claim 1, wherein said method is capable of detecting 1 to 5 RNA or DNA molecules per cell.

### Claim 22

A method for assaying target biopolymers in a celluar specimen, said method comprising the steps of:
contacting a cellular specimen with a fixation medium comprising at least one agent that is a precipitating agent or a cross linking agent to yield a fixed spedmen having therein cells with substantially intact membranes;
incubating said fixed Specimen with a hybridization solution comprising a denaturing agent, a hybrid stabilizing agent, a buffering agent, a membrane pore-forming agent and at least two probes each specific for different target biopolymers to be detected with each such probe having a detectable label, said incubating being under hybridizing conditions for not less than five minutes so that cellular polynucleotides of said cellular specimen have shifted to about 70% single-strandedness, wherein each detectable label is selected to be distinguishable from each other detectable label; and
detectingduplexes formed between each said probe and each said different target biopolymer in said cells with substantially intact membranes by means of said detectable labels;
wherein at least two target biopolymers are simultaneously assayed in a cell of the cellular specimen, and
wherein the time for performing said steps of contacting, incubating, and detecting does not exceed about four hours;
whereby said target biopolymers are detected in cells of said cellular specimen;
wherein said shift under hybridizing conditions to about 70% single-stranded cellular polynucleotides while maintaing cellular spatial relationships is deteminable by a process comprising the following four steps:
(a) staining cells with a stain able to stain polynucleotides within said cells to yield a different emission spectrum upon ultraviolet excitation of stained double-stranded and stained single-stranded polynucleotides;
(b) determining the total amount of emitted fluorescence of a cell sample not treated under hybridizing conditions;
(c) determining the total amount of emitted fluorescence of cell samples treated by various fixation and hybridization procedures and times;
(d) assessing when the nmottnt of fluorescence corresponding to the amount of double-stranded nucleic acid has decreased about 70%.

### Claim 23 (depends on 22)

The method of claim 22, wherein:
said target biopolymers comprise:
a target nucleotide sequence, and
a target biopolymer that is not a nucleotide sequence; and wherein;
said probes specific for different target biopolymers to be detected and each bearing a detectable label comprise:
at least one first probe having a nucleotide sequence at least substantially complementary to a specific target nucleotide sequence to be detected, and
at least one second probe having specificity for a target biopolymer that is not a nucleotide sequence;
detecting duplexes formed between said first probe and said target nucleotide sequence and duplexes formed between said second probe and said target biopolymer that is not a polynucleotide by means of said detectable labels;
wherein a target nucleotide sequence and a target biopolymer that is not a polynucleotide are simultaneously assayed in a cell of said fixed cellular specimen, and
wherein said contacting said fixed cellular specimen, said incubating and said detecting is done within four hours.

### Claim 24 (depends on 22)

The method of claim 22, wherein said detectable labels are directly detectable labels.

### Claim 25 (depends on 22)

The method of claim 22, wherein said step of detecting duplexes is accomplished by means of detectable labels which are added after fomation of duplexes is complete.

### Claim 26 (depends on 22)

The method of claim 22, wherein said detectable labels are selected from the group consisting of: radioactive labels, fluorescers, chemilnminescers, and enzyme labels.

### Claim 27 (depends on 22)

The method of claim 22, wherein one of said detectable labels is selected from avidin or streptavidin.

### Claim 28 (depends on 22)

The method of claim 22, wherein said precipitating agent is selected from the group consisting of: ethanol, methanol, acetone, combinations thereof, and combinations of any of the foregoing with formaldehyde.

### Claim 29 (depends on 22)

The method of claim 22, wherein said cross-linking agent is selected from the group consisting of: paraformaldehyde, formaldehyde, dimethylsuberimidate and ethyl-dimethylamino-propylcarbodimide.

### Claim 30 (depends on 22)

The method of claim 22, wherein said denaturing agent is selected from the group consisting of: formarmide, urea, sodium iodide, thiocyanate, guanidine, perchlorate, trichloroacetate and tetramethylamine.

### Claim 31 (depends on 30)

The method of claim 30, wherein said denaturing agent is formamide.

### Claim 32 (depends on 31)

The method of claim 31, wherein said denaturing agent is formamide at a concentration of 20% to 80%.

### Claim 33 (depends on 22)

The method of claim 22, wherein said hybrid stabilizing agent is selected from the group consisting of: sodium chloride, lithium chloride, magnesium chloride and ferric sulfate.

### Claim 34 (depends on 22)

The method of claim 22, wherein said pore-forming agent is a selective pore-forming agent.

### Claim 35 (depends on 22)

The method of claim 22, wherein said pore-forming agent is selected from the group consisting of: polyoxyethylene(23) lauryl ether, polyoxyethylene(20) cetyl ether, 3-((3-cholamidopropyl)-dimethyl amoniuo)-1-propanesulphonate, octylphenoxypolyethoxyethanol, sodium deoxycholate and sodium dodecyl sulfate.

