Microfluidic technologies for rapid, high-throughput screening and selection of monoclonal antibodies from single cells
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....
| Main Author: | |
|---|---|
| Language: | English |
| Published: |
University of British Columbia
2012
|
| Online Access: | http://hdl.handle.net/2429/43575 |
| id |
ndltd-UBC-oai-circle.library.ubc.ca-2429-43575 |
|---|---|
| record_format |
oai_dc |
| spelling |
ndltd-UBC-oai-circle.library.ubc.ca-2429-435752018-01-05T17:26:13Z Microfluidic technologies for rapid, high-throughput screening and selection of monoclonal antibodies from single cells Singhal, Anupam 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 2012-11-09T19:17:36Z 2013-04-30 2012 2012-11 Text Thesis/Dissertation http://hdl.handle.net/2429/43575 eng Attribution-NonCommercial-NoDerivatives 4.0 International http://creativecommons.org/licenses/by-nc-nd/4.0/ University of British Columbia |
| collection |
NDLTD |
| language |
English |
| sources |
NDLTD |
| 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. === Applied Science, Faculty of === Chemical and Biological Engineering, Department of === Graduate |
| author |
Singhal, Anupam |
| spellingShingle |
Singhal, Anupam Microfluidic technologies for rapid, high-throughput screening and selection of monoclonal antibodies from single cells |
| author_facet |
Singhal, Anupam |
| author_sort |
Singhal, Anupam |
| title |
Microfluidic technologies for rapid, high-throughput screening and selection of monoclonal antibodies from single cells |
| title_short |
Microfluidic technologies for rapid, high-throughput screening and selection of monoclonal antibodies from single cells |
| title_full |
Microfluidic technologies for rapid, high-throughput screening and selection of monoclonal antibodies from single cells |
| title_fullStr |
Microfluidic technologies for rapid, high-throughput screening and selection of monoclonal antibodies from single cells |
| title_full_unstemmed |
Microfluidic technologies for rapid, high-throughput screening and selection of monoclonal antibodies from single cells |
| title_sort |
microfluidic technologies for rapid, high-throughput screening and selection of monoclonal antibodies from single cells |
| publisher |
University of British Columbia |
| publishDate |
2012 |
| url |
http://hdl.handle.net/2429/43575 |
| work_keys_str_mv |
AT singhalanupam microfluidictechnologiesforrapidhighthroughputscreeningandselectionofmonoclonalantibodiesfromsinglecells |
| _version_ |
1718583597299924992 |