Volume 9 Number 71981
Nucleic Acids Research
Monoclonal antibodies showing sequence specificity in their interaction with single-stranded DNAs
J.S.Lee, J.R.Lewis, A.R.Morgan, T.R.Mosmann and B.Singh
Departments of Biochemistry and Immunology, University of Alberta, Edmonton, Alberta
T6G 2H7, Canada
Received 20 January 1981
ABSTRACT
Six hybridona cell lines which secrete monoclonal
antibodies binding to nucleic acids were produced from
autoimmune NZB/NZW nice. Four of the antibodies were IgG's and
the other two were IgM's. Using a solid phase radioiramunoassay
(SPRIA) the binding of the antibodies to over thirty different
nucleic acids was estimated. All the antibodies were extremely
specific. There was no detectable interaction with various
RNAs , and single-stranded PNAs bound more antibodies than
duplex or multi-stranded DNAs. In every case the antibodies
also showed considerable sequence preferences. For example one
monoclonal antibody bound to d(TTC) n but not to d(TCC) n while
another interacted strongly with d(TG) n and d(CA) n but not
with d(TC) n , d(GA) n or homopolymers. In other cases the
patterns of sequence specificity were extremely difficult to
interpret although it seems clear that monoclonal antibodies
have the potential to distinguish between any two nucleic acids
however similar.
INTRODUCTION
Antibodies to nucleic acids are of interest for several
reasons. First they play a significant role in autoimmune
diseases such as Systemic Lupus Erythenatosus (SLE) (1).
Second, they can provide a model system for protein recognition
of nucleic acids in general. Finally, they can be used as
probes for particular nucleic acid sequences or structures in
more conplex systens such as ribosomes or chromosomes (2,3).
Before the introduction of the hybridoma technology, antibodies
were obtained directly from immunized animals. These antibodies
often show a wide range of specificities which in some cases
limits their usefulness. Moreover, the degree of specificity of
individual members of these heterogeneous antibody populations
© IRL Press Limited, 1 Falconberg Court. London W1V 5FG, U.K.
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Nucleic Acids Research
is not yet c l e a r - For example it has b e e n
shown previously
a r e v i e w see 4) that structural features of nucleic
be distinguished
demonstrated
attempt
(e.g. duplex
or t r i p l e x ) but it has yet to be
that sequence specificity
can also be a c h i e v e d . To
to a n s w e r this question we h a v e produced
h y b r i d o m a cell lines which secrete m o n o c l o n a l
bind
to nucleic
specificities
MATERIALS
Four
(for
acids can
a c i d s , and have investigated
several
antibodies
which
their
in d e t a i l .
AND M E T H O D S
6 1/2 month old fenale N Z B / N Z W m i c e , (a gift of D r .
A.S.
R u s s e l l , Dept . of M e d i c i n e , TIniversity of Alberta) were
used
for a single cell
develop
fusion. At this age the nice b e g i n to
an a u t o i n m u n e disease resembling
systemic
lupus
e r y t h e m a t o s u s and antibodies against v a r i o u s nucleic
be detected
in the sera
of l i p o p o l y s a c c h a r i d e
acids can
( 5 ) . Two m i c e were injected with
25ug
(LPS) and two w i t h 50pg and _then 48 h o u r s
later the spleen cells were harvested
for fusion with clone 43
of the mouse m y e l o m a MOPC 315 ( 7 ) . This cell line
but does not secrete Ig light chains
synthesizes
( 6 , 7 ) . LPS t r e a t m e n t , in
g e n e r a l , leads to a larger n u m b e r of v i a b l e hybridomas and will
b e described
in detail elsewhere
( 8 ) . The cell
t e c h n i q u e , limiting dilutions and cloning
performed
e s s e n t i a l l y as described
days w h e n a total of a p p r o x i m a t e l y
could be v i s u a l i z e d , screening
acid b i n d i n g
Polyvinyl
aciHs
1.4 mM KH2PO4
h o u r s . Dsing
1,000 individual
using a Solid
Inc.) were coated
clones
Phase
microtiter
with v a r i o u s
acids it was found
that
Ing of nucleic acid b e c o n e s p e r m a n e n t l y
clones two g r o u p s of antigens were
attached
of the
used.
