[CANCER RESEARCH 37, 1480-1485, May 1977]
Procedures for Radioimmunoassay of the Mouse Mammary Tumor
Virus
Joel B. Sheffield,2 Thomas Daly,2 Arnold S. Dion, and Nancy Taraschi2
Institute for MedicalResearch,Camden,NewJersey08103
SUMMARY
1251
A procedure for radioimmunoassay
protein
antigen
derived
from
munine
of the major glyco
mammary
tumor
virus
is described . The assay is sensitive to 0.05 ng of antigen and
is highly reproducible. The antigen, gp55, has been found
tO be group specific and will detect viruses in 13 separate
mouse strains, as well as from continuous cell lines. Factors
affecting the assay have been examined.
INTRODUCTION
For studies of the distribution and synthesis of the mouse
MuMTV,2 we have found it necessary to detect small quanti
ties of virus-related materials in a large background of unne
lated materials. There have been several reports of madioim
munoassays for MuMTV, based either on whole (1) on dis
nupted (19) virus and purified components (11, 12). In our
studies of these assay techniques, we have attempted to
characterize the reaction in detail and to define the paname
tens for optimum sensitivity. We have concentrated on a
single antigen assay, using a highly purified MuMTV glyco
protein, gp55. We have also investigated possible sources
of errors in the assay and have obtained information that
will, we feel, be of value to others who are developing such
systems.
MATERIALS AND METHODS
The virus used in this study was obtained from the milk of
females of various mouse strains at 3rd on greaten parity and
purified by a combination of rate zonal and isopyknic cen
tnifugation (14). The purity of the virus was monitored by
electron microscopy. One ml of milk yielded, on the aver
age, 0.25 mg of virus protein. Specific saccharides were
provided by L. Warren of the Wistar Institute, Philadelphia,
Pa. The saccharides used were: a-methyl-D-galactoside; amethyl-O-glucoside; methyl-f3-D-xylopymanoside; methyl-aD-glucopynanoside; O-nitnophenyI-@3-D-thiofucoside; p-ami
nophenyl-f3-D-thiogalactoside; and a-methyl-D-mannoside.
I This
work
was
supported
by
Contract
N01-CP-53561
within
The
Virus
Cancer Program and Grant CA-08740 from the National Cancer Institute.
2 Present
address:
Hahnemann
Medical
College,
230
N.
Broad
St.,
Phila
delphia, Pa. 19102, where requests for reprints can be sent.
3 The
abbreviations
used
are:
MuMTV,
munine
mammary
tumor
sodium dodecyl sulfate.
Received November 19, 1976; accepted February 16, 1977.
1480
virus;
SDS,
was
obtained
from
Amersham/Seanle,
Arlington
Heights, Ill., as the iodide.
Purification of gp55 from Rlll-MuMTV. The technique for
the purification of gp55 has been described in detail (13).
Briefly, density-banded vinions were disrupted (3 mg viral
protein/mI)
in 0.2 M sodium phosphate buffercontaining
0.01 M EDTA, 0.2% (v/v) j3-mercaptoethanol, and 1.0% (v/v)
Nonidet P-40. Before chromatography, the solubilized virus
was diluted10-foldwith DEAE-cellulosebuffer(0.22M
Tnis-HCI, pH 7.2, 0.2% @3-mercaptoethanol,0.2% Nonidet P
40, and 30% glycerol) and applied to a DEAE-cellulose
column equilibrated with the same buffer. Under these con
ditions, gp55 is not adsorbed to the column and is re
covered in the application and wash fractions. For final
purification of the highly enriched gp55 resulting from this
step, the application and wash fractions were pooled, dia
lysed overnight at 4°against 0.1 M ammonium acetate,
concentrated by dialysis in a vacuum in collodion bags (No.
100; Carl Schleicher and Schuell, Keene, N. H.), and applied
to a Sephadex G-100 column (2 x 34 cm) equilibrated with
0.1 M ammonium acetate. Effluent fractions were monitored
with an LKB Uvicord (280 nm), and individual fractions were
monitored by SDS-polyacmylamide gel electrophonesis. Fi
nally, fractions containing only gp55 were pooled, lyophi
lized,
and resuspendedinsterile
water.
