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Patent 1266769 Summary

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(12) Patent: (11) CA 1266769
(21) Application Number: 595288
(54) English Title: MAGNETIC PARTICLES FOR USE IN SEPARATIONS
(54) French Title: PARTICULES AIMANTEES POUR EMPLOI DANS LES SEPARATIONS
Status: Deemed expired
Bibliographic Data
(52) Canadian Patent Classification (CPC):
  • 31/121
(51) International Patent Classification (IPC):
  • H01F 1/00 (2006.01)
  • B01D 15/08 (2006.01)
  • B01J 20/32 (2006.01)
  • B03C 1/01 (2006.01)
  • C12Q 1/42 (2006.01)
  • C12Q 1/54 (2006.01)
  • G01N 33/543 (2006.01)
  • C12Q 1/68 (2006.01)
(72) Inventors :
  • CHAGNON, MARK S. (United States of America)
  • GROMAN, ERNEST V. (United States of America)
  • JOSEPHSON, LEE (United States of America)
  • WHITEHEAD, ROY A. (United States of America)
(73) Owners :
  • CHIRON DIAGNOSTICS CORPORATION (United States of America)
(71) Applicants :
(74) Agent: OSLER, HOSKIN & HARCOURT LLP
(74) Associate agent:
(45) Issued: 1990-03-20
(22) Filed Date: 1984-04-30
Availability of licence: Yes
(25) Language of filing: English

Patent Cooperation Treaty (PCT): No

(30) Application Priority Data:
Application No. Country/Territory Date
493,991 United States of America 1983-05-12

Abstracts

English Abstract




ABSTRACT
A process is provided for the preparation of
magnetic particles to which a wide variety of molecules may
be coupled. The magnetic particles can be dispersed in
aqueous media without rapid settling and conveniently
reclaimed from media with a magnetic field. Preferred
particles do not become magnetic after application of a
magnetic field and can be redispersed and reused. The
magnetic particles are useful in biological systems involving
separations.


Claims

Note: Claims are shown in the official language in which they were submitted.



-56-
The embodiments of the invention in which an exclu-
sive property or privilege is claimed are defined as follows:

1. A magnetically-responsive particle comprising a
magnetic metal oxide core generally surrounded by a silane
coat to which molecules can be covalently coupled, a mass of
the particles being dispersible in aqueous media to form an
aqueous dispersion having
(a) a fifty-percent-turbidity-decrease settling
time of greater than about 1.5 hours in the
absence of a magnetic field; and
(b) a ninety-five-percent-turbidity-decrease sepa-
ration time of less than about 10 minutes in
the presence of a magnetic field;
the magnetic field being applied to the aqueous dispersion by
bringing a vessel containing a volume of the dispersion into
contact with a pole face of a permanent magnet, the permanent
magnet having a volume which is less than the volume of the
aqueous dispersion in the vessel.

2. The magnetically-responsive particle of claim 1
wherein the metal oxide core includes a group of superpara-
magnetic crystals.

3. The magnetically-responsive particle of claim 2
wherein the superparamagnetic crystals are comprised of iron
oxide including divalent and trivalent iron cations.

4. The magnetically-responsive particle of claim 1
wherein the silane coat generally surrounding the core com-
prises a bifunctional silane polymeric material bearing a
first set of functionalities capable of adsorptively or
covalently binding to the metal oxide core and a second set
of functionalities capable of covalently coupling to organic
molecules.



-57-
5. The magnetically-responsive particle of claim 4
wherein the silane polymeric material bear organofunctional-
ities selected from the group consisting of aminophenyl,
amino, carboxylic acid, hydroxyl, sulfhydryl, phenolic, ali-
phatic, hydrophobic and amphipathic moieties.

6. The magnetically-responsive particle of claim 4
wherein the silane polymeric material is formed from silane
monomers selected from the group consisting of p-aminophen-
yltrimethoxysilane, 3-aminopropyltrimethoxysilane, N-2-amino-
ethyl-3-aminopropyltrimethoxysilane, n-dodecyltrie-thoxysilane
and n-hexyltrimethoxysilane.

7. The magnetically-responsive particle of claim 4
wherein the mean diameter thereof as measured by light scat-
tering is between about 0.1 µ and about 1.5 µ.

8. The magnetically-responsive particle of claim 7
wherein the surface area thereof as measured by nitrogen gas
adsorption is at least about 100 m2/gm.

9. A magnetically-responsive particle comprising a
superparamagnetic iron oxide core generally surrounded by a
coat of polymeric silane to which molecules can be covalently
coupled, the iron oxide core including a group of crystals of
iron oxide, the particle having a mean diameter as measured
by light scattering between about 0.1 µ and about 1.5 µ and a
surface area as measured by nitrogen gas adsorption of at
least about 100 m2/gm, a mass of the particles being dispers-
ible in aqueous media to form an aqueous dispersion having
(a) a fifty-percent-turbidity-decrease settling
time of greater than about 1.5 hours in the
absence of a magnetic field; and
(b) a ninety-five-percent-turbidity-decrease sepa-
ration time of less than about 10 minutes in
the presence of a magnetic field;



-58-
the magnetic field being applied to the aqueous dispersion by
bringing a vessel containing a volume of the dispersion into
contact with a pole face of a permanent magnet, the permanent
magnet having a volume which is less than the volume of the
aqueous dispersion in the vessel.

10. A magnetically-responsive particle comprising a
ferromagnetic metal oxide core generally surrounded by a coat
of polymeric silane to which molecules can be covalently
coupled, the metal oxide core including a group of crystals
of metal oxide, the particle having a mean diameter as
measured by light scattering between about 0.1 µ and about
1.5 µ and a surface area as measured by nitrogen gas
adsorption of at least about 100 m2/gm, a mass of the
particles being dispersible in aqueous media to form an
aqueous dispersion having
(a) a fifty-percent-turbidity-decrease settling
time of greater than about 1.5 hours in the
absence of a magnetic field; and
(b) a ninety-five-percent-turbidity-decrease sepa-
ration time of less than about 10 minutes in
the presence of a magnetic field;
the magnetic field being applied to the aqueous dispersion by
bringing a vessel containing a volume of the dispersion into
contact with a pole face of a permanent magnet, the permanent
magnet having a volume which is less than the volume of the
aqueous dispersion in the vessel.

11. A process for preparing magnetically-responsive
particles with mean diameters between about 0.1 µ and about
1.5 µ as measured by light scattering, which process com-
prises:
(a) precipitating divalent and trivalent transition
metal salts in base;
(b) washing the precipitate to approximate neu-
trality;



-59-
(c) washing the precipitate in an electrolyte;
(d) resuspending the precipitate in a solution of
silane monomer capable of forming a polymeric
coat adsorptively or covalently bound to the
precipitate to which molecules can be
covalently coupled; and
(e) causing the silane polymeric coat to become
adsorptively or covalently bound to the pre-
cipitate.

12. A process for preparing superparamagnetic par-
ticles with mean diameters between about 0.1 µ and about
1.5 µ as measured by light scattering, which process com-
prises:
(a) precipitating divalent and trivalent iron
cations of divalent and trivalent iron salts in
base;
(b) washing the precipitate in water to approximate
neutrality;
(c) washing the precipitate in an electrolyte; and
(d) coating the washed precipitate with an adsorp-
tively or covalently bound silane polymer.

13. The process of claim 12 wherein the divalent
and trivalent iron salts are FeCl2 and FeCl3.

14. The process of claim 12 wherein the divalent
and trivalent iron cations are used in an Fe2+/Fe3+ ratio of
about 4/1 to about 1/2.

15. The process of claim 12 wherein the washings
are performed by redispersing the precipitate in water and
electrolyte and magnetically collecting the precipitate
between washings.

16. The process of claim 12 wherein the precipitate



-60-
is coated with the silane polymer by deposition of said
silane polymer from acidic aqueous solution.

17. The process of claim 12 wherein the precipitate
is coated with the silane polymer by deposition of said
silane polymer from acidic organic solution.

18. The process of claim 17 wherein the deposition
of silane polymer from acidic organic solution comprises:
(a) suspending the washed precipitate in an organic
solvent containing about 1% (V/V) water;
(b) adding an acidic solution of a silane monomer;
(c) homogenizing the precipitate at high speed;
(d) mixing the precipitate with a wetting agent,
which agent is miscible in the organic solvent
and water;
(e) heating to a temperature sufficient to
evaporate water and organic solvent; and
(f) washing the wetting agent from the precipitate.

19. The process of claim 18 wherein the organic
solvent is methanol and the wetting agent is glycerol.

20. The process of claim 18 wherein the solution of
silane monomer is made acidic with orthophosphorous acid or
glacial acetic acid.

21. The process of claim 12 wherein said silane
polymer is formed from a silane monomer selected from the
group consisting of p-aminophenyltrimethoxysilanel 3-amino-
propyltrimethoxysilane, n-dodecyltriethoxysilane and n-hexyl-
trimethoxysilane.

22. A process for preparing magnetically-responsive
particles comprising superparamagnetic iron oxide cores gen-
erally surrounded by a coat of polymeric silane to which



-61-
molecules can be covalently coupled, which particles have
mean diameters between about 0.1 µ and about 1.5 µ as
measured by light scattering, a mass of the particles being
dispersible in aqueous media to form an aqueous dispersion
having
(a) a fifty-percent-turbidity-decrease settling
time of greater than about 1.5 hours in the
absence of a magnetic field; and
(b) a ninety-five-percent-turbidity-decrease sepa-
ration time of less than about 10 minutes in
the presence of a magnetic field;
the magnetic field being applied to the aqueous dispersion by
bringing a vessel containing a volume of the dispersion into
contact with a pole face of a permanent magnet, the permanent
magnet having a volume which is less than the volume of the
aqueous dispersion in the vessel, which process comprises:
(a) precipitating FeCl2 and FeCl3 in an Fe2+/Fe3+
ratio of about 2/1 with sodium hydroxide;
(b) washing the precipitate in water to approximate
neutrality by redispersing and magnetically
separating the precipitate;
(c) washing the precipitate in a sodium chloride
solution by redispersing and magnetically
separating the precipitate;
(d) suspending the washed precipitate in methanol
containing about 1% (V/V) water;
(e) adding an acidic solution of a silane monomer
to the suspension of precipitate;
(f) homogenizing the precipitate at high speed;
(g) mixing the precipitate with glycerol;
(h) heating the precipitate and glycerol to a
temperature in the range from about 160° to
about 170°C; and
(i) washing glycerol from the precipitate.

23. The process of claim 22 wherein the silane


-62-
monomer is selected from the group consisting of p-aminophen-
yltrimethoxysilane, 3-aminopropyltrimethoxysilane, N-2-amino-
ethyl.-3-aminopropyltrimethoxysilane, n-dodecyltriethoxysilane
and n-hexyltrimethoxysilane.


Description

Note: Descriptions are shown in the official language in which they were submitted.


.

l. IELD OF T~E I ~ NTION

This invention relates to ~agnetically re~ponsive
parti~les and to their use in 6y~tem~ i.n which the
separation of certain molecules fr~m the ~urrounding
medium i necessary or desirable. ~ore! particularly, the
invention relat*s to ~ethods ~or the preparation of
~agnetically responsive particle~ comprising a metal oxine
core surrou~ded b~ a stable ~ilane coating to ~hich a wide
variety of organic ~nd/or biological ~olecules may be
: coupled. The particles (coupled or uncoupled) c~n be
disper~ed in aqueous media without r~pid gravitational
settling and conveniently reclaimed from the media with a
magnetic field. Preferably, the proce~s provided herein
yields particles that are superpar~m~gnetic; that is, they
do not become permanently magnetized after application of
a magnetic field. Thi~ property permi~s the particles ~o
be redispersed without magnetic aggregate formation.
~ence the particles may be reused or recycled. Stability
of the ~ilane coating and the covalent attacbent of
molecules thereto ~lso contribute to particle use an~
xeuse.
~ he magnetically respon~ive particles of this
invention may b~ coupled to ~iologi~al or organic
molecules with affinity ~or or the ~bility to adsorb or
which interact with certain other biological or organic
molecules. Particle~ ~o coupled may be used in a var iety
of in vitro ~r in vivo &y~tems involving separation ~teps
or the directed movement of coupled ~olecules to
par~icular site~, including, bue not limited to,
immunologic~l ass~ys, oth~r biological assays, bioche~ical
or enzy~atic reactions, ~ffinity chro~atogr~ph~c
purification~, cell ~orting and diagnostic and therapeutic
uses.
3~

~a

7~


2. BACKGROUND 0~ T~E INVENT~ON
2.1. MAG~FTIC SEPARA~IONS IN ~I~LOGICAL
STE~S-_ G~NE M L CDN5lD-~A5-0:-

The u~e of ~agneti~ separationl~ ~n biol~gicalsystems a~ ~n alternative to gravitational or centrifugal
~eparations ha~ be~n revie~ed lB.L~ ~irlschbein et al.~
Chemtech, March 19~2sl7~ 179 ~1982); ~. Pourfarzaneh, The
Ligand Quarterly 5tl~:41-47 ~1982); ~nd POJ. ~alling and
tO ~. Dunnill, En~yme Microb. ~echnol. 2s~ 10 (1980)~o
5everal advantayes of using magn~tically separable
particle~ as supports for biological molecule~ 6uch as
enzymes, antibodies and other bioaffinity adsorbent~ are
generally recognized. ~or in~t~nce, when ~agnetic
parSicles are u~ed as 601id phase 8upport& in imMobili~ed
enzyme systems Isee, e.~., P.J. Robinson e~ al., Biotech.
Bioeng.~ XV:60~ 6Q6 (1973)], the enzyme may be selectively
recovered from media, including media conta1ning ~u6pended
~olids, allowing recycling in enzyme re~ctor~. When used
as solid ~upports in immunoassays or other competitive
binding assays, magnetic particle~ permit homogene~us
reaction ~onditions twhich promote optimal binding
ki~etic~ and minimally alter analyt~ ads~rbent
equilibrium) and facilitate separation of ~ound from
unbound analyte, compared to centri~uga~ion. Centrifu~al
separations are ~im~ consuming, require expensive and
energ~ consumin~ equipment and po e radiological,
biolvgical and physical haz~rdc. ~agnetic separations, on
the other hand, are relatively ~apid ~nd e~sy, ~e~ui~ing
simple equipmentO Finally, the u~e of no~ porou~
~dssÆbent-coupled ~agnetic particle~ in af~-inity
chromatography ystems allows better ~ss ~ran~fe~ ~nd
result~ in les. fouling than in co~ventional ~f~inity
chro~atography systems.

