Magnetic particles for use in separations

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.

TABLE OF CONTENTS 
1. Field of the Invention 
2. Background of the Invention 
2.1. Magnetic Separations in Biological Systems: General Considerations 
2.2. Separations in Radioimmunoassays 
2.3. Application of Magnetic Separations in Other Biological Systems 
3. Nomenclature 
4. Summary of the Invention 
5. Brief Description of the Figures 
6. Detailed Description of the Invention 
6.1. Magnetic Particle Preparation 
6.2. Silane Coupling Chemistry 
6.3. Use of Magnetic particles in Biological 
6.4. Use of Magnetic Particles in Immobilized Enzyme Systems 
6.5. Use of Magnetic Particles in Affinity Chromatography 
7. Examples 
7.1. Preparation of Metal Oxide 
7.2. Silanization 
7.3. Physical Characteristics of Silanized Magnetic Particles 
7.4. Coupling of Aminophenyl Magnetic Particles to Antibodies to Thyroxine 
7.5. Magnetic Particle Radioimmunoassay for Thyroxine 
7.6. Magnetic Particle Radioimmunoassay for Theophylline 
7.7. Effect of Variation of Fe.sup.2+ /Fe.sup.3+ Ratio of Magnetic 
Particles on T.sub.4 Radioimmunoassay 
7.8. Coupling of Carboxylic Acid-Terminated Magnetic Particles to B.sub.12 
Binding Protein 
7.8.1. Preparation of Carboxylic Acid-Tersinated Magnetic Particles 
7.8.2. Carbodiimide Coupling of B.sub.12 Binding Protein and Human Serum 
Alubmin to Carboxylic Acid-Terminated Particles 
7.9. Magnetic Particle Competitive Binding Assay for Vitamin B.sub.12 
7.10. Coupling of Magnetic particles Coated with 3-Aminopropyl or 
N-2-Aminoethyl-3-Aminopropyl Silane to Proteins 
7.10.1. Coupling of N-2-Aminoethyl-3-Aminopropyl Magnetic Particles to 
Antibodies to Triiodothyronine 
7.10.2. Coupling of N-2-Aminoethyl-3-Aminopropyl Magnetic Particles to 
Antibodies to Thyroid Stimulating Hormone 
7.11 Magnetic Particle Radioimmunoassay for Triiodothyronine 
7.12. Magnetic Particle Radioimmunoassay for Thyroid Stimulating Hormone 
7.13. Coupling of Magnetic Particles Coated with 
N-2-Aminoethyl-3-Aminopropyl Silane to Enzymes by Use of Glutaraldehyde 
1. FIELD OF THE INVENTION 
This invention relates to magnetically responsive particles and to their 
use in systems in which the separation of certain molecules from the 
surrounding medium is necessary or desirable. More particularly, the 
invention relates to methods for the preparation of magnetically 
responsive particles comprising a metal oxide core surrounded by a stable 
silane coating to which a wide variety of organic and/or biological 
molecules may be coupled. The particles (coupled or uncoupled) can be 
dispersed in aqueous media without rapid gravitational settling and 
conveniently reclaimed from the media with a magnetic field. Preferably, 
the process provided herein yields particles that are superparamagnetic; 
that is, they do not become permanently magnetized after application of a 
magnetic field. This property permits the particles to be redispersed 
without magnetic aggregate formation. Hence the particles may be reused or 
recycled. Stability of the silane coating and the covalent attachent of 
molecules thereto also contribute to particle use and reuse. 
The magnetically responsive particles of this invention may be coupled to 
biological or organic molecules with affinity for or the ability to adsorb 
or which interact with certain other biological or organic molecules. 
Particles so coupled may be used in a variety of in vitro or in vivo 
systems involving separation steps or the directed movement of coupled 
molecules to particular sites, including, but not limited to, 
immunological assays, other biological assays, biochemical or enzymatic 
reactions, affinity chromatographic purifications, cell sorting and 
diagnostic and therapeutic uses. 
2. BACKGROUND OF THE INVENTION 
2.1. Magnetic Separations in Biological Systems: General Considerations 
The use of magnetic separations in biological systems as an alternative to 
gravitational or centrifugal separations has been reviewed [B. L. 
Hirschbein et al., Chemtech, March 1982:172-179 (1982); M. Pourfarzaneh, 
The Ligand Quarterly 5(1):41-47 (1982); and P. J. Halling and P. Dunnill, 
Enzyme Microb. Technol. 2:2-10 (1980)]. Several advantages of using 
magnetically separable particles as supports for biological molecules such 
as enzymes, antibodies and other bioaffinity adsorbents are generally 
recognized. For instance, when magnetic particles are used as solid phase 
supports in immobilized enzyme systems [see, e.g., P. J. Robinson et al., 
Biotech. Bioeng., XV:603-606 (1973)], the enzyme may be selectively 
recovered from media, including media containing suspended solids, 
allowing recycling in enzyme reactors. When used as solid supports in 
immunoassays or other competitive binding assays, magnetic particles 
permit homogeneous reaction conditions (which promote optimal binding 
kinetics and minimally alter analyte-adsorbent equilibrium) and facilitate 
separation of bound from unbound analyte, compared to centrifugation. 
Centrifugal separations are time-consuming, require expensive and 
energy-consuming equipment and pose radiological, biological and physical 
hazards. Magnetic separations, on the other hand, are relatively rapid and 
easy, requiring simple equipment. Finally, the use of non porous 
adsorbent-coupled magnetic particles in affinity chromatography systems 
allows better mass transfer and results in less fouling than in 
conventional affinity chromatography systems. 
Although the general concept of magnetizing molecules by coupling them to 
magnetic particles has been discussed and the potential advantages of 
using such particles for biological purposes recognized, the practical 
development of magnetic separations has been hindered by several critical 
properties of magnetic particles developed thus far. 
Large magnetic particles (mean diameter in solution greater than 10 
microns(.mu.)) can respond to weak magnetic fields and magnetic field 
gradients; however, they tend to settle rapidly, limiting their usefulness 
for reactions requiring homogeneous conditions. Large particles also have 
a more limited surface area per weight than smaller particles, so that 
less material can be coupled to them. Examples of large particles are 
those of Robinson et al. [supra] which are 50-125.mu. in diameter, those 
of Mosbach and Anderson [Nature, 270:259-261 (1977)] which are 60-140.mu. 
in diameter and those of Guesdon et al. [J. Allergy Clin. Immunol. 
61(1):23-27 (1978)] which are 50-160.mu. in diameter. Composite particles 
made by Hersh and Yaverbaum [U.S. Pat. No. 3,933,997] comprise 
ferromagnetic iron oxide (Fe.sub.3 O.sub.4) carrier particles. The iron 
oxide carrier particles were reported to have diameters between 1.5 and 
10.mu.. However, based on the reported settling rate of 5 minutes and 
coupling capacity of only 12 mg of protein per gram of composite particles 
[L. S. Hersh and S. Yaverbaum, Clin. Chim. Acta, 63:69-72 (1975)], the 
actual size of the composite particles in solution is expected to be 
substantially greater than 10.mu.. 
The Hersh and Yaverbaum ferromagnetic carrier particles of U.S. Pat. No. 
3,933,997 are silanized with silanes capable of reacting with anti-digoxin 
antibodies to chemically couple the antibodies to the carrier particles. 
Various silane couplings are discussed in U.S. Pat. No. 3,652,761, which 
is hereby incorporated by reference. That the diameters of the composite 
particles are probably greater than 10.mu. 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 in Enzymology, K. Mosbach (ed.), 44:134-148, 140 
(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 solutions with acid [H. W. Weetall, in: Methods in Enzymology, 
supra, p. 139 (1976)] have also been used. Each 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, including, 
e.g., cross linking between particles by siloxane formation, van der Waals 
interactions or physical adhesion between adjacent particles. This 
interparticle bonding yields covalently or physically bonded aggregates of 
silanized carrier particles of considerably larger diameter 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. 
Small magnetic particles with a mean diameter in solution less than about 
0.03.mu. can be kept in solution by thermal agitation and therefore do not 
spontaneously settle. However, the magnetic field and magnetic field 
gradient required to remove such particles from solution are so large as 
to require heavy and bulky magnets for their generation, which are 
inconvenient to use in bench top work. Magnets capable of generating 
magnetic fields in excess of 5000 Oersteds are typically required to 
separate magnetic particles of less than 0.03.mu. in diameter. An 
approximate quantitative relationship between the net force (F) acting on 
a particle and the magnetic field is given by the equation below 
(Hirschbein et al., supra): 
EQU F=(X.sub.v -X.sub.v .degree.)VH(dH/dx), 
where X.sub.v and X.sub.v .degree. are the volume susceptibilities of the 
particle and the medium, respectively, V is the volume of the particle, H 
is the applied magnetic field and dH/ox is the magnetic field gradient. 
This expression is only an approximation because it ignores particle shape 
and particle interactions. Nevertheless, it does indicate that the force 
on a magnetic particle is directly proportional to the volume of the 
particle. 
