U.S. patent application number 10/449355 was filed with the patent office on 2003-10-30 for increased separation efficiency via controlled aggregation of magnetic nanoparticles.
Invention is credited to Liberti, Paul A., Rao, Galla Chandra, Terstappen, Leon W.M.M.
Application Number | 20030203507 10/449355 |
Document ID | / |
Family ID | 27804987 |
Filed Date | 2003-10-30 |
United States Patent
Application |
20030203507 |
Kind Code |
A1 |
Liberti, Paul A. ; et
al. |
October 30, 2003 |
Increased separation efficiency via controlled aggregation of
magnetic nanoparticles
Abstract
Compositions and methods are disclosed which enhance the
microscopic observation and analysis of biological entities such as
cells, bacteria and viruses by eliminating interfering magnetic
clusters created by naturally occurring aggregators of colloidal
magnetic particles. Additionally means for significantly enhancing
the magnetic isolation of low antigen density target cells from
biological samples are disclosed.
Inventors: |
Liberti, Paul A.; (Naples,
FL) ; Rao, Galla Chandra; (Princeton, NJ) ;
Terstappen, Leon W.M.M; (Huntingdon Valley, PA) |
Correspondence
Address: |
Jared Mayes
IMMUNICON CORPORATION
Suite 100
3401 Masons Mill Road
Huntingdon Valley
PA
19006
US
|
Family ID: |
27804987 |
Appl. No.: |
10/449355 |
Filed: |
May 30, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10449355 |
May 30, 2003 |
|
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09351515 |
Jul 12, 1999 |
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Current U.S.
Class: |
436/526 |
Current CPC
Class: |
G01N 33/54326 20130101;
Y10T 436/108331 20150115; Y10T 436/25375 20150115; Y10S 435/967
20130101; Y10S 436/805 20130101; Y10S 436/824 20130101 |
Class at
Publication: |
436/526 |
International
Class: |
G01N 033/553 |
Claims
What is claimed is:
1. A method for isolating a target bioentity from a biological
sample by means of colloidal magnetic particles with reduced
aggregation of said magnetic particles, comprising: a) obtaining a
biological specimen suspected of containing said target bioentity
together with non-target bioentities and endogenous aggregating
factors; b) contacting said biological specimen with a reagent
effective to inactivate any endogenous aggregating factors present
in said specimen; c) preparing an immunomagnetic suspension
comprising a mixture of said specimen and colloidal, magnetic
particles coupled to a biospecific ligand having binding affinity
for at least one characteristic determinant present on said target
bioentity; and d) subjecting said immunomagnetic suspension to a
magnetic field to obtain a target bioentity- enriched fraction.
2. A method as claimed in claim 1, further comprising the steps of
e) purifying said target bioentity from said enriched fraction; and
f) analyzing said purified target bioentity.
3. A method as claimed in claim 1, further comprising the steps of:
e) adding to said target bioentity enriched fraction at least one
biospecific reagent which has binding affinity for at least one
additional characteristic determinant on said target bioentity; f)
separating said target bioentity in a magnetic field to remove
unbound biospecific reagent from said enriched fraction.
4. A method as claimed in claim 3, further comprising the steps of:
g) purifying said separated target bioentity; and h) analyzing said
purified target bioentity.
5. A method as claimed in claim 3, further comprising the steps of:
g) adding a non-cell exclusion agent to said separated target
bioentities to allow exclusion of non-nucleated entities present in
the sample; h) purifying said target bioentity; and i) analyzing
said purified target bioentities.
6. A method as claimed in claim 1, wherein said target bioentity is
selected from the group consisting of tumor cells, virally infected
cells, fetal cells in maternal circulation, virus particles,
bacterial cells, white blood cells, myocardial cells, epithelial
cells, endothelial cells, proteins, hormones, DNA, and RNA.
7. A method as claimed in claim 2, wherein said target bioentities
are analyzed by a process selected from the group consisting of
multiparameter flow cytometry, immunofluorescent microscopy, laser
scanning cytometry, bright field base image analysis, capillary
volumetry, manual cell analysis and automated cell analysis.
8. A method as claimed in claim 1, said aggregation inhibiting
agent being at least one selected from those consisting of a
reducing agent, an animal serum protein, an immune-complex, a
carbohydrate, a chelating agent, an unconjugated ferrofluid, and
diamino butane.
9. A method as claimed in claim 5, wherein said aggregation
inhibiting agent is a reducing agent selected from the group
consisting of mercapto ethane sulfonic acid [MES], mercapto propane
sulfonic acid [MPS] and dithiothreitol [DTT].
10. A method as claimed in claim 1, wherein said biospecific ligand
is a monoclonal antibody.
11. A method as claimed in claim 9, wherein said biospecific ligand
is a monoclonal antibody having affinity for epithelial cell
adhesion molecule.
12. A method for isolating target bioentities from a biological
sample by means of colloidal magnetic particles with controlled
aggregation of said magnetic particles, comprising: a) obtaining a
biological specimen suspected of containing said target bioentities
together with non-target bioentities and endogenous aggregating
factors; b) contacting said biological specimen with a reagent
effective to inactivate any endogenous aggregating factors present
in said specimen; c) preparing an immunomagnetic suspension
comprising a mixture of colloidal, magnetic particles coupled to a
biospecific ligand having affinity for at least one characteristic
determinant present on said target bioentity, said magnetic
particles being further coupled to a first exogenous aggregation
enhancing factor which comprises one member of a specific binding
pair; d) adding a second exogenous aggregation enhancing factor to
said immunomagnetic suspension to increase aggregation of said
particles, said second aggregating enhancing factor comprising the
other member of said specific binding pair; and e) subjecting said
sample to a magnetic field to obtain a target bioentity-enriched
fraction.
13. A method as claimed in claim 12, further comprising the steps
of: f) adding to said immunomagnetic suspension at least one
biospecific reagent having binding affinity for at least one
additional characteristic determinant on said target bioentity; g)
separating said target bioentities in a magnetic field to remove
unbound biospecific reagent; and h) adding a non-cell exclusion
agent to said separated bioentities to allow exclusion of
non-nucleated entities present in the sample; i) purifying said
target bioentities; and j) analyzing said separated and purified
target bioentities.
14. A method as claimed in claim 12, further comprising examining
said purified target bioentity-enriched fraction to determine the
degree of aggregation mediated by said first and second members of
said specific binding pair.
15. A method as claimed in claim 12, wherein one or the other
member of said specific binding pair is added to said purified
bioentity fraction to reverse aggregation of said sample, thereby
facilitating analysis of said target bioentities.
16. A method as claimed in claim 12, wherein said specific binding
pair is selected from the group consisting of biotin-streptavidin,
antigen-antibody, receptor-hormone, receptor-ligand,
agonist-antagonist, lectin-carbohydrate, Protein A-antibody Fc, and
avidin-biotin, biotin analog-streptavidin, biotin analog-avidin,
desthiobiotin-streptavidin, desthiobiotin-avidin,
iminobiotin-streptavidin, and iminobiotin-avidin.
17. A method as claimed in claim 12, wherein said biospecific
ligand is a monoclonal antibody.
18. A method as claimed in claim 17, wherein said biospecific
ligand is an antibody having affinity for epithelial cell adhesion
molecule.
19. A method as claimed in claim 12 wherein said at least one
biospecific reagent is selected from the group of consisting of
monoclonal antibodies, polyclonal antibodies, detectably labeled
antibodies, antibody fragments, and single chain antibodies.
20. A method as claimed in claim 12 wherein said target bioentities
are analyzed by a process selected from the group consisting of
multiparameter flow cytometry, immunofluorescent microscopy, laser
scanning cytometry, bright field base image analysis, capillary
volumetry, manual cell analysis and automated cell analysis.
21. A method as claimed in claim 12, wherein said immunomagnetic
suspension is incubated for less than 30 minutes.
22. A method as claimed in claim 12, wherein said purification of
said sample is performed in a magnetic field gradient of less than
6.3 kGauss/cm.
23. A method as claimed in claim 12, wherein said colloidal
magnetic particle concentration in said immunomagnetic suspension
is less than 10 .mu.g per milliliter.
24. A method for isolating low antigen density tumor cells from a
biological sample by means of colloidal magnetic particles with
controlled aggregation of said magnetic particles, comprising: a)
obtaining a biological specimen suspected of containing said tumor
cells together with non-tumor cells and endogenous aggregating
factors; b) preparing an immunomagnetic suspension comprising a
mixture of said specimen and colloidal, magnetic particles coupled
to a biospecific ligand having binding affinity for at least one
characteristic determinant present on said tumor cell, said
magnetic particles being further coupled to a first exogenous
aggregation enhancing factor, said factor comprising one member of
a specific binding pair; c) adding a second exogenous aggregating
enhancing factor to said immunomagnetic suspension to increase
aggregation of said particles, said second aggregating enhancing
factor comprising the other member of said specific binding pair;
and d) purifying said sample in a magnetic field to obtain a tumor
cell-enriched fraction.
25. A method as claimed in claim 24, further comprising the steps
of: e) adding to said fraction, at least one biospecific reagent
having binding affinity for at least one additional characteristic
determinant on said tumor cell; f) separating said tumor cells in a
magnetic field to remove unbound biospecific reagent; g) adding a
non-cell exclusion agent to said separated cells to allow exclusion
of non-nucleated entities present in the sample; and h) analyzing
said separated tumor cells to assess at least one of tumor cell
number and type.
26. A method as claimed in claim 25, wherein a member of said
specific binding pair is added to the separated cells to reverse
aggregation of said sample, thereby facilitating analysis of said
cells.
27. A method as claimed in claim 24, wherein said specific binding
pair is selected from the group consisting of biotin-streptavidin,
antigen-antibody, receptor-hormone, receptor-ligand,
agonist-antagonist, lectin-carbohydrate, Protein A-antibody Fc, and
avidin-biotin, biotin analog-avidin, desthiobiotin-streptavidin,
desthiobiotin-avidin, iminobiotin-streptavidin, and
iminobiotin-avidin.
28. A method as claimed in claim 24, wherein said biospecific
ligand is a monoclonal antibody.
29. A method as claimed in claim 28, wherein said biospecific
ligand is an antibody having binding affinity for epithelial cell
adhesion molecule.
30. A method as claimed in claim 25, wherein said at least one
biospecific reagent is selected from the group of consisting of
monoclonal antibodies, polyclonal antibodies, detectably labeled
antibodies, antibody fragments and single chain antibodies.
31. A method as claimed in claim 25 wherein said tumor cells are
analyzed by a process selected from the group consisting of
multiparameter flow cytometry, immunofluorescent microscopy, laser
scanning cytometry, bright field base image analysis, capillary
volumetry, manual cell analysis and automated cell analysis.
32. A method as claimed in claim 24 wherein said biological sample
is pretreated with an aggregation inhibiting agent to inactivate
endogenous aggregation factors present in the sample prior to the
preparation of said immunomagnetic suspension.
33. A method as claimed in claim 25, wherein said purified tumor
cell-enriched fraction is examined to determine the degree of
aggregation mediated by said first and second members of said
specific binding pair.