-


Although the gener~l concept of ~agnetizing
molecules by coupling them to magne~ic particle6 has been
discussed and the potential ~dvantage~ of usin~ Quch
particles for biological purpose~ recogni~ed, the
practical development of magnetic separations ha~ been
hindered by several critical propertie~ of magnetic
particle~ developed thus ~ar.
; Large magnetic particles ~mear~ diameter in
~olution greater th~n 10 microns~)) can re pond to weak
g~ magnetic fie7ds ~nd magnetic field gradients; however,
they tend to ~ettle rapidly, limiting their u~efulne~s for
reactions requiring homogeneous condition~. Large
particles also have a more limited surf~ce area per weight
than smaller particles, so that less ~aterial can be
c~upled to them. Examples of large particles are those of
Robin~on et al. l~E~ which are 5~ 125 ~ in diameter,
those of Mosb~ch and Anderson tNature~ 270:25~ 261 (1977)1
,~ which are 6~ 140 ~ in diameter a~d th~se of Guesdon et al.
[~. Allergy Clin~ Immunol. 61~1)-2~ 27 ~1578)1 which are
5~ 160 ~ in diameter. Composite particles m~de by ~ersh
and Yaverbaum lU.S. Pat. No. 3,933,997~ compri~e
ferromagn~tic iron oxi~e ~Fe304) carrier particlesO
The iron oxiue carri~r particles were reported to have
diameters between 1.5 ~nd 10 ~. However, based on the
reported settling ra~e of 5 ~inu~es and coupling capacity
of only 12 mg of protein per gram of composite particles
~L.~. ~ersh ~nd S. Yaverbaum, Clin. Chim. Acta, 63:6~ 72
(1975)], the actual size of the compositQ particles in
olution is expected to be substanti~lly greater than 10 ~.
The ~ersh and Yaverbaum ferromagnetic carrier
par~icles o U.S. ~. No~ 3~933,997 are ~ilanized with
~ilane~ capable Df reacting wi~h anti-digoxin ~ntibodi~s
to chemically couple the antibodie~ to ~he c~rrier
particles. ~ariou~ ~ilane couplin~s are di6cu~ed in U.S.
,

-- -7- ~



Pat. No. 3,652,761. That the diameters of the composite
particles are probably greater than 10 ~ may be explained,
at least in part, by the method of silanization employed in
the Hersch and Yaverbaum patent. Procedures for
silanization known in the art generally differ from each
other in the media chosen for the polymerization of silane
and its deposition on reactive surfaces. Organic solvents
such as toluene [H.W. Weetall, in: Methods is Enzymology,
K. Mosbach (ed.), 44:134-148, 140 (1976~], methanol ~U.S.
pat. No. 3,933,997] and chloroform EU~S~ Pat. No. 3,652,761]
have been used. Silane depositions from aqueous alcohol and
a~ueous solution with acid [H.W. Weetall, in: Methods in
15 Enzymology, su~, p. 139 (1976)], methanol ~U.S. Pat. No.
3,933,997] and chloroform [U.S. Pat. No. 3,652,761] have
been used. Silane depositions from aqueous alcohol and
aqueous solution with acid [H.W. Weetall, in: Methods in
Enzymology, supra, p. 139 (1976)] have also been used. Each
20 of these silanization procedures employs air and/or oven
drying in a dehydration step. When applied to silanization
of magnetic carrier particles such dehydration methods allow
the silanized surfaces of the carrier particles to contact
each other, potentially resulting in interparticle bonding,
25 including, e.g., cross-linking between particles by siloxane
formation, van der Waals interactions or physical adhesion
between adjacent particles. This interparticle bonding
yields convalently or physically bonded aggregates of
silanized carrier particles of considerably larger diameter
30 than individual carrier particles. Such aggregates have low
surface area per unit weight and hence, a low capacity for
coupling with molecules such as antibodies, antigens or
enzymes. Such aggregates also have gravitational
settling times which are too short for many applications.
35 Small magnetic particles with a mean diameter in
solution less than about 0.03 ~ can be kept in solution
by thermal agitation and therefore do not spontaneously

~6~
-8-

~ettle. ~owev~r, the ~agnetlc field and ~agnetic field
gr~dient required to r*~ove such particle~ from solution
are ~o large as to reguire heavy and bulky ~agnets for
their gener~tion, which are inconvenient to use ~n benc~
~ top work. ~agnets capable of geneEating ~gnetic ~ield~
in exce~s of 5000 Oer~ted~ are typi~lly required to
~eparate ~agneti~ particlel3 c3f 1~!S6 than 0. 03 1~ in
diameter. An approximate quantitative relation~hip
between the net force ~) acting on a particle and the
magnetic field ifi given by the eguatiorl b~low (~irschbein
et al-J ~
F~(Xv-X~)VH(d~/dx),
where Xv and Xv are the volume susceptibilities of
tbe particle and the medium, respectiYely, V is the volume
of the particle, ~ i8 the applied ~agnetic ~ield and dH/ax
i~ the magnetic field gradient. Th$s expression is only
an approximAtion bec~use it ignores particle shape and
particle interaction~. Nevertheless~ it does i~dicate
that the force on a magnetic particl~ i~ directly
proportional to the volume o~ the particle.
Magnetic parti~les of less th~n 0.03 ~ are used
in s~ c~lled ferro~luids, which ~re described, for
example, in U.S. patent No. 3,531,413. Ferr~fluids have
numero~s applications, but ar~ impracti~al for
appli~ations requiring ~eparation of the magnetic
particles Prom ~urrounding ~edia beGau~e of the large
magnetic fields and ~agneti~ ~ield gradients re~uired to
effec~ the separ~tion
Ferromagne~ic ~ateri~l~ in ge~eral become
permanently ~agne~i2ed in repon~e to ~agnet~c field~.
Material~ termed ~cuperparamagne~ic~ experience a force in
a ma~netic field gr~dient, bu~ ~o n~ b~co~e per~anently
magnetiz~d. Cry tal~ o~ ~agnetic iron oxide~ ~ay be
either ~errvm3gnetic or auperparamagnetic, depending on

7~



the size o~ the crystals. Superpara~gn~tic oxides s~f
iron generally result when the cry~tal i~ le~ ~han about
300 A(0.03 Il) in diameter; larger cry~tal~ g~rlerally h~ve
a ferromagnetic character. Following in~tia~ exposur2 to
21 magnetic field, erromagnèti~ partic:lles tend to
aggr~gate be~ause of magnetic ~ttra~tion between the
per~anently magnetized particl~, a~ ha,s b~en noted by
Robin~on et ~1. 1.~1 and by E~er~h ~n~ Yaverb~um f~].
Di~persible magnetic irt~n oxi~e part$cle
repc>Etedly having 300 A diameters and ~urface amine groups
were prepared by base precipi t~tion of ;Eerrous chlc>ride
and ferric chlor~de ~Fe2 /Fe3 ~1) in the presence of
polyethylene iminet according to Rembaum in ~.S. Pat. No.
4,267,234. Reportedlyt these particles w~re expo ed to a
magnetic field three times during preparation and were
described as redispersible. The magnetiG particles were
mixed with a glutaraldehyde suspension polymerization
sy tem to form magnetic polyglut~raldehyde microspheres
with reported diamet~r~ of 0.1 ~. Polyglutaraldehyde
microspheres have coniugated aldehyde groups on the
sur~ace which can form bonds to ~min~ containing molecules
~uch as protei~. Howevert in ~neral, only compounas
which are capable of reacting with aldehyde groups can be
directly linked to the ~urface o~ polygluthraldehyde
microspheres. ~oreover, ~agne~ic polyglutarald~hyde
microspheres are not ~ufficie~tly ~table for ~ertain
applicati~ns.
f




2 . 2 0 SEPARATIONS IN RADIOI~MUNOASS~r S

Radioimmunoassay (PsIA) i~ a term used to describe
nleth~ds for ~nalyzing . he concen'crations o~ 8ub8tances
involving ~ radioacti~ely l~beled ~ubstance which binds ~o
35 an ~ntibody. The amount of radioactivity bound i altered

7~
- 1~

by the presence ~f an unl~beled test substance c~pable of
binding to the ~me Dntibody. The unlDbeled ~ubstanre, i
present, compete~ for binding si~es wi~h ~he l~bel~d
~ubstance ~nd thu~ decre~6e6 the amount o~ r~dioac~ivi~y
bound to t~e ~ntibody. ~be decrea64 ~n bou~d
radioactivity can be correlated to the ccncentrati~n of
the unlabeled test substance by mean~ of a standard
curve. An escenti~l ~tep of RIA i~ the ~eparat$on of
bound and free label which must be accompli6hed 1n order
to quantitate the bound ~r~tlon.
~ vari~ty o~ c~nventional ~eparation app~oaches
have been applied to radioimmunoa~says (RIA) including
coatsd tube~, p~rticulate 6y~te~6, and double ~ntibody
separation methods. Coated tubes, such as described in
U.S. Pat. No. 3,646~346~ allow sep~ration of bound and
free label without centrifugation but suffer from two
major disadvanta~es. Fir~t, the surface of the tube
limits the amount of ~ntibody that can be employed in the
reaction. Second the ~ntiboay i~ far remo~ed ~as much as
:2 0.5 cm) from some antigen~ ~lowing the reaction betwee~
the antibody and antigen lG.~9 Pars~n~d in: Method~ in
Enzymology, J. Langone (ed.) 73:225 ~1981): and P.N.
Nayak, The Ligand Quarterly 4t4):34 51981)~.
Antibodies h~ve been attached to particulate
systems to facilitate ~eparation [~ee, e.g., U.S. Pats.
;~os. 3,65~,761 and 3,555,143~. Such ~ystems have large
surface areas permitting nearly unlimited amounts of
antibody to be used, but the particulates frequently
3D settle during th~ ass~y. The tub~ requently mu~t be
agitated to w hieve even partial homogeneity ~P.~. Jacobs,
The Lig2nd Quarterly, 4~4):2~ 33 tl981)J. Centri~ugation
is ~till required to ef~ect complete separation o bound
and ~ree label.


&~
,.~ --11--



Antibodies may react with labeled and unlabeled
molecules followed by separation using a second antibody
raised to the first antibody ~Id.]. The technique, termed
the double antibody method, achieves homogenPity of antibody
during reaction with label but requires an incubation period
for reaction of first and second antibodies followed by a
centrifugation to pellet the antibodies.
Antibodies have been attached to magnetic supports
in an effort to eliminate the centrifugation steps in
radioimmunoassays for nortriptyline, methotrexate, digoxin,
thyroxine and human placental lactogen [R.S. Kamel et al.,
Clin. Chem., 25(1Z):1997-2002 (1979); R.S. Kamel and J.
Gardner, Clin~ Chim. Acta, 89:363-370 (1978); U.S. Pat. No.
3,933,997; C. Dawes and J. Gardner, Clin. Chim. Acta,
86:353-356 (1978); D.S. Ithakissios _ al., Clin. Chim.
Acta, 84:69-84 (1978); D.S. Ithakissios and D.O.
Kubiatowicz, Clin. Chem. 23~11):2072-2079 (1977); and
L. Nye et al., Clin. Chim Acta, 69:387-396 ~1976),
respectively]. Such methods su~fer from large particle
size (10-100 ~ in diameter) and require agitation to keep
the antibody dispersed during the assay. Since substantial
separation occurs from spontaneous settling in the absence
of a magnetic field these previous methods are in fact
only magnetically assisted gravimetric separations. The
problem of settling was addressed by Davies and Janata
whose approach in U.S. Pat. No. 4,177,253 was to employ
magnetic articles comprising low density cores of
30 materials such as hollow glass or polyproplyene (4-10 ~ in
diameter) with magnetic coatings (2 m~-10 ~ thick)
covering a proportion of the particle surface. Anti-
estradiol antibodies were coupled to such particles and
their potential usefulness in estradiol RIAs
36

-12- ~6~

W~5 demonstrated. While thi~ spproach may have overcome
the problem o~ ~ettling, the particle 6ize and tbe
magnetic co~ting nonethele~ present limit~tions on
~urface area and h~nce li~ata~ion~ on the availability of
~it~B for antibody couplinq.