Group A: - lug/ral r U n , lpg/ml d T n , lug/nl
1708
plates
nucleic
(2.7 nM K C 1 , 137 nM NaCl . 10 mM
by some unknown m e c h a n i s m . For the initial screening
heat-denatured
14
final pH of 7.2) at 4° for at least 24
^F-lahelleH nucleic
approximately
After
below.
chloride 96-well f l a t - b o t t o m e d
in 50ul of PBS buffer
were
(6,7,9).
for the production of nucleic
(SPRIA) described
(Dynatecb L a b o r a t o r i e s
Na2HPO4,
previously
antibodies was initiated
PadioImmunoAssay
fusion
in agarose
ribosomal RNA f r o n J2_«_ coli and
lug/nl
Nucleic Acids Research
heat-denatured
calf thymus DNA.
firoup B: - lug/ml R17 R N A ,
thymus T)NA, lug/nl r U n • d A n • dT,,
specificity
lug/nl r A n ' d T n . For the
tests the wells were coated with
the a p p r o p r i a t e nucleic
tines with
and
lug/ml native calf
lOOul of PBST
remove unbound
50ul of lug/ml of
acid as a b o v e . A f t e r washing
three
(PBS buffer with 0.05% T w e e n ) to
nucleic acid, 25ul of PBST w a s added
followed
by
20-100ul of the sample to be t e s t e d . A f t e r incubation at
20°C
for 2 h o u r s the wells were again washed
lOOul
of PBST to remove unbound
goat anti-mouse
further
IgG
three times with
(10) was then added
and
incubated
2 h o u r s . Thus the 1*25 reagent bound
mouse a n t i b o d i e s which were bound
for a
to any monoclonal
to the nucleic
p l a t e . The plate was finally washed
PBS to remove excess
1125-labelled
a n t i b o d i e s . 30-50ul of
acids on the
three tines with
lOOul of
1*25 r e a g e n t . The wells w e r e excised
a h o t - w i r e cutter and counted
in a L K B - W a l l a c
1270
with
Backgamma
I I , u s u a l l y for one m i n u t e .
The source of the nucleic
been described
previously
acids used
in this study
( 1 1 , 1 2 ) . In general
p y r i m i d i n e DNAs were prepared by d e p u r i n a t i o n
w h e r e a s single-stranded
exonuclease
Gel exclusion
purine DNAs were prepared
chromatopraphy was performed
•growth medium was precipitated
resuspended
of the d u p l e x ,
by
III digestion of the a p p r o p r i a t e duplex
column of Sephadex C - 2 0 0 . A p p r o x i n a t e l y
with 50Z
fractions were monitored
assay described
(12,13).
on a
10ml of the
(NH4)2SO4
in 1 ml of the eluting buffer
T r i s - H C l , pH R.0) The sample was eluted
have
single-stranded
140ml
hybridoma
and
(1 M N a C l ,
at 9 ml/hour
at 280 nn as well as by the
100 mM
and
SPRIA
above*
RESULTS
The fused
cells fron the spleens of the two groups of nice
were plated out into
192 individual wells to allow clones to
d e v e l o p . A f t e r a p p r o x i m a t e l y one week i n d i v i d u a l
be directly v i s u a l i z e d
well
clones
could
and there were on a v e r a g e about five per
(2.0 m l ) . There was no significant d i f f e r e n c e in the
number of c l o n e s originating
receiving d i f f e r e n t
from the two groups of n i c e
doses of L P S . A f t e r
ten days the
SPRIA
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Nucleic Acids Research
a s s a y w a s used
to i d e n t i f y t h o s e wells w h i c h w e r e
antibodies
which bound
wells were
found
the
to b e p o s i t i v e
SPRIA a s s a y w h i c h
This was repeated
medium
fron
to e i t h e r
each w e l l
the B g r o u p
Moreover
as g i v i n g
or m o r e a b o v e
using
one third
31 w e l l s were s t i l l
were also positive
the i n t e r a c t i o n
than
conparing
were
and c l o n i n g
hybridomas
secreting
antibodies
DNAs . Finally after
in a g a r o s e
continued
obtained
obtained
from
from
HEd 9 and 10 w e r e d e r i v e d
By far the m o s t
produced
technology
on the b a s i s
column
with
50ug L P S .
immunoglobulin
for I g G ) using
elution
weights
gel filtration
profiles
eluted
in the e x c l u d e d
on
a r e s h o w n in
protein
case
p e a k and
as an IgM, as is HEd 5. For HEd 7 on
the antibodies were partially
included
on the
and t h u s HEd 7 is a n IgG as are HEd 6, HEd 8 and HEd 1 0 .
