CA2100363A1 - Agglutination reaction device utilizing porous absorbent material - Google Patents
Agglutination reaction device utilizing porous absorbent materialInfo
- Publication number
- CA2100363A1 CA2100363A1 CA 2100363 CA2100363A CA2100363A1 CA 2100363 A1 CA2100363 A1 CA 2100363A1 CA 2100363 CA2100363 CA 2100363 CA 2100363 A CA2100363 A CA 2100363A CA 2100363 A1 CA2100363 A1 CA 2100363A1
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- Prior art keywords
- layer
- chamber
- well
- liquid
- slot
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-
- 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
- G01N33/5302—Apparatus specially adapted for immunological test procedures
- G01N33/5304—Reaction vessels, e.g. agglutination plates
-
- 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
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- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Food Science & Technology (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
- Physical Or Chemical Processes And Apparatus (AREA)
- Automatic Analysis And Handling Materials Therefor (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
Disclosed is an improved device (10) for performing agglutination reactions having at least one chamber (10) having a reaction zone and a distal zone, in which chamber agglutination reactions can be performed. The improvement comprises utilization in the device of means for controlling the overall rate of liquid flow through the chamber (22), the means comprising a layer of liquid absorbant material (22) selectively impregnated with a substance to form an impregnated region and a non-impregnated region wherein the non-impregnated region is liquid absorbant and the impregnated region is liquid-occlusive. The non-impregnated region is in liquid communication with the distal zone of the chamber (10).
Description
W092/08973 PCT/US91/0~573 l ~100363 IMPROVED AGGLUTINATION ,REACTION DEVICE
UTILIZING POROUS ABSORBENT MATERIAL
BACKGRO~ND OF THE INVENTION
The present invention is directed to a device for performing an agglutination reaction of immunochemical particles. The agglutination reaction device is designed to provide a convenient means for performing and reading the results of an agglutination reaction.
Agglutination reactions and their procedures are generally well known in the art. A typical agglutination reaction consists of the clumping together (or aggregation) in suspension of antigen- or antibody-bearing cells, microorganisms, or particles in the presence of specific analytes. This clumping or agglutination of particles is then monitored to determine the absence or presence of an analyte sought to be detected.
One method for reacting immunochemical particle reagents is to place liquid reagents on a glass slide and generally rock or swirl the slide back and forth to cause the reagents to mix and form agglutinations. Methods have also been developed to avoid the necessary swirling of the particle reagents in order to visualize the agglutinations.
For example, U.S. Patent 4,596,695 discloses an agglutination reaction chamber for reacting immunochémical particle reagents. The chamber includes a first transparent panel having a first surface and a second panel having a second surface spaced apart from the first surface to define a chamber inbetween. The chamber intrinsically causes immunochemical particle reagents to flow by capillary action without an external motion imparted to the chamber during which fIow the immunochemical particle reagents can react.
An object of the present invention is to provide SUBS T ITUTE SHEI~
W092/0~973 PCT/US91/0~573 2~1~0~3 2 a device that can be easily adapted for use in the automated diagnosis of a plurality of samples. Another object of the present invention is to provide a device ~apable of performing multiple, highly sensitive, diagnostic tests simultaneously on a single sample in a single device. In particular, the present invention is directed toward a device that can be used in an automated fashion where the reaction can be rapidly performed and monitored with a minimum of sample material. In another aspect, the present invention is directed toward a device having multiple channels radiating from a central well where multiple reactions on a single sample can be rapidly performed and monitored with a minimum of sample material with the results of such reactions being easily, visibly observable. In particular, the present invention is directed to an improved device for performing agglutination reactions which device has means for controlling the overall rate of liquid flow through the device comprising a porous absorbent material in liquid communication with a capillary chamber in the device.
SUMMARY OF THE INVENTION
The present invention is directed to a device having an agglutination reaction chamber for performing agglutination immunoassay reactions. In one aspect, the device comprises a first wettable (hydrophilic) layer and a parallel second layer wherein the first layer has channels such that when the first and second layers are brought into contact with each other an aggl1~tination reaction chamber is formed for conducting fluid by capillary action. The agglutination reaction chamber can additionally include a sample recei~ing well contiguous with the ingress of the chamber formed by the first and W~92/08973 PCT/US91/~8573 (.. .. .
