CA2084342A1 - Self-contained assay assembly and apparatus - Google Patents
Self-contained assay assembly and apparatusInfo
- Publication number
- CA2084342A1 CA2084342A1 CA002084342A CA2084342A CA2084342A1 CA 2084342 A1 CA2084342 A1 CA 2084342A1 CA 002084342 A CA002084342 A CA 002084342A CA 2084342 A CA2084342 A CA 2084342A CA 2084342 A1 CA2084342 A1 CA 2084342A1
- Authority
- CA
- Canada
- Prior art keywords
- reaction
- plate
- well
- assembly
- reagent
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
- C12Q1/682—Signal amplification
Abstract
A self-contained assembly (22) for assaying an analyte in a liquid sample. The pair of disc-like rotatable plates (24, 28) forming the assembly are relatively rotatable to align reagent reservoirs (92, 94, 96, 98) in one plate (28) with a reaction well (66) in the other plate (24), for sequential addition of multiple reagents, either in liquid or solid form, to the reaction well (66). In one embodiment, the reaction well (66) includes solid-phase particles which can be transferred from the reaction well (66) to spaced wells (74a, 74b, 74c) in the assembly by a combination of relative movement of the plate (28), and rotation of the entire assembly (22).
Description
1/19~o~ r~ a~/v _u 2~34~
SELF-CONTAINED ASSAY ASSEMBLY AND APPARATUS
1. Field of the Invention The present invention rel~tes to an asse~bly for assaying an analyte in a liquid sample, and in parti~ular, to a self-oontained assembly which provideg prepacked reagents which can be added to the sample in a de~ined seguence.
.
~. BackqroU~d_of the Invention Many analyte assays involve a sequen~ of reaction steps in which an analyte is reacted sequentially with two or more reagents. Typically in this type o~ assay, a re-agent added and the mixture is allowed to react for a preselected tim~ befor~ addition of the next reagent.
Where the assay involves analyt~ binding to a solid support, the re~ction chamber containing~analyte bound to the support may b~ washed between reagent-a~di~i~n~steps.
Ideally,:it is d~sired to carry out multiple-~ddition assays of this:type in a simple self-contained de~ice w~ich : ca~ be ~dap4ecl for automated or semi-automated operation in : a ciinical setti~, or alte~natively, ~ay be reliably :
:
.
, -, : .
2 20~3~2 practiced in a home or doctor's office setting by simple assay procedures.
Heretofore, a variety of self-contained diagnos~ic devices which provide multiple, prepackaged reagents have 5 been proposed. U.S. Patent No. 4,806,316 d~scribes a disposable diagnostic device in which a liquid sample is distri~uted to multiple reaction wells which may contain prepackaged reagents for reacti~n with the analyte. The device may be adapted ~or sequential-reaction assays by transferrin~ each sample succe5sively through two or more wells, where the different wells contain the desir~d prepackaged reagents.
U.S. Patent No. 4,519,981 shows a rotary diagnostic device containing a series of side-by side, radially overlapping reaction chambers, each of which may contain a prepackag2d reagent. The cells are interconnected to allow sample ~luid flow from through each chamber to:a su~cessive chamber by centrifugal motion alternately in opposite directions. T~at is, fluid sample is moved from a first chamber to a second, side-adjacent chamber ~y rotating th~
device in one direction, and after a selected reaction time, the sample fluid is moved ~rom the second-chamber to a side-adjacent third chamber by rotary motion in the opposite direction.
Another multi-reagent rotary device is disclosed in U.S. Patent No. 4,390,499. The device includes a s~ries of compartments separated by rupturable seals. In operation, pneumatic and centrifugal ~orces ar~ used to break the seals betwe~n compartments, for sequential ~ixing of the reagent material with the #ample .
U.S. Patont No. 4,469,793 discloces a rotary diagnos-tic device in which a sample fluid is deposited in a radially inner well, and distribut8d in a predetermined volume to eac~ of a plurality of outer receptor cells under c2ntrifugal iorces applied by~spinning the device in one -3 20~3~2 direction to ~orce the sample ~irst into overflow wells, then in th2 opposite direction to dispPnse the predeter-mined volumes of sample into the outer cells. The compart-ments o~ the cells may be supplied with di~ferent liquids for use in simultaneous analyse~3. Also disclosed in this patent is a solid reactor bead which is designed to pick up succes5ively, a quantity of the analyte being ~easured, then a ~uantity of a reagent containing a biological indicator.
10 The aboveodescribed assay devices all involve rela-tively complicated pressure and/or centrifugal force mechanism for releasing prepacka~ed reagents into a sample or for transferring the sample from one reagent chamb r to another. For this reason, the devices may be relatively expensive in manufacture, and als~ may be unreliable in operation, due to variations in centripetal or pneumatic force applie~ to the device during operation, or variations in the force needed to rupture seals or advance the sa~ple between chambers, and/or variable volume losses which occur as a liquid is transferred from cha~ber to chamber.
3. Summarv of the Invention The inV~ntion includes, in one aspect, a self-con tained assembly for detecting an analyte ligand by detect-able, analyte specific binding to a solid support. The as-sembly includes a reaotion plate having a well ~ontaining the solid support, and a transfer plate containing first and second reagents contained in separate first and second reservoirs- The transfer platP is mounted on the reaction plate for movement thereon to a sample-addition position, at which sample can be added to the well, one reagent-transfer position at which one reservoir is ali~n~d wi~h the well, a wash position at which wa~h solution can be introdu~ed into the well, and another reagent-tran~fer position at which the second reservoir is aligned with the ~, . .
.: . ' . " " . . ' . . . ' . , ~ , .' .: :. .: '- '., ' " .: .. '.. ... :.. . . . .. .. , . '. '.. . . .: .
SELF-CONTAINED ASSAY ASSEMBLY AND APPARATUS
1. Field of the Invention The present invention rel~tes to an asse~bly for assaying an analyte in a liquid sample, and in parti~ular, to a self-oontained assembly which provideg prepacked reagents which can be added to the sample in a de~ined seguence.
.
~. BackqroU~d_of the Invention Many analyte assays involve a sequen~ of reaction steps in which an analyte is reacted sequentially with two or more reagents. Typically in this type o~ assay, a re-agent added and the mixture is allowed to react for a preselected tim~ befor~ addition of the next reagent.
Where the assay involves analyt~ binding to a solid support, the re~ction chamber containing~analyte bound to the support may b~ washed between reagent-a~di~i~n~steps.
Ideally,:it is d~sired to carry out multiple-~ddition assays of this:type in a simple self-contained de~ice w~ich : ca~ be ~dap4ecl for automated or semi-automated operation in : a ciinical setti~, or alte~natively, ~ay be reliably :
:
.
, -, : .
2 20~3~2 practiced in a home or doctor's office setting by simple assay procedures.
Heretofore, a variety of self-contained diagnos~ic devices which provide multiple, prepackaged reagents have 5 been proposed. U.S. Patent No. 4,806,316 d~scribes a disposable diagnostic device in which a liquid sample is distri~uted to multiple reaction wells which may contain prepackaged reagents for reacti~n with the analyte. The device may be adapted ~or sequential-reaction assays by transferrin~ each sample succe5sively through two or more wells, where the different wells contain the desir~d prepackaged reagents.
U.S. Patent No. 4,519,981 shows a rotary diagnostic device containing a series of side-by side, radially overlapping reaction chambers, each of which may contain a prepackag2d reagent. The cells are interconnected to allow sample ~luid flow from through each chamber to:a su~cessive chamber by centrifugal motion alternately in opposite directions. T~at is, fluid sample is moved from a first chamber to a second, side-adjacent chamber ~y rotating th~
device in one direction, and after a selected reaction time, the sample fluid is moved ~rom the second-chamber to a side-adjacent third chamber by rotary motion in the opposite direction.
Another multi-reagent rotary device is disclosed in U.S. Patent No. 4,390,499. The device includes a s~ries of compartments separated by rupturable seals. In operation, pneumatic and centrifugal ~orces ar~ used to break the seals betwe~n compartments, for sequential ~ixing of the reagent material with the #ample .
U.S. Patont No. 4,469,793 discloces a rotary diagnos-tic device in which a sample fluid is deposited in a radially inner well, and distribut8d in a predetermined volume to eac~ of a plurality of outer receptor cells under c2ntrifugal iorces applied by~spinning the device in one -3 20~3~2 direction to ~orce the sample ~irst into overflow wells, then in th2 opposite direction to dispPnse the predeter-mined volumes of sample into the outer cells. The compart-ments o~ the cells may be supplied with di~ferent liquids for use in simultaneous analyse~3. Also disclosed in this patent is a solid reactor bead which is designed to pick up succes5ively, a quantity of the analyte being ~easured, then a ~uantity of a reagent containing a biological indicator.
10 The aboveodescribed assay devices all involve rela-tively complicated pressure and/or centrifugal force mechanism for releasing prepacka~ed reagents into a sample or for transferring the sample from one reagent chamb r to another. For this reason, the devices may be relatively expensive in manufacture, and als~ may be unreliable in operation, due to variations in centripetal or pneumatic force applie~ to the device during operation, or variations in the force needed to rupture seals or advance the sa~ple between chambers, and/or variable volume losses which occur as a liquid is transferred from cha~ber to chamber.
3. Summarv of the Invention The inV~ntion includes, in one aspect, a self-con tained assembly for detecting an analyte ligand by detect-able, analyte specific binding to a solid support. The as-sembly includes a reaotion plate having a well ~ontaining the solid support, and a transfer plate containing first and second reagents contained in separate first and second reservoirs- The transfer platP is mounted on the reaction plate for movement thereon to a sample-addition position, at which sample can be added to the well, one reagent-transfer position at which one reservoir is ali~n~d wi~h the well, a wash position at which wa~h solution can be introdu~ed into the well, and another reagent-tran~fer position at which the second reservoir is aligned with the ~, . .
.: . ' . " " . . ' . . . ' . , ~ , .' .: :. .: '- '., ' " .: .. '.. ... :.. . . . .. .. , . '. '.. . . .: .
4 2~342 well. The transfer and reaction plates are designed t~
prevent release of reagents ~rom their corresponding reservoirs until the associatecl reservoir is aligned with the well.
In one embodiment, the reagent is in the form of a liquid solution, the reservoir containing the solution includes a channel formed in the plate, and the channel is sPaled at its opPning in the transfer plate. Preferably the channel and seal are formed by an elastomeric sleeve held in a channel within the transfer plate, and the confronting planes of the transfer and reaction plates are spaced, adjacent the region of the sleeve, to ~orm a capillary lock, to prevent liquid ~rom leaking from the reservoir by capillarity. Also in a preferred embodi~ent, the transfer plate includes a chamfered opsning communicat-ing a reservoir in the transfer plate with the well in the rei~ction plate.
In another general embodiment, at least one of the rei~gents i~cludes a pelletized form of the reayent, and the pelletized reagent is deliver~d ~rom the associated reagent reservoir to the well by gravity, when the reagent reser-voir is aligned with the reaction well.
~T~e reaction well in the reaction plate may be formed by an elongate channel containing a solid-phase assay support. In this embodiment, the transfer plate may include ports which are alignable wit~ spaced area~ in the channel, when the transfer plate is moved to a wash position, ~ormi~g an enclosed passageway for pa5sing a solution through the channel.
In one~ me$hod, the assembly is used for detecting a nucleic acid with a known tar~et sequence. Here the solid-phase support in the reaction is coated with immobilized nucleir acid fragments, and the assembly includes r~agent reservoirS for sequential addition to the reaction channel of ~1) a probe e~fecti~e to hybridize with both th~ target .
.
2 ~ 3 ~ 2 sequence and the immobilized fragments, and (2) a reporter molecule ~ffective to bind, dir~ctly or indirectly, to the probe.
In another method, the assemhly is used in an immuno-assay for detection of a ligand effective to bind immun~-speci~ically to the solid support. Here the ~ssembly includes reagent reservoirs for sequential addition to the reaction channel of tl) an ant:iligand reagent capable to binding immunospecifically with the ligand, when the ligand is bound t~ the support, and (2) a reagent capable reacting with th~ antiligand reagent to produce a detsct-able sighal on the solid support.
In another aspect, the invention includes apparatus for detecking an analyte ligand by detectable, ligand-spe~ific binding to a solid support~ The apparatusincludes a self-contained assemb~y of the type des~ribed above~ and a device ~or holding the reaction plate, and rotating ~aid transfer plate to its various positions.