2.3. APPLICATION O~ MAGNETIC SEPARATIONS
IN QT~ER BIOLOGICAL SYSTEMS
~ agnetic ~epar~ions hav~ been ~pplied in other
biological ~yctems beside~ RIA~ SeYeral noni~otop~c
im~uno~ssays, such as ~luoroi~munoassay~ (FIA) and
enzym~ immunoassay~ (EIA~ have been develop~d which employ
antibod~ coupled (or ~ntige~ couplffd) magnetic particles.
The principle of competitive binding i~ the ~ame in FIA
and EIA as in RIA except that fluorophores and enzymes,
respectively, are ~ubstituted for radioisot~pes as l~bel.
~y way of illu~tration, ~. Pourfarzaneh et al. 2nd R.S.
Kamel et al. developed magnetiz~ble 801i~ pha~e FIAs for
cortisol and phenytoin, r~pectively, utilizing
f~rr~magne~ic~cellulose/iron oxid~ particles to which
~nt~bodie~ were coupled by cyanogen bromide activ~tion l~-
Pourfarzaneh et al., Clin, Ch~m., 26(6):73~ 733 ~1980);
R.Sc Kamel et al., Clin. Che~., 26(9):1281-1284 (1980)~.
A no~ c~mpetiti~e ~olid pha~e ~andwich technique
EIA ~or the measurement of Ig~ was d~scribed by J.-L.
Guesdon e al~ ~. Allergy Clin. Immunol,, 61(1):2~ 27
~1978)~. By this method, anti~IgE antibodie~ coupl~d by
glutaraldehyde activ~tion to magnetic polya~rylamid~
agaro~e be~ds ~re incubated with a ~e~t ~ample containing
IgE to allow binding. Bound IgE i~ quantitate~ by adding
a s~c~nd ~nti-~gE ~ntibody labeled with eith~r alk~line
phosphatase or ~ galacto~id~e. ~he enzy~ l~beled
second ~ntibody ~o~plexe~ with IgE bound to the fir~t
an~ibody, forming ~he ~andwich, and the part$cle are

-13-


separated magnetically. Enzyme activity associated with the
particles, which is proportional to bound IgE is then
measured permitting IgE quantitation.
A magnetizable solid phase non-immune radioassay
for vitamin Bl2 has been reported by D~So Ithakissios and
D.O. Kubiatowicz [Clin. Chem. 23(11):2072-2079 (1977)]. The
principle of competitive binding in non--immune radioassays
is the same as in RIA with both assays employing
radioisotopic labels. However, while RIA is based on
antibody-antigen binding, non-immune radioassays are based
on the binding or interaction of certain biomolecules like
vitamin Bl2 with specific or non-specific binding, carrier,
or receptor proteins~ The magnetic particles of Ithakissios
and Kubiatowicz were composed of barium ferrite particles
embedded in a water-insoluble protein matxix.
In addition to their use in the solid phase
biological assays just described, magnetic particles have
been used for a variety of other biological purposes.
Magnetic particles have been used in cell sorting systems to
20 isolate select viruses, bacteria and other cells from mixed
populations [U.S. Pats. Nos. 3,970,518; 4,230,685; and
4~267~234]o They have been used in affinity chromatography
` systems to selectively isolate and purify molecules from
solution and are particularly advantageous for
25 purifications from colloidal suspensions ~K.N Mosbach and L.
Anderson, Nature 170:259-261 ~1977)]. Magnetic particles
have also been used as the solid phase support in
immobilized enzyme systems. Enzymes coupled to magnetic
particles are contacted with substrates for a time
30 sufficient to catalyze the biochemical reaction.
Thereafter, the enzyme can be magnetically separated from
products and unreac~ed substra~e and potentially can be




, .. . ::

-14~


reused. Magnetic particles have been used as supports for
-chymotrypsin, R-galactosidase [U.S. Pat. No. 4,152,210],
and glucose isomerase [U.S. Pat. No. 4,343,901], in
immobilized enzyme systems.

3. Nomenclature

The term "magnetically responsive particle" or
"magnetic particle " is defined as any particle
dispersible or suspendable in aqueous media without
significant gravitational settling and separable from
suspension by application of a magnetic field, which
particle comprises a magnetic metal oxide core generally
surrounded by an adsorptively or covalently bound sheath
or coat bearing organic functionalities to which bioaffinity
adsorbents may be covalently coupled. The
term "magnetocluster" is a synonym of "magnetically
responsive particle" and "magnetic particle n .
The term "metal oxide core" is defined as a
crystal or group (or cluster) of crystals of a transition
metal oxide having ferrospinel structure and comprising
trivalent and divalent cations of the same or different
transition metals~ By way of illustration, a metal oxide
25 core may be comprised of a cluster of superparamagnetic
crystals of an iron oxide, or a cluster of ferromagnetic
crystals of an iron oxide, or may consist of a single
ferromagnetic crystal of an iron oxide.
The term "bioaffinity adsorbent" is defined
30 as any biological or other organic molecule capable of
specific or nonspecific bindinq or interaction with
another biological molecule, which binding or interaction
may be referred to as "ligand/ligate" binding or
interaction and is exemplified by, but not limited to,


7~
~ 1~

antibody/antigen, antib~dy/hapten, enzyme/substrflt~,
enzyme/inhibitor, enzy~e/co~actor, binding
pro~ein/~ubstrate, c~ri~r protei~/ ub~tr~te9
lectin/~arbohydrate~ receptor/hor~one, receptor/effector
or rep~e~sor/inducer binding~ or ~nteraction~O
The term acoupl~d ~agnetic~lly re~pon~ive
particle~ or Ucoupled magnetic particle~ i8 de~ined a~ any
magnetic particle to which one or more types of
bioaffinity ad~orbent~ are coupled by Govaleflt bonds;
which covalent bonds ~ay be amide, e~ter~ ether
~ulfonamide, di~ulfide, ~o or other ~uitable organic
linkages depending ~n the function~lities availAble for
bonding on both the coat of the magnetic particle and the
bio~ffinity adsorbent(~).
The term ~Eilan~ refers to any bifunctional
organosilane and is de~ined ~s ~n U.S~ Pat. No. 3,652,761
~Is an organofunction~l and ~ilico~ functional ~ilicon
compound characterized in that the ilicon portion of the
molecule has an ~ffinity for inorganic materials while the
organic portion of the molecule i~ tailor~d to combine
wi~h organicsO Silanes are ~uitable coating materi~l~ for
metal oxide core~ by virtue o~ t~e~r
silico~ function21ities and c~n be coupled to bioaffinity
adsorbent~ through their organofunctionalities.
~he ter~ ~superparamagnetism~ is defined a~ that
~agnetic behavior exhibited by iron oxides w~th ry6tal
size le~s th~n about 300 ~, which behAvior is
characterized by ~e~pon~iveness to a magnetic field
without resultant permanent ~agnetization.
The ~erm ~erromagnet~m~ i8 defined ~s that
magnetic beha~ior exhibit~d by ir~n oxides wi~h cry~tal
8ize greater than ~bout 500 A, whi~h beha~ior i~
characteri2ed by responsiven~ to fl ~agnetic ~ield with
resultant per~anent ~agnetization.


1~

The term ~errofluid~ is defin~d as a liquid
comprising a colloid~l disper~ion o~ finely divided
magnetic particles of ~ubdom~in ~ize, usually 5~ 500 ~, in
a c~rrier liquid ~nd ~ ~urfactant m~teri~l, whi~h
particles r~ain sub6tantially uniformly di~per~ed
throughout the }iquid ca~r~er even in the Rresence of
~agnetic fi~lds of up to about 5000 Oersteds.
~ ~he term ~immunoassay~ i8 de~ined as a~y method
for mea~uring the ~oncentration or a~ount of ~n analyte in
1~ a ~olution based on the immunolog$cal ~inding or
interaction of a polyclonal or monoclonal ~tibody and ~n
antigen, which ~ethod (a) require~ ~ ~eparation of bound
from unbound analyt~; ~b) employs a radioisotopiG~
fluorometric, enzymatic, che~ilumine~cent or other label
~s the me~n~ for measuring the boun~ and/or u~bound
analy~e: and ~c) may be described as ~competi~ive~ if the
amount o bound ~easurable label is generally inver6ely
proportional to the ~mount of analyte originally in
solution or ~no~ competitive~ i~ the amount of bound
29 measurable label is generally directly proportional to the
amount of analyt~ originally in solution. Tabel may be in
the antigen, the anti~ody, or in d~uble ~ntibody methods,
the ~econd antibody. Immunoassays ~e exemplified by~ but
are not limited to, radioimmunoassays (RIA),
2 immunoradio~etric ~ssay~ ~IRMA), fluoroimmunoassays (FIA),
enzyme im~unoassays ~EI~ nd ~andwich method
immunoa~s~ys.
The ~erm ~bi~ding assaya or ~no~ immune assay~ i~
defined as any ~ethod f~r measuring the concentratiGn ~r
amount o~ an ~nalyte i~ ~olution based on the speci~ic or
nonspecific bi~ding or interaction, other than
antibody/antigen biading or interaotion, ~ ~ biodf~inity
~d~orbent ~nd ~n~ther biological or organic mol~cule,
which method (a) requiEes a ~eparati~n o~ bound ~rom


:~z~

- 1~

unbound analyte; (b) employ6 B radioi~otopic,
fluorometric~ enzymatic, chem~luminescent or other label
a~ the mean~ ~or meaEuring the bound an~/or unbound
~nalyte; and (c) ~ay be de~cribed a~ acompe~ ve~ if the
~mount of bound mea~urable label i~ gen~rally ~nversely
proportional to the amount of analyte origin~lly in
~olution or ~no~ competitive~ if the amount o~ bound
measur~ble label i8 gener~lly directly proportional to the
amount of anaIyte originally in ~olution.
The term "immobili~ed enzy~e r~action~ ifi ~efined
as any enzymatically catalyzed ~iochemical conversion or
~ynthesis or degradation wherein the enzyme molecule or
active ~ite thereof i8 ~ot freely 601uble but is
~dsorp~ively or covalently bound to a solid phase suppor~,
which support is ~uspended in or contacted with the
surr~unding medium and which may be reclaimed or ~eparated
from said medium.
The term "affinity chromatography~ i5 defined as
a metho~ ~or s@parating, isolating, ~ndjor puri~ying a
~elected molecule from its surrounding medium on the ba~is
of it~ binding or interaction with ~ bioaffinity adsorbent
adsorptively or covalently bound to ~ ~olid phase ~upport,
which support i8 6uspended in or cont~cted with the
surrounding medium and which may be reclaimed or ~eparated
from said medium.
4. SUMMARY OF T~E INVENTION
This invention provide~ novel magnetic particles
use~ul in biologic~l application~ involving the separation
of molecules rom or the di~ected ~ovement of molecules in
the surrounding medium. ~ethods and compo~i~ion~ ~or
preparing and u~ing the magnetic particle~ ~re provided.



-18-


The magnetic particles particles comprise a
magnetic metal oxide core generally surrounded by an
adsorptively or covalently bound silane coat to which a
wide variety of bioaffinity adsorbents c:an be covalently
bonded through selected coupling chemist:ries. The magnetic
metal oxide core preferably includes a group of
superparamagnetic iron-oxide crystals, the coat is
preferably a silane polymer and the coupling chemistries
include, but are not limited to, diazotization, carbodiimide
and glutaraldehyde couplings.
The magnetic particles produced by the method
described herein can remain dispersed in an aqueous
medium for a time sufficient to permit the particles to be
used in a number of assay procedures. The particles are
preferably between about 0.1 ~ and about 1.5 ~ in diameter.
Remarkably, preferred particles of the invention with mean
diameters in this range can be produced with a surface
~; area as high as about 100 to 150-m2/gm, which provides a
high capacity for bioaffinity adsorbent coupling.
Magnetic particles of this size range overcome the rapid
settling problems of larger particles, but obviate the
need for large magnets to generate the magnetic fields
and magnetic field gradients re~uirèd to separate smaller
particles. Magnets used to effect separations for the
magnetic particles of this invention need only generate
magnetic fields between about 100 and about 1000 Oersteds.
Such fields can be obtained with permanent magnets
which are preferably smaller than the container which
30 holds the dispersion of magnetic particles and thus~ may be
suitable for benchtop use. Although ferromagnetic particles
may be useful in certain applications of the invention,
particles with superparamagnetic behavior are usually
preferred since superparamagnetic particles do not exhibit
35 the magnetic aggregation associated with ferromagnetic
particles and permit redispersion and reuse.

~2Ç~6~7~
, .

