Download Development of Mouse Hybridomas by Fusion of Myeloma Cells

HYBRIDOMA AND HYBRIDOMICS
Volume 22, Number 5, 2003
© Mary Ann Liebert, Inc.
Short Communication
Development of Mouse Hybridomas by Fusion of
Myeloma Cells with Lymphocytes Derived from
Spleen, Lymph Node, and Bone Marrow
AYNUR BAŞALP and FATIMA YÜCEL
ABSTRACT
Since its discovery by Kohler and Milstein in 1975, hybridoma technology has found a wide use in almost
every field of biology and medicine. A general and simple approach for developing monoclonal antibodies is
to use splenocytes from immunized mice. In the present study, 10 fusion experiments were carried out to analyze the hybridization efficiencies of mouse myeloma cells with lymphocytes derived from spleen, lymph node,
and bone marrow and we found a higher yield of antigen specific antibody producing hybridoma lines when
the lymph nodes were used.
INTRODUCTION
K
MILSTEIN ’S revolutionary work—hybridoma
technology—made it possible to fuse mouse myeloma cells
with B lymphocytes from immunized mice, thus resulting in antibody-producing somatic cell hybrids.(1) Since then, hybridoma
technology has become widely adopted, and monoclonal antibodies (MAbs) have provided the solution to many biological
problems. The most interesting and unique characteristics of a
MAb is its specificity, not just for the immunizing protein but for
a particular epitope region on it. In the past two decades, MAbs
specific for numerous antigens (e.g., bacteria, viruses, parasites,
tumor antigens, receptors, hormones, enzymes) have been generated, and MAbs have found widespread use in many areas of medical research, diagnostics, and therapeutics.(2–4) Although MAbs
are powerful scientific tools and diagnostic reagents, there are still
some unsolved practical problems. These technical problems include obtaining mAbs with high affinity and adequate specificity
to very weak immunogens and antigens that are available in only
very small amounts.
In the traditional hybridoma technology, mice are immunized with desired antigen and once the animals are making
OHLER AND
a good antibody response, MAbs are generated by fusing the
mouse splenocytes with a B cell myeloma from the same
strain. In the investigation reported below, we used lymphocytes derived from spleen, lymph nodes, and bone marrow to
enhance the chance of obtaining MAbs to weak or rare antigens.
MATERIALS AND METHODS
Antigens
Hepatitis B surface antigens (HBsAg; Chemicon AG-850,
AG-852, Temecula, CA), Verticillium dahliae, vascular endothelial growth factor–bovine serum albumin mixture (VEGFBSA; National Cancer Institute, Biological Resources Branch,
Bethesda, MD), and anti-HBV IgG antibody were used as the
immunogen. All antigens were emulsified with equal volume
of Freund’s complete and incomplete mineral oil adjuvant, respectively, and immunizations were performed intraperitonally.
Booster immunizations were done in phosphate buffered saline
(PBS) by systemic routes.
Research Institute for Genetic Engineering and Biotechnology (RIGEB), TÜBITAK, Gebze, Kocaeli, Turkey.
329
330
BAŞALP AND YÜCEL
TABLE 1.
COMPARISON
OF THE
NUMBER OF TOTAL HYBRID CELLS AND SPECIFIC ANTIBODY -PRODUCING HYBRID CELLS
OBTAINED FROM SPLEEN AND LYMPH NODES
Spleen
Lymph node
Number of
total hybrid
Number of
specific hybrid
Number of
total hybrid
Number of
specific hybrid
HBsAg
Fusion 1
Fusion 2
210
426
1
—
83
51
2
—
V. dahliae
Fusion 1
Fusion 2
715
112
1
—
370
15
1
—
VEGF-BSA a
Fusion 1
Fusion 2
Fusion 3
262
146
179
—
—
—
31
125
39
1
4
1
Anti-HBV antibody
Fusion 1
Fusion 2
Fusion 3
576
84
57
1
1
1
31
2
436
1
—
3
Antigen
aAntibodies
specific for VEGF and BSA.
Fusion
Fusion was carried out by some modifications of classical
fusion protocols.(5) Briefly, lymphocytes from spleen, lymph
nodes (bronchial, axillar, inguinal, popliteal, and intraperitonal),
and bone marrow were fused with F0 (ATCC CRL 1646) mouse
myeloma cells, and PEG 4000 was used as the fusing agent.
