Download Use of magnetic beads for isolation of antigen

Customer Report
DYNALogue 02/2001
Use of magnetic
beads for
isolation of
antigenspecific human
B cells
producing
monoclonal
antibodies
Ekaterina Sidorova
Institute for Viral
Preparations of Russian
Academy of Medical
Sciences, Moscow,
Russia and
Anders Rosén
Linköping University,
Dept of Biomedicine
and Surgery, Division of
Cell Biology, Linköping,
Sweden.
References
1. Milstein C. With the
benefit of hindsight.
Immunol. Today 2000;
21(8); 359-363.
2. Herzenberg L.A., De Rosa
S.C. and Herzenberg L.A.
Monoclonal antibodies and
the FACS: complementary
tools for immunobiology
and medicine.
Immunol.Today 2000; 21(8);
383-390.
Introduction
Methods
Monoclonal antibodies play an enormous role in the
development of highly specific tests for
disease-associated marker biomolecules. Mainly
murine monoclonal antibodies from immune
splenocytes fused with myeloma cells have been
used. These antibodies are now being used for
therapy (1-4) but for many reasons, human or
humanised monoclonals are more effective in
activating human effector functions, to eliminate
antigens and also avoid reactions against murine
antibodies.
Antigens
Obtaining human monoclonal antibodies is
important in cases where in vivo immune response
is very weak or when animals do not respond to the
antigen. For example, EBNA-1 of Epstein-Barr virus
(EBV) is low immunogenic in mice. This is explained
by its structural composition (numerous Gly-Ala
repeats), which does not fit into MHC class 1 groove.
After prolonged infection there is formation of
EBNA-1 antibodies in infected people (5), thus
human monoclonal antibodies to EBNA-1 may be
obtained. To date, one exceptional anti-EBNA-1
antibody with specificity against residues 408-446
outside the repeat region is described (6).
Many different approaches have been used to
develop human monoclonal antibodies. The most
successful approach so far involved B lymphocyte
transformation from infected seropositive
patients by EBV (7) with subsequent homo- or
hetero-hybridisation to develop long-lived
antibody-producing hybridomas. However, this
procedure involves the use of infectious material
requiring special safety laboratories for infections
like HIV.
To avoid contact with the infectious material,
immunisation in vitro was suggested (8). This
procedure is sophisticated and the number of
resulting antibody-producing cells is relatively low
(9).
3. van Spriel A.B., van Ojik
H.H. and van de Winkel
J.G.J. Immunotherapeutic
perspectivefor bispecific
antibodies. Immunol. Today
2000; 21(8); 391-3974.
We have developed a novel approach based on the
positive selection of B cells specific for
antigens from normal human tissue or from blood
with the subsequent transformation of isolated cells
by EBV.
4. Fisk R.B. Anti-IgE as
novel therapy for the
treatment of asthma. Curr.
Opin. Pulm. Med. 1999;
5(1); 76-80.
To isolate B cells bearing those antigen-specific
receptors, magnetic beads (Dynal Biotech), were
used.
6
Synthetic peptides MN-24 corresponding to V3 loop
of gp120 of HIV-1 strain (residues 302-322) and
peptide 107 (p107) from EBNA-1 were
dissolved in phosphate-buffered saline (PBS),
sterilised and stored at -20°C. The Po/myelin
protein was isolated from bovine peripheral nerve
tissue.
Preparation of sensitised magnetic beads
Dynabeads ® M-450 Tosylactivated were used to
couple viral peptides and Po protein. Briefly,
magnetic beads were washed in 0.1 M phosphatebuffered saline (PBS), pH 7.4 and incubated for 24
hours at 22°C with 200 mg of peptides or Po protein
(1 ml), rotating end-over-end. After removal of
supernatant, magnetic beads were washed three
times, last time overnight at 4°C using buffer
containing stabilising/blocking protein, suspended
in the same buffer and stored at 4°C.
Culture media and lymphocyte preparation
For B cell culture RPMI 1640 medium mixed (1:1)
with OptiMem medium, or Iscove medium mixed
(1:1) with OptiMem medium (GibcoTM, Invitrogen,
Sweden) and supplemented with 10% of fetal calf
serum (FCS), glutamin and antibiotics (complete
media) were used.