- 1~

~ he method for preparing the ~agnetic particleæ
may co~prise precipit~ting met~l ~alts i~ ba~e to for~
~ine magn~tic metal oxide cry6~al~, redisp~r~ing and
washing the cry~t~l~ in wat~r and in an elec~rolyte.
~agnetic ~epar~tion~ may be us~d to colle~t the crystal~
between washe~ if the cry~tal are 6uperpara~agnetic. The
crystals may then be coated with a m~terial c~pable of
ad orptively os covalently bonding to the metal oxide ~nd
bearing orga~ic func ionalitie~ for ~oupling with
~ bioa~finity a~sorbents.
In one embodiment the coating aroun~ the metal
oxide core i~ a poly~er o~ ~ilane. ~he s~lanization ~ay
be performed by r~dispersing the magnetic ~etal oxide
crystals in ~n acidic organic solution, ~dding an
organo~ilane, dehydra~ing by heating in the presence of a
wetting agent mi6cible b~th in water and the organic
~olution, and washing the resulting m~gnetic ~ilanized
metal oxides. Alternatively, silanization may be
performed in acidic agueous ~olution.
2~ The magnetic particles of thi~ invention can be
covalently bonded by conventional coupling chemi~tries to
bi~a~finity ad~orbent~ including, but not limited ~o,
antibodies, antigens and 6pecif~c binding protein~, whi~h
coupled magnetic particle6 an be u~ed in immunoassays or
2~ other binding asEays ~or the measurement o~ analytes in
~olution. Such a~says preerably compris~ mixing ~ s~mple
conta~ning ~n unknown concentr~tion of analyte with a
known a~ount of labeled analy~e in the presence of
~agnetic particle6 coupled to a bio~ffini~y ~dsorbent
capable o~ binding to or interacting with both unlabeled
and labeled an~lyte, Allowing the ~inding or interaction
to occur, magnetically ~ep~rating the par~icles, measuring
the amoun o~ label associated with the magnetic p~rticles
~nd~or the amount o~ l~bel ~ree in ~olu~ion ~d

correl~ting ~he amount of label to a ~tandard curve
constructed similarly to determ~ne the concentratisn of
analyte in the ~ampleO
~ he ~agnetic particles o~ thiE~ invention Are
~ui~able for use in ~o~ ed ~nzyme ~y~tems,
particularly wher~ enzyme recycling 1~ desir~d. ~nzymatic
reactions ~re pr~fer~bly carried out by disperging
Ynzym~ coupled magnetic particles in a reaction mixture
containing sub~trate~), allowing the enzymatic reaction
to occur, m~gnetically ~eparating the ~nzym~ coupled
magn~tic particle fro~ the reaction mixture containing
produc~( ) and unreacted ~ubstra~e~ ndt if de~ired,
redispersing the par~icles ~n fre~h ~ubstra~e( ) thereby
reusing enzyme.
Affinity chromatography ~eparations and cell
~orting car be performed using lthe magnetic particle~ of
this invention, preferably by disper~ing bioaffinity
ad~orbent-coupled magnetic particles in solutionæ or
~uspension~ containinq molecules or cell~ to be i~olated
2 and/or purified, ~llowing the bioaffinity ~dsoxbent ~nd
the desired molecule~ or cells to interact, magnetically
separ~ting the partlcle~ from the ~olutions or suspension
and r~covering the isolated molecule~ or cell~ from the
magnetic particles.
~t i~ furth~r c~ntempl~ted that the m~gnetic
particles of this inventlon c~n be u~ed i~ ~n vivo æystems
for the diagnostic ~ocaliz~tion of ~ell8 or tissu~s
recognized by t~e particul~r ~ioaffinity ad~orbent coupled
to the particle and ~l~o ~or magnetically directed
deli~ery o~ ther~peutic age~s coupled to th~ particles to
pathologi~l si~e~.
The magnetic particle~ of this invention overcome
problems associated wi~h the 6ize, ~urf~ce area~
gravitation~l ~e~tling rate and magnetic character of


-2.1-

previously developed magnetic particles. Gravitational
set~ling ti~es in exce~s of ~bout 1.5 hourB Gan be
aehieved with magnetic particles of the inv~ntion, where
the grav~tational ~ettling time is defin~d to be the ti~e
for the t~rbidity ~ a di~per~l~n of p~lr~icle~ of the
invention in be absence o~ ~ ~agnetic field to fall by
~ifty perc~nt. Magnetic ceparation times ~f le~s than
~bout ten minutes can be ~chiev~d with magnetic particies
of the in~ention by contacting ~ ~es~el cont~ining
disper6ion of the p~rticles with ~ pole face of ~
permanent magnet no larger in volu~e tban the volume of
: the ve~sel, where the magnetic separation ti~e i~ de:Eined
to b~ ~he time ~or the turbidity o~ the dispersion to fall
by 95 per~ent. Furthermore, the us~ of ~ilane as the
coating surrounding the metal oxide ~ore of the ~agnetic
particles described herein makes po~sible the coupling of
wide variety of molecules under ~n equally wide variety
of coupling conditions compared to other magnetic particle
coatings known in th~ art with more limited c~upling
functionalitie~.
Preferred magnetically responsive particles of
the invention have ~etal oxide cores comprised of clusters
of superparamagnetic crystals, af~ording effic~en~
separatio~ o~ tbe particles ln ~ow ~agnetic fields
(10~ 1000 Oers ed~ while maintaining ~uperpar~magnetic
properties~ ~ggregati~n of particles i~ controlled during
particle ~ynthesis ko produce particles which are
preferab}y small enough to ~void substantial gravitational
settling ov~r ti~e~ ~uf~icient to permit disper~ion~ of
~he particle~ to be used in ~n intended blological ~ssay
or other ~pplication. The advantage of having
superparamagnet$c cores in m~gnetically ~e~pon6i~e
particles i~ that such particle can be repea~edly exposed
3 to ~agnetic fields. Because they do not be~ome

~67~9
2~

permanently ~agnetized and therefore do not ~agnetically
aggreg~te, the particle~ can be redispersed ~nd reused.
Even after ~ niz~tion, preferred particle~ of the
invention having cores made up of clu~ter~ of cry~tals
exhibit a remarkably high 6urfæce area per uni~ weight and
a generally correspondingly high ~oupli.ng capacity, which
indicates that ~uch particles have an open or porou~
structure.
None of the prior art magnetic particle~ used in
1~ the biologic~l ~ystems described in Section 2 ~boYe have
the same composition, ~ize, ~urfa~e are~, coupling
versatility, ~ettlig propertles ~nd magne~ic behavior as
the magnetic particles of the invention. The magnetic
particles of this invention are suit~ble for many o~ the
assays, enzyme immobili~ation, cell 60rting and a~finity
chromatography procedures reported in the literature and,
in fact, overcome many of the problems associated with
particle ~ettling and reuse experienced in the past with
- such procedures.

5~ BRIEF DESCRIPTION OF T~E FIGURES

FIG. 1 is ~ graphical representation of the
change in turbidity (4 concentration) of a ~uspension of
2~ magnetic particles in the presence and ab~ence of a
magnetic fiela ~5 ~ function of time.
FIG. 2 i~ a photomicrograph o~ ~uperp~ramagnetic
particles ~ilanized with ~ aminopropylSrimethoxy ~ila~e.





-2~

6. DETAILED DESCRIP~ION 0~ T~E INYENTION

6.1~ M~GNETIC PARTICLE PRE~

Preferred magnetic particles of the invention may
be made in two steps. ~ir~t, superpara,magnetic iron
oxides are made by precipitation of divalent (Fe~ and
trival2nt (Pe3~ iron ~lt~, e.g., ~eCl~ and ~eC13,
in ba~e. Secondly an organosil~ne coating i8 ~pplied to
_the iron oXiQ~.
The ratio o~ Fe2~ and ~e3+ can be varied
without ~ubstantial changes in the ~inal product by
increasing the amount of Fe2 while maintaining a
~onstant molar amount of iron. The preferred
Fe2+/~e3+ ratio is 2/1 but an Fe2+/~e3+ ratio o~
4/1 al~o works ~ati~factorily in the procedure of Section
7.1 (See also Section 7~7). An ~e2~/Fe3~ ratio of 1/2
produces magnetic particles of ~lightly inferior guality
to those resulting from the higher ~e2+~Fe3~ ratiOC7
This magnetic oxide tends to ~bleeda or become oluble
durinq the rinsing procedure of Section 7.1 and the
pazticle aize s more heterogeneous than t~e resulting
from Fe2+/~e~ o~ 2/1 or 4/1. Nevertheless, it can be
~ilanized to yiel~ a usable magnetic particle as
demonstrated in Section 7.7.
Aqueous ~olutions o the iron sal~s are mixed in
a base such as sodium hydrsxide which results ~n the
formation of a cry~alline precipitate of
superparamagnetic i~n oxide. The precipit~te i8 wsshed
r~pe3ted1y wi~h *a~er by magnetic211y ~eparating it ~nd
redispersing it until a neu~al pH i~ re~ched. The
precipitate i~ then wa~hed once in an electrolytic
solution, e.q. ~ ~odium chloride ~olu~ion. ~he
electrolyte wash ~tep i~ important to in~ure fineness o~



-2

the iron oxide cry~tal~. Fin211y the precipitate i~
washed with meth~nol until a re~idue of 1.0~ (V/V3 water
is left.
The repeated u~e of ~agnetic field~ to ~eparate
the iron oxide from su~pen~ion dur~ng l:he ~shing ~eps is
facilitated by ~uperparamagnetiæm. R~gardless of how ~any
tlmes the parti~les are ~ubjected ko magnetic fields, ~hey
neve~ become permanently ~agnetized ancl consequently can
be redi6per~ed by mild agitat~on. Permanently magneti~ed
(ferromagnetic~ ~et~l oxide& cannot be prepared by thi~
washing procedure as they tend to magnetically a~gregate
after exposure to magnetic fields and cannot be
homogeneous1y redi~persed.
Other divalent tran~ition metal salt~ such as
magne~ium, mangane~e, cobalt, nickel, zinc and copper
~alts may be substituted for iron (II) salts in the
precipitation procedure to yield magne~ic ~e~al oxides.
For example, the substitution o~ divalent cobalt chloride
~CoC12) for FeC12 in the procedure o Section 7.1
produced ferr0magnetic metal oxide particles.
Ferromagnetic ~etal oxides Luch a~ that produced with
CoC12, ~ay be washed in ~he ab~ence of magnetic fields
by employing conventional technigues of centrifugation or
filtration between *~ hings to avoid ~agnetizing the
particles. As long as the re6ulting ferromagnetic metal
oxides are of su~fi~ien~ly s~all di~meter ~o remain
disper~ed in aqueous ~ediat they ~ay also be il~nized and
coupled ~o bio~ffinity ~dsorbents for use in fiystems
reguiring a single magnetic separation, e~g. certain
radioi~munoa~ay~ Ferrom3gneti6m limits particle
use~ulnes~ in tho~e applic~tion~ requir$ng redisper~ion or
reu~e.
Magnetic metal oxide~ produced by ba e
3 precip~ta~ion ~ay be coated by ~ny one of several suitable

-25-


silanes. The silane coupling materials have two features:
They are able to adsorptively or covalently bind to the
metal oxide and are able to form covalent bonds with
bioaffinity adsorbents through organofunctionalities.
When silanization is used to coat the metal oxide
cores of the magnetic particles of this invention,
organosilanes of the general formula R~Si~OX)3 may be used
wherein ~OX)3 represents a trialkoxy group, typically
trimethoxy or triethoxy, and R represents any aryl or alkyl
or aralkyl group terminating in aminophenyl, amino,
hydroxyl, sulphydryl, aliphatic, hydrophobic or mixed
function (amphipathic) or other organic group suitable for
covalent coupling to a bioaffinity adsorbent. Such
organosilanes include, but are not limited to, p-amino-
phenyltrimethoxysilane~ 3-aminopropyltrimethoxysilane,
triaminofunctional silane (H2NCH2CH2-NH-CH2CH2-NH-CH2CH2-
CH2-Si-(OCH3)3, n-dodecyltriethoxysilane and n-hexyltri-
methoxysilane. lFor other possible silane coupling agents
see U.S. Pat. No, 3,652,761]. Generally, chlorosilanes
cannot be employed unless provision is made to neutralize
the hydrochloric acid evolved.
In one embodiment, the silane is deposited on the
metal oxide core from acidic organic solution. The
silanization reaction occurs in two steps. First, a
trimethoxysilane is placed in an organic solvent, such as
methanol, water and an acid, e.g., phosphorous acid o~
glacial acetic acid. It condenses to form silane polymers;



R-Si(OCH ) ~ HO ~ Si - O - Si - O -

~H OH


-26- ~26~7~


Secondly, these polymers associate with the metal oxide,
perhaps by forming a covalent bond with surface OH groups
through dehydration:




OH OH
¦ ¦ R R
+ HO -- Si -- O ~ Si --
OH OH

- H20
R R
HO ~ O -- Si -- O
1 O


Adsorption of silane polymers to the metal oxide is also
20 possible.
An important aspect of the acidic organic
silanization procedure of this invention is the method
of dehydration used to effect the adsorption or covalent
binding of the silane polymer to the metal oxide. This
25 association is accomplished by heating the silane
polymer and metal oxide in the presence of a wetting agent
miscible in both the organic solvent and water. Glycerol,
with a boiling poir.t of about 290C, is a suitable wetting
agent. Heating to about 160-170C in ~he presence of
D glycerol serves two purposes. It insures the evapora~ion of
water, the or~anic solvent (which may be e.g., methanol,
ethanol, dioxane, acetone or other moderately polar
solvents) and any excess silane monomer. Moreover~
the presence of glycerol prevents the aggregation or
35 clumping and potential cross-linking, of particles that is

-27-


an inherent problem of other silanization techniques known
in the art wherein dehydration is brought about by heating
to dryne 5 5 .
In another embodiment an acidic aqueous
silanization procedure is used to deposit a silane polymer
on the surface of the metal oxide core. Here, the metal
oxide is suspended in an acidic ~pH approximately 4.5)
solution of 10% silane monomer. Silanization is achieved by
heating for about two hours at 90-95C. Glycerol
dehydration is again used.
The presence of silane on iron oxide particles was
confirmed by the following observations. First, after
treatment with 6N hydrochloric acid, the iron oxide was
dissolved and a white, amorphous residue was left which is
not present if unsilanized iron oxide is similarly digested.
The acid insoluble residue was silane. Secondly, the
diazotization method of Section 7.4 permits the attachment
of antibodies to the particles. Diazotization does not
promote the attachment of unsilanized particles. Finally,
the attachment of antibody is extremely stable, far more
stable than that resulting from the adsorption of antibodies
to metal oxide~.

