U.S. patent number 4,623,627 [Application Number 06/524,596] was granted by the patent office on 1986-11-18 for monoclonal antibody having specificity for the double-stranded conformation of native dna and diagnostic methods using same.
This patent grant is currently assigned to Cetus Corporation. Invention is credited to Stanley N. Cohen, Chun-Ming Huang.
United States Patent |
4,623,627 |
Huang , et al. |
November 18, 1986 |
Monoclonal antibody having specificity for the double-stranded
conformation of native DNA and diagnostic methods using same
Abstract
Monoclonal antibodies having conformation-dependent specificity
for native dsDNA, as exemplified by the IgM antibody produced by
murine hybridoma ATCC No HB 8329. These antibodies are used to
detect DNA duplex formation in DNA hybridization tests.
Inventors: |
Huang; Chun-Ming (Cupertino,
CA), Cohen; Stanley N. (Portola Valley, CA) |
Assignee: |
Cetus Corporation (Emeryville,
CA)
|
Family
ID: |
24089879 |
Appl.
No.: |
06/524,596 |
Filed: |
August 19, 1983 |
Current U.S.
Class: |
435/333;
435/6.14; 435/7.9; 435/948; 435/962; 436/548; 530/388.21;
530/388.9; 530/809; 530/864 |
Current CPC
Class: |
C07K
16/18 (20130101); C12Q 1/6804 (20130101); G01N
33/5308 (20130101); Y10S 530/864 (20130101); Y10S
435/948 (20130101); Y10S 530/809 (20130101); Y10S
435/962 (20130101) |
Current International
Class: |
C07K
16/18 (20060101); C12Q 1/68 (20060101); G01N
33/53 (20060101); C12N 005/00 (); C12N 015/00 ();
C12Q 001/68 (); G01N 033/577 () |
Field of
Search: |
;435/6,7,68,172.2,240,948,810 ;436/508,513,540,548,815,501
;260/112B,112R ;935/78,95,103,104,105,110 ;530/387,809 ;536/27 |
References Cited
[Referenced By]
U.S. Patent Documents
|
|
|
4358535 |
November 1982 |
Falkow et al. |
|
Foreign Patent Documents
Other References
Hahn, B. H., et al, Arthritis and Rheumatism (1980) 23:942-945.
.
Tron, F., et al, J Immun (1980) 125:2805-2809. .
Gilliam, A. C., et al, J. Immun (1980) 125:874-885. .
Andrzejewski, Jr., C., et al, J Immun (1980) 124:1499-1502. .
Ballard, D. W., et al, Molecular Immunology (1982) 19:793-799.
.
Koike, T., et al, Immunology Letters (1982) 4:93-97. .
Lafer, E. M., et al, PNAS (U.S.A.) (1981) 78:3546-3550. .
Moller, A., et al, J Biol Chem (1982) 251:12081-12085. .
Ballard, Fed. Proc., 40 (3): 975, Abst. 4221, Mar. 1, 1981. .
Tron et al, Clin. Immunol. Immunopathol., 24 (3): 351-360, (1982),
Abstract. .
Tron et al, Clin. Immunol. and Immunopath., 24:351-360 (1982).
.
Ballard et al, Molec. Immunol., 19 (6): 793-799 (1982). .
Kohler et al, Nature, 256: 495-497 (1975)..
|
Primary Examiner: Kepplinger; Esther M.
Attorney, Agent or Firm: Halluin; Albert P. Ciotti; Thomas
E. Hasak; Janet E.
Claims
We claim:
1. Hybridoma ATCC HB 8329.
Description
DESCRIPTION
1. Technical Field
The invention is in the fields of immunochemistry, nucleic acid
chemistry, and immunodiagnostics.
