HYBRIDOMA

Volume 13, Number 1, 1994

Mary Ann Liebert, Inc., Publishers

Influence of Priming and Inoculation Dose on the Production of

Monoclonal Antibodies in Two Age Groups of BALB/c Mice

R. DE DEKEN, J. BRANDT, F. CEULEMANS, S. GEERTS, and R. BEUDEKER

ABSTRACT

An experiment was set up to assess the influence of some parameters on the production of a.sei tes and monoclonal
antibodies against circulating excretory-secretory antigens of Taenia s agina ta cysticerci in mice. The following
parameters were examined: time lapse between priming and the inoculation of hybridoma cells, age and body
weight of the mice at the time of inoculation, number of cells injected IP, and the resulting antibody titers of the
ascites. In this experiment the method used to prime the mice was the only factor having an influence on the
amount of ascites produced. Injection of a higher number of hybridoma cells (2-4 x 10'' cells) coincided with
higher antibody titers and resulted in an earlier ascites production. The antibody titer of the ascites was
increasing with time.

INTRODUCTION sis/6' about 80 different antibody-secreting hybridomas were

(mAbs) by
THEhybridoma
PRODUCTION OF MONOCLONAL ANTIBODIES
cells in vitro, apart from being expensive and
time consuming, usually yields lower titers than those produced
in vivo, i.e., through injecting intraperitoneally (IP) hybridoma
in BALB/c mice and collecting ascites afterwards. Even though
in vivo production of monoclonal antibodies appears to be more
rewarding, the method in itself appears to be easily affected by
many variables.
Several studies are reported on the influence of, among other
things, the number of hybridoma cells injected and the kind of
chemical to prime the mice. Timing and quantity of the prime-
injection, age or body weight, immune status, and sex of the
mice have to be taken into consideration/1-5' There is no doubt
that yielding antibodies through ascites puts a severe strain on
the animals. Therefore, a procedure has to be selected that aims
at reducing both the strain on each animal and the number of
animals involved. Hence, the stress imposed on the receptor
mouse and its survival, the quantity of ascites produced, the titer
of antibody present in it, and, to some extent, the time required
for sufficient production are all important factors in determining
anoptimal procedure.
To develop a sandwich ELISA for the detection of circulating
excretory-secretory antigens in Taenia saginata cysticerco-
injected into some 300 BALB/c mice. With some of these
hybridomas, sufficient ascites were produced, whereas with
others the results were poor.
From experience gained during this work and from reports
elsewhere/5' it seemed that more ascites could be obtained when
mice were primed with 0.1 ml of Freund's incomplete adjuvant
(FLA) rather than with 0.5 ml of pristane. Equally, the volume in
which hybridoma cells were suspended was apparently impor-
tant, i.e., the same quantity of cells in 0.5 ml of medium instead
of using a smaller volume (0.2 or 0.3 ml) increased the
production.
To optimize ascites production of those hybridomas giving a
poor harvest, an experiment was set up to examine the following
parameters: (i) the time lapse and frequency between priming
and the inoculation of hybridoma cells; (ii) the age and body
weight of the mice at the time of inoculation; and (iii) the number
of cells injected IP.
MATERIAL AND METHODS
Mice
Ninety-six female BALB/c mice, equally divided into two
groups of 2 and 4 months of age, were obtained from Charles

Institute of Tropical Medicine, 2000 Antwerp, Belgium.

53
54 DE DEKEN ET AL.

River Wiga (Sulzfeld 1, Germany). Care of the animals
accordance with institutional guidelines.
Priming
All mice were primed by IP injection of 0.1 ml of FIA (Sigma
# F-5506) and divided into three groups of 32 each (groups A,
was in Next, the individually collected ascites of each mouse were
centrifuged at 206 x g and the volume of supernatant was
measured. This volume was considered to be the ascites produc-
tion of that particular mouse on that day.
Subsequently, ascites productions collected on the same day
were pooled, centrifuged again and stored at 19°C for deter-
mination of the antibody titers.
—

B, and C) according to the frequency and timing of priming.

