U.S. patent application number 12/857506 was filed with the patent office on 2011-02-24 for screening assays and methods.
Invention is credited to J. Christopher Love, Hidde L. Ploegh, Jehnna Ronan.
Application Number | 20110046008 12/857506 |
Document ID | / |
Family ID | 37889414 |
Filed Date | 2011-02-24 |
United States Patent
Application |
20110046008 |
Kind Code |
A1 |
Love; J. Christopher ; et
al. |
February 24, 2011 |
SCREENING ASSAYS AND METHODS
Abstract
Screening assays and methods of performing such assays are
provided. In certain examples, the assays and methods may be
designed to determine whether or not two or more species can
associate with each other. In some examples, the assays and methods
may be used to determine if a known antigen binds to an unknown
monoclonal antibody.
Inventors: |
Love; J. Christopher;
(Somervile, MA) ; Ploegh; Hidde L.; (Brookline,
MA) ; Ronan; Jehnna; (Chester, NH) |
Correspondence
Address: |
Mintz Levin Cohn Ferris Glovsky and Popeo, P.C.
One Financial Center
Boston
MA
02111
US
|
Family ID: |
37889414 |
Appl. No.: |
12/857506 |
Filed: |
August 16, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11523124 |
Sep 18, 2006 |
7776553 |
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12857506 |
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60717976 |
Sep 16, 2005 |
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Current U.S.
Class: |
506/9 ;
435/283.1; 435/7.1; 506/10; 506/14 |
Current CPC
Class: |
G01N 33/505 20130101;
B01J 2219/0074 20130101; B01L 2300/0819 20130101; B01J 2219/00675
20130101; B01L 3/50853 20130101; G01N 21/6452 20130101; G01N 33/531
20130101; B01J 19/0046 20130101; Y10S 436/809 20130101; B01L
3/50853 20130101; B01L 2300/0819 20130101; B01L 2300/12 20130101;
G01N 21/6452 20130101; Y10T 436/24 20150115; G01N 33/5052 20130101;
G01N 33/543 20130101; B01L 2200/025 20130101; B01J 2219/00585
20130101; B01J 2219/00317 20130101; B01J 2219/00725 20130101; B01J
2219/00722 20130101; G01N 33/6854 20130101; B01L 3/50853 20130101;
G01N 33/577 20130101; B01J 2219/00527 20130101; B01L 2300/0819
20130101; B01J 2219/00734 20130101; B01L 2200/12 20130101; B01L
3/50853 20130101; G01N 33/5052 20130101; G01N 33/577 20130101; G01N
33/54366 20130101; B01L 2300/0819 20130101; G01N 21/6452 20130101;
B82Y 30/00 20130101; B01L 2200/12 20130101; B01L 2200/12 20130101;
B01L 2300/12 20130101; B01L 2200/025 20130101 |
Class at
Publication: |
506/9 ; 435/7.1;
435/283.1; 506/10; 506/14 |
International
Class: |
C40B 30/04 20060101
C40B030/04; G01N 33/53 20060101 G01N033/53; C12M 1/00 20060101
C12M001/00; C40B 30/06 20060101 C40B030/06; C40B 40/02 20060101
C40B040/02 |
Goverment Interests
STATEMENT AS TO FEDERALLY SPONSORED RESEARCH
[0002] This invention was funded in part by the U.S. Government
under grant numbers NAKFI Nano08 awarded by the National Academy of
Sciences and/or grant 5R01AI034893-1 awarded by the National
Institutes of Health. The Government has certain rights in the
invention.
Claims
1.-25. (canceled)
26. A method of screening a secreted product comprising: sealing a
moldable slab to a substrate, said moldable slab comprising at
least one microwell of less than 100 micrometers in diameter, each
of said microwells comprising a single cell that produces a
secreted product in a volume of 10 nanoliters or less of fluid in
said microwell, allowing said cell to secrete said product in said
volume; exposing said volume of said product to at least one
defined ligand, and determining if said secreted product binds to
said ligand, wherein the steps of the method of screening secreted
products are performed in less than one day.
27. The method of claim 26, wherein the method of screening is
performed in less than 6 hours.
28. The method of claim 26, wherein the substrate is pretreated
with the defined ligand.
29. The method of claim 26, wherein the substrate is pretreated
with a secondary antibody.
30. The method of claim 26, wherein the moldable slab comprises a
microwell array, wherein at least one microwell of the microwell
array comprises the cell that produces the secreted product.
31. The method of claim 30, wherein the microwell array is
incubated for less than 24 hours while contacting the
substrate.
32. The method of claim 30, wherein the microwell array is
incubated for less than 5 hours while contacting the substrate.
33. The method of claim 30, wherein the microwell array is
incubated for less than 2 hours while contacting the substrate.
34. The method of claim 23, wherein the secreted product and the
defined ligand associate on the substrate forming a microarray.
35. The method of claim 34, wherein the microarray on the substrate
is removed from the moldable slab.
36. The method of claim 35, wherein cells in the microwells are
maintained in the medium and the microwell array on the moldable
slab contacts a new substrate and forms at least one new
microarray.
37. The method of claim 36, wherein between 5 and 100 new
microarrays are formed.
38. The method of claim 26, wherein the secreted bound to the
defined ligand is detected.
39. The method of claim 26, wherein said cell is a primary
cell.
40. The method of claim 26, wherein said cell is a cancer cell.
41. The method of claim 26, wherein said cell is of a tissue type
selected from the group consisting of heart, brain, liver prostate,
breast, kidney, and colon.
42. The method of claim 26, wherein said cell is an immune
cell.
43. The method of claim 42, wherein said immune cell is a T cell or
a B cell.
44. The method of claim 23, wherein said secreted product comprises
a cytokine, chemokine, antibody, or growth factor.
45. The method of claim 44, wherein said secreted product is
selected from the group consisting of tumor necrosis factor alpha
(TNF-.alpha.), immunoglobulin E (IgE), interferon-.gamma.
(IFN-.gamma.), interleukin-1 (IL-1), IL-2, IL-4, IL-10, and
IL-13.
46. The method of claim 26, wherein said secreted product is a
recombinant polypeptide or a recombinant immunoglobulin.
47. The method of claim 26, wherein said secreted product comprises
a bacteria, yeast, parasite, or virus.
48. The method of claim 26, wherein said volume is 1 picoliter to 1
nanoliter.
49. The method of claim 26, wherein said determining step is
carried out by fluorescence detection, mass spectrometry, surface
plasmon resonance, or a colorimetric assay.
50. The method of claim 26, wherein secreted product-ligand binding
is detected using a fluorescence microscope, fluorimeter, or
camera.
51. The method of claim 26, wherein said secreted product-ligand
binding is detected using an instrument comprising a charge coupled
device, a photomultiplier tube, or a diode array.
52. The method of claim 26, wherein the steps of the method of
screening are performed in less than 12 hours.
53. The method of claim 30, wherein the monoclonal antibody
associates with the substrate to form a microarray.
54. The method of claim 38, wherein the secreted product bound to
the defined ligand is identified using a detectable marker.
55. The method of claim 54, wherein the detectable marker comprises
a fluorescent label, a colorimetric label, or a radiolabel.
56. The method of claim 54, wherein the detectable marker is a
radiolabel.
57. The method of claim 56, wherein the radiolabel comprises
.sup.3H, .sup.14C, .sup.32P, .sup.33P, .sup.35S or .sup.125I.
58. The method of claim 26, wherein the cell is retrieved from the
moldable slab.
59. The method of claim 26, further comprising characterization of
said cell after the capture of said secreted product to determine a
phenotype of said cell.
60. The method of claim 59, wherein said phenotype is determined
using immunofluorescence labeling or genetic sequencing.
Description
RELATED U.S. APPLICATION
[0001] This application claims priority to U.S. Ser. No. 60/717,976
filed Sep. 16, 2005, which is incorporated herein by reference in
its entirety.
FIELD OF THE TECHNOLOGY
[0003] The invention relates to screening assays and methods to
identify secreted products.
BACKGROUND
[0004] Assays exist to identify compounds or molecules of interest
that may be involved in a disease process or other condition or in
treating a disease process or condition. Existing assays have some
drawbacks. One significant drawback is the time required to screen
for many compounds or molecules. Another drawback is that it may
not be possible to recover the compound or molecule post-screening.
There remains a need for better screening assays and methods.
SUMMARY
[0005] The invention provides a printed microarray of unknown
cell-derived products. Each position of the printed array comprises
a deposit, which is less than 100 micrometers in diameter, and
corresponds to a secretion of a single cell. The deposit is a
secreted product such as an antibody, cytokine, chemokine, or
inflammatory mediator or another cellular component, e.g., DNA,
RNA, or a lipid, which is liberated from an intact cell upon lysis
or permeabilization of the cell. Optionally, the printed microarray
comprises a capture ligand, e.g., which binds to a single
immunoglobin isotype or a chemical capture moiety, e.g., a support
derivatized to retain a class of secreted products.
[0006] An engraving plate includes a plurality of wells, each of
the wells is less than 100 micrometers in diameter and comprises a
single cell. Preferably, the number of cells is less than 5 cells.
The engraving plate is a gas-permeable conformable composition. The
plate has an elastic modulus (Young's Modulus) in the range of
200-2000 Kilopascal (kPa). The composition of the plate is
preferably poly(dimethylsiloxane). The wells of the plate contain
at least one cell. That cell is an immune cell, an
antibody-producing cell, a hybridoma cell, a T cell, or other cell
from the blood or a tissue. The function or secretory profile of
the cell or cells is unknown. The cell produces a recombinant
secreted polypeptide, the polypeptide has an amino acid target
sequence for a chemical modification or a recombinant
immunoglobulin chain that has an amino acid sequence of an enzyme
cleavage site.
[0007] This invention also provides a method of screening by
disposing a conformable support comprising a plurality of
uncharacterized secretory cells on a substrate, e.g., a glass
slide, a plastic slide or a bead and exposing a first species
comprising an unknown cell-derived, e.g., secreted, product,
transferred from the conformable support to the substrate, to a
second species to determine if the first species and the second
species associate. The second species is a known target ligand,
e.g., a defined antigen of a pathogenic organism. For example, the
first species is an antibody and the second species is a defined
target antigen for which antibody binding is sought.
[0008] Another method comprises depositing a suspension of cells
onto a moldable slab containing at least one microwell that forms a
microwell array that allows the suspension of cells to settle where
at least one cell settles into the at least one microwell of the
microwell array. The microwell array then contacts a substrate,
which is pretreated with a first species. The microwell array is
then incubated, for about 1, 5, 10, 20, 30, 40, 50 min but less
than 24 hours, and allowing at least one cell to secrete a second
species. After incubating, the first species and the second species
form an association of the substrate, which is where the microarray
is formed. The microarray is then removed from the moldable slab,
which still contains cells in the microwells and is placed in a
reservoir containing a medium. The cells in the microwells can be
maintained in the medium and the microwell array can contact a new
substrate and form more than a new microarray, whereby the
microwell array can "stamp" more than one microarrays, where about
5 to about 100 microarrays are formed. The association is then
detected between the first species and the second species on the
microarray.
[0009] The second species, which is secreted by the cells, is a
monoclonal antibody or a cytokine and the first species is a
secondary antibody, wherein a labeled antigen, or fragment thereof,
can associate with the monoclonal antibody. Or the first species is
an antigen and the second species is a monoclonal antibody, wherein
a labeled secondary antibody can associate with the monoclonal
antibody. The label is a fluorescent label, a colorimetric label or
a radio label. The association on the microarray can be detected
with at least one labeled species. The cell is a bacterial cell or
a hybridoma, which then is retrieved from the moldable slab if an
association occurs between the first species and the second
species. The cell is challenged with an antigen prior to disposal
of the moldable slab on the substrate. The second species also
comprises a catalyst, which is an enzyme and the first species is a
potential enzyme substrate or a potential enzyme substrate
analog.
[0010] The moldable slab is fabricated by soft lithography and
replica molding and is of a biocompatible material, which is not
toxic and gas permeable. The moldable slab, made of
poly(dimethylsiloxane), can compress against the substrate to form
a tight, but reversible seal with the substrate. The microwell
array comprises a block of wells where a well has a diameter of
about 50 .mu.m and a depth of about 50 .mu.m and the wells are
separated by about 50 .mu.m or a well has a diameter of about 100
.mu.m and a depth of about 100 .mu.m and the wells are separated by
about 100 .mu.m. The wells are sized to retain about 1 nanoliter or
less of fluid.
[0011] The invention provides a method of screening a monoclonal
antibody by contacting a moldable slab with a substrate, which has
at least one secondary antibody. The moldable slab contains at
least one microwell and at least one hybridoma that secretes a
monoclonal antibody in the at least one microwell. The monoclonal
antibody is then exposed to at least one antigen to determine if
the monoclonal antibody can bind to the antigen. The method is
performed in less than about one day or about 6 hours.
[0012] A method of screening a monoclonal antibody by contacting a
moldable slab to a substrate, where the moldable slab has at least
one microwell and at least one hybridoma that secretes a monoclonal
antibody in the microwell. The substrate has at least one antigen
or at least one secondary antibody on a surface of the substrate.
The microarray formed is then used to detect if the monoclonal
antibody can bind to the at least one antigen or the at least one
antibody on the surface of the substrate. The method of screening
the monoclonal antibody is performed in less than about one day or
about 6 hours.
[0013] A kit is assembled that comprises a substrate, a moldable
slab configured to receive the substrate and to provide a fluid
tight seal between the moldable slab and the substrate, and
instructions for using the conformable support and the substrate to
identify species that may associate. The moldable slab or the
substrate or both of the kit comprises one or more materials
selected from the group consisting of glass, plastic, polystyrene,
polycarbonate, poly(dimethylsiloxane), nitrocellulose,
poly(vinylidene fluoride), or a metal. The metal is one or more of
gold, palladium, platinum, silver, steel or alloys or mixtures
thereof. The substrate is a glass slide, a plastic slide or a bead
and the moldable slabs contains a microwell array.
