Note: Descriptions are shown in the official language in which they were submitted.
-- 1 --
HYBRIDIZATION ASSAY WITH IMMOBILIZATION
OF HYBRIDS BY ANTI-HYBRID BINDING
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
2a medium contàining 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
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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 suGh 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.
INFORMATION DISCLOSURE
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
hybridlzation assay techniques generally in~olve
2Q immobilization of the sample nucleic acid on a
solid support. Hybridization between particular
base sequ~nces or genes of interest in the sample
nucleic acids and a labeled form of the probe
nucleic acid is determined by separating the solid
support from the remainder of the reaction mixture
which contains the 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
3~ assay poses two si~nificant problems. Firstly, the
procedures required to accomplish immobilization
are generally time consuming and add a step which
MS-1362
~ _ 3 ~ 3~3~
is undeslrable 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 immobil-
ized probe and label the sample nucleic acids in situ
or one can use a dual hybridization technique requir-
ing two probes, one of which is immobilized and the
other labeled [Methods in Enzymology 65:468tl968) 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 hiyh degree of
technical skill which is not routinely found in clin-
ical technicians and there are no simple, reliable
methods Eor monitoring the labeling yield, which
can be a significant problem if the labeling media
con-tain variable amounts of inhibitors of the labeling
reaction. The dual hybridization technique has the
disadvan-tages of requiring an additional reagent
and incubation step and the kinetics of the hybridi-
zation reaction can be slow and inefficient. The
accuracy of -the assay can also be variable if the
complementarity of the two probes wi-th the sample
sequence is variable.
The use of immobilized RNA probes and detec-
tion of resulting immobilized DNA-RNA or RNA-RNA
hybrids by labeled antibodies selective for such re-
spective hybrids is described in Canadian Patent Appli-
cation No. 481,294, filed May 10, 1985. This method
-- 4
eliminates the need to immobilize or label sample
nucleic acids, however, the probe strand involved
in hybridization is immobilized which can
significantly decrease the rate at which the probe
and the sequence of interest can reanneal.
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 ~alloran r "Nucleic Acids in
Immunology", ed. Plescia and Braun,
Springer-Verlag, NY (1969) pp. 13 e~ seq]. Some
success has been achieved in generating antibodies
that will bind RNA DNA 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)]. 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. 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
3Q microscopy with fluorescein-labeled antibody to
RNA-DNA.
Early procedures for detection of specific DNA
sequences followed solution-phase formats wherein
hybridization involved soluble forms of both the
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.Y~3~
- 5
sample DNA and a radiolabeled RNA probe. The
hybrid ~ormed in the solution was isolated by
density-gradient centrifugation [Hall and
Spiegelmann (1961) Proc. Natl. Acad. Sci. 47:137].
The centrifugation was too slow and impractical for
analysis of large numbers of samples~ This
solution hybridization method was made more
practical by the discovery that nitrocellulose
filters strongly adsorb single-stranded DNA along
with any hybridized RNA [Nygaard and Hall (19643 J.
Mol. Biol. 9:125]. The labeled RNA probe binds
very weakly to the filters and the method was
improved by exposing the filters to ribonuclease
which hydrolyzes the single stranded probe but not
the RNA-~NA hybrid. The method is limited to
hybridizations of DNA samples with RNA probes and
the digestion with ribonuclease has to be
controlled carefully; therefore, solid-phase
hybridization methods became more important.
Accordingl~, there is a great need for a
nucleic acid hybridization assay which does not
require the immobilization of either probe or
sample nucleic acids and which overccmes the
limitations of the known solution-phase
hybridization techniques. 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
3a principal objectives of the present invention.
SUMMARY OF THE INVENTION
The present invention provides a nucleic acid
hybridization assay wherein the hybrid formed
MS-1362
between the particular polynucleotide sequence to
be detected and a nucleic acid probe becomes
immobilized by binding to an immobilized or
immobili~able form of an antibody reagent selective
for binding to such hybrids. According to
preferred embodiments, this permits separation of
hybridized and unhybridized probe if a labeled
probe is employed or separation of hybrid bound
from free labeled second antibody reagent if a
labeled form of anti-hybrid reagent is used. The
present method thus dispenses completely with the
need for immobilization of either sample or probe
nucleic acids and allows hybridization to proceed
in solution where the rate of hybridi~ation is
rapid and more efficient.
In general, the method of the present
invention comprises contact of a test sample
containing single stranded nucleic acids with the
probe which has a single stranded base sequence
that is substantially complementary to the sequence
to be detected. This hybridization step will
proceed under conditions that are favorable to
hybridization between th~ probe and the sequence to
be detected. The resulting hybrids can then be
detected by addition of the antibody reagent which
is selective for binding duplexes formed by
hybridization between the complementary probe
seque~ce and the sequence to be detected. At this
stage of the assay, the antibody reagent will
3~ normally be in an immobilized state, such as being
fixed chemically or physically to a solid support.
Alternatively, the anti-hybrid can be contacted
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3~
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with the formed hybrids in a soluble form and
thereafter rendered immobilized such as by contact
with an immobilized form of an antibody raised
against the anti-hybrid or by employing a
ligand-modified anti-hybrid and addition of an
immobilized form of a binding partner for the
ligand.
The resulting duplexes that become bound to
the immobilized antibody reagent can be determined
in a variety of manners. Normally, one will use a
labeled probe or a labeled form of a second
anti-hybrid reagent. In the first instance,
hybridized labeled probe that becomes associated
with the immobilized phase is separated from
unhybridized probe that remains in solution and
either the label associated with the immobilized
phase or the label in the remaining unhybridized
probe is measuredO Where a labeled anti-hybrid is
used, that which becomes associated wlth the
immobilized phase by binding to formed hybrid is
separated from that which remains in solution and
again the label that either has become associated
with the immobile phase or remains with unbound
labeled anti-hybrid is measured. A variety of
labels can be used including radiolabe~s, and
preferably, nonradioisotopic labels such as
en~ymes, fluorescers, ligands, and the like.
The present invention is characterized by a
nu~er of significant advantages.
First, the hybridization of sample nucl~ic
acids with a probe can be conducted in solution
where the reaction rates are fastest. Typical
solid-phase hybridization rates are limited by
diffusion of the labeled probe to the s~mple
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4 t~ ~3f~
-- 8 --
nucleic acid lmmobilized on the solid surface.
This limitation does not occur with solution
hybridization.
Second, nonspecific binding of a labeled probe
to the solid phase used to immobilize the sample
nucleic acid is a problem in solid-phase methods.
Typically, the sample nucleic acids are adsorbed
onto the solid phase; therefore, the solid must be
a substance which binds nucleic acids with high
lQ affinity, but this property becomes a problem in
the subsequent hybridization step where nonspecific
adsorption of the labeled probe is to be minimized.
In the present invention, the solid-phase for
anti-hybrid immobilization can be chosen from
materials which do not adsorb nucleic acids.
Also, most hybridization methods require
immobilization of sample nucleic acids by
adsorption to a solid surface or by covalent
coupling to a solid. Proteins and other materials
2Q in the sample interfere with the immobilization;
therefore, the sample has to be purified by methods
such as phenol extraction. The present solution
hybridization method immobilizes the hybxids with
antibodies which have high affinity and specificity
for the hybrid and are not subject to interference
by proteins~ carbohydrates and lipids in the
sample.
Further, when sample nucleic acids are
immobilized by adsorption or covalent coupling,
3a there is no convenient means to insure that the
nucleic acids are immobilized in high and
reproducible yieldsO Furthermore, the adsorption
process makes part ~f the polynucleotide sequence
unavailable for hybridization. For instance, less
MS-13~2
3~
than half of the DNA adsorbed onto a nitrocellulose
men~rane is available for hybridization. Thus, the
detection limits of the assay are not optimal and
the yield of hybrid can be variable. Hybridization
in solution as permitted by the present invention
allows the sample nucleic acids to form hybrids
with maximum efficiency.
