Note: Descriptions are shown in the official language in which they were submitted.
- I - 1 339 J ~ ~
P~OCESS FOR PRODUCING MAGNETICALLY RESPONSIVE
POLYMER PARTICLES AND APP~ICATIONS T~EREOF
Field of the Invention
This invention relates to a process to make magnetically
responsive polymer particles and their use in immunoassays,
biomedical and industrial applications.
8ackground of the Invention
Many biological techniques, such as immunoassays, affinity
purification etc., require the separation of bound from free
fractions. Magnetic particles have been used to facilitate the
desired separation.
Magnetic particles have been formed from a variety of
particulate and magnetic matter, using a variety of processes,
having different characteristics. For example, Ikeda et al. U.S.
Patent No. 4,582,622, discloses a magnetic particle comprised of
gelatin, water-soluble polysaccharide, sodium phosphate and
ferromagnetic substances; U.S. Patent Nos. 4,628,037 and 4,554,088
discloses magnetic particles comprised of a magnetic metal oxide
core surrounded by a coat of polymeric silane; U.S. Patent No.
_ 4,452,773 discloses discrete colloidal sized particles having a core
of ferromagnetic iron oxide (Fe304) which is coated with a
water-soluble polysaccharide or a derivative thereof having
functional groups; and Mansfield U.S. Patent No. 4,297,337 discloses
magnetic glass- or crystal-containing material as a particulate
carrier.
Summary of the Invention
The present invention provides a novel process for producing
magnetically responsive polymer particles, hereinafter referred to as magnetic
particles, from polymeric particles with average size from about 1 to 100
5 microns in diameter regardless of shape and composition. The magnetic
particles of this invention may be prepared by first producing magnetically
responsive metal oxide, hereinafter referred to as metal oxide, with average size
of about 1 micron or less and thin coating a polymeric core particle with a layer
of polymer containing metal oxide. The surface of these magnetic particles can
1 o be coated further with another layer of polymer or functionalized polymer to provide the desired surface characteristics.
The magnetic particles produced by the present invention are
monodispersed in size with rough surface and have a magnetic metal oxide
content of from about 5% to 0%, preferably from 10% to 25%. Particles with
these characteristics have been found to be useful in immunoassays and a wide
variety of biomedical applications. These magnetic particles can be used for
passive or covalent coupling of biological material such as antigens, antibodies,
enzymes or DNA/RNA and used as solid phase for various types of
immunoassays, DNA/RNA hybridization assays, affinity purification, cell
2 o separation and other biomedical applications. The magnetic particles can also
be used for industrial application such as treatment of industrial waste.
According to an aspect of the invention, a process to determine the
presence or concentration of an analyte comprises:
contacting magnetic particles of a uniform size distribution and magnetic
25 content comprising:
an inner core polymer particle able to absorb a monomer:
. .
-A
2 a 13395~5
an inner core polymer particle able to absorb a monomer;
a first coating on said inner core polymer particle wherein said first
coating evenly covers said inner core polymer particle, said first coating
comprising magnetically responsive metal oxide particles and a first polymer,
wherein said first polymer is prepared from monomers which are adsorbable by
the inner core polymer particle, said magnetic particles additionally comprising an outer layer selected from the group consisting of:
(i) a layer of functionalized polymer evenly covering the first coating,
and
0 (ii) an outer polymer covering the first coating and a layer of
functionalized polymer evenly covering the outer polymer coating,
said magnetic particles having a nucleic acid complementary to said
nucleic acid sequence of said target molecule, attached to said functionalized
polymer,
with a fluid specimen to form a suspension;
incubating said suspension under hybridizing conditions for a period of
time sufficient to permit hybridization;
separating said magnetic particle from said suspension;
adding a labelled nucleic acid sequence complementary to said nucleic
2 o acid sequence of said target molecule to form a second suspension;
incubating said second suspension under hybridizing conditions for a
period of time sufficient to permit hybridization;
separating said magnetic particle from said second suspension; and
detecting duplex formation on said magnetic particle by means of said
2 5 label.
In accordance with a further aspect of the invention, a process for
isolating a biosubstance comprises:
contacting magnetic particles of a uniform size distribution and magnetic
content comprising:
;h~
13~9 ..1~
2 b
an inner core polymer particle able to absorb a monomer; a first coating
on said inner core polymer particle wherein said first coating evenly covers said
inner core polymer particle, said first coating comprising magnetically
responsive metal oxide particles and a first polymer, wherein said first polymeris prepared from monomers which are adsorbable by the inner core polymer
particle, said magnetic particles additionally comprising an outer layer selected
from the group consisting of:
(i) a layer of functionalized polymer evenly covering the first coating,
and
0 (ii) an outer polymer covering the first coating and a layer of
functionalized polymer evenly covering the outer polymer coating,
said magnetic particles having a ligand specific for said biosubstance;
with a fluid specimen to form a suspension;
incubating said suspension until sufficient biosubstances have reacted with
said ligand;
separating said magnetic particles from said suspension; separating said
magnetic particles from said biosubstance; and
obtaining essentially pure biosubstance.
According to a further aspect of the invention, a process for removing
2 0 an unwanted biosubstance comprises: contacting magnetic particles of a
uniform size distribution and magnetic content comprising: an inner core
polymer particle able to absorb a monomer; a first coating on said inner core
polymer particle wherein said first coating evenly covers said inner core
polymer particle, said first coating comprising magnetically responsive metal
2 5 oxide particles and a first polymer, wherein said first polymer is prepared from
monomers which are adsorbable by the inner core polymer particle, said
magnetic particles additionally comprising an outer layer selected from the
group consisting of:
1339~
2 c
a first coating on said inner core polymer particle wherein said first
coating evenly covers said inner core polymer particleJ said first coating
comprising magnetically responsive metal oxide particles and a first polymer,
wherein said first polymer is prepared from monomers which are adsorbable by
the inner core polymer particle, said magnetic particles additionally comprisingan outer layer selected from the group consisting of:
(i) a layer of functionalized polymer evenly covering the first coating, and
(ii) an outer polymer coating covering the first coating and a layer of
functionalized polymer evenly covering the outer polymer coating,
said magnetic particles having a ligand specific for said analyte attached
to said functionalized polymer,
with a fluid specimen to form a suspension;
incubating said suspension until sufficient analyte has reacted with said
specific ligand;
separating said magnetic particles from said suspension;
adding a labelled ligand specific for said analyte to said separated
magnetic particles to form a second suspension;
incubating said second suspension until sufficient analyte has reacted
with said labelled ligand specific for said analyte;
2 o separating said magnetic particles from said second suspension;measuring the amount of labelled ligand associated witll said magnetic
particles;
relating the amount of labelled ligand measured with the amount of
analyte measured for a control sample.
2 5 According to another aspect of the invention, a process to determine the
presence or concentration of specific nucleic acid sequences in nucleic acid
target molecules comprises:
contacting magnetic particles of a uniform size distribution and magnetic
content comprising:
,
A
1339545
2 d
(i) a layer of functionalized polymer evenly covering the first coating,
and
(ii) an outer polymer covering the first coating and a layer of
functionalized polymer evenly covering the outer polymer coating,
said magnetic particles having a ligand specific for said biosubstance;
with a fluid specimen to form a suspension;
incubating said suspension until sufficient biosubstances has reacted with
said ligand;
separating said magnetic particles from said suspension; and
0 obtaining suspension free of unwanted biosubstance.