[0014] A kit comprising a substrate, a moldable slab having a
plurality of microwells and configured to receive the substrate and
to provide a fluid tight seal between the moldable slab and the
substrate, and instructions for using the moldable slab and the
substrate to identify species that may associate.
[0015] A test apparatus comprising a moldable slab comprising at
least one microwell that forms a microwell array that contacts a
substrate in a manner to provide a fluid tight seal between the
moldable slab and the substrate. The apparatus puts one species,
generally a cell, in at least one well of the microwell array and
the microwells of the moldable slab are sized and arranged to
retain about one nanoliter or less of fluid volume. The analytical
methods described herein offer numerous advantages over prior
methods, including time-saving and cost effectiveness.
[0016] All publications, patent applications, patents, and other
references mentioned herein are incorporated by reference in their
entirety. In addition, the materials, methods, and examples are
illustrative only and not intended to be limiting.
BRIEF DESCRIPTION OF THE FIGURES
[0017] FIG. 1A is a top view and FIG. 1B is a cross-section of a
schematic along section line 1B-1B of a moldable slab, in
accordance with certain examples.
[0018] FIG. 1C is a top view of an insert and FIG. 1D is a side
view of an insert in contact with a moldable slab, in accordance
with certain examples.
[0019] FIGS. 2A-2D are schematics of a method for transferring
material secreted by a cell to a substrate, in accordance with
certain examples.
[0020] FIGS. 3A-3D are schematics of a method for detecting
association of two species, in accordance with certain
examples.
[0021] FIGS. 4A-4D are schematics of a membrane and its use with
one or more substrates, in accordance with certain examples.
[0022] FIG. 5 is a photograph of Hyb9901 cells (anti-ovalbumin)
disposed in 100 micron diameter microwells molded in
poly(dimethylsiloxane), in accordance with certain examples.
[0023] FIGS. 6A-6E are schematics of a method for immobilizing
antibodies on a substrate, in accordance with certain examples.
[0024] FIG. 7 is a graph showing the percentages of wells filled by
one or more cells versus the concentration of cells, in accordance
with certain examples.
[0025] FIG. 8 is a graph showing the average number of cells per
well versus the concentration of cells, in accordance with certain
examples.
[0026] FIG. 9 is a bar graph showing the number of cells counted
per well for two different concentrations of cells, in accordance
with certain examples.
[0027] FIG. 10 is a graph showing the percent of viable cells as a
function of incubation time, in accordance with certain
examples.
[0028] FIG. 11A, is a micrograph of Hyb9901 cells disposed in 100
micron diameter wells composed of polydimethylsiloxane and FIG. 11B
is a micrograph of the slide, on which the array of microwells of
FIG. 11A were disposed for 4 h, with physisorbed ovalbumin that
associated with mouse anti-ovalbumin and was probed with a
fluorescent secondary antibody (Goat-anti Mouse), in accordance
with certain examples.
[0029] FIG. 12A is a micrograph of Hyb9901 cells disposed in 100
micron diameter wells composed of polydimethylsiloxane and FIG. 12B
is a micrograph of physisorbed Protein G and secondary Ab
(Goat-anti Mouse) on a glass slide, incubated with microwells of
FIG. 12A for 4 hours (anti-ovalbumin hybridomas), after the glass
slide was probed with fluorescent antigen (Ovalbumin-Alexa 488), in
accordance with certain examples.
[0030] FIGS. 13A-D is a schematic diagram depicting method for
preparation of engraved arrays of secreted products from a mixture
of cells.
[0031] FIGS. 14A-E is a schematic displaying two methods for
detection of antibodies on the surface of a substrate after
microengraving.
[0032] FIGS. 15A-B are fluorescence micrographs of two microarrays
prepared sequentially using the same array of microwells.
[0033] FIG. 16A is a fluorescence micrograph of a region of a
microarray generated from a polyclonal mixture of cells and a phase
contrast micrograph.
[0034] FIG. 16B is an autoradiograph of .sup.35S-labelled
H-2K.sup.b immunoprecipitated using supernatants from cultures
containing Hyb 099-01, Y3, and four clones.
[0035] FIG. 16C is a fluorescence micrograph of a region of a
microarray showing conjugation of captured antibody.
[0036] FIG. 17A is a micrograph showing spots generated from a
polyclonal mixture of hybridomas that are reactive with
fluorescently labeled H-2K.sup.b tetramers.
[0037] FIG. 17B is a micrograph showing spots generated from one
expanded hybridoma (clone 136) that are reactive with fluorescently
labeled H-2K.sup.b tetramers (red) and goat-anti-mouse IgG
(green).
[0038] FIG. 18 is a drawing of a schematic illustration of proposed
method for identifying antibodies that bind surface-expressed
epitopes on a pathogen.
[0039] FIG. 19A is a graphic of the capture of different serotypes
of the same microbe.
[0040] FIG. 19B is a drawing of the capture of altered microbe
(genetic mutant, drug, enzyme-treated) to discover rare
epitopes.
[0041] FIG. 20 is a fluoresence micrograph of human IgG captured by
microengraving with EBV-transformed B cells and labeled with Alexa
488-goat-anti-human IgG.
[0042] FIG. 21 is a drawing of a scheme for generating a single
assay to measure both the phenotype and secretory function of
individual cells.
[0043] FIG. 22 is a drawing of a schematic diagram depicting the
process for depositing cells from a suspension into microwells and
capturing antibodies on a solid support.
[0044] FIG. 23 is a fluorescent micrograph of an engraved
microarray stained for captured IFN.gamma. (green) and IL-4 (red,
indicated with arrowheads).
[0045] FIG. 24 is a drawing of a graphical abstract of antibodies
engineered to allow enzymatic installation of a specific chemical
moiety that can react with a functionalized organic surface
designed to resist non-specific adsorption of proteins.
[0046] FIG. 25 is a drawing of a method for attaching antibodies
modified with an analog of biotin to a SAM bearing a reactive
hydrazide moiety.
[0047] It will be recognized by the person of ordinary skill in the
art, given the benefit of this disclosure, that the examples shown
in the figures are not necessarily drawn to scale. Certain features
or components may have been enlarged, reduced or distorted to
facilitate a better understanding of the illustrative aspects and
examples disclosed herein. In addition, the use of shading,
patterns, dashes and the like in the figures is not intended to
imply or mean any particular material or orientation unless
otherwise clear from the context.
DETAILED DESCRIPTION
[0048] The methods of the invention allow identification and
characterization of previously unknown or undefined cell-derived
compositions. The compositions are secreted such as antibodies or
cytokines or compositions that are released upon lysis or
permeabilization of the cell such as cytoplasmic, nuclear, or cell
membrane components. Any cellular composition including biopolmers,
e.g., DNA and RNA, as well as lipids and glycolipida, is captured
by this method provided that the solid support (substrate) is
appropriately functionalized (e.g., poly dT nucleotides for RNA).
In addition to immune cells, other eukaryotic cells are
interrogated. For example, single cell suspensions from normal or
cancerous tissue of tissue type, e.g., heart, brain, liver,
prostate, breast, or colon. The systems are also useful to
characterize whole cells and secreted products from their cell
types (bacteria, yeast, small parasites (malaria)).
[0049] In addition to analysis of secreted cell-derived products,
the method encompasses characterization of the cells remaining in
the wells after the capture of the secreted products (e.g., by
immunofluoresence, genetic sequencing). In conjunction with
identification and characterization of secreted products, the cells
in corresponding wells are retrieved and matched with the materials
secreted.
[0050] The methods, apparatus and kits disclosed herein are used to
identify antibodies and other cell secreted products. Hybridomas
may be identified using these methods. Hybridomas are made using
known methods, for example, a mouse is immunized with an antigen or
a plurality of antigens. The spleen of the mouse is removed and
broken up to form a suspension. The suspended spleen cells are
fused with mouse myeloma cells, e.g., in polyethylene glycol. The
cells are then cultured over several days in media containing
hypoxanthine, aminopterin and thymidine (HAT media) such that any
unfused spleen cells die. Unfused myeloma cells also are killed in
the HAT media because they lack the enzyme hypoxanthine ribosyl
transferase (HPRT) and thus cannot produce inosine from
hypoxanthine, and the aminopterin prevents the myeloma cells from
using an alternative pathway to produce inosine from thymidine.
Because inosine is a precursor to many nucleic acid pyrimidines,
the myeloma cells die because they are unable to produce nucleic
acids. In contrast, the hybridomas have the HPRT and can use the
hypoxanthine and the thymidine in the HAT media to produce nucleic
acids.
[0051] Using the methods, apparatus and kits disclosed herein, a
single, or a few hybridomas are placed in a microwell of a moldable
slab and are allowed to produce monoclonal antibodies for a few
hours, e.g., 4-8 hours. Secreted monoclonal antibodies are
immobilized on a substrate and exposed to known antigen(s) to
determine which monoclonal antibodies binds to the known antigen.
Results lead to a rapid high-throughput screening of hybridoma
cells that product antibodies against specific antigens and also
allows for the identification and expansion of cells producing
monoclonal antibodies.
[0052] Another application for the method, apparatus and kits
disclosed herein is to identify personalized antibodies raised
against tissue samples from an individual. For example, the
methods, apparatus and kits disclosed herein may be used to raise
antibodies against a tumor in an individual. A sample or biopsy of
the individual's tumor may be taken and injected into a mouse.
After several weeks, the mouse's spleen cells may be removed and
monoclonal antibodies may be identified. Production of such
monoclonal antibodies may provide for treatment of the individual's
tumor by injection or administration of the produced monoclonal
antibodies, e.g., site-specific delivery of the monoclonal
antibodies. Fully humanized antibodies containing a human constant
region (Fc) would be ideal for this application.
[0053] Another application for the method, apparatus and kits
disclosed herein is to determine the efficacy of an immune response
related to a particular immunotherapy such as a vaccine. For
example, the number of lymphocytes secreting specific molecules
indicative of proliferation (e.g., TNF-alpha, IL-1, IgE) and the
overall output levels of these markers from individual cells may be
assayed. The assay may require antibodies against each secreted
factor immobilized on the substrate to capture molecules produced
by individual cells and a second specific antibody with a
fluorescent marker to screen for the amount of each marker captured
on the substrate. This assay produces a new type of personalized
medicine for evaluating the responsiveness of individuals to
selected immunotherapies.
[0054] The advent of antimicrobial drugs and vaccines using
inactivated or attenuated microorganisms has had a remarkable
impact on the overall health of the world population in the last
150 years, especially in first-world countries. In the present era,
however, there has been a resurgence of infectious diseases thought
to be under control or eradicated. New outbreaks of lethal
infectious diseases that have been suppressed in recent times by
the administration of antibiotics or first generation vaccines
(staphylococcus, rubella, mumps) underscore this observation.
Evolutionary pressure applied to various microorganisms by the use
of vaccines and antimicrobial drugs has resulted in increasing
numbers of strains resistant to current therapeutics. These factors
combined with both existing and potential epidemic diseases--HIV,
malaria, influenza--for which treatments are limited, if available
at all, suggest a need for new therapies--either vaccines or
drugs.
[0055] Two current strategies for developing new therapies for
infectious diseases are: 1) rational design of vaccines, and 2)
generation of therapeutic monoclonal antibodies (mAb). The
knowledge of the genomes for common pathogens, and the advent of
computational tools for mining these data, has enabled a more
rational approach for determining immunogenic factors than
empirical data from inactivated materials. Although polyclonal sera
containing antibodies have been used clinically for passive
immunization since the late 1800s, their use in modern medicine has
been limited by the advent of antimicrobial drugs. The development
of hybridomas and related technologies in the 1970s and 1980s has
led to monoclonal therapies, but economic and political suasions
have focused these treatments largely on cancer and autoimmune
disorders. The threat of bioterrorism has renewed interest in
identifying antibodies against potential biological agents, e.g.,
anthrax.
[0056] At least two factors continue to hinder progress in
identifying surface-expressed, immunogenic epitopes on pathogens
and subsequently, new therapies. First, genomic analysis has
yielded some leads for formulating new vaccines, but it can not
predict other factors that can enhance (or mask) the immunogenicity
of an epitope--post-translational modifications, conformational
variations, non-proteinaceous materials, or genetic variation among
serotypes. Second, for therapies based on neutralizing antibodies,
it is likely that single mAbs for infectious diseases will be
insufficient; a cocktail of mAbs recognizing a range of epitopes
should be more effective than a single mAb. Libraries of suitable
mAbs from which to create such cocktails are small or non-existent.
Thus, new tools for the rapid identification of antigens that evoke
robust and protective immune responses for a large number of
infectious agents would greatly assist in the design of vaccines
against epidemic infectious diseases (malaria, HIV, influenza) and
prophylactic treatments for others (small pox, anthrax). This
screening technology enables high-throughput and rapid analysis of
large polyclonal populations of immortalized B cells (>100,000
cells in <12 h) to identify clones producing antibodies reactive
against the surface epitopes of a pathogen.
[0057] There is growing evidence that common phenotypic markers
(e.g., CD4+) can encompass a number of subsets of cells with
diverse functions, indicated by the types of cytokines secreted. A
simple analytical tool that provides information about both
phenotypic markers and secreted factors for individual primary
cells (without extended ex vivo culturing) facilitates studies in
cell biology, especially immunobiology. The methods disclosed make
it possible to retrieve the cells for subsequent culture or genetic
analysis, applications include i) profiling immunological responses
to administered vaccines, allergic reactions, or foreign pathogens,
and ii) extending understanding of the cell biology of cancers and
autoimmune disorders.
[0058] Also, the disclosed method, apparatus and kits is a simple
analytical assay for individual primary cells that allows both
determination of their phenotypes (expressed surface or
intracellular proteins) and direct measurement of their functional
behaviors (secreted cytokines, antibodies, growth factors). More
specifically, the technology rapidly correlates surface-displayed
phenotypes with functional secretory behaviors of individual
primary cells, preferably without extended culture or other
manipulations that could modify the expression of markers or the
behavior. Application of the methods, apparatus and kits make it
possible to have a platform for the systematic analysis of an
immune response to various diseases, allergies, and treatments
(e.g., vaccines) or a systematic analysis of immune responses for
individuals to various diseases using limited sizes of samples, and
should facilitate the transition of clinical medicine towards
predictive, personalized healthcare.
