Note: Descriptions are shown in the official language in which they were submitted.
2~33Q~
S -1-
METHOD FOR SEPARATING COMPONENTS IN A MIXTURE
BACKGROUND OF T~E INVENTION
1. Field of the Invention
This invention relates to methods for separating a
component, which may or may not be particulate, from
other components in a mixture. The invention has
particular application to separation, for example, for
the purpose of assaying cells from biological fluids such
as blood, lymphatic fluid, urine, cell cultures,
suspended feces etc., microorganisms, organelles,
molecular aggregates, nucleic acids, and ligands.
Detection and identification of microorganisms,
particularly in biological samples and food and drug
products, are required for clinical diagnosis of disease,
prevention of disease transmis8ion, and food quality.
Traditionally, bacteria and fungi are detected and
identified by culture methods, which are notoriously slow
and tedious. More recently, immunoassays for microbial
antigens and nucleic acid probe assays have been
developed. However, these methods usually only permit
detection of a specific organism. In determination of
septicemia, an assay for a general indicator of growth,
such as carbon dioxide, is often used. This is followed
by culture methods to identify the organism and then by
antibiotic susceptibility testing. There is a need to
obtain test results more rapidly with less labor.
7444M 26950-FF
-2- ~3~9~
Several techniques are known for carrying out
separations. For example, one may employ centrifugation
and washing; differential migration of bound and free
fractions, e.g., chromatoelectrophoreses, gel filtration,
etc.; chemical precipitation of the bound or free
fraction, e.g., by meanR of organic solvents, salts,
acids, etc. followed by filtration or centrifugation;
immunological precipitation of the bound fraction, e.g ,
by double antibody technique followed by filtration or
centrifugation; absorption of the bound or free fraction
onto selective sorbing media, e.~., charcoal, silicates,
resins, etc.; magnetic separation techniques, and the
like.
Many of the separation techniques, including those
used in immunoassays, are relatively long and complicated
procedures. Such procedures reduce operator efficiency,
decrease throughput, and increase the costs of tests.
Other separation techniques which are rapid and simple do
not adequately distinguish between the bound and free
fractions and therefore are unsuited for immunoassays or
can only be utilized in a limited number of tests.
2. Des~ tion of the Rel~d~
Recently, Microdrop Co. published a method for
converting an entire samp~e of urine into small beads of
agarose or other gel such that each bead traps at most
one organism ~Weaver, et al., Bio/Te~hnology (1988)
:1084-1089). The beads included a culture medium and
were dispersed in oil. Growth was directly detected by
the formation of an indicator dye in the infected
particles. The method offers the ability to detect,
sort, count, and study individual organisms. However,
the method is cumbersome and separation of the bacteria
from growth inhibiting substances in the sample occurs
only by dilution and diffusion.
7444M 26950-FF
-3- ~3~
Cells are commonly separated by centri~ugation in
high density or high viscosity media, and lysosomes that
have incorporated gold particles have been isolated by
virtue of their increased density using sucrose density
gradient velocity centrifugation (~enning, et al,
Biochim. Biophys. Acta. 354 (1974) 114-120. Also, Dorn
et al., J. ~lin. Micro 3 (1976) 251-257 demonstrated that
bacteria along with insoluhle cellular debris can be
separated from the soluble components of a ly~ed blood
sample by spinning the suspension with an underlying
layer of sucrose solution. This is the basis of a
product from I.E. DuPont de Nemours Company called the
ISOLATOR. Further, Leduc et al. (Biochim. Bio~hys. Acta.
885 (1986) 248) separated polynucleosome~ bearing
poly-ADP-ribose synthetase on their surface from unbound
polynucleosomes by causing specific antibodies to the
synthetase to bind, combining the mixture with
gold-labelled protein A and separating by sucrose
gradient velocity sedimentation whereupon the gold bound
polynucleosomes separated more rapidly. Courtoy, et al.
(J. Cell ~ioloey, 98 ~1984) 870-876) have described the
shift of equilibrium density induced by
3,3 -diaminobenzidine cytochemistry in a procedure for
the analysis and purification of peroxidase-containing
organelles.
SUMMAR~ OF THE INVENTION
The method of the present invention is directed to
the separation of a material of interest from a mixture
containing the material of interest and other insoluble
and/or soluble components. The method comprises binding
in a first liquid medium the material of interest to a
bead (MI-bead) and providing in a liquid system the
MI-bead wherein the MI-bead has a density greater than
the liquid system, which is comprised of the liquid
7444M 26950-FF
-4- 2033~ ~
medium~ The density of at least a portion of the liquid
system is greater than the other insoluble components in
the liquid system. Next, the liquid system is subjected
to a centrifugal force sufficient to separate the MI-bead
from the insoluble components. When the material of
interest is to be separated from other soluble components
a second liquid medium is layered with the first liquid
medium.
The method of the present invention has particular
application to the separation of organic and biochemical
materials of interest from, for example, cell cultures,
body fluids and the like.
One embodiment of a method in accordance with the
present invention is a method for separating a material
of interest from a mixture containing the material of
interest and other components. The method comprises
causing the material of interest to become bound to a
bead (MI-bead) in an aqueous medium. The aqueous medium
has, or subsequently is caused to have, a density less
than that of the MI-bead but greater than that of any of
the other components which are insoluble in the medium.
The medium is subjected to centrifugation to cause the
MI-bead to collect in an area within or outside the
medium separated from the other components with the
proviso that, where the other components are soluble in
the aqueous medium, the MI bead i8 caused to collect only
out 8 i de the medium.
Another embodiment of a method in accordance with
the present invention is a method for separating a
microorganism from a mixture containing the microorg~nism
and other components. The method comprises binding the
microorganism to beads in an aqueous medium to ~orm
microorganism-bead. The beads have a density greater
than 1.2 g/cm and an average diameter of about 1 to
- 35 50,000 nm. Next, the medium is subjected to
7444M 26950-FF
2~33~
centrifugation to concentrate the microorganism-bead. In
another embodiment of the above, the density of the
aqueous medium is adjusted to be greater than that of any
of the other components which are in~oluble in the medium
but less than that of the microorganism-bead. In another
embodiment of the above, the centrifugation cause3 the
microorganism-bead to collect in an area outside of the
medium. The area is separated from that of the other
components. In another embodiment of the a~ove, the
aqueoug medium is layered on a medium of higher viscosity
than the aqueous medium prior to subjecting the aqueous
medium to centrifugation.
Another embodiment of the present invention involves
a method for separating a microorganism from a mixture
containing the microorganism and other components. The
method comprises binding the microorganism to a
multiplicity of beads in a first aqueous medium to form
microorganism-bead. The beads have a density greater
than 10 g/cm3 and an average diameter of about 1 to
50 nm. The first medium i8 contacted with a second
medium having a density greater than the first medium but
less than that of the microorganism bead. The contacted
first and second media are subjected to centrifugation to
cause the microorganism-bead to collect in an area
outside of the first medium. The area is separated from
that of other components.
In another embodiment of the above, the first
aqueous medium and the second medium are combined and
layered on a third medium of a viscosity higher than the
combined first and second media.
Another embodiment of the present invention involves
a method for separating microorganisms from an aqueous
medium, which method includes the step of centrifugation,
and wherein the improvement comprises (a) providing an
7444M 26950-FF
-6- 2~
aqueous medium denser than said microorgani~ms and (b)
prior to said centrifugation, causing said microorganism3
to bind to beads to form aggregates consisting of a
single microorganism and a multiplicity of beads. Said
method may also include layering said aqueous medium over
a second liquid medium.
Another aspect of the present invention is a method
for conducting an assay for an analyte. The method
comprises causing the analyte, or a substance whose
presence is related to the presence of the analyte, to
become bound to a bead (A-bead) in an aqueous medium.
The aqueous medium has, or subsequently is caused to
have, a density less than that of the A-bead but greater
than that of any of the other components which are
insoluble in the medium. The medium is subjected to
centrifugation to cause the A-bead to collect in an area
within or outside the medium separated from the other
components with the proviso that, where the other
components are soluble in the medium, the A-bead is
caused to collect outside the medium. The analyte or
other substance is then detected.
The invention further includes compositions and kits
for conducting the methods and assays of the invention.
BRIEF DESC~IPTION OF ~T ~AWINGS
Fig. 1 shows a density calibration of a Percoll
gradient.
Fig. 2 shows a gravimetric density calibration of
the Percoll-diatrazoate gradient.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
The present invention relates generally to a method
of separating a material of interest (MI) from other
components in a sample. The MI to be separated will be
7444M 26950-FF
--7--
caused to bind to a bead (MI-bead). The preerred
approach for achieving binding between the beads and the
MI is charge interactions or ligand-receptor binding.
The MI may also be bound to the beads by virtue of
nucleic acid hybridization. The MI-bead has a density
greater than that of a liquid medium in which it is
suspended. The density of at least a portion of the
liquid medium is greater than the density of other
insoluble components in the medium. The medium is
subjected to a centrifugal force sufficient to spacially
separate the MI-bead from the other components. The
separated MI-bead can be washed further and examined by
physical or chemical methods. The MI-bead can also be
treated to reverse the binding with MI. Reversal of
binding can be followed by separation of the free beads
to provide a means of separating the MI from the beads.
The present method ha~ wide application in the field
of the separation and assay of a material of interest
from other components in a sample, particularly for
geparating biological materials, such as particulate
material, e.g., cells, microorganisms, such as bacteria
and fungi, organelles, molecular aggregates, and the like
and non-particulates, e.g., ligands and nucleic acids.