Before
dilutions
testing
the s p e c f i c i t y
of the a n t i b o d i e s ,
of the v a r i o u s m e d i a w e r e m a d e in o r d e r
the g r e a t e s t
dilution which
response. The results
25 a n d t h e f o l l o w i n g
1710
25ug L P S , and
c l o n e s H E d 9 and HEd 7. In the first
thus HEd " is i d e n t i f i e d
the o t h e r hand
of
performed
a r e IgM and I g G . T h e s e can
of their m o l e c u l a r
G200 column. Typical
were
antibodies
hybridomas.
with
the m i c e i n j e c t e d
a Sephadex
the a n t i b o d i e s
below were
the m i c e i n j e c t e d
probable classes
from
dilutions
acid b i n d i n g
described
f o r IgM and 1 5 0 , 0 0 0
1 for m e d i a
for
limiting
these six m o n o c l o n a l
(1,000,000
Fig.
of the
(Mosmann, T.R., unpublished
from
b y the h y b r i d o m a
be distinguished
s u g g e s t s , on
the m a j o r i t y
in l a r g e v o l u n e s . T h i s p a t t e r n of
o b s e r v a t i o n s ) . The experiments
HEd 5-8 w e r e
as judged
(")) to e n s u r e m o n o c l o n a l ity six
is not u n e x p e c t e d
on the m e d i u m
p o s i t i v e for
significantly
specific
using
to s e c r e t e nucleic
and c o u l d b e c u l t u r e d
regression
found
for the B g r o u p . T h i s i m m e d i a t e l y
sinp,le-s t r a n d e d
occasion
for the A g r o u p .
the two g r o u p s of a n t i g e n s , that
hybrldomas
the v o l u m e of
w i t h the A group a n t i g e n s
from the CPM in the SPRIA assay w e r e a l w a y s
greater
in
significantly
c a s e s all w e l l s which w e r e
antigens
a result
background).
for the S P R I A a s s a y . On this
o n l y 21 of the o r i g i n a l
p o s i t i v e . In b o t h
(defined
was two-fold
two days l a t e r
producing
the A or B g r o u p a n t i g e n s . 31
ranged
s t i l l gave
to a s c e r t a i n
rise to a m a x i m u m
from d i l u t i o n s
SPRIA a s s a y s w e r e
serial
of 1 in 10 t o 1 in
performed
at
these
Nucleic Acids Research
-1800
25
30
35
FRACTION
Figure 1. Gel exclusion chroraatography of nedla from
hybridoraas (a) HEd 9 and (b) REd 7 on a Sephadex G200
colunn. The fractions eluting from the column were
monitored at 280 nm (
) and also tested with the SPRIA
assay using (a) dT n or (b) heat-denatured calf thynus DNA
as the antigen (
O
) • Bromophenol blue and blue
dextran as molecular weight markers showed that the first
protein peak was excluded while the second was largely
included.
dilutions. The results were very reproducible (±20Z) and in
general only one measurement was performed for each antigen. A
false positive was only recorded on one occasion. Since
heat-denatured calf thymus DNA and dT n were always run for
comparative purposes they also served as a check on the
internal consistency of the assay. The specific activity of
various preparations of the 1*25 goat anti-mouse IgG was such
that with heat-denatured calf thymus DNA or dT n as antigen
(see below) the CPM measured in the SPRIA assay were always in
excess of 1,000 (usually 2,000-10,000) compared to a background
(i.e. in the absence of added antigen) of less than 50 CPM.
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Table I presents
against
Except
the individual
the r e s u l t s
for HEd 9 the preferred
thymus DNA which i s a r b i t r a r i l y
preferred
antigen
This confirms
antigen in a l l
there
antigen
set at
i s dTn and t h i s
the six hybridomas
tested
i s heat-denatured
calf
100Z. For HEd 9 the
is a r b i t r a r i l y
the o r i g i n a l observation
that
cases i s a single-stranded
i s no detectable
was confirmed
for
nucleic acids comprising groups A and B.
the
set
at 100%.
preferred
DNA. Surprisingly,
binding to single-stranded
RNAs. This
using competition experiments in which the
antibodies were premixed with 0.1 ug of ribosomal RNA before
HEd 5
HEd 6
HEd 7
HEd 8
HEd 9
HEd 10
Table I. Results of the SPRIA assay for the six monoclonal antibodies
tested against various RNA and DNA polymers. All are normalized against
heat-denatured calf thymus DNA at 100% except for HEd 9, where dTn is used
as the standard at 100%.
