United States Patent [19]
`Stuart et al.
`
`[11] Patent Number:
`[45] Date of Patent:
`
`4,732,847
`Mar. 22, 1988
`
`[54] MONOCLONAL ANTIBODIES FOR
`DNA-RNA HYBRID COMPLEXES AND
`THEIR USES
`
`[75] Inventors: W. Dorsey Stuart, Honolulu, Hi;
`-
`Mark B. Frank, Irvine, Calif.
`[73] Assignee: University of Hawaii, Honolulu, Hi.
`[21] Appl. No.: 454,317
`[22] Filed:
`Dec. 29, 1982
`
`[63]
`
`Related US. Application Data
`Continuation of Ser. No. 271,769, Jun. 9, 1981, aban
`doned.
`
`[51] Int. Cl.‘ ................... .. C12Q 1/68; GOlN 33/577;
`C07K 15/04
`[52] U.S. Cl. ......................................... .. 435/6; 435/7;
`435/68; 435/ 172.2; 435/240.27; 435/188;
`435/810; 436/501; 436/503; 436/504; 436/518;
`436/527; 436/ 543; 436/ 548; 436/ 804; 436/ 808;
`530/387; 530/389; 530/809; 935/104; 935/ 108;
`935/110
`[58] Field of Search ............. .. 436/548, 501, 503, 504,
`436/518, 527, 543, 548, 94, 804, 808, 815, 823;
`424/1, 85, 88, 177, 251, 253; 435/4, 6, 7, 21, 25,
`28, 68, 70, 91, 172, 240, 259, 270, 810, 948, 188;
`260/112 R; 23/915; 530/387, 809, 389;
`935/108, 110, 104
`
`[56]
`
`References Cited
`FOREIGN PATENT DOCUMENTS
`0144914 6/1985 European Pat. Off. .
`0146039 6/1985 European Pat. Off. .
`
`OTHER PUBLICATIONS
`Andrezejewski, C. et al., J. of Immunology, 126(1):
`226-231, (1-1981).
`
`Lerner, E. A. et al., Proceedings of the National Acad
`emy of Sciences, U.S.A., 78: 2737-2741, (5-1981).
`Raap, A. K. et al., Histochemistry, 81:517-520, (1984).
`Boguslawski, A. J. et al., J. Immunol. Methods, 89(1):
`123-130, (1986).
`Stollar, B. D., Science 160:609-611, (1970).
`Andrews, B. S. et al., Journal of Experimental Medi
`cine, 148:1198-1215, (ll-1978).
`Eilat, D. et al., Journal of Immunology, 124(2):766-768,
`(2-1980).
`Andrzejewski, C. et al., Journal of Immunology, 124:
`1499-1502, (3-1980).
`Stuart, W. D. et al., Proc. National Academy of Sci
`ences, vol. 78(6), pp. 3751-3754, (6-1981), (Applicants’
`Publication).
`Frank, M. B. et al., Genetics 94, S33-S34, (1980), ab
`stract (Applicants’ Publication).
`Stuart, W. D. and Porter, D. L., Experimental Cell
`Research, vol. 113, pp. 219-222, (1978).
`Rudkin, G. T. and Stollar, B. D., Nature, vol. 265, pp.
`472-473, (2-1977).
`Primary Examiner-Margaret Moskowitz
`Attorney, Agent, or Firm-Bertram I. Rowland
`[s7]
`ABSTRACI‘
`Monoclonal antibodies are provided capable of distin
`guishing DNA-RNA hybrid complexes from single
`stranded DNA and RNA and double stranded DNA
`and RNA. The antibodies ?nd particular use in deter
`mining the presence of a speci?c nucleic acid sequence
`on a solid surface. Single stranded polynucleotide is
`?xed to a solid (gel) surface and then hybridized with
`the complementary probe. The hybrid complex speci?c
`monoclonal antibody is then added to bind to any hy
`brid complexes which have formed. By appropriate
`label, the hybrid complex may be visualized in a variety
`of ways.
`
`15 Claims, N0 Drawings
`
`Enzo Exhibit 2024
`BD v. Enzo
`Case IPR2017-00172
`
`Exhibit 2024 Page 1
`
`

`

`1
`
`4,732,847
`
`MONOCLONAL ANTIBODIES FOR DNA-RNA
`HYBRID COMPLEXES AND THEIR USES
`
`CROSS REFERENCE TO RELATED
`APPLICATIONS
`This is a continuation application of pending prior
`application Ser. No. 271,769, ?led June 9, 1981, now
`
`abandoned.