UTILIZING POROUS ABSORBENT MATERIAL
BACKGRO~ND OF THE INVENTION
The present invention is directed to a device for performing an agglutination reaction of immunochemical particles. The agglutination reaction device is designed to provide a convenient means for performing and reading the results of an agglutination reaction.
Agglutination reactions and their procedures are generally well known in the art. A typical agglutination reaction consists of the clumping together (or aggregation) in suspension of antigen- or antibody-bearing cells, microorganisms, or particles in the presence of specific analytes. This clumping or agglutination of particles is then monitored to determine the absence or presence of an analyte sought to be detected.
One method for reacting immunochemical particle reagents is to place liquid reagents on a glass slide and generally rock or swirl the slide back and forth to cause the reagents to mix and form agglutinations. Methods have also been developed to avoid the necessary swirling of the particle reagents in order to visualize the agglutinations.
For example, U.S. Patent 4,596,695 discloses an agglutination reaction chamber for reacting immunochémical particle reagents. The chamber includes a first transparent panel having a first surface and a second panel having a second surface spaced apart from the first surface to define a chamber inbetween. The chamber intrinsically causes immunochemical particle reagents to flow by capillary action without an external motion imparted to the chamber during which fIow the immunochemical particle reagents can react.
An object of the present invention is to provide SUBS T ITUTE SHEI~
W092/0~973 PCT/US91/0~573 2~1~0~3 2 a device that can be easily adapted for use in the automated diagnosis of a plurality of samples. Another object of the present invention is to provide a device ~apable of performing multiple, highly sensitive, diagnostic tests simultaneously on a single sample in a single device. In particular, the present invention is directed toward a device that can be used in an automated fashion where the reaction can be rapidly performed and monitored with a minimum of sample material. In another aspect, the present invention is directed toward a device having multiple channels radiating from a central well where multiple reactions on a single sample can be rapidly performed and monitored with a minimum of sample material with the results of such reactions being easily, visibly observable. In particular, the present invention is directed to an improved device for performing agglutination reactions which device has means for controlling the overall rate of liquid flow through the device comprising a porous absorbent material in liquid communication with a capillary chamber in the device.
SUMMARY OF THE INVENTION
The present invention is directed to a device having an agglutination reaction chamber for performing agglutination immunoassay reactions. In one aspect, the device comprises a first wettable (hydrophilic) layer and a parallel second layer wherein the first layer has channels such that when the first and second layers are brought into contact with each other an aggl1~tination reaction chamber is formed for conducting fluid by capillary action. The agglutination reaction chamber can additionally include a sample recei~ing well contiguous with the ingress of the chamber formed by the first and W~92/08973 PCT/US91/~8573 (.. .. .
3 210~ 3 second layers. The chambers have means for controlling the flow of liquid (fluid) when a predetermined amount of reagent for performing an assay is dispersed in them, for example in a well. The means for controlling the overall rate of liquid flow thro~gh the chamber comprises utilizin~
a porous absorbent material such as an absorbent paper in liquid communication with the chamber, typically at the distal end of the chamber.
In the agglutination reaction chamber of the present invention the reagent can be present in dried spots or strips. It is also possible to suspend the reagent in a water-soluble polymer.
A copending United States Patent Application, Serial Number 07/138,253, filed on December 23, 1987, entitled "Agglutination Reaction Device" (the disclosure of which is hereby specifically incorporated herein by reference), teaches an agglutination reaction chamber which is constructed to be very small in size to accommodate automated and efficient use of sample and reagents.
Typically, the length of such a chamber is from about 10 to about 75 millimeters (mm), the channels are from about 0.01 to about 5.0 mm in depth and from about 0.1 to about 10.0 mm in width. A typical overall size for such an agglutination reaction device having four chambers and a sample receiving well is about 37.5 mm x 12.5 mm x 1.5 mm (1 x w x h).
