U.S. patent application number 12/140691 was filed with the patent office on 2009-03-12 for apparatus and method for conducting assays.
Invention is credited to John Francis Gordon.
Application Number | 20090068064 12/140691 |
Document ID | / |
Family ID | 10801090 |
Filed Date | 2009-03-12 |
United States Patent
Application |
20090068064 |
Kind Code |
A1 |
Gordon; John Francis |
March 12, 2009 |
APPARATUS AND METHOD FOR CONDUCTING ASSAYS
Abstract
A multi-well assay plate structure (54) and assay apparatus and
a method for performing chemical biochemical assays is described.
The multi-well assay plate structure (54) defines a relatively
shallow substantially enclosed space (71) above a plurality of
wells (76), with the enclosed space (71) having an inlet (72) and
an outlet (22) separate from the inlet. Fluid introduced via the
inlet (72) flows into the space (71) and/or wells (76) by
displacing air. Withdrawal of the fluid via the inlet (72) or
outlet leaves fluid in the wells (76) allowing various tests to be
performed. Various embodiments of the structure are described. The
preferred arrangement embodies the structure on a transparent
plastic disk which can be used with automatic fluid handling
apparatus (80) and the results assessed using optical assessment
apparatus (81). The apparatus can be used to perform a variety of
assays but, in particular, biochemical/chemical assay, immunoassays
and genetic (DNA) assays and it can be used in a laboratory for
multiple sample testing or at a point-of-care, i.e. in a surgery or
clinic.
Inventors: |
Gordon; John Francis;
(Glasgow, GB) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
2040 MAIN STREET, FOURTEENTH FLOOR
IRVINE
CA
92614
US
|
Family ID: |
10801090 |
Appl. No.: |
12/140691 |
Filed: |
June 17, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09284421 |
Jun 11, 1999 |
7387898 |
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PCT/GB97/02708 |
Oct 8, 1997 |
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12140691 |
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Current U.S.
Class: |
422/82.05 ;
422/400 |
Current CPC
Class: |
B01L 3/5085 20130101;
B01L 2400/0406 20130101; B01L 3/5027 20130101; B01L 2200/025
20130101; B01L 2300/0803 20130101; G01N 33/5304 20130101; B01L
2300/021 20130101; Y10T 436/111666 20150115; Y10T 436/2575
20150115; G01N 2035/0451 20130101; B01L 3/5088 20130101; B01L
2300/165 20130101; B01L 2200/0642 20130101; B01L 3/50855
20130101 |
Class at
Publication: |
422/82.05 ;
422/102 |
International
Class: |
G01N 21/75 20060101
G01N021/75; B01L 3/00 20060101 B01L003/00 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 10, 1996 |
GB |
9020934.1 |
Aug 10, 1996 |
GB |
9620934.1 |
Claims
1. An assay plate structure for use in conducting optical assays of
a fluid analyte, the plate structure comprising: a disc for
rotation about a central axis, the disc having upper and lower
plates. spaced apart a distance to facilitate the flow of a fluid
between said plates by capillary action and a plurality of
substantially radially extending walls disposed between the plates,
said walls subdividing the disc into a plurality of disc sectors;
and a plurality of disc inserts arranged to be received by
respective disc sectors and to be retained therein, the structure
further including a plurality of openings through the upper plate,
at least one opening above each disc sector for introducing a
liquid analyte into the sector space between the upper plate and
the disc insert, the upper surface of each disc insert and the
opposed surface of the upper plate being substantially planar, and
the flow of fluid between the upper plate and the disc insert being
facilitated by capillary action.
2. The assay plate structure of claim 1 wherein a vent opening is
provided for each disc segment around the periphery thereof,
between the radially outer edge of the upper plate and each disc
insert.
3. The assay plate structure of claim 1 wherein the plate structure
is provided in the form of a disc and includes digitally encoded
address information.
4. The assay plate structure of claim 1 wherein the plate structure
is transparent for an optical inspection of said wells from outside
structure.
5. A chemical/biochemical assay apparatus comprising an assay plate
structure in accordance with claim 1 and having a plurality of
wells for receiving samples to be. assayed, said assay apparatus
further including: fluid handling means for introducing and
removing fluid reagents into said assay plate structure to allow a
fluid reagent mixture to be retained in each well, and optical
assessment means for measuring optical result of the reaction in
each well.
6. An assay structure for use in conducting optical assays of a
fluid analyte, the structure comprising, a disc for rotation about
a central axis, the disc including optically readable digitally
encoded address information provided therein for optical inspection
thereof from externally of said disc and said disc have one or more
disc insert receiving sectors; one or more disc inserts to be
received by a respective disc sectors and to be retained therein,
the structure further including one or more openings within the
disc to provide, at least one opening relative a disc sector for
fluid communication with a disc insert received in said sector.