2. Background Art
Many biomedical research and recombinant DNA procedures involve the
use of single-stranded DNA (ssDNA) probes to identify particular
DNA molecules. The nucleotide sequence of the probe is
complementary to part or all of the nucleotide sequence of a strand
of the DNA molecule to be identified and the procedures involve
hybridizing (annealing) the probe to a denatured (single-stranded)
form of the DNA to be identified. Resulting duplexes are detected
directly via components (e.g., radioisotopes) of the probe that are
per se detectable or indirectly via reactive components of the
probe (e.g., biotin or derivatives thereof) that form detectable
derivatives. Examples of such procedures are described in U.S. Pat.
No. 4,358,535 and European Patent Application No. 82301804.9
(publication no 0063879). A major practical problem in this DNA
probe technology lies in the difficulty and expense of
incorporating moieties such as radioisotopes or biotin into the
complementary ssDNA probe. A main object of the present invention
is to avoid this problem and provide a technique for detecting DNA
duplexes that does not involve the inclusion of such moieties in
the complementary ssDNA probe.
The present invention provides an immunochemical that specifically
recognizes the doublestrandedness of native DNA duplexes, namely a
monoclonal antibody that has conformation-dependent specificity for
native double-stranded DNA (dsDNA). By using this antibody in DNA
probe technology, the requisite that the probe DNA itself contain
an added detection component is eliminated. The only requisite of
the probe DNA is complementarity to the DNA to be identified.
Accordingly, a major feature of this invention is that it allows
the probe DNA to be produced by cloning single-stranded DNA (ssDNA)
using vectors such as M13. Cloning the probe with such
single-stranded phage vectors allows excess probe to be used,
thereby increasing the sensitivity of duplex detection.
As re.g.ards the monoclonal antibody of the invention, there are
numerous prior reports of murine monoclonal antibodies against
native DNA. See Arthritis and Rheumatism (1980) 23:942-945; J Immun
(1980) 125:2805-2809; J Immun (1980) 125:824-885; J Immun (1980)
124:1499-1502; Molecular Immunology (1982) 19:793-799; and
Immunology Letters (1982) 4:93-97. These antibodies were generated
by hybridomas made by fusing available murine plasmacytomas with
spleen cells from mice (e.g., F.sub.1 hybrid New Zealand
Black/White mice) that normally produced high titers of anti-dsDNA
antibodies. All of the prior anti-DNA monoclonal antibodies have
either bound ssDNA preferentially or bound both ssDNA and dsDNA.
Since these prior antibodies are not exclusively specific for dsDNA
they are unsuitable for detecting duplexes in the above described
DNA probe technology.
Polyclonal and monoclonal antibodies against the Z form of DNA have
been reported. PNAS (USA) (1981) 78:3546-3550 and J Biol Chem
(1982) 251:12081-12085.
DISCLOSURE OF THE INVENTION
One aspect of the invention is a monoclonal antibody that has
exclusive specificity for the double-stranded conformation of
native DNA. This antibody conjugated to a label or a chromatography
support are variations of this aspect of the invention.
Another aspect of the invention is hybridoma ATCC HB 8329 which
produces a monoclonal (IgM light chain) antibody that has exclusive
specificity for the double-stranded conformation of native DNA.
Other aspects of the invention are immune complexes that comprise
the above described monoclonal antibody bound to dsDNA. Embodiments
of such complexes are binary complexes of dsDNA and labeled
conjugates of the monoclonal antibody and ternary complexes of
dsDNA, the monoclonal antibody and a labeled antibody against the
monoclonal antibody.
Various immunodiagnostic methods are additional aspects of the
invention. The common steps in these methods are (1) binding the
monoclonal antibody to dsDNA and (2) detecting the resulting
complex via a label conjugated to the monoclonal antibody or a
label conjugated to an immunochemical bound directly or indirectly
to the monoclonal antibody. When used in DNA probe technology to
detect a given DNA sequence in a sample, such as a sequence that
characterizes a genetic disorder, pathogenic disease, or other
medical condition, the binding reaction (step (1) above) will be
preceded by the steps of treating the sample to denature any dsDNA
contained in it and hybridizing the denatured (single-stranded) DNA
with ssDNA probe that is complementary to the given DNA
sequence.