The material of interest may be present in biological
samples ~uch as cell cultures, body fluids, and the like;
food products, drug products, and so forth. Generally,
the material of interest is present with a wide range of
particulate materials that are present in biological
fluids or solubilized biological solids and other soluble
components. The invention provides a separation meth-)d
which is more convenient and rapid than mere
centrifugation, filtration, and prior separation methods
and is particularly applicable to the pretreatment of
suspensions where it is desired to carry out an analysis
of a material of interest separated from other
7444M 26950-FF
components. The invention has application to the assay
of an analyte in a sample where a separation step is
required.
Before proceeding further with a description of the
specific embodiments of the present invention, a number
of terms will be defined.
Material of interest (MI) -- the compound or
composition to be separated. The material of interest
can be non-particulate or particulate. Non-particulate
MI can be comprised of a member of a specific binding
pair (sbp) and may be a ligand, which is mono- or
polyvalent, usually antigenic or haptenic, and is a
single compound or plurality of compounds which share at
lea~t one common epitopic or determinant site. The MI
can also be a particle. Exemplary o~ a MI that is a
particle is a cell bearing a blood group antigen such as
A, B, D, etc., or an HLA antigen, or a microorganism such
as a bacterium, fungus, protozoan, or virus.
The MI can be an analyte, either monovalent
~monoepitopic) or polyvalent (polyepitopic). The
polyvalent ligand analyte will normally be poly(amino
acids), i.e., polypeptides and proteins, polysaccharides,
lipids, nucleic acids, and combinations thereof. Such
combinations include components of bacteria, fungi,
viruses, chromosomes, genes, mitochondria, nuclei, cell
membranes and the like.
For the most part, the polyepitopic ligand analytes
to which the subject invention can be applied will have a
molecular weight of at least about 5,000, more usually at
least about 10,000. In the poly(amino acid) categorv.
the poly(amino acids) of interest will generally be from
about 5,000 to 5,000,000 molecular weight, more usually
from about 20,000 to 1,000,000 molecular weight; among
the hormones of interest, the molecular weights will
usually range from about 5,000 to 60,000 molecu~ar weight.
7444M 26950-FF
2~33~1~
g
A wide variety of proteins may be considered as to
the family of proteins having similar structural
features, proteins having particular biological
functions, proteins related to specific microorganisms,
particularly disease causing microorganisms, etc.
The monoepitopic ligand analytes will generally be
from about 100 to 2,000 molecular weight, more usually
from 125 to 1,000 molecular weight. The analytes include
drugs, metabolites, pesticides,. pollutants, and the
like. Included among drugs of interest are the
alkaloids. Among the alkalo~ds are morphine alkaloids,
which includes morphine, codeine, heroin,
dextromethorphan, their derivatives and metabolites;
cocaine alkaloids, which include cocaine and benzoyl
ecgonine, their derivatives and metabolites, ergot
alkaloids, which include the diethylamide of lysergic
acid; steroid alkalo~ds; iminazoyl alkaloids; quinazoline
alkaloids, isoquinoline alkaloids; quinoline alkaloids,
which include quinine and quinidine; diterpene alkaloids,
their derivatives and metabolites.
The next group of drugs includes steroids, which
includes the estrogens, androgens, adrenocortical
ste~oids, bile acids, cardiotonic glycosides and
aglycones, which includes digoxin and digoxigenin,
saponins and sapogenins, their derivatives and
metabolites. Also included are the steroid mimetic
substances, such as diethylstilbestrol.
The next group of drugs is lactams having from 5 to
6 annular members, which include the barbiturates, e.~.
phenobarbital and secobarbital, diphenylhydantonin,
primidone, ethosuximide, and their metabolites.
The next group of drugs is aminoalkylbenzenes, with
alkyl of from 2 to 3 carbon atoms, which includes the
amphetamines, catecholamines, which includes ephedrine,
7444M 26950-FF
-lo- 2~3~
L~dopa, epinephrine, narceine, papaverine, and their
metabolites.
The next group of drugs is benzheterocyclics which
include oxazepam, chlorpromazine, tegretol, imipramine,
their derivatives and metabolites, the heterocyclic rings
being azepines, diazepines and phenothiazines.
The next group of drugs is purines, which includes
theophylline, caffeine? their metabolites and derivatives.
The next group of drugs includes those derived from
marijuana, which includes cannabinol and
tetrahydrocannabinol.
The next group of drugs includes the vitamins such
as A, B, e.g. B12, C, D, E and K, folic acid, thiamine.
The next group of drugs is prostaglandins, which
differ by the degree and sites of hydroxylation and
unsaturation.
The next group of drugs is antibiotics, which
include penicillin, chloromycetin, actinomycetin,
tetracycline, terramycin, the metabolites and derivatives.
The next group o~ drug8 i8 the nucleosides and
nucleotides, which include ATP, NAD, FMN, adenoslne,
guanosine, thymidine, and cytidine with their appropriate
sugar and phosphate substituents.
The next group of drugs i8 miscellaneous individual
drugs which include methadone, meprobamate, serotonin,
meperidine, amitriptyline, nortriptyllne, lidocaine,
procaineamide, acetylprocaineamide, propranolol,
griseofulvin, valproic acid, butyrophenones,
antihistamines, anticholinergic drugs, such as atropine,
their metabolites and derivatives.
Metabolites related to diseased states include
spermine, galactose, phenylpyruvic acid, and porphyrin
Type 1.
The next group of drugs is aminoglycosides, such as
gentamicin, kanamicin, tobramycin, and amikacin.
7444M 26950-FF
203~0~
Among pe3ticides of intere~t are polyhalogenated
biphenyls, phosphate esters, thiophosphates, carbamates,
polyhalogenated sulfenamides, their metabolites and
derivatives.
For receptor analytes, the molecular weights will
generally range from 10,000 to 2X108, more usually from
10,000 to 106. For immunoglobulins, IgA, IgG, IgE and
IgM, the molecular weights will generally vary from about
160,000 to about 106. Enzymes will normally range from
about 10,000 to 1,000,000 in molecular weight. Natural
receptors vary widely, generally being at least about
25,000 molecular weight and may be 106 or higher
molecular weight, including such materials as avidin,
DNA, RNA, thyroxine binding globulin, thyroxine binding
prealbumin, transcortin, etc.
7444M 26950-FF
20~Q~
-12-
Illustrative microorganisms include:
Corvnebacteria
9Corynebacterium diphtheria
Pneumococci
Diplococcus pneumoniae
Streytococci
Streptococcus pyrogenes
Streptococcus salivarus
Staphylococci
AStaphylococcus aureus
Staphylococcus albus
6eria
Neisseria meningitidi6
Neisseria gonorrhea
Enterobacteriaciae
Escherichia coli
Aerobacter aerogenes The coliform
19Klebsiella pneumoniae bacteria
Salmonella typhosa
Salmonella choleraesuis The Salmonellae
Salmonella typhimurium
Shigella dysenteria
Shigella schmitzii
Shigella arabinotarda
The Shigellae
20Shigella flexneri
Shigella boydii
Shigella sonnei
Other enter~c bacilli
Proteus vulgaris
Proteus mirabilis Proteus species
Proteus morgani
25Pseudomonas aeruginosa
Alcallgenes faecalis
Vibrio cholerae
7444M 26950-FF
-13- 2 ~ 3 0~
Hemophilus-Bordetella group Rhizopus oryzae
Hemophilus influenza, H. ducryi Rhizopus arrhizua Phycomycetes
Hemophilufi hemophilus Rhizopus nigricans
Hemophilus aegypticus Sporotrichum schenkii
Hemophi lu6 parainfluenza Flonsecaea pedrosoi
Bordetella pertussis Fonsecacea compact
5Pasteurellae Fonsecacea dermatidis
Pasteurella pestis Cladosporium carrionii
Pasteurella tulareusis Phialophora verrucosa
Brucellae Aspergillus nidulans
Brucella melitensis Madurella mycetomi
Brucella abortus Madurella grisea
Brucella suis Allescheria boydii
,Aerobic Spore-forming Bacilli Phialophora jeanselmei
VBacillus anthracis Microsporum gypseum
Bacillus subtilis Trichophyton mentagrophytes
Bacillus megaterium Keratinomyces ajelloi
Bacillus cereus Microsporum canis
obic Spore-formi~Bacilli Trichophyton rubrum
Clostridium botulinum Microsporum adouini
Clostridium tetani Viruses
Clostridium perfringens Adenoviruses
Clostridium novyi herpes Viruses
Clostridium septicum Herpes simplex
Clostridium histolyticum Varicella (Chicken pox)
Clostridium tertium Herpes Zoster (Shingles)
Clostridium bifermentans Virus B
Clostridium sporogenes Cytomegalovirus
2,~Mvcobacteria Pox Viruse6
VMycobacterium tuberculosis hominis Variola (smallpox)
Mycobacterium bovis Vaccinia
Mycobacterium avium Poxvirus bovis
Mycobacterium leprae Paravaccinia
Mycobacterium paratuberculo6is Molluscum contagiosum
Actinomvce~es (fungus-like bacteria) PicQrnaviruses
Actinomyces Isaeli Poliovirus
25Actinomyces bovis Coxsackievirus
Actinomyces naeslundii Echoviruses
Nocardia asteroides Rhinoviruses
Nocardia brasiliensis Myxoviruse~
~hL~i~gh~ Influenza(A, B, and C)
Treponema pallidum Spirillum minus Parainfluenza (1-4)
Treponema pertenue Streptobacillus Mumps Virus
monoiliformis Newcastle Disease Virus
Treponema carateum Measles Virus
Borrelia recurrent is Rinderpest Virus
Leptospira icterohemorrhagiae Canine Distemper Virus
Leptospira canicola Respiratory Syncytial Virus
Trvpsnasomes Rubella Virus
Mvcoplasmas Arboviruses
Mycoplasma pneumoniae
7444M 26950-FF
-14- 2 ~ 33 ~ ~ ~
Other pathogens Eastern Equine Eucephalitis Virus
Listeria monocytogenes Western Equine Eucephalitis Viru6
Erysipelothrix rhusiopathiae Sindbls Viru6
Streptobacillus moniliformis Chikugunya Virus
Donvania granulomatis Semliki Forest Virus
Bartonella bacilliformis Mayora Virus
~Rickettsiae (bacteria-like parasites) St. Louis Encephaliti6 Virus
Rickettsia prowazekii California Encephalitis Virus
Rickettsia mooseri Colorado Tick Fever Virus
Rickettsia rickettsii Yellow Fever Virus
Rickettsia conori Dengue Virus
Rickettsia australis Reoviruses
Rickettsia sibiricus Reovirus Types 1-3
Retroviruses
ORickettsia akari Human Immunodeficiency Viruses (HIV)
Rickettsia tsutsugamushi Human T-cell Lymphotrophic
Virus I & II (HTLV)
Rickettsia burnetti Hepatitis
Rickettsia quintana Hepatitis A Virus
Chlamydia (unclassifiable parasite6 Hepatitis B Virus
bacterial/viral) Hepatltis nonA-nonB Virus
15ChlamYdia agents (naming uncertain) Tumor Viruses
E~ngi Rauscher Leukemia Virus
Cryptococcus neoformans Gross Virus
Blastomyce~ dermatidis Maloney Leukemia Virus
Hisoplasma capsulatum
Coccidioides immitis ~uman Papilloma Virus
Paracoccidioides brasiliensis
2OCandida albicang
Aspergillus fumigatus
Mucor corymbifer (Absidia corymbifera)
7444M 26950-FF
2~33~
--15--
Member of a specific binding pair ("sbp
member~ -one of two different molecules, having an area
on the surface or in a cavity which specifically binds to
and is thereby defined as complementary or reciprocal
with a particular spatial and polar organization of the
other molecule. The members of the specific binding pair
are referred to a~ ligand and receptor (antiligand).
