Surface immobilization of magnetically collected materials

A method for determining the presence and/or concentration of a target substance e.g. protein, nucleic acid, bioparticle etc. in a fluid sample is provided. The method disclosed combines elements of immunoassays, coated cup assays and magnetic particle separation to effect the quantitation and recovery of an analyte in solution. Also the method ensures the non-reorientation of magnetically collected material by linking the magnetic particles to a collection surface via a specific binding pair. This linkage immobilizes the magnetic-analyte-containing material and thus allows for vigorous washing and reagent addition without significant redistribution or displacement. Thus the assay of this invention offers the speed of diffusion controlled kinetics as in a ferrofluid assay, the speed of collection of labeled target substance as in a magnetic assay as well as the ability to magnetically monolayer the ferrofluid, all of which is combined with the ease of washing and signal detection found in a coated cup assay.

FIELD OF THE INVENTION 
The present invention is directed to a method of immobilization of 
magnetically collected material. This method of immobilization is useful 
in a variety of applications, including immunoassay, nucleic acid 
detection, drug testing, gene therapy, in-vitro fertilization, cell 
analysis and food testing. The invention described herein enables the 
determination of the concentration of an analyte of biological or medical 
significance with improved precision due to an increased signal to noise 
ratio at the low end of analyte concentration and an increased total 
signal at the high end of analyte concentration. The improved assay 
combines elements of a magnetic immunoassay together with elements of a 
coated cup-type assay. 
BACKGROUND OF THE INVENTION 
Magnetic gradients have been used to separate magnetically responsive 
material from non-magnetically responsive materials for hundreds of years. 
Historically, the major industrial application was in the field of mining, 
from which derive some of the oldest inventions and basic patents. 
Magnetics have evolved as applied to separations, as well as in numerous 
other well-known applications, such as the magnetic recording industry, 
the medical sciences, electronics and other industrial uses. 
The science of separations involving the isolation of molecules has evolved 
independently. Separations initially involved principles of solubility, 
advancing to methods based on gross physical properties of molecules, such 
as size and shape. With the introduction of chromatography, separation 
techniques were developed based initially on chemical interactions which 
were not well defined. This approach generally led to employing surface 
charge density as a basis for one form of separation. These and other 
methods based on similar principles achieved separation typically on the 
basis of one property or characteristic of a molecule and it became, and 
in many instances still is, customary to employ several techniques 
sequentially in order to isolate some specific compound of interest from a 
complex mixture. 
With the emergence of modern biological science, the principle of affinity 
reactions (recognition at the molecular level via molecular "locks" and 
"keys") became known and was embodied in technology such as immunoassay, 
DNA/RNA hybridization techniques and, more recently, mammalian cell 
isolation. In such methods where a separation is performed, means for 
retrieving the bound element of a binding pair reaction (lock or key) was 
required. Methods involving "anchoring" one element evolved from simple, 
physically removable surfaces to various beads which could be retrieved by 
centrifugation. With this evolution came the appreciation that small beads 
have higher surface to mass ratios and that other forces, such as 
magnetics, could be conveniently used for separation. 
In order for magnetic particle-based separation technology to become a 
reality, materials had to be developed with a property commonly referred 
to as superparamagnetism, which is the property whereby a magnetic 
particle exhibits a magnetic dipole while in a magnetic field and loses 
the dipole upon removal of the field. This property allows such materials 
to be resuspendable following a magnetic collection, which is essential 
for most separation applications. Given the various developments which had 
to occur, it is only in the last three decades or so that magnetic 
separations have been introduced into medical science and technology as a 
means for selecting out of complex mixtures material to be examined or 
analyzed, or alternatively as a means for performing a "bound/free" 
separation, as in the case of immunoassay. In view of the wide-scale use 
of magnetics in this and other clinically relevant testing, this step is 
critical for almost all such applications. With the extensive use of 
automation, it has become clear that there is a considerable need for 
improvement as regards the collection of magnetic materials. The present 
invention addresses this need. 
Immunoassays are widely used in the clinical laboratory for determining the 
concentration of an analyte of biological or medical significance. The 
principles of immunoassays are reasonably well understood. Generally 
speaking, there are two categories of immunoassays: competitive assays and 
sandwich assays. For low molecular weight analytes such as drugs or 
metabolites, it is customary to perform competitive immunoassays. 
Typically, a fixed, limited quantity of specific antibody is allowed to 
incubate with a known concentration of labeled analyte and test sample 
containing some unknown concentration of the analyte of interest. The 
quantity of label bound to antibody is inversely proportional to the 
amount of analyte in the test sample. For quantitation, it is customary to 
perform a bound/free separation so that labeled analyte associated with 
the antibody can be detected. Assays which employ such a separation are 
termed heterogeneous. There are numerous ways for performing the 
bound/free separation which include adsorbing or covalently linking 
specific antibody to the inside of a tube (coated tube assay) or onto 
beads which can either be centrifuged or separated with filters or by 
magnets. Typically, a separation system should have the characteristics 
that the separation can easily be performed, excess reagent can be removed 
simply and non-specifically bound analyte can be washed free of the 
immobilized antibody with its specifically bound, labeled analyte. For 
analytes which have at least two characteristic antigenic determinants, a 
simpler and more precise approach is to perform a sandwich immunoassay, 
which uses two antibodies. One of the antibodies is directed to one 
antigenic binding site as a capture antibody while the other antibody is 
directed at the second binding site as the signal generating antibody. 
Thus, if the capture antibody is separated from solution, or bound on some 
solid support, the only way in which signal generating antibody can be 
bound to solid support or separated from solution is via binding to 
analyte. The advantages of sandwich assay are that: (1) signal is directly 
proportional to analyte concentration on the low end of the analyte curve; 
(2) extreme sensitivity can be obtained on the low concentration end; (3) 
sandwich assays are assays of "excesses" since capture antibody and label 
antibody are typically in excess of analyte, and so error is mainly 
related to accuracy of sample input; and (4) a wide dynamic analyte 
detection range (as much as 4-5 logs) is possible. Sandwich assay 
techniques, like competitive assays, employ a wide range of systems for 
performing bound/free separations. 
Considerable effort has been devoted to the development of assays capable 
of being performed as quickly and as simply as possible. Several 
inventions are based on the principle of covalently attaching a fixed 
quantity of antibody to a well-defined region on a solid support where the 
latter has reasonable capillary action. See, for example, U.S. Pat. Nos. 
5,126,242; 4,517,288; 4,786,606; 4,774,174; 4,906,439; 5,364,796; 
4,446,232; and 4,752,562. Typically in such assays, specimen and labeled 
analyte are placed with great precision on such a solid support so as to 
permit competitive binding on the bound antibodies to occur. Next, 
solution is added which causes unbound labeled analyte to be carried from 
the binding region via capillary action. If the analyte is enzyme labeled, 
and if the liquid employed to "chromatograph" away unbound labeled analyte 
contains excess substrate, then a color is developed which will be 
proportional to the quantity of enzyme specifically bound. Another type of 
assay operates on a different principle, which effects "bound/free" 
separation by positioning solid phase antibody in some fraction of the 
total volume of the system. If the fractional volume in which specific 
binding takes place can be partitioned from the remainder of the system, 
then it will be possible to quantitate bound signal in the presence of an 
equilibrium quantity of "free" analyte, but the amount of "free" analyte 
in the detection region will be reduced by the volume element of the 
immobilized antibody regions divided by the total volume of the system. 