7. The assay structure of claim 6 wherein a vent opening is
provided in said disc for a disc sector.
8. The assay structure of claim 6 wherein the one or more disc
inserts are snap-fitted to the disc.
9. The assay structure of claim 6 wherein the disc inserts and the
disc include lock and key portions to allow the inserts to be
snap-fitted to the disc in a correct orientation only.
10. The assay structure of claim 6 wherein the disc and inserts are
made of optically transmissive plastic.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to apparatus and to a method
for conducting assays and, in particular, to multi-well plate
structures for receiving and holding, in separate wells, volumes of
liquid for the purpose of conducting chemical or biochemical
assays. Multi-well trays or plates having a 2-dimensional array of
small wells are commonly used in medicine and science to facilitate
testing of a liquid analyte. One particular area of use is blood
screening where blood or blood products are introduced into the
wells to test for viruses such as HIV, hepatitis etc.
[0003] 2. Description of the Related Art
[0004] Such tests (immunoassays) typically involve an
antigen-antibody interaction, where the surfaces of the wells are
coated with specific antigen itself. This approach detects
circulating antibodies to that specific antigen. Alternatively the
wells can be coated with a specific antibody which captures
circulating antigen which is, in turn, identified by a second
antibody directed against a second epitope on the captured antigen.
These two approaches are just two of the large number of variants
developed in immunoassay (review Principles and Practice of
Immunoassay Price & Newman 1997 ISBN 1-56159-145-0).
[0005] In an immunoassays sample must be applied and in most cases
subsequent addition of reagents or washing buffer is required.
Typically the well is exposed to blood or blood product and the
well is rinsed clean and a further reactant, which binds either to
exposed antibodies or captured antigens is introduced into the
wells, to create an observable reaction. These reactions may
produce a color or some other observable change. This enables the
wells containing specific antigen antibody reactions to be
identified and the extent of these reactions quantified.
[0006] It is often necessary to fill each well of a multi-well tray
with a precisely defined volume of analyte. This is normally
achieved using a single or multi-headed micro-pipette. However,
this process is often time consuming and, particularly where a
large number of wells are to be filled, can lead to a number of
wells being missed.
[0007] In certain circumstances it is necessary that the wells of a
tray be contained within a substantially closed container, e.g. to
avoid the risk of contamination of the wells and of leakage of
contaminated material. With trays such as this, it may be difficult
or impossible to gain access to the wells to enable them to be
filled using a micro-pipette.
SUMMARY OF THE INVENTION
[0008] It is an object of the present invention to overcome or at
least mitigate the disadvantages of known multi-well trays.
[0009] This is achieved by providing a multi-well assay plate
structure which defines a relatively shallow substantially enclosed
space above a plurality of wells, with the enclosed space having an
inlet and an outlet separate from the inlet. Fluid introduced via
the inlet flows into the space, and covers the wells, by displacing
air. Withdrawal of the fluid from the space via the inlet or outlet
leaves fluid in the wells allowing various tests to be
performed.
[0010] According to a first aspect of the present invention there
is defined a multi-well assay plate structure comprising: [0011] a
first upper surface, [0012] a second lower surface having a
plurality of wells disposed therein, [0013] the first and second
surfaces defining a chamber having an inlet and an outlet, the
inlet and outlet allowing fluid to be introduced and withdrawn from
the chamber, the wells being proportioned and dimensioned to retain
a volume of fluid in each well following withdrawal of the
liquid.
[0014] Preferably, the chamber is shallow enough to allow fluid to
fill the wells and the chamber. The wells are deep enough to retain
a volume of fluid following withdrawal of fluid in the space above
the wells.
[0015] The plate structure can be of any convenient shape but,
advantageously, is sector-shaped with a detachable handle at the
longer arc-portion to facilitate locating the sector on a disc.
Conveniently, a plurality of sector-shaped structures are located
on the disc.
[0016] Conveniently, also the sectors and discs are made of plastic
and the sectors can be snap-fitted onto the disc. The sectors and
the disc include lock and key portions to allow the sectors to be
snap-fitted in the correct orientation only.
[0017] Alternatively, a disc with a plurality of separate sections
can be manufactured or molded in one piece instead of snap-in
sectors.
[0018] The composite structure may be snap-fitted onto a compact
disk.
[0019] The disk structure may have a circumferential gutter
extending around its periphery to facilitate collection of fluid
following fluid introduction/withdrawal from the chamber.
[0020] The wells are dimensioned and proportioned in terms of
diameter and depth to receive and retain fluid containing the
analyte or part of the reagent under test. The exact dimensions are
a matter of choice and depend on a number of parameters such as the
type of material of the surfaces of the chamber and wells;
viscosity of the fluid and the depth (height) of the space between
the first and second surfaces.