These will usually be members o~ an immunological pair
such as antigen-antibody, although other specific binding
pairg such as biotin-avidin, hormones-hormone receptors,
lectin-carbohydrate, nucleic acid duplexes, IgG-protein
A, DNA-DNA, DNA-RNA, and the like are not immunological
pairs but are included in the invention,
Ligand-any organic compound for which a receptor
naturally exists or can be prepared.
Ligand analog--a modified ligand which can compete
with the analogous ligand for a receptor, the
modification usually providing means to join a ligand
analog to another molecule. The ligand analog will
usually differ from the ligand by more than replacement
of a hydrogen with a bond which links the ligand analog
to a hub or label, but need not. The ligand analog can
bind to the receptor in a manner similar to the ligand.
The analog could be, for example, an antibody directed
against the idiotype of an antibody to the ligand.
Receptor ("antiligand")--any compound or composition
capable of recognizing a particular spatial and polar
organization of a molecule, e.g., epitopic or determinant
site. Illustrative receptors include naturally occurring
receptors, e.g., thyroxine binding globulin, antibodies,
enzymes, Fab fragments, lectins, nucleic acids, protein
A, complement component Clq, and the li~e.
Particulate MI -- the particulate MI are generally
at least about 0.1 microns and not more than about 100
microns in diameter, usually 0.5 to 25 microns. The
7444M 26950-FF
-16- 2~3~
particulate MI may be organic or inorganic, swellable or
non-swellable, porous or non-porous, usually of a density
approximating the density of water, generally from about
0.7 to about 1.5 g/ml. Usually the particulate MI will
have a charge, either positive or negative, and may have
sbp members on their surface. Normally, the particulate
MI will be biologic materials such as cells such as e.g.,
erythrocytes, leukocytes, lymphocytes, hybridomas,
microorganisms such as, e.g., streptococcus,
staphylococcus aureus, E. coli, viruses; organelles, such
as, e.g. nuclei, mitochondria, nucleosomes; and the
like. The particulate MI can also be particles comprised
of organic and inorganic polymers, liposomes, latex
particles, phospholipid vesicles, chylomicrons,
lipoproteins, and the like.
The polymers will normally be either addition or
condensation polymers. Particulate MI derived therefrom
will be adsorptive or functionalizable so as to bind,
either directly or indirectly, an sbp member.
The particulate MI may be an analyte or may have an
analyte bound thereto, or an analyte may become bound
thereto before or during the separation. The particulate
MI may be formed ~rom particles not initially bound to
the analyte and derived from naturally occurring
~5 materials, naturally occurring materials which axe
synthetically modified and synthetic materials. Among
organic polymers of particular interest are
polysaccharides, particularly cross-linked
polysaccharides, such as agarose, which is available as
Sepharose, dextran, available as Sephadex and Sephac t-vl,
cellulose, starch, and the like; addition polymers, such
as polystyrene, polyvinyl alcohol, homopolymers and
copolymers of derivatives of acrylate and methacrylate,
particularly esters and amides having free hydroxyl
functionalities, and the like.
7444M 26950-FF
2~3~,~a
-17-
The particles for use in assay3 will usually be
polyfunctional and will have bound to or be capable of
specific non-covalent binding to an sbp member, such as
antibodies, avidin, biotin, lectins, protein A, and the
like. A wide variety of func~ional groups are available
or can be incorporated. Functional groups include
carboxylic acids, aldehydes, amino groups, cyano groups,
ethylene groups, hydroxyl groups, mercapto groups and the
like. The manner of linking a wide variety of compounds
to particles is well known and is amply illustrated in
the literature. See for example Cuatrecasas, J. Biol.
Chem., 245 3059 (1970). The length of a linking group
may vary widely, depending upon the nature of the
compound being linked, the effect of the distance between
the compound being linked and the particle on the binding
of sbp members and the analyte and the like.
The particulate MI may have an electronic charge,
either positive or negative. The particle can be
inherently charged or can be treated chemically or
phygically to introduce a charge. For example, groups
such as carboxyl, sulfonate, phosphate, amino, and the
like can be chemically bound to or formed on the
particles by techniques known in the art. Cells are
normally negatively charged due to the presence of sialic
acid residues on the cell surface. Latex particles can
be positively or negatively charged but normally will
have a negative charge as a result of the introduction of
functional groups or absorption of charged polymers such
as polypeptides, proteins, polyacrylate, and the like.
The particles can be colored, fluorescent or
non-fluorescent, usually non-fluorescent, but when
fluorescent can be either fluorescent directly or by
virtue of fluorescent compounds or fluorescers bound to
the particle in conventional ways.
7444M 26950-FF
-18- 2~3~
Label--A member of a signal producing system that
may be bound to, or caused to bind to, an MI including
analytes, or to a substance whose presence is related to
the presence of the MI. The label can be isotopic or
non-isotopic, usually non-isotopic, including catalysts
such as an enzyme, a chromogen such as a fluorescer, dye
or chemiluminescer, a particle, and so forth.
Signal Producing ~ystem--The signal producing system
may have one or more components, at least one component
being a label. The signal producing system generates a
signal that relates to the presence or amount of MI, or a
substance whose presence is related to the presence of a
MI, in a sample. The signal producing system includes
all of the reagents required to produce a measurable
signal. Other components of the signal producing system
can include substrates, enhancers, activators,
chemiluminescent compounds, cofactors, inhibitors,
scavengers, metal ions, specific binding substances
required for binding of signal generating substances, and
the like. Other components of the signal producing
system may be coenzymes, substances that react with
enzymic products, other enzymes and catalysts, and the
like. The signal producing system provides a signal
detectable by external means, preferably by measurement
of the degree of aggregation of particles or by use of
electromagnetic radiation, desirably by visual
examination.
A large number of enzymes and coenzymes useful in a
signal producing system are indicated in U.S. Patent No.
4,275,149, columns 19 to 23, and U.S. Patent No.
4,318,980, columns 10 to 14, which disclosures are
incorporated herein by reference. A number of enzyme
combinations are set forth in U.S. Patent no. 4,275,149,
columns 23 to 28, which combinations can find use in the
7444M 26950-FF
% ~
-19-
subject invention. This disclosure is incorporated
herein by reference.
~ eads--particles that are generally at least about 1
to 50,000 nm, usually at least about 20 to 10,000 nm.
~eads of density less than 8g/cm3 are preferably 100 to
50,000 nm average diameter. Denser beads are usually 5
to 30 nm, preferably from about 1 to 50 nm in average
diameter. The bead may be organic or inorganic,
swellable or non-swellable, porous or non-porous. The
beads can be composed of material that is transparent,
partially transparent, or opaque. The beads may have any
shape suitable to their use such as round, oval,
irregular, etc.
The beads will be capable of binding the MI (whether
particulate or not). Therefore, the beads will usually
have on their surface an agent that will afford such a
binding capability. The binding can be specific or
non-specific, covalent or non-covalent. The agent can
include an sbp member such as a specific receptor or
nucleic acid. For example, the beads could have
anti-immunoglobulin on their surface and antibodies to a
microorganism could be utilized to effect binding.
Alternatively, the agent could be a lectln or a polyionic
reagent, particularly where the MI is a microorganism.
The beads may have a charge, either positive or negative,
and may also have sbp members on their surface.
The beads are selected to have a higher density than
any other insoluble component in the mixture from which
the MI is to be separated. Usually, bead densities of at
least 1.2 g/cm3 are required, and much higher densities
usually at least 4g/cm3 are preferred, particularly
where particulate MI is to be separated and the beads are
smaller than the particulate MI. The main requirement is
that the aggregate of one or a multiplicity of beads
bound to the MI must exceed the density of other
7444M 26950-FF
-20-
insoluble components in the mixture from which the MI is
to be separated.