`
`'
`
`10
`
`BACKGROUND OF THE INVENTION
`1. Field of the Invention
`The discovery of monoclonal antibodies created the
`opportunity to prepare compositions which could bind
`to a speci?c polar and spatial organization, referred to
`as an epitopic site or determinant site. Polyclonal anti
`bodies had previously found a broad spectrum of appli
`cations, particularly in the diagnostic area. The hetero
`geneity of the polyclonal antisera was conceived to 20
`provide advantages and disadvantages to the speci?city
`of the antisera. The polyclonal antibodies provided an
`average response to a particular structure, which could
`have the cumulative effect of high speci?city, high
`binding constant and high titer. However, monoclonal
`antibodies could provide a number of unique opportuni
`ties with antigenic compositions which have a plurality
`of determinant sites. By being able to select for a spe
`ci?c determinant site, rather than having a mixture of
`antibodies capable of recognizing a plurality of determi
`nant sites, new approaches to the detection of a variety
`of antigens became possible.
`For the most part, the use of monoclonal antibodies
`has been directed to the detection of antigens, macro
`molecular proteins having a plurality of determinant
`sites. The potential for using monoclonal antibodies in
`other situations has not received attention.
`2. Description of the Prior Art
`Stuart and Porter (1978) Exp. Cell Res. 113, 219-222
`describe the production of antibodies to RNA-DNA
`hybrid duplexes for forming in situ hybrids on polytene
`40
`chromosomes as an antigenic test system. Frank et al.
`(1980) Genetics 94, s33-s34 (abstr.) reported the prepa
`ration of monoclonal antibodies in progress. Rudkin and
`Stollar (1977) Nature (London) 265, 472-473 describe
`polyclonal antibody binding to polytene chromosomes.
`Stollar (1970) Science 169, 609-611 describes the prepa
`ration of rabbit antibodies to poly(rA).poly(dT) hy
`brids.
`
`2
`subject method can be used for the determination of a
`particular DNA or RNA sequence using a variety of
`techniques and labels. The subject method can ?nd
`application in hybrid DNA technology, disease diagno
`sis, gene identi?cation, DNA and RNA isolation, etc.
`The monoclonal immunoglobulins may be any of the
`conventional immunoglobulins or fragments thereof.
`Included among the immunoglobulins are IgA, IgD,
`IgE, IgG and IgM. For the most part, the immunoglob
`ulins of interest will be IgG and IgM. In some instances,
`it may be desirable to use fragments of immunoglobu
`lins, particularly Fab and F(ab’)2 or other convenient
`fragment which maintains the desired speci?city.
`The monoclonal antibodies can be prepared by the
`15 method of 0i and Herzenberg (1980) in Selected Meth
`ods in Cellular Immunology, eds. Mishell and Shiigi
`(Freeman, San Francisco) pp. 351-372 While any verte
`brate may be used, for the most part mice ?nd use.
`Conveniently, the mouse is immunized against the du
`plex by having DNA-RNA hybrid duplexes bound to
`an antigen and the antigen injected into the mice in
`accordance with conventional ways. After a sufficient
`time, the spleen is removed from the mouse and the
`splenocytes crossed with an appropriate myeloma fu
`sion partner to produce hybridomas. The hybridomas
`may then be used for injection into the peritoneal cavity
`of a mouse for production of ascites ?uid, which may be
`extracted and used as an enriched source of the desired
`monoclonal antibodies.
`The subject method may be used for the detection of
`speci?c sequences of nucleic acids from a wide variety
`of sources. Normally, a unique sequence will require at
`least 12. nucleotides, more usually about 18 nucleotides
`and the complementary strand or probe may be of any
`size. As a matter of convenience, usually the comple
`mentary strand or probe will be less than about 10,000
`nucleotides, more usually less than about 5,000 nucleo
`tides, but this is primarily a matter of convenience and
`not critical to the operability of the subject invention.
`The source of the DNA may be single stranded or dou
`ble stranded DNA. The DNA may be chromosomal
`DNA, including nuclear, mitochondrial, plastid e. g.