Each of the three groups consisted of an equal number of mice
from each age class (Table 1). Group A was injected twice, 30
and 4 days prior to the inoculation of the hybridoma cells; groups
B and C were primed, respectively, 12 and 5 days before
inoculation.
Inoculation of hybridoma cells
Hybridoma cells were cloned after a fusion between SP2/0
myeloma cells and popliteal lymphocytes. In a preliminary trial,
hybridoma 10E11 A, used in this experiment and secreting IgM,
was shown to be a poor ascites producer (in total, 3 ml of ascites
in 4 mice). Twenty-four groups of 4 mice, equally divided
between the two age classes and three priming methods, were
injected IP with 0.5, 1, 2, or 4 million hybridoma cells,
suspended in 0.5 ml of RPMI-1640 (GIBCO BRL # 041-
02400). All mice were inoculated on the same day.
Determination of the antibody titer
Ascites and supernatant of the hybridoma culture were seri-
ally diluted from 40,000 to 640,000 times and from 20 to 40,960
times, respectively. The quantity of mAbs in these dilutions was
compared by ELISA using homologous antigen (excretion-
secretion products of Cysticercus bovis). ES diluted 1/10 in
coating buffer was coated onto polystyrene plates. Rabbit
anti-mouse IgG alkaline phosphatase conjugate (Sigma #
A-1902) was diluted 1/500 and used with 4-nitrophenyl phos-
phate (Janssen Chimica # 12.88682) as substrate. The test was
repeated once for each dilution and the mean of the two readings
was taken as the final result.
mAbs in the ascites and serum of some mice primed similarly
as the mice of group B and inoculated with 0.5 or 4 million cells
were also quantified to compare the antibody titer between
serum and ascites of individual mice.

Collection of ascites
Ascites were collected six times a week in 10-ml sterile tabes
Statistical interpretation of the results
containing a drop of liquemine Roche. For ethical reasons, all Because the results obtained for most of the quantitative
animals were anesthetized with ether during sampling and variables showed no normal distribution, the Kruskall-Wallis
euthanized after the second harvest; 5 mice died before the test was used. Results for qualitative variables were analyzed by
second collection. For these mice the mean amount of ascites the chi-square method.
produced by the other 3 mice in the group was determined.
On each collection, presence of palpable tumors or hemor-
rhagic ascites was recorded separately. These variables were RESULTS
assessed qualitatively as follows: 0 absence of blood in the
=

ascites, abdomen without tumor, 0.5 presence of blood

=
All ascites were collected between 9 and 24 days after
(palpable tumors) at one of both ascites collections, 1 blood
=
inoculation of the hybridoma cells. The total amount of recuper-
(palpable tumors) at all collections. ated fluid was 265.5 ml. The ascites production in the different
groups of mice, according to priming, age-class, or inoculation
Experiment dose of hybridoma cells, is shown in Table 2.
Table 1. Summary of the Planning of the
The statistical processing of the results is shown in Table 3.
Number of cells On the basis of these results, it can be concluded that in this
a
Time of priming inoculated experiment the method used to prime the mice was the only
(days before inoculation) (million cells) Number of mice factor playing a role in the amount of ascites produced. Age only
affected the amount of blood in the ascites or the degree of tumor
Group A: 30 + 4 0.5 8b
1 8 development: 2-month-old mice produced larger tumors,
2 8 whereas 4-month-old mice had more hemorrhagic ascites.
4 8 A significant negative correlation could be observed between
the presence of palpable tumors in the abdomen of the mice and
Group B: 12 0.5 8
1 8
collection of hemorrhagic ascites (r =
—0.345, p < 0.001),
whereas a positive correlation existed between the presence of
2 8
4 8 palpable tumors in the abdomen and the day on which the ascites
could be collected for the first time (r= +0.553,p< 0.001). On
Group C: 5 0.5 8 the other hand, hemorrhagic ascites was negatively correlated
1 8 with the day of first ascites collection, but this correlation was
2 8
4
not significant (r =
-0.193, p =
0.06). The number of
hybridoma cells injected was inversely related to tumor devel-
aPriming, 0.1 ml HA IP. opment and to the number of days before the first ascites could
b8, 4 mice 2 months old and 4 mice 4 months old. be collected (Table 3).
MAß PRODUCTION IN BALB/c MICE 55

Table 2. Amount (ml) of Ascites Produced (after Centrifugation)

Age of mice Millions of
(in months) cells injected Group A Group B Group C Total
0.5 13.8 15.2 7.3 36.3
1 13.1 11.3 8.9 33.3
2 13.2 12.7 9.6 35.5
4 12.3 12.2 6.8 31.3
0.5 13.0 12.8 9.0 34.8
1 13.4 9.3 10.7 33.4
2 11.2 9.0 8.6 28.8
4 13.3 11.4 7.4 32.1
Subtotals: 103.3 93.9 68.3
Total ml of ascites produced: 265.5