[0021] Advantageously, the dimensions of the structure are such
that the wells fill to retain sufficient fluid the space is flooded
and withdrawal to allow a measurable reaction to be measured within
an individual well without contribution from adjacent wells. The
overall process of sequential steps of flood and fill is
advantageous in that it allows both discrete measurements within
individual wells when filled and efficient washing of an array of
wells (flood) which is useful in multistep procedures, such as
immunoassays, which requires sequential application of reagents
interspersed with rigorous washing steps. This permits the wells to
be cleaned or rinsed in the same way as filling to allow subsequent
tests to be carried out within an individual well whilst avoiding
cross-contamination between adjacent wells.
[0022] The structure is preferably made of transparent or otherwise
optically transmissive plastic to facilitate optical reading of the
wells to determine the results of the tests. Conveniently, the
structure is integrated with automatic fluid handling apparatus and
an optical reader to allow automatic fluid handling and optical
assessment of the results of the reactions. Alternatively, fluid
handling can be manually controlled and the results of the
reactions within the structure can be assessed by an optical reader
or be scored by visual assessment.
[0023] According to a second aspect of the present invention there
is provided a multi-well assay structure comprising an upper
surface and a lower closely spaced opposed surface, said upper and
lower surfaces defining a relatively shallow space therebetween,
the lower surface having a plurality of wells therein, at least two
spaced apart openings providing access to said space from an
external location, wherein a fluid introduced into said space
through one of said openings fills substantially all of the space
and covers of the wells and said fluid, when subsequently withdrawn
through the same or the other opening, leaves the wells filled with
liquid.
[0024] The volume of fluid introduced into each well when using the
structure of the present invention is substantially defined by the
volume of the well. The accuracy and precision with which the wells
can be filled is therefore defined by the accuracy and precision
with which the wells can be fabricated and which is generally high.
Furthermore, the multiplicity of wells can be filled by way of a
single injection and withdrawal of fluid through an opening into
the space containing the wells, so that the wells can be filled
extremely rapidly.
[0025] The structure of the present invention provides for the
filling of a plurality of wells m a substantially closed chamber,
the only openings into that container being the fluid injection
opening and a second `vent` opening.
[0026] The structure of the present invention simplifies the
process of cleaning or rinsing previously filled wells as this can
be achieved by repeatedly injecting and withdrawing fluid through
one of said openings.
[0027] Conveniently, the spacing between said upper and lower
surfaces is sufficiently small to facilitate the flow of fluid m
said space by capillary or capillary like action. Typically, the
spacing is less than 1 mm and preferably less than 0.5 mm.
[0028] Preferably, said upper and lower surfaces are substantially
planer.
[0029] The wells may have any suitable geometry. For example, the
wells may be provided m said lower surface by blind circular holes
with a semi-spherical termination. Alternatively, the wells may
have substantially straight sidewalls, e.g. so that the sidewalls
extend substantially vertically and terminate m a flat base
Vertical sidewalls assist m preventing the transfer of fluid
between adjacent wells.
[0030] The surfaces may be provided by respective upper and lower
plates which are spaced apart by one or more spacer walls.
[0031] Preferably, the opening through which fluid is introduced
into said space is provided through either the upper or lower
surface and, more preferably, through the upper surface. The
additional opening may be provided through said upper or lower
surface or through a side surface.
[0032] Preferably, said opening for introducing a fluid comprises a
relatively small opening arranged to receive the end of a syringe
or similar liquid injecting device, where the opening forms a
substantially air-tight seal around said end.
[0033] Preferably, said lower surface of the container is treated
to increase the hydrophobicity to facilitate smooth flow of liquid
across the sector and hydrophilicity to aid movement of liquid into
desired locations, e.g. wells. This helps to, prevent the formation
of air pockets in the space and aids filling of the wells. The
treatment may comprise for example exposing the surface to a
wetting agent, e.g. poly-1-lysine, or exposing the surface to a gas
plasma.
[0034] In one embodiment of the present invention, the multi-well
structure is embodied in a disc. The disc effectively comprises
upper and lower circular plates, the internal surfaces of which
respectively define said upper and lower opposed surfaces.
Preferably, said opening for introducing liquid into the space is a
hole passing through the upper circular plate. Preferably, the
second opening is provided at the peripheral edge of the disc. The
space between the upper and lower plates is subdivided, by one or
more dividing walls, to provide a plurality of multi-well plates in
which case each space is provided with an opening and a vent to
enable each space to be independently filled. The dividing walls
may extend radially and/or may be concentric to one another.
[0035] Preferably, at least one of the upper and lower plates
forming the container are transparent to enable optical inspection
of the wells from outside the container. The other of the upper and
lower plates may comprise a reflecting surface so that radiation
entering into the container through the transparent plate
transverses the container in both directions, resulting in an
improved signal detection for optical inspection.