`chloroplast, viral, phage, or the like. Alternatively, the
`DNA may be from an extrachromosomal source, such
`as plasmids, double minules, transposons, ars containing
`fragments, or the like.
`In addition to elements which may be stably main
`tained in a host cell, DNA fragments of varying size
`may also be detected. The fragments may be naturally
`occurring or synthetic, may involve wild type, allelic or
`mutant genes, including structural genes, regulatory
`' genes, multimers, inverted repeat features, or the like.
`RNA can come from various sources, both natural or
`synthetic. Various types of RNA include messenger
`RNA, ribosomal RNA, nucleolar RNA, transfer RNA,
`viral RNA and heterogeneous nuclear RNA, or the
`like. In addition, whole naturally occurring entities or
`fragments thereof may be involved.
`To form the hybrid complex, DNA or RNA sequen
`ces complementary to the sequence of interest will be
`employed. These can be derived from naturally occur
`ring sequences, employing the entire sequence or frag
`ment thereof. Single strands will be employed which
`will normally be of at least 12 nucleotides, more usually
`of at least about 18 nucleotides, and can be of 30 nucleo
`tides or greater. Particularly, where the nucleotide se
`quence is obtained from a naturally occurring source,
`
`25
`
`30
`
`35
`
`45
`
`SUMMARY OF THE INVENTION
`Monoclonal antibodies highly speci?c for hybrid
`DNA-RNA duplexes are provided. The monoclonal
`antibodies may be labeled directly or indirectly to pro
`vide means for detecting DNA or RNA, which may be
`a fragment or a portion of a much larger polynucleo
`tide, such as a chromosome. By in situ denaturation and
`hybridization of double stranded DNA, the single
`strands may be affixed to a solid support, then com
`plexed with RNA and detected using labeled mono
`clonal antibody, labeled either directly or indirectly,
`with a label which provides a detectable signal.
`
`55
`
`DESCRIPTION OF THE SPECIFIC
`EMBODIMENTS
`The subject invention is concerned with the prepara
`tion and use of monoclonal antibodies speci?c for hy
`brid DNA-RNA duplexes in detecting the presence of a
`speci?c sequence employing in situ hybridization. The
`
`65
`
`Exhibit 2024 Page 2
`
`

`

`20
`
`25
`
`4,732,847
`4
`3
`Hybridization will be carried out under conditions
`large sequences can be obtained as naturally occurring
`which favor hybrid DNA complexing between RNA
`e.g. messenger RNA, as fragments by mechanical shear
`and DNA. Various hybridization buffer solutions may
`ing or restriction enzyme cleavage of nucleic acids,
`be employed as described in Wahl et al, supra; Stuart
`such as chromosomes, or the like. Single strands can be
`and Porter, supra Normally, solutions of saline sodium
`isolated by denaturation of double stranded DNA under 5
`citrate containing from about 30 to 60% formamide ?nd
`elevated temperature conditions in an aqueous medium,
`normally having such additives as formamide, salts, or
`use. Other additives may include sodium dodecylsul
`fate, ethylene diamine tetraacetic acid, ?coll (about
`the like to enhance the rate of melting of the RNA and
`300-500 kdaltons), polyvinyl pyrrolidone (about
`maintain its single stranded character.
`In many cases it will be convenient and desirable to 10
`250-500 kdaltons), serum albumin, dextran sulfate
`(about 10,000 to 1,000,000 kdaltons), glycine (about
`use messenger RNA as a probe. The messenger RNA
`may be used intact or may be cleaved to provide smaller
`0.5~2% wt/vol), and about 0.5—5 mg/ml of sonicated
`fragments. Messenger RNA may be isolated from other
`denatured DNA e.g. calf thymus or salmon sperm.
`RNA by passage through an oligo-dT column. Where
`The temperature for the hybridization will generally
`the sequence of interest is a major expression product, 15
`vary from about room temperature to up to about 70°
`the messenger RNA is particularly useful. However,
`C., more usually from about 30° to 50° C.
`where the DNA has introns, the use of messenger RNA
`The hybridization can be terminated by cooling, fol
`may not prove useful. In many situations, therefore,
`lowed by washing or by washing at a temperature at a
`synthetic sequences may be prepared or once a se
`range of about 20° to 75° C. with an appropriate buff
`quence is determined, it may be readily cloned and used
`ered solution, such as phosphate buffered saline or sa
`repetitively.