The antibody titer in ascites collected on the different days of DISCUSSION

the experiment is shown in Fig. 1. It could be concluded from the
ELISA results that ascites had a much higher titer of antibodies
than corresponding supernatants from an in vitro culture and that
the titer of antibodies in ascites was increasing with time: ascites
collected on a more advanced stage of the experiment showed
higher titers (Fig. 1).
Yields of mAbs in ascites may vary according to the hybri-
doma injected into the abdomen. In our experience with mono-
clonal antibodies against circulating ES antigens in T. saginata
cysticercosis, only a minority of hybridomas lead to a sufficient
production of ascites, without any apparent link with the isotype

Table 3. Statistical Interpretation of the Results

Number of
Parameters Group Age cells injected

Amount of ascites produced:

p < 0.001 N.S. N.S.
A B: N.S.
C:p < 0.001
-

A
B C: p 0.004
-

Amount of ascites/collection:
-

p 0.0025
=
N.S. N.S.
A B: N.S.
C:p 0.001
-

A =

B C: p 0.007
-

Day of the first collection:

-

N.S. N.S. p < 0.001

0.5 A.p < 0.001
1 4:p < 0.001
-

0.5 2:p < 0.001

-

1 2:p < 0.001

-

0.5 1:N.S.
-

2-4: N.S.
-

The presence of many blood cells in the ascites:

p 0.009 = 0.03 N.S.
A B: N.S.
C: p < 0.01
-

A
-

B C:p<0.01
The presence of palpable tumors in the abdomen:
-

N.S. p =
0.013 p < 0.001
0.5 A:p 0.003 =

1 4: p 0.003
-

0.5 2: p = 0.02
-

0.5 1:N.S.
-

1 2: N.S.
-

2-4: N.S.
-

N.S., Not significant.

56 DE DEKEN ET AL.

Optical Density

4,9 5,2 5,5 6,1

Log Dilution Factor

Day 9 - *-- Day 10 Day 12 -o- Day 13

Day 16 -«-- Day Day 19 -*•-
17 Day 20
FIG. 1. ELISA results for dilutions of ascites produced on different days after inoculation of hybridoma cells.

of the antibody involved. More specifically, only 16 out of 81
monoclonals gave yields of minimum 2 ml of ascites per mouse
and, of these 16, only three were non-IgMs. A similar propor-
tion between isotypes was found in the other group. Amongst
these hybridomas, 10E11 A, a typically poor producer of ascites,
was selected to study some parameters affecting ascites produc-
tion.
Priming the mice with 0.1 ml of FLA twice, i.e., 30 and 4 days
(group A), or once, i.e., 12 days (group B), prior to inoculation
results in a significanüy higher production of ascites than
priming once at day 5 (group C) before inoculation. This is in
contradiction with the observation of Mueller/5' where priming
with FIA could be done until 1 or 2 days before inoculation of the
hybridoma cells without a concomitant decrease of ascites
production. On the other hand, ascites produced in mice of
group C, although less voluminous, contained significantly less
blood than in the other groups. Less hemorrhagic ascites was
also observed more frequently in mice aged 2 months than those
aged 4 months. The presence of erythrocytes in the ascites of
older mice may lead to an overestimation of its volume, and
probably offers an explanation for the higher volume of ascites
in older mice reported by Brodeur'2' and Jones ,<4) although these
authors do not mention whether blood cells were present in the
ascites or not.
Mice without blood in their ascites were more prone to the
development of large abdominal tumors after inoculation. The

Optical Density

3,3 3,6 3,9 4,2 4,5 4,8 5,1 5,4

Log Dilution Factor
•»- S 1 day 15 • »
S 1 day 12 A 1 day 12 A 1 day 15
S 2 day 10 • • A 2 day 10 S 2 day 12 •- A 2 day 12

Mouse 1 : 0.5 million Cells l.p.

