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BACKGROUND OF THE INVENTION
This invention relates to a system, apparatus and method for processing
samples and more particularly to performing (1) analytical procedures on
samples based on complexation reaction principles and (2) affinity
separations. Encompassed within this category of analytical procedures are
tests based on immunoassay principles, nucleic acid hybridization
principles, and affinity complexing principles. The complexation
technology is applicable to a wide range of analytical applications
including the detection of soluble antigens, cells, cellular fragments,
microorganisms and their products. Encompassed within the category of
affinity separations are the separations of biomolecules from complex
mixtures.
Generally, the types of test within this category of analytical procedures
are procedurally complex, frequently they involve multiple reagent
additions, extensive wash steps, and prolonged incubation times. These
procedural complexities diminish the testing convenience. Furthermore,
such tests must afford high sensitivity. In order to achieve this
sensitivity, long reagent equilibration times are required in order to
capture the minute quantities of analytes in samples.
Analytically, complexation based test can be applied to a wide variety of
analytes which are of diagnostic importance to clinical medicine and of
investigative importance to life science research. The complexation tests
are inherently sensitive and specific. However, as noted above, such tests
can be labor intensive and require hours of analysis time to perform.
For example, a typical test can comprise the following generalized
procedural steps. In the first step, sample fluids containing the analyte
for testing is contacted with a solid phase "capture reagent"--prepared
with a complexing reagent specific for the analyte of interest. The sample
fluid is then equilibrated with the solid phase reagent for sufficient
time to enable analyte attachment to the support. Following equilibration
with the capture reagent, the sample fluid is removed and the solid phase
reagent rinsed to remove excess sample materials. During this process the
analyte attached to the capture support remains fixed on the surface of
the capture support.
As a general rule for the analytical purposes the attached analyte cannot
be detected directly. A second "amplifying complexing reagent" often must
therefore be added and equilibrated with the solid phase support in order
to detect or visualize the presence of analyte. This equilibration must
also be carried out for sufficient time to insure effective reaction of
analyte with the solid phase surface. Following equilibration, the excess
"free" amplifying reagent must be separated from the "bound" amplifying
reagent. This is accomplished by washing the solid phase support to
eliminate the unfixed active elements. At this stage, depending on the
nature of the amplifying reagent a direct reading of the active elements
on the solid phase support can be taken. If a test is preferred using an
enzyme or other tracer to amplify the test signal, one or more substrate
reagents must first be added. Following an additional incubation period,
color development can then be observed.
Unfortunately, the numerous manipulations required in performing the
tests--notably during the successive reagent additions, rinsings, and
incubations entail risk of errors in timing, reagent measurement, specimen
identification, risk of user infection, and accidental loss of test
samples. This is particularly true when the large numbers of samples are
batched together at the same time. The problems encountered by diagnostic
laboratories in carrying out these types of tests are thus many. Clinical
laboratories must cope with large throughput of samples, interpret the
significance of the results, provide a wide range of determinations,
return results quickly, and ensure that each assay test is performed
accurately. This must be done economically in spite of the difficulties
encountered with techniques that are labor intensive, and complex when
compared with other tests performed in clinical laboratory. (British
Medical Bulletin, 30, 38-43, 1974).
While there are many variations by which such tests can be performed, a
review of the practical and theoretical constraints are well documented
and beyond the scope of this invention. To overcome these difficulties,
numerous devices and automation approaches have been described in the
prior art.
Currently, there are a number of automated machines which are commercially
available. These types of machines make use of multi-well consumables such
as microtiter plates or variations thereof. In general, these machines are
useful in tests where quantitative information on analyte concentrations
are required. Generally, these machines are complex and expensive.
To accommodate all of the testing functions mentioned above, the instrument
designs make use of a multiple modules such as a sample application
module, wash module, and plate reader or detector. Frequently, these
systems are not fully automated requiring operator involvement to move the
multi-well plate from one module to the next.
The most serious drawback to these types of systems results from their
prolonged testing time and limited range of analytical application. Tests
can require hours to days to perform. Prolonged equilibration times are
required since the microtiter test wells provide relatively small surface
area--consequently limiting the amount of complexing reagent available for
reaction with the analyte. Furthermore, these systems tend to rely on
simple diffusion or mechanical vibration to bring the analyte in contact
with the surface of the test wells. As a consequence, equilibration
generally requires hours to accomplish. Test results are therefore not
readily available in case of medical emergency or during the
patient/physician interview.
