U.S. patent application number 16/816494 was filed with the patent office on 2020-07-16 for methods for assaying cellular binding interactions.
This patent application is currently assigned to The University of British Columbia. The applicant listed for this patent is The University of British Columbia. Invention is credited to Daniel J. Da Costa, Carl L. G. Hansen, Charles A. Haynes, John W. Schrader, Anupam Singhal.
Application Number | 20200225227 16/816494 |
Document ID | / |
Family ID | 45467283 |
Filed Date | 2020-07-16 |
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United States Patent
Application |
20200225227 |
Kind Code |
A1 |
Singhal; Anupam ; et
al. |
July 16, 2020 |
Methods for Assaying Cellular Binding Interactions
Abstract
There are provided methods, and devices for assaying for a
binding interaction between a protein, such as a monoclonal
antibody, produced by a cell, and a biomolecule. The method may
include retaining the cell within a chamber having an aperture;
exposing the protein produced by the cell to a capture substrate,
wherein the capture substrate is in fluid communication with the
protein produced by the cell and wherein the capture substrate is
operable to bind the protein produced by the cell; flowing a fluid
volume comprising the biomolecule through the chamber via said
aperture, wherein the fluid volume is in fluid communication with
the capture substrate; and determining a binding interaction
between the protein produced by the cell and the biomolecule.
Inventors: |
Singhal; Anupam;
(Mississauga, CA) ; Hansen; Carl L. G.;
(Vancouver, CA) ; Schrader; John W.; (Vancouver,
CA) ; Haynes; Charles A.; (Vancouver, CA) ; Da
Costa; Daniel J.; (Pitt Meadows, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The University of British Columbia |
Vancouver |
|
CA |
|
|
Assignee: |
The University of British
Columbia
Vancouver
CA
|
Family ID: |
45467283 |
Appl. No.: |
16/816494 |
Filed: |
March 12, 2020 |
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Application
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16746540 |
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16816494 |
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16579561 |
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16129555 |
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14879791 |
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13184363 |
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9188593 |
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61365237 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01L 2300/0867 20130101;
B01L 2400/0481 20130101; G01N 33/6854 20130101; B01L 2200/0668
20130101; B01L 3/502761 20130101; G01N 33/577 20130101; G01N
33/56966 20130101; B01L 2300/0681 20130101; B01L 2300/0864
20130101; G01N 33/582 20130101; B01L 3/502738 20130101; B01L
2300/0861 20130101 |
International
Class: |
G01N 33/569 20060101
G01N033/569; G01N 33/68 20060101 G01N033/68; B01L 3/00 20060101
B01L003/00; G01N 33/58 20060101 G01N033/58; G01N 33/577 20060101
G01N033/577 |
Claims
1.-45. (canceled)
46. A method of assaying for a binding interaction between a
secreted monoclonal antibody produced by a single antibody
producing cell (APC) and an antigen, the method comprising:
retaining the single APC within a chamber having a volume of from
100 pL to 100 nL, a solid wall, and an aperture that defines an
opening of the chamber; incubating the single APC within the
chamber to produce a secreted monoclonal antibody; exposing the
secreted monoclonal antibody to a first removeable capture
substrate bound to an antigen, wherein the antigen is capable of
capturing the secreted monoclonal antibody, incubating the secreted
monoclonal antibody with the first removeable capture substrate to
produce a bound antibody; measuring a binding interaction between
the bound antibody and the antigen; lysing the single APC and
capturing the nucleic acids of the single APC on a second
removeable capture substrate.
47. The method of claim 46, wherein the single APC is a primary B
cell or a memory B cell.
48. The method of claim 46, wherein the single APC is a primary
plasma cell.
49. The method of claim 46, wherein the single APC is from a human,
a rabbit, a rat, a mouse, a sheep, an ape, a monkey, a goat, a dog,
a cat, a camel, or a pig.
50. The method of claim 46, wherein the antigen is fluorescently
labeled.
51. The method of claim 46, wherein the second removeable capture
substrate comprises an oligo(dT) mRNA capture bead capable of
binding mRNA from the single APC.
52. The method of claim 50, wherein the second removeable capture
substrate comprises an oligo(dT) mRNA capture bead capable of
binding mRNA from the single APC.
53. The method of claim 46, wherein the second removeable capture
substrate is capable of binding nucleic acids encoding the variable
regions of the secreted monoclonal antibody and capturing the
nucleic acids comprises capturing nucleic acids encoding the
variable regions of the secreted monoclonal antibody.
54. The method of claim 50, wherein the second removeable capture
substrate is capable of binding nucleic acids encoding the variable
regions of the secreted monoclonal antibody and capturing the
nucleic acids comprises capturing nucleic acids encoding the
variable regions of the secreted monoclonal antibody.
55. The method of claim 46, further comprising washing the second
removeable capture substrate after lysing.
56. The method of claim 50, further comprising washing the second
removeable capture substrate after lysing.
57. The method of claim 51, further comprising washing the second
removeable capture substrate after lysing.
58. The method of claim 52, further comprising washing the second
removeable capture substrate after lysing.
59. The method of claim 53, further comprising washing the second
removeable capture substrate after lysing.
60. The method of claim 46, further comprising recovering the first
removeable capture substrate and the second removeable capture
substrate.
61. The method of claim 46, further comprising recovering the
second removeable capture substrate.
62. The method of claim 50, further comprising recovering the
second removeable capture substrate.
63. The method of claim 51, further comprising recovering the
second removeable capture substrate.
64. The method of claim 53, further comprising recovering the
second removable capture substrate.
65. The method of claim 64, further comprising sequencing the
nucleic acids encoding the variable regions of the secreted
monoclonal antibody.
66. The method of claim 46, wherein measuring the binding
interaction comprises measuring an antigen-antibody binding kinetic
property between the antigen and the bound antibody.
67. The method of claim 66, wherein the antigen-antibody binding
kinetic property is a K.sub.on rate; a K.sub.off rate, a
dissociation constant, or a combination thereof.
68. The method of claim 46, wherein measuring the binding
interaction comprises measuring the affinity of the bound antibody
and the antigen.
69. The method of claim 46, wherein measuring the binding
interaction comprises measuring the avidity of the bound antibody
and the antigen.
70. The method of claim 46, wherein the antigen is a cell fragment,
a bacterium, a virus, a viral fragment, or a protein.
71. The method of claim 46, wherein the aperture serves as the
inlet and the outlet of the chamber.
72. The method of claim 46, wherein measuring the binding
interaction comprises fluorescence imaging of the secreted
monoclonal antibody binding to the antigen.
73. The method of claim 46, wherein measuring the binding
interaction is carried out via surface plasmon resonance (SPR)
spectroscopy, fluorescence anisotropy, interferometry or
fluorescence resonance energy transfer (FRET).
74. The method of claim 46, further comprising performing a reverse
transcription polymerase chain reaction (RT-PCR) on the nucleic
acids of the single APC to amplify the heavy and light chain genes
of the secreted monoclonal antibody.
75. The method of claim 50, further comprising performing a reverse
transcription polymerase chain reaction (RT-PCR) on the nucleic
acids of the APC to amplify the heavy and light chain genes of the
secreted monoclonal antibody.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Patent Application Ser. No. 61/365,237 entitled "METHODS FOR
ASSAYING CELLULAR BINDING INTERACTIONS" filed on 16 Jul. 2010,
which is incorporated herein by reference in its entirety.
FIELD OF INVENTION
[0002] This invention relates to the field of microfluidics and
protein binding, more specifically, binding interaction between
biomolecules.
BACKGROUND
[0003] Antibodies are defense proteins produced by the vertebrate
adaptive immune system for the purposes of binding and targeting
for clearance of a diverse range of bacteria, viruses, and other
foreign molecules (collectively referred to as antigens) (see, for
e.g., Abbas et al. (1997), Cellular and Molecular Immunology, 3rd
Ed., Chapter 3, pp. 37-65). As a result of their ability to bind
target antigens selectively and with high affinity, antibodies are
useful tools for protein purification, cell sorting, diagnostics,
and therapeutics.
[0004] Conventional antibody production has involved the
immunization of animals (i.e., mice) with a target antigen, such as
a virus, bacteria, foreign protein, or other molecule. The
immunized mice produce on the order of 10.sup.4-10.sup.5 antibody
secreting cells (ASCs), each with the capacity to produce a unique
(monoclonal) antibody specific to the target antigen (see, for
e.g., Poulson et al. (1997), J. Immunol. 179: 3841-3850; and
Babcock et al. (1996), Proc. Natl. Acad. Sci. USA 93:
7843-7848).
[0005] The ASCs are then harvested from the immunized animals and
screened in order to select which cells are producing antibodies of
desired affinity and selectivity to the target antigen. Since
single ASCs do not produce antibodies in sufficiently large
quantities for binding affinity measurements, each ASC is clonally
expanded. Primary ASCs do not grow efficiently in laboratory tissue
cultures; thus, clonal expansion may be achieved by fusing ASCs to
murine myeloma (cancer) cells to produce immortalized,
antibody-secreting (hybridoma) cells (see, for e.g., Kohler, G. and
Milstein, C. (1975), Nature 256: 495-497). Using this method,
expansion of each successfully created hybridoma then produces a
monoclonal antibody in sufficiently high concentrations to measure
its affinity and selectivity to a target antigen.
[0006] It has been recognized that a limitation of hybridoma
technology is the low efficiency of the fusion process. For
example, whereas an immune response may produce on the order of
10.sup.4-10.sup.5 antibody secreting cells, a typical fusion will
yield less than 100 viable hybridomas. (see, for e.g., Kohler, G.
and Milstein, C. (1975), Nature 256: 495-497; Karpas et al. (2001),
Proc. Natl. Acad. Sci. USA 98: 1799-1804; and Spieker-Polet et al.
(1995), Proc. Natl. Acad. Sci. USA 92: 9348-9352). Therefore,
fusions from hundreds to thousands of animals are required to fully
sample the diversity of antibodies produced in an immune response,
making the hybridoma approach both time-consuming and expensive.
Attempts to circumvent hybridoma generation by immortalizing
antibody-producing cells using viral transformations have resulted
in modest gains in the efficiency of ASC immortalization. However,
these approaches still require costly and time-consuming clonal
expansion in order to produce sufficient quantities of monoclonal
antibodies to screen for affinity and selectivity to target
antigens (see for e.g., Pasqualini, R. and Arap, W. (2004), Proc.
Natl. Acad. Sci. USA 101: 257-259; Lanzavecchia et al. (2007),
Current Opinion in Biotechnology 18: 523-528; and Traggiai et al.
(2004), Nat Med 10: 871-875).
[0007] Devices have been developed to estimate the equilibrium
dissociation constants of antibodies secreted from single
antibody-secreting cells (Story, C. M. et al. Proc. Natl. Acad.
Sci. U.S.A. (2008) 105(46):17902-17907; and Jin, A. et al. Nat.
Med. (2009) 15(9):1088-1092), but do not measure antibody-antigen
binding kinetics using antibodies secreted from single cells.
SUMMARY
[0008] In a first embodiment, there is provided a method of
assaying for a binding interaction between a protein produced by a
cell and a biomolecule: (a) retaining the cell within a chamber
having an inlet and an outlet; (b) exposing the protein produced by
the cell to a capture substrate, wherein the capture substrate is
in fluid communication with the protein produced by the cell and
wherein the capture substrate is operable to bind the protein
produced by the cell; (c) flowing a first fluid volume comprising
the biomolecule through the inlet into the chamber and out the
outlet, wherein the first fluid volume is in fluid communication
with the capture substrate; and (d) determining binding
interactions between the protein produced by a cell and the
biomolecule.
[0009] The cell may be an antibody producing cell (APC), the
protein produced by the cell is an antibody and the biomolecule is
an antigen. The cell may be a single cell. The biomolecule may be a
fluorescently labeled antigen. The determining binding interactions
may be a measure of antigen-antibody binding kinetics. The
determining the antigen-antibody binding kinetics may include
fluorescence imaging of antigen-antibody binding. The determining
the binding interactions may be by one or more of the following
techniques: surface plasmon resonance (SPR) spectroscopy,
fluorescence anisotropy, interferometry, or fluorescence resonance
energy transfer (FRET). The determining of the binding interaction
may be by a nanocalorimeter or a nanowire nanosensor. The measure
of antigen-antibody binding kinetics may be the K.sub.on rate. The
measure of antigen-antibody binding kinetics may be the K.sub.off
rate. The measure of antigen-antibody binding kinetics may be the
both the K.sub.on rate and the K.sub.off rate. The protein produced
by the cell may be an antibody. The antibody may be a monoclonal
antibody. The protein produced by the cell may be an antigen. The
biomolecule may be an antigen. The biomolecule may be selected from
one of the following: an antibody, a whole cell, a cell fragment, a
bacterium, a virus, a viral fragment, and a protein. The protein
produced by the cell may not be secreted by the cell, and the
method may further include a step of cell lysis prior to exposing
the protein produced by the cell to the capture substrate. The
protein produced by the cell may not be secreted by the cell, and
the method may further include a step of cell lysis after exposing
the protein produced by the cell to the capture substrate. The
capture substrate may be a removable capture substrate. The
removable capture substrate may be an anti-Ig bead. The removable
capture substrate may be an anti-Ig bead and/or oligo (dT) bead.
