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FIELD OF THE INVENTION
This invention relates to nucleic acid hybridization assay methods and
reagent systems for detecting specific polynucleotide sequences. The
principle of nucleic acid hybridization assays was developed by workers in
the recombinant DNA field as a means for determining and isolating
particular polynucleotide base sequences of interest. It was found that
single stranded nucleic acids, e.g., DNA and RNA, such as obtained by
denaturing their double stranded forms, will hybridize or recombine under
appropriate conditions with complementary single stranded nucleic acids.
By labeling such complementary probe nucleic acids with some readily
detectable chemical group, it was then made possible to detect the
presence of any polynucleotide sequence of interest in a test medium
containing sample nucleic acids in single stranded form.
In addition to the recombinant DNA field, the analytical hybridization
technique can be applied to the detection of polynucleotides of importance
in the fields of human and veterinary medicine, agriculture, and food
science, among others. In particular, the technique can be used to detect
and identify etiological agents such as bacteria and viruses, to screen
bacteria for antibiotic resistance, to aid in the diagnosis of genetic
disorders such as sickle cell anemia and thalassemia, and to detect
cancerous cells. A general review of the technique and its present and
future significance is provided in Biotechnology (August 1983), pp.
471-478.
BACKGROUND INFORMATION
The following information is provided for the purpose of making known
information believed by the applicant to be of possible relevance to the
present invention. No admission is necessarily intended, nor should be
construed, that any of the following information constitutes prior art
against the present invention.
The state-of-the-art nucleic acid hybridization assay techniques generally
involve immobilization of the sample nucleic acid on a solid support.
Hybridization between particular base sequences or genes of interest in
the sample nucleic acid is determined by separating the solid support from
the remainder of the reaction mixture which contains unbound labeled
probe, followed by detection of the label on the solid support.
The need to immobilize sample nucleic acids in order to conduct the
state-of-the-art hybridization assay poses two significant problems.
Firstly, the procedures required to accomplish immobilization are
generally time consuming and add a step which is undesirable for routine
use of the technique in a clinical laboratory. Secondly, proteins and
other materials in the heterogeneous sample, particularly in the case of
clinical samples, can interfere with the immobilization of the nucleic
acids.
As alternatives to immobilizing sample nucleic acids and adding labeled
probe, one can use an immobilized probe and label the sample nucleic acids
in situ, or one can use a dual hybridization technique requiring two
probes, one of which is immobilized and the other labeled [Methods in
Enzymology 65:468(1968) and Gene 21:77-86(1983)]. The former alternative,
however, is even less desirable since the in situ labeling of the sample
nucleic acids requires a high degree of technical skill which is not
routinely found in clinical technicians and there are no simple, reliable
methods for monitoring the labeling yield, which can be a significant
problem if the labeling media contain variable amounts of inhibitors of
the labeling reaction. The dual hybridization technique has the
disadvantages of requiring an additional reagent and incubation step and
the kinetics of the hybridization reaction can be slow and inefficient.
The accuracy of the assay can also be variable if the complementarity of
the two probes with the sample sequence is variable.
Techniques for directly detecting the polynucleotide duplex formed as the
product of hybridization between the sample and probe polynucleotides, and
thereby dispensing with the chemical labeling and immobilization of sample
or probe polynucleotides, have been generally unsatisfactory. Attempts to
generate antibodies which will selectively bind double stranded DNA.DNA
hybrids over single stranded DNA have failed [Parker and Halloran,
"Nucleic Acids in Immunology", ed. Plescia and Braun, Springer-Verlag,
N.Y. (1969) pp. 18 et seq]. Some success has been achieved in generating
antibodies that will bind DNA.RNA mixed hybrids or RNA.RNA hybrids and
have low affinity for the single stranded polynucleotides [see, for
example, Rudkin and Stollar, Nature 265:472(1977)]. Rudkin and Stollar
fixed whole cells on microscope slides and exposed the DNA in the nucleus.
It was hybridized with an RNA probe and the hybrid was detected by
fluorescence microscopy with fluorescein-labeled antibody to DNA.RNA.