[0036] In an alterative embodiment of the present invention there
is provided a disc arranged to receive a plurality of sector (pie)
shaped inserts each of which comprises a generally planar upper
surface having a plurality of wells provided therein. For each
insert, the disc comprises a substantially planar surface arranged,
in use, to oppose said substantially planar insert surface and
means for retaining the insert in position so that the respective
planar surfaces are in closely spaced opposition to one another,
and said at least two openings.
[0037] Preferably, the opening for filling the container is
provided through the planar surface of the disc. The vent opening
is preferably provided at, or adjacent to, the peripheral edge of
the disc.
[0038] The disc preferably comprises upper and lower circular
plates separated by radially extending spacers. The spacers define
slots between the plates for receiving said inserts. Preferably,
said planar surface of each insert comprises upstanding walls
around at least a portion of its periphery for the purpose of
sealing the inner edges of the insert to the opposed planar surface
of the disc, thereby to prevent seepage of liquid around the
insert.
[0039] According to a third aspect of the present invention there
is provided a method of filling the wells of the multi-well
structure of the above first aspect of the present invention, said
method comprising the steps of: [0040] introducing a fluid into
said chamber through one of said openings to substantially flood
the chamber; and [0041] subsequently withdrawing the fluid from the
chamber through the same or the other opening to leave liquid in
the wells.
[0042] Preferably, the method further includes the step of forming
an air tight seal between the fluid inlet and an end region of a
syringe or similar liquid injecting device, and injecting fluid
through the opening into the chamber and subsequently sucking
liquid out of the space through the opening.
[0043] According to a fourth aspect of the present invention there
is provided a method of conducting a chemical or biochemical assay
said method comprising the steps of: [0044] providing a surface
within a substantially enclosed chamber having a plurality of wells
at spaced locations sufficient to allow a reaction at each well
location, [0045] treating each well with a first reagent, flooding
the enclosed chamber and covering the wells with a fluid carrying
at least a second reagent, [0046] removing excess fluid from said
chamber to leave a mixture of said first and second reagents in
each well, and [0047] optically assessing each well and determining
if a reaction occurred and correlating the reaction results to
provide an assay of the chemical or biochemical reactions under
test.
[0048] Preferably, the step of optical assessment is carried out
automatically using optical reading apparatus.
[0049] Preferably also, the surfaces with the wells having first
fluid carrying reagents are prior prepared for loading into the
structure.
[0050] Conveniently, the fluid carrying at least the second reagent
is introduced into the structure and withdrawn from the structure
using suitable automatic fluid handling apparatus.
[0051] Conveniently also, after optical assessment of the results
of the assay, the automated fluid handling apparatus is used to
inject and withdraw rinsing fluid a predetermined number of times
from the well tray to clean the wells for receiving subsequent
samples for assay.
[0052] According to a fifth aspect of the present invention, there
is provided chemical/biochemical assay apparatus comprising [0053]
an assay plate structure defined in said first aspect and having a
plurality of wells for receiving samples to be assayed, [0054]
fluid handling means for introducing and removing fluid reagents
into said assay plate structure to allow a fluid reagent mixture to
be retained in each well, and [0055] optical assessment means for
measuring optical result of the reaction in each well.
[0056] Preferably, the fluid handling means and the optical
assessment means are automated.
[0057] According to a sixth aspect of the present invention there
is provided an assay plate structure for use in conducting optical
assays of a fluid analyte, the plate structure comprising: [0058] a
disc for rotation about a central axis, the disc having upper and
lower plates and a plurality of substantially radially extending
walls disposed between the plate, wherein said walls sub-divide the
disc into a plurality of disc sectors; and [0059] a plurality of
disc inserts arranged to be received by respective disk sectors and
to be retained therein, [0060] the structure further having a
plurality of openings through the upper surface, at least one
opening above each disc sector for introducing a liquid analyte
into the sector space between the plate and the disc insert.
[0061] Preferably, the disc further comprises a lower plate, spaced
apart from said upper plate by said radially extending walls. More
preferably, the upper and lower plates are circular.
[0062] Preferably, the upper surface of each disc insert and the
opposed surface of the plate are substantially planar, and, more
preferably, are in a closely spaced arrangement.
[0063] Preferably, a vent opening is provided for each disc segment
around the periphery thereof, between the radially outer edge of
the upper plate and each disc insert.