[0059] Antibodies are ubiquitous reagents in biology for
applications that range from basic biochemistry to clinical
diagnostics. They are commonly immobilized on surfaces to retrieve
other biopolymers from a surrounding solution (sera, culture
supernatant). Examples of supports used include beads/resins,
microarrays, and nanoparticles. The most frequent methods for
immobilizing antibodies are i) physical adsorption onto a
hydrophobic substrate, ii) covalent attachment at reactive sites on
the protein, or iii) non-covalent interactions between an
immobilized receptor (streptavidin, protein A or G, anti-Ig) and an
antibody or its derivative multiply decorated with ligands. These
strategies do not induce a specific orientation on every antibody
immobilized--for example, with the binding region positioned away
from the underlying surface. Though random orientation of a protein
on surfaces may be sufficient in many assays designed to determine
unknown protein-protein interactions, the development of
miniaturized biological assays that incorporate micro- and
nanoscale components (with limited surface areas) motivates the
need for new strategies to attach antibodies (and other proteins)
on surfaces that preserve function in high-density. The sensitivity
of detection and the yield of capture depend on the number of
binding sites available at the interface between the supporting
surface and the surrounding solution. Two key parameters
influencing this value are: i) the density of molecules and ii) the
accessibility of the binding region on the immobilized molecule to
other molecules at the interface. Orientation of an immobilized
molecule on the surface is, therefore, important for improving
accessibility.
[0060] The miniaturization of microarrays on planar surfaces can
improve the density of information available in a single
experiment, and the incorporation of nanoparticles in biological
assays can improve the sensitivity of diagnostics. Both
applications require functional organic surfaces capable of binding
specific molecules from a surrounding solution, but in both
instances, the available surface area per feature or particle is
limited. This characteristic suggests that the overall limits of
detection afforded by these methods will depend on the number of
functional receptors presented at the interface between the solid
support and the surrounding environment. Although antibodies are
used routinely in immunochemical assays for detecting specific
analytes, most techniques for immobilizing them on surfaces do not
favor a particular orientation of the molecules. This example
outlines an approach for engineering full-length antibodies to
carry a specific chemical functionality at the C-terminus of the
heavy chains of the immunoglobulin. The addition of a short peptide
sequence recognized by an enzyme, BirA ligase from Escherichia
coli, makes it possible to incorporate an unnatural analog of
biotin containing a ketone to the antibody. Reaction of this moiety
with an organic surface designed to resist non-specific adhesion of
other proteins improves the orientation of the modified antibodies
attached to the surface. This general approach extends to other
proteins of interest (fragments of antibodies, recombinant
enzymes).
[0061] Examples of the technology disclosed herein may be used to
identify unknown species disposed on a substrate that can associate
with a known species. For examples, the methods disclosed herein
are used to identify an antibody that binds to or interacts with a
desired target antigen. The exact nature of the assays depends, for
example, on the selected species, the selected slab or substrate
and the information desired from the assay. Certain embodiments of
the technology disclosed herein provides significant advantages
including, for example, (1) a single array of microwells can
contain greater than 625 wells per square inch compared to about 1
well per square inch for a conventional 96-well plate; (2) the
dilution of a single cell per well makes it possible to identify
cells producing antigen-specific antibodies in a single screen
compared to iterative testing required for assays using
conventional methods, such as a 96- or 384-well plates; (3) the
limited volume of the microwells (about 1 nanoliter or less)
permits sufficient concentrations (e.g., about 1 .mu.M) of antibody
to be reached within a few hours instead of 5-7 days; (4) an assay
for positive antibodies may be integrated into a method and does
not require any additional manipulations to array or test the
secreted antibodies for specificity; and (5) the screening methods,
apparatus and kits can be multiplexed to screen simultaneously for
many different cells producing antibodies against different
antigens.
[0062] In accordance with certain examples, the methods, apparatus
and kits disclosed herein may be used in determining whether or not
two species associate. As used herein, the term "associate" refers
to interactions such as binding, adsorption, ionic attraction or
some other type of interaction between the two species. In some
examples, species that associate preferably bind to each other with
an association constant of at least about 10.sup.9 M.sup.-1 or
larger. Species which bind to each other with such association
constants allow for easy distinction between species that associate
and those that do not associate.
[0063] In accordance with certain examples, a moldable slab may be
used in the methods and kits described herein. As used herein
"moldable slab" refers to an apparatus which can flex, move or
distort, at least in one dimension, when placed in contact with a
substrate. For example, in certain configurations the moldable slab
may include a material, e.g., an elastomeric material, such that as
the moldable slab is placed in contact with a substrate, a
substantially fluid tight seal may be formed between the moldable
slab and the substrate to retard or to prevent any fluid in the
moldable slab from escaping or leaking.
[0064] Protocols for identifying cells from a single colony that
serve as sources of monoclonal antibodies rely on limited dilution
of candidate cells into microtiter plates. The cells are diluted
into 96-well plates or 384-well plates, and expanded for 5-7 days.
At that time, aliquots of the media from each well may be tested to
identify positive wells producing the desired antibody. For
example, an immunoassay such as enzyme-linked immunosorbant assay
(ELISA) may be used to identify positive wells producing the
desired antibody. The contents of the positive well are then
diluted again into a microtiter plate and the process is repeated
until the entire plate is derived from a single colony. This serial
process typically requires 2-3 months to complete and usually
allows only about 1000 different types of cells to be screened for
the desired functionality (e.g., producing a specific
antibody).
[0065] Two factors determine the time required to isolate a single
monoclonal hybridoma by this method. First, the sensitivity of the
assay used to detect antibodies of interest sets the frequency at
which cloned cells can be tested for specificity--for example,
sufficient concentrations of antibodies that can be detected by
enzyme-linked immunosorbant assays (ELISA) are achieved seven to
ten days after seeding individual cells into a microtiter plate.
Second, the total number of manipulations limits the number of
clones that can be screened efficiently in any single round of
selection (10-100 plates/screen).
[0066] Two alternatives for sorting cells into microtiter plates at
limiting dilutions include picking clones from semi-solid media,
and fluorescence-activated cell sorting (FACS). Cells plated in
agar or other hydrogels are challenged to survive and grow slowly,
and the correlation between cells that stain positive in FACS and
those that readily secrete products is not straightforward. Both
methods have improved the efficiency of screening by serial
dilution, but the resulting cultures usually require additional
independent testing by ELISA or equivalent methods to verify
secretion and specificity. Other methods have been developed for
the analysis of individual cells in large numbers, such as
microfluidic devices, cell-based microarrays, ELISPOT and hemolytic
plaque assays, but these methods do not allow both high-throughput
analysis of a secreted product and the recovery of living cells for
clonal expansion.
[0067] Generally, arrays of antibodies with known specificities are
used to detect the presence or absence of specific analytes in an
unknown mixture, which is used as an analytical tool for detecting
known antigens with known antibodies; it is not designed for
discovering new types of antibodies. Arrays of microwells are also
used for screening for hybridomas producing antigen-specific
antibodies but then are subsequently, not in parallel, screened by
traditional immunoassays (flow cytometry, ELISA) for antigen
specificity.
[0068] In contrast, examples of the methods, apparatus and kits
described herein may use a moldable array of microwells or chambers
(e.g., .about.50-100 microns in diameter) to retain one (or a few)
cells in each microwell. The array is placed in physical contact
with a substrate in such a manner that the microwells become closed
containers or a test apparatus. Incubation of this system allows
the cells to produce products, such as, antibodies, cytokines and
other secreted products, that are then immobilized on the substrate
in the regions contacted by the microwells. In this manner, a
microarray of the cellular products from each microwell is
produced. After incubation of the system for a suitable time, e.g.,
1, 5, 30, 40, 50 minutes to a few hours, the microwell array is
removed from the substrate, and the immobilized cellular products
on the substrate, the microarray or microengraving, may be screened
with a known species to determine whether or not the immobilized
cellular product(s) associate with the known species. The method
disclosed herein represents a novel approach by combining detection
of species present in an unknown mixture and, in a parallel and
efficient manner, screening for hybridomas producing
antigen-specific antibodies. Additional uses for screening
non-cellular products using the methods, apparatus and kits are
also described.
[0069] The soft lithographic technique is used to microengrave a
dense array of microwells (0.1-1 nL each) containing individual
cells to print a corresponding array of the molecules secreted by
each cell. The cells remain in culture in a microwell after the
engraving, and the microarrays are interrogated in a manner similar
to commercial microarrays of proteins or antibodies--for example,
by use of fluorescently labeled reagents and laser-based
fluorescence scanners. This method, therefore, enables rapid
identification of those cells that exhibit desired properties, such
as secretion of an antigen-specific antibody, and their subsequent
recovery from individual wells for clonal expansion.
[0070] Referring to FIGS. 1A and 1B, a top view of a moldable slab
100 is shown. The moldable slab 100 comprises a plurality of
microwells or chambers, such as microwells 110, 120 and 130. In the
illustration shown in FIG. 1A, each of the microwells is shown as
having substantially the same cross-sectional shape. For example
and also referring to FIG. 1B, which shows a cross-section through
line 1B-1B in FIG. 1A, the cross-section of the moldable slab 100
shows microwells 110, 120 and 130 as being circular when viewed
from the top and generally cylindrical when viewed from a side. The
exact number, dimensions, shape and the like of each microwell of
the moldable slab 100 may vary. For example, when viewed from the
top, the cross-section of each microwell may be circular, square,
elliptical, toroidal, rhomboid or other selected shape. In
addition, any particular microwell of the moldable slab may be a
different shape than another microwell in the moldable slab.
[0071] In certain configurations, each microwell of the moldable
slab may be sized and arranged to retain or to hold a single cell
or a few cells (e.g., 3-5 cells), such as a bacterial cell, a
hybridoma or other selected cell. In some examples, the diameter of
each microwell of the moldable slab may vary from about 10 microns
to about 100 microns, more particularly, about 25 microns to about
100 microns, e.g., about 50-100 microns. Additionally each
microwell is separated from another by a length similar to the
depth and/or height. The size of any selected microwell may vary
depending on the size of the cell, or cells, to be retained by the
microwell. In certain examples, the microwell is sized to be large
enough so that the cell may remain viable but is not so large that
any products produced by the cell will be diluted by a large fluid
volume. For example, the volume of each microwell of the moldable
slab is large enough to retain a cell and to provide a buffer,
nutrients, etc. to keep the cell alive, but the volume of the
microwell is not so large that any desired screening products will
be diluted by solvent or buffer to a non-detectable level. In
certain configurations, the volume of the microwell varies from
about 1 picoliter to about 100 nanoliters, more particularly about
10 picoliters to about 10 nanoliters, e.g., about 100 picoliters to
about 1 nanoliter.
[0072] The exact number of the wells or chambers in the moldable
slab may vary. In some examples, the moldable slab may include a
single large microwell where a single species may be screened. For
example, a moldable slab may include a single type of cell,
catalyst or other selected species that may be screened. In
configurations where the moldable slab is configured as an array,
the number of individual microwells may vary from about 24, 48, 96,
384, 1024, 2048, 5096 or more or any value in between these
illustrative values. As material in the moldable slab is
transferred to the substrate, an array of disposed material forms
on the substrate which reflects the material present in the
microwell or microwells of the moldable slab. One or more of the
microwells in the moldable slab may be blocked or prevented from
transferring material to the substrate using an insert or device
placed between a particular microwell in the moldable slab and the
substrate. This feature provides for selective disposition of
arrays of material on a substrate.
[0073] The moldable slab may be configured in a variety of manners.
For example, the moldable slab is configured as a plate comprising
one or more microwells. The moldable slab may also be configured as
a bead comprising one or more microwells or a bead configured to
retain material on its surface. Any particular configuration for a
moldable slab may be used provided that material on the moldable
slab, or products produced by material on the moldable slab, may be
transferred, at least to some extent, to a substrate.
[0074] The moldable slab is configured to receive an insert
comprising one or more openings. In certain examples, the insert
comprises a plurality of openings. Referring now to FIGS. 1C and
1D, a moldable slab 150 comprises an insert 160 which may be
disposed on top of the moldable slab 150. The insert 160 may be
permanently fixed or may be removable. In this illustration, the
insert 160 comprises a plurality of openings, such as opening 162.
As the moldable slab 150 is brought into contact with a substrate,
the openings in the insert 160 allow for transfer of material from
the moldable slab 150 to certain areas on the substrate. As
material is transferred from the moldable slab 150 to the substrate
through openings in the insert 160, an array is formed on the
substrate which reflects the number of openings, and spacing of the
openings, of the insert 160. This configuration permits the
moldable slab 150 to take the form of a single large microwell
which may be used to hold material such as, for example, a
hybridoma, a bacterial cell and the like. The moldable slab
contains one or more inserts and the insert is produced by using
the same or similar materials that are used to produce the moldable
slab.
[0075] The material or materials used to produce the moldable slab
include polymeric and metallic compositions. In certain examples,
the moldable slab may be made from two or more materials, only one
of which may impart the moldable properties to the slab. In other
examples, two or more materials which are elastomeric may be used.
Illustrative materials include, but are not limited to, glass,
plastic (including both rigid and soft materials), polystyrene,
polycarbonate, poly(dimethylsiloxane) (PDMS), nitrocellulose,
poly(vinylidene fluoride) (PVDF), metals such as gold, palladium,
platinum, silver and alloys thereof, steel and mixtures of any of
these materials. The rigidity of some materials, such as
polystyrene, would not allow for conformal contact, and thus
sealing, of the microwells against a substrate for testing the
specificity of the antibodies produced in a parallel. PDMS,
however, is a suitable material for this technique because it is
not toxic, it is gas permeable, and it is easily compressed to form
a tight, but reversible, seal with a rigid substrate.