BRIEF DESCRIPTION OF THE DRAWING~
Figs. 1 and 2 are schematic illustrations of
lQ preferred methods for performing the present
invention. These methods are described in detail
below.
D~SCRIPTION OF THE PREFERRED EMBODIMENT~
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
2Q complementary single strands of DNA resulting from
this "melting" or "denaturation" will reassociate
(sometimes reerred to as reannealing or
hybridization) to reform the duplex structureO 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 li.e., "homologous with") a second
single stranded nucleic acid under appropriate
conditions, will result in the formation of
MS-1362
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DNA'DNA, RNA-DNA, or RNA RNA hybrids, as the case
may be.
The Probe
The probe will comprise at least one single
stranded base sequence substantially complementary
to or homologous with 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 nonhomologous sequences. These
nonhomologous sequences can be linear, or they can
be self-complementary and form hairpin loops. In
addition, the homologous region of the probe can be
flanked at the 3'- and 5'-termini by nonhomologous
sequences, such as those comprising the DNA or RNA
of a vector into which the homologous 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 duple~ed with a complementary
polynucleotide strand or strands, provided that the
cri~ical homologous segment or segments are in
single stranded form and available for
hybridization w.ith sample DNA or RNA. It will
generally be preferred to employ probes which are
substantially in single stranded form. The
preparation of a suitable probe for a particular
assay is a matter of routine ~kill in the art.
MS-1362
The Antibody Reagent
A critical feature of the present invention is
the ability to immobilize selectively hybrids
formed under the favorable kinetics of interaction
of the probe and sequences to be detected in
solution. In contrast with prior art methods, the
present invention uniquely provides for such
immobilization by binding of formed hybrids to an
antibody reagent which is added to the reaction
la medium in an immobilized state or which can be
rendered immobilized in a subsequent step by
conventional methods.
As used herein, antibody reagent refers to an
immunologically derived binding substance having
anti-hybrid binding activity and can be whole
antibodies or fragments thereof, or aggregates or
conjugates thereof, of the conventional polyclonal
or monoclonal variety. Pre~erred antibody reagents
will be those that are selective for binding double
stranded nucleic acids over single stranded nucleic
acids, e.g., those which selectively bind (i)
DNA-RNA or RNA-RNA hybrids or lii) intercalation
complexes.
It is currently known that antibodies can be
stimulated which are selective for DNA-RNA or
RNA-RNA hybrids over the single stranded nucleic
acids, however, it is presently considered
infeasible to generate such selectivity in the case
of DNA-DNA hybrids. To the extent that selective
3~ DNA-DNA antibodies are developed in the future,
they will clearly be applicable to the present
invention. Antibodies to DNA-RNA or RNA-RNA
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t''q,~ ~3~
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hybrids can be used where -the probe is RNA and the
sample nucleic acids are DNA or RNA or where the
probe is DNA and the sample ~NA.
Fur~her, it should be understood that in
referring to an RNA probe used with an anti-DNA RNA
or anti-RNA-RNA reagent, it is contemplated herein
that not all nucleotides comprised in the probe be
ribonucleotides, i.e., bearing-a 2'-hydroxyl group.
The fundamental feature of an RNA probe as used
herein is that it be sufficiently non-DNA in
character to enable the stimulation of antibodies
to DNA-RNA or RNA-RNA hybrids comprising an RNA
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 in the deo~y form
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, an RNA probe can comprise nucleotides
having other 2'-modifications, or in general any
other modification along its ribose phosphate
backbone provided there is not 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
3~ 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
MS 1362
3~
~ 13
intended to be detected. Preferably, the modified
strand in the immunogen would be identical to the
modified strand in an RNA probe. An example of an
immunogen is the hybrid poly(2'-0-methyladenylic
acid) poly(2'-deoxythymidylic acid3. Another would
be polyt2'-0-ethylinosinic acid)-poly(ribo~ytidylic
acid~. The following are further examples of
modified nucleotides which could be comprised in an
RNA probe: 2'-0-methylribonucleotide, 2-0
a -ethylribonucleotide, 2'-azidodeoxyribonucleotide,
2' chlorodeoxyribonucleotide, 2'-0
-acetylribonucleotide, and the methylphosphonates
or phosphorothiolates of ribonucleotides or
deoxyribonucleotides. Modified nucleotides can
appear in RNA probes as a result of introduction
during enzymic synthesis of the probe from a
template. For example, adenosine
5'-0-(1-thiotriphosphate) (ATP~S) and dATP~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'-0-acetylated with acetic anhydride under
mild conditions in an aqueous ~olvent.
Immunogens for stimulating antibodies specific
for RNA-DNA hybrids can comprise homopolymeric or
heteropolymeric polynucleotide duplexes. Among the
possible homopolymer duplexes, particularly
preferred is poly(rA)-poly(dT) [Kitagawa and
3~ 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 ~X174 virion DNA with
R~A polymerase [Nakazato (1980) Biochem. 19:2835].
MS-1362
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 in-
fects 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.
Further information regarding antibodies to RNA-DNA
and RNA-RNA hybrids is provided in Canadian
Patent Application No. 481,294, filed May 10,
1985.
Antibodies to intercalation complexes can
be prepared against an immunogen which will usually
comprise an ionic complex between a cationic protein
or protein derivative (e.g., methylated bovine serum
albumin) and the anionic intercalator~nucleic acid
complex. Ideally, the intercalator will be covalent-
ly coupled to the double stranded nucleic acid. Al-
terna-tively, the intercala-tor-nucleic acid conjugate
can be covalently coupled to a carrier protein. The
nucleic acid porticn of the immunogen can comprise
the specific paired sequences found in the assay
hybrid or can comprise any other desirable sequences
since the specificity of the antibody will generally
not be dependent upon the particular base sequences
involved. Further information regarding antibodies
to in-tercalation complexes is provided in Canadian
Patent Applieation No. 469,908, Eiled Deeember
12, 1984.
As stated above, the antibody reagent can
consist of whole antibodies, antibody fragments,
polyfunctional antibody aggregates, or in general
any substance comprising one or more speeifie
binding sites from an antibody. 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 whieh retains specifie binding affinity
for the hybridized probe ean also be employed, for
instance, the fragments of IgG conven-tionally known
as Fab, F(ab'), and F(ab')2. In addition, aggregates,
polymers, derivatives and conjugates of immunoglobu-
lins 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 monoelonal teeh-
niques. Antiserum ean be obtained by well-established
teehniques involving immunization of an animal, sueh
as a mouse, rabbit, guinea pig or goat, with an
appropriate immunogen. The immunoglobulins ean also
be obtained by somatie eell hybridiza-tion -teehniques r
sueh resulting in what are eommonly referred to as
monoelonal antibodies, also involving the use of an
appropriate immunogen.
In those instanees where an antibody reagent
seleetive for interealation complexes is employed as
the anti-hybrid, a varie-ty of interealator eompounds
ean be involved. In general it can be said that
the intercalator compound preferably is a
~ - 16 -
~ ~ ~5 ~
low molecular weight, planar, usually aromatic but
sometimes polycyclic, molecule capable of binding
with double stranded nucleic acids, e.g., DNA'DNA,
DNA-RNA, or RNA RNA duplexes, usually by insertion
between base pairs. The primary binding mechanism
will usually be noncovalent, with covalent binding
occurring as a second step where the intercalator
has reactive or activatable chemical groups which
will form covalen-t bonds with neighboring chemical
groups on one or both of the intercalated duplex
s'~rands. The result of intercalation is the
spreading of ad~acent base pairs to about twice
their normal separation distance, leading to an
increase in molecular length of the duplex.
Further, unwinding of the double helix of about 12
to 36 degrees must occur in order to accommodate
the intercalator. General reviews and further in-
formation can be obtained from Lerman, J. Mol.