BRIEF DESCRIPTION OF THE DRAWINGS
[0064] These and other aspects of the present invention will now be
described with reference to the accompanying drawings, in
which:
[0065] FIG. 1 is a diagrammatic representation of a multi-well
assay plate structure according to a first embodiment of the
present invention;
[0066] FIGS. 2a to 2c illustrate the steps involved in filling the
wells of the container of FIG. 1;
[0067] FIG. 2d is an enlarged detail of part of the structure of
FIGS. 2a to 2c;
[0068] FIG. 3 shows a multi-well assay plate structure according to
a second embodiment of the present invention;
[0069] FIG. 4a shows a third embodiment of a disc-style structure
for conducting multi-tests;
[0070] FIG. 4b shows an enlarged cross-sectional detail of FIG. 4a
to allow snap-fitting of the plates in the disc sectors;
[0071] FIG. 4c is a fourth embodiment of a disc-style structure for
conducting multi-tests;
[0072] FIG. 4d shows a modification of the outer disc with hinged
sectors and which is applicable to the previous embodiments;
[0073] FIG. 5 depicts chemical/biochemical assay apparatus for
conducting an assay on reactions carried out using the multi-well
assay plate structures shown in FIG. 3 or FIGS. 4a, b, c and d,
and
[0074] FIGS. 6a and 6b depict the data and graphs respectively of
antigen/antibody biochemical assays carried out using the apparatus
of FIG. 5 on the assay plate shown in FIGS. 4a, b, c and d.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0075] Reference is first made to FIG. I which shows a multi-well
assay plate, generally indicated by reference numeral 10, having a
box-like construction with a rectangular cross-section. The assay
plate 10 comprises an upper plate 12, a lower plate 14, and side
and rear spacers 16,18,20 all of which are made of a transparent
polycarbonate. The front of the box, indicated generally by the
reference numeral 22, is open to the surrounding space.
[0076] The spacers 16,18,20 are dimensioned to produce a space 21
of uniform spacing d between the opposed inner surfaces 12a, 14a of
the upper and lower plates 12,14. Spacing d is chosen such that a
selected liquid is able to flow through the space 21 between the
upper and lower plates 12,14 in a controlled manner by capillary or
capillary-like action. Generally, d is less than 0.5 mm.
[0077] A small opening 23 extends through the upper plate 12 to
communicate the inner space 21 with the exterior space surrounding
the container. Opening 23 is located close to the rear wall 20 in
order to prevent air-locks forming in the container during filling
as will be described in more detail below.
[0078] A regular array of wells or depressions 24 are formed in the
upper surface 14a of the lower plate 14. Typically, the
polycarbonate assay plate with wells 24 is produced by suitably
molding the lower plate 14 or by etching or pressing. The wells 24
are 2 mm in diameter and 1 mm deep and typically have a volume of 5
.mu.l and any suitable number of wells may be provided. The wells
are spaced 4 mm apart (center to center).
[0079] FIGS. 2a to 2c illustrate the process by which the wells 24
of the assay plate 10 are filled with a liquid analyte 25. The end
26 of a syringe 28 containing the liquid analyte 25 is pressed into
the opening 23 provided in the upper plate 12 of the container 10
(FIG. 2a) so as to form an air-tight seal between the periphery of
the syringe and the inner surface of the opening 23. The plunger 30
of the syringe 28 is then depressed to force the liquid 25 through
the opening 23 into the space 21 within the plate 10. As best seen
in FIG. 2b, due to the capillary or capillary like flow of liquid
through the space 21, the entire space 21 is filled and wells 24
are covered before liquid 25 begins to flow through the front open
face 22 of the container 10. When it is observed that all of the
space 21 is filled and the wells 24 are covered with liquid, and
preferably prior to liquid flowing out through the front face 22,
the plunger 30 of the syringe 28 is withdrawn. This action empties
the space 21 of liquid, but results in the wells 24 being filled
with liquid 25 as shown in FIG. 2c. FIG. 2d shows an enlarged
cross-sectional view through part of the assay plate structure and
showing how liquid is retained in wells 24 up to the meniscus. As
with the filling process, liquid flows from the space 21 in a
controlled manner. No puddles or drops of liquid remain in the
space 22, other than in the wells 24.
[0080] It will be appreciated that prior to introducing the liquid
analyte 25 into the space 21, for example during the manufacture of
the assay plate 10, the wells 24 of the plate 10 may be coated with
an appropriate reactant. For example, if it is desired to conduct
antigen-antibody reactions, the wells 24 are coated with an
antigen. The remainder of the surface 14a is coated with a blocking
agent to prevent antigen and antibodies from binding to surface
14a. Once the wells 24 have been filled with the liquid analyte 25,
any antibodies present in the liquid analyte 25 will bind with the
antigens contained in the wells 24. There is no binding of the
antibodies to surface 14a. If it is necessary to conduct a further
reaction in the wells 24, e.g. to bind a colored or fluorescent
label to the bound antibodies or exposed antigens, it is possible
to repeat the steps of FIGS. 2a to 2c in order to introduce the
labelled components into the wells 24. Prior to introducing the
labelled components, if it is necessary to rinse the wells 24 and
the inner surfaces 12a, 14a of the plate 10, this is again easily
achieved by repeating steps 2a to 2c with the syringe 28
containing, for example, distilled water.
