Magnetic separation device and methods for use in heterogeneous assays

Magnetic separation devices are described for use in immunoassay or hybridization assay procedures. The most preferred embodiments comprise a defined relationship between microtube and microtiter plate receiving orifices and rare earth cobalt magnets having predetermined magnetic field orientations.

FIELD OF THE INVENTION 
This invention relates to devices useful in performing heterogeneous type 
assays and more particularly describes new magnetic separation devices for 
use with immunoassay and hybridization type assays utilizing micromagnetic 
or ferrous particles. 
BACKGROUND OF THE INVENTION 
In recent years, health care has improved dramatically, in large measures 
due to the availability and improvement in assays. Assays detect 
particular chemical constituents, i.e., ligands, which have been 
correlated or are associated with various disease conditions. Ligands are 
detected primarily through a binding reaction with a ligand binding 
specific substance which preferentially binds to the ligand and not to 
other chemical constituents which may be present in the sample to be 
tested. Through a variety of imaginative techniques, the presence or 
absence of such a binding reaction can be detected, and thus in turn the 
presence or absence of the ligand in the sample determined. 
There are two major types of assays to which this claimed invention relates 
and these include immunoassays and hybridization assays. Immunoassays have 
been in existence longer and are based upon the specificity of the 
reaction between an antibody and an antigen for which the antibody is 
specific. Antibodies initially used where of polyclonal origin, e.g., they 
were produced in animals following a challenge by the antigen for which 
the antibodies were desired. Of late, following the innovative 
developments by Koller and Millstein in 1975 (Nature 225:1061), monoclonal 
antibodies have been preferred since they can be more easily produced and 
allow exquisite selection by affinity and avidity. 
There further exists a variety of techniques for labeling the antibodies 
and/or antigens including the employment of isotopic labels, fluorescent 
molecules, chemiluminescent molecules, enzymes, light scattering 
particles, energy transfer, schemes between pairs of spectrally matched 
molecules, and the like. These techniques are well known in the art and 
need not be reviewed in detail here. It is worthy, however, to distinguish 
between two diverse types of assays; homogenous and heterogeneous assays. 
Homogeneous are most desired from an operational standpoint since the 
entire reaction, and an addition of reagents for the performance of the 
assay, take place in a single solution along with the final detection 
step. Accordingly, mechanical manipulations which are time consuming and 
can cause errors are avoided, however, the technical aspects of developing 
such an assay with the desired sensitivity are substantial. In contrast, 
numerous assays are performed on a heterogeneous basis in that certain 
steps are performed in one solution which generally includes some type of 
solid phase material. The reaction to be detected takes place either in 
solution or on the solid phase and is then followed by a separation step 
whereby unreacted components, and thus contaminating influences, may be 
effectively removed. The result is generally a higher level sensitivity at 
the expense of additional mechanical manipulations. Conventional 
heterogeneous assays have employed dipsticks which may be easily removed 
from the solution by hand, or large beads which similarly allow facile 
transfer. Smaller beads, generally of latex or similar materials, have 
been employed and have relied upon filter and/or centrifugal methods for 
their sequestration from the fluids. The concept of employing large 
magnetic particles has also been explored and is described by Smith et al. 
in U.S. Pat. Nos. 4,272,510 and 4,292,920. Specifically, Smith et al. 
describe the use of BB type particles and their removal from solution by 
the employment of electromagnetic energized nails for removing the solid 
phase from container to container. While somewhat inelegant, the Smith et 
al. method does have an advantage in that the container walls which often 
provide a contaminating influence are eliminated assuming, of course, the 
detectable reaction has taken place on the solid phase. The methods, 
however, suffer from substantial risk of loss of accuracy due to the 
failure to remove and/or transfer all solid phase particles to another 
container, particularly as would be the case with micromagnetic or ferrous 
particles. Such microparticles would be greatly preferred over the large 
beads described by Smith et al. because the present has a far greater 
surface area upon which reactions may take place. Sensitivity is 
accordingly dramatically improved. 