According to a further aspect of the invention, a process for industrial
waste purification comprises removing unwanted substances from industrial
waste, the use of magnetic particles of a uniform size distribution and magneticcontent comprises; an inner core polymer particle able to absorb a monomer; a
first coating on said inner core polymer particle wherein said first coating
evenly covers said inner core polymer particle, said first coating comprising
magnetically responsive metal oxide particles and a first polymer, wherein said
first polymer is prepared from monomers which are adsorbable by the inner
core polymer particle, said magnetic particles addltionally comprising an outer
2 o layer selected from the group consisting of:
(i) a layer of functionalized polymer evenly covering the first coating, and
(ii) an outer polymer covering the first coating and a layer of functionalized
polymer evenly covering the outer polymer coating.
In accordance with another aspect of the invention, a process to
2 5 determine the presence or concentration of an analyte comprises:
contacting magnetic particles of a uniform size distribution and magnetic
content comprising:
an inner core polymer particle able to absorb a monomer:
13395~5
a coating on said inner core polymer particle wherein said coating
evenly covers said inner core polymer particle, said coating comprising
magnetically responsive metal oxide particles and a functionalized polymer,
wherein said functionalized polymer is prepared from functionalized monomers
5 which are adsorbable by the inner core polymer particle said magnetic particles
having a ligand specific for said analyte attached to said functionalized
polymer, with a fluid specimen to form a suspension;
incubating said suspension until sufficient analyte has reacted with said
specific ligand;
separating said magnetic particles from said suspension;
adding a labelled ligand specific for said analyte to said separated
magnetic particles to form a second suspension;
incubating said second suspension until sufficient analyte has reacted
with said labelled ligand specific for said analyte;
separating said magnetic particles from said second suspension;
measuring the amount of labelled ligand associated with said magnetic
particles;
relating the amount of labelled ligand measured with the amount of
analyte measured for a control sample.
2 o Objectives and Advanta~es
It is the objective of an aspect of this inveniton to:
Develop a process of producing magnetically responsive polymer
particles easily from readily available polmer particles.
Develop a process of producing magnetically responsive polymer
2 5 particles with moderate sedimentation and fast magnetic separation.
Develop a process of producing magnetically responsive polymer
particles with various surface charges, and functional groups for passive
adsorption or covalent coupling of biological material.
. = . . "~, .
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- 3 1 3 3 ~ ~4 5
Develop medical, biological, diagnostic and industrial
applications using these magnetically responsive polymer particles.
The advanta~es of various aspects of this invention include:
A wide variety of polymeric core particles with size from about
1 to 100 microns can easily be transformed to magnetically
responsive particles.
The metal oxide content can be varied according to the
applications.
The surface can be derivatized into a wide variety of functional
groups for covalent coupling.
A wide variety of monomer can be used for the final coating to
provide different surface characteristics of the resulting polymer.
Both crosslinked and noncrosslinked magnetically responsive
polymer particles can be produced.
Monodispersed magnetically responsive polymer particles can be
produced.
Detailed Description of the Invention
The magnetic particles of this invention may be prepared by
first producing metal oxide with average size of about 1 micron or
less. The metal oxide is produced by heating and precipitating a
mixture of divalent and trivalent metal salt, preferably a mixture
of ferrous and ferric sulfate or chloride with sodium hydroxide
solution. The molar ratio of divalent to trivalent metal salt can
be varied from 0.5 to 2.0, preferably 0.5 to 1.0, to obtain the
desirable size and magnetic characteristics of metal oxide. It is
observed that the molar ratio of divalent to trivalent metal salt
affects the size of the metal oxide: the smaller the molar ratio of
divalent to trivalent metal salt, the smaller the size of metal
oxide. The molar ratio of divalent to trivalent metal salt also
affects the color of the resulting magnetic particles: the smaller
the molar ratio, the lighter the brownish color of the resulting
magnetic particles. Preferably, the metal oxide is either
superparamagnetic or paramagnetic although ferromagnetic metal oxide
can also be used, provided centrifugation instead of magnetic
separation is used during the clean up.
1339~S
-- 4 --
Other divalent transition metal salts such as manganese, magnesium,
cobalt, nickel, zinc, and copper salts may be substituted for
ferrous salt.
After the metal oxide has been precipitated, it is washed
several times with centrifugation at 250xg until the supernatant is
neutral in pH. The metal oxide is resuspended in deionized water
and mechanically stirred at high speed to break down the aggregate
of metal oxide crystals. Further centrifugation at 250xg will not
pellet all of the metal oxide. The supernatant which contain
smaller size metal oxide crystals is collected and the pellet is
resuspended in deionized water. This process is repeated for at
least three times or until most of metal oxide can no longer be
pelleted at 250xg. The metal oxide obtained this way usually has
size less than 2.0 micron. Low speed centrifugation at 100xg to
remove largers crystals will reduce the size to less than 0.8 micron.
The metal oxide with average size of 1.0 micron or less is mixed
with monomer and coated onto the polymeric core particles,
preferably polystyrene particles, with size of 1 to 100 microns in
the presence of initiator. Addition of a small quantity of
emulsifier will help prevent the particles from agglomerating. The
magnetic particles are then coated with a protective layer of
polymer, preferably polystyrene, to prevent the metal oxide from
falling off. If functionalized magnetic particles are desired the
magnetic particles can be coated further with another layer of
functionalized polymer to provide functional groups such as
carboxyl, amino or hydroxyl for covalent coupling of biological
material.
The magnetic particles prepared according to this invention can
be illustrated in Figure I, where A represents the core particle, B
represents the metal oxide/polymer coating, C represents the
protective polymer coating and D represents the functionalized
polymer coating. Figure II shows the transmission electron
micrograph of 0.08 to 0.1 micron slice of a magnetic particle
1 339~45
5/6/7/8
prepared according to this invention. Figure III shows a scanning
electron micrograph of 6.8 micron magnetic particles, prepared
according to this invention. Figure III a is at 1000x and Figure
III b is at 5000x magnification.
13~9S45
g
The polymeric core particles useful in this invention may be of
any polymer which can be obtained as a dispersion of small particles
and which can absorb a monomer thereby causing the metal oxide and
monomer mixture to coat onto the surface of the corè particles. ~he
core particles may be of any size and shape, preferably of l to 100
microns in size and spherical in shape. When monodispersed core
particles are used the resulting magnetic particles will also be
monodispersed in size. The core particles may be obtained by
emulsion polymerization, suspension polymerization or other means of
polymerization with or without a crosslinking agent such as divinyl
benzene or the like. Among the monomers which can be used to
prepare core particles are styrene, methyl methacrylate,
vinyltoluene and the like. A mixture of the monomers can also be
used. The monomer used for magnetic metal oxide coating or
protective coating may or may not be the same type as the core
particles. The weight ratio of monomer used for metal oxide coating
to core particles may be from 0.1 to 12, preferably from 0.2 to 6,
depending upon the thickness of metal oxide/polymer layer desired.