### Claim 36 (depends on 22)

The method of claim 22, wherein said hybridization is carried out at a temperature from 15.degree. C. to 80.degree. C.

### Claim 37 (depends on 36)

The method of claim 36, wherein said temperature is from 50.degree. C. to 55.degree. C.

### Claim 38 (depends on 22)

The method of claim 22, wherein the formamide concentration is 50% and the NaCl concentration is 0.75M.

### Claim 39 (depends on 22)

The method of claim 22, wherein the formamide concentration is 50%, the NaCl concentration is 0.75M, and the sodium citrate concentration is 0.075M.

### Claim 40 (depends on 22)

The method of claim 22, wherein said method is capable of detecting 1 to 5 molecules of a first target biopolymer per cell and 1 to 5 molecules of a second target biopolymer per cell.

### Claim 41

A method of for assaying target polynucleotides in peripheral blood or bone marrow cells, said method comprising the steps of:
depositing a specimen of blood or bone marrow on a solid support;
contacting a cellular specirnen with a fixation medium to yield a fixed specimen having therein cells with substantially intact membranes, said fixation medium comprising at least one agent that is a precipitating agent or a cross-linking agent, said agent being an agent that allows double-stranded cellular polynucleotides to shift unnder hybridizing conditions to about 70% single-stranded cellular polynucleotides while maintaining cellular spatial relationships;
incubating said fixed specimen with a hybridization solution comprising about 20% to 80% formnmide, about 5 times concentrated SSC, about 0.1M tris(hydroxymethyl)aminomethane-HCl , pH 7.4, about 0.1% of octylphenoxypolyethoxyethanol and a photobiotinylated single-stranded anti-sense RNA probe having 75-150 bases at least substantially complementary to a specific target nucleotide sequence to be detected said incubating being under hybridizing conditions so that double-strained cellular polynucleotides shift to about 70% single-stainded cellular polynucleotide, and
adding an agent selected from avidin and streptavidin bearing a detectable label, said agent being at a concentration sufficient to bind said agent to said hybridized probe within a at least five minutes to form a probe-bound agent;
washing said labeled specimen with a solution containing 0.1% octylphenoxypolyethoxyethanol to remove unbound agent; and
detecing probe-bound agent in said cells with substantially intact membranes;
wherein said shift under hybridizing conditions to about 70% single-stranded cellular polynucleotides while maintaining cellular spatial relationships is determinable by a process comprising the following four steps:
(a) staining cells with a stain able to stain polynucleotides within said cells to yield a different emission spectrum upon ultraviolet excitation of stained double-straned and stained single-stranded polynucleotides;
(b) determing the total amount of emitted fluorescence of a cell sample not treated under hybridizing conditions;
(c) determining the total amount of emitted fluorescence of cell samples treated by various fixation and hybridization procedures and times;
(d) assessing when the amount of fluorescence corresponding to the amount of double-stranded polynucleotides has decreased about 70%.

### Claim 42 (depends on 41)

The method of claim 41, wherein said target polynucleotide is an oncogene.

### Claim 43 (depends on 41)

The method of claim 41, wherein said target polynucleotide is a viral gene.

### Claim 44 (depends on 41)

The method of claim 41, wherein said probe comprises a mixture of different probes, each to a different region of said target polynucleotide.

### Claim 45 (depends on 41)

The method of claim 41, wherein said method is accomplished within about 4 hours.

### Claim 46 (depends on 41)

The method of claim 41, wherein said detecting of said bound detectable label is quantitative.

### Claim 47

A method for assaying a target polynucleotide in tissue specimens, said method comprising the steps of:
depositing a tissue specimen on a solid support;
contacting said tissue specimen with a fixation medium to yield a fixed specimen having therein cells with substantially intact membranes while maintaining cellular spatial relationships;
wherein said contacting is performed for at least 20 minutes at a temperature from -20.degree. C. to 50.degree. C., wherein said fixation medium comprises 50% methanol/50% acetone, so as to allow about a 70% shift of double-stranded to single-stranded cellular polynucleotides;
incubating said fixed specimen with a hybridization solution comprising about 20% to 80% formamide, about 5 times concentrated SSC, about 0.1M sodium phosphate, pH 7.4, about 0.1% of octylphenoxypolyethoxyethanol, 20 mM vanadyl ribonucleoside complexes, a photobiotinylated single-stranded anti-sense RNA probe having 75-160 bases at least substantially complementary to a specific target nucleotide sequence to be detected, and low molecular weight denatured DNA at a concentration 100 times greater than the probe concentration said incubating being under hybridizing conditions so that the double-stranded cellular polynucleotides have shifted to about 70% single-strandedness, and
adding a detectably labeled agent selected from avidin and streptavidin at a concentration sufficient to bind to duplexes formed between said probe and said target polynucleotide within at least 5 min to form bound detectably labeled agent;
washing said labeled specimen with a solution containing 0.1% octylphenoxypolyethoxyethanol to remove unbound detectably labeled agent; and
detecting said bound detectably labeled agent in said cells having substantially intact membranes, wherein:
said shift under hybridizing conditions to about 70% single-stranded cellular polyucleotides while maiutnining cellular spatial relationships is determinable by a process comprising the following four steps:
(a) staining cells with a stain able to stain polynucleotides within said cells to yield a different emission spectrum upon ultraviolet excitation of stained double-stranded and stained single-stranded polvnucleotides;
(b) determining the total amount of emitted fluorescence of a cell sample not treated under hybridizing conditions;
(c) determining the total mount of emitted fluorescence of cell snmples treated by various fixation and hybridization procedures and times;
(d) assessing when the amount of fluorescence corresponding to the amount of double-stranded nucleic acid has decreased about 70%.