Corning et al. has made available commercially a magnetic separation device 
which is intended for use with large, e.g., 12 mm by 75 mm, test tubes 
containing the assay reagent mixtures and the magnetic particle solid 
phase component. The Corning device has horizontal molded ridges for 
receiving the test tube whereby the particles become attracted to a side 
of the test tube allowing removal of the liquids. The design of the 
Corning device is not, however, optimized for use with small sample 
volumes and cannot be made optimal for such application therefore limiting 
its utility. 
It is an object of the present invention to provide devices for use with 
assays employing magnetic microparticles which are capable of use with 
small volume assays or &gt;60 at a time. 
It is another object of the present invention to provide devices which may 
be employed with microtiter type trays whereby a plurality of assays may 
be performed simultaneously. 
It is yet another object of the present invention to provide an 
autoclavable separation devices for use with ferrous solid phase 
materials. 
It is a still further object of the present invention to provide a magnetic 
separation device which is readily adaptable to automated pipetting 
systems. 
It is still yet a further object of the present invention to provide a 
magnetic separation device which may be adapted to provide one, two or 
four ferrous particle attraction positions within each sample container. 
Another class of assays of more recent vintage are those relying upon the 
hybridization of nucleic acid probes with target nucleic acids. The target 
nucleic acid is generally that associated with an infective organism, 
e.g., bacteria virus and like, although the detection of specific cellular 
genomes is also contemplated. By greatly simplified explanation, 
hybridization assays rely upon the greatly preferred pairings between 
complementary nucleotide bases. Specifically, the preferred pairings are 
between adenine and thymidine, guanine and cytidine. Each strand of 
deoxyribonucleic acid (DNA) is comprised of a series of the foregoing 
bases, while its complementary strand comprises a matching but 
complementary series of DNA bases. Thus, one can disassociate the 
double-stranded nucleic acid into single-strands and with probes comprised 
of nucleic acids having complementary sequences, one may produce 
double-stranded nucleic acid wherein the probe is hybridized to the target 
nucleic acid only at complementary positions. 
DNA is transcribed into ribonucleic acid (RNA) which is also comprised of 
ribonucleotides of the same four bases with the exception that uracil is 
substituted for thymidine. Because of the manner of transcription, the RNA 
sequence is also complementary to the DNA sequence and accordingly 
comprises the same basic identifying information. Thus, one may similarly 
detect RNA of a microorganism or cell by hybridizing the target RNA to a 
probe comprising complementary sequence. The production and formulation of 
nucleic acid probes while a comparatively recent development, are still 
arts well known and well described in the literature. A helpful reference 
in this regard is Maniatis et al., a cloning manual, the relevant portions 
of which along with references referred to therein are incorporated herein 
by reference. 
As may be readily appreciated, many of the same techniques employed with 
immunoassays are the labeling, and heterogeneous/homogenous schemes are 
applicable to hybridization assays. In particular, the employment of solid 
phase materials in heterogeneous assays are techniques which make 
hybridization assays especially useful. The utilization of micromagnetic 
particles is not, however, commonplace primarily in large measure due to 
the inapplicability of the present magnetic separation devices to 
hybridization assays. 
It is, therefore, another object of the present invention to provide 
suitable magnetic separation devices useful with hybridization assays and 
ferrous solid phase particles. 
SUMMARY OF THE INVENTION 
In accordance with the principles and objects of the present invention, 
there are provided magnetic separation devices for use in heterogeneous 
immunoassay and/or hybridization assays which comprise a base having a 
plurality of orifices for receiving nonferrous containers which hold the 
sample and assay components including ferrous particles which may or may 
not exhibit a natural magnetism. Each of the orifices is surrounded by 
preferably, a plurality of magnetics, most preferably four, which are 
spaced, most preferably equidistant, about the peripheral of the orifice. 
