Particle separation method

A method is disclosed for separating a substance from a liquid medium. The method comprises combining the liquid medium containing the substance with magnetic particles under conditions for non-specific chemical binding of the magnetic particles. Thereafter, the medium is subjected to a magnetic field gradient to separate the particles from the medium. The preferred non-specific binding is achieved as the result of charge interactions between the particles usually by means of a polyionic reagent. The method of the invention has particular application to the separation of cells and microorganisms from aqueous suspensions and also to the determination of an analyte in a sample suspected of containing the analyte. The analyte is a member of a specific binding pair (sbp). The sample is combined in an assay medium with magnetic particles and a sbp member complementary to the analyte. Magnetic or non-magnetic particles capable of specific binding to the analyte or its complementary sbp member must be included in the assay medium. The combination is made under conditions for non-specifically aggregating the magnetic particles or coaggregating the magnetic and non-magnetic particles when non-magnetic particles are present. The assay medium is subjected to a magnetic field gradient to separate the aggregated particles from the medium. Then, the medium or the particles are examined for the presence or amount of the analyte or an sbp member, the binding of which is affected by the presence of the analyte.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention relates to methods for separating a substance, usually 
particulates, from a fluid medium by use of a magnetic field gradient. The 
invention has particular application to separation of cells from 
biological fluids, such as blood, lymphatic fluid, urine, cell cultures, 
etc. 
Numerous techniques are known for determining the presence or amount of an 
analyte in a sample, such as a biological fluid, for example, serum or 
urine. An in vitro assay procedure is the most common of these techniques. 
Many of these techniques involve competitive binding of the analyte to be 
determined and a labeled analog of such analyte to binding sites on a 
specific receptor, for example, an antibody. Many of these techniques 
involve a separation step where the unbound labeled analog is separated 
from bound labeled analog and either the bound or unbound analog is 
examined for the signal produced by the label. The signal is produced in 
relation to the amount of analyte in the sample. 
Several techniques are known for separating bound and unbound fractions. 
For example, one may employ differential migration of the bound and the 
free fractions, e.g., chromatoelectrophereses, gel filtration, etc.; 
chemical precipitation of the bound or free fraction, e.g., by means of 
organic solvents, salts, acids, etc. followed by filtration or 
centrifugation; immunological precipitation of the bound fraction, e.g., 
by double antibody technique followed by filtration or centrifugation; 
absorption of the bound or free fraction onto selective sorbing media, 
e.g., charcoal, silicates, resins, etc.; magnetic separation techniques, 
and the like. 
Magnetic separations generally fall into two general categories. There are 
those separations in which the material to be separated is intrinsically 
magnetic. On the other hand, one or more components of a mixture can be 
rendered magnetic by the attachment of a magnetically responsive entity. 
In biochemical separations, materials of interest are generally not 
sufficiently magnetic and thus magnetic particles bound to antibodies, 
lectins, and other targeting molecules have been used for isolating many 
of these materials. Magnetic particles targeted for specific molecules 
have also been used in a variety of immunoassays. 
Many of the separation techniques used in immunoassays are relatively long 
and complicated procedures. Such procedures reduce operator efficiency, 
decrease thoughput, and increase the costs of tests. Other separation 
techniques which are rapid and simple do not adequately distinguish 
between the bound and free fractions and therefore are unsuited for 
immunoassays or can only be utilized in a limited number of tests. 
2. Description of the Related Art. 
A method for determining the concentration of substances in biological 
fluids (e.g., drugs, hormones, vitamins and enzymes) wherein magnetically 
responsive, permeable, solid, water insoluble, micro particles are 
employed is disclosed in U.S. Pat. No. 4,115,534. Functional magnetic 
particles formed by dissolving a mucopolysaccaride such as chitosan in 
acidified aqueous solution containing a mixture of ferrous chloride and 
ferric chloride is disclosed in U.S. Pat. No. 4,285,819. The microspheres 
may be employed to remove dissolved ions from waste aqueous streams by 
formation of chelates. U.S. Pat. No. 3,933,997 describes a solid phase 
radio immunoassay for digoxin where anti-digoxin antibodies are coupled to 
magnetically responsive particles. Small magnetic particles coated with an 
antibody layer are used in U.S. Pat. No. 3,970,518 to provide large and 
widely distributed surface area for sorting out and separating select 
organisms and cells from populations thereof. U.S. Pat. No. 4,018,886 
discloses small magnetic particles used to provide large and widely 
distributed surface area for separating a select protein from a solution 
to enable detection thereof. The particles are coated with a protein that 
will interact specifically with the select protein. U.S. Pat. No. 
4,070,246 describes compositions comprising stable, water insoluble 
coatings on substrates to which biologically active proteins can be 
covalently coupled so that the resulting product has the biological 
properties of the protein and the mechanical properties of the substrate, 
for example, magnetic properties of a metal support. A diagnostic method 
employing a mixture of normally separable protein-coated particles is 
discussed in U.S. Pat. No. 4,115,535. Microspheres of acrolein 
homopolymers and copolymer with hydrophilic comonomers such as methacrylic 
acid and/or hydroxyethylmethacrylate are discussed in U.S. Pat. No. 
4,413,070. U.S. Pat. No. 4,452,773 discloses magnetic iron-dextran 
microspheres which can be covalently bonded to antibodies, enzymes and 
other biological molecules and used to label and separate cells and other 
biological particles and molecules by means of a magnetic field. Coated 
magnetizeable microparticles, reversible suspensions thereof, and 
processes relating thereto are disclosed in U.S. Pat. No. 4,454,234. A 
method of separating cationic from anionic beads in mixed resin beds 
employing a ferromagnetic material intricately incorporated with each of 
the ionic beads is described in U.S. Pat. No. 4,523,996. A magnetic 
separation method utilizing a colloid of magnetic particles is discussed 
in U.S. Pat. No. 4,526,681. UK Patent Application GB No. 2,152,664A 
discloses magnetic assay reagents. 
An electron-dense antibody conjugate made by the covalent bonding of an 
iron-dextran particle to an antibody molecule is reported by Dutton, et 
al. (1979) Proc. Natl. Acad. Sci. 76:3392-3396. Ithakissios, et al. 
describes the use of protein containing magnetic microparticles in 
radioassays in Clin. Chem. 23:2072-2079 (1977). The separation of cells 
labeled with immunospecific iron dextran microspheres using high gradient 
magnetic chromotography is disclosed by Molday, et al. (1984) FEBS 
170:232-238. In J. Immunol. Meth. 52:353-367 (1982) Molday, et al. 
describe an immuno specific ferromagnetic iron-dextran reagent for the 
labeling and magnetic separation of cells. An application of magnetic 
microspheres in labeling and separation of cells is also disclosed by 
Molday, et al. in Nature 268:437-438 (1977). A solid phase 
fluoroimmunoassay of human albumin and biological fluids is discussed by 
Nargessi, et al. (1978) Clin. Chim. Acta. 89:455-460. Nye, et al. (1976) 
Clin. Chim. Acta. 69:387-396 discloses a solid phase magnetic particle 
radioimmunoassay. Magnetic fluids are described by Rosenweig (1983) Scien. 
Amer. 10:136-194. Magnetic protein A microspheres and their use in a 
method for cell separation are disclosed by Widder, et al. (1979) Clin. 
Immunol. and Immunopath. 14:395-400. 
SUMMARY OF THE INVENTION 
The method of the present invention is directed to the separation of a 
substance from a liquid medium by causing the binding of the substance to 
very small magnetic particles. Where the substance is present as a 
non-particulate solute, it will normally bind to the magnetic particles 
through specific ligand-receptor binding. Where the substance is present 
as non-magnetic particles, binding may also be specific but will usually 
be non-specific such as through electrostatic or hydrophobic interactions. 
Chemical means is then provided to non-specifically bind the magnetic 
particles to each other and usually to the non magnetic particles and to 
cause aggregation or coaggregation of the particles. Next the medium is 
subjected to a magnetic field gradient to separate the particles from the 
medium. Preferably the non specific binding is achieved through charge 
interactions and is reversible. 
The method of the present invention has particular application in the assay 
of organic and biochemical analytes particularly those analytes of 
interest in the analysis of body fluids. Of special interest are assays 
where the analyte is a member of a specific binding pair (sbp) that is 
bound, or can become bound, to the surface of a particle. Where the 
analyte is a surface component or becomes bound to a non-magnetic 
particle, the method involves combining in an assay medium the sample, the 
non-magnetic particle when the analyte becomes bound to such particle, and 
magnetic particles under conditions for binding non-magnetic and magnetic 
particles and chemically inducing non-specific agglutination of the 
magnetic particles. The non-magnetic particle or the magnetic particle is 
usually bound to an sbp member. If the sbp member on the non-magnetic 
particle is not complementary to the analyte, then a complementary sbp 
member is also added. Next, the assay medium is subjected to a magnetic 
field gradient to separate the particles from the medium. After the 
separation, the medium or the particles are examined for the presence or 
amount of an sbp member, which is affected by the presence of analyte in 
the sample. Normally, the sbp member is detected by virtue of a signal 
created by the use of a signal producing system that generates a signal in 
relation to the amount of the analyte in the sample. The particles 
separated from the medium can be washed prior to their examination. 
Furthermore, the particles can subsequently be treated after separation 
from the medium to reverse their non-specific binding. 
The method of the invention provides a way of separating non-magnetic 
particles from a medium by virtue of the chemically controlled 
non-specific reversible binding of such particles to magnetic particles. 
Because of the small size of the magnetic particles, it also provides for 
very rapid binding of a substance to be separated. By then aggregating the 
particles there is provided a much more rapid and complete magnetic 
separation than has been achieved by previous methods. 
The invention includes compositions and kits for conducting the method of 
the invention, particularly for conducting an assay for determining an 
analyte in a sample suspected of containing the analyte. 
DESCRIPTION OF THE SPECIFIC EMBODIMENTS 
The present invention relates to a method of separating substances bound to 
suspended particles from a liquid medium. The method involves magnetic 
particles and chemically controlled non-specific binding of the magnetic 
particles to each other. Usually the substance to be separated will be 
bound to, or will be caused to bind to, non-magnetic particles. The 
preferred approach for achieving non-specific binding between the 
non-magnetic particles and the magnetic particles or between the magnetic 
particles themselves is charge interactions. The bound particles are 
separated from the medium by the use of a magnetic field gradient. The 
separated particles can be washed and examined by physical or chemical 
methods. The particles can also be treated to reverse the non-specific 
binding. Where non-magnetic particles are used, reversal of binding can be 
followed by separation of the free magnetic particles to provide a means 
of separating the non-magnetic particles from the magnetic particles. 