34. A kit for inhibiting endogenous aggregation of colloidal
magnetic particles in processing of biological material for
isolation of target bioentities from such materials, comprising: a)
coated magnetic nanoparticles comprising a magnetic core material,
a protein base coating material, and an antibody that binds
specifically to a first characteristic determinant of said target
bioentity, said antibody being coupled, directly or indirectly, to
said base coating material; b) at least one antibody having binding
specificity for a second characteristic determinant of said target
bioentity; c) an endogenous aggregation inhibiting factor; and d) a
non-cell exclusion agent for excluding non-nucleated cells from
analysis.
35. A kit for isolating low antigen density tumor cells from a
biological sample with controlled aggregation of colloidal magnetic
particles, comprising: a) a reagent effective to inactivate
endogenous aggregating factors; b) coated magnetic nanoparticles
comprising a magnetic core material, a protein base coating
material, and an antibody that binds specifically to a first
characteristic determinant of said tumor cell, said antibody being
coupled, directly or indirectly, to said base coating material;
said magnetic particles being further coupled to a first exogenous
aggregation enhancing factor, said factor comprising one member of
a specific binding pair; c) at least one antibody having binding
specificity for a second characteristic determinant of said tumor
cell; d) a second exogenous aggregation enhancing factor, said
second aggregation factor comprising the other member of said
specific binding pair; and e) a non-cell excluding agent for
excluding non-nucleated cells from analysis.
36. A kit as claimed in claim 35, wherein said specific binding
pair is selected from the group consisting of biotin-streptavidin,
antigen-antibody, receptor-hormone, receptor-ligand,
agonist-antagonist, lectin-carbohydrate, Protein A-antibody Fc, and
avidin-biotin, biotin analog-avidin, desthiobiotin-streptavidin,
desthiobiotin-avidin, iminobiotin-streptavidin, and
iminobiotin-avidin.
37. A kit as claimed in claim 35, said kit further comprising a
reagent for reversing the effect of said exogenous aggregation
factor.
38. A kit as claimed in claim 35, said reagent effective to inhibit
endogenous aggregation factor being at least one selected from the
group consisting of a reducing agent, an animal serum protein, an
immune-complexes, a carbohydrate, a chelating agent, an
unconjugated ferrofluid, and diamino butane.
39. A method as claimed in claim 38, wherein said said endogenous
aggregation factor inhibiting reagent is a reducing agent selected
from the group of agents consisting of mercapto ethane sulfonic
acid [MES], mercapto Propane Sulfonic acid [MPS] and dithiothreitol
[DTT].
Description
PRIORITY INFORMATION
[0001] The present application is a continuation of U.S.
application Ser. No. 09/351,515 filed on Jul. 12, 1999.
FIELD OF THE INVENTION
[0002] This invention relates to the fields of bioaffinity
separations and diagnostic testing of biological samples. More
specifically, the invention provides compositions and methods
which, may be used in magnetic separation assays and enrichment
procedures for controlling endogenous magnetic particle aggregation
factors which, if uncontrolled, would obscure visualization of
isolated entities. Also provided are methods for constructing and
synthesizing reversible aggregation factors and the resulting
compositions which simultaneously enhance recovery of rare
biological substances while facilitating observation of substances
so isolated.
BACKGROUND OF THE INVENTION
[0003] Several publications are referenced in this application by
numerals in parentheses in order to more fully describe the state
of the art to which this invention pertains. The disclosure of each
of these publications is incorporated by reference herein.
[0004] Many laboratory and clinical procedures employ bio-specific
affinity reactions. Such reactions are commonly utilized in
diagnostic testing of biological samples, or for the separation of
a wide range of target substances, especially biological entities
such as cell, viruses, proteins, nucleic acids and the like.
Various methods are available for analyzing or separating the
above-mentioned target substances based upon complex formation
between the substance of interest and another substance to which
the target substance specifically binds. Separation of complexes
from unbound material may be accomplished gravitationally, e.g. by
settling, or, alternatively, by centrifugation of finely divided
particles or beads coupled to the target substance. If desired,
such particles or beads may be made magnetic to facilitate the
bound/free separation step. Magnetic particles are well known in
the art, as is their use in immune and other bio-specific affinity
reactions. See, for example, U.S. Pat. No. 4,554,088 and
Immunoassays for Clinical Chemistry, pp. 147-162, Hunter et al.
eds., Churchill Livingston, Edinborough (1983). Generally, any
material which facilitates magnetic or gravitational separation may
be employed for this purpose. However, in the past 20 years the
superiority of magnetics for performing such separations has led to
its use in many applications.
[0005] Magnetic particles generally fall into two broad categories.
The first category includes particles that are permanently
magnetizable, or ferromagnetic. The second category comprises
particles that demonstrate bulk magnetic behavior only when
subjected to a magnetic field. The latter are referred to as
magnetically responsive particles. Materials displaying
magnetically responsive behavior are sometimes described as
superparamagnetic. However, materials exhibiting bulk ferromagnetic
properties, e.g., magnetic iron oxide, may be characterized as
superparamagnetic only when provided in crystals of about 30 nm or
less in diameter. Larger crystals of ferromagnetic materials, by
contrast, retain permanent magnet characteristics after exposure to
a magnetic field and tend to aggregate thereafter due to strong
particle-particle interactions. Magnetic particles can be
classified as large (1.5 to about 50 microns), small (0.7-1.5
microns), and colloidal or nanoparticles (<200 nm). The latter
are also called ferrofluids or ferrofluid-like and have many of the
properties of classical ferrofluids. Liberti et al pp 777-790, E.
Pelizzetti (ed) "Fine Particles Science and Technology" Kluwer
Acad. Publishers, Netherlands, 1996.
[0006] Small magnetic particles are quite useful in analyses
involving bio-specific affinity reactions, as they are conveniently
coated with biofunctional polymers (e.g., proteins), provide very
high surface areas and give reasonable reaction kinetics. Magnetic
particles ranging from 0.7-1.5 microns have been described in the
patent literature, including, by way of example, U.S. Pat. Nos.
3,970,518; 4,018,886; 4,230,685; 4,267,234; 4,452,773; 4,554,088;
and 4,659,678. Certain of these particles are disclosed to be
useful solid supports for immunologic reagents.
[0007] In addition to the small magnetic particles mentioned above,
there are a class of large magnetic particles ranging in size from
approximately 1.5-50 microns, which also have superparamagnetic
behavior. Typical of such materials are those invented by Ugelstad
(U.S. Pat. No. 4,654,267) and manufactured by Dynal, (Oslo,
Norway). The Ugelstad process involves the synthesis of polymer
particles which are caused to swell and magnetite crystals are
embedded in the swelled particles. Other materials in the same size
range are prepared by synthesizing the particle in the presence of
dispersed magnetite crystals. This results in the trapping of
magnetite crystals in a polymer matrix, thus making the resultant
materials magnetic. In both cases, the resultant particles have
superparamagnetic behavior, which is manifested by the ability to
disperse readily upon removal of the magnetic field. Unlike
magnetic colloids or nanoparticles, these materials, as well as
small magnetic particles, are readily separated with simple
laboratory magnetics because of the mass of magnetic material per
particle. Thus, separations are effected in gradients from as low
as a few hundred gauss/cm on up to about 1.5 kilogauss/cm.
Colloidal magnetic particles, (below approximately 200 nm),on the
other hand, require substantially higher magnetic gradients because
of their diffusion energy, small magnetic mass per particle and
Stokes drag.
[0008] U.S. Pat. No. 4,795,698 to Owen et al. relates to
polymer-coated, colloidal, superparamagnetic particles. Such
particles are manufactured by precipitation of a magnetic species
in the presence of a biofunctional polymer. The structure of the
resulting particles, referred to herein as single-shot particles,
has been found to be a micro-agglomerate in which one or more
ferromagnetic crystallites having a diameter of 5-10 nm are
embedded within a polymer body having a diameter on the order of 50
nm. The resulting particles exhibit an appreciable tendency to
remain in aqueous suspension for observation periods as long as
several months. U.S. Pat. No. 4,452,773 to Molday describes a
material similar in properties to those described in Owen et al.,
which is produced by forming magnetite and other iron oxides from
Fe.sup.+2/Fe.sup.+3 via base addition in the presence of very high
concentrations of dextran. Materials so produced have colloidal
properties and have proved to be very useful in cell separation.
This technology has been commercialized by Miltenyi Biotec,
Bergisch Gladbach, Germany.
[0009] Another method for producing superparamagnetic colloidal
particles is described in U.S. Pat. No. 5,597,531. In contrast to
the particles described in the Owen et al. patent, these latter
particles are produced by directly coating a biofunctional polymer
onto pre-formed superparamagnetic crystals which have been
dispersed, e.g., by sonic energy into quasi-stable crystalline
clusters ranging in size from about 25-120 nm. The resulting
particles, referred to herein as direct coated (DC) particles,
exhibit a significantly larger magnetic moment than Owen et al. or
Molday nanoparticles of the same overall size and can be separated
effectively in magnetic gradients greater than about 6
kGauss/cm.
[0010] Magnetic separation techniques are known wherein a magnetic
field is applied to a fluid medium in order to separate
ferromagnetic bodies from the fluid medium. In contrast, the
tendency of colloidal superparamagnetic particles to remain in
suspension, in conjunction with their relatively weak magnetic
responsiveness, requires the use of high-gradient magnetic
separation (HGMS) techniques in order to separate such particles
from a fluid medium in which they are suspended. In HGMS systems,
the gradient of the magnetic field, i.e., the spatial derivative,
exerts a greater influence upon the behavior of the suspended
particles than is exerted by the strength of the field at a given
point.
[0011] High gradient magnetic separation is useful for separating a
wide variety of magnetically labeled biological materials,
including eukaryotic and prokaryotic cells, viruses, nucleic acids,
proteins, and carbohydrates. In methods known heretofore,
biological material has been separable by HGMS, provided at least
one characteristic determinant is present on the material, which is
capable of being specifically recognized and bound to a receptor,
such as an antibody, antibody fragment, specific binding protein
(e.g., protein A, streptavidin), lectin, and the like.
[0012] HGMS systems can be divided into two broad categories. One
such category includes magnetic separation systems which employ a
magnetic circuit that is entirely situated externally to a
separation chamber or vessel. Examples of such external separators
(or open field gradient separators) are described in U.S. Pat. No.
5,186,827. In several of the embodiments described in the '827
patent, the requisite magnetic field gradient is produced by
positioning permanent magnets around the periphery of a
non-magnetic container such that the like poles of the magnets are
in a field-opposing configuration. The extent of the magnetic field
gradient within the test medium obtainable in such a system is
limited by the strength of the magnets and the separation distance
between the magnets. Hence, there exists a finite limit to
gradients that can be obtained with external gradient systems. In a
co-pending application Ser. No. 60/098,021, means for maximizing
radial gradients and methods for maximizing separation efficiency
via novel vessel designs are disclosed.