However, these methods are described, as in the case of the hybridization
techniques discussed above employing labeled probes, as requiring
immobilization of the sample nucleic acids. Immobilization of cellular DNA
for in situ hybridization is particularly tenuous because the DNA must
remain fixed to delicate cell residues during the hybridization and
immunochemical detection steps. The results observed by fluorescence
microscopy do not give quantitative data on the amount of hybrid formed.
Accordingly, there is an established need for a nucleic acid hybridization
assay which does not require the immobilization or labeling of sample
nucleic acids, and which does not require dual probes. Further, such
technique should allow the use of a variety of labels, particularly of the
nonradioisotopic type. A nucleic acid hybridization assay method and
reagent system having these and other advantages are principal objectives
of the present invention.
SUMMARY OF THE INVENTION
A nucleic acid hybridization assay method has now been devised which
eliminates the need to immobilize or label sample nucleic acids and which
requires but a single probe element. The present invention provides a
method for determining a particular polynucleotide sequence in an
appropriate test medium containing single stranded nucleic acids. The test
medium is combined with an immobilized or immobilizable polynucleotide
probe, comprising at least one single stranded base sequence which is
substantially complementary to the sequence to be determined, under
conditions favorable to hybridization between the sequence to be
determined and the complementary probe sequence. The complementary probe
sequence will be selected to be substantially composed of RNA when the
sequence to be determined is RNA or DNA, that is, such probe sequence can
be selected to be RNA whether the sample sequence of interest is RNA or
DNA. Alternatively, when the sample sequence of interest is RNA, the
complementary probe sequence can be selected to be substantially composed
of either DNA or RNA. Thus, hybrids resulting from hybridization between
the probe and the sample sequence will be DNA.RNA or RNA.RNA duplexes.
The resulting hybrids can then be detected, after or simultaneously with
immobilization of the probe where such was combined with the test medium
in an immobilizable form, by addition of an antibody reagent capable of
binding to the DNA.RNA or RNA.RNA duplexes formed and determining the
antibody reagent that becomes bound to such duplexes. A variety of
protocols and reagent combinations can be employed in order to carry out
the principles of the present method. Important features of the present
invention are that the sample nucleic acids are not immobilized or
required to be labeled before contact with the probe.
The antibody reagent is the key to specific and sensitive detection of
hybridization between the probe and sample nucleic acids. Of course, whole
antibodies or appropriate fragments and polyfunctional forms thereof can
be used as described more fully below, and it will be understood that,
when used in this disclosure and the claims which follow, the term
antibody reagent will mean whole antibodies and their polyfunctional and
fragmented forms as well, unless otherwise noted.
Determination of binding of the antibody reagent to hybridization duplexes
can be accomplished in any convenient manner. It is preferred that the
antibody reagent be labeled with a detectable chemical group such as an
enzymatically active group, a fluorescer, a chromophore, a luminescer, a
specifically bindable ligand, or a radioisotope, the nonradioisotopic
labels being especially preferred. The labeled antibody reagent which
becomes bound to resulting immobilized hybrid duplexes can be readily
separated from that which does not become so bound and the detectable
chemical group or label is measured in either separated fraction, usually
the former.
By eliminating the need to immobilize or label the sample nucleic acids,
the present invention provides a highly advantageous hybridization assay
technique. The analyst is not required to have the high level of skill or
to take the requisite time to perform the immobilization or labeling
procedures. Moreover, there is complete elimination of the potential for
sample interferences with the immobilization procedure. The test kit
provided to the clinical user would include the probe already immobilized
or in a readily immobilizable form such as by binding to an immobilized
binding partner. In the prior art systems, interferences from extraneous
proteins and other materials in the sample can be a serious problem
whether the sample nucleic acids to be immobilized are RNA or DNA.
In the prior art methods, immobilization is accomplished by adsorption onto
a microporous membrane, such as nitrocellulose, or by covalent bonding to
reactive sites on a solid support. In the first case, proteins from the
sample can coat the surface and block the adsorption of nucleic acids.
Furthermore, many procedures require baking at elevated temperatures,
commonly higher than 80.degree. C., in vacuo to fix adsorbed nucleic acids
to the support. If mucus or other materials endogeneous to the sample are
present, they can become dried to the support to form a film that can
adsorb the labeled probe during hybridization and increase the background
signal and consequently decrease sensitivity. Also, if an enzyme or other
protein is involved in the detection of the label, it can often bind
nonspecifically to the film and contribute even further to the background
problem. If covalent immobilization is employed, proteins and other
materials from the sample can be expected to have available reactive
groups which will engage in the coupling reaction and neutralize the
coupling of the desired nucleic acids.