The present method has wide application in the field of the separation of 
suspended particles from a medium, particularly for separating biological 
materials, such as cells and microorganisms, and in the fields of 
immunoassays and blood typing. The invention provides a separation method 
which is more convenient and rapid than centrifugation, filtration, and 
prior magnetic separation methods and is particularly applicable to the 
pretreatment of suspensions where it is desired to carry out an analysis 
of either the particle-free liquid medium or the separated particles. The 
invention also has application to the assay of an analyte in a sample 
where a separation step is required. 
Before proceeding further with the description of the specific embodiments 
of the present invention, a number of terms will be defined. 
Analyte--the compound or composition to be measured, the material of 
interest. The analyte can be a member of a specific binding pair (sbp) and 
may be a ligand, which is mono- or polyvalent, usually antigenic or 
haptenic, and is a single compound or plurality of compounds which share 
at least one common epitopic or determinant site. The analyte can also be 
a component of a particle or can become bound to a particle during an 
assay. Exemplary of an analyte that is a component of a particle is an 
antigen on the surface of a cell such as a blood group antigen (A, B, AB, 
O, D, etc.) or an HLA antigen. Exemplary of an analyte becoming bound to a 
particle during an assay is an sbp member where a complementary sbp member 
is bound to a particle, glycoprotein or glycolipids where a lectin is 
bound to a particle, antibodies where protein A is bound to a particle, 
and the like. The binding involved when an analyte becomes bound to a 
particle can be specific or non-specific, immunological or 
non-immunological. 
The polyvalent ligand analytes will normally be poly(amino acids), i.e., 
polypeptides and proteins, polysaccharides, nucleic acids, and 
combinations thereof. Such combinations include components of bacteria, 
viruses, chromosomes, genes, mitochondria, nuclei, cell membranes and the 
like. 
The precise nature of some of the analytes together with numerous examples 
thereof are disclosed in U.S. Pat. No. 4,299,916 to Litman, et al., 
particularly at columns 16 to 23, the disclosure of which is incorporated 
herein by reference. 
For the most part, the polyepitopic ligand analytes employed in the subject 
invention will have a molecular weight of at least about 5,000, more 
usually at least about 10,000. In the poly(amino acid) category, the 
poly(amino acids) of interest will generally be from about 5,000 to 
5,000,000 molecular weight, more usually from about 20,000 to 1,000,000 
molecular weight, among the hormones of interest, the molecular weights 
will usually range from about 5,000 to 60,000 molecular weight. 
A wide variety of proteins may be considered as to the family of proteins 
having similar structural features, proteins having particular biological 
functions, proteins related to specific microorganisms, particularly 
disease causing microorganisms, etc. 
The monoepitopic ligand analytes will generally be from about 100 to 2,000 
molecular weight, more usually from 125 to 1,000 molecular weight. The 
analytes of interest include drugs, metabolites, pesticides, pollutants, 
and the like. Included among drugs of interest are the alkaloids. Among 
the alkaloids are morphine alkaloids, which includes morphine, codeine, 
heroin, dextromethorphan, their derivatives and metabolites; cocaine 
alkaloids, which include cocaine and benzoyl ecgonine, their derivatives 
and metabolites, ergot alkaloids, which include the diethylamide of 
lysergic acid; steroid alkaloids; iminazoyl alkaloids; quinazoline 
alkaloids, isoquinoline alkaloids; quinoline alkaloids, which include 
quinine and quinidine; diterpene alkaloids, their derivatives and 
metabolites. 
The next group of drugs includes steroids, which includes the estrogens, 
estogens, androgens, andreocortical steroids, bile acids, cardiotonic 
glycosides and aglycones, which includes digoxin and digoxigenin, saponins 
and sapogenins, their derivatives and metabolites. Also included are the 
steroid mimetic substances, such as diethylstilbestrol. 
The next group of drugs is lactams having from 5 to 6 annular members, 
which include the barbituates, e.g. phenobarbital and secobarbital, 
diphenylhydantonin, primidone, ethosuximide, and their metabolites. 
The next group of drugs is aminoalkylbenzenes, with alkyl of from 2 to 3 
carbon atoms, which includes the amphetamines, catecholamines, which 
includes ephedrine, L-dopa, epinephrine, narceine, papaverine, and their 
metabolites. 
The next group of drugs is benzheterocyclics which include oxazepam, 
chloropromazine, tegretol, imipramine, their derivatives and metabolites, 
the heterocyclic rings being azepines, diazepines and phenothiazines. 
The next group of drugs is purines, which includes theophylline, caffeine, 
their metabolites and derivatives. 
The next group of drugs includes those derived from marijuana, which 
includes cannabinol and tetrahydrocannabinol. 
The next group of drugs includes the vitamins such as A, B, e.g. B.sub.12, 
C, D, E and K, folic acid, thiamine. 
The next group of drugs is prostaglandins, which differ by the degree and 
sites of hydroxylation and unsaturation. 
The next group of drugs is antibiotics, which include penicillin, 
chloromycetin, actinomycetin, tetracycline, terramycin, the metabolites 
and derivatives. 
The next group of drugs is the nucleosides and nucleotides, which include 
ATP, NAD, FMN, adenosine, guanosine, thymidine, and cytidine with their 
appropriate sugar and phosphate substituents. 
The next group of drugs is miscellaneous individual drugs which include 
methadone, meprobamate, serotonin, meperidine, amitriptyline, 
nortriptyline, lidocaine, procaineamide, acetylprocaineamide, propranolol, 
griseofulvin, valproic acid, butyrophenones, antihistamines, 
anticholinergic drugs, such as atropine, their metabolites and 
derivatives. 
Metabolites related to diseased states include spermine, galactose, 
phenylpyruvic acid, and porphyrin Type 1. 
The next group of drugs is aminoglycosides, such as gentamicin, kanamicin, 
tobramycin, and amikacin. 
Among pesticides of interest are polyhalogenated biphenyls, phosphate 
esters, thiophosphates, carbamates, polyhalogenated sulfenamides, their 
metabolites and derivatives. 
For receptor analytes, the molecular weights will generally range from 
10,000 to 2.times.10.sup.8, more usually from 10,000 to 10.sup.6. For 
immunoglobulins, IgA, IgG, IgE and IgM, the molecular weights will 
generally vary from about 160,000 to about 10.sup.6. Enzymes will normally 
range from about 10,000 to 1,000,000 in molecular weight. Natural 
receptors vary widely, generally being at least about 25,000 molecular 
weight and may be 10.sup.6 or higher molecular weight, including such 
materials as avidin, DNA, RNA, thyroxine binding globulin, thyroxine 
binding prealbumin, transcortin, etc. 
Ligand analog or analyte analog--a modified ligand or ligand surrogate or 
modified analyte or analyte surrogate which can compete with the analogous 
ligand or analyte for a receptor, the modification providing means to join 
a ligand analog or analyte analog to another molecule. The ligand analog 
or analyte analog will usually differ from the ligand or analyte by more 
than replacement of a hydrogen with a bond which links the ligand analog 
or analyte analog to a hub or label, but need not. The term ligand 
surrogate or analyte surrogate refers to a compound having the capability 
of specifically binding a receptor complementary to the ligand or analyte. 
Thus, the ligand surrogate or analyte surrogate can bind to the receptor 
in a manner similar to the ligand or analyte. The surrogate could be, for 
example, an antibody directed against the idiotype of an antibody to the 
ligand or analyte. 
Poly(ligand analog)--a plurality of ligand analogs joined together 
covalently, normally to a hub nucleus. The hub nucleus is a polyfunctional 
material, normally polymeric, usually having a plurality of functional 
groups, e.g., hydroxyl, amino, mercapto, ethylenic, etc. as sites for 
linking. The hub nucleus may be water soluble or insoluble, preferably 
water soluble, and will normally be at least about 30,000 molecular weight 
and may be 10 million or more molecular weight. Illustrative hub nuclei 
include polysaccharides, polypeptides (including proteins), nucleic acids, 
anion exchange resins, and the like. Water insoluble hub nuclei can also 
include walls of containers, e.g. glass or plastic, glass beads, addition 
and condensation polymers, Sephadex and Agarose beads and the like. 
Member of a specific binding pair ("sbp member")--one of two different 
molecules, having an area on the surface or in a cavity which specifically 
binds to and is thereby defined as complementary with a particular spatial 
and polar organization of the other molecule. The members of the specific 
binding pair are referred to as ligand and receptor (antiligand). These 
will usually be members of an immunological pair such as antigen-antibody, 
although other specific binding pairs such as biotin-avidin, 
hormones-hormone receptors, nucleic acid duplexes, IgG-protein A, DNA-DNA, 
DNA-RNA, and the like are not immunological pairs but are included in the 
invention. 
Ligand-any organic compound for which a receptor naturally exists or can be 
prepared. 
Receptor ("antiligand")--any compound or composition capable of recognizing 
a particular spatial and polar organization of a molecule, e.g., epitopic 
or determinant site. Illustrative receptors include naturally occurring 
receptors, e.g., thyroxine binding globulin, antibodies, enzymes, Fab 
fragments, lectins, nucleic acids, protein A, complement component Clq, 
and the like. 
Non-magnetic particles--diamagnetic or paramagnetic particles usually with 
a magnetic susceptibility (X) of less than 1.times.10.sup.-5 
emu/Oecm.sup.3. The non-magnetic particles are generally at least about 
0.02 microns and not more than about 100 microns, usually at least about 
0.05 microns and less than about 20 microns, preferably from about 0.3 to 
10 microns diameter. The non-magnetic particle may be organic or 
inorganic, swellable or non-swellable, porous or non-porous, preferably of 
a density approximating water, generally from about 0.7 to about 1.5 g/ml, 
and composed of material that can be transparent, partially transparent, 
or opaque. Usually the non-magnetic particles will have a charge, either 
positive or negative, and may have sbp members on their surface. Normally, 
the non-magnetic particles will be biologic materials such as cells and 
microorganisms, e.g., erythrocytes, leukocytes, lymphocytes, hybridomas, 
streptococcus, staphylococcus aureus, E. coli, viruses, and the like. The 
non-magnetic particles can also be particles comprised of organic and 
inorganic polymers, liposomes, latex particles, phospholipid vesicles, 
chylomicrons, lipoproteins, and the like. 
The polymers will normally be either addition or condensation polymers. 
Non-magnetic particles derived therefrom will be readily dispersible in 
the assay medium and may be adsorptive or functionalizable so as to bind, 
either directly or indirectly, an sbp member or a magnetic particle. 