6.2. SILANE COUPLING CHEMISTRY
- - -
An initial consideration for choosing a silane
coating and the appropriate chemistry for coupling
bioaffinity adsorbents to magnetic particles is the natur~
of the bioaffinity adsorbent itself, its susceptibilities to
such factors as pH and temperature as well as the
availability of reactive groups on the molecue for
coupling. For instance, if an antibody is to be coupled to
the magnetic particle, the coupling chemistry should be


-28- ~2~7~

nondestructive to the immunoglobulin protein, the covalent
linkage should be formed at a site on the protein molecule
such that the antibody/antigen interaction will not be
blocked or hindered, and the resulting linkage should be
stable under the coupling conditions chosen. Similarly, if
an enzyme i5 to be coupled to the magnetic particle, the
coupling chemistry should not denature the enzyme protein
and the covalent lin~age should be formed at a site on the
molecule other than the active or catalytic site or other
sites that may interfere with enzyme/substrate or enzyme/
cofactor interaction.
A variety of coupling chemistries are known in
the art and have been described in U.S. Patent No. 3,652,761.
By way of illustration, diazotization can be used to couple
p-aminophenyl terminated silanes to immunoglobulins. Coup-
ling of immunoglobulins and other proteins to 3-aminopropyl~
terminated and N-2-aminoethyl-3-aminopropyl-terminated sil-
anes has been accomplished by the use of glutaraldehyde. The
procedure consists of two basic steps: l) activation of the
particle by reaction with glutaraldehyde followed by removal
of unreacted glutaraldehyde followed by removal and 2) reac-
tion of the proteins with the activated particles followed
by removal of the unreacted proteins. The procedure is
widely used for the immobilization of proteins and cells lA.M.
Klibanov, Science, 219:722 (1983)]. If the magnetic particles
are coated by carboxy-terminated silanes, bioaffinity adsor-
bents such as proteins and immunoglobulins can be coupled to
them by first treatlng the particles with 3-(3-dimethyl-
aminopropyl)carbodiimlde. Generally, magnetic particles
coated with silanes bearing certain organofunctionalities
can be modified to

-2~

substitu~e msre desirable functionali~e~ for tho e
already pr~ent on the ~urface. ~o~ exampleO di~20
der~vative~ can be prepared ~ram ~ a~inopropyltriethox~
silane by reactlon wi~h p ni~r~ benzoic ~G~, reduction of
the nitro group ~o ~n amine ~nd then diazot~zat$on with
nitrou~ ~cid. ~he same ~ilane can be con~erted to the
~sQthiocyanoalky1silane derivative by rleaction of the
~min~ function group with thiophosgene.
To effect coupling to the ~3qnletic particle, an
0 ~queous ~olution of a bioaf~inity ~d~orbent can b~
con~acted with ~he ~ilane coa~ed par~icle at or below room
temperature, Wl~en a protein (or im~u~oglobulin) is to be
coupled, generaIly a r~tio of 1:10 -1:30, mg protein: mg
particle i~ used. Contact periods of between about 3 to
24 hours ~re u~ually ~ufficient f~r coupling. During thi~
period, the pH is maintained at a v~lue that will not
denature the bio~ffinity adsorbent and which best suits
the type of linka~e being ormed, e.g. ~or azo link~ses, a
p~ of ~ 9.
It has been observed that ~fter coupling of
antibodies to ~ ne co~ted magnet~c particles by either
the diazoti2ation, carbodii~ide, or glutaraldehyde methods
de cribed in greater detail in Section 7.5, 7.8 an~ 7~10,
respectiv~ly, the anti~odie~ re~ain magnetic even after
2~ the following rigorous t~eatment6s 24 hours at 50C in
phosphate buffered saline ~B5)~ ~1 days at 37C in PBS~
30 minu~e~ at 23~C in IM sodium chloride, and repeated
rinses in ethanol or meth~nol at r~om temperature.
~ntibodieS adsorbed to iron oxide. ~re subst~ntially
3D detached by any o~ the e treat~ent~. The~e resul~s
indicate that ~he silan~ i8 ~ery tightly ~ssoci~ted with
the ~etal oxide ~nd th~ ~h~ co~pllng o~ antibody to the
p~rt~cle re~ults f~om an e6~entially ~rreversible covalent
coupling. The tight a~sociation of ~he ~ n~ ~o the

-

~ 9
-3

~etal oxide together with the cov~lent coupling o~
bioaffinity ad~rbents (e.g~, ~ntibodie~ ~re feature~
: which impart 6tability onto coupled ~agnetic p~rticles,
commercially important attribute.
s




6.3. ~SE OF MA~NE~IC PRRTICLE~
IN ~IO~OGICAL ASSAYS
, The ~agnetic particles of thi~i invention ~ay be
used in immunoassays ~nd other binding a~says as defined
~n ~ection 3. The mo~t preYalent type~; ~f a~ays used for
diagno~tic and research purpos~e are r~dioim~unoassay~,
~luoroimmunoassays, enzym~ immunoassay~ d no~ i~mune
r3dioassays, based on the principle of ~o~pe~i~ive
binding. Basically, a ligand, such a~ an antibody or
specific binding protein, d~r~cted against a ligate, ~uch
as an antigen, i~ ~aturated with an exce~s of labeled
ligate (*ligate). lAlternatively, competi~ve a~says may
be ru~ with labeled ligand and unlabeled lig~te.
No~ co~petitive a~says, s~ ~lled sandwich ~ssays, are
al~o widely e~ployed.l By the method o~ this lnvention,
:~ the ligand i~ coupled to a ~agnet~c particl~. ~xamples of
`:~ labels are radioisotope~: tritiumr 14_c~rbon,
57-cobalt and, preferablyt 125-iodine; ~luoro~etric
label~: rhodamine o~ fluoresceln isothiocyan~te; ~nd
en~y~es (generally chosen fo~ the ea~e with which the
enzym~tic reactlon c~n be ~ea~ured): ~lkaline phosphata~e
or ~ D~galactosida ~. If nonlabel~d l~g~te i~ added to
ligand along with *lig~te, le~s *ligate will be ~ound in
the ligan~ ligate complex ~ the ratio of unlabeled to
labeled li~At~ incr~se~ the ligan~ *ligate complex
can b~ phy~ically ~ep~rated ro~ ~ligate~ the amount of
unlabeled lig~te in ~ te~t ~ubstance ~n b~ de~ermined.



7~ '
.~
-31

To ~easure unlab~led liga~e, ~ s~andard urve
- must be constructed. ~hi~ i~ done by ~ixing ~ fixed
amount of ligand and ~ligate ~nd ~dding ~ known ~mount of
unlabeled ligate to each. When the reaction Is complete,
the ligan~ ~ligate is ~epar~ted from ~l.igate. A graph i~
then made that relates the label in the ~ollected
ligan~ *li~ate co~plex to the ~mount of added unlabeled
l'igate, ~o determine th~ a~ount of unl~beled lig~te in an
experimental ~ample, ~n 31~uot of the ~sa~ple is added to
the ~ame ligan~ *ligate mixture u~ed to obtain the
standard curve. The ligan~ ~ligate complex i~ collected
and the label measured, ~nd the a~ount of unl~beled ligand
is read from the ~tandard curve. This is possible with
any ~mple, no ~atter how complex, as long as nothing
interferes wi~h the ligan~ ~ligate interaction. ~y the
method of this`invention, ~he ligan~ *ligate complex is
separated ~agneti~ally from ree ~ligate.
This general ~ethodology can be applied in assays
for the measurement of a wide variety of compounds
including hor~ones, phar~acologic agents~ vitamin and
cofactor~ hematological ~ub~tance~ viru~ ~ntigens,
nucleic acid~, nucleotides~ glycosides and sugars. By way
of illustration, th~ compounds li~ted in T~ble I ~re all
measurable by magn~tic particles i~munoas~ays and binding
assays lsee D. Frei~elder, Phy6ical ~ioche~istry:
~pplication~ to Biochemi~try and Mole~ul~r ~iology, p.
254, W.~. Freeman and Company~ San Prancis~o ~1976~.





6~
-3

TABLE I


~ormone6:
Thyroid hormone~ Prolactin
~thyroxine~ tr~iod~
thyronine, thyroid Thyrocalcitonin
binding globuli~,
thyroi~ ~timulati~g Parathyroid hormone
hormone, thyr~globulin)
~uman chorionic gonadotrophin
Gastrointe~tin~l hor~nes
(glucagon, g~strin, ~u~an placental lactogen
enteroglucagon,
~ecretin, p~ncreoz~ Po~terior pituitary peptides
min, vasoactiv~ (oxyto~in, vasopre~sin,
intestinal peptide, neurophysin)
gastric inhibitory pep
tide, motilin, in~ulin) Bradykinin
Follicl~ ~timulating hormone Cortisol
Leutenizing ~ormone Corticotrophin
Progesterone Human growth hormone
T~stosterone
E6triol
Estr~diol
~ :
Digoxin Tetrahyd~ocannabinol
Theophylline Barbi~ur~tes
Morphine and opi~te Nicotine and met~bolic
3D alkaloid~ product~
Cardiac glycoside~ Pheno~hi~zine
Prostaglandins Amphetamines
Lysergic acid and deri~ativ~
-




... .

-33~ 7~9


TABLE I (cont.)

Vitamins and cofactors:
D, B12, folic acid, cyclic AMP

Hematolo~ical substances:
Fibrinogen, fibrin,
and fibrinopeptides Prothrombin
Plasminogen and plasmin Transferrin and ferritin
Antihemophilic factor Erthropoietin

Virus antigens: --
Hepatitis antigen Polio
Herpes simplex Rabies
Vaccinia Q fever
Several Groups A Psittacosis group
arboviruses
~ ~0
Nuclei acids and nucleotides:
DN~, RNAr cytosine derivatives
:
25 6.4. USE OF MAGNETIC PARTICLES IN
IMMOBILIZED ENZYME SYSTEMS
... .. _ .

Enzymes may be coupled to the magnetic particles of
this invention by the methods described in Sec~ion 6.2.
They may be used in immobilized enzyme systems, particularly
in batch reactors or continuous-flow stirred-tank reactors
(CSTR), to facilitate separation of enzyme from product
after the reaction has occurred and to permit enzyme reuse
and recycle. A method for using enzyme-coupled magnetic
particles in biochemical reactions was described by
Dunnill and Lilly in U.S. Pat. No. 4,152,210. The
magnetic particles of this invention may be advantageously

~2~
,.............................................................. .

-34

~ubstituted or ~hose o Dunnill ~d Lilly to ~Yoid
problem of ~ettling ~d to allow enzy~e recycle.
Brie~ly, sub~tr~tes are contacted with 2n2ym~ coupled
~agnetic particle~ in a reactor under condition~ of pH,
temper~ture ~nd ~ub~tr~te concentration that best promote
the re~ction. After completion of the reaction the
~articles are ~agnetically ~eparated from the bulk liquid
(which may be a solution or ~uspen~ion) from which product
can be r~trieved free of enzy~eO The enzym~ coupled
1~ ~agnetic particle~ can then ~e reused. I~mobiliz~d
enzymes (coupled to no~ ~agnetic support~) have b~en used
in a number o~ industri~lly important enzy~tic reac~ions,
some of which ~re li~ted in Table II. ~he m~gnetic
particlee of this invention can be substituted ~or the
no~ magnetic ~olid phases prev~ously employed wh~ch
include glass, ceramics, polyacrylamide, DEA~ cellulose,
chitin, porous ~ilica, cellulose beads and
alumin~ silicates.

~L~ TI
INDUSTRIALLY IMPORTANT IMMOBILIZED
EN~ ME REACTIONS
. _ _
~, ~
Amyl~ glucosidase ~alto~e~Glucose
Glucoee Oxidase Glucose/gluc~nic ~eid
Glucoamylase Star~hJglucose, Dextrin/glucose
~ Amyla e St~rch/malto~e
Invertase Sucrose/glucose
Glucose ~o~erase Glucose/f~ucto~e




;7
- 3

Lact~se l,a~tos~e~gluco~e
Trypsill Protein~ ino acid~
h~lno~cyl~e W~ acetyl- DIr ~ethionine/~ethionirle
Ly60zyme Lysi~ of ~ Y ~deikticu8

I~a AFFI~IqY
~0 .,
Tbe proeess of ~ff~nity ~hromatogr~phy enable~
the efficient iiol~tion o~ molecules by ~aking ul3e o~
eatures unique to ~he ~ol@cule: the ability to secognize
or be recognized wi'ch ~ high degree of 6electivity by
15 bioaffini~cy ad~orbent ~uch a~ an eE~zyme or ~ntibody and
the abiliky to bind or adsorb therQto. The process of
af~inity chromatogr~phy s~mply involv~s placing a
~elective bloaffinity ~d~orbent or ligand in contact with
a ~olution cont~ining several kinds o~ substances
20 including the desired 6pecie~, th~ ligate. ~h~ liyate is
selectively ad~orbed to tbe li~and, wbic:h i~ coupled to an
insoluble support or ~atrix. The nonbinding species ~re
removed by wa~hing. The ligate i~ then recovered by
eluting with a ~peciPie~ de~orbing ~n, e.g. ~ buf~er ~t
25 a p~ or ionic tr~ngkh ~h~t will cau~e detachment o the
adsorbed ligate.
By the method o~ this invent~on, ~3gnetic
par~icles may be u~ed ~18 th~ insoluble ~uppor~ to which
the ligand i8 coupled. The particl~ ~ay be ~uspended in
3~ b~tch reactor~ containing the liga~e ~o be ~ol~ted. The
par~icles with bound li~ate may be ~eparated magnetically
froan the bulk fluid and wa~hed, ~th ~nagne~c sep~r3ti~næ
betweesl wa6hes. Iiinally, the lig~-te can be rocovered $rom
the particle by de~rptlon. The magnetic p~rticle~ of
this invention m~y be u~d in a variety o~ inity
~y6tem~; exempli~ied by ~ho~e listed in T~ble III.

