U.S. patent application number 09/302920 was filed with the patent office on 2001-07-19 for multiplex flow assays preferably with magnetic particles as solid phase.
Invention is credited to EDWARDS, RICHARD B., WATKINS, MICHAEL I..
Application Number | 20010008217 09/302920 |
Document ID | / |
Family ID | 25519815 |
Filed Date | 2001-07-19 |
United States Patent
Application |
20010008217 |
Kind Code |
A1 |
WATKINS, MICHAEL I. ; et
al. |
July 19, 2001 |
MULTIPLEX FLOW ASSAYS PREFERABLY WITH MAGNETIC PARTICLES AS SOLID
PHASE
Abstract
Heterogeneous assays for different analytes in a single
biological sample are performed simultaneously in a multiplexed
assay that combines flow cytometry with the use of magnetic
particles as the solid phase and yields an individual result for
each analyte. The particles are distinguishable from each other by
characteristics that permit them to be differentiated into groups,
each group carrying an assay reagent bonded to the particle surface
that is distinct from the assay reagents of particles in other
groups. The magnetic particles facilitate separation of the solid
and liquid phases, permitting the assays to be performed by
automated equipment. Assays are also disclosed for the simultaneous
detection of antibodies of different classes and a common antigen
specificity or of a common class and different antigen
specificities. Each type is accomplished by immunological binding
at the surfaces of two distinct solid phases in a sequential manner
with dissociation of the binding and washing of the solid phase in
between the binding steps.
Inventors: |
WATKINS, MICHAEL I.;
(VACAVILLE, CA) ; EDWARDS, RICHARD B.; (VACAVILLE,
CA) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW
TWO EMBARCADERO CENTER
EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Family ID: |
25519815 |
Appl. No.: |
09/302920 |
Filed: |
April 30, 1999 |
Current U.S.
Class: |
210/222 ; 435/5;
435/7.1; 436/518; 436/526 |
Current CPC
Class: |
Y10T 428/2984 20150115;
G01N 33/54333 20130101; Y10S 435/973 20130101; G01N 33/56994
20130101; G01N 33/545 20130101; G01N 33/6854 20130101; G01N
33/54326 20130101; G01N 33/582 20130101; Y10T 428/2982 20150115;
Y10T 428/2991 20150115; Y10T 428/2989 20150115; Y10T 428/2987
20150115; G01N 33/537 20130101 |
Class at
Publication: |
210/222 ;
436/518; 435/5; 435/7.1; 436/526 |
International
Class: |
C12Q 001/70; G01N
033/53 |
Claims
We claim:
1. A method for individually detecting a plurality of analytes in a
single fluid biological sample by assays that include the binding
of species in said biological sample to a solid phase that is in
contact with a liquid medium in which said solid phase is insoluble
and the separation of said solid phase from said liquid medium,
said method comprising: using as said solid phase a plurality of
microparticles of magnetically responsive material each with an
assay reagent coupled thereto that is selectively active in an
assay for one of said analytes, said microparticles classifiable
into groups differing by the value of a selected differentiation
parameter whose value is detectable, each group distinguishable
from other groups by the value of said parameter and by the assay
reagent coupled to the microparticles of said group; magnetically
separating microparticles in all of said groups from said liquid
medium; and defining said liquid medium as a first liquid medium,
suspending said microparticles separated therefrom in a second
liquid medium, analyzing said microparticles in said second liquid
medium in accordance with both the value of said selected
differentiation parameter and said plurality of assays, thereby
achieving individual detection of said analytes in said biological
sample.
2. A method in accordance with claim 1 in which said selected
differentiation parameter is particle size and differentiation
among said groups is performed by flow cytometry.
3. A method in accordance with claim 1 in which one of said assay
reagents coupled to said solid phase is a binding protein specific
for one of said analytes, said method includes adding to said first
liquid medium a detectable label that binds to said solid phase,
and said magnetic separation comprises separating detectable label
that is bound to said solid phase from unbound detectable label
that is suspended in said first liquid medium.
4. A method in accordance with claim 3 in which said detectable
label is added to said first liquid medium as a conjugate with an
additional quantity of said one analyte, causing said conjugate and
said one analyte in said sample to compete for said binding protein
in a competitive assay.
5. A method in accordance with claim 3 in which said detectable
label is added to said first liquid medium or said second liquid
medium as a conjugate with an additional quantity of said one
analyte after a first incubation period, causing said conjugate to
react with those sites of said binding protein not occupied by said
one analyte in said sample in a sequential assay.
6. A method in accordance with claim 3 in which said binding
protein is defined as a first binding protein, and said detectable
label is added to said first liquid medium as a conjugate with a
second binding protein that is also specific for said one analyte,
causing said one analyte to bind to both said first binding protein
and said conjugate in a sandwich assay.
7. A method in accordance with claim 6 in which said one analyte is
an antigen, said first binding protein is a first antibody to said
antigen, and said second binding protein is a second antibody to
said antigen.
8. A method in accordance with claim 1 in which one of said assay
reagents is an antigen bound to said solid phase and specific for
one of said analytes in said first liquid medium which is an
antibody of a selected immunoglobulin class, and said separation
comprises separating said microparticles with said one analyte
bound thereto from antibodies of said selected immunoglobulin class
other than those specific for said bound antigen, in said first
liquid medium prior to contacting said microparticles with a second
liquid medium containing a detectable label that binds to all
members of said selected immunoglobulin class.
9. A method in accordance with claim 1 in which analysis of said
microparticles is performed by fluorescence detection.
10. A method in accordance with claim 1 in which said analytes in
said assays are detected by the use of a phycoerythrin-labeled
binding member and analysis of said microparticles is performed by
detection of fluorescence of said phycoerythrin label.
11. A method in accordance with claim 2 in which said range is a
diameter from about 0.3 micrometers to about 100 micrometers.
12. A method in accordance with claim 2 in which said range is a
diameter of from about 0.5 micrometers to about 40 micrometers.
13. A method in accordance with claim 2 in which the standard
deviation of the particle diameters of each of said subranges is
less than one-third of the separation of the mean diameters of
adjacent subranges.
14. A method in accordance with claim 1 in which said selected
differentiation parameter is fluorescence decay time, and
differentiation among said groups is performed by time-resolved
fluorescence detection.
15. A method in accordance with claim 1 in which said selected
differentiation parameter is degree of light scatter.
16. A method in accordance with claim 1 in which said selected
differentiation parameter is intensity of fluorescence.
17. A method in accordance with claim 1 in which said selected
differentiation parameter is a combination of forward light
scatter, lateral light scatter, and fluorescence intensity at a
plurality of wavelengths.
18. A method in accordance with claim 1 in which said selected
differentiation parameter is absorbance.
19. A method in accordance with claim 1 in which said selected
differentiation parameter is the relative quantities of particles
in each group.
20. A method in accordance with claim 1 in which said
microparticles are comprised of a combination of a polymer and a
paramagnetic substance.
21. A composition comprising a plurality of solid-phase assay
reagents selectively active in a plurality of assays each for a
different analyte, each said solid-phase assay reagent comprising a
binding species that is selective active in a single assay and
coupled to one of a plurality of microparticles of magnetically
responsive material, the sizes of said microparticles varying in
size over a range that is an aggregate of a plurality of subranges,
each subrange distinguishable from other subranges of said
aggregate by flow cytometry and by the binding species coupled
thereto.
22. A composition in accordance with claim 21 in which said range
is a diameter from about 0.3 micrometers to about 100
micrometers.
23. A composition in accordance with claim 21 in which said range
is a diameter of from about 0.5 micrometers to about 40
micrometers.
24. A composition in accordance with claim 21 in which the standard
deviation of the particle diameters of each said subrange is less
than one third of the separation of the mean diameters of adjacent
subranges.
25. A composition in accordance with claim 21 in which said
microparticles have a porosity substantially less than
macroporous.
26. A composition in accordance with claim 21 consisting
essentially of from two to 100 binding species, each selectively
active in a single assay relative to the remaining binding
species.
27. A composition in accordance with claim 21 in which said
microparticles are comprised of a combination of a polymer and a
paramagnetic substance.