Biol. 3:18(1961); Bloomfield et al, "Physical
Chemistry of Nucleic Acids", Chapter 7, pp. 429-
476, Harper and Rowe, NY(1974); Waring, Nature
219:1320 (1968); Hartmann et al, Angew. Chem.,
Engl. Ed. 7:693(1968); Lippard, Accts. Chem. Res.
11:211(1978); Wilson, Intercalation Chemistry(1982),
445; and Berman et al, Ann. Rev. Biophys. Bioeng.
20:87(1981), as well as from the above-referenced
Canadian Application No. 469,908. Exemplary of
intercalators are acridine dyes, e.g., acridine
orange, the phenanthridines, e.g.~ ethidium, the
phenazines, furocoumarins, phenothiazines, and
quinolines.
The intercalation complexes are formed in
the assay medium during hybridization by use of a
probe which has been modified in its complementary,
~ i .
3~
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single stranded region to have the intercalator
chemically linked thereto such that upon
hybridization the intercalation complexes are
formed. Essentially any convenient method can be
useq to accomplish such linkage. Usually, the
linkag~ is formed by effecting intercalation with a
reactive, preferably photoreactive intercalator,
followed by the linking reaction. A particularly
useful method involves the azidointercalators.
Upon exposure to ultraviolet or visible light, the
reactive nitrenes are readily generat~d. The
nitrenes of arylazides prefer insertion reactions
over their rearrangement products [see White et al,
Methods in Enzymol. 46:6~4(1977)]. Representative
azidointercalators are 3-azidoacridine,
9-azidoacridine, ethidium monoazide, ethidium
diazide, ethidium dimer azide [Mitchell et al, JACS
104:4265(1982)], 4-azido-7-chloroquinoline, and
2-azidofluorene. Other useful photoreactable
2Q intercalators are the urocoumarins which form
[2+2] cycloadducts with pyrimidine residues.
Alkylating agents can also be used such as
bischloroethylamines and epoxides or aziridines,
e.g~, aflatoxins, polycyclic hydrocarbon epoxides,
mitomycin, and norphillin A. The
intercalator-modified duplex is then denatured to
yield the modified single stranded probe.
While as stated above preferred anti-hybrids
for use in the present method will discriminate in
their binding between double stranded and single
skranded nucleic acids, it is not critical to have
such selectivity for all embodiments. Where such
selectivity is not critical, a variety of
additional approaches for obtaining anti-hybrid
MS-1362
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become available. These include anti-DNA-DNA
antibodies which are essentially nonspecific for
DNA whether single or double stranded, antibodies
raised against chemically modified nucleic acids
such as cis-diaminedichloroplatinium-DNA adducts,
antibodies to 0~-04 oxidized DNA such as
anti-thymine glycol, antibodies to antibiotic-DNA
complexes such as anti-distamycin A-DNA complex or
anti-netropsin-DNA complex and antibodies to
modified bases such as anti-5-bromouracil and
anti-6-methyladenosine. The criticality of double
stranded selectivity exists whenever the format of
the assay involves the labeling of single stranded
nucleic acid and thus requires discrimination
between hybridized and unhybridized labeled nucleic
acid. Such a situation is presented when either a
probe or single stranded sample nucleic acids are
labeled. On the other hand, when a labeled form of
a second anti-hybrid reagent is employed, it is
only necessary that one or the other of the
ultimately immobilized anti-hybrid and the labeled
anti-hybrid have selectivity for double stranded
nucleic acids over single stranded nucleic acids.
While the present method has been particularly
described as involving the immobilization of
hybrids by the hinding of antibody reagents, it
will be recognized that essentially any substance
which has the ability to selectively bind the
nucleic acid hybrids formed during the assay may be
likewise used. This applies both to those assay
3a formats requiring selectivity for double stranded
nucleic acids as well as where double versus single
stranded selectivity is not critical as discussed
above. Equivalents of the antibody reagent of the
MS-1362
~2~ 3~
19 -
present invention which can be used without
departing from the present inventive concept will
normally exhibit a highly specific noncovalent
binding, as in ligand-receptor and immunoglobulin
binding. Materials available now or in the future
having such characteristic binding are contemplated
as equivalents to the present antibody reagent.
The antibody reagent is brought into contact
with the formed hybrids either in an immobilized
lQ form or an immobilizable form, i.e., can thereafter
be rendered immobilized by conventional methods.
It will generally be preferably to employ
immobilized forms of the anti-hybrid. A large
variety of methods are known for immobilizing
proteins on solid supports and most of the methods
are applicable to antibodies [see Methods in
Enzymology, Vol. 44 (1976~]. Antibodies are
commonly immobilized either by covalent coupling or
by noncovalent adsorption. Noncovalent methods
2Q frequently emp~oyed are nonspecific adsorption to
polystyrene beads or microparticles and to
polyvinylchloride surfaces. Many covalent methods
are used and a few include cyanogen bromide
activated agaroses and dextrans; glutaraldehyde
activated nylons and polyacrylamides; and epoxides
on acrylic and other supports. Antibodies of the
IgG class can also be immobilized by the binding to
immo~ilized forms of proteil~ A. Where protein A is
used for ~his immobilization and labeled
anti-hybrid will be used in the assay, care will be
taken to prevent the labeled anti-hybrid from also
being bound by the immobilized protein A such as by
saturating the protein A binding sites or utilizing
a labeled anti-hybrid whic~ does not comprise the
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~2~ 3~
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protein A receptor site, e.g., by using Fab or Fab
or an anti-hybrid from an immunoglobulin class
other than IgG.
The primary purpose of immobilization is to
enable physical manipulation and isolation or
separation of hybrids formed in the assay in order
to measure them by the response or signal produced
by the label. Thus, immobilization can occur
subsequent to the binding of anti-hybrid to hybrids
formed in the assay. A variety of ways to
accomplish this are apparent.
One can employ immobilized anti-tanti-hybrid)
antibodies, taking care that if labeled anti-hybrid
is to be used in the assay that the immobilized
antibodies do not have significant affinity for the
labeled reagent. This can be accomplished by using
antigenically distinguishable immunoglobulins for
the two anti-hybrid reagents such as using
anti-hybrids from different immunoglobulin classes.
Immobilized protein A can also be employed with the
same precautions as discussed above regarding the
use of protein A and labeled anti-hybrid.
Particularly useful are schemes involving the
chemical modification of the anti-hybrid by
conjugation to one member of a specific binding
pair and the use of an immobilized form of the
other member of the pair. Useful binding pairs
from which to choose include biotin/avidin, haptens
and antigensJantibodies, carbohydrates/lectins,
enzymes/inhibitors, and the like. It is preferable
to modify the anti~hybrid with biotin or a hapten
and employ an immobilized form of avidin or
anti-hapten antibody, respectively.
MS-1362
5~1 ~ 3J~t,~
-- 21 --
De tectior~ La~e ~s
The hybrids of interest that ultimately become
associated with the immobilized antibody reagent
can be determined in a variety of ways. It will
generally be preferred to employ a labeled probe or
a labeled form of a second antibody reagent capable
of binding the formed hybrid. The latter is
particularly preferred because neither the probe
nor the sample nucleic acids need be chemically
modified such as by being labeled in order to
conduct the assay. One can simply contact the
hybrids with the immobilized or immobilizable first
antibody reagent and with the labeled second
antibody reagent and measure the label that becomes
associated with the immobilized phase or
alternatively measure the label remaining as
unbound labeled second antibody reagent. It is
possible to use the same substance as the first and
second antibody reagents. For instance, one can
use anti-DNA-RNA where DNA RNA hybrids are formed
and one fraction of such anti-hybrids can be
immobilized and another fraction labeled. The
hybrids will have multiple epitopes for
anti-DNA-RNA to allow for binding of both labeled
and immobilized anti-hybrid. AlternatiYely,
different anti-hybrids can be used, for example,
where monoclonal antibodies are used one can employ
differen~ hybridomas for different binding
specificities and/or affinities.