100
100
100
100
<5
100
<5
58
83
49
100
48
Ribosomal
RNA
<5
<5
<5
<5
<5
<5
rU
<5
<5
<5
<5
<5
<5
Native Calf
Thymus DNA
86
50
60
58
<5
37
r
<5
8
<5
22
<5
23
dT .dA .dT
n
n n
22
32
<5
53
<5
50
dA .dT
n
n
<5
8
<5
15
<5
17
d(TTG) n .d(CAA) n
<5
<5
<5
<5
<5
<5
8
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
<5
Heat-Denatured
Calf-Thymus DNA
dT
n
n
VdTn
d(TC) .d(GA)
n
n
Blank w e l l
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adding t o the p l a t e s .
heat-denatured
represents
this
calf
at a l l ,
that
the antibodies
test
t h i s hypothesis
antibodies
the
three
response shown by
to dAn-dTn may be due to binding to dTn
by opening up the duplex. In a l l
if
other
duplex polymers. The small
the antibodies
detectable,
all
25% drop in the
change. Also shown in Table I i s
the six types of antibody with
repeat ing-sequence
some of
in an approximately
to the SPRIA assay while for
of
this
excess of RNA. For HEd 8 and HEd 10
resulted
there was no significant
interaction
1 ng of
thymus DNA was bound to the plates
a hundred-fold
competition
response
Since approximately
cases the binding i s
which would tend to confirm
prefer
the
single-stranded
results
DNAs. To
for various
barely
the view
further
single-stranded
purine and pyrimidine DNAs are shown in Tables I I and
III
Table I I . Results of the SPRIA assay for the six monoclonal antibodies
tested against various polypurine DNAs.
o
m
00
HEd
HEd
HEd
HEd
HEd
HEd
rH
100
100
100
100
<5
100
dT
n
<5
58
83
49
100
48
dA
n
<5
<5
<5
<5
<5
<5
dl
n
<5
55
<5
<5
<5
<5
dG
n
7
17
<5
<5
<5
<5
d(GAA)
n
7
14
<5
8
<5
9
d(GA) n
<5
<5
<5
<5
<5
<5
d(GGA)
n
<5
<5
<5
<5
<5
<5
d(Gn2A)
n
<5
<5
<5
<5
<5
<5
d(Gm6A)
n
<5
<5
<5
<5
<5
<5
Heat-Denatured
Calf-Thymus DNA
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Nucleic Acids Research
HEd 7
HEd 8
HEd 9
HEd 10
100
100
100
100
<5
100
dT
n
<5
58
83
49
100
48
d(TTC) n
<5
26
68
25
96
35
d(TC) n
<5
66
39
67
21
64
d(TCC)n
<5
59
<5
46
<5
70
dC
<5
<5
<5
<5
<5
<5
HEd 5
HEd 6
Table i n . Results of the SPRIA assay for the six monoclonal antibodies
tested against various polypyrimldine DNAs.
Heat-Denatured
Calf Thymus DNA
n
respectively. Surprisingly, except for HEd 6 binding to dl n ,
the interaction with polypurine DNAs is limited. Recently it
was shown (12) that repeating-sequence polypurines containing
hypoxanthine or guanine form stable self-structures which are
probably tetraplexes. However, dAn and d(Gm^A) n are truly
single-stranded at neutral pH (12) and therefore the limited
binding to polypurines cannot be interpreted simply as being
due to the formation of DNA self-structure.
Repeating-sequence pyrinidine DNAs on the other hand, show
significant binding to these antibodies except for HEd 5 (Table
III). Surprisingly, in no case was there significant binding to
dC n but for HEd 6, HEd 8 and HEd 10 there was a strong
interaction with d(TCC) n . For HEd 7 and HEd 9 the results are
presented graphically in Figure 2. It would appear that two
consecutive thytnines are required for a strong interaction. As
far as we are aware, this is the first demonstration of
sequence specificity in the Interaction of antibodies with
nucleic acid polymers, although specificity has previously been
demonstrated with deoxyribodi- and tri-nucleotides (14,15).