28. A composition in accordance with claim 27 in which said
paramagnetic substance is a metal oxide.
29. A composition in accordance with claim 27 in which said polymer
is formed from monomers including carboxylate groups to permit
covalent bonding of assay binding members at the microparticle
surface.
30. A method for the analysis of a sample to simultaneously yet
individually detect antibodies of different classes that have a
single common antigen specificity, said method comprising:
contacting said sample with a first solid phase coated with said
antigen to cause antibodies in said sample to bind to said antigen;
washing said first solid phase to isolate said first group from
unbound species; suspending said first solid phase thus washed in a
liquid suspending medium and releasing said antibodies from said
antigen into said medium to form a supernatant containing unbound
multiple-class antibodies specific to said antigen; isolating said
supernatant from said first solid phase and contacting said
supernatant with a second solid phase, said second solid phase
comprising one or more portions each coated with an immunological
binding member with specific binding affinity for a single antibody
class, and each portion being capable of differentiation from other
such portions; and individually detecting the occurrence of
immunological binding between said antibody classes and said
immunological binding members while differentiating between said
portions.
31. A method in accordance with claim 30 in which said first solid
phase is a plurality of microparticles.
32. A method in accordance with claim 30 in which said second solid
phase is a plurality of microparticles.
33. A method in accordance with claim 30 in which said first and
second solid phase s are each a plurality of microparticles.
34. A method in accordance with claim 30 in which said occurrence
of immunological binding is detected using labeled class-specific
antibodies.
35. A method in accordance with claim 30 in which said occurrence
of immunological binding is detected using phycoerythim-labeled
class-specific antibodies and individual detection is achieved by
fluorescence detection.
36. A method in accordance with claim 30 in which said occurrence
of immunological binding is detected using labeled analogs of said
antigen.
37. A method in accordance with claim 30 in which said occurrence
of immunological binding is detected using phycoerythrin-labeled
analogs of said antigen and individual detection is achieved by
fluorescence detection.
38. A method in accordance with claim 31 in which said
microparticles are of magnetically responsive material, and said
washing is facilitated by magnetically separating said first group
of microparticles from said sample.
39. A method in accordance with claim 31 in which said
microparticles are of magnetically responsive material, and both
said washing of said first group of microparticles and said
isolating of said supernatant from said first solid phase are
facilitated by magnetically separating said microparticles from
said sample and said supernatant.
40. A method in accordance with claim 32 in which said portions are
subgroups of microparticles differing from each other by particle
size, and differentiation between said subgroups is achieved by
flow cytometry.
41. A method for the analysis of a sample to simultaneously yet
individually detect antibodies of different antigen specificities
that are of a single common immunoglobulin class, said method
comprising: contacting said sample with a first solid phase coated
with class-specific antibody having specific binding affinity for a
single selected class of antibodies to cause antibodies in said
sample of said single selected class to be immobilized to said
first solid phase through immunological binding to said
class-specific antibody coated thereon; washing said first solid
phase to isolate said first solid phase from unbound species;
contacting said first solid phase thus washed with a liquid medium
and releasing said antibodies of said single selected class from
said first solid phase by dissociating said immunological binding
to form a supernatant containing antibodies of a said selected
class but of multiple antigen specificities; isolating said
supernatant from said first solid phase and contacting said
supernatant with a second solid phase, said second solid phase
comprising one or more portions each coated with an immunological
binding member with specific binding affinity for antibodies of a
single antigen specificity that is distinct from antigen
specifities of other portions, and each portion being capable of
differentiation from other such portions; and individually
detecting the occurrence of immunological binding between said
antibodies and said immunological binding members while
differentiating between said portions.
42. A method in accordance with claim 41 in which said first solid
phase is a plurality of particles.
43. A method in accordance with claim 41 in which said second solid
phase is a plurality of particles.
44. A method in accordance with claim 41 in which said first and
second solid phases are each a plurality of particles.
45. A method in accordance with claim 42 in which said
microparticles are of magnetically responsive material, and said
washing is facilitated by magnetically separating said
microparticles from said sample.
46. A method in accordance with claim 42 in which said
microparticles are of magnetically responsive material, and both
said washing of said microparticles and said isolating of said
supernatant from said microparticles are facilitated by
magnetically separating said first group of microparticles from
said sample.
47. A method in accordance with claim 43 in which said portions
differ from each other by particle size, and differentiation
between said portions is achieved by flow cytometry.
48. A method in accordance with claim 43 in which said individual
detection of the occurrence of immunological binding is achieved by
the use of fluorophore-labeled binding members.
49. A method in accordance with claim 48 in which said
fluorophore-labeled binding members are phycoerythrin-labeled
binding members.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation-in-part of co-pending
application Ser. No. 08/972,563, filed Nov. 18, 1997, the contents
of which are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] This invention resides in the field of clinical assays
indicative of biological conditions, and is of interest in the
technology of binding assays for analytes in biological fluids for
purposes of diagnosis, monitoring, or other clinical functions.
[0004] 2. Description of the Prior Art
[0005] Since the initial disclosure of radioimmunoassays in 1961, a
wide variety of in vitro assays using affinity-type binding have
been developed. Variations include the type of binding (for
example, specific vs. non-specific, and immunological vs.
non-immunological), the type of detection (including the use of
labels such as enzyme labels, radioactive labels, fluorescent
labels, and chemiluminescent labels), methods of detecting whether
or not binding has occurred (including methods in which bound
species are separated from unbound species and methods that do not
include such separation), and various other aspects of the assay
procedure. The technology is currently used for the detection and
quantitation of countless species, and serves as an analytical tool
in the detection and monitoring of many physiological conditions
and functions and the diagnosis and treatment of many diseases.
[0006] Improvements in the efficiency and reproducibility of these
assays have been made by various developments including improved
labels, methods of detection, automation, and systems for multiplex
analyses. Each procedure however requires a sequence of steps, and
any means of shortening the sequence, increasing the number of
analyses that can be performed within a given period of time, or
improving the reproducibility and versatility of the assay will
benefit the purpose of the assay.
[0007] Many binding assays are heterogeneous assays, which rely in
part on the transfer of analyte from a liquid sample to a solid
phase by the binding of the analyte during the assay to the surface
of the solid phase. At some stage of the assay, whose sequence
varies depending on the assay protocol, the solid phase and the
liquid phase are separated and the determination leading to
detection and/or quantitation of the analyte is performed on one of
the two separated phases. One type of solid phase that has been
used are magnetic particles, which offer the combined advantages of
a high surface area and the ability to be temporarily immobilized
at the wall of the assay receptacle by imposition of a magnetic
field while the liquid phase is aspirated, the solid phase is
washed, or both. Descriptions of such particles and their use are
found in Forrest et al., U.S. Pat. No. 4,141,687 (Technicon
Instruments Corporation, Feb. 27, 1979); Ithakissios, U.S. Pat. No.
4,115,534 (Minnesota Mining and Manufacturing Company, Sep. 19,
1978); Vlieger, A. M., et al., Analytical Biochemistry 205:1-7
(1992); Dudley, Journal of Clinical Immunoassay 14:77-82 (1991);
and Smart, Journal of Clinical Immunoassay 15:246-251 (1992).
[0008] Of further possible relevance to this invention is the state
of the art relating to the use of flow cytometry for the detection
and analysis of particles and species bound to the particles. Flow
cytometry has been disclosed for use in the detection and
separation of antigens and antibodies by Coulter Electronics Inc.,
United Kingdom Patent No. 1,561,042 (published Feb. 13, 1980); and
for quantitation of PCR (Polymerase Chain Reaction) products by
Vlieger, A. M., et al., Analytical Biochemistry 205:1-7 (1992).
Flow cytometry has been limited in the analysis of biological
samples. The sensitivity of those assay formats that do not require
separation of free from bound species (i.e., sandwich and
competitive assays) is adversely affected by the increased
background signal noise caused by the unbound label.
Antigen-capture antibody assays require the removal of non-specific
immunoglobulin before the addition of class-specific labeled
anti-Ig. Samples containing particulates (such as stool samples,
for example) require the removal of this debris which would
otherwise interfere with the flow cytometric measurement.