Since the present invention provides the probe in preferred embodiments in
an already immobilized form or in a form which is readily immobilized by
binding to an immobilized binding partner, the inefficiencies inherent in
the prior art immobilization procedures are overcome and thus the
detection limits of the assay are maintained. A further advantage is that
nonspecific binding of sample RNA or DNA to the solid support will not be
recognized by the antibody reagent. Therefore, the background signal will
be low and the detection limit accordingly improved. Relative to the dual
hybridization method which uses both a labeled probe and an immobilized
probe, the labeled nucleotide can bind nonspecifically to the solid
support and contribute background signal. This is not a possibility in the
present method since no labeled probe is involved.
BRIEF DESCRIPTION OF THE DRAWINGS
The drawings are schematic representations of preferred methods for
performing the present invention. The use of nucleic acid hybridization as
an analytical tool is based fundamentally on the double stranded, duplex
structure of DNA. The hydrogen bonds between the purine and pyrimidine
bases of the respective strands in double stranded DNA can be reversibly
broken. The two complementary single strands of DNA resulting from this
melting or denaturation of DNA will associate (also referred to as
reannealing or hybridization) to reform the duplexed structure. As is now
well known in the art, contact of a first single stranded nucleic acid,
either DNA or RNA, which comprises a base sequence sufficiently
complementary to a second single stranded nucleic acid under appropriate
solution conditions, will result in the formation of DNA.DNA, DNA.RNA, or
RNA.RNA hybrids, as the case may be.
In the embodiment depicted in FIG. 1 of the drawings, the single stranded
sample nucleic acids are brought into contact with the immobilized probe
under favorable hybridization conditions. The resulting immobilized,
hybridized duplexes, optionally after separating such duplexes from the
remainder of the reaction mixture, are contacted with a labeled form of
antibodies specific for the DNA.RNA or RNA.RNA duplexes. After washing to
remove unbound labeled antibody, the label present on the solid support is
measured.
In the embodiment depicted in FIG. 2 of the drawings, the single stranded
sample nucleic acids are contacted with a soluble form of the probe which
has been appropriately chemically modified to comprise bindable biotin
moieties. To the resulting soluble hybrids that are formed is added an
immobilized form of avidin, a binding partner for biotin, resulting in
formation of immobilized hybrids. The thus immobilized duplexes,
optionally after separating them from the remainder of the reaction
mixture, are contacted with labeled anti-hybrid antibodies, and after
washing, the label present on the solid support is measured as above.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The Probe
The probe will comprise at least one single stranded base sequence
substantially complementary to the sequence to be detected. However, such
base sequence need not be a single continuous polynucleotide segment, but
can be comprised of two or more individual segments interrupted by
noncomplementary sequences. These nonhybridizable sequences can be linear,
or they can be self-complementary and form hairpin loops. In addition, the
complementary region of the probe can be flanked at the 3'- and 5'-termini
by nonhybridizable sequences, such as those comprising the DNA or RNA of a
vector into which the complementary sequence had been inserted for
propagation. In either instance, the probe as presented as an analytical
reagent will exhibit detectable hybridization at one or more points with
sample nucleic acids of interest. Linear or circular single stranded
polynucleotides can be used as the probe element, with major or minor
portions being duplexed with a complementary polynucleotide strand or
strands, provided that the critical homologous segment or segments are in
single stranded form and available for hybridization with sample DNA or
RNA, and provided that the antibody reagent selected for use with the
probe does not significantly crossreact with the double stranded regions
in the probe (e.g., where the antibody reagent is specific for DNA.RNA
hybrids and the probe comprises RNA.RNA double stranded regions, or vice
versa). The complementary probe sequence can be of any convenient or
desired length, ranging from as few as a dozen to as many as 10,000 bases,
and including oligonucleotides having less than about 50 bases.
The RNA or DNA probe can be obtained in a variety of conventional manners.
For example, in the case of RNA probes, RNA can be isolated as the natural
products of cells, such as 5s, 16s and 23s ribosomal RNAs from bacteria or
cellular transfer RNAs. It is also practical to isolate specific messenger
RNAs from cells which specialize in production of large amounts of a
protein for which the messenger codes.