The invention also contemplates kits for carrying out such
immunodiagnostic methods.
Methods and kits for isolating and/or identifying dsDNA molecules,
such as plasmids after denaturing, are also part of the
invention.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A, 1B, and 2 are graphs of the results of blocking
radioimmunoassay tests described in the examples.
MODES FOR CARRYING OUT THE INVENTION
The invention antibody recognizes the double-stranded conformation
of native DNA as the immunodominant feature of the dsDNA molecule.
In other words, the specificity of the determinant(s) the antibody
recognizes is dictated by the double-stranded conformation of
native dsDNA rather than by any sequence of nucleotides. The
antibody recognizes native DNA duplexes re.g.ardless of the species
of the DNA. Thus, as used herein the term "native" refers to dsDNA
that occurs in nature and includes, without limitation, dsDNA
derived from microorganisms, viruses, plants, fish, avians, and
mammals or synthetic dsDNA, such as the duplexes in DNA probe
technology, that is homologous or substantially homologous to
naturally occurring dsDNA. The antibody does not bind to some
synthetic dsDNA made from complementary homopolymeric strands of
DNA.
While the monoclonal antibody that is specifically exemplified
herein is a murine IgM, the invention is not limited to any
particular species, class or subclass of immunoglobulin. Monoclonal
antibodies of mouse, rat and human origin are currently preferred
because of the availability of suitable mouse, rat and human cell
lines to make the hybridomas that produce the antibody. Monoclonal
antibodies of the same class or other classes such as IgG
(including IgG subclasses such as IgG1, IgG2A, IgG3, etc), IgA, and
IgD that are functionally equivalent to the murine IgM specifically
exemplified herein may be made and identified by following the
hybridoma synthesis and screening techniques described herein. As
used herein the term "functional equivalent" is intended to mean a
monoclonal antibody other than the exemplified murine IgM that also
is native dsDNA specific. Preferred functional equivalents
recognize the same determinant and cross-block the exemplified
murine IgM.
If desired, the monoclonal antibodies may be derivatized (labeled)
using conventional labeling reagents and procedures. As used herein
the term "label" is intended to include both moieties that may be
detected directly, such as radioisotopes or fluorochromes, and
reactive moieties that are detected indirectly via a reaction that
forms a detectable product, such as enzymes that are reacted with
substrate to form a product that may be detected
spectrophotometrically.
The antibodies may also be covalently coupled to chromatography
supports (e.g., the surfaces of tubes or plates or the surfaces of
particulate bodies such as beads) using available bifunctional
coupling agents, such as carbodiimides, to make effective
adsorbents for affinity purifying dsDNA. Such adsorbents may be
used to isolate plasmids in the following manner. Bacteria
containing the desired plasmid are lysed and their DNA is
denatured. Lysing and denaturing may be done in a single step by
suspending the cells in buffer at a high pH, e.g., 12.5. The
resulting solution is then neutralized by adding cold buffer at pH
7-7.5. This will cause DNA duplexes to reform. Cellular debris is
then removed from the solution by filtering or centrifugation. The
filtrate/supernatant is then passed through a column containing the
monoclonal antibody fixed to a support. Duplex plasmid DNA will be
retained by the column and may be eluted therefrom with an
appropriate elutant.
Kits for isolating plasmid DNA will contain the monoclonal antibody
conjugated to the support, buffer for lysing bacterial cells and
denaturing the DNA, a neutralizing buffer and an elution
reagent.