Magnetic particles of less than 0.03.mu. are used in so-called ferrofluids, 
which are described, for example, in U.S. Pat. No. 3,531,413. Ferrofluids 
have numerous applications, but are impractical for applications requiring 
separation of the magnetic particles from surrounding media because of the 
large magnetic fields and magnetic field gradients required to effect the 
separations. 
Ferromagnetic materials in general become permanently magnetized in reponse 
to magnetic fields. Materials termed "superparamagnetic" experience a 
force in a magnetic field gradient, but do not become permanently 
magnetized. Crystals of magnetic iron oxides may be either ferromagnetic 
or superparamagnetic, depending on the size of the crystals. 
Superparamagnetic oxides of iron generally result when the crystal is less 
than about 300 .ANG.(0.03.mu.) in diameter; larger crystals generally have 
a ferromagnetic character. Following initial exposure to a magnetic field, 
ferromagnetic particles tend to aggregate because of magnetic attraction 
between the permanently magnetized particles, as has been noted by 
Robinson et al. [supra] and by Hersh and Yaverbaum [supra]. 
Dispersible magnetic iron oxide particles reportedly having 300 A diameters 
and surface amine groups were prepared by base precipitation of ferrous 
chloride and ferric chloride (Fe.sup.2+ /Fe.sup.3+ =1) in the presence of 
polyethylene imine, according to Rembaum in U.S. Pat. No. 4,267,234. 
Reportedly, these particles were exposed to a magnetic field three times 
during preparation and were described as redispersible. The magnetic 
particles were mixed with a glutaraldehyde suspension polymerization 
system to form magnetic polyglutaraldehyde microspheres with reported 
diameters of 0.1.mu.. Polyglutaraldehyde microspheres have conjugated 
aldehyde groups on the surface which can form bonds to amino containing 
molecules such as proteins. However, in general, only compounds which are 
capable of reacting with aldehyde groups can be directly linked to the 
surface of polyglutaraldehyde microspheres. Moreover, magnetic 
polyglutaraldehyde microspheres are not sufficiently stable for certain 
applications. 
2.2. Separations in Radioimmunoassays 
Radioimmunoassay (RIA) is a term used to describe methods for analyzing the 
concentrations of substances involving a radioactively labeled substance 
which binds to an antibody. The amount of radioactivity bound is altered 
by the presence of an unlabeled test substance capable of binding to the 
same antibody. The unlabeled substance, if present, competes for binding 
sites with the labeled substance and thus decreases the amount of 
radioactivity bound to the antibody. The decrease in bound radioactivity 
can be correlated to the concentration of the unlabeled test substance by 
means of a standard curve. An essential step of RIA is the separation of 
bound and free label which must be accomplished in order to quantitate the 
bound fraction. 
A variety of conventional separation approaches have been applied to 
radioimmunoassays (RIA) including coated tubes, particulate systems, and 
double antibody separation methods. Coated tubes, such as described in 
U.S. Pat. No. 3,646,346, allow separation of bound and free label without 
centrifugation but suffer from two major disadvantages. First, the surface 
of the tube limits the amount of antibody that can be employed in the 
reaction. Second the antibody is far removed (as much as 0.5 cm) from some 
antigen, slowing the reaction between the antibody and antigen [G. M. 
Parsons, in: Methods in Enzymology, J. Langone (ed.) 73:225 (1981); and P. 
N. Nayak, The Ligand Quarterly 4(4):34 (1981)]. 
Antibodies have been attached to particulate systems to facilitate 
separation [see, e.g., U.S. Pat. Nos. 3,652,761 and 3,555,143]. Such 
systems have large surface areas permitting nearly unlimited amounts of 
antibody to be used, but the particulates frequently settle during the 
assay. The tube frequently must be agitated to achieve even partial 
homogeneity [P. M. Jacobs, The Ligand Quarterly, 4(4):23-33 (1981)]. 
Centrifugation is still required to effect complete separation of bound 
and free label. 
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 homogeneity 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(12):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 et 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), repectively, hereby incorporated by 
reference]. Such methods suffer from large particle sizes (10-100.mu. 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 particles comprising low density cores of materials 
such as hollow glass or polyproplyene (4-10.mu. in diameter) with magnetic 
coatings (2 m.mu.10.mu. thick) covering a proportion of the particle 
surface. Anti-estradiol antibodies were coupled to such particles and 
their potential usefulness in estradiol RIAs was demonstrated. While this 
approach may have overcome the problem of settling, the particle size and 
the magnetic coating nonetheless present limitations on surface area and 
hence limitations on the availability of sites for antibody coupling. 
2.3. Application of Magnetic Separations in other Biological Systems 
Magnetic separations have been applied in other biological systems besides 
RIA. Several nonisotopic immunoassays, such as fluoroimmunoassays (FIA) 
and enzyme-immunoassays (EIA) have been developed which employ 
antibody-coupled (or antigen-coupled) magnetic particles. The principle of 
competitive binding is the same in FIA and EIA as in RIA except that 
fluorophores and enzymes, respectively, are substituted for radioisotopes 
as label. By way of illustration, M. Pourfarzaneh et al. and R. S. Kamel 
et al. developed magnetizable solid-phase FIAs for cortisol and phenytoin, 
respectively, utilizing ferromagnetic cellulose/iron oxide particles to 
which antibodies were coupled by cyanogen bromide activation [M. 
Pourfarzaneh et al., Clin. Chem., 26(6):730-733 (1980); R. S. Kamel et 
al., Clin. Chem., 26(9):1281-1284 (1980)]. 
A non-competitive solid phase sandwich technique EIA for the measurement of 
IgE was described by J.- L. Guesdon et al. [J. Allergy Clin. Immunol., 
61(1):23-27 (1978)]. By this method, anti-IgE antibodies coupled by 
glutaraldehyde activation to magnetic polyacrylamideagarose beads are 
incubated with a test sample containing IgE to allow binding. Bound IgE is 
quantitated by adding a second anti-IgE antibody labeled with either 
alkaline phosphatase or .beta.-galactosidase. The enzyme labeled second 
antibody complexes with IgE bound to the first antibody, forming the 
sandwich, and the particles are 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 B.sub.12 has 
been reported by D. S. 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 B.sub.12 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 matrix. 
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 isolate select viruses, bacteria and other cells from mixed 
populations [U.S. Pat. Nos. 3,970,518; 4,230,685; and 4,267,234, hereby 
incorporated by reference]. They have been used in affinity chromatography 
systems to selectively isolate and purify molecules from solution and are 
particularly advantageous for purifications from colloidal suspensions [K. 
Mosbach and L. Anderson, Nature 170:259-261 (1977), hereby incorporated by 
reference]. 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 sufficient to catalyze 
the biochemical reaction. Thereafter, the enzyme can be magnetically 
separated from products and unreacted substrate and potentially can be 
reused. Magnetic particles have been used as supports for 
.alpha.-chymotrypsin, .beta.-galactosidase [U.S. Pat. No. 4,152,210, 
hereby incorporated by reference] and glucose isomerase [U.S. Pat. No. 
4,343,901, hereby incorporated by reference] 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". 
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 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 as any biological or other 
organic molecule capable of specific or nonspecific binding 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, antibody/antigen, antibody/hapten, 
enzyme/substrate, enzyme/inhibitor, enzyme/cofactor, binding 
protein/substrate, carrier protein/substrate, lectin/carbohydrate, 
receptor/hormone, receptor/effector or repressor/inducer bindings or 
interactions. 
The term "coupled magnetically responsive particle" or "coupled magnetic 
particle" is defined as any magnetic particle to which one or more types 
of bioaffinity adsorbents are coupled by covalent bonds, which covalent 
bonds may be amide, ester, ether sulfonamide, disulfide, azo or other 
suitable organic linkages depending on the functionalities available for 
bonding on both the coat of the magnetic particle and the bioaffinity 
adsorbent(s). 
The term "silane" refers to any bifunctional organosilane and is defined as 
in U.S. Pat. No. 3,652,761 as an organofunctional and silicon functional 
silicon compound characterized in that the silicon portion of the molecule 
has an affinity for inorganic materials while the organic portion of the 
molecule is tailored to combine with organics. Silanes are suitable 
coating materials for metal oxide cores by virtue of their 
silicon-functionalities and can be coupled to bioaffinity adsorbents 
through their organofunctionalities. 
The term "superparamagnetism" is defined as that magnetic behavior 
exhibited by iron oxides with crystal size less than about 300.ANG., which 
behavior is characterized by responsiveness to a magnetic field without 
resultant permanent magnetization. 
The term "ferromagnetism" is defined as that magnetic behavior exhibited by 
iron oxides with crystal size greater than about 500.ANG., which behavior 
is characterized by responsiveness to a magnetic field with resultant 
permanent magnetization. 
The term "ferrofluid" is defined as a liquid comprising a colloidal 
dispersion of finely divided magnetic particles of subdomain size, usually 
50-500 .ANG., in a carrier liquid and a surfactant material, which 
particles remain substantially uniformly dispersed throughout the liquid 
carrier even in the presence of magnetic fields of up to about 5000 
Oersteds. 