In vitro synthesis of RNA probes can be accomplished with a vector which
contains the very active Salmonella typhimurium bacteriophage SP6
transcription promoter [Green et al (1983) Cell 32:681]. A vector with
multiple restriction endonuclease sites adjacent to the promoter is
available from Promega Biotec, Madison, Wis. A DNA probe is cloned into
the vector which is then propagated in a bacterial host. Multiple RNA
copies of the cloned DNA probe can be synthesized in vitro using DNA
dependent RNA polymerase from bacteriophage SP6.
DNA probes can be prepared from a variety of sources. An entire bacterial
genome can be immobilized for a hybridization assay designed to detect
bacteria in a typically sterile sample. The assay would be capable of
detecting abundant bacterial RNAs such as ribosimal RNAs and transfer
RNAs. Alternatively, specific DNA sequences complementary to cellular RNAs
can be cloned into well known plasmid or viral vectors and used as
hybridization probes.
It should be understood that in using the expressions "RNA probe" and "DNA
probe" herein, it is not implied that all nucleotides comprised in the
probe be ribonucleotides or 2'-deoxyribonucleotides. The fundamental
feature of an RNA or DNA probe for purposes of the present invention is
that it be of such character to enable the stimulation of antibodies to
DNA.RNA or RNA.RNA hybrids comprising an RNA or DNA probe which do not
crossreact to an analytically significant degree with the individual
single strands forming such hybrids. Therefore, one or more of the
2'-positions on the nucleotides comprised in the probe can be chemically
modified provided the antibody binding characteristics necessary for
performance of the present assay are maintained to a substantial degree.
Likewise, in addition or alternatively to such limited 2'-deoxy
modification, a probe can have in general any other modification along its
ribose phosphate backbone provided there is no substantial interference
with the specificity of the antibody to the double stranded hybridization
product compared to its individual single strands.
Where such modifications exist in an RNA or DNA probe, the immunogen used
to raise the antibody reagent would preferably comprise one strand having
substantially corresponding modifications and the other strand being
substantially unmodified RNA or DNA, depending on whether sample RNA or
DNA was intended to be detected. Preferably, the modified strand in the
immunogen would be identical to the modified strand in an RNA or DNA
probe. An example of an immunogen is the hybrid poly(2'-0-methyladenylic
acid).poly(2'-deoxythymidylic acid). Another would be
poly(2'-O-ethylinosinic acid).poly(ribocytidylic acid). The following are
further examples of modified nucleotides which could be comprised in a
modified probe: 2'-O-methylribonucleotide, 2'-O-ethylribonucleotide,
2'-azidodeoxyribonucleotide, 2'-chlorodeoxyribonucleotide,
2'-O-acetylribonucleotide, and the phosphorothiolates or
methylphosphonates of ribonucleotides or deoxyribonucleotides. Modified
nucleotides can appear in probes as a result of introduction during
enzymic synthesis of the probe from a template. For example, adenosine
5'-O-(1-thiotriphosphate) (ATP.alpha.S) and dATP.alpha.S are substrates
for DNA dependent RNA polymerases and DNA polymerases, respectively.
Alternatively, the chemical modification can be introduced after the probe
has been prepared. For example, an RNA probe can be 2'-O-acetylated with
acetic anhydride under mild conditions in an aqueous solvent [Steward, D.
L. et al, (1972) Biochim. Biophys. Acta 262:227].
The critical property of an RNA or DNA probe for use herein is that
antibodies raised against the probe duplexed with a complementary RNA or
DNA strand, as desired, will discriminate in their binding properties
between the duplexed form of the probe and single stranded nucleic acids.
It is this property which enables detection of hybridized probe in the
assay mixture without significant background binding to the unhybridized
single stranded form of the probe or any nonspecifically bound single
stranded sample nucleic acids. While as described above certain
modifications along the ribonucleotide or deoxyribonucleotide strand can
be tolerated without loss of antibody discrimination of the duplex from
single strands, it will generally be preferable to employ RNA probes which
are composed entirely of ribonucleotides when the sample polynucleotide is
RNA or DNA. DNA probes can be used advantageously when the sample is RNA.