[0081] There is illustrated in FIG. 3 a second embodiment of the
present invention which depicts a multi-well assay plate in the
form of a disk 32 designed for use with a rotating scanning device
having a CD player type format. One such device is described for
example in WO96/09548. The disk 32 shown in FIG. 3 comprises a pair
of upper and lower circular plates 34,36 sandwiched together to
provide a cylindrical space 38 therebetween. This space 38 is
divided into eight sectors 40 by radially extending spacers 42. A
plurality of wells 44 are provided in each sector 40 (one set of
which is shown in broken outline) by forming the upper surface 36a
of the lower circular plate 36 as described with reference to FIG.
1. The wells 44 are of the same size and are spaced as for FIG.
1.
[0082] Each sector 40 provides a chamber or space 46 which can be
filled independently via openings 48 provided through the top
surface of each sector 40. The peripheral edge 50 of each sector 40
is open to the surrounding space to provide a vent for the sector
40 to allow liquid to flow through the space or chamber 46 by
displacing air therefrom.
[0083] In order to enable the disk 32 to be compatible with
scanning devices such as are described in WO 96/09548, the upper
and/or lower plates 34,36 are made of transparent polycarbonate to
enable a light beam to be scanned across the disk surface. The disk
32 is provided with a central hole 52 to enable the disk 32 to be
mounted on a rotatable shaft.
[0084] As is described in WO 96/09548, one of the surfaces of the
upper or lower plates 34, 36 may be provided with digitally encoded
address information which can be read by the scanned light beam.
This information may be encoded by way of "pits" and "lans" pressed
or molded into one of the plates. This address information can be
used to provide accurate location information on the part of the
disk which is begin scanned by the light beam.
[0085] There is shown in FIG. 4 a third embodiment of a disk assay
plate 54 which comprises upper and lower circular transparent
polycarbonate plates 56,58 which are spaced apart by a number of
radially extending spacer walls 60 to create a plurality of disk
sectors 62. The inner surfaces 56a, 58a of the circular plates 56,
58 are both planar.
[0086] Each disk sector 62 is arranged to receive a sector plate
insert 64 which is a transparent polycarbonate plate with a
detachable handle 66 on the outer side to facilitate entry and
removal of the plate insert 64 in the sector 62. The plate insert
64 and spacer wall 60 have respective recesses/projections (not
shown in the interest of clarity) which allow the assay plate 64 to
be inserted only in the correct orientation. The plate 64 has a
groove 68, as shown in FIG. 4b for example, which allows the inset
to be snap-fitted over a projection 70 upstanding from plate 58
into the sector. The thickness of the sector insert plate 64 is
marginally less than the spacing provided between the upper and
lower plates 56,58 so that the plate insert 64 can be
pressed/fitted into one of the disk sector 62 to define a liquid
receiving chamber or space 73 between the upper surface 64a of the
insert plate 64 and the lower surface 56a of the upper disk plate
56. Openings 72 are provided through the upper plate 56 into each
disk sector 64 whilst the space 70 between the radially outermost
peripheral edge 74 of the insert plate 64 and the upper plate 56
provides a further vent or filling opening into the disk sector
62.
[0087] The surface 64a of the insert plate 64 is provided with a
plurality of wells 76 as described with respect to FIG. 1. The
wells are 2 mm in diameter, 1 mm in depth and 4 mm apart (spaced
between centers). These wells are filled by introducing liquid into
the disk sector 64 through the upper opening 72 to fill space 70
and subsequently withdrawing the liquid through the same opening as
previously described.
[0088] Reference is now made to FIG. 5 of the drawings which
depicts assay apparatus for conducting an assay on reactions
carried out using the assay plate structures of the already
described embodiments. However, for convenience, the assay
apparatus will be described in combination with the preferred
embodiment shown in FIGS. 4a, b with like numerals referring to
like parts.
[0089] In this case the plate 54 is mounted on a shaft 74 carried
by a turntable 77. The apparatus includes a suitable automatic
fluid filling/withdrawal system, generally indicated by reference
numeral 80, which operates a syringe 82 to dispense/retrieve fluid
from a reservoir 84 via the openings 72 into the space 70 between
the plate surface 56a and the surface 64a of each sector plate 64.
The fluid can of course be dispensed and retrieved manually if
desired. This is achieved for each sector by rotating the disk
plate 54 to a suitable position to allow fluid filling/withdrawal.
It will be appreciated that the plates are pre-prepared with
various reagents, e.g. antigens, and they are inserted in the
appropriate wells 76, as described with reference to FIGS. 4a, 4b.
The plates are first flooded with fluid carrying antibodies and
withdrawal of the fluid leaves the antibody/antigen reagents
filling the wells 76 resulting in a reaction.