[0013] Another type of HGMS separator utilizes a ferromagnetic
collection structure that is disposed within the test medium in
order to: (1) intensify an applied magnetic field; and (2) produce
a magnetic field gradient within the test medium. Previously
disclosed internal HGMS systems comprise fine steel wool or gauze
packed within a column that is situated adjacent to a magnet. The
applied magnetic field is concentrated in the vicinity of the steel
wires so that suspended magnetic particles will be attracted
toward, and adhere to, the surfaces of the wires. The gradient
produced on such wires is inversely proportional to the wire
diameter whereas the magnetic "reach" decreases with diameter.
Hence, very high gradients can be generated.
[0014] One major drawback of internal gradient systems is that the
use of steel wool, gauze material, steel microbeads or the like,
may entrap non-magnetic components of the test medium by capillary
action in the vicinity of intersecting wires or within interstices
between intersecting wires. Various coating procedures have been
applied to such internal gradient columns (U.S. Pat. Nos.
5,693,539; 4,375,407), however, the large surface area in such
systems still creates recovery problems due to absorption. Hence,
internal gradient systems are not desirable, particularly when
recovery of very low frequency captured entities is the goal of the
separation. Further, these systems make automation difficult and
costly.
[0015] On the other hand, HGMS approaches using external gradients
for cell separation provide a number of conveniences. Firstly,
simple laboratory tubes such as test tubes, centrifuge tubes or
even vacutainers (used for blood collection) can be employed. When
external gradients are of the kind in which separated cells are
effectively monolayered, as is the case with quadrupole/hexapole
devices (U.S. Pat. No. 5,186,827) or the opposing dipole
arrangement described in U.S. Pat. No. 5,466,574, washing of cells
or subsequent manipulations are facilitated. Further, recoveries of
cells from tubes or similar containers is a simple and efficient
process. This is particularly the case when compared to recoveries
from high gradient columns. Such separation vessels also provide
another important feature which is the ability to reduce volume of
the original sample. For example, if a particular human blood cell
subset, (e.g. magnetically labeled CD 34+ cells), is isolated from
blood diluted 20% with buffer to reduce viscosity, a 15 ml conical
test tube may be employed as the separation vessel in an
appropriate quadrupole magnetic device. After appropriate washes
and/or separations and resuspensions to remove non-bound cells,
CD34+ cells can very effectively be resuspended in a volume of 200
.mu.l.
[0016] This can be accomplished, for example, by starting with 12
ml of solution (blood, ferrofluid and dilution buffer) in a 15 ml
conical test tube, performing a separation, discarding the
"supernatant" and subsequent wash "supernatants" and resuspending
the recovered cells in 3 ml of appropriate cell buffer. A second
separation is then performed which may include additional
separation/wash steps (as might be necessary for doing
labeling/staining reactions) and finally the isolated cells are
easily resuspended in a final volume of 200 .mu.l. By reducing
volume in this sequential fashion, and employing a vortex mixer for
resuspension, cells adhered to the tube above the resuspension
volume are recovered into the reduced volume. When done carefully
and rapidly in appropriately treated vessels, cell recovery is
quite efficient, ranging between 70-90%.
[0017] The efficiency with which magnetic separations can be done
and the recovery and purity of magnetically labeled cells will
depend on many factors. These include such considerations as the
number of cells being separated, the receptor density of such
cells, the magnetic load per cell, the non-specific binding (NSB)
of the magnetic material, the technique employed, the nature of the
vessel, the nature of the vessel surface, the viscosity of the
medium and the magnetic separation device employed. If the level of
non-specific binding of a system is substantially constant, as is
usually the case, then as the target population decreases so does
the purity. As an example, a system with 0.2% NSB that recovers 80%
of a population which is at 0.25% in the original mixture will have
a purity of 50%. Whereas if the initial population were at 1.0%,
the purity would be 80%. Not as obvious is the fact that the
smaller the population of a targeted cell, the more difficult it
will be to magnetically label and to recover. Furthermore, labeling
and recovery will markedly depend on the nature of magnetic
particle employed. For example, when cells are incubated with large
magnetic particles, such as Dynal beads, the cells are labeled
through collisions created by mixing of the system as the beads
tend to be too large to diffuse.
[0018] Thus, if a cell were present in a population at a frequency
of 1 cell/ml of blood or even less, as could be the case for tumor
cells in very early cancers, then the probability of labeling
target cells will be related to the numbers of magnetic particles
added to the system and the length of time of mixing. Since mixing
of cells with such particles for substantial periods of time will
be deleterious, it becomes necessary to increase particle
concentration as much a possible. There is, however, a limit to the
quantity of magnetic particle that can be added to the system, in
that one can substitute a system comprising a rare cell mixed in
with other blood cells with one comprising a rare cell mixed in
with large quantities of magnetic particles upon separation, in
which case the ability to enumerate the cells of interest or to
examine them is not markedly improved.
[0019] There is another drawback to the use of large particles to
isolate cells having rare frequencies (1-50 cells/ml of blood).
Despite the fact that large magnetic particles allow the use of
external gradients of very simple design and relatively low
magnetic gradient, large particles tend to cluster around cells in
a cage-like fashion making them difficult to "see" or to analyze.
Hence, the particles must be released before analysis, and
releasing the particles often introduces other complications.
[0020] In theory, colloidal magnetic particles, used in conjunction
with high gradient magnetic separation, should be the method of
choice for separating a cell subset of interest from a mixed
population of eukaryotic cells, particularly if the subset of
interest comprises only a small fraction of the entire population.
With appropriate magnetic loading, sufficient force is exerted on a
cell, facilitating its isolation even in a media as viscous as
moderately diluted whole blood. As noted, colloidal magnetic
materials below about 200 nanometers will exhibit Brownian motion
which markedly enhances their ability to collide with and
magnetically label rare cells. This is demonstrated in U.S. Pat.
No. 5,541,072, where results of very efficient tumor cell purging
experiments are described employing 100 nm colloidal magnetic
particles (ferrofluids). Just as importantly, colloidal materials
at or below the size range noted do not generally interfere with
viewing of cells. Cells so retrieved can be examined by flow
cytometry with minimal forward scattering effects or by microscopy
employing visible or fluorescent techniques. Because of their
diffusive properties, such materials, in contrast to large magnetic
particles, readily "find" and magnetically label rare biological
entities such as tumor cells in blood.
[0021] There is, however, a significant problem which arises in the
use of ferrofluid-like materials for cell separation in external
field gradient systems which, for reasons given above, is the
device design of choice. Direct monoclonal antibody conjugates of
Owen et al. materials or Molday nanoparticles, such as those
produced by Miltenyi Biotec, do not have sufficient magnetic moment
for use in cell selection employing the best available external
magnetic gradient devices, such as the quadrupole or hexapole
magnetic devices described in U.S. Pat. No. 5,186,827. When used
for separations in moderately diluted whole blood, they are even
less effective. Using similar materials, which are substantially
more magnetic, as described in U.S. Pat. No. 5,698,271, more
promising results have been obtained. In model spiking experiments,
it has been found that SKBR3 cells (breast tumor line), which have
a high EpCAM (epithelial cell-adhesion molecule) determinant
density, are efficiently separated from whole blood with direct
conjugates of anti EpCAM MAb ferrofluids even at very low spiking
densities (1-5 cells/ml blood). On the other hand, PC3 cells (a
prostate tumor line) which have low antigen density are separated
at significantly lower efficiency. Most likely this is a
consequence of inadequate magnetic loading onto these low density
receptor cells.
[0022] From the foregoing discussion, it would be advantageous to
provide a magnetic separation system which combines the beneficial
properties of both colloidal magnetic materials and large magnetic
particles (e.g., diffusion based labeling and large magnetic
moment, respectively) for separations involving rare events or for
cells with very low density receptors. One could envision starting
a separation process with a magnetic colloidal or nanoparticle
which, due to their Brownian motion, would rapidly find and label
cells in rare numbers or cells with very low density receptors.
Once that labeling is achieved, it would be desirable to convert
the magnetic moment of the nanoparticle to a value similar to that
of a large magnetic particle. In that way, magnetically labeled
entities could be separated in the kinds of gradient fields used
for larger particles, e.g., a simple external field gradient
separator. In the case of very low density receptor cells, which
are recovered inefficiently even in high gradient external field
separators, use of such a principle would clearly increase the
efficiency of separation. In applications where cells are to be
analyzed or used for some biological purpose following separation,
it would also be very desirable to be able to convert the magnetic
moment of the labeled entity back to that of its original colloidal
magnetic labeling density. This approach would permit separation
from excessive magnetic material, which would facilitate subsequent
analysis or use.
[0023] U.S. Pat. No. 5,466,574 to Liberti et al., describes a
system which has some of the foregoing features regarding "loading
on" of magnetic materials onto cells. It was discovered that when
cells were first labeled with specific monoclonal antibodies (with
or without biotinylation) followed by magnetic labeling with goat
anti-mouse ferrofluid or with streptavidin-ferrofluid
(respectively), separation was enhanced in the presence of excess
monoclonal antibody. The unique ability of ferrofluids to create
this "no wash" enhancing procedure is due to immunochemical
crosslinking of free ferrofluid in solution to ferrofluid-bound
target cells. Ferrofluid bound to monoclonal antibody on cells, in
turn, binds to free ferrofluid in solution via free monoclonal
antibody. This results in immunochemical clusters of monoclonal
antibody/ferrofluid "growing" off of monoclonal antibody labeled
cell determinants (referred to as chaining). Thus, magnetic colloid
is "artificially" loaded onto cells making them more magnetic and
easier to separate. The phenomenon was found to obey immunochemical
rules, in that a high excess of monoclonal antibody resulted in a
decrease in chaining (monoclonal excess zone) and a loss of
separation efficiency. Similarly high levels of ferrofluid also
reduced chaining (ferrofluid excess zone). Chaining has been found
to be useful for purging unwanted cells, e.g. tumor cells, in bone
marrow or peripheral blood "grafts." By this method, very high
levels of magnetic material (visible brown rims around cells, as
observed via microscopy) can be loaded onto target cells giving
rise to very efficient separation in high gradient fields of only
8-12 kGauss/cm gradients. On the other hand, cells labeled with
"monomeric" ferrofluid were found to separate less efficiently in
the same gradient.
[0024] In attempts to use chaining for isolating rare cells from
whole blood, several problems have been encountered. First,
although spiked cells are, indeed, efficiently recovered, they are
so densely covered with ferrofluid (chaining) that the ability to
analyze them is markedly reduced. Hence this approach is not ideal
for applications wherein the positively selected cells are to be
observed via microscopy or flow cytometry. Additionally, chaining
seems to promote non-specific binding. In summary, designing a
chaining-based assay where the level of chaining simultaneously
gives rise to separation enhancement, non-obstructed viewing of the
isolated cells and acceptable levels of non-specific binding is
extraordinarily difficult. The chaining reaction is difficult to
control because it requires immunochemical stoichiometry. For
example, most (>99%) of the added monoclonal antibody (or
tagging ligand) will always be free in solution regardless of the
affinity of the antibodies. Hence, the amount of ferrofluid
required to achieve immunochemical equivalence (where the best
separations take place via chaining) generally leads to more
chaining than is desired, particularly in the case where the
selected cell is to be viewed and/or further studied. Chaining can
be lessened by concurrent decreases in labeling monoclonal antibody
and added ferrofluid, however this results in a sacrifice of
separation efficiency. Another drawback to the use of chaining to
enhance separation is the inability to, in some practical manner,
reverse chaining. If chaining could be reversed and the concomitant
increase in non-specific binding decreased, the phenomenon would
provide a viable approach to enabling the desired "loading on" of
magnetic material. Another disadvantage of this method is that a
two step reaction is required, i.e., reaction of targets with
primary monoclonal antibody in a first step followed by repetition
with ferrofluid specific for primary monoclonal antibody in the
second step. This approach cannot be used in assays where primary
antibody is directly conjugated to ferrofluid.