In one embodiment, the device is effective to rotate the assembly as a unit, the reaction well includes a radially extending channel, the solid support surface includes at laast two solid-phase particles carried in the channel for movement therein ~rom inner toward outer radial positions, and the transfer plate includes a receiving slot into which the particles can be received, when the p~rti-cles in the channel are subjected to an outward force, and the channel is aligned with said slot. The reaction pIate includes particle-receiving wells into which particl~s in the slot can be deposited, wh~n the r~agent plat~ co~tain-ing deposited particles is rotated with respect to theraaction plate.
In an ~mbodiment o~ the device designed ~or heating the liquid con~ents of the well, the assembly further includes structure for sealing the well whsn heatang is applied. The assembly includes a cover plate attached to , .
:.
' .
J ~ ~ v`~--u ~
2~3'~2 6 v the reaction plate, and the sealing structure includes (a) a sealing pad carried in the tra:nsfer plate for floating in a direction normal to the plane o~ the plate, and (b) a cam surface in the cover plate for biasing the pad against the S well platform, when the tran~fer plate is moved to a heating position.
Also in an embodiment of the device, the solid support includes a bead in the well, ,and the device includes a detector having a light sensor, and a tube extending from the light senso~ and positionable to encompass a spherical ~urface portion of the bead.
These and objects and features of the invention will become more fully apparent when the ~ollowinq detailed description of the invention i5 read in conjunction with the accompanying drawings.
Brief Descri~tion of the Drawin~s Figure l is an exploded perspective view of the components in an assay apparatus constructed according to the present invention; -Figure 2 is an enlarged, partially cutaway perspectiveview of an assay assembly constructed according to the invention;
Fi~ure 3 is a plan view of an cover plate in the Figure 2 assembly;
Figure 4 is a plan view of a transfer plate in the - Fi~ure 2 assembly;
Figure 5 is a plan view of a reaction plate in the Figure 2 assembly;
Figures 6A and 6B are fragmentary seGtional views taken at relati~e plate position in the Figure 2 assembly at which a reagent re5exvoir in the transf er d is~ in th~
assembly ~s isolated from a reaction channel in th~
reaction pla~a (6A), and the reservoir is in a:transfer position with resp~ct to the channel (68);
7 2~ ~3 ~2 Figures 7A and 7B are enlarged, fragmentary sectional views of res~rvoir regions seen along lines A-A in Figure 6A and along line B-B in Figure 6B, respectively;
Figure ~ is an enlarged, fragmentary sectional view of sealed well in the assembly, during a heating step in the operation of the assembly;
Figures 9A and 9B are s~ctional views similar to 6A
and 6B, respectively, but illustrating an embodiment of an assembly i~ whi~h the reagent in the reservoir i~ in dried particle form;
Figure 10 is an enlarged, fragmentary sectional view of the assembly showing the assembly in a positio~ for irrigating the assembly well;
Figure 11 is an enlarged sectional view of a well region of the assembly, showing detector struckure ~ox detecting an optical signa~ from a bead in the well;
Figures 12A-12C illustrate the steps in trans~erring solid-phase particles from the reaction channel in the reaction plat~ into a slot ~ormed in the transfer plate ~12A, 12B), and fro~ this slot into separate wells in the reaction plate ~12C~;
Figure 13 is a flow diagram of the steps carried out by the apparatus in executing an exemplary analyte assay;
and Figures 14A-14D are schematic illustrations of the sequential nu~leic a~id binding reactions in a DNA analyte assay which can be carried out in an automated ~ashion by the apparatus of the invention.
Detailed Des~rlption of the Invention A. i~Ll~LJ~ D ~lY~ e~e9-Figures 1 illustrates, in exploded view, an assay apparatu5 20 constructed according to the present inven-tion. The app ratus includas an assembly 22 whi~h also ~orms part of th3 invention and which will be detailed .
, . ~ . , ,~ . , ' :
20~3~
below with r~ference to Figures :2-12. Briefly, the assem-bly is formed of reaction and cover plat~s 24, 26, respec-tively which are joined together at their outer ed~es, a~d an intermediate transfer plate 2B which can be rotated relative to the two outer plates about a oentral axis 30.
To ~his end, the transfer plate includes a hub 32 having an elongate opening 34 (Figure 2) which is engageable for rotating the transfer plate, with the reaction and cover plates supported in a stationary position.
A control device 36 in the apparatus includes a base 38 whicih defines an annular sea~ 40 in which the a5sembly is supported d~lring an assay operation. The base provides five pins, such as pins 42, arranged asymmetrically about the seat, as shown. These pins engage corresponding holes in the bottom of the assembly, described below, to immobi-lize the cover and re~ction plates against rotation in the seat.
The bas~ is supported on and attached to a drive motor which is indicated by a drive shaft 44 in the figure, the motor itself being contained with a motor housing 45. A
three-arm support 46 attached to the end of the drive shaft has a projection 48 designed to engage the opening in hub 32, with the reaction surface of the assembly resting on the support arms, as can be appreciated in the ~igure.
Thus, when the assembly is received and immobilized in seat 40, with projection 48 received.in opening 34 in the hub, the intermediate transfer plate in the assembly can b~
rotated to 5elected positions with respect to the cover and reaction plates by the drive motor.
The ~otor in the control device can be moved ~rom a lowered transfer position in whioh the assembly i5 i~mob~-lized in seat 40, as just described, to a raised free-rotation position in which the assembly is disengaged ~rom the seat and is ~ree to rotate on~drive ~otor 44. Because o~ a rel tlv-ly high coeffi=ient of internal (transfer .
20~42 plate) movement, the assembly can be rotated as a unit by the drive motor, such that the assembly and motor in a free-rotation position can function as a centrifuge to force material radially outwardly in the assembly, for a purpose to be described. The assembly can al~o be agitat ed, for mixing reaction components, by oscillating the as-sembly on the motor in a free-rotation positiDn.
A wash unit 50 in the device is designed to circulate wash solution(s) through the assembly, as will be described below with respect to Figure 10. Briefly, when the assem-bly is placed in a wash condition, openings 52a, 52b formed in cover plate of the assembly form the ends of a closed passageway whioh includes a portion of the reaction well in the assembly- ~nit 50, which is mounted on base 38 as indicated, in~ludes a vertically positionable arm 54 which holds a pair of tubes 56a, 56b, for insertion into openings 52a, 52b, respectively, when the arm is moved to a lowered, wash positinn. In a wash operation, wash solution is supplied through tube 56a under pressure, and is removed through tub~ 56b.
Also shown in Fi~ure 1 is a heating unit 62 in the apparatus used for heating a reaction well in the region in the assembly to selected reaction temperatures. The unit is attached to the lower side of base 38, as indicated, to position a heating eleme~t 64 in the unit dire~tly below the reaction well in the assemb~y, with the assembly seated on the base.
Although not shown in Figure 1, device 3Ç ~ay also include an optical sensing unit for detecting analyte in 30 the assembly- One preferred unit will be described below :~
with respect to ~igure 11.
The as5ay assembly of the invention is illustrated particularlY with respect to Figures 2-12. Reaction plats 24 in the assembly (Fiqures 2 and 5) provide~ an elongate, radially extonding channel or well 66 which ~erves a~ the :
: ~ ' , .
2~3~2 reaction chamber in the assembly, i.e., the chamber where the analyte and various reagents required for analyte binding and detection are brought into liquid contact with a solid-phase support containE!d in the well. In the S embodiment shown, the well is formed in a metal, e.g., aluminum, insert 166 which is attached to the lower side of a disk-like plate member 168. The insert is carried below a platform 170 defined by the upper surface of plate member 168, above the insert. Formed in the platform are radially spaced openings 1~2a and 172b adjacent opposite end reyions o~ the channel, and a central opening 1~2c. Openings 172a and 172b are align~d with openings 52a and 52b in plate 26, for a purpose to be described.
Details o~ the well construction in the plate are seen in Figure 7B which shows an enlarged cross-section of t~e well region in plate 24 and o~erlying regions of plates 26, 28. The channel or well formed i~ the insert has a sub-stantially U-shaped cross-section extending between oppo-site e~ds, corresponding to the regions below openin~s 172a and 172b in Figure 5, with the outer radial end region of the channel forming a tapered ramp 70, as seen in Figure 10. The insert is raceived in a cavity 174 for~ed in the lower region of plate member 168, and attached to the plate member by flanges, such as seen a~ 176. Eaoh opening in platform 170, such as opening 172a shown in Figure 7~, communicates with the well, and includes a short ~ylindri c l-bore section 178 and a chamfered section 180. This construction serves to draw liquid into and through the opening from a reser~oir in plate 28 into the channel in the transfer plate, as will be described below.
Carried in the channel are one or more solid-phase beads or partlcles, su~h as particlas 68a, 68b, 68c, which form solid phase supports in the assay reaction. In ~he e~bodim nt des~ribed in Seotion C below for solid-phase DNA
determination, the hree pareicl-s ar- ~or ~1) positiYe `
~ U Y l/ IY~ " ~, ,, ~
11 2~3 ~2 control, ~2) negative control, and (3) analyte binding.
The channel and particles are s;hown in cross section in Figures 10 and 12A. The invention also contemplates a rea~tion format containing only a ~ingle solid-phase support.
Located just beyond the outer end o~ the channel in plate 24 is an arcuate groove 72 which serves as a guide for the particles, also for a purpose to be described.
Adjacent this groove are three wells 74a, 74b, 74c into which the three paxticles may be transferred from channel 66 after the chemical reaction, also as will be described below. The three wells may contain a detection solution for detecting the presence of reporter molecules bound to the solid particles.
The reaction plate is secured to the cover plate by fasteners, such a~ rivets 75 ~Figure 2), connecting the outer annular rim regions of the two plates. The fasteners are received throuqh holes, such as holes 76, formed in th~
plate . Also f ormed in the reaction plate is a central opening 82 through which projection 48 i~ re~eived when the assembly is seated on the control device. ~ -Cover plate 26 in the assembly is illustrated in plan view in Figure 3. The plate is a circular disc havinS~ the-same diametç~r as the reaction plate. Formed in an outer 25 annular rim region 83 of the plake are holes, such as holes 84, for fastening an upper and reaction plates (dotted line 85 in Figure 3 indicates the outer edge of transfer plate 28 in the assembly). Also formed in the cover plate are: :
(i) a central opening 86 through which the cover portion o~ .-30 hub 32 is received, ~s seen in Figure 2; (ii) a series of windows B8a, 88b, 88c which are aligned with wells 74a, 74b, 74c, respectively, in the reaction plate; (ili~ above described openings 52a, 52b, which are aligned with openings 172a, 172b, respectively, for cir~ulating wash solut-on through the channel; and (iv) an opening 52~
: .
. '! .' , ' . ' ~ , , ' . ' ' ' ' ' ,. ' , ' . , , ~ ' ~ . . .
20~43~2 between ports 52a and 52b, in alignment with port 172c.
With reference now to Figures 2 and 4, transfer plate 28 include~ a plat~ member 190 having a central opening 90 in which hub 32 is fixed for rotation with the plat~. The S plat~ includes four reagent relservoirs 92, ~4, 96, and 98 which tak~ the form of cyli;n~rical channels extending through the plate. With refere~ce particularly to Figure 7A, reservoir 92, which is rapresentative, is fo~med of a sleeve 100 which extendc through plate member 184~ extend-ing beyond the plane of the plate member on both upper and lower sides of the plate member. In the completed asse~-bly, the sleeves forming the several reservoirs are axi~lly compressed, forming liquid-tight seals against confronting faces of the cover plate and reaction plate, as illustrated in Figure 7~- Th~ sleeves are preferably formed of Teflon or polyethylene- The sealing ends of the sleeves are also referred to herein as means f or preventing release o~
liquid reagent in a reservoir u~til the reservoir is aligned with one of the openings in the trans~er plate communicating w~th channel 66.
The spacing between the plate member f orming pla~e 2 8 and the confronting 5urfaces of the cover and reaction plates in the assembly function to prevent liquid contained in each reservoir from being drawn by capillarity into th~
gap between the two plates. That is, the gap between con~ronting pla~e surfaces, such as gap 186, is too great to produce capillary flow between the two surfacesO The gap in the ~mbodiment shown is provided by the r~latively greater lengths of the sleeves forming the reservoirs compared with the thickness of plate member 184. Alterna-:
tlYely, the region of the transfer plate member im~ediately adjacent each sleeve may be recessed to for~ a capillary-block gap around each sleeve.
As just indicated, ~ach of the reservoir~ in plate 28 35 i6 alignable with one of the openings 172a-172c in the . :
`
13 2 0~3~l2 reaction plate communicating with channel 66, for deliver-ing fluid reagent5 to the reaction chamber, to be described below with reference to Fi~ures 6A, 6B and 7B. In particu-lar, it is noted that some o~ the reservoirs, such as reservoirs 94~ 96, and 98 are radially spaced, preventing cross-contaminatiOn o~ liquid reagents in the two raser-voirs, as the reservoirs are moved relative to the cover and reaction plates.