The beads can be derived from naturally occurring
materials, naturally occurring materials which are
synthetically modified and synthetic materials. One
group of beads are those comprised of a heavy metal,
i.e., a metal of atomic number greater than 20 such as a
Group IB metal, e.g., gold or silver. The heavy metal
may be in the form of a metal sol (colloidal suspensions
of silver, gold, mercury, lead, palladium, etc.), or may
be a metal salt such as an oxide, sulfide, insoluble
phosphate or sulfate, of a heavy metal, e.g. lead,
barium, calcium, titanium, etc. Another group of beads
are polyol beads, i.e., bead~ comprised of polymeric
compounds having more than one hydroxyl group. Among
organic polymers of particular interest are
polysaccharides, particularly cross-linked
polysaccharides, such as agarose, which is available as
Sepharose, dextran, available as Sephadex and Sephacryl,
cellulose, starch, and the like; addition polymers, such
as polystyrene, polyvinyl alcohol, homopolymers and
copolymers of derivatives of acrylate and methacrylate,
particularly esters and amides having free hydroxyl
functionalities, and the like. Other examples of beads
that can be used in the present invention by way of
illustration and not limitation are silica beads
comprised of glass, latex, polyacrylamide, carbon, boron
nitride, carborane, silicon carbide, metal silicates and
the like.
The relatively large size of cells and
microorganisms requires the binding of particularly lli~h
density beads, when the beads are substantially smaller
than the cells or microorganisms. Preferred beads
contain heavy metals, and are insoluble and colloidally
stable, preferably group lB metal sols, particularly gold
7444M 26950-~
2~33~0
-21-
and metal oxides and sulfides, and insoluble sulfates
such as those of barium and calcium of 5 - 70 nm in
diameter that have densities above 10 g/cm3.
Alternatively larger beads can be used whereupon they may
be comprised of latex, silica, glass, agarose, cellulose,
sephadex, etc., provided only that they are insoluble in
and denser than the liquid medium when bound to the
material of interest.
~igher rates and efficiency of binding of beads to
the material of interest to be separated are achieved
with higher concentrations of beads. When it is desired
to avoid excessively bulky sediments and still maximize
concentration of beads, it i8 preferable to use small
extremely dense beads, usually in the range of 1 - 50 nm
having specific gravities of 10 - 20 g/cm3.
The beads will usually be polyfunctional and will
have bound to, or be capable of specific non-covalent
binding to, an sbp member, such as antibodies, avidin,
biotin, lectins, protein A, and the like. A wide variety
of functional groups are available or can be
incorporated. Functional groups include carboxylic
acids, aldehydes, amino groups, cyano groups, ethylene
groups, hydroxyl groups, mercapto groups and the like.
The manner of linking a wide variety of compounds to
particles is well known and is amply illustrated in the
literature. See for example Cuatrecasas, J. Biol. Chem.,
2~ 3059 (1970). The length of a linking group may vary
widely, depending upon the nature of the compound being
linked, the effect of the distance between the compound
being linked and the particle on the binding of sbp
me~bers and the analyte and the like.
The bead may have an electronic charge, either
positive or negative. The beads can be inherently
charged or can be treated chemically or physically to
introduce a charge. For example, groups such as
7444M 26950-FF
-22- 2~33~
carboxyl, sulfonate, phosphate, amino, and the like can
be chemically bound to or formed on the particles by
techniques known in the art. Latex particles can be
positively or negatively charged but normally will have a
negative charge as a result of the introduction of
functional groups or absorption of charged polymers such
as polypeptides, proteins, polyacrylate, and the like.
The beads can be fluorescent or non-fluorescent,
u~ually non-fluorescent, but ~hen fluorescent can be
either fluorescent directly or by virtue of fluorescent
compounds or fluorescers bound to the bead in
conventional ways. The fluorescers will usually be
dissolved in or bound covalently or non-covalently to the
bead and will frequently be substantially uniformly bound
through the bead. Fluoresceinated latex particles are
taught in U.S. Patent No. 3,853,987.
The fluorescers of interest will generally emit
light at a wavelength above 350nm, usually above 400nm
and preferably above 450nm. Desirably, the fluorescers
have a high quantum efficiency, a large Stokes shift and
are chemically stable under the conditions of their
conjugation and use. The term fluorescer is intended to
include ubstances that emit light upon activation by
electromagnetic radiation or chemical activation and
includes fluorescent and phosphorescent substances,
scintillators, and chemiluminescent substances.
Fluorescers of interest fall into a variety of
categories having certain primary functionalities. These
primary functionalities include 1- and
2-aminonaphthalene, pyrenes, quaternary phenanthridine
salts, 9-aminoacridines, p,p~-diaminostilbenes, imines.
anthracenes, oxacarbocyanine, merocyanine,
3-aminoequilenin, perylene, bis-benzoxazole,
bis-p-oxazolyl benzene, 1,2-benzophenazine, retinol,
bis-3-aminopyridinium salts, hellebrigenin, tetracycline,
7444M 26950-FF
-23- 2 ~ 0
æterophenol, benzimidazolylphenylamine, 2-oxo-3-chromen,
indole, xanthene, 7-hydroxycoumarin, 4,5-benzimidazoles,
phenoxazine, salicylate, strophanthidin, porphyrins,
triarylmethanes, flavin and rare earth chelates oxides
and salts. Exemplary fluorescers are enumerated in U.S.
Patent No. 4,318,707, columns 7 and 8. Squaraine dyes
described in U. S. Patent No. 4,806,488 are also useful
as fluorescers.
Additionally, light absorbent beads can be employed
which are solid insoluble particles.
The beads are sometimes referred to herein as
particles when a more generic term is appropriate in
describing the present invention.
Specific binding -- the specific recognition of one
of two different molecules for the other to the exclusion
of other molecules. Generally, the molecules have an
area on the surface or in a cavity giving rise to
specific recognition between the two molecules. The
primary binding influence arises from hydrogen bonding.
Exemplary of specific binding are antibody-antigen
interactions, enzyme - substrate interactions, and so
forth.
Non-specific binding--non-covalent binding between,
for example, particulate MI and beads, that is relatively
independent of specific surface structures. Such
non-specific binding will usually result from charge or
electronic interactions between oppositely charged
particles or between particles having the same charge
where a polyionic reagent having a charge opposite
thereto is employed. Non-specific binding may also
result from hydrophobic interactions of molecules or
particles with a surface.
Polyionic reagent--a compound, composition, or
material, either inorganic or organic, naturally
occurring or synthetic, having at least two of the same
7444M 26950-FF
-24- 2~3~
charge, either polyanionic or polycationic, preferably at
least ten of the same charge; e.g., a polyelectrolyte.
Exemplary of polycationic reagents are polyalkylene
amines such as polyethyleneimine and polypropyleneimine
and their lower alkyl ammonium salts such as polybrene
(CH3)2CH2CH2N (C~3)2CH2c~I2C~I2CH2-) ,
metal ions such as calcium and barium ion, aminodextrans,
protamine, positively charged liposomes, polylysine, and
the like.
Exemplary of polyanionic reagents are heparin,
dextran sulfate, negatively charged phospholipid
vesicles, polycarboxylic acids, such as polyacrylate,
polyglutamate and the like. The above materials and
their preparation or isolation are well known in the art
and many are commercially available.
Releasing agent--a compound, composition, or
material, either naturally occurring or synthetic,
organic or inorganic, capable of reversing the
non-specific or specific binding between MI and beads,
i.e., dissociating MI from the beads. The releasing
agent acts upon the specific or non-specific bond between
the MI and the beads. For example, where non-specific
binding results from charge interactions, the releasing
agent can act to change the p~ of the medium to one which
is unfavorable or incompatible with the charge
interactions. The releasing agent can, therefore, be an
acid such as a mineral acid or an organic acid or a base
such as a mineral base or an organic base.
Alternatively, the releasing agent can act to shield
ionic interactions and thus can be a high ionic strer-~h
solution or a solution of a neutral polymer such as
dextran. Alternatively, the releasing agent can have a
charge which disrupts the non-specific binding between
the particulate MI and the beads. Exemplary of the
latter are polyelectrolyte salts such as citrate,
7444M 26950-~F
2~3~0
-25-
polyacrylate, dextran sulfate, and the like. Where the
particles are bound by a polyionic bridge, the releasing
agent can be a polyionic agent o:E opposite charge or can
be a reagent which depolymerizes the polyionic reagent.
Where the MI and beads are of opposite charge, other
positively or negatively charged polyelectrolytes or high
ionic strength solu.tions may be used.
Where specific binding is involved the releasing
agent can be one that disrupts the specific interactions
such as, for example, an excess of ligand or receptor or
a ligand or receptor mimic where ligand receptor binding
is involved; chelating agents, such as ethylenediamine-
tetraacetate (EDTA), which can disrupt metal ligand
bonding; reducing agents such as mercaptoethanol, which
can diarupt disulfide bonds; nucleophilic reagents such
as hydroxylamine, which can disrupt esters; and the
like.
Insoluble components -- components in a mixture
other than MI generally arising from the biological
sample or food or drug product under investigation.
These components are usually particulate material and may
be, for example, cellular debri~, nuclei, mitochondria,
nucleosomes, chylomicrons, low density lipoproteins
(LDL), and so forth. The method of the present invention
is particularly useful for blood and may entail
pretreatment of samples by use of solubilizing agents and
agents for lysis of cells. These processes may include
the use of mild detergents, cellular lysis by osmotic
shock, sonication, antibodies and complement, pressure,
etc. The thus prepared suspensions will usually cont~;n
soluble contaminants and cellular debris as well as the
material of interest to be separated.