The north-south field orientation of each magnet is preferably orientated 
so that it is coplanar with a cross-sectional plane through the receiving 
orifice and accordingly impinges upon the nonferrous container in a 
direction that is defectively perpendicular to the generally aligned axes 
of of the container. Ideally, the north-south field direction orientation 
of the magnetics about the periphery is in the same direction regardless 
of its position about the periphery of the orifice. Thus, all magnetics in 
this preferred embodiment are aligned in a single particular direction in 
relation to the base. More preferably, the north-south magnetic field 
orientation of the magnetics about the periphery of the receiving orifice 
are alternating 180.degree. in direction. Thus, reviewing the field 
orientation of the magnetics by preceding in a common direction about the 
periphery of the orifice, e.g., clockwise or counterclockwise for each 
orifice, results in the first magnetic having a north-south field 
direction which is 180.degree. opposing that of the next magnet which in 
turn is 180.degree. opposing that of the following and so on. Thus, every 
other magnet about the periphery has a substantially identical field 
orientation. 
Most preferred embodiments of the device further comprise a guided 
pipetting means capable of pipetting fluids to or from a plurality of the 
nonferrous containers simultaneously. Other embodiments further comprise 
means for agitating the reagents within each of the nonferrous containers 
and for incubating the nonferrous containers and a transfer slide adapted 
to engage the base means for mating the plurality of nonferrous containers 
to the receiving orifices and for removing same. 
Other embodiments of the present invention comprise receiving 
orifice-magnet orientations which provide for one or two spot attraction 
sites within the nonferrous container while the most preferred embodiment 
above, having a receiving orifice surrounded by four magnets results in 
four spot attraction sites. 
Novel processes are provided comprised of the magnetic separation devices 
of the present invention in immunoassay or hybridization assays for a 
ligand in a fluid sample.

DETAILED DESCRIPTION AND BEST MODE 
Figure one shows a preferred embodiment of the magnetic separation device 
of the present invention. It advantageously accommodates microtubes such 
as the Micronic.RTM. type tube and most preferably it is made to 
accommodate 96 such tubes simultaneously in 12 rows by 8 rows or channels. 
Such an orientation is similar to that of a common usage with respect to 
Microtiter.RTM. type trays which provide 96 wells. Other manufacturers 
have made accessory devices for use with such trays, such as for example, 
the Propet.RTM. pipetting system from Cetus, Emeryville, Calif. By 
advantageously providing the 96 sample format, the device of the present 
invention could potentially be used with such automated devices already 
present in the laboratory. 
FIG. 1 shows the base 1 of the separator device having a plurality of 
receiving orifice 3 for receiving the microtubes (See FIG. 2, number 20). 
Preferably, the base will be comprised of a nonferrous or nonmagnetic 
material and may be advantageously machined out of a metal such as 
aluminum or molded out of suitable plastic. Most preferably, the materials 
shall be selected so that they may withstand typical sterilizing 
procedures such as autoclaving. Surrounding the periphery of orifice 3 are 
machined orifice 6 (or molded orifice 6) for receiving magnets 5. Having a 
north-south field orientation which is coplanar with the surface of the 
base 1 and a cross-section of receiving orifice 3. 
If the Neodymium Iron Boron Magnets all aligned in the same "North" 
direction as shown in FIG. 8, it was discovered that this results in a 
field strength within the receiving orifice of approximately 500-600 
Gauss. If, however, the magnets have an alternate "North" pattern as shown 
in FIG. 9, e.g., as one proceeds around the clockwise direction of the 
periphery of each orifice, the magnetic field alternates 180.degree. in 
direction, it was surprisingly discovered that such an orientation tends 
to focus the field lines within the receiving orifice and the magnetic 
field increased to approximately 1400-1600 Gauss. As a result of this 
surprisingly orientation, a dramatic increase in the separation of 
magnetic microparticles, such as those available from Advanced Magnetics, 
Inc., occurs within the solution. As may be apparent, separation results 
in the localization of four areas or spot attraction sites within the 
microtube. Less preferred embodiments of the magnetic separation device of 
the present invention utilize fewer magnets such as 24, or 59, evenly 
dispersed between receiving orifices 3 in base 1 whereby 1, or 2 
respectively spot attraction sites result within each microtube. 