[0025] U.S. Pat. No. 5,108,933 to Liberti et al. discloses the use
of weakly magnetic colloidal materials such as those described by
Owen et al. or Molday in immunoassays employing external field
magnetic separators. Such materials are described therein as
agglomerable and resuspendable colloidal magnetic materials which
remain substantially undisturbed in an external magnetic field
system, for example, those commercially available at that time
(Ciba Corning, Wampole, Mass.; Serono Diagnostics, Norwell, Mass.).
By contrast, materials made by the process disclosed in the '531
patent being substantially more magnetic, as noted above, will
separate in those separators. In the '933 patent means for
converting the colloid to an agglomerate are disclosed so as to
make them separable in those separators. Thus, such materials could
be used for performing the bound/free separation step of
immunoassays. There is no mention in '933 for the need of, or
methods for reversing agglomeration reactions.
[0026] In light of the foregoing and recent discoveries of
naturally occurring ferrofluid aggregation factors, the present
inventors have recognized the need for compositions and methods for
controlling aggregation of ferrofluid by endogenous factors during
the isolation and immunochemical characterization of rare target
bioentities. Such compositions and methods may be used to advantage
to facilitate analysis and observation of bioentities so isolated.
Further, this invention also permits the use of substantially less
magnetic reagent as well as the opportunity to use lower magnetic
gradients. In the case of a fixed gradient, the invention provides
for the capture or isolation of entities which might have otherwise
had insufficient magnetic labeling to be captured.
SUMMARY OF THE INVENTION
[0027] In accordance with the present invention, methods,
compositions and kits are provided for controlling the aggregation
of ferromagnetic nanoparticles by endogenous aggregation factors.
Ferrofluid aggregation often presents problems during subsequent
viewing of the isolated targets. The methods of the invention
facilitate visualization of the isolated bioentities by allowing
the investigator to control the level of aggregation. In one
embodiment of the invention, a method is provided for inhibiting
the aggregation of magnetic nanoparticles on the surface of
isolated target entities. The method comprises obtaining a
biological specimen suspected of containing a target bioentity.
Next, immunomagnetic suspensions are prepared by mixing the
specimen with colloidal, magnetic particles coupled to a
biospecific ligand having affinity for at least one characteristic
determinant of the target bioentity. The immunomagnetic suspension
is thereafter subjected to a magnetic field to obtain target
bioentity enriched fractions. optionally, the fractions are then
examined to determine the characteristics of the target bioentity
so isolated. Inhibition of ferrofluid aggregation facilitates
subsequent analysis of cells as aggregates of ferrofluid on the
cell surface are eliminated. The absence of such aggregates is
important for several types of analyses including, for example,
flow cytometry and immunofluorescence microscopy.
[0028] The reagents provided herein efficiently inhibit or remove
endogenous aggregation factors. The factor removal or inhibition
step may be performed before or simultaneously with the addition of
ferrofluid to the biological specimen for separation and
enrichment.
[0029] To further characterize target bioentities isolated using
the methods of the invention, the method optionally includes the
steps of adding to the target bioentity enriched fraction at least
one biospecific reagent which recognizes and effectively labels at
least one additional characteristic determinant on said target
bioentity. The labeled target bioentities are then separated in a
magnetic field to remove unbound biospecific reagents. A non-cell
exclusion agent is added to the separated bioentities to allow
exclusion of non-nucleated components present in the sample. After
purifying the target bioentity, it is then ready for analysis using
a variety of different analysis platforms. Target bioentities
include, without limitation, tumor cells, virally infected cells,
fetal cells in maternal circulation, virus particles, bacterial
cells, white blood cells, myocardial cells, epithelial cells,
endothelial cells, proteins, hormones, DNA, and RNA. Target
bioentities may be analyzed by a process selected from the group
consisting of multiparameter flow cytometry, immunoflourescent
microscopy, laser scanning cytometry, bright field base image
analysis, capillary volumetry, manual cell analysis and automated
cell analysis. Aggregation inhibiting agents suitable for use in
the methods of the present invention, include, but are not limited
to reducing agents, animal serum proteins, immune-complexes,
carbohydrates, chelating agent, unconjugated ferrofluid, and
diamino butane. In the case where the endogenous aggregation factor
is of the IgM class and reactive with ferrofluids, preferred
aggregation inhibiting agents are reducing agents, such as Mercapto
ethane sulfonic acid [MES], Mercapto Propane Sulfonic acid [MPS]
and dithiothreitol [DTT]. In a particularly preferred embodiment,
the biospecific ligand is a monoclonal antibody having affinity for
an epithelial cell adhesion molecule.
[0030] In an alternative and preferred embodiment of the invention,
a method is provided for isolating target bioentities from a
biological sample by controlling aggregation of magnetic
nanoparticles. The method entails obtaining a biological specimen
suspected of containing said target bioentity and contacting the
biological specimen with a reagent effective to inactivate any
endogenous aggregating factors present. Immunomagnetic suspensions
are then prepared wherein the specimen is mixed with colloidal,
magnetic particles coupled to a biospecific ligand having affinity
for at least one antigen present on the target bioentity, the
magnetic particles being further coupled to a first exogenous
aggregation enhancing factor which comprises a first member of a
specific binding pair. A second multivalent exogenous aggregation
enhancing factor is then added to the immunomagnetic suspension to
increase aggregation of the particles, the second aggregating
enhancing factor comprising the second member of the specific
binding pair, which reversibly binds to the magnetically labeled
target bioentity. The sample is then subjected to a magnetic field
to obtain a target bioentity enriched fraction. This preferred
embodiment takes advantage of the fact that aggregating ferrofluid
onto target entities in a controlled and reversible fashion results
in substantially improved isolation efficiency.
[0031] In a further embodiment, the above described method further
comprises the steps of adding at least one biospecific reagent
which recognizes and labels at least one additional characteristic
determinant on said target bioentity. The target bioentity so
labeled is then separated in a magnetic field to remove unbound
biospecific reagent. A non-cell exclusion agent is added to the
separated bioentities to allow exclusion of non-nucleated
components present in the sample. The target bioentity is then
purified and further analyzed. In order to reverse the aggregation
mediated by the exogenous aggregation factors, a member of the
specific binding pair may be added in excess to the purified
bioentity to reduce ferrofluid aggregation on the surface of cells,
thereby facilitating viewing of the cells, e.g. in a microscope.
Suitable specific binding pairs for this purpose include, without
limitation, biotin-streptavidin, antigen-antibody,
receptor-hormone, receptor-ligand, agonist-antagonist,
lectin-carbohydrate, Protein A-antibody Fc, avidin-biotin, biotin
analog-streptavidin, biotin analog-avidin,
desthiobiotin-streptavidin, desthiobiotin-avidin,
iminobiotin-streptavidi- n, and iminobiotin-avidin. Preferably, the
biospecific ligand is a monoclonal antibody having affinity for
epithelial cell adhesion molecule. Exemplary biospecific reagents
include monoclonal antibodies, polyclonal antibodies, detectably
labeled antibodies, antibody fragments, and single chain
antibodies. Isolated target bioentities may be analyzed by a
process selected from the group consisting of multiparameter flow
cytometry, immunofluorescent microscopy, laser scanning cytometry,
bright field base image analysis, capillary volumetry, manual cell
analysis and automated cell analysis.
[0032] In accordance with the present invention, controlling
aggregation of ferrofluid in a sample has several unexpected
benefits previously noted, e.g. increasing efficiency of separation
of some particular entity. It has been discovered that addition of
an exogenous aggregation enhancing factor gives rise to increased
magnetic loading, resulting in increased separation efficiency
while reducing the amount of ferrofluid required to isolate the
target bioentity. The increased magnetic loading also allows for
reduced incubation periods and facilitates isolation of the target
bioentity in the presence of a suboptimal magnetic field.
[0033] In an additional embodiment of the present invention a kit
is provided which facilitates the practice of the methods described
herein. An exemplary kit for isolating target bioentities includes
i) coated magnetic nanoparticles comprising a magnetic core
material, a protein base coating material, and an antibody that
binds specifically to a first characteristic determinant of said
target bioentity, said antibody being coupled, directly or
indirectly, to said base coating material; ii) at least one
antibody having binding specificity for a second characteristic
determinant of said rare biological substance; iii) an aggregation
inhibiting factor; and iv) a non-cell exclusion agent for excluding
non-nucleated sample components other than said target bioentity
from analysis.
[0034] A kit for improving the isolation efficiency of certain
biological entities, such as might be required for isolating low
antigen density tumor cells from a biological sample, is also
provided in accordance with the present invention. This kit
utilizes controlled and reversible aggregation of magnetic
nanoparticles to achieve such improvement. Such a kit includes i) a
reagent effective to inactivate endogenous aggregating factors; ii)
coated magnetic nanoparticles comprising a magnetic core material,
a protein base coating material, and an antibody that binds
specifically to a first characteristic determinant of said tumor
cell, the antibody being coupled, directly or indirectly, to the
base coating material; the magnetic particles being further coupled
to a first exogenous aggregation enhancing factor, the factor
comprising one member of a specific binding pair; iii) at least one
antibody having binding specificity for a second characteristic
determinant of said tumor cell; iv) a second exogenous aggregation
enhancing factor, the second aggregation enhancing factor
comprising the second member of the specific binding pair; and v) a
non-cell exclusion agent for excluding non-nucleated sample
components other than the tumor cells from analysis. The kit may
optionally include a reagent for reversing the exogenous
aggregation factor. Specific binding pairs useful in such a kit,
include without limitation, biotin-streptavidin, antigen-antibody,
receptor-hormone, receptor-ligand, agonist-antagonist,
lectin-carbohydrate, Protein A-antibody Fc, and avidin-biotin,
biotin analog-avidin, desthiobiotin-streptavidin,
desthiobiotin-avidin, iminobiotin-streptavidin, and
iminobiotin-avidin. Reagents effective to inactivate endogenous
aggregating factors include reducing agents, animal serum proteins,
immune-complexes, carbohydrates, chelating agent, unconjugated
ferrofluid, and diamino butane.
[0035] The methods, compositions and kits of the invention provide
the means for controlling the aggregation of magnetic
nanoparticles, thus facilitating the isolation, visualization and
characterization of rare biological substances or cells from
biological specimens.