The removable capture substrate may include a capture substrate
capable of capturing both nucleic acids and antibodies. The
removable capture substrate may include a capture substrate capable
of capturing nucleic acids and a capture substrate capable of
capturing antibodies. The removable capture substrate may include a
capture substrate capable of capturing nucleic acids. The binding
of the antibodies may be further tested by viral inactivation. The
binding of the antibodies may be further tested by bacterial
inactivation. The binding of the antibodies may be further tested
by cell inactivation. The method may further include adding the
cell to a reverse transcription polymerase chain reaction (RT-PCR)
reaction to amplify the heavy and light chain genes. The
amplification may be performed in a number of ways. For example, 1)
the cells may be eluted into RT-PCR mix containing primers for both
heavy and light chain genes for multiplex amplification of both
genes in a single reaction. Alternatively, the cells may be eluted
into RT-PCR mix without primers, the mix may then be split into two
equal volume aliquots and the respective heavy and light chain
primers may be added to the two aliquots for single-plex
amplification. Both methods have been shown to work to amplify the
heavy and light chains from a single cell. The exposing the protein
produced by the cell to the capture substrate may include flowing a
removable capture substrate into the chamber. The method may
further include washing the cell prior to flowing a removable
capture substrate into the chamber. The protein produced by the
cell may be an antigen and the biomolecule may be an antibody. The
antibody may be a monoclonal antibody. The biomolecule may be a
fluorescently labeled antibody. The fluorescently labeled antibody
may be a monoclonal antibody. The determining binding interactions
may be a measure of antigen-antibody binding kinetics. The measure
of antigen-antibody binding kinetics may be any one or both of: a
K.sub.on rate; and a K.sub.off rate. The APC may be from one of the
following: a human, a rabbit, a rat, a mouse, a sheep, an ape, a
monkey, a goat; a dog, a cat, a camel, or a pig. The removable
capture substrate may be a carboxylic acid (COOH) functionalized
bead. The removable capture substrate may be capable of binding the
protein produced by the cell and the nucleic acids encoding the
protein produced by the cell. The method may further include
washing the cell prior to exposing the protein produced by the cell
to a capture substrate. The APC may be selected from one of the
following: a primary B cell and a memory B cell.
[0010] In a further embodiment, there is provided a cell assay
method, the method including: distributing an antibody producing
cell (APC) to a chamber, wherein the APC is in a first fluid;
replacing the first fluid with a second fluid while maintaining the
APC in the chamber; placing the antibodies produced by the APC in
fluid communication with an antigen; and determining the
antigen-antibody binding kinetics of the antibodies produced by the
APC with the antigen.
[0011] In a further embodiment, there is provided a method of
assaying for a binding interaction between a protein produced by a
cell and a biomolecule, the method including: (a) retaining the
cell within a chamber having an aperture; (b) exposing the protein
produced by the cell to a capture substrate, wherein the capture
substrate is in fluid communication with the protein produced by
the cell and wherein the capture substrate is operable to bind the
protein produced by the cell; (c) flowing a fluid volume comprising
the biomolecule through the chamber via said aperture, wherein the
fluid volume is in fluid communication with the capture substrate;
and (d) determining a binding interaction between the protein
produced by the cell and the biomolecule.
[0012] The measure of antigen-antibody binding kinetics may be the
K.sub.on rate. The measure of antigen-antibody binding kinetics may
be the K.sub.off rate. The measure of antigen-antibody binding
kinetics may be the both the K.sub.on rate and the K.sub.off rate.
The binding of the antibodies may be further tested by viral
inactivation. The binding of the antibodies may be further tested
by bacterial inactivation. The binding of the antibodies may be
further tested by cell inactivation. The determining of
antigen-antibody binding kinetics may be by one or more of the
following techniques: surface plasmon resonance (SPR) spectroscopy,
fluorescence anisotropy, interferometry, or fluorescence resonance
energy transfer (FRET). The determining of antigen-antibody binding
kinetics may be by a nanocalorimeter or a nanowire nanosensor. The
method may further include adding the cell to a reverse
transcription polymerase chain reaction (RT-PCR) reaction to
amplify the heavy and light chain genes. The placing the antibodies
produced by the APC in fluid communication with an antigen may
include flowing a removable capture substrate into the chamber. The
method may further include washing the cell prior to flowing a
removable capture substrate into the chamber. The APC may be from
one of the following: a human, a rabbit, a rat, a mouse, a sheep,
an ape, a monkey, a goat; a dog, a cat, a camel, or a pig. The
removable capture substrate may be a carboxylic acid (COOH)
functionalized bead. The removable capture substrate may be capable
of binding the protein produced by the cell and the nucleic acids
encoding the protein produced by the cell. The method may further
include washing the cell prior to exposing the protein produced by
the cell to a capture substrate. The APC may be selected from one
of the following: a primary B cell and a memory B cell. The method
may further include adding a removable capture substrate to the
chamber to capture the antibodies produced by the APC prior to
placing the antibodies produced by the APC in fluid communication
with an antigen. The placing of the antibodies produced by the APC
in fluid communication with an antigen may include flowing a
fluorescently labeled antigen through the chamber. The method may
further include collecting the mRNA from the cell for a reverse
transcription polymerase chain reaction (RT-PCR) reaction to
amplify the heavy and light chain genes. The determining the
antigen-antibody binding kinetics may include fluorescence imaging
of antigen-antibody binding.
[0013] In a further embodiment, there is provided a microfluidic
device for assaying for a binding interaction between a protein
produced by a cell and a biomolecule, the device comprising: a
chamber, having: (i) at least one inlet; (ii) at least one outlet;
and (iii) a reversible trap having spaced apart structural members
extending across the chamber to separate the at least one inlet and
at least one outlet wherein the spaced apart structural members are
operable to allow fluid flow through the chamber from the inlet to
the outlet while providing size selection for a particle within the
fluid flow.
[0014] In a further embodiment, there is provided a microfluidic
device for assaying for a binding interaction between a protein
produced by a cell and a biomolecule, the device comprising: a
chamber, having: (i) at least one inlet; (ii) at least one outlet;
and (iii) a reversible trap, wherein the reversible trap is a
narrowing of the chamber from to allow fluid flow through the
chamber from the inlet to the outlet while providing size selection
for a particle within the fluid flow.
[0015] In a further embodiment, there is provided a microfluidic
device for assaying a binding interaction between a protein
produced by a cell and a biomolecule, the device including: a
chamber having an aperture and a channel for receiving a flowed
fluid volume through the chamber via said aperture, the channel
providing size selection for a particle within said fluid
volume.
[0016] In a further embodiment, there is provided a microfluidic
device for assaying a binding interaction between a protein
produced by a cell and a biomolecule, the device including: a
chamber having an aperture; a reversible trap having spaced apart
structural members extending across the chamber, the structural
members being operable to allow a fluid volume to flow through the
chamber while providing size selection for a particle within said
fluid volume.
[0017] The distance between the spaced apart structural members may
be less than or equal to about 4.6 microns. The distance between
the spaced apart structural members may be less than or equal to
about 4.5 microns. The distance between the spaced apart structural
members may be less than or equal to about 4.4 microns. The
distance between the spaced apart structural members may be less
than or equal to about 4.3 microns. The distance between the spaced
apart structural members may be less than or equal to about 4.2
microns. The distance between the spaced apart structural members
may be less than or equal to about 4.1 microns. The distance
between the spaced apart structural members may be less than or
equal to about 4.0 microns. The distance between the spaced apart
structural members may be less than or equal to about 3.9 microns.
The distance between the spaced apart structural members may be
less than or equal to about 3.8 microns. The distance between the
spaced apart structural members may be less than or equal to about
3.7 microns. The distance between the spaced apart structural
members may be less than or equal to about 3.6 microns. The
distance between the spaced apart structural members may be less
than or equal to about 3.5 microns. The distance between the spaced
apart structural members may be less than or equal to about 3.4
microns. The distance between the spaced apart structural members
may be less than or equal to about 3.3 microns. The distance
between the spaced apart structural members may be less than or
equal to about 3.2 microns. The distance between the spaced apart
structural members may be less than or equal to about 3.1 microns.
The distance between the spaced apart structural members may be
less than or equal to about 3.0 microns. The distance between the
spaced apart structural members may be less than or equal to about
2.9 microns. The distance between the spaced apart structural
members may be less than or equal to about 2.8 microns. The
distance between the spaced apart structural members may be less
than or equal to about 2.7 microns. The distance between the spaced
apart structural members may be less than or equal to about 2.6
microns. The distance between the spaced apart structural members
may be less than or equal to about 2.5 microns. The distance
between the spaced apart structural members may be less than or
equal to about 2.4 microns. The distance between the spaced apart
structural members may be less than or equal to about 2.3 microns.
The distance between the spaced apart structural members may be
less than or equal to about 2.2 microns. The distance between the
spaced apart structural members may be less than or equal to about
2.1 microns. The distance between the spaced apart structural
members may be less than or equal to about 2.0 microns. The
distance between the spaced apart structural members may be less
than or equal to about 1.9 microns. The distance between the spaced
apart structural members may be less than or equal to about 1.8
microns. The distance between the spaced apart structural members
may be less than or equal to about 1.7 microns. The distance
between the spaced apart structural members may be less than or
equal to about 1.6 microns. The distance between the spaced apart
structural members may be less than or equal to about 1.5 microns.
The distance between the spaced apart structural members may be
less than or equal to about 1.4 microns. The distance between the
spaced apart structural members may be less than or equal to about
1.3 microns. The distance between the spaced apart structural
members may be less than or equal to about 1.2 microns. The
distance between the spaced apart structural members may be less
than or equal to about 1.1 microns. The distance between the spaced
apart structural members may be less than or equal to about 1.0
microns. The distance between the spaced apart structural members
may be less than or equal to about 0.9 microns. The distance
between the spaced apart structural members may be less than or
equal to about 0.8 microns. The distance between the spaced apart
structural members may be less than or equal to about 0.7 microns.
The distance between the spaced apart structural members may be
less than or equal to about 0.6 microns. The distance between the
spaced apart structural members may be less than or equal to about
0.5 microns. The spaced apart structural members may be posts. The
spaced apart structural members may be between 5 to 30 microns in
width. The spaced apart structural members may be between 10 to 20
microns in width. The spaced apart structural members may be
between 5 to 30 microns in width. The spaced apart structural
members may be between 5 to 20 microns in width. The spaced apart
structural members may be between 5 to 10 microns in width.
[0018] The narrowing of the chamber may be from greater than about
10 microns to less than about 5.0 microns. The narrowing of the
chamber may be from greater than about 10 microns to less than
about 4.9 microns. The narrowing of the chamber may be from greater
than about 10 microns to less than about 4.8 microns. The narrowing
of the chamber may be from greater than about 10 microns to less
than about 4.7 microns. The narrowing of the chamber may be from
greater than about 10 microns to less than about 4.6 microns. The
narrowing of the chamber may be from greater than about 10 microns
to less than about 4.5 microns. The narrowing of the chamber may be
from greater than about 10 microns to less than about 4.4 microns.
The narrowing of the chamber may be from greater than about 10
microns to less than about 4.3 microns. The narrowing of the
chamber may be from greater than about 10 microns to less than
about 4.2 microns. The narrowing of the chamber may be from greater
than about 10 microns to less than about 4.1 microns. The narrowing
of the chamber may be from greater than about 10 microns to less
than about 4.0 microns.
[0019] It will be appreciated by a person of skill in the art that
the distance between the spaced apart structural members and the
narrowing of the chamber to produce the reversible trap, will
depend on the size of the cells being assayed and the size of the
removable capture substrate, and the flow velocity through the
chamber, whereby the cell and the removable capture substrate are
retained in the chamber at a first flow velocity and whereby the
removable capture substrate is retained in the chamber and the cell
is able to deform and fit through the reversible trap at a second
flow velocity. Alternatively, there may be different sized
removable capture substrates and some may be permitted to pass
through the reversible trap, while other may be retained.
Furthermore, there may be further flow velocities possible with a
given device, whereby the reversible trap may deform to allow the
removable capture substrates to pass through the chamber.
Alternatively, the chamber may be pierced to remove the removable
substrate and/or cells. The narrowing of the chamber may correspond
to the channel size selection.
[0020] The particle may be selected from one or more of the cell,
the biomolecule, the protein, the protein bound to a removeable
capture substrate, and the removeable capture substrate. The size
selection of the reversible trap may prevent the cell and the
removeable capture substrate from passing through the reversible
trap, and may allow the biomolecule and the protein to pass through
the reversible trap at a first flow velocity, and the size
selection of the reversible trap may prevent the removeable capture
substrate from passing through the reversible trap, while allowing
the cell, the biomolecule and the protein to pass through the
reversible trap at a second flow velocity. The outlet may be a
sieve valve and the flow velocity through the chamber when the
valve is in an open position may be sufficient to allow the cell to
deform and pass through the reversible trap. The device may be
operable to provide two or more flow velocities through the
chamber. The device may be operable to provide two flow velocities
through the chamber. The device may be operable to provide three
flow velocities through the chamber. The device may be operable to
provide four flow velocities through the chamber. The microfluidic
device may be operable to allow for removal of the removeable
capture substrate. The microfluidic device may be operable to allow
for removal of the cell.
[0021] The cells get trapped in the chambers when the sieve valves
are closed. However, as with the posts, the cells deform when the
sieve valve is opened and there is increased flow through the
chambers. Both implementations of the reversible trap have worked,
but the bead post design is slightly more robust at retaining the
beads. The cells being used in the present experiments are about 10
microns in diameter, the beads are 5 microns in diameter, and the
space between the posts is less than 3 microns.