The monoclonal antibodies of the invention may be made using the
somatic cell hybridization procedure first described by Kohler, G.
and Milstein, C., Nature (1975) 256:495-497. The tumor cell lines,
reagents, and conditions used in this procedure are well known and
have been reviewed extensively in the literature. (Somatic Cell
Genetics (1979) 5:957-972 and Monoclonal Antibodies (1980) Plenum
Press). Basically the procedure involves fusing an appropriate
tumor cell line with cells (typically spleen cells) that produce
the antibody of interest using a fusogen such as polyethylene
glycol. Antibody-producing cells are typically made by immunizing a
host with the immunogen of interest. In this regard the common
source of anti-DNA antibodies are animals that have or are prone to
have systemic lupus erythematosus (SLE) or similar diseases. Such
animals spontaneously produce antibodies against DNA. Normal
animals immunized with DNA are generally not considered to be good
sources of anti-DNA antibody-producing cells. In making the murine
IgM specifically exemplified herein, however, a normal mouse
immunized with synthetic polynucleotide homopolymers (e.g.,
poly(dA.multidot.dT).multidot.poly(dA.multidot.dT),
poly(dI.multidot.dC).multidot.poly(dI.multidot.dC), and
poly(dG.multidot.dC).multidot.poly(dG.multidot.dC)) and boosted
with foreign native DNA were used as a source of anti-DNA
antibody-producing spleen cells. These spleen cells are fused with
a compatible myeloma line, such as line FO (Transplantation Proc
(1980) Vol XII, No 3:447-450), that gives a high fusion frequency.
After the fusion the conventional procedures of growing the fusion
product in a selective growth medium, such as HAT medium, to
eliminate unhybridized myeloma and spleen cells is followed. Clones
having the required specificity are identified by assaying the
hybridoma culture medium for the ability to bind to dsDNA and
ssDNA. dsDNA+/ ssDNA- clones may be further characterized by
further specificity testing. Hybridomas that produce antibodies
having conformation-dependent specificity for dsDNA may be
subcloned by limiting dilution techniques and grown in vitro in
culture medium or injected into host animals and grown in vivo. The
antibodies may be separated from resulting culture medium or body
fluids by conventional antibody fractionation procedures such as
ammonium sulfate precipitation, DEAE cellulose chromatography,
affinity chromatography and the like. The antibody may be further
purified, if desired, by ultracentrifugation and
microfiltration.
A principal use of the above-described anti-dsDNA monoclonal
antibodies is to detect DNA duplex formation in diagnostic DNA
hybridization tests similar to those described in U.S. Pat. No.
4,358,535. These methods are used in the field of medical
diagnostics to determine the presence or quantity of a specific DNA
molecule in a sample that characterizes a particular organism such
as pathogenic bacteria, fungi, yeasts, or viruses or a genetic
disorder such as sickle cell anemia or thalassemia. Such
determinations permit the diagnosis of diseases, infections or
disorders of the patient from which the sample is taken. They are
also used to screen bacteria to determine antibiotic resistance and
in gene mapping.
In these hybridizations a single-stranded polynucleotide probe is
prepared that is complementary to a strand of the DNA of interest
(e.g., the DNA that characterizes or differentiates the organism,
disorder, condition, etc). This polynucleotide probe is then
applied under hybridizing conditions to a sample suspected of
containing the DNA of interest which has been treated so as to
denature dsDNA in the sample and immobilize the resulting ssDNA.
Immobilization is usually achieved by applying the sample to an
insoluble material that has a high affinity for DNA, such as a
nitrocellulose filter or other such inert porous support.
Denaturation may be accomplished thermally or by treating the
sample with a DNA denaturing agent. Alternatively, the
complementary ssDNA probe could be immobilized and the denatured
sample applied to the immobilized probe. The surface may be
postcoated with an inert (nonhaptenic) material such as albumin to
avoid nonspecific binding of other reagents to the support.
Unhybridized materials are then removed and the hybridizate is
assayed for the presence of nucleic acid duplexes. The absence of
duplexes indicates the absence of the DNA of interest; positive
detection of duplexes indicates the presence of the nucelic acid
sequence of interest. In quantitative hybridization techniques, the
amount of duplex formation is determined and is proportional to the
amount of the DNA of interest in the sample.
The particular hybridization technique that is used is not critical
to this invention. Examples of current techniques are those
described in PNAS (USA) (1975) 72:3961-3965, PNAS (USA) (1969)
63:378-383; Nature (1969) 223:582-587 and the patent literature
mentioned under "Background Art" above. The invention may be used
with such procedures, and other existing hybridization procedures,
as well as with hybridization procedures that are developed in the
future.