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Nucleic Acids Research
i
1
i
wo
1
1
-
\
HEd 9
\
\
\
\
1L
\
80
-
\
\
b
\
\
\ \
\\
60 _
7.
_
\
(CPM)
V\
>*
40 -
D HEd 7
\i
L
\
20
\
\
V
,
i
0
I
dTn
\
1
d(TTC) n
dCTC),,
d(TCC) n
Figure 2. Effect of b a s e c o m p o s i t i o n on the b i n d i n g of HEd
9 (.0) and HEd 7 ( Q ) to v a r i o u s s i n g l e - s t r a n d e d
pyrimidine DNAs . The CPM fron the SPRIA assay are shown as
a p e r c e n t a g e of that found for d T n .as the a n t i g e n .
The i n t e r a c t i o n
substituted
of the a n t i b o d i e s with DNAs
pyrimidines
containing
is shown in Table IV. Except
for HEd 5
(which doesn't bind
to d T n ) the binding of the a n t i b o d i e s
dUn
weaker
is c o n s i d e r a b l y
important
further i n v e s t i g a t e d
bromine
or iodine
halogenated
with
non-halogenated
dAn-dBrUn
of p y r i m i d i n e s
suggest
either
in the 5 p o s i t i o n . Very s u r p r i s i n g l y
(even d B r C n ) gave strong
since
give b a c k g r o u n d
in itself
all
responses
(even HEd 5 which does not bind
pyrimidine). Halogenation
for binding
an
and this w a s
p y r i m i d i n e DNAs c o n t a i n i n g
p y r i m i d i n e DNAs
with all the a n t i b o d i e s
sufficient
than to d T n . This would
role for the 5 p o s i t i o n
to
to any
is not
the duplexes d ( B r U G ) n • d ( C A ) n and
readings.
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Nucleic Acids Research
Table IV. Results of the SPRIA assay for t h e s i x monoclonal antibodies
tested against various DNAs containing a l t e r n a t i n g pyrimidine-purine
sequences or s u b s t i t u t e d pyrimidines.
o
i-i
HEd
HEd
HEd
HEd
HEd
HEd
GO
100
100
100
100
<5
100
<5
58
83
49
100
48
<5
23
<5
9
<5
11
108
113
99
78
95
103
dIU
n
107
144
191
99
111
107
dBrCn
111
226
99
126
135
124
d(BrUG) .d(CA)
n
n
<5
<5
<:5
<5
<5
<5
dA .dBrU
n
n
<5
<5
<5
<5
<5
<5
d(AT)n
<5
<5
<5
6
<5
6
d(TG)
n
50
85
35
66
108
56
d(CA)n
68
8
<5
<5
137
<5
d(TG) .d(CA)
n
n
<5
<5
<5
<5
<5
<5
Heat-Denatured
Calf-Thymus DNA
dT
n
dU
n
dBru
n
Finally, in Table IV are shown the results for duplex and
single-stranded alternating pyrimidine/purine DNAs. HEd 5 binds
well to both d(TG)n and d(CA)n suggesting that in the absence
of halogenated bases the preferred antigen contains alternating
pyrimidines and purines since the interaction with virtually
a l l other single-stranded DNAs is negligible (Tables I, II and
III).
Thus this is another example of sequence specificity.
Whereas a l l the antibodies bind to some extent to d(TG)n and/or
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Nucleic Acids Research
d ( C A ) n the Interaction with
the duplex d ( T G ) n • d ( C A ) n Is In all
cases I n s i g n i f i c a n t . This serves to emphasize
these antibodies for single-stranded
the preference of
DNAs.
DISCUSSION
We have described
the p r o d u c t i o n
together w i t h an investigation
m o n o c l o n a l antibodies
of six hybridomas
of the binding
of their
to v a r i o u s nucleic a c i d s . All of the six
types of antibody bind exclusively
to DNAs
for s i n g l e - s t r a n d e d DNAs) and there is no
(with a preference
detectable
i n t e r a c t i o n with R N A s . Two p r e v i o u s studies with
hybridomas
from a u t o i m m u n e NZB/NZW mice produced
antibodies
showing specificity
Thus
for ribosomal RNA and duplex DNA
It seems possible that
antibodies
monoclonal
the selection
of m o n o c l o n a l
specific for s i n g l e - s t r a n d e d DNAs
be an artefact
in this study may
of the Initial s c r e e n i n g procedure where
g r o u p s of antigens were u s e d . H o w e v e r
it is clear that
type of d e t a i l e d analysis may be extremely
of the aetiology
Close
(16,17).