[0022] A chamber may be in fluid communication with a first
auxiliary chamber, wherein there is may be a valve between the
chamber and the first auxiliary chamber. The first auxiliary
chamber may be in fluid communication with a second auxiliary
chamber, wherein there is a valve between the first and second
auxiliary chambers, wherein the valve has an open position to allow
fluid flow from the first auxiliary chamber to the second auxiliary
chamber and a closed position to prevent fluid flow from the first
auxiliary chamber to the second auxiliary chamber. The first
auxiliary chamber may be in fluid communication with a second
auxiliary chamber and the second auxiliary chamber is in fluid
communication with a third auxiliary chamber, wherein there is a
valve between the first and second auxiliary chambers, wherein the
valve has an open position to allow fluid flow from the first
auxiliary chamber to the second auxiliary chamber and a closed
position to prevent fluid flow from the first auxiliary chamber to
the second auxiliary chamber, wherein there is a valve between the
second and third auxiliary chambers, wherein the valve has an open
position to allow fluid flow from the second auxiliary chamber to
the third auxiliary chamber and a closed position to prevent fluid
flow from the second auxiliary chamber to the third auxiliary
chamber. The volumes of the first second and third auxiliary
chambers relative to the chamber may be such that fluid may be
flowed into these chambers such that subsequent RT and PCR or other
reactions may be carried out without exchanging the fluid (for
example, where a first outlet is in a closed position).
[0023] The volume of the auxiliary chambers may be expandable. The
volume of the chamber may be between 0.1 nL to 100.0 nL. The
unexpanded volume of the expandable the chamber may be between 0.1
nL to 100.0 nL. The volume of the chamber may be 0.6 nL. The
unexpanded chamber may be 0.6 nL. The effective volume of a given
chamber may be increased by expanding the initial chamber or by
opening a valve to provide fluid flow into one or more auxiliary
chambers. The ratio between the second auxiliary chamber and the
first auxiliary chamber may be 5:1. The ratio between the second
auxiliary chamber and the first auxiliary chamber may be at least
5:1. The ratio between the expanded chamber and the unexpanded
chamber may be 5:1 or the ratio between the expanded first
auxiliary chamber unexpanded first auxiliary chamber may be 5:1.
The ratio between the expanded chamber and the unexpanded chamber
may be at least 5:1 or the ratio between the expanded first
auxiliary chamber unexpanded first auxiliary chamber may be at
least 5:1. The ratio between the second auxiliary chamber and the
first auxiliary chamber, or between the expanded chamber and the
unexpanded chamber, or between the expanded first auxiliary chamber
unexpanded first auxiliary chamber may vary depending on the
reaction mixtures chosen, the concentrations of the components of
the mixture and the concentration of the material being assayed.
Alternatively, the chamber may be between 0.05 nL and 100.0 nL.
Alternatively, the chamber may be between 0.05 nL and 90.0 nL.
Alternatively, the chamber may be between 0.1 nL and 95.0 nL.
Alternatively, the chamber may be between 0.1 nL and 90.0 nL.
Alternatively, the chamber may be between 0.1 nL and 85.0 nL.
Alternatively, the chamber may be between 0.1 nL and 80.0 nL.
Alternatively, the chamber may be between 0.1 nL and 75.0 nL.
Alternatively, the chamber may be between 0.1 nL and 70.0 nL.
Alternatively, the chamber may be between 0.1 nL and 65.0 nL.
Alternatively, the chamber may be between 0.1 nL and 60.0 nL.
Alternatively, the chamber may be between 0.1 nL and 55.0 nL.
Alternatively, the chamber may be between 0.1 nL and 50.0 nL.
Alternatively, the chamber may be between 0.1 nL and 45.0 nL.
Alternatively, the chamber may be between 0.1 nL and 40.0 nL.
Alternatively, the chamber may be between 0.1 nL and 35.0 nL.
Alternatively, the chamber may be between 0.1 nL and 30.0 nL.
Alternatively, the chamber may be between 0.1 nL and 25.0 nL.
Alternatively, the chamber may be between 0.1 nL and 20.0 nL.
Alternatively, the chamber may be between 0.1 nL and 15.0 nL.
Alternatively, the chamber may be between 0.1 nL and 10.0 nL.
Alternatively, the chamber may be between 0.1 nL and 9.0 nL.
Alternatively, the chamber may be between 0.1 nL and 8.0 nL.
Alternatively, the chamber may be between 0.1 nL and 7.0 nL.
Alternatively, the chamber may be between 0.1 nL and 6.0 nL.
Alternatively, the chamber may be between 0.1 nL and 5.0 nL.
Alternatively, the chamber may be between 0.1 nL and 4.0 nL.
Alternatively, the chamber may be between 0.1 nL and 3.0 nL.
Alternatively, the chamber may be between 0.1 nL and 2.0 nL.
Alternatively, the chamber may be between 0.1 nL and 1.0 nL.
[0024] In a further embodiment, there is provided a method of
assaying for a protein of interest produced by a cell, the method
comprising: incubating the cell with a removable capture substrate
in a buffer, wherein the removable capture substrate is capable of
binding the protein of interest and nucleic acids encoding the
protein of interest; and screening the bound removable capture
substrate to determine whether the cell produces the protein of
interest.
[0025] In a further embodiment, there is provided a method of
assaying for a protein of interest produced by a cell, the method
comprising: incubating the cell with a removable capture substrate
in a buffer, wherein the removable capture substrate is capable of
binding the protein of interest; and screening the bound removable
capture substrate to determine whether the cell produces the
protein of interest.
[0026] In a further embodiment, there is provided a method of
identifying a monoclonal antibody of interest, the method
comprising: incubating an APC with a removable capture substrate in
a suitable buffer, wherein the removable capture substrate is
capable of binding the monoclonal antibody produced by the APC and
nucleic acids encoding the variable regions of the monoclonal
antibody; and screening the bound removable capture substrate to
determine whether the APC produces the monoclonal antibody of
interest.
[0027] In a further embodiment, there is provided a cell assay
method, the method comprising: distributing an APC to a chamber,
wherein there is on average one APC in the chamber, wherein the APC
is incubated with a removable capture substrate in a first
solution, and wherein the removable capture substrate is capable of
binding an antibody of interest produced by the APC and nucleic
acids encoding the variable regions of the antibody of interest;
replacing the first solution with a second solution while
maintaining the APC in the chamber; placing the antibody of
interest produced by the APC in fluid communication with an
antigen; and screening the bound removable capture substrate to
determine whether the APC produces the antibody of interest.
[0028] In a further embodiment, there is provided a method of
assaying for a chemical interaction between a protein produced by a
cell and a biomolecule, the method comprising: distributing the
cell to a chamber, wherein the cell is in a first solution;
replacing the first solution with a second solution while
maintaining the cell in the chamber; placing the protein in fluid
communication with the biomolecule; and testing the chemical
interaction of the protein produced by the cell with the
biomolecule.
[0029] In a further embodiment, there is provided a method of
identifying a monoclonal antibody of interest, the method
comprising: incubating an antibody producing cell (APC) with a
removable capture substrate in a suitable buffer, wherein the
removable capture substrate is capable of binding the monoclonal
antibody produced by the APC and nucleic acids encoding the
variable regions of the monoclonal antibody; and screening the
bound removable capture substrate to determine whether the APC
produces the monoclonal antibody of interest.
[0030] In a further embodiment, there is provided a cell assay
method, the method comprising: distributing an antibody producing
cell (APC) to a chamber, wherein there is on average one APC in the
chamber, wherein the APC is incubated with a removable capture
substrate in a first solution, and wherein the removable capture
substrate is capable of binding an antibody of interest produced by
the APC and nucleic acids encoding the variable regions of the
antibody of interest; replacing the first solution with a second
solution while maintaining the APC in the chamber; placing the
antibody of interest produced by the APC in fluid communication
with an antigen; and screening the bound removable capture
substrate to determine whether the APC produces the antibody of
interest.
[0031] In a further embodiment, there is provided a method of
assaying for a protein of interest produced by a cell. The method
involves incubating the cell with a removable capture substrate in
a suitable buffer, wherein the removable capture substrate is
capable of binding the protein of interest; and screening the bound
removable capture substrate to determine whether the cell produces
the protein of interest.
[0032] The method may involve determining the binding affinity of
the protein of interest. The method may involve determining a
dissociation rate; and association rate and dissociation rate. The
method may involve lysing the cell prior to incubation with the
removable capture substrate, wherein the protein of interest is not
secreted by the cell.
[0033] In a further embodiment, there is provided an device for
selecting a cell that produces a protein having a binding affinity
for a biomolecule. The device may include a microfluidic device as
described herein operably configured to hold an aliquot, wherein
the aliquot on average contains one cell, and wherein the protein
produced by the cell is in fluid communication with the
biomolecule; and a detector for detecting the binding affinity of
the protein produced by the cell.
[0034] The device may include a detector that is a fluorescence
imager for detecting the binding affinity. The device may include a
detector that is a surface plasmon resonance (SPR) spectroscopy
apparatus, or a fluorescence anisotropy apparatus, or an
interferometry apparatus, or a FRET apparatus. Further, the device
may include a detector that is a nanocalorimeter or a nanowire
nanosensor.
[0035] In a further embodiment, there is provided a kit for
identifying a cell that produces antibodies having a binding
affinity for an antigen. The kit includes a microfluidic device as
contemplated herein; and a removable capture substrate. The kit may
include the removable capture substrate being capable of binding
proteins, or nucleic acids, or proteins and nucleic acids. The kit
may include the removable capture substrate being a microsphere.
Further, the kit may include the microsphere being a polystyrene
bead or a silica bead. Further, the kit may include the microsphere
being a carboxylic acid (COOH) functionalized bead.
[0036] The kit may include an antigen label. The kit may include an
antigen label that is a fluorescent label. Further, the kit may
include instructions for the use of the device contemplated herein
to identify a cell that produces proteins having a desired binding
affinity. Further, the kit may include instructions for immunizing
an animal and collecting APCs. Further, the kit may include an
antigen.
BRIEF DESCRIPTION OF THE DRAWINGS
[0037] FIG. 1 shows a microfluidic device and schematics for
bead-based measurements of antibody-antigen binding kinetics. Panel
(A) is an illustration of a microfluidic device containing control
channels for individually selecting six reagent inlets and
actuating sieve valves on the reagent outlet channel. Panel (B)
shows a microscopic image of the device with food coloring to
visualize distinct reagent inlets (as shown) and control channels
(as shown) (5.times. magnification); Top inset depicts a close-up
of beads trapped using sieve valves (20.times. magnification;
Bottom inset depicts fluorescence image of beads during binding
kinetic measurements (100.times. magnification). Panel (C) shows a
schematic of a bead assay for direct measurement of association and
dissociation kinetics of immobilized mAbs and fluorescently-labeled
antigen. Panel (D) shows a variation of a bead assay for indirect
measurement of dissociation kinetics of immobilized mAbs and
unlabeled antigen molecules.
[0038] FIG. 2 shows a schematic diagram of an embodiment of a
microfluidic device for the detection of antibody secreted from
single cells. (A) Hydraulic pressure is applied to valves
(fully-closing) and sieve valves (partially-closing) formed by the
intersection of actuation control channels with rounded- or
square-profile flow channels, respectively. (B) An expanded view of
an embodiment of a microfluidic device for the detection of
antibody secreted from single cells. (1) chip is flushed with
1.times.PBS; (2) antibody-secreting cells and antibody-capture
beads are loaded into chambers; (3) cells are incubated for one
hour to allow for antibody secretion; (4): mix valve is opened to
allow for secreted antibody to bind to beads; (5) beads and cells
are captured against sieve valve and unbound antibody is washed
out; (6) chambers flushed with fluorescently-labeled antigen; image
and measure antibody-antigen association kinetics; and (7) flushed
out unbound antigen with 1.times.PBS; image and measure
antibody-antigen dissociation kinetics.
[0039] FIG. 3 shows plots of microfluidic bead-based measurements
of antibody-antigen binding kinetics. Direct fluorescent
measurements of association and dissociation kinetics of (A) D1.3
mAb and HEL-Dylight488 conjugate, (B) HyHEL-5 mAb and
HEL-Dylight488 conjugate, (C) LGB-1 mAb and enhanced green
fluorescent protein (EGFP) are demonstrated. (D) Indirect
measurement of dissociation kinetics of D1.3 mAb and HEL using
HEL-Dylight488 conjugate is demonstrated.
[0040] FIG. 4 shows simultaneous measurement of multiple
antibody-antigen binding kinetics using optical and spatial
multiplexing. (A) Plots measured association and dissociation
kinetics of 3 distinct mAbs (HyHEL-5, D1.3, and LGB-1 mAb)
interacting with 2 different antigens (HEL-Dylight633 conjugate and
EGFP) is demonstrated. (B) A micrograph showing false-coloured,
overlay of images taken with distinct fluorescence filter cubes to
identify anti-lysozyme mAbs and anti-EGFP mAbs is demonstrated.
[0041] FIG. 5 shows plots of sensitivity and detection limit of
antibody-antigen binding kinetics measurements. (A) Measured
association kinetics of D1.3 mAb-Dylight488 conjugate on rabbit
anti-mouse pAb coated beads is demonstrated. Inset demonstrates a
schematic of bead assay for measuring binding kinetics of
fluorescently-labeled mouse mAb and rabbit anti-mouse pAb coated
beads. (B) Association kinetics of HEL-Dylight488 conjugate on
beads with varying amounts of immobilized D1.3 mAb is demonstrated.