When the metal oxide prepared from a mixture of ferrous and ferric
salts is used for coating it is preferred to use a monomer to core
particle weight ratio of about 0.1 to 0.5. However when the metal
oxide prepared from a mixture of manganese (II) and ferric salts is
used for coating the weight ratio of monomer to core particles may
be from 0.1 to 12. As a result when crosslinked magnetic particles
which are inert to common organic solvent are desired, it is
prefered to use the metal oxide prepared from a mixture of manganese
(II) and ferric salts with monomer cotaining 2% to 10%, preferably
8% to 10% by weight of crosslinking agent and a monomer to core
particle weight ratio of 3 to 12, preferably 4 to 6. When lower
monomer to core particle weight ratio (i.e. 0.1 to 0.5) is used
during the metal oxide/polymer coating it is preferred to overcoat
the resulting magnetic particles with a protective layer of polymer
coating to further adhere the metal oxide to the surface of the
magnetic particles. However, when higher monomer to core particle
lo 1339~
ratio (i.e. 3 to 12) is used no protective polymer coating is
necessary. The polymerization temperature may be from 50~C to 90~C,
preferably 55~C to 65~C. The polymerization initiator may either be
water soluble such as potassium persulfate and the like or water
insoluble such as benzoyl peroxide and the like. Other means of
polymerization initiation such as radiation, ionization or the like
may also be used. It is found unexpectly that magnetic particles
can be produced without using any emulsifier when the metal oxide
prepared from a mixture of manganese ( II) and ferric salts is used
for coating. However, a small amount of emulsifier such as sodium
dodecylsulfate, Aerosol 22, Tween 20 or Nonidet P-40 (NP 40) is
found to be useful in preventing the particles from extensive
aggregation during the metal oxide/polymer coating when the metal
oxide prepared from a mixture of ferrous and ferric salts is used
for coating. Other emulsifiers with the same capability may also be
used. The magnetic metal oxide content can be varied from 5% to
50%, preferably from 10% to 25% by using different amount of metal
oxide during the metal oxide/polymer coating. ~lutiple metal
oxide/polymer coatings can also be employed to increase the metal
oxide content. Other ingredients commonly used in polymerization
may also be added as long as magnetic particles with desirable
characteristics can be obtained. The ingredients for metal
oxide/polymer coating may be added all at once at the beginning of
metal oxide/polymer coating process or added stepwise. When the
metal oxide prepared from a mixture of ferrous and ferric salt is
used, it is preferred to add the ingredients stepwise. The
ingredients may be mixed by mechanic stirring, tumbling or other
means of agitation under vacuum or inert gas such as argon. The
functional groups can be incorporated onto the surface of the
magnetic particles by either using a mixture of monomer and
functionalized monomer during the metal oxide/polymer coating or
overcoating the magnetic particles with a thin layer of
functionalized monomer at the end. The functionalized monomer used
may be selected from one or a mixture of the following:
2-hyroxyethyl methacrylate, 2-aminoethyl methacrylate,
- ll 1339~4~
trimethylammoniumethyl methacrylate methosulfate, dimethylaminoethyl
methacrylate, methacrylic acid, undecylenic acid, methyl propene
sulfonic acid, undecyleny1 alcohol, oleyl amine, glycidyl
methacrylate, acrolein, glutaraldehyde and the like. The magnetic
particles can also be overcoated with a layer of different polymer
than the one used for metal oxide/polymer coating or protective
coating to take up the surface characteristics of that polymer.
Applications of Magnetic Particles
The uses of a wide variety of magnetic particles as solid phase
for various applications such as fluorescence immunoassays,
radioimmunoassays, enzyme immunoassays, cell separations, enzyme
immobilizations and affinity purificatins have been reviewed in
literature as examplified by the following articles: Hirschbein et
al, Chemical Technology, March 1982, 172-179 (1982); Pourfarzaneh,
The Ligand Ouarterly, 5(1): 41-47 (1982); Halling and Dunnill,
Enzyme ~licrobe Technology, 2: 2-10 (1980); ~losbach and Anderson,
Nature, 270: 259-261 (1977); Guesdon et al, J. Allergy Clinical
immunology, 61(1), 23-27 (1978). Some applications have also been
disclosed in the U.S. Patent Nos. 4,152,210 and 4,343,901 for enzyme
immobilizatins; U.S. Patent Nos. 3,970,518, 4,230,685, and
4,267,2343 for cell separations; U.S. Patent Nos. 4,554,088,
4,628,037, and 3,933,997 for immunoassays.
Some magnetic particles may be useful in one application, but
not in another application. For example, the magnetic particles
disclosed in U.S. Patent No. 4,554,088 and 4,628,037, which comprise
a superparamagnetic metal oxide core generally surrounded by a coat
of polymeric silane, may be useful in immunoassay and affinity
purification, due to the large surface area and slower settling
rate, but are not suitable in cell separation application such as
bone marrow purging. Due to the small size of the magnetic
particles, disclosed in these two patents, it is very difficult to
remove all of the magnetic particles from the cell suspension
effectively. ~oreover, the nonspecific binding of smaller magnetic
particles to normal cells would be much higher. In using magnetic
particles for bone marrow purging, the magnetic particles are coated
133954~
- 12 -
with antibody, such as sheep anti-mouse IgG, and the bone marrow is
treated with a mixture of several monoclonal antibodies against the
cancer cell surface antigens. The magnetic particles will bind only
to the cancer cells and cause them to be separated from normal cells
by passing them through a strong magnetic field. The cleansed cells
are then put back into the patient.
By using the processes of this invention magnetic particles can
be optimized in terms of size, surface area, metal oxide content and
surface characteristics for a wide variety of biomedical
applications. The magnetic particles produced by this invention can
be used as solid phase for enzyme irmunoassay, fluorescence
immunoassay, radioimmunoassay, DNA/RNA hybridization assay, and
other disgnostic applications. Immunoassays can be performed by
using various configurations such as sandwich assays and competitive
binding assays etc., which are obvious to those skilled in the art.
The DNA/RNA hybridization can also be performed by using various
configurations such as solid phase hybridization or liquid phase
hybridization. In solid phase hybridization configuration a DNA or
RNA probe (catcher probe) is immobilized on the magnetic particle
first. The immobilized catcher probe is then used to hybridize with
complimentary strand of DNA from the sample (sample DNA). Finally
another probe (signal probe) which is labeled with fluorescent,
radioactive or enzyme tracer and capable of hybridizing with another
part of the sample DNA is used for signal generation. In liquid
phase hybridization configuration the catcher probe and signal probe
are allowed to hybridize with the sample DNA in the liquid phase
first and then immobilized to the magnetic particles.
Alternatively, the signal probe can also be labelled with one or
several biotin groups and the signal is detected by binding the
biotin groups with avidin labelled fluorescent, radioactive or
enzymatic tracer to enchance the sensitivity of the assay.