Such assays are referred to as "curtain assays" as this large fraction of 
unbound analyte and signal is effectively hidden behind a curtain. 
Each of the above-described analytical systems suffers from its own 
peculiar deficiencies. In the case where capillary action is employed to 
chromatograph away unbound signal, non-specific binding of signal 
producing agent to the matrix can result in substantial background. In the 
case where signal producing agent includes labeling antibody or some part 
thereof as in the case of sandwich assays, non-specific binding becomes a 
significant concern. Thus, sandwich assays where medium to high 
sensitivity is required cannot be performed. In the curtain type assays, 
there is a finite limit on the smallness of the fractional volume where 
antibody can be bound. Hence, free signal analyte in that region results 
in low-end sensitivity problems. 
Perhaps the most common and most sensitive method of assay heretofore in 
use involves covalently immobilizing a capture agent (e.g. monoclonal 
antibody) a solid support, such as a microtiter well, a cup or a tube. 
Such coated cup assays are well-known in the art. They have been used 
since the 1960's with radioimmunoassays, and remain common in many of the 
clinical analyzers in laboratories today, such as the Amerlite, 
Cyber-fluor, Delfia, and the ES-300 systems. See also U.S. Pat. No. 
4,376,110. Advantages of coated cup assays include that they provide a 
homogeneous and single layer of analyte for analysis. Although coated cup 
technology is currently used in various immunoassay formats, the time 
required for the assay components to diffuse to the coated wall is 
excessive. Heating and constant shaking can reduce incubation times, but 
sensitive assays such as TSH and CEA still require 30-60 minutes for the 
incubation of analyte, signal producing agent and immobilized capture 
antibody. Additionally, the assay is highly dependent on the manufacture 
of cups with an evenly distributed coating of capture antibody on the cup 
surface. 
Another type of immunoassay involves attachment of the capture antibody to 
a mobile solid phase, such as latex or other polymeric microbeads, some of 
which are magnetically responsive. Centrifugation, settling, filtration, 
or magnetic means are used to accomplish the bound/free separation. See 
U.S. Pat. Nos. 5,242,837; 5,169,754; 4,988,618; 5,206,159; 4,343,901; and 
4,267,235. In certain cases, the act of binding to the particle, 
introduces a change in a property of the particle, which can be detected. 
While the benefits of a mobile solid phase include increased surface area 
and therefore decreased incubation times, problems with these assays 
include clogging of tubing, aggregation, settling, and in the cases where 
centrifugation is used, extremely complicated automation procedures. Most 
of the magnetic particles are large (1000-5000 nm in diameter,) so the 
problems of clogging and settling are particularly prevalent, and must be 
engineered around. 
Recently, a class of magnetic materials appropriately referred to as 
ferrofluids have been introduced into immunoassay technology. Ferrofluids 
are nanosized crystals or crystal clusters of magnetite which are coated 
with materials that act as surfactants. Historically, most surfactants 
were, indeed, detergents; more recently, polymers or proteins have been 
used in that role. Ferrofluids have a variety of unique properties, among 
which is that thermodynamically they act as solutes. Like lyophilic 
colloids, they interact strongly with solvent and exhibit a variety of 
most unusual phenomena. With the availability of polymer/protein coated 
ferrofluids and the use of appropriate coupling chemistries, immunoassays 
in which ferrofluids have been used to perform bound/free separations have 
been devised. See U.S. Pat. Nos. 4,795,698; 4,965,007; 5,283,079; and 
5,238,811. Ferrofluids have a decided advantage when compared to other 
capture systems, particularly those that employ relatively large magnetic 
particles (greater than 0.5 microns), which is attributable to 
translational and rotational diffusion. Thus, by employing ferrofluids in 
immunoassays, binding reactions proceed at diffusion controlled rates and 
do not require the constant mixing necessary when larger particles are 
used. 
For polymer/protein coated ferrofluids wherein the crystal core is composed 
of magnetite clusters, the magnetic gradient required to effect separation 
is inversely related to the numbers of crystals in the clusters. 
Typically, crystal sizes are in the range of about 4-12 nm, while after 
coupling of bioligand, sizes range from about 20 nm to as large as 300-400 
nm. Materials synthesized from crystal clusters up to about 120 nm that 
are well coated with polymer/protein will exhibit colloidal stability for 
long periods (such materials typically show no signs of settling for up to 
one month, or longer). As the size decreases within the optimal range for 
this bioligand-coupled material, which is 60 to 150 nm, such materials 
become more difficult to separate magnetically. Materials in the 20 nm 
range are difficult to effectively separate, even in high gradient 
magnetic separation devices employing very fine stainless steel wires 
capable of generating gradients of 150-200 kGauss/cm. Materials of 40-60 
nm, which appear to be composed of cores having a cluster of three to six 
magnetite crystals, can be effectively collected with such gradients. 
The above class of materials are particularly useful in performing 
bioanalytical separations, due principally to the ability of such 
materials to diffuse and to be magnetically immobilized. Since diffusion 
constants are inversely related to colloid size, the smaller 
bioligand-coupled ferrofluids have notable advantage over larger size 
materials. Further, in that smaller diameter materials have greater 
surface area per unit mass, such materials provide additional advantages 
over larger size materials when used in binding reactions. For example, 
less material is required to be introduced into the system; i.e., the 
binding particles represent a smaller volume fraction. As documented in 
commonly owned U.S. Pat. No. 5,466,574, due to their surface-to-mass 
ratio, the quantities of these ferrofluid particles can be manipulated, so 
as to be deposited on a collection surface in a substantially uniform 
thickness, which may aptly be characterized as a monolayer. This property 
makes possible quantitative signal detection while the magnetic particles 
are immobilized on wires, microtiter cups, rods, sheets, or other solid 
supports. However, the experience gained with forming such monolayers in 
external collection devices of the type described in U.S. patent 
application Ser. No. 08/006,071, has shown that while monolayers can be 
regularly and reproducibly formed in the apparatus disclosed in that 
application, as well as related U.S. Pat. No. 5,186,827 and commonly owned 
U.S. patent application Ser. No. 08/424,271, these monolayers are not 
sufficiently stable to withstand repeated and vigorous washing without 
constructing wash devices which are highly controlled, insofar as the rate 
of addition of wash solution and shearing force of solution removal are 
concerned. When too vigorously washed, the shearing force of the meniscus 
as it travels past the monolayer distorts the monolayer such that 
multilayering occurs at the bottom of the cup. Additionally, prolonged 
exposure of the monolayer to a high gradient magnetic field which is not 
radially symmetrical will result in lateral movement of the particles on 
the collection surface resulting in clumping in the region of highest 
field gradient. This can occur in radial fields where relatively small 
inhomogeneities exist. Thus, any inhomogeneities will result in undesired 
clumping and resultant distortion of the monolayer. In automated magnetic 
separation devices in which space is limited, the constraints imposed 
often detract from the ability of the device to produce highly symmetrical 
gradients. Consequently, the accumulation of particles in a clump 
precludes the possibility of reading signal generated by the labeled 
specific receptor while the analyte is magnetically immobilized on the cup 
wall. Such particle clumping also causes unbound signal to be trapped, 
which can result in higher background signal. Resuspension is, therefore, 
required in the washing and signal detection stages, which is undesirable 
for many reasons, including, above all, that resuspension is an extra step 
requiring time and manipulation, which to be reproducible on an automated 
machine would require considerable additional engineering and programming. 