Immobilization of the Probe
As described previously, the probe will be presented for hybridization with
sample nucleic acids in either an immobilized or an immobilizable form. An
immobilizable form of the probe will be one in which the probe can be
conveniently rendered immobilized subsequent to the hybridization
reaction. The means by which the probe is ultimately immobilized is not
critical to the present invention and any available approach can be taken
so long as hybrids formed between the probe and the sequence of interest
are rendered immobilized through a property of the probe. Thus, sample
nucleic acids are not subjected to direct immobilization.
When presented to the hybridization reaction in an immobilized form, the
probe can be in any appropriate form that enables the probe, and any
components of the reaction mixture that have become associated therewith
by hybridization and/or by binding of the anti-hybrid reagent, to be
subsequently isolated or separated from the remaining mixture such as by
centrifugation, filtration, chromatography, or decanting. A variety of
compositions and configurations of an immobilized probe will thus be
evident and available to the worker in the field. Essentially any form of
the probe that is insoluble in the reaction mixture can be used. For
example, the probe can be aggregated or otherwise precipitated, attached
to an insoluble material, polymer, or support, or entrapped in a gel such
as agarose or polyacrylamide [see Meth. Enzymol. 12B:635(1968) and PNAS
67:807(1970)]. It is particularly preferred to employ a solid support to
which the probe is attached or fixed by covalent or noncovalent bonds, the
latter including adsorption methods that provide for a suitably stable and
strong attachment. The solid support can take on a variety of shapes and
compositions, including microparticles, beads, porous and impermeable
strips and membranes, the interior surface of reaction vessels such as
test tubes and microtiter plates, and the like. Means for attaching a
desired reaction partner to a selected solid support will be a matter of
routine skill to the worker in the field.
One method for adsorbing the probe onto nitrocellulose membranes involves
saturating a solution of probe with sodium iodide and spotting or
filtering aliquots onto the membrane [Bresser et al (1983) DNA 2:243]. The
sodium iodide facilitates denaturation of the probe and enhances
adsorption onto the membrane. Alternatively, the probe can be treated with
glyoxal, usually at concentrations around 1 molar(M), and then adsorbed
onto the membrane. The probe is fixed by baking at around 80.degree. C.
under vacuum for a period in the range of 2-4 hours. [Thomas, P. S.,
(1983) Meth. in Enzymol. 100:255].
Covalent immobilization of RNA or DNA probes can also be accomplished. A
wide variety of support materials and coupling techniques can be employed.
For example, the probe can be coupled to phosphocellulose through
phosphate groups activated by carbodiimide or carbonyldiimidazole [Bautz,
E. K. F., and Hall, B. D., (1962) Proc. Nat'l. Acad. Sci. USA 48:400-408;
Shih, T. Y., and Martin, M. A., (1974) Biochem. 13:3411-3418]. Also, diazo
groups on m-diazobenzoyloxymethyl cellulose can react with guanine and
thymidine residues of the polynucleotide [Noyes, B. E., and Stark, G. R.,
5:301-310; Reiser, J., et al, (1978) Biochem. Biophys. Res. Commun.
85:1104-1112]. Polysaccharide supports can also be used with coupling
through phosphodiester links formed between the terminal phosphate of the
polynucleotide and the support hydroxyls by water soluble carbodiimide
activation [Richwood, D., (1972) Biochim. Biophys. Acta 269:47-50; Gilham,
P. T., (1968) Biochem. 7:2809-2813], or by coupling nucleophilic sites on
the polynucleotide with a cyanogen bromide activated support [Arndt-Jovin,
D. J., et al, (1975) Eur. J. Biochem. 54:411-418; Linberg, U., and
Eriksson, S., (1971) Eur. J. Biochem. 18:474-479]. Further, the
3'-hydroxyl terminus of the probe can be oxidized by periodate and coupled
by Schiff base formation with supports bearing amine or hydrazide groups
[Gilham, P. T., (1971) Method. Enzymol. 21:191-197; Hansske, H. D., et al,
(1979) Method. Enzymol. 59:172-181]. Supports having nucleophilic sites
can be reacted with cyanuric chloride and then with the polynucleotide
[Hunger, H. D., et al, (1981) Biochim. Biophys. Acta 653:344-349].