The immunoassays and DNA/RNA hybridization assays can be used to
measure a wide variety of compounds such as drugs, hormones,
antibodies, peptides, DNA, RNA, nucleotides, viral antigens, and
carbohydrates in biological samples.
1339545
- 13 -
The magnetic particles produced by this invention can also be
used for affinity purification, cell separation, enzyme
immobilization and other biomedical applications. In cell
separation the magnetic particles are used to either remove unwanted
cells (negative selection) or enrich the wanted cells (positive
selection) through immunological reactions or nonimmunological
reactions. This principle can be used to remove cancer cells from
bone marrow (bone marrow purging), purify cell populations through
either positive or negative selection for tissue culture and perform
various cellular immunoassays etc. In affinity purification the
magnetic particles are used in place of conventinal solid phase such
as polyacrylamide gels, sepharose gels or other cellulose beads to
purify a wide variety of biological materials such as antibodies,
antigens, enzymes. inhibitors, cofactors, single stranded DNA,
binding proteins, haptens and carbohydrates etc. In another
application similar to the affinity purification, the magnetic
particles can be used to cross adsorb and remove unwanted protein
components from the antisera or clinical samples. In enzyme
immobilization the enzyme is immobilize onto the magnetic particles
through various means of coupling so as to preserve the enzyme
activity and to permit the reuse of immobilized enzyme. The
magnetic particles with immobilized enzyme can be used to replace
other solid phases such as glass beads, controled pore glass, silica
gels and cellulose beads etc., which are commonly used in
immobilized enzyme systems to produce a wide variety of materials
such as carbohydrates, amino acids, and proteins, etc.
The magnetic particles produced by this invention can be used
for industrial applications like the treatment of industrial waste,
to remove harmful chemicals, i.e. organic or inorganic solvents from
industrial material.
These applications are all facilitated by the ease of
separation, fast reaction rate and large surface area common to most
of magnetic particles. The following examples are provided to
further illustrate the versatility and advantages of this ivnention,
13395~
- 14 -
the details thereof are not to be construed as limitations, for it
will be apparent that various equivalents, changes and modifications
may be resorted to without departing from the spirit and scope
thereof and it is understood that such equivalent embodiments are
intended to be included therein.
General Procedures for the Preparation of Metal Oxide
Example 1
In a three-necked round bottom flask equipped with mechanical
stirrer, condenser, thermometer, dropping funnel and heating mantle
was placed a mixture containing 0.361 mol of ferrous sulfate and
0.369 mol of ferric sulfate (Fe /Fe ratio = 1) in 400 ml of
deionized water. The mixture was heated to 85 to 90 ~C with
stirring and added dropwise 850 ml of 6 N sodium hydroxide over a
period of 90 minutes. The mixture was stirred at 85 to 90 ~C for
one more hour and cooled to room temperature. The metal oxide
precipitates were centrifuged at 250xg for 10 minutes. The clear
supernatant was decanted and the pellet was resuspended in 900 ml of
deionized water using mechanical stirrer. This cleaning process was
repeated six times or until the supernatant was almost neutral in
pH. The supernatant was decanted and resuspended in 200 ml of
deionized water. Further centrifugation at 250xg will not pellet
all of the metal oxide precipitates. The supernatant which
contained smaller size metal oxide crystals was collected and the
pellet was resuspended in 200 ml of deionized water. This process
was repeated for at least three times or until most of metal oxide
can no longer be pelleted at 250xg. The metal oxide obtained this
way usually has size less than 2.0 micron. The combined metal oxide
suspension was centrifuged at 100xg for 10 minutes. The supernatant
was collected to give 800 ml of 8.6 ~ w/v magnetic metal oxide
suspension having the size less than 0.8 microns.
Example 2
Same procedures as described in Example 1 were followed except
0.235 mol of ferrous sulfate, 0.297 mol of ferric sulfate
(Fe /Fe ratio = 0.79) in 400 ml of deionized water and 480
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- 15 -
ml of 6 N sodium hydroxide were used to give 2000 ml of 2.86 % w/v
suspension of magnetic metal oxide.
Example 3
Same procedures as described in Example 1 were followed except
0.178 mol of ferrous sulfate, 0.298 mol of ferric sulfate
(Fe /Fe ratio = 0.59) in 400 ml of deionized water and 520
ml of 6 N sodium hydroxide were used to give 1500 ml of 2.98 % w/v
suspension of magnetic metal oxide.
Example 4
Same procedures as described in Example 1 were followed except
0.15 mol of ferrous sulfate, 0.276 mol of ferric sulfate
(Fe /Fe ratio = 0.54) in 400 ml of deionized water and 520
ml of 6 N sodium hydroxide were used to give 700 ml of 6.88 % w/v
suspension of magnetic metal oxide.
Example 5
Same procedures as described in Example 1 were followed except
0.116 mol of manganese sulfate, 0.146 mol of ferric sulfate
(Mn /Fe ratio = 0.79) in 225 ml of deionized water and 240
ml of 6 N sodium hydroxide were used to give 1700 ml of 1.8 Z w/v
suspension of magnetic metal oxide.
Preparation of Magnetic Particles
Example 6
A mixture containing 600 ml of deionized water, 6 ml of styrene
and 80 ml of 8.6 ~ w/v magnetic metal oxide prepared as described in
Example 1, was placed in a sealed bottle. The bottle was evacuated
and rotated at about 60 rpm in a 55 ~C oven for one hour. To the
mixture were added 12 9 of potassium persulfate and 850 ml of 5 %
w/v, 4.0 micron polystrene particles. The bottle was resealed,
evacuated and rotated for one hour and added 50 ml of 2 Z sodium
dodecylsulfate. After five more hours 6 ml of styrene and 10 9 of
potassium persulfate were added to the mixture. The mixture was
rotated for another fifteen hours, filtered through two layers of
cheese cloth, separated magnetically and washed several times with
deionized water until the supernatant was clear. The resulting
- 16 - 1339~ .5
magnetic particles were resuspended to 1.6 l with deionized water to
give a 2.5 % w/v suspension with about 11 % magnetic metal oxide
content and 4.3 micron average size.
Example 7
The magnetic particles, 1.6 l of 2.5 % w/v, prepared as
described in Example 6, were carboxylated by adding 1 9 of sodium
dodecylsulfate, 10 9 of potassium persulfate and a solution
containing 0.98 ml of undecylenic acid and 0.02 ml of divinyl
banzene in 4 ml of methanol. The mixture was placed in a sealed
bottle, evacuated and rotated at about 60 rpm in a 55 ~C oven for 5
hours. The resulting carboxyl magnetic particles were separated
magnetically and washed several times with deionized water until the
supernatant was clear. The carboxyl magnetic particles were
resuspended to 680 ml with deionized water to give a 5.8 Z w/v
suspension with about 11 % magnetic metal oxide content and 4.3
micron average size.