Traditional separation techniques, such as filtration or
centrifugation would be successful in removing unbound label or
non-specific Ig but would fail to remove interfering particulates
from the patient sample. In addition, these traditional separation
techniques are difficult and/or costly to automate. The use of
magnetic particles and magnetism is a well known method and has
been shown to be both efficient and cost-effective in automated
diagnostic systems.
[0009] Of still further possible relevance to this invention is the
state of the art relating to the detection of antibodies of
distinct classes but with a single common antigen specificity. The
detection of antibodies of a particular class separately from those
of other classes (i.e., IgG as distinct from IgA, or IgM) is
relevant to various diagnostic determinations. For example, during
the course of infection by an antigen, the different antibody
classes are raised at different times, the IgM antibodies generally
arising first, and as the infection progresses, the IgM and IgA
antibodies dropping in concentration while the IgG antibodies
arise. Determination of the relative amounts of these antibody
classes for a specific antigen can thus be used as a measure of the
stage of the infection and of how recently the infected person has
been exposed to a particular disease, which information is of value
in deciding how best to treat the disease. Differentiation among
the antibody classes can also serve as an indication of whether or
not a particular disease is active at the time of the assay.
Existing assays capable of this type of differentiation have
involved the use of multiple fluorophores that are excited at a
common wavelength but emit at different wavelengths, achieved for
example through energy transfer. Unfortunately, the utility and
accuracy of this approach is limited by overlapping emission
spectra and imperfect energy transfer. The approach also suffers
from a lack of sensitivity. Other approaches involve the use of
blocking agents which often give rise to false positives and
similar problems.
[0010] A related type of diagnostic assay is one that detects
antibodies of a single class but of multiple antigen specificities.
This type of assay is useful in various types of screening tests
and in generalized determinations which offer the useful
information of whether exposure has occurred and when, without
distinguishing in terms of the particular nature of the exposure.
In formation of this type is useful in determining whether a
subject is susceptible to infection in general, and can generate
this type of information from a single patient sample rather than
requiring multiple samples. Existing assays for this type of
differentiation use blocking agents to inactivate antibodies of
particular classes. The unfortunate result of these blocking agents
is that they often give rise to false positive results.
SUMMARY OF THE INVENTION
[0011] The present invention resides in various ways of performing
multiplex assays that are capable of differentiating between
analytes.
[0012] In one aspect, this invention combines multiplexing of
heterogeneous binding assays of a single fluid sample by flow
cytometry with the use of solid magnetic particles as the solid
phase to facilitate the separation of solid and liquid phases. The
magnetic particles have sizes that are microscopic (and hence
termed "microparticles") and that are classifiable into groups
according to distinguishable characteristics or differentiation
parameters. The groups are substantially discrete (nonoverlapping),
with the mean values of the distinguishing characteristics of
adjacent groups sufficiently far apart to permit differentiation of
each group from the others by conventional automated detection
methods. An assay reagent is bonded to each particle, with
substantially all particles within each group bearing the same
assay reagent and with different assay reagents from one group to
the next. The groups are thus distinguishable not only by their
distinctive differentiation parameters for purposes of
differentiation but also by the assay reagents bonded to the
particles such that all particles in each group take part in a
distinct binding assay, and do so in a selective manner relative to
the assay reagents bonded to particles in other groups.
[0013] This aspect of the invention further resides in a
combination of solid particles for use in the multiplex assay
described in the preceding paragraph, the particles being of
magnetically responsive material and having a particular detectable
parameter that encompasses a range of values that differentiate the
particles into two or more substantially discrete groups that are
distinguishable by automated detection methods that are appropriate
for the particular parameter. The particles bearing assay reagents
bonded to their surfaces, with a distinct assay reagent for each
group.
[0014] The magnetic character of the particles permits the
automated separation of solid phase from liquid phase at a point in
the sequence of the assay prior to the stage at which the particle
groups are differentiated according to the differentiation
parameter. The separation can serve any of a variety of purposes,
including the removal of sample debris from the assay components,
the removal of sample components that would otherwise contribute
significantly to the background noise at the detection stage, the
removal of competing binding members that are not the subject of
any of the assays but would otherwise interfere with the results,
and the removal of bound from unbound species such as labels,
analytes, analyte binding members, and label-binding member
conjugates. The particular function in any given assay or
combination of assays will depend on the nature of the assay and
the assay protocol.
[0015] In another aspect, this invention provides methods for the
simultaneous yet individual detection of antibodies of different
immunogloblin classes (IgG, IgA, IgM, etc.) that have a single
common antigen specificity, and alternatively the simultaneous yet
individual detection of antibodies of different antigen
specificities that are of a single immunoglobulin class. Each of
these methods involves immunological binding at the surfaces of two
distinct solid phases with an intervening dissociation of the
binding and appropriate washing steps. When detecting antibodies of
different classes but the same antigen specificity, the first solid
phase is coated with the common antigen, and the second solid phase
is coated with an immunological binding member that is specific for
one of the various antibody classes of interest. When detecting
antibodies of different antigen specificities but the same class,
the first solid phase is coated with the antibody that is specific
for the antibody class of interest, and the second solid phase is
coated with an immunological binding member that is specific for
one of the various antigen specificities of interest. In each case,
the second solid phase can be uniform in its reactivity and
specificity, but may alternatively consist of two or more subgroups
that are capable of differentiation from each other and each of
which has a different specificity. This permits multiple
determinations as well as differentiation. Thus, the various
subgroups of the second solid phase can differ in terms of the
antibody class that they are specific for (in the case of the first
type of determination) or in terms of the antigen specificities of
the antibodies that they capture. The differentiation may be
according to any of various differentiation parameters.
[0016] These and other features and advantages of the invention
will be more readily understood by the description that
follows.
DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS
[0017] The term "magnetically responsive material" is used herein
to denote a material that responds to a magnetic field.
Magnetically responsive materials of interest in this invention
include paramagnetic materials, ferromagnetic materials,
ferrimagnetic materials, and metamagnetic materials. Paramagnetic
materials are preferred. Examples are iron, nickel, and cobalt, as
well as metal oxides such as Fe.sub.3O.sub.4, BaFel.sub.2O.sub.19,
CoO, NiO, Mn.sub.2O.sub.3, Cr.sub.2O.sub.3, and CoMnP. Rather than
constituting the entire microparticle, the magnetically responsive
material is preferably only one component of the microparticle
whose remainder consists of a polymeric material to which the
magnetically responsive material is affixed and which is chemically
derivatized to permit attachment of an assay reagent.
[0018] In aspects and embodiments of this invention that involve
the use of solid magnetic microparticles, the quantity of
magnetically responsive material in the microparticle is not
critical and can vary over a wide range. The quantity can affect
the density of the microparticle, however, and both the quantity
and the particle size can affect the ease of maintaining the
microparticle in suspension for purposes of achieving maximal
contact between the liquid and solid phase and for facilitating
flow cytometry. Furthermore, an excessive quantity of magnetically
responsive material in the microparticles will produce
autofluorescence at a level high enough to interfere with the assay
results. It is therefore preferred that the concentration of
magnetically responsive material be low enough to minimize any
autofluorescence emanating from the material. With these
considerations in mind, the magnetically responsive material in a
microparticle in accordance with this invention preferably ranges
from about 1% to about 75% by weight of the particle as a whole. A
more preferred weight percent range is from about 2% to about 50%,
a still more preferred weight percent range is from about 3% to
about 25%, and an even more preferred weight percent range is from
about 5% to about 15%. The magnetically responsive material can be
dispersed throughout the polymer, applied as a coating on the
polymer surface or as one of two or more coatings on the surface,
or incorporated or affixed in any other manner that secures the
material in the polymer matrix.
[0019] When a solid phases consisting of microparticles is used,
the polymeric matrix that forms the microparticle can be any
material that can be formed into a microparticle and that bears
certain other characteristics that make it useful in these assays.