Mouse 2 : 4 million Cells l.p.
Cut-off neg. Ascites : 0.039 (p-0.01)
FIG. 2. mAb titer in serum (S) and ascites (A) of individual mice sampled twice.
mAb PRODUCTION IN BALB/c MICE
lower the number of hybridoma cells inoculated the higher the
probability of this tumor development and the longer the time
required before the first ascites could be collected. This last
observation is in agreement with the findings of Brodeur.'2'
Early ascites production would thus require the injection of a
large number of hybridoma cells. However, this procedure
needs caution; in this experiment, 3 out of 5 mice, which died
before the second ascites collection, had been injected with the
highest number (4 million) of hybridoma cells. The ascites of
these mice had been very hemorrhagic and the ensuing anemia
caused their death.
In the present study, mice of different weights had been
randomly divided between the different groups but the differ-
ence in body weight between the heaviest and lightest mice was
not more than 10%. This weight difference did not significantly
influence any of the parameters studied. Nevertheless, Bro-
deur'3' succeeded in increasing ascites production significantly
by using, instead of BALB/c mice, hybrids of BALB/c and
Swiss Webster/HPB, which were about 40% heavier.
The ELISA results showed that ascites had a much higher titer
of antibodies than corresponding supernatants from an in vitro
culture and that the titer of antibodies in ascites was increasing
with time (Fig. 1). While in this experiment ascites of one
particular day could be mixture of ascites collected from mice
sampled for a second time and mice producing their first ascites
in a later stage of the experiment, it was not clear whether this
increase was due to differences in antibody production between
subsequent ascites collections or to higher antibody production
in mice producing ascites in a later stage of the experiment.
Determination of antibodies in ascites of individual mice inocu-
lated with 0.5 or 4 million cells and sampled on two occasions
(Fig. 2) indicated that the increase was mainly due to the
antibody titer of the second ascites sample being higher than that of
the first. This phenomenon is equally reported by Brodeur.'2' It was
also observed that inoculation of 4 million hybridoma cells gave
rise to a higher antibody titer than 0.5 million cells (Fig. 2).
Antibody titers in serum or ascites of hybridoma-inoculated
mice are supposed to be almost identical.'7' This was also true
for the two mice shown in Fig. 2, although ascites collected for
the second time seemed to contain a slightly higher titer of
antibodies than serum.
CONCLUSION
From the results of this experiment it can be concluded that:
1. Priming mice by IP injection with 0.1 ml of FIA is preferably
done twice (at day 30 and day 4 prior to inoculation) or once
57
12 days before inoculation instead of once 5 days before
inoculation.
2. Because the antibody titer is increasing with time, collecting
ascites repeatedly from the same animal can be advanta-
geous, although not advisible for ethical reasons.
3. It is recommended to inject a higher number of hybridoma
cells (2-4 million cells), because this coincides with higher
antibody titers and results in an earlier production of ascites.
However, in this experiment it was observed that a fatal
anemia due to the production of hemorrhagic ascites can
occur soon after injection of 4 million hybridoma cells.
4. Mice aged 2 months are preferred over older animals,
because the ascites production is almost indifferent, and in
the former it is less hemorrhagic.
REFERENCES
1. Adrion RF: Optimization of in vivo production of monoclonal
antibodies. In: Experimental Design in Biotechnology. Haaland PD
(Ed.), Marcel Dekkerlnc, New York, 1989, pp. 19-35.
2. Brodeur BR, Tsang P, and Larose Y: Parameters affecting tumor-
formation in mice and monoclonal antibody production. J Immunol
Methods 1984;71:265.
3. Brodeur BR and Tsang PS: High yield monoclonal antibody produc-
tion in ascites. J Immunol Methods 1986;86:239.
4. Jones SL, Cox JC, and Pearson JE: Increased monoclonal antibody
ascites production in mice primed with Freund's incomplete adju-
vant. J Immunol Methods 1990;129:227.
5. Mueller UW, Harves CS, and Jones WR: Monoclonal antibody
production by hybridoma growth in Freund's adjuvant primed mice.
J Immunol Methods 1986;87:193.
6. BrandtJRA, GeertsS, DeDeken R, KumarV, Ceulemans F, BrijsL,
and Falla N: A monoclonal antibody-based Elisa for the detection of
circulating excretory-secretory antigens in Taenia saginata cysticer-
cosis. Int J Parasitai 1992;22:471.
7. Goding JW: Monoclonal antibodies: Principles and Practice. Aca-
demic Press Inc., London, 1983.
Address reprint requests to:
Dr. R. De Deken
Institute of Tropical Medicine
Nationalestraat 155
B-2000 Antwerpen
Belgium
Received for publication: 6/30/93
Accepted after revision: 10/20/93