[0090] The following example of an assay within the embodiment
shown in FIG. 4b is described to provide a better understanding of
the steps involved:
Multi-Antigen Elisa Using Sectors
[0091] 1. The underside of upper surface (56a) of is coated with
silicone spray to aid fluid movement. Sector plates 64 are also
coated including wells 76. Any excess silicone is removed.
[0092] 2. Sectors wells 76 are loaded by hand with a panel of seven
antigens--Human Serum Albumin, Antitrypsin, Macroglobulin,
Antithrombin III, Catalase, Antichymotrypsin and Plasminogen at a
concentration of 20 .mu.g/ml in PBS and a volume of 2 .mu.l/well.
Control wells contain PBS only. Antigens can be arranged in blocks
of the same on the sector plate 64 in a series giving a panel of
tests evenly distributed over the sector. Incubate at room
temperature for 15 minutes.
[0093] 3. Wash with 0.05% PBS-Tween using flood/fill technique--1
ml is flooded across the sector plate via holes 72 in the top plate
using a 1 ml pipette. This pipetted up and down three times then
withdrawn and the washing discarded. This repeated a further three
times to ensure complete washing.
[0094] 4. Blocking is carried out to prevent reactions occurring
other than at well sites with 50 mg/ml Bovine Serum Albumin (BSA)
(in PBS) using flood/fill. 1 ml of BSA/PBS is flooded across the
sector, pipetted up and down three times, withdrawn and discarded.
This allows all wells 76 to be filled simultaneously. Incubate for
15 minutes at room temperature.
[0095] 5. Wash as before.
[0096] 6. Primary antibodies are applied to the sector plate 64 as
a mixture using flood/fill with each individual antibody at the
following concentrations: anti-Human Serum Albumin 1/1000,
anti-Antitrypsin 1/2000, anti-Macroglobulin 1/2000,
anti-Antithrombin III 1/1000, anti-Catalase 1,1000,
anti-Antichymotrypsin 1/1000, anti-Plasminogen 1/1000. Antibodies
are diluted in 0.5 mg/ml BSA/PBS. Incubate for 10 minutes at room
temperature.
[0097] 7. Wash as before.
[0098] 8. Second antibody is Amdex anti-IgG (peroxidase conjugate)
at a concentration of 1/1000 in 0.5 mg/ml BSA/PBS. After washing
this is applied to the sector using flood/fill. Incubate at room
temperature for 10 minutes.
[0099] 9. Wash as before.
[0100] 10. The substrate is insoluble Tetramethylbenzidine (TMB).
This reacts with the peroxidase on the second antibody to produce
an intense blue color. After washing this is applied to the sector
plate 64 by flood/fill but is left flooded across the sector plate
64 after pipetting up and down several times. Incubate for 10
minutes at room temperature.
[0101] 11. Remove TMB and discard. Wash out the wells with
distilled water four times by flood/fill. A blue precipitate will
be evident in wells with a positive reaction. No color is produced
in negative wells. Store sections in dark as TMB will slowly fade
in daylight.
[0102] The data for the above assay is shown in FIG. 6a and is
graphically represented in FIG. 6b which is reproducible and is
representative of a large number of experiments (712).
[0103] It will be seen that there is a significant measurable
change for each antibody/antigen reaction compared with the
background level. The reaction results in an optical change, from
transparent to colored (blue) and which is measured using an
optical detector which measures light transmissivity through the
disk and wells. In this case optical assessment was carried out
using the apparatus as shown in FIG. 5 by locating the plate 64 in
a light transmissive microscope 80 (Zeiss Axiophot fitted with a
JVC video camera 83 (Model No. TK-1280E)) and sensing the change in
optical signal. The output of the video camera is connected to
Macintosh IICx 85 with video frame capture. The results can be
displayed via the Mac display 87 or a hard copy provided by printer
86. Analysis was carried out by measuring mean grayscale values in
center of wells quantified by NIH Image software. Background levels
taken from sectors which had not been exposed to immunochemicals or
chromogen were subtracted from all experimental wells. Experimental
wells contained array or seven separate antigens listed above. In
addition, experimental controls were carried out in which specific
antigen was omitted wells and wells exposed to the same regime of
blocking, antibody binding and exposure to chromogenic substrate.
The average reading from these experimental controls minus mean
reading from the sector alone was defined as the background level
of staining. Experimental readings from the seven specific antigens
providing signals of approximately five to six times greater than
this background. It will be observed that there is no
cross-contamination between wells 76 because of the efficiency of
withdrawal and because the substrate in this case is insoluble.
However, this assay would also work satisfactorily for soluble
substrates because of fluid withdrawal from the sector plate 64
leaving fluid in the wells 76 only, not on surface 64a.