Labeling Procedures. Purified MuMTV component gp55
was labeled with 1251by the Chloramine-T method (6) with
some modifications. Initially, 5 @.tg
of protein in 20 pi of 0.25
M phosphate
buffer
1251- in NaOH
at 0.1
(pH 7.5) were
mCi/@l.
Ten
mixed
with 10 to 30
@.tlof Chlomamine-T
(250
@l
of
p.g/
ml) were then added and allowed to react with gentle mixing
for 1 mm, at which time 10 @l
of sodium metabisulfite (750
@tg/ml)and 50 @tlof 0.1 M potassium iodide were added to
terminatethe reaction.
The totalreactionvolume was di
luted to 1.0 ml with 0.05 M phosphate buffer, pH 7.5, con
taming 0.25% gelatin and 0.5% Nonidet P-40 (radioimmune
buffer), and dialyzed against several changes of a 10@ex
cess of buffer. Upon removal from the dialysis bag, the
activity was determined , and the material was diluted and
separated into aliquots for storage at —70°.
Labeled mate
nial was dialyzed every 2 weeks to limit the effects of degra
dation. Purity and integrity of the protein preparations were
monitored by SDS-polyacrylamide gel electmophoresis and
radioautography. We generally obtained specific activity of
1 to 3 x 10@cpm/ng.
Antisera. For most of the experiments, we used polyspe
cific antisera generated against isopyknically banded
MuMTV extracted from the milk of RIll mice after their 3rd
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MuMTV Radioimmunoassay
litter or later. The sera were made by repeated i.m. inocula
tions of whole or ether-extracted RIll virus emulsified in
Freund's complete adjuvant, into rabbits. Since these p0lyspecific semareacted with C57 virus-free milk, absorption
was necessary. Two sequential protocols were tested. For
in vitro absorption, 1 volume of serum was mixed with 2
volumes of skimmed C5'7 milk (5 mm at 250 x g) and
incubated at 37°for 0.5 hm,after which it was centrifuged for
0.5 hr at 100,000 x g . One-half ml of in vitro absorbed serum
was inoculated i.p. into C57 female mice, and the mice were
exsanguinated after 18 hr. The resulting in vivo and in vitro
absorbed serum, although diluted by passage through the
mouse, still bears reactivity against MuMTV.
Immunological Procedures. The system used was a 2step precipitation assay (15). An antibody dilution series
was first performed with labeled antigens by combining
30,000 cpm of antigen
@
@
in 100
@lof radioimmune
buffer,
100 @tI
of serially diluted anti-MuMTV, and 100 @l
of radioim
mune buffer. The reaction mixture was agitated by inversion
and incubated at 37°for 2 hr, after which 100 of appropni
ately diluted goat anti-rabbit immunoglobulin (Miles Labo
ratories, Elkhart, Ind.) were added to each tube, mixed, and
incubatedat4°
overnight.
The immune precipitate
was then
centrifuged .at 1000 x g in an International Model PR-6
centrifuge for 15 mm, and 120 @l
of the supemnatant were
withdrawn and counted. After determination of the 50%
binding antibody dilution (with our current serum, 1:1350),
competition inhibition assays were performed, substituting
the competing antigen for the 100
madioimmune buffer.
We compared direct competition experiments, in which
labeled and test antigen were added simultaneously, with a
binding site occupation assay, in which the test antigen was
added to the antibody and incubated for 2 hr at 37°,after
which labeled antigen was added for another 2 hr before
adding the goat anti-rabbit sena. All antigen dilution series
were performed in madioimmune buffer and were run in
parallel with standardized amounts of unlabeled protein.
Normal rabbit serum controls were run, as well as nonanti
genic samples.
A major complication
in developing
this assay was the
difficulty of pipetting constant volumes of the reagent solu
tions. The main reason for this was the presence of deter
gent, which caused the solution to wet the tips of most of
the microlitem pipetting devices, leading to significant vania
tions. In order to obtain values which were accurate to 1%,
it was necessary
to use positive
displacement
pipetting
systems. We found the Kimble Model D-73 dispenser satis
factory for dispensing reagents and the Scientific Man ufac
turing Industries (Ememyville, Calif.) Micro/pettor an excel
lent unit for sampling. Any system that used the displace
ment of a column of air was unsatisfactory.