In prior DNA hybridization procedures duplex detection was done by
incorporating detectable moieties such as radioisotopes or biotin
in the complementary polynucleotide probe. When the probe was
hybridized with denatured sample DNA, the moieties in turn were
incorporated into the resulting nucleic acid duplexes, making
duplex detection possible. The monoclonal antibodies of the
invention make it possible to avoid incorporating detectable
moieties in the complementary polynucleotide probes. Correlatively
they permit the polynucleotide probes to be made by cloning with
single-stranded phage vectors such as bacterio-phages of the Ff
group (e.g., M13, fd, fl) and .phi.X 174, and derivatives thereof.
Cloning with M13 or other single-stranded phage vectors provides
the polynucleotide probe in large quantity (at least about 0.5 mg/L
of culture) and high quality (pure with no breaks or ends). Use of
higher concentrations of probe in the hybridization increases the
sensitivity of duplex detection. This cloning procedure is commonly
used to make ssDNA for use in the Sanger chain-termination method
of DNA sequencing. These vectors and DNA cloning procedures
employing them are described in The Single-Stranded DNA Phages
(1978) Cold Spring Harbor Laboratory. Alternative methods for
obtaining the complementary probe are available but they are not as
efficient as cloning with single-stranded phage vectors. For
instance dsDNA of proper sequence could be denatured beforehand or
in situ, followed by restriction if necessary, and used in the
hybridization.
The use of monoclonal antibodies in the detection phase of the DNA
hybridization tests makes that phase equivalent to an
immunoassay--with the duplex being the antigen to be detected.
Accordingly, a variety of conventional immunoassay procedures may
be used. Since the duplex will already typically be immobilized on
a solid insoluble support, the antibody may be applied to the
support, incubated under conditions that allow immune complex
formation between the antibody and any immobilized duplex on the
support, and the support washed to remove unbound antibody.
Temperature, pH, and duration are the most important conditions in
the incubation. The temperature will usually range between
5.degree. C. and 40.degree. C., the pH will usually range between 6
and 9 and the binding reaction will usually reach equilibrium in
about 1 to 18 hr. Antibody will normally be used in excess. In
instances where the antibody is labeled directly immune complexes
may be detected via the label on the antibody. A more common and
preferred procedure is to use unlabeled monoclonal antibody and
incubate the immobilized dsDNA-monoclonal antibody complex with an
enzyme-conjugated antibody against the monoclonal antibody. The
same incubation conditions as were used in the initial incubation
may be used. The resulting ternary complex may be treated with
substrate and detected spectrophotometrically via the
enzyme-substrate reaction. By using conventional procedures in
which the detection means is bound indirectly to the
dsDNA-monoclonal antibody via one or more layers of immunochemical
it may be possible to amplify the detection signal to improve the
sensitivity or the detection limit of the procedure.
The kits for carrying out the above described preferred
hybridization tests will normally contain a DNA immobilizing
material, a hybridization solution such as those described at
column 5, lines 8-24 of U.S. Pat. No. 4,358,535, the ssDNA probe
(either separate or precoated onto the immobilizing material), the
monoclonal antibody, enzyme-conjugated antibody against the
monoclonal antibody and an appropriate substrate. The kits may also
contain a suitable buffer for dilution and washing, a dsDNA
denaturing agent such as dilute aqueous NaOH, a post-coating
preparation such as bovine serum albumin and directions for
carrying out the tests. These components may be packaged and stored
in conventional manners.
The following examples illustrate various aspects of the invention.
These examples are not intended to limit the invention in any
way.