Frequently, the non-magnetic particles will be an analyte, be bound to an 
analyte, or will become bound to an analyte during an assay. The 
non-magnetic particles not initially bound to the analyte can be derived 
from naturally occurring materials, naturally occurring materials which 
are synthetically modified and synthetic materials. Among organic polymers 
of particular interest are polysaccharides, particularly cross-linked 
polysaccharides, such a agarose, which is available as Sepharose, dextran, 
available as Sephadex and Sephacryl, cellulose, starch, and the like; 
addition polymers, such as polystyrene, polyvinyl alcohol, homopolymers 
and copolymers of derivatives of acrylate and methacrylate, particularly 
esters and amides having free hydroxyl functionalities, and the like. 
The non-magnetic particles for use in assays will usually be polyfunctional 
and will have bound to or be capable of specific non-covalent binding to 
an sbp member, such as antibodies, avidin, biotin, lectins, protein A, and 
the like. A wide variety of functional groups are available or can be 
incorporated. Functional groups include carboxylic acids, aldehydes, amino 
groups, cyano groups, ethylene groups, hydroxyl groups, mercapto groups 
and the like. The manner of linking a wide variety of compounds to 
particles is well known and is amply illustrated in the literature. See 
for example Cautrecasas, J. Biol. Chem., 245 3059 (1970). The length of a 
linking group may vary widely, depending upon the nature of the compound 
being linked, the effect of the distance between the compound being linked 
and the particle on the binding of sbp members and the analyte and the 
like. 
The non-magnetic particle will normally have an electronic charge, either 
positive or negative. The particle can be inherently charged or can be 
treated chemically or physically to introduce a charge. For example, 
groups such as carboxyl, sulfonate, phosphate, amino, and the like can be 
chemically bound to or formed on the particles by techniques known in the 
art. Cells are normally negatively charged due to the presence of sialic 
acid residues on the cell surface. Latex particles can be positively or 
negatively charged but normally will have a negative charge as a result of 
the introduction of functional groups or absorption of charged polymers 
such as polypeptides, proteins, polyacrylate, and the like. 
The non-magnetic particles can be fluorescent or non-fluorescent, usually 
non-fluorescent, but when fluorescent can be either fluorescent directly 
or by virtue of fluorescent compounds or fluorescers bound to the particle 
in conventional ways. The fluorescers will usually be dissolved in or 
bound covalently or non-covalently to the non-magnetic particle and will 
frequently be substantially uniformly bound through the particle. 
Fluoresceinated latex particles are taught in U.S. Pat. No. 3,853,987 and 
are available commercially as Covaspheres from Covalent Technology Corp. 
The fluorescers of interest will generally emit light at a wavelength above 
350 nm, usually above 400 nm and preferably above 450 nm. Desirably, the 
fluorescers have a high quantum efficiency, a large Stokes shift and are 
chemically stable under the conditions of their conjugation and use. The 
term fluorescer is intended to include substances that emit light upon 
activation by electromagnetic radiation or chemical activation and 
includes fluorescent and phosphorescent substances, scintillators, and 
chemiluminescent substances. 
Fluorescers of interest fall into a variety of categories having certain 
primary functionalities. These primary functionalities include 1- and 
2-aminonaphthalene, p,p-diaminostilbenes, pyrenes, quaternary 
phenanthridine salts, 9-aminoacridines, p,p'-diaminostilbenes, imines, 
anthracenes, oxacarbocyanine, merocyanine, 3-aminoequilenin, perylene, 
bis-benzoxazole, bis-p-oxazolyl benzene, 1,2-benzophenazine, retinol, 
bis-3-aminopyridinium salts, hellebrigenin, tetracycline, sterophenol, 
benzimidazolylphenylamine, 2-oxo-3-chromen, indole, xanthene, 
7-hydroxycoumarin, 4,5-benzimidazoles, phenoxazine, salicylate, 
strophanthidin, porphyrins, triarylmethanes, flavin and rare earth 
chelates oxides and salts. Exemplary fluorescers are enumerated in U.S. 
Pat. No. 4,318,707, columns 7 and 8, the disclosure of which is 
incorporated herein by reference. Squaraine dyes described in U.S. patent 
application Ser. No. 773,401, filed September 6, 1985 (the relevant 
disclosure of which is incorporated by reference) are also useful as 
fluorescers. 
Additionally, light absorbent non-magnetic particles can be employed which 
are solid insoluble particles of at least about 10 nm in diameter. 
Many different types of particles may be employed. Of particular interest 
are carbon particles, such as charcoal, lamp black, graphite, colloidal 
carbon and the like. Besides carbon particles metal sols may also find 
use, particularly of the noble metals, gold, silver, and platinum. 
Label--A member of the signal producing system that is conjugated to an sbp 
member. The label can be isotopic or non-isotopic, usually non-isotopic, 
including catalysts such as an enzyme, a chromogen such as a fluorescer, 
dye or chemiluminescer, a radioactive substance, a particle, and so forth. 
Signal Producing System--The signal producing system may have one or more 
components, at least one component being a label. The signal producing 
system generates a signal that relates to the presence or amount of 
analyte in a sample. The signal producing system includes all of the 
reagents required to produce a measurable signal. When the label is not 
conjugated to an sbp member analogous to the analyte, the label is 
normally bound to an sbp member complementary to an sbp member that is 
analogous to the analyte. Other components of the signal producing system 
can include substrates, enhancers, activators, chemiluminiscent compounds, 
cofactors, inhibitors, scavengers, metal ions, specific binding substances 
required for binding of signal generating substances, and the like. Other 
components of the signal producing system may be coenzymes, substances 
that react with enzymic products, other enzymes and catalysts, and the 
like. The signal producing system provides a signal detectable by external 
means, preferably by measurement of the degree of aggregation of particles 
or by use of electromagnetic radiation, desirably by visual examination. 
For the most part, the signal producing system will involve particles, 
such as fluorescent particles or other light absorbing particles, a 
chromophoric substrate and enzyme, where chromophoric substrates are 
enzymatically converted to dyes which absorb light in the ultraviolet or 
visible region, phosphors, fluorescers or chemiluminescers. 
The signal-producing system can include at least one catalyst, usually an 
enzyme, and at least one substrate and may include two or more catalysts 
and a plurality of substrates, and may include a combination of enzymes, 
where the substrate of one enzyme is the product of the other enzyme. The 
operation of the signal producing system is to produce a product which 
provides a detectable signal related to the amount of analyte in the 
sample. 
A large number of enzymes and coenzymes useful in a signal producing system 
are indicated in U.S. Pat. No. 4,275,149, columns 19 to 23, and U.S. Pat. 
No. 4,318,980, columns 10 to 14, which disclosures are incorporated herein 
by reference. A number of enzyme combinations are set forth in U.S. Pat. 
No. 4,275,149, columns 23 to 28, which combinations can find use in the 
subject invention. This disclosure is incorporated herein by reference. 
Of particular interest are enzymes which involve the production of hydrogen 
peroxide and the use of the hydrogen peroxide to oxidize a dye precursor 
to a dye. Particular combinations include saccharide oxidases, e.g., 
glucose and galactose oxidase, or heterocyclic oxidases, such as uricase 
and xanthine oxidase, coupled with an enzyme which employs the hydrogen 
peroxide to oxidize a dye precursor, that is, a peroxidase such as horse 
radish peroxidase, lactoperoxidase, or microperoxidase. Additional enzyme 
combinations may be found in the subject matter incorporated by reference. 
When a single enzyme is used as a label, other enzymes may find use such 
as hydrolases, transferases, and oxidoreductases, preferably hydrolases 
such as alkaline phosphatase and .beta.-galactosidase. Alternatively, 
luciferases may be used such as firefly luciferase and bacterial 
luciferase. 
Illustrative coenzymes which find use include NAD[H]; NADP[H], pyridoxal 
phosphate; FAD[H]; FMN[H], etc., usually coenzymes involving cycling 
reactions, see particularly U.S. Pat. No. 4,318,980. 
The product of the enzyme reaction will usually be a dye or fluorescer. A 
large number of illustrative fluorescers are indicated in U.S. Pat. No. 
4,275,149, columns 30 and 31, which disclosure is incorporated herein by 
reference. 
Magnetic particles--particles that are intrinsically magnetically 
responsive or have been rendered magnetic by, for example, attachment to a 
magnetically responsive substance or by incorporation of such substance 
into the particles. The magnetic particles can be paramagnetic, 
ferromagnetic, or superparamagnetic, usually paramagnetic and will have 
magnetic susceptibilities (X) of at least 5.times.10.sup.-5 
emu/Oecm.sup.3, usually at least 4.times.10.sup.-4 emu/Oecm.sup.3. The 
diameter of the particles should be small, generally in the range from 
about 5 nm to 1 micron, preferably from about 10 to 250 nm, more 
preferably from about 20 to 100 nm, most preferably colloidal. 
Exemplary of the magnetic component of particles that are intrinsically 
magnetic or magnetically responsive are complex salts and oxides, borides, 
and sulfides of iron, cobalt, nickel and rare earth elements having high 
magnetic susceptibility, e.g. hematite, ferrite. The magnetic component of 
other such particles includes pure metals or alloys comprising one or more 
of these elements. 
For the most part the magnetic particles will contain a core of the 
magnetic component with surface functional groups such as hydroxyl, 
silicate, carboxylate, sulfate, amino, phosphate and the like. Frequently, 
an additional surface coating will be employed that is covalently or 
non-covalently bound to the surface. The surface coating can be an anionic 
or cationic detergent, usually anionic; or the coating can be a protein 
such as albumin, immunoglobulin, avidin, fetuin or the like; or it can be 
a carbohydrate such as dextran, chitosan, amylose and the like, or 
combinations or these substances in their native form or functionalized so 
as to control their charge and hydrophilicity. Alternatively, the 
particles can be coated with other amphiphilic substances such as 
lipopolysaccharides, octyl glucoside, etc. 
Alternatively, the magnetic component can be incorporated into a particle 
such as, for example, impregnating the substance in a polymeric matrix. 
However, this procedure frequently gives particles larger than the 
magnetic particles of this invention. For a more in-depth discussion of 
the preparation of magnetic particles by this method, see Whitesides, et 
al. (1983) Trends in Biotechnology, 1(5):144-148 and references cited 
therein. 
Preferred magnetic particles of less than a hundred nanometers in diameter 
can be made by precipitating iron oxides in the presence or absence of a 
coating such as a polysaccharide or protein. Magnetic particles of a few 
microns diameter can also be made by a ball milling process and removing 
material which is not in the size range of interest. Typically, magnetic 
particles formed by this latter process are quite polydisperse, and not as 
generally useful. More useful monodisperse metal oxide suspensions can be 
prepared by careful control of pH, temperature and concentrations during 
the precipitaion process. Coating the magnetic particles with 
macromolecules can increase their colliodal stability. This can be done by 
direct adsorption of high molecular weight polymers or by functionalizing 
the surface of the particle and then binding macromolecules to the 
functional groups. Emulsion polymerization and grafting techniques provide 
a means for coating magnetic particles with polymers. 