of autoimmune
mixed
this
useful in the study
diseases.
scrutiny of the binding
data for HEd 8 and HEd 10
show that their properties are very s i m i l a r . For e x a m p l e , they
are both positive
different a n t i g e n s
for the same group of 16 antigens
tested. M o r e o v e r , p r e l i m i n a r y
focusing e x p e r i m e n t s
of the 33
isoelectrlc
show that the heavy and light chains of
HEd 8 and HEd 10 are not r e s o l v a b l e . Thus both hybridomas may
be secreting
identical a n t i b o d i e s , yet these two hybridomas are
derived from different a n i m a l s . A l t h o u g h
syngeneic
this result might suggest
rather-limited
these mice are
that there is a
set of potential autoimmune
from the i m m u n o l o g l c a l point
of v i e w the discovery
and IgG classes of antibodies suggests
disease progresses
through
a n t i b o d i e s . Finally
that
this
of both IgM
autoimmune
the same stages as other
immune
responses.
The p a t t e r n s of specificity
shown by the v a r i o u s
a n t i b o d i e s are extremely difficult
to i n t e r p r e t . For example
HEd 9 in its interaction with p y r i m i d i n e DNAs appears to show a
clear p r e f e r e n c e for two consecutive
does not bind significantly
thymines
(Fig. 2) and it
to d G n , d A n or d C n . Yet it shows a
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very
strong
complexity
response
could accommodate
varying
and
amounts
deoxyrlbose
binding
to both d ( T G ) n
is not s u r p r i s i n g
contribution
combination
extremely
s e v e r a l b a s e s , and t h e s e
influenced
(X-ray
short
suggesting
binding
problem
the n u m b e r
nature
is p r o b a b l y
be
best
the p r e f e r r e d
calf
that
determined
of defined
antigen has been
HEd 7, a p p a r e n t l y
A summary
is s h o w n
bound
to d C n and only weak b i n d i n g
this s t r o n g
well
due t o the v e r y
in the b a c k g r o u n d
s h o w n ) . Thus
polymers
with
polarizable
1718
at r e g i o n s
binding
nature
of
that
these
the
to a l a r g e
of the l ! 2 5 g o a t
anti-mouse
antibody
(data n o t
to the h a l o g e n a t e d
(e.g. the Fc p o r t i o n ) not d i r e c t l y
site. This
dIUn
of any of
leads
of added D N A b i n d i n g
binding
and t h e i r
possible
c o m e s from the fact
the a n t i b o d i e s m a y be b o u n d
the a n t i g e n
this
DNAs m a y be a
p r e s e n c e of t h e s e p o l y m e r s on the PVC p l a t e s
IgG in the a b s e n c e
of
to d B r U n ,
binding
t o d U n . It seems
to halogenated
for this v i e w
polymers
of the h y b r i d o m a s
extremely
that
polymers. Support
antigen
in T a b l e V.
them
effect
(the
individually.
may be a s l a r g e as
sequence
there w a s no d e t e c t a b l e
binding
binds
of the p r e f e r r e d
recognized
( 1 8 ) , and repeating
though
inhibition
s e q u e n c e . It
t h y m u s D N A , of a l l the a n t i g e n s
of b a s e s b e i n g
specificities
extent
by d i r e c t
this D N A c o n t a i n s a s e q u e n c e
A l l of the a n t i b o d i e s
increase
antibodies
of the
s t u d i e s , or by
oligonucleotldes
a r e not a v a i l a b l e .
non-specific
patterns would
of the a n t l g e n i c
in the i d e n t i f i c a t i o n
or even m o r e
even
latter
or
s i t e ) w h i c h h a s not b e e n tested
is that
and d B r C n
If this
than one b a s e
of a l l six m o n o c l o n a l
in a l l c a s e s . F o r e x a m p l e
Another
length
by m o r e
crystallographic)
to h e a t - d e n a t u r e d
apparent
c o n t r i b u t i o n by
by t h e b a s e s e q u e n c e
T h e exact
is a l s o u n c l e a r w h e t h e r
tested,
of the
D N A a n t i g e n , and so a l l six are to s o m e
of b i n d i n g w i t h
preferred
phosphate
a m a j o r part
be r e q u i r e d .