One such characteristic is that the matrix be inert to the
components of the biological sample and to the assay reagents other
than the assay reagent that is affixed to the microparticle. Other
characteristics are that the matrix have minimal autofluorescence,
that it be solid and insoluble in the sample and in any other
solvents or carriers used in the assay, and that it be capable of
affixing an assay reagent to the microparticle. Examples of
suitable polymers are polyesters, polyethers, polyolefins,
polyalkylene oxides, polyamides, polyurethanes, polysaccharides,
celluloses, and polyisoprenes. Crosslinking is useful in many
polymers for imparting structural integrity and rigidity to the
microparticle. These considerations are also applicable to aspects
and embodiments of this invention in which a solid phase other than
microparticles is used.
[0020] Functional groups for attachment of the assay reagent can be
incorporated into the polymer structure by conventional means,
including the use of monomers that contain the functional groups,
either as the sole monomer or as a co-monomer. Examples of suitable
functional groups are amine groups (--NH.sub.2), ammonium groups
(--NH.sub.3.sup.+or --NR.sub.3+), hydroxyl groups (--OH),
carboxylic acid groups (--COOH), and isocyanate groups (--NCO).
Useful monomers for introducing carboxylic acid groups into
polyolefins, for example, are acrylic acid and methacrylic
acid.
[0021] Attachment of the assay reagent to the solid phase surface
can be achieved by electrostatic attraction, specific affinity
interaction, hydrophobic interaction, or covalent bonding. Covalent
bonding is preferred. Linking groups can be used as a means of
increasing the density of reactive groups on the solid phase
surface and decreasing steric hindrance to increase the range and
sensitivity of the assay, or as a means of adding specific types of
reactive groups to the solid phase surface to broaden the range of
types of assay reagents that can be affixed to the solid phase.
Examples of suitable useful linking groups are polylysine,
polyaspartic acid, polyglutamic acid and polyarginine.
[0022] In embodiments in which microparticles are used as the solid
phase and detection is performed by flow cytometry, care should be
taken to avoid the use of particles that emit high autofluorescence
since this renders them unsuitable for flow cytometry. Particles
created by standard emulsion polymerization techniques from a wide
variety of starting monomers generally exhibit low
autofluorescence. Conversely, particles that have been modified to
increase porosity and therefore surface area (such particles are
referred to in the literature as "macroporous" particles) exhibit
high autofluorescence. Autofluorescence in such particles further
increases with increasing size and increasing percentage of
divinylbenzene monomer.
[0023] Within these limitations, the size range of the
microparticles can vary and particular size ranges are not critical
to the invention. In most cases, the aggregated size range of the
microparticles lies within the range of from about 0.3 micrometers
to about 100 micrometers in particle diameter, and preferably
within the range of from about 0.5 micrometers to about 40
micrometers.
[0024] Multiplexing with the use of microparticles in accordance
with this invention is achieved by assigning the microparticles to
two or more groups, each group performing a separate assay and
separable from the other group(s) by a "differentiation parameter,"
which term is used herein to denote a distinguishable
characteristic that permits separate detection of the assay result
in one group from that in another group. One example of a
differentiation parameter that can be used to distinguish among the
various groups of particles is the particle size. The groups in
this example are defined by nonoverlapping subranges of size. The
particles fall into two or more such subranges, and in most cases
the subranges will number from two to 100, each selectively active
in a single assay and inert relative to the other assays
simultaneously being performed or detected.
[0025] The widths of the size subranges and the spacing between
mean diameters of adjacent subranges are selected to permit
differentiation of the subranges by flow cytometry, and will be
readily apparent to those skilled in the use of and instrumentation
for flow cytometry. In this specification, the term "mean diameter"
refers to a number average diameter. In most cases, a preferred
subrange width is about .+-.5% CV or less of the mean diameter,
where CV is the coefficient of variation and is defined as the
standard deviation of the particle diameter divided by the mean
particle diameter times 100 percent. The minimum spacing between
mean diameters among the various subranges can vary depending on
the microparticle size distribution, the ease of segregating
microparticles by size for purposes of attaching different assay
reagents, and the type and sensitivity of the flow cytometry
equipment. In most cases, best results will be achieved when the
mean diameters of different subranges are spaced apart by at least
about 6% of the mean diameter of one of the subranges, preferably
at least about 8% of the mean diameter of one of the subranges and
most preferably at least about 10% of the mean diameter of one of
the subranges. Another preferred subrange width relation is that in
which the standard deviation of the particle diameters within each
subrange is less than one third of the separation of the mean
diameters of adjacent subranges.
[0026] Another example of a differentiation parameter that can be
used to distinguish among the various groups of particles is
fluorescence. Differentiation is accomplished by incorporating
various fluorescent materials in the particles, the various
fluorescent materials having different fluorescent emission spectra
and being distinguishable on this basis.
[0027] Fluorescence can in fact be used both as a means of
distinguishing the groups from each other and as a means of
detection for the assay performed on the particle. The use of
fluorescent materials with different emission spectra can be used
as a means of distinguishing the groups from each other and as a
means of distinguishing the group classification from the assay
detections. An example of a fluorescence substance that can be used
as a means of distinguishing groups is fluorescein and an example
of a substance that can be used for the assay detection is
phycoerythrin. Different particle groups are dyed with differing
concentrations of fluorescein and assay-specific reporters are
labeled with phycoerythrin.
[0028] Still other examples of a differentiation parameter that can
be used to distinguish among the various groups of particles are
light scatter, light emission, or combinations of light scatter and
emission. Side angle light scatter varies with particle size,
granularity, absorbance and surface roughness, while forward angle
light scatter is mainly affected by size and refractive index.
Thus, varying any of these qualities can serve as a means of
distinguishing the various groups. Light emission can be varied by
incorporating fluorescent materials in the microparticles and using
fluorescent materials that have different fluorescence intensities
or that emit fluorescence at different wavelengths, or by varying
the amount of fluorescent material incorporated. By using a
plurality of fluorescent emissions at various wavelengths, the
wavelength difference can be used to distinguish the particle
groups from each other and also to distinguish the labels
indicating the occurrence of binding reactions in the assay from
the labels that identify the particle groups.
[0029] In a preferred embodiment, the microparticles will have two
or more fluorochromes incorporated within them so that each
microparticle in the array will have at least three distinguishable
parameters associated with it, i.e., side scatter together with
fluorescent emissions at two separate wavelengths. For example, the
microparticle can be made to contain a red fluorochrome such as Cy5
together with an orange fluorochrome such as Cy5.5. Additional
fluorochromes can be used to further expand the system. Each
microparticle can thus contain a plurality of fluorescent dyes at
varying wavelengths.
[0030] Still another example of a differentiation parameter that
can be used to distinguish among the various groups of particles is
absorbance. When light is applied to microparticles the absorbance
of the light by the particles is indicated mostly by the strength
of the laterally (side-angle) scattered light while the strength of
the forward-scattered light is relatively unaffected. Consequently,
the difference in absorbance between various colored dyes
associated with the microparticles is determined by observing
differences in the strength of the laterally scattered light.
[0031] A still further example of a differentiation parameter that
can be used to distinguish among the various groups of particles is
the number of particles in each group. The number of particles of
each group in an assay is varied in a known way, and the count of
particles having various assay responses is determined. The various
responses are associated with a particular assay by the number of
particles having each response.
[0032] As the above examples illustrate, a wide array of parameters
or characteristics can be used as differentiation parameters to
distinguish the microparticles of one group from those of another.
The differentiation parameters may arise from particle size, from
particle composition, from particle physical characteristics that
affect light scattering, from excitable fluorescent dyes or colored
dyes that impart different emission spectra and/or scattering
characteristics to the microparticles, or from different
concentrations of one or more fluorescent dyes. When the
distinguishable microparticle parameter is a fluorescent dye or
color, it can be coated on the surface of the microparticle,
embedded in the microparticle, or bound to the molecules of the
microparticle material. Thus, fluorescent microparticles can be
manufactured by combining the polymer material with the fluorescent
dye, or by impregnating the microparticle with the dye.
Microparticles with dyes already incorporated and thereby suitable
for use in the present invention are commercially available, from
suppliers such as Spherotech, Inc. (Libertyville, Ill., USA) and
Molecular Probes, Inc. (Eugene, Oreg., USA). A list of vendors of
flow cytometric products can be found on the Internet at
www.molbio.princeton.edu/facs/FCMsites.html.