SUMMARY OF THE INVENTION 
The present invention provides a way of ensuring the non-reorientation of 
magnetically collected material, e.g., during the processing steps of an 
assay method which may include removal of the collection surface from the 
magnetic field, washing of the collected material, reagent addition, 
buffer or solvent changes, sequential reactions, heating, drying, 
irradiation, and sonication. Non-reorientation is achieved by linkage of 
the magnetic particle to the collection surface via a specific binding 
pair. The reaction of the specific binding pair may be either simultaneous 
with or prior to magnetic particle collection. Optionally, the specific 
binding pair reaction is reversible. The present invention may also be 
practiced so as to facilitate the formation of a monolayer of 
analyte-bearing magnetic microparticles upon collection. 
The instant invention also provides an immunoassay which combines 
operational elements of a coated cup assay with operational elements of a 
magnetic bead or ferrofluid assay. By providing a collection surface upon 
which is coated one member of a specific binding pair, the other member of 
which pair is borne by the magnetic material, a substantially uniform 
thickness or layer of magnetic particles, and thus, analyte can be 
obtained. This layer of particles and analyte in the immobilized state can 
then be maintained in place throughout the rigors of reagent addition and 
repeated washings without appreciable displacement or redistribution. 
Thus, the assay of this invention offers the speed of a 
diffusion-controlled kinetics, as in a ferrofluid assay, the speed of 
collection of labeled target substance, as in a magnetic assay, as well as 
the ability to magnetically monolayer ferrofluid in the manner described 
in commonly owed U.S. Pat. No. 5,466,574, all of which is combined with 
the ease of washing and signal detection found in a coated cup assay. 
In one embodiment, the method of the present invention is applied to the 
determination of the presence or quantity of an analyte having at least 
one characteristic determinant, in a test sample. Such an assay is carried 
out by adding to the test sample a signal producing agent comprising a 
binding substance that binds specifically to a characteristic determinant 
of the analyte and a capture agent comprising a specific binding substance 
which binds specifically to the other binding site of the analyte and 
which is directly bonded to magnetic particles or adapted to be bonded to 
magnetic particles provided in the test sample, the magnetic particles 
bearing one member of a specific binding pair for collection. The test 
sample is then subjected to conditions causing complex formation between 
the analyte, the signal producing agent and the capture agent. The test 
sample is then contacted with a collection surface on which is affixed the 
other member of the specific binding pair for collection, and subjected to 
the influence of a magnetic field to promote binding interactions between 
the specific binding members and cause the complex-bound magnetic 
particles to collect on the collection surface. The uncomplexed signal 
producing agent is thereafter separated from the complexed signal 
producing agent, and signal is detected in the separated, complexed signal 
producing agent, or optionally from the separated uncomplexed original 
producing agent, from which the presence or quantity of analyte in the 
test sample can be determined. The magnetic particles are typically 
present in an amount in excess of that required to bind all of the formed 
complexes. 
According to its broader aspects, the present invention affords an 
improvement in analytical methods involving collection of analyte-bearing 
magnetic particles from a test sample on a collection surface associated 
with a receptacle, wherein a treatment solution is deposited in the 
receptacle in an amount sufficient to submerge the collected 
analyte-bearing magnetic particles and the treatment solution is evacuated 
from the receptacle, but without appreciable displacement caused by the 
addition or removal of the treatment solution. The improvement lies in 
directly or indirectly bonding to the magnetic particles one member of a 
specific binding pair, directly or indirectly affixing to the collection 
surface the other member of the specific binding pair and contacting the 
collection surface with the test sample under the influence of a magnetic 
field to promote binding interaction between the specific binding pair 
members and cause binding of the analyte-bearing magnetic particles to the 
collection surface, whereby the analyte-bearing magnetic particles are 
maintained in place on the collection surface. In this way, the 
analyte-bearing magnetic particles are maintained in place on the 
collection surface, without appreciable displacement or reorientation 
caused by addition or removal of the treatment solution. Representative 
examples of treatment solutions used in this method are signal producing 
agents, wash solutions, physiological buffers, biological fluids, such as 
serum or plasma, various formulations of tissue culture media, such as 
DMEM, RPMI, Hams f12, distilled water, or any other substance or solutions 
that may be required in conducting the assays described herein. 
In a further embodiment of the present invention, the presence or quantity 
of analyte of the type previously described is determined in a two stage 
assay. The first stage of the assay comprises the steps of mixing together 
the test sample and a capture agent which contains one member of a first 
specific binding pair for capture, and a binding substance that binds 
selectively to one of the first or second binding sites on the analyte. 
The test sample containing the capture agent is then exposed to conditions 
allowing complex formation to occur between the analyte and capture agent. 
Formed complexes (capture agent and analyte) are contacted with an excess 
of magnetic particles bearing the other member of the first specific 
binding pair, and one member of a second specific binding pair for 
collection, under conditions causing binding of the members of the first 
specific binding pair. The test sample is then placed into a receptacle 
containing a collection surface to which is affixed the other member of 
the second specific binding pair, under the influence of a magnetic field 
to promote binding interaction between complex-bound, magnetic particles 
and the other member of the second specific binding pair, causing the 
complex-bound magnetic particles to be collected on the collection 
surface. Complex bound magnetic particles are separated from the test 
sample, e.g. via washing with a wash solution or simple aspiration. The 
second stage of the assay comprises the steps of contacting the 
complex-bound magnetic particles with a signal producing agent comprising 
a specific binding substance that binds specifically to the other of the 
binding sites on the analyte under conditions causing binding of the 
signal producing agent to analyte contained in the complex-bound magnetic 
particles. The unbound signal producing agent is then separated from 
complex-bound signal producing agent, e.g. by washing or aspiration. 
Signal is then detected from the bound signal producing agent, from which 
the presence and/or quantity of said analyte in said test sample is 
determined. 
In yet another embodiment of the present invention the presence or quantity 
of analyte in a test fluid is determined by means of a "phasing" 
phenomenon. In this embodiment the assay is again carried out in two 
stages, the first stage being substantially as described immediately 
above. The second stage of this method comprises the steps of placing the 
separated complex-bound magnetic particles in a non-magnetic fluid phase 
and then contacting the complex-bound magnetic particles on the collection 
surface with a solution containing a non-magnetic signal producing agent, 
which comprises a specific binding substance that binds specifically to 
the other binding site on the analyte, and a quantity of colloidal 
magnetic particles. The added solution forms a distinct phase in the 
non-magnetic fluid phase, with the magnetic and non-magnetic components of 
the solution being stably confined within the distinct phase. A magnetic 
field is imposed in the vicinity of the collection surface, having a 
region of sufficiently high intensity to cause the distinct phase to be 
positioned adjacent to the collection surface. The specific binding 
substance of the signal producing agent then binds the analyte present in 
the complex-bound magnetic particles. Unbound signal producing agent is 
separated from analyte bound signal producing agent and the presence 
and/or quantity of the analyte in the test sample is determined. 