Electrophoresis. During this study, several electrophore
sis conditions were used. Acrylamide gel slabs (7.5%) were
prepared using 0.1 M phosphate buffer (pH 7.8), 0.1% SDS,
and 0.1% 2-mercaptoethanol, and run as described previ
ously (16). Samples were dissociated in 0.01 M phosphate,
pH 7.8, 1% SOS, and 1% 2-mercaptoethanol at 60°for 30
mm. Gels were stained with Coomassie Blue G-250, as
described previously.
Acrylamide gel slabs (10%) were prepared in an E-C verti
cal gel apparatus (EC Apparatus Corp. , St. Petersburg,
Fla.), using a modification of the methods described by
Laemmli (9). The 10% separation gel buffer contained 0.375
M Tnis (pH 8.8), 0.06 M HCI, and 0.1%
SDS. The 3% stacking
gel buffer contained 0.06 M Tnis (pH 6.8), 0.06 M HCI, and
0.1% SDS. Samples were dissociated in 0.06 M Tnis (pH 6.8),
0.06 M HCI, 2% SDS, and 5% 2-memcaptoethanol at 60°for
30 mm, followed by the addition of bromphenol blue in
100% glycerol,
to a final glycerol
concentration
of 10%. The
electrode buffer contained 0.025 M Tnis (pH 8.3), 0.192 M
glycine, and 0.1% SDS. Gels were run at 200 V until the
bromphenol blue had migrated 9 to 10 cm through the
separation gel.
Slabs were fixed overnight in 7% acetic acid-5% metha
nol. Gels containing 1251-labeledprotein were then rinsed in
distilled water, dried in vacuo with heat onto Whatman No. 1
filter paper, and pressed with Kodak RP Royal-X-Omat film
and exposed at —70°
for an appropriate length of time.
Densitometry was performed with a Model EC91O densi
tometer (EC Apparatus Co.) with filters at 650 nm for Coo
massie Blue-stained gels and nadioautognams. Sensitivity
settings were identical for matched gels.
RESULTS
Chart lb shows a radioautognam of the iodinated gp55
after SDS-polyacnylamide gel electrophonesis compared
with a Coomassie Blue-stained gel of the starting material
(Chart la). Only a single labeled component is visible.
An antibody precipitation curve is presented in Chart 2.
The antiserum is able to precipitate greaten than 80% of
gp55. Immune precipitation of purified iodinated gp55 is not
affected by either of the absorption protocols used.
Kinetics of Binding to Antibody. The time curve of associ
ation of antigen with antibody was determined by adding
the goat anti-rabbit serum at various times after the start of
the reaction. The curve is presented in Chart 3. It can be
seen that 30% of the antigen is bound within 2 hr at a 1:4000
dilution, and 50% is bound at an antibody dilution of 1:800.
This information' was used in determining conditions for
a binding site occupation assay in which the test antigen
was added to the antibody and incubated before the addi
tion of labeled antigen. For practical reasons, we allowed
the test antigen to interact with the antibody for 2 hr before
adding the labeled antigen for the same time period. Under
these conditions, 50% of the labeled antigen would be
bound to the antibody in the absence of competition. The
results of such an assay, compared with a direct competi
tion assay, are given in Chart 4. The binding site occupation
assay is more sensitive than the other by a factor of 4.
Inhibition Curves. The inhibition of the precipitation of
iodinated gp55 by disrupted virus and by purified gp55 can
be seen in Chart 5. In this case, the curve appears simple
with a linear region from 0.1 to 1.5 ng of gp55 and 0.5 to 10
ng of RIll virus. It is significant that the amount of gp55
detected in the disrupted vinion is less than predicted by
Sarkar and Dion (13) who indicated that gp55 represents
27% of the total protein. In repeated experiments, a 10-fold
excess of viral protein is required to displace gp55, indicat
ing that gp55 is 10% of the virus. Munine Rauscher leukemia
MAY 1977
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1481
J_ B. Sheffield et at.