(C) Equilibrium bead fluorescence varies linearly with the amount
of immobilized D1.3 mAb. Inset shows a close-up of the graph to
highlight detection limit of 1% bead coverage. (D) Direct
measurement of equilibrium dissociation constants by measuring
equilibrium bead fluorescence using immobilized D1.3 mAb and
varying concentrations of HEL-Dylight488.
[0042] FIG. 6 shows antibody-antigen binding kinetics measured
using antibodies secreted from a single cell. (A) Microscope image
of D1.3 hybridoma cell loaded into a microfluidic device adjacent
to rabbit anti-mouse pAb coated beads trapped using a sieve valve
is shown. (B) "Single-cycle" binding kinetics from a single bead
containing D1.3 mAbs secreted from a single cell and subject to
increasing concentrations of HEL-Dylight488 conjugate is
demonstrated.
[0043] FIG. 7 shows the effect of fluorophore stability on measured
antibody-antigen binding kinetics. (A) Photobleaching rates of
fluorescent dye molecules under 100 W Hg lamp illumination using
100.times.oil-immersion objective (NA 1.30) are plotted. (B) Effect
of fluorescent exposure times on measured association kinetics of
D1.3 mAb and HEL-Dylight488 are plotted.
[0044] FIG. 8 shows the effect of different bead immobilization
chemistries on measured antibody-antigen binding kinetics. Measured
kinetics are unaffected by bead composition (silica or polystyrene)
or by different polyclonal capture antibodies (rabbit or goat
pAbs).
[0045] FIG. 9 shows a plot of measured dissociation kinetics of
mouse mAb from antibody capture beads. No dissociation of D1.3
mAb-Dylight488 conjugate from Rabbit anti-Ms pAb coated beads was
observed over 3 days.
[0046] FIG. 10 shows a plot of the effect of antigen re-binding on
measured antibody-antigen dissociation kinetics. Dissociation
kinetics of D1.3 mAb and HEL-Dylight488 conjugate were unaffected
by the presence of a large concentration of competitive antigen (2
mg/mL HEL).
[0047] FIG. 11 shows a plot of the effect of mass transport on
measured antibody-antigen binding kinetics. Association and
dissociation kinetics of D1.3 mAb and HEL-Dylight488 conjugate were
unaffected by varying flow rates over a range of .about.3-15
.mu.L/hr.
[0048] FIG. 12 shows representative microscopic images of primary
ASCs in a microfluidic chamber in fluid communication with antibody
capture beads and oligo(dT) beads.
[0049] FIG. 13 shows an image of an ELISPOT control assay
confirming that the cells depicted in FIG. 12 are ASCs. The left
image represents cells that secreted any antibody; the right image
represents only those cells that secreted HEL-specific
antibodies.
[0050] FIG. 14 shows a scheme for preparing dual-capture (i.e.,
dual-function) beads using carbodiimide chemistry.
[0051] FIG. 15 shows images of dual-function beads. Polystyrene
COOH beads were conjugated with rabbit anti-mouse pAb and amine
functionalized oligo(dT).sub.25 using carbodiimide chemistry. (A)
Brightfield image of dual-function beads trapped using microfluidic
sieve valve. (B) Fluorescence image of synthetic single-stranded
DNA molecules captured on dual-function beads. Synthetic DNA
molecules are labeled with Cy5 fluorophore for visualization and
also contain a poly(A) tail that binds to the oligo(dT) on the bead
surface. (C) Fluorescence image of mouse D1.3 monoclonal antibody
(mAb) captured on dual-function beads. D1.3 mAbs are labeled with
Dylight488 fluorophore for visualization and bind to the Rabbit
anti-Mouse pAb on the bead surface.
[0052] FIG. 16 shows a microscopic image (A) and antibody-antigen
binding kinetics (B) as determined from a microfluidic device for
dual purpose beads.
[0053] FIG. 17 depicts (A) K.sub.d, (B) K.sub.on, and (C) K.sub.off
rates determined from specific eluted chambers according to Example
9 herein.
[0054] FIG. 18 shows representative fluorescence intensity data
over time for specific eluted chambers according to Example 9
herein. (A) depicts data for ROOC04; (B) depicts data for
R04C06.
[0055] FIG. 19 shows Kappa chain results from the first round of
RT-PCR are shown in FIG. 19, Panel A. Kappa chain results from the
second round of RT-PCR are shown in FIG. 19, Panel B. Heavy chain
results from the first round of RT-PCR are shown in FIG. 19, Panel
C. Heavy chain results from the second round of RT-PCR are shown in
FIG. 19, Panel D.
[0056] FIG. 20 shows a microfluidic device according to an
embodiment of the invention described herein, showing a reversible
trap. (A) brightfield image at 20.times. magnification; (B)
brightfield image at 40.times. magnification.
[0057] FIG. 21 shows a schematic whereby a microfluidic device
according to an embodiment of the invention described herein is
used as described herein. (1) Flush chip with 1.times.PBS; (2) Load
antibody-secreting cells into chambers; (3) Load antibody-capture
beads into inlet channel; (4) Load antibody-capture beads into
chamber against bead filter; (5) Incubate cells for 1 hour to allow
antibody secretion and capture on beads; (6) Wash out unbound
antibody; (7) Load fluorescently-labeled antigen into inlet
channel; (8) Flush chambers with fluorescently-labeled antigen;
image and measure antibody-antigen association kinetics; (9) Flush
our unbound antigen with 1.times.PBS; image and measure
antibody-antigen dissociation kinetics; and (10) Open sieve valve
and flush cell out of the chamber to the elution port for recovery
from device.
[0058] FIG. 22 shows a schematic diagram of an alternative
embodiment of the microfluidic device for assaying binding
interactions.
DETAILED DESCRIPTION
[0059] A binding interaction, as referred to herein, includes a
molecular interaction. A molecular interaction is commonly
understood as referring to a situation when two or more molecules
are attracted to one another by a force, where the force could be
for example, electrostatic, dipole-dipole, hydrogen bonding,
covalent, or hydrophobic in nature. A binding affinity is commonly
understood as referring to an average strength of a molecular
interaction. Similarly, "avidity" is used to describe the combined
strength of multiple interactions. When used in the present
application, "affinity" is meant to encompass one or more
interactions, including avidity. The methods described herein may
involve determining the binding affinity of the protein of
interest. The methods described herein may also involve determining
a dissociation rate; and association rate and dissociation rate.
Alternatively, the methods described herein may include determining
binding kinetics.
[0060] The method may involve testing the antigen binding affinity
by fluorescence imaging. The method may involve testing the antigen
binding affinity using any of the following techniques plasmon
resonance (SPR) spectroscopy, fluorescence anisotropy, or
interferometry. These techniques are understood to measure
antibody-antigen binding kinetics, including, but not limited to
surface plasmon resonance (SPR) spectroscopy, fluorescence
anisotropy, interferometry, or fluorescence resonance energy
transfer (FRET). See, for e.g., Bornhop et al. (2007), Science 317:
1732-1736; Homola et al. (1999) Sensors and Actuators B: Chemical
54: 3-15; and Xavier, K. A. and Willson, R. C. (1998), Biophys. J.
74: 2036-2045. Further, the method may involve testing the antigen
binding affinity by a nanocalorimeter or a nanowire nanosensor.
See, for e.g., Wang et al. (2005), Proc. Natl. Acad. Sci. USA 102:
3208-3212 and Lee et al. (2009) Proc. Natl. Acad. Sci. USA 106:
15225-15230. Another method that could be employed would be to use
a technique such as dark-field microscopy and use antigens or
antibodies labeled with gold nanoparticles. This could be used to
detect single molecules and generate on/off rates by counting the
molecules. See, for e.g., Ueno et al. (2010) Biophysical J. 98:
2014-2023; Raschke et al. (2003) Nano Letters 3: 935-938; and
Sinnischen et al. (2000) App. Phys. Letters 77: 2949-2951. Further
methods for labeling and detecting binding events and/or binding
kinetics would be known to a person of skill in the art. For
example, binding assays may include determining the number of
binding events.
[0061] A protein, as referred to herein, refers to organic
compounds made of amino acids, including both standard and
non-standard amino acids. Standard amino acids include the
following: alanine, cysteine, aspartic acid, glutamic acid,
phenylalanine, glycine, histidine, isoleucine, lysine, leucine,
methionine, asparagine, proline, glutamine, arginine, serine,
threonine, valine, tryptophan, and tyrosine. An example of a
protein is an antibody.
[0062] A biomolecule, as referred to herein, may include, but is
not limited to, an antibody, or an antibody fragment, or a whole
cell, or a cell fragment, or a bacterium, or a virus, or a viral
fragment, a nucleic acid or a protein.
[0063] A "chamber", as used herein, refers to an enclosed space
within a microfluidic device in which a cells may be retained. Each
chamber may have at least one inlet for permitting fluid, including
fluid containing a cell, to enter the chamber, and at least one
outlet to permit fluid and/or the cell to exit the chamber
(depending on the design of the chamber and/or the flow through the
chamber). Persons skilled in the art will understand that an inlet
or an outlet can vary considerably in terms of structure and
dimension, and may be characterized in a most general sense as an
aperture that can be reversibly switched between an open position,
to permit fluid to flow into or out of the chamber, and a closed
position to seal the chamber and thereby isolate and retain its
contents. Alternatively, the aperture may also be intermediate
between the open and closed positions to allow some fluid flow or
may be a sieve valve that allows for fluid flow out of the cell,
but not other particles (for example, the cell, the beads etc.). A
chamber, as referred to herein, refers to a portion of a
microfluidic device which is designed to hold, for example, a cell.
As used herein, the chamber is of an exceptionally small and
discrete sizing. Typical volumes are in the range of .about.100 pl
to .about.100 nl. For example, a chamber can be designed with a
volume of approximately 500 pL (less than 1 nL), with dimensions of
approximately 100 microns (width), 500 microns (length), and 10
microns (height).
[0064] The direction of fluid flow through the chamber dictates an
"upstream" and a "downstream" orientation of the chamber.
Accordingly, an inlet will be located at an upstream position of
the chamber, and an outlet will be generally located at a
downstream position of the chamber. A person skilled in the art
will understand, however, that a single aperture could function as
both an inlet and an outlet.
[0065] An "inlet" or an "outlet", as used herein, may include any
aperture whereby fluid flow is restricted through the inlet or
outlet. There may be a valve to control flow, or flow may be
controlled by separating the channels with a layer which prevents
flow (for example, oil). Alternatively, an aperture may serve as
both an inlet and outlet. Furthermore, an aperture (i.e. inlet or
outlet) as used herein is meant to exclude the surface opening of a
microwell.
[0066] A "microfluidic device", as used herein, refers to any
device that allows for the precise control and manipulation of
fluids in a geometrically constrained structure. For example, where
at least one dimension of the structure (width, length, height) is
less than 1 mm.
[0067] A solution, as referred to herein, may include, but is not
limited to, a solution that can maintain the viability of a cell.
Further, the solution may include a suitable buffer that can both
retain the viability of a cell such that binding interactions can
be obtained or allow for an effective lysis of the cell to obtain
nucleic acids from the cell and/or antibodies or other proteins
depending on the application. Alternatively, the solution may be
suitable for performing an assay.
[0068] A capture substrate, as referred to herein, is meant to
encompass a wide range of substrates capable of capturing a protein
or biomolecule of interest. These substrates may be modified to
alter their surface (internal and external) properties depending on
the desired use. For example, a substrate may be bound to
antibodies or antigens to capture an antibody of interest. A
capture substrate may be, for example, a microsphere or a
nanosphere or other microparticles including, but not limited to a
polystyrene bead or a silica bead (for example, antibody capture
beads and oligo(dT) mRNA capture beads). In an alternate
arrangement, instead of modifying the beads with oligo(dT),
specific primers could be utilized instead. Optionally, the
microsphere may be a carboxylic acid (COOH) functionalized bead.
Beads which make use of alternate chemical interactions can fall
within this definition. See: for e.g., G. T. Hermanson (2008),
Bioconjugate Techniques, 2nd Edition, Published by Academic Press,
Inc. For example, an alternate scheme for preparing these beads
would be to use streptavidin coated beads and to mix these beads
with biotinylized rabbit anti-mouse pAbs and biotinylated
oligo(dT). A capture substrate can also be an anti-Ig bead which
binds an antibody to the capture substrate. A capture substrate can
be modified such that it binds multiple biomolecules of interest,
for example both mRNA and protein. Alternately, each capture
substrate could be limited to a particular biomolecule, for
example, one capture substrate being limited to binding mRNA and a
second capture substrate being limited to binding a protein.
Capture substrates are commercially available or may be made de
novo and/or modified as needed for the particular application.
Capture substrates may be removable, as in the case of beads.
However, capture substrates may also be fixed (and thus,
non-removable).
[0069] Nucleic acids, as referred to herein, include macromolecules
composed of chains of monomeric nucleotides. Common examples of
nucleic acids include deoxyribonucleic acid (DNA) and ribonucleic
acid (RNA).
[0070] In a further embodiment, a cell assay method is provided.
The method involves distributing an antibody producing cell (APC)
to a chamber, wherein the APC is in a first solution, and wherein
there is on average one APC in the chamber; replacing the first
solution with a second solution while maintaining the APC in the
chamber; placing the antibodies produced by the APC in fluid
communication with an antigen; and testing the binding of the
antibodies produced by the APC with the antigen. Optionally, the
method may involve adding anti-Ig beads to the chamber to capture
the antibodies produced by the APC. Optionally, the method may
involve lysing the APC to capture antibodies produced by the APC
wherein the antibodies are not secreted by the APC.