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
According to a preferred embodiment, the present invention provides an 
assay method having the distinctive capability of immobilizing and 
preventing the reorientation or lateral movement of magnetic 
microparticles after magnetic collection of such particles upon a 
collection surface. This capability is highly advantageous in many 
analytical applications. The immobilization is achieved with the coating 
of one member of a specific binding pair to the collection surface. The 
other member of the specific binding pair is borne by the magnetic 
microparticle. In the process of magnetic collection of the magnetic 
microparticle, the binding pairs react to form a stable bond, optionally 
covalent. This bond must be strong enough to withstand whatever forces 
will be operative upon the collection surface and the immobilized 
microparticles. Depending on the requirements of the system, these forces 
may include removal of the collection surface from the magnetic field, 
washing of the microparticles, reagent addition, buffer or solvent 
changes, sequential reactions, heating, drying, irradiation, and 
sonication. 
The terms "analyte" or "target substance" as used herein, refer to a wide 
variety of substances of biological or medical interest which are 
measurable individually or as a group. Examples include hormones, 
proteins, peptides, oligonucleotides, drugs, chemical substances, 
macromolecules (e.g., nucleic acids-RNA, DNA) and particulate analytes, 
which include bioparticles such as cells, viruses, bacteria and the like. 
The term "determinant," when used in reference to any of the foregoing 
analytes or target substances, means that portion of the target surface 
involved in and responsible for selective binding to the specific binding 
substance, the presence of which is required for selective binding to 
occur. In fundamental terms, determinants are the molecular contact 
regions on analytes or target substances that are recognized by receptors 
in specific binding pair reactions. The term "specific binding pair" as 
used herein includes antigen-antibody, receptor-hormone, receptor-ligand, 
agonist-antagonist, lectin-carbohydrate, nucleic acid (RNA or DNA) 
hybridizing sequences, Fc receptor of mouse IgG-protein A, avidin-biotin, 
streptavidin-biotin and virus-receptor pairs. Various other 
determinant-specific binding substance combinations are determinable using 
the methods of this invention, as will be apparent to those skilled in the 
art. The term "antibody" as used herein includes immunoglobulins, 
monoclonal or polyclonal antibodies, immunoreactive immunoglobulin 
fragments, single chain antibodies, and peptides, oligonucleotides or a 
combination thereof which specifically recognize determinants with 
specificity similar to traditionally generated antibodies. The term 
"monolayer" as used herein describes a relatively thin layer of analyte or 
target substance-bound magnetic particles collected in substantially 
uniform thickness, such that essentially no interfering substances may be 
entrapped within the layer. 
The preferred magnetic particles for use in carrying out this invention are 
particles that behave as colloids. Such particles are characterized by 
their sub-micron particle size, which is generally less than about 200 
nanometers (nm) (0.20 microns), and their stability to gravitational 
separation from solution for extended periods of time. Suitable materials 
are composed of a crystalline core of superparamagnetic material 
surrounded by molecules which are bonded, e.g. physically absorbed or 
covalently attached, to the magnetic core and which confer stabilizing 
colloidal properties. The size of the colloidal particles is sufficiently 
small that they do not contain a complete magnetic domain, and their 
Brownian energy exceeds their magnetic moment. As a consequence, North 
Pole, South Pole alignment and subsequent mutual attraction/repulsion of 
these colloidal magnetic particles does not appear to occur even in 
moderately strong magnetic fields, contributing to their solution 
stability. Accordingly, colloidal magnetic particles are not readily 
separable from solution as such even with powerful electromagnets, but 
instead require a magnetic gradient to be generated within the test medium 
in which the particles are suspended in order to achieve separation of the 
discrete particles. 
Magnetic particles having the above-described properties can be prepared as 
described in U.S. Pat. No. 4,795,698, and commonly owned U.S. application 
Ser. Nos. 397,106, and 08/482,448. 
With the proper collection vessels and magnetic fields, the colloidal 
magnetic particle or ferrofluids described above can be manipulated to 
form a relatively thin, substantially uniform layer of particles such that 
no interfering substances may be entrapped within such layer. These thin 
layers are believed to be monolayers of magnetic material bearing target 
substance which is bound to the magnetic material. The manner in which the 
methods of the present invention facilitate such monolayer formation 
constitutes a notable improvement over existing magnetic immunoassays and 
coated cup-type assays. Since the specific binding reaction can probably 
not occur if the collected particles are layered one on top of the other, 
due to impeded contact between the two members of the specific binding 
pair, any layer of magnetic particles substantially in excess of a 
monolayer would not be immobilized on the collection surface. However, 
upon change in the magnetic field, washing, reagent addition or any other 
disturbance of the environment of the collection surface, the collected 
multiple layers of magnetic particles may be dislodged from their 
collection site. These particles may then migrate to a new location on the 
collection surface not occupied by magnetic particles, and the binding 
pair member borne by such particles could then react with the binding pair 
member affixed to the collection surface at the new location. 
In some situations, it may be desirable to obtain a monolayer of magnetic 
particle-bound target substance, but other constraints may not allow for 
realization of the specialized conditions required to form such layers. 
The present invention can be supplemented so as to initially form 
multilayers of magnetic particles, perhaps with a magnetic field that is 
not optimal for the formation of monolayers or with excessive magnetic 
material collected upon a surface, i.e. magnetic particle excess. One such 
embodiment involves collection with a monopole. The bottom-most layer of 
magnetic particles would form an immobilized monolayer via the specific 
binding pair reaction, but the upper layers would not. Repositioning the 
collection vessel, such that a new portion of the vessel wall is exposed 
to the region of highest magnetic field will result in a newly exposed 
"collection surface." The upper layers of the magnetic particles would 
cascade over to the newly exposed collection surface, where immobilization 
would occur. This procedure could be repeated until all of the magnetic 
particles have been immobilized in a monolayer. Alternately, excess 
magnetic particles could be added to a system, followed by binding pair 
reaction and immobilization of the analyte-bearing magnetic particles on 
the collection surface with unbound components discarded, typically by 
removal from the magnetic field. It will be appreciated that by limiting 
the area of the collection surface on which the specific binding pair 
member is affixed, a pre-determined quantity of magnetic material or 
target substance can be immobilized. This capability may be beneficially 
used in the collection of some fixed number of mammalian cells in a 
monolayer on a microscope slide for further analysis. As a practical 
example, this aspect of the present invention should enable the critical 
examination of 100,000 CD4+ cells. Magnetically bound cells would be 
magnetically immobilized upon a collection surface, the binding pair 
reaction would take place, and excess cells removed in the absence of a 
magnetic field. 