The term "immunoassay" is defined as any method for measuring the 
concentration or amount of an analyte in a solution based on the 
immunological binding or interaction of a polyclonal or monoclonal 
antibody and an antigen, which method (a) requires a separation of bound 
from unbound analyte; (b) employs a radioisotopic, fluorometric, 
enzymatic, chemiluminescent or other label as the means for measuring the 
bound and/or unbound analyte; and (c) may be described as "competitive" if 
the amount of bound measurable label is generally inversely proportional 
to the amount of analyte originally in solution or "non-competitive" if 
the amount of bound measurable label is generally directly proportional to 
the amount of analyte originally in solution. Label may be in the antigen, 
the antibody, or in double antibody methods, the second antibody. 
Immunoassays are exemplified by, but are not limited to, radioimmunoassays 
(RIA), immunoradiometric assays (IRMA), fluoroimmunoassays (FIA), enzyme 
immunoassays (EIA), and sandwich method immunoassays. 
The term "binding assay" or "non immune assay" is defined as any method for 
measuring the concentration or amount of an analyte in solution based on 
the specific or nonspecific binding or interaction, other than 
antibody/antigen binding or interaction, of a bioaffinity adsorbent and 
another biological or organic molecule, which method (a) requires a 
separation of bound from unbound analyte; (b) employs a radioisotopic, 
fluorometric, enzymatic, chemiluminescent or other label as the means for 
measuring the bound and/or unbound analyte; and (c) may be described as 
"competitive" if the amount of bound measurable label is generally 
inversely proportional to the amount of analyte originally in solution or 
"non-competitive" if the amount of bound measurable label is generally 
directly proportional to the amount of analyte originally in solution. 
The term "immobilized enzyme reaction" is defined as any enzymatically 
catalyzed biochemical conversion or synthesis or degradation wherein the 
enzyme molecule or active site thereof is not freely soluble but is 
adsorptively or covalently bound to a solid phase support, which support 
is suspended in or contacted with the surrounding medium and which may be 
reclaimed or separated from said medium. 
The term "affinity chromatography" is defined as a method for separating, 
isolating, and/or purifying a selected molecule from its surrounding 
medium on the basis of its binding or interaction with a bioaffinity 
adsorbent adsorptively or covalently bound to a solid phase support, which 
support is suspended in or contacted with the surrounding medium and which 
may be reclaimed or separated from said medium. 
4. SUMMARY OF THE INVENTION 
This invention provides novel magnetic particles useful in biological 
applications involving the separation of molecules from or the directed 
movement of molecules in the surrounding medium. Methods and compositions 
for preparing and using the magnetic particles are provided. 
The magnetic 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 can be covalently bonded through 
selected coupling chemistries. 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.mu. 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 m.sub.2 /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 required to separate 
smaller particles. Magnets used to effect separations of 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 
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 the 
magnetic aggregation associated with ferromagnetic particles and permit 
redispersion and reuse. 
The method for preparing the magnetic particles may comprise precipitating 
metal salts in base to form fine magnetic metal oxide crystals, 
redispersing and washing the crystals in water and in an electrolyte. 
Magnetic separations may be used to collect the crystals between washes if 
the crystals are superparamagnetic. The crystals may then be coated with a 
material capable of adsorptively or covalently bonding to the metal oxide 
and bearing organic functionalities for coupling with bioaffinity 
adsorbents. 
In one embodiment the coating around the metal oxide core is a polymer of 
silane. The silanization may be performed by redispersing the magnetic 
metal oxide crystals in an acidic organic solution, adding an 
organosilane, dehydrating by heating in the presence of a wetting agent 
miscible both in water and the organic solution, and washing the resulting 
magnetic silanized metal oxides. Alternatively, silanization may be 
performed in acidic aqueous solution. 
The magnetic particles of this invention can be covalently bonded by 
conventional coupling chemistries to bioaffinity adsorbents including, but 
not limited to, antibodies, antigens and specific binding proteins, which 
coupled magnetic particles can be used in immunoassays or other binding 
assays for the measurement of analytes in solution. Such assays preferably 
comprise mixing a sample containing an unknown concentration of analyte 
with a known amount of labeled analyte in the presence of magnetic 
particles coupled to a bioaffinity adsorbent capable of binding to or 
interacting with both unlabeled and labeled analyte, allowing the binding 
or interaction to occur, magnetically separating the particles, measuring 
the amount of label associated with the magnetic particles and/or the 
amount of label free in solution and correlating the amount of label to a 
standard curve constructed similarly to determine the concentration of 
analyte in the sample. 
The magnetic particles of this invention are suitable for use in 
immobilized enzyme systems, particularly where enzyme recycling is 
desired. Enzymatic reactions are preferably carried out by dispersing 
enzyme-coupled magnetic particles in a reaction mixture containing 
substrate(s), allowing the enzymatic reaction to occur, magnetically 
separating the enzyme-coupled magnetic particle from the reaction mixture 
containing product(s) and unreacted substrate(s) and, if desired, 
redispersing the particles in fresh substrate(s) thereby reusing enzyme. 
Affinity chromatography separations and cell sorting can be performed using 
the magnetic particles of this invention, preferably by dispersing 
bioaffinity adsorbent-coupled magnetic particles in solutions or 
suspensions containing molecules or cells to be isolated and/or purified, 
allowing the bioaffinity adsorbent and the desired molecules or cells to 
interact, magnetically separating the particles from the solutions or 
suspension and recovering the isolated molecules or cells from the 
magnetic particles. 
It is further contemplated that the magnetic particles of this invention 
can be used in in vivo systems for the diagnostic localization of cells or 
tissues recognized by the particular bioaffinity adsorbent coupled to the 
particle and also for magnetically directed delivery of therapeutic agents 
coupled to the particles to pathological sites. 
The magnetic particles of this invention overcome problems associated with 
the size, surface area, gravitational settling rate and magnetic character 
of previously developed magnetic particles. Gravitational settling times 
in excess of about 1.5 hours can be achieved with magnetic particles of 
the invention, where the gravitational settling time is defined to be the 
time for the turbidity of a dispersion of particles of the invention in 
the absence of a magnetic field to fall by fifty percent. Magnetic 
separation times of less than about ten minutes can be achieved with 
magnetic particles of the invention by contacting a vessel containing a 
dispersion of the particles with a pole face of a permanent magnet no 
larger in volume than the volume of the vessel, where the magnetic 
separation time is defined to be the time for the turbidity of the 
dispersion to fall by 95 percent. Furthermore, the use of silane as the 
coating surrounding the metal oxide core of the magnetic particles 
described herein makes possible the coupling of a wide variety of 
molecules under an equally wide variety of coupling conditions compared to 
other magnetic particle coatings known in the art with more limited 
coupling functionalities. 
Preferred magnetically responsive particles of the invention have metal 
oxide cores comprised of clusters of superparamagnetic crystals, affording 
efficient separation of the particles in low magnetic fields (100-1000 
Oersteds) while maintaining superparamagnetic properties. Aggregation of 
particles is controlled during particle synthesis to produce particles 
which are preferably small enough to avoid substantial gravitational 
settling over times sufficient to permit dispersions of the particles to 
be used in an intended biological assay or other application. The 
advantage of having superparamagnetic cores in magnetically responsive 
particles is that such particles can be repeatedly exposed to magnetic 
fields. Because they do not become permanently magnetized and therefore do 
not magnetically aggregate, the particles can be redispersed and reused. 
Even after silanization, preferred particles of the invention having cores 
made up of clusters of crystals exhibit a remarkably high surface area per 
unit weight and a generally correspondingly high coupling capacity, which 
indicates that such particles have an open or porous structure. 
None of the prior art magnetic particles used in the biological systems 
described in Section 2 above have the same composition, size, surface 
area, coupling versatility, settling properties and magnetic behavior as 
the magnetic particles of the invention. The magnetic particles of this 
invention are suitable for many of the assays, enzyme immobilization, cell 
sorting and affinity chromatography procedures reported in the literature 
and, in fact, overcome many of the problems associated with particle 
settling and reuse experienced in the past with such procedures.

6. DETAILED DESCRIPTION OF THE INVENTION 
6.1. Magnetic Particle Preparation 
Preferred magnetic particles of the invention may be made in two steps. 
First, superparamagnetic iron oxides are made by precipitation of divalent 
(Fe.sup.2+) and trivalent (Fe.sup.3+) iron salts, e.g., FeCl.sub.2 and 
FeCl.sub.3, in base. Secondly an organosilane coating is applied to the 
iron oxide. 
The ratio of Fe.sup.2+ and Fe.sup.3+ can be varied without substantial 
changes in the final product by increasing the amount of Fe.sup.2+ while 
maintaining a constant molar amount of iron. The preferred Fe.sup.2+ 
/Fe.sup.3+ ratio is 2/1 but an Fe.sup.2+ /Fe.sup.3+ ratio of 4/1 also 
works satisfactorily in the procedure of Section 7.1 (See also Section 
7.7). An Fe.sup.2+ /Fe.sup.3+ ratio of 1/2 produces magnetic particles of 
slightly inferior quality to those resulting from the higher Fe.sup.2+ 
/Fe.sup.3+ ratios This magnetic oxide tends to "bleed" or become soluble 
during the rinsing procedure of Section 7.1 and the particle size is more 
heterogeneous than the resulting from Fe.sup.2+ /Fe.sup.3+ of 2/1 or 4/1. 