6769
~ 36-


AFFINInr SY STEM~
S




Li~and, imlTobile enti~
Inhibitor, cofactor, prosthetic Enzymes; apoenzymes
gr~up, polymeric sub~trate
Enzyme Polymeric inhibito~s
:: 10 Nueleic acid, ~ingle str~nd ~ucleic acid, cGmplementar~
strand
~: ~apten; antigen Antibody
Antibody ~ Proteins; polysaccharides
t5 ~ono~acc~laride; polysaccharide Lectins; receptor~
Lectin Gly~oproteins; receptors
Small target compounds ~3inding ~rDteins
Bindiny Protein Small target compounds

7. EXA~PLES
7 .1. P~aEPARATION OF METAL OX IDE

~he me'cal oxide particle~ were prepared by mixing
a solution of iron~II) (F~2~) ~nd iron(III) (Fe3~J
salts wi~h base as follow~: a solution that i~ 0~ 5M
ferrous chloride (FeC12) and 0.25M gerric chloride
(~eC13) (200 mls~ wa mixed with 5M ~odium hydroxide
3~ (NaO~) (200 mls) ~t 60C by pour ing both solutions into a
500 ml beaker con~aining 100 mls vf distilled water. All
steps were pergor~ed ~t room ltemperA'cure unle~ o~cherwi~e
indic:at@d,. The ~ixture wa~ ~tirred ~or 2 ~inutes during
which time a b~ack, magne~cic precipitate i~orroedO After


7~;~
- 3

~ettling~ the volume of the 6ettled precipitate wa~
approximately 175 ml~. The concentratisn o iron oxide in
he precipitate wa~ about 60 ~g/ml (ba~ed on ~ y~eld of
11.2 ~ms of iron oxid~ ~ determined in~ra)~ ~hi~ i5 in
~ontrhst to ~ommercially available ~agnlet$~ ~ron oxides,
such a~ Pfizer ~228 yPe20~ (Pflzer ~ineræl~, Pigment~
and Metals Division, New York, Ne), thle standard magnetic
~xide for recording t~pe~, which ~an ~ttain concentration~
of about 700 mg/ml in aqueou~ slurry. Irhe compari~on i8
included to emphasize the f~neness o ehe parti~les ~ade
by thi~ method. Very Pine particles are incapable of
dense packing and ~ntrsin ~he ~o~t w~ter. L~rger and
denser particlest on ~he other h~nd, pack ~e~ely,
excluding the ~08t wa~er.
~he precipitate was then wash~d with water until
a pH of ~ 8 was rea~Aed ~ determ~ned by p~ paperD The
following washing technique was employed:

The particles were su~pended in 1.8 liters o water in a 2
liter beaker and ~ollected by magnetic extraction. The
beaker was placed on top o~ a ring ~agnet, 1/2 inch hiyh
~nd 6 inche~ in di~meter, which caused the magnetic
p~rticle~ to ettle. The water wa~ poured off withou~ the
1088 of particle~ by holding the ~agnet to the bottom o~
; ~ the beaker while decanting. A ~imilar w2shing technigue
was employed fo~ ~11 washes through~ut, except that
volumes were adju~t~d as ne~e sary. Typi~ally, three
washes were ufficient to ~chieve neutral p~. The
magnetic oxide w~ then wa~hed once with 1.0 li~er of
0.02M ~odium chloride ~Cl) in ~he ~me be~ker.
~ he water WDS then ~eplaced w$th methanol,
lea~ing ~ trace of ~ater to cataly~e hydroly~i~ of ~he
methoxy silane l~ee S~c~i~n 7.2.)~ Thi~ was acco~plished
by aspirat~ng B00 ~1 of 0.2M NaCl and bringing the total

~2~ ~'69
-3~

volu~e to 1 liter with met~anol~ The ~ateri~l w~s
resuspended, and ~agnetically ~xtracted; 800 ml~ of
supernatant wer~ removed, and another 800 ~1~ o~ ~ethanol
wer~ added. Ater three additions of ~ethanol, the oxide
was ready for 6il~niz~tion in ~ ~olutilDn wb~ch w~
approximately 1% ~Y~V) water. A portion of the
precipitat~ wa~ dried at 7~C for 24 hours ~nd weighed;
11.2 gram~ of magnetic iron oxide were ~ormed.
It i~ to be noted that throughout this proGedure
1~ the ~agnetic iron oxid~ particl~, because of their
superparamagnetic properties, never became permanently
~agn~tized despite repeated exposure to magnetic fields.
Consequ~ntly, on~y mi}d ~gitation was reyuired to
resuspend the particles during the water wafihings ~nd
methanol replacement treatment.

7.2. SILANIZATION

~ he magnetic iron oxide particles (~ee S~ction
7.1.) 6u&pended in 250 mls of methanol containing
approxim~tely 1% (V/Y) water were placed i~ ~ Virti~ 23
homogenizer ~Virtis Company, Inc., Gardiner, N~). Two
grams of orthopho~phorous acid (~isher Scientific Co.,
Pittsubrgh, PA) and 10 ~1~ of ~ aminopheny1trime~hox~
~5 ~ilane (Ar7025, Petrarch Systems, Inc. t Bristol, PA) were
added. In an ~lternative protocol, 5 ~1~ of glacial
acetic acid hase been 6ubstituted for the 2 gms of
orthophosphorGus acid. The mixture was homogenized at
23,00~ rpm f~r 10 minutes and at 9,0~ rpm for 120
~inu~es. The contents were poured into a 500 m~ glass
b~aker cont~ining 200 ml~ of gly~erol and heated on a hot
pla~e un~il a ~emperature o~ 1~0-~70~C wa~ roached. ~he
mixture w~s allowed to cool ~o room temperature. Both the
heating and cooling ~tep~ were perfor~ed under nitrogen

1~36676~
., ~
-3~

with stirring. ~he glycerol particle slur~y (about 200
mls in volume~ wa~ poured ~nto 1.5 liter~ of water in a 2
liter beaker; the particle~ wer~ washed exhaustively
tusu~lly ~our ti~es) with water ~ccording to the technique
described in ~ection 7.1.
Thi~ ~ilani2ation pr~cedure ~as perormed with
~ther ~ilane6, including ~ ~minopropyltrimethox~
~ilane, ~ ~ a~inoethyl- ~ aminopropyltrimethoxy~ilane,
~ d~decyltriethoxy6ilane a~d ~ hexyltrimethoxysil~ne
(~ 0800, ~ 0700, ~ 6224 and ~ 7334, respect~vely, Petrarch
Sy tem~, Inc., Bri~tol, PA).
As an alternative to the ~bove ~ilanization
procedure, ~ilane h~s also been ~epo~ited on
~uperparamagne~ic iron oxide (as prepared in Section 7.1)
~rom acidic aqueous solution. Superparamagnetic iron
oxide with Fe2 /Fe3 ratio o$ 2 was washed w~th water
as descri~ed ~n Section 7.1. The transfer to methanol was
omitted. One gram o~ particles t~b~ut 20 ~ls ~f settled
particle~) wa~ ~ixed with 1~0 ml~ o~ A 10~ ~olu~ion of
: 20 ~ aminopropyltrimethoxy~ilane in water. The p~ was
~djusted to 4.5 with glacial acetic acid. The mixture was
heated at 9~ 95C ~or 2 hours while mixing with a ~etal
~tirblade attached to an electric motor. After cooling,
the par~icles were washed 3 time~ with wa~er (100 mls), 3
25 times with ~ethanol (100 ml~ ~nd 3 times with water ~100
ml~), and the pre~ence of fiilane on ~he p~rticle8 was
confir~ed.

7.3. P~3YSICAL CHARACTERIST~CS OF
S I L~N I Z ED MAG NETIC PARTICLES
The mean particle diameter a~ ~easured ~y light
; sc~ttering ~nd the surf~ce area p2r gr~m as mea6ured by
nitrogen gas ad~orption for E~aminophenyl ~ niz~d,


-40-



3-aminopropyl silanized, and N-2-aminoethyl-3-aminopropyl
silanized particles are summarized in Table IV. The
particle surface area is closely related to the capacity
of the particles to bind protein; as much as 300 mg/gm of
protein can be coupled to the N-2-aminoethyl-3-aminopropyl
silanized particle, far higher than previously reported
values of 12 mg protein/gm of particles [Hersh and
Yaverbaum, Clin. Chem. Acta 63:69 (1975)]. For
comparison, the surface areas per gram for two hypo-
thetical spherical particles of silanized magnetite are
listed in Table IV. The density of the hypothetical
particles was taken to be 2.5 gm/cc, an estimate of the
density of silanized magnetite particles. The diameter
of each hypothetical particle was taken to be the mean
diameter of the particles of the invention next to which
entries for the hypothetical particle is listed. Observe
that the surface area per gram of the particles of the
invention as measured by nitrogen gas absorption is far
gre~ter than the calculated surface area per gram for
perfect spheres of silanized magnetite of the same diameter.
The greater surface area per gram of the particles of the
invention indicates that the particles of the invention have
a porous or otherwise open structure. Hypothetical perfect
spheres of silanized magnetite having a diameter of 0.01
have calculated surface axea per gram of about 120 m2/gm.




~'~6~7~
--41-

T~L.E_ IV

CHARACTERIS~CS OF SILANI~ED ~ETIC PAR ~CLES

Mea~ured l~ypoth.
~ean Dizlm.l Surf. Ar~2 Surf. Area3
Si 1~ne ~ _ (m2/~ (m2/gm~
t~ 2 amlnoethyl- 0.561 140 4.3

minopropyl

p- aminopbenyl 0.803 N~4 --

3- aminopropyl 0.612 122 3.g
:
-- . .

Dian,eter (in mic:rons) was measured by light
~cattering ~n a Coulter N~ 4 Particle Size Analyzer.
2 Surface area was mea~ured by 2i~2 ~as adsorption.
3 Calculated Eurf~ce area per gralD for a perf~ct phe~e
with 2~ denslty at 2.5 gm/cc:.
4 Not Measured.
`- 25
~`
- Becau~e the~ mean diam~ter~; of the ~ilar~i~ed
magnetic particles produced by the proced~res of ~eotions
7.1 and 7.2 ar~ considerably ~ ller than the diameters of
30 oth~r magnetic particles de~cribed in the liter~ture, they
exhibit ~slower graviJDetric 6ettling times th~n tho~e
previou~ly reported. }'or instance, the settling ti~e of
the particles d~scribe~ her~in i5 ~pproa~ ely 150
minu~ ir c~ntr~t to ~;ettl~ng ti~es o~O A) 5 ~inute6 for
35 the particles of ~ersh and Yaverbaum [Clin. Che~. Acta 63:
69 (1975) 1, estiJnated to b~ greate~ than 10 lJ in diameter;
and b) le58 than 1 minu~e for the particle~ of Robinson et
al. [Biotech. ~io~nS~. XV:603 (1973)1 which are 50~160 )I in

6~
-4~

The ~ilanized magne~ic particl~ o~ thi~ invention
~re char~cter$~ed ~y very ~low r~ o~ grav~m@tr~c
se~ling ~ a result o their ~ize and compo~ition; never-
theless they re~pond promptly ~o we~k ~agnetic field~.
This ifi depict~a ln PIG. 1 where the ch~nge in turbidity
over time of ~ 6u~pen ion of ~ilan~zed magnetic particles
re~ulting from spontaneou particle ~ettl~ng in the absence
of a magnetic field i~ comp3red So the change in the
turbidity produced in ~he pre~enc~ of a ~mari~ cobalt
tO ~agnet. It c~n be ~een that after 30 minute~ the turbidity
of the 6uspension ha~ changed only ~lightly more than 104
in the absence of a ~agnetic field. ~o~ever, in the
pr~ence o~ a we~k magnetic field, the turbidity o~ the
particle su~pen ion drops by more than 95~ of it~ original
value within 6 minutes. In ~nother experiment, ~ decrease
in ~urbi~ity of only about 4~ in 30 minutes was observed.
A photomicrograph of ~uperp~ramagnetic particles
silanized with ~ aminotrimethox~ilanes ~6IN" particles)
is ~hown in F~G. 2. It can be ~en tbat the particles vary
2~ in ~hape and 8ize and that they are made up of a groups or
~lu~ters of indi~idual superparamagnetic cry~t~ls (le~s
than 300 A) which appear roughly ~pherical in ~hape.