Also formed in trans~er plate is an openings 102a-102c 10 alignable with opening 172a-172c, respectively, in the reaction plate and at the same time with openings S2a-52c in the cover plate. Any of th three sets o~ alignabl~
openings are referred to herein collectively as means for introduci~g sample into the assembly's reaction region.
lS Other openings formed in the transfer plate include three windows 104a, 104b, 104c which are alig~able with windows 88a, 88b, 88c, respectively, for viewin~ wells 74a~
74b, 74c, respectively, when the two sets of windows are aligned. In addition, the tra~5fer plate provides three sets of radially spaced openings, such as openin~s 106a, 106b, which are alignable, at one o~ three difXerent plate positions, with openings 172a, 172b, respectively, in the reaction plate, at the same time, with ports 52a, 52b in . -the cover plate, for forming a continuous passageway 108 lFigure 10) for circulating wash solution through the reaction region.
The transfer plate also includes a plurality of floating sealing pads, such as pads 192, 194, which are designed for sealing the channel openings in the reaction plate, when the liquid contenk5 of the xeaction plate are heated. Details of the operation of the pads i~ ~ heati~g operation are seen in Figure 8. Shown here are an enlarged sectional view t ken through the assembly in the r~io~ of the reaction channel, and a portion ~f heater ele~ent 64 use~ in heating the liquid contents in the ~hannel~ As 14 20~s3~?~
seen, the heating element makes contact with the lower side of insert 166 during heating.
Pad 192, which is r~presentative, is an elo~gate elastomeric pad, such as formed from polyethylene or other compressible, low-friction polymer material, which is dimensioned to cover openings 1.72a-172c, when the pad is positioned on platform 170, as i.ndicated ïn Figure 8. The pad is carried in a radially extending slot 194 formed in the transfer plate member, ~or floating in a direction normal to the plane of the plate. The pad's h~lyht dimension allows the pad to ~e ~oved easily, a~d without pad compressio~, betw2en the cover and rea~tion plates, as the transfer plate is rotated relative to the other two plates. However, as the pad is moved toward a sealing position above th~ reaction well, it makes contact with a radially extending cam member 195 formed on khe lower side of the cover plate, immediately above openings 172a-172c.
As can be appreciated from Figure 8, this conta~t acts to bias, i.e., compress, the pad against the plat~orm open-ings, sealing the openings to the reaction well.
~ he transfer plate also provides an L-shap~ slot 112 ~ormed in its lower side, i.e., the side confronting the reaction plate. The slot include~--radially and circum-~erentially extending portions 112a, 112b which are alignable with the outer e~d r gion of channel 66 and groove 72 in the reaction plate, respecti~ely. The slot is positioned and dimension~d to receive the parti~les from channel 66 (Figure 12A) as will be described in Seotion B.
The plates in the assembly are preferably formed by injection molding of a suitable plastic. For formi~g the trans~er platle, plate member 184 can be inje~t~d molded ~bout the four sleeves forming the four reservoirs in the transfer plate, and hub 32, and subsequently ~itted with the four sealing:pads. F9r forming the reactio~ pl~te, plate member :L68 can be injected molded about insert 166.
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With the transfer plate placed between the cover and reaction plates, the two outer plate5 are secured together, such as by rivets, glue or heat welding. As noted above, constru~tion and plate attachment is such as to bias the cover and reaction plates firmly against con~ronting sides ~f the transfer plate, to compress the elastomeric seals in the assembly. Liquid reagent can be added to the reser-voirs and wells by suitable positioning of the transfer plate a~ter assembly.
In another general embodiment of the assembly, the reagent reservoirs are designed for delivery of a dehydrat-ed reagent particle composed of the dried reagent, pre~era-bly formulated in pelletized form. This embodiment is illustra~ed in Figures 9A and 9B which shows a fragmentary portion of an assay assembly 120, with oover plate $22, .-.
transfer plate 124, and reaction plate 126. The cover and reaction plates in the assembly are substantially identical to those in assembly 22. The transfer plate includes a plurality of reservoirs, such as res~rvoir 128, which co~municate with the reaction plate, but not the cover plate.
Each reservoir contains a dried reagent particle, such as particle 130 in reservoir~I28, which is held within the reservoir, ~or deposit into reaction channel 132 in the reaction plate, when the reservoir is aligned with the transfer plate, as seen in Figure 9B. The lower end of the reservoir is sealed by the slaeves forming the re~gent reservoirs, to prevent exposure of the r~agent to moisture or contamination by other reagents.
The pelletized reagent may be formulated, if dasired, with a variety of water-soluble bulking agents, such as water-soluble polymers, ac~ording to well-known methods.
Where the assembly includes one or more solid-phase particles, th~ese are added to the re~ction channel in the ~ :-reaction~plate before the txansfer and reactio~ plates ~re 16 2~ 4 joined.
The asse~blies described ind illustrated above are designed particularly ~or use in a solid-phase DNA analyt~
assay involving (i) at lea5t one solid-phase particle, (ii~
~our separate li~uid and/or solid reagents which are added sequentially, (iii) a heating step following eaoh reag~nt addition, and (iv) three individlaal wash steps which follow the addition of the second, third, and ~ourth reagents, as will be dDscri~ed below.
A variety of alternative assembly configurations are contemplated in the present invention. For example, the assay ~ay involve sequential addition o~ two reagents, requiring only two reservoirs, cr two or more of the re-agents may be added to the reaction region simultaneously, in a configuration in which th2 reservoirs are carri~d along a common radial line.
~ urther, the solid-phase particles in the reaction may be replaced by one or more separate solid-phase supports formed on the channel wall, and/or the solid ph~se parti-cles may be carried within separated wells in the reac~ionchamber, obYiatin~ the need for particle transfer after the assay reaction. ~lternatively, t~e assay r~action may occur in a liquid phase, either in a free solution or in a absorbent filter, eliminating wash steps. In t~e case of an absorbent-filter type reaction region, a liquid reagent may be drawn onto the filter by wicking or capillarity, eliminating the need for venting liquid-carrying reser-voirs.
B. oPe~ation of the Assembly and A~aratus In operation, the assembly is placed in the seat o~
the control device, with the pins o~ the devic~ being received in the corresponding holes in the as~embly reaction plate, and with proje~tion 48 being rec~ived in hub 32, as desc~ib-d above. Hub 32 is initially or~ented .
~ U Y l / l ~ J 7 ~ ~ ~ u 17 ~ O~3'~
with respect to the pins such that the trans~er plate is in a home position at which all of the reservoirs and the reaction region are sealed.
The following exemplary operation will be described 5 with referenCe to Fiyure 13, wh:ich is a flow diagram of the assay steps in the DNA-analyte assay described in section C below. The stQps shown in solid-line boxes are prefera-bly executed in an automated fashion by a suitable micro-processor control unit ~not shown) in the control device.
Th~ sample and/or wash-solution addition steps shown in the dashed-line boxes may be performed either manually or under the control of the apparatus.
The control device is first actuated to move the transfer plate in a clockwise direction in Figure 2, to align opening 102a in the transfer plate with opening 52a in the cover plate and opening 172~ in the react~on plate, and a liquid sample is introduced through the aligned opening into the reaction channel. The analyte sampIe may b~ any fluid sample, such as a blood, serum, or plasma sample, in a suitable amount, typicaily between about 10-200 ~1.
The transfer plat~ is rotated ~urther in the same direction to align reservoir 92 with opening 172a in the reaction plate, as shown in Figures 6B and 7B, to transfer the liquid reagent in:the reservoir into the channel. As the reservoir seal first overIaps the edge of opening 172a, the fluid seal in the reservoir is broken, allowing fluid to ~low readlly out of the reservoir. As discussed above, the capillary lock feature of the transfer plate prevents the fluid from ~lowing by capillarity out of the reservoir into th~ region b~tween transfe~ and reaction plates. :The chamfered opening 172a serves to direct fluid :from the reservoir into the well by a co~bination of capillarity, ~provided by narrower upper section of t~e opening, and ':
, 18 2~ 3~
rapid fluid flow provided by the lower cha~fered'section of the opening.
As ~entioned above, in an alternative embodiment o~
the invention, the reservoir may contain a dehydrated, pelletized reagent which i5 deposited by gra~ity into the reaction channel, as indicated in Figure 9B. To mix thQ
reagent with the li~uid sample, the transfer plate may be rotated back to a channel sealing position, the assembly lifted from the seat in the control device, by raisin~ the drive motor to a free-rotation position, and the as~embly is oscillated slowly as a unit by the motor.
A~ter introducing the liquid (or solid) raa5ent into the reaction well, the transfer plate i5 rokated slightly to position pad 192 over the reaction well. As described with respect to Figure 8, the pad at this position is compressed between the cover and reaction plates, forming a tight seal about the reaction well. The heating element in the device is th~n activated to heat the well for a selected reaction period. During the heating cycle, heated fluid from the reaction mixture is prevented from escaping from ~he well by the seal over openings 172a-172c. :
The transfer plate is now rotated to transf~r the content5 o~ re5ervoir 94 into the channel, and the reaction mixture is again ~ixed and reacted for a given reaction period at a selected temperatur~ As can be appreciat~d wlth referenCe to Figure 4, continued movement of the transfer plate in a cloc~wise direction aligns the first set of wash-solution ports, indicated at 106a, 106b with openings 52a, 52b, respectively, in the cover plate, and openings 172a, 172b, respectively in the reaction plate.
When this alignment is achieved, the wash unit is lower~d to insert the wash-solution tubes into the aligned port , and wash solution is oirculatPd through the reaction channel as illus~rated in Figure 10, to wash the initial ample and first two reagents from the reaction ~hamber.
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The above steps are repeated to (i) transfer, mix andincubate a third reagent with the washed solid-phase particles in the reaction chamber, (ii) remove the third reagent by washing, (iii) trans~er, mix and incubate a fourth reagent with the washad particles, and (iv) remove the fourth reagent by wa~hing. These steps are shown in Figure 13, where the three separate wash steps are indicat-ed at (1), (2), and (3).
After completing the rea~tion, an analyte-dependent signal on the solid-phase surface, i.e., the solid-phase beads, may be read with the beads in the reaction well. In order to minimize optical-reading artifacts ~rom the other beads in the reaction well, the signal detector in the device (or in a s~parate optical reading devi~e~ preferably - 15 has a construction like that shown for detector 196 in Figure 11, allowing direct reading of each bead. The detector includes an optical sensor 198 and a refractory tube 200 dimensioned to cover a segment of ~he sphere as indicated, when the tube is lowered through an cpening in the assembly (formed by aligned opening in the cover, transfer, and reaction plates) into the reaction well.
After each bead is read, the tube is raised and then -- lowered into another assembly opening to read the next bead.
Alternatively, the beads may be distributed into separate readin~ chambers, according to the operation following described wi~h respect to Figures 12A-12C.
Briefly, the transfer plate is rotated to th2 position shown in Figure 12B to align the radial portion of slot 112 with the reaction channel. With the drive motor moved to its raised position, the assembly is now rotated at a speed sufficient to force the particles in the reaction channel radially outwardly into the slot, as shown in ~igure 12A.
The particles are forced ultimately into the cir~umferen-tial portion o~ the slct, as shown in Figure 12B. The .
2~ 3~ 2 particles are captured in groove 72 (Figure 5) at this position, to prevent their return to the reaction channel at the end of centrifugation.
To distribute the particle~ into the respective wells in the reaction plate, the motor is moved to its lowered position, the assembly reseated on the control device, and the transferred plate rotated i.n a clockwise direction, i.e., in a direction which tends to force the particles toward the back of the slot (the particle positions shown in Figure 12B). As the slot is moved over each well in the reaction plate, the f orwardmost partiole in the slot drops into that well, until particle transfer into all three wells is completed.
The amount of analyte associated with each o~ the solid phase particles may be determined by a variety of known methods. Typically, the analyte, which is specifical-ly bound to the particle, itself binds a label pro~e (reporter molecule) which contains fluorescen~, or enzyme reporter moieties which can be detected and/or quantitated by standard photodetector or spectrophotometric means. As indicated above, th~ wells may contain a detection solution which is reactive with the reporter moieties to produce the desired detection signal.
One advantage of the particle transfer feature of the invention is that several solid-phase p~rticles can be reacted under identical conditions in a single cha~ber, then isolated for detection. The separation o~ the particles for detection allows accurate detection by chemilumineSCenCe, fluor~scence, or enzyme activity which is not possible when the particles are closely space.d andlor in the 5ame reaction cham~er. However, it will be recog~ized that the invention is also advantageous for carrying out a solid-phase reaction employing o~e or more beads, or oth~r solid-support surf aces in the reaction 3S well, and r~ading the solid surf aces in the well, as ~1 20~43~
described with respect to Figure ll.