[0076] In other examples, sols or gels, e.g., agar, a hydrogel,
matrigel, etc. may be used in the moldable slab. In some examples,
the material to be assayed, or cells which secrete a material to be
assayed, may be embedded, impregnated in or injected into the
moldable slab. For example, a monolayer of cells is cultured on the
moldable slab. In some examples, cells are embedded in a hydrogel
which is used to produce, or is coated on, the moldable slab.
Additional materials suitable for use in the moldable slabs will be
readily selected by the person of ordinary skill in the art, given
the benefit of this disclosure.
[0077] The moldable slab may include additives, fillers,
insulators, growth factors and the like. For example, the moldable
slab may include one or more materials which act as an insulator to
assist in keeping a substantially constant temperature for the
materials, e.g., cells, in the moldable slab. The moldable slab may
also include antibiotics or other compounds which can reduce or
prevent growth of unwanted organisms such as, for example, bacteria
or fungus. Such additional materials may be included in the
moldable slab, coated on the moldable slab, e.g., only in the
microwells or on the entire moldable slab, or may otherwise be
impregnated in the moldable slab to provide a desired result.
[0078] The moldable slab may be cast in a mold using suitable
materials described above. Photolithographic and replica molding
techniques may be used. The material is coated on a master mold,
vapor deposed on a master mold or added onto or in a master mold to
provide a moldable slab comprising a desired number of chambers. In
some examples, a moldable slab configured as an array is produced
using photolithography and replica molding, from monolithic slabs
of poly(dimethylsiloxane) (PDMS). For example, a layer of a
photoresist is patterned on a suitable substrate, such as a 3 inch
silicon wafer, to produce a master with a positive relief pattern
of the moldable slab. A suitable material (e.g., PDMS) is cast onto
the master, cured and peeled away to provide the moldable slab. In
other examples, a moldable slab is produced by casting a material
and using a press, plate, punch, air or the like to provide
depressions in the surface of the cast material prior to curing or
hardening of the cast material. Generally, at least 50 replicas are
created from a single mold with minimal wear. Additional methods
for producing moldable slabs suitable for use with the methods,
apparatus and kits disclosed herein will be readily selected by the
person of ordinary skill in the art, given the benefit of this
disclosure. Illustrative methods for producing moldable slabs are
described in more detail in U.S. Pat. No. 6,180,239 and U.S. Pat.
No. 6,776,094, the entire disclosure of each of which is hereby
incorporated herein by reference in its entirety.
[0079] A substrate may be used with the moldable slab to provide a
test apparatus. The configuration of the substrate may vary and
typically the substrate is selected such that it can "mate" with
the moldable slab to provide a substantially fluid tight test
apparatus. The ability of the moldable slab to flex, distort, bend,
conform, etc. to a surface of the substrate assists in providing a
substantially fluid tight test apparatus. In certain examples, the
substrate may be a solid substrate, such as a glass or plastic
slide, which may be placed on or in contact with the moldable slab.
Generally, the array was designed to fit within the boundaries of a
1''.times.3'' glass slide--a common format for microarray readers;
variations in shape and spacing of individual wells were used to
encode their specific location within the array.
[0080] For example and referring to FIGS. 2A-2D, a moldable slab
200 may include a material, such as a cell 210 in a fluid medium
220. A substrate 230 (FIG. 2B) may be disposed on the moldable slab
200 to form test apparatus 250 (FIG. 2C). The test apparatus 250 is
substantially fluid tight such that the test apparatus 250 may be
oriented in any direction without substantial loss of fluid. For
example, the test apparatus may be flipped over (FIG. 2D) to permit
products secreted or produced by the cell 210 to settle, adsorb,
become immobilized, etc. on a surface of the substrate 230 under
gravitational force. While FIGS. 2A-2D are shown with a cell 210 in
the moldable slab 200, non-cellular species, such as proteins,
catalysts, nanomaterials, etc. could instead be disposed in the
moldable slab such that the proteins, catalysts, nanomaterials,
etc. could be transferred to the substrate 230. Additional
materials that may be used in the methods, apparatus and kits
disclosed herein will be readily selected by the person of ordinary
skill in the art, given the benefit of this disclosure.
Illustrative materials that may be used to provide a substrate
include, but are not limited to, glass, plastic (including both
rigid and soft materials), polystyrene, polycarbonate,
poly(dimethylsiloxane), nitrocellulose, poly(vinylidene fluoride)
(PVDF), metals such as gold, palladium, platinum, silver and alloys
thereof, steel, mixtures of any of these materials, and other
materials that may be used to provide a moldable slab.
[0081] The substrate may be coated with a composition or compound
that acts to retain material transferred from the moldable slab. In
some examples, an entire surface of the substrate may be coated
such that material may be retained on the entire surface. In other
examples, select areas of a surface may include a composition or
compound that acts to retain material transferred from the moldable
slab. Selective coating may assist in formation of a plurality of
"spots" or "patterns" on the substrate that can be assayed. For
example, a coating that acts to retain material may be disposed
over a mask which has openings spaced a suitable distance from each
other to provide an array.
[0082] As material is transferred from the moldable slab, the
material may be retained by the array coating and can be
subsequently screened against one or more species. In certain
examples, the composition or compound may act to adsorb the
material, e.g., by trapping some portion of the material in a
matrix by physisorption of material on the substrate may occur.
Such physisorption may be, for example, adsorption of antibodies
directly onto the substrate, adsorption of proteins recognizing the
constant region of the antibody's structure (Fc portion), e.g.,
Protein A or G, adsorption of secondary antibodies recognizing the
constant region of the antibody's structure (Fc portion), e.g.,
Goat anti-Mouse, or combination of proteins and antibodies, e.g.,
Protein G and secondary antibodies. Overall, the region of
microwells on the PDMS slab matched regions of the microarray and
the specificity of the antibody produced by the individual cells in
wells could be determined from the microarray. Materials suitable
for retaining a desired material on a substrate will be readily
selected by the person of ordinary skill in the art, given the
benefit of this disclosure.
[0083] In other examples, the substrate may include a linking group
which can react with a portion of the material to assist in
retaining material on the substrate. In some examples, the surface
of the substrate may be chemically modified. For example, the
surface of the substrate may include silanes on glass. Modification
of the surface with silanes containing free amine or carboxylic
acid groups for linking secondary antibodies to surface by
NHS-ester activation and subsequent amide linkages may be used, for
example. Modification of surface with silanes containing a free
nitrile group for electrostatic capture of primary or secondary
antibodies (See Bioconj. Chem., 1999, vol 10 pp 346-353) may also
be used. Thiols on a metal, such as gold, palladium, silver or
platinum, may be used. For example, modification of the surface
with thiols containing free amine or carboxylic acid groups for
linking secondary antibodies to surface by NHS-ester activation and
subsequent amide linkages may be used. Modification of a surface
with thiols containing a free nitrile group for electrostatic
capture of primary or secondary antibodies may be used. Covalent
modification of exposed functional groups on a polymeric surface to
cross-link secondary antibodies to surface may be performed.
Additional methods and compositions for chemically modifying a
surface of the substrate will be selected by the person of ordinary
skill in the art, given the benefit of this disclosure.
[0084] The material in the moldable slab may be attached to the
substrate using moieties or tags appended to secreted molecules.
For example, covalent modification of secondary antibodies with a
chemical moiety recognized by a second chemical moiety immobilized
on the surface of the substrate may be used to immobilize the
secondary antibodies to the substrate. In an illustrative example,
biotinylated antibodies along with streptavidin immobilized on a
surface of the substrate may be used. Peptide sequences or proteins
may be appended to the secreted molecule and used to retain the
secreted molecule on a surface of the substrate. Addition of
appended moieties to the secreted molecules is performed in a
manner to avoid or minimize disruption of the native structure of
the molecule to provide a secreted molecule that has a structure as
close as possible to the native structure of the molecule. Moieties
or tags are selected for appending to secreted molecules or other
molecules disposed in a moldable slab.
[0085] Material is transferred from the moldable slab to the
substrate such that a suitable amount is present to detect
association. An effective amount of material will vary for
different materials disposed on the substrate depending, for
example, on binding constants, temperature, ionic strength,
concentration, pattern size, etc. In some examples, an effective
amount provides at least a detectable signal after a labeled
species associates with the material disposed on the substrate. A
detectable signal may vary depending on the technique or method
used to detect association. For example, more material may be
disposed on the substrate where colorimetric methods are used for
detection, while less material may be disposed on the substrate
where mass spectroscopy is used for detection. In certain examples,
a sufficient amount of material is disposed to provide a
concentration of about 1 attomole/cm.sup.2 to about 1
micromole/cm.sup.2, more particularly about 1 femtomole/cm.sup.2 to
about 100 nanomoles/cm.sup.2, e.g., about 10 femtomoles/cm.sup.2 to
about 10 nanomoles/cm.sup.2.
[0086] In certain configurations which involve a cell or a few
cells disposed in a microwell of the moldable slab, the total time
required for performing an assay is less than about 24-48 hours,
more particularly less than a day, e.g., less than about 12 hours.
For example, because the volume of each microwell may be a few
nanoliters or less, the time required for a cell to express and/or
secrete a detectable amount of material may be only a few hours,
e.g., less than about 8-12 hours. Expression and/or secretion of
the material is typically the rate limiting step in performing
assays involving monoclonal antibodies and other materials. In many
instances, association of the species, even after time is allowed
to reach an equilibrium state, proceeds rapidly when compared to
the time required to express the material. The significant time
savings provided by the methods, apparatus and kits disclosed
herein provides for a substantial increase in throughput to screen
large numbers of unknown species against a known species. It is a
significant advantage that certain embodiments of the methods,
apparatus and kits disclosed herein can reduce screening time from
months to less than a day.
[0087] The moldable slab may be loaded with a selected material by
placing the material in a fluid, such as water, a buffer, a solvent
or the like, and adding a suitable amount of the dissolved or
suspended material to the moldable slab. Some material disposed in
the moldable slab will be retained in the wells or chambers of the
moldable slab. Additional material may be suspended in fluid on top
of the moldable slab, e.g., not in the wells of the moldable slab.
Such additional material may optionally be wicked away or removed
using, for example, micropipets, filter paper, drying agents, air
streams and the like, such that only the chambers or wells of the
moldable slab retain material, such as cells, catalysts, etc. This
process assists in distinguishing which wells in the moldable slab
secreted a molecule or compound disposed on the substrate that
associated with a known species, and provides for rapid recovery
and further analysis of such molecule or compound from the moldable
slab.
[0088] The methods, apparatus and kits described herein may be used
to provide a material on a substrate that can be tested for a
desired binding specificity. The species which the material on the
substrate may be exposed to can vary depending on the nature of the
material disposed on the substrate. For example, where the material
disposed on the substrate is a monoclonal antibody, the monoclonal
antibody may be exposed to an antigen, or series or antigens, to
determine whether or not the monoclonal antibody may associate with
the antigen. Where the material disposed on the substrate is a
catalyst, a reactant may be exposed to the catalyst to determine
whether or not the catalyst may associate with the reactant. Where
the material disposed on the substrate is an enzyme, a potential
enzyme substrate, or a potential enzyme substrate analog, may be
exposed to the substrate to determine whether or not the enzyme and
the potential enzyme substrate, a potential enzyme substrate
analog, can associate with the enzyme disposed on the
substrate.
[0089] Where the material disposed on the substrate is a cellular
product produced only in a bacterial cell after successful
transformation, the cellular product may be disposed on the
substrate and screened with a known substance that binds to the
cellular product to distinguish between bacterial cells that have a
transformation product, e.g., a plasmid, phasmid, etc., and those
that do not. In some examples, a cell, or a cell in a moldable
slab, may be exposed to an antigen and any resulting cytokine or
cytokines, e.g., monokines, lymphokines, etc., can be disposed on a
substrate and screened against a species to determine if a
particular type of cytokine is produced in response to antigen
exposure. It will be within the ability of the person of ordinary
skill in the art, given the benefit of this disclosure, to select
suitable species to test for association with a material disposed
on a substrate.
[0090] Referring to FIGS. 3A and 3B where a single microwell or
chamber 305 of a moldable slab 300 is shown, substrate 310 may be
placed in fluid communication with the moldable slab 300 such that
a substantially fluid tight seal is formed to retain cell 330
within test apparatus 305. Cell 330 secretes cellular products 342
and 344 which become adsorbed to the substrate 310. Subsequent to
adsorption of the cellular products 342 and 344, one or more known
species, such as species 352 and species 354, may be exposed to the
cellular products 342 and 344 for a sufficient period to allow
association between the species and the cellular products if such
association may occur. In this simple illustration, species 352
associates with cellular product 342, while species 354 does not
associate with either of cellular products 342 or 344. Prior to
detection, an optional washing step may occur to remove any excess
species that do not associate with the products disposed on the
substrate 310. Species 352 may contain a label, such as a
fluorescent label, colorimetric label, etc. such that detection of
the product 342--species 352 complex may be performed. While the
illustrative example shown in FIGS. 3A-3D is shown with reference
to a cell, non-cellular species, such as catalysts, nanomaterials,
etc. may also be identified using similar methods.
[0091] One particularly useful application for the methods,
apparatus and kits disclosed herein is to provide an array of
unknown monoclonal antibodies disposed on a substrate. The
generation of monoclonal antibodies have significant utility as
research tools and are also highly valuable as potential drug
candidates. Efficient screening methods have limited the number of
candidate antibodies produced for therapeutic purposes. The uses of
the methods, apparatus and kits disclosed herein can be extended,
however, to use the array of secreting cells to assay the response
of cells on a solid substrate and include screening any secreted
material of interest from any cell type.
[0092] Examples of secreted materials include, for example,
cytokines, chemokines, and pathogens such as viruses. Examples of
cell types that could be used include all classes of lymphocytes
(e.g., B cells, T cells) and other cells specializing in secretion
(for example, liver or kidney cells). Known antigens may be added
to the array to determine which monoclonal antibodies could bind to
the antigen. The known antigen may be added to each member of the
array or different antigens may be added to some members in the
array. In a typical configuration, more than one type of monoclonal
antibody may be present at each array member such that the
monoclonal antibodies may be screened batchwise to determine if any
of the monoclonal antibodies in any particular array member can
associate with the antigen. Such batchwise screening provides for
rapid testing of a plurality of monoclonal antibodies. If one or
more monoclonal antibodies in any particular array member do bind
to the antigen, the monoclonal antibodies can be singled out
individually for further testing to determine which particular
monoclonal antibody, or monoclonal antibodies, in the array member
can bind to the antigen.