100
80
•p140—ø..-—----—@
ID
pt@J@@―
60
LI,
gp68—e--
1
2
3
4
5
6
7
8
TIME(HR)
KINETICS
OFANTIBODY-ANTI6EN
REACTION
Chart 3. Kinetics of antibody-antigen reaction, with gp55, and antiserum
dilution of 1:4000 (0) and 1:800 (•).
gp3ô —@
@
p25—@
100
90
80
10
a
60
p12*..-
A
B
1O@2
Chart 1. SDS-polyacrylamide gels of RIII MuMTV, stained with Coomassie
Brilliant Blue (A) and iodinated gp55 (B), a nadioautogram.
10_i
MG PROTEIN
COtIPARISOII
OF SEQUENTIAL
ADDITION
ANDDIRECTCOli@ETITION
ASSAYS
10
10
9
20
8
@
@
Chart 4. Comparison of sequential addition (C) and direct competition (U)
assays. In this experiment, disrupted RIII virus was used as the antigen.
7
3O,@
6
4o:@
5
50
4
6O@
e
70
3
a
80
sb
i@o 450
1350 40@0 i@ö
1/ ANTISERUMDILUTION
Na
a
Chart 2. Precipitation of gp55 (U) by milk-absorbed antiserum. The data
are presented both as cpm/120 pi of supernatant and as percentage of total
precipitated. The reaction mixture contained 400 @I.
There were 33,000 cpm
of gp55 added to the assay mixture.
$16 PROTEIN
virus
and
C57
mouse
milk
at high
concentrations
do
not
cause any displacement.
Factors Affecting the Assay.. The influence of KCI con
centration on the immune precipitation reaction was deter
mined. As shown in Chart 6, surprisingly low amounts of
KCI caused significant displacement of antigen from the
immune complex. This occurs with both antigen systems. In
order for us to obtain meaningful results, we have found it
1482
INHIBITIONOFP@CIPITATI0N
OFSF55
Chart 5. Inhibition of precipitation of gp55 by gp55 (U), RIII MuMTV (C),
RLV (0), and C57 milk (•).The data are presented both as cpm in 120 @iI
of
supennatant and as percentage inhibition. There were 22,000 cpm of gp55
added to each reaction mixture. Total reaction volume was 400 @.il.
necessary to maintain the salt concentration at or below
0.10 M. Otherwise, the salt-induced displacement would be
interpreted as a false positive.
CANCER RESEARCHVOL. 37
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MuMTV Radioimmunoassay
Glycerol, which is used as a stabilizing factor in virus
protein purification procedures (4), also has a similar effect
on the immune reaction (Chart 7). Presumably, this is due to
additional viscosity which interferes either with successful
centnifugation of the precipitate or reduces the collision
frequency of the reactants.
Proteolytic release of counts from the labeled antigen is a
possible souce of false readings. This possibility remains
even though the reaction mixture contains 2.5 mg of gelatin
per ml which should overwhelm such activity. We have
adopted 2 controls for proteolytic activity. First, the immune
reaction mixture is resuspended and allowed to incubate for
an additional 24 hr, and the supernatant is examined for
additional release of material. Second, an aliquot of the
supemnatant is precipitated with 5% tnichloroacetic acid to
detect any soluble counts. With these tests we have not yet
encountered any evidence of such activity in our samples.
Studies on the Nature of the Reactive Sites of gp55. In a
recent publication (18), we discussed the sensitivity of iodi
nated gp55 to trypsin and demonstrated that up to 30% of
the immune reactivity was sensitive to this protease, while
the remaining 70% consisted of several antigenic fragments
of molecular weights from 36,000 to 12,000 daltons. We
have attempted further characterization of the reactivity of
this antigen.
Attempts to inhibit the precipitation of iodinated gp55
with the saccharides listed under “Materials
and Methods―
in concentrations of up to 10 @g/mlwere unsuccessful.
Similarly, treatment of the antigen with f3-galactosidase or
galactose oxidase had no effect; neither did pemiodatetreat
ment.