BRIEF DESCRIPTION OF THE DRAWING
[0036] FIGS. 1A-1H are a series of micrographs depicting what is
observed in a microscope in samples derived from blood donors with
high levels of endogenous aggregating factors versus those with low
levels of endogenous aggregation factors. Breast cancer cells were
spiked into whole blood and selected using EPCAM colloidal magnetic
particles and stained in suspension. FIG. 1A, transmitted light
only, low aggregation; FIG. 1B, transmitted light only, high
aggregation; FIG. 1C, cells stained with Hoechst nuclear stain, low
aggregation; FIG. 1D, cells stained with Hoechst nuclear stain,
high aggregation; FIG. 1E, cells stained with the epithelial cell
marker cytokeratin Alexa 488, low aggregation; FIG. 1F, cells
stained with the epithelial cell marker cytokeratin Alexa 488, high
aggregation; FIG. 1G, cells stained with tumor cell receptor marker
erb2-conjugated to phycoerythrin, low aggregation; FIG. 1H, cells
stained with tumor cell receptor marker erb2-conjugated to
phycoerythrin, high aggregation.
DETAILED DESCRIPTION OF THE INVENTION
[0037] The term "target bioentities" as used herein refers to a
wide variety of materials of biological or medical interest.
Examples include hormones, proteins, peptides, lectins,
oligonucleotides, drugs, chemical substances, nucleic acid
molecules, (e.g., RNA and/or DNA) and particulate analytes of
biological origin, which include bioparticles such as cells,
viruses, bacteria and the like. In a preferred embodiment of the
invention, rare cells, such as fetal cells in maternal circulation,
or circulating cancer cells may be efficiently isolated from
non-target cells and/or other bioentities, using the compositions,
methods and kits of the present invention. The term "biological
specimen" includes, without limitation, cell-containing bodily,
fluids, peripheral blood, tissue homogenates, nipple aspirates, and
any other source of rare cells that is obtainable from a human
subject. An exemplary tissue homogenate may be obtained from the
sentinel node in a breast cancer patient. The term "determinant",
when used in reference to any of the foregoing target bioentities,
may be specifically bound by a biospecific ligand or a biospecific
reagent, and refers to that portion of the target bioentity
involved in, and responsible for, selective binding to a specific
binding substance, the presence of which is required for selective
binding to occur. In fundamental terms, determinants are molecular
contact regions on target bioentities that are recognized by
receptors in specific binding pair reactions. The term "specific
binding pair" as used herein includes antigen-antibody,
receptor-hormone, receptor-ligand, agonist-antagonist,
lectin-carbohydrate, nucleic acid (RNA or DNA) hybridizing
sequences, Fc receptor or mouse IgG-protein A, avidin-biotin,
streptavidin-biotin and virus-receptor interactions. Various other
determinant-specific binding substance combinations are
contemplated for use in practicing the methods of this invention,
such as will be apparent to those skilled in the art. The term
"antibody" as used herein, includes immunoglobulins, monoclonal or
polyclonal antibodies, immunoreactive immunoglobulin fragments, and
single chain antibodies. Also contemplated for use in the invention
are peptides, oligonucleotides or a combination thereof which
specifically recognize determinants with specificity similar to
traditionally generated antibodies. The term "detectably label" is
used to herein to refer to any substance whose detection or
measurement, either directly or indirectly, by physical or chemical
means, is indicative of the presence of the target bioentity in the
test sample. Representative examples of useful detectable labels,
include, but are not limited to the following: molecules or ions
directly or indirectly detectable based on light absorbance,
fluorescence, reflectance, light scatter, phosphorescence, or
luminescence properties; molecules or ions detectable by their
radioactive properties; molecules or ions detectable by their
nuclear magnetic resonance or paramagnetic properties. Included
among the group of molecules indirectly detectable based on light
absorbance or fluorescence, for example, are various enzymes which
cause appropriate substrates to convert, e.g., from non-light
absorbing to light absorbing molecules, or from non-fluorescent to
fluorescent molecules. A nucleic acid dye or other reporter
molecule, sometimes referred to herein as a non-cell exclusion
agent, which is capable of identifying both target bioentities and
certain non-target bioentities, such as intact nucleated cells, is
added to the sample to allow exclusion of any residual
non-nucleated cells or other potentially interfering sample
components prior to analysis by flowcytometry, microscopy, or other
analytical platforms. Non-cell exclusion agents may be reactive
with DNA, RNA, protein, or lipids such that the amount of signal
obtained is typical of that obtained for cells or the image
obtained reveals typical features of a cell, such as cell and
nuclear membranes, nucleus, and mitochondria.
[0038] The term "optimal" used herein describing ferrofluid
concentration, magnetic field strength, or incubation time refers
to the conditions used in a standard, unmodified assay, separation,
isolation, or enrichment.
[0039] The term "sub-optimal" used herein describing ferrofluid
concentration, magnetic field strength, or incubation time refers
to the conditions used in an assay, separation, isolation, or
enrichment which would produce inferior results, as compared to
results obtainable under optimal conditions.
[0040] The term "ferrofluid" as used herein refers to magnetic
nanoparticles in suspension. The terms ferrofluid and magnetic
nanoparticles are used interchangeably herein.
[0041] Endogenous ferrofluid aggregation factors are those present
in a sample isolated from a test subject. Exogenous aggregation
factors are those provided herein which are added and/or reversed
as desired by the investigator.
[0042] The preferred magnetic particles for use in this invention
are particles that behave as colloids and are superparamagnetic.
The colloids are characterized by their size, i.e., smaller than
200 nm or of a size which doesn't interfere with the analysis. The
superparamagnetic particles become magnetic only when they are
subjected to a magnetic field gradient and do not become
permanently magnetic. The colloidal superparamagnetic particles do
not separate or settle from aqueous solution for extended periods
of time. These particles are composed of either single crystals of
iron oxides or agglomerates of such crystals surrounded by
molecules either physically adsorbed or covalently attached. The
colloidal magnetic particles with the above characteristics can be
prepared as described in U.S. Pat. Nos. 4,795,698; 5,512,332 and
5,597,531. A monoclonal antibody, which recognizes a specific
subset of cells, conjugated to magnetic particles is preferred for
use in this invention.
[0043] In the course of studies on rare cell isolation, a factor
present in the blood of certain patients was discovered which
effectuates aggregation of magnetic nanoparticles. Further, this
enhanced aggregation effect appears to be readily reversible. Thus,
rare cells having low receptor densities can be isolated more
efficiently from moderately diluted whole blood in external field
quadrupole or hexapole separators by "loading on" more ferrofluid
than that which is specifically bound to a characteristic
determinant of the rare cell of interest. When isolated cells are
examined by microscopy, ferrofluid clusters are present on the cell
membrane. Reversal of the enhancing effect deaggregates the
clusters, facilitating microscopic analysis of the cells. This
endogenous enhancing effect exists in varying levels in about 90%
of apparently normal donor blood samples. By manipulating assay
conditions, the resultant ferrofluid clusters can be dispersed
without damaging the cells of interest. Thus, the present invention
provides methods for eliminating the effects of endogenous
ferrofluid aggregation factors and where desirable, means for
constructing agents capable of controlled and reversible
aggregation of ferrofluid. This permits effective and efficient
isolation and enrichment and subsequent analysis of rare cells, and
other biological entities such as viruses and bacteria.
[0044] The endogenous ferrofluid aggregating substance found in
blood has the following characteristics: (1) it is present in
plasma or serum; (2) it is sensitive to millimolar concentrations
of dithiothreitol; and (3) it reacts with "bare" crystalline
regions on direct coated ferrofluid, as described in U.S. Pat. No.
5,597,531. Control experiments revealed that the factor is not
ferritin, transferrin, fibrinogen, C1q, human anti-mouse or
anti-BSA antibodies. The aggregating substance is also not of the
IgG subclass since IgG-depleted plasma also caused aggregation of
the ferrofluid. Ferrofluid aggregation appears to be correlatable
with ferrofluid or serum concentration. The dependency of
aggregation on concentration of either component is similar to that
observed with precipitin curves and based on the observations noted
above, it was postulated that the substance is an IgM. This was
conclusively proven by removing the IgM from the plasma samples via
immunoaffinity purification. The resulting IgM depleted plasma did
not effectuate ferrofluid aggregation.
[0045] Control experiments, where the same plasma samples were
absorbed through a BSA affinity media, did show ferrofluid
aggregation. Further study demonstrated that extensive adsorption
of sera containing the aggregating factor with ferrofluids only
removed a small amount of the total IgM and that purified human IgM
causes ferrofluid aggregation. These observations along with
identity studies of adsorbed aggregating factor and the ability to
cause aggregation with adsorbed/desorbed material led to the
conclusion that the aggregation factor in these experiments is,
indeed, a highly specific IgM. Based on the inability to inhibit
aggregation by any component used in forming the ferrofluid except
for magnetite crystals poorly coated with protein or magnetite
crystals partially coated with detergent, it is believed that the
epitope recognized by the IgM is present on the magnetite
crystalline surface. The role of this specific IgM is not known but
it is present in a significant portion of the human population and
at varying levels. It is possible that this antibody plays some
role in iron metabolism.
[0046] Methods of manipulating endogenous enhancing factors are
described herein which permit patient-to-patient comparison of
isolated cells in a meaningful manner. Because the endogenous
aggregation enhancing factor concentration varies in patient
populations, it is difficult to create a standard procedure for
separating rare circulating cells, be they tumor cells, fetal cells
in maternal blood or virally infected cells. Recent discoveries in
a co-pending application Ser. No. 09/248,388) have demonstrated
that the magnitude of the number of circulating cells is directly
related to tumor burden and stage of disease in breast cancer
patients. Similarly, viral burden has been shown to be of
significance in the prognosis of HIV infection. The need for
accurate quantitation of such entities is becoming ever more
important. The methods disclosed herein are conveniently practiced
by means of test kits which may be used to advantage in the
clinical setting.
[0047] Having discovered the presence of this ferrofluid
aggregation factor (FFAF) in donor sera, careful studies confirmed
that its presence markedly improves the retrieval of spiked low
density receptor tumor cells using monoclonal antibody conjugated
ferrofluid. In these studies, cells spiked at levels of 1 cell/2
mls of blood containing FFAF are routinely retrieved at greater
than 70% efficiency. In contrast, in donor blood where FFAF is
absent, efficiency of retrieval is reduced to approximately 15-25%.
From those experiments, the following observations were made:
[0048] 1. low density receptor cells are often isolated less
efficiently than cells with high density receptors;
[0049] 2. separation efficiency varies considerably for low density
receptor cells and this variation is dependent on the blood
donor;
[0050] 3. when either cell type (high or low density characteristic
determinant cells) was examined via microscopy following
ferrofluid/magnetic isolation from the blood of different donors,
ferrofluid was aggregated in about 90% of patient samples; and
[0051] 4. tumor cells retrieved had differing degrees of visible
ferrofluid aggregated on their surfaces.
[0052] Moreover, since the aggregation material is sensitive to
DTT, retrieved cells can be readily visualized by microscopy for
morphological characteristics by reduction of the ferrofluid
aggregates prior to observation.
[0053] In comparing the phenomena of FFAF and chaining (see U.S.