Preparation of Monoclonal Antibody
A nine week-old female BALB/C mouse was immunized as follows:
______________________________________ Day Inoculant (administered
ip) ______________________________________ 0 100 .mu.g poly(dA-dT)
+ 100 .mu.g mBSA in CFA 14 100 .mu.g poly(dI-dC) + 100 .mu.g mBSA
in ICFA 23 50 .mu.g poly(dA-dT) + 50 .mu.g EcoRI digested pBR322 +
100 .mu.g mBSA in PBS 37 50 .mu.g poly(dA-dT) + 50 .mu.g EcoRI
digested pBR322 + 100 .mu.g mBSA in PBS
______________________________________ mBSA = methylated bovine
serum albumin CFA/ICFA = complete/incomplete Freund's adjuvant PBS
= phosphate buffered saline (0.14 M NaCl, 10 mM sodium phosphate,
pH 7.0)
All polynucleotides used in the inoculation were puchased from P.
L. Biochemicals, Inc., Milwaukee, Wis.
The mouse's spleen was removed on day 40. Spleen cells
(1.12.times.10.sup.8) were fused with FO murine myeloma cells
(1.12.times.10.sup.8) obtained originally from Dr. S. Fazekas de
St. Groth, Basel Institute for Immunology, using the fusion and
selection procedures described by Oi and Herzenberg in Selected
Methods in Cellular Immunology, pp. 351-372.
Culture supernatants from wells containing surviving cells were
screened for antibodies having conformation-dependent specificity
for dsDNA using a solid phase enzyme-linked immunosorbent assay
(ELISA) designed to evaluate binding to ssDNA and dsDNA. pBR322
restricted with EcoRI was used as the dsDNA and M13 single-stranded
phage DNA was used as ssDNA. One hundred .mu.l of solutions of
these DNAs (10 .mu.g/ml) in 0.1 M carbonate buffer, pH 9.8, were
added to Immulon microtiter plates and incubated therein for 1-2 hr
at room temperature. The plates were emptied and washed
(3.times.200.mu.l) with PBS containing 0.05% Tween surfactant
(PBS-Tween) and post-coated with a 1% solution of BSA in PBS for 10
min. One hundred .mu.l of hybridoma culture supernatant was added
to each well and incubated for one hr at room temperature. The
plates were emptied and washed (3.times.200.mu.l) with PBS-Tween
and 100 .mu.l of goat anti-mouse Ig conjugated to alkaline
phosphatase (Zymed Laboratories, South San Francisco, Calif.) was
added to each well. After standing at room temperature for one hr,
the wells were emptied and filled with 100 .mu.l of one mg/ml
p-nitrophenylphosphate in 10% diethanolamine buffer. The plates
were incubated at 37.degree. C. for about one hr. Absorbance
(optical density, OD) at 405 nm was read using a microtiter plate
reader (Micro ELISA Autoreader, Dynatech). OD readings higher than
five times the control reading (medium instead of culture
supernatant) are considered positive.
1459 Culture supernatants were screened in the above manner.
Supernatant from one well, designated CH26-1352, reacted with the
dsDNA but not with the ssDNA. The cells was expanded and subcloned
by limiting dilution. A cloned sample of hybridoma CH26-1352 was
deposited in the American Type Culture Collection, 12301 Park Lawn
Drive, Rockville, Md. 20852, USA on August 3, 1983. This sample was
assigned ATCC No HB 8329. Isotype analysis of the monoclonal
antibody produced by hybridoma CH26-1352 indicated it is an
IgM.
CH26-1352 Cells were injected intraperitoneally into
Pristane-primed BALB/C mice. After 5-10 days, ascites were
harvested from these mice. Both purified antibody from ascites and
from culture supernatant were used in the specificity tests
described below. The antibody was purified by dialyzing the
ascites/supernatant against distilled water followed by
centrifugation.
Additional Specificity Testing of Monoclonal Antibody Produced by
Hybridoma CH26-1352
The specificity of monoclonal antibody CH26-1352 for the
double-stranded conformation of DNA was confirmed by testing it in
the above-described ELISA using another single-stranded phage DNA
(.phi.X 174, New England Biolab), and the following dsDNAs:
replicative form (RF)I .phi.X 174, RFII .phi.X 174, calf thymus
DNA, RF M13, and pBEU27. The antibody did not bind to ss.phi.X 174
but bound to all five of the dsDNAs.