In general, the magnetic particle that is best for a given task will be 
determined primarily by the size and properties of the particles to be 
separated. For immunoassays or the isolation of cells, the magnetic 
particles preferably should be readily suspendable, form stable, 
preferably colloidal, suspensions, and have high magnetic susceptibility. 
Where an sbp member is bound to the surface, its ability to bind to a 
complementary sbp should be retained and should be stable with time. 
Small (&lt;100 nm) magnetic particles are advantageously used in immunoassays 
and cell separation procedures. These particles preferably have a 
homogenous core of metal, metal oxide or other metal compound. When 
colloidally stable, small particles can be suspended for long periods of 
time. Their large surface to volume ratio and relatively higher rates of 
diffusion enable them to quickly bind molecules and particles that are 
dispersed in the medium. Small magnetic particles are also less 
susceptible than large magnetic particles to aggregation due to residual 
magnetic moments after they have been exposed to a large applied magnetic 
field. Also, methods are known for colloidally stabilizing such small 
particles. 
Magnetic particles of an intermediate size (100-1000 nm) can also be 
employed. They can be suspended readily and require a lower surface charge 
density to prevent spontaneous aggregation than do smaller particles. 
Magnetic particles of this size range can be created by emulsion 
polymerization and have the advantage of a surface that is easily modified 
whether by grafting or the covalent bonding of macromolecules to their 
surface. However, they remain suspended for shorter times and their lower 
surface to volume ratio decreases the rate of binding to the substance to 
be separated. 
Magnetic fluid--a colloidal suspension of magnetic particles in a liquid 
carrier that are not readily separated by a magnetic field. The resulting 
liquid has the bulk properties of a magnetic material. The fluid becomes 
spontaneously magnetized in the presence of an external magnetic field. 
The liquid also acts as a fluid and is capable of assuming the shape of 
its container, of flowing, and of moving around obstacles. Exemplary of a 
magnetic fluid is a ferrofluid where the suspended particles are 
ferromagnetic particles (see, for example, Rosenweig, supra, and U.S. Pat. 
No. 4,019,994, the disclosure of which is incorporated herein by 
reference, and Khalafolla, et al. (1980) IEEE Transactions on Magnetics, 
MAG-16:178-183). 
The colloidal magnetic particles can be coated with protein material, e.g., 
a serum protein such as albumin, gammaglobulin, etc., and the like. The 
colloidal magnetic particles can be mixed with an aqueous buffered 
solution of protein to prepare the protein-coated colloidal magnetic 
particles. The coating of the magnetic particles with protein can be 
accomplished by physical (e.g., absorption) or chemical binding. 
Non-specific binding--non-covalent hinding between particles that is 
relatively independent of specific surface structures. Such non-specific 
binding will usually result from charge or electronic interactions between 
oppositely charged particles or between particles having the same charge 
where a polyionic reagent having a charge opposite thereto is employed. 
Non-specific binding may also result from hydrophobic interactions between 
particles. 
Polyionic reagent--a compound, composition, or material, either inorganic 
or organic, naturally occurring or synthetic, having at least two of the 
same charge, either polyanionic or polycationic, preferably at least ten 
of the same charge; e.g., a polyelectrolyte. 
Exemplary of polycationic reagents are polyalkylene amines such as 
polyethyleneimine and polypropyleneimine and their lower alkyl ammonium 
salts such as polybrene (--N(CH.sub.3).sub.2 CH.sub.2 CH.sub.2 
N(CH.sub.3).sub.2 CH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 --).sub.n, metal 
ions such as calcium and barium ion, aminodextrans, protamine, positively 
charged liposomes, polylysine, and the like. 
Exemplary of polyanionic reagents are heparin, dextran sulfate, negatively 
charged phospholipid vesicles, polycarboxylic acids, such as polyacrylate, 
polyglutamate and the like. The above materials and their preparation or 
isolation are well known in the art and many are commercially available. 
Releasing agent--a compound, composition, or material, either naturally 
occurring or synthetic, organic or inorganic, capable of reversing the 
non-specific binding between particles, i.e., dissociating such particles. 
The releasing agent acts upon the non-specific bond between the particles. 
For example, where the non-specific binding results from charge 
interactions, the releasing agent can act to change the pH of the medium 
to one which is unfavorable or incompatible with the charge interactions. 
The releasing agent can, therefore, be an acid such as a mineral acid or 
an organic acid or a base such as a mineral base or an organic base. 
Alternatively, the releasing agent can act to shield ionic interactions 
and thus can be a high ionic strength solution or a solution of a neutral 
polymer such as dextran. Alternatively, the releasing agent can have a 
charge which disrupts the non-specific binding between the particles and 
the magnetic particles. Exemplary of the latter are be polyelectrolyte 
salts such as citrate, polyacrylate, dextran sulfate, and the like. Where 
the particles are bound by a polyionic bridge, the releasing agent can be 
a polyionic agent of opposite charge or can be a reagent which 
depolymerizes the polyionic reagent. Where the particles and magnetic 
particles are of opposite charge, other positively or negatively charged 
polyelectrolytes or high ionic strength solutions may be used. 
Ancillary Materials-Various ancillary materials will frequently be employed 
in an assay in accordance with the present invention. For example, buffers 
will normally be present in the assay medium, as well as stabilizers for 
the assay medium and the assay components. Frequently, in addition to 
these additives, additional proteins may be included, such as albumins, or 
surfactants, particularly non-ionic surfactants, binding enhancers, e.g., 
polyalkylene glycols, or the like. 
As mentioned above, the present invention involves a method for separating 
a substance from a liquid medium. The method comprises combining a liquid 
medium containing the substance with magnetic particles, preferably 
dispersed as a magnetic liquid, under conditions for binding the substance 
to the magnetic particles and non-specifically binding and aggregating the 
magnetic particles where a chemical means is used to cause the suspended 
magnetic particles to non-specifically bind to one another. The substance 
to be separated will frequently be a non-magnetic particle or will be 
bound to a non-magnetic particle. The non-specific binding is usually 
conveniently obtained as the result of charge interactions, which can also 
serve to non-specifically bind non magnetic particles to the magnetic 
particles. For example, the non-magnetic particles and the magnetic 
particles can have opposite electronic charges and non-specific binding 
will occur spontaneously. Where the particles and the magnetic particles 
have the same charge, a polyionic reagent having an opposite charge can be 
added to the medium to cause non-specific binding between the non-magnetic 
particles and the magnetic particles and between the magnetic particles. 
After the above combination is formed, the medium is subjected to a 
magnetic field gradient to separate the particles from the medium. 
In carrying out the method, a liquid, usually aqueous, medium will be 
employed. Other polar solvents may also be employed, usually oxygenated 
organic solvents from one to six, more usually from one to four, carbon 
atoms, including alcohols, ethers, and the like. Usually these cosolvents 
will be present in less than about 40 weight percent, more usually in less 
than about 20 weight percent. The pH for the medium will usually be 
selected to promote non-specific binding and aggregation of the magnetic 
particles prior to separation. Where the particles are negatively charged, 
increasing the pH will tend to increase the charge and prevent spontaneous 
aggregation caused by non-specific hydrophobic and Van der Waals 
interactions. The converse applies to positively charge particles. Where 
an oppositely charged polyelectrolyte is added to cause aggregation, 
changes in pH that increase the charge of the polyelectrolyte will often 
decrease the charge of the particles and an optimum pH must be selected 
that will avoid the use of excessive amounts of this reagent. Generally, a 
pH range of 5 to 10, more usually 6 to 9, will be used. For assays, other 
considerations with respect to pH are to maintain a significant level of 
binding of sbp members while optimizing signal producing proficiency. In 
some instances, a compromise will be made between these considerations. 
Various buffers may be used to achieve the desired pH and maintain the pH 
during the determination. Illustrative buffers include borate, phosphate, 
carbonate, Tris, barbital, and the like. The particular buffer employed is 
not critical to this invention; however, in individual separations or 
individual assays, one buffer may be preferred over another. When 
non-magnetic particles are involved, a reagent that promotes reversal of 
the binding of the particles and the magnetic particles can be added after 
the separation has been accomplished. 
Moderate temperatures are normally employed for carrying out the method and 
usually constant temperatures during the period for conducting the method. 
Generally, the temperatures will be chosen to promote non-specific binding 
of the particles prior to separation. The temperature for the method, 
particularly involving an assay, will generally range from about 0.degree. 
to 50.degree. C., more usually from about 15.degree. to 40.degree. C. 
Again, after the separation is accomplished a temperature that promotes 
reversal of the binding of the particles and the magnetic particles can 
then be chosen. 
The concentration of the magnetic particles in the medium will depend on 
the amount of the substance in the medium that is to be separated and 
whether or not it is particulate, the rate of separation that is desired, 
the magnetic field gradient and field strength, the magnetic 
susceptability of the magnetic particles and the like. In general, higher 
concentrations of magnetic particles provide more efficient and rapid 
separations but too high a concentration can cause excessive entrainment 
of the medium. The concentration is normally determined empirically and 
will generally vary from about 0.1 to 1000 .mu.g/ml, more usually from 
about 0.5 to 200 .mu.g/ml, frequently from about 1 to 50 .mu.g/ml. 
Where non-magnetic particles are to be separated from a medium, the 
concentration of the non-magnetic particles can vary widely depending upon 
the need. For example, in separation of cells from blood, the cell volume 
may represent 50% of the total volume of the blood. By contrast, it may be 
desired to separate as few as 1000 bacteria/ml from a sample of water. 
When it is necessary to obtain non-magnetic particles that are relatively 
free of the medium as in an assay, usually the total volume of the 
non-magnetic particles should be less than 5% of the medium. In an assay 
where the analyte is a component of a particle or becomes bound to a 
particle, the analyte will generally vary from about 10.sup.-4 to 
10.sup.-14 M, more usually from about 10.sup.-6 to 10.sup.-12 M. Where 
non-magnetic particles other than natural particles associated with the 
analyte are added to the medium, their concentration will depend on 
numerous factors such as particle size and surface area, concentration of 
the analyte, desired rate of reaction with the analyte or complementary 
sbp and the like. In general, added non-magnetic concentrations will be 
about 0.01 to 100 .mu.g/ml, more usually from about 0.1 to 20 .mu.g/ml. 
Considerations such as the concentration of the analyte, non-specific 
binding effects, desired rate of the reaction, temperature, solubility, 
viscosity, and the like will normally determine the concentration of other 
assay reagents. 