`line sodium citrate, containing minor amounts of vari
`The probe may be a single composition or a mixture
`ous other additives such as sodium dodesyl sulfate, and
`of compositions which may be segregated by molecular
`the like.
`weight range, migratory aptitude in an electric ?eld,
`The identi?cation of the presence of the hybrids may
`density, or combinations thereof. Various techniques
`now be achieved by employing monoclonal antibodies
`can be used for the puri?cation of sequences or probes,
`speci?c for the hybrid complex. Detection can be
`such as electrolphoresis, sucrose or cesium chloride
`achieved by labeling either the monoclonal antibody
`gradient fractionation, treatment with a speci?c binding
`speci?c for the hybrid DNA-RN A complex, hereinafter
`column, such as oligo-dT for messenger RNA, combi
`referred to as “anticomplex” or by employing labeled
`nations thereof, or the like.
`antibodies which bind to the anticomplex. For example,
`The single stranded polynucleotide will be ?xed to a
`where the monoclonal antibody is derived from a
`solid support, either covalently or non-covalently. By
`mouse, antibodies to mouse antibodies e.g. rabbit anti(
`?xed is intended that under the conditions of the hybrid
`mouse IgG), could be labeled so as to bind to any an
`ization and assay, there will be no signi?cant migration
`ticomplex bound to the complex bound to the solid
`of the polynucleotide. By solid support is intended both
`support.
`solid and semi-solid supports. Supports can include
`A wide variety of labels have been used in other
`glass slides, container walls, membranes, paper, gels,
`environments which would be applicable here. One of
`and the like. The solid supports may be coated or func
`the more common labels is radionuclides, which can be
`tionalized to provide better adherence of the single
`used with autoradiography to visualize the areas of
`stranded polynucleotide to the support.
`binding. Another label is a fluorescer e.g. fluorescein,
`Various techniques may be employed for preparation
`merocyanine, rhodamine, etc., which by irradiation
`of the sample for in situ hybridization. In one technique,
`with light of excitation, the presence of fluorescence
`particularly with chromosomes or other large polynu
`can be monitored. Alternatively, an enzyme can be used
`cleotides, the DNA is ?xed with a mildly acidic aqueous
`which results in a product which can be detected and
`solution e.g. from about 25 to 75% of a carboxylic acid
`localized in the area of the enzyme. A large number of
`and then frozen. Post ?xing involves employing a
`dyes or metals capable of reduction can be employed to
`mildly acidic alkanolic solution e.g. ethanol-acetic acid,
`provide detection. Common enzymes include horserad
`followed by storage in an alkanol, e. g. ethanol. The
`ish peroxidase, glucose oxidase, ,B-galactosidase, or the
`usual treatment involves an alkanolic-carboxylic acid
`like. The particular label or manner in which the detect
`mixture which may vary from about l:3—3:l, followed
`able signal is observed is not critical to this invention.
`by freezing and then storage in an alkanol e.g. 95%
`Evidently, by employing antibodies to the anticomplex,
`ethanol.
`the number of labels associated with a particular bind
`Alternatively, colony hybridization may be em
`ing of the anticomplex to the complex can be greatly
`ployed as described in the now classical paper by Grun
`ampli?ed.
`stein and Hogness, Proc. Nat. Acad. Sci. USA. 72, 5S
`The following examples are offered by way illustra
`396l~3965 (1975). Colonies are formed on ?lters with
`tion and not be way of limitation.
`replica plating on an agar plate. Solutions are applied to
`the underside of the ?lter to diffuse into the colony for
`lysis employing strong base, followed by proteolysis
`with an appropriate protease and heating at an elevated 60
`temperature to fix the DNA.
`An alternative technique may be employed using a
`gel for separation of the nucleotides by electrophoretic
`mobility. For RNA, the RNA is normally pretreated to
`remove interfering reagents. The RNA or DNA con- 65
`tained in the gel is then transferred to diazo-substituted
`paper. See Wahl et al. Proc. Natl. Acad. Sci. USA 76,
`3683-3687 (1979).