Cross-Reaction between Inbred Mouse Strains. We have
compared the displacement of gp55 by MuMTV from several
sources and observe essentially the same degree of dis
placement for all virus samples tested . However, the relative
content of gp55 varies extensively, as can be seen in Chart
8. The data are summarized
in Table 1 . It appears from these
data that gp55 is a group-specific antigen, confirming the
observations of others (8, 10, 15), but that conversion from
antigen content to virus content may not be strictly linear. In
fact, the specific gp55 content of various “purified―
virus
samples may vary over a 10-fold mange.This may be due to a
combination of factors. Electron microscope observations
in this laboratory have demonstrated a considerable vania
tion in the vimion:nonvimion material ratio in preparations of
virus from different sources. Studies are now in progress to
determine whether the degree of impurity as determined by
50
0
a
40
a
30
0
MOUSE
STRAIN
a
0 GR
20
0
a
0 BALB/crC3H
A OBA
10
EG PROTEIN
0.01
0.05 0.1
KC.CONC.
(MOLES)
EFFECT
OFSALTOn IPViIJNE
PRECIPITATION
Chart 6. Effect of salt on the immunoprecipitation
reaction.
INHIBITION
OF 6P55PRECIPITATION
BY DIFFERENT
VIRUSPREPARATIONS
Chart 8. InhibItion of precipitatIon of gp55 by different virus preparations.
0, GA; S, A; 0, BALB/cfC3H; t@,DBA. All values are based on sg of protein
in the virus preparation.
Table 1
gp55 content of viruses isolated from milk of different mouse
strains
50
Values
milk.Mouse
are cornected
to 1 ml oforiginal
covered
0
gp55 re
re
40
gp55/mg
covered
30
milk)A
20
GA
0.46
180
82.8
DBA/2
BALB/cfC3H
C3H
AIII
C3HfC57
0.39
0.51
0.18
0.24
0.11
11
33
80
100
35
4.3
16.8
14.4
24.0
3.8
AfC57
0.15
DD0.39
0.0977
strainProtein(mg/mI
milk)@Lg
proteinTotal
(@g/ml
a
‘4
10
7.5
GLYCEROL
15
22.5
CONCENTRATION
30
1 w/v
Chart 7. Effect of glycerol on the Immune precipitation reaction.
MAY 1977
3
6630.0
0.5
5.9
1483
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J. B. Sheffield et at.
@
@
electron microscopy correlates with that calculated from
the nadioimmunoassay. An additional source of variation
may be that the vinions have different specific contents of
gp55; as indicated by Sankar and Dion (13), virus from strain
30
A mice has a larger specific
20
content
of the major glycopro
tein. Also, as discussed below, gp55 is readily lost from the
surface of vinions and remains soluble. It is possible that the
envelopes of the different strains of MuMTV have different
affinities for gp55 and so retain different amounts through
thepurification.
Application of the Assay. The gp55 assay has, because of
its antigenic simplicity and high sensitivity, been used for
our routine assaying of virus production and antigenic con
tent. Because of the cross-reaction between gp55 (spike
protein) of virus from different sources, it has been possible
to use the assay for the detection and quantitation of virus
antigen in the milk of various mouse strains and for the
routine screening of mouse milk for evidence of infection.
The nadioimmunoassayhas been compared totheimmuno
diffusion assay, which is in extensive use in this laboratory
(2), and has been found to be in essentially
complete
10
1
2
3
4
5
6
TIME OF INCUBATION AT 37' (HR)
@LEASE
OFSOLUBLE
ANTIGEN
F00I@
PURIFIED
Rill Mti@TV
Chart 10. Release of soluble gp55 antigen from purified RIII MuMTV. Virus
was incubated for the times indicated at 37°,and the soluble antigen was
separated from the insoluble by filtration through an O.1-@mpore size nu
cleopore filter. The virus bound to the filter was eluted with radioimmune
buffer, and both samples were tested by nadioimmunoassay.
agree
ment, but with the added advantage of quantitation. We
have observed that the virus content of infected mouse milk
is not a strict function of the original inoculating dose, nor
does it represent a 1-step increase from a low to a high
value. Our results indicate a broad distribution of antigen
content, as indicated in Chart 9.