A further limitation of these system is that they tend to be limited to
immunoassay applications and not really adaptable to the newer
complexation test based on DNA and RNA hydridization principles.
In order to overcome these limitations, a number of processor systems which
make use of alternate test devices have been recently reported in the
patent literature. For example, U.S. Pat. No. 4,071,315 issued Jan. 31,
1978 to G. Chateau disclosed a processor concept which makes use of a
complexing capture reagent attached to roll of porous film which is fed
sequentially through a series of operational modules.
The Chateau system suffers from many of the same disadvantages as mentioned
above. The system is complex depending on the function of multiple
independent modules and relies on simple diffusion to effect mixing and
accomplish analyte equilibration with the film reagent. A further
disadvantage of this system is that the system does not provide rapid
turnaround time for test results. However, once engaged, large numbers of
samples can be run with high throughput.
U.S. Pat. No. 4,225,558 issued Sept. 30, 1980 to Peterson et al. describes
a centrifugal technique in which a plurality of fluid test cells arranged
on the periphery of a motor driven rotor. The fluids to be tested and
respective reagents are introduced separately into corresponding test
cells and are subsequently mixed in a reaction chamber for analysis.
Introduction of the fluids is accomplished by the use of vacuum and the
fluids are mixed by centrifugal force. This system has a disadvantage of
requiring the use of centrifugal force which reduces the throughput of the
system and renders the system unnecessarily complicated.
Another system is described in U.S. Pat. No. 4,424,279 issued Jan. 3, 1984
to Bohn et al. and U.S. Pat. No. 4,458,020 issued July 3, 1984 to Bohn et
al. These patents, both assigned to Quidel, describe an apparatus for
processing a cylindrical tube having an open end into which a plunger
filter assembly is fitted. Beads sensitized with complexing reagent such
as an antibody can be used in conjunction with the device. The operation
is centered around a plunger which is depressed to mix the sample with the
sensitized beads. Thereafter the reagent is added and the plunger is
raised to clear the chamber of the fluids. The beads are washed in much
the same way by raising and lowering the plunger. Although the system is
simple in design, it suffers from the requirement that the reagents and
wash fluids be added manually and that a four chambered dispensing unit
and the filter tube assembly be manually moved from one position to the
next in order to accomplish the assay. Operator involvement is extensive.
Furthermore since the tube and filter assembly remain open during
handling, the devices are subject to spillage and thus subject the user to
potential contact with infectious materials.
Michael Cais and Moshue Shimoni, in Analytical Biochemistry 18, 324-329
(1981) describes a tube device for performing immunoassays in which
separation of "free" and "bound" analyte is claimed to be rapidly and
safely accomplished by liquid extraction techniques. While it is not
explicitly described they indicate that a simple automated instrument has
been developed which processes up to 40 assay tubes simultaneously. This
device suffers from the disadvantage of being complex in design requiring
high precision parts, and is limited to application to only those analytes
which can be separated by solvent partioning.
A device for separating plasma from a centrifugal blood sample is described
in U.S. Pat. No. 4,483,825 issued Nov. 20, 1984 to Fatcher. This device
includes a pipette having a filter disposed over one of the two open ends.
The filter end is inserted into the tube holding the blood sample and
operated like a piston to force the plasma through the filter into the
pipette. Such device has not been used for complexation type testing.
Furthermore, being open at both ends, the device would expose the operator
to biohazardous materials.
In these prior art systems, auxiliary apparatus and equipment has been
typically employed for the sequential exposure, equilibration and washing
of the solid phase capture reagent. For example, a vibrator or shaker is
useful to both maintain controlled uniform exposure of the reaction fluids
with the capture reagent and to hasten the rate of analyte interaction
with the support. In addition a centrifuge is useful and effective in the
aggregation of suspended solid phase reagents following equilibration. An
aspirator can then be used to facilitate the decantation of the reaction
fluids from solid phase reagents. Peristaltic pumps or automated syringes
are useful to add reagents and/or wash solutions. These many separate
functions have not readily lent themselves to an uncomplicated single
automation concept, at least in a single instrument.
A number of systems have been reported which make use of chromatographic
principles to increase the efficiency of analyte capture and thus
shortening the analysis time. German Patent Application DE No. 3217-032-A
describes an immunoassay separation process using dry chromatographic
column materials. In this type of application, no automated system is
described.