`
`EXPERIMENTAL
`Chromosomal Preparations
`Salivary glands from larvae were excised in Grace’s
`insect medium (GIBCO), ?xed for 8-10 min. in me
`thanol/acetic acid, 3:1 (vol/ vol), placed on acid washed
`slides with one drop of 45% acetic acid, and squashed
`under siliconized coverslips. The preparations were
`held at —20° C. for at least 30 min and then immersed in
`liquid nitrogen. After 1 min. the slides were withdrawn
`and the coverslips were removed by inserting a no. 10
`
`30
`
`35
`
`45
`
`50
`
`Exhibit 2024 Page 3
`
`

`

`4,732,847
`5
`surgical blade between the slide and the coverslip. The
`slides were immediately immersed in 95% ethanol. The
`slides remained in ethanol for at least 4 hr. prior to the
`in situ hybridization. Preparations may be stored in
`ethanol for up to 2 weeks without deterioration or
`structure of hybridization results.
`
`6
`Antibody Production
`Rabbit antibody to poly(rA).poly(dT) hybrids com
`plexed to methylated bovine serum albumin was pre
`pared by the method of Stollar, (1970) Science 169,
`609-611. Mouse monoclonal hybrid cell cultures to the
`same antigen were prepared by the method of Oi and
`Herzenberg, supra. immunized spleen cells were fused
`with P3-NS-l BALB/c myeloma cells (originally de
`rived from the BALB/c-MOPC-2l cell line) provided
`by Douglas Vann. Positive fusion cultures were cloned
`by the limiting dilution method and a number of hy
`bridomas were obtained. One positive clone (FS.B12
`G10) was chosen for use in this study.
`Identi?cation of Hybrids
`Regions of in situ hybridization were detected by
`secondary immuno?uorescence as reported by Stuart
`and Porter, supra. Goat anti-rabbit and goat anti-mouse
`Ig antisera conjugated with ?uorescein isothiocyanate
`were purchased from ‘Antibodies, Inc. Preparations
`were examined under a Zeiss dark-?eld transmitted
`?uorescence system (quartz/halogen source, KP 500
`excitation ?lter (Zeiss), and 530 nm barrier ?lter). Pho
`tomicrographs were taken with Tri-X Pan ?lm (Kodak
`ASA 400).
`The rabbit antiserum and mouse hybridoma antibod
`ies were tested for speci?city. When no RNA probe
`was present during the hybridization and the prepara
`tion was redigested with RNase A, no secondary ?uo
`rescent bands were observed. When the RNA normally
`present in the polytene preparation was not predigested
`with RNase A, multiple ?uorescent bands were ob
`served after hybridization. This evidences that the anti
`bodies were not reactive with chromosomal DNA but
`were able to bind to RNA-DNA duplexes. Absorption
`of antibody reagents with single stranded poly(rA)
`methylated albumin complexes with poly(dT)
`methylated albumin complexes did not reduce immuno
`?uorescent visualization of in situ RNA-DNA hybrids.
`Absorption of antibody reagents with poly(rA)
`poly(dT) duplexes complexed to methylated albumin
`removed all activity responsible for the secondary im
`muno?uorescent bands. Therefore, the antibodies rec
`ognized RNA-DNA hybrids, but not other forms of
`RNA or DNA.
`Several hundred preparations from both D. silvesteris
`and D. heteroneura were examined for localization of
`.the 18/288 rRNA locus. After the ?uorescent regions
`were hybridized, a phase condenser was inserted into
`the system and phase-contrast photomicrographs were
`taken. The coverslip was then removed and the prepa
`ration stained with acetoorcein. A fresh coverslip was
`then added to the stained preparation, producing a con
`ventionally banded aspect. This procedure allowed the
`placement of the in situ hybrid band uneqivocally at the
`chromosome three locus.
`In mature larval cells the nucleolus showed little or
`no ?uorescence. In cells obtained from younger larvae
`?uorescent staining of the nucleolus was very strong.
`This indicates that either the DNA sequences for
`18/288 rRNA are present in the nucleolus during early
`larval development but are not available for hybridiza
`tion at the third-instar larval stage immediately prior to
`pupation or the subject method is not capable of visual
`izing the sequences in the enlarged nucleolus. Hybridi
`zation of 28S rRNA to the young larval cells identi?ed
`with mouse monoclonal antibodies was evidenced by
`
`20
`
`35
`
`40
`
`RNA Probe
`RNA was isolated from larvae quick-frozen in liquid
`nitrogen and stored at —70° C. for up to 2 weeks. The
`isolation method of Kirby ((1968) in Methods Enzymol.