7.40 COUPLI~G OF AM~NOP~NYL M~GNETIC
PARTICLES TO A~TIBODIES TO T~ ROXINE
Fir~t~ thyroxine (~4) anti~erum w~s prepared as
follow~: 5.0 ml~ of serum of ~heep immunized with T~
(obtained feom R~dioa~s~y Sy~tem~ ~aboratorie~, Inc.,
Carson, C~) were added ~o a 50 ml ce~trifuge ~ube. ~wo
5.0 ~1 aliquots of phosphate buffered 6~1~ne (PBS) were
~dded to the ~ube ~ollowed by 15 ~1~ of 80~ ~aturated
a~monium ~ulfate, p~ 7.4, at ~C. A~t2r ~ixing, the tube
was stored at 4~C for 90 mi~utes. The ~ix~ur~ was then


centrifuged at 3,000 rpm for 30 minu~es a~ ~C. The
~upernatant fraction wa~ decanted and ~he pellet
resu~pended and di~olv~d ~o cl~ri~y ~ 5.0 ~1~ of PBS.
The T~ ~nti~erum pr~paration (1:2 in PE~S3 wa6 di~lyzed
again~t ~BS, tranæferred fro~ the dialy is tubin0 t~ a 50
~1 ce~rifuge tube to which ~0 ml6 of ~BS were added,
bringing the tot~l volume to 50 ml~. The T4 antiserum
preparation ~1~10 in ~BS) wa~ refrigerated until u~ed for
coupling.
To 1740 mg o~ p aminophenyl ~ilanized particles
in 100 Ml o~ lN hydrochloric acid (~1), 25 ~1~ of 0.6M
3sdium nitrite (~a~021 were added. The NaN02 was
~dded ~lowl~ below the sur~ace of the particle/HCl mixture
while ~aintaining th~ temperature between 0 and 5C with
care taken to ~void freezing. After 10 minutes, the
mixture was brought to pH 7.~ 8.5 by addition of 65 l~ls of
1.2M NaO~ and 18 mls of I~ sodium bicarbonate (Na~C03),
8till maintaining temperature a~ 0 to 5C~ . Then, 50
mls of PBS containing 100 mg of the gamma globulin
fraction of 6heep ~erum containing ~ntibodie~ to thyroxine
(the T~ anti~eru~ prepar~tion describ~d ~ ) were
added. Th@ p~ was maintained between 7.~ 8.5 while the
mixture was incubated ~or 18 hour~ at OD to 5C. The
antibod~ coupled particle~ were washed exhau~tively with ,
O.lM ~odium phosphate bu~fer, p~ 702 (3 timefi), LM NaCl,
methanol, lM NaCl and O.lM 60dium phosphate buffer again.
Wash ~teps were repeat2d twice or ~ore. All washe were
. perf~rmed by di~per~ing ~he partisles and magnetical7y
sep~rating them as described in sec~ion 7.1. After
washing, the par~icle~ wer~ resuspended in P~S ~nd
incubated overnight at 50C. The partiele~ were wa~h2d in
methan~l, lM ~aCl and 0.1~ sodium pho~phate buer a~
before, ~nd ~wlce in Free T4 Tracer Buffer. The
particles w~re resuspended in Free T4 Tr~cer Buf~er and
~tored at ~C u~til u~ed ~or radioimmunoass~y.

~ .~ 2~6~6~3
4~

7n50 ~AGNETIt: PAP~TICLE RADIOI3~MUNOASSA!Ir FOR THYROXINE

The quantity of antibod~ ~oup:led ~agnetic
part~cle~ t~ be u~ed in the thyroxine radioimmuno~scay
~RIA) wa8 determ~ned empiric~lly using the following RIA
pracedur~:

Ten microliter~ (~lsJ of ~t~ndard were pipetted into
12x75mm polypropylene tubes followed by 500 yls of tracer
and 100 ~ls of magnetic particles. After vort~xing, the
~ixture wa~ incubated ~t 37~C for 15 ~inutes ~fter which
time the tubes were pl~ced on ~ magnetic ra~k for io
minutes. The r~ck consisted of a te~t tube holder with a
cylindrical ~button~ magnet (Incor 18, IndianR General
Magnetic Product~ Corp., Valpar~iso, IN) a~ the bottom of
~ach tube. The magnetic particles with antibody and bound
tracer were pulled to the ~ottom o~ ~he tubes allowing the
~nbound tracer to be removed by inverting the rack and
pouring off supernat~nts. Radioactivity in th~ pellet wa~
2~ determined on a Tracor 1290 Gamma CounteE (Trazor
Analytic, Inc., Elk Gro~e Vill~ge, lL).
The reagents u~ed in the as~ay were a~ follows~

Standards were p~epared by adding T4 to ~4-~ree human
Qerum. T4 w~ removed fr~m the ~erum by incubation of
serum wi~h activa~ed ch~rcoal followed by filtration to
remove the charGo21 a~cording ~o the method of Carter
~Clin. Chem 24, 362 ~1978)~. The tracer w~s
5I-thyroxine pureha~ed from Cambridge ~edical
3D Diagnostics (~155) and was diluted into O.OLM ~ris buffer
cont~ining 100 ~g/ml bovin~ ~ru~ ~lbumin, 10 ~
~alicyl~te, 50 ~g/ml ~ ~milinonaphthalen~ ~ ~u~fonic ~cid
at pH 7.4. Magnetic par~icles ~t Yari~u~ conccn~rations
in phosph~te buffered ~aline (PBS~ with 0.1~ bovine serum

7'~
.
~ 45-

albu~nin WR e used $n the RIA to determine a suitable
concentration of part~cle~ for T4 measurement~. A
quantity of ~agnetic particle6 of ~pprvxim~tely 50 ~9 per
tube was c:hosen or t:he ~IA. Thi~ amount permitted good
di placement oiE tracer f~om the ~ltib~dy fc~r the de~ired
c:oncentration range of ~,~ (~ 32 l~g/dl) I
E~ving thu~ determined the opti~aal quantity, the
P~IA procedure de~cribed ~ was performed using
approximately 50 1~9 per tube of ~nagneltic particles l:o
1~ construct a radioilluDunoas&ay ~tandard curve for T4. The
data obtained ~rom th RIA ~ pre~ented in Table Y.

~BLE V

RIA STANDARD CURVE FOR T
~4
T4 Concentration cpm (average o:E 2 tubes~
_.. ..
O 1~g/dl 36763
2 lJgtdl 24880
~ 4 ~g/dl 18916
8 l~g/dl 13737
16 ~g/dl lû159
32 llg/dl 7632
~o'cal 69219

.




7 . 6 . ~NE:TIC PARTICLE RADIOI~ aOASSI~
OR THEOPHY LLINE
Rabbit anti- theophylline antibodies we~ prepared
and t:oupled to p aminophenyl silanized parti~les 3ccording
to methods si~nilar to tho~e de~cribed in Sec~tion 7~ 4 . The


7~


~nti~ tlleophyllirle ai3tilbody~- coupled magnetic particles were
used in ~ radioimmuno~ssay with the followirlg p~otocol: 20
~15 of theophylline ~tzlndard ~obtained ~7y adding
theophylline to theophyll1n~ free ~uman ~erura), 100 ~1~ of
125I- the~phylline tracer tobtain~d rollD C11ilical A~says,
Cambrid~ nd 1 r~l of ~ntibvd~ coupled ~agnetic
particles wsre vort~xed. After ~ 15 mi.nu'ce incub~tion at
~oom tempe~e~tu~e, a 10 minute magnetic separation was
employed. A standard curve was c~n~tructed 3nd the data
10 obt~ined are ~hown in Table VI~

q A13LE VI

RIA STANDARD CURVE FOR T~EOP~ LLINE

The~ph,~lline Concentrzltlon ~of 2 tubes)

O l~g/dl
2 ~g/dl 28217
8 l~g/al 19797
20 ~g~dl 1335Z
6û l~g/dl 814
Total 52461



7, 7. EFFECT OF VARIATION OF Fe2~/Fe3~ RATIO
OF M~AGNETIC PAR~ICLES ON T4 RADIOIMMUN0AS5P~
~
l~agnetic iron oxides were ~nade according to t!he
cry6tallization procedur~ of Section 7.1 b~ maintaining
c:onstant 7nolar a~ount~ o~ iron bl3t v~rying th~
Fe2+/Fe3+ ratao ~rom 4 to 9.5,. These p~rticles wese


~2~
. .
- 4~

~ilaniæ~?à, coupl~d to ~nti~ T4 antibodies and used in the
T,~, RIA, a~ in ~eGtior3~ 7.2, 7.4 and 7O5~ re~pectively.
The vari~qtion of Fe2 /Fe3~ ratio did no'c 6ub~tan'ci~slly
5 affect ~he performance of the~e magn~t~c p~rticlea in the
T4 RIA a~ ~hown ~t Table VI I .

T~LE VI I




T4 RIA STANDARD CURV:E:S U~SING ~NETIC
PARTICLES WIT~3 VARIED Fe ~3+ RA~IOS
T4 CDnCent:ration CDISl ~veraae of 2 tubeæ)
~e2~3~ ~,4Fe2-t~3+ ~n 5

0 ~g/dl 3563335642
1 ~Ig~dl 316Rl- 33139
2 ~g/dl 3057230195
4 ll9/dl 2470225543
~ I~g/~l 1868019720
16 I~g/dl 128û3 11625
32 ~g/dl 10C12 B005
~otal 77866 75636

~ . . _ , _

7.8. COUPLXt~G OF CARBOX~LIC ACID- TERMINATED
MA~NETlC PARTICLES 13D f31~ BINDIP~; PROTEIN
7.8.1. PREPARATIOM OF CARBO~YLIC ACID-
TERMINATED M~;NETIC PARTICLES
30 - ~ - - - . . .
A ~uperparamagne~ic aron oxide was made by the
procedule described ir Sec~ion 7~1 ~nd ~i.lanized ~s in
Se~tion 7.2 wi~h 3- amiraopropyl~rimethoxysilang in~tead of
the ~minophenyl 6il~ne. ~he alaino group of the ~ ne was


then reacted with gll2taric ~nhydrid2 to ¢onvert the
terminal:ion from ~n amine to c~rboxylic ~cid~, The
conversion of the termination w~s accoD~pliE;hed ~
follows: five grams of amin~propyl ~ilanized particles in
5 water were washed four tlmes ~th 1.5 l~ters of O,hM
NaHC03 using ~ehe washing procedure of Sec:tion 7~1. The
volume was ~djusted to 100 mls arad 2.Bri gm ~lutaric
anhydride was added. ~he particles were washed two times
and the reaction with glutaric ~nhydrid~ was repeated.
10 The carboxylic ~c~d- ter~inated ~agnetic particles were
washed f ive ~cime-~ with water to prepare them ~or re~ction
with p~te in .

7. 8. 2. C~RBODIIMID C:OUP~ OF B12 BINDI~æ PROTEIN
AND ~UMAN SE~RU~ AL13UMIN .10 CARBO~Y ~IC
ACID- ~ERMINATED M~ NE_C PART ICLES__ __
To 50 mg of carboxy- ter3r.inated magnetic particles
in 1 ml of water were added 4 mq~ of 3- (3 dime~hylamin~
propyl)-carbodiimide. After mixing by shaking for 2
minutes, 0.05~mg of B12 binding ~rotein (intrinsic
factor [IP~ from hog gut obtained fr~m Dr. Ro~ Allen,
Denver; CO) and 0.75 mg Qf human ~eru~ albumin (~SA,
obtained from Sigma Ch~mical Co., ~ 8763) were addea to
~30 ml in water. The p~ wa~ adju&ted to pH 5.6 ~nd
maintained by the addi~ion of 0.1~ ~Cl or 0.1N NaO~ for
three hour~. The p~rticle~ w~re then washed with 10 ml~
~f O.lM Borate with 0.5M NaCl p~ 8.3, 10 ml~ of phosphate
buf~ered ~alin~ (PBS) with 0.1~ ~SA, and 10 mls of
distilled water empl~ying the ~agnetic ~eparation
techni~ue a in ~c~ion 7.1. P~rticles were washed three
times with PBS and ~t~red in PBS unt~l u~e.





71>9~ ETIC PARl`ICLE COMPETITIVE
~1~
Usi1ng the ~P- ~nd ~ collpled ~agnetic particles
made by the method of Bection 7.7, a ti'cering of the
particles wa~ performed to ~cert~in the quantity of
particle~ neede~ in a competitive binding ass~y for
vitamin B12 (B12). The following ~s~ay prs:>tocol was
u ~ed:
l00 I!ls ~f standard and 1900 IJls or' tracer buffer were
added to 12x7~mm polypropyl~ne tubesc The mixture~ were -
placed into a boilin~ water bath for l~ minutes to effect
denatur~tion of binding proteins in hulaan ~erum samples.
15 Then l00 l l~ of v~rious concentrations of magnetic
part~cles in phosphate buffer were ~dded to determine the
optimal quantity of particles for assaying B12
conc~ntrations between 0 and 2000 pico~ram/ml (pg/~l).
After incubation of the mixtues for 1 bour at room
temperatur~y a magnetic separation of bound and free B12
was performed acc~rding to the procedur~ of ~nd ~sing the
magnetic rack described in Section 7O5~ Radioactivity in
the pellet~ was then counted on a Tracor 1290 Gamma
Counter ~Tracor ~nalytic, Inc. ~ Elk ~rove Village, IL).
~be reagents u~ed in the ~ssay were a~ ~oll~ws:
B12 standaras were obtained from Corning ~edical ~nd
Scientific, Divi~ion o~ Corn~ng Gla~ Work~, Medfield, ~A
~474267. They ~re made with B12-free human ~erum
albumin in PBS ~nd sodium azide ~dded a~ a preservative.
3D Th~ tracer was 57C~ B12 (vita~in Bl~ tagged with
radioactiv~ ~obalt~ ~r~m Cornang Medic~l and ~ientific~
Division of Corning ~la~ ~ork~, ~edfield, MA, ~47~2B7.
The tracer i5 in a borate buf~er p~ 9.2, containing 0.001
potassium cyanide and ~odium ~zide. Magnetic particles


&~7'~
- 5~

were diluted in PBS ~t var~vuE; concentr~tions ~ determine
~che quan~i'cy o~ par~icl~s need~d l:o mea~ure B12
concentration~ be'cween O ~nd 2000 p~/mlD
~ quantity of magnetic par~icle~ o~E approximately
50 l~g/tube wa~ selected and wa~ us~d in the Bl~
competi~ivc binding ~s~ay ~ 'co con~ltruct a ~andard
c:urve; the da~a are presented in ~able VIII.