[0093] Instead of using a moldable slab in the methods, apparatus
and kits disclosed herein, alternative compositions may be used.
For example, a membrane comprising a plurality of openings is
placed on top of a substrate such as a glass or plastic slide, a
metal plate, a porous filter material, or other rigid slab on which
the membrane is in contact. In certain examples, the membrane may
be disposed on the substrate and a substantially fluid tight seal
may be formed between the membrane and the substrate. Referring to
FIG. 4A, a membrane 400 is shown comprising a plurality of
openings. In certain examples, the membrane may have a thickness
410 (FIG. 4B) of about 0.01 mm to about 1 mm, more particularly
about 0.02 mm to about 0.2 mm, e.g., 0.03 mm to about 0.1 mm. In
certain examples, the holes of the membrane may be sized and
arranged to retain one or a few cells. In other examples, the holes
may be sized and arranged such that each hole may retain a few
nanoliters of fluid volume. This configuration would allow use in a
manner similar to the wells of the moldable slab, but would have
the advantage that two surfaces could be patterned or spotted
simultaneously, or if the substrate surface was porous, the
continuous exchange of nutrients through the back surface of the
pattern transfer element may be permitted. Referring to FIG. 4C, a
membrane 430 may be placed in contact with a substrate 440. Cells,
such as hybridomas, bacterial cells, etc., may be disposed in the
openings of the membrane 430, and products secreted by the cells
may be adsorbed to the surface of substrate 440. Referring to FIG.
4D, a membrane 450 may be placed between a first substrate 460 and
a second substrate 470. Cells within openings of membrane 450 may
secrete products which can be disposed on a surface of substrate
460 and a surface of substrate 470. In embodiments where two or
more substrates are simultaneously patterned using a membrane, it
may be desirable to turn, invert or agitate the
substrate-membrane-substrate assembly periodically to assist
disposition of material on both substrate surfaces.
[0094] One or more of the substrates may include a compound or
molecule that acts to retain secretion products on a surface of the
substrate. One of the two substrates may also have a compound or
molecule disposed on its surface that is capable of activating or
modulating the secretion products of a cell or cells contained in
the wells. Additional alternative configurations for disposing
and/or transferring material to a substrate will be readily
selected by the person of ordinary skill in the art, given the
benefit of this disclosure. In some examples, each hole or opening
of the membrane may be the same size and configuration as the other
holes or openings in the membrane. In other examples, the holes or
openings in the membrane may be sized differently such that
differential disposition of material is patterned onto the
substrate. In certain examples, the membrane may be produced using
materials such as, for example, rubber, Teflon.RTM. polymer,
polycarbonate, polydimethylsiloxane, and thermoset polymers.
Additional materials, such as the moldable slab materials discussed
herein, may also be used.
[0095] Identification of positive associations is performed using
numerous suitable techniques including, for example, fluorescence,
mass spectrometry, or other analytical methods used in traditional
immunoassays (e.g., colorimetric methods). For example, the species
added to the substrate may include a fluorescent label such that if
the labeled species added to the substrate associates with the
material disposed on the substrate, fluorescence emission may
occur. Illustrative fluorescent labels include, for example,
fluorescein isothiocyanate, didansyl chloride, lanthanides and
lanthanide chelates, Alexafluor.RTM. dyes, inorganic semiconductor
nanocrystals (e.g., quantum dots composed of II/VI or III/V
semiconductors), and similar labels. Any fluorescence emissions may
be detected visually or may be detected using suitable instruments,
such as fluorescence microscopes, fluorimeters, cameras, or
instruments that include a charge coupled device, a photomultiplier
tube, a diode array and the like. Other labels that emit light,
e.g., phosphorescent labels, chemiluminescent labels, etc., may
also be used and detected using similar techniques as those used in
connection with fluorescence detection.
[0096] The detectable moiety added to the substrate may include a
colorimetric label such that if the labeled species added to the
substrate associates with the material disposed on the substrate,
and after addition of a suitable enzyme substrate, a color may
result. For example, a colorimetric label is an enzyme, such as
horseradish peroxidase. After an enzyme substrate, such as, for
example, o-phenylenediamine dihydrochloride, is added to the enzyme
a colored product is produced if the colorimetric label is present.
The colored product may be detected visually or may be detected
using suitable instruments such as, for example, UV/Visible
instruments, plate readers, etc. In some examples, the colorimetric
label may be a dye, e.g., an organic or an inorganic dye, such that
if association occurs, the well or chamber remains colored, whereas
if no association occurs, the well or chamber is clear and
colorless. For example, if no association occurs the well appears
clear or nearly colorless after unassociated labeled species are
removed by washing.
[0097] Other detectable markers include a radiolabel. The
radiolabel may be integrated into the species or may be added as a
tag to the species. When a radiolabel is used, it may be desirable
to construct the substrate with an absorbing material between array
members to prevent or reduce crosstalk between the various members
disposed on the substrate. Illustrative radiolabels include, but
are not limited to, .sup.3H, .sup.14C, .sup.32P, .sup.33P, .sup.35S
and .sup.125I. The species disposed on the substrate may be
radiolabeled, and upon association, any radioactive emission from
the species may be quenched by a molecule or group which associates
with the species disposed on the substrate. Suitable radiolabels
for use in the methods, apparatus and kits disclosed herein will be
readily selected by the person of ordinary skill in the art, given
the benefit of this disclosure.
[0098] Binding may be measured using mass spectroscopy. For
example, the species may be allowed a sufficient time to associate
and the contents (after optional washing steps) of a particular
array member, or a selected array on a substrate, may be removed
and analyzed using mass spectroscopy. Numerous different mass
spectroscopic techniques may be used. For example, matrix-assisted
laser desorbed ionization (MALDI), electrospray ionization (ESI),
fast atom bombardment (FAB), time of flight (TOF), MALDI/TOF,
ESI/TOF, chemical ionization (CI), liquid secondary ion mass
spectroscopy (LSIMS) or other mass spectroscopic techniques may be
used. In some examples, tandem mass spectroscopy may be performed.
Mass spectroscopic techniques are useful for distinguishing between
association and non-association. In examples where mass
spectroscopy is used, an array may be generated on an appropriate
substrate (e.g., a metal plate for MALDI). The entire array may be
probed with a mixture of proteins used for immunization (e.g.,
entire cell lysates) and then characterized by mass spectrometry.
Identification of proteins, lipids, or carbohydrates which are
bound by specific antibodies may be accomplished, for example, by
comparing the spectrometry data against databases of known
biomolecules or compounds.
[0099] A proximity assay may also be used. For example, the species
disposed on the substrate may be labeled with a radioactive label
prior to transfer to the substrate. The species added to the
transferred species on the substrate may include a fluorescent
label, such that if association of the two species occurs,
radioactive emission will excite the fluorescent label, and
fluorescence emission may be detected as a positive indicator of
association. Because this energy transfer process requires the
radioactive label and the fluorescent label to be close, e.g.,
within a few microns, fluorescently labeled species that remain
free in solution would not emit light. Such proximity methods have
the added benefit that no washing steps or separation steps are
required to determine if association occurs. Any fluorescence
emission may be detected using the illustrative techniques
disclosed herein, e.g., plate readers, flourimeters, etc. Suitable
fluorescent and radioactive labels for performing proximity assays
to assess association will be readily selected by the person of
ordinary skill in the art, given the benefit of this
disclosure.
[0100] A significant advantage of the methods, apparatus and kits
disclosed herein that if association of the species does occur,
then the material in the moldable slab, e.g., the cell or cells in
a particular well or chamber, may be recovered and used for further
analysis. In contrast, many existing methods do not permit recovery
of cells for further use and/or analysis but instead require
re-isolation and/or regrowth of the cells. For example, because the
methods, apparatus and kits disclosed herein permit the cells to
remain alive in the wells of the moldable slab, it is possible to
produce multiple arrays for screening by stamping or patterning the
array onto multiple substrates, and to retrieve positively
identified cells for clonal expansion by micropipetting or similar
techniques.
[0101] Cells confined in microwells and sealed against a glass
slide (such that the total media available was limited to the
volume of the microwell) may be maintained for about 5 hours and up
to 24-48 hours, e.g., 1, 2, 6, 12, 18, 24, or 36 hours without a
significant loss to viability. (FIG. 10). When a moldable slab is
removed from a substrate and immersed in media, the cells remained
loosely adhered to the bottom of the wells; vigorous rinsing or
intentional extraction of the cells was required to dislodge them
from the wells. Because the cells in the microwells remain viable
after printing, the same set of microwells could produce multiple
copies of an engraved microarray at different time points, by
Elispot, FACS, ELISA, or other immunochemical methods is
challenging. The resulting microarrays have a high degree of
similarity, but are not identical; variations between specific
spots may result from either fluctuation in the rate of secretion
related to various stages of cell division or cell death.
[0102] The species (e.g., secreted cellular product), and/or cell
producing the species, may be retrieved from the moldable slab
using numerous techniques. For example, a micropipette may be used.
An array of wells in the moldable slab can be coded or addressed to
identify specific wells in the array to match positive hits from
the substrate. Methods for coding the system include adjusting the
spacing between wells, the shape of the wells, and the size of the
wells. After removing the array of microwells from the substrate or
the moldable slab, the wells can be immersed in appropriate cell
culture media to maintain the viability of the cells until testing
and/or characterization is complete. Extraction of the desired
cell(s) from the identified wells can be performed with a
micropipette. The extracted cell(s) may be expanded to generate a
stable cell line, e.g., a stable hybridoma cell line. Expansion may
be carried out in a microtiter plate (e.g., 96 or 384-well plate)
containing suitable media to sustain cell viability. For example,
in the case of a hybridoma cell line, a population of adherent
feeder cells (e.g., fibroblasts) may be present to condition the
media and provide a surface for cell-cell contact. Culturing the
cells in the microwells for 1-3 days prior to extraction by
micropipette would allow a few cycles of cell division within the
microwells and make it possible to extract a few copies of the
desired cell rather than a single copy. The exact conditions
required to sustain the cells will vary depending on the cell type,
and it will be within the ability of the person of ordinary skill
in the art, given the benefit of this disclosure, to select
suitable media and growth conditions to promote cell viability.
[0103] Microengraving offers three primary advantages over
traditional screening by serial dilution and ELISA. First,
microengraving allows the identification and segregation of the
cells that secrete antigen-specific antibodies from a polyclonal
mixture early in the screening process. The ability to isolate
those cells should preserve slow-growing clones and rare clones
(e.g., those that recognize particular epitopes of interest);
traditional screening by serial dilution tends to favor
fast-growing clones because the time required to attain detectable
levels of antibodies is sufficiently long to allow outgrowth of the
population tested. Second, segregation of cells early in the
screening process reduces the labor and time required to maintain
many individual clones while characterizing the antibodies produced
for appropriate reactivity in immunochemical assays. Table 1,
below, summarizes the significant differences in materials and time
required for cloning by microengraving and serial dilution. Table
2, below, summarizes the cost analysis for microengraving and
limited serial dilution. Third, microengraving simplifies the
requirements for screening polyclonal populations to identify
clones with different specificities. A single microarray can be
screened with multiple, differentially labelled antigens, or
fragments of antigens; equivalent screens by ELISA would require
independent assays for each condition tested. The ability to
immunize mice with mixtures of antigens furthers improve the rate
of selection. A current disadvantage of microengraving is the
manual retrieval of cells from the microwells. Existing or modified
instruments for picking mammalian cells from colonies are useful
for automated retrieval of cells, thereby further reducing the time
involved in the screening process.
TABLE-US-00001 TABLE 1 Comparison of Time and Resources Required to
Clone Hybridomas Limiting Serial Dilution Microengraving Method
Time after fusion until first screen 7-13 days 7-10 days Number of
wells (per plate) 96-384 wells 25,000-100,000 wells Percent wells
filled (average) 33-100% (1-2 cells/well) 50-80% (1-3 cells/well)
Number of cells (per plate) ~32-384 cells ~12,500-80,000 cells
Typical number of plates per screen 10-100 1-10 Total volume of
fluid per well 75-200 .mu.L 0.1-1 nL Time till detectable levels of
antibody 2-7 days 2-4 h are produced Antigen required per screen
(per plate) 24-48 .mu.g ~0.75 .mu.g Use different antigens in a
single No Yes, multiple labels screen? Time per screen (culture +
assay) 7-10 days ~10 h Minimum screens to determine 2-3 rounds 1-2
rounds monoclonality Minimum time for screening 19-30 days 1-2 days
Time required to maintain cultures 5 min (every 5 min between
screens (per plate) 2 days) Expected yield of desired hybridomas
0-1 0->>1 (per plate)
TABLE-US-00002 TABLE 2 Cost Analysis for Microengraving Limiting
Serial Dilution Microengraving Method Cost for Immunization of
Mouse (1 antigen $120 (6 wks) $120 (6 wks) requires ~1 mg total
material) Time after fusion until first screen 7-13 days 7-10 days
Cost of screening/plate.dagger. Labor (sorting and screening) 1 hr
FTE 1 hr FTE Actual Time (culture + assay) 5-7 days ~10 h Supplies
(plates and tips) $8 $12* Reagents (antibodies & proteins)
$6.50 + 24-48 mg Ag $0.50 + 1-7 mg Ag Media $12 $6 Total cost per
plate ($) $26.50 $18.50 Number of wells (per plate) 96-384 wells
25,000-100,000 wells Percent wells filled (average) 33-100% (1-2
cells/well) 50-80% (1-3 cells/well) Number of cells (per plate)
~32-384 cells ~12,500-80,000 cells Cost per cell screened
$0.07-$0.83 $0.0002-$0.001 Minimum screens to determine 2-3 rounds
1-2 rounds monoclonality Typical number of plates per screen 10-100
1-10 Total cost for cloning ONE hybridoma $530-$7,950 $18.50-$370
Minimum time for screening 19-30 days 1-10 days (Cost should scale
as ~1/n with increasing numbers of recovered clones per screen)
Expected yield of desired hybridomas (per 0-1 0->>1 plate)
Maximum yield of unique monoclonal 1 ??? (>1) hybridomas (per
plate) Test different antigens in a single screen? No Yes, multiple
labels (2-16)
[0104] Microengraving enables similar assays for detecting a
variety of secretions from large numbers of individual cells in a
(semi)quantitative manner. The captured secretions represent the
amounts produced over a fixed period of time, and analysis does not
require monitoring a short-lived event such as calcium flux. The
method is not limited to screening hybridomas derived from mouse
splenocytes, but also can be applied to Epstein-Barr
virus-transformed human B cells, and in principle, primary cells
from peripheral blood or tissue. The methods are useful in
measuring other secreted products, such as cytokines, for
monitoring an immunological response and for measuring the
frequency of cells within a population that produce a specific
secreted factor in diagnostic assays. Multiplexed labeling, and the
ability to generate multiple engravings from the same array of
microwells, thereby allowing testing for the presence of multiple
secreted products from single cells.