While the concentrations of the various reagents will generally be 
determined by the concentration range of interest of the particles to be 
separated or of the concentration range of the analyte in an assay, the 
final concentration of each of the reagents will normally be determined 
empirically to optimize the rate and extent of separation of the particles 
and, in the case of an assay, the sensitivity and specificity of the assay 
over the range of interest. 
Chemical means for forming non-specific bonds between the particles will 
usually be included in the liquid medium. Except where non-magnetic 
particles are to be separated that have an opposite charge to the magnetic 
particles, this chemical means is usually a polyionic reagent having a 
charge opposite to that of the particles. The amount of polyionic reagent 
added should be sufficient so that substantially all of the particles 
become aggregated or coaggregated. This concentration must be determined 
empirically. Excess reagent must generally be avoided because this 
interferes with complete aggregation of the particles. Generally, the 
polyionic reagent will have a concentration in the liquid medium 
sufficient to provide a number of ions associated with the polymer and 
equal to the total number of charges of opposite sign on all the particles 
in the medium. Where non-magnetic particles are to be separated that have 
an opposite charge to the magnetic particles, the chemical means for 
forming non-specific bonds between the particles will frequently be a low 
ionic strength buffer. 
In an assay, the aqueous medium can also contain one or more members of a 
single producing system. As mentioned above the concentration of the 
various members of the single producing system will vary and be dependent 
upon the concentration range of interest of the analyte and the type of 
measurement or assay involved. As a general point, the concentration of 
the various members of the signal producing system will be selected to 
optimize the signal produced in relation to the concentration range of 
interest of the analyte. 
The binding of non-magnetic particles to magnetic particles or of magnetic 
particles to each other is affected by pH. The binding is also affected by 
other factors such as ionic strength and the presence of ionic and 
non-ionic polymers. Generally, where non-specific binding is due to charge 
interactions the ionic strength should be chosen initially to facilitate 
the binding between the particles. For this purpose the ionic strength is 
generally low and can be in the range of 0.001 to 0.5M, preferably 0.005 
to 0.1M. After the separation has been completed, the ionic strength can 
be adjusted upward to facilitate the reversal of the coupling of the 
particles and the magnetic particles. For this purpose, the ionic strength 
of the medium will normally be from about 0.1 to 3M, preferably from about 
0.15 to 1M. The principles for causing particles to aggregate or to remain 
suspended are well known in the field of colloid science. 
After the magnetic particles have been combined in the liquid medium for 
the purpose of an assay where specific binding to the magnetic particles 
is required, the liquid medium is then held for a period of time 
sufficient for the binding to occur. Normally this requires 0.5-120 
minutes, more frequently 1-60 min. The subsequent chemically induced 
non-specific aggregation of the magnetic particles, and the non-specific 
coaggreation of particles when only non-specific binding of particles is 
required, will occur essentially instantaneously, and it is usually 
sufficient to allow the mixture to stand for 60 sec., frequently less than 
15 sec.; preferably the magnetic field is applied immediately after 
mixing. The extent of binding between the particles and the magnetic 
particles or between magnetic particles controls the efficiency of the 
magnetic separation. 
After aggregation of the particles, a magnetic field is applied to achieve 
a separation of the particles from the medium. The application of a 
magnetic field to the medium can be carried out in a conventional manner 
that provides for a high magnetic field gradient. Normally, the method is 
conducted in a container made of non-magnetic material, for example, glass 
or plastic. In applying the magnetic field, the reaction container can be 
placed in close proximity to an electromagnet or permanent magnet, 
preferably permanent, which has a geometry to maximize the field intensity 
and gradient within the suspension. The higher the strength of the 
magnetic field and the higher the gradient, the faster the separation. 
Normally, it will be convenient to carry out the separation in a tube of 
diameter from about 2 to 50 mm, preferably from about 3 to 15 mm, with one 
or more permanent magnets mounted as close to the tube as practical to 
provide field strengths of at least about 200 Oe and preferably at least 
about 1KOe with magnetic field gradients usually at least about 20 KOe/cm. 
The magnetic field is applied for a sufficient period of time to provide 
the desired degree of separation of the particles from the medium. 
Depending on the geometry, field strength, magnetic susceptibility of the 
particle and the like, the magnetic field is applied for a period of about 
2 seconds to 1 hour, preferably about 5 seconds to 60 seconds. 
Once the particles have been concentrated to one part of the container, the 
suspending liquid medium can be separated from the particles by any 
convenient means such as, for example, decantation, pipeting, and the 
like. 
The particles separated from the liquid medium can be treated to reverse 
the non-specific binding between the particles by suspending the particles 
in a liquid medium with reagents added to facilitate reversal of the 
binding. In one approach, where the particles are bound by ionic 
interactions, ionic strength and the pH of the medium can be adjusted to 
facilitate reversal of the binding. Generally, increasing the ionic 
strength will reverse electrostatic binding. Where the particles are 
negatively charged, a decrease in pH will lower the charge and reduce 
binding interactions. Alternatively, if a polycationic aggregating agent 
is used, increasing the pH can neutralize the charge and reverse binding. 
Thus, it may be desirable to change the pH to as high or low value as 
allowed by the stability of the reagents, usually no less than pH 4 or 
greater than pH 10. 
The reversal of the binding between the particles and the magnetic 
particles is dependent upon the nature of the non-specific binding between 
the particles. Where non-specific binding results from charge interaction, 
an agent can be added to reverse the charge interactions responsible for 
the non-specific binding. For example, a releasing agent can be added. 
Where binding results from aggregation of particles with opposite charges, 
either a polycationic or polyanionic polyelectrolyte can be used. Where 
the particles have like charges and an oppositely charged polyelectrolyte 
was the chemical means for binding the particles, a polyelectrolyte of the 
same charge as on the particles can be used to dissociate the particles. 
The polyelectrolytes can be, for example, polyanions such as dextran 
sulfate, heparin, polyglutamate, polyacrylate, phospholipid vesicles, 
carboxymethyedextran. Aminodextran, chitosan, polybrene, 
polyethyleneimine, and cationic liposomes are exemplary of polycations 
that can be employed. 
Where a polycation was used to initiate non-specific binding between the 
particles and the magnetic particles, or between the magnetic particles, a 
polyanion can be employed to reverse the binding. Alternatively, where a 
polyanion was used to form the non-specific binding between the particles 
and the magnetic particles or between the magnetic particles, a polycation 
can be used to reverse the binding. For example, where polycations such as 
polybrene or barium ion have been employed, the releasing agent can be a 
polyanion such as citrate or sulfate. Detergents can act as a releasing 
agent for liposomes and when particles are non specifically aggregated 
primarily through hydrophobic interactions. 
The concentration of the releasing agent should be sufficient to result in 
substantial or complete reversal of the non-specific binding between the 
particles. The concentration of the releasing agent is generally dependent 
upon the nature of binding between the particles and the magnetic 
particles and the nature of the particles. Generally, the concentration of 
the releasing agent will be at least equal to the concentration of ionic 
or hydrophobic sites on the particles, preferably at least 10 fold higher. 
It is important to choose the releasing agent with regard to the nature of 
the particles in the aggregate so as to minimize or avoid damage to the 
particles after the release from the aggregate. 
Once the particles have been separated from the aggregate, they may be used 
as desired. For example, in an assay the separated particles can be 
examined for the presence of a detectable signal in relation to the amount 
of an analyte in the sample. The separated particles can be cells which 
can be used as desired. For example, the separated particles can be red 
blood cells. 
In a preferred embodiment of the invention, the magnetic particles are 
provided as a magnetic liquid, e.g., ferrofluid. The particles to be 
separated are combined with the magnetic liquid. 
An important application of the present method is the removal of cells from 
a sample containing cells such as, for example, removal of red blood cells 
from whole blood. In the method, using whole blood by way of example and 
not by way of limitation, a whole blood sample is combined in a liquid 
medium with charged magnetic particles under conditions for non-specific 
binding of the magnetic particles to the cells. The cells will usually 
have a negative charge by virtue of sialic acid residues or the like on 
the surface of the cells. The magnetic particles can be positively 
charged, resulting in direct non-specific binding between the cells and 
the magnetic particles. Preferably, the magnetic particles have a negative 
charge. In this latter instance a polycationic reagent is included in the 
medium to provide conditions for non-specific binding between the cells 
and the magnetic particles. Useful polycationic reagents in this method 
can be, for example, polybrene, polyalkyleneimines, aminodextran, 
chitosan, and positively charged liposomes. The preferred polycationic 
reagent for removing cells from whole blood is polybrene or 
polyethyleneimine. 
Next, the medium can be subjected to a magnetic field gradient to separate 
the cells from the medium. Application of the magnetic field results in 
concentration of the cell-magnetic particle aggregate to one portion of 
the container, which permits its removal of the residual cell-free medium 
by, for example, decantation, pipetting, etc. 
The separated cell-magnetic particle aggregate can then be treated to 
release the cells from the aggregate as described above. Where polybrene 
or polyethyleneimine is employed as a polycationic binding agent, 
preferred releasing agents are citrate or polyacrylate. 
The present method provides particluar advantages for automated blood 
typing procedures by providing a way to prepare blood plasma without 
centrifugation. It is also useful in the Coombs antiglobulin test where 
immunoglobulin-containing plasma is first combined with test cells and 
must then be fully removed in order to determine if antibodies from the 
plasma have bound to the cells. In this procedure magnetic particles and a 
non-specific binding agent are added to the mixture of plasma and test 
cells and the subsequent separated cells are resuspended with the help of 
a releasing agent. Moreover, the present method can be employed in 
immunoassays wherein an spb member is bound to a particle and it is 
desired to separate and wash the particles without centrifugation; the 
particles can be magnetic or non-magnetic. 
The present invention has application to assays for an analyte in a sample 
suspected of containing the analyte. The analyte is an spb member. In the 
assay the sample is combined in an assay medium with an spb member 
complementary to the analyte wherein at least one of the analyte or the 
complementary spb member is associated with the surface of a non-magnetic 
particle, usually a cell, latex particle, or a magnetic particle. The 
present invention offers the improvement of combining charged magnetic 
particles with the medium under conditions for non-specific binding and 
aggregation of the magnetic particles. Frequently, the conditions for 
non-specific binding include combining a polyionic reagent to cause 
non-specific binding between the magnetic particles. The assay will 
normally involve a signal producing system for producing a detectable 
signal in relation to the amount of analyte in the sample. The signal 
producing system usually includes a labeled sbp member. The medium may be 
further combined with none, one or more members of the signal producing 
system. The medium is subjected to a magnetic field gradient to separate 
aggregates comprising the magnetic particles from the medium. The 
separated aggregates or the medium can be examined for the presence of a 
detectable signal. Such a determination can require the addition of any 
remaining members of the signal producing system not added above. The 
separated aggregates can be treated according to the above conditions to 
separate non-magnetic particles from the magnetic particles prior to 
examining for the presence of a detectable signal. After the non-magnetic 
particles have been separated from the magnetic particles, the non 
magnetic particles may be examined for the presence of a detectable signal 
produced in relation to the amount of analyte in the sample. For this 
purpose they can be combined with any remaining members of the signal 
producing system not added above in order to generate a detectable signal. 