Pat. No. 5,466,574) to enhance cell recoveries, some interesting
conclusions can be drawn. When FFAF is present in blood in
exceedingly low concentrations, as is the case in many individuals,
excellent recoveries of low density receptor spiked cells are
obtained, e.g., 1-5 cells/ml blood. The quality of the recovered
cells, as assessed by the level of magnetic material on their
surfaces is quite suitable for morphological examination or further
manipulation. When FFAF is in high concentrations, recoveries are
also excellent but the quality of the recovered cells is not
acceptable as the agglomerates on the cells obscures their viewing.
Hence, unlike the components of chaining, i.e. monoclonal
antibody-ferrofluid "chains", which are limited by concentration,
there is a level of FFAF which can lead to very effective cell
separation, thus providing an isolated population of cells which
can be studied effectively. In certain embodiments of the
invention, endogenous FFAF's in the samples are inactivated at the
outset so that aggregation can be controlled by the investigator by
adding the compositions described herein. In this way, aggregation
can be enhanced or inhibited as desired.
[0054] FFAF and any similar substances must be evaluated in assays
to isolate rare cells. Such factors must be controlled in order to
develop a test which functions reliably for every patient. For
example, many individuals have anti-rodent antibodies, or
antibodies to components, such as carbohydrates, which might be
found on the surface of some magnetic nanoparticles. Other
potential aggregating substances normally present in blood include
C1q, rheumatoid factor, and blood clotting proteins. Such reactions
must either be controlled, so as to make them constant from
specimen to specimen, or be eliminated altogether.
[0055] In the case of anti-rodent antibodies, this can be
accomplished by adding rodent proteins to the system so as to
inhibit their aggregating effects. This addition is quite different
from adding such components to immunoassay systems. In the latter
case, anti-rodent antibodies generally enhance isolation but also
tend to increase false positives as they link captured antibody
with labeling antibody in sandwich-type reactions. In contrast,
additional antibodies used in the methods of the present invention
enhance the recovery of low density receptor cells. Thus, in one
case aggregating factors can artificially elevate false positives,
and in another facilitate isolation of the target bioentity.
[0056] Besides competitively inhibiting such factors, they may be
disabled or adsorbed from solution, or the determinant on the
magnetic colloid to which such factors bind may be eliminated. FFAF
activity may be inhibited by formulating a special buffer that
contains optimal amounts of additives such as mouse and other
animal serum proteins, immune-complexes, carbohydrates, chelating
agents which inhibit various activation systems, including
complement, and compounds which specifically inhibit the
interaction of C1q with reacted antibody, such as diamino butane.
In the case where FFAF is an IgM, reducing agents effectively
disable the FFAF without affecting the ligands used for labeling
cells. Thus, such factors could be selectively disabled chemically
or enzymatically. Adsorption with appropriate materials, e.g.,
unconjugated ferrofluid, provides another route for removing
aggregating factors from the sample.
[0057] It may also be possible to make the amount of aggregator
constant from patient to patient so as to always have identical
levels of enhancement. This may be accomplished in different ways.
For example, all endogenous factors may be reduced to the same
level in all patient samples. However this seems a difficult and
impractical solution. As an alternative, a two step process may be
employed where, as a first step, all endogenous FFAF's are disabled
without affecting specific ligand binding of the ferrofluid to its
target or subsequent cell analysis. The second step entails a
controlled aggregation reaction. Colloidal magnetic materials
conjugated to two kinds of ligands may be used to practice the two
step method described above. One ligand, such as monoclonal
antibody would be directed to cell surface determinants and
effectively labels cells. The secondary ligand would have no
reactivity with any component of blood yet have binding affinity
for a multivalent component which would be added following binding
of the primary ligand. Thus, additional magnetic colloid would bind
to colloid already bound to cells, thereby enhancing their magnetic
load just as FFAF does. Similarly, it would be preferable if the
reaction of the secondary ligand and its multivalent component were
reversible. By selecting the right components of the secondary
reactions and their concentrations, it should be possible to add
ferrofluid and the aggregation factor simultaneously.
[0058] Therefore, FFAFs are provided which facilitate controlled
aggregation of ferrofluids, thereby enhancing recoveries of rarely
occurring biological entities. The ideal aggregating factor is one
which mediates a reversible aggregating effect. Reversal of
aggregation eliminates magnetic nanoparticle clusters so as to
facilitate visualization of isolated cells. The factors of the
invention operate in a manner similar to the ideal magnetic
particle conditions described above i.e., by converting colloid
nanoparticles which are bound to target into large particles with
the added ability to readily reverse that process.
[0059] The identification and elucidation of an endogenous factor
present in the blood of most normal donors which enhances the
efficiency of isolation of low density receptor rare cells is
described herein.
[0060] Preferable FFAFs include specific multivalent substances
which recognize determinant(s) on ferrofluid magnetic particles
thereby crosslinking the particles. This factor may naturally occur
in plasma or may be an exogenously added reagent. Several types of
exogenous reagents are suitable for this purpose and include, but
are not limited to, IgG, dimeric IgG, IgM, Streptavidin, Avidin,
Protein A, Protein G, dimeric or tetrameric poly-A or poly-T, or
specific oligonucleotide sequences. The secondary ligand can be
introduced onto ferrofluid and is recognized by FFAF. There are
several types of secondary ligands which may be selected such as
hapten, biotin, biotin analogues (iminobiotin, desthiobiotin),
sheep IgG, goat IgG, rat IgG, poly-A or poly-T or oligonucleotide.
FFAF-secondary ligand interaction may be either reversible or
irreversible but a reversible interaction is preferred. There are
several reagents which may be used to reverse FFAF-secondary ligand
interaction such as reducing agents, excess of haptens, excess of
hapten analogues, excess of analogues of the secondary reagent,
change of salt concentration, change of pH or change of
temperature.
[0061] When an assay or separation is performed under optimal
conditions, the percent recovery of the target cells is maximized.
However, the ability to modify the conditions is apparent when
exogenous aggregation is induced. In the case of high density
surface receptor target cells, the amount of ferrofluid needed for
maximum recovery is quite high without exogenous aggregation, to
ensure that each target is magnetically responsive by saturating
all the available binding sites. One benefit of this invention is
that less total ferrofluid is needed for separation, as it does not
need to saturate all the available binding sites. This is due to
the ability to form crosslinks of magnetic particles, mediated by
the exogenous aggregation factor and increase the magnetic mass per
binding site. Instead of rendering cells magnetically responsive by
the use of many single magnetic particles per cell, i.e. one
particle binds one cell surface antigen, the aggregates of multiple
particles can bind the single antigen and will maintain the same
magnetic force as optimally captured cells. Each particle now has
the capability to bind a cell surface antigen or another particle.
In other words, even though less total ferrofluid is used, each
target cell will have the same magnetic responsiveness, and the
resulting separation efficiency will be comparable to that obtained
under optimal conditions. Optimally, 10 .mu.g of ferrofluid is used
per 1 ml of sample. This concentration may be reduced 10-fold in
accordance with the present invention.
[0062] In addition to reducing the amount of ferrofluid needed, the
magnetic strength as well as the incubation time can be reduced.
Because the aggregates of the invention are larger than individual
magnetic particles in a non-aggregating separation, a lesser
magnetic field strength can still move the aggregates. Promoting
exogenous aggregation creates temporary and reversible large
magnetic particles from small magnetic particles. Indeed, the
benefits of large particles, including the ability to use weak
magnetic fields for separation, can be applied to the present
invention. The quadrupole magnetic separators typically used
maintain a field gradient strength of 6.3 kGauss/cm at the vessel
surface. Magnetic arrangements, such as dipoles, which have almost
half the magnetic field strength at the vessel surface may be
utilized in practicing the methods of the present invention.
[0063] In the non-aggregating system, longer incubation times are
required to increase the number of magnetic particles per cell for
effective separation. In this system, one magnetic particle will
bind one cell surface antigen. However, in an exogenously-induced
aggregation system, the same number of particles per cell can be
achieved by causing multiple particles per antigen, via the induced
aggregates. This allows the incubation time to be shortened because
not all of the available binding sites will need to be bound to
magnetic particles. By adding this second binding pair member, the
particles now can bind to other particles, instead of only being
capable of binding to free cell surface antigens. Therefore, as
explained above, the binding sites do not need to be saturated to
have the same magnetic responsiveness as optimal conditions, which
allows the reduction of incubation time. The minimum time for
magnetic incubations is 30 minutes. Using the present invention,
these times may be reduced up to 3-fold. However, it is not
intended that both ferrofluid concentration be reduced and
incubation time be shortened simultaneously. Variations of these
steps should be exploited independently of one another in order to
maintain maximal recovery of target bioentities.
[0064] A secondary ligand which is recognized by an exogenous
ferrofluid aggregation factor will be coupled to ferrofluid in
addition to the above monoclonal antibody by standard coupling
chemistry. The secondary ligand may be a small molecule such as
hapten or biotin analogue or a big molecule such as an antibody or
a specific protein or a polymer such as polypeptides or
polyoligonucleotides. A biotin analogue such as desthiobiotin is
preferred for conjugation to magnetic particles as a secondary
ligand in this invention as it exhibits a lower affinity
(Ka=10.sup.6 M.sup.-1 for streptavidin, as compared to native
biotin (Ka=10.sup.15 M.sup.-1).
[0065] The interaction between streptavidin and desthiobiotin can
be easily disrupted by the addition of excess biotin. The
combination of desthiobiotin and avidin has been used to remove
magnetic particles or insoluble phase from the target substances
(PCT/US94/10124 and U.S. Pat. No. 5,332,679). In this invention,
that combination is used only to aggregate and disaggregate
magnetic particles and not to remove magnetic particles from the
target substance.
[0066] The reaction vessel for use in this invention may be either
glass or plastic, however, plastic tubes are preferred. The bottom
of the tube may be round or conical in shape. Tubes with different
lengths or diameters may be used to process different volumes of
samples. For example, in some instances a 50 ml conical tube may be
used to process 20 ml of blood or more. In one embodiment of this
invention, a 12.times.75 mm polystyrene tube or 15 ml conical tube
is used. The reaction vessel used during incubation with magnetic
particles and the vessel used during magnetic separation does not
necessarily need to be the same. Two different types of vessels may
be used, one type for incubation and another type for magnetic
separation. However, it is preferred to use only one vessel in both
cases. The magnetic separation vessel may be a tube or a
flow-through chamber or some other device.
[0067] The test medium used in practicing the present invention may
be any liquid or solution which contains the target substance and
is preferably blood. A test sample in the reaction vessel is
incubated with a ferrofluid conjugated to antibodies specific for a
target substance and a secondary ligand specific for FFAF.
Additionally, an exogenous FFAF is added simultaneously with
ferrofluid to the test sample or after binding of ferrofluid to
target substance. Optionally, a reagent which inhibits or disables
naturally occurring aggregating factor may be added simultaneously
with ferrofluid, or added prior to ferrofluid addition. After an
optimum incubation time, magnetically labeled targets are separated
from the rest of the test medium in a magnetic separator. The
magnetic separator and separation time are selected based upon test
medium and reaction vessel. It is preferred to use high-gradient
magnetic separation devices such as those described in U.S. Pat.