Section C below describes an exemplary assay method for detection of analyte ~NA by a solid-phase reaction method. The method is illustrates various advantages o~
S the assembly and apparatus, int:luding (i) the ability to carry out complex, ~ultiple reagent assays in a simple, selfocontained assembly, (ii) quantitative reagent transfer from the transfer plate to the reaction plate, (iii) the abi~ity to intersperse reaction steps with wash cycles, lo ~iv) tha ability to carry out heated incubation steps without liquid loss, and (v) the ability to conduct multiple solid pha~e rea~tions in a single chamber, then separate the solid-phase supports for analyte detection.
Although the DNA ~nalyte assay is illustrative of one type of assay which may be carried out in the assembly and apparatus of the invention, it will be understood that the ~nvention is readily adapted to a wide variety of assay procedures in which solid or liquid reagents are to be added to a reaction region, preferably sequentially, for 29 detection or quantitation of an analyte in a reaction zone.
In particular, the invention contemplates detecting a ligand analyte in which the solid-support surface in the reaction well includes molecules which bind specifically with the ligand, or whi~h~ can bind speci~ically to th~
ligand through a bivalent intermediate molecule provided by way of one of the added reagentsO I~ a pre~erred for~at, the ligand is an antigen, and the ligand-binding molecule carried on the solid support is a ligand-specific antibody.
After addition of a ligand-containing sample to the solid 30: support, and a wash step to remove unbound sample material, a first reporter reagent is added to the well. This reagent contzlins~ a reporter-labeled molecule capable o~
binding specifically to t~e ligand, with such bound to the solid support. Following a second wash step to remove 35 . unbound material, a second reagent containing a sub trate 22 2~3'~12 for detection of the bo~nd reporter is added t~ the reaction well. Preferably the reporter is an enzyme, such as alkaline phosphata5e or ~eroxidase, and the second reagent contains substr~te capable of reacting with the enzyme reporter to produce a dek~ctable color reaction in the reaction well.
In both the DNA probe fo:rmat, and the ligand ~ormat just described the multiple reagents carried in the assem bly for producing analyte-specific binding to the solid support, and detectable signal of the bound analyte are referred to herein, collectively, as reaction reagents required for bindiny of analyte to the solid support in detectable form.
It will be appreciated that the assembly of the invention may be manually operated, particularly where the assembly has a simplified format in which reagents are added t~ a liquid r~action chamber, for production of an analyte-dependent signal in a liquid-phase. He2e the user can re~dily manipulate the assembly to its reagent tran~fer positions, and perform any necess~ry mixing by manual shaking.
C. Solid-Phase Assay for DNA -.
Figures 14A-14D illustrate schem~tically the sequence of reaction steps in a solid-phaqe DNA analyte assay carried out using the assembly and apparatus of the invention.
Considering first the solid-phase particle5 and reagents whiçh are included in the assembly, the -three solid-phase particles: serve as pos,itive and negative controls (particles a and:b) and for specifi~ a~alyte assay (particle c). The negative-control partiGle is un~oated ~ and the positive-control and analyte particle~ are coated with single-stranded DNA fragments which are ~o~plementary , '.:
7 ~J 7 1 ~
23 20~ 2 to positive-control and analyte-specific capturing probes, respectively. The nature of these probes is described below. The positive-control and analyte particles are prepared by derivatizing polymer or glass beads with the selected DNA fragments, according to standard coupling methods. The analyte solid-phase particle is indicated at ~30 in Figures 14A-14D, an~ the DNA ~ragments coating this particle which are complementary to the analyte probes are indicated by dashed lines, such as at 13Z.
A fir~t liquid reagent, contained in reservoir 92 in the assembly, includes a denaturation agent, such as NaO~, positive-control and analyte-specific capturing probes, a positive-control DNA, and positive-control and speci~ic amplifier prDbes. The positive-control DNA is a duplex DNA
fragment which has a number of ~nown se~uence regions which are unique to that fragment, i.e., not present in th~
analyte DNA. The amplifier probes include a series of probes having a fir5t region which is complementary to one o~ several different sequences in the positive control DNA
and in the analyte nucleic acid, and a second, common region which is complementary to a se~uen~e in a branched DNA contained in~the third reagent. In the amplifier probe illustrated at 134 in Figure 14A~ a first region which is complementarY tQ a sequence in an analyte nucl~ic a~id ic indicated by a square-wave pattern at 136, and a second common region, indicated by coiled line 138, which is complementarY to the sequence in the DNA on a branched DNA.
The positive-control captur~ng probes include a series of probes which have a common first-sequence which is complementarY t~ a sequen~e in the DNA fragment carried on the positive control particle, and differ~nt second sequen~es which re ~omplementary to different known sequence regio~s in the positive-~ontrol DNA. The ana~yte ~apturing prQ~s similarly include a series of probes which h2ve a co~mo~l first-~equence which is complem@ntary to a .
, :
20~ ~3~2 seguence in the DNA fragment carried on the analyte particle, and different second se~uences which are common to the different ~nown sequence regions in th~ analyte nucleic acid. One such analyte capturing probe is illus-trated at 140 in Figure 14A, whi~h shows a first region,indicated by a sawtooth pattern at 142, which is complemen-tary to a sequence in an analyte nucleic acid, and a second common region, indicated by dashed line 144, which is complementarY to the sequence in the DNA carried on the lo analyte particle.
A second liquid reagent, contained in reservoir 94, includes an annealing agent or buffer which allows DNA
annsaling, such as by neutralizing a base denaturation reagent.
A third liquid reagent, carried in reservoir 96, includes the above-mentioned branched DNA molecule, which is indicated at 146 in Figure5 14~14D. The mole~ule includes a sequence indicated by coiled regi~n 148, whi~h is complementary to region 138 of th~ binding pro~es, and a group of branched-chain seguences, such as that indicated by dotted line 150, which are complementary to a label . probe sequence. In this embodiment, the branched DNA is - adapted ~or indirect binding to analyte and positive control nucleic acid,: i.e., through the amplifier probes.
AlternativelY, the branched DN~ may be desi~ned for direct hybridization to sequences in the analyte and positive-control nucleic acid.
A fourth liquid reagent, contained in reservoir 98, includes a labeling probe, indicated at 152 in Figures 14C-30 14D, w~ich includes a DNA fragment moiety 154 with a nucleotide sequence complementary to sequence 150 in the :branched DNA molecule, and a reporter moiety 154 which i~
used for signal detection.
. The reporter moiety in the present assay asse~bly is preferably an enzy~e, such as alkaline phosphatase, whieh ~ 2~.3~
can generate a chemiluminescent signal in a suitable detection solution.
Compl~ting the description of the chemical components of the assembly, the wells in the assembly are partially filled with a detection solution which are reactive with the reporter m~iety to generate a dete~table signal. For example, a solution containing clox~tane is reactive with an alkaline phosphatase reporter moiety to generate a c~emi-luminescent signal which can be detected and quantitated with a photomultiplier conventionally.
Initially the assembly is positioned ~or sample addition and a sample containing a DNA analyte 160 is added to the assembly's reaction chamber. As indicated above, the analyte includes multiple sequences, such as sequence ~5 162,~which are complementary to regions of the analyte capturin~ probes in the assay, and one or more sequences~
such as sequen~e 164, which are complementary to the binding probes in the assay.
After sample addition, the assembly is manipulated to add and ~ix the first reagent, then incubated for 10 minutes at 65C to denature the analyte. With addition of the annealing buffèr, mixing, and further incubation for 10 minutes, the followi~g probe-mediated bindi~g reactions occur: tl) positive-control capturing probes hybridize with the positive-control particle and with the positive-~ontrol DNA to link this DNA to the pasitive-control particle; (2) amplifier probes hybridize with the positive control DNA; ~3) analyte capturing probes hybridize with ths analyte particle and with the analyte to link analyte DNA to the particle; and (4) ampli~ier probes hybridize with the analyte DNA. Steps (3) and (4) are illustrated i~
Figure 14B.
A~ter the an~ealiny reaction, the assembly reaction chamber is manipulated for one or more wash step~ i~ which ' ' :: :
wash solution is circulated through the rea~tion channel t~
remove unbound reagents- The third rea~ent is now added, mixed, and allowed to anneal at 55C, to hybridize the branched DNA with particle-bound amplifier probes, as is illustrated in Figure 14C. Following annealiny, unbound branched DNA is removed by a serond wash step.
The final labeling probe is added to the particles, mixed and allowed to anne21 with the bound branched DNA, to bind reporter molecules to the positive-control and analyte particles. Binding of th~ labeling probe molecules to the analyte particle is illustrated in Figure 14D. Unbound labeling probe is then removed by a khird wash step, as indicated.
The assembly is now manipulated by the step5 shown at the right in Figure 10, and descri~ed above, to transfer the three particles into the respective wells for signal detection. Reaction of the bound reporter with ~he detection fluid in the wells produces a detect~ble chemi luminescent signal which is measured and converted to a quantitative as5ay value whi~h is displayed. In the three particle assay, the positive-control value is used to calculate a standard curve o~ analyte concentration as a function o~ concentration, with background (negative control) subtraction. Analyte concentration is determined from the standard curve, after background subtra~tion.
The assembly and apparatus provid~ se~eral advantages in a solid-phase DNA assay, as can be appreciated. The assay can be carried out in a substantially ~utomated fashion, and without addition of r~ayents from external sources. Accordingly, a minimum of laboratory training and user manipulation5 are requir d.
The ability to carry out multiple solid-pha~e reaG-tions in a sin~le chamber minimizes variations in quantita-tive analyke measurements due to variations in reaction 20~ A~
conditions, allowing self-corrected analyte determinations based on a standard curve with background subtraction.
The following example illu~;trates the use of the appa-ratus for detection of hepatitis B virus (HBV) nucleic S acid. In this example, an HBV assay similar to the one described with raspect to Figure 14 was carried out, and a similar manual assay using a co,nventional microtitre plate ~ormat was carried out in parallel. The sample was an ~iBV-containing plas~id, at a plasmid concentration o~ 10 atto-moles/~l. Positive beads (having a capture ~ound to theirsurfaces), and control bPad5 (lacking the capture sequence) were run in the same chamber, i.e., either microtitre plate or self-contained apparatus, and the microtitre plate assay was run in triplicate. Table 1 b~low shows th~ r~sults for two different runs. As seenl the self-cont ined assay for-mat gave about the s~me positive/control (-cignal/noise) ratis as the mean microtitre assay format in the first run, and a substantially greater positive/~ontrol ratio in the second run.
~3 __ _ _ --=_ _ Mierot t~r ..... Cnrtridqo __._ _~ _ . . . __ Pos Cion S/N Po~ on . __ __ __ . _ Run 1 76 59 i~l 100.3 23.88 _ _ _ . . _ _ __ __ Mean 93.23 18.63 4 95 11.8' i~:~ ~.13 I . . . . __. _ 2 5 Ru rl 2 . 18 . 2 8 la~ 13.2g 92 . 69 ~ 1~ .
_ __ . __ __ ean ~;L 14 . 57 6 . 7? ~ 0 64 . ~ __ , _ , _ = _ _ ., _, ===: _~
In thæ cur~ent Yin~e cartridqe orocessor it_i~ Qrl~po3~ible to . .
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20~3~2 The results demonstrate that a multi-step DNA hybrid-ization assay can be run in an automated format, using the apparatus of the present invention, with a signaljnoise ratio which is comparable to that of a manual, microtitre plate format.
Although the invention has been described with respect to certain embodiments, configurations, and applications, it will be apparent to those skilled in the art that various changes and modifications may ~e made without departing from the invention.
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prevent release of reagents ~rom their corresponding reservoirs until the associatecl reservoir is aligned with the well.
In one embodiment, the reagent is in the form of a liquid solution, the reservoir containing the solution includes a channel formed in the plate, and the channel is sPaled at its opPning in the transfer plate. Preferably the channel and seal are formed by an elastomeric sleeve held in a channel within the transfer plate, and the confronting planes of the transfer and reaction plates are spaced, adjacent the region of the sleeve, to ~orm a capillary lock, to prevent liquid ~rom leaking from the reservoir by capillarity. Also in a preferred embodi~ent, the transfer plate includes a chamfered opsning communicat-ing a reservoir in the transfer plate with the well in the rei~ction plate.
In another general embodiment, at least one of the rei~gents i~cludes a pelletized form of the reayent, and the pelletized reagent is deliver~d ~rom the associated reagent reservoir to the well by gravity, when the reagent reser-voir is aligned with the reaction well.