3Q Labeled probes can be used in the present
invention in several ways. In one case, a single
labeled probe is used and the resulting labeled
hybrids are separated from the free labeled probe
for detection by binding to immobilized or
immobilizable anti-hybrid. Alternati~ely, one can
M~-1362
s~
- 22 -
employ a dual hybridization approach involving two
probe segments which are complementary to mutually
exclusive segments on the sequence to be detected.
One of the probe segments will be labeled and the
other will form epitopes for anti-hybrid upon
hybridization with the sequence to be detected.
A variety of other labeling schemes will be
evident. For instance, although significantly less
desirable one can introduce the label by labeling
the single stranded sample nucleic acids in situ
thus yielding labeled hybxids upon hybridization
with the unlabeled probe. Also, as will be
discussed in more detail below, the component to be
labeled can be linked directly, e.g., by covalent
bonds, to the substance that provides the
ultimately detectable signal or by indirect
linkages such as by incorporation of the ultimately
det~ctable substance in a microcapsule or liposome
which in turn is linked to the component to be
labeled. Further, it will be understood that one
can label with a specifically bindable ligand,
e.g., a hapten or biotin, and bring in the
ultimately detectable substance at any desirable
time in the assay by addition of a binding partner
for the ligand, e.g., an antibody or avidin, to
which the detectable species is linked. When using
immobilized and labeled anti-hybrids, they can be
added to the reaction mixture simultaneously;
however, depending on the particular system,
3~ performance might be optimized by addin~ one of the
- components a predetermined period of time before
the other.
Labels of various types can be used to label
probes, second anti-hybrid, and so forth as
MS-1362
; ?3 ~;
- 23 -
described above. The label will be a substance
which has a detectable physical, chemical, or
electrical property. Such materials have been
well-developed in ~he 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)22:1232, U.S. Reissue
Pat. 31,006r and UK Pat. 2,019,408), enzyme
lQ substrates ~see British Pat. Spec. 1,548,741~,
coenzymes (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 Clin.
Chem.(1979)25:512, and ibid, 1531); specifically
bindable ligands such as biotin (see European Pat.
Spec. 63,879) or a hapten (see PCT Publ. 83-2286);
and radioisotopes such as 3H, 35S, 32p, 125I, and
14C. Such labels 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,
coenzymes and inhibitors). For example, a
cofactor-labeled species can be detected by adding
the enzyme (or enzymes where a cycling system is
used) for which the label is a cofactor and a
substrate or substrates for the enzyme. A hapten
or ligand (e.g., biotin) labeled species can be
3~ 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
MS 1362
~2~ 2
- 24 -
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 limited
to, ~-galactosidase, alkaline phosphatase and
peroxidase.
Where a second antibody reagent is used to
measure the duplexes that become associated with
the immobilized first antibody reagent, such second
antibody can be detected based on a native property
such as its own antigenicity. A labeled
anti-(antibody) antibody will bind to the second
antibody reagent where the label for the second
antibody is any conventional label as above.
Further, antibody can be detected by complement
fixation or the use of labeled protein A, as well
as other techniques known in the art for detecting
antibodies. Where labeled protein ~ is used, the
immobilized antibody reagent will be in a form that
2a lacks the protein A binding si-te (e.g., one can use
immobilized Fab or Fab').
Methods for preparing the labeled components
used in the preferred embodiments of the present
invention are readily available from the prior art.
Most preferred are those assay formats which
involve labeled probes or labeled second
anti-hy~rid~ The labels described above can be
used for these embodiments. When labeling probes
one will employ synthetic approaches which are
3~ effective for modifying nucleic acids without
substantially interfering with the ability of the
labeled probe to participate in hybridization, and
will select labels which are sufficiently stable
MS-136,.
3~
- 25 -
under the conditions to be used for hybridization
to enable their subsequent detection.
By way of example, the following approaches
can be used in labeling probes. Radiolabeled
nucleotides can be incorporated into DNA probes by
methods such as nick translation, terminal labeling
with terminal deoxynucleotidyl transferase, and in
vivo labeling. Radiolabeled nucleo~ides can be
incorporated into RNA probes during in vitro
synthesis with DNA dependent RNA polymerase from
bacteriophage SP6 using the Riboprobe M DNA
template system from Promega Biotec~ Madison, WI.
The method of Langer et al [(1981) Proc. Nat'l.
Acad. Sci., 78:6633] can be used to couple biotin
to the primary amine of 5-(3-amino)allyluridine and
deoxyuridine triphosphates. These biotinylated
nucleotides can be incorporated into double
stranded DNA by nick translation or added to the
3'-OH terminus with terminal deoxynucleotidyl
transerase. Biotin can also be attached to the
3'-O~ terminus of RNA through polyamine ~Broker,
T.R., ~1978) Nucl. Acids Res. 4:363] and cytochrome
C bridges [Sodja, A. and Davidson, N. (1978) Nucl.
Acids. Res. 5:385]. Direct coupling of protein
labels to probes can be accomplished by the method
of Renz [(1983) EMBO Journal, 2:817~ who coupled
125I-histones to denatured DNA with glutaraldehyde.
Enzymes such as peroxidase and alkaline phosphatase
can be linked to DNA probes by means of similar
3~ chemistry [Renz and Kurz (1984) Nucl. Acids Res.
12:3435]. Other chemistries for end-labeling DNA
probes include that described by Eshaghpour et al
[(1979~ Nucl. Acids Res. 7:1485]~ One or more
4-thiouridine residues can be introduced on the
MS-1362
- 26 -
3'-OH ends of DNA and the thiols reacted with
various electrophilic low molecular weight
reagents. This chemistry can be used to attach
various haptens to DNA probes. Labeling with the
hapten N-acetoxy-N-2-acetylaminofluorene is
described by Tchen et al [(1984~ Proc. Nat'l. Acad.
Sci. 81:3466]. DNA and RNA probes can be reacted
with N-acetoxy-N-2-acetylaminofluorene to yield an
adduct having N-2-acetylaminofluorene residues
attached at the 8-carbon of guanine. The
covalently modified DNA can be detected with
antibody raised against the
N-acetoxy-N-2-acetyl-aminofluorene residue. The
method of Hu and Messing ~(1982) Gene, 17:271] can
be used for adding labels to probes cloned into
single stranded M13 vectors. A universal primer,
complementary to the region 5' to the cloning site,
initiates DNA synthesis complementary to the M13
strand downstream from the probe sequence. Since
the DNA polymerase will incorporate radioactive
nucleotide triphosphates and biotin
5-(3-aminoallyl) deoxyuridine triphosphate into the
new strand, those labels can be attached to the
vector away from the probe sequence. The double
stranded portion can also be modified by reaction
with 8-azidoethidium.
The preparation of labeled antibodies is
described extensively in the literature.
Incorporation of 1 5I-label can be accomplished by
the method of Balton and Hunter (1973) Biochem. JO
133:529. Ishikawa et al (1983~ J. Immunoassay
4:209 have outlined several different methods for
coupling various enzymes to antibodies. Yoshitake
et al (1979~ Eur. J. Biochem. 101:395 have
MS~1362
- 27 -
describPd a method for using maleimides to couple
glucose oxidase to antibody. Alkaline phosphatase
can be coupled to antibody with glutaraldehyde
~Voller et al (1976~ Bull. World Health Organ.,
53:55]. Antibodies can be labeled with fluorescein
by the metho~ of Blakeslee and Baines (1976) J.
Immunol. Meth., 13:305. Chemiluminescent labels
can be introduced by the method of Schroeder et al
(1981) Clin. Chem. 27:1378.
lQ With reference to the drawings and the
examples which follow, a few specific embodiments
of the present assay can be described.