[0105] A protein or antigen is immobilized on a substrate, and then
a species secreted by a cell is added to determine whether or not
such species associates with the protein or antigen that is
immobilized on the substrate. A labeled secondary antibody may then
be added to probe for positive capture of the species. The protein
or antigen is disposed on the substrate by transferal from a
moldable slab or by disposal using conventional techniques such as,
for example, pipetting. The labeled secondary antibody may be
labeled with any of the illustrative labels disclosed herein, e.g.,
fluorescent labels, radioactive labels, colorimetric labels, etc.
Detection may be accomplished using any of the methods disclosed
herein, such as, for example, fluorescence, mass spectroscopy,
etc., depending on the selected label.
[0106] The methods, apparatus and kits disclosed herein may be used
to immobilize antibodies on monolayers of cells. For example,
secretion of antibodies from wells of a moldable slab onto
monolayers of fixed and permeabilized cells followed by treatment
with a fluorescently labeled secondary antibody can provide a
method to characterize antibodies by structural labeling within the
cell. Optical microscopy would allow identification of antibodies
labeling specific organelles. For example, organelles such as
cytoskeleton, endoplasmic reticulum, Golgi apparatus or other
organelles may be labeled.
[0107] The species that can be identified using the methods,
apparatus and kits disclosed herein may be further characterized
using conventional techniques, such as immunoprecipitation, Western
blots, or other biochemical analysis to identify the specific
species. For example, the monoclonal antibodies may be sequenced.
X-ray crystallography, NMR analysis and the like may also be
performed to characterize the structure of the identified species.
Additional suitable techniques for characterizing the species
identified using the technology disclosed herein will be readily
selected by the person of ordinary skill in the art, given the
benefit of this disclosure.
[0108] Engineered organisms may be used with the methods, apparatus
and kits disclosed herein. For example, a modified Ig locus may be
introduced into an animal (e.g., a D.sup.h-/- or J.sup.h-/- mouse)
that includes a genetic encoded chemical structure for attaching
antibodies secreted to the solid slab. For example, a recombinant
Ig molecule includes a sortase signal peptide sequence onto the end
of the Ig locus. This addition would produce antibodies with the
sortase signal peptide expressed on the Fc portion of the antibody.
Incubation of the cells in a media containing recombinant sortase
enzymes on top of a substrate containing immobilized peptidoglycans
allows covalent attachment of the antibody to the peptidoglycans
via transpeptidation. Another illustrative example would include
addition of a fragment of a ubiquitin-specific protease that
includes the active catalytic site onto the end of the Ig locus.
This addition would produce antibodies with a known reactive
oligopeptide sequence expressed on the Fc portion of the antibody.
Incubation of cells producing these labeled antibodies on top of a
substrate containing an immobilized electrophile (e.g., vinyl
methyl ester) would allow covalent attachment of the antibody to
the surface. Such modifications may also be performed in bacterial
cells, viruses, insect cell lines and the like.
[0109] Certain specific examples are described below to illustrate
further the novel and inventive subject matter disclosed herein.
The following materials and methods were used to demonstrate the
compositions and analytical techniques described herein.
[0110] The examples using known hybridomas suggested that it should
be possible to identify rapidly cells producing antigen-specific
antibodies within a polyclonal mixture and retrieve them for clonal
expansion. Mice with peptide-loaded H-2K.sup.b/streptavidin
tetramers, and generated hybridomas by fusion of splenocytes with
NS-1 cells were immunized according to standard protocols.
Following bulk selection of the fused cells, polyclonal mixtures
were loaded into arrays of microwells and incubated on glass slides
coated with goat-anti-mouse IgG antibodies. The resulting
microarrays were stained using tetramers of H-2K.sup.b prepared
with fluorescently-labeled streptavidin (FIG. 16A and FIGS. 17A-B).
Approximately 200,000 cells from the mixtures were screened across
ten microarrays. 50 cells were arbitrarily selected to extract for
expansion from the .about.4,300 positive spots identified on the
microarrays. The cells expanded in the microwells for four days
(doubling every .about.12-24 h) before being extracted using a
micromanipulator and deposited into a 96-well plate. 42 clones
survived extraction and subsequent expansion; of these clones, the
supernatants of 17 of which showed strong responses by indirect
ELISA relative to a control antibody (AF6-88.5).
[0111] The specificity of the antibodies from four clones (c113,
c127, c128, and c136) was tested further by the immunoprecipitation
of properly assembled H-2K.sup.b molecules from detergent extracts
prepared from .sup.35S-methonine/cysteine-labeled EL4 cells (FIG.
16B). Three of the four clones recovered H-2K.sup.b. No detectable
amounts of IgG were present in the supernatants of the clone that
did not recover H-2K.sup.b (c113), suggesting that this clone may
represent a false positive selected from the combination of the
microarray screen and indirect ELISA, or that it had lost its
ability to produce the antibody of interest (chromosome loss or
epigenetic change). The antibodies produced by clones 127, 128, and
136 were all IgG.sub.1.gamma.. SDS-PAGE of denatured
.sup.35S-labeled antibodies produced by clones 127 and 136 showed
single bands that migrated differently for both the heavy and light
chains; these data suggest that the hybridomas produce unique
antibodies. To further evaluate clone 136, the cells were expanded,
frozen, revived, and then used to prepare an engraved microarray
(FIG. 16C and FIGS. 17A-B). Antibodies were captured on a slide
coated with goat-anti-mouse IgG, and the array was probed with both
fluorescent antigen (H-2K.sup.b) and a fluorescent antibody
(goat-anti-mouse IgG). This array showed that all cells in the
population tested produced IgG that was also specific to
H-2K.sup.b.
[0112] Cell culture and purification methods are more specifically
described herein. Cell types EL4, NS-1 (ATCC), HYB 099-01 (Statens
Serum Institut, Copenhagen, Denmark) and Y3 cells were grown in
Dulbecco's Modified Eagle Medium (DMEM) (Gibco, Grand Island, N.Y.)
supplemented with 10% (v/v) fetal calf serum (FCS, Hyclone, Logan,
Utah), 50 units penicillin/50 .mu.g streptomycin, 20 mM HEPES, 50
.mu.M 2-mercaptoethanol, 1 mM sodium pyruvate, and 0.1 mM
nonessential amino acids (Gibco, Grand Island, N.Y.) at 37.degree.
C. (5% CO.sub.2). The cells were split every 2 to 3 days under
sterile conditions.
[0113] Peptides, having the sequences SIINFEKL (SEQ ID NO.:1) and
SIYRYYGL (SEQ ID NO.:2), were synthesized by standard Fmoc-based
solid-phase peptide chemistry, confirmed by MALDI-MS and LC/MS
analysis, and were used directly, without further purification.
Murine .beta..sub.2-microglobulin as well as a fusion protein of
murine class I MHC heavy chain having a C-terminal biotinylation
sequence were individually expressed, purified and reconstituted to
H-2K.sup.b complexes. The recombinant proteins were expressed in
Escherichia coli employing the
isopropyl-.beta.-D-thiogalactopyranoside (IPTG)-inducible pET
vector system (Novagen) and BL21(DE3) as an expression host. The
recombinant proteins were isolated as inclusion bodies and
dissolved under denaturing conditions (8 M of urea). Reconstitution
was performed under dilute conditions in the presence of a large
excess of the appropriate peptide. Subsequently, the monomeric,
soluble H-2K.sup.b-peptide complexes were appended with a biotin
moiety under the agency of BirA biotin ligase (Avidity) and
purified by size exclusion chromatography (Superdex 75, Amersham
Biosciences) to remove aggregates and free biotin. For
immunization, tetrameric complexes were produced through stepwise
addition of the appropriate soluble MHC class I complex to
Streptavidin (Invitrogen) to a final molar ratio of 4:1. For
interrogation of the microarray, tetrameric complexes were formed
in a similar manner using Streptavidin Alexa 546 (Invitrogen) or
Streptavidin Alexa 647 (Invitrogen).
[0114] Balb/c female mice (8 weeks-old, Taconic) were immunized
once every two weeks subcutaneously with an emulsified 1:1 mixture
of antigen (25-35 .mu.g dissolved in 100 .mu.L PBS) and complete
Freund's adjuvant (first immunization only) or incomplete Freund's
adjuvant (Sigma-Aldrich, St. Louis, Mo.). Three days prior to
tissue harvest, antigen (100 .mu.g H-2K.sup.b/tetramers in PBS) was
injected intraperitoneally. The fusion of splenocytes with NS-1
cells was performed according to standard protocols. Media used for
bulk selection were supplemented with 20% (v/v) FCS (Hyclone,
Logan, Utah) and 10% (v/v) Hybridoma Cloning Factor (Bioveris,
Gaithersburg, Md.), hypoxanthine aminopterin thymidine (HAT; ATCC,
Manassas, Va.), hypoxanthine thymidine (HT; ATCC, Manassas, Va.),
and 50% PEG (Sigma-Aldrich).
Example 1
Microengraving
[0115] To prepare the microwells for engraving, cells were
deposited on the surface of the PDMS slab at an appropriate
dilution and allowed to settle before removing excess media, as
shown in FIGS. 13A-B. The number of cells deposited per well
depended on the concentration of cells, the volume applied, the
time allowed for the cells to settle, the size of the microwells,
and the size of the PDMS slab. (FIG. 6A). For slabs of PDMS
.about.25.times.50.times.5 mm.sup.3 containing 25,000 wells (100
.mu.m diameters separated by 100 .mu.m), 0.5 mL of a cell
suspension, ranging from 1.times.10.sup.5 to 5.times.10.sup.5
cells/mL, deposited for three to five minutes yielded one to three
cells in .about.50-75% of the wells (FIG. 5). The percentage of
wells filled (FIG. 7) and the average number of cells per well
(FIG. 8) were determined by counting the amount of cells in
randomly determined fields with a 10.times. lens and averaging the
data collected from each microwell array. Referring to FIG. 7, as
the concentration of cells present increased, the percentage of
wells in the microarray filled by cells also increased. Referring
to FIG. 8, as the concentration of cells present increased, the
average number of cells per well also increased. Referring to FIG.
9, the results observed were consistent with using higher
concentration of cells to increase the average number of cells in
each well and using lower concentrations of cells to favor one or a
few cells in each well. Cells confined in microwells and submerged
in a large reservoir of culture media divided normally every 12-24
h for more than one week in culture.
[0116] Given the established rate of immunoglobulin (Ig) secretion
for plasma cells and their derivatives (5,000 molecules/s), a
single cell confined in a small volume (.about.0.1-1 nL) produced
detectable concentrations of antibodies (.about.0.1-1 .mu.M) in
less than 5 h. Two established hybridomas--Hyb 099-01, anti-chicken
ovalbumin, and Y3, anti-mouse H-2K.sup.b (major histocompatibility
complex (MHC) class I)--were used to test the feasibility of the
method for engraving microarrays of secreted antibodies on a glass
slide. The antibodies secreted were detected in two ways (FIGS.
14A-E). In the first approach, secondary antibodies, goat
anti-mouse Ig (gamma), were immobilized covalently on
epoxide-functionalized slides; these slides were then incubated
with an array of cells for 2 h, and interrogated with a mixture of
fluorescently labeled antigens. Correlating the region of
microwells on the PDMS slab with the matching region of the
microarray data showed that (1) the fluorescent spots corresponded
to the wells containing cells, (2) the level of non-specific
binding of the fluorescently labeled proteins was low in regions
where there were empty wells, and (3) the specificity of the
antibody produced by the individual cells in wells could be
determined from the microarray. (FIGS. 14A-C and FIGS. 12A-C).
[0117] In a second approach similar to indirect ELISA, antigen--in
this case, ovalbumin--was immobilized on slides by covalent
attachment or by non-specific adsorption, the primary antibody was
captured from cells contained in microwells, and the microarray was
stained with a fluorescently labeled secondary antibody (goat
anti-mouse IgG) (FIGS. 14D-E and FIGS. 11A-B). This format of the
assay showed greater sensitivity to variations in the number of
cells per well, and the amount of antibody secreted by individual
cells, than the one using labeled antigens (FIG. 14A). The presence
(or absence) of successful complexes formed between antibody and
specific antigens was determined, and the relative rates of
production by individual cells was assessed.
[0118] Cells confined in microwells and sealed against a glass
slide (such that the total media available was limited to the
volume of the microwell) showed little or no loss in viability for
up to 5 h. (FIG. 10). When a PDMS slab was removed from a glass
slide and immersed in media, the cells remained loosely adhered to
the bottom of the wells; vigorous rinsing or intentional extraction
of the cells was required to dislodge them from the wells.