`12, 94-95) was used with the following modi?caticns,
`sodium triisopropylnaphthalenesulfonate was substi
`tuted for sodium p-toluenesulfonate; the phenol/cresol
`extraction was repeated twice; and 3 M NaCl was sub
`stituted for 3 M sodium acetate to remove glycogen and
`tRNA. The high molecular weight RNA salt precipi
`tate was resuspended and reprecipitated with 3 M NaCl
`a total of three times. The ?nal precipitate was resus
`pended and made 0.12 M in NaCl, 0.01 M in Tris.HCl at
`pH 7.6, 1 mM in EDTA, and 0.02% in sodium dodecyl
`sulfate at an RNA concentration of 2 mg/ml and ap
`plied to an oligo(dT)-cellulose column to remove
`polyadenylated RNA species (Mezl and Hunt (1978)
`Biochem. J. 141, 617-625). RNA not adsorbed to the
`25
`column was collected and precipitated with 2.5 vol of
`ethanol at —20° C. overnight. The precipitate was col
`lected by centrifugation and resuspended at a concen
`tration of 800 ug/ml in 0.01 M Tris.HCl, pH 7.4/1 mM
`EDTA/0.l M NaCl. At this point the RNA sample was
`30
`further puri?ed by either (a) sucrose gradient fraction
`ation or (b) preparative electrophoresis as follows.
`(a) A 500 pl sample of RNA was heated to 38° C. for
`5 min. and applied directly to a 17 ml linear sucrose
`gradient (5—20%) made in the same buffer. Heating to
`65° C. was avoided because this treatment results in the
`denaturation of the 28S rRNA species found in insects
`and the denatured RNA comigrates with the 18S spe
`cies during centrifugation. The gradient was centri
`fuged in a Beckman instrument using an SW27.1 rotor
`at 25,000 rpm for 21.5 hr at 4° C. Fractions were col
`lected and those containing 28S RNA were pooled,
`precipitated, and resuspended in hybridization solution
`(1:1 mixture of fornamide and 0.6 M NaCl/0.06 M Na
`citrate, pH 7).
`45
`(b) RNA (230 pg) was applied in 2.5 ml of sample
`buffer to a 6 M urea/ 1% agarose gel (53><l20 mm)
`formulated by the method of Long and David (1980)
`Cell 18, 1185-1196. The gel was run for 8 hr at 95 V(l00
`mA) and then stained for 15 min. with ethidium bro
`mide. The portion of gel containing the large fragment
`of the denatured 28S rRNA was placed in a dialysis bag
`with 3 ml of gel buffer and electroeluted from the aga
`rose for 3 hr. The eluted 28S fragment was precipitated
`with ethanol and resuspended in hybridization solution.
`RNA puri?ed by either method gave similar hybridi
`zation results.
`
`50
`
`55
`
`In Situ Hybridization
`Hybrids were prepared as reported by Stuart and
`Porter, supra. Estimates of hybridization rates were
`obtained by incubating preparations (sealed with rubber
`cement) at 70° C. for 1 hr and then immediately shifting
`the preparations to 40° C. by immersion in a thermostat
`ted water bath. C,t values were calculated as initial
`concentration of the RNA probe in mol per liter multi
`plied by time(sec) of incubation at 40“ C. The hybridiza
`tion was terminated by immersion in an ice-water bath.
`
`60
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`4,732,847
`8
`7
`secondary immuno?uorescence with counterstaining
`3. Mouse IgM monoclonal antibodies according to
`by ?uorescence microscopy with 1500 magni?cation.
`claim 1. 1.
`4. Monoclonal antibodies according to claim 1 la
`Hybridization to a single band in the genome is visible.
`Hybridization was performed at C,t values ranging
`beled with a member of the group consisting of radionu
`from 1X 10-4 to 4x101 to determine if the rate of hy
`clides, ?uorescers or enzymes.