[0033] The type of assay reagent attached to the microparticle
surface for any single group of microparticles will vary depending
on both the analyte and the type of assay. The assay reagent can be
a binding agent with specific affinity for the analyte, or a
binding agent with affinity for a narrow range of species that
includes the analyte but excludes other analytes whose assays are
performed by contact with other microparticle subranges, or any
binding species in general that will selectively engage in the
assay for a single analyte to the exclusion of the others. Examples
of assay reagents are antibodies, antigens or haptens, and other
types of proteins with binding specificity such as avidin and
biotin.
[0034] Another type of assay reagent that can be attached to the
microparticle surface for any single group of microparticles is the
analyte itself. In the analysis, the attached analyte will compete
with a narrow range of species in the sample that also includes
analyte. Examples of these assay reagents are antibodies, antigens
and haptens.
[0035] The assay performed at the surfaces of microparticles within
a single group of particles can be any type of heterogeneous assay
that yields a result differentiating a certain analyte from others
in the sample.
[0036] Competitive assays, for example, can be performed by using
magnetically responsive microparticles to which are bound molecules
of a binding protein (such as an antibody) specific for the
analyte. During the assay, the sample and a quantity of labeled
analyte, either simultaneously or sequentially, are mixed with the
microparticles. By using a limited number of binding sites on the
microparticles, the assay causes competition between the labeled
analyte and the analyte in the sample for the available binding
sites. After a suitable incubation period, the mixture of liquid
and solid is placed under the influence of a magnetic field,
causing the microparticles to adhere to the walls of the reaction
vessel, and the liquid phase is removed. The microparticles, still
adhering to the vessel wall, are then washed to remove any
remaining unbound analyte and label, and resuspended in a carrier
liquid for introduction into a flow cytometer where the
microparticles are classified by size and the label detected. An
example of an analyte that is readily detected in this manner is
vitamin B.sub.12. A useful particle-bound assay reagent for this
analyte is intrinsic factor, and a competing label-bound analyte is
B.sub.12 covalently linked to phycoerythrin.
[0037] Immunometric or sandwich assays, as another example, are
performed by using magnetically responsive microparticles to which
are bound antibodies to the analyte. In this case, the bound
antibodies are present in excess relative to the suspected quantity
range of the analyte so that all of the analyte binds. The
microparticles are placed in contact with the sample, and
simultaneously or sequentially, a second antibody to same analyte
is added, again in excess relative to the analyte, the first and
second antibodies binding different epitopes on the analyte in a
non-interfering manner, and the second antibody being conjugated to
a detectable label. After a suitable incubation period, the liquid
mixture with microparticles suspended therein is placed under the
influence of a magnetic field, causing the microparticles to adhere
to the walls of the reaction vessel, and the liquid phase is
removed. The microparticles, still adhering to the vessel wall, are
then washed to remove excess amounts of the second, labeled
antibody that have not become bound to the immobilized analyte, and
the microparticles are then resuspended in a carrier liquid for
introduction into a flow cytometer where they are sorted by size
and the label detected. An example of an analyte that is readily
detected in this manner is thyroid stimulating hormone (TSH). The
label on the second antibody can again be phycoerythrin.
[0038] This invention can also be applied to assays that do not
separate bound label from unbound label but nevertheless require
separation of the solid from the liquid phase at some point in the
assay. Examples are assays for identifying antibodies to infectious
disease agents. The analyte antibodies are members of one of
several possible immunoglobulin classes, and it is medically useful
to know which class is present among the antibodies to the target
antigen. The analyte antibodies bind to particle-bound antigen in
the assay, and are followed by labeled antibodies to the human
immunoglobulin class of interest. In order to prevent such labeled
antibodies from reacting with members of the immunoglobulin class
that are not directed at the disease antigen, it is necessary to
remove the sample from the particles thereby removing the
non-specific immunoglobulins. The magnetic particles serve this
purpose as in the assays described in the preceding paragraphs, and
this is performed before the label is added.
[0039] A different type of serological assay for antibodies are a
further example, performed by using magnetically responsive
microparticles to which are bound antibodies to the immunoglobulin
class of the antibody analyte. The microparticles are placed in
contact with the sample. After a suitable incubation period, the
liquid mixture with suspended microparticles is placed under a
magnetic field to adhere the microparticles to the reaction vessel
walls, and the liquid phase is removed. Labeled antigen is then
added to the vessel containing the microparticles, the antigen
being the one that the analyte antibodies are directed towards and
that is conjugated to a detectable label or is attached through
other binding pairs. After a suitable incubation period, this new
liquid mixture is introduced into a flow cytometer where the
microparticles are classified by the differentiation parameter and
the label detected. An example of an analyte susceptible to this
type of assay is human anti-Rubella IgM. The particle-bound reagent
is anti-human IgM antibody and the labeled reagent is Rubella
antigen.
[0040] The multiple assays that can be performed on a single fluid
sample in accordance with this invention can be all of the same
type (i.e., all competitive, all immunometric, all serological,
etc.) or a combination of different types. Examples of combinations
of assays that can be performed by this method are:
[0041] (1) Assays for thyroid stimulating hormones and either free
T.sub.4 or total T.sub.4;
[0042] (2) Assays for vitamin B.sub.12 and folate; and
[0043] (3) ToRCH assays, detecting serum IgG antibodies to
Toxoplasma gondii, Rubella virus, Cytomegalovirus, and Herpes
Simplex Virus Types 1 and 2.
[0044] Methods of and instrumentation for flow cytometry are known
in the art, and those that are known can be used in the practice of
the present invention. Flow cytometry in general resides in the
passage of a suspension of the microparticles as a stream past a
light beam and electro-optical sensors, in such a manner that only
one particle at a time passes through the region. As each particle
passes this region, the light beam is perturbed by the presence of
the particle, and the resulting scattered and fluorescent light are
detected. The optical signals are used by the instrumentation to
identify the subgroup to which each particle belongs, along with
the presence and amount of label, so that individual assay results
are achieved. Descriptions of instrumentation and methods for flow
cytometry are found in the literature. Examples are McHugh, "Flow
Microsphere Immunoassay for the Quantitative and Simultaneous
Detection of Multiple Soluble Analytes," Methods in Cell Biology
42, Part B (Academic Press, 1994); McHugh et al.,
"Microsphere-Based Fluorescence Immunoassays Using Flow Cytometry
Instrumentation," Clinical Flow Cytometry, Bauer, K. D., et al.,
eds. (Baltimore, Md., USA: Williams and Williams, 1993), pp.
535-544; Lindmo et al., "Immunometric Assay Using Mixtures of Two
Particle Types of Different Affinity," J. Immunol. Meth. 126:
183-189 (1990); McHugh, "Flow Cytometry and the Application of
Microsphere-Based Fluorescence Immunoassays," Immunochemica 5: 116
(1991); Horan et al., "Fluid Phase Particle Fluorescence Analysis:
Rheumatoid Factor Specificity Evaluated by Laser Flow
Cytophotometry," Immunoassays in the Clinical Laboratory, 185-189
(Liss 1979); Wilson et al., "A New Microsphere-Based
Immunofluorescence Assay Using Flow Cytometry," J. Immunol. Meth.
107: 225-230 (1988); Fulwyler et al., "Flow Microsphere Immunoassay
for the Quantitative and Simultaneous Detection of Multiple Soluble
Analytes," Meth. Cell Biol. 33: 613-629 (1990); Coulter Electronics
Inc., United Kingdom Patent No. 1,561,042 (published Feb. 13,
1980); and Steinkamp et al., Review of Scientific Instruments
44(9): 1301-1310 (1973).
[0045] Similarly, methods of and instrumentation for applying and
removing a magnetic field as part of an automated assay are known
to those skilled in the art and reported in the literature.
Examples of literature reports are the Forrest et al. patent, the
Ithakissios patent, the Vlieger et al. paper, the Dudley paper and
the Smart paper, all referenced above in the Description of the
Prior Art. All of the citations in this and the preceding paragraph
are incorporated herein by reference.
[0046] As noted above, one aspect of this invention resides in
assays for the simultaneous yet individual detection of antibodies
of different classes that have a single common antigen specificity.