An example of an assay embodying the method of this invention is a sandwich 
assay employing two monoclonal antibodies directed towards different 
regions of an analyte of clinical significance, such as human chorionic 
gonadotropin (hCG) or thyroid stimulating hormone (TSH.) One antibody may 
be labeled with a signal generating enzyme such as horseradish peroxidase 
(HRP) or alkaline phosphatase (AP), and the other antibody could be 
conjugated with biotin or a hapten which can be immunologically recognized 
without difficulty, to facilitate analyte capture. A magnetic ferrofluid 
particle could be coated with avidin, streptavidin, or anti-hapten, as the 
case may be, and optionally coated with two such types of materials. The 
assay could be performed in a microtiter cup or other similar receptacle, 
which had been coated with either the same specific binding pair member to 
which the second monoclonal antibody is conjugated, or a member of some 
other specific binding pair which is bound either directly, or indirectly, 
e.g. via a protein or other polymer coating, to the collection surface. 
The assay could proceed by the formation of the sandwiches resulting from 
incubation of the monoclonal antibodies with the sample in the coated cup. 
This first incubation might take 2-10 minutes. A subsequent addition of 
coated ferrofluid would require 2-10 minutes. Magnetic collection of the 
analyte-bound ferrofluid using the appropriate magnetic design would 
remove all ferrofluid and all labeled analyte from solution. Preferably, 
the magnetic array would be designed such that the magnetic material would 
be attracted to the collection surface evenly, such that a layer of 
substantially uniform thickness would be formed. Optionally, by 
controlling the amount of ferrofluid in the assay as described in commonly 
owned U.S. Pat. No. 5,466,574, a substantially thin layer or a monolayer 
could be obtained. Magnetic collection might require 30 seconds-5 minutes. 
Excess signal producing antibody and unbound sample would be washed out 
using a compatible wash buffer, but the magnetically collected material 
would remain as uniformly deposited on the collection surface because it 
is bound to said surface via the specific binding pair reaction. After the 
addition of enzyme substrate, the signal could be detected, optionally 
with the collection surface remaining in the magnetic field. 
The results obtained from assays conducted as generally described above, 
and more specifically in the examples that follow, are significantly 
better than either coated cup assays or magnetic ferrofluid assays. The 
improvement over coated cup assays is realized in the significantly 
shorter time needed for incubations. For example, in a coated cup assay, 
if a biotinylated capture antibody is used with a streptavidin- or 
avidin-coated cup, at least two kinds of reactions occur. Capture antibody 
can react with the cup first, with the sandwich reaction taking place 
thereafter. Alternatively, sandwich can form in solution and diffuse to 
the wall to initiate the capture reaction. Such reactions require 
considerable time. On the other hand, if a sandwich is allowed to form in 
solution and ferrofluid is subsequently added to the system so as to 
capture the sandwiches, and this step is followed by magnetic separation, 
the kinetics of the latter procedure are significantly quicker. 
The improvement over magnetic assays is realized in the enhanced (higher) 
signal at the "high end" and the increased signal-to-noise ratio at the 
"low end." The ability to avoid resuspension of the magnetic material in 
connection with washing or signal detection operations also results in 
easier automation and less restrictive magnetic arrays. The improvement 
over previous monolaying techniques, such as described in U.S. Pat. No. 
5,466,574 is realized in that simpler wash schemes can be employed, as 
even vigorous washing will not distort or displace the monolayer. 
Furthermore, material so collected can be removed from the magnetic field 
and still exist as a monolayer for washing or signal detection. Finally, 
improved washing can decrease background signal, increasing 
signal-to-noise ratios and reproducibility. 
Of course the methods of the present invention can also be utilized in 
performing assays other than the sandwich assay described above. The 
present invention is also applicable in competitive assays in which there 
is commonly only one characteristic determinant on the target substance, 
as well as dual stage assays for more complex molecules. Some of the dual 
stage assays are used to differentiate acute infection from chronic 
infection by determining the presence of patient IgM or IgG specific 
antibodies, respectively. 
The use of the present invention in dual stage assays provides particular 
benefits not previously obtainable. In a dual stage assay, a mixture of 
substances is removed from solution in the first stage with a specific 
binding substance. This mixture is further subdivided by the second stage 
incubation with a different specific binding substance, which also 
comprises a detectable label. The ease of washing afforded by the methods 
of the present invention is important in a dual stage assay. However, the 
second incubation is constrained by the immobilization of the analyte of 
interest on the collection surface. However, inasmuch as the first stage 
was a magnetic assay, the second stage can be accelerated by using the 
same magnetic fields. For example, the non-magnetic, labeled, specific 
binding substance used in the second stage could be provided in a solution 
which also contains a quantity of small magnetic particles capable of 
forming ferrophases, as described in commonly owned U.S. application Ser. 
No. 08/228,818. If this magnetic ferrophase were added to a non-magnetic 
liquid phase in the collection chamber, the ferrophasing phenomenon could 
be utilized to transport both the magnetic and the non-magnetic components 
of the newly added solution to a region of the collection vessel very 
close to the collection surface. Since the specific binding material would 
be so close to the immobilized substances, the reaction would proceed at a 
rate significantly faster than the reaction rate of an immobilized 
substance with a second substance in solution. The small magnetic 
particles which are collected during the second incubation can be easily 
washed away upon removal of the collection surface from the influence of 
the magnetic field, as they have no affinity for the collection surface. 
Alternatively, by choosing a very small or weakly magnetic ferrofluid to 
create the ferrophase none of it would collect given the magnetic field 
gradient strength normally employed for separating the ferrofluid 
previously collected and immobilized. 
An alternative method for a dual stage assay is also provided when the 
instant invention is combined with the teaching of commonly owned U.S. 
application Ser. No. 08/395,967. In this embodiment of the invention, the 
specific binding pair that immobilizes the magnetic particle on the 
collection surface forms a dissociable bond. For example, the binding pair 
could comprise a poly-A tail coating on the magnetic particle and a poly-T 
tail affixed to the collection surface. Removal from the magnetic field, 
combined with a slight increase in temperature would dissociate the bond 
between this specific binding pair, thereby releasing the magnetic 
material from the collection surface. Therefore, the second stage of the 
dual stage reaction with the labeled, specific binding substance could 
proceed in solution, limited only by diffusion-controlled kinetics, which 
are significantly faster than the kinetics of a reaction with an 
immobilized substance. After completion of the second stage reaction 
magnetic collection would separate the complexed magnetic material from 
the non-magnetic, uncomplexed material and then the temperature would be 
lowered and re-immobilization would occur via hybridization. The 
uncomplexed material would be washed away with a compatible buffer, and 
the detection of the labeled substance could proceed. 