The method illustrated in Fig. 1 involves the
use of an unlabeled polynucleotide probe which is
either RNA or DNA when the sample sequence of
interest is RNA or is RNA when the sample sequence
is DNA. Two populations of anti hybrid antibodies
(Ab) are employed which can comprise the same or
different polyclonal or monoclonal immunoglobulins
selective for RNA-RNA or RNA DNA hybrids, as the
case may be. The first anti-hybrid reagent is
presented in an immobilized form and the second is
labeled with a fluorescent substance. Upon
formation o hybrids between the sequence of
interest and the probe, binding sites for the
anti-hybrid reagents are formed. As a result, the
hybrids become immobilized through binding of the
immobilized anti-hybrid and fluorescently labeled
through binding of the labeled anti-hybrid. The
3Q resulting immobilized and labeled hybrids are
separated from unbound labeled anti-hybrid and the
amount of fluorescence produced by the immobile
species is measured and correlated with the
MS-1362
- 28 -
presence or amount of the sequence of interest in
the sample.
In the method shown in Fig. 2, the probe is
doubly modified, both with a biotin label (bio) and
a chemically linked intercalator (I~. An antibody
(Ab) to intercalation complexes serves as the
critical anti-hybrid reagent and is presented in an
immobilized form. The biotin-label is detected by
use of avidin (Av) labeled with a detectable
enzyme. Upon completion of the hybridi~ation step,
the resulting immobilized and labeled hybrids are
separated from unbound labeled avidin and the
en~yme activity of the immobile species is measured
and related to tne presence of the sequence of
interest.
Reac-tion Mixture
The test sample to be assayed can be any
medium of interest, and will usually be a liquid
sample of medical, veterinary, environmental,
nutritional, or industrial significance. Human and
animal specimens and body fluids particularly can
be assayed by the present method, including urine,
blood (serum or plasma), milk, cerebrospinal fluid,
sputum, fecal matter, lung aspirates, throat swabs,
genital swabs and exudates, rectal swabs, and
nasopharnygal aspirates. Where the test sample
obtained from the patient or other source to be
tested contains principally double stranded nucleic
acids, such as contained in cells, the sample will
3Q be treated to denature the nucleic acids, and if
necessary first to release nuclei- acids from
cells. Denaturation of nucleic acids is preferably
MS-1362
3 .~
- 29
accomplished by heating in boiling water or alkali
treatment ~e.g., 0.1 N sodium hydroxide), which if
desired, can simultaneously be used to lyse cells.
Also, release of nucleic acids can, for example, be
obtained by mechanical disruption (freeze/thaw,
abrasion, sonication), physical/chemical disruption
(detergents such as Triton, Tween, sodium
dodecylsulfate, alkali treatment osmotic shock, or
heat3, or enzymatic lysis (lysozyme, proteinase K,
pepsin). The resulting test medium will contain
nucleic acids in single stranded form which can
then be assayed according to the pxesent
hybridization method. In those situations where
RNA-DNA hybrids are to be detected with labeled
antibody reagents, mRNA and rRNA in the sample can
be removed from participating in the hybridization
reactions by conventional methods such as treatment
with alkaline conditions, e.g., the same conditions
used to denature the nucleic acids in the sample.
As is known in the art, various hybridization
conditions can be employed in the assay.
Typically, hybridization will proceed at slightly
elevated temperatures, e.g., between about 35 and
75DC and usually around 65C, in a solution
comprising buffer at pH between about 6 and 8 and
with appropriate ionic strength (e.g.~ 5XSSC where
lXSSC = a .15M s~dium chloride and 0.015M sodium
citrate, pH 7.0) and optionally protein such as
bovine serum albumin, and a denatured foreign DNA
3a such as from cal thymus or salmon sperm. In cases
where lower hybridization temperatures are
desirable, hydrogen bonding reagents such as
dimethyl su'foxide and formamide can be included.
The degree of complementarity between the sample
* Txade Mark
MS-1362
- 30 -
and probe strands required for hyhridization to
occur depends on the stringency of the conditions.
Factors which determine stringency are known in the
art.
Normally, the temperature conditions selected
for hybridization will be incompatible with the
binding of the anti-hybrid reagent to formed hyrids
and detection of the label response. Accordingly,
the anti-hybrid binding step and label detection
lQ step will proceed after completion of the
hybridization step. The reaction mixture will
usually be brought to a temperature in the range of
from about 3C to about 40C and the binding and
detection steps then performed. Dilution of the
hybridization mixture prior to addition of
anti-hybrids is desirable when the salt and/or
formamide concentrations are high enough to
interfere significantly with the antibody binding
reaction.
Reagent System
The present invention additionally provides a
reagent system, i.e., reagent combination or means,
comprising all of the essential elements required
to conduct a desired assay method. The reagent
system is presented in a commercially packaged
form, as a composition or admixture where the
compatability of the reagents will allow, in a test
device configuration, or more usually as a test
kit, i.e., a packaged combination of one or more
containers, devices, or the like holding the
necessary reagents, and usually including written
instructions for the performance of assays.
MS-1362
- 31 -
Reagent systems of the present invention include
all configurations and compositions for performing
the various hybridization formats described herein.
In all cases, the reagent system will comprise
ll~ a nucleic acid probe as described herein, and
l2) the anti-hybrid reagent. A test kit form of
the system can additionally include ancillary
chemicals such as the components of the
hybridization solution and denaturation agents
capable of converting double stranded nucleic acids
in a test sample into single stranded form.
Preferably, there is included a chemical lysing and
denaturing agent, e.g., alkali, for treating the
sample to release single stranded nucleic acid
therefrom.
The present invention will now be illustrated,
but is not intended to be limited, by the following
examples.
MS-1362
32
Examp~e I
Hybridization Assay Using Immobilized
and Labeled Anti-Hybrids
A. Preparation of an RNA probe for cytomegalovirus
A DNA fragment from the genome of
cylomegalovirus is cloned into a vector containing
the promoter for DNA dependent RNA polymerase from
bacteriophage SP6 that infects SaZmone~a
typh~murium LT2 cells. The clon~d sequence is
lQ transcribed by the polymerase to produce an RNA
probe.
Cytomegalovirus DNA is digested with EcoRI
restriction endonuclease and the fragments are
cloned into the plasmid pACYC 184 [Tamashiro et al
(1982) J. Virology 42:547-557]. The plasmids are
propagated in E. coli strain HB101 and the EcoRI e
fragment defined in the Tamashiro reference is used
for preparation of the probe. The purified plasmid
is dige~ted with EcoRI restriction endonuclease and
the insert is isolated by agarose gel
electrophoxesis [Maniatis et al ~1982) "Molecular
Cloning", Cold Spring Harbor Laboratory]. The
cytomegalovirus EcoRI e fragment is cloned into the
EcoRI site of the pSP65 vector available from
Promega Biotec, Madison, WI.
The pSP65 vector with the EcoRI e insert is
propagated in E. coli JM103 cells at 37C. The
cells are lysed and the cellular DNA is isolated by
phenol/chloroform extractions. The plasmid is
3~ separated from the chromosomal DNA by
MS-1362
:~2~
~ 33 -
centrifugation in a cesium chloride-ethidium
bromide gradient [Maniatis et al, supra].
The cesium chloride and ethidium hromide are
separated from the plasmid by gel filtration on
Sephadex G-50 (Pharmacia Fine Chemicals,
Piscataway, NJ) in 10 mM Tris-hydrochloride buffer,
pH 7.5, containing 50 mM NaCl and 1 mM EDTA. The
effluent containing DNA is collected and the DNA is
precipitated with cold ethanol. The precipitate is
dissolved in 10 mM Tris-hydrochloride buffer, pH
7.5, containing 50 mM NaCl, 10 mM MgCl~ and 1 mM
dithiothreitol and digested for 1 hour at 37C with
1 unit Hind III restriction endonuclease per
microgram DNA. The mixture is extracted once with
phenol/chloroform and the DN~ is precipitated with
cold ethanol. The precipitate is dissolved in 10
mM Tris-hydrochloride buffer, pH 7.4, to give 0.5
mg DNA/mL.