Hybrid cells were selected by incubating cells in HAT medium,
and positive clones were selected with ELISA. (6) Cloning and
subcloning were performed as described.(7)
ELISA
Antigen-specific immune activity was determined by ELISA.
Microtiter ELISA wells were coated with HBsAg (400 ng/mL),
VEGF-BSA (400 ng/mL), Goat anti-HBV polyclonal antibody
(1 mg/mL), and Verticillium dahliae (surface antigens, 1 mg/mL)
in PBS at 4°C by overnight incubation, and unbound antigens
were removed by washing with PBS 1 0.05% Tween-20. Free
sites were blocked with 200 mL of 1% BSA or powdered milk
for 1 h at 37°C and wells thoroughly washed. The diluted mouse
serum or hybrid cell supernatant was added to each well and incubated for 1 h at 37°C. After washing, alkaline phosphatase
conjugated goat antimouse IgG/IgM and polyvalent antibody
(Sigma, St. Louis, MO, diluted as 1/4000, 1/1000 and 1/1000,
respectively) were added. After 1 h of incubation at 37°C, the
plates were washed, and PNPP (para nitrophenyl phosphate) at
1 mg/mL in substrate buffer (0.1 M glycine, pH 10.4, 1 mM
ZnCl2, and 1 mM MgCl2) was added. The absorbance at 405
nm was read on a BioTec (Winooski, VT) EIA reader.
RESULTS AND DISCUSSION
Production of MAbs involves labor- and time-intensive procedures, lengthening their production time and ultimately their
cost. Most laboratories engaged in the production of MAbs currently use lymphocytes from spleen. In the present study, a total of 10 fusions were carried out using lymphocytes from
spleen and lymph nodes separately after immunizing animals
with different antigens. Table 1 shows the number of total hybrid cells and antigen specific antibody producing hybrid cells
from spleen and lymph nodes, respectively. When we compared
the yield, the number of total hybrid cells seem higher in fusions performed using splenocytes. On the contrary, the number of hybridomas producing antibody of interest using lymph
nodes was about two times higher than that of hybridomas with
the conventional method using mouse spleen cells—four versus 10.
The efficient use of lymph nodes for generating monoclonal
antibodies with high specificity and affinity to antigens was also
shown by other investigators.(8) Bynum and co-workers reported making high-affinity monoclonal antibodies by somatic
TABLE 2. COMPARISON OF THE NUMBER OF TOTAL HYBRID
CELLS AND SPECIFIC ANTIBODY -PRODUCING HYBRID CELLS BY
USING BONE MARROW
Bone marrow
Number of
total hybrid
Number of
specific hybrid
V. dahliae
Fusion 1
280
—
Anti-HBV antibody
Fusion 1
Fusion 2
Fusion 3
134
280
238
1a
—
—
Antigen
aAntibody
has cross-reactivity.
MOUSE HYBRIDOMAS WITH LYMPHOCYTES FROM SPLEEN, LYMPH NODE, AND BONE MARROW
fusion of lymphocytes isolated from peripheral lymph nodes.(9)
Sado et al. also used iliac lymph nodes for cell fusion and have
shown that the production frequency of target hybridomas was
about 10 times higher than that of a conventional spleen
method.(10) Kimura et al. showed that fusion of popliteal lymph
node lymphocytes produced hybridomas secreting antibodies to
the peptide moiety of erythrocyte membrane protein. On the
other hand, splenic lymphocyte fusion produced hybridomas secreting antibodies to the carbohydrate moiety of the same antigen.(11) Our data shown here is consistent with the previously
reported results and reveal the generation of MAbs to antigens
with higher specificity using lymph nodes.
It is well established that, in mammals, lymphocytes circulate between bone marrow, spleen and lymph nodes and these
organs are the major sites of antibody production in secondary
immune response and thus, can be used as a source of activated
B lymphocytes. Lymph nodes are small noduler aggregates of
lymphoid tissue situated along lymphatic channels throughout
the body and form part of a network that filters antigens from
the interstitial tissue fluid.(12) Different classes of lymphocytes
(T and B cells) and nonlymphoid accessory cells (dendritic
cells, macrophages) are sequestered in particular areas of the
node. Follicles are the B cell–rich areas of lymph nodes and T
cells are located in parafollicular areas. Most of these T cells
are CD4 T helper cells interacting with CD8 T cytotoxic cells.
