| Summary: | This
thesis
describes
the
development
of
novel
microfluidic
technologies
for
rapid,
high-‐throughput
screening
and
selection
of
monoclonal
antibodies
(mAbs)
from
single
cells.
Microfluidic
devices
were
used
to
compartmentalize
single
antibody-‐
secreting
cells
(ASCs)
in
small-‐volume
chambers
(i.e.
hundreds
of
picoliters
to
nanoliters)
in
order
to
concentrate
secreted
mAbs
for
measurement
of
antigen
binding
kinetics
and
affinities
using
a
novel
microfluidic
fluorescence
bead
assay.
Microfluidic
single-‐cell
antibody
screening
was
performed
on
ASCs
harvested
from
antigen-‐
immunized
mice
and
purified
by
fluorescence-‐activated
cell
sorting
(FACS).
Following
microfluidic
selection
of
ASCs
producing
antigen-‐specific
mAbs,
ASCs
were
sequentially
recovered
from
the
microfluidic
device
and
subjected
to
single-‐cell
RT-‐PCR
to
amplify
the
antibody-‐encoding
heavy
and
light
chain
genes.
Antibody
genes
for
selected
high-‐
affinity
mAbs
are
sequenced
and
cloned
into
expression
vectors
for
recombinant
production
in
mammalian
cell
lines.
Nearly
200
high-‐affinity
mouse
mAbs
to
the
model
antigen
hen
egg
lysozyme
(HEL)
were
selected
as
a
validation
of
this
technology,
representing
a
ten-‐fold
increase
in
the
number
of
high
affinity
anti-‐HEL
mAbs
previously
selected
using
single-‐cell
micro-‐technologies
and
the
traditional
hybridoma
approach.
Microfluidic
single-‐cell
mAb
screening
also
yielded
important
insights
into
affinity
maturation,
immuno-‐dominance,
and
antibody
stereotypy
in
the
adaptive
immune
system.
By
circumventing
time-‐consuming
limiting
dilution
and
clonal
expansion
in
the
hybridoma
approach,
microfluidic
single-‐cell
screening
will
enable
selection
of
mAbs
from
other
animal
species
(e.g.
rabbits,
humans)
for
both
therapeutic
and
research
applications. === Applied Science, Faculty of === Chemical and Biological Engineering, Department of === Graduate
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