The invention further comprises a composition comprising an aggregate of 
(a) non-magnetic particles to which are bound an sbp member and that are 
non-specifically electrostatically bound to (b) magnetic particles. The 
composition may further comprise a polyionic reagent of opposite charge to 
the magnetic particles and the non-magnetic particles when the 
non-magnetic particles and magnetic particles have the same charge. The 
aggregate of the composition is generally capable of being disassociated 
into its component particles by employing a releasing agent. 
Alternatively, the composition of the invention can comprise magnetic 
particles to which are bound an sbp member and a polyionic reagent wherein 
the magnetic particles are non-specifically bound to one another. 
As a matter of convenience, the reagents for conducting an assay can be 
provided in a kit in package combination in predetermined amounts for use 
in assaying for an analyte. The kit can comprise (a) an sbp member 
complementary to the analyte, (b) an sbp member bound to a charged 
particle if neither the analyte nor the complementary sbp member is bound 
to a charged particle and (c) charged magnetic particles where the 
particles are not magnetic. The kit can also include reagents for 
generating a signal in relation to the amount of analyte in the sample. 
Furthermore, the kit can comprise a polyionic reagent having a charge 
opposite to that of the particles when all the particles have the same 
charge. Additionally, the kit can further comprise a releasing agent for 
reversing the binding between the particles. Ancillary agents can be 
included as necessary.

EXAMPLES 
The invention is described further by the following illustrative examples. 
All parts and percentages herein are by volume unless otherwise indicated. 
Temperatures are in degrees Centigrade (.degree.C.). 
EXAMPLE 1 
Preparation of Plasma 
Uncoagulated whole blood (480 .mu.l, 16 mg/ml) and a ferrofluid (250 .mu.l, 
4.5 mg Fe/ml) were sequentially added to a container placed in a magnetic 
field produced by a permanent magnet. The magnitude of the magnetic field 
was 4.0 Kgauss. The erythrocyte-particle aggregates moved towards the 
magnet poles and greater than 99% of erythrocytes present in blood were 
removed. The clear plasma was transferred to another container by 
decantation. Results were obtained on blood from 175 subjects; the time 
for complete separation varied from 15-85 sec. The ferrofluid comprised 
iron magnetic particles coated with succinylated bovine serum albumin. The 
ferrofluid was prepared according to Example 4. The succinylated bovine 
serum albumin was prepared as described in Example 4. 
EXAMPLE 2 
Assay for Anti-Rh Antibody 
To the plasma prepared in Example 1 were added polyacrylic acid (10 .mu.l, 
2 mg/ml) and Rh positive test cells stained with a squarate dye. Fifty 
.mu.l squarate dye (10.sup.-4 M, dissolved in DMF) was added to a 
suspension of 1 ml of erythrocytes. (The squarate dye was 
2-(p-dibutyl-amino-m-hydroxyphenyl)-4-(4-diethylimmonio-2-hydroxy-2,5-cycl 
ohexadienylidene)-3-oxo-1-cyclobutenolate and was prepared by condensing 
squaric acid with 3-N,N-dibutyl-aminophenol in n-butanol:toluene (2:1) 
followed by azeotropic removal of water.) The mixture was incubated for 8 
min. at 37.degree. C. Buffer (16 gm/l glycine, 0.03M NaCl, 0.015M 
phosphate pH 6.7) (500 .mu.l), ferrofluid, and polybrene (10 .mu.l, 16 
mg/ml) were then sequentially added and separation of test cells occurred 
in the presence of a magnetic field having the same intensity as described 
above. The test cells (held via the aggregation in accordance with the 
present invention against the sides of the container) were washed twice 
with buffer (a low ionic strength saline solution, phosphate (0.003M), pH 
6.7 containing 0.24M glycine and 0.03M NaCl). Next polyacrylic acid (10 
.mu.l, 2 mg/ml) followed by antihuman immunoglobulin containing 1% 
polyvinylpyrrolidone (PVP) were added. After a 3 min. incubation at 
25.degree. C., the reaction mixture was diluted with citric acid (800 
.mu.l, 0.2M) and analyzed by a fiber optic particle cytometer method 
described by Briggs, J., et al, J. Immunol. 81, 73-81 (1985) and in U.S. 
patent application Ser. No. 397,285 filed July 12, 1982, the disclosure of 
which is incorporated herein by reference in its entirety. 
Briefly, in U.S. Ser. No. 397,285, method and apparatus are provided for 
determining the presence of particles in a dispersion in relation to the 
detection of the presence or amount of a material of interest. An optical 
fiber is used to define a relatively small volume from which fluorescent 
light can be received and analyzed. The volume is related to the volume in 
which there is likely to be only a single particle that results in a 
predetermined fluctuation. By employing a variety of techniques that allow 
for changes in fluorescence fluctuations in relation to the presence of an 
analyte in a sample, the amount of analyte present may be determined. The 
fluctuations are observed over a period of time in a static mode or by 
sampling a plurality of volumes in the sample. By comparing the observed 
results with results obtained with assay solutions having a known amount 
of analyte, the amount of analyte can be quantitatively determined. 
The results from the above experiment are summarized in the following 
table: 
TABLE 1 
______________________________________ 
Sample Signal.sup.a 
______________________________________ 
Control plasma 30 .+-. 4 
Control plasma spiked with anti-Rh Ab.sup.b 
80 .+-. 9 
______________________________________ 
.sup.a A signal greater than 5 SD (standard deviation) from control plasm 
was regarded as positive. 
.sup.b Enough antiRh antibody was added to give a 1.sup.+ score (scale 
1.sup.+ to 4.sup.+) with a commercially available antibody screen test 
using conventional antihuman serum. 
The results demonstrate that a sensitive assay for anti-Rh antibody can be 
carried out in accordance with the present invention. 
EXAMPLE 3 
Separation of Beads Labeled with Anti-triiodothyronine (T3) 
A. Reagents and Abbreviations 
1. PB-T3(.sup.125 I)=carboxysubstituted polystyrene heads labeled with 
anti-T3 antibodies (radioiodinated) by EDAC coupling. 
2. MP=magnetic particles a) PGA=magnetite derivatized with glucuronic acid 
through phosphate (0.2-0.8 .mu.m). b) CM-Dex.sub.3 
("ferrofluid")=carboxymethyl dextran - magnetite (0.030-0.45 .mu.m) 
prepared according to a procedure similar to that described in U.S. Pat. 
No. 4,452,773. c) M4100 BioMag-COOH (0.2-1.0 .mu.m) M4100 BioMag particles 
from Advanced Magnetics Inc.; succinylated on free amine groups of 
particles. 
3. Polybrene=Hexadimethrine Bromide obtained from Sigma Chem. Co. 
4. Assay buffer=PBS/0.1% BSA. 
5. Normal human serum. 
B. Procedure 
To 250 .mu.l of PB-T3(.sup.125 I), containing 55 .mu.g bead, was added 50 
.mu.l Assay Buffer or normal human serum. After incubating this mixture 
for 20 minutes at room temperature, 100 l of MP was added (PGA, 
CM-Dex.sub.m or BioMag-COOH) containing 0.2 mg Fe. To this reaction 
mixture, 50 .mu.l of polybrene (at varying concentration) was added. After 
agitating for about 3 minutes, the tubes were placed in a magnetic field 
having an intensity of 2.6 Kgauss for five minutes. After separation, the 
supernates were decanted and the pellets counted using Beckman gamma 5500 
counter. 
C. Results 
TABLE 2 
______________________________________ 
Concentration Amount of Polystyrene 
Magnetic of Polybrene Removed from Reaction 
Particles 
(mg/ml) Mixture (%) 
Used buffer serum buffer serum 
______________________________________ 
PGA 0.14 0.56 92 73 
CM-Dex.sub.m 
0.4 0.4 87 84 
Biomag- 0.035 0.14 88 77 
COOH 
______________________________________ 
Removal of 0.26 .mu.m polystyrene beads, coated with antiT.sub.3 
antibodies, from reaction mixture by coaggregating them with negatively 
charged magnetic particles using polybrene. 
TABLE 3 
______________________________________ 
Concentration Amount of Polystyrene 
Magnetic of Polybrene Removed from Reaction 
Particles 
(mg/ml) Mixture (%) 
Used buffer serum buffer serum 
______________________________________ 
PGA 0.035 0.14 &gt;99 &gt;99 
CM-Dex.sub.m 
0.4 0.4 &gt;99 &gt;99 
BioMag- 0.035 0.14 &gt;99 &gt;99 
COOH 
______________________________________ 
Removal of 0.51-1.2 micrometer polystyrene beads, coated with antiT.sub.3 
antibodies, from reaction mixture, by coaggregating them with negatively 
charged magnetic particles using polybrene. 
TABLE 4 
______________________________________ 
Concentration Amount of Polystyrene 
Magnetic of Polybrene Removed from Reaction 
Particles (mg/ml) Mixture (%) 
Used buffer assay buffer serum 
______________________________________ 
PGA 0.035 0.14 92 92 
CM-Dex.sub.m 
0.4 0.4 98 98 
BioMag-COOH 
0.035 0.14 90 94 
______________________________________ 
Removal of succinylated 0.26 .mu.m polystyrene beads, coated with 
antiT.sub.3 antibodies, from reaction mixture, by coaggregating them with 
negatively charged magnetic particles using polybrene. 
TABLE 5 
______________________________________ 
NaCl CPM.sup.a removed from Reaction (%) 
(moles/l) PGA CM-Dex.sub.3 
______________________________________ 
0 95 82 
0.058 93 80 
0.086 90 88 
0.141 94 90 
0.252 88 32 
0.474 5 4 
______________________________________ 
CPM = counts per minute 
TABLE 6 
______________________________________ 
Polybrene CPM removed from reaction (%) 
(mg/ml) PGA CM-Dex.sub.3 
______________________________________ 
0 19 2 
0.025 20 2 
0.07 90 3 
0.14 94 5 
0.28 93 55 
0.56 90 90 
1.12 88 92 
2.24 85 93 
4.48 87 91 
8.96 90 82 
17.92 56 16 
______________________________________ 
Discussion 
We have demonstrated that polystyrene beads coated with anti-T.sub.3 
antibodies can be effectively removed from a reaction mixture by 
coaggregating them nonspecifically with negatively charged magnetic 
particles, using polybrene and a magnetic field. 