Other systems have been developed including those using manifold systems
such as those offered by J. T. Baker and Waters, Inc. These systems are
designed to speed up the flow and collection of column eluants by the use
of vacuum. As such, these systems depend upon open columns in which fluid
flows in only one direction. The processor consists simply of a vacuum
source to suck the wash solution through the immunoreagents support. As a
result, the test requires multiple tubes and considerable operator
intervention to complete the test procedure.
Many flow-through type systems making use of a chromatographic type column
bed for analyte capture and processing are well known in the immunoassay
and bioseparation literature. However, the flow-through systems have the
disadvantage that they make use columns which must be carefully packed in
order to avoid both fluid channeling and the inclusion of trapped gas
which may reduce fluid contact with the supports. This adds to the
difficulty and cost to the manufacture of the column device. Furthermore
during the process, analyte capture must be accomplished in a single pass
necessitating a highly efficient column. This adds back pressure to the
flow system frequently necessitating positive displacement pumps to
compensate increased back pressure and insure positive fluid flow.
Furthermore the simultaneous processing of multiple samples is not
possible. In addition, during consecutive cycles, these systems are
subject to contamination and plugging due to entrapment of particles and
debris. Furthermore, reduced activity of capture reagent in the subsequent
reprocessing is also observed. Sample and reagent mixing prior to
equilibration with the capture support must be carried out prior to the
reagents reaching the test device. This is frequently accomplished by
using a long connecting tube which tends to result in contamination and
requires considerable lengths of time for liquid to reach the reaction
container.
Japanese Patent Application J58,223,758 published Dec. 26, 1983, filed in
the name of Kokaietal, describes a flow-through system and circulatory
reaction device which claims to overcome many of the disadvantages of
flow-through systems. The reaction tube is an open tube, having an opening
at both ends. A filter is used to provide a support for immobilization of
antibodies or antigens in a nozzle. Positive pressure is used to force the
material through the cell and to wash the solid phase capture reagents.
The system is mechanically complex--requiring operation of numerous
syringe pumps. The system is operated at positive pressure and therefore
subject to emission or release of toxic and infectious aerosols.
Furthermore, since the system makes use of a packed capture bed with fluid
flow in one direction, diminished flow and increased back pressure will
result from plugging.
SUMMARY OF THE INVENTION
It is therefore an object of this invention to provide an automated system
which can perform many of the procedural steps required in tests based on
complexation reactions (for example, antigen-antibody binding, nucleic
acid hybridization, and surface adherence). Specifically, it is an object
of this invention (1) to achieve test operations involving sample removal,
sample equilibration with solid phase reagents, wash, reagent additions,
and color development steps with no operator assistance; (2) to provide a
means of actively promoting the interaction between analyte and complexing
reagent. In this way, testing time can be substantially reduced without
comprising the ultimate detection limit or sensitivity of the assay; (3)
to provide for enhanced user safety by using a negative pressure closed
system in which biohazardous materials can be collected and decontaminated
without operator contact with sample materials; and (4) to provide an
inexpensive test system which enables multiple tests to be performed
simultaneously.
This invention finds use in a system for processing samples disposed in a
fluid receptacle having an open end and containing an analyte to be
processed and a capture reagent on a solid support, the system including
means for subjecting the analyte to the capture reagent. The invention
comprises the improvement in which: the system includes a manifold and
receptacle defining a closed chamber and first and second ports
communicating with the chamber, means to place the open end of the fluid
receptacle in fluid communication with the first port, and means for
selectively introducing to and removing from the chamber and hence the
receptacle through the second port vacuum, air, reagents, and wash fluids,
thereby to effect rapid interaction between the capture reagent and
analyte, efficient washing of the capture reagent, and detection of the
analyte.
The system of this invention applies coordinated pulses of vacuum and air
to the chamber and hence to the receptacle. This permits fluids to be
consecutively added to the receptacle and withdrawn. The system also
provides for recycling the fluids within the receptacle back and forth
through the capture reagent thus affording a means of rapid interaction
between the capture reagent and the sample fluids. Once the analyte is
attached to the capture reagent, the wash fluids may be introduced in the
same manner into the receptacle and withdrawn. In a similar manner
developing agents may be introduced into the receptacle and withdrawn
again.
In one embodiment, the manifold has plural receptacle ports located at
equal distances from the second port. In a preferred embodiment of the
invention, the receptacle may be elongated and closed at one end and open
at the other, with the open end being in fluid communication with the
chamber. A porous retainer is positioned in the open end so as to retain
the capture reagent trapped within the compartment. Although the capture
reagent typically is disposed upon a solid support this solid support may
be either particulates (beads) or it may be attached to the porous
retainer itself or to the walls of the receptacle. In a preferred
embodiment of the invention, the receptacle itself is collapsible.