Ancillary Materials--Various ancillary materials
will frequently be employed in a separation in accordance
with the present invention. For example, buffers will
7444M 26950-FF
2 ~
-26-
often be present in the liquid medium, a~ well as
stabilizers for the liquid medium and the other
components. Frequently, in addition to these additives,
additional proteins may be included, such as albumins, or
surfactants, particularly non-ionic surfactants, binding
enhancers, e.g., polyalkylene glycols, or the like.
As mentioned above, the present invention involves a
method for separating a MI from a mixture containing MI
and insoluble components. The method comprises providing
in an aqueous medium MI bound to a bead (MI-bead). The
MI-bead has a density greater than the medium. The
medium is more dense than the insoluble components in the
mixture. The MI to be separated will be bound to the
beads by specific or non-specific binding depending on
the nature of the MI. Specific binding is more
frequently employed for the binding of non-particulate MI
to beads to form MI-beads. However, specific binding may
also be involved in binding particulate MI to beads.
Usually, ligand-receptor binding is employed. The beads
can have bound thereto an sbp member complementary to the
non-particulate MI. Non-specific binding is usually
con~eniently employed for particulate MI and is
preferably obtained as the result of charge
interactions. For eæample, the particulate MI and the
beads can have opposite electronic charges and
non-specific binding will occur spontaneously. Where the
particulate MI and the beads have the same charge, a
polyionic reagent having an opposite charge can be added
to the medium to cause non-specific binding between the
particulate MI and the beads.
Next, the medium is subjected to a centrifugal force
sufficient to separate the insoluble components and the
MI-bead.
In carrying out the method, a liquid medium that is
comprised of an aqueous medium is employed. The term
7444M 26950-FF
2~3~
27-
"aqueous medium~ as used herein includes media wherein
the only solvent present is ~ater or media containing
water and other polar solvents usually oxygenated organic
solvents from one to six, more usually from one to four,
carbon atoms, including alcohols, ethers, and the like.
Usually, when present, these cosolvents will be present
in less than about 40 weight percent, more usually in
less than about 20 weight percent. The p~ for the medium
will usually be selected to promote specific or
non-specific binding of the MI to the beads prior to
separation. Where the beads are negatively charged,
increasing the pH will tend to increase the charge and
prevent spontaneous aggregation caused by non-specific
hydrophobic and Van der Waals interactions. The converse
applies to positively charge beads. Where an oppositely
charged polyelectrolyte is added to cause binding,
changes in pH that increase the charge of the
polyelectrolyte will often decrease the charge of the
beads and an optimum pH must be selected that will avoid
the use of excessive amounts of this reagent. Generally,
a pH range of 5 to 10, more usually 6 to 9, will be
used. For ligand - receptor binding other considerations
with respect to pH are to maintain a significant level of
binding of sbp members, for example, or nucleic acids.
Various buffers may be used to achieve the desired pH and
maintain the pH during the determination. Illustrative
buffers include borate, phosphate, carbonate, Tris,
barbital, and the like. The particular buffer employed
is not critical to this invention; however, in individual
separations, one buffer may be preferred over anothel
When particulate MI is involved, a reagent that promotes
reversal of the binding of the particulate MI and the
beads can be added after the separaticn has been
accomplished.
7444M 26950-FF
Moderate temperatures are normally employed for
carrying out the method and usually constant temperatures
during the period for conducting the method. Generally,
the temperatures will be chosen to promote specific or
non-specific binding of the MI to the beads prior to
separation. The temperature for the method, will
~enerally range from about 0 to 50C., more usually from
about 15 to 40C. Again, after the separation is
accomplished a temperature that promotes reversal of the
binding of the MI and the beads can then be chosen.
The concentration of the beads in the medium will
depend on the amount of MI in the medium that is to be
separated and whether it is particulate or
non-particulate, the size and binding capacity of the
beads, the rate of separation that is desired, the
strength of the centrifugal force, and the relative
density of the MI-bead, the medium, and the insoluble
components. In general, higher concentrations of beads
provide more rapid binding to the MI, and when the MI is
particulate and the beads are smaller than the MI, higher
concentrations of beads can al~o provide more efficient
and rapid separations. ~owever, too high a concentration
of beads can cause excessive entrainment of the medium.
The concentration is normally determined empirically and
will generally vary from about 104 to over 1014 per
ml, more usually from about 106 to 1012 per ml,
frequently from about 107 to 101 per ml.
Where particulate MI is to be separated from a
medium, the concentration of the particulate MI can vary
widely depending upon the need. For example, in
separation of blood cells from plasma, the cell volume
may represent 50% of the total volume of the blood. By
contrast, it may be desired to separate as few as one
bacterium/ml from a sample of water. When it is
necessary to obtain particulate MI that is relatively
7444M 26950-FF
2133~
-29-
free of the aqueous medium as in an assay, usually the
total volume of the particulate MI should be less than
20% of the medium. Where the MI i3 non-particulate and
becomes bound to a bead, the MI will generally vary from
about 10 4 to 10 14M, more usually from about 10 6
to 10 12M. Considerations such as the concentration of
the MI, specific and non-specific hinding effects,
desired rate of the reaction, temperature, solubility,
relative densities, centrifugal force, and the like will
normally determine the concentration of other reagents.
While the concentrations of the various reagents
will generally be determined by the concentration range
of interest of the MI to be separated, the final
concentration of each of the reagents will normally be
determined empirically ~o optimize the rate and extent of
separation of the MI.
Where non-specific binding is involved, chemical
means for forming non-specific bonds between particulate
MI and the beads will usually be included in the aqueous
medium. Except where particulate MI are to be separated
that have an opposite charge to the beads this chemical
means is usually a polyionic reagent having a charge
opposite to that of the beads. The amount of polyionic
reagent added should be suf~icient so that substantially
all of the particulate MI becomes bound to the beads.
This concentration should be determined empirically.
Excess reagent should generally be avoided where it
interferes with complete binding between particulate MI
and the beads. Generally, the polyionic reagent will
have a concentration in the liquid medium sufficient ~-0
provide a number of ions associated with the polymer ~nd
equal to the total number of charges of opposite sign on
all the particles in the medium. Where particulate MI
are to be separated that have an opposite charge to the
beads, the chemical means for forming non-specific bonds
7444M 26950-FF
-30-
between the particles will frequently be a low ionic
strength buffer.
The binding of MI or particulate MI to beads may be
affected by pH. The binding may also be affected by
other Eactors such as ionic strength and the presence of
ionic and non-ionic polymers. Generally, where
non-specific binding is due to charge interactions, the
ionic strength should be chosen initially to facilitate
the binding between the particles. For this purpose the
ionic strength is generally low and can be in the range
of 0.001 to 0.5M, preferably 0.005 to O.lM. After the
separation has been completed, the ionic strength can be
adjusted upward to facilitate the reversal of the
coupling of the particulate MI and the beads. For this
purpose, the ionic strength of the medium will normally
be from about 0.1 to lOM, preferably from about 0.15 to
lM. The principles for causing particles to aggregate or
to remain suspended are well known in the field of
colloid science. Where specific binding is involved the
ionic strength is generally not critical and will usually
be in the range of 10-3 to lOM or greater.
After the beads have been added to the aqueous
medium and where specific binding of MI to the beads is
utilized, the aqueous medium i8 then held for a period of
time sufficient for this binding to occur. Normally this
requires 0.1-120 minutes, more frequently 1-60 min.
Where chemically induced non-specific binding of
particulate MI to beads is involved, such binding will
usually occur very rapidly, and it is usually sufficlent
to allow the mixture to stand for only a few minutes
often for only 60 sec., frequently less than 15 sec.:
preferably the centrifugal force is applied immediately
after adding the beads and other reagents to the aqueous
medium. The extent of binding between the MI, or the
7444M 26950-FF
2 ~
-31-
particulate MI, and the beads is one factor in
controlling the efficiency of the separation.
The density of at least a portion of a liquid medium
that is comprised of the aqueous medium is greater than
that of the insoluble components in the mixture. The
liquid medium may be a homogenous aqueous medium, an
aqueous suspension of particles capable of providing a
density gradient on centrifugation, an a~ueous medium
layered with a second, usually more viscous, aqueous or
non-aqueous liquid medium. The second medium will be at
least as dense, and usually denser than, the aqueous
medium. Generally, the density of at least a portion of
the liquid medium, preferably the aqueous medium, is at
least 1.05 times the density of the insoluble
components. Generally, the density of the insoluble
components will be about 0.7 to 1.2 g/cm3, usually 1.0
to 1.15 g/cm3. Of course, as mentioned above, the
density of the liquid medium should be less than that of
the MI-bead.
Relatively higher densities of aqueous media can be
achieved in a number of different ways. For example, a
high density solute can be added to the aqueous medium to
achieve the desired density. Preferably, the high
density solute will not readily cross cell membranes
(where cells are to be separated) nor add substantially
to the ionic strength of the medium. Exemplary of such
high density solutes are nonionic or neutral water
soluble, polyiodinated organic solutes, which are
commercially available under the tradename XYPAQUE~ and
NYCODENZ~. Other high density solutes include high
molecular weight salts, i.e., salts composed of atoms of
atomic number higher than 34, such as cesium chloride~
etc.; sucrose, and the like. The use of high molecular
weight salts can be less desirable where viable
microorganisms are to be separated.
7444M 26950-FF
2~Q~9
~32-
Another approach in preparing a higher den~ity
aqueous medium is the addition to the medium of
particulate suspensions such as silica particle
suspensions (available commercially under the tradename
PERCOLL~ and so forth. ~igh density non-aqueous media
may be comprised of organic or inorganic liquids such as
halocarbons, silicon fluids and the like.