Nevertheless, it can be silanized to yield a usable magnetic particle as 
demonstrated in Section 7.7. 
Aqueous solutions of the iron salts are mixed in a base such as sodium 
hydroxide which results in the formation of a crystalline precipitate of 
superparamagnetic iron oxide. The precipitate is washed repeatedly with 
water by magnetically separating it and redispersing it until a neutral pH 
is reached. The precipitate is then washed once in an electrolytic 
solution, e.g. a sodium chloride solution. The electrolyte wash step is 
important to insure fineness of the iron oxide crystals. Finally the 
precipitate is washed with methanol until a residue of 1.0% (V/V) water is 
left. 
The repeated use of magnetic fields to separate the iron oxide from 
suspension during the washing steps is facilitated by superparamagnetism. 
Regardless of how many times the particles are subjected to magnetic 
fields, they never become permanently magnetized and consequently can be 
redispersed by mild agitation. Permanently magnetized (ferromagnetic) 
metal oxides cannot be prepared by this washing procedure as they tend to 
magnetically aggregate after exposure to magnetic fields and cannot be 
homogeneously redispersed. 
Other divalent transition metal salts such as magnesium, manganese, cobalt, 
nickel, zinc and copper salts may be substituted for iron (II) salts in 
the precipitation procedure to yield magnetic metal oxides. For example, 
the substitution of divalent cobalt chloride (CoCl.sub.2) for FeCl.sub.2 
in the procedure of Section 7.1 produced ferromagnetic metal oxide 
particles. Ferromagnetic metal oxides such as that produced with 
CoCl.sub.2, may be washed in the absence of magnetic fields by employing 
conventional techniques of centrifugation or filtration between washings 
to avoid magnetizing the particles. As long as the resulting ferromagnetic 
metal oxides are of sufficiently small diameter to remain dispersed in 
aqueous media, they may also be silanized and coupled to bioaffinity 
adsorbents for use in systems requiring a single magnetic separation, e.g. 
certain radioimmunoassays. Ferromagnetism limits particle usefulness in 
those applications requiring redispersion or reuse. 
Magnetic metal oxides produced by base precipitation may be coated by any 
one of several suitable 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).sub.3 may be used wherein (OX).sub.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-aminophenyltrimethoxysilane, 3-aminopropyltrimethoxysilane, 
N-2-aminoethy-3-aminopropyltrimethoxysilane, triaminofunctional silane 
(H.sub.2 NCH.sub.2 CH.sub.2 --NH--CH.sub.2 CH.sub.2 --NH--CH.sub.2 
CH.sub.2 --CH.sub.2 --Si--(OCH.sub.3).sub.3, n-dodecyltriethoxysilane and 
n-hexyltrimethoxysilane. [For other possible silane coupling agents see 
U.S. Pat. No. 3,652,761, incorporated by reference, supra]. 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 or glacial acetic 
acid. It condenses to form silane polymers; 
##STR1## 
Secondly, these polymers associate with the metal oxide, perhaps by 
forming a covalent bond with surface OH groups through dehydration: 
Adsorption of silane polymers to the metal oxide is also possible. 
An important aspect of the acidic organic silanization procedure of this 
invention is the method of dehydration used to effect the adsorptive or 
covalent binding of the silane polymer to the metal oxide. This 
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 of about 290.degree. C., is a suitable 
wetting agent. Heating to about 160.degree.-170.degree. in the presence of 
glycerol serves two purposes. It insures the evaporation of water, the 
organic 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 clumping and 
potential cross linking, of particles that is an inherent problem of other 
silanization techniques known in the art wherein dehydration is brought 
about by heating to dryness. 
In another embodiment an acidic aqueous silanization procedure is used to 
deposit a silane polymer on 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.degree.-95.degree. C. 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 oxides. 
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 
nature of the bioaffinity adsorbent itself, its susceptibilities to such 
factors as pH and temperature as well as the availability of reactive 
groups on the molecule for coupling. For instance, if an antibody is to be 
coupled to the magnetic particle, the coupling chemistry should be 
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 is to be coupled to the magnetic particle, the coupling chemistry 
should not denature the enzyme protein and the covalent linkage 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 
interactions. 
A variety of coupling chemistries are known in the art and have been 
described in U.S. Pat. No. 3,652,761 incorporated by reference, supra. By 
way of illustration, diazotization can be used to couple 
p-aminophenyl-terminated silanes to immunoglobulins. Coupling of 
immunoglobulins and other proteins to 3-aminopropyl-terminted and 
N-2-aminoethyl3-aminopropyl-terminated silanes has been accomplished by 
the use of glutaraldehyde. The procedure consists of two basic steps: (1) 
activation of the particle by reaction with glutaraldehyde followed by 
removal of unreacted glutaraldehyde and (2) reaction 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 [A. 
M. Klibanov, Science, 219:722 (1983), hereby incorporated by reference]. 
If the magnetic particles are coated by carboxy-terminated silanes, 
bioaffinity adsorbents such as proteins and immunoglobulins can be coupled 
to them by first treating the particles with 3-(3-dimethylaminopropyl) 
carbodiimide. 
Generally, magnetic particles coated with silanes bearing certain 
organofunctionalities can be modified to substitute more desirable 
functionalities for those already present on the surface. For example, 
diazo derivatives can be prepared from 3-aminopropyltriethoxysilane by 
reaction with p-nitro-benzoic acid, reduction of the nitro group to an 
amine and then diazotiation with nitrous acid. The same silane can be 
converted to the isothiocyanoalkylsilane derivative by reaction of the 
amino-function group with thiophosgene. 
To effect coupling to the magnetic particle, an aqueous solution of a 
bioaffinity adsorbent can be contacted with the silane coated particle at 
or below room temperature. When a protein (or immunoglobulin) is to be 
coupled, generally a ratio of 1:10-1:30, mg protein: mg particle is used. 
Contact periods of between about 3 to 24 hours are usually sufficient for 
coupling. During this period, the pH is maintained at a value that will 
not denature the bioaffinity adsorbent and which best suits the type of 
linkage being formed, e.g. for azo linkages, a pH of 8-9. 
It has been observed that after coupling of antibodies to silane coated 
magnetic particles by either the diazotization, carbodiimide, or 
glutaraldehyde methods described in greater detail in Section 7.5, 7.8 and 
7.10, respectively, the antibodies remain magnetic even after the 
following rigorous treatments: 24 hours at 50.degree. C. in phosphate 
buffered saline (PBS), 21 days at 37.degree. C. in PBS, 30 minutes at 
23.degree. C. in 1M sodium chloride, and repeated rinses in ethanol or 
methanol at room temperature. Antibodies adsorbed to iron oxides are 
substantially detached by any of these treatments. These results indicate 
that the silane is very tightly associated with the metal oxide and that 
the coupling of antibody to the particle results from an essentially 
irreversible covalent coupling. The tight association of the silane to the 
metal oxide together with the covalent coupling of bioaffinity adsorbents 
(e.g., antibodies) are features which impart stability onto coupled 
magnetic particles, a commercially important attribute. 
6.3. Use of Magnetic Particles in Biological Assays 
The magnetic particles of this invention may be used in immunoassays and 
other binding assays as defined in Section 3. The most prevalent types of 
assays used for diagnostic and research purposes are radioimmunoassays, 
fluoroimmunoassays, enzyme-immunoassays, and non-immune radioassays, based 
on the principle of competitive binding. Basically, a ligand, such as an 
antibody or specific binding protein, directed against a ligate, such as 
an antigen, is saturated with an excess of labeled ligate (*ligate). 
[Alternatively, competitive assays may be run with labeled ligand and 
unlabeled ligate. Non-competitive assays, so-called sandwich assays, are 
also widely employed.] By the method of this invention, the ligand is 
coupled to a magnetic particle. Examples of labels are radioisotopes: 
tritium, .sup.14 - carbon, .sup.57 -cobalt and, preferably, .sup.125 - 
iodine; fluorometric labels: rhodamine or fluorescein isothiocyanate; and 
enzymes (generally chosen for the ease with which the enzymatic reaction 
can be measured): alkaline phosphatase or .beta.-D-galactosidase. If 
nonlabeled ligate is added to ligand along with *ligate, less *ligate will 
be found in the ligand-ligate complex as the ratio of unlabeled to labeled 
ligate increases. If the ligand-*ligate complex can be physically 
separated from *ligate, the amount of unlabeled ligate in a test substance 
can be determined. 
To measure unlabeled ligate, a standard curve must be constructed. This is 
done by mixing a fixed amount of ligand and *ligate and adding a known 
amount of unlabeled ligate to each. When the reaction is complete, the 
ligand-*ligate is separated from *ligate. A graph is then made that 
relates the label in the collected ligand-*ligate complex to the amount of 
added unlabeled ligate. To determine the amount of unlabeled ligate in an 
experimental sample, an aliquot of the sample is added to the same 
ligand-*ligate mixture used to obtain the standard curve. The 
ligand-*ligate complex is collected and the label measured, and the amount 
of unlabeled ligand is read from the standard curve. This is possible with 
any sample, no matter how complex, as long as nothing interferes with the 
ligand-*ligate interaction. By the method of this invention, the 
ligand-*ligate complex is separated magnetically from free *ligate. 