It will be appreciated by those skilled in the art that the range of 
applications of the instant invention is not limited to the examples 
provided herein. The specific binding pairs listed above could include an 
extensive list of haptens, antigen/antibody pairs, or other specific 
binding pairs. The present invention provides for both the use of one 
binding pair in two different situations, but could also include two 
totally different binding pairs, or a single binding pair, using two 
different analogs of a substance. For example, a magnetic particle coated 
solely with avidin could be used, while the coating on the collection 
surface and the capture agent could comprise biotin. In the second case, 
if two binding pairs were to be used, poly-A/poly-T could be used to coat 
the collection surface and the magnetic particle, respectively. The same 
magnetic particle could also have a streptavidin coating as a specific 
binding pair member for capture, to bind a biotinylated capture monoclonal 
antibody. It is also possible that the two binding pairs could share a 
common member. For example, the magnetic particle could be coated solely 
with streptavidin, but the capture monoclonal antibody could be 
biotinylated and the collection surface could be coated with 
anti-streptavidin. In the third case, the coating on the magnetic particle 
could comprise solely streptavidin, but the capture monoclonal antibody 
could be biotinylated and the collection surface could have iminobiotin 
affixed thereon. It will be understood by those skilled in the art that 
the specific binding pairs may be provided differently, even if they 
comprise the same substance. For example, the capture antibody could be 
biotinylated, but the biotin on the collection surface need not be 
provided on an antibody, or even a protein. Coating a synthetic polymer 
with biotin may prove more advantageous, depending on the desired 
application. 
It may also be advantageous in certain applications to separate the 
magnetic collection from the binding pair reaction. This variation is also 
within the scope of the present invention. For example, it may be 
advantageous to use a magnetic particle which has been coated with the 
specific receptor for the analyte or target substance. It is also possible 
that in such situations long incubations are required, such that the 
competing reaction of the binding pair member on the magnetic particle 
with the binding pair member on the collection surface proceeds to a 
substantial extent and therefore the main reaction for the capture of the 
target substance would be impaired. To be able to use magnetic particles 
directly coated with antibody, for example, which may be useful for other 
reasons, the binding pair reaction could be engineered to occur either 
immediately before or after magnetic separation. One example of such a 
reaction can be envisioned where the specific binding pair reaction 
requires the addition of a reagent to occur. For example, the collection 
surface and the magnetic particle might both be coated with biotin. 
Immediately after the magnetic separation, streptavidin or avidin could be 
added to immobilize the magnetic particle by linking the particle to the 
collection surface. Another example might be to have a first hapten coated 
on the magnetic particle and a second hapten coated upon the collection 
surface. A bifunctional antibody with specificity to the two haptens could 
be added either immediately before or just after magnetic separation and 
would immobilize the magnetic particles on the collection surface. An 
alternative approach would be to a use a binding pair which requires a 
co-factor or certain solution conditions to become active. For example, 
calcium-dependent antibody-determinant reactions could be employed for 
this purpose. (See Protein-Metal Interactions, Chapter 9.) One of the 
reactants could be used to coat the collection surface or the magnetic 
particle. After the reaction with the target substance has occurred, 
calcium could be added to the test sample, which could be placed in the 
magnetic field and the magnetic collection could occur. Also, manipulation 
of pH, temperature, ionic strength, or other conditions may be employed to 
change the conformation of a member of the specific binding pair. Yet 
another approach is to use temperature control, e.g. to regulate the 
binding of DNA or RNA probes. For example, two nucleic acid probes could 
be borne upon the magnetic particle. One probe would be complementary to 
the target substance, and one complementary to a probe affixed to the 
collection surface. These probes would be of sufficiently different 
length, that the so-called "melting temperature" (T.sub.m) of the two 
hybridization partners is significantly different. Preferably, the target 
substance probe would be longer and therefore have a higher T.sub.m. 
Therefore, the initial incubation with the target substance could be 
conducted at a temperature that would allow the annealing of the probe 
borne upon the magnetic particle with the target nucleic acid. However, 
this temperature would be too high to allow the annealing of the probe to 
the collection surface to occur. This second annealing reaction would only 
be allowed to occur after the temperature had been reduced, optionally 
after the solution had been exposed to a magnetic field gradient for 
collection. 
Although the present invention is described herein primarily with reference 
to immunoassay applications, the invention is not limited to immunoassay. 
Nucleic acid separation and detection are also within the scope of this 
invention. Thus the methods described herein may be used in sample 
preparation for polymerase chain reaction (PCR) or other applications 
involving recognition and separation of a unique segment of nucleic acid 
with a low copy number. For example, a mixture of DNA, which contains only 
a few DNA molecules of interest could be selected from solution by means 
of a biotinylated probe complementary to a unique segment of nucleic acid 
of interest. Collection and immobilization in accordance with the present 
invention would allow for vigorous washing, which would enable the removal 
of all contaminating nucleic acid from the collection surface. The 
resultant DNA could be released from the collection surface as described 
hereinabove, or as described in commonly owned U.S. application Ser. No. 
08/395,967 for use in PCR or other target amplification systems. 
Alternately, the DNA could be detected while immobilized on the collection 
surface by the use of a second probe which may be enzymatically labeled, 
or have some other detectable characteristic. 
Various signal producing substances may be used for generating a detectable 
signal. These include substances selected from the group consisting of 
molecules or ions directly or indirectly detectable based on light 
absorbance, fluorescence, phosphorescence, or luminescence (e.g. 
chemiluminescence or electrochemiluminescence) properties; molecules or 
ions detectable by their radioactive property; and molecules or ions 
detectable by their nuclear magnetic resonance or paramagnetic properties. 
A wide range of fluorophores, enzymes, or various molecules that can be 
made to emit light upon excitation could be used to produce the signals, 
all of which are well known in the art. 
The collection surface for the assays and other methods described herein 
may include microtiter cups, coated tubes, microtiter plates, capillary 
vessels, microscope slides, or other small or large chambers. 
The methods described herein lend themselves nicely to microfabricated 
chips or biosensors, since the present invention provides for the 
construction of arrays of captured molecules, cells, or combinations 
thereof, the size of which is limited only by engineering constraints.

The following examples will serve to illustrate the principles of this 
invention; however, these examples should not be construed as limiting the 
scope of this invention. 
EXAMPLE 1 
Increase in Signal in a Monolayered Nanoparticle Assay 
A sandwich type immunoassay was used for all of the following examples set 
forth herein except where otherwise noted. Briefly, the capture monoclonal 
antibody was biotinylated, the signal monoclonal antibody was conjugated 
to peroxidase enzyme, the analyte was thyroid stimulating hormone (TSH) 
and the ferrofluid was prepared as described in commonly owned U.S. 
application Ser. No. 08/482,448 and covalently coupled to streptavidin. 
The assay method of this invention affords advantages over conventional 
immunomagnetic assays in which the test receptacle is not coated with a 
"collection" antibody, as illustrated by the following experiment 
comparing the two methods. 
Coated cups were prepared by coating with BSA, biotinylated by the 
following procedure. Bovine serum albumin (BSA) was obtained from Intergen 
(Purchase, N.Y.) and biotinylated with an excess of biotin ester 
(succinimidyl 6-(biotinamido)hexanoate from Molecular Probes (Eugene, 
Oreg.) After quenching, the biotinylated BSA was purified over a Sephadex 
G25 column. To coat the cups, 200 .mu.l of biotin-BSA at 1 mg/ml in 50 mM 
bicarbonate buffer pH 8.5 was added to polystyrene cups and incubated at 
4.degree. C. overnight. After overnight incubation, each cup was washed 
with 300 .mu.l of wash buffer (20 mM ionic strength buffer pH 8.5, 
containing 0.05% non-ionic detergent) to remove unbound biotin-BSA. 