These assays are conducted by first contacting the sample with a
solid phase whose surface bears the antigen whose specificity is
common to the antibodies being detected. This will result in the
capture of all classes of antibodies with specific binding affinity
towards that antigen. The solid phase is then separated from the
sample and washed to remove unbound materials, which may include
antibodies with other specificities and/or other extraneous sample
components. The washed solid phase is then placed in contact with a
liquid medium and the captured antibodies are released into the
medium by deactivation of the immunological binding. A supernatant
is thereby formed that contains antibodies of different
immunoglobulin classes but the same antigen specificity. The
supernatant is then placed in contact with a second solid phase.
The second solid phase is in one or more portions, each portion
bearing antibodies on its surface that are specific to a particular
immunoglobulin class. Thus, one portion bears anti-IgG antibodies
on its surface, another bears anti-IgM antibodies, and still others
bear anti-IgA antibodies and/or antibodies specific for other
classes. The solid phase may consist of only one of these portions
or two or more. When two or more such portions are present, the
portions are distinguishable from each other in a manner permitting
differentiation between them so that independent determinations can
be made without separating the portions. This is accomplished by
using any of the "differentiation parameters" described above in
connection with other aspects of this invention. Once these
antibodies are captured by the second solid phase, the presence of
the antibodies on the second solid phase surface is detected by
contacting the second solid phase with labeled binding members that
have a specific binding affinity toward the captured antibodies.
Thus, class-specific antibodies, for example IgG-specific,
IgA-specific, and IgM-specific antibodies, all labeled, can be
used, or alternatively, labeled antigen can be used. The presence
of the label and, if desired, its amount can then be determined by
conventional means.
[0047] As further noted above, the invention also resides in assays
for the simultaneous yet individual detection of antibodies of
different antigen specificities but a common immunoglobulin class.
These assays are begun by contacting sample with a solid phase
coated with an antibody that is specific for the immunoglobulin
class of interest. This will result in the capture of antibodies of
that immunoglobulin class regardless of their antigen specificity.
The solid phase is then separated from the sample and washed to
remove unbound materials, which may include antibodies of other
immunoglobulin classes and/or extraneous sample components. The
washed solid phase is then placed in contact with a liquid medium
and the captured antibodies are released into the medium by
deactivation of the immunological binding. The supernatant thus
contains antibodies of the single selected class but different
antigen specificities. The supernatant is then placed in contact
with a second solid phase, which is in one or more portions, each
bearing immmunological binding members on its surface that are
specific for antibodies of a single antigen specificity. These
immunological binding members are preferably the antigens
themselves. Thus, when more than one portion is used, different
portions will bear different antigens on their surfaces. Likewise,
when more than one portion is used, the portions will be
distinguishable from each other in a manner permitting
differentiation between them so that independent determinations can
be made without separating the portions. Here again, any of the
"differentiation parameters" listed above may be used. Once the
antibodies are captured by the second solid phase, their presence
on the surface of the second solid phase is detected by contacting
the second solid phase with a labeled antibody that is specific for
the common immunological class.
[0048] In the assays described in the preceding two paragraphs,
either or both of the first and second solid phases of each assay
may be particles or any other size, shape or configuration of solid
material, including test tube walls, dipsticks and the like.
Microparticles are of particular interest, and magnetic
microparticles are preferred. Microparticles offer various options
for the differentiation parameter as described above and are
readily analyzed and differentiated by flow cytometry. Magnetic
microparticles further provide the capability of separation from
the liquid phases by magnetic fields. Magnetic separation is useful
in the washing stage, and also in the recovery of the supernatant
from the first solid phase after the captured antibodies have been
released.
[0049] In both assays, the release of the captured antibodies from
the antigen or antibody coating on the first solid phase is
achieved by conventional means that will dissociate the binding
interaction without irreversibly denaturing the antibody. This may
be achieved for example by the use of a weak acid such as 0.1 M
acetic acid. Other acids and methods will be readily apparent to
those skilled in the art.
[0050] The detection steps in each of the various assays addressed
by this invention may use any of the wide variety of detection
methods used in immunological assays. Fluorescence by the use of
fluorophore labels conjugated to one of the binding members is one
means of detection that is widely used and applicable to these
assays as well. When fluorophores are used, it is preferable to
select a fluorophore that contributes as little autofluorescence as
possible. The fluorophore phycoerythrin is preferred in this
regard, since its extinction coefficient and quantum yield are
superior to those of other fluorophores.
[0051] This invention is applicable to the analysis of biological
fluids, notably physiological fluids such as whole blood, serum,
urine, spinal fluid, saliva, and stool samples.
[0052] The following examples are offered strictly for purposes of
illustration.
EXAMPLE 1
[0053] This example illustrates the attachment of viral antigen
(Rubella (RUB), Cytomegalovirus (CMV) and Herpes Simplex Virus 2
(HSV2)) to magnetic beads.
[0054] Three types of magnetic beads were used:
[0055] SPHERO.TM. Carboxyl Magnetic particles, from Spherotech,
Inc., Libertyville, Illinois, USA --poly(styrene/acrylic acid
particles), 4.35 micrometers (.mu.m) in diameter, density 1.17
g/cc, containing 12% magnetite (by weight)
[0056] SPHERO.TM. Carboxyl Magnetic particles, from Spherotech,
Inc., Libertyville, Ill., USA--poly(styrene/acrylic acid
particles), 3.18 .mu.m in diameter, density 1.17 g/cc, containing
12% magnetite (by weight)
[0057] SINTEF Applied Chemistry, Trondheim,
Norway--poly(styrene/divinylbe- nzene) particles, 10 .mu.m in
diameter, density 1.23 g/cc, containing 17.9% magnetite/maghemite
(by weight)
[0058] Table I lists the amounts of each of the materials used in
this preparation:
1TABLE I Amounts Used Volume of Viral Amount Weight of Volume of
Phosphate Buffer Bead Antigen of Beads Viral Antigen Viral Antigen
(100 mM) 4.35 .mu.m CMV 10 mg 225.8 .mu.g 322.6 .mu.L 677.4 .mu.L
3.18 .mu.m HDV2 5 mg 163.0 .mu.g 815.0 .mu.L 185.0 .mu.L 10 .mu.m
RUB 5 mg 5.2 .mu.g 104.0 .mu.L 896.0 .mu.L
[0059] The beads in each case were placed in test tubes and washed
multiple times with 100 mM phosphate buffer, pH 6.8. The washed
beads were then suspended in the volume of phosphate buffer listed
in Table I, and respective antigen solution was added (CMV antigen
from Chemicon International Incorporated, Temecula, Calif., USA;
HSV2 antigen from Ross Southern Labs, Salt Lake City, Utah, USA;
and RUB antigen from Viral Antigens, Memphis, Tenn., USA) in the
amount listed in the Table. The test tubes were then rotated in
end-over-end fashion overnight at room temperature. The tubes were
then placed on a magnetic separator and the supernatant was drawn
off and discarded. The resulting beads were washed with a wash
buffer consisting of 50 mM phosphate buffer, pH 7.4, 0.01% Tween
20, 1% bovine serum albumin, 0.1% sodium azide, 150 mM sodium
chloride, then again subjected to magnetic separation, and
suspended in a storage buffer consisting of 50 mM phosphate buffer,
pH 7.4, 5% glycerol, 1% bovine serum albumin, 0.1% sodium azide,
150 mM sodium chloride.
EXAMPLE 2
[0060] This example illustrates the use of the CMV-coated magnetic
beads of Example 1 in a flow cytometric immunoassay.
[0061] Procedure:
[0062] 1. 100 .mu.L of Bio-Rad CMV IgG Immunoassay positive and
negative controls (Bio-Rad Laboratories, Inc., Hercules, Calif.,
USA, diluted 1:10 in wash buffer) were added to 12.times.75 mm
polypropylene test tubes.
[0063] 2. To each tube was added 100 .mu.L of the CMV
antigen-coated particles (described in Example 1) diluted 1:1000 in
wash buffer.
[0064] 3. The tubes were vortexed at ambient temperature for 30
minutes.
[0065] 4. After vortexing, 800 .mu.L of wash buffer was added to
each tube.
[0066] 5. The tubes were placed in a magnetic separator for 3
minutes and the liquid phase removed.