TA13LE ~JI I I
lû -
B12 C~MPETITIVE BINDING RSS~__S A DARD CURVE

BConcentr~tion ~GQ9~e~es)
--12

0 pg~ml 552 3
lûO pg/ml 5220
250 pg/ml 4169
500 pg/ml 3295
1000 pg~ml 2278
2000 pg/ml 1745
Tot~l 16515



7 .10. COUPLIPæ OF P~NETIC PARTICLES COATED W~T~
AMINOETHY Ir 3- AMINOPROE~ L SILANE TO PROTEINS
7.,10.1. CO~lPLI~æ OP' N- 2- AMINO~T~ I.- 2- AMINOPROFYL ~AGNETIC
PARTICI,ES T{~ ANTIBt)DIES ~0 T~RIIODOTHY RONINE
~0 .
Six- tenth~ of a ç~r~m of N- 2- aminoethyl- 3-
aminopropyl magnetic p~rticle~ (~bbreviated "DINR
particles for ~dinitrogen", ~ignifying that th~ particles
have a N/Si ra'cio of 2) prepared a~ in Section '7. 2. were


~6~7~
w51-

resuspended in water. The particle~ were w~shed once in
water and ~hen twice with 30 mls of O.lM pho~phate buffer,
pB 7 . 4 with ~agnetic ~eparation~ between wa~hing~. After
su~pending the washed p~rticle6 ~n 15 ~1~ of 0.1
phosphate, 15 ~1~ of a 5% (V/V) ~olution of
glutaraldehyde, formed by dilutin~ 25~ glutar&ldehyde
(G-5~82, Sigma Chemical Co., St. Loui~, ~0) with D~lM
phosphate, were added. The particles were mixed for 3
hour~ ~t room temperature by gently rotating the reaction
1~ vessel. Unreacted glutaraldehyde wa~ washed ~way with 5
additions of 30 ~1~ of O.LM pho phate bu~fer. The
glutaraldehyde activated particle~ were th~n resuspended
in 15 mls of 0.LM pho phate.
Trii~dothyronine ~T ) ~nti~erum ~1-6 ml8
~5 3
obtained by immunizing rabbit~ with T3-BSA conjugate's)
w~s added to ~he activated particle~ and ~tirred on a
wheel mixer at room temperature for 16 to 24 hours. The
~3 antibod~ coupled particles were washed once with 3a
ml~ of 0~1~ pho~phate and suspended in 15 mls of ~.2~
glycine solution in order to react any unreacted aldehyde
groups. The su pension was mixed by ~hak ing ~or 25
minutesO The antibod~ coupl~d particles were wa~hed with
30 mls of 0.1 phosphate~ 30 mls of ethanol ~nd twice with
150 mls of PBS with 0.1~ bovine serum albumin (BSA~. They
were resu~pended in P8S, 1% BSA and stored at ~C until
used f~r RI~ for T3.

7.10.20 COUPLING OF ~ ~ AMINO~T~ ~ ~ AMINOPROPYL
MAGN~TIC PARTICLES ~0 ANTI~ODIES TO
3n T~YROID STIMULATING HORMONE
The coupling procedure of Section 6.1Q.l ~a~
followed with minor ~odificatio~sO Twenty gr~8 of DIN
particle~ were wa~hed ~hree times with 1.5 liter~ of
methanol prior to glutaraldehyd~ ~tiv~tion. Glut~r-


769
- 5~
/



aldehyde ~ctivation w~s perforll~ed ~ in ~ection 7,1U.l.
with adj~stment~ for sc2ll1e.
A goat gam~a glDbulin fra::tion c:vntalning
antibodles to human thyroid stimulating hormone (TSH) wa6
5 coupled ~o the DIN particle~ r~her thlan who~e ~nti~era.
Fractionation wa~ accompli~hed by pr~c~pitatiorl of
gammaglobulin$ ~ith 404 ammoni~ ulfa1ke followed by
dialy~is agains~c 2BS. Approximately 4 grams of ~rotein
(200 mls at 20 mg/ml) were coupled. Complete attachment
10 of protein was evident by the ab~ence o~ optical density
at 280 nm in the 6upernatant after coupling. This
indicated the attachment of about 20 mg of protein per
gram o particl~s. Tbe particles were then washed three
times with 1. 5 liter~ of lM NaCl, three times with PBS and
15 incuba'ced at 50C overnight. Par~icle~ were ~hen washed 3
more times in PBS/BSA and titered for use in the TS~ ass~y.

7011~ ~NETIC ~P~RTICLE RAD~OIMMUNOASS~
FOR TRI IODOTHY P~ONINE _ _
The quantity of particles to be used in the T3
~IA was determined in the f~llowing a~say:

Stanaards were prep~r~d by adaing T3 to T3- free bu~nan
25 6erum a with T4 (see Section 7.5~)

Tracer was 125IT3 ~rom Corning ~edical and Scientific,
Divi6ion of Corning Glass Works, Medfield~ ~A (#471û6).

3~ Magnetic par~icles were diluted ~o var ious concentr~tions
in P~S~ BSA to deteEmine the quantity of particles needed.

The a ~ay protocol was ~s ~ollow6: 5û ~1~ o~ ~tandard,
100 ~lls of tracer ~nd B00 ~ of D~ 7agnetic particle~
~5


were pipet ed into 12a;:75mm polypropylene tubec. After
vortexing" the tubes were incuba'ced :Eor 2 hour~ at room
temperature. The ~ssay was îerminat~d by IlDa~n~tic
~epar~tion. By titering the quantity of particles in the
assay wlth ~ 0 ng/ml 6tand~rd/ ~ quan~i.ty of ~0 ~g/tube
was deemed to be optimal or the as~ay protc>cvl~ Table ~X
shows the T3 P~IA ~tand~rd curve data obtaill~d with ~chese
par t icles .

~ . IW3L~ IX

RIA STANDARD CV3~ OR T3

T3 Conc e n t r a t i on 9~e s )

0. O ng/ml 17278
0. 25 ng/ml 1~034
0 . 50 ng/ml 13456
1. ûO ng/ml 12127
2~ 20 00 ng~ml 8758
4 . 00 ng~ml S776
8 . 00 ng/ml 3~97
~otal 26946
~5
. . .

7 .1~ ~ ~NETIC PARTICLE RADIOIMMUPIOASS~
FOR THY ROID ;E;TIMULATIt~ HVRMONE
3~
The ~auanti~y of particle~ to be~ usedl in the TSH
RIA was determined in the following ~ssay:

Stan~ards w~re in normal human ser~ ~Corning ~edical and
35 Scientiic, ~47186, Medfield, ~).

f~


Tracer was 125I-r~bbit ~nti-TS~ ~ntibody in PBS ~corning
~edical and Scientific, ~4741~5~ Medfield, MA).

Magnetic particle~ w~re diluted to ~eriou~ concentration~
in PB~ BSA to determine the ~uantity of particle~ need~d.

The assay protocol was as follow~: 100 yl~ of ~tandaxd
and 100 ~1~ of tracer were pipetted ints 12x75 ~m
polypropylene tube i vort~xed, and incubated for 3 hours
1~ ~t room tempera~ure~ ~agnetic par~icle~ ~500 ~lsl w~re
added and the mixture was ~ortexe~ and inçubated for ~
hour at room temperatule~ 500 ~15 of water were adaed and
the usual magnetic separation was e~ployed to ~epirate
bound from unbound tracer. In the presence of TS~, a
~andwich is for~2d bet~een magnetic antibody (goat
anti-TS~ antibody, ~ee Secti~n ~lO~lo) TS~ and tracer
5I-antibody (r~bbit ~nti TS~ antibody~. Thu~,
increasing concentrations of ana~yte (TS~) increase the
amount of bound radioactivity. Table X ~hows the T~H RIA
~tandard rurve data obtain~d by thi~ proc~dure~




TABLE X




~IA STANDARD CURVE FOR TS~




TS~ Concentration cpm




O ~IU/ml* 1615


~ U~ 309


3D 3O0 ~IU~l* 3014




6~0 ~IU/~l~ ~448


15~0 ~ 7793



30.0 ~XU/ml* 11063


60.0 ~IU/mln 15030


~o~al 45168




~IU=micro Int~r~ational Unit~




y` ~
^ ~


7013. CQUPLING VF MRGNETIC PARTI~LES CO~TED
WIT~ ~ ~ ~MIN~ET~Y ~ ~ AMINOPRO~ L ~ILANE
~0 EN~ MES ~ USE ~ G LUTA~ALDE~ DE
Magnetic particleæ ~1 gm3 were ~ct~v~ted with
glutaraldehyde as in Sectisn 7.1001. After washing, the
particles were resu~pended in 15 mls of PBS. Then 3 mls
o particle~ ~2 gm) were ~ixed with 5 ~ of ~lkaline
1~ phosphat~se (Sigma Che~ical Company, ~ 9761~ or 5 ~g of
galactosida~e ~Sigma Chemical Company, 5635) di~solved
in 2.0 ml~ of PBS. The coupl~d particles were washed with
glyci~e an~ then w~shed 5 times with PBS and re ~æpended
in PBS with 0.14 ~SA.
Enzyme as~ays for magnetic alkaline phosphatase
activity was perfoxmed as follows:
To a 3 ~1 cuvette 3 ~læ of 0.05~ Tri~ ~C.l were
added, pH B.O, with 3mM p nitrophenyl-phosphate~ Then 100
~ls of diluted magnetic particles with ~oupled alkaline
phosphatase were added. The increase in optical density
at 410 nm wa~ record~d.
Enzyme assay for magnetic ~ galactosida~e
actiYity wa~ performed as follows:
To a 3ml cuvett~ 3 ml~ of O.lM phosphate were
adde~, p~ 7.4, ~ith O.OlM ~erc~ptoethanol and 0.005
~ ~itrophenyl~ ~ ~ galactopyrano~id~. ~hen 100 ~ls o~
diluted magnetic p2rticl~6 coupled to ~ galactosidase were
added. The increase in optical density at 410 ~m was
recorded 7
It is apparent ~ha~ many modification~ and
variations of this invention a hereinabo~e ~et forth may

be made without departing from the spirit ~nd 8cope
thereofD ~h~ ~p~cific emb~di~ent~ de cribed are given by
way nf example only and ~he invention i~ lim~ted only by
the terms of th~ appended clai~s.

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Administrative Status

Title Date
Forecasted Issue Date 1990-03-20
(22) Filed 1984-04-30
(45) Issued 1990-03-20
Deemed Expired 2007-03-20
Correction of Expired 2012-12-05

Abandonment History

There is no abandonment history.

Payment History

Fee Type Anniversary Year Due Date Amount Paid Paid Date
Registration of a document - section 124 $0.00 1984-08-31
Application Fee $0.00 1989-03-30
Maintenance Fee - Patent - Old Act 2 1992-03-20 $100.00 1992-02-03
Maintenance Fee - Patent - Old Act 3 1993-03-22 $100.00 1993-03-22
Maintenance Fee - Patent - Old Act 4 1994-03-21 $100.00 1994-02-22
Maintenance Fee - Patent - Old Act 5 1995-03-20 $150.00 1995-02-02
Maintenance Fee - Patent - Old Act 6 1996-03-20 $150.00 1996-02-06
Maintenance Fee - Patent - Old Act 7 1997-03-20 $150.00 1997-03-20
Registration of a document - section 124 $50.00 1998-03-19
Maintenance Fee - Patent - Old Act 8 1998-03-20 $150.00 1998-03-19
Maintenance Fee - Patent - Old Act 9 1999-03-22 $150.00 1999-02-03
Maintenance Fee - Patent - Old Act 10 2000-03-20 $200.00 2000-03-02
Maintenance Fee - Patent - Old Act 11 2001-03-20 $200.00 2001-03-05
Maintenance Fee - Patent - Old Act 12 2002-03-20 $200.00 2002-03-05
Maintenance Fee - Patent - Old Act 13 2003-03-20 $200.00 2003-03-05
Maintenance Fee - Patent - Old Act 14 2004-03-22 $250.00 2004-03-04
Maintenance Fee - Patent - Old Act 15 2005-03-21 $450.00 2005-03-04
Owners on Record

Note: Records showing the ownership history in alphabetical order.

Current Owners on Record
CHIRON DIAGNOSTICS CORPORATION
Past Owners on Record
ADVANCED MAGNETICS, INC.
CHAGNON, MARK S.
GROMAN, ERNEST V.
JOSEPHSON, LEE
WHITEHEAD, ROY A.
Past Owners that do not appear in the "Owners on Record" listing will appear in other documentation within the application.
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Drawings 1993-09-18 2 94
Claims 1993-09-18 7 236
Abstract 1993-09-18 1 15
Cover Page 1993-09-18 1 23
Description 1993-09-18 52 2,534
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Fees 1998-03-19 1 38
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