[0119] Determining the time-dependent variability of secretion from
single cells by Elispot, FACS, ELISA, or other immunochemical
methods is challenging. Because the cells in the microwells remain
viable after printing, the same set of microwells were used to
produce multiple copies of an engraved microarray at different time
points. A set of microwells were incubated on a glass slide and was
coated with secondary antibodies for 2 h. The microwells were
removed and rinsed gently with fresh media, and the microwells were
incubated on a second glass slide for another 2 h. (FIGS. 15A-B).
The minimum incubation period is about 1 min to about 10 min, e.g.,
30, 45 sec, 1, 2, 3, 5, 8 min. Qualitative inspection of the
resulting microarrays indicates that they have a high degree of
similarity, but are not identical; variations between specific
spots may result from either fluctuation in the rate of secretion
related to various stages of cell division or cell death. The total
number of replicas that can be made is about 100, more particularly
about 4 to about 10 replicas using the same set of cells.
[0120] Microarray fabrication and methods for rapid high-throughput
screening are described herein. The microwell arrays were
fabricated in poly(dimethylsiloxane) (PDMS, Sylgard 184, Dow
Corning) using photolithography and replica molding. One layer of
photoresist (SU-8-100, Microchem, Newton, Mass.) was patterned on a
3 inch silicon wafer using a transparency photomask (CAD/Art
Services, Bandon, Oreg.) to produce a master with a positive relief
pattern of the microwell array. To facilitate removal of PDMS in
subsequent steps, the masters were silanized by treatment with
(tridecafluoro-1,1,2,2-tetrahydrooctyl)-1-trichlorosilane (UCT,
Bristol, Pa.) in a vacuum desiccator for 1 h. PDMS was cast onto
the master, cured for 2 h at 60.degree. C., and peeled away. The
PDMS was allowed to swell in hexanes for 24 h, deswell in acetone
for 24 h, and then dry in an oven at 130.degree. C. for 24 h to
remove low-molecular weight oligomers. The microwell array was
treated with an oxygen plasma (PDC-32G, Hayrick, Ithaca, N.Y.) for
20 seconds; this process also sterilizes the array. The
plasma-treated device was immersed in a solution of 10% w/v Bovine
Serum Albumin (BSA) (0.01% sodium azide) for 1 h at room
temperature and then rinsed with sterile phosphate buffered saline
(PBS, Gibco, Grand Island, N.Y.). The estimated variation in height
is less than 5%, the wells in the center of the array are taller
than those on the outer edge of the array. The phase contrast
images indicate the lateral dimensions vary less than 2%, the top
of a single well is wider than the bottom of the same cell.
[0121] Glass slides (1''.times.3'', VWR brand) were prepared and
cleaned in "piranha" solution (conc. H.sub.2SO.sub.4:30%
H.sub.2O.sub.2, 7:3) at 70.degree. C. for at least 1 hour. The
slides were thoroughly rinsed with deionized water (Millipore, 18
M.OMEGA.) and immersed in an ethanolic solution containing
3-glycidoxypropyltrimethoxysilane (95% ethanol, 1% v/v silane, pH
adjusted to 4.5 using glacial acetic acid) at room temperature. The
slides were removed after 1 hour, rinsed twice in 95% ethanol, and
dried in an oven at 130.degree. C. for 12 h. A solution of antigen
(10-100 .mu.g/mL ovalbumin (Sigma-Aldrich) or K.sup.b-streptavidin
tetramers,) or secondary antibody (200 .mu.g/mL Goat anti-mouse Ig
(gamma) Zymed, San Francisco, Calif.) in PBS was deposited on the
surface of a slide under a coverslip (LifterSlip, Erie Scientific
Company, Portsmouth, N.H.) and incubated overnight at 4.degree. C.
Following incubation, the slides were immersed in blocking buffer
(PBS, 0.01% w/v NaN.sub.3, 1% w/v BSA) for 1 h at 25.degree. C. or
stored overnight at 4.degree. C. The slides were rinsed in
PBS/Tween 20 (0.05% w/v, PBST), PBS, and then deionized water. They
were spun dry for 5 min at 750 rpm immediately before being sealed
against the microwell arrays.
[0122] A suspension of cells was then diluted to 1.times.10.sup.5
cells/mL in serum-containing media and 0.5-1 mL was pipetted onto
the surface of the microwell array. The cells were allowed to
settle for 3 minutes. The surface of the array was dewetted by
applying a piece of extra thick filter paper (Bio-Rad, Hercules,
Calif.) or by vacuum aspiration at one edge of the array while
tilting the array. The percentage of wells filled and the average
number of cells per well were determined by counting the number of
cells in randomly-determined viewing fields with a 10.times. lens
and averaging data collected from multiple microwell arrays.
[0123] To engrave the microarray, an array of microwells filled
with cells and dewetted of excess media was placed well-side-down
on the surface of a treated, dry glass slide. The combination of
the array and glass slide was sandwiched together in a
hybridization chamber (DT-1001, Die-Tech, San Jose, Calif.); the
screws used to clamp the chamber together were tightened just until
finger-tight. The entire assembly was incubated at 37.degree. C.
for 2-4 h. After incubation, the treated glass slide was removed
from the surface of the microwell array and immediately immersed in
blocking buffer (1% BSA/0.05% Tween 20/PBS), and agitated for 1 h
at room temperature. After placing the glass slide in blocking
buffer, the microwell array was quickly immersed in a bath of
pre-warmed media before media contained in the microwells
completely evaporated. (FIGS. 6A-E)
[0124] After blocking in preparation for interrogation of the
microarray, glass slides were rinsed with PBST, PBS, and then
deionized water for 5 min each; the slides were spun dry for 5 min
at 750 rpm. A solution of either goat anti-mouse secondary antibody
(Alexa Fluor 488 or 532, Invitrogen) or fluorescent antigen (e.g.
10 .mu.g/mL Ovalbumin-Alexa Fluor 488 or 555 conjugate (Invitrogen)
or K.sup.b tetramers prepared using streptavidin-Alexa 546 or 647
(Invitrogen) in PBS (80 .mu.l)) was deposited on the surface of a
slide under a coverslip (LifterSlip, Erie Scientific Company,
Portsmouth, N.H.) and incubated in the dark for 1 h at room
temperature. The slides were rinsed with PBST, PBS, and deionized
water, and spun dry for 5 min at 750 rpm. The slides were imaged
with a GenePix 4000B microarray scanner (Molecular Devices,
Sunnyvale, Calif.) using 532 and 635 nm lasers and
factory-installed emission filters. The lasers were used at 100%
power and the PMT gain was set between 600 and 900 to maximize the
dynamic range of the detector without saturation. Images of the
microarrays were analyzed using GenePix Pro 6.1 (Molecular Devices,
Sunnyvale, Calif.). Color ratio images were generated in GenePix
and saved in the red and green channels of a 24-bit TIFF file.
Background intensities were subtracted using median values measured
in regions between individual spots of the array. The
signal-to-noise ratio for a given positive spot in the array to
negative (or background) spots within the same subarray was
calculated by dividing the sum of the median intensity values from
each channel for a given spot by the average sum of median
intensity values for the negative spots determined from at least 20
negative spots. The mean values reported are the average of at
least 128 positive spots from more than 12 subarrays.
[0125] In order to perform microscopy and micromanipulation, phase
contrast images were acquired using Metamorph software (v6.3r3,
Molecular Devices, Sunnyvale, Calif.) and an inverted microscope
(Nikon Eclipse TE2000-E) equipped with a Hamamatsu ORCA AG camera.
Cells were retrieved from individual wells using a micromanipulator
(IM-9A, Narishige, Tokyo, Japan) fitted with hand-drawn capillaries
(GC-1). To withdraw the contents of a well, the array of microwells
was positioned on the microscope under a layer of media (.about.1
mL), and a capillary with an outer diameter of 100 .mu.m (inner
diameter .about.50 .mu.m) was positioned directly over the top of
an appropriate well. A small volume (.about.1-5 .mu.L) was
withdrawn with the affixed syringe until the cells were removed
from the well successfully. The tip was then transferred into a
well of a 96-well plate containing 200 .mu.L media (10% hybridoma
cloning factor) and the cell(s) expelled into the volume. Both
extraction from the microwell and deposition of the cells into
another container (96-well plate) were monitored visually to ensure
the transfer of the cells into and out of the tip.
Example 2
Identification of Antibodies Specific for Surface Epitopes of
Infectious Agents
[0126] The methods described herein are useful to identify
antibodies reactive against surface-exposed antigens present on
infectious agents (bacteria, viruses, fungi), e.g., i) to identify
new therapeutic agents for use in passive immunizations and ii) to
discover candidate antigens for the development of new vaccines
intended to invoke a protective humoral immune response. Memory B
cells from convalescent human patients for other diseases, e.g.,
Aspergillus and malaria, are immortalized and their secreted
products are screened using the engraved microarray. A schematic
illustration (FIG. 18) of the method for identifying antibodies
that bind surface-expressed epitopes on a pathogen. B cells are
derived from an inoculated animal or a blood sample from a
convalescent patient and screening with different serotypes or
mutants of the pathogens should bias the screen for specific types
of antibodies--for example, serotype-independent ones.
[0127] Microarrays of antibodies from a polyclonal mixture of cells
are screened using whole pathogens; competitive assays using
multiple serotypes or genetic variants allow systematic analysis of
a range of pathogens (bacteria, fungi, viruses). Subsequent
characterization of the antigens recognized by identified
antibodies can provide a catalog of candidate antigens for
developing a vaccine against the agent. The antibodies themselves
are useful for passive immunization strategies. For example, target
antigens are identified using a mouse model and a human pathogen,
e.g., opportunistic fungal pathogen (Cryptococcus neoformans) that
affects immunocompromised humans. Another strategy utilizes
screening transformed populations of memory B cells from
convalescent individuals. Translational research is used to assess
value of identified antibodies or epitopes for passive immunization
or development of vaccines.
[0128] Using a mouse model for human disease, mice are immunized
with fixed (or heat-inactivated) pathogens or inoculated with
sub-lethal dosages by appropriate routes (e.g., inhalation,
sub-cutaneous injection). Hybridomas are prepared from splenocytes
by poly(ethylene-glycol)-mediated fusion with mouse myeloma cells
(NS-1, Sp2/0). After bulk chemical selection of the surviving
hybridomas, the growing polyclonal populations are loaded into
microwells and used to engrave arrays of polyclonal antibodies
(FIGS. 13A-D). A unique advantage that engraved microarrays afford
over other methods for screening antibodies against antigens
(enzyme-linked immunosorbant assays, flow cytometry) is the ability
to design the probes for the array in a manner that biases the
search for specific reactivity (FIG. 19A). On arrays generated
using hybridomas prepared from mice challenged with C. neoformans,
differentially-labeled variants of C. neoformans is used to
identify two types of antibodies: 1) serotype-independent
antibodies, and 2) antibodies recognizing antigens other than the
glucuronoxylomannan layer (GXM) that masks much of the exposed
surface on C. neoformans. For the first type, there are three
serotypes of C. neoformans known to cause human disease (A, B, and
C). Each variant is labeled with a different fluorophore (e.g.,
N-hydroxyl-succinimide Alexafluor 488, 532, or 647) and the array
is incubated with a suspension of these yeast, which lead to some
antibodies capable of binding all three serotypes.
[0129] In a second approach, drug-treated wildtype variants are
used to identify antibodies recognizing epitopes on the surface
other than GXM. Common anti-fungal agents, echinocandins, can
disrupt the formation of the GXM layer, and allow access to the
underlying cellular surface. (FIG. 19B). Such antibodies could
supplement the use of drugs to treat an infection by improving the
ability of the host's immune system to clear cells lacking mannan
layers.
[0130] Antigen-specific clones are identified and retrieved by
micromanipulation. The clonality and diversity of the antibodies
produced are characterized by isotype, by SDS-PAGE gel
electrophoresis of immunoprecipitated .sup.35S-labeled antibodies,
and by genetic sequencing. Also, in vitro assays are used to
determine the extent to which individual monoclonal antibodies, or
oligoclonal mixtures, confer protection via enhanced opsonization
and phagocytosis of C. neoformans by macrophages or dendritic
cells. A transgenic mouse that cannot produce circulating
immunoglobulins (AID-/-, .mu.S-/-) provides a background for
measuring the usefulness of passive immunization in vivo without
convolving a host-derived antibody response.
[0131] The disclosed method, apparatus and kits identifies epitopes
recognized by neutralizing antibodies. The selected antibodies are
those recovered antibodies that recognize a specific protein from
the pathogen by immunoprecipitation or immunoblotting
detergent-solubilized lysates of the bacteria, virus, or fungus.
Specific staining with the monoclonal antibody and subsequent
analysis by mass spectrometry allows identification of the protein
recognized. Additional analysis using synthetic peptide libraries
based on the sequence of the protein likely is used to refine the
identity of a specific epitope, i.e., fine mapping.
[0132] In the event that an antibody binds the intact pathogen, but
does not immunoprecipitate or stain a protein from a lysate, the
identity of the antigen is determined as follows. First, for
viruses and yeast, the whole microbe and corresponding lysates are
treated with glycosidases, and subsequently, treated with the
antibody to determine if binding is conserved after
deglycosylation. This approach has led to successful recognition of
carbohydrate epitopes essential for binding anti-HIV antibodies
specific for gp120 (2G12 epitope). Second, for bacteria,
identification of non-proteinaceous epitopes would require
disruption of pathways involved in surface-expression of
glycolipids or other cell-wall components. For many bacterial
pathogens (e.g., Salmonella enteritidis), a range of mutant strains
exist that are used to deduce the component recognized by an
antibody.
[0133] Though mouse and humanized antibodies have had utility as
therapeutic agents, it is increasingly understood that fully human
antibodies induce fewer side effects than chimeric or transgenic
ones. The method, described herein to identify and retrieve
transformed human memory B cells producing human antibodies. Blood
samples from convalescent individuals recovered from infectious
diseases, e.g., Aspergillus, malaria, and influenza and transform
the populations of human memory B cells in these samples by
incubation with Epstein-Barr virus and a cocktail of suitable
stimulants (CpG DNA, cytokines). Secreted human IgG by
microengraving from such cells can be detected using the disclosed
method, apparatus and kits (FIG. 20). These methods identify
antibodies for use in passive immunizations, and identify what
epitopes on a pathogen instigate a humoral immune response.