~T~e reaction well in the reaction plate may be formed by an elongate channel containing a solid-phase assay support. In this embodiment, the transfer plate may include ports which are alignable wit~ spaced area~ in the channel, when the transfer plate is moved to a wash position, ~ormi~g an enclosed passageway for pa5sing a solution through the channel.
In one~ me$hod, the assembly is used for detecting a nucleic acid with a known tar~et sequence. Here the solid-phase support in the reaction is coated with immobilized nucleir acid fragments, and the assembly includes r~agent reservoirS for sequential addition to the reaction channel of ~1) a probe e~fecti~e to hybridize with both th~ target .
.
2 ~ 3 ~ 2 sequence and the immobilized fragments, and (2) a reporter molecule ~ffective to bind, dir~ctly or indirectly, to the probe.
In another method, the assemhly is used in an immuno-assay for detection of a ligand effective to bind immun~-speci~ically to the solid support. Here the ~ssembly includes reagent reservoirs for sequential addition to the reaction channel of tl) an ant:iligand reagent capable to binding immunospecifically with the ligand, when the ligand is bound t~ the support, and (2) a reagent capable reacting with th~ antiligand reagent to produce a detsct-able sighal on the solid support.
In another aspect, the invention includes apparatus for detecking an analyte ligand by detectable, ligand-spe~ific binding to a solid support~ The apparatusincludes a self-contained assemb~y of the type des~ribed above~ and a device ~or holding the reaction plate, and rotating ~aid transfer plate to its various positions.
In one embodiment, the device is effective to rotate the assembly as a unit, the reaction well includes a radially extending channel, the solid support surface includes at laast two solid-phase particles carried in the channel for movement therein ~rom inner toward outer radial positions, and the transfer plate includes a receiving slot into which the particles can be received, when the p~rti-cles in the channel are subjected to an outward force, and the channel is aligned with said slot. The reaction pIate includes particle-receiving wells into which particl~s in the slot can be deposited, wh~n the r~agent plat~ co~tain-ing deposited particles is rotated with respect to theraaction plate.
In an ~mbodiment o~ the device designed ~or heating the liquid con~ents of the well, the assembly further includes structure for sealing the well whsn heatang is applied. The assembly includes a cover plate attached to , .
:.
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2~3'~2 6 v the reaction plate, and the sealing structure includes (a) a sealing pad carried in the tra:nsfer plate for floating in a direction normal to the plane o~ the plate, and (b) a cam surface in the cover plate for biasing the pad against the S well platform, when the tran~fer plate is moved to a heating position.
Also in an embodiment of the device, the solid support includes a bead in the well, ,and the device includes a detector having a light sensor, and a tube extending from the light senso~ and positionable to encompass a spherical ~urface portion of the bead.
These and objects and features of the invention will become more fully apparent when the ~ollowinq detailed description of the invention i5 read in conjunction with the accompanying drawings.
Brief Descri~tion of the Drawin~s Figure l is an exploded perspective view of the components in an assay apparatus constructed according to the present invention; -Figure 2 is an enlarged, partially cutaway perspectiveview of an assay assembly constructed according to the invention;
Fi~ure 3 is a plan view of an cover plate in the Figure 2 assembly;
Figure 4 is a plan view of a transfer plate in the - Fi~ure 2 assembly;
Figure 5 is a plan view of a reaction plate in the Figure 2 assembly;
Figures 6A and 6B are fragmentary seGtional views taken at relati~e plate position in the Figure 2 assembly at which a reagent re5exvoir in the transf er d is~ in th~
assembly ~s isolated from a reaction channel in th~
reaction pla~a (6A), and the reservoir is in a:transfer position with resp~ct to the channel (68);
7 2~ ~3 ~2 Figures 7A and 7B are enlarged, fragmentary sectional views of res~rvoir regions seen along lines A-A in Figure 6A and along line B-B in Figure 6B, respectively;
Figure ~ is an enlarged, fragmentary sectional view of sealed well in the assembly, during a heating step in the operation of the assembly;
Figures 9A and 9B are s~ctional views similar to 6A
and 6B, respectively, but illustrating an embodiment of an assembly i~ whi~h the reagent in the reservoir i~ in dried particle form;
Figure 10 is an enlarged, fragmentary sectional view of the assembly showing the assembly in a positio~ for irrigating the assembly well;
Figure 11 is an enlarged sectional view of a well region of the assembly, showing detector struckure ~ox detecting an optical signa~ from a bead in the well;
Figures 12A-12C illustrate the steps in trans~erring solid-phase particles from the reaction channel in the reaction plat~ into a slot ~ormed in the transfer plate ~12A, 12B), and fro~ this slot into separate wells in the reaction plate ~12C~;
Figure 13 is a flow diagram of the steps carried out by the apparatus in executing an exemplary analyte assay;
and Figures 14A-14D are schematic illustrations of the sequential nu~leic a~id binding reactions in a DNA analyte assay which can be carried out in an automated ~ashion by the apparatus of the invention.
Detailed Des~rlption of the Invention A. i~Ll~LJ~ D ~lY~ e~e9-Figures 1 illustrates, in exploded view, an assay apparatu5 20 constructed according to the present inven-tion. The app ratus includas an assembly 22 whi~h also ~orms part of th3 invention and which will be detailed .
, . ~ . , ,~ . , ' :
20~3~
below with r~ference to Figures :2-12. Briefly, the assem-bly is formed of reaction and cover plat~s 24, 26, respec-tively which are joined together at their outer ed~es, a~d an intermediate transfer plate 2B which can be rotated relative to the two outer plates about a oentral axis 30.
To ~his end, the transfer plate includes a hub 32 having an elongate opening 34 (Figure 2) which is engageable for rotating the transfer plate, with the reaction and cover plates supported in a stationary position.
A control device 36 in the apparatus includes a base 38 whicih defines an annular sea~ 40 in which the a5sembly is supported d~lring an assay operation. The base provides five pins, such as pins 42, arranged asymmetrically about the seat, as shown. These pins engage corresponding holes in the bottom of the assembly, described below, to immobi-lize the cover and re~ction plates against rotation in the seat.
The bas~ is supported on and attached to a drive motor which is indicated by a drive shaft 44 in the figure, the motor itself being contained with a motor housing 45. A
three-arm support 46 attached to the end of the drive shaft has a projection 48 designed to engage the opening in hub 32, with the reaction surface of the assembly resting on the support arms, as can be appreciated in the ~igure.
Thus, when the assembly is received and immobilized in seat 40, with projection 48 received.in opening 34 in the hub, the intermediate transfer plate in the assembly can b~
rotated to 5elected positions with respect to the cover and reaction plates by the drive motor.
The ~otor in the control device can be moved ~rom a lowered transfer position in whioh the assembly i5 i~mob~-lized in seat 40, as just described, to a raised free-rotation position in which the assembly is disengaged ~rom the seat and is ~ree to rotate on~drive ~otor 44. Because o~ a rel tlv-ly high coeffi=ient of internal (transfer .
20~42 plate) movement, the assembly can be rotated as a unit by the drive motor, such that the assembly and motor in a free-rotation position can function as a centrifuge to force material radially outwardly in the assembly, for a purpose to be described. The assembly can al~o be agitat ed, for mixing reaction components, by oscillating the as-sembly on the motor in a free-rotation positiDn.
A wash unit 50 in the device is designed to circulate wash solution(s) through the assembly, as will be described below with respect to Figure 10. Briefly, when the assem-bly is placed in a wash condition, openings 52a, 52b formed in cover plate of the assembly form the ends of a closed passageway whioh includes a portion of the reaction well in the assembly- ~nit 50, which is mounted on base 38 as indicated, in~ludes a vertically positionable arm 54 which holds a pair of tubes 56a, 56b, for insertion into openings 52a, 52b, respectively, when the arm is moved to a lowered, wash positinn. In a wash operation, wash solution is supplied through tube 56a under pressure, and is removed through tub~ 56b.
Also shown in Fi~ure 1 is a heating unit 62 in the apparatus used for heating a reaction well in the region in the assembly to selected reaction temperatures. The unit is attached to the lower side of base 38, as indicated, to position a heating eleme~t 64 in the unit dire~tly below the reaction well in the assemb~y, with the assembly seated on the base.
Although not shown in Figure 1, device 3Ç ~ay also include an optical sensing unit for detecting analyte in 30 the assembly- One preferred unit will be described below :~
with respect to ~igure 11.
The as5ay assembly of the invention is illustrated particularlY with respect to Figures 2-12. Reaction plats 24 in the assembly (Fiqures 2 and 5) provide~ an elongate, radially extonding channel or well 66 which ~erves a~ the :
: ~ ' , .
2~3~2 reaction chamber in the assembly, i.e., the chamber where the analyte and various reagents required for analyte binding and detection are brought into liquid contact with a solid-phase support containE!d in the well. In the S embodiment shown, the well is formed in a metal, e.g., aluminum, insert 166 which is attached to the lower side of a disk-like plate member 168. The insert is carried below a platform 170 defined by the upper surface of plate member 168, above the insert. Formed in the platform are radially spaced openings 1~2a and 172b adjacent opposite end reyions o~ the channel, and a central opening 1~2c. Openings 172a and 172b are align~d with openings 52a and 52b in plate 26, for a purpose to be described.
Details o~ the well construction in the plate are seen in Figure 7B which shows an enlarged cross-section of t~e well region in plate 24 and o~erlying regions of plates 26, 28. The channel or well formed i~ the insert has a sub-stantially U-shaped cross-section extending between oppo-site e~ds, corresponding to the regions below openin~s 172a and 172b in Figure 5, with the outer radial end region of the channel forming a tapered ramp 70, as seen in Figure 10. The insert is raceived in a cavity 174 for~ed in the lower region of plate member 168, and attached to the plate member by flanges, such as seen a~ 176. Eaoh opening in platform 170, such as opening 172a shown in Figure 7~, communicates with the well, and includes a short ~ylindri c l-bore section 178 and a chamfered section 180. This construction serves to draw liquid into and through the opening from a reser~oir in plate 28 into the channel in the transfer plate, as will be described below.
Carried in the channel are one or more solid-phase beads or partlcles, su~h as particlas 68a, 68b, 68c, which form solid phase supports in the assay reaction. In ~he e~bodim nt des~ribed in Seotion C below for solid-phase DNA
determination, the hree pareicl-s ar- ~or ~1) positiYe `
~ U Y l/ IY~ " ~, ,, ~
11 2~3 ~2 control, ~2) negative control, and (3) analyte binding.
The channel and particles are s;hown in cross section in Figures 10 and 12A. The invention also contemplates a rea~tion format containing only a ~ingle solid-phase support.
Located just beyond the outer end o~ the channel in plate 24 is an arcuate groove 72 which serves as a guide for the particles, also for a purpose to be described.
Adjacent this groove are three wells 74a, 74b, 74c into which the three paxticles may be transferred from channel 66 after the chemical reaction, also as will be described below. The three wells may contain a detection solution for detecting the presence of reporter molecules bound to the solid particles.
The reaction plate is secured to the cover plate by fasteners, such a~ rivets 75 ~Figure 2), connecting the outer annular rim regions of the two plates. The fasteners are received throuqh holes, such as holes 76, formed in th~
plate . Also f ormed in the reaction plate is a central opening 82 through which projection 48 i~ re~eived when the assembly is seated on the control device. ~ -Cover plate 26 in the assembly is illustrated in plan view in Figure 3. The plate is a circular disc havinS~ the-same diametç~r as the reaction plate. Formed in an outer 25 annular rim region 83 of the plake are holes, such as holes 84, for fastening an upper and reaction plates (dotted line 85 in Figure 3 indicates the outer edge of transfer plate 28 in the assembly). Also formed in the cover plate are: :
(i) a central opening 86 through which the cover portion o~ .-30 hub 32 is received, ~s seen in Figure 2; (ii) a series of windows B8a, 88b, 88c which are aligned with wells 74a, 74b, 74c, respectively, in the reaction plate; (ili~ above described openings 52a, 52b, which are aligned with openings 172a, 172b, respectively, for cir~ulating wash solut-on through the channel; and (iv) an opening 52~
: .
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20~43~2 between ports 52a and 52b, in alignment with port 172c.
With reference now to Figures 2 and 4, transfer plate 28 include~ a plat~ member 190 having a central opening 90 in which hub 32 is fixed for rotation with the plat~. The S plat~ includes four reagent relservoirs 92, ~4, 96, and 98 which tak~ the form of cyli;n~rical channels extending through the plate. With refere~ce particularly to Figure 7A, reservoir 92, which is rapresentative, is fo~med of a sleeve 100 which extendc through plate member 184~ extend-ing beyond the plane of the plate member on both upper and lower sides of the plate member. In the completed asse~-bly, the sleeves forming the several reservoirs are axi~lly compressed, forming liquid-tight seals against confronting faces of the cover plate and reaction plate, as illustrated in Figure 7~- Th~ sleeves are preferably formed of Teflon or polyethylene- The sealing ends of the sleeves are also referred to herein as means f or preventing release o~
liquid reagent in a reservoir u~til the reservoir is aligned with one of the openings in the trans~er plate communicating w~th channel 66.