[0071] A cell as referred to herein includes an antibody producing
cell (also referred to herein as an "APC"). An APC refers to a cell
that can produce an antibody. An antibody producing cell is not
limited to cells that secrete antibodies, which are also referred
to herein as antibody secreting cells (also referred to herein as
an "ASC"). For example, it will be understood from the relevant art
that memory B cells, without stimulation, do not normally secrete
antibodies. See, for e.g., Abbas et al. (1997), Cellular and
Molecular Immunology, 3.sup.rd Ed., pp. 22-23). Examples of
antibody producing cells (APCs) include B cells, memory B cells,
primary B cells (which are also known in the art as naive B cells),
and B cell hybridomas. A primary B cell can be harvested from the
spleen, blood, or bone marrow of an animal, for example from a
mouse, by FACS sorting for a cell surface marker, for example, the
CD138+ marker (See: for e.g., Smith et al. (1996) Eur. J. Immunol.
26: 444-448).
[0072] Antibodies are defense proteins produced by the vertebrate
adaptive immune system for the purposes of binding and targeting
for clearance a diverse range of bacteria, viruses, and other
foreign molecules (antigens). As a result of their ability to bind
target antigens selectively and with high affinity, antibodies are
invaluable tools for protein purification, cell sorting, and
diagnostics. Antibodies are produced by B cells and are secreted by
activated B cells. (See generally, for e.g., Abbas et al. (1997),
Cellular and Molecular Immunology, 3.sup.rd Ed., Chapter 3, pp.
37-65). Antibodies are also referred to herein as immunoglobulin
(also referred to herein as Ig). An antibody, as referred to
herein, can include, but is not limited to polyclonal antibodies
and monoclonal antibodies. Unlike polyclonal antibodies, monoclonal
antibodies are monospecific antibodies that are the same because
they are made by one type of immune cell that are all clones of a
unique parent cell. A single APC or ASC can serve as the source of
a monoclonal antibody. Antibodies are not limited to a specific
isotype and can include, but are not limited to the following
isotypes: IgM, IgG, IgD, IgE, and IgA. Typically, it is understood
that antibodies are comprised of light and heavy chains that have
variable and constant regions therein (see generally, for e.g.,
Abbas et al. (1997), Cellular and Molecular Immunology, 3.sup.rd
Ed., Chapter 3, pp. 37-65).
[0073] In a further embodiment, a method of identifying a
monoclonal antibody of interest is provided. The method involves
incubating an APC with a removable capture substrate (RCS) in a
suitable buffer, wherein the removable capture substrate is capable
of binding the monoclonal antibody produced by the APC and nucleic
acids encoding the variable regions of the monoclonal antibody; and
screening the bound removable capture substrate to determine
whether the APC produces the monoclonal antibody of interest.
[0074] In a further embodiment, a cell assay method is provided.
The method involves distributing an APC to a chamber, wherein there
is on average one APC in the chamber, wherein the APC is incubated
with a removable capture substrate in a first solution, and wherein
the removable capture substrate is capable of binding an antibody
of interest produced by the APC and nucleic acids encoding the
variable regions of the antibody of interest; replacing the first
solution with a second solution while maintaining the APC in the
chamber; placing the antibody of interest produced by the APC in
fluid communication with an antigen; and screening the bound
removable capture substrate to determine whether the APC produces
the antibody of interest.
[0075] In a further embodiment an apparatus for selecting a cell
that produces a protein having a binding affinity for a biomolecule
is provided. The apparatus includes a microfluidic device operably
configured to hold an aliquot, wherein the aliquot on average
contains one cell, and wherein the protein produced by the cell is
in fluid communication with the biomolecule; and a detector for
detecting the binding affinity of the protein produced by the cell.
However, the microfluidic device may also hold more than one cell,
particular in an assay where the antigen or biomolecule of interest
is a cell, or a cell fragment. Similarly, the antigen may be a
virus or a bacterial cell.
[0076] In a further embodiment, a kit for identifying a cell that
produces antibodies having a binding affinity for an antigen is
provided. The kit includes a microfluidic device and a removable
capture substrate.
[0077] An antigen, as referred to herein, refers to a molecule
recognized by the immune system. As such, an antigen can include a
molecule that can elicit an immune response in an organism,
including in an animal. Examples of antigens include, but are not
limited to bacterial antigens and viral antigens.
[0078] A method is provided for identifying antibody secreting
cells (ASCs) that produce antibodies having a particular binding
affinity for an antigen or functional attributes. The method
involves distributing an ASC within a discrete aliquot wherein
there is on average one ASC in the aliquot, placing the antibodies
in fluid communication with the antigen; and testing the antigen
binding affinity of the antibodies produced by the ASC. The method
is based in part on the discovery that a single ASC, without clonal
expansion, is capable of producing enough antibodies to test
binding affinity for an antigen or to test other functional
attributes. Furthermore, the method is also based, in part, on the
discovery that clonal expansion via the production of hybridomas is
not required for larger scale production of monoclonal antibodies,
whereby the variable regions for the antibodies of interest may be
sequenced from an ASC of interest or collected with antibodies.
[0079] By way of example, a sensitive, low-cost microfluidic
bead-based fluorescence assay is described herein for measuring
antibody-antigen binding kinetics within low abundance samples.
Direct measurements of antibody-antigen binding kinetics may be
made by time-course fluorescence microscopy of antibody-conjugated
beads retained in microfluidic chambers and subject to a series of
wash cycles with fluorescently-labeled antigen and buffer. A
variation of the bead-based assay may include measuring the
dissociation kinetics of unlabeled antibody and antigen molecules.
As disclosed herein, multiple antibody-antigen interactions were
measured spanning nearly four orders of magnitude in equilibrium
binding affinity. The rate constants measured by way of the assay
disclosed herein were validated with previously published values
using SPR spectroscopy.
[0080] The methods provided herein are also contemplated for being
used to screen mutagenic B cell lines. Further, the methods
provided herein are contemplated for being used to screen the
selectivity and specificity of antibodies to multiple different
antigens.
Antibody Binding Kinetics
[0081] The affinity or binding strength of an antibody for its
target antigen is an important parameter when selecting an antibody
for a given application. Although the affinity of an
antibody-antigen interaction is typically quantified by an
equilibrium binding constant (K.sub.d), which describes the dynamic
equilibrium between binding and unbinding events, the kinetic rate
constants (k.sub.on and k.sub.off) provide a more complete
characterization of an antibody-antigen interaction. Two antibodies
with identical K.sub.d values may exhibit dramatically different
binding kinetics which, in turn, will determine their respective
suitability for a given application. For instance, antibodies with
rapid association and dissociation kinetics may be desirable for
sensing applications, whereas antibody-antigen interactions with
very long half-lives may be critical for histological staining,
enzyme-linked immunosorbent assays (ELISA), and Western blotting.
Similarly, therapeutic antibodies that bind their target antigens
with long half-lives could, in principle, be administered in lower
dosages, reducing the cost and side-effects of these therapies.
Direct measurement of binding kinetic constants can be a critical
factor for selecting antibodies for both clinical and research
applications. Examples of kinetic assays include, but are not
limited to viral and other pathogenic neutralization, cell
signaling and growth inhibition, modulation of enzymatic activity
(inhibit or enhance).
Microfluidics
[0082] Microfluidics refers to a multidisciplinary field dedicated
to the design of systems in which small volumes of fluids will be
used for a variety of purposes, including lab-on-a-chip technology.
See: for e.g., Squires and Quake (2005), Reviews of Modern Physics
77: 977-1026. Microfluidic technologies enable small-scale
(picoliter to nanoliter) fluid handling operations for
high-throughput biochemical analyses with low reagent costs and
rapid analysis times. In particular, microfluidic devices
fabricated from a silicone rubber, polydimethylsiloxane (PDMS), can
be designed and fabricated in 24-48 hours, enabling rapid
prototyping of devices. See: for e.g., McDonald, J. C. et al.
(2000), Electrophoresis 21: 27-40. Microfluidic devices that
integrate valves into pumps, mixers, fluidic multiplexers (MUXes),
and other fluid-handling components have been successfully applied
for protein crystallization, chemical synthesis, protein and DNA
detection and single cell analysis. See, for e.g., Thorsen et al.
(2002), Science 298: 580-584; Hansen, et al. (2002), Proc. Natl.
Acad. Sci. USA 99: 16531-16536; Maerkl, S. J. & Quake, S. R.
(2007) Science 315: 233-237; Hansen et al. (2004), Proc. Natl.
Acad. Sci. USA 101, 14431-14436; Huang, B. et al. (2007) Science
315, 81-84; and Cai et al. (2006) Nature 440: 358-362. Microfluidic
devices, as described herein, can include chambers of varying
sizes. For example, chambers can be designed with a volume of
approximately 500 pL (less than 1 nL), with dimensions of
approximately 100 microns (width), 500 microns (length), and 10
microns (height).
[0083] As disclosed herein, antibody-antigen binding kinetics were
measured with approximately 4.times.10.sup.4 antibody molecules
(.about.66 zeptomoles) immobilized on a single bead and less than
2.times.10.sup.6 antibodies (.about.3 attomoles) loaded into the
microfluidic device. This represents a reduction of greater than
four orders of magnitude in both detection limit and sample
consumption compared to SPR spectroscopy and a recently reported
microfluidic fluorescence assay for measuring protein-protein
binding kinetics. See, for e.g., Bates, S. R.; Quake, S. R. (2009),
Appl. Phys. Lett. 95, 073705. Since each antibody-antigen
interaction can be characterized on a single bead, millions of
distinct antibody-antigen interactions can be characterized with a
single lot of commercially available beads (i.e., 1 mL at
10.sup.7-10.sup.8 beads/mL). By using the bead surface rather than
the chip surface as the sensor, a single microfluidic device may be
re-used indefinitely and may be imaged using a standard inverted
fluorescence microscope. However, a person of skill in the art
could also apply the basic methods described herein to a
microfluidic system having antigen and/or antibodies bound to the
surface of a chip. It is further shown herein that an assay
applying a method described herein may be used to perform
simultaneous kinetic measurements of multiple antibody-antigen
interactions using spatial and optical multiplexing. By comparison,
characterization of each antibody-antigen interaction using SPR
spectroscopy requires specialized instrumentation and a unique flow
cell on comparatively expensive sensor chips. The low detection
limit of the microfluidic bead assay coupled with small volume
compartmentalization was exploited in order to measure the antigen
binding kinetics of antibodies secreted by a single ASC. It is
contemplated that the microfluidic bead assay described herein
could be used for measuring antibody-antigen binding kinetics from
rare blood samples, for screening scarce antibodies produced by
primary plasma cells from immunized animals, as well as for
selecting clones for recombinant protein production. Additionally,
it is contemplated that in addition to its utility for measuring
antibody-antigen binding kinetics, the microfluidic bead-based
assay described herein can be used for measuring other
protein-protein and biomolecular interactions with a wide range of
binding affinities, such as protein-carbohydrate binding,
protein-DNA (i.e., transcription factor binding) and protein-RNA
interactions. It is also contemplated that upon identifying an ASC
that secretes antibodies which are optimal for a particular
purpose, the ASC in question can be cloned by reverse-transcriptase
PCR and standardized cloning techniques.
EXPERIMENTAL METHODS
Microfluidic Device Fabrication and Control
[0084] All microfluidic devices were fabricated using multilayer
soft lithography (see, for e.g., Unger, M. A. et al. (2000),
Science, 288: 113-116 and Thorsen, T. et al. (2002), Science 298:
580-584. Devices were composed of two layers of
poly(dimethylsiloxane) (PDMS) elastomer (GE RTV 615) bonded to No
1.5 glass coverslips (Ted Pella, Inc.). The devices were designed
in AutoCAD software (Autodesk) and printed on high resolution
(20,000 dpi) transparency masks (CAD/Art Services). Master molds
were fabricated in photoresist on silicon wafers (Silicon Quest) by
standard optical lithography. The control master molds were
fabricated out of 20-25 .mu.m high SU-8 2025 photoresist
(Microchem). The flow master molds were fabricated with 12 .mu.m
rounded SPR220-7.0 photoresist channels (Rohm and Haas) and 6 .mu.m
SU-8 5 photoresist (Microchem) channels with rectangular
cross-section. Microfluidic valves were actuated at 30 psi pressure
which was controlled using off-chip solenoid valves (Fluidigm Corp)
controlled using LabView 7.1 software and a NI-6533 DAQ card
(National Instruments). Compressed air (3-4 psi) was used to push
reagent solutions into the device.
Reagent Preparation
[0085] Protein A-coated 5.5 .mu.m diameter polystyrene beads (Bangs
Labs) were incubated with 1 mg/mL solutions of Rabbit anti-mouse
polyclonal antibodies (pAbs) (Jackson Immunoresearch). All antibody
and antigen solutions were prepared in PBS/BSA/Tween solution
consisting of 1.times.PBS, pH 7.4 (Gibco) with 10 mg/mL BSA (Sigma)
and 0.5% Polyoxyethylene (20) sorbitan monolaurate (similar to
Tween-20, EMD Biosciences). Lysozyme from chicken egg white (HEL)
was purchased from Sigma, and the D1.3 and HyHEL-5 mouse monoclonal
antibodies to lysozyme were generously provided by Dr. Richard
Willson (University of Houston). The anti-GFP mouse monoclonal
antibody (LGB-1) was purchased from Abcam. Fluorescent protein
conjugates were prepared using Dylight488 and Dylight633 NHS esters
(Pierce) and were purified using Slide-A-Lyzer dialysis cassettes
(Pierce). The concentration of fluorescent conjugates was measured
by spectrophotometry (Nanodrop). In order to minimize protein
denaturation, fluorescent HEL conjugates were labeled at
dye-to-protein (D/P) ratios of less than 1, whereas the
D1.3-Dylight488 conjugate was prepared at a D/P ratio of
.about.5.