This pSP65 ~ector with the EcoRI e insert is
2Q transcribed with SP6 DNA dependent RNA polymerase
starting at the promoter and ending at the site cut
by Hind III restriction endonuclease. Most of the
DNA in the transcribed region is the EcoRI e
insert.
The transcription reaction mixture (500 ~L)
has the following compositionso 50 ~g of the Hind
III digested DNA; 40 mM Tris-hydrochloride buffer~
pH 7.5; 6 mM MgC12; 2 mM spermine; 0.5 mM ATP, CTP,
UTP and GTP; 10 mM dithiothreitol; 500 units RNasin
3Q (Promega Biotec) and 50 units of SP6 RNA polymerase
(Promega Biotec). The reaction is allowed to
proceed for 1 hour at room temperature and then an
additional 50 units of RNA polymerase is added and
reacted for one hour.
MS-1362
- 34 -
DN~ in the mixture is destroyed by digestion
for 10 minutes at 37C with 10 ~g of RNase-free
DNase. The reaction mixture is extracted with
phenol/chloroform and chromatographed on Sephadex
G-50 in 10 mM Tris-hydrochloride buffer, pH 7.4,
0.1 M NaCl. The RNA is collected and precipitated
with cold ethanol. The precipitate is dissolved in
50 mM sodium acetate buffer, pH 6.0, containing 1
mM EDTA.
B. Preparation of monoclonal antibody to RNA-DNA
1. Preparation of RNA-DNA hybrid - The
hybrid i5 prepared by transcription of ~X174 virion
DNA with DNA dependent RNA polymerase from E. coli.
The procedure is described by Nakazato (1980)
Biochem 19O2835.
2. Preparation of methylated thyroglobulin -
Bovine thyroglobulin ISigma Chemical Co., St.
Louis, MO), 100 mg, is combined with 10 ml of
anhydrous methanol and 400 ~L of 2.5 M HCl in
2Q methanol. The mixture is allowed to react for 5
days on a rotary mixer at room temperature. The
precipitate is collected by centrifugation and
washed twice with methanol and twice with ethanol.
Then it is dried under vacuum overnight.
3. Immunization of mice - One hundred fifty
micrograms of RNA-DNA hybrid in 250 ~L of 20 mM
Tris-hydrochloride buffer, pH 7.4, 1 mM EDT~ is
combined with 150 ~g of methylated thyroglobulin in
250 ~L water. A precipitate forms and 2.5 ml of
the Tris buffer is added. The entire mixture is
MS-1362
,'3~
- 35
emulsified with an equal volume of Freunds
adjuvant. BALB/c mice are each immunized with 0.5
ml of the suspension. Booster immunizations are
given 3 weeks later and at one week intervals
thereafter. Blood is taken at two week intervals
beginning one week after the first boost.
Antibody titers in the serums are measured by
an enzyme labeled immunosorbent assay method.
Immulon II (Dynateck, Alexandria, VA) microtiter
wells are coated with RNA DNA by placing 50 ~L of a
5 ~g/ml solution in each well. The RNA-DNA is in
0.015 M sodium citrate buffer, pH 6.8, containing
0.15 M NaCl. When the solutions have stood at room
temperature for 2 hours, the wells are washed with
0.02 M sodium phosphate buffer, pH 7.4, containing
5 mg bovine serum albumin/mL and 0.5% Tween 20
detergent (v/v). Appropriate dilutions of
antiserums are added to the wells to allow binding
of antibodies to the in~obilized RNA DNA. Then the
bound antibodies are detected with enzyme labeled
antl-mouse IgG by well known procedures. Spleen
cells from a mouse with a high serum titer to
RNA-DNA but low titer to single stranded DNA are
fused with myeloma cells to produce hybridomas
[Stuart et al (1981) Proc. Natl. Acad. Sci. USA.
78:3751, Galfre and Milstein (1981) Meth. in
Enzymol. 73:1].
Cloned hybridomas are grown intraperitoneally
in mice to produce adequate quantities of antibody
3Q for further work. Albumin is removed from the
ascites fluid by chromatography on Affigel-Blue~
resin (Bio-Rad Laboratories, Richmond, CA)
equilibrated with 10 mM Tris-hydrochloride buffer,
pH 8.0, 0.15 M NaCl. The column effluent
MS-1362
~5~ 3~
- 36 -
containing antibody is chromatographed on
DEAE-Sepharose ~Pharmacia Fine Chemicals,
Piscataway, NJ) using a linear gradient from 10 mM
Tris-hydrochloride buffer, pH 8.0, to this buffer
containing 0.2 M NaCl. The major peak of eluted
protein contains the monoclonal antibody free of
transferrin and albumin.
C. Immobilization of monoclonal antibody
to RNA DNA.
The monoclonal antibody to RNA DNA is
immobilized on magnetizable microparticles, Act
-Magnogel AcA44, available from LKB Instruments,
Gaithersburg, MD. The particles are porous
polyacrylamide-agarose beads containing 7% iron
oxide and this resin is activated with
glutaraldehyde. Coupling o the antibody to
Ac~-Magnogel AcA44 is carried out according to the
manufacturer's instructions~
Do Fluorescein labeled antibody to RNA-DNA
2Q Purified antibody to RNA-DNA is dialyzed into
0.1 M sodium borate buffer, pH 9.0, and 0.5 ml
containing 5 mg antibody is combined with 0.5 ml of
5-~4,6-dichlorotriazin-2-yl)-aminofluorescein
(Molecular Probes, Inc., Junction City, OR) in the
borate buffer. The mixture is allowed to react for
1 hour at 25C and then chromatographed on a 1 x 25
cm Biogel P-6DG (Bio-Rad Laboratories) column with
0.1 M Tris-hydrochloride buffer, pH 8.0, as eluent.
~bsorbances at 280 nm of 1.0 ml fractions are
3Q monitored and those comprising the first peak are
pooled. The fluorescein/protein ratio is
MS-1362
~ ~r~ .3;~"
determined by the method of The and Feltkamp (1970
Immunology 18:865.
E. Hybridization assay for cytomegalovirus
in urine
This assay involves the separation of
cytomegalovirus from urine by centrifugation and
hybridization of viral DNA with the RNA probe in
solution. The amount of hybrid formed is
determined immunochemically with the immobilized
antibody to RNA DNA and the fluorescein labeled
antibody.
Cells and cellular debris are removed from
urine by centxifugation in a Sorvall GLC-3
centrifuge at 3000 rpm for five minutes. Ten
milliliters of supernatant is placed in a
polyallomer ultracentrifuge tube and run at 25,000
rpm in a Beckman Ti50 rotor for 75 minutes. The
supernatant is removed and the pellet is dissolved
in 0.05 mL of 0.1 M NaOH and incubated at 37C for
30 minutes.
One hundred fifty microliters of the ollowing
solution is added: 0,1 M sodium phosphate buffer,
pH 6.0; 1.8 M NaCl; 0.1% sodium dodecylsulfate
(w/v) and 1 ~M EDTA. Then 20 ~L of the RNA probe
(20 ng) is added and the mixture is incubated at
65C for 10 hours, The reaction is cooled to room
temperature and 650 ~L of 0.1 M sodium phosphate
buffer, pH 7.4, containing 2 mM MgCl2 and 5.0 mg
bovine serum albumin/ml is added. Then 50 ~L cf
the immobilized antibody is added and the mixture
is agitated for 30 minutes in such a way as to keep
the solid-phase antibody in suspension. Fifty
MS~1362
iY~3
- 38 -
microliters of fluorescein labeled antibody is
added and the agitation is continued for 1 hour,
The concentrations of the immobilized antibody and
the fluorescein labeled antibody reagents are
optimized in preliminary experiments to provide
antibody levels in large excess over those required
to bind all of the RNA-DNA hybrid present in the
assays.