This general methodology can be applied in assays for the measurement of a 
wide variety of compounds including hormones, pharmacologic agents, 
vitamins and cofactors, hematological substances, virus antigens, nucleic 
acids, nucleotides, glycosides and sugars. By way of illustration, the 
compounds listed in Table I are all measurable by magnetic particles 
immunoassays and binding assays [see D. Freifelder, Physical Biochemistry: 
Applications to Biochemistry and Molecular Biology, p. 259, W.H. Freeman 
and Company, San Francisco (1976)]. 
TABLE I 
______________________________________ 
SUBSTANCES MEASURABLE IN MAGNETIC 
TICLE ASSAYS 
______________________________________ 
Hormones: 
Thyroid hormones Prolactin 
(thyroxine, triiodo- 
Thyrocalcitonin 
thyronine, thyroid 
Parathyroid hormone 
binding globulin, Human chorionic gonadotro- 
thyroid-stimulating 
phin 
hormone, thyroglobulin) 
Human placental lactogen 
Gastrointestinal hormones 
Posterior pituitary peptides 
(glucagon, gastrin, 
(oxytocin, vasopressin, 
enteroglucagon, neurophysin) 
secretin, pancreozy 
Bradykinin 
min, vasoactive Cortisol 
intestinal peptide, 
Corticotrophin 
gastric inhibitory pep- 
Human growth hormone 
tide, motilin, insulin) 
Follicle-stimulating hormone 
Leutenizing Hormone 
Progesterone 
Testosterone 
Estriol 
Estradiol 
Pharmacologic agents: 
Digoxin Tetrahydrocannabinol 
Theophylline Barbiturates 
Morphine and opiate 
Nicotine and metabolic 
alkaloids products 
Cardiac glycosides 
Phenothiazines 
Prostaglandins Amphetamines 
Lysergic acid and derivatives 
Vitamins and cofactors: 
D, B12, folic acid, cyclic AMP 
Hematological substances: 
Fibrinogen, fibrin, 
Prothrombin 
and fibrinopeptides 
Transferrin and ferritin 
Plasminogen and plasmin 
Erthropoietin 
Antihemophilic factor 
Virus antigens: 
Hepatitis antigen Polio 
Herpes simplex Rabies 
Vaccinia Q fever 
Several Groups A Psittacosis group 
arboviruses 
Nucleic acids and nucleotides: 
DNA, RNA, cytosine derivatives 
______________________________________ 
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 Section 6.2. They may be used in immobilized enzyme 
systems, particulary 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, incorporated by 
reference, supra. The magnetic particles of this invention may be 
advantageously substituted for those of Dunnill and Lilly to avoid 
problems of settling and to allow enzyme recycle. Briefly, substrates are 
contacted with enzyme-coupled magnetic particles in a reactor under 
conditions of pH, temperature and substrate concentration that best 
promote the reaction. After completion of the reaction the particles are 
magnetically separated from the bulk liquid (which may be a solution or 
suspension) from which product can be retrieved free of enzyme. The 
enzyme-coupled magnetic particles can then be reused. Immobilized enzymes 
(coupled to non-magnetic supports) have been used in a number of 
industrially important enzymatic reactions, some of which are listed in 
Table II. The magnetic particles of this invention can be substituted for 
the non-magnetic solid phases previously employed which include glass, 
ceramics, polyacrylamide, DEAE-cellulose, chitin, porous silica, cellulose 
beads and alumino-silicates. 
TABLE II 
______________________________________ 
INDUSTRIALLY IMPORTANT IMMOBILIZED 
ENZYME REACTIONS 
Enzyme Reactant/Product 
______________________________________ 
Amylo-glucosidase 
Maltose/Glucose 
Glucose Oxidase 
Glucose/gluconic acid 
Glucoamylase Starch/glucose, Dextrin/glucose 
.beta.-Amylase 
Starch/maltose 
Invertase Sucrose/glucose 
Glucose isomerase 
Glucose/fructose 
Lactase Lactose/glucose 
Trypsin Proteins/amino acids 
Aminoacylase N--acetyl-DL-methionine/methionine 
Lysozyme Lysis of M. lysodeikticus 
______________________________________ 
6.5. Use of Magnetic Particles in Affinity Chromatography 
The process of affinity chromatography enables the efficient isolation of 
molecules by making use of features unique to the molecule: the ability to 
recognize or be recognized with a high degree of selectivity by a 
bioaffinity adsorbent such as an enzyme or antibody and the ability to 
bind or adsorb thereto. The process of affinity chromatography simply 
involves placing a selective bioaffinity adsorbent or ligand in contact 
with a solution containing several kinds of substances including the 
desired species, the ligate. The ligate is selectively adsorbed to the 
ligand, which is coupled to an insoluble support or matrix. The nonbinding 
species are removed by washing. The ligate is then recovered by eluting 
with a specific desorbing agent, e.g. a buffer at a pH or ionic strength 
that will cause detachment of the adsorbed ligate. 
By the method of this invention, magnetic particles may be used as the 
insoluble support to which the ligand is coupled. The particles may be 
suspended in batch reactors containing the ligate to be isolated. The 
particles with bound ligate may be separated magnetically from the bulk 
fluid and washed, with magnetic separations between washes. Finally, the 
ligate can be recovered from the particle by desorption. The magnetic 
particles of this invention may be used in a variety of affinity systems 
exemplified by those listed in Table III. 
TABLE III 
______________________________________ 
AFFINITY SYSTEMS 
Ligand, immobile entity 
Ligate, soluble entity 
______________________________________ 
Inhibitor, cofactor, prosthetic 
Enzymes; apoenzymes 
group, polymeric substrate 
Enzyme Polymeric inhibitors 
Nucleic acid, single strand 
Nucleic acid, complementary 
strand 
Hapten; antigen Antibody 
Antibody (IgG) Proteins; polysaccharides 
Monosaccharide; polysaccharide 
Lectins; receptors 
Lectin Glycoproteins; receptors 
Small target compounds 
Binding Proteins 
Binding Protein Small target compounds 
______________________________________ 
7. EXAMPLES 
7.1. Preparation of Metal Oxide 
The metal oxide particles were prepared by mixing a solution of iron(II) 
(Fe.sup.2+) and iron(III) (Fe.sup.3+) salts with base as follows: a 
solution that is 0.5 M ferrous chloride (FeCl.sub.2) and 0.25 M ferric 
chloride (FeCl.sub.3) (200 mls) was mixed with 5 M sodium hydroxide (NaOH) 
(200 mls) at 60.degree. C. by pouring both solutions into a 500 ml beaker 
container 100 mls of distilled water. All steps were performed at room 
temperature unless otherwise indicated. The mixture was stirred for 2 
minutes during which time a black, magnetic precipitate formed. After 
settling, the volume of the settled precipitate was approximately 175 mls. 
The concentration of iron oxide in the precipitate was about 60 mg/ml 
(based on a yield of 11.2 gms of iron oxide as determined infra). This is 
in contrast to commercially available magnetic iron oxides, such as Pfizer 
#2228 .gamma.Fe.sub.2 O.sub.3 (Pfizer Minerals, Pigments and Metals 
Division, New York, NY), the standard magnetic oxide for recording tapes, 
which can attain concentrations of about 700 mg/ml in aqueous slurry. The 
comparison is included to emphasize the fineness of the particles made by 
this method. Very fine particles are incapable of dense packing and 
entrain the most water. Larger and denser particles, on the other hand, 
pack densely, excluding the most water. 
The precipitate was then washed with water until a pH of 6-8 was reached as 
determined by pH paper. The following washing technique was employed: The 
particles were suspended in 1.8 liters of water in a 2 liter beaker and 
collected by magnetic extraction. The beaker was placed on top of a ring 
magnet, 1/2 inch high and 6 inches in diameter, which caused the magnetic 
particles to settle. The water was poured off without the loss of 
particles by holding the magnet to the bottom of the beaker while 
decanting. A similar washing technique was employed for all washes 
throughout, except that volumes were adjusted as necessary. Typically, 
three washes were sufficient to achieve neutral pH. The magnetic oxide was 
then washed once with 1.0 liter of 0.02 M sodium chloride (NaCl) in the 
same beaker. 
The water was then replaced with methanol, leaving a trace of water to 
catalyze hydrolysis of the methoxy silane (see Section 7.2.). This was 
accomplished by aspirating 800 mls of 0.2 M NaCl and bringing the total 
volume to 1 liter with methanol. The material was resuspended, and 
magnetically extracted; 800 mls of supernatant were removed, and another 
800 mls of methanol were added. After three additions of methanol, the 
oxide was ready for silanization in a solution which was approximately 1% 
(V/V) water. A portion of the precipitate was dried at 70.degree. C. for 
24 hours and weighed; 11.2 grams of magnetic iron oxide were formed. 