Microscopy
[0086] The microfluidic devices were imaged on a Nikon TE200
Eclipse inverted epifluorescence microscope equipped with green
(470/40 nm excitation, 535/30 nm emission) and red (600/60 nm
excitation, 655 nm long-pass emission) fluorescence filter cubes
(Chroma Technology). Fluorescence images were taken using a 16-bit,
cooled CCD camera (Apogee Alta U2000) and a 100.times.oil immersion
objective (N.A. 1.30, Nikon Plan Fluor). The sensitivity of the
fluorescence measurements was tuned by binning pixels on the CCD
detection camera and modulating the fluorescence exposure times (20
ms-1 s) with a computer-controlled mechanical shutter (Ludl).
Cell Culture
[0087] Mouse D1.3 hybridoma cells were cultured in RPMI 1640 media
(Gibco) with 10% FCS. Prior to loading into microfluidic devices,
cells were washed by centrifugation at 1500 rpm and re-suspended in
fresh media in order to remove antibodies secreted in the cell
media.
Microfluidic Bead-Based Fluorescence Assay
[0088] A microfluidic device was designed and fabricated to perform
bead-based fluorescence measurements of antigen-antibody binding
kinetics (see FIGS. 1A and B herein). The device consists of six
fluidic inlets, each used for loading a distinct reagent and
controlled with an independent control valve, which join into a
common fluidic outlet. The fluidic outlet can be partitioned into
discrete .about.200 pL chambers by actuating a set of microfluidic
"sieve" valves which, when actuated, act as filters to immobilize
large particles (>1 micron) while still allowing fluid exchange.
See, for e.g., Marcus, J. S. et al. (2006) Analytical Chemistry 78:
3084-3089.
[0089] At the start of the experiment, the fluidic outlet was
flushed with a PBS/BSA/Tween solution from the top and bottom
fluidic inlets in order to pre-coat channel walls and reduce
nonspecific binding. Next, 5.5 .mu.m diameter Protein A beads
coated with Rabbit anti-mouse pAb were loaded through the device to
the fluidic outlet. The microfluidic sieve valves were then
actuated and the fluidic outlet was again washed with PBS/BSA/Tween
solution to immobilize the beads against the traps and wash out any
free rabbit pAb in solution. The beads were then washed with the
mouse antibody selected for kinetic characterization. Again, free
mouse antibody was washed out of the fluidic outlet using
PBS/BSA/Tween. Finally, the beads were washed with
fluorescently-labeled antigen and fluorescently imaged at defined
time intervals to measure the rate of antibody-antigen association
(see FIG. 1C herein). When chemical equilibrium between the
antibody and antigen was reached, as detected by a plateau in bead
fluorescence, the beads were flushed with PBS buffer and imaged to
measure the rate of antibody-antigen dissociation. The process was
repeated with varying concentrations of fluorescently-labeled
antigen, each loaded onto the microfluidic device from a separate
fluidic inlet.
[0090] A second version of the microfluidic bead assay was
implemented to indirectly measure dissociation kinetics between
antibodies and unlabeled antigen molecules by displacement with
fluorescently labeled antigen (see FIG. 1D herein). In this assay,
after the antibody of interest was captured on Rabbit anti-mouse
pAb-coated Protein A beads, beads were washed with unlabeled
antigen at high concentration (>1 M) to saturate all antibody
binding sites. Beads were then washed with fluorescently labeled
antigen while imaging at defined time intervals. Dissociation of
the unlabeled antigen was then inferred by accumulated fluorescence
on the beads.
[0091] In order to measure the antigen binding kinetics from
antibodies secreted from single cells, Protein A beads coated with
Rabbit anti-mouse pAb were first immobilized in the fluidic outlet
channel using the microfluidic sieve valves. A solution of
RPMI-1640 media containing 10.sup.5 hybridoma cells/mL was then
loaded into the device from a separate fluidic inlet and the
control valve was momentarily opened to allow for a single
hybridoma cell to be brought in close proximity with beads
immobilized in the first sieve trap in the fluidic outlet channel.
The hybridoma cell was then allowed to incubate next to the beads
for 1 hour, and subsequently washed with PBS/BSA/Tween buffer to
wash out any free antibody in solution and halt antibody secretion
from the cell. Kinetic measurements of antigen binding were then
performed in the same manner as with purified antibodies.
[0092] FIG. 2 shows a schematic diagram of a microfluidic device
operable for detecting antibody secreted from antibody secreting
cells. The steps utilized are, for example, as follows: (1) flush
microfluidic channels with cell culture media; (2) load channels
with antibody secreting cells (top) and capture beads (bottom); (3)
incubate cells with beads to capture secreted antibody; (4) trap
cells and beads with sieve valves and flush out unbound antibody by
blowing buffer over cell-bead mixture; (5) flow
fluorescently-labeled antigen over trapped cells and beads; and (6)
flush out unbound antigen by blowing buffer over trapped cells and
beads and image fluorescent beads.
Data Analysis.
[0093] Fluorescent images were analyzed using MaximDL 4 imaging
software. Fluorescent intensities were measured by selecting line
profiles through the beads and recording the maximum intensity at
the bead surface. During protein binding experiments, line profiles
were constructed through the same beads at each measurement time
point in order to avoid any systemic variations caused by
differences in bead-to-bead binding capacity, variation in position
in the flow channel and non-uniform illumination over the field of
view. The measured fluorescence bead intensities were assumed to be
proportional to the concentration of antibody-antigen complex
([AbAg]) and were fit to the following first-order, mass action and
Langmuir isotherm equations using nonlinear least squares
minimization:
F ( t ) = ( F m ax - F 0 ) [ Ag ] o [ Ag ] o + K d ( 1 - e - ( k on
[ Ag ] 0 + k off ) t ) + F 0 ( equation 1 ) F ( t ) = ( F ma x - F
0 ) [ Ag ] o [ Ag ] o + K d e - k off t + F 0 ( equation 2 ) F ( t
) = ( F ma x - F 0 ) [ Ag ] o [ Ag ] o + K d + F 0 ( equation 3 )
##EQU00001##
where F(t) represents the measured bead fluorescence at time t,
F.sub.0 and F.sub.max represent the background and maximum bead
fluorescence, respectively, [Ag].sub.0 represents the solution
concentration of antigen (in M), and, k.sub.f and k.sub.r represent
the intrinsic association and dissociation rate constants, in units
of M.sup.-1s.sup.-1 and s.sup.-1, respectively.
Examples
[0094] The following examples describe embodiments of the invention
detailed herein.
Example 1. Measurement of Antibody-Antigen Binding Kinetics on
Beads
[0095] The binding kinetics of the D1.3 mouse monoclonal antibody
(mAb) to fluorescently-labeled hen egg lysozyme (HEL) was measured
using the methodologies and techniques described herein. See: FIG.
3A and Table 1 herein.
TABLE-US-00001 TABLE 1 Antibody-antigen binding kinetics measured
using the microfluidic fluorescence bead assay. Antibody/Antigen
interaction k.sub.on (M.sup.-1s.sup.-1) k.sub.off (s.sup.-1)
K.sub.d D1.3 mAb/HEL-Dylight488 1.87 .+-. 0.48 .times. 10.sup.6
2.10 .+-. 0.25 .times. 10.sup.-3 1.20 .+-. 0.42 nM D1.3
mAb/HEL-Dylight633 1.27 .+-. 0.22 .times. 10.sup.6 2.15 .+-. 0.23
.times. 10.sup.-3 1.75 .+-. 0.46 nM HyHEL-5 mAb/HEL-Dylight633 5.75
.+-. 0.71 .times. 10.sup.6 1.69 .+-. 0.30 .times. 10.sup.-4 30.0
.+-. 7.4 pM LGB-1 mAb/EGFP 5.00 .+-. 0.72 .times. 10.sup.4 5.15
.+-. 0.89 .times. 10.sup.-3 106 .+-. 28 nM
[0096] The measured association and dissociation rate constants for
the D1.3/HEL interaction were 1.87.+-.0.48.times.10.sup.6
M.sup.-1s.sup.-1 and 2.10.+-.0.25.times.10.sup.-3 s.sup.-1,
respectively, and were consistent with values of
1.0-2.0.times.10.sup.6 M.sup.-1s.sup.-1 and
1.15-3.04.times.10.sup.-3 s.sup.-1 previously measured using
surface plasmon resonance (SPR) spectroscopy, stopped-flow
fluorescence quenching, and competitive ELISA. See, for e.g.,
Batista, F. D. and Neuberger, M. S. (1998), Immunity 8: 751-759 and
Ito, W. et al. (1995), Journal of Molecular Biology 248: 729-732. A
ten-fold smaller association rate constant previously reported for
the D1.3/HEL interaction (1.67.times.10.sup.5 M.sup.-1s.sup.-1) can
likely be attributed to differences between the full D1.3 mAb used
in our microfluidic bead-based measurements and the recombinant
single-chain antibody fragment used by Bedouelle and coworkers
(England et al. (1999) J. Immunol. 162: 2129-2136).
[0097] Additionally, indirect, label-free measurements of the D1.3
mAb/HEL dissociation rate constant using a variation of our
microfluidic bead assay were performed using the methodologies and
techniques described herein. See: FIGS. 1D and 3D herein. In this
assay, D1.3 mAbs immobilized on beads were first saturated with
unlabeled HEL and subsequently washed with fluorescently-labeled
HEL. Measurements of the accumulated bead fluorescence faithfully
reflected the D1.3/HEL dissociation kinetics provided the labeled
HEL was at a sufficiently high concentration to ensure that
dissociation was rate-limiting (i.e. k.sub.on[Ag]>k.sub.off, or,
equivalently, [Ag]>K.sub.d). Using this method, the dissociation
rate constant of D1.3 and unlabeled HEL was measured to be
1.45.+-.0.30.times.10.sup.-3 s.sup.-1, in close agreement with
direct dissociation measurements between D1.3 and
fluorescently-labeled HEL. See Table 1 herein.
[0098] The microfluidic bead assay was used to measure the binding
kinetics of HEL and HyHEL-5, a distinct mouse mAb with
significantly stronger binding affinity to HEL than D1.3. In
comparison to the D1.3 mAb, HyHEL-5 bound HEL with a nearly
four-fold larger association rate constant
(5.75.+-.0.71.times.10.sup.6 M.sup.-1s.sup.-1) and ten-fold smaller
dissociation rate constant (1.69.+-.0.30.times.10.sup.-4 s.sup.-1).
See: FIG. 3B herein. Thus, HyHEL-5 bound HEL with a .about.40-fold
smaller equilibrium dissociation constant than D1.3 (30 pM vs. 1.2
nM). See: Table 1 herein. Compared with the microfluidic bead
assay, previous measurements of the HyHEL-5/HEL interaction using
solution-phase fluorescence anisotropy resulted in a similar
dissociation rate constant (2.2.times.10.sup.-4 s.sup.-1), but a
three- to five-fold larger association rate constant
(1.5-3.3.times.10.sup.7 M.sup.-1s.sup.-1). See, for e.g., Xavier,
K. A. and Willson, R. C. (1998) Biophys. J. 74: 2036-2045. Since
HyHEL-5 mAb binds HEL with near diffusion-limited association
kinetics, immobilization of the mAb in the microfluidic bead assay
could potentially result in slower association kinetics when
compared with solution-phase fluorescence anisotropy measurements.
However, since the diffusion constant of HEL is approximately three
times larger than that of the mAb, immobilization of the HyHEL-5
mAb would reduce the effective diffusion coefficient
(D.apprxeq.D.sub.mAb+D.sub.HEL) and, hence, the apparent
association rate constant by at most 25%. See, for e.g., Tyn, M. T.
and Gusek, T. W. (1990), Biotechnology and Bioengineering 35:
327-338 and He, L. and Niemeyer, B. (2003), Biotechnol. Prog. 2003,
19: 544-548. Therefore, the difference in measured and reported
association rate constants is likely a result of different buffer
solutions, as the HyHEL-5 and HEL binding interaction is known to
be very sensitive to solution pH and buffer salt concentration.
See, for e.g., Xavier, K. A. and Willson, R. C. (1998) Biophys. J.
74: 2036-2045 and Dlugosz et al. (2009), The Journal of Physical
Chemistry 113: 15662-15669.
[0099] The binding kinetics of a commercially available mouse
monoclonal antibody (LGB-1, Abcam) to enhanced green fluorescent
protein (eGFP) were also measured using the methodologies and
techniques described herein. See: FIG. 3 herein. This binding
interaction was chosen to demonstrate that the bead-based assay can
be used to measure binding kinetics of a previously uncharacterized
antibody without optimizing the bead immobilization chemistry. In
this instance, native eGFP fluorescence was measured without an
exogenous fluorescent label. The measured association and
dissociation rate constants for the LGB-1/eGFP interaction were
5.00.+-.0.72.times.10.sup.4 M.sup.-1s.sup.-1 and
5.15.+-.0.89.times.10.sup.-3 s.sup.-1, respectively. See: Table 1
herein.