Example 8
A mixture containing 600 ml of deionized water, 6 ml of styrene
and 80 ml of 8.6 % w/v magnetic metal oxide prepared as described in
Exmaple 1, was placed in a sealed bottle. The bottle was evacuated
and rotated at about 60 rpm in a 55 ~C oven for one hour. To the
mixture were added 12 9 of potassium persulfate and 850 ml of 4.78 %
w/v, 6.1 micron polystrene particles. The bottle was resealed,
evacuated, rotated for five hours and added 6 ml of styrene and lO g
of potassium persulfate. The mixture was rotated for another
fifteen hours, filtered through two layers of cheese cloth,
separated magnetically and washed several times with deionized water
until the supernatant was clear. The resulting magnetic particles
were resuspended to 1.5 l with deionized water and carboxylated by
adding l 9 of sodium dodecylsulfate, 10 9 of potassium persulfate
and a solution containing 0.98 ml of undecylenic acid and 0.02 ml of
divinyl benzene in 4 ml of methanol. The mixture was placed in a
sealed bottle, evacuated and rotated at about 60 rpm in a 55 ~C oven
for 5 hours. The resulting carboxyl magnetic particles were
separated magnetically and washed several times with deionized water
- 17 13 39 5~ ~
until the supernatant was clear. The carboxyl magnetic particles
were resuspended to 800 ml with deionized water to give a 4.3 qO
suspension with about 11.6 % magnetic metal oxide content and 6.8
micron average size.
Example 9
A mixture containing 600 ml of deionized water, 6 ml of styrene
and 60 ml of 8.6 % w/v magnetic metal oxide prepared as described in
Example 1, was placed in a three-necked round bottom flask and
stirred at 67 ~C for one hour under argon. To the mixture were
added 12 9 of potassium persulfate and 470 ml of 5 % w/v, 2.7 micron
polystrene particles. The mixture was stirred at 67 ~C for one hour
and added 30 ml of 2 % sodium dodecylsulfate. After stirring at 67
~C under argon for five more hours 6 ml of styrene and 6 9 of
potassium persulfate were added to the mixture. The mixture was
stirred at 67 ~C under argon for another fifteen hours, filtered
through two layers of cheese cloth, separated magnetically and
washed several times with deionized water until the supernatant was
clear. The resulting magnetic particles were resuspended to 900 ml
with deionized water and carboxylated by adding 0.6 9 of sodium
dodecylsulfate, 10 9 of potassium persulfate and a solution
containing 0.598 ml of undecylenic acid and 0.012 ml of divinyl
benzene in 2.4 ml of methanol. The mixture was placed in a sealed
bottle, evacuated and rotated at about 60 rpm in a 55 ~C oven for 5
hours. The resulting carboxyl magnetic particles were separated
magnetically and washed several times with deionized water until the
supernatant was clear. The carboxyl magnetic particles were
resuspended to 500 ml to give a 6.5 % w/v suspension with about 14 %
magnetic metal oxide content and 4.0 micron average size.
Example 10
A mixture containing 600 ml of deionized water, 6 ml of styrene
and 60 ml of 8.6 % w/v magnetic metal oxide prepared as described in
Example 1, was placed in a sealed bottle. The bottle was evacuated
and rotated at about 60 rpm in a 55 ~C oven for one hour. To the
mixture were added 12 9 of potassium persulfate and 470 ml of 5 %
w/v, 2.7 micron polystyrene particles. The bottle was resealed,
1339545
- 18 -
evacuated and rotated for one hour and added 30 ml of 2 % sodium
dodecylsulfate. After five more hours 6 ml of styrene and 10 9 of
potassium persulfate were added to the mixture. The mixture was
rotated for another fifteen hours, filtered through two layers of
cheese cloth, separated magnetically and washed several times with
deionized water until the supernatant was clear. The resulting
magnetic particles were resuspended to 500 ml with deionized water
to give a 6.8 % w/v suspension with about 14 % magnetic metal oxide
content and 4.0 micron average size.
Example 11
A mixture containing 180 ml of deionized water, 2 ml of styrene
and 20 ml of 8.6 % w/v magnetic metal oxide, prepared as described
in Example 1, was placed in a sealed bottle. The bottle was
evacuated and rotated at about 60 rpm in a 55 ~C oven for one hour.
To the mixture were added 4 9 of potassium persulfate and 160 ml of
6.8 X w/v magnetic particles (3.0 micron, 14 % metal oxide content),
prepared as described in Example 10. The bottle was resealed,
evacuated and rotated for one hour and added 10 ml of 2 % sodium
dodecylsulfate. After 5 more hours 2 ml of styrene and 2 9 of
potassium persulfate were added to the mixture. The mixture was
rotated for another fifteen hours, filtered through two layers of
cheese cloth, separated magnetically and washed several times with
deionized water until the supernatant was clear. The resulting
magnetic particles were resuspended to 160 ml with deionized water
to give a 7.78 % w/v suspension with about 19 % metal oxide content
and 4.2 micron average size.
Example 12
A mixture containing 90 ml of deionized water, 1 ml of styrene
and 10 ml of 8.6 X w/v magnetic metal oxide, prepared as described
in Example 1, was placed in a sealed bottle. The bottle was
evacuated and rotated at about 60 rpm in a 55 ~C oven for one hour.
To the mixture were added 1 9 of potassium persulfate and 80 ml of
7.78 % w/v magnetic -particles (3.2 micron, 19 % metal oxide
content), prepared as described in Example 11. The bottle was
resealed, evacuated and rotated for four hour and added 5 ml of 2 %
1~39~
- 19.-
sodium dodecylsulfate. After 5 more hours l ml of styrene and 1 9of potassium persulfate were added to the mixture. The mixture was
rotated for another fifteen hours, filtered through two layers of
cheese cloth, separated magnetically and washed several times with
deionized water until the supernatant was clear. The resulting
magnetic particles were resuspended to 160 ml with deionized water
to give a 4.5 Z w/v suspension with about 23 % metal oxide content
and 4.5 micron average size.
Example 13
A mixture containing 400 ml of deionized water, 1.92 ml of
styrene, 0.08 ml of divinyl benzene, 4 9 of potassium persulfate, 20
g of 200 - 400 mesh 4 % divinyl benzene cross linked polystyrene
beads and 10 ml of 8.6 % w/v magnetic metal oxide, preapred as
described in Example 1, was placed in a sealed bottle. The bottle
was evacuated and rotated at about 60 rpm in a 55 ~C oven for 15
hours. The mixture was allowed to settle and the supernatant was
decanted. The resulting magnetic beads were resuspended in 200 ml
of deionized water and allowed to settle again. This process was
repeated several times until the supernatant was clear. The
resulting magnetic beads were resuspended in 200 ml of deionized
water and added 0.1 9 of sodium dodecyl sulfate, 2.0 9 of potassium
persulfate, 0.48 ml of styrene, and 0.02 ml of divinyl benzene. The
bottle was resealed, evacuated and rotated at about 60 rpm in a 55
~C over for one hour and added a solution containing 0.098 ml of
undecylenic acid and 0.002 ml of divinyl benzene in 0.4 ml of
methanol. The mixture was rotated for four more hours and cleaned
up by gravitational sedimentation as described previously. The
water was removed by filtration and the carboxyl magnetic beads were
dried to give 20 9 of 200 - 400 mesh carboxyl magnetic beads.