It is to be noted that throughout this procedure the magnetic iron oxide 
particles, because of their superparamagnetic properties, never became 
permanently magnetized despite repeated exposure to magnetic fields. 
Consequently, only mild agitation was required to resuspend the particles 
during the water washings and methanol replacement treatment. 
7.2. Silanization 
The magnetic iron oxide particles (see Section 7.1.) suspended in 250 mls 
of methanol containing approximately 1% (V/V) water were placed in a 
Virtis 23 homogenizer (Virtis Company, Inc., Gardiner, NY). Two grams of 
orthophosphorous acid (Fisher Scientific Co., Pittsubrgh, PA) and 10 mls 
of p-aminophenyltrimethoxysilane (A-7025, Petrarch Systems, Inc., Bristol, 
PA) were added. In an alternative protocol, 5 mls of glacial acetic acid 
have been substituted for the 2 gms of orthophosphorous acid. The mixture 
was homogenized at 23,000 rpm for 10 minutes and at 9,000 rpm for 120 
minutes. The contents were poured into a 500 ml glass beaker containing 
200 mls of glycerol and heated on a hot plate until a temperature of 
160.degree.-170.degree. C. was reached. The mixture was allowed to cool to 
room temperature. Both the heating and cooling steps were performed under 
nitrogen with stirring. The glycerol particle slurry (about 200 mls in 
volume) was poured into 1.5 liters of water in a 2 liter beaker; the 
particles were washed exhaustively (usually four times) with water 
according to the technique described in section 7.1. 
This silanization procedure was performed with other silanes, including 
3-aminopropyltrimethoxysilane, 
N-2-aminoethyl-3-aminopropyltrimethoxysilane, n-dodecyltriethoxysilane and 
n-hexyltrimethoxysilane (A-0800, A-0700, D-6224 and H-7334, respectively, 
Petrarch Systems, Inc., Bristol, PA). 
As an alternative to the above silanization procedure, silane has also been 
deposited on superparamagnetic iron oxide (as prepared in Section 7.1) 
from acidic aqueous solution. Superparamagnetic iron oxide with Fe.sup.2+ 
/Fe.sup.3+ ratio of 2 was washed with water as described in Section 7.1. 
The transfer to methanol was omitted. One gram of particles (about 20 mls 
of settled particles) was mixed with 100 mls of a 10% solution of 
3-aminopropyltrimethoxysilane in water. The pH was adjusted to 4.5 with 
glacial acetic acid. The mixture was heated at 90.degree.-95.degree. C. 
for 2 hours while mixing with a metal stirblade attached to an electric 
motor. After cooling, the particles were washed 3 times with water (100 
mls), 3 times with methanol (100 mls) and 3 times with water (100 mls), 
and the presence of silane on the particles was confirmed. 
7.3. Physical Characteristics of Silanized Magnetic Particles 
The mean particle diameter as measured by light scattering and the surface 
area per gram as measured by nitrogen gas adsorption for p-aminophenyl 
silanized, 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 hypothetical 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 for greater 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.mu. have calculated surface area per gram of about 120 
m.sup.2 /gm. 
TABLE IV 
______________________________________ 
CHARACTERISTICS OF SILANIZED 
MAGNETIC TICLES 
Measured Hypoth. 
Mean Diam..sup.1 
Surf. Area.sup.2 
Surf. Area.sup.3 
Silane (.mu.) (m.sup.2 /gm) 
(m.sup.2 /gm) 
______________________________________ 
N--2 aminoethyl- 
0.561 140 4.3 
3-aminopropyl 
p-aminophenyl 
0.803 NM.sup.4 -- 
3-aminopropyl 
0.612 122 3.9 
______________________________________ 
.sup.1 Diameter (in microns) was measured by light scattering on a Coulte 
N4 Particle Size Analyzer. 
.sup.2 Surface area was measured by N.sub.2 gas adsorption. 
.sup.3 Calculated surface area per gram for a perfect sphere with a 
density at 2.5 gm/cc. 
.sup.4 Not Measured. 
Because the mean diameters of the silanized magnetic particles produced by 
the procedures of Sections 7.1 and 7.2 are considerably smaller than the 
diameters of other magnetic particles described in the literature, they 
exhibit slower gravimetric settling times than those previously reported. 
For instance, the settling time of the particles described herein is 
approximately 150 minutes in contrast to settling times of: (a) 5 minutes 
for the particles of Hersh and Yaverbaum, [Clin. Chem. Acta 63: 69 
(1975)], estimated to be greater than 10.mu. in diameter; and (b) less 
than 1 minute for the particles of Robinson et al. [Biotech. Bioeng. 
XV:603 (1973)] which are 50-160.mu. in diameter. 
The silanized magnetic particles of this invention are characterized by 
very slow rates of gravimetric settling as a result of their size and 
composition; nevertheless they respond promptly to weak magnetic fields. 
This is depicted in FIG. 1 where the change in turbidity over time of a 
suspension of silanized magnetic particles resulting from spontaneous 
particle settling in the absence of a magnetic field is compared to the 
change in the turbidity produced in the presence of a samarium-cobalt 
magnet. It can be seen that after 30 minutes the turbidity of the 
suspension has changed only slightly more than 10% in the absence of a 
magnetic field. However, in the presence of a weak magnetic field, the 
turbidity of the particle suspension drops by more than 95% of its 
original value within 6 minutes. In another experiment, a decrease in 
turbidity of only about 4% in 30 minutes was observed. 
A photomicrograph of superparamagnetic particles silanized with 
3-aminotrimethoxysilanes ("SIN" particles) is shown in FIG. 2. It can be 
seen that the particles vary in shape and size and that they are made up 
of a groups or clusters of individual superparamagnetic crystals (less 
than 300 A) which appear roughly spherical in shape. 
7.4. Coupling of Aminophenyl Magnetic Particles to Antibodies to Thyroxine 
First, thyroxine (T.sub.4) antiserum was prepared as follows: 5.0 mls of 
serum of sheep immunized with T.sub.4 (obtained from Radioassay Systems 
Laboratories, Inc., Carson, CA) were added to a 50 ml centrifuge tube. Two 
5.0 ml aliquots of phosphate buffered saline (PBS) were added to the tube 
followed by 15 mls of 80% saturated ammonium sulfate, pH 7.4, at 4.degree. 
C. After mixing, the tube was stored at 4.degree. C. for 90 minutes. The 
mixture was then centrifuged at 3,000 rpm for 30 minutes at 4.degree. C. 
The supernatant fraction was decanted and the pellet resuspended and 
dissolved to clarity in 5.0 mls of PBS. The T.sub.4 antiserum preparation 
(1:2 in PBS) was dialyzed against PBS, transferred from the dialysis 
tubing to a 50 ml centrifuge tube to which 40 mls of PBS were added, 
bringing the total volume to 50 mls. The T.sub.4 antiserum preparation 
(1:10 in PBS) was refrigerated until used for coupling. 
To 1740 mg of p-aminophenyl silanized particles in 100 mls of 1N 
hydrochloric acid (HCl), 25 mls of 0.6 M sodium nitrite (NaNO.sub.2) were 
added. The NaNO.sub.2 was added slowly below the surface of the 
particle/HCl mixture while maintaining the temperature between 0.degree. 
and 5.degree. C. with care taken to avoid freezing. After 10 minutes, the 
mixture was brought to pH 7.5-8.5 by addition of 65 mls of 1.2 M NaOH and 
18 mls of 1 M sodium bicarbonate (NaHCO.sub.3), still maintaining 
temperature at 0.degree. to 5.degree. C. Then, 50 mls of PBS containing 
100 mg of the gamma globulin fraction of sheep serum containing antibodies 
to thyroxine (the T.sub.4 antiserum preparation described supra) were 
added. The pH was maintained between 7.5-8.5 while the mixture was 
incubated for 18 hours at 0.degree. to 5.degree. C. The antibody-coupled 
particles were washed exhaustively with 0.1 M sodium phosphate buffer, pH 
7.2 (3 times), 1 M NaCl, methanol, 1 M NaCl and 0.1 M sodium phosphate 
buffer again. Wash steps were repeated twice or more. All washes were 
performed by dispersing the particles and magnetically separating them as 
described in section 7.1. After washing, the particles were resuspended in 
PBS and incubated overnight at 50.degree. C. The particles were washed in 
methanol, 1 M NaCl and 0.1 M sodium phosphate buffer as before, and twice 
in Free T.sub.4 Tracer Buffer. The particles were resuspended in Free 
T.sub.4 Tracer Buffer and stored at 4.degree. C. until used for 
radioimmunoassay. 