[0100] Collectively, the measured binding kinetics of the
anti-lysozyme and anti-eGFP mAbs span nearly four orders of
magnitude in equilibrium dissociation constants (30 pM-100 nM),
with association rate constants varying from
5.times.10.sup.4-10.sup.6 M.sup.-1s.sup.-1 and dissociation rate
constants ranging from 10.sup.-3-10.sup.-4 s.sup.-1. See: Table 1
herein. In principle, the microfluidic bead-based assay can be used
to characterize stronger antibody-antigen interactions than the
HyHEL-5/HEL interaction; however, binding interactions with
dissociation rate constants lower than 10.sup.-4s.sup.-1 require
measurements to be taken over several days or weeks. On the other
hand, the bead-based assay can be readily used to measure binding
interactions weaker than the LGB-1/eGFP interaction. Using this
assay, the practical upper limit in measurable dissociation rate
constants is approximately 10.sup.-1s.sup.-1, as a result of the
time required to exchange solutions in the microfluidic device.
Thus, the microfluidic bead-based assay should enable
characterization of antibody-antigen interactions that span greater
than six orders of magnitude in equilibrium binding affinity.
Example 2. Simultaneous Measurement of Multiple Antibody-Antigen
Binding Kinetics Using Optical and Spatial Multiplexing
[0101] The binding kinetics of multiple antibody-antigen
interactions were measured simultaneously using both optical and
spatial multiplexing of the bead-based assay using the
methodologies and techniques described herein. Each antibody was
immobilized on a distinct population of beads and, subsequently,
beads from each population were sequentially trapped using sieve
valves on the microfluidic device. Since beads trapped by the sieve
valves remain immobilized throughout the duration of each
experiment, the spatial address of beads was tracked in order to
identify each antibody. Subsequently, the trapped beads were washed
with a mixture of antigens, each labeled with a spectrally distinct
fluorophore. The beads were then imaged with different fluorescence
filter sets designed to coincide with each fluorescent antigen. In
this manner, the binding kinetics of 3 different monoclonal
antibodies (D1.3, HyHEL-5 and LGB-1) to two different fluorescent
antigens (HEL-Dylight488 and eGFP) were simultaneously measured.
See: FIG. 4 herein. By employing this strategy, it was possible to
spectrally distinguish which beads were coated with anti-lysozyme
mAbs or anti-eGFP mAbs, whereas the two anti-lysozyme mAbs (D1.3
and HyHEL-5) were discriminated based on their unique binding
kinetics for HEL. In addition, the fluorescence intensities of
HyHEL-5 coated beads were significantly higher than the D1.3 coated
beads, consistent with the fact that HyHEL-5 binds HEL with a
significantly lower equilibrium dissociation constant than D1.3.
See: FIG. 4 herein. This technique can be extended to measure any
combination of m.times.n antibody-antigen interactions in which m
antibodies are immobilized on different beads and exposed to a
solution of n antigens, each with a spectrally-resolvable
fluorescent label. In practice, several hundred antibody-antigen
interactions could be measured simultaneously by imaging up to 100
beads in a single field of view with five to six spectrally
distinct fluorophores. Multiplexed bead measurements could be used
for simultaneously analyzing the binding kinetics and binding
specificities of a panel of mAbs to multiple different antigens in
serum and other complex mixtures.
Example 3. Microfluidic Bead-Based Fluorescence Measurements
Reflect Intrinsic Antibody-Antigen Binding Kinetics
[0102] A series of experiments were performed using the
methodologies and techniques described herein to verify that
bead-based fluorescence measurements reflected intrinsic
antibody-antigen binding kinetics, and were unaffected by artifacts
arising from fluorescent labeling of the antigen, antibody
immobilization, diffusion limitation or mass transport effects.
Fluorescent labeling of HEL did not alter the intrinsic D1.3/HEL
binding kinetics, as indicated by the agreement between
microfluidic bead-based measurements using fluorescently labeled
HEL and previously reported measurements using SPR spectroscopy
with unlabeled HEL. See, for e.g., Batista, F. D. and Neuberger, M.
S. (1998), Immunity 8: 751-759 and Ito, W. et al. (1995), Journal
of Molecular Biology 248: 729-732. Moreover, no differences were
observed in bead-based kinetic measurements of the D1.3 mAb binding
to HEL labeled with two different fluorophores, Dylight488 and
Dylight633 (Pierce). See: Table 1 herein. It was ensured that
photobleaching of fluorophores did not affect the measured binding
kinetics by measuring the photobleaching rates of the of the
fluorescent dyes used in this study (Dylight488, Dylight633, and
eGFP) and selecting fluorescence exposure times of less than 100
ms, such that each measurement resulted in less than 5% reduction
in bead fluorescence. See: FIG. 7A herein. Indeed, measured binding
kinetics were consistent over a large range of fluorescence
exposure times (ms), whereas exposure times of greater than is
resulted in substantial photobleaching and an artificial increase
in measured association and dissociation binding kinetics when
compared to intrinsic kinetics. See FIG. 7B herein.
[0103] To examine the effect of different antibody bead
immobilization chemistries, we verified that measured association
and dissociation rate constants for the D1.3/HEL interaction were
the same when captured on silica or polystyrene beads coated with
either rabbit or goat anti-mouse polyclonal antibody. See: FIG. 8
herein. It was further verified that multivalent binding between
the rabbit anti-mouse pAbs and fluorescently-labeled D1.3 mAb
resulted in no detectable dissociation over the course of 3 days,
which would otherwise artificially accelerate the measured
antibody-antigen binding kinetics. See: FIG. 9 herein. The nearly
irreversible bond between rabbit pAb and the mouse mAbs was
critical to successful antibody-antigen binding kinetic
measurements as attempts to measure D1.3/HEL binding kinetics using
Protein A beads without Rabbit anti-mouse pAbs were unsuccessful
due to rapid dissociation (and low affinity) of protein A/mouse mAb
complexes.
[0104] Several experiments were also conducted to verify that
diffusion limitation and mass transport did not affect bead-based
measurements of antibody-antigen binding kinetics. In the
diffusion-limited regime, antibodies adjacent on the bead surface
would compete for fluorescent antigen, thus reducing the apparent
association rate constant. Similarly, the apparent rate of
antibody-antigen dissociation would be reduced due to antigen
rebinding to adjacent antibodies. See, for e.g., Berg, H. C. and
Purcell, E. M. (1977), Biophys. J. 20: 193-219 and Lauffenburger,
D. A. and Linderman, J. (1965) Receptors: Models for Binding,
Trafficking, and Signaling; Oxford University Press. Nearly
identical association and dissociation kinetics for the D1.3-HEL
interaction was measured by varying the amount of bead-immobilized
D1.3 mAb over two orders of magnitude. See FIG. 10B herein.
Dissociation kinetics of the D1.3 antibody and fluorescently
labeled HEL were also similar both in the presence and absence of a
high concentration (.about.2 mg/mL) of competitive unlabeled HEL
antigen. See: FIG. 10 herein. Thus, there was no observable
competition between antibodies adjacent to one another on the beads
and, hence, no diffusion limitation. It was also confirmed that the
association and dissociation rate constants of the D1.3-HEL
interaction remained constant over a range of flow rates from 3-15
.mu.L/hr, suggesting no effect of mass transport on the measured
kinetics. See: FIG. 11 herein.
Example 4. Bead-Based Kinetic Measurements Exhibit Low Detection
Limits and Minimal Sample Consumption
[0105] To quantify the detection limit and minimal sample
consumption required for microfluidic bead-based measurements of
antibody-antigen binding kinetics, antibody-antigen binding
kinetics were measured using varying amounts of bead-immobilized
mAb along with the methodologies and techniques described herein.
The association rate constant of fluorescently-labeled D1.3 mAb
binding to Rabbit anti-mouse pAb coated Protein A beads was
measured. See: FIG. 5A herein. Using the measured kinetic on-rate
constant for this interaction (k.sub.on=1.10.+-.0.11.times.10.sup.6
M.sup.-1s.sup.-1) and modulating the loading time of D1.3 mAb, the
amount of bead-immobilized D1.3 mAb was varied over two orders of
magnitude. Then, the antibody-antigen binding kinetics with as
little as 1% of the bead surface covered with D1.3 mAb was
successfully measured. See: FIG. 5B herein. Using the
manufacturer's specifications as well as steric considerations, a
single 5.5 micron bead can bind 4.times.10.sup.6 antibody molecules
(.about.6.6 amol); therefore, it was estimated that the detection
limit of our microfluidic fluorescence bead assay is to be
.about.4.times.10.sup.4 antibodies or .about.66 zeptomoles. See:
FIG. 5C herein. In contrast, SPR spectroscopy requires at least 200
pg (.about.109 molecules) of immobilized antibody in order to
generate a detectable refractive index change. See, for e.g.,
Biacore Life Sciences--Biacore 3000 System Information. Website:
http://www.biacore.com/lifesciences/products/systems_overview/3000/system-
_information/index.html. Additionally, D1.3/HEL binding kinetics
were successfully measured by loading less than 2 million D1.3 mAb
molecules (.about.3 attomoles) into the microfluidic device. In
theory, the minimum sample consumption of the microfluidic bead
assay could be reduced even further by reducing losses associated
with channel dead volumes and optimizing the capture efficiency of
antibodies on beads, as well as using microfluidic pumps to achieve
flow rates less than 1 .mu.L/hr. Thus, when compared with
alternative techniques and SPR spectroscopy, our microfluidic
bead-based assay can measure antigen-antibody binding kinetics with
a reduction in both detection limit and sample consumption by four
orders of magnitude.
Example 5. Measurement of Binding Kinetics of Antigen and Antibody
Secreted from Single Cells
[0106] Based on the low detection limit of the bead-based assay and
in an effort to measure the antigen binding kinetics of antibodies
secreted from single antibody secreting cells, single D1.3
hybridoma cells were loaded adjacent to Rabbit anti-Mouse pAb
coated Protein A beads captured in the microfluidic device, and
then co-incubated the cells and beads for 1 hour at room
temperature. See: FIG. 6 herein. Subsequently, antibody-antigen
binding kinetics were measured by recording the fluorescence of a
single bead washed with buffer and successively higher
concentrations of fluorescent antigen, in a manner analogous to the
single-cycle kinetics technique used with SPR spectroscopy. See,
for e.g., Biacore Life Sciences--Single-Cycle Kinetics. Website:
http://www.biacore.com/lifesciences/technology/introduction/data_interact-
ion/SCK/index.html and Abdiche et al. (2008) Analytical
Biochemistry 377: 209-217. Using the methodologies and experimental
techniques described herein, the association and dissociation rate
constants for the D1.3/HEL interaction were successfully measured
using antibodies secreted by a single D1.3 hybridoma cell, which
were consistent with measurements on purified antibodies. See: FIG.
6 and Table 1 herein.
[0107] Antibody-secreting cells are known to secrete thousands of
antibodies per second at 37.degree. C., and would, therefore,
secrete enough antibodies in approximately one hour to saturate the
surface of a single 5.5 .mu.m bead with maximum binding capacity of
.about.4.times.10.sup.6 antibody molecules. See, for e.g., Niels
Jerne (1984) The Generative Grammar of the Immune System and
McKinney et al. (1995) Journal of Biotechnology 40: 31-48. While it
is reasonable to suspect that the hybridoma cells secrete
antibodies at a reduced rate when incubated at room temperature;
nonetheless, single D1.3 hybridoma cells secreted sufficient
antibody within 1 hour at room temperature for complete kinetic
characterization. However, based on the incubation time and
detection limit of the assay (.about.4.times.10.sup.4 antibodies),
it can be inferred that single hybridoma cells secreted greater
than 10 antibodies/second when incubated at room temperature in the
microfluidic device.
[0108] Examples 1-5 show that the methods described herein are
suitable for measuring antibody-antigen kinetics in a microfluidic
environment from a single cell.
Example 6. Dual Function Beads
[0109] An overview of a scheme for preparing dual-capture (i.e.,
dual function) beads using carbodiimide chemistry is shown in FIG.
14. A representative experiment utilizing dual function beads is
shown in FIG. 15. Briefly, polystyrene COOH beads (Bangs Labs) were
conjugated with Rabbit anti-mouse pAb (Jackson ImmunoResearch) and
amine-functionalized oligo(dT).sub.25 (Genelink) using carbodiimide
chemistry. In FIG. 14(A), a brightfield image of dual-function
beads trapped using a microfluidic sieve valve is shown. In FIG.
14(B), a fluorescence image of synthetic single-stranded DNA
molecules captured on dual-function beads is shown. Synthetic DNA
molecules are labeled with Cy5 fluorophore for visualization and
also contain a poly(A) tail that binds to the oligo(dT) on the bead
surface. In FIG. 14(C), a fluorescence image of mouse D1.3
monoclonal antibody (mAb) captured on dual-function beads. D1.3
mAbs are labeled with Dylight488 fluorophore for visualization and
bind to the Rabbit anti-Mouse pAb on the bead surface.
[0110] It will be understood that while carboxylic acid (COOH)
beads are disclosed herein, other beads, which make use of
alternate chemical interactions, could also be used, See: for e.g.,
G. T. Hermanson (2008), Bioconjugate Techniques, 2nd Edition,
Published by Academic Press, Inc. For example, an alternate scheme
for preparing the beads would be to use streptavidin coated beads
and to mix these beads with biotinylized rabbit anti-mouse pAbs and
biotinylated oligo(dT).