[0067] 6. Steps 4 and 5 are repeated but with 1000 .mu.L of wash
buffer.
[0068] 7. 200 .mu.L of a 1:100 dilution of anti human
IgG-phycoerythrin conjugate (Chemicon International Inc., Temecula,
Calif., USA) is added.
[0069] 8. The tubes were vortexed at ambient temperature for 30
minutes.
[0070] 9. After this time, the samples are injected into the flow
cytometer (Bryte HS, Bio-Rad Laboratories, Inc., Hercules, Calif.,
USA) equipped with a Xenon arc lamp.
[0071] Results:
[0072] Positive and negative CMV controls exhibited fluorescent
peaks corresponding to 898 and 60 relative linear fluorescence
units, respectively. As expected, the positive control gave
significantly elevated signal relative to that of the negative
control.
EXAMPLE 3
[0073] This example illustrates the use of the CMV, HSV2 and
RUB-coated magnetic particles of Example 1 in a simultaneous
multi-analyte flow cytometric immunoassay.
[0074] Procedure:
[0075] 1. 100 .mu.L of patient samples (diluted 1:10 in wash
buffer), of known CMV, HSV2 and RUB antibody status, were added to
12.times.75 mm polypropylene test tubes.
[0076] 2. To each tube was added 100 .mu.L of a mixture of CMV,
HSV2 and RUB antigen-coated particles (described in Example 1)
diluted in wash buffer.
[0077] 3. The tubes were vortexed at ambient temperature for 15
minutes.
[0078] 4. After vortexing, 800 .mu.L of wash buffer was added to
each tube.
[0079] 5. The tubes were placed in a magnetic separator for 5
minutes and the liquid phase removed.
[0080] 6. Steps 4 and 5 are repeated but with 1000 .mu.L of wash
buffer.
[0081] 7. 200 .mu.L of a 1:300 dilution of anti-human
IgG-phycoerythrin conjugate (Chemicon International Inc., Temecula,
Calif., USA) is added.
[0082] 8. The tubes were vortexed at ambient temperature for 15
minutes.
[0083] 9. After this time, the samples are injected into the flow
cytometer (Bryte HS, Bio-Rad Laboratories, Inc., Hercules, Calif.,
USA) equipped with a Xenon arc lamp.
[0084] Results:
[0085] The results are summarized in Table II below:
2TABLE II Test Results Antibody Status Relative Linear Fluorescence
Units Sample CMV HSV2 RUB CMV HSV2 RUB CN6 + - + 14 7 155 CN8 + - +
16 6 181 CN12 - - + 5 7 240 CN15 - - + 5 6 329 23 - + - 5 45 43
[0086] The data in Table II show that positive samples have
substantially increased fluorescence relative to the negative
samples.
EXAMPLE 4
[0087] This example illustrates the covalent attachment of rubella
(RUB) antigen to magnetic beads.
[0088] The magnetic particles were SPHERO.TM. Carboxyl Magnetic
particles, from Spherotech, Inc., Libertyville, Ill., USA
--poly(styrene/alkylenic acid particles), 25 mg/mL, 7.1 micrometers
in diameter, density 1.097 g/cc, containing 5% magnetite (by
weight).
[0089] 1.13 mL of beads were placed in a test tube and washed
multiple times with 50 mM 2-(N-morpholino)ethanesulfonic acid (MES)
buffer, pH 5.5. The washed beads were then suspended in 1.25 mL of
2 mg/mL polylysine (MW 18,000) in 50 mM MES buffer, pH 5.5. To the
resulting solution was added 125 .mu.L of 20 mg/mL
1-ethyl-3-(3-dimethyl-aminopropy- l)carbodiimide (EDC) in water.
The solution was placed on an end-over-end rotator at ambient
temperature for 18 hours. After this time the solution was
separated from the particles and discarded. The particles were then
washed multiple times with 0.2M borate buffer, pH 8.5. The
particles were resuspended in 2.5 mL of borate buffer, pH 8.5. To
this solution was added 50 mg of succinic anhydride. The solution
was then placed on an end-over-end rotator at ambient temperature
for 4 hours. After this time the solution was separated from the
particles and discarded. The particles were then washed multiple
times with 50 mM MES, pH 5.5. The particles were washed twice with
0.1M carbonate buffer, pH 9.6 and then 3 times with 20 mM phosphate
buffer, pH 4.5. The particles were finally suspended in 1 mL of 20
mM phosphate buffer, pH 4.5. To this solution was added 1 mL of 20
mg/mL EDC in 20 mM phosphate buffer, pH 4.5. The solution was then
placed on an end-over-end rotator at ambient temperature for 4
hours. After this time the solution was separated from the
particles and discarded. The particles were washed 3 times with 20
mM phosphate buffer, pH 4.5. Afterwards the beads were suspended in
2 mL of 0.2M borate buffer, pH 8.5. To this was added 0.5 mL of 0.2
mg/mL of rubella antigen from Viral Antigens Incorporated, Memphis,
Tennessee, USA in 0.2M borate buffer, pH 8.5, 2 mg/mL
3-[(3-cholamidopropyl)dimethylammo- nio]-1-propane-sulfonate
(CHAPS), 0.1% sodium azide. The test tube were then rotated in
end-over-end fashion overnight at room temperature. The next day,
100 .mu.L of 0.25M hydroxylamine in 0.2M borate buffer, pH 8.5 was
introduced. The solution was then placed on an end-over-end rotator
at ambient temperature for 1 hour. After this time the solution was
separated from the particles and discarded. The particles were
washed 3 times with wash buffer (see Example 1). The resulting
beads were taken up in 2.5 mL of wash buffer and placed on an
end-over-end rotator at ambient temperature for 1 hour.
[0090] After this time the solution was separated from the
particles and discarded. The particles were washed 3 times with
storage buffer (see Example 1). Finally the particles were
suspended in 1 .mu.L of storage buffer and placed at 4.degree.
C.
EXAMPLE 5
[0091] This example illustrates the use of magnetic particles with
covalently attached rubella antigen of Example 4 in a quantitative
flow cytometric immunoassay.
[0092] Procedure:
[0093] 1. 100 .mu.L of Bio-Rad RUB IgG Immunoassay standards, high
positive, low positive and negative controls (Bio-Rad Laboratories,
Inc., Hercules, Calif., diluted 1:30 in wash buffer), were added to
12.times.75 mm polypropylene test tubes.
[0094] 2. To each tube was added 100.mu.L of the RUB antigen-coated
particles (described above) diluted 1:200 in wash buffer.
[0095] 3. The tubes were vortexed at ambient temperature for 15
minutes.
[0096] 4. After vortexing, 750 .mu.L of wash buffer was added to
each tube.
[0097] 5. The tubes were placed in a magnetic separator for 1
minute and the liquid phase removed.
[0098] 6. Steps 4 and 5 are repeated two more times but with 1000
.mu.L of wash buffer.
[0099] 7. 200 .mu.L of a 1:300 dilution of anti human
IgG-phycoerythrin conjugate (Chemicon International Inc., Temecula,
Calif., USA) is added.
[0100] 8. The tubes were vortexed at ambient temperature for 15
minutes.
[0101] 9. The samples are then injected into the flow cytometer
(Bryte HS, Bio-Rad Laboratories, Inc., Hercules, Calif., USA)
equipped with a Xenon/Mercury arc lamp.
[0102] Results:
[0103] Table III contains data generated by following the above
protocol. The standards were fitted to a 4-parameter logistic
equation. The concentrations of all samples were calculated from
this curve. The values for the controls are similar to the values
assigned by the Bio-Rad ELISA technique.
3TABLE III Test Results Relative Linear Fluorescence Observed [RUB]
Reported [RUB] Sample Units (IU/mL) (IU/mL) Standard 0 21 0 0
Standard 1 101 6 8 Standard 2 225 30 30 Standard 3 464 115 96
Standard 4 652 216 240 Standard 5 1171 623 614 High Positive 580
174 135 Low Positive 175 18 14 Negative 33 0.3 0.5
EXAMPLE 6
[0104] This example illustrates the simultaneous yet individual
detection of antibodies of two different immunoglobulin classes,
both having a common antigen specificity. The antigen specificity
is Rubella, and the two immunoglobulin classes are IgG and IgM.