### Claim 48 (depends on 47)

The method of claim 47, wherein said target polynucleotide is an oncogene.

### Claim 49 (depends on 47)

The method of claim 47, wherein said target polynucleotide is a viral gene.

### Claim 50 (depends on 47)

The method of claim 47, wherein said probe comprises a mixture of different probes, each to a different region of said target pelynucleotide.

### Claim 51 (depends on 47)

The method of claim 47, wherein said method is accomplished within about 4 hours.

### Claim 52 (depends on 47)

The method of claim 47, wherein said detecting of said detectable label is quantitative.

### Claim 53 (depends on 2)

The method of claim 2, wherein said method is capable of detecting as few as one copy of said target nucleotide sequence per cell.

### Claim 54

A method for selection of a fixative and determination of the optimum hybridization conditions for conducting an assay for a target polynucleotide in a cellular specimen, said assay comprising the steps of:
(A) contacting a cellular specimen with a fixation medium to yield a fixed specimen having therein cells with substantially intact membranes, said fixation medium compriagent or a cross-linking agent, said precpitating agent or a cross-linking agent, said agent being an agent that allows double-stranded cellular polynucleotides to shift under hybridizing conditions to about 70% single-stranded cellular polynucleotides while maintaining cellular spatial relationships;
(B) incubating said fixed specimen with a hybriclization solution comprising a denaturing agent, a hybrid stabilizing agent, a buffering agent, a membrane pore-forming agent and at least one probe having a nucleotide sequence at least substantially complementary to a specific target nucleotide sequence to be detected, said incubating being under hybridizing conditions so that double-stranded cellular polynucleotides skift to about 70% single-stranded cellular polynucleotides, and
(C) detecting duplexes formed between said probe and said specific target nucleotide sequence in said cells with substantially intact membranes;
wherein said method is capable of detecting as few as five polynucleotide molecules per cell;
wherein selection of a fixstive comprises the steps of:
(a) harvesting cells from a specimen;
(b) applying a fixative;
(c) incubating said cells with said fixative;
(d) comparing the degree of preservation of cellular morphology after fixation to the morphology prior to fixation;
(e) rating the fixation according to the retention of visual morphology;
(f) rating the fixation according to the preservation of cellular antigens; and
(g) selecting a fixative which scores satisfactorily in terms of preservation of cellular morphology and/or preservation of cellular antigenicity, and
wherein determination of the optimum hybridization conditions comprises the steps of:
(h) contacting cells from a specimen with said fixative selected in step (g);
(i) incubating said fixed specimen with said hybridization solution, said contacting being under hybridizing conditions at a temperature from 15 to 80 degrees Celsius;
(j) determining the shift of double-strandedness to single-strandedness by the cellular polynucleotides in the cells after step (i) has been performed at a desired temperature for a plurality of lengths of time by a process comprising the following four steps:
(I) staining cells with a stain able to stain polynucleotides within said cells to yield a different emission spectrum upon ultraviolet excitation of stained double-stranded and stained single-stranded polynucleotides;
(II) determining the total amount of emitted fluorescence of a cell sample not treated under hybridizing conditions;
(III) determining the total amount of emitted fluorescence of cell samples treated by various fixation and hybridization procedures and times;
(IV) assessing when the svnount of fluorescence corresponding to the amnount of double-stranded nucleic acid has decreased about 70%; and
(k) Selecting the time for the incubating step (B) of the assay method as the time when about 70% of said cellular polynucleotide has shifted to single-stranded in step (j) and selecting the fixation medium of step (A) as the fixative selected in step (g) or, if about 70% of said cellular polynucleotide has not shifted to single-stranded after a desired length of time in step (j), selecting a different fixative in step (g) and performing steps (h) through (k) again.

### Claim 55 (depends on 54)

The method d claim 54, wherein the fixative in step (b) is selected from the group consisting of: 95% ethanol/5% acetic acid, 75% ethanol/20% acetic acid, 50% methanol/50% acetone and 10% formaldehyde/90% methanol.

### Claim 56 (depends on 55)

The method of claim 55, wherein step (i) is performed at a plurality of temperatures in the recited range, whereby the desired temperature for step (j) is determined.

### Claim 57 (depends on 56)

The method of claim 56, wherein step (j) is perfomed for lengths of time from 5 to 120 minutes.