The results presented in Tables 2, 3, and 4 indicate that larger 
polystyrene beads can be removed from the reaction mixture more 
effectively than small beads. Also, the difference of removal efficiency 
between succinylated and nonsuccinylated polystyrene beads indicates that 
the charge distribution on the surface of microparticles contributes to 
the coaggregation and hence to the removal efficiency. 
The results showing the effect of ionic strength and polystyrene 
concentration on the separation of polystyrene beads is presented in 
Tables 5 and 6. At high NaCl concentrations, the coaggregation of 
particles, and hence the removal of the polystyrene beads, was 
substantially reduced. This demonstrates that coaggregation was based on 
interactions between negatively and positively charged groups. Table 6 
shows that there is an optimum concentration of polybrene and too high or 
two low concentrations have reduced ability to cause aggregation. 
It was demonstrated that the polystyrene beads coated with anti-T3 
antibodies can be effectively removed from the reaction mixture within one 
minute (including aggregation time and magnetic separation time). 
The present method can be an attractive approach for removing a bound 
fraction from a reaction mixture in heterogenous immunoassays where, e.g., 
microbeads, labeled with antibodies or antigens, are employed. 
EXAMPLE 4 
Preparation of Ferrofluid 
A. Preparation of Colloidal Magnetic Iron Oxide (Ferrofluid) 
A solution of 20 ml 2M FeCl.sub.3, 10 ml 2M FeCl.sub.2, and 20 ml 1M HCl 
was added dropwise over five minutes with stirring to a solution of 25 ml 
concentrated NH.sub.4 OH in 500 ml water. The precipitate settled out, and 
the supernatant was decanted. The residue was stirred for two minutes with 
500 ml 2M HC10.sub.4 and again allowed to settle out. The supernatant was 
decanted, and the residue was taken up in water and dialyzed against 10 mM 
HC10.sub.4. The resulting colloid had a volume of 80 ml and an iron 
content of 28 mg/ml. The average particle size as determined by dynamic 
light scatter was 60 nm. Literature reference--R. Massart, C. R. Acad. 
Sci. Paris, 291C, 1 (1980). 
B. Coating of Colloidal Magnetic Iron Oxide with Proteins 
Rabbit Serum Albumin (RSA): A solution (2 ml) of 11 mg/ml RSA was added to 
2 ml of a 1:4 dilution into water of the colloidal magnetic iron oxide 
from above. After five minutes, 0.50 ml of 550 mM Tris-HCl, pH 8.0, was 
added. The resulting colloid had no visible particulate matter. 
Succinylated Bovine Serum Albumin (sBSA): A solution of 105 ml of 9.5 mg/ml 
sBSA (prepared by treatment of 5.0 g BSA in 250 ml 0.1M sodium phosphate, 
pH 8.0 with 0.20 g succinic anhydride) in water was adjusted to pH 3.38 
with 0.1M HC10.sub.4. A solution of 30 ml of 35 mg/ml colloidal magnetic 
iron oxide in 10 mM HC10.sub.4 was diluted with 75 ml water and added to 
the sBSA solution. The pH of the solution was then adjusted to pH 9.06 
with 1M NMe.sub.4 OH. The average particle size in the resulting colloid 
was determined by dynamic light scatter to be 63 nm. 
EXAMPLE 5 
Coagglutination of Ferrofluid and Latex Beads 
Into a series of test tubes was pipetted 100 .mu.l DC.sub.16 AS 
(1,3-bis[4-(dihexadecylamino)]-2,4-dihydroxycyclobutene diylium 
dihydroxide, bis (inner salt) dyed carboxylic latex beads (OD 0.455 .mu.. 
est. about 1.5.times.10.sup.8 beads/ml, prepared as described in Example 
7, Part A) in PBS buffer, 700 .mu.l diluted freon-treated normal human 
serum (2.5% in PBS), and 100 .mu.l of freshly diluted commercial, aqueous 
based ferrofluid EMG 805 200 g (Ferrofluidics Corp., Nashua, NH; 10% in 
PBS, the iron content was determined as .apprxeq.17 mg/ml). One hundred 
.mu.l of 0.5% polybrene in PBS containing 0.011M .beta.-CD 
(.beta.-cyclodextran) was then added on vortex. Immediately, the test tube 
was placed into a Corning magnetic separator with a magnetic field 
intensity of 2.6 Kgauss. At different separation times, 500 .mu.l aliquots 
of the separated liquid was then taken from a test tube, diluted with an 
equal volume of PBS buffer and the fluorescence measured as described in 
Example 2. As a control, total fluorescence was determined, using no 
magnetic particle and was found to be 60420 KH.sub.z. The results are 
summarized in Table 7. 
TABLE 7 
______________________________________ 
Separation Time Fluorescence 
(sec) (KHz) % 
______________________________________ 
60 526 0.87 
30 727 1.2 
20 933 1.5 
10 1197 2.0 
0 60420 100.0 
______________________________________ 
The results indicated that the rate of polybrene coagglutination in 
accordance with the present invention is greater than 99% in 1 minute. 
EXAMPLE 6 
Effect of Concentration of Latex Beads on Coagglutination of Ferrofluid and 
Latex Beads 
A similar protocol as in Example 5 was used. Various concentrations of 
latex bead suspensions were prepared about (10.sup.7 to 10.sup.10 
beads/ml). A hundred .mu.l of each stock suspension was taken into a test 
tube. To each test tube was then added 700 .mu.l diluted serum (2.5% in 
PBS) and 100 .mu.l diluted ferrofluid. After addition of 100 .mu.l of 0.5% 
polybrene in PBS containing 0.011M .beta.-CD, the mixture was vortexed 
(.apprxeq.3 sec) and preincubated to a total of 10 seconds before 
inserting into the magnetic separator. At exactly 1 minute after polybrene 
addition (magnetic separation time 50 sec), 500 .mu.l aliquot of the 
separated liquid was taken out, diluted to 1 ml, and fluorescence was 
determined. The controls used no magnetic particles and were diluted to 
proper concentration before measurement. 
The results are summarized in Table 8. 
TABLE 8 
______________________________________ 
Bead Stock 
Fluorescence 
(beads/ml) 
Total Remaining (%) 
______________________________________ 
1.2 .times. 10.sup.10 
3.41 .times. 10.sup.5 
33060 (0.97) 
6.3 .times. 10.sup.9 
1.86 .times. 10.sup.5 
18516 (1.0) 
1.2 .times. 10.sup.9 
5.43 .times. 10.sup.4 
5218 (0.96) 
7.9 .times. 10.sup.8 
2.71 .times. 10.sup.4 
1713 (0.63) 
1.6 .times. 10.sup.8 
5.89 .times. 10.sup.3 
732 (1.2) 
7.9 .times. 10.sup.7 
3.21 .times. 10.sup.3 
509 (1.6) 
1.0 .times. 10.sup.7 
4.60 .times. 10.sup.2 
361 (7.8) 
______________________________________ 
The above example demonstrates that up to 10.sup.10 beads/ml concentration 
of latex beads can be efficiently removed in less than 1 minute in 
accordance with the present invention. 
EXAMPLE 7 
Assay for Hepatitis B Surface Antigen (HBsAg) 
Before describing the assay a number of terms will be defined: 
RT--room temperature 
EDAC--1-ethyl-3-(3-Dimethylaminopropyl)carbodiimide 
PBS--phosphate buffered saline 
DTE--dithiothrietol 
EDTA--ethylenediaminetetraacetate, sodium salt 
BSA--bovine serum albumin 
IgM--immunoglobulin M 
IgG--immunoglobulin G 
NHS--N-hydroxysuccinimide 
MP--magnetic particles 
LISS--glycine 18 g/liter, potassium phosphate 230 mg/liter, sodium 
phosphate 
squaraine dye--DC.sub.16 AS 
A. Preparation of squaraine dyed latex beads 
Carboxylated polystyrene particles (beads) of uniform 0.716 micron diameter 
were purchased from Duke Scientific Corp. of Palo Alto. The particles are 
manufactured by Dow Chemical Co. and are packaged in 15 ml vials 
containing 10% by weight of suspended solids in deionized water with trace 
amounts of nonionic surfactants. 
The beads were prepared for dyeing by centrifugation (15,000 rpm for 10 
min) and decantation of the supernatant fluid. The pellet was resuspended 
in ethylene glycol to the same volume as before centrifugation. 
The squaraine dye was prepared by condensing squaric acid with 
dihexadecylphenyl amine (2:1 molar ratio) in refluxing n-butanol-benzene 
with azeotropic removal of water. 
Five hundred micrograms of squaraine dye was dissolved in 0.5 ml hot benzyl 
alcohol in a small tube or vial (with magnetic stir bar) clamped in an oil 
bath maintained at 140.degree.. The dye solution was slowly diluted with 1 
ml ethylene glycol. 
One milliliter of the ethylene glycol bead suspension was added dropwise to 
the hot dye solution while stirring vigorously. Stirring was continued for 
15 minutes; then the mixture was pipetted into 5 to 10 ml of 70% ethanol 
in water. The dyed beads were centrifuged and washed twice in 70% ethanol 
and then sonicated to disperse the beads after centrifugation. The beads 
were washed twice in deionized water and then stored in deionized water at 
a concentration not exceeding 100 mg solids per ml. 
B. Attachment of antibody to squaraine dyed latex beads via avidin biotin 
interaction: 
1. Covalent attachment of avidin to squaraine latex beads. 
Squaraine dyed latex beads (0.85 ml, 2.3.times.10.sup.11 beads/ml, 0.716 
.mu.m diameter) were suspended in 2 ml dist. water and the carboxyl groups 
were activated by reaction with EDAC (sigma, 18.75 mg added to the bead 
suspension) for 3 to 4 min. at room temperature. The activated beads were 
then added to avidin D solution (Vector, 1.5 mg in 3 ml 0.1M NaCl) and the 
reaction was carried out over night at room temperature with occasional 
sonication. The beads were washed by centrifugation and coated with BSA by 
suspension in buffer (0.17M glycine, 0.1M NaCl, pH 9.2) containing 1% BSA 
(Sigma, RIA grade). The beads were washed by centrifugation, following 
incubation at room temperature for 1 hour, and resuspended in the same 
buffer without BSA (3 ml). 
Biotin binding capacity was determined with .sup.14 C-biotin and was shown 
to be 77 pmol per 6.times.10.sup.8 beads. 