According to the method of this invention, samples having an analyte are
processed by placing the sample in a closed receptacle, which has an
access port, together with a capture reagent for the analyte. A porous
retainer may be positioned in the receptacle to prevent the escape of the
capture reagent. The method includes the steps of subjecting the access
port and hence the receptacle to a vacuum and thereafter selectively
subjecting the receptacle to reagent, vacuum, air and/or wash fluids
thereby to capture, wash, and/or develop the analyte. Typically the
receptacle is alternately subjected to vacuum and air to recycle the
analyte and reagents back and forth across the capture reagent.
The method, apparatus and system of this invention are designed to perform
a variety of tests or separations based on complexation reactions while
enabling user safety is to be enhanced, operator involvement to be
minimized and testing times to be substantially reduced. Complexation
reactions include, antigen-antibody binding, nucleic acid hybridization
and surface adherence. The test processes include sample removal, sample
equilibration with a solid phase reagent, washing reagent additions, and
color development steps. Testing time is reduced by providing a means of
actively promoting the interaction between analyte and the complexing
reagent. User safety is enhanced by employing a closed system in which
biohazardous materials can be collected and decontaminated without
operator contact with the sample materials. Test throughput is also
enhanced by providing a system and apparatus that permits multiple tests
to be performed simultaneously. The system described is one that is
readily adaptable to a variety of different tests and analytical
applications. These include immunoassays, DNA hybridization and affinity
staining tests. Finally the method and system of this invention is adapted
to perform automatically many of the tests and separations involving
complexation reactions with minimal operator intervention.
These types of tests tend to be procedurally complex involving staged
introduction and equilibration of sample, wash fluids, and amplifying and
visualization reagents for a solid phase "capture" reagent. Some means for
agitation, vibration or sonication is frequently employed during the
equilibration steps to enhance the efficiency and rate of analyte
interaction with the solid phase capture reagents.
Several embodiments of the receptacle are possible. They may take the form
of either collapsible and non-collapsible types. In one form, the
receptacle may constitute a pipette like device having a bulbous
compartment at one end connected with an elongated tube. A porous plug can
be positioned within the tube so as to retain certain types of solid phase
capture reagents within the receptacles. Alternately the capture reagents
may be attached to the porous plug itself, to particulates within the
receptacle, to a packed bed disposed within the tubular portion of the
receptacle, or to the walls of the tubular portion. In other forms of the
invention the receptacle may constitute a hollow tube closed at one end or
a flexible tube-like enclosure sealed at one end.
It has been found that the configuration and position of the porous septum
in the rapid capture device is surprisingly important to achieve automated
capture of sample analyte. A packed column configuration is ideally suited
for maximum rate of analyte capture since molecular diffusion distances
are minimized. In opposition, however, the back pressure and restricted
flow created by the packed bed represents an important obstacle to
effective movement of fluids and air in and out of the receptacle. A
packed bed, thus, prolongs the time for recycling the sample through the
capture beads. Further a packed bed hinders the rapid and efficient wash
of the capture beads since the fluids cannot be effectively removed
without air being in the pressure reservoir.
By placing the porous septum in close proximity to the pressure reservoir,
i.e. so that the internal volume of the bulbous portion and the elongated
passageway leading to the access port each exceed the volume of the test
fluids, then the recycling process can be carried out totally within the
confines of the receptacle. Furthermore the obstructions to fluid and air
flow through the device are greatly reduced. This results from the fact
that during the process cycle fluid moving into the device will lift and
tend to break up the bed of packed capture reagent. The capture
particulates therefore contribute little to the back pressure, and most
importantly do not restrict the flow of air into the device. Furthermore,
the capture efficiency achieved from the packed bed configuration tends to
be retained, since as the fluid is withdrawn from the receptacle the loose
capture particulates are quickly swept into the column chamber, and
repacked, thus establishing an effective column bed. In this way, both
restriction to flow is greatly reduced and the conditions for optimum
analyte capture retained. The geometry of the bulbous portion also
facilitates rapid funneling of the loose capture back into the column
chamber, thus further facilitating the rapid information of a packed
column by the capture beads during the recycling process.
In another embodiment, multiple different capture reagent supports can be
integrated into the test receptacles. These can be used to simultaneously
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