In any of the ahove preparations, the aqueous medium
may be agitated, where necessary, after addition of the
appropriate agent and prior to subjecting the medium to a
centrifugal force, to ensure uniform distribution of the
agent in the aqueous medium, thus achieving uniform
density in the aqueous medium. Of course, the agitation
should not be so great as to cause harm to the MI
especially particulate MI such as cells or microorganisms.
In an alternate embodiment the aqueous medium can be
layered with a second medium that is at least as dense as
the aqueous medium and of a higher viscosity than the
aqueous medium to minimize mixing of the layers. The
second medium is aqueous or nonaqueous and, when aqueous,
may include polar solvents as described above for the
aqueous medium.
The bulk viscosity of the second medium is at least
1.5 times the viscosity of the aqueous medium, usually
about 2 to 100 times the viscosity, preferably about 2.5
to 75 times the viscosity, of the aqueous medium.
Generally, the viscosity of the aqueous medium iæ in the
range of 0.6 to 1.3 and that of the second medium is 1.5
to 100 cp.
A higher viscosity of the second medium can be
achieved by including as part of the medium a solute ~.hat
will increase bulk viscosity, for example, a polyol sllch
as glycol and polyvinylalcohol, a saccharide, such as
mono- and polysaccharides including, by way of example
and not limitation, mannitol, glucose, agar, agarose,
7444M 26950-FF
~33~
-33-
sucrose, starch, dextran, or hydrophilic polymers such as
polyethylene glycol, polyacrylate, polyvinylpyrrolidone;
and the like. The viscosity of the second medium can be
controlled not only by the concentration and nature of
the solute but also by temperature where it is desirous
to achieve such control. The viscosity of the second
medium can be controlled by adjusting temperature where
an increase in temperature will usually cause the medium
to have a lower viscosity. Thus layers can be created at
one temperature where the lower layer is a gel or is
relatively viscou~ or a solid, and the centrifugation can
be carried out at another, usually higher, temperature
where the viscosity is lower or the medium becomes fluid
and the beads pass more rapidly through the layer.
Separation of the layers can then be facilitated by
returning the lower layer to the original temperature.
Next, a centrifugal force is applied to the aqueous
medium, either alone or layered with a second medium, as
mentioned above. The intensity of the centrifugal force
is sufficient to separate the MI-bead from the insoluble
components. The application of a centrifugal force to
the medium can be carried out in any conventional manner
that provides for a centrifugal force such as by
conventional centrifugation or spinning the vessel
containing the medium on its axis. The ~ethod can be
conducted in a container made of a material such as, for
example, glass or plastic. In applying the centrifugal
force, the reaction container can be placed in a
centrifuge. The greater the centrifugal force, the
faster the separation of the MI-bead and the insolubl~
components. Preferably, the container will be spun ()--
its axis whereupon it will be convenient to carry out the
separation in a tube of diameter from about 1 to 10,
preferably from about 1.5 to 5cm. The required strength
of the centrifugal force that is, the rate and ratios of
7444M 26950-FF
2~3~0
-34-
rotation, will vary based on the buoyant density of the
MI-beads in the liquid medium, the viscosity of the
liquid medium, and the desired separation time. The
centrifugal force is applied for a sufficient period of
time to provide the desired degree of separation of the
MI-bead and the insoluble components.
Where the aqueous medium is denser than the
insoluble components, the MI-bead and the insoluble
components become separated under application of a
centrifugal force by virtue of the MI-bead and the
insoluble components moving in opposing directions in the
medium. The more dense MI-beads will migrate outward in
the medium, for example, to the bottom of a container
containing the medium, whereas the insoluble components
migrate to the upper part of the aqueous medium or toward
the centrifugal axis.
Where a second medium is employed the MI-beads will
migrate into the second medium, under the appropriate
centrifugal force, whereas the insoluble components will
remain in the aqueous medium.
In another embodiment of the above, the aqueous
medium, rather than be treated to adjust density, can be
layered with a medium having a density less than that of
the MI-bead but greater than that of any of the other
insoluble components in the aqueous medium. In this
embodiment, application of a centrifugal force of
~ufficient strength will cause the MI-bead to migrate
into the more dense medium while the insoluble
components, and soluble components, remain in the a~ueous
medium-
For example, a microorganism may be separated from amixture containing the microorganism and other
components, by binding the microorganism to a
multiplicity of beads in a first aqueous medium to form
7444M 26950-FF
2~33~
-35-
microorganism-bead. The first medium is contacted with a
second medium having a density greater than the first
medium but less ~han that of the microorganism-bead. The
contacted first and second media are subjected to
cen~rifugation to cause the microorganism-bead to collect
in an area outside of the first medium. The area is
separated from that of other components.
In another embodiment of the abo~e, the first
aqueous medium and the second medium are combined and
layered on a third medium of a viscosity higher than the
combined first and second media. In one example, the
third medium contains a saccharide and said layers a~e
formed at a temperature wherein said third medium of
higher viscosity is a gel and at least a portion of the
centrifugation is carried out at a temperature wherein
said medium of higher viscosity is not a gel. Following
said portion of the centrifugation, the medium of higher
viscosity may be returned to a gel and the layers may be
separated.
In another embodiment of the above, the aqueous
medium is denser than the insoluble components and is
layered with a second medium of a higher viscosity and at
least equal density to the aqueous medium. This latter
higher viscosity medium will have characteristics similar
to that described earlier for the second medium.
Application of a centrifugal force of sufficient strength
will cause the MI-bead to migrate into the second medium
whereas the insoluble components, as well as soluble
components, will remain in the aqueous medium.
Once the MI-bead has separated from the insolub l P
components, the MI-bead can be separated f~om the medium
and the insoluble components by any convenient means such
as, for example, decantation, pipetting, and the like. -
The MI-bead can be separated from the second medium,
where a second medium is employed, by any convenient
7444M 26950-FE
~ ~3 ~
-36-
method such as, for example, filtration, decantation or
pipetting. The separated MI-bead can be treated to
reverse the binding between the MI and beads. The
reversal of the binding between the MI, whether
particulate or non-particulate, and the beads is
dependent upon the nature of the binding between the
particles. For example, the MI-bead can be suspended in
a liquid medium with reagents added to facilitate
reversal of the binding. In one approach, where
particulate MI is bound to beads by ionic interactions,
ionic strength and the pH of the medium can be adjusted
to facilitate reversal of the binding. Generally,
increasing the ionic strength will reverse electrostatic
binding. Where the particulate ~I and the beads are
oppositely charged, a change in pH can neutralize one of
the charges and reduce binding interactions.
Alternatively, if a polycationic binding agent is used to
bind negatively charged particulate MI to negatively
charged beads, decreasing the p~ can neutralize the
charge and reverse binding. Th~s, it may be desirable to
change the pH to as high or low value as allowed by the
stability of the reagents, usually no less than pH 4 or
greater than pH 10.
Where non-specific bindi~g results from charge
interaction, an agent other than acid or base can be
added to reverse the charge interactions responsible for
the non-specific binding. For example, a releasing agent
can be added. Where the particles have like charges and
an oppositely charged polyelectrolyte was the chemical
means for binding the particles, a polyelectrolyte o~ ~he
same charge as on the particles can be used to disso-iate
the particles. The polyelectrolytes can be, for example,
polyanions s~ch as dextran sulfate, heparin,
polyglutamate, polyacrylate, phospholipid vesicles,
carboxymethyldextran and citrate. Aminodextran,
744hM 26950-FF
2 ~
-37-
chitosan, polybrene, polyethyleneimine, and cationic
liposomes are exemplary of polycations that can be
employed.
Where a polycation was usecl to initiate non-specific
binding between the particulate MI and the beads, a
polyanion can be employed to reverse the binding.
Alternatively, where a polyanion was used to form the
non-specific binding between particles, a polycation can
be used to reverse the binding. For example, where
polycations such as polybrene or barium ion have been
employed, the releasing agent can be a polyanion such as
citrate or sulfate. Detergents can act as a releasing
agent for liposomes and when particles are
non-specifically aggregated primarily through hydrophobic
interactiOns
For particulate or non-particulate MI that is
specifically bound to the beads (to form MI-beads~,
through ligand-receptor binding, the free ligand or
receptor can act as a releasing agent. Where binding is
covalent, a hydrolytic or redox reagent that can disrupt
the covalent binding will usually be used.
The concentration of the releasing agent should be
sufficient to result in substantial or complete reversal
of the binding between particulate MI and the beads. The
~5 concentration of the releasing agent is generally
dependent upon the nature of binding between the
particulate MI and the beads and the nature of the MI.
Generally, for particulate MI the concentration of the
releasing agent will be at least equal to the
concentration of ionic or hydrophobic sites on the
particulate MI ? preferably at least 10 fold higher.
It is important to choose the releasing agent with
regard to the nature of the MI so as to minimize or avoid
damage to the MI after the release from the MI-bead, ~
7444M 26950-FF
2 ~
-3~-
especially where particulate MI such as cells or
microorganisms are involved.
Once the MI has been released from the MI-bead, it
may be used as desired. For example, the released MI can
be examined for the presence of a detectable signal in
relation to the amount of an analyte in the sample.
Generally, a label is employed, either initially or in a
subsequent detection step. Released particulate MI can
be cells which can be used as desired. For example, the
released particulate MI can be red blood cells or
microorganisms. If detection of cells or microorganisms
is desired, one may utilize an agent that will
incorporate into the membrane of the material to be
detected, such as a dye. For example, intercalation dyes
such as squarate dyes or cyanine dyes, and the like, can
be employed.