In general, any method can be employed for immobilizing the probe, provided
that the complementary single stranded sequence is available for
hybridization to sample nucleic acids. Particular methods or materials are
not critical to the present invention.
A particularly attractive alternative to employing directly immobilized
probe is to use an immobilizable form of probe which allows hybridization
to proceed in solution where the kinetics are more rapid. Normally in such
embodiment, one would use a probe which comprises a reactive site capable
of forming a stable covalent or noncovalent bond with a reaction partner
and obtain immobilization by exposure to an immobilized form of such
reaction partner. Preferably, such reactive site in the probe is a binding
site such as a biotin or hapten moiety which is capable of specific
noncovalent binding with a binding substance such as avidin or an antibody
which serves as the reaction partner.
Essentially any pair of substances can comprise the reactive site/reactive
partner pair which exhibit an appropriate affinity for interacting to form
a stable bond, that is a linking or coupling between the two which remains
substantially intact during the subsequent assay steps, principally the
separation and detection steps. The bond formed may be a covalent bond or
a noncovalent interaction, the latter being preferred especially when
characterized by a degree of selectivity or specificity. In the case of
such preferred bond formation, the reactive site on the probe will be
referred to as a binding site and the reaction partner as a binding
substance with which it forms a noncovalent, commonly specific, bond or
linkage.
In such preferred embodiment, the binding site can be present in a single
stranded hybridizable portion or in a single or double stranded
nonhybridizable portion of the probe or can be present as a result of a
chemical modification of the probe. Examples of binding sites existing in
the nucleotide sequence are where the probe comprises a promoter sequence
(e.g., lac-promoter, trp-promoter) which is bindable by a promoter protein
(e.g., bacteriophage promoters, RNA polymerase), or comprises an operator
sequence (e.g., lac operator) which is bindable by a repressor protein
(e.g., lac repressor), or comprises rare, antigenic nucleotides or
sequences (e.g., 5-bromo or 5-iododeoxyuridine, Z-DNA) which are bindable
by specific antibodies [see also British Pat. Spec. 2,125,964]. Binding
sites introduced by chemical modification of the polynucleotide comprised
in the probe are particularly useful and normally involve linking one
member of a specific binding pair to the probe nucleic acid. Useful
binding pairs from which to choose include biotin/avidin (including egg
white avidin and streptavidin), haptens and antigens/antibodies,
carbohydrates/lectins, enzymes/inhibitors, and the like. Where the binding
pair consists of a proteinaceous member and a nonproteinaceous member, it
will normally be preferred to link the nonproteinaceous member to the
probe since the proteinaceous member may be unstable under the denaturing
conditions of hybridization of the probe. Preferable systems involve
linking the probe with biotin or a hapten and employing immobilized avidin
or anti-hapten antibody reagent, respectively.
When the probe is presented for hybridization with the sequence of interest
in an immobilizable form, the subsequent steps of immobilization of the
formed duplexes through a property of the probe and addition of the
anti-hybrid antibody reagent can proceed in any desired order.
Immobilization and anti-hybrid addition can be accomplished by
simultaneous addition of the involved reagents and materials, or one can
precede the other, with or without intervening wash or separation steps,
in either order. Where ordered additions are followed, of course one will
take into account the concentrations of the added reagents so as not to
oversaturate the formed hybrids and inhibit interaction therewith of the
second added materials.
Although immobilized probes or immobilizable probes which become bound to
solid supports by specific binding processes described above are
preferred, immobilizable probes can be bound to supports by processes with
relatively low specificity. In this case the support would bind the
hybridized probe but not the unhybridized form. Then the amount of hybrid
would be measured with the antibody reagent. An example of a support of
this type is hydroxyapatite which binds DNA.RNA and RNA.RNA duplexes but
not the single stranded species [Brenner and Falkow, Adv. in Genet.,
16:81(1973)].
Also, a chemically active or activatable group can be introduced into the
probe and allowed to react with the solid support following the
hybridization. This system would give a covalently immobilized probe and
the amount of hybrid coupled to the support can be determined with the
antibody reagent.