The assay has also been used to analyze virus antigen
released from continuous cell lines in vitro (J. B. Sheffield,
T. M. Daly, A. M. McCaffrey, and N. Tanaschi, studies in
progress). In these studies, we have noticed a considerable
amount of soluble antigen similar to that described by Kim
ball et at. (5, 7, 8). We have been able to reproducibly sepa
rate the soluble antigen from the virus particles by filtration
through a 0.1 j@mnucleopore filter. Chart 10 shows the
progressive release of soluble gp55 antigen upon incuba
tion of RIll MuMTV in 0.01 M phosphate, pH 4-0.15 M NaCI at
37°.The initial virus was obtained
by the standard
proce
dune of centrifugal purification and presumably had no sol
uble antigen after the final centnifugation. Nevertheless,
during the overnight resuspension at 4°,7% of the viral
antigen became soluble. Incubation at 37°accelerated the
process so that 25% of the antigen was soluble within 4 hr.
It is not yet clean whether this is due to selective solubiliza
tion of gp55 on to the gradual destruction of the vinions.
Moore and Sarkam(10) showed several years ago that virus
incubated at 37°was rapidly degraded morphologically.
These studies have emphasized the need to define our
assay carefully, since soluble antigen is thus not necessarily
an indication
of particulate
virus.
DISCUSSION
We have described the features of a radioimmunoassay
for MuMTV. We have found that the use of detergent (Noni
det P-40) in the reaction mixture confers greater specificity
by reducing nonspecific trapping and also ensures the solu
a
I.
bility
I-
0
a
I-
p4
MTIGENICCONTENT
OFSAMPLES
OFMILKFROIINuPITY
INFECTED
C57MIcE
Chart 9. Antigenic content of samples of milk from MuMTV-infected C57
mice. The milk samples were diluted 1:20 before testing. The standard curve
presents the ability of RIII virus to inhibit precipitation of gp55. Each point on
the samples represents a different mouse.
1484
of the reacting
materials.
The difficulties
we have
encountered with pipetting have been overcome by using
positive displacement systems. The use of a high concen
tration of nonspecific protein (gelatin) reduces nonspecific
adsorption of labeled antigen and minimizes the effects of
proteases. By measuring the supemnatant after immune pre
cipitation, we can eliminate the problems of successive
washing of the pellet.
The gp55 assay is highly specific for a single protein . In its
optimum form, it is able to detect as little as 50 pg of
purified gp55. We have not encountered the conformation
shift problems mentioned by Parks et at. (12). Since the
gp55 we use as our test antigen cross-reacts with virus from
all sources tested, we have used the assay to monitor virus
production by cells derived from tumors of BALB/cfC3H,
GR, A, and RIlI strains.
Our attempts to characterize the antibody-reactive sites of
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MuMTV Radioimmunoassay
the antigen suggest that the particular serum we use reacts
with protein portions of the molecule, since reagents that
react with polysacchamides do not have any effect on the
immune precipitation (17). The failure to inhibit precipita
tion with various sugars is suggestive, but it is inappropriate
to draw absolute conclusions on the basis of a limited num
ben of saccharides. The ci'oss-meaction of gp55 from dif
ferent mouse strains, however, is further evidence that the
protein moiety contains the pertinent antigenic sites, since
the protein appears to be conserved among strains while
variation in glycosylation has been postulated (3). Other
sera may react more strongly with the sugar portion of the
molecule, but we have not thus far encountered this.
Our experience with unexpected sources of error such as
salt concentration and the observation of a significant de
gree of solubilization of antigen during incubation empha
size a need to define and carefully control the conditions of
the assay. Once the conditions are understood, however,
we feel that such assay procedures can give reproducible
and accurate results.
ACKNOWLEDGMENTS
The authors wish to thank Harold Ott, for excellent photographic assist
ance, and Alice Smith, for hen patience in typing this manuscript. We also
thank Dr. Canole A. Long of the Institute for Medical Research, for her critical
reading of the manuscript, and Dr. Paul LoGerfo of Francis Delafield Hospital
in New York, for his assistance in the initial phases of this work.
REFERENCES
1. Cardiff, A. D. Quantitation of Mouse Mammary Tumor virus (MTV) Vini
one by Radloimmune Assay. J. Immunol., 111: 1722-1729, 1973.
2. Charney. J., Pullingen, B. D., and Moore, D. H. Development of an
Infectivity Assay for Mouse Mammary Tumor Virus. J. NatI. Cancer Inst.,
43: 1289-1296,1969.