of b a s e s , the s p e c i f i c i t y
determinant, however,
identified
extra
would
be p r o v i d e d
sequence-specific.
five
could
site
contribute
contribute
could
single-stranded
could
moieties
interactions
is p r o f o u n d l y
This
combining
e n e r g y . For e x a m p l e ,
complex. The binding
structural
a n d to d ( C A ) n »
the a n t i b o d y
to t h e b i n d i n g
e n e r g y , a n d only a s m a l l
base-specific
best
since
requires
further
involved
Nucleic Acids Research
Table V. Summary of the apparent s p e c i f i c i t i e s and class of antibodies
from the s i x hybridomas. Halogenated polymers and native calf thymus DNA
have not been Included in the consideration of apparent s p e c i f i c i t i e s .
See Tables 1 to 4 and refer to the text for further d e t a i l s .
Hybridoma
(Laboratory
Designation)
Apparent Specificity
Preferred
Antigen
Other Strongly-Bound
Antigens
Class
of
Antibody
HEd 5
Heat-denatured
Calf Thymus
d(TG) n , d(CA) Q
IgM
HEd 6
Heat-denatured
Calf Thymus
dl n , Single-stranded pyrimidine DNAs and d(TG) n
IgG
HEd 7
Heat-denatured
Calf Thymus
dT n , d(TTC) n
IgG
HEd 8
Heat-denatured
Calf Thymus
Single-stranded pyrlmidine
DNAs and d(TG) n
IgG
HEd 9
dT
n
dCTTC) n , d(TG) n , d(CA) n
IgM
HEd 10
Heat-denatured
Calf Thymus
Single-stranded pyrimidlne
DNAs and d(TG)
n
IgG
investigation.
Throughout this report i t has been generally assumed that
a l l antigens are bound to the PVC plates In approximately equal
amounts. Some experiments with labelled antigens were performed
to show that approximately 1 ng was bound though this test has
not been possible with a l l antigens because of the lack of
labelled polymers. There is l i t t l e doubt however that there
will be variations in the amount of antigen bound. For example
heat-denatured calf thymus DNA has a complex secondary
structure and is of a significantly higher molecular weight
than the synthetic polymers. This may lead to more of i t being
bound to the plates which in turn might explain why for five of
the six hybridomas this was the preferred antigen (excluding
the halogenated polymers). However these potential problems
with the SPRIA assay do not affect the general conclusions of
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Nucleic Acids Research
this study. First, the different types of antibodies in many
cases complement one another. For example, it cannot be argued
that the lack of a response of HEd 9 to d(TCC) n (Fig. 2) is due
to lack of polymer being bound to the plate since HEd 6, HEd 8
and HEd 10 all give very significant results in the presence of
this polymer. Second, in the case of the antibodies interacting
with RNA and of HEd 9 with dC n the negative result was
directly confirmed using competition experiments. Finally other
authors have used a similar SPRIA assay for antibodies binding
to single-stranded and duplex DNAs (17,18); and in this study
it was shown (by the fact that antibodies were bound) that the
majority of the antigens tested were to some extent bound to
the plates. Thus the binding of nucleic acids to the PVC plates
is a widespread phenomenon and there is no good reason for
believing that a particular nucleic acid antigen will not be
bound.
The general conclusion of this study is that considerable
specificity can be realized in the interaction of antibodies
with nucleic acids. First the antibodies exhibit a structural
specificity exemplified by their preference for single-stranded
DNAs. Second they show an absolute requirement for a
deoxyribose backbone since there is no detectable binding to
RNA. Finally, they also demonstrate sequence specificity since
they recognize differences in the nucleic acid bases. Therefore
every basic feature of nucleic acid structure is being
recognized simultaneously by the antibodies and consequently
they have the potential for distinguishing any two nucleic
acids however similar. It seems clear that monoclonal
antibodies will find widespread use in many areas of nucleic
acid research.
Abbreviations Used
HEd x - Hybridoma from Edmonton Hx
PVC - Polyvinyl chloride
m^A - 6-methylamino adenosine
n^A - 2-amino adenosine
SPRIA - Solid Phase Radioimmunoassay
LPS - Lipopolysaccharide.
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ACKNOWLEDGEMENTS
We would like to thank C. L e e for excellent technical
a s s i s t a n c e . This work was supported by the Medical Research
Council of Canada.
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