7.5. Magnetic Particle Radioimmunoassay for Thyroxine 
The quantity of antibody-coupled magnetic particles to be used in the 
thyroxine radioimmunoassay (RIA) was determined empirically using the 
following RIA procedure: 
Ten microliters (.mu.ls) of standard were pipetted into 12.times.75 mm 
polypropylene tubes followed by 500 .mu.ls of tracer and 100 .mu.ls of 
magnetic particles. After vortexing, the mixture was incubated at 
37.degree. C. for 15 minutes after which time the tubes were placed on a 
magnetic rack for 10 minutes. The rack consisted of a test tube holder 
with a cylindrical "button" magnet (Incor 18, Indiana General Magnetic 
Products Corp., Valparaiso, IN) at the bottom of each tube. The magnetic 
particles with antibody and bound tracer were pulled to the bottom of the 
tubes allowing the unbound tracer to be removed by inverting the rack and 
pouring off supernatants. Radioactivity in the pellet was determined on a 
Tracor 1290 Gamma Counter (Tracor Analytic, Inc., Elk Grove Village, IL). 
The reagents used in the assay were as follows: 
Standards were prepared by adding T.sub.4 to T.sub.4 -free human serum. 
T.sub.4 was removed from the serum by incubation of serum with activated 
charcoal followed by filtration to remove the charcoal according to the 
method of Carter [Clin. Chem 24, 362 (1978)]. The tracer was .sup.125 
I-thyroxine purchased from Cambridge Medical Diagnostics (#155) and was 
diluted into 0.01 M Tris buffer containing 100 .mu.g/ml bovine serum 
albumin, 10 .mu.g/ml salicylate, 50 .mu.g/ml 
8-amilinonaphthalene-8-sulfonic acid at pH 7.4. Magnetic particles at 
various concentrations in phosphate buffered saline (PBS) with 0.1% bovine 
serum albumin were used in the RIA to determine a suitable concentration 
of particles for T.sub.4 measurements. A quantity of magnetic particles of 
approximately 50 .mu.g per tube was chosen for the RIA. This amount 
permitted good displacement of tracer from the antibody for the desired 
concentration range of T.sub.4 (0-32 .mu.g/dl). 
Having thus determined the optimal quantity, the RIA procedure described 
supra was performed using approximately 50 .mu.g per tube of magnetic 
particles to construct a radioimmunoassay standard curve for T.sub.4 . The 
data obtained from the RIA is presented in Table V. 
TABLE V 
______________________________________ 
RIA STANDARD CURVE FOR T.sub.4 
T4 Concentration 
cpm (average of 2 tubes) 
______________________________________ 
0 .mu.g/dl 36763 
2 .mu.g/dl 24880 
4 .mu.g/dl 18916 
8 .mu.g/dl 13737 
16 .mu.g/dl 10159 
32 .mu.g/dl 7632 
Total 69219 
______________________________________ 
7.6. Magnetic Particle Radioimmunoassay for Theophylline 
Rabbit anti-theophylline antibodies were prepared and coupled to 
p-aminophenyl silanized particles according to methods similar to those 
described in Section 7.4. The anti-theophylline antibody-coupled magnetic 
particles were used in a radioimmunoassay with the following protocol: 20 
.mu.ls of theophylline standard (obtained by adding theophylline to 
theophylline-free human serum), 100 .mu.ls of .sup.125 I-theophylline 
tracer (obtained from Clinical Assays, Cambridge, MA), and 1 ml of 
antibody-coupled magnetic particles were vortexed. After a 15 minute 
incubation at room temperature, a 10 minute magnetic separation was 
employed. A standard curve was constructed and the data obtained are shown 
in Table VI. 
TABLE VI 
______________________________________ 
RIA STANDARD CURVE FOR THEOPHYLLINE 
Theophylline Concentration 
cpm (average of 2 tubes) 
______________________________________ 
0 .mu.g/dl 35061 
2 .mu.g/dl 28217 
8 .mu.g/dl 19797 
20 .mu.g/dl 13352 
60 .mu.g/dl 8148 
Total 52461 
______________________________________ 
7.7. Effect of Variation of Fe.sup.2+ /Fe.sup.3+ Ratio of Magnetic 
Particles on T4 Radioimmunoassay 
Magnetic iron oxides were made according to the crystallization procedure 
of Section 7.1 by maintaining constant molar amounts of iron but varying 
the Fe.sup.2+ /Fe.sup.3+ ratio from 4 to 0.5. These particles were 
silanized, coupled to anti-T.sub.4 antibodies and used in the T.sub.4 RIA, 
as in Sections 7.2, 7.4 and 7.5, respectively. The variation of Fe.sup.2+ 
/Fe.sup.3+ ratio did not substantially affect the performance of these 
magnetic particles in the T.sub.4 RIA as shown in Table VII. 
TABLE VII 
______________________________________ 
T.sub.4 RIA STANDARD CURVES USING MAGNETIC 
TICLES WITH VARIED Fe.sup.2+ /Fe.sup.3+ RATIOS 
cpm (average of 2 tubes) 
T4 Concentration 
Fe.sup.2+ /Fe.sup.3+ = 4 
Fe.sup.2+ /Fe.sup.3+ = 0.5 
______________________________________ 
0 .mu.g/dl 35633 35642 
1 .mu.g/dl 31681 33139 
2 .mu.g/dl 30572 30195 
4 .mu.g/dl 24702 25543 
8 .mu.g/dl 18680 19720 
16 .mu.g/dl 12803 11625 
32 .mu.g/dl 10012 8005 
Total 77866 75636 
______________________________________ 
7.8. Coupling of Carboxylic Acid-Terminated Magnetic Particles to B.sub.12 
Binding Protein 
7.8.1 Preparation of Carboxylic Acid-Terminated Magnetic Particles 
A superparamagnetic iron oxide was made by the procedure described in 
Section 7.1 and silanized as in Section 7.2 with 
3-aminopropyltrimethyoxysilane instead of the aminophenyl silane. The 
amino group of the silane was then reacted with glutaric anhydride to 
convert the termination from an amine to carboxylic acid. The conversion 
of the termination was accomplished as follows: five grams of aminopropyl 
silanized particles in water were washed four times with 1.5 liters of 0.1 
M NaHCO.sub.3 using the washing procedure of Section 7.1. The volume was 
adjusted to 100 mls and 2.85 gm glutaric anhydride was added. The 
particles were washed two times and the reaction with glutaric anhydride 
was repeated. The carboxylic acid-terminated magnetic particles were 
washed five times with water to prepare them for reaction with protein. 
7.8.2. Carbodiimide Coupling of B.sub.12 Binding Protein and Human Serum 
Albumin to Carboxylic Acid-Terminated Magnetic Particles 
To 50 mg of carboxy-terminated magnetic particles in 1 ml of water were 
added 4 mg of 3-(3 dimethylaminopropyl)-carbodiimide. After mixing by 
shaking for 2 minutes, 0.05 mg of B.sub.12 binding protein (intrinsic 
factor (IF) from hog gut obtained from Dr. R. H. Allen, Denver, CO) and 
0.75 mg of human serum albumin (HSA, obtained from Sigma Chemical Co., 
A-8763) were added to 0.30 ml in water. The pH was adjusted to pH 5.6 and 
maintained by the addition of 0.1 N HCl or 0.1 N NaOH for three hours. The 
particles were then washed with 10 mls of 0.1 M Borate with 0.5 M NaCl pH 
8.3, 10 mls of phosphate buffered saline (PBS) with 0.1% HSA, and 10 mls 
of distilled water employing the magnetic separation technique as in 
Section 7.1. Particles were washed three times with PBS and stored in PBS 
until use. 
7.9. Magnetic Particle Competitive Binding Assay for Vitamin B.sub.12 
Using the IF- and HSA-coupled magnetic particles made by the method of 
Section 7.7, a titering of the particles was performed to ascertain the 
quantity of particles needed in a competitive binding assay for vitamin 
B.sub.12 (B.sub.12). The following assay protocol was used: 
100 .mu.ls of standard and 1000 .mu.ls of tracer buffer were added to 
12.times.75 mm polypropylene tubes. The mixtures were placed into a 
boiling water bath for 15 minutes to effect denaturation of binding 
proteins in human serum samples. Then 100 .mu.ls of various concentrations 
of magnetic particles in phosphate buffer were added to determine the 
optimal quantity of particles for assaying B.sub.12 concentrations between 
0 and 2000 picogram/ml (pg/ml). After incubation of the mixtues for 1 hour 
at room temperature, a magnetic separation of bound and free B.sub.12 was 
performed according to the procedure of and using the magnetic rack 
described in Section 7.5. Radioactivity in the pellets was then counted on 
a Tracor 1290 Gamma Counter (Tracor Analytic, Inc., Elk Grove Village, 
IL). 
The reagents used in the assay were as follows: 
B.sub.12 standards were obtained from Corning Medical and Scientific, 
Division of Corning Glass Works, Medfield, MA #474267. They are made with 
B.sub.12 -free human serum albumin in PBS and sodium azide added as a 
preservative. The tracer was .sup.57 Co-B.sub.12 (vitamin B.sub.12 tagged 
with radioactive cobalt) from Corning Medical and Scientific, Division of 
Corning Glass Works, Medfield, MA, #474287. The tracer is in a borate 
buffer pH 9.2, containing 0.001% potassium cyanide and sodium azide. 
Magnetic particles were diluted in PBS at various concentrations to 
determine the quantity of particles needed to measure B.sub.12 
concentrations between 0 and 2000 pg/ml. 