The paracortex contains many interdigitating antigen presenting cells (APC) expressing high levels of MHC class II receptors. When a foreign antigen is transported to the lymph node,
T cells encounter the antigen and also dendritic cells, as well
as other APCs, present the antigen to the T cell. Thus, the lymph
nodes are the sites where T cell responses to lymph borne protein antigens are initiated. Bone marrow is a rich source of a
variety of soluble factors (IL-1, IL-2, IL-6, IL-7, GMCSF, and
IFN) and represents a special environment that supports and facilitates antibody production. Dilosa et al. have shown that germinal center B cells migrate to the bone marrow and produce
large quantities of specific antibody associated with secondary
immune response.(13) In four seperate fusions, bone marrow was
isolated from immune animals, and lymphocytes were fused
with mouse myeloma cells. Although primary hybridomas
could be established from fusions with bone marrow–derived
cells, only one out of 932 hybrid cells was production registered (Table 2).
The results of this study indicate that the use of spleen, lymph
nodes, and bone marrow in the same fusion after immunizing
mice, especially with the rare or less immunodominant antigens, maximizes the chance of generating MAbs with the fewest
attempts. This approach is a time- and labor-saving technique
for making monoclonal antibodies.
331
ACKNOWLEDGMENTS
This work was partly supported by TÜBITAK grant SBAG
MAM1 102 S005 and SBAG 2496.
REFERENCES
1. Kohler G, and Milstein C: Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 1975;256:
495–497.
2. Reichert JM: Monoclonal antibodies in the clinic. Nat Biotech
2001;19:819–822.
3. Glennie MJ, and Johnson PM: Clinical trials of antibody therapy.
Immunol Today 2000;21:403–410.
4. Vora S: Monoclonal antibodies in enzyme research: present and
potential applications. Anal Biochem 1985;144:307–318.
5. Galfre G, and Milstein C: Preperation of monoclonal antibodies:
Strategies and procedures. Methods Enzymol 1983;73:3–46.
6. Doulliard JY, and Hoffman T: Enzyme-linked immunosorbent assay for screening monoclonal antibody production using enzymelabeled second antibody. Methods Enzymol 1983;92:168–174.
7. Reid LCM: Cloning. Methods Enzymol 1979;58:152–164.
8. Jahn S, Grunow R, Kiessig ST, Specht U, Matthes H, Hiepe F, and
Hlin Von Baehr R: Establishment of human Ig producing heterohybridomas by fusion of mouse myeloma cells with human lymphocytes derived from peripheral blood, bone marrow, spleen,
lymph node and synovial fluid. Effect of polyclonal prestimulation
and cryopreservation. J Immunol Methods 1988;107:59–66.
9. Bynum J, Andrews JL, Ellis B, Kull FC, Austin EA, and Kilpatrick
K: Development of class-switched, affinity-maturated monoclonala ntibodies following a 7-day immunization schedule. Hybridoma
1999;8:407–411.
10. Sado Y, and Okigaki T: A novel method for production of monoclonal antibodies. Evaluation and expectation of the rat lymph node
method in cell and molecular biology. Cell Biol Int 1996;20:7–14.
11. Kimura A, Nakashima R, Inoeu H, Yasuda S, Ikemoto E, and Nishimoto TT: Differential production of monoclonal antibodies to carbohydrate moity or peptides moiety of glycoproteins by different
routes of immunization. Hybrdioma 1998;17:245–250.
12. Abbas AK, Lichtman AH, and Pober JS: Cellular and Molecular
Immunology. Saunders, Philadelphia, 2000, pp. 17–38.
13. Dilosa RM, Kunihiko M, Masuda A, Szakal AS, and Tew JG: Germinal center B cells and antibody production in the bone marrow.
J Immunol 1991;146:4071–4077.
Adress reprint requests to:
Aynur Başalp, Ph.D.
TÜBÌTAK—RIGEB
41470 Gebze, Kocaeli, Turkey
E-mail: [email protected]
Received for publication June 16, 2003. Accepted for publication July 21, 2003.