At the end of the reaction period the solid
support is attracted to one wall of the reaction
tube with a magnet and the liquid is removed by
aspiration. The solid phase is washed twice, 1 mL
each, with 0.1 M sodium phosphate buffer~ pH 7.4
containing 2.0 mM MgC12 and 0.1% Tween 20 (v/v~.
The fluorescein labeled antibody bound to the
solid phase is dissociated by addition of lo0 mL of
0.1 M NaO~. The suspension is agitated for 5
minutes and the solid phase is attracted to the
bottom of the tube with a magnet. The fluorescence
2Q of the solution is measured with 495 nm light for
excitation and 520 nm for emission.
A control assay is run in parallel with a
urine sample which is known to be free of
cytomegalovirus. Fluorescence signals generated
with the infected urine specimens will be higher
than those for the controls.
MS-1362
s~
-- 39 --
Examp Z e II
Hybridization Assay Using Immobilized
Anti-Hybrid and Labeled Probe
A. Preparation of a labeled probe for
cytomegolovirus DNA
The EcoRI e fragment from cytomegolovirus
described in Example I above is cloned into the
M13mp8 vector available from New England Biolabs,
Beverly, MA. -The recombinant ViïUS is propagated
lQ in the E. coli K12JM101 host. The virus is
separated from the culture fluid by precipitation
with polyethyleneglycol and the single stranded
virion DNA is isolated by phenoltchloroform
extraction.
A 17 base oligodeoxynucleotide primer with the
sequence GTAAAACGACGGCCAGT is complementary to a
segment of M13mp8 close to the 3'-OH end of the
EcoRI e insert. This primer will initiate DNA
synthesis by the Klenow fragment o-f DNA polymerase
I from E. coli and the replication is directed
through the EcoRI e insert. The new DNA probe is
labeled with biotin by including Bio-11-dUTP
~available from En~o Biochem, Inc., New York, NY)
in the reaction mixture in place of the usual dTTP
[Leary et al (1983) Proc. Natl. Acad. Sci.
80:4045].
The purified M13mp8 DNA with the EcoRI e
insert is combined with a molar excess of the 17
base primer (New England Biolabs~ in 20 mM Tris-HCl
3~ buffer, pH 8.0, containing 10 mM MgCl2 (final
concentrations) [Bankier and Barrell, Techniques in
MS-1362
3~2
- 40 -
Nucleic Acid Biochemistry, Elsevier, Ireland]. The
mixture is incubated at 55C for 45 minutes to
anneal the primer to the M13mp8 DNA. The reaction
mixture is made 15 mM in dATP, dGTP, dCTP and
Bio-ll-dUTP and finally the Klenow fragment of DNA
polymerase I is added. This reaction is incubated
at 25C for a period determined for each set of
reagents. The reaction time is optimized to give
newly synthesized DNA fragments somewhat larger
than the EcoRI e insert. To determine the optimum
time, samples are taken from the reaction mixture
at various times and electrophoresed in denaturing
al~aline agarose gel~ ~Maniatis et al, supr~].
This procedure will provide biotin-label~d DNAs
with some variations in length; however, extension
of the labeled DNA beyond the EcoRI e insert into
the M13 sequence is acceptable for the present
purpose.
The DNA is purified by phenol/chloroform
2Q extraction and precipitated with ethanol. It is
dissolved in 20 mM Tris-hydrochloride buffer, pH
8.0, and ethidium residues are introduced as
covalent intercalation complexes. For this purpose
8-azidoethidium is prepared and purified as
2~ described by Graves et al ~1977) BiochimO Biophys.
Acta 479:98. (Our studies show that this procedure
gives a mixture of 3- and 8-azidoethidium isomers.)
A solution o~ the biotin labeled DNA complexed
to the M13 template (about 50 ~g DNA/mL) is made
3~ 0.5 mM in 8-azidoethidium and photolyzed for 1 hour
at a distance 10 to 20 cm from a 150 watt outdoor
spotlight. The D~A solution is stirred during the
photolysis in a glass reaction tube which is in a
glass water bath. The glass absorbs ultraviolet
MS-1362
3~
~ 41 -
radiation and the water bath is used to keep the
reaction temperature between 20 and 30~C.
At the end of the photolysis the noncovalently
bound ethidium photolysis products are removed by
10 successive extractions with water saturated -
n-butanol. Then the DNA is precipitated with
ethanol and dissolved in the Tris buffer. The
amount of covalently bound DNA is estimated by
means of optical absorption measurements and the
extinction coefficients of E490 ~ 4 x 103 M CM 1
fcr photolyzed ethidium azide~ the relationship
between A260 and A490 for photolyzed ethidium bound
to DNA [A26~ = (A490 x 2.3)-0.011] and E260 - 1.3 x
104 M 1 cm for the DNA base pair concentration.
The ethidium will be covalently bound to the
DNA almost exclusively in the double stranded
region where intercalation complexes can form. The
goal is to introduce one ethidium residue per lO to
50 base pairs. The photolysis reaction goes nearly
2Q to completion during khe one hour reaction period
and the incorporation of ethidium can be decreased
by reducing the photolysis time or reducing the
ethidium concentration. Incorporation can be
increased by repeating the photolysis with fresh
8-azidoethidium.
The final step is the separation of the biotin
labeled and intercalator-modified probe from the
Ml3 mp8 template DNA. The separation is
accomplished by electrophoresis in alkaline agarose
gel [Maniatis et al, supra]. The DNA bands are
detected in the gel by fluorescent staining ~ith
ethidium bromide. The biotin labeled DNA is
smaller than the M13 mp8 template strand and
therefore migrates faster. Gel containing the
M~-1362
~'~5~3~
- 42 -
biotin labeled DNA is excised and the DNA is
recovered by electroelution as described in the
Maniatis reference.
B. Preparation of monoclonal antibody to
ethidium intercalated DNA
l. Preparation of covalent ethidium-DNA
complexes
About 250 mg of salmon sperm DNA (Sigma
Chemical Co., St. Louis, MO) i5 dissolved in 40 ml
of 50 mM NaCl and sheared by five passages through
a 23 gauge needle. The sheared DNA is placed in a
250 mL flask and diluted with an additional 160 mL
of buffer. One hundred forty-five microliters (145
~L~ of S1-nuclease, 200,000 units per mL (Pharmacia
P-L Biochemicals, Piscataway, NJ), is added and the
mixture is incubated at 37C for 50 minutes.
Then the reaction mixture is extracted twice
with phenol:chloroform, once with chloroform and
the DNA is precipitated twice with ethanol
2~ [Maniatis et al, s~p~]. The final precipitate is
dissolved in 70 mL of 20 mM Tris hydrochloride
buffer, pH 800.
This DNA is reacted with 8-azidoethidium under
the following conditions. The reaction mixture is
prepared with 33 ml of 2.7 mj DNA/mL, 13.5 mL of
4.95 mM 8~azidoethidium, 13.5 mL of 0.2 M
Tris-hydrochloride buffer, pH 8.0, 0.2 M NaCl, and
76 mL water. The mixture is placed in a 250 mL
beaker with a water jacket maintained at 22C. The
3~ mixture is stirred and illuminated for 60 minutes
by a 150 watt spotlight at a distance of 10 cm.
MS-1362
~,J~ X9
- 43 -
This photolysis is repeated with an identical
reaction mixture.
The photolyzed reaction mixtures are combined
and extracted 10-times with an equal volume each
time of n-butanol saturated with 20 mM
Tris-hydrochloride buffer, pH 8.0, 0.2 M NaCl. The
extracted DNA solution is combined with 23 mL of
4.95 mM 8-azidoethidium and 77 mL of 20 ~M
Tris-hydrochloride buffer, pH 8.0, 0.2 M NaCl.