Blocking Radioimmunoassay (RIA)
Blocking RIAs were also carried out to illustrate the specificity
of monoclonal antibody CH26-1352. Flexible polyvinylchloride
microtiter plate wells were filled with 50 .mu.l of a 25 .mu.g/ml
solution of the purified monoclonal antibody in carbonate buffer,
0.1 M, pH 9.8 and incubated for 2 hr at room temperature. The
solution was then aspirated from the wells, the wells were washed
(2.times.200 .mu.l) with PBS-Tween, and post coated with 1% BSA in
PBS (hereafter RIA buffer) for 10 min. Twenty-five .mu.l of serial
diluted DNA test samples (EcoRI-digested pBR322; pBEU27;
ClaI-digested RFM13; M13; .phi.X 174 RF.phi.X174; .lambda.DNA; M13
containing a 1 kb pBR322 insert; M13 containing a 2.5 kb chlamydia
insert; E. coli RNA; calf thymus DNA; and salmon testes DNA) in RIA
buffer were added to the wells and the plates were shook for about
one min. Twenty-five .mu.l of .sup.32 P-labeled pBR322 prepared by
nick translation was added to the wells and the contents were
incubated for one hr at room temperature. The wells were then
emptied and washed (3.times.200 .mu.l) with PBS-Tween. The wells
were cut and read with a scintillation counter. % Binding was
calculated from the cpm readings using the formula: ##EQU1##
FIGS. 1A and 1B are graphs (% binding vs. dilution) of the results
of these RIAs. As shown, no significant blocking of the .sup.32
P-labeled pBR322 was observed with the ssDNA and RNA samples.
Blocking was observed with all dsDNA samples.
Blocking RIAs were also carried out using the above procedure on
heat-denatured and nondenatured dsDNAs. The denatured dsDNA was
made up by boiling 40 .mu.g/ml solutions of the dsDNA in PBS for 15
min at 100.degree. C. The boiled DNA was quick chilled in ice water
and diluted 1/2.times. with cold RIA buffer. Serial dilutions of
the denatured DNA were made as above. The DNA samples tested were:
EcoRI digested pBR322, boiled; EcoRI digested pBR322, not boiled;
EcoRI digested pBEU27, boiled; EcoRI digested pBEU27, not boiled;
calf thymus DNA boiled; calf thymus DNA, not boiled; M13 013; M13
011. The results of these tests are reported in FIG. 2.
Blocking RIAs using the above procedure were carried out using
various synthetic nucleic acid polymers as samples. The following
polymers did not block .sup.32 P-labeled pBR322 binding:
______________________________________ poly(dA-dT).poly(dA-dT)
poly(dG) poly(dA-dU).poly(dA-dU) poly(dC) poly(dI-dC).poly(dI-dC)
poly(I) poly(dA).poly(dT) poly(dA) poly(dA).poly(dU) poly(dT)
poly(dI).poly(dC) ______________________________________
Positive reactivity was shown by poly(dG).multidot.poly(dC),
poly(I).multidot.poly(dC), and poly(rG).multidot.poly(dC) and weak
positive reactivity by poly(dA-dC).multidot.poly(dT-dG).
Solid Phase DNA/DNA Hybridizations Using M13 Probes and Monoclonal
Antibody CH26-1352.
M13 containing fragments of pBR322 and .lambda. DNA were used as
probes. These probes were made by the "shotgun" M13 cloning
procedure described in the BRL Instruction Manual for M13
Cloning/Dideoxy Sequences. See also PNAS (USA) (1977) 74:3642-3646
and Nature (London) (1978) 272:375-377. In this procedure pBR322
and .lambda. DNA are restricted with a restriction enzyme that has
a restriction site in the lacZ gene of mp9 (an M13 derivative).
RFM13 is restricted with the same enzyme leaving it with compatible
ends with those generated in the pBR322 and .lambda. DNA fragments.