Example 14
A mixture containing 100 ml of deionized water, 0.5 ml of
styrene, 2 9 of potassium persulfate, 75 ml of 5 % w/v 4.0 micron
polystrene particles and 10 ml of 6.88 % w/v magnetic metal oxide,
prepared as descrived in Example 4, was placed in a sealed bottle.
The bottle was evacuated and rotated at about 60 rpm in a 55 ~C oven
1339~5
- 20 -
for fifteen hours. The mixture was filtered through two layers of
cheese cloth, separated magnetically and washed several times with
deionized water until the supernatant was clear. The resulting
magnetic particles were resuspended to 150 ml with deionized to give
a 2.5 Z w/v suspension with about 14 % metal oxide content and 4.3
micro~ average size.
Example 15
Same procedures as described in Example 14 were fol1Owed except
20 ml of 6.88 % w/v magnetic metal oxide, prepared as described in
Example 4, was used to give 160 ml of 2.5 % w/v suspension with
about 18 % metal oxide content and 4.3 micron average size.
Example 16
A mixture containing 2000 ml of deionized water, 13 ml of
styrene and 550 ml of 2.98 % w/v magnetic metal oxide prepared as
described in Example 3, was placed in a sealed bottle. The bottle
was evacuated and rotated at about 60 rpm in a 55 ~C oven for one
hour. To the mixture were added 20 9 of potassium persulfate and
950 ml of 10 % w/v, 3.0 micron polystyrene particles. The bottle
was resealed, evacuated and rotated for one hour and added 60 ml of
2 % sodium dodecylsulfate. After five more hours 8 ml of styrene
and 10 9 of potassium persulfate were added to the mixture. The
mixture was rotated for another fifteen hours, filtered through two
layers of cheese cloth, separated magnetically and washed several
times with deionized water until the supernatant was clear. The
resulting magnetic particles were resuspended to 3000 ml with
deionized water to give a 3.38% w/v suspension with about 12 %
magnetic metal oxide content and 3.Z micron average size.
Example 17
A mixture containing 150 ml of magnetic particles (3.2 micron,
3.38 % w/v with 12 % metal oxide content) prepared as described in
Example 16, 2 ml of 1 % NP 40, 0.5 ml of methyl methacrylate or
styrene, 1 9 of potassium persulfate and 2 ml of functionalized
monomer, trimethylammoniumethyl methacrylate methosulfate (40 %
aqueous solution), was placed in a sealed bottle. The bottle was
rotated at about 60 rpm in a 55 ~C oven for four hours. The mixture
- 21 - 13~954~
was filtered through two layers of cheese cloth, separated
magnetically and washed several times with deionized water until the
supernatant was clear. The resulting magnetic particles were
resuspended to 200 ml with deionized water to give a 2.5 % w/v
suspension of magnetic particles with trimethylammonium functional
groups on the surface.
Example 18
Same procedures as described in Example 17 were followed except
1 ml of functionalized monomer, 2-aminoethyl methacrylate, was used
to give 200 ml of 2.5 % w/v suspension of magnetic particles with
amino groups on the surface.
Example 19
Same procedures as described in Example 17 were followed except
1 ml of functionalized monomer, 2-hydroxyethyl methacrylate, was
used to give 200 ml of 2.5 % w/v suspension of magnetic particles
with hydroxyl groups on the surface.
Example 20
Same procedures as described in Example 17 were followed except
1 ml of monomer, 1-vinyl-2-pyrrolidinone, was used to give 200 ml of
2.5 % w/v suspension of magnetic particles with
polyvinylpyrrolidinone on the surface.
Example 21
Same procedures as described in Example 17 were followed except
1 9 of functionalized monomer, methyl propene sulfonic acid, was
used to give 200 ml of 2.5 % w/v suspension of magnetic particles
with sulfonic acids groups on the surface.
Example 22
Same procedures as described in Example 17 were followed except
1 ml of functionalized monomer, dimethylaminoethyl methacrylate, was
used to give 200 ml of 2.5 % w/v suspension of magnetic particles
with dimethylamino groups on the surface.
Example 23
A mixture containing 20 ml of 7.0 Z w/v, 2.11 micron polystyrene
particles, 100 ml of 1.8 % w/v metal oxide prepared as described in
Example 5, 50 ml of deionized water and a solution containg 0.15 9
13395~.~
- 22 -
of benzoyl peroxide in 7.5 ml of styrene was placed in a sealed
bottle. The bottle was evacuated and rotated at about 60 rpm in a
55 ~C oven for fifteen hours. The mixture was filtered through two
layers of cheese cloth, separated magnetically and washed several
times with deionized water until the supernatant was clear. The
resulting magnetic particles were resuspended to 200 ml with
deionized water to give 5.0 % w/v suspension with about 16.8 % metal
oxide content and 3.6 micron average size.
Examp1e 24
A mixture containing 20 ml of 7.0 % w/v, 2.11 micron polystyrene
particles, 100 ml of 1.8 % w/v metal oxide prepared as described in
Example 5, 50 ml of deionized water and a solution containing 0.15 9
of benzoyl peroixde and 0.75 ml of divinyl benzene in 6.75 ml of
styrene was placed in a sealed bottle. The bottle was evacuated and
rotated at about 60 rpm in a 55 ~C oven for fifteen hours. The
mixture was filtered throught wo layers of cheese cloth, separated
magnetically and washed several times with deionized water until the
supernatant was clear. The resulting crosslinked magnetic particles
were resuspended to 200 ml with deionized water to give 5.0 % w/v
suspension with about 16.8 % metal oxide content and 3.6 micron
average size. The crosslinked magnetic particles prepared this way
were found to be uniform in size and inert to common organic
solvents such as acetone, acetonitrile and dimethyl fonmamide.
Example 25
A mixture containing 20 ml of 7.0 % w/v, 2.11 micron polystyrene
particles, 150 ml of 1.8 % w/v metal oxide prepared as described in
Example 5 and a solution containing 0.15 9 of benzoyl peroxide, 0.75
ml of divinyl benzene in 6.75 ml of styrene was placed in a sealed
bottle. The bottle was evacuated and rotated at about 60 rpm in a
55 ~C oven for fifteen hours. The mixture was filtered through two
layers of cheese cloth, separated magnetically and washed several
times with deionized water until the supernatant was clear. The
resulting crosslinked magnetic particles were resuspended to 200 ml
with deionized water to give 5.4 % w/v suspension with about 23
metal oxide content and 4.0 micron average size. The crosslinked
- 23 - 13 39 5 4 ~
magnetic particles prepared this way were found to be uniform in
size and inert to common organic solvents such as acetone,
actonitrile and dimethyl formamide.