Most preferably, the magnets 5 employed are permanent magnets, most 
preferably possessing a strong magnetic field. The stronger the magnetic 
field, the more effective the separation and the faster such separation is 
effective. Most preferred magnetics are rare earth Neodymium Iron Boron 
magnetics available from IG Technologies, Valparaiso, Indiana. Magnets of 
0.13".times.0.13".times.0.5" were advantageously used as possessing the 
necessary strength and size requirements so that they could be suitably 
installed in orifice 6 between receiving orifice 3 which accommodate the 
microtubes commercially available. Orifice 6, in microtiter plate 
geometrics can be installed from the top or bottom of the microtiter plate 
and is based on the manufacturers plate geometry. 
While the magnets 5 may be press fit into machine orifice 6, other 
production techniques may be employed. For example, base 1 could be 
manufactured by investment casting method whereby magnet orifice 6 
possesses a size and shape more closely matching that of magnet 5. 
Alternately, base 1 may be produced from plastic such as by an injection 
molding process which also allows for close tolerances in form fitting 
magnet receiving orifice 6. 
FIG. 2 shows a transfer tray for use with the magnetic separation device 
shown in FIG. 1. Transfer tray 21 possess tube receiving orifice 23 for 
receiving microtubes 20. Tube transfer tray 21 further comprises tray legs 
22 which serve to support to transfer tray 21 a sufficient distance of a 
surface to allow loading of microtubes 20 and also serve to provide 
alignment and a vertical height adjustment with tray leg receiving orifice 
7 (FIG. 1) upon mateable engagement of the microtubes 20 with receiving 
orifice 3 (FIG. 1). Proper vertical height alignment is required in the 
magnetic separator for optimum separation efficiencies. 
Receiving tray 21 further assists the operator during the performance of 
the assay to transfer microtubes 20 to agitation devices for physically 
urging separated microparticles into solution from their spot attraction 
sites. The tube transfer tray 21 further assists in transferring the 
microtubes to an incubator block for providing a temperature controlled 
environment as required for the particular assay being performed. 
FIG. 3 shows a perspective view of the most preferred embodiment wherein 
receiving base 31, made from a nonferrous metal such as aluminum, is 
machined to accommodate cylinders 33. preferably again aluminum, 
dimensioned to accommodate the microtubes. The machined out area of 
receiving base 31 is also dimensioned to accommodate magnets 35 such that 
each receiving tube 33 is surrounded by four magnets 35. Most preferably, 
the magnetic field orientation of magnets 35 are arranged in a alternate 
pattern as previously discussed. Magnets 35 and tubes 33 are held in place 
by base cover 32 which may be permanently attached to receiving base 31 by 
means of screws, adhesives, ribbons and the like. While this most 
preferred embodiment is shown with nine receiving orifice 37, it will be 
readily appreciated that this construction embodiment may be scaled up to 
the 96 receiving orifice (or more as desired) shown in FIG. 1. 
FIG. 4 shows a perspective view of receiving based/separator 41 with tube 
transfer tray 42 mateably engaged therewith with microtubes 43 installed 
therein. A guided channel pipetter 44 is employed to pipette fluids to 
and/or from the microtubes through fluid connection tube 47 to the fluid 
pump mechanism (not shown). The pipetter 44 with individual pipettes for 
each microtube within a particular row or column engages receiving 
base/separator 41 by mateable engagement of alignment pegs 45 with 
alignment holes 46. 
FIG. 5 shows the results of an experiment directed to measuring the speed 
of particle movement vs. magnetic orientation as determined by light 
intensity measured in a spectrophotometer. As will be clearly understood 
upon study of the figure, it is apparent that a tremendous, surprising and 
unexpected increase in speed of particle movement is observed in the 
device of the invention when alternating magnetic poles are utilized. 
Nonetheless, unit directional poles will generally be adequate for most 
samples, however, the alternating poles will clearly be preferred with 
respect to viscous samples such as those containing blood and the like. 
Accordingly, the preferred embodiment of the present inventions will 
utilize alternating magnetic poles as shown in area 9 of FIG. 1. 