[0104] In a modification, if it was unnecessary to withdraw all of
the liquid to leave a film on surface 64, the assay would still
work with an insoluble substrate in each well; cross-contamination
would still not occur. However, this arrangement would be
unsatisfactory for soluble substrates in the wells as the film
could cause dispersal to other locations and provide contamination
of other wells.
[0105] With the embodiment shown in FIGS. 4a, 4b the disk sector
plate 54 is more suitable for conducting a variety of different
assays, e.g. antigen/antibody assays for different patients, i.e.
one patient/sector.
[0106] It will be appreciated that modifications may be made to the
above described embodiments without departing from the scope of the
present invention. For example, the opening through which the
liquid analyte is introduced may be provided through the lower
plate of the multi-well container. More than one opening, can be
used for faster flooding. This opening may be arranged to receive
the tip of a syringe needle. The vent opening may also be provided
in any one of the walls of the container although it is preferably
provided in a peripheral wall. The opening 22 may be provided by a
single opening 22 or by a series of openings or vents as shown in
FIG. 4d for example. A laser may be used with CD optics instead of
the microscope and video camera for the embodiment of FIG. 4. The
top plate in the embodiment of FIGS. 3 and 4 may be snap-fitted to
the lower plate and may be snap-fitted onto a CD base plate which
would receive sections and provide the advantage of positioned
information. As shown in FIG. 4c the upper planar surface 56 can
have sector covers connected to a lower surface or central boss by
a hinge, for example integrated living hinge 90 at the inner radius
to allow each disk sector 62 to be pivotably raised and lowered and
allow sector plates 64 to be inserted into each sector. The well
size and spacing may be varied as required, for example the wells
could be 3 mm in diameter; 1.5 mm apart and spaced 5.5 mm between
center. The exact size and spacing is a matter of choice consistent
with the requirement that fluid is retained in the wells after
withdrawal as described above. However, the wells could also be
filled during flooding of the space depending on the well size,
type of plastic and fluid properties. However, liquid will still be
retained in the wells upon withdrawal of the liquid. Also, the
structure and inserts made may be of any suitable optically
transmissive plastic, such as polystyrene or perspex.TM.. The
handle 66 may be integrated with or detachable from plate 64. As
shown in FIG. 4a the radially extending ribs may have radial
shoulders 92 to define a recess 94 for receiving the plate 64 also
defining the spacing height between the surface 64a of the plate 64
and the underside 56a for receiving the liquid. Suitable materials
may be used to coat the interior of the sectors to aid fluid
movement as described with reference to silicone above. This may be
applied to the underside of the top surface and to the top surface
of the plate as for the other embodiments. Suitable materials may
be used to increase the hydrophobicity of liquid across the sector
and hydrophilicity to and movement of liquid into the desired
location, e.g. wells. The wells may be coated with a suitable
optical reflective material to enhance the reflection of light and
observation of reactions occurring within the wells and, similarly,
lenses 90 may be located in the top or bottom light transmissive
plates 12 and 14 as seen in FIG. 8, to improve optical assessment
of the reaction. These lenses may be moulded into the upper or
lower plates of the exemplary embodiments during the manufacture as
is well known in plastic molding processes. Separate optical
elements may be used instead, if appropriate.
[0107] In a modification to the embodiments described, the wells
are absent from the upper surface of the plate and that plate
retains its planar surface to enable a thin, uniform layer of
liquid to be introduced into the space between the upper disk plate
and the insert plate. An insoluble substrate with reagent or
reagents (e.g. an antigen) may be applied directly to the planar
surface of the insert plate by for example applying spots of
reagent thereto.
[0108] For certain applications, it may be appropriate to provide
each insert with a lid which can be slid into the space between the
insert and the upper plate 22 of the disk following filling of the
wells. The lower surface of the lid may be arranged to be flush
with the surface of the insert so as to close off each well. This
prevents liquid from being thrown out of the wells during spinning
of the disk during automated reading and analysis. The invention
has use in immunoassay applications including tests for sexually
transmitted diseases, parasites, allergens, cancer markers and
cardiac markers, either in laboratories or at point-of-care
locations, for example medical practitioners offices or the like.
Other applications of the invention are in chemical and biochemical
assays. Examples of such assays include immunoassay, clinical
biochemistry tests, nucleic acid analysis and receptor ligand
interactions. Examples of clinical biochemistry uses would be in
measurement of serum analytes such as glucose, urea, creatinine and
enzymes such as alkaline phosphatase. Immunoassay application
include tests designed to detect infectious organisms, viruses,
parasites as well as endogenous analytes such as circulating
hormone levels and cancer markers. Examples of chemical analysis
include measure of phosphate and nitrate levels in water,
environmental and industrial monitoring including potable and waste
water and process monitoring. The system could be used in a variety
of settings including clinical laboratories, doctor's and
veterinary surgeries as well as industrial and research
laboratories.
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