Example 26
A mixture containing 15 ml of 9.16 % w/v, 3.2 micron polystyrene
particles, 100 ml of 1.8 % w/v metal oxide prepared as described in
Example 5, 55 ml of deionized water and a solution containing 0.15 9
of benzoyl peroxide and 0.75 ml of divinyl benzene in 6.75 ml of
styrene was placed in a sealed bottle. The bottle was evacuated and
rotated at about 60 rpm in a 55 ~C oven for fifteen hours. The
mixture was filtered through two layers of cheese cloth, separated
magnetically and washed several times with deionized water until the
supernatant was clear. The resulting crosslinked magnetic particles
were resuspended to 200 ml with deionized water to give 4.7 % w/v
suspension with about 16.8 % metal oxide content and 5.5 micron
average size., The crosslinked magnetic particles prepared this way
were found to be uniform in size and inert to common organic
solvents such as acetone, actonitrile and dimethyl formamide.
Example 27
A mixture containg 30 ml of 4.5 % w/v, 4.1 micron polystyrene
particles, 100 ml of 1.8 % w/v metal oxide prepared as described in
Example 5, 40 ml of deionized water and a solution containing 0.15 9
of benzoyl peroxide and 0.75 ml of divinyl benzene in 6.75 ml of
styrene was placed in a sealed bottle. The bottle was evacuated and
rotated at about 60 rpm in a 55 ~C oven for fifteen hours. The
mixture was filtered through two layers of cheese cloth, separated
magnetically and washed several times with deionized water until the
supernatant was clear. The resulting crosslinked magnetic particles
were resuspended to 200 ml with deionized water to give 4.5 % w/v
suspension with about 16.9 % metal oxide content and 6.7 micron
average size. The crosslinked magnetic particles prepared this way
were found to be unform in size and inert to common organic solvents
such as acetone, acetonitrile and dimethyl formamide.
13395~
- 24 -
Example 28
~ mixture containing 20 ml of 7.0 % w/v, 2.11 micron polystyrene
particles, 100 ml of 1.8 % w/v metal oxide prepared as described in
Example 5, 50 ml of deionized water and a solution containing 0.15 9
of benzoyl peroixde, 0.75 ml of undecylenyl alcohol and 0.75 ml of
divinyl benzene in 6 ml of styrene was placed in a sealed bottle.
The bottle was evacuated and rotated at about 60 rpm in a 55 ~C oven
for fifteen hours. The mixture was filtered through two layers of
cheese cloth, separated magnetically and washed several times with
deionized water until the supernatant was clear. The resulting
crosslinked hydroxyl magnetic particles were filtered and dried to
give 9 9 of powder with about 16.8 % metal oxide content and 3.9
micron average size. The crosslinked hydroxyl magnetic particles
prepared this way were found to be uniform in size and inert to
common organic solvents such as acetone, acetonitrile and dimethyl
formamide.
Coupling Biological Materials to ~agnetic Particle
Example 29
In a 80 ml bottle was place 30 ml of 4.3 micron, 5.0 % w/v
carboxyl magnetic particles prepared as described in Example 7. The
particles were separated magnetically and resuspended in 50 ml of
phosphate buffer (0.1 M. pH 5.5). To the particle suspension were
added 20 mg of bovine serum albumin and 100 mg of
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC). The mixture
was rotated end to end at room temperature for two hours and
separated magnetically. The particles were washed once with 80 ml
of phosphate buffer and resuspended to 75 ml with phosphate buffered
saline (0.1 M, pH 7.0) to give a 2.0 X w/v suspension.
To couple bovine serum albumin to magnetic particles by passive
adsorption the same procedures were followed except no EDC was used.
Example 30
In a 4 ml vial was placed 1 ml of 4.3 micron, 5.0 X w/v carboxyl
magnetic particles prepared as described in Example 7. The
particles were separated magnetically and washed once with 2 ml of
1~39~45
- 25 -
phosphate buffer (0.1 M, pH 5.5) and resuspended to 2 ml with the
same buffer. To the particles suspension were added 140 ml of 1.4
mg/ml Goat (Gt) anti Mouse (Ms) IgG and 10 mg of 1-ethyl-3
-(3-dimenthylaminopropyl) carbodiimide. The vial was rotated end to
end at room temperature for two hours. The particles were separated
magnetically, washed once with 2 ml of phosphate buffer and
resuspended to 2 ml with phosphate buffered saline (0.1 M, pH 7.0)
to give a 2.5 X w/v Gt anti Ms Ig~ coated magnetic particles. Other
kind of antibody either monoclonal or polyclonal could also be
coupled to carboxyl magnetic particles by using the same procedures.
To couple Gt anti Ms IgG or other kind of antibody to the
magnetic particles by passive adsorption the same procedures were
followed except no EDC was used.
Example 31
In a 4 ml vial was placed a 2.5 ml of bovine serum albumin
coated magnetic particles (4.3 micron, 2 % w/v) prepared as
described in Example 29. The particles were separated magnetically
and resuspended to 2 ml with phosphate buffer (0.1 M, pH 5.5). To
the mixture were added 10 ul of Ms anti B red cells surface antigen
(20 mg/ml) and 1 mg of 1-ethyl-3-(3-dimethylaminopropyl)
-carbodiimide. The mixture was rotated end to end at room
temperature for two hours. The particles were separated
magnetically, washed once with phosphate buffer and resuspended in 2
ml of phosphate buffered saline (0.1 M, pH 7.0) to give a 2.5 % w/v
suspension.
Example 32
Same procedures as described in Example 31 were followed except
using 40 ul of Ms anti A red cells surface antigen (5 mg/ml) to give
2 ml of 2.5 X w/v suspension.
Blood Typing Using Magnetic Particles
Example 33
In a 5mm x 65rm test tube labeled A was placed 25 ul of 2.5 7,
w/v Ms anti A coated magnetic particles prepared as described in
Example 32. To the other test tube labeled B was placed 25 ul of
133954~
- 26 -
2.5 7, wtv Ms anti B coated magnetic particles prepared as described
in Example 31. To both test tubes was added 50 ul of 1 ~ packed red
blood cells prepared by 1 to 100 dilution of packed red blood cells
in isotonic buffered saline. The test tubes were shaked by finger
tapping for several times and placed on the top of a magnet. The
results were summarized as follows:
BLOOD TYPE
A B O AB
TUBE A + - - +
TUBE B - + - +
Where + represent a positive reaction, meaning the red cells
were agglutinated by the corresponding antibody coated magnetic
particles as a result the supernatant in the test tube was clear
after magnetic separation. On the other hand the supernatant of a
negative reaction would remain cloudy after magnetic separation due
to the absence of agglutination between the red cells and the
antibody coated magnetic particles.
Immunoassays llsing ~lagnetic Particles
Example 34
In a 2 ml microcentrifuge tube was placed 1 ml of 6 % w/v, 3
micron carboxyl magnetic particles. The particles were centrifuged
for 3 minutes at 10000 rpm. The supernatant was aspirated and the
particles were resuspended by vortexing with 1 ml of 5 to 100 ug/ml
recombinant HBcAg in acetate buffer. The tube was rotated at toom
temperature for two hours and centrifuged as described before. The
supernatant was aspirated and the particles were resuspended in 1 ml
of overcoat solution containing acetate buffer and 2 to 10 % of
normal animal serum. The tube was rotated at room temperature for 2
to 16 hours and centrifuged as described before. The supernant was
aspirated and the particles were washed three times with 1 ml of
isotonic buffered slaine (IBS) by centrifugation and resuspension.