A quantity of magnetic particles of approximately 50 .mu.g/tube was 
selected and was used in the B.sub.12 competitive binding assay supra to 
construct a standard curve; the data are presented in Table VIII. 
TABLE VIII 
______________________________________ 
B.sub.12 COMPETITIVE BINDING ASSAY STANDARD CURVE 
B.sub.12 Concentration 
cpm (average of 2 tubes) 
______________________________________ 
0 pg/ml 5523 
100 pg/ml 5220 
250 pg/ml 4169 
500 pg/ml 3295 
1000 pg/ml 2278 
2000 pg/ml 1745 
Total 16515 
______________________________________ 
7.10. Coupling of Magnetic Particles Coated With Aminoethyl-3-Aminopropyl 
Silane to Proteins 
7.10.1. Coupling of N-2-Aminoethyl-2-Aminopropyl Magnetic Particles to 
Antibodies to Triiodothyronine 
Six-tenths of a gram of N-2-aminoethyl-3-aminopropyl magnetic particles 
(abbreviated "DIN" particles for "dinitrogen", signifying that the 
particles have a N/Si ratio of 2) prepared as in Section 7.2. were 
resuspended in water. The particles were washed once in water and then 
twice with 30 mls of 0.1 M phosphate buffer, pH 7.4 with magnetic 
separations between washings. After suspending the washed particles in 15 
mls of 0.1 M phosphate, 15 mls of a 5% (V/V) solution of glutaraldehyde, 
formed by diluting 25% glutaraldehyde (G- 5882, Sigma Chemical Co., St. 
Louis, MO) with 0.1 M phosphate, were added. The particles were mixed for 
3 hours at room temperature by gently rotating the reaction vessel. 
Unreacted glutaraldehyde was washed away with 5 additions of 30 mls of 0.1 
M phosphate buffer. The glutaraldehyde activated particles were then 
resuspended in 15 mls of 0.1 M phosphate. 
Triiodothyronine (T.sub.3) antiserum (1.6 mls, obtained by immunizing 
rabbits with T.sub.3- BSA conjugates) was added to the activated particles 
and stirred on a wheel mixer at room temperature for 16 to 24 hours. The 
T.sub.3 antibody-coupled particles were washed once with 30 mls of 0.1 M 
phosphate and suspended in 15 mls of 0.2 M glycine solution in order to 
react any unreacted aldehyde groups. The suspension was mixed by shaking 
for 25 minutes. The antibody-coupled particles were washed with 30 mls of 
0.1 phosphate, 30 mls of ethanol and twice with 150 mls of PBS with 0.1% 
bovine serum albumin (BSA). They were resuspended in PBS, 1% BSA and 
stored at 4.degree. C. until used for RIA for T.sub.3. 
7.10.2. Coupling of N-2-Aminoethyl-3-Aminopropyl Magnetic Particles to 
Antibodies to Thyroid Stimulating Hormone 
The coupling procedure of Section 6.10.1 was followed with minor 
modifications. Twenty grams of DIN particles were washed three times with 
1.5 liters of methanol prior to glutaraldehyde activation. Glutaraldehyde 
activation was performed as in Section 7.10.1. with adjustments for scale. 
A goat gamma globulin fraction containing antibodies to human thyroid 
stimulating hormone (TSH) was coupled to the DIN particles rather than 
whole antisera. Fractionation was accomplished by precipitation of 
gammaglobulins with 40% ammonium sulfate followed by dialysis against PBS. 
Approximately 4 grams of protein (200 mls at 20 mg/ml) were coupled. 
Complete attachment of protein was evident by the absence of optical 
density at 280 nm in the supernatant after coupling. This indicated the 
attachment of about 20 mg of protein per gram of particles. The particles 
were then washed three times with 1.5 liters of 1 M NaCl, three times with 
PBS and incubated at 50.degree. C. overnight. Particles were then washed 3 
more times in PBS/BSA and titered for use in the TSH assay. 
7.11. Magnetic Particle Radioimmunoassay for Triiodothyronine 
The quantity of particles to be used in the T.sub.3 RIA was determined in 
the following assay: 
Standards were prepared by adding T.sub.3 to T.sub.3 -free human serum as 
with T.sub.4 (see Section 7.5.) 
Tracer was .sup.125 IT.sub.3 from Corning Medical and Scientific, Division 
of Corning Glass Works, Medfield, MA (#47106). 
Magnetic particles were diluted to various concentrations in PBS-BSA to 
determine the quantity of particles needed. 
The assay protocol was as follows: 50 .mu.ls of standard, 100 .mu.ls of 
tracer and 800 .mu.ls of DIN magnetic particles were pipetted into 
12.times.75 mm polypropylene tubes. After vortexing, the tubes were 
incubated for 2 hours at room temperature. The assay was terminated by 
magnetic separation. By titering the quantity of particles in the assay 
with a 0 ng/ml standard, a quantity of 30 .mu.g/tube was deemed to be 
optimal for the assay protocol. Table IX shows the T.sub.3 RIA standard 
curve data obtained with these particles. 
TABLE IX 
______________________________________ 
RIA STANDARD CURVE FOR T.sub.3 
T.sub.3 Concentration 
cpm (average of 2 tubes) 
______________________________________ 
0.0 ng/ml 17278 
0.25 ng/ml 15034 
0.50 ng/ml 13456 
1.00 ng/ml 12127 
2.00 ng/ml 8758 
4.00 ng/ml 5776 
8.00 ng/ml 3897 
Total 26946 
______________________________________ 
7.12. Magnetic Particle Radioimmunoassay for Thyroid Stimulating Hormone 
The quantity of particles to be used in the TSH RIA was determined in the 
following assay: 
Standards were in normal human serum (Corning Medical and Scientific, 
#47186, Medfield, MA). 
Tracer was .sup.125 I-rabbit anti-TSH antibody in PBS (Corning Medical and 
Scientific, #474185, Medfield, MA). 
Magnetic particles were diluted to various concentrations in PBS-BSA to 
determine the quantity of particles needed. 
The assay protocol was as follows: 100 .mu.ls of standard and 100 .mu.ls of 
tracer were pipetted into 12.times.75 mm polypropylene tubes, vortexed, 
and incubated for 3 hours at room temperature. Magnetic particles (500 
.mu.ls) were added and the mixture was vortexed and incubated for 1 hour 
at room temperature. 500 .mu.ls of water were added and the usual magnetic 
separation was employed to separate bound from unbound tracer. In the 
presence of TSH, a sandwich is formed between magnetic antibody (goat 
anti-TSH antibody, see Section 7.10.1.) TSH and tracer .sup.125 I-antibody 
(rabbit anti-TSH antibody). Thus, increasing concentrations of analyte 
(TSH) increase the amount of bound radioactivity. Table X shows the TSH 
RIA standard curve data obtained by this procedure. 
TABLE X 
______________________________________ 
RIA STANDARD CURVE FOR TSH 
TSH Concentration cpm 
______________________________________ 
0 .mu.IU/ml* 1615 
1.5 .mu.IU/ml* 2309 
3.0 .mu.IU/ml* 3014 
6.0 .mu.IU/ml* 4448 
15.0 .mu.IU/ml* 7793 
30.0 .mu.IU/ml* 11063 
60.0 .mu.IU/ml* 15030 
Total 45168 
______________________________________ 
*.mu.IU = micro International Units 
7.13. Coupling of Magnetic Particles Coated with 
N-2-Aminoethyl-3-Aminopropyl Silane to Enzymes by Use of Glutaraldehyde 
Magnetic particles (1 gm) were activated with glutaraldehyde as in Section 
7.10.1. After washing, the particles were resuspended in 15 mls of PBS. 
Then 3 mls of particles (2 gm) were mixed with 5 mg of alkaline 
phosphatase (Sigma Chemical Company, p-9761) or 5 mg of 
.beta.-galactosidase (Sigma Chemical Company, 5635) dissolved in 2.0 mls 
of PBS. The coupled particles were washed with glycine and then washed 5 
times with PBS and resuspended in PBS with 0.1% BSA. 
Enzyme assays for magnetic alkaline phosphatase activity was performed as 
follows: 
To a 3 ml cuvette 3 mls of 0.05 M Tris-HCl were added, pH 8.0, with 3 mM 
p-nitrophenyl-phosphate. Then 100 .mu.ls of diluted magnetic particles 
with coupled alkaline phosphatase were added. The increase in optical 
density at 410 nm was recorded. 
Enzyme assay for magnetic .beta.-galactosidase activity was performed as 
follows: 
To a 3 ml cuvette 3 mls of 0.1 M phosphate were added, pH 7.4, with 0.01 M 
mercaptoethanol and 0.005 M O-nitrophenyl-.beta.-O-galactopyranoside. Then 
100 .mu.ls of diluted magnetic particles coupled to .beta.-galactosidase 
were added. The increase in optical density at 410 nm was recorded. 
It is apparent that many modifications and variations of this invention as 
hereinabove set forth may be made without departing from the spirit and 
scope thereof. The specific embodiments described are given by way of 
example only and the invention is limited only by the terms of the 
appended claims.