The spacing between the plate member f orming pla~e 2 8 and the confronting 5urfaces of the cover and reaction plates in the assembly function to prevent liquid contained in each reservoir from being drawn by capillarity into th~
gap between the two plates. That is, the gap between con~ronting pla~e surfaces, such as gap 186, is too great to produce capillary flow between the two surfacesO The gap in the ~mbodiment shown is provided by the r~latively greater lengths of the sleeves forming the reservoirs compared with the thickness of plate member 184. Alterna-:
tlYely, the region of the transfer plate member im~ediately adjacent each sleeve may be recessed to for~ a capillary-block gap around each sleeve.
As just indicated, ~ach of the reservoir~ in plate 28 35 i6 alignable with one of the openings 172a-172c in the . :
`
13 2 0~3~l2 reaction plate communicating with channel 66, for deliver-ing fluid reagent5 to the reaction chamber, to be described below with reference to Fi~ures 6A, 6B and 7B. In particu-lar, it is noted that some o~ the reservoirs, such as reservoirs 94~ 96, and 98 are radially spaced, preventing cross-contaminatiOn o~ liquid reagents in the two raser-voirs, as the reservoirs are moved relative to the cover and reaction plates.
Also formed in trans~er plate is an openings 102a-102c 10 alignable with opening 172a-172c, respectively, in the reaction plate and at the same time with openings S2a-52c in the cover plate. Any of th three sets o~ alignabl~
openings are referred to herein collectively as means for introduci~g sample into the assembly's reaction region.
lS Other openings formed in the transfer plate include three windows 104a, 104b, 104c which are alig~able with windows 88a, 88b, 88c, respectively, for viewin~ wells 74a~
74b, 74c, respectively, when the two sets of windows are aligned. In addition, the tra~5fer plate provides three sets of radially spaced openings, such as openin~s 106a, 106b, which are alignable, at one o~ three difXerent plate positions, with openings 172a, 172b, respectively, in the reaction plate, at the same time, with ports 52a, 52b in . -the cover plate, for forming a continuous passageway 108 lFigure 10) for circulating wash solution through the reaction region.
The transfer plate also includes a plurality of floating sealing pads, such as pads 192, 194, which are designed for sealing the channel openings in the reaction plate, when the liquid contenk5 of the xeaction plate are heated. Details of the operation of the pads i~ ~ heati~g operation are seen in Figure 8. Shown here are an enlarged sectional view t ken through the assembly in the r~io~ of the reaction channel, and a portion ~f heater ele~ent 64 use~ in heating the liquid contents in the ~hannel~ As 14 20~s3~?~
seen, the heating element makes contact with the lower side of insert 166 during heating.
Pad 192, which is r~presentative, is an elo~gate elastomeric pad, such as formed from polyethylene or other compressible, low-friction polymer material, which is dimensioned to cover openings 1.72a-172c, when the pad is positioned on platform 170, as i.ndicated ïn Figure 8. The pad is carried in a radially extending slot 194 formed in the transfer plate member, ~or floating in a direction normal to the plane of the plate. The pad's h~lyht dimension allows the pad to ~e ~oved easily, a~d without pad compressio~, betw2en the cover and rea~tion plates, as the transfer plate is rotated relative to the other two plates. However, as the pad is moved toward a sealing position above th~ reaction well, it makes contact with a radially extending cam member 195 formed on khe lower side of the cover plate, immediately above openings 172a-172c.
As can be appreciated from Figure 8, this conta~t acts to bias, i.e., compress, the pad against the plat~orm open-ings, sealing the openings to the reaction well.
~ he transfer plate also provides an L-shap~ slot 112 ~ormed in its lower side, i.e., the side confronting the reaction plate. The slot include~--radially and circum-~erentially extending portions 112a, 112b which are alignable with the outer e~d r gion of channel 66 and groove 72 in the reaction plate, respecti~ely. The slot is positioned and dimension~d to receive the parti~les from channel 66 (Figure 12A) as will be described in Seotion B.
The plates in the assembly are preferably formed by injection molding of a suitable plastic. For formi~g the trans~er platle, plate member 184 can be inje~t~d molded ~bout the four sleeves forming the four reservoirs in the transfer plate, and hub 32, and subsequently ~itted with the four sealing:pads. F9r forming the reactio~ pl~te, plate member :L68 can be injected molded about insert 166.
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2 0 ~
With the transfer plate placed between the cover and reaction plates, the two outer plate5 are secured together, such as by rivets, glue or heat welding. As noted above, constru~tion and plate attachment is such as to bias the cover and reaction plates firmly against con~ronting sides ~f the transfer plate, to compress the elastomeric seals in the assembly. Liquid reagent can be added to the reser-voirs and wells by suitable positioning of the transfer plate a~ter assembly.
In another general embodiment of the assembly, the reagent reservoirs are designed for delivery of a dehydrat-ed reagent particle composed of the dried reagent, pre~era-bly formulated in pelletized form. This embodiment is illustra~ed in Figures 9A and 9B which shows a fragmentary portion of an assay assembly 120, with oover plate $22, .-.
transfer plate 124, and reaction plate 126. The cover and reaction plates in the assembly are substantially identical to those in assembly 22. The transfer plate includes a plurality of reservoirs, such as res~rvoir 128, which co~municate with the reaction plate, but not the cover plate.
Each reservoir contains a dried reagent particle, such as particle 130 in reservoir~I28, which is held within the reservoir, ~or deposit into reaction channel 132 in the reaction plate, when the reservoir is aligned with the transfer plate, as seen in Figure 9B. The lower end of the reservoir is sealed by the slaeves forming the re~gent reservoirs, to prevent exposure of the r~agent to moisture or contamination by other reagents.
The pelletized reagent may be formulated, if dasired, with a variety of water-soluble bulking agents, such as water-soluble polymers, ac~ording to well-known methods.
Where the assembly includes one or more solid-phase particles, th~ese are added to the re~ction channel in the ~ :-reaction~plate before the txansfer and reactio~ plates ~re 16 2~ 4 joined.
The asse~blies described ind illustrated above are designed particularly ~or use in a solid-phase DNA analyt~
assay involving (i) at lea5t one solid-phase particle, (ii~
~our separate li~uid and/or solid reagents which are added sequentially, (iii) a heating step following eaoh reag~nt addition, and (iv) three individlaal wash steps which follow the addition of the second, third, and ~ourth reagents, as will be dDscri~ed below.
A variety of alternative assembly configurations are contemplated in the present invention. For example, the assay ~ay involve sequential addition o~ two reagents, requiring only two reservoirs, cr two or more of the re-agents may be added to the reaction region simultaneously, in a configuration in which th2 reservoirs are carri~d along a common radial line.
~ urther, the solid-phase particles in the reaction may be replaced by one or more separate solid-phase supports formed on the channel wall, and/or the solid ph~se parti-cles may be carried within separated wells in the reac~ionchamber, obYiatin~ the need for particle transfer after the assay reaction. ~lternatively, t~e assay r~action may occur in a liquid phase, either in a free solution or in a absorbent filter, eliminating wash steps. In t~e case of an absorbent-filter type reaction region, a liquid reagent may be drawn onto the filter by wicking or capillarity, eliminating the need for venting liquid-carrying reser-voirs.
B. oPe~ation of the Assembly and A~aratus In operation, the assembly is placed in the seat o~
the control device, with the pins o~ the devic~ being received in the corresponding holes in the as~embly reaction plate, and with proje~tion 48 being rec~ived in hub 32, as desc~ib-d above. Hub 32 is initially or~ented .
~ U Y l / l ~ J 7 ~ ~ ~ u 17 ~ O~3'~
with respect to the pins such that the trans~er plate is in a home position at which all of the reservoirs and the reaction region are sealed.
The following exemplary operation will be described 5 with referenCe to Fiyure 13, wh:ich is a flow diagram of the assay steps in the DNA-analyte assay described in section C below. The stQps shown in solid-line boxes are prefera-bly executed in an automated fashion by a suitable micro-processor control unit ~not shown) in the control device.
Th~ sample and/or wash-solution addition steps shown in the dashed-line boxes may be performed either manually or under the control of the apparatus.
The control device is first actuated to move the transfer plate in a clockwise direction in Figure 2, to align opening 102a in the transfer plate with opening 52a in the cover plate and opening 172~ in the react~on plate, and a liquid sample is introduced through the aligned opening into the reaction channel. The analyte sampIe may b~ any fluid sample, such as a blood, serum, or plasma sample, in a suitable amount, typicaily between about 10-200 ~1.
The transfer plat~ is rotated ~urther in the same direction to align reservoir 92 with opening 172a in the reaction plate, as shown in Figures 6B and 7B, to transfer the liquid reagent in:the reservoir into the channel. As the reservoir seal first overIaps the edge of opening 172a, the fluid seal in the reservoir is broken, allowing fluid to ~low readlly out of the reservoir. As discussed above, the capillary lock feature of the transfer plate prevents the fluid from ~lowing by capillarity out of the reservoir into th~ region b~tween transfe~ and reaction plates. :The chamfered opening 172a serves to direct fluid :from the reservoir into the well by a co~bination of capillarity, ~provided by narrower upper section of t~e opening, and ':
, 18 2~ 3~
rapid fluid flow provided by the lower cha~fered'section of the opening.
As ~entioned above, in an alternative embodiment o~
the invention, the reservoir may contain a dehydrated, pelletized reagent which i5 deposited by gra~ity into the reaction channel, as indicated in Figure 9B. To mix thQ
reagent with the li~uid sample, the transfer plate may be rotated back to a channel sealing position, the assembly lifted from the seat in the control device, by raisin~ the drive motor to a free-rotation position, and the as~embly is oscillated slowly as a unit by the motor.
A~ter introducing the liquid (or solid) raa5ent into the reaction well, the transfer plate i5 rokated slightly to position pad 192 over the reaction well. As described with respect to Figure 8, the pad at this position is compressed between the cover and reaction plates, forming a tight seal about the reaction well. The heating element in the device is th~n activated to heat the well for a selected reaction period. During the heating cycle, heated fluid from the reaction mixture is prevented from escaping from ~he well by the seal over openings 172a-172c. :
The transfer plate is now rotated to transf~r the content5 o~ re5ervoir 94 into the channel, and the reaction mixture is again ~ixed and reacted for a given reaction period at a selected temperatur~ As can be appreciat~d wlth referenCe to Figure 4, continued movement of the transfer plate in a cloc~wise direction aligns the first set of wash-solution ports, indicated at 106a, 106b with openings 52a, 52b, respectively, in the cover plate, and openings 172a, 172b, respectively in the reaction plate.
When this alignment is achieved, the wash unit is lower~d to insert the wash-solution tubes into the aligned port , and wash solution is oirculatPd through the reaction channel as illus~rated in Figure 10, to wash the initial ample and first two reagents from the reaction ~hamber.
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, .-' 19 2 0 ~ ~ 3 ~ ~
The above steps are repeated to (i) transfer, mix andincubate a third reagent with the washed solid-phase particles in the reaction chamber, (ii) remove the third reagent by washing, (iii) trans~er, mix and incubate a fourth reagent with the washad particles, and (iv) remove the fourth reagent by wa~hing. These steps are shown in Figure 13, where the three separate wash steps are indicat-ed at (1), (2), and (3).
After completing the rea~tion, an analyte-dependent signal on the solid-phase surface, i.e., the solid-phase beads, may be read with the beads in the reaction well. In order to minimize optical-reading artifacts ~rom the other beads in the reaction well, the signal detector in the device (or in a s~parate optical reading devi~e~ preferably - 15 has a construction like that shown for detector 196 in Figure 11, allowing direct reading of each bead. The detector includes an optical sensor 198 and a refractory tube 200 dimensioned to cover a segment of ~he sphere as indicated, when the tube is lowered through an cpening in the assembly (formed by aligned opening in the cover, transfer, and reaction plates) into the reaction well.
After each bead is read, the tube is raised and then -- lowered into another assembly opening to read the next bead.
Alternatively, the beads may be distributed into separate readin~ chambers, according to the operation following described wi~h respect to Figures 12A-12C.
Briefly, the transfer plate is rotated to th2 position shown in Figure 12B to align the radial portion of slot 112 with the reaction channel. With the drive motor moved to its raised position, the assembly is now rotated at a speed sufficient to force the particles in the reaction channel radially outwardly into the slot, as shown in ~igure 12A.
