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UBC Theses and Dissertations
Microfluidic technologies for rapid, high-throughput screening and selection of monoclonal antibodies from single cells Singhal, Anupam
Abstract
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.
Item Metadata
| Title |
Microfluidic technologies for rapid, high-throughput screening and selection of monoclonal antibodies from single cells
|
| Creator | |
| Publisher |
University of British Columbia
|
| Date Issued |
2012
|
| Description |
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.
|
| Genre | |
| Type | |
| Language |
eng
|
| Date Available |
2013-04-30
|
| Provider |
Vancouver : University of British Columbia Library
|
| Rights |
Attribution-NonCommercial-NoDerivatives 4.0 International
|
| DOI |
10.14288/1.0071837
|
| URI | |
| Degree | |
| Program | |
| Affiliation | |
| Degree Grantor |
University of British Columbia
|
| Graduation Date |
2012-11
|
| Campus | |
| Scholarly Level |
Graduate
|
| Rights URI | |
| Aggregated Source Repository |
DSpace
|
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Item Citations and Data
Rights
Attribution-NonCommercial-NoDerivatives 4.0 International