Washing was repeated four more times. After final wash, all the wash 
buffer was removed and the cups were stored at 4.degree. C. until use. 
The assay was performed in cups coated as described above by incubating 80 
.mu.l of TSH serum standard with 40 .mu.l of monoclonal antibody (MoAb) 
mixture (biotinylated capture MoAb at 1.75 .mu.g/ml and MoAb-peroxidase at 
0.75 .mu.g/ml) in 0.1M ionic strength buffer, pH 7.5 containing 5% BSA in 
a cup at 37.degree. C. for 10 minutes to form capture 
MoAb-TSH-MoAb-peroxidase sandwiches. Then 40 .mu.l of streptavidin 
ferrofluid (S.Av. F.F.) at 0.0125 mg/ml in ferrofluid dilution buffer 
(Immunicon Corp., Huntingdon Valley, Pa.) was added to the above reaction 
mixture and incubated for another 3 minutes to bind all capture 
MoAb-biotin. The cups were then transferred to a quadrupole magnetic nest 
as described in U.S. Pat. No. 5,186,827, the entire disclosure of which is 
incorporated by reference herein, to separate ferrofluid from unreacted 
MoAb-peroxidase. After 2 minutes of separation, the supernatant was 
removed and 300 .mu.l of wash buffer (20 mM ionic strength buffer, pH 8.5 
containing 0.05% non-ionic detergent) was added to the cups. This washing 
procedure was repeated three more times. 
The experiment was duplicated in uncoated cups with resuspension of the 
ferrofluid. After the final wash step, all the supernatant was removed, 
the cups were removed from the magnetic nest and 200 .mu.l of 
chemiluminescence signal reagent containing luminol was added. The 
ferrofluid was resuspended by mixing signal reagent with a pipet many 
times and the signal was read 5 minutes after the addition of the signal 
reagent using a chemiluminescence plate reader. 
The procedure used on the cups coated with biotinylated BSA using 
immobilized ferrofluid, was as follows. After the final wash step, all the 
supernatant was removed and 200 .mu.l of chemiluminescence signal reagent 
was added to the cup in the magnetic nest. The signal was read 5 minutes 
after signal reagent addition using a chemiluminescence plate reader with 
the ferrofluid immobilized on the cup in the magnetic nest. The data for 
these tests are compiled in table I below. 
TABLE I 
______________________________________ 
Signal from 
Signal from 
ferrofluid 
TSH STD resuspended 
immobilized on the 
(.mu.IU/ml) ferrofluid 
cup 
______________________________________ 
0.0 3.0 2.7 
0.13 11.9 16.7 
0.75 127 162.5 
4.0 900.5 991 
20 4053 4505 
110 16433 17644 
______________________________________ 
all signal values are in CHL units 
Note that at the low end, the signal-to-noise ratio has dramatically 
improved with the ferrofluid immobilized. The signal-to-noise ratio 
improved from 11.9:3.0 (or 4.0) to 16.7:2.7 (or 6.2). At the high end, the 
total signal has increased approximately 7% as well. 
EXAMPLE 2 
Signal Due to Ferrofluid Particles Reacting with the Coated Cup Without 
Magnetic Collection 
The amount of signal generated by magnetic particles reacting with the 
coated cup in the absence of magnetic separation was experimentally 
determined, so as to assess the advantages magnetics bring to this system. 
In carrying out this experiment, 80 .mu.l of TSH serum standard (110 
.mu.IU/ml) was incubated with 40 .mu.l of monoclonal antibody mixture as 
described in example 1, above, in a biotin-coated cup at 37.degree. C. for 
10 minutes to form capture MoAb-TSH-MoAb-peroxidase sandwiches. Then 40 
.mu.l of S.Av.F.F. in ferrofluid dilution buffer (Immunicon Corp., 
Huntingdon Valley, Pa.) was added to the resulting mixture and incubated 
for another 8 minutes to bind all capture MoAb-biotin. After ferrofluid 
incubation, but without magnetic separation, the supernatant was removed 
and 300 .mu.l of wash buffer (20 mM ionic strength buffer pH 8.5 
containing non-ionic detergent) was added to the cup. This was repeated 
three more times. Then 200 .mu.l of signal reagent was added to the cup 
and chemiluminescence signal was read at 5 minutes using a 
chemiluminescence plate reader. 
Table II shows the signal obtained from coated cups without magnetic 
collection. For comparison, the signal with magnetic collection in coated 
cups is also shown. The signal obtained by performing magnetic collection 
was used as 100%. 
TABLE II 
______________________________________ 
Signal 
% signal 
______________________________________ 
Coated cup without 
680 4.5% 
magnetic collection 
Coated cup with 15097 100% 
magnetic collection 
______________________________________ 
Note that less than 5% of the signal is obtained if a magnetic collection 
is not done. Although this signal could be increased without magnetic 
collection by increasing the incubation time, to obtain a full signal, an 
extremely long assay would be required. These data clearly demonstrate the 
advantage of combining a liquid stage ferrofluid assay, coated well 
methodology and magnetically assisted collection. 
EXAMPLE 3 
Signal Improvement Not Due to Protein Coating or Non-Specific Interactions 
The improvement in signal readout is due to the creation of a stable 
monolayer as illustrated in the following example. A comparison experiment 
was performed as described in example 1, above, using uncoated cups, cups 
coated with biotinylated BSA and BSA only. In the case of BSA only, two 
effects can be anticipated. These are that the monolayer can be distorted 
such that signal will be lost via quenching in multilayered ferrofluid, or 
ferrofluid could be lost during the wash steps. Therefore, the assay of 
example 1 was repeated with cups treated as described above, some with 
resuspension of the magnetic material. The incubation times were 10 
minutes for the antibody incubation, 5 minutes for the streptavidin 
ferrofluid incubation, and 3 minutes for the magnetic collection. The 
analyte concentration was 110 .mu.IU/ml. The results are tabulated in 
Table III below. Signal from resuspended ferrofluid in an uncoated cup is 
set as 100% 
TABLE III 
______________________________________ 
Signal 
% signal 
______________________________________ 
Ferrofluid resuspended 
16732 100% 
into solution in an 
uncoated cup 
Ferrofluid not 13881 83% 
resuspended in an 
uncoated cup 
Ferrofluid not 13718 82% 
resuspended in BSA 
coated cup 
Ferrofluid not 18077 108% 
resuspended in biotin- 
BSA coated cup 
______________________________________ 
Note that not resuspending the ferrofluid in an uncoated cup results in the 
loss of about 17% of the signal. In this case, the monolayer has been 
destroyed by the repeated washings and reagent addition, and a multilayer 
of magnetic material has reduced the available signal. Coating the cup 
with BSA has no appreciable effect on this result. However, not 
resuspending the magnetic material in a cup which has been coated with one 
member of a specific binding pair, with the other member borne upon the 
magnetic material, results in higher signal than the control. It this 
case, effective washing and monolayered analyte combine to increase the 
signal available for analysis. 