While the present invention has been specifically described with reference 
to Micronic.RTM. type microtubes, it will be readily appreciated that the 
principles of the invention may be readily applied to microtiter type tray 
embodiments. Specifically, obvious mechanical alterations to the 
embodiment shown in the figures will be required in order to adequately 
receive the microtiter trays and place the magnets in juxtaposition with 
the wells. Preferably, such placement will involve the utilization of at 
least four magnets, advantageously spaced evenly about the periphery of 
each well. Most preferably, the magnetic field orientation will alternate 
approximately 180.degree. C. with each magnet as one uses the magnets in a 
clockwise or counterclockwise direction about the periphery of the orifice 
or other area receiving the well of a microtiter tray. Actual physical 
embodiments of the base for receiving the microtiter tray may involve 
exposure of coated magnets whereby the placement of the microtiter tray on 
top of the base allows the magnets to protrude upwardly between the wells 
of the microtiter tray. Alternatively, and particularly with respect to 
that type of microtiter tray which has a solid bottom, the base will 
simply accommodate the microtiter tray and a cover comprising downwardly 
protruding magnets will mateably engage with the top of a microtiter tray 
whereby the magnets protrude downwardly between the microtiter wells. 
Holes are provided in the top plate for fluid removal. Given the 
disclosure and particularly the accompanying figures, one skilled in the 
art will readily determine suitable physical constructions for 
accommodating the microtiter tray of choice. 
Employment of the device will become clear upon review of its use in the 
following hybridization assay example. Study of this example with the 
foregoing description and accompanying drawings will make its similar 
employment in immunoassays obvious to and well within the skill of one of 
ordinary skill in the art. 
EXAMPLE 1 
Detection of Listeria Monocytogenes 
A DNA probe assay for Listeria monocytogenes was performed using 
0.5-1.5.mu. diameter magnetic particles (Advanced Magnetics, Inc.) with 
oligo dT14 covalently coupled to the surface of the magnetic particles. A 
base was comprised of 117 magnets glued into a plate which mates with the 
chosen microtiter plate (Titertek/96 well plates). When the microtiter 
plate of this assay is mated with the base, each well in the microtiter 
plate has a magnet at each quadrant, thereby effecting separation of the 
magnetic particles from the supernatant as described earlier. 
An overnight culture of Listeria was grown in brain heart infusion broth at 
37.degree. C. and 1 ml of this sample culture was added to 1 ml of a 
processing buffer as defined below and a mixture of 70 ul total was added 
to each microtiter plate well. 
PROCESSING BUFFER 
5M GuSCN (guanidine isothiocyanate) 
0.30M Tris-HCl, pH 7.5 (Tris-[hydroxymethyl]aminomethane) 
0.10M Na.sub.2 EDTA (ethylene diaminetetraacetic acid) 
20% dextran sulfate (MW 5000) (wt/vol.) 
2.0 ul of a 35 mer oligonucleotide of DNA tailed with 160 dA residues (35 
ng/ul in 2.5M GuSCN, 10 mM EDTA, pH 7.5) was added to each well and 
incubated at 37.degree. C. for 15 minutes. 140 ul dT14 derivatized 
magnetic beads (dA50 binding capacity of 5 ug/ml) in a bead reagent buffer 
(as defined below) was added to each well: 
BEAD REAGENT BUFFER 
Tris-HCl 0.1M, pH 7.4 
acetylated BSA 0.5% (Bovine Serum Albumin) 
10 ug/ml sonicated calf thymus DNA 
10 mM EDTA 
4% saponin 
0.5% sarkosyl 
0.5M NaCl 
0.1% azide 
0.01% silicone antifoaming agent 
Magnetic beads were separated by mating the microtiter plate with the base 
containing 117 magnetics and supernatant removed, and 0.1 ml of wash 
buffer 1 was added to each well at room temperature, and the beads 
resuspended after unmating the plate from the base containing 117 magnets. 
WASH BUFFER 1 
1M GuSCN 
Tris-HCl 0.1M, pH 7.4 
acetylated BSA 0.5% 
10 ug/ml calf thymus DNA 
EDTA 10 mM 
0.1% sodium azide 
sarkosyl 0.5% 
saponin 1% 
antifoam Magnetic beads were reseparated by mating the microtiter plate 
with the base containing 117 magnet, supernatant removed, 0.1 ml of wash 
buffer 1 added at room temperature, and the beads resuspended after 
unmating the plate from the base containing 117 magnets. 