`5. A method for detecting the presence of a nucleo
`bridization was consistent with . that expected for
`tide sequence which comprises:
`18/28S rRNA. Results have been reported by Szabo et
`a1 (1977) J. Mol. Biol. 115, 539-563, reporting C,t
`fixing a DNA or RNA sequence to a solid support as
`a single strand;
`curves and rate constants for both 58 and l8/28S rRNA
`hybridizing said ?xed single stranded nucleic acid
`of D. melanogaster hybridized in situ under conditions
`similar to those used here. Faint ?uorescence of both
`sequence with a sequence of the other type of nu
`cleic acid having a predetermined nucleic acid
`the nucleolus and the chromosome 3 band at a C,t value
`sequence to bind to any homologous ?xed se
`of 1X 10"-2 was observed.
`quence to form a hybrid duplex;
`The subject invention provides for an accurate and
`adding to said hybrid duplex mouse IgG or IgM
`simple technique for detecting the presence of a particu
`lar nucleic acid sequence by hybridization in situ. The
`monoclonal antibodies which speci?cally bind to
`DNA-RNA hybrid complexes, whereby said
`use of monoclonal antibodies substantially precludes
`signi?cant binding to single stranded nucleic acids for
`monoclonal antibodies bind to hybrid duplexes
`bound to said solid support; and
`double stranded DNA or RNA. Thus, by employing a
`determining the presence of monoclonal antibody
`probe having a complementary sequence to the alterna
`tive type of nucleic acid e.g. DNA with RNA, the
`bound to said solid support by means of a label
`providing a detectable signal, which label is bound
`presence of the sequence either by itself or as part of a
`directly or indirectly to said monoclonal antibody.
`much larger sequence may be readily detected. Further
`6. A method according to claim 5, wherein said ?xed
`more, by appropriate use of hybridization conditions
`nucleotide sequence is DNA.
`and rates, one can readily determine the proportionate
`homology of the sample to the probe. The subject
`7. A method according to claim 6, wherein said ?xed
`method and compositions therefore provides great ?exi
`nucleotide sequence is chromosomal.
`8. A method according to claim 5, wherein said ?xed
`bility in being able to determine the presence of a partic
`ular nucleic acid sequence, having application for the
`nucleotide sequence is RNA.
`detection of genes, structural, regulatory or expressing
`9. A method according to claim 8, wherein said RNA
`RNA the presence of mutations, and the like.
`is messenger RNA.
`Cell lines identi?ed in the present application as G10
`10. A method according to any one of claims 5, 6 or
`H4, G11 K3, and A6 P3 have been deposited with the
`8, wherein said label is bound to an antibody capable of
`speci?cally binding to said monoclonal antibody bound
`American Type Culture Collection, 12301 Parklawn
`to said hybrid complex.
`Drive, Rockville, Md., 20852, where they have respec
`tively been granted the accesion numbers HB 8076, HB
`11. A method according to claim 10, wherein said
`8077, and HB 8078.
`label is a radionuclide.
`Although the foregoing invention has been described
`12. A method according to claim 10, wherein said
`some detail by way of illustration and example for pur
`label is a ?uorescer.
`poses of clarity of understanding, it will be obvious that
`13. A method according to claim 10, wherein said
`certain changes and modi?cations may be practiced
`label is an enzyme.
`within the scope of the appended claims.
`14. A kit for determining the presence in situ of a
`hybrid DNA-RNA complex which comprises primary
`Although the foregoing invention has been described
`IgM or IgG mouse monoclonal antibodies having the
`in some detail by way of illustration and example for
`property of speci?cally binding to said complex but not
`purposes of clarity of understanding, it will be obvious
`that certain changes and modi?cations may be practiced
`to isolated DNA or RNA and secondary antibodies
`having the property speci?cally binding to said primary
`within the scope of the appended claims.
`What is claimed is:
`antibodies, said secondary antibodies having a label
`capable of providing a detectable signal.
`1. IgM or IgG mouse monoclonal antibodies which
`speci?cally bind to DNA-RNA hybrid complexes but
`15. A kit according to claim 14, wherein said label is
`selected from the group consisting of radionuclides,
`which do not bind to isolated RNA or DNA.
`2. Mouse IgG monoclonal antibodies according to
`?uorescers or enzymes.
`claim 1.
`* * Ill
`
`5
`
`25
`
`40
`
`45
`
`50
`
`55
`
`65
`
`4‘
`
`it
`
`Exhibit 2024 Page 5
`
`