The particles are forced ultimately into the cir~umferen-tial portion o~ the slct, as shown in Figure 12B. The .
2~ 3~ 2 particles are captured in groove 72 (Figure 5) at this position, to prevent their return to the reaction channel at the end of centrifugation.
To distribute the particle~ into the respective wells in the reaction plate, the motor is moved to its lowered position, the assembly reseated on the control device, and the transferred plate rotated i.n a clockwise direction, i.e., in a direction which tends to force the particles toward the back of the slot (the particle positions shown in Figure 12B). As the slot is moved over each well in the reaction plate, the f orwardmost partiole in the slot drops into that well, until particle transfer into all three wells is completed.
The amount of analyte associated with each o~ the solid phase particles may be determined by a variety of known methods. Typically, the analyte, which is specifical-ly bound to the particle, itself binds a label pro~e (reporter molecule) which contains fluorescen~, or enzyme reporter moieties which can be detected and/or quantitated by standard photodetector or spectrophotometric means. As indicated above, th~ wells may contain a detection solution which is reactive with the reporter moieties to produce the desired detection signal.
One advantage of the particle transfer feature of the invention is that several solid-phase p~rticles can be reacted under identical conditions in a single cha~ber, then isolated for detection. The separation o~ the particles for detection allows accurate detection by chemilumineSCenCe, fluor~scence, or enzyme activity which is not possible when the particles are closely space.d andlor in the 5ame reaction cham~er. However, it will be recog~ized that the invention is also advantageous for carrying out a solid-phase reaction employing o~e or more beads, or oth~r solid-support surf aces in the reaction 3S well, and r~ading the solid surf aces in the well, as ~1 20~43~
described with respect to Figure ll.
Section C below describes an exemplary assay method for detection of analyte ~NA by a solid-phase reaction method. The method is illustrates various advantages o~
S the assembly and apparatus, int:luding (i) the ability to carry out complex, ~ultiple reagent assays in a simple, selfocontained assembly, (ii) quantitative reagent transfer from the transfer plate to the reaction plate, (iii) the abi~ity to intersperse reaction steps with wash cycles, lo ~iv) tha ability to carry out heated incubation steps without liquid loss, and (v) the ability to conduct multiple solid pha~e rea~tions in a single chamber, then separate the solid-phase supports for analyte detection.
Although the DNA ~nalyte assay is illustrative of one type of assay which may be carried out in the assembly and apparatus of the invention, it will be understood that the ~nvention is readily adapted to a wide variety of assay procedures in which solid or liquid reagents are to be added to a reaction region, preferably sequentially, for 29 detection or quantitation of an analyte in a reaction zone.
In particular, the invention contemplates detecting a ligand analyte in which the solid-support surface in the reaction well includes molecules which bind specifically with the ligand, or whi~h~ can bind speci~ically to th~
ligand through a bivalent intermediate molecule provided by way of one of the added reagentsO I~ a pre~erred for~at, the ligand is an antigen, and the ligand-binding molecule carried on the solid support is a ligand-specific antibody.
After addition of a ligand-containing sample to the solid 30: support, and a wash step to remove unbound sample material, a first reporter reagent is added to the well. This reagent contzlins~ a reporter-labeled molecule capable o~
binding specifically to t~e ligand, with such bound to the solid support. Following a second wash step to remove 35 . unbound material, a second reagent containing a sub trate 22 2~3'~12 for detection of the bo~nd reporter is added t~ the reaction well. Preferably the reporter is an enzyme, such as alkaline phosphata5e or ~eroxidase, and the second reagent contains substr~te capable of reacting with the enzyme reporter to produce a dek~ctable color reaction in the reaction well.
In both the DNA probe fo:rmat, and the ligand ~ormat just described the multiple reagents carried in the assem bly for producing analyte-specific binding to the solid support, and detectable signal of the bound analyte are referred to herein, collectively, as reaction reagents required for bindiny of analyte to the solid support in detectable form.
It will be appreciated that the assembly of the invention may be manually operated, particularly where the assembly has a simplified format in which reagents are added t~ a liquid r~action chamber, for production of an analyte-dependent signal in a liquid-phase. He2e the user can re~dily manipulate the assembly to its reagent tran~fer positions, and perform any necess~ry mixing by manual shaking.
C. Solid-Phase Assay for DNA -.
Figures 14A-14D illustrate schem~tically the sequence of reaction steps in a solid-phaqe DNA analyte assay carried out using the assembly and apparatus of the invention.
Considering first the solid-phase particle5 and reagents whiçh are included in the assembly, the -three solid-phase particles: serve as pos,itive and negative controls (particles a and:b) and for specifi~ a~alyte assay (particle c). The negative-control partiGle is un~oated ~ and the positive-control and analyte particle~ are coated with single-stranded DNA fragments which are ~o~plementary , '.:
7 ~J 7 1 ~
23 20~ 2 to positive-control and analyte-specific capturing probes, respectively. The nature of these probes is described below. The positive-control and analyte particles are prepared by derivatizing polymer or glass beads with the selected DNA fragments, according to standard coupling methods. The analyte solid-phase particle is indicated at ~30 in Figures 14A-14D, an~ the DNA ~ragments coating this particle which are complementary to the analyte probes are indicated by dashed lines, such as at 13Z.
A fir~t liquid reagent, contained in reservoir 92 in the assembly, includes a denaturation agent, such as NaO~, positive-control and analyte-specific capturing probes, a positive-control DNA, and positive-control and speci~ic amplifier prDbes. The positive-control DNA is a duplex DNA
fragment which has a number of ~nown se~uence regions which are unique to that fragment, i.e., not present in th~
analyte DNA. The amplifier probes include a series of probes having a fir5t region which is complementary to one o~ several different sequences in the positive control DNA
and in the analyte nucleic acid, and a second, common region which is complementary to a se~uen~e in a branched DNA contained in~the third reagent. In the amplifier probe illustrated at 134 in Figure 14A~ a first region which is complementarY tQ a sequence in an analyte nucl~ic a~id ic indicated by a square-wave pattern at 136, and a second common region, indicated by coiled line 138, which is complementarY to the sequence in the DNA on a branched DNA.
The positive-control captur~ng probes include a series of probes which have a common first-sequence which is complementarY t~ a sequen~e in the DNA fragment carried on the positive control particle, and differ~nt second sequen~es which re ~omplementary to different known sequence regio~s in the positive-~ontrol DNA. The ana~yte ~apturing prQ~s similarly include a series of probes which h2ve a co~mo~l first-~equence which is complem@ntary to a .
, :
20~ ~3~2 seguence in the DNA fragment carried on the analyte particle, and different second se~uences which are common to the different ~nown sequence regions in th~ analyte nucleic acid. One such analyte capturing probe is illus-trated at 140 in Figure 14A, whi~h shows a first region,indicated by a sawtooth pattern at 142, which is complemen-tary to a sequence in an analyte nucleic acid, and a second common region, indicated by dashed line 144, which is complementarY to the sequence in the DNA carried on the lo analyte particle.
A second liquid reagent, contained in reservoir 94, includes an annealing agent or buffer which allows DNA
annsaling, such as by neutralizing a base denaturation reagent.
A third liquid reagent, carried in reservoir 96, includes the above-mentioned branched DNA molecule, which is indicated at 146 in Figure5 14~14D. The mole~ule includes a sequence indicated by coiled regi~n 148, whi~h is complementary to region 138 of th~ binding pro~es, and a group of branched-chain seguences, such as that indicated by dotted line 150, which are complementary to a label . probe sequence. In this embodiment, the branched DNA is - adapted ~or indirect binding to analyte and positive control nucleic acid,: i.e., through the amplifier probes.
AlternativelY, the branched DN~ may be desi~ned for direct hybridization to sequences in the analyte and positive-control nucleic acid.
A fourth liquid reagent, contained in reservoir 98, includes a labeling probe, indicated at 152 in Figures 14C-30 14D, w~ich includes a DNA fragment moiety 154 with a nucleotide sequence complementary to sequence 150 in the :branched DNA molecule, and a reporter moiety 154 which i~
used for signal detection.
. The reporter moiety in the present assay asse~bly is preferably an enzy~e, such as alkaline phosphatase, whieh ~ 2~.3~
can generate a chemiluminescent signal in a suitable detection solution.
Compl~ting the description of the chemical components of the assembly, the wells in the assembly are partially filled with a detection solution which are reactive with the reporter m~iety to generate a dete~table signal. For example, a solution containing clox~tane is reactive with an alkaline phosphatase reporter moiety to generate a c~emi-luminescent signal which can be detected and quantitated with a photomultiplier conventionally.
Initially the assembly is positioned ~or sample addition and a sample containing a DNA analyte 160 is added to the assembly's reaction chamber. As indicated above, the analyte includes multiple sequences, such as sequence ~5 162,~which are complementary to regions of the analyte capturin~ probes in the assay, and one or more sequences~
such as sequen~e 164, which are complementary to the binding probes in the assay.
After sample addition, the assembly is manipulated to add and ~ix the first reagent, then incubated for 10 minutes at 65C to denature the analyte. With addition of the annealing buffèr, mixing, and further incubation for 10 minutes, the followi~g probe-mediated bindi~g reactions occur: tl) positive-control capturing probes hybridize with the positive-control particle and with the positive-~ontrol DNA to link this DNA to the pasitive-control particle; (2) amplifier probes hybridize with the positive control DNA; ~3) analyte capturing probes hybridize with ths analyte particle and with the analyte to link analyte DNA to the particle; and (4) ampli~ier probes hybridize with the analyte DNA. Steps (3) and (4) are illustrated i~
Figure 14B.
A~ter the an~ealiny reaction, the assembly reaction chamber is manipulated for one or more wash step~ i~ which ' ' :: :
wash solution is circulated through the rea~tion channel t~
remove unbound reagents- The third rea~ent is now added, mixed, and allowed to anneal at 55C, to hybridize the branched DNA with particle-bound amplifier probes, as is illustrated in Figure 14C. Following annealiny, unbound branched DNA is removed by a serond wash step.
The final labeling probe is added to the particles, mixed and allowed to anne21 with the bound branched DNA, to bind reporter molecules to the positive-control and analyte particles. Binding of th~ labeling probe molecules to the analyte particle is illustrated in Figure 14D. Unbound labeling probe is then removed by a khird wash step, as indicated.
The assembly is now manipulated by the step5 shown at the right in Figure 10, and descri~ed above, to transfer the three particles into the respective wells for signal detection. Reaction of the bound reporter with ~he detection fluid in the wells produces a detect~ble chemi luminescent signal which is measured and converted to a quantitative as5ay value whi~h is displayed. In the three particle assay, the positive-control value is used to calculate a standard curve o~ analyte concentration as a function o~ concentration, with background (negative control) subtraction. Analyte concentration is determined from the standard curve, after background subtra~tion.
The assembly and apparatus provid~ se~eral advantages in a solid-phase DNA assay, as can be appreciated. The assay can be carried out in a substantially ~utomated fashion, and without addition of r~ayents from external sources. Accordingly, a minimum of laboratory training and user manipulation5 are requir d.
The ability to carry out multiple solid-pha~e reaG-tions in a sin~le chamber minimizes variations in quantita-tive analyke measurements due to variations in reaction 20~ A~
conditions, allowing self-corrected analyte determinations based on a standard curve with background subtraction.
The following example illu~;trates the use of the appa-ratus for detection of hepatitis B virus (HBV) nucleic S acid. In this example, an HBV assay similar to the one described with raspect to Figure 14 was carried out, and a similar manual assay using a co,nventional microtitre plate ~ormat was carried out in parallel. The sample was an ~iBV-containing plas~id, at a plasmid concentration o~ 10 atto-moles/~l. Positive beads (having a capture ~ound to theirsurfaces), and control bPad5 (lacking the capture sequence) were run in the same chamber, i.e., either microtitre plate or self-contained apparatus, and the microtitre plate assay was run in triplicate. Table 1 b~low shows th~ r~sults for two different runs. As seenl the self-cont ined assay for-mat gave about the s~me positive/control (-cignal/noise) ratis as the mean microtitre assay format in the first run, and a substantially greater positive/~ontrol ratio in the second run.
~3 __ _ _ --=_ _ Mierot t~r ..... Cnrtridqo __._ _~ _ . . . __ Pos Cion S/N Po~ on . __ __ __ . _ Run 1 76 59 i~l 100.3 23.88 _ _ _ . . _ _ __ __ Mean 93.23 18.63 4 95 11.8' i~:~ ~.13 I . . . . __. _ 2 5 Ru rl 2 . 18 . 2 8 la~ 13.2g 92 . 69 ~ 1~ .