Magnetic beads were reseparated by mating the microtiter plate with the 
base containing 117 magnet, supernatant removed, 0.1 ml of wash buffer 1 
added at room temperature, and the beads resuspended after unmating the 
plate from the base containing 117 magnets. 
65 ul of chemical eluant (defined below) was added to each well, the 
constructs mixed and incubated for two minutes at 37.degree. C. 
CHEMICAL ELUANT 
2.5M GuSCN 
Tris-HCl 0.1M, pH 7.4 
10 ug/ml sonicated calf thymus DNA 
acetylated BSA 0.5% 
EDTA 10 mM 
1.0% saponin 
0.5% sarkosyl 
Magnetic beads were separated by mating the microtiter-plate with the base 
containing 117 magnets and the eluate from each well transferred to a 
fresh well containing ul of a 14 ng/ml concentration of riboprobe cloned 
from the 3'-end of E. coli. The mixture was incubated at 37.degree. C. for 
four minutes. 140 ul of beads in reagent (dA50 binding capacity of &gt;5 
ug/ml), was added to each well and incubated for two minutes at 37.degree. 
C., the beads magnetically separated by mating the microtiter plate with 
the base containing 117 magnets at 37.degree. C. 
The supernatant was removed and 0.1 ml of wash buffer 2 (defined below) 
added to each well and the beads resuspended at 37.degree. C. after 
unmating the base from the microtiter plate. 
WASH BUFFER 
Tris 0.1M, pH 7.4 
10 ug/ml E. coli DNA or t-RNA 
EDTA 10 mM 
acetylated BSA 0.5% 
0.5M NaCl 
0.5% sarkosyl 
Magnetic beads were separated by mating the microtiter plate with the base 
containing 117 magnets, supernatant removed, 0.1 ml of wash buffer 2 added 
at 37.degree. C., and the beads resuspended after unmating the plate from 
the base containing 117 magnets. 
Separate magnetic beads by mating the microtiter plate with the base 
containing 117 magnets, supernatant removed, 0.1 ml of wash buffer 2 added 
at 37.degree. C., and the beads resuspended after unmating the plate from 
the base containing 117 magnets. 
Magnetic beads were separated by mating the microtiter plate with the base 
containing 117 magnets, supernatant removed, 0.1 ml of wash buffer 2 added 
at 37.degree. C., and the beads resuspended after unmating the plate from 
the base containing 117 magnets. 
Separate magnetic beads by mating the microtiter plate with the base 
containing 117 magnets, supernatant removed, 0.1 ml of wash buffer 2 added 
at 37.degree. C., and the beads resuspended after unmating the plate from 
the base containing 117 magnets. 
Magnetic beads were separated by mating the microtiter plate with the base 
containing 117 magnets, supernatant removed, 0.1 ml of wash buffer 2 added 
at 37.degree. C., and the beads after unmating the plate from the base 
containing 117 magnets. 
Separate magnetic beads by mating the microtiter plate with the base 
containing 117 magnets, supernatant removed, 0.1 ml of wash buffer 2 added 
at 37.degree. C., and the beads resuspended after unmating the plate from 
the base containing 117 magnets. 
Magnetic beads were separated by mating the microtiter plate with the base 
containing 117 magnets, supernatant removed, 0.1 ml of wash buffer 2 added 
at 37.degree. C., and the beads resuspended after unmating the plate from 
the base containing 117 magnets. 
100 ul wash buffer 2 was added for each well and incubated at 68.degree. C. 
for two minutes. 
Beads were magnetically separated by mating the microtiter plate with the 
base containing 117 magnets and supernatant removed and transferred to 
scintillation fluid and then the amount of P.sup.32 in each vial was 
determined by use of a Beckman 1800 scintillation counter. The presence of 
Listeria was confirmed by P.sup.32 counts that were more than an order of 
magnitude greater than when Listeria was absent in control wells.