One important aspect of the method in accordance
with the present invention is that it is possible to
separate one or more of a plurality of materials of
interest from insoluble components in a medium. This can
be accomplished by employing sets of beads of different
sizes or of different densities, or both. Each set of
beads has a binding affinity, whether specific or
non-specific as described above, for one of the materials
of interest. The method i8 performed as above described
and the different sets of beads each form a separate
discrete aggregate in one or more media employed in the
separation. The discrete aggregates are separated and
treated as mentioned above for a single material of
interest.
One application of the present method is the rem~)val
of microorganisms from solid debris such as, for examPle,
removal of microorganisms from a cervical mucus
specimen. In the method, using cervical mucus by way of
example and not by way of limitation, a cervical mucus
7444M 26950-FF
2~3~
-39-
sample is combined in an aqueous medium with beads under
conditions ~or specific binding of the beads to the
microorganisms of interest to form microorganism-beads.
The microorganisms have an antigen on their surface. The
beads have an antibody for such antigen bound on their
surface resulting in specific binding between the
microorganisms and the beads. The aqueous medium is
selected to be less dense than the microorganism-beads
but of greater density than other insoluble components in
the medium.
Next, the medium is subjected to a centrifugal force
to cause the microorganism-beads to separate from the
insoluble components in the medium thereby permitting
separation from the insoluble compounds by, for example,
decantation, pipetting, etc.
The separated microorganism-bead can then be treated
to release the microorganism from the bead as described
above.
The present invention has application to assays for
an analyte in a sample suspected of containing the
analyte. The analyte is an sbp member. The analyte may
be a particulate or non-particulate MI and can form an
MI-bead by binding to a bead by means of a complementary
sbp member that i8 or becomes bound to the bead. Either
the analyte, or a substance whose presence is related to
the presence of the analyte, such as, e.g., an antigen
removed from the surface of a cell, an sbp member
reciprocal to the analyte or the like, can be the MI. In
the assay the sample is combined in an aqueous medium
usually with an sbp member complementary to the ana]v~ f` .
At least one of the analyte or the complementary sbp
member is an MI capable of binding to beads that are
added to the medium to form MI-bead.
Preferably, prior to contacting with the sample, the
aqueous medium may be treated to adjust its density and
7444M 26950-FF
~ g~ 3 ~ ~
-40-
thereupon, or subsequent to contact with the sample, the
aqueous medium is contacted with a second medium of
higher viscosity than the aqueous medium in accordance
with the present invention. Following binding of MI to
the beads the medium is then subjected to a centrifugal
force sufficient to separate the MI-bead from other
components in the sample. The assay will normally
involve a signal producing system for producing a
detectable signal in relation to the amount of analyte in
the sample. The signal producing system usually includes
a labeled sbp member. The MI-bead, usually after
separation from that portion of the medium containing
other components, may be further combined with none, one
or more members of the signal producing system. The
latter can be examined for the presence of a detectable
signal. Such a determination can require the addition of
any remaining members of the signal producing system not
added above. Alternatively, the MI-bead can be treated
to separate MI from the bead prior to examining for the
presence of a detectable signal. After the MI has been
separated from the beads, the MI may be examined for the
presence of a detectable signal produced in relation to
the amount of analyte in the sample. This latter
approach is most applicable to particulate MI. For this
purpose the MI can be combined with any remaining members
of the signal producing system not added above in order
to generate a detectable signal.
The method permits rapid concentration and
æeparation of substances to be assayed or detected from
fluids that can inhibit growth or otherwise interfere
with detection. Other methods for separation as for
example, (1~ separation of microorganisms from solid
debris by binding to beads and centrifugation without a
high density medium or, (2) separation of non-particulate
materials from other solutes by binding of the material
7444M 26950-FF
-``` 2~3~
-41-
to beads and centrifuging without the use of a second
liquid layer that is free of the sample, fail to achieve
good separation. The method offers an advantage not only
for the purpose of carrying out an assay for the material
to be separated but also for the separation of one cell
type from a mixture of cells such as, for example, a
hybridoma producing a specific antibody from other
hybridomas for the purpose of preparing pure antibodies.
Thus, by the use of heavy beads that are not
substantially larger than the cells to be separated such
as gold sols or silica particles, the non-specific
binding usually encountered in separation of cells by
panning can be minimized. The method may also have use
in removing metastatic cells and T-cells from bone marrow
cells for transplantation or to prevent tumor recurrence
or graft versus host disease.
The invention further comprises a composition
comprising (1) an aqueous medium containing beads to
which are bound a material of interest (MI-bead) wherein
the medium has a aensity less than the MI-bead and
greater than other insoluble components in the medium.
This composition can further include a second medium of
at least equal density and higher viscosity than the
aqueous medium. Alternatively, the composition may
comprise (1) an aqueous medium containing MI-bead and (2)
a second liquid medium layered with the aqueous medium
wherein the second medium has a density less than the
MI-bead but greater than other insoluble components in
the aqueous medium. Alternatively, in the composition of
the invention the beads can have a MI bound to an sbs
member bound thereto or the beads can be bound to MI ~,y
virtue of a polyionic reagent.
As a matter of convenience, the reagents for
conducting a separation in accordance with the present
invention can be provided in a kit in packaged
7444M 26950-FF
2~33 ~.3~
-42-
combination in predetermined amounts for use in such
separation. The components of the kit can be packaged
separate or one or more components of the kit can be
packaged together depending on the interreactivity of
such components. The kit can comprise beads, means for
providing a high density liquid medium, and means for
providing a medium of higher viscosity than the liquid
medium. The kit can further include means for causing
the beads to bind to a material of interest. For
example, the beads can have an sbp member bound to the
surface. The sbp member is complementary to a MI and
provides means for binding MI to the beads. The kit can
include a polyionic reagent for binding a particulate MI
to the beads. Additionally, a releasing agent can be
included for releasing particulate MI from the beads.
For assays the kit can comprise (a) an sbp member
complementary to the analyte, (b) beads having an sbp
member bound thereto, which sbp member is complementary
to the sbp member or to the analyte. Alternatively, the
kit can comprise charged beads and a polyionic reagent
having a charge opposite to that of the particulate MI
when all the particles have the same charge.
Additionally, the kit can further comprise a releasing
agent for reversing the binding between the particles.
2S The kit can also include reagents for generating a signal
in relation to the amount of analyte in the sample.
Ancillary agents can be included as necessary.
EXAMPLES
The invention is described further by the followi ng
illustrative examples. All parts and percentages heleln
are by volume unless otherwise indicated. Temperatures
are in degrees Centigrade (~C).
7444M 26950-FF
2 ~ t~ ~~3
-~3-
MaTERIALS AND MET~ODS
Escherichia coli, strain K-12, ATCC 10798 was used
for all experiments described. Bacteria were grown on
Tryptic Soy agar (TSA) plates overnight at 370C in a 5%
C2 incubator. Bacteria were suspended in 1 mL
phosphate buffered saline (PBS), p~ 7.5, and washed by
centrifugation in a Microfuge for 2 minutes at room
temperature The bacterial pellet was resuspended at
109 CFU/mL in PBS, p~ 7.5, containing 1% BSA (W/V) and
50 ~g/mL polyclonal rabbit anti-E. coli (DAKO), and the
cells were incubated for 30 minutes at RT. The bacteria
were centrifuged as described above and washed 3x using
PBS-1% BSA, pH 7.5, to remove excess unbound rabbit
antibody.
Colloidal gold bind~n~ - The antibody-coated
bacteria were incubated in 100 ~L of PBS - 1% BSA,
pH 7.5, containing Goat-anti-rabbit IgG-Colloidal Gold
conjugates (10l2 to 10l3 particles/mL) of various
sizes (5, 20, and 30 nm particle sizes). After
resuspending the bacteria in the colloidal gold reagent,
the cells were incubated at RT for 30 to 45 minutes.
4 ~ 9ilica Particle Binding: - The
antibody-coated bacteria were incubated in 50 ~L of
PBS-1% BSA, pH 7.5 containing 4 ~m goat-anti-rabbit
IgG-silica particles (3.6~107/mL). After suspending
the bacteria in the silica particle suspension, the
sample was mixed on a rotary shaker at RT for 15 to 60
minutes.
Stock Percoll~ (SIGMA Chemical Company,
St. Louis, MO) was made by diluting the commercially
available Percoll using 2.5 M sucrose in a ratio 9:1
This stock Percoll was pipetted into Oak Ridge tubes .~nd
the gradients were centrifuged in a Sorvall RC-5B
centrifuge at 12,500 rpm for 30 minutes at 200C. Density
Marker beads ~Pharmacia, Piscataway, N.J.) were used to
7444M - 26950-FF
-44- 2~3~ ~
calibrate the gradients visually. For direct gradient
density measurements, immediately after the
centrifugation, 0.25 mL ali~uots were removed
sequentially from the top of the gradient using a
positive displacement pipette and weighed on a Mettler
balance. The pipette was calibrated using distilled
water and densities were computed directly.
Stock Nycodenz (Nycomed, Oslo, Norway) was made by
dissolving the solid in distilled water and adjusting the
pH to 7.5. The stock was diluted to 55.5% (w/v) to
produce solutions of varying densities (1.2 to 1.3 g/mL)
and polyvinylpyrrolidone (PVP) (9OK) was added to it at a
final concentration of 1 mg/mL. 2 mL of the Nycodenz/PVP
solution was added to the bottom of Oak Ridge tubes and
the gamples containing particles (gold or silica) and
bacteria were overlayered in 0.5 mL of PBS and the tube
was centrifuged as described above for the Percoll
gradients.
For gradients wherein the viable bacteria were
estimated in the different fractions of the gradient,
1 mL fractions were removed sequentially from the top of
the gradient tube, serially diluted in PBS (10-fold
dilutions), if need be, and 1 mL samples were plated on
previously dried TSA plates. The plates were incubated
at 37C overnight and visible colonies were counted the
next day to determine colony-forming units (CFU).