No. 5,186,827. After aspirating the uncollected liquid, the
collected cells may be resuspended in an isotonic buffer or
permeabilizing solution to permeabilize cells for intracellular
staining. The magnetically labeled cells are reseparated
magnetically to remove permeabilizing reagents. The collected cells
are resuspended in a small volume of cell buffer for staining with
labeling substances. The volume of the buffer may be from 100-300
.mu.l. Optionally the cell buffer may contain staining antibodies.
Additionally, the cell buffer may contain a disaggregating reagent
as described above, e.g., biotin. The final concentration of biotin
may be from 1-5 mM. The incubation time for antibody staining or
for ferrofluid disaggregation with disaggregating reagent may be
from 10-60 minutes and is preferably 15 minutes. After optimum
staining with antibodies or disaggregation of ferrofluid, excess
reagents may be removed from cells by another magnetic separation.
After aspirating the uncollected liquid, the collected cells are
resuspended in a small volume of isotonic buffer. The volume of
this buffer may be from 100-500 .mu.l. The ferrofluid labeled cells
may be further processed or analyzed by flowcytometry or
microscopy.
[0068] While magnetic particles conjugated to antibody only have
been described above, other types of conjugated magnetic particles
are contemplated for use in the present invention. Magnetic
particles conjugated to proteins other than antibodies may be used.
For example, streptavidin conjugated magnetic particles may be used
to bind target cells which are labeled with antibody-biotin
conjugates. Following labeling of target cells, excess unbound
antibody-biotin may be removed by a wash step using a centrifuge.
The target cells labeled with antibody-biotin are then incubated
with streptavidin ferrofluid for magnetic labeling of cells.
Desthiobiotin conjugated to any polymer or protein (aggregating
factor) will be added to the test medium to aggregate ferrofluid.
Aggregating factor may be added simultaneously with magnetic
particles or after the magnetic particles bind the target cells.
The number of desthiobiotins per polymer or protein should be more
than one to aggregate ferrofluid. Preferably desthiobiotin
conjugated to bovine serum albumin (BSA) may be used. The number of
desthiobiotins on BSA may be 2-10. Such desthiobiotin/protein
conjugates may be synthesized as set forth hereinbelow.
[0069] Although the present invention is described herein primarily
with reference to tumor cell selection from blood, the invention is
not limited to tumor cell selection. Other cell types present in
blood, leukophoresis or bone marrow, such as CD34, CD4, and fetal
cells may be selected. The antigenic determinants on those cells
may be low to high. More generally, the invention applies to the
isolation of any cell which requires magnetic enhancement for its
efficient isolation.
[0070] The following examples are provided to illustrate various
embodiments of the invention. These examples are not intended to
limit the scope of the invention in any way.
EXAMPLE I
[0071] The following data illustrate the effects of the FFAF of the
present invention on the recovery of low and high density receptor
tumor cells spiked into blood samples. An exemplary FFAF has been
identified as a specific IgM present in the blood samples of most
donors. Reducing agents such as dithiothreitol (DTT) and
mercaptoethane sulfonic acid (MES) which cleave disulfide linkages,
prevented ferrofluid aggregation in blood by converting pentameric
IgM to its monomeric form. DTT is not a preferred reagent for use
in the methods of the present invention, as high concentrations
alter cellular morphology, and are toxic to target cells and
leukocytes.
[0072] In this example, the effect of MES on ferrofluid aggregation
and tumor cell recovery of both high and low antigen density tumor
cells from spiked blood is described. The protocol used for this
study was as follows. Blood (2 ml) was placed in a 12.times.75 mm
polystyrene tube and 1 ml of Immunicon dilution-wash buffer was
added to dilute the blood. Next, 100 .mu.l of cell buffer (isotonic
7 mM phosphate, pH 7.4 with 1% BSA and 50 mM EDTA) containing
approximately 1000 SKBR3 or PC3 cells was added. Increasing volumes
of MES (not exceeding 150 .mu.l) were added to the blood samples to
obtain different concentrations of reducing agent. After mixing,
EpCAM MAb (GA73.3; 50 .mu.l) conjugated ferrofluid magnetic
particles were added to the sample. The final concentration of
magnetic particles was 5 .mu.g/ml. The blood sample was mixed well
and incubated for 15 minutes at room temperature. After the
incubation, the tube containing the blood sample was placed in a
quadrupole magnetic separation device. Magnetic separation was
performed for 10 minutes. The supernatant was aspirated and the
tube was removed from the magnetic device. The magnetically
collected cells were resuspended in 1 ml of dilution-wash buffer
and reseparated in a quadrupole magnetic separation device for 5
minutes. The supernatant was discarded and after removal from the
quadrupole device, the target cells were resuspended in 150 .mu.l
of dilution wash buffer. A portion of this sample (5 .mu.l) was
spotted on a microscope slide. The recovered cells were then
photographed using a microscope with a digital camera attached to
it.
[0073] The remaining sample was subjected to flowcytometry analysis
to assess the recovery of tumor cells using the following
procedure. Phycoerythrin (PE)-conjugated MAb (5 .mu.l) specific for
tumor cells (Neu 24.7) and 5 .mu.l of peridinin chlorophyll protein
(PerCP)-conjugated CD45 monoclonal antibody were added to the
sample which was then incubated for 15 minutes. After the
incubation, 1 ml of dilution-wash buffer was added and a magnetic
separation was performed in order to remove excess staining
antibodies. The magnetically collected cells were resuspended in
500 .mu.l of dilution-wash buffer. Nucleic acid dye (10 .mu.l) and
5 .mu.l of 3 mM sized fluorescent beads (5000) were added to this
sample. The sample was then analyzed on a FACSCalibur flowcytometer
(Becton Dickinson) using FL1 as threshold. The fraction of the
fluorescent beads acquired in the flowcytometer was used to
determine the amount of sample analyzed by flowcytometry which, in
turn, facilitates calculation of the recovery of spiked tumor
cells.
1 Recovery of tumor cells (%) Concentration of MES (mM) SKBR3 PC3 0
77 46 20 80 50 50 80 31 75 82 27 100 70 17
[0074] When viewed by microscopy, the final sample showed free
ferrofluid aggregates and ferrofluid aggregates on tumor cells in
the absence of MES. As the concentration of MES was increased,
ferrofluid aggregates decreased and no aggregates were seen at
higher concentrations of MES. These visual results were then
compared with tumor cell recovery as measured by flowcytometry. The
addition of MES had no significant effect on recovery of SKBR3
cells (high antigen density) although microscopy revealed that it
decreased ferrofluid aggregation in solution and on cell surfaces.
In contrast, MES had a significant effect on the recovery of PC3
cells (low antigen density). As the concentration of MES was
increased from 0-100 mM, recovery was decreased from 47% to 17%.
This decrease in recovery of PC3 cells in the presence of
increasing concentrations of MES was due to inhibition of
ferrofluid aggregation and not due to any side effects of MES on
cells, as MES did not decrease the recovery of same spiked PC3
cells from lysed blood samples. Lysed blood is obtained by lysing
red blood cells with ammonium chloride followed by wash step which
removes plasma and ammonium chloride. Lysed blood samples contain
only leukocytes (white cells) whereas whole blood also contains
erthyrocytes and plasma. No ferrofluid aggregation is observed with
lysed blood. Moreover, MES has no significant effect on cell
morphology. These data show that low antigen density cells were
isolated less efficiently than high antigen density cells and
inhibition of ferrofluid aggregation dramatically affects the
isolation of low antigen density cells. Ferrofluid aggregation was
also not observed when washed blood (blood cells with plasma
removed) samples were utilized Therefore, washed blood samples were
used as a control for no aggregation. Just as with whole blood, the
recovery of SKBR3 cells was not decreased with washed blood. PC3
cell recovery on the other hand was decreased significantly (2 to
5-fold) when washed blood was used. This data clearly shows that
ferrofluid aggregation does not have any effect on SKBR3 cells
recovery but has a major effect on PC3 cells recovery.
[0075] In summary, aggregation of tumor specific ferrofluid with
the plasma component (IgM) present in blood of many patients at
varying levels has a significant effect on recovery of low antigen
density cells. Recovery of such cells is affected by the extent of
ferrofluid aggregation and increases with increasing aggregation.
Ferrofluid aggregation increases the recovery of low antigen
density cells by increasing magnetic load on the cells. Ferrofluid
aggregation can vary from one blood donor to another depending upon
the concentration of the aggregating factor or aggregator. As a
result, the recovery of tumor cells will vary from person to person
even though they may possess the same number of circulating tumor
cells. It is also possible that the concentration of the aggregator
present in the blood from the same person can vary with time thus
altering the extent of ferrofluid aggregation and recovery of tumor
cells. The best way to prevent this variation is to prevent
naturally occurring ferrofluid aggregation. However, this gives
rise to a decrease in the efficiency of tumor cells isolation and
detection. One means to increase tumor cell recovery under these
circumstances will be to improve the magnetic device with a higher
gradient which can pull weakly magnetic labeled cells effectively
and increase their recovery. The other way to increase the recovery
of tumor cells will be to mimic natural ferrofluid aggregation with
an exogenous reagent. This reagent can be a specific multivalent
reagent which can recognize ferrofluid and can be added to the
blood and ferrofluid. The specific reagent will aggregate
ferrofluid similarly to IgM but under a controlled reaction.
Controlled aggregation will have two advantages: (1) the percentage
of tumor cells recovered will increase; and (2) the percentage of
tumor cells recovered will not vary from patient to patient and
will not vary with time from the same patient when the samples have
the same number of tumor cells.
EXAMPLE II
[0076] Preparation of Desthiobiotin/EpCAM MAb Ferrofluid for
Controlled Aggregation.
[0077] A base ferrofluid was made as described in U.S. Pat. No.
5,698,271. Monoclonal antibody to the epithelial cell adhesion
molecule (EPCAM) was conjugated to base material by standard
coupling chemistry, as used in U.S. patent application Ser. No.
09/248,388. EpCAM MAb ferrofluid was then resuspended in 20 mM
HEPES, pH 7.5 for conjugation to desthiobiotin using
N-hydroxysuccinimide-DL-desthiobiotin (NHS-desthiobiotin) (Sigma,
Cat.# H-2134). A stock solution of NHS-desthiobiotin was made in
DMSO at 1 mg/ml. NHS-desthiobiotin (19 .mu.g) was added to 1 mg of
EpCAM MAb ferrofluid and incubated at room temperature for 2 hours.
Unreacted NHS-desthiobiotin was removed by washing 3 times with 20
mM HEPES, pH 7.5 containing 1 mg/ml BSA, 0.05% ProClin 300 using a
high gradient magnet. After the final wash, desthiobiotin/EpCAM MAb
ferrofluid was resuspended in Immunicon ferrofluid storage buffer
and filtered through a 0.2 .mu.m syringe filter.
EXAMPLE III
[0078] Increase of Recovery of Low Antigen Density PC3 Tumor Cells
From Spiked Blood by Aggregation of Desthiobiotin/EpCAM Ferrofluid
with Streptavidin.