2. Preparation of biotinylated anti-HBsAg monoclonal antibody: 
Anti-HBsAg monoclonal antibody IgGl (from Celtek or Royal Free Hospital, 
purified by protein-A affinity chromatography, 1.0 mg/ml in 0.1M phosphate 
buffer, pH 8.2) was reacted with 25 fold molar excess biotinyl-NHS (Sigma, 
3.4 mg/ml in DMF) for 4 hours at room temperature. 
3. Immobilization of biotinylated antibody on avidin squaraine latex beads: 
Biotinylated anti-HBsAg monoclonal anitbody (0.4 mg, 0.1M phosphated 
buffer, pH 8.2) was incubated with avidin latex beads 
(1.25.times.10.sup.10 beads) for 2 hours at room temperature. The beads 
were washed by centrifugation and resuspended in 0.01M glycine, 0.01M NaCl 
pH 8.2, 0.2% BSA. 0.05% Tween 20 (final bead concentration 
6.25.times.10.sup.9 /ml). 
C. Preparation of succinylated magnetic particles: 
Two hundred (200) mg magnetic particles (Advanced Magnetic, BioMag 4100, 4 
ml) were washed by magnetic separation (3.times.40 ml 0.1M phosphate 
buffer, pH 7.0) and resuspended in 15 ml of the above buffer. The 
particles were reacted with succinic anhydride (5 ml of 1M in DMF) by 
addition of 5 aliquots over 2 hours (the pH was adjusted to 7.0 following 
each addition). The succinylated particles were washed by magnetic 
separation (3.times.40 ml 0.1M phosphate buffer, pH 7.0, and 2.times.40 ml 
LISS), resuspended in 20 ml LISS and stored at 4.degree. C. with 0.02% 
azide. 
F. Assay Protocol: 
Reagents: 
1. Anti-HBsAg IgGl monoclonal antibody (from Celtek or Royal Free Hospital, 
London) covalently attached to squarate dyed-latex beads (0.716.mu. 
diameter, preparation detailed above) (squaraine-latex beads-anti-HBsAg). 
2. HBsAg (Abbott positive control, ASUZYME II, 6 ng/ml). 
3. Magnetic particles: Succ-BioMag (100 mg/ml) prepared as described above. 
4. Polybrene (Sigma, av. MW 5000) 10 mg/ml in LISS. 
5. 0.2M citrate, pH 8.2. 
6. IgM anti-HBsAg monoclonal antibody (Celtek) 0.5-1.0 mg/ml in 2X PBS. 
G. Assay Procedure: 
1. Squaraine-latex beads-anti HBsAg (5 .mu.l containing 3.times.10.sup.7 
beads) was added to 100 .mu.l sample [50% serum in LISS, with or without 
antigen (1.5 or 3 ng/ml)] and incubated for 8 min at RT. 
2. Coaggreation of latex beads with magnetic particles was achieved by 
addition of 10 .mu.l of succ. BioMag followed by 10 .mu.l of polybrene. 
3. Magnetic separation of latex-MP coaggregates was achieved in a magnetic 
field gradient of 2.3 Kgauss (1 min). The results are found below in Table 
9. 
4. Dissociation of the latex-MP coaggregates was achieved in 50 .mu.l 
citrate. 
5. Addition of IgM anti-HBsAg (5 .mu.g) and incubation for 5 min at RT for 
antigen dependent agglutination. 
6. Magnetic separation of succ. BiogMag from latex beads 
7. Dilution with 0.2M citrate, pH 8.2 and measurement of squaraine latex 
agglutination by laser light scattering (Nicomp HN5-90). The results are 
set forth below in Table 10. 
H. Results 
1. Efficiency of separation of anti-HBsAg-squaraine latex beads from 50% 
serum: 
The amount of anti-HBsAg-squaraine latex beads remaining in the serum 
following magnetic separation was assessed by fluorescence 
spectrophotometry. 
TABLE 9 
______________________________________ 
Fluorescence of supernate 
With HBsAg 
(1.5 ng/ml in serum) 
Without HBsAg 
______________________________________ 
3.0 3.9 
3.0 3.0 
2.2 5.7 
5.8 1.9 
______________________________________ 
(Total fluorescence units of antiHBsAg-squaraine dyed latex beads assay: 
98) 
The above results demonstrate that an effective separation of dyed latex 
beads from the medium was achieved in accordance with the present 
invention. 
2. The results of the assay for HBsAg are summarized in the following 
table: 
TABLE 10 
______________________________________ 
Average Diameter (nm) 
With HBsAg 
(1.5 ng/ml in serum) 
Without HBsAg 
______________________________________ 
2770 881 
2760 795 
______________________________________ 
The above results indicate that a sensitive assay for HBsAg can be carried 
out utilizing a separation in accordance with the present invention. A 
substantially higher level of agglutination was observed when the HBsAg 
was present in the medium. 
EXAMPLE 8 
Assay for Thyroid Stimulating Hormone 
A. Abbreviations and some materials: 
TSH--thyroid stimulating hormone, human 
MP--magnetic particles 
FF--ferrofluid from Ferrofluidics Corporation (EMG 805 200 g) 
LC--long chain 
11C6 or 9D7--monoclonal antibody to the .alpha.-subunit of hTSH 
% B--%.sup.125 I-TSH bound (specific bound) 
% NSB--% non-specific bound 
BMP--Biomag particles from Advanced Magnetics Inc. 
Buffer A--PBS +0.1% BSA +0.05% Tween 20, pH 7.4 
PB--polybrene 
r.t.--room temperature 
Ab--antibody 
Ag--antigin 
Serum--TSH free serum from Immuno-search Inc. 
B. Binding OF .sup.125 I-TSH to FF-avidin 
One hundred (100) .mu.l FF-avidin, prepared by adsorption of avidin on FF, 
and 100 .mu.l biotin-LC-11C6 in Buffer A (.apprxeq.1 .mu.g antibody), and 
100 .mu.l .sup.125 I-TSH (.apprxeq.1 ng/ml) in Buffer A or serum were 
incubated at r.t., 15 min. (buffer) or 25 min. (serum). 
Fifty (50) .mu.l of polybrene in PBS was added; in buffer, PB=1.6 mg/ml, 
held for 1 min.; in serum, PB=25 mg/ml, held for 3 min. 
The material was subjected to a magnetic field of 2.1-2.6 Kgauss in a 
Corning magnetic separator for 3 min. The material was washed 1 time with 
0.5 ml PBS +0.05% Tween 20, and the MP were counted. The results are 
summarized in Table 11. 
TABLE 11 
______________________________________ 
% B 
MP in buffer 
in serum 
______________________________________ 
FF-avidin 51(1)* 60(1)* 
FF-avidin 50(5)* 65(1)* 
BMP-avidin (control) 
N.D.** 68(2)* 
______________________________________ 
*% NSB in parenthesis 
**N.D. not determined 
The above results indicate that ferrofluids coated with avidin can be 
separated in accordance with the present invention by combining with 
biotin bound to antibody and adding polybrene to non-specifically 
agglutinated the particles. 
C. Binding of .sup.125 I-TSH to FF with addition of avidin 
Fifty (50) .mu.l avidin (2 .mu.g), 100 .mu.l .sup.125 I-TSH in buffer A or 
TSH free serum, and 100 .mu.l biotin-LC-11C6 in buffer A were incubated at 
r.t. for 15 min. 
Fifty (50) .mu.l ferrofluid (containing .apprxeq.200 .mu.l Fe) was added 
followed by 50 .mu.l polybrene; for the assay in buffer: 1.6 mg/ml, for 
the assay in serum: 12.5 mg/ml. 
The material was subjected to magnetic field of 2.1-2.8 Kgauss in a Corning 
magnetic separator for 3 min., washed 1 time, and counted. The results are 
summarized in Table 12. 
TABLE 12 
______________________________________ 
% B 
particles in buffer 
in serum 
______________________________________ 
FF 53(10)* 48(3)* 
FF-avidin (control**) 
51(11)* 49(2)* 
BMP-avidin (control**) 
42(35)* 52(4)* 
______________________________________ 
*% NSB in parenthesis 
**In accordance with Section B above 
The above results indicate that ferrofluids can be separated in accordance 
with the present invention by combining with avidin and biotin bound to 
antibody and adding polybrene to non-specifically agglutinated the 
particles. 
D. Competitive TSH assay with FF-avidin 
Fifty (50) .mu.l TSH at 0, 200 ng, 2 .mu.g, 20 g and 200 g/ml in serum 
(i.e. 0, 10 ng, 100 ng, 1 .mu.g and 10 g/assay), 50 .mu.l .sup.125 I-TSH 
(2 ng/ml, 0.1 ng/assay) in serum, 100 .mu.l biotin-LC-9D7 (1 g Ab/assay) 
in buffer A, and FF-avidin prepared as described above were combined and 
incubated at r.t. for 15 min. Fifty (50) .mu.l polybrene at 12.5 mg/ml was 
was added. After 3 min. the material was subjected to a magnetic field of 
2.1-2.6 Kgauss, separated, washed and counted as above. 
The results are summarized in Table 13. 
TABLE 13 
______________________________________ 
TSH (ng/assay) % B 
______________________________________ 
0 49 
10 49 
100 48 
1000 21 
10000 3.3 
______________________________________ 
The above results indicate that an assay for TSH can be carried out 
utilizing a separation in accordance with the present invention. A 
substantially lower percent of binding was observed when TSH was present 
in the medium. 
E. Competitive TSH assay with FF and addition of avidin 
Fifty (50) .mu.l TSH at 0, 200 ng, 2 g, 20 g and 200 g/ml in serum (i.e. 0, 
10 ng, 100 ng, 1 g and 10 g/assay), 50 l .sup.125 I-TSH (2 ng/ml, 0.1 
ng/assay) in serum, 100 .mu.l biotin-LC-9D7 (1 g Ab/assay) in buffer A, 
and 50 .mu.l avidin (2 g/assay) were incubated at r.t. for 15 min. Then, 
50 .mu.l FF (containing 200 g Fe) was added and after 5 min. 50 .mu.l 
polybrene at 12.5 mg/ml was added. After 3 min. the material was subjected 
to a magnetic field of 2.1-2.6 Kgauss separated, washed and counted as 
above. 
The results are summarized in Table 14. 
TABLE 14 
______________________________________ 
TSH (ng/assay) % B 
______________________________________ 
0 46 
10 46 
100 42 
1000 20 
10000 2.5 
______________________________________ 
The above results indicate that an assay for TSH can be carried out 
utilizing a separation in accordance with the present invention. A 
substantially lower percent of binding was observed when TSH was present 
in the medium. 
Although the foregoing invention has been described in some detail by way 
of illustration and example for purposes of clarity and understanding, it 
will be obvious that certain changes or modifications may be practiced 
within the scope of the appended claims.