Total lymphocytes were prepared from freshly
removed tonsils obtained from University
Hospital, Linköping, ENT-Department or from blood
of a patient with polyneuropathy associated with
monoclonal gammopathy of uncertain significance
(PN-MGUS). Mononuclear cells were separated by
density centrifugation in Ficoll-Paque (Amersham
Pharmacia Biotech, Sweden). The interface fraction
was collected, washed and
depleted for
macrophages (in the case of blood cells) by
adherence-to-plastic for 1 hour at 37°C or for T cells
(SRBC-rosetting) and macrophages (in the case of
tonsil cells).
Selection of B lymphocyte specific for MN-24
and p107 peptides and to Po protein
Magnetic beads coated by viral peptides (107 beads/
ml) were washed just before the cell separation by
PBS-bovine serum albumin and then by RPMI 16401% FCS (3 times each).
Human B cells (108/ml) were mixed (10:1) with the
washed magnetic beads and incubated for 2 hours
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at 4°C on a rotator. After incubation, the target cells
with 3-5 attached magnetic beads (rosetted cells)
were separated from non-rosetted cells using a
magnetic device (Dynal MPC®-1), washed five times
with cold complete medium, counted and used for
transformation. Normal B lymphocytes untouched
by magnetic beads were used as a control.
Determination of human immunoglobulins and
antibodies
Human immunoglobulins (Ig), anti-peptide and
anti-Po/myelin antibodies were determined by ELISA
(10).
Results
Incubation of 100 x 106 of tonsil B lymphocytes with
magnetic beads coated by MN-24 or by p107 peptides
resulted in 365 x 103 and 270 x 103 rosetted cells,
respectively. Incubation of 38 x 106 or 8.4 x 106 of
blood B lymphocytes with magnetic beads coated
by Po protein yielded 0.8 x 106 and 1 x 105 rosettes,
respectively.
In all plates, the first growing clones became
visible after one week. In a month, practically all
wells contained transformed B cell clones.
Antibodies to MN-24 peptide were produced in 52
cultures of 384 in MN-24 master plates, and
antibodies to p107 in 24 cultures of 192 in p107
master plates. None of growing clones in control
plates containing unselected B lymphocytes were
positive for antibodies to either MN-24 or p107.
Antibodies to Po/myelin were produced in all cell
cultures from preselected cells.
All secreted antibodies were of IgM isotype. It was
not unexpected, since it is known that EBV
transforms mainly B lymphocytes expressing
surface IgM.
DYNALogue 02/2001
Of all colonies obtained, the most positive for
anti-MN-24, anti-p107 and anti-Po protein (one for
each) were selected and expanded. Cells have
continuously secreted antibodies during 3 years.
It was shown that initially clones of M2D6 cells
(selected by MN-24-beads), E2C5 cells (selected
by p107-beads) and TJ99D cells (selected by Po
protein-beads) were CD5-positive, thus belonging to
the B1-lymphocytes subpopulation. After cultivation
during 4 months, cells gradually lost CD5
expression.
Antibodies produced by the B cell clones M2D6 and
E2C5 were monoclonal but polyreactive, whereas antiPo/myelin antibodies were monospecific.
Discussion
The results obtained in this study confirm and
expand our previous data [11], and prove that
isolation of B lymphocytes bearing specific Igreceptors with antigen-coated magnetic beads is a
convenient and simple method for developing
human antibody-producing cultures for human
monoclonal antibodies.
The method described here permits specific B
lymphocyte isolation from normal human tissues in
quantities sufficient for subsequent transformation
and culturing.
Conclusion
An approach was developed for the isolation of
human B cells based on the positive selection of B
cells bearing specific Ig-receptors with magnetic
beads coated by respective antigens.
This approach seems to be more economic, timesaving and safe compared to more traditional
methods.
Ordering Information
Product
Product No.
Content
Dynabeads® M-450 Tosylactivated
Dynabeads® M-450 Tosylactivated
140.03
140.04
2 ml
10 ml
www.dynalbiotech.com
7
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Athlin L., Rosén A. and
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method using antigencoated magnetic beads for
specific B cell isolation
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