[0079] In this example, prostate carcinoma cells (PC3) which have a
low EpCAM antigen density were spiked into normal blood and used as
a model system to assess recovery of those spiked cells. A known
number of PC3 cells (.about.5000) in 50 .mu.l of buffer (isotonic 7
mM phosphate, pH 7.4 with 1% BSA and 50 mM EDTA) were spiked into 1
ml of normal blood without plasma in a 12.times.75 mm polystyrene
tube. Blood without plasma was used in these experiments to prevent
any interference of plasma components in the selection of target
cells and it was prepared by centrifugation of blood. 500 .mu.l of
Immunicon dilution-wash buffer and 15 .mu.l of streptavidin at
different concentrations in PBS were added to aliquots of the blood
sample. After mixing the sample, desthiobiotin/EpCAM MAb ferrofluid
(25 .mu.l) from Example 1 was added to the sample, mixed well and
incubated at room temperature for 15 minutes. The final
concentration of ferrofluid was 5 .mu.g/ml. After the incubation,
the tube containing the blood sample was placed in a quadrupole
magnetic separator for 10 minutes for collection of magnetically
labeled cells. The uncollected sample was aspirated and the tube
was removed from the magnetic separator. The magnetically collected
cells were resuspended in 750 .mu.l of dilution-wash buffer and
reseparated in a magnetic separator for 5 minutes. The uncollected
sample was discarded again and the collected cells were resuspended
in 150 .mu.l of dilution-wash buffer after removal of the tube from
the magnetic separator.
[0080] The sample was then stained with antibodies to determine the
recovery of tumor cells by flowcytometry as follows. 5 .mu.l of
phycoerythrin (PE)-conjugated MAb specific for tumor cells (Neu
24.7) and 5 .mu.l of peridinin chlorophyll protein
(PerCP)--conjugated CD45 MAb were added-to the sample and incubated
for 15 minutes. After the incubation, 1 ml of dilution-wash buffer
was added and a magnetic separation was performed for 5 minutes in
order to remove excess staining antibodies. The magnetically
collected cells were resuspended in 500 .mu.l of dilution-wash
buffer. Nucleic acid dye (10 .mu.l) and 5 .mu.l of 3.mu.M
fluorescent beads (5000) were added to this sample. The sample was
then analyzed on a FACSCalibur flowcytometer (Becton Dickinson)
using FL1 as threshold. The fraction of the fluorescent beads
acquired in the flowcytometer was used to determine the amount of
sample analyzed by flowcytometry which was then used to calculate
the recovery of spiked tumor cells. The percent recovery of tumor
cells are shown in the following Table.
2 Concentration of Aggregator, Tumor Cells (PC3) Streptavidin
(.mu.g/ml) % recovery 0.0 14 0.2 60 0.5 74 2.0 80 5.0 75
[0081] The samples which were left after the flowcytometry analysis
were divided into two parts. Biotin from a stock solution in PBS
was added to one part of the sample to final concentration of 2 mM
and incubated at room temperature for 15 minutes to disaggregate
streptavidin-mediated ferrofluid aggregates. These samples (5
.mu.l) were spotted on a microscope slide and photographs of the
recovered cells were taken using a microscope with a digital camera
attached to it.
[0082] The data indicate that the recovery of tumor cells (PC3) was
increased significantly as the concentration of aggregator
(streptavidin) was increased, reaching a maximum at a 2 .mu.g/ml
concentration of streptavidin. These results were correlated to
free ferrofluid aggregates in solution and ferrofluid aggregates on
cells as observed with microscopy. There were no ferrofluid
aggregates at 0 .mu.g/ml of streptavidin and ferrofluid aggregates
were increased as the concentration of streptavidin was increased.
Streptavidin causes aggregation of ferrofluid by multivalent
binding of streptavidin to desthiobiotin on different ferrofluid
particles. All these ferrofluid aggregates were reversibly
disaggregated by the addition of excess biotin. The principle of
this disaggregation of ferrofluid by excess biotin was due to
displacement of desthiobiotin from streptavidin as biotin has a
higher affinity than desthiobiotin for streptavidin.
EXAMPLE IV
[0083] Recovery of Spiked Low and High EPCAM Antigen Density Cells
from Blood With and Without Aggregation of Desthiobiotin/EpCAM MAb
Ferrofluid.
[0084] Breast carcinoma cells (SKBR3) have about 7-times higher
EPCAM antigen density compared to PC3 cells and were chosen as the
model high antigen density tumor cells for this example. A known
number of SKBR3 or PC3 cells in cell buffer were spiked into 1 ml
of blood without plasma separately in a 12.times.75 mm tube.
Ferrofluid dilution-wash buffer (500 .mu.l) and 15 .mu.l of PBS
containing streptavidin were added to the sample. After mixing the
sample, 25 .mu.l of desthiobiotin/EpCAM MAb ferrofluid was added
and the blood sample mixed well and incubated for 15 minutes. After
incubation, the tube was placed in a quadrupole magnetic separator
for 10 minutes to collect magnetically labeled cells. The
magnetically isolated cells were analyzed for recovery of tumor
cells by flowcytometry and for observation of cells by microscopy
as described in Example II.
3 Concentration of aggregator, PC3 cells SKBR3 cells Streptavidin
recovery recovery (.mu.g/ml) (%) (%) 0 23 91 2 77 98
[0085] The data reveal a significant difference in recovery of
tumor cells between low and high antigen density cells when the
ferrofluid aggregator, streptavidin, was not added to the blood
sample. There were also no ferrofluid aggregates in solution or on
cell surfaces without streptavidin as observed with microscopy.
Addition of streptavidin to the blood sample increased the recovery
of low antigen density PC3 cells significantly (3-fold) with a
commensurate increase of ferrofluid aggregation in solution and on
the cells. On the other hand, there was only a small difference in
recovery of high antigen density SKBR3 cells with and without
streptavidin present in the blood sample. There were enough
ferrofluid particles on SKBR3 cells even without ferrofluid
aggregation to collect them effectively and to recover all of them.
Ferrofluid aggregates in solution and on cells were completely
disaggregated by the addition of excess biotin to the sample. In
the case of low antigen density cells, there were not enough
ferrofluid particles on cells to be collected effectively by
magnetic methods. Ferrofluid aggregation by streptavidin increased
the number of particles on these cells facilitating collection,
effectively resulting higher recovery. It is also noteworthy that
aggregation of ferrofluid increased the recovery of low antigen
density cells close to that obtained with the high antigen density
cells. In other words, there was no significant difference in
recovery of low and high antigen density tumor cells upon addition
of reversible ferrofluid aggregator to the blood sample.
EXAMPLE V
[0086] Inhibition of Ferrofluid Aggregation by Endogenous
Aggregation Factors and Creation of Controlled Ferrofluid
Aggregation with an Exogenous Aggregation Factor.
[0087] In this example, a method is provided to inhibit all
endogenous ferrofluid aggregation factors and to create controlled
ferrofluid aggregation by addition of an exogenous aggregation
factor. The endogenous ferrofluid aggregation factors present in
the sample can be inhibited by adding a variety of inhibitors to
the sample. These inhibitors will act on different endogenous
aggregation factors and prevent them from either crosslinking or
binding to ferrofluid to cause aggregation. Inhibition will
eliminate any variations in ferrofluid aggregation from sample to
sample as endogenous aggregation factors are present at different
concentrations in different samples. Once endogenous
factor-ferrofluid aggregation is prevented, ferrofluid aggregation
can be promoted by adding an exogenous aggregation factor which can
enhance the recovery of targets efficiently. The exogenous
aggregation can be controlled consistently with all the samples and
it can be readily reversed.
[0088] The blood sample is preincubated with a buffer containing
inhibitors to inhibit endogenous ferrofluid aggregation factors
before ferrofluid is added to the blood. The antibody coupled
ferrofluid contains bovine serum albumin and streptavidin on the
surface of ferrofluid particles in addition to antibody. Therefore,
the possible ferrofluid aggregation factors can be IgM (specific
for crystal surface), human-anti-mouse antibody (HAMA),
human-anti-bovine serum albumin antibody (HABAA),
human-anti-streptavidin etc. If any of the above aggregation
factors are present in plasma, they will recognize and bind to
ferrofluid and cause ferrofluid to aggregate. It is already known
that some patient plasma samples have HAMA and HABAA present
therein. Clearly, any other components used to manufacture
ferrofluids could also be targets for aggregation and would needs
to be dealt with accordingly.
[0089] One of the inhibitors can be a reducing agent, such as
mercaptoethane sulfonic acid at 100 mM, which can disable
IgM-induced aggregation without affecting the ligands used for
labeling cells. The reducing agent can be added as a single reagent
to the blood or could be placed in a blood collection tube. The
second inhibitor can be bovine serum albumin, which can be included
in the buffer at 10 mg/ml, and will neutralize any HABAA. The third
inhibitor can be nonspecific mouse antibody, in particular, the
appropriate isotype which matches the antibody on the ferrofluid.
This can be included in the buffer at a concentration of 0.5-5
mg/ml to neutralize even the most severe HAMA. The fourth inhibitor
can be Streptavidin to be included in the buffer, if necessary, to
neutralize any anti-streptavidin antibody present in plasma.
However, there is no any information regarding the existence of
anti-streptavidin antibody in plasma at this date.
[0090] The pre-treatment of blood with the above buffer and
reducing agent can be from 15-30 minutes to neutralize all
endogenous aggregation factors. After all endogenous aggregation
factors are neutralized, an exogenous ferrofluid aggregation factor
is added to the sample, followed by ferrofluid. The ferrofluid is
coupled to an antibody specific for targets, as well as to another
ligand specific for the exogenous aggregation factor. After optimum
labeling of target cells with ferrofluid and induced aggregation of
ferrofluid with exogenous aggregation factor, the sample is
subjected to magnetic separation to enrich targets. After removing
all non-targets, magnetically-labeled targets and free ferrofluid
are resuspended in a small volume of buffer. The
magnetically-labeled targets, such as cells, can be permeabilized
to stain intracellular antigens. The sample is then incubated with
different staining reagents depending upon the desired analysis
method, including flow cytometry or fluorescent or bright field
microscopy. After optimum incubation time, the excess staining
reagents are removed by wash step using magnetic separation. The
magnetically-labeled cells are then resuspended in a small volume
of buffer. The final sample contains free ferrofluid aggregates and
aggregates on target cells. The final sample without any further
treatment can be used for flowcytometry analysis, as ferrofluid
aggregation on cell surface does not interfere with the analysis.
However, ferrofluid aggregation on cell surface interferes with
microscopy analysis.
[0091] In such cases, exogenous mediated-ferrofluid aggregation
should be reversed. This can be achieved by resuspending the final
sample in a buffer containing a disaggregation factor which binds
to exogenous aggregation factor. The disaggregation factor
disaggregates all ferrofluid aggregates, leaving cells easy to view
and analyze. These methods permit effective target recovery and
visualization for morphology studies.
[0092] Several patents and pending U.S. patent applications are
referred to in the present specification. The entire disclosures of
each of these patents and patent applications are incorporated by
reference herein.
[0093] While certain of the preferred embodiments of the present
invention have been described and specifically exemplified above,
it is not intended that the invention be limited to such
embodiments. Various modifications may be made thereto without
departing from the scope and spirit of the invention, as set forth
in the claims.
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