Anti-Hybrid Antibody Reagent and Detection Schemes
The antibody reagent of the invention is principally characterized by its
ability to bind the DNA.RNA or RNA.RNA hybrids formed between the probe
and complementary sample nucleic acids to the significant exclusion of
single stranded polynucleotides. As stated previously above, the antibody
reagent can consist of whole antibodies, antibody fragments,
polyfunctional antibody aggregates, or in general any substance comprising
one or more specific binding sites from an antibody for RNA.RNA or
DNA.RNA, as the case may be. When in the form of whole antibody, it can
belong to any of the classes and subclasses of known immunoglobulins,
e.g., IgG, IgM, and so forth. Any fragment of any such antibody which
retains specific binding affinity for the hybridized probe can also be
employed, for instance, the fragments of IgG conventionally known as Fab,
F(ab'), and F(ab').sub.2. In addition, aggregates, polymers, derivatives
and conjugates of immunoglobulins or their fragments can be used where
appropriate.
The immunoglobulin source for the antibody reagent can be obtained in any
available manner such as conventional antiserum and monoclonal techniques.
Antiserum can be obtained by well-established techniques involving
immunization of an animal, such as a mouse, rabbit, guinea pig or goat,
with an appropriate immunogen. The immunoglobulins can also be obtained by
somatic cell hybridization techniques, such resulting in what are commonly
referred to as monoclonal antibodies, also involving the use of an
appropriate immunogen.
Immunogens for stimulating antibodies specific for DNA.RNA hybrids can
comprise homopolymeric or heteropolymeric polynucleotide duplexes. Among
the possible homopolymer duplexes, particularly preferred is
poly(rA).poly(dT) [Kitagawa and Stollar (1982) Mol. Immunol. 19:413].
However, in general, heteropolymer duplexes will be preferably used and
can be prepared in a variety of ways, including transcription of .phi.X174
virion DNA with RNA polymerase [Nakazato (1980) Biochem. 19:2835]. The
selected RNA.DNA duplexes are adsorbed to a methylated protein, or
otherwise linked to a conventional immunogenic carrier material, such as
bovine serum albumin, and injected into the desired host animal [see also
Stollar (1980) Meth. Enzymol. 70:70].
Antibodies to RNA.RNA duplexes can be raised against double stranded RNAs
from viruses such as reovirus or Fiji disease virus which infects sugar
cane, among others. Also, homopolymer duplexes such as poly(rI).poly(rC)
or poly(rA) poly(rU), among others, can be used for immunization as above.
The binding of the antibody reagent to the hybridized probe duplex
according to the present method can be detected by any convenient
technique. Advantageously, the antibody reagent will itself be labeled
with a detectable chemical group. Such detectable chemical group can be
any material having a detectable physical or chemical property. Such
materials have been well-developed in the field of immunoassays and in
general most any label useful in such methods can be applied to the
present invention. Particularly useful are enzymatically active groups,
such as enzymes (see Clin. Chem. (1976)22:1243, U.S. Pat. No. 31,006 and
UK Pat. 2,019,408), enzyme substrates (see U.S. Pat. No. 4,492,751,
cofactors (see U.S. Pat. Nos. 4,230,797 and 4,238,565), and enzyme
inhibitors (see U.S. Pat. No. 4,134,792); fluorescers (see Clin. Chem.
(1979)25:353); chromophores; luminescers such as chemiluminescers and
bioluminescers (see U.S. Pat. No. 4,380,580); specifically bindable
ligands such as biotin (see European Pat. Spec. 63,879) or a hapten (see
PCT Publ. 83-2286); and radioisotopes such as .sup.3 H, .sup.35 S, .sup.32
P, .sup.125 I, and .sup.14 C. Such labels and labeling pairs are detected
on the basis of their own physical properties (e.g., fluorescers,
chromophores and radioisotopes) or their reactive or binding properties
(e.g., enzymes, substrates, cofactors and inhibitors). For example, a
cofactor-labeled antibody can be detected by adding the enzyme for which
the label is a cofactor and a substrate for the enzyme. A hapten or ligand
(e.g., biotin) labeled antibody can be detected by adding an antibody to
the hapten or a protein (e.g., avidin) which binds the ligand, tagged with
a detectable molecule. Such detectable molecule can be some molecule with
a measurable physical property (e.g., fluorescence or absorbance) or a
participant in an enzyme reaction (e.g., see above list). For example, one
can use an enzyme which acts upon a substrate to generate a product with a
measurable physical property. Examples of the latter include, but are not
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