[0105] A 10-.mu.L sample was placed in a tube, and 100 .mu.L of
Rubella antigen-coated magnetic particles (0.9 tm in diameter) were
added. The mixture was incubated for 16 minutes on a vortexer at
room temperature. The particles were then washed once with 300
.mu.L, and twice with 400 .mu.L portions of a wash buffer, each
time followed by vortexing, magnetic separation and aspiration.
Acetic acid (100 .mu.L, 0.1 M) was then added, and the mixture was
incubated for 16 minutes on a vortexer at room temperature.
Magnetic separation was again performed for one minute, and the
supernatant was transferred to another tube. Potassium hydrogen
phosphate (K.sub.2HPO.sub.4, 50 .mu.L, 0.6 M) was then added,
followed by 50 .mu.L of a mixture of 7.1 .mu.m diameter goat
anti-hIgM-coated magnetic particles and 4.35 .mu.m diameter
magnetic particles coated with goat anti-hIgG (Fc specific,
F(ab').sub.2 fragment) in Neonatal Calf Serum. The resulting
mixture was incubated for 16 minutes on a vortexer at room
temperature, followed by washing once with 200 .mu.L, then twice
with 400 .mu.L portions of wash buffer. After a one-minute magnetic
separation and aspiration, labeled class-specific antibodies were
added, 100 .mu.L of a mixture of anti-hIgM-phycoerythrin and
anti-hIgG-phycoerythrin. The mixture was incubated for 16 minutes
on a vortexer at room temperature, then washed once with 300 .mu.L,
then twice with 400 .mu.L portions of wash buffer, vortexed and
magnetically separated (one minute), then aspirated. The particles
were suspended in wash buffer (150 .mu.L), then read on the flow
cytometer of the preceding examples.
[0106] The results in relative linear fluorescence units are listed
in Table IV below.
4TABLE IV Test Results for Rubella Antibodies IgG and IgM 4.35
.mu.m Particle 7.10 .mu.m (IgG) Particle (IgM) IgG Value IgM Value
Relative Linear Relative Linear Sample (IU/mL) (signal/-cutoff)
Units Units GW917 29.07 2.38 410.5 396.0 GW22 253 0.947 403.2 89.0
66S 0 0.534 80.2 63.5 Diluent 5.1 6.9 Particles only 2.2 3.2
[0107] The data in Table IV demonstrate that the positive samples
(GW917 and GW22) gave significantly greater signals than the
negative sample (66S).
EXAMPLE 7
[0108] This example illustrates the simultaneous yet individual
detection of antibodies of a distinct antigen specificity and a
single immunoglobulin class. The antigen specificity is the
Rubella, and the immunoglobulin class is IgM.
[0109] A 10-.mu.L sample was placed in a tube, and 100 .mu.L of
anti-human IgM-coated magnetic particles (0.9 .mu.m in diameter)
were added. The mixture was incubated for 16 minutes on a vortexer
at room temperature. The particles were then washed once with 300
.mu.L, and twice with 400 .mu.L portions of a wash buffer, each
time followed by vortexing, magnetic separation and aspiration.
Acetic acid (100 .mu.L, 0.1 M) was then added, and the mixture was
incubated for 16 minutes on a vortexer at room temperature.
Magnetic separation was again performed for one minute, and the
supernatant was transferred to another tube. Potassium hydrogen
phosphate (K.sub.2HPO.sub.4, 50 .mu.L, 0.6 M) was then added,
followed by 50 AL of a Rubella antigen-coated 7.1-.mu.m diameter
magnetic particles diluted 1:35 in Neonatal Calf Serum. The
resulting mixture was incubated for 16 minutes on a vortexer at
room temperature, followed by washing once with 200 .mu.L, then
twice with 400 .mu.L portions of wash buffer. After a one-minute
magnetic separation and aspiration, 100 .mu.L of anti-hIgM was
added. The mixture was incubated for 16 minutes on a vortexer at
room temperature, then washed once with 300 .mu.L, then twice with
400 .mu.L portions of wash buffer, vortexed and magnetically
separated (one minute), then aspirated. The particles were
suspended in wash buffer (150 .mu.L), then read on the flow
cytometer of the preceding examples.
[0110] The results (in relative linear fluorescence units) are
listed in Table V below.
5TABLE V Test Results for Rubella IgM Antibodies IgM Value Relative
Sample (signal/-cutoff) Linear Units GW22 0.947 10.5 GW917 2.98
145.9 GW35 3.469 25.9 66S 0.534 8.7
[0111] The data in Table V demonstrate that the positive samples
(GW917 and GW35) were well separated from the negative sample
(66S), and that the equivocal sample (GW22) gave a higher signal
than the negative sample (66S).
EXAMPLE 8
[0112] This example illustrates the advantage of the use of
phycoerythrin as a fluorophore, by comparing phycoerythrin to
various other fluorophores in terms of the signal-to-noise ratio,
i.e., the signal of the positive serum to that of the negative
serum. This ratio is a measure of the ability of a fluorophore to
produce an analyte-specific signal.
[0113] In these experiments, a serum sample (100 .mu.L of a 1 :30
dilution) containing HSV2 was contacted with SINTEF particles (100
.mu.L, 10 .mu.m in diameter, magnetic and porous) coated with HSV2
antigen, and the particles are then washed. The washed particles
were then resuspended, and anti-human IgG labelled with fluorophore
(200 .mu.L) was added. The particles were then read on a flow
cytometer. Three groups of comparisons were made, and the results
are shown in Table VI below.
6TABLE VI Fluorophore Comparisons Comparison No. Fluorophore
Signal/Noise Ratio 1 fluorescein 1.8 phycoerythrin 9.4 2 bodipy
TMR-X 1.9 tetramethylrhodamine 1.7 phycoerythrin 10.7 3 oregon
green 1.8 PyMPO 1.4 phycoerythrin 11.5
[0114] The higher signal-to-noise ratio of phycoerythrin relative
to each of the other fluorophores is clear from each
comparison.
EXAMPLE 9
[0115] This example illustrates the relationship between various
characteristics of the particle and the degree of autofluorescence
created by the particle. The particles were
poly(styrene-co-divinylbenzen- e) and the characteristics that were
varied include porosity vs. smoothness, diameter, percent
divinylbenzene (DVB) content, and percent magnetite content. The
results in terms of relative linear fluorescence units (RLFU) are
listed in Table VII below.
7TABLE VII Autofluorescence vs. Various Particle Parameters
Porous/- Diameter Particle No. smooth (.mu.m) % DVB % Magnetite
RLFU 1 porous 10.0 n/a 17.9 13.6 2 smooth 10.0 n/a 0 6.9 3 smooth
7.0 5 0 6.6 4 smooth 7.0 40 0 6.3 5 smooth 7.0 60 0 6.3 6 porous
4.2 37 0 6.9 7 porous 4.2 50 0 9.9 8 porous 15.0 50 0 27.6 9 porous
15.0 80 0 61.2 10 smooth 7.0 2 0 4.0 11 smooth 7.1 n/a 5 10.0 12
smooth 2.8 n/a 24 33.5 13 smooth 4.5 n/a 24.5 63.8 14 porous 4.5
n/a 10.5 30.9 15 smooth 1.9 n/a 20 7.1 16 smooth 3.18 n/a 12 8.2 17
smooth 4.35 n/a 12 10.0 18 smooth 7.1 n/a 5 16.7
[0116] These data indicate that smooth particles exhibit less
autofluorescence than porous particles. Furthermore, for smooth
particles there is little difference in autofluorescence with
increasing amounts of divinylbenzene, whereas for porous particles,
autofluorescence increases with increasing amounts of
divinylbenzene. Magnetic particles exhibit higher autofluorescence
than non-magnetic particles, and for smooth particles increasing
the particles size produces an increase in autofluorescence.
[0117] The foregoing is offered primarily for purposes of
illustration. It will be readily apparent to those skilled in the
art that the operating conditions, materials, procedural steps and
other parameters described herein may be further modified or
substituted in various ways without departing from the spirit and
scope of the invention.
* * * * *
References