The restricted DNAs are separated from the digest by phenol,
phenol:CHCl.sub.3 and CHCl.sub.3 extraction followed by ethanol
precipitation and are ligated. Recombinant DNA is distinguished
from recircularized RFM13 by insertional inactivation of the lacZ
gene due to the presence of the insert. In the presence of
isopropyl-.beta.-D-thiogalactopyranoside, recircularized RFM13 will
form blue plaques on indicator plates containing X gal agar,
whereas insert-containing M13 will form white (colorless) plaques.
The following M13 probes were made: M13 containing a .about.1/kb
BamHI-PvuI pBR322 fragment (M13(pBR322)); M13 containing a 565 bp
HindIII .lambda. fragment (M13(.lambda.#2)); and M13 containing a
2322 bp HindIII .lambda. fragment (M13(.lambda.#7)). M13 without
any insert was also used as a control.
Immulon microtiter plate wells were filled with 100 .mu.l of the
M13 probe diluted (10 .mu.g/ml) in 0.1 M carbonate buffer and
incubated for 2 hr at room temperature. Wells filled with M13 were
used as controls. The wells were emptied and washed (2.times.200
.mu.l) with PBS-Tween and post-coated (2.times.200 .mu.l) with RIA
buffer at 37.degree. C., 5 min each coating. pBR322 DNA and
.lambda. DNA were digested with HaeIII and HincIII, respectively
and diluted at 10 .mu.g/ml in 6.times.SSC hybridization buffer
(6.times.SSC =0.02% Ficol, 0.02% polyvinyl pyrrolidine, 0.10% BSA,
and 0.25% sodium dodecyl sulfate). The digested DNA was boiled for
10 minutes and diluted 1/2.times. in hot (.about.90.degree. C.)
6.times.SSC buffer. Serial dilutions of the heat-denatured pBR322
and .lambda. DNA samples (100 .mu.l) were added to the wells, the
plate was sealed and the sealed plate was incubated overnight at
68.degree. C. The wells were then emptied and washed (3.times.200
.mu.l) with PBS-Tween. One hundred .mu.l of culture supernatant
from hybridoma CH26-1352 was added to each well and incubated for 1
hr at room temperature. The wells were emptied and washed
(3.times.200 .mu.l) with PBS-Tween. One hundred .mu.l of goat
anti-mouse Ig conjugated to alkaline phosphatase was added to each
well and incubated for 1 hr. One hundred .mu.l of
p-nitrophenylphosphate in 10% diethanolamine buffer was then added
and the wells' contents were incubated at 37.degree. C. for 1/2-1
hr. Absorbance at 405 nm was read as described above.
Specific hybridization between denatured .lambda. DNA and M13
(.lambda.#2) probe down to a dilution of 15.6 ng/ml denatured
.lambda. DNA was observed. Specific hybridizations between the
denatured .lambda. DNA and M13(.lambda.#7) were observed down to
3.9 ng/ml denatured DNA. The greater sensitivity observed with the
M13 (.lambda.#7) probe is believed to be attributable to the fact
that the insert of that probe is larger than the insert of
M13(.lambda.#2). Hybridizations between M13(pBR322) probe and
denatured pBR322 DNA was observed down to about 3.9 ng/ml denatured
DNA. No hybridization was observed between M13(pBR322) and
denatured .lambda. DNA. Some nonspecific binding between the
M13(.lambda.) probes and denatured pBR322 DNA occurred but the
absorbance readings between these probes and denatured .lambda. DNA
were significantly higher than the readings between these probes
and denatured pBR322 DNA. Some nonspecific binding between M13
probe and denatured pBR322 were observed but again, absorbance
readings were significantly higher with the M13(pBR322) probe. No
nonspecific hybridization between M13 probe and .lambda. DNA was
observed.
Modifications of the above described modes for carrying out the
invention that are obvious to those of skill in the fields of
immunochemistry, nucleic acid chemistry, immunodiagnostics, and
related fields are intended to be within the scope of the following
claims.
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