Example 7. Multiplex RT-PCR of the Antibody Heavy and Light Chain
Genes
[0111] Results from a multiplex RT-PCR of the antibody heavy and
light chain genes indicated that a gene product coinciding with the
proper molecular size was obtained. Briefly, D1.3 hybridoma cells
were lysed using a nonionic detergent (1% NP-40 in 1.times.PBS) and
the lysate was then mixed with rabbit anti-mouse pAb,
oligo(dT)-conjugated dual-capture beads for mRNA capture.
Generally, a gentle lysis buffer is preferred for cell lysis and
can include, in addition to the foregoing: 0.5% NP-40 in
1.times.PBS or DI water or 0.5% Tween-20 in 1.times.PBS or DI
water. Generally, it is preferable and within the knowledge of
those persons skilled in the art to use lysis buffers that can
sufficiently lyse the outer membrane of the cell in question, while
keeping the nucleus intact. RT-PCR was performed using degenerate
primers for both heavy and light chain genes and resulted in bands
of the expected size for antibody heaby and light chains (date not
shown). The results suggest that dual purpose RNA and antibody
beads are capable of capturing RNA suitable for amplification. For
comparison, RT-PCR of antibody genes was performed using
commercially available oligo(dT) beads and dual-capture beads. The
methodology utilized herein is generally as follows: [0112] 1)
Capture oligo(dT) beads in microfluidic chambers using sieve
valves. [0113] 2) Load cells in microfluidic chambers. [0114] 3)
Load antibody-capture beads in chambers. [0115] 4) Incubate cells
and beads. [0116] 5) Measure antibody-antigen binding kinetics.
[0117] 6) Lyse cells using either a) 1% NP-40 in 1.times.PBS, or b)
alkaline lysis solution (100 mM Tris-HCl, pH 7.5, 500 mM LiCl, 10
mM EDTA, pH 8.0 1% LiDS, 5 mM dithiothreitol (DTT)). During lysis,
the cell lysate is flushed over the stack of trapped oligo(dT)
beads. The oligo(dT) beads and alkaline lysis solution are from the
Dynabead mRNA direct kit developed by Invitrogen, but alternatives
to these reagents exist. [0118] 7) Wash beads with 1.times.PBS to
remove lysis solution. [0119] 8) Open sieve valves. [0120] 9) Open
microfluidic chamber valves and send beads to an output port (one
chamber at a time). [0121] 10) Recover beads from output port using
a pipette. [0122] 11) Pipette beads into 50 microL of one-step
RT-PCR mix. [0123] i) dNTPs; [0124] ii) mixture of RT and DNA
polymerase enzymes; [0125] iii) degenerate primers for both heavy
and light chain genes (PCR reagents from a One-Step RT-PCR kit
developed by Qiagen, but could also be prepared ourselves); [0126]
12) Perform RT and Touchdown PCR using the following protocol:
[0127] i) RT at 50.degree. C. for 30 min; [0128] ii) 95.degree. C.
for 15 min to inactivate RT enzyme and activate DNA polymerase
[0129] iii) First ten cycles of Touchdown PCR: [0130] a) 94C for
30s; [0131] b) 55.degree. C. for 1 min (decrease by IC each cycle,
until 45C); [0132] c) 72.degree. C. for 1 min. [0133] iv) 30 cycles
of PCR [0134] a) 94C for 30s; [0135] b) 45.degree. C. for 1 min;
[0136] c) 72.degree. C. for 1 min. [0137] 13) Visualize RT-PCR
amplicons on 0.5% DNA agarose gel using SYBRsafe fluorescent dye.
[0138] 14) Extract amplicons from gel and purify using standard gel
extraction kit (Qiagen). [0139] 15) Sequence samples.
Example 8. Microfluidic Antibody-Antigen Binding Kinetics Measured
Using Dual Function Beads and Antibodies Secreted by Single
Hybridoma Cells
[0140] Microscope image of D1.3 hybridoma cell adjacent to Rabbit
anti-Mouse pAb, oligo(dT)-conjugated polystyrene beads trapped by a
microfluidic sieve valve. After a 2 hour incubation the beads with
the D1.3 cell, antibody-antigen binding kinetics were measured
using fluorescently labeled HEL-Dylight488 conjugate. These results
are highlighted in FIG. 16 and show that dual purpose beads are
suitable for testing antibody-antigen binding kinetics.
Example 9. Mouse Experiment: Antibody Binding Kinetics and
Whole-Cell Heavy Chain RT-PCR with Beads
[0141] These experiments were designed to detect antibodies from
primary splenocytes harvested from BALB/c immunized mice. The cells
were eluted and whole-cell single-plex RT-PCR was performed of
heavy and light chain antibody genes. Thereafter, binding kinetics
of the antibodies were measured.
[0142] Chip.
[0143] Bead v6.6 chip with .about.3 micron high sieve channels, 2
micron gratings (fabricated: May 29, 2011 with RTV615).
[0144] Reagents.
[0145] The following reagents were used herein: 1.times.PBS for
reagent flush; FACS-sorted CD138+ primary splenocytes in
RPMI-10-2-ME media; 4.9 micron Rabbit anti-Mouse Protein A beads; 5
microL of stock bead solution resuspended in 100 .mu.L of
RPMI-10-2-ME media; and 214 ng/mL HEL488 in 1.times.PBS.
[0146] Experimental Protocol.
[0147] The following experimental protocol was followed: washed
chip with 1.times.PBS; closed sieve valves; spun down primary cells
and decanted .about.400 of 500 .mu.L of media; and re-suspended
cells in remaining media.
[0148] Thereafter, the cells were loaded in all chambers
sequentially with deliberate negative controls included (e.g.,
R1C14 and ROC02); load 4.9 micron Rabbit anti-Mouse Protein A beads
in all chambers sequentially; incubate cells and beads for 1 h 20
min; and wash all chambers with 214 ng/mL HEL488 for 5 min.
Thereafter, chamber intensities were analyzed using Image Analysis.
Positive chambers were determined as follows: ROC04, ROC08, R2C02,
R2C04, R2C07, R2C12, R2C13, R3C06, R4C01, R4C03, R4C05, R5C08,
R5C09, R5C12, R5C14, R6C01, R6C02, R6C09, R6C10, R6C11, and
R7C05.
[0149] RT-PCR mix without primers was prepared from a One-Step
RT-PCR kit (Qiagen). The mix comprised: 10 .mu.L 5.times.RT-PCR
buffer.times.16=160 .mu.L; 21 .mu.L RNase-free water.times.16=336
.mu.L; 2 .mu.L dNTPs.times.16=32 .mu.L; 2 .mu.L Enzyme
mix.times.16=32 .mu.L. The RT-PCR mix without primers was aliquoted
into 8 tubes of 70 .mu.L each (2 reaction volumes, not including
primer volume).
[0150] Prepared primer solutions to be mixed with RT-PCR after cell
elution were as follows: Heavy chain--7.5 .mu.L 8 .mu.M 3' IgH
first primer.times.8=60 .mu.L; and 7.5 .mu.L 8 .mu.M 5' IgH first
primer.times.8=60 .mu.L. Kappa chain--7.5 .mu.L 8 .mu.M 3' IgK
first primer.times.8=60 .mu.L; and 7.5 .mu.L 8 .mu.M 5' IgK first
primer.times.8=60 .mu.L. 15 .mu.L of primer mixes were aliquoted
into each of 8 tubes. The primers used herein were selected based
on what is taught in Table II of Tiller et al. (2009) J. Immunol.
Methods 350: 183-193, which is incorporated herein by reference.
Those persons skilled in the art that variants to the primers
defined in Table II could be used under certain circumstances
including, for example, the primers in Table III in Tiller et al.
(2009).
[0151] Eight (8) of the brightest chambers [ROC04, R2C04, R2C07,
R3C06, R5C12, R5C14, R6C01, R6C10] were eluted. The eluted cell
samples were pipetted directly into 70 .mu.L RT-PCR mix without
primers. The RT-PCR/cell mix was split into two (2) equal parts of
35 .mu.L and mixed with kappa and heavy chain primers,
respectively. RT-PCR was performed using a thermal cycler. Briefly,
the "NESTIST5" protocol was used for the kappa chain reactions,
comprising: RT step: 50.degree. C. for 30 min; Hotstart/RT
inactivation: 95.degree. C. for 15 min; and 50 Cycles
(denaturation: 940C for 30s; anneal: 500C for 30s; and extension:
720C for 55s). Then, there was a final extension: 720C for 10 min.
Heavy chain reactions performed using the "NEST1H" protocol,
comprising: RT step: 500C for 30 min; Hotstart/RT inactivation:
950C for 15 min; and 50 Cycles (denaturation: 940C for 30s; anneal:
560C for 30s; extension: 720C for 55s). Then, there was a final
extension: 720C for 10 min.
[0152] Thereafter, kinetics were measured on each of the eluted
chambers. A summary of the kinetics data is shown in FIG. 17.
Representative kinetics sample data is shown in each of FIGS. 18A-E
herein. Dissociation kinetics were measured and then association
kinetics were measured using freshly loaded HEL488. Thereafter, a
second round of single-plex RT-PCR was performed using nested
second round primers. The heavy chain mix comprised of: 10 .mu.L
5.times.RT-PCR buffer.times.8=80 .mu.L; 21 .mu.L RNase-free
water.times.8=168 .mu.L; 2 .mu.L dNTPs.times.8=16 .mu.L; 2 .mu.L
Enzyme mix.times.8=16 .mu.L; 7.5 .mu.L 8 .mu.M 3' IgH second
primer.times.8=45 .mu.L; and 7.5 .mu.L 8 .mu.M 5' IgH second
primer.times.8=45 .mu.L. The kappa chain mix comprised of: 10 .mu.L
5.times.RT-PCR buffer.times.8=80 .mu.L; 21 .mu.L RNase-free
water.times.8=168 .mu.L; 2 .mu.L dNTPs.times.8=16 .mu.L; 2 .mu.L
Enzyme mix.times.8=16 .mu.L; 7.5 .mu.L 8 .mu.M 3' IgK second
primer.times.8=45 .mu.L; and 7.5 .mu.L 8 .mu.M 5' IgK second
primer.times.8=45 .mu.L. Thereafter, 3.5 .mu.L of template from
each of the first round reactions was added and RT-PCR was
performed on a thermal cycler. Kappa chain reactions were performed
using the "NEST2K" protocol. Briefly, there was no RT step;
hotstart/RT inactivation was at 95.degree. C. for 15 min followed
by 50 cycles (denaturation: 94.degree. C. for 30s; anneal:
45.degree. C. for 30s; and extension: 72.degree. C. for 55s). Then,
there was a final extension: 72.degree. C. for 10 min.
[0153] Heavy chain reactions performed using the "NEST2H" protocol.
Briefly, there was no RT step; hotstart/RT inactivation: 95.degree.
C. for 15 min, followed by 50 cycles (denaturation: 94.degree. C.
for 30s; anneal: 60.degree. C. for 30s; and extension: 72.degree.
C. for 55s). Then, there was a final extension: 72.degree. C. for
10 min. The RT-PCR products for both first and second round
reactions were run on a gel. Kappa chain results from the first
round are shown in FIG. 19A. Kappa chain results from the second
round are shown in FIG. 19B. Heavy chain results from the first
round are shown in FIG. 19C. Heavy chain results from the second
round are shown in FIG. 19D. The gel products were sequenced by
standard procedures known to those skilled in the art. Based on the
sequence data generated, variants in antibody sequences were
detectable. As a representative example, mutations in the ROOC04
sample are shown in Table 2 herein.
TABLE-US-00002 TABLE 2 R00C04 (9 non-synonymous mutations) Position
Situation Germline Ab R00C04 Ab (from IMGT) (from IMGT) residue
residue L-36 CDR1-L S N L-92 FR3-L S T H-17 FR1-H A D H-36 CDR1-H S
R H-40 FR2-H H L H-64 CDR2-H N K H-65 CDR2-H T S H-83 FR3-H S I
H-94 FR3-H P L
Example 10. Microfluidic Device
[0154] A microfluidic device has been developed for assaying a
binding interaction between a protein produced by a cell and a
biomolecule. The device has a chamber having an aperture and a
channel for receiving a flowed fluid volume through the chamber via
said aperture. The channel provides for size selection for a
particle within the fluid volume. Alternately, another embodiment
of the microfluidic device has a chamber having an aperture and a
reversible trap. The reversible trap has spaced apart structural
members extending across the chamber. The structural members are
operable to allow a fluid volume to flow through the chamber while
providing size selection for a particle within the fluid volume.
See, for example: FIG. 20 herein.
[0155] Although various embodiments of the invention are disclosed
herein, many adaptations and modifications may be made within the
scope of the invention in accordance with the common general
knowledge of those skilled in this art. Such modifications include
the substitution of known equivalents for any aspect of the
invention in order to achieve the same result in substantially the
same way. Numeric ranges are inclusive of the numbers defining the
range. The word "comprising" is used herein as an open-ended term,
substantially equivalent to the phrase "including, but not limited
to", and the word "comprises" has a corresponding meaning. As used
herein, the singular forms "a", "an" and "the" include plural
referents unless the context clearly dictates otherwise. Thus, for
example, reference to "a thing" includes more than one such thing.
Citation of references herein is not an admission that such
references are prior art to the present invention. The invention
includes all embodiments and variations substantially as
hereinbefore described and with reference to the examples and
drawings. Further, citation of references herein is not an
admission that such references are prior art to the present
invention nor does it constitute any admission as to the contents
or date of these documents.
* * * * *
References