_ __ . __ __ ean ~;L 14 . 57 6 . 7? ~ 0 64 . ~ __ , _ , _ = _ _ ., _, ===: _~
In thæ cur~ent Yin~e cartridqe orocessor it_i~ Qrl~po3~ible to . .
' ' .
- . . : , . .
20~3~2 The results demonstrate that a multi-step DNA hybrid-ization assay can be run in an automated format, using the apparatus of the present invention, with a signaljnoise ratio which is comparable to that of a manual, microtitre plate format.
Although the invention has been described with respect to certain embodiments, configurations, and applications, it will be apparent to those skilled in the art that various changes and modifications may ~e made without departing from the invention.
~: .
...
,.~
,: .
.- .. . . :~ , . ;, . , . , :
Claims (20)
1. A self-contained assembly for detecting an analyte ligand by detectable, analyte-specific binding to a solid support, comprising a reaction plate containing such solid support and defining a well for holding liquid reagents in contact with the support, a transfer plate containing first and second reagent reservoirs, carried in said first and second reservoirs, first and second reaction reagents required for binding of ligand in detectable form to the solid support, means mounting the transfer plate on the reaction plate for movement thereon to a sample-addition position, at which sample can be added to said well, a first reagent-transfer position at which the first reservoir is aligned with said well, a wash position at which wash solution can be introduced into the well, and a second reagent-transfer position at which the second reservoir is aligned with said well, and means for preventing release of first and second reagents from their reservoirs until the associated reservoir is aligned with said well.
2. The assembly of claim 1, wherein at least one of said reagents is in the form of a liquid solution, the reservoir containing the solution includes a channel formed in the plate, and said preventing means includes an elastomeric seal between the end of the reservoir and the confronting surface of the reaction plate.
3. The assembly of claim 2, wherein said reservoir and seal are formed by an elastomeric sleeve held in a channel within the transfer plate, and the confronting planes of the transfer and reaction plates are spaced in the region of the sleeve, to form a capillary lock, to prevent material from liquid from leaking from the reser-voir by capillarity.
4. The assembly of claim 3, which further includes a cover plate secured to the reaction plate, and having ports which are alignable with said reservoirs, in such transfer positions, and said sleeve is effective to form a seal between the transfer plate and both the reaction and cover plates.
5. The assembly of claim 1, wherein the transfer plate includes a chamfered opening communicating a reser-voir in the transfer plate with the well in the reaction plate.
6. The assembly of claim 1, wherein said well in a radially extending channel, said transfer plate includes a plurality of chamfered openings spaced along the channel, in a radial direction, and said reservoirs are radially offset to communicate with different ones of said chamfered openings.
7. The assembly of claim 1, for use with a heater effective to heat the liquid contents of the well, which further includes means for sealing for sealing said well when said heater is applied.
8. The assembly of claim 7, which further includes a cover plate attached to said reaction plate, and wherein said sealing means includes (a) a sealing pad carried in said transfer plate for floating in a direction normal to the plane of the plate, and (b) means on said cover plate for biasing the pad against said well, when the transfer plate is moved to a heating position.
9. The assembly of claim 1, wherein at least one of said reagents includes a pelletized form of the reagent, and the pelletized reagent is delivered from the associated reagent reservoir to said well by gravity, when that reagent reservoir is aligned with the reaction well.
10. The assembly of claim 1, wherein said reaction well in the reaction plate is formed by an elongate channel containing a solid-phase assay support, and said transfer plate includes ports which are alignable with spaced areas in the channel, when the transfer plate is moved to a wash position, forming an enclosed passageway for passing a solution through the channel.
11. The assembly of claim 1, wherein the reaction and transfer plates are mounted for rotation relative to one another.
12. The assembly of claim 1, for use in a DNA probe assay for detection of a nucleic acid with a known target sequence, wherein the solid-phase support in the reaction is coated with immobilized nucleic acid fragments, and said assembly includes reagent reservoirs for sequential addition to the reaction channel of (1) a probe effective to hybridize with both the target sequence and the immobi-lized fragments, and (2) a reporter molecule effective to bind, directly or indirectly, to said probe.
WO 91/?950?
WO 91/?950?
13. The assembly of claim 12, wherein the reagent reservoirs for addition of the probe include one reservoir containing the probe in a denaturation agent and a second reservoir containing an annealing buffer for addition to the reaction reservoir after addition of the denaturation buffer.
14. The assembly of claim 13, wherein the reagent reservoirs for addition the reporter molecule includes one reservoir containing a branched nucleic acid fragment effective to bind directly or indirectly to said analyte, molecule, and a second reservoir containing a reporter-labeled molecule effective to bind to the branched frag-ment.
15. The assembly of claim 1, for use in an immunoas-say for detection of a ligand effective to bind immunoaspe-cifically to the solid support, wherein said assembly includes reagent reservoirs for sequential addition to the reaction channel of (1) an antiligand reagent capable to binding immunospecifically with such ligand, when the ligand is bound to the support, and and (2) a reagent capable to reacting with the antiligand reagent to produce a detectable signal on the solid support.
16. A self-contained assembly for assaying, in a liquid sample, an analyte nucleic acid having a known target sequence, comprising a reaction plate containing a reaction well formed by an elongate, radially extending channel and a solid-phase surface contained in the well and coated with immobilized nucleic acid fragments, a cover plate secured to the reaction plate, and having ports which are alignable with spaced areas of said slot, WO 91/?950?
an intermediate transfer plate which includes four reagent reservoirs containing (1) a denaturation agent and a probe effective to bind to the analyte DNA and to said immobilized fragments, (2) an annealing buffer, (3) branched nucleic acid fragments effective to bind, directly or indirectly, to said analyte, and (4) reporter-labeled fragments effective to bind to the branched fragments, where the four reagent reservoirs are designed for sequen-tial addition to said reaction reservoir, said transfer plate further having ports which are alignable with the ports in the cover plate, when the transfer plate is moved to a wash position, providing an enclosed passageway for circulating a solution through the reaction slot, means mounting the transfer plate between the reaction and cover plates for relative rotation with respect thereto, from a home position, at which four reservoirs are isolated from said well, to first-fourth transfer positions at which the first-fourth reservoirs are aligned with said well, allowing sequential transfer of the first-fourth reagents, respectively, to the well, and means for preventing release of first and second reagents from their reservoirs until the associated reservoir is aligned with said well.
an intermediate transfer plate which includes four reagent reservoirs containing (1) a denaturation agent and a probe effective to bind to the analyte DNA and to said immobilized fragments, (2) an annealing buffer, (3) branched nucleic acid fragments effective to bind, directly or indirectly, to said analyte, and (4) reporter-labeled fragments effective to bind to the branched fragments, where the four reagent reservoirs are designed for sequen-tial addition to said reaction reservoir, said transfer plate further having ports which are alignable with the ports in the cover plate, when the transfer plate is moved to a wash position, providing an enclosed passageway for circulating a solution through the reaction slot, means mounting the transfer plate between the reaction and cover plates for relative rotation with respect thereto, from a home position, at which four reservoirs are isolated from said well, to first-fourth transfer positions at which the first-fourth reservoirs are aligned with said well, allowing sequential transfer of the first-fourth reagents, respectively, to the well, and means for preventing release of first and second reagents from their reservoirs until the associated reservoir is aligned with said well.
17. Apparatus for detecting an analyte ligand by detectable, ligand-specific binding o a solid support, comprising a self-contained assembly composed of (a) a reaction plate containing such solid support and defining a well for holding liquid reagents in contact with the support, (b) a transfer plate containing first and second reagent reser-voirs, (c) carried in said first and second reservoirs, first and second reaction reagents required for binding of ligand in detectable form to the solid support, (d) means mounting the transfer plate on the reaction plate for WO 91/?950?
movement thereon to a sample-addition position, at which sample can be added to said well, a first reagent transfer position at which the first reservoir is aligned with said well, a wash position at which wash solution can be introduced into the well, and a second reagent-transfer position at which the second reservoir is aligned with said well, and (e) means for preventing release of first and second regents from their reservoirs until the associated reservoir is aligned with said wall, and a device for holding said reaction plate, and rotating said transfer plate to its various positions.
movement thereon to a sample-addition position, at which sample can be added to said well, a first reagent transfer position at which the first reservoir is aligned with said well, a wash position at which wash solution can be introduced into the well, and a second reagent-transfer position at which the second reservoir is aligned with said well, and (e) means for preventing release of first and second regents from their reservoirs until the associated reservoir is aligned with said wall, and a device for holding said reaction plate, and rotating said transfer plate to its various positions.
18. The apparatus of claim 17, wherein the device is effective to rotate the assembly as a unit, said the reaction well includes a radially extending channel, said solid support surface includes at least two solid-phase particles carried in the channel for movement therein from inner toward outer radial positions, said transfer plate includes a receiving slot into which such particles can be received, when the particles in the channel are subjected to an outward force, and said slot is aligned with said slot, and said reaction plate includes particle-receiving wells into which particles in said slot can be deposited, when the reagent plate containing deposited particles is rotated with respect to the reaction plate.
19. The apparatus of claim 18, wherein said device has a heater for heating the liquid contents of said cell, wherein said assembly further includes means for sealing for sealing said well when said heater is applied, said sealing means including a cover plate attached to said reaction plate, and wherein said sealing means includes (a) a sealing pad carried in said transfer plate for floating in a direction normal to the plane of the plate, and (b) means on said cover plate for biasing the pad against said well, when the transfer plate is moved to a heating position.
20. The apparatus of claim 19, wherein said solid support includes a bead in said well, and said device includes detection means for detecting analyte bound to the bead, said detection means including a light detector, and a tube extending from the light detector and positionable to encompass a spherical surface portion of the bead.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US53900290A | 1990-06-15 | 1990-06-15 | |
| US539,002 | 1990-06-15 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2084342A1 true CA2084342A1 (en) | 1991-12-16 |
Family
ID=24149337
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002084342A Abandoned CA2084342A1 (en) | 1990-06-15 | 1991-06-13 | Self-contained assay assembly and apparatus |
Country Status (13)
| Country | Link |
|---|---|
| US (1) | US5310523A (en) |
| EP (1) | EP0533801B1 (en) |
| JP (1) | JPH05507878A (en) |
| KR (1) | KR0182619B1 (en) |
| AT (1) | ATE109685T1 (en) |
| AU (1) | AU8050591A (en) |
| CA (1) | CA2084342A1 (en) |
| DE (1) | DE69103420T2 (en) |
| ES (1) | ES2057904T3 (en) |
| IE (1) | IE65253B1 (en) |
| PT (1) | PT98009B (en) |
| TW (1) | TW197495B (en) |
| WO (1) | WO1991019567A1 (en) |
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-
1991
- 1991-06-13 WO PCT/US1991/004207 patent/WO1991019567A1/en active IP Right Grant
- 1991-06-13 ES ES91911636T patent/ES2057904T3/en not_active Expired - Lifetime
- 1991-06-13 AT AT91911636T patent/ATE109685T1/en not_active IP Right Cessation
- 1991-06-13 AU AU80505/91A patent/AU8050591A/en not_active Abandoned
- 1991-06-13 CA CA002084342A patent/CA2084342A1/en not_active Abandoned
- 1991-06-13 DE DE69103420T patent/DE69103420T2/en not_active Expired - Fee Related
- 1991-06-13 EP EP91911636A patent/EP0533801B1/en not_active Expired - Lifetime
- 1991-06-13 JP JP91511515A patent/JPH05507878A/en active Pending
- 1991-06-13 KR KR1019920703240A patent/KR0182619B1/en not_active IP Right Cessation
- 1991-06-14 IE IE204991A patent/IE65253B1/en not_active IP Right Cessation
- 1991-06-17 PT PT98009A patent/PT98009B/en not_active IP Right Cessation
- 1991-07-12 TW TW080105430A patent/TW197495B/zh active
- 1991-08-27 US US07/750,325 patent/US5310523A/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| ES2057904T3 (en) | 1994-10-16 |
| PT98009B (en) | 1998-12-31 |
| EP0533801B1 (en) | 1994-08-10 |
| JPH05507878A (en) | 1993-11-11 |
| EP0533801A1 (en) | 1993-03-31 |
| IE65253B1 (en) | 1995-10-18 |
| PT98009A (en) | 1993-08-31 |
| US5310523A (en) | 1994-05-10 |
| ATE109685T1 (en) | 1994-08-15 |
| WO1991019567A1 (en) | 1991-12-26 |
| TW197495B (en) | 1993-01-01 |
| DE69103420D1 (en) | 1994-09-15 |
| IE912049A1 (en) | 1991-12-18 |
| DE69103420T2 (en) | 1994-12-01 |
| AU8050591A (en) | 1992-01-07 |
| KR0182619B1 (en) | 1999-05-01 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| FZDE | Discontinued |