Example 3
Simultaneous Determination of Phenotype and Functional Behavior of
Individual Primary Cells
[0134] Another application for the disclosed method, apparatus and
kits is the determination of the phenotype of a cell and its
functional behavior (e.g., secretion of extracellular factors)
using soft lithography for measuring secreted factors from large
numbers of single cells (>100,000) and for correlating them with
the phenotypic markers displayed on the producing cell.
Traditionally, these characteristics for cells extracted from
tissue or blood are established by independent methods--for
example, flow cytometry and immunosorbant assays--that provide
information about either phenotype or function, but rarely both.
Furthermore, existing methods for measuring functional behaviors,
such as secretion of cytokines, often require additional
manipulations and culturing to generate sufficient material from a
bulk population of cells for detection. The graphical abstract
(FIG. 21) suggests how a soft lithographic technique could provide
both characteristics for a large number of cells. Comparison of the
frequency of specific cells present after onset of a disease or
administration of a vaccine would provide a profile of how an
individual is responding relative to others. Extraction of specific
cells by micromanipulation would allow genetic sequencing of unique
markers (e.g., B cell or T cell receptors). Allowing a single assay
to measure both the phenotype and secretory function of individual
cells
[0135] The method, apparatus and kits described may be extended for
identifying and retrieving monoclonal hybridomas secreting
antigen-specific antibodies so that a highly multiplexed platform
for profiling large numbers (>100,000) of primary cells is
generated on the basis of both secreted factors and surface markers
(FIG. 22). Minimally, four unique secreted factors and phenotypic
markers, are realized and detected with an aim to detect at least
10. The experiments described here establish the method using T and
B cells from various tissues of mice, which can be extended to
analyze human lymphocytes derived from blood samples.
[0136] The method described herein utilizes the microengraving
method, apparatus and kits described, however, the immunosorbant
capture of cytokines on the glass slide likely will require a
sandwich-style format in which an antibody immobilized on the
surface captures the factor of interest and a second antibody,
reactive against a different epitope, is used for labeling. Though
cytokines are typically secreted from cells at a rate that is
1000-fold less than that for antibodies produced by hybridomas or
plasma cells, a single cell confined in a microwell of
50.times.50.times.50 .mu.m.sup.3 generates sufficient cytokines for
a concentration of 25 ng/mL within 4 h. The nature of the assay
requires immobilization of N different capture antibodies uniformly
on the surface of the slide, where N is the number of secreted
factors one aims to detect. This constraint may reduce the signal
available from each secretion by a factor equal to the number of
different cytokines probed. For two capture antibodies, the
available surface area and reactive antibodies is sufficient for
detection (FIG. 23). The composition of deposition buffers and
testing alternative immobilization strategies can be altered to
maximize the number of available sites for capture.
[0137] Multiplexed detection using fluorescent materials is limited
by the number of emitted wavelengths that can be cleanly
distinguished from one another. For traditional organic dyes and
standard optical filters, the limit is approximately four. Two ways
to extend this range are 1) the use of quantum dots as labels, and
2) spectral imaging (or deconvolution of overlapping signals).
Quantum dots provide the optimal material for detecting a multitude
of cytokines. Conjugated polyclonal, affinity-purified antibodies
are directed against different cytokines to commercially-available
quantum dots of various colors (Invitrogen or Evident Technologies)
using N-hydroxylsuccinimide esters. Optimization of the labeling
conditions and subsequent analysis of cross-reactivity is tested
using spotted arrays of recombinant cytokines captured on glass
slides slabing appropriate mixtures of capture antibodies.
IFN.gamma. (Th1 response), IL-2 (activated lymphocytes), IL-13 (Th2
response), and IL-10 (regulatory cells) are measured.
[0138] Both hybridomas and mononuclear cells from blood remain
loosely adhered to the bottom surface of the poly(dimethylsiloxane)
(PDMS) microwells during manipulations of the wells. Because the
cells can remain in the wells, standard protocols are used for
immunofluorescence to analyze extra- or intracellular proteins
present on the cells. Scanning arrays of microwells containing
labeled cells will require a microscope equipped with an automated
stage and focus. Commercial instruments for laser-scanning
cytometry on glass slides is suitable for the analysis, however,
custom-designed optics can be tailored to allow the greatest degree
of flexibility in analysis.
[0139] An exemplary analysis is carried out as follows. Cells,
e.g., splenocytes from a mouse (e.g., C57B1/6) are obtained and the
number of cells with specific phenotypic markers and their
secretion patterns determined by microengraving. For the evaluation
of immune responses, the focus is initially on T cell markers (CD4
and CD8), and two chemokines (IFN.gamma. and IL-13). FACS analysis
provides confirmation of the frequency of cells. Intracellular
staining for IFN.gamma. and IL-13 gives an indication of the
relative number of cells producing each of those cytokines.
Splenocytes taken from transgenic OTII mice, which have CD4+ T
cells expressing a T cell receptor specific for a peptide fragment
derived from ovalbumin are used as an example. Immunization of
these mice with ovalbumin, or cell cultures stimulated in vitro
with the antigen and .alpha.IFN.gamma. antibodies, skew the immune
response to develop a Th1 or Th2 response.
[0140] The example experiment allows confirmation of a hypothesis
suggested by Sallusto et al. that the age of a mature dendritic
cell determines the likelihood of an interacting CD4+ T cell to
polarize into Th1- or Th2-type. (Langenkamp, A., Messi, M.,
Lanzavecchia, A. & Sallusto, F. Kinetics of dendritic cell
activation: impact on priming of TH1, TH2 and nonpolarized T cells.
Nat. Immunol. 1, 311-316 (2000)). Dendritic cells from a donor
mouse are isolated and exposed to ovalbumin in the presence of
lipopolysaccharide for 8-48 h. After loading with antigen, the
cells are transferred into an OTII mouse by intravenous tail
injection. After 1-2 days, the frequency of Th1- and Th2-polarized
T cells present in the lymph nodes and the spleen are measured as a
function of the maturation time of the injected DCs. The DCs
matured more than 12 h should induce a greater percentage of
Th2-polarized or non-polarized T cells than DCs matured for less
than 12 h.
[0141] The data demonstrated the development and verification of
the technique for connecting phenotype with functional responses
for large numbers of individual primary cells. The application of
the technology is useful to profile immunological responses as a
function of disease. It may be used in the context of transgenic
mice with model diseases (diabetes, mouse pathogens), and to
evaluate immune responses to human diseases, e.g., opportunistic
fungal pathogens (e.g., Aspergillus), malaria, influenza, and
diabetes using blood samples. Because the cells can be retrieved
from the microwells by micromanipulation, the technique may be
extended to include genetic sequencing of unique features from
single cells identified in a screen, e.g., B cell or T cell
receptors.
[0142] The application of the disclosed methods, apparatus, and
kits will provide a tool for monitoring an immune response directly
by determining the efficacy of a vaccine or correlating the
frequencies of particular types of cells upon onset of a certain
disease. The ability to use limited numbers of cells from a blood
sample is particularly suited for use as a diagnostic tool to
detect early genetic defects in pediatrics (e.g.,
immunodysregulation, polyendocrinopathy, and enteropathy, X-linked
syndrome (IPEX)--a deficiency of CD4+CD25+ regulatory T cells). The
relative ease of processing a sample by this method allows
inexpensive diagnostic applications in clinical microlabs and/or
third-world countries. The methods are useful to characterize
cellular identity and behavior in patient-derived samples from
individuals suffering from or at risk of developing infectious
diseases, cancer, or neurological disorders.
Example 4
Engineering Antibodies to Improve their Orientation on Surfaces
[0143] To direct the orientation of cell secreted products relative
to the surface on which they are printed, a nucleic acid sequence
encoding a short peptide recognition sequence (5-15 residue, e.g,
8-10 residue sequence) is incorporated into a gene encoding the
secreted polypeptide, e.g., the heavy and/or light chain of an
immunoglobulin chain. An enzymatic or other chemical reaction
installs a unique (orthogonal) chemical moiety on the short peptide
sequence that would provide a specific site for attaching the
secreted product, e.g., antibody, to a solid support or other
scaffold (e.g., another molecule, enzyme, or polymer matrix). The
enzyme inducing the conversion or chemical modification is encoded
in the cell itself or is provided externally, e.g., extracellularly
in the culture medium. A transgenic mouse carrying this
modification is used to produce hybridomas containing this
"chemical handle" without additional cloning or reengineering.
[0144] One example of such a peptide recognition sequence for a
selected enzyme and the enzyme (BirA ligase). Another example is
sortase, a transpeptidase encoded by many bacteria such as
Staphylococcus aureus. Sortases recognize a 5-amino acid peptide
motif, LPXTG (SEQ ID NO.: 3). Two other classes of enzymes that
recognize short peptide motifs are transglutaminases and lipoic
acid ligases. The modifications are made to either terminus of
either the heavy or light chains, e.g., the C-terminus of the heavy
or light chains contains the chemical tag.
[0145] Immunoglobulins (Ig) are engineered to contain specific
chemical sites for the purpose of improving orientation and
accessibility of binding domains in surface-based and
nanoparticle-based assays. Transgenic mice that contain the
sequence encoding the sites for chemical modification are useful in
making hybridomas that produce antibodies containing appropriate
sites for defined ligation to surface-based assays or other
molecular constructs (drugs, labels) without requiring additional
cloning (FIG. 24).
[0146] The methods, apparatus and kits disclose a method for
attaching antibodies to surfaces using a chemical functional group
installed at the C-terminus of the heavy chain of an Ig-gamma (IgG)
(FIG. 25). Attaching an antibody modified in this manner to a
surface directs its orientation in a predictable manner, and thus
improves functionality. For example, molecular cloning is used to
insert a 15-amino acid peptide sequence (GLNDIFEAQKIEWHE (SEQ ID
NO.: 4)) at the C-terminus of a secreted product such as an
antibody. An enzyme, biotin ligase (BirA), produced by Escherichia
coli ligates either biotin or a related ketone analog at the lysine
residue in the peptide. Modification of the Ig heavy chain with the
ketone analog will allow site-specific attachment using a hydrazide
moiety present on the surface. This approach is used for
immobilizing antibodies on self-assembled monolayers (SAMs)
supported on gold or palladium.
[0147] A stably-transfected cell line, is generated that expresses
an antibody with an appended amino-acid sequence at the C terminus
of the heavy chain. The variable regions of the heavy and light
chains (IgG1, .kappa.) expressed by a mouse hybridoma, Hyb 9901
(anti-chicken ovalbumin), from mRNA are then cloned. These
fragments are subcloned into mammalian expression vectors
containing the constant regions for the heavy and light chains. At
the 3' end of the sequence for the heavy chain a sequence encoding
a 7 amino-acid epitope is inserted, which recognized by tobacco
etch virus (TEV) protease (ENLYFQ/S (SEQ ID NO.: 5) where/indicates
cleavage site) followed by the epitope recognized by the BirA
ligase (GLNDIFEAQKIEWHE (SEQ ID NO.: 6)). Expression of the
antibody is induced by transfection of the two plasmids into 293T
or CHO cells. Proper assembly of secreted antibodies is verified by
ELISA using immobilized ovalbumin.
[0148] BirA ligase from E. coli cultures is expressed and purified
and then an analog of biotin containing a ketone is synthesized
according to reported protocols. To ligate the analog to the
modified antibodies collected from the cell cultures, a buffered
solution of the antibodies is incubated with BirA, biotin analog,
and ATP. Successful modification of the antibodies is confirmed by
Western blot analysis using anti-mouse IgG to identify the heavy
chain and anti-biotin antibodies (or streptavidin).
[0149] SAMs are prepared on thin films (20 nm) of gold or palladium
using a mixture of two thiols, (1-Mercapto-11-undecyl) tri(ethylene
glycol) and a hydrazide-terminated derivative,
HS(CH.sub.2).sub.11(OCH.sub.2CH.sub.2).sub.6OCH.sub.2CO.sub.2NHNH.sub.2.
The density of sites for attachment are controlled by varying the
molar ratio of the two thiols used to prepare the SAM. Exposure of
the surface to a buffered solution of the modified antibodies will
lead to ligation (FIG. 25). The relative density of functional
antibodies immobilized on the SAMs is measured by surface plasmon
resonance using SAMs with antibodies immobilized by standard
protein coupling methods (e.g., EDC-NHS ester) as controls.
[0150] The example described here establishes a method for
attaching antibodies to surfaces using a specific chemical ligation
at the C-terminus of the heavy chain. Using this example, the
modification of gold nanoparticles or quantum dots with the
engineered antibodies may be conducted. A transgenic mouse
incorporating the peptide tail at the C terminus of the IgG1
constant region is generated. Thus, all IgG1 antibodies generated
by hybridomas from these mice incorporate the specific handle for
oriented attachment without additional cloning or modifications.
Such antibodies would have an intrinsic site for subsequent
modifications (orthogonal labeling, monovalent ligation of a drug
or enzyme).
[0151] When introducing elements of the examples disclosed herein,
the articles "a," "an," "the" and "said" are intended to mean that
there are one or more of the elements. The terms "comprising,"
"including" and "having" are intended to be open ended and mean
that there may be additional elements other than the listed
elements. It will be recognized by the person of ordinary skill in
the art, given the benefit of this disclosure, that various
components of the examples can be interchanged or substituted with
various components in other examples. Should the meaning of the
terms of any of the patents or publications incorporated herein by
reference conflict with the meaning of the terms used in this
disclosure, the meaning of the terms in this disclosure are
intended to be controlling.
[0152] Although certain aspects, examples and embodiments have been
described above, it will be recognized by the person of ordinary
skill in the art, given the benefit of this disclosure, that
additions, substitutions, modifications, and alterations of the
disclosed illustrative features, aspects, examples and embodiments
are possible.
* * * * *