EXAMPLE 4 
Direct Conjugated Ferrofluid Shows Higher Signal Generation in the 
Resuspended State Than in the Multilayered State 
To demonstrate that a monolayer phenomenon induced by specific interaction 
between ferrofluid and cup is required even if ferrofluid having capture 
antibody directly bonded thereto is employed, the following experiments 
were done. A monoclonal antibody (anti-TSH) with no affinity for biotin 
was directly coupled to ferrofluid. It was prepared by the following 
procedure: BSA ferrofluid was prepared as described in commonly owned U.S. 
patent application Ser. No. 08/482,448. The BSA ferrofluid was activated 
using N-succinimidyl-4-(N-maleimido methyl)cyclohexane-1-carboxylate 
(SMCC) (Pierce, Rockford, Ill.) TSH capture monoclonal antibody was 
activated with Traut's reagent (Pierce, Rockford, Ill.) and purified over 
a PD-10 column (Pharmacia Biotech, Uppsala, Sweden.) The activated 
ferrofluid was mixed with the activated monoclonal antibody and allowed to 
react at room temperature for 1 hour followed by overnight reaction at 
4.degree. C. After quenching, the ferrofluid was washed and a final 0.2 
micron filtration was performed. 
For the experiment, 80 .mu.l of TSH serum standard (110 .mu.IU/ml) was 
incubated with 40 .mu.l of MoAb-peroxidase (0.75 .mu.g/ml) in 0.1M ionic 
strength buffer pH 7.5 containing 5% BSA and 40 .mu.l of anti-TSH capture 
MoAb coupled-ferrofluid at 0.018 mg/ml in ferrofluid dilution buffer 
(Immunicon Corp., Huntingdon Valley, Pa.) in a microtiter cup at 
377.degree. C. for 15 minutes to form ferrofluid-TSH-MoAb-peroxidase 
sandwiches. The cup was then transferred to a quadrupole magnetic nest, as 
previously noted, to separate ferrofluid from unreacted MoAb-peroxidase. 
After 3 minutes of separation, the supernatant was removed and 300 .mu.l 
of wash buffer was added to the cup. This was repeated three more times. 
After the final wash step, all the supernatant was removed and 200 .mu.l 
of chemiluminescence signal reagent was added to the cup in the magnetic 
nest. In one set of experiments the magnetic material was resuspended and 
in the other the material remained immobilized. Then signal was read at 5 
minutes using the chemiluminescence plate reader. These experiments were 
done in a biotin-BSA coated cup and in an uncoated cup and results are 
shown in Table IV. 
TABLE IV 
______________________________________ 
Signal 
% Signal 
______________________________________ 
Ferrofluid not 17895 76% 
resuspended in biotin- 
BSA coated cup 
Ferrofluid resuspended 
23402 100% 
in uncoated cup 
______________________________________ 
In this experiment, a non-specific receptor was coated onto the ferrofluid. 
Therefore, there was no specific interaction between the collection 
surface and the magnetic particle. These results clearly show that there 
is higher signal with an antibody coupled-ferrofluid in the resuspended 
state. In other words, magnetically immobilized materials result in 
quenching of signal compared to the resuspended material. Although the 
monolayered collection should improve signal, the monolayer is damaged by 
repeated washing, resulting in the pile up of ferrofluid during the wash 
and consequent signal loss. 
EXAMPLE 5 
Decrease in Non-Specific Signal of a Ferrofluid Specifically Bound to Cup 
Surface vs. Magnetically Immobilized Ferrofluid 
This experiment shows that when ferrofluid is collected uniformly and kept 
immobilized during washing, the trapping of free signal antibody was 
significantly decreased. 
80 .mu.l of the zero TSH serum standard (0 .mu.IU/ml) was incubated with 40 
.mu.l of monoclonal antibody mixture in 0.1M ionic strength buffer pH 7.5 
containing 5% BSA in a cup at 37.degree. C. for 10 minutes to form capture 
MoAb-TSH-MoAb-peroxidase sandwiches. Then 40 .mu.l of St.A.F.F. at 18.75 
.mu.g/ml in ferrofluid dilution buffer (Immunicon Corp., Huntingdon 
Valley, Pa.) was added to the above reaction mixture and incubated for 
another 5 minutes to bind all capture MoAb-biotin. The cup was then 
transferred to a quadrupole magnetic nest, as previously mentioned, to 
separate ferrofluid from unreacted MoAb-peroxidase. After 3 minutes of 
separation, the supernatant was removed and 300 .mu.l of wash buffer (20 
mM ionic strength buffer pH 8.5 containing non-ionic detergent) was added 
to the cup. This was repeated three more times. 
After the final wash step, all the supernatant was removed and 200 .mu.l of 
chemiluminescence signal reagent was added to the cup in the magnetic 
nest. Then signal was read at 5 minutes using the chemiluminescence plate 
reader. These experiments were done in biotin-BSA coated cups and in 
uncoated cups, and the results are shown in Table V. Six replicates were 
included in this experiment and the raw data as well as the averages and 
coefficients of variance (C.V.) are reported. 
TABLE V 
______________________________________ 
Non 
specific 
Average 
signal 
(C.V.) 
______________________________________ 
Ferrofluid immobilizized on a 
3.02 3.26 (9.0%) 
coated cup 3.38 
3.29 
2.83 
3.38 
3.66 
Ferrofluid resuspended in an 
6.22 4.66 (27.8%) 
uncoated cup 4.57 
3.11 
6.22 
3.75 
4.11 
______________________________________ 
As can be seen from Table V, lower non-specific signal is consistently 
obtained with ferrofluid collected and washed in accordance with the 
present invention, compared to the material collected and washed while 
immobilized solely by magnetic means. This result could be explained by 
surface tension "scrubbing effects" acting on ferrofluid rigidly held onto 
a surface versus ferrofluid which could reorient as meniscus passes by. It 
is worthwhile noting that this invention results in decreased non-specific 
binding as well as increased signal output of specifically bound material 
when the latter is compared with resuspended ferrofluid, see Example 1. 
These data suggest that, in addition to creating stable monolayers, the 
specific binding pair reaction orients ferrofluid particles such that 
sandwiches are facing towards the center of the reaction vessel, thus 
resulting in higher levels of signal output compared with randomly 
oriented ferrofluids. Hence, it appears likely that, as applied to these 
kinds of assays, the method of the invention produces increased 
signal-to-noise ratios by two independent mechanisms. 
The disclosures of each of the aforementioned, commonly owned U.S. patent 
applications are incorporated by reference in the present specification, 
as set forth herein in full. 
While various aspects of the present invention have been described and 
exemplified above in terms of certain preferred embodiments, various other 
embodiments will be apparent to those skilled in the art. For example, the 
method of the invention may be used in processes involving immobilization 
of cells, cell components, organelles, viruses or any biological or any 
other substance having at least one characteristic determinant. Thus the 
present invention could be beneficially used in the fields of diagnostic 
and therapeutic medicine, drug testing, gene therapy, in-vitro 
fertilization, forensic science, as well as food and environmental 
testing. The invention is, therefore, not limited to the embodiments 
specifically described and exemplified above, but is capable of variation 
and modification without departing from the spirit of the invention, the 
full scope of which is delineated by the appended claims.