For DNA analysis, samples were lysed in 1% SDS
containing 25 mM Tris, pH 8.0, 10 mM EDTA, 250 ~g/mL
Proteinase K and incubated at 50C for 30 minutes. This
solubilized most of the cellular debris~ and DNA was
measured by the bibenzimidazole method using a Hoefe- TKO
Mini fluorimeter, Cesarone, et al. Anal. Bioch~m.
100:188-197 (1979). Uncut Lambda DNA was used as a
standard for the calibration of the fluorimeter.
7444M 26950-FF
2~3~
-45-
Whole blood which was fairly fresh (<1 week old)
was lysed using a ISOLATOR~ (duPont) for 5-10 minutes
and EDTA added to a final concentration of 5 mM. This
lysed blood sample was used for experiments in which the
separation of bacteria from lysed blood components was
checked. Typically, in such cases 50% (v/v) of the
sample consisted of lysed blood.
RESULTS
The Densitv of Bacteria is Increased bv Colloidal Gold
Bindi~g
A. Percoll Gradientæ
E. coli cells were coated with rabbit antibody
and treated with colloidal gold particles bearing Goat
anti-rabbit Ig&; 5, 20 and 30 nm colloidal gold particles
were used to enhance the density of the bacteria, and
Percoll gradients (starting density = 1.124 g/mL) were
used to separate the excess colloidal gold particles from
the ~. ~Qli coated with colloidal gold. The Percoll
gradients were examined visually. Several points are
noteworthy.
1. The den~ity of bacteria is increased by
the binding of colloidal gold particles to the cells. E.
coli which were either not treated with the colloidal
gold conjugate or did not have rabbit antibody bound on
their surface band on top of the gradient while colloidal
gold binding caused the bacteria to band at positions
within the gradient.
2. As the size of the colloidal gold
particle binding to the bacteria was increased, the
apparent density of the bacteria was also increased; 5nm
particle-coated-bacteria appeared as a heterogenous ~and
with densities ranging from 1.119 to 1.121 g/mL; 20 nm
particle-coated-bacteria appeared as a band at 1.152
while 30 nm particle-coated-bacteria appeared as a band
7444M 26950-FF
2 ~
-46-
at >1.16 g/mL. Excess colloidal gold particles were
pulled to the bottom of the tubes during the
centrifugation.
3. A density calibration of the Percoll
gradient was developed and it showed that the 5 nm
particle coated bacteria were in a shallow region of the
gradient while the 20 and 30 nm particle-coated-bacteria
were towards the bottom of the gradient. (Fig. 1)
B. Percoll-diatrazoate Gradients
To obtain more accurate density determinations
of the E. coli coated with 20 and 30 nm colloidal gold
particles, separations were done in Percoll containing
sodium diatrazoate (Hypaque) of starting density 1.19
g/mL. As the size of the colloidal gold increased,
density increased. E. coli coated with 5 nm particles
appeared as a band at 1.1377 while 20 nm particles
provided densities of 1.1907 and 1.2023 g/ml; 30 nm
particleæ provided densities of 1.1963 to 1.2173 g/mL.
The gravimetric density calibration of the
Percoll-diatrazoate gradients i8 shown in Fig. 2.
C. PE~COLL GRA~IENTS
E. coli cells were coated with rabbit antibody and
treated with gold particles bearing Goat anti-rabbit
IgG. Percoll gradients (starting density = 1.124 g/ml)
were used to separate the free bacteria. The gradients
were fractionated and the fractions were plated to
determine the position of the bacteria. The results ~re
shown in Table 1.
7444M 26950-FF
2~3~
-47-
TABLE 1
BA~_ RIAL CAPTURE IN BUFFER
S~Ee~ IL~BY PER~OLL GRADIENTS
% BACTERIA
FRACTION +AB:+GOLD-AB:+GOLD
A (TOP) 0 69.6
B 0 17.2
C 0 1.2
D 8.4 5.3
E (PELLET) 91.6 7.3
~5
-
N = 2
AVERAGES OF DUPLICATES
T~E ~NSITY OF BACTERIA IS INCREASE~ BY 4 um SILICA
P~RTICLE BINDING
A. P~RCOLL GRADI~NTS
E.coli cells were coated with rabbit antibody and treated
with silica particles bearing goat anti-rabbit IgG.
Percoll gradients (starting density = 1.124 g/ml) were
used to separate the free bacteria from silica particles
(density ~ 1.4 g/mL). The gradients were fractionated
and the fractions were plated to determine the position
of the bacteria. The results are shown in Table 2.
The density of bacteria was increased by bindin~ lo
silica particles. ~. coli which were either not treated
with the silica or did not have rabbit antibody bound on
their surface banded on top of the gradient while silica
7444M 26950-FF
-48~ 3 ~3 ~ ~
binding caused the bacteria to pellet to the bottom of
the gradient.
TABL~ 2
BACTERIAL CAPTUR~ USIN~ SILICA
PERCOLL SEPARATION
% BACTERIA
FRACTION NO. +A~ -AB _ _
1 ~TOP) 10.7 80.0
2 6.8 13.0
3 0.7 2.0
4 5.7 0
5 (BOTTOM) 76.0 4.4
Liquid Lavers of Density >1.23 Float Cellular Debris
Derived From Blood
The data obtained above shows that labelling of
E. coli with 30 nm colloidal gold particles can increase
the bacterial density to >1.2 g/mL.
Whole blood was lysed using an ISOLATOR (duPont de
Nemours Company, Wilmington, Delaware) tube and the lysed
blood was overlayered on top of a Nycodenz cushion of an
equal volume of different varying densities. These tubes
were then subjected to centrifugation and the appearance
of the tubes was e~amined. Centrifugation of lysed blood
over a cushion of density 1.0 resulted in a large pellet
of blood-der;ved cellular debris. Increasing the density
of the liquid medium to >1.23 caused the cellular
debris to remain on top of the cushion. DNA analysis of
the pellet showed that >9S% of the DNA present in the
7444M 26950-FF
-49- 2~J~3~
initial sample was absent in the pellet after
centrifugation in the medium of density .>1.23 g/ml.
LIQUID LAYERS ~F DENSITY 1.2 ALLOW GOLD-LABELLFD BACTERIA
TO BE PELLETED: NYCODENZ CUS~IONS
E. 501i cells were coated with rabbit antibody and
treated with colloidal gold particles (30 nm) bearing
goat anti-rabbit IgG. Nycodenz cushions were uæed to
separate the free bacteria from colloidal gold
particles. The Nycodenz gradient was fractionated and
the fractions were plated to determine the position of
the bacteria. The results are sho~n in Table 3.
The density of bacteria was increased by binding to
30 nm colloidal gold particles. E. coli which were
either not treated with the colloidal gold or did not
have rabbit antibody bound on their surface did not enter
the Nycodenz cushion while colloidal gold binding caused
the bacteria to pellet to the bottom of the gradient;
>93% of the bacteria were captured and effectively
separated to the bottom of the tube (Table 3).
7444M 26~50-FF
-50-
TABLE 3
BACTERIAL CAPTURE IN BUFFER
SEPARATION BY NYCODENZ CUSHIONS
% BACTERIA
FRACTION +~B:+GOLD +AB:+GOLD -AB:+&OLD -AB:+GOLD
A(TOP) 1.5 0.5 76 76
B 0.4 3.7 18.2 18.4
C 1.1 0 1.8 2.1
D 3.4 0.5 0 0.8
E(PELLET) 93.5 95.3 3.9 2.9
4 mL NYCODENZ CUS~IONS (d = 1.2g/ml)/lmg/ml PVP (9OK)
SAMPLE IN 1 mL
LIQUID LAY~RS OF ~ENSITY 1.3 ALLOW SILICA ~OUND BACTERIA
TO_BE P~LLET~D: NYCO~ENZ CUS~IONS
E. coli cells were coated with rabbit antibody and
treated with silica particles bearing goat anti-rabbit
IgG in the presence and absence of 50% lysed blood.
Nycodenz cushions were used to separate the free bacteria
from silica particles (density ~1.4 g/mL). The
Nycodenz gradient was fractionated and the ~ractions were
plated to determine the position of the bacteria. The
results are shown in Table 4.
The density of bacteria was increased by bindin~ ~:o
silica particles. E. coli which were either not tre~.ed
with th silica or did not have rabbit antibody bound on
their surface did not enter the Nycodenz cushion (d=1.3
g/ml) while silica binding caused the bacteria to pellet
to the bottom of the gradient. As shown in Table 4,
7444M 26950-FF
2 O ~
-
> 98% of the bacteria were captured by silica and
separated from the blood components using Nycodenz,
d = 1.3.
TABL~ 4
~ACTERIAL CAPTURE ~SING SILICA
50/0 LYSED BLOOD NYCODENZ SEPARATION
% BACTERIA
FRQ~ION +AB:+SILICA +AB:+SILICA -AB:+SILICA -AB:+SILICA
l(TOP) 0.4 1 90.7 85.2
2 0.4 0.5 5.3 4.1
3(BOTTOM) 99.2 98.6 4 10.7
The invention described herein provides for a
simple, effective, and complete separation of a material
of interest from other components in a sample. A
significant advantage of the present invention is that
washing of the material of interest free of other
components can occ.ur during passage from one medium to a
higher density and/or higher viscosity medium.
Although the foregoing invention has been described
in some detail by way of illustration and example for the
purposes of clarity and understanding, it will be obvious
that certain changes or modifications may be practiced
within the scope of the appended claims.
7444M 26950-FF