This solution is photolyzed for 60 minutes as
described above. The reaction products are
extracted 10 times with buffer saturated butanol as
described above and the DNA i5 precipitated with
ethanol. The precipitate is dissolved in 10 mM
Tris-hydrochloride buffer, pH 8.0, 1 mM EDTA and
the absorbances at 260 and ~90 nm are recorded.
~ . Preparation of covalent ethidium-DNA
methylated thyroglobulin complex.
Methylated thyroglobulin (5 mg) prepared as
described in Example I, part B-2 above is dissolved
in 1.0 mL water and 5.6 ml of a 2.2 mg/mL covalent
ethidium DNA solution is added. A precipitate
forms immediately and the suspension is diluted to
62.5 mL with O.lS M NaCl. This suspension is
emulsified with an equal volume of Freunds adjuvant
and each BALB/c mouse is immunized subcutaneously
with 0.5 mL of the mixture. Booster immunizations
are given at 2 week intervals with the same
mixture. I'est bleedings are taken one week after
3Q each immunization beginning after the third boost.
Antibody titers are assayed by standard enzyme
label immunoadsorbant procedures. Covalently
MS-1362
~C ~ 3~
- 44 -
intercalated DNA, double stranded DNA and single
strandPd DNA are coated on microtiter wells as
described in Example I, part B-3 a~ove. Diluted
antiserums are incubated in the coated wells and
antibody bound to the immobilized antigens are
detected as outlined above. Antiserums are
screened for those which have low titers to single
and double stranded DNA but high titers to
covalently intercalated DNA.
In an additional experiment, antibody titers
to noncovalent ethidium-DNA intercalation complex
~ are measured. For this purpose, double stranded
DNA is coated on the wells and 100 ~M ethidium
bromide is included in ths diluted antiserum and
all subsequent binding and wash solutions, but it
is not included in the substrate solution used for
measurement of enzyme activity.
Antiserums typically give higher titers to
covalently intercalated DNA than to the nonco~alent
2Q complex. This difference is due to the presence of
antibodies to the ethidium residue which becomes
covalently bound in low yield to the
phosphate-ribose structure of double stranded DNA.
Therefore animals with high titers to noncovalently
intercalated DNA are chosen for preparation of
hybridomas.
Spleen cells from the selected mice are fused
with myeloma cells to produce hybridom~s [Stuart et
al, sup~a; Gilfre and Milstein, sup~a~. The
3~ hybridoma cells are cloned in selective medium and
those which produce antibody to noncovalently
intercalated DNA are selected for antibody
production intraperitoneally in mice.
MS-1362
`~
- 45 -
The antibody is isolated from the ascites
fluid as described in Example I, part B-3 above.
C. Preparation of ~-Galactosidase-Streptavidin
Conjugate
Sulfhydryl residues on ~-galactosidase are
exposed by reduction with dithiothreitol.
~-Galactosidase (30,000 units, grade VIII, Sigma
Chemical Co.) in 2 mL of 0.1 M
N-2-hydroxyethyl-piperazine-N'-2-ethane sulfonate
lQ buffer (HEPES), pH 7.0, 0.09 M NaCl, is combined
with 3.5 ~mol of dithiothreitol and allowed to
stand at room temperature for 4 hours. The
dithiothreitol is removed by chromatography on a
2.5 x 80 cm column of Sepharose 6B Cl (Pharmacia
Fine Chemicals) in the buffer described above.
Fractions containing protein are combined into a
pool. The number of moles of sulfhydryl groups per
mole of enzyme are measured by the method of Ellman
(1959) Arch. Biochem. Biophys. 82:70.
Succinimidyl-4-(N-maleimidomethyl)cyclohexane
1-carboxylate ISMCC) (Pierce Chemical Co.,
Rockford, IL), 5.3 mg, is dissolved in 250 ~L of
anhydrous N,N-dimethylformamide and a 40 ~L aliquot
is combined with 3 mL of 0.1 M HEPES buffer, p~
7.0, 0.15 M NaCl. A 25 ~L aliquot of this aqueous
solution is added to 825 llL of HEPES/NaCl buffer
and 100 ~L of 1 mM glutathione Ireduced form).
When this reaction mixture has stood at room
temperature for 15 minutes, the unreacted
glutathione is determined by Ellman's method. The
results are used to calculate the SMCC
concentration.
MS-1362
23~
- 46 -
Streptavidin obtained from Bethseda Research
Laboratories, Gaithersburg, MD, is exchanged into
0~1 M HEPES buffer, pH 7.0, 0.15 M NaCl by gel
exclusion chromatography in Biogel P-6DG.
Following exchange, 1.75 ml of 3.7 mg/mL
streptavidin is combined with 17.6 ~L of 61 mM SMCC
and allowed to react for one hour at 30C. The
reaction mixture is chromatographed on a 1 x 25 cm
column of Biogel P-6DG in the HEPES buffer. The
fractions corresponding to the first effluent peak
with absorbance at 280 nm are pooled and assayed
for maleimide content by back titration of
glutathione as outlined above.
Since the maleimide i5 subject to hydrolysis,
coupling to ~-galactosidase is initiated as soon as
possible. Activated streptavidin, 3.9 mg, in 3.3
ml of the HEPES buffer is added to 32 mg of reduced
~-galactosidase to give a reaction volume of 9.3
ml. After 4 hours at 25C the reaction is ~uenched
2Q by adding 800 ~L of 1 mM glutathione and incubated
an additional 30 minutes at 25C. Then the
reaction mixture is chromatographed on a 1.5 x 110
cm column of Biogel A-1.5 m ~Bio-Rad Laboratories)
and developed with 0.1 M HEPE5 buffer, pH 7.0, 0.15
M NaClO Two milliliter fractions are collected and
absorbances at 280 nm are recorded. The first peak
is pooled for further study.
MS-1362
- 47 -
D. Immobilization of antibody to ethidium
intercalated DNA.
The monoclonal antibody to ethidi~m
intercalated DNA is immobilized on Act-Magnogel
AcA44 as described in Example 1, part C above.
E. Hybridization assay for cytomegalovirus in
urine.
Processing of urine specimens and
hybridization reactions are carried out as
described in Example I, part E above except that
the biotin labeled DNA probe ~section A) is used in
place of the RNA probe. Following the
hybridization reaction, 650 ~L of 0.1 M sodium
phosphate buffer, pH 7.4, containing 2 mM MgCl2,
5.0 mg bovine serum albumin/ml and the immobilized
antibody to ethidium intercalated DNA (Part D
above~ is added. The mixture is agitated for 30
minutes in such a way as to keep the solid-phase
antibody in suspension. Then 50 ~L of
~-galactosidase labeled streptavidin is added and
the agitation is continued for one hour.
When the incubation is completed, the solid
phase is attracted to one wall of the reaction tube
with a magnet and the liquid is removed by
aspiration. The solid phase is washed twice, l mL
each, with 0.1 M sodium phosphate buffer, pH 7.4,
containing 2.0 mM MgCl2 and 0.1~ Tween 20 (v/v).
The enzyme activity associated with the solid
phase is measured by addition of 1.0 ml of 0.1 M
3a sodium phosphate buffer, pH 7.4, containing 800 ~M
MS-1362
3~
- ~8 -
7 ~-galactosyl-3-[6-aminohexylcarboxamide] coumarin
[Worah et al (1981) Clin. ChemO 27:673]. At the
end of this incubation the solid phase particles
are attracted to the bottom of the cuvette with a
magnet and the fluorescence of the solution is
recorded using 400 nanometers (nm) excitation and
450 nm emission.
A control assay is run in parallel with a
urine sample which is known to be free of
cytomegalovirus. The fluorescence signals
generated with the infected urine specimens will be
higher than the controls.
MS-1362