3. Dion, A. 5., and Moore, D. H. Cation Requirement for RNA and DNA
Templated Polymerase Activities of B-type Oncogenic RNA Viruses
MAY 1977
(MuMTV). In: D. H. Russell (ed), Polyamines in Normal and Neoplastic
Growth, pp. 323-334. New York: Raven Press, 1973.
4. Dion, A. S., Vaidya, A. B., Fout, G. S., and Moore, D. H. Isolation and
Characterization of RNA-directed DNA Polymenase from a B-type ANA
Tumor Virus. J. Virol., 14: 40-46, 1974.
5. Fine, D. L., Plowman, J. K., Kelley, S. P., Arthur, L. A., and Hillman, A. E.
Enhanced Production of Mouse Mammary Tumor Virus in Dexamethasone
treated 5-Iododeoxyunidine-stlmulated Mammary Tumor Cell Cultures. J.
NatI. Cancer Inst., 52: 1881-1886, 1974.
6. Greenwood, F. C., Hunter, W. , and Glover, J. S. The Preparation of ‘31Ilabeled Human Growth Hormone. Biochem. J., 89: 114-123, 1963.
7. Kimball, P. C., Michalides, A., Colcher, D., and Schlom, J. Characteniza
tion of Mouse Mammary Tumor Viruses from Primary Cell Cultures. II.
Biochemical and Biophysical Studies. J. NatI. Cancer Inst., 56:119-124,
1976.
8. Kimball, P. C., Schochetman, G., Boehm-Tnuitt, M., and Schlom, J. Char
actenization of Mouse Mammary Tumor Viruses from Primary Tumor Cell
Cuftures. I. Immunological and Structural Studies. J. NatI. Cancer Inst., 56:
111—117,
1976.
9. Laemmli, U. K. Cleavage of Structural Proteins during the Assembly of
the Head of Bacteriophage T4. Nature, 227: 680-686, 1970.
10. Moore, D. H., Charney, J., Kramarsky, B., Lasfargues, E. Y., Sarkar, N.,
Brennan, M. J. , Burrows, J. H., Sirsat, S. M., Paymaster, J. C., and
Vaidya, A. B. Search for a Human Breast Cancer Virus. Nature, 229: 611614, 1971.
11. Noon, M. C., Wolfond, A. G., and Parks, W. P. Expression of Mouse
Mammary Tumor Viral Polypeptides in Milks and Tissues. J. Immunol.,
115:653—657,
1975.
12. Parks, W. P., Howk, A. S., Scolnick, E. M., Oroszlan, S., and Gilden, R.
V. Immunochemical Characterization of Two Major Polypeptides from
Munine Mammary Tumor Virus. J. Virol., 13: 1200-1210, 1974.
13. Sarkar, N. H., and Dion, A. S. Polypeptides of the Mouse Mammary
Tumor Virus: Characterization of Two Group-Specific Antigens. Virol
ogy, 64: 471-491 , 1975.
14. Sarkan, N. H., and Moore, D. H. Separation of B and C-type Vinions by
Centnifugation in Gentle Density Gradients. J. Virol., 13: 1143—1147,
1974.
15. Scolnick, E., Parks, W. P., and Livingston, D. M. Radioimmunoassay of
Mammalian Type C Viral Proteins. J. Immunol., 109: 570-577, 1972.
16. Sheffield, J. B., and Daly, T. M. Extrinsic Labeling of MuMTV with a
Galactose Oxidase-tnitiated Borohydnide Method. Virology, 70: 247-250.
1976.
17. Sheffield, J. B., and Emmelot, P. Studies on Plasma Membranes. XVI.
Tissue-specific Antigens in the Liver Cell Surface. Exptl. Cell Res., 71:
97-105, 1972.
18. Sheffield, J. B., Zachanchuk, C. M., Taraschi, N., and Daly, T. M. Effect
ofTrypsin on Mouse Mammary Tumor Virus. J. Virol., 19: 255-266, 1976.
19. Verstnaeten, A. A., Hageman, P. C. , and Kwa, H. G. A Radioimmune
Assay
for MTV Antigens.
European
J. Cancer,
9: 155—157, 1973.
1485
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Procedures for Radioimmunoassay of the Mouse Mammary
Tumor Virus
Joel B. Sheffield, Thomas Daly, Arnold S. Dion, et al.
Cancer Res 1977;37:1480-1485.
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