Finally, the particles were resuspended with 1 ml of IBS and stored
at 2 to 8~C.
1339~4~
- 27 -
Example 35
To the first two columns of a 96-well microtiter plate was
placed 20 ul of 0.25~ w/v heptitis B core antigen (HBcAg) coated
magnetic particles prepared as described in Example 34. Sample
preparation consisted of various dilutions of a HBcAb positive serum
into a negative plasma, followed by a 1:100 dilution of each sample
into specimen dilution buffer (SDB). The SDB contained phosphate
buffer, protein stabilizers, detergentand antimicrobial agents. To
the wells containing the particles were added 50 ul of each final
sample dilution. After 30 minutes incubation at 37~C, the particles
were separated for two minutes on a magnetic separator and washed
three times with 200 ul wash buffer containing salts and detergent.
To each well containing the particles was added 50 ul of goat
antihuman IgG-B-D-galactosidase conjugate (0.5 ug/ml) in diluent
containing salts, protein stabilizers, glycerol, detergent and
antimicrobial agents. After 15 minutes incubation at 37~C the
particles were separated and washed three times as described above
and resuspended in 30 ul of IBS. The particles were transferred to
the first two columns of a black microtiter plate (Dynatech). To
each well containing the particles was added 100 ul of a solution
containing 4-methylumbelliferyl-B-galactopyranoside (~lUG, Sigma).
The plate was incubated at 37~C and the fluorescence intensity was
measured by using a Fluorescence Concentration Analyzer (FCA,
Pandex) equipped with 365 nm excitation and 450 nm emmision filters
at five minutes interval and 10 X gain setting. The increase in
fluorescence intentsity in a five minutes interval was recorded in
arbitrary fluorescence unit (AFU) and presented in Table I.
13 3954 ~-
- 28 -
TABLE I
Dilution of Positive AFU (5 Minutes)
Specimen Average of Two Wells
1:100 22687
1:1000 5933
1:5000 1516
1:8000 835
1:10000 639
1:15000 495
1:20000 427
1:25000 307
Example 36
The coupling of mouse antiHBsAg to carboxyl magnetic particles
was similar to Example 30.
To the wells of a black 96-well microtiter plate (Dynatech) were
added 20 ul of 0.25% w/v, 3.2 micron, mouse antiHBsAg coated
carboxyl magnetic particles in duplicate. To the wells containing
the magnetic particles was added 100 ul of neat plasma containing
various amounts of HBsAg or a HBsAg-negative plasma. After 30
minutes incubation at 37~C, the particles were separated for two
minutes on a magnetic separator and washed once with 100 ul of wash
buffer containing salts and detergent. To each will containing the
particles was added 20 ul of mouse antiHBsAg--B-galactosidase
conjugate in diluent containing salts, protein stabilizers,
glycerol, detergent and antimicrobial agents. After 15 minutes
incubation at 37~C, the particles were separated and washed five
times as described above. To each will containing the particles was
aaded 50 ul of a solution containing 4-methylumbelliferyl-B-D
-galactopyranoside (MUG, Sigma). The plate was incubated at 37~C
and the fluorescence intentisty was measured by using a Fluorescence
Concentration Analyzer (FCA, Pandex) equipped with 365 nm excitation
13395~.~
- 29 -
and 450 nm emission filters ar five minutes interval and 10 X galn
setting. The increase in fluorescence intentsity in a five minutes
interval was recorded in arbitrary fluorescence unit (AFU) and
presented in Table II.
TABLE II
HBsAg Conc. AFU (5 Minutes)
(nano gm) Average of Two Wells
1.0 1149
0.5 455
0.25 218
0.125 118
neg. 14
Example 37
The HIV-1 antigens from HTLV-IIIB/H-9 cells (Gallo Strain) were
coupled to 3.6 micron carboxyl magnetic particles by using similar
procedures as described in Example 34.
To the wells of a 96-well microtiter plate were added 20 ul of
0.25g w/v of HIV coated magnetic particles in duplicate. To the
wells containing the particles were added 50 ul of positive,
borderline and negative specimens diluted 1:100 in specimen dilution
buffer (SDB) containing phosphate buffer, protein stabilizers,
detergent and antimicrobial agents. After 30 minutes incubation at
37~C, the particles were separated for two minutes on a magnetic
separator and washed three times with 100 ul of washed buffer
containing salts and detergent. To each well containing particles
was added 50 ul of goat antihuman-B-galactosidase (approximately 0.5
ug/ml) conjugate in diluent containing salts, protein stabilizers,
glycerol, detergent and antimicrobial agents. After 15 minutes
incubation at 37~C, the particles were washed four times as
described above. The particles were transferred to the black
microtiter plate (Dynatech). To each well containing particles was
13395~
- 30 -
added 100 ul of a solution containing 4-methylumbelliferyl-B-D
-galactopyranoside (MUG, Sigma). The plate was incubated at 37~C
and the fluorescence intensity was measured by using a Fluorescence
Concentration Analyzer (FCA, Pandex) equipped with 365 nm excitation
and 450 nm emission filters at five minutes intervals and 25 X gain
setting. The increase in fluorescence intensity in a five minutes
interval was recorded in arbitrary fluorescence unit (AFU) and
presented in Table III.
TABLE III
Anti-HIV AFU (5 minutes)
Specimens Average of Two Wells
Positive Control 9462
Borderline Specimen 527
Negative Control 86
Cell Separation Using Magnetic Particles
Example 38
The 4.3 micron carboxyl magnetic particles prepared as described
in Example 7 were washed and sonicated in phosphate buffered saline
(PBS, pH 7.7), sterilized in 70% ethanol for 10 minutes, washed
three times in PBS and incubated for 48 hours at 4~C with
affinity-purified sheep anti-mouse immunoglobulin antibody (SAM) at
0.5 mg/ml and a ratio of 3.3 mg antibody/lOOmg particles. Before
use, the antibody coated magnetic particles were washed in PBS and
resuspend at the desired concentration in PBS.
Human tissue culture cALLa-positive NAL~-16 leukemia cells were
washed and suspended in PBS. One fraction was not treated with
antibody (-Mo~b). The other fraction was treated with two anti-CD10
and one anti-CD9 monoclonal antibodies (+MoAb) for 30 minutes at
4~C, washed in PBS and adjusted to 3.5 x 106 cells/ml on PBS. To
two tubes, one containing the antibody treated cells (+MoAb), the
other containing untreated cells (-~loAb) were added SA~ coated
magnetic particles at a particle to starting cell ratio of 45. The
133~515
- 31 -
tubes were rotated at 4~C for 30 ~inutes. The particles were
separated with a magnetic separator. The supernatant was collectéd
and centrifuged to collect the remaining cells. The pellet was
resuspended in 100 ul of trypan blue and total cell count was made.
The results were presented in Table IV.
TABLE IV
Particle/cell Cells Cells %
Ratio +/- MoAb Received Depletion
0 + 7.62x105 0 (Control)
+ 2.89x104 96.2
- 7.33x105 4.6
3n