The aforesaid copending United States Patent Application also generally discloses a means for controlling the flow of fluid in an agglutination reaction chamber involving the configuration of the channel or geometric formations within the channel such as ridges, particularly ridges formed in the channel which extend across the entire width of the channel and for at least a portion of the len~th of the channel. The aforesaid copending United States Patent Application also discloses SU8~3TITUTE SHEET
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WO9~/08973 PCT/~JS91/~8573 2~ ~363 another means for controlling the flow of fluid in the chamber, namely utilization of a water-soluble material, such as a water-soluble polymer, (e.g., polyvinylpyrrolidone, polyvinylalcohol, gelatin, or bovine serum albumin) dried in portions of the channel.
However, it has been found that such expedients, while useful in helping to control the overall rate of liquid (fluid) flow in the channels, can be difficult to employ so as to obtain consistently uniform results. For example, where a water-soluble polymer such as polyvinlypyrolidone is utilized, it has been found that it can be difficult to obtain dried coatings of the polyvinylpyrrolidone so as to obtain consistent stability of overall flow of liquid in the channels. Also, there are advantages with respect to the ease of manufacture of devices utilizing a porous absorbent medium such as paper compared to utili~ation of coatings such ae dried polyvinylpyrrolidone.
The present invention is directed to devices for performing agglutination reactions having impro~ed properties including improved means for controlling the overall rate of liquid flow through the agglutination chamber. The present invention also is directed to such devices constructed in the form of convenient, disposable structures, such ~s disposable, laminated cards, optionally mounted in disposable rigid containers.
BRIEF DESCRIPTION OF THE DR~WINGS
Figure 1 is a top perspective view of one embodiment of the present agglutination reaction device showing a rec~angular sample receiving well and four reaction chambers.
Figure 2 is a top perspective view of one SUE~STITUTE 5HIEE:T
~.~
'2~00363 embodiment of a bottom surface of the present agglutination reaction device showing a rectangular sample receiving well and four channels ~for chambers) having strips of porous material at their distal ends.
Figure 3 is an exploded, top perspective view of another embodiment showing a three layer structure comprising a first or base layer, a second layer showing a cutout for a round receiving well and a generally straight agglutination chamber, a strip of porous absorbent material, and a third or top layer.
Figure 4 is an exploded, top perspective view of another embodiment showing a three layer structure comprising a first or base layer, a second layer showing a cutout for a round receiving well and an agglutination chamber having a flared portion at its distal end, a strip of porous absorbent material, and a third or top layer.
Figure 5 is an exploded, top perspective view of another embodiment showing a three layer structure comprising a first or base layer, a second layer showing a cutout for a round receiving well and a generally straight agglutination chamber with an integral porous absorbent strip in the second layer at the distal end, and a third or top layer.
Figure 6 is an exploded, top perspective view of another embodiment showing a three layer structure comprising a first or base layer, a second layer showing a cutout for a round receiving well and an agglutination chamber having a flared portion at its distal end with an integral porous absorbent strip in the second layer at the distal end of the chamber, and a third or top layer.
Figure 7 is a top plan view of another embodiment showing the parts of a laminated structure comprising a base layer, a second layer~having a cutout for a round receiving well and multiple radiating agglutination chambers having flared distal zones, an annular structure SUBSTITUTE SHEET
- .
... ~.. . . . .... . .. . . .
WO9~/08973 PCT/US~1/08573 2~003~3 6 ,' having alternating liquid absorbent regions (4) and liquid-occlusive regions (26), and another round layer which in cooperation with the annular structure forms the top layer.
Fi~ure 8 is a schematic diagram illustrating regions of different flow rate per unit area outward from the receiving well for an agglutination chamber having a flared distal end, and illustratiny a band of agglutinated particles in the flared distal end.
Figure 9 is a schematic diagram illustrating regions of different flow rate per unit area outward from the receiving well for an agglutination chamber having semicircular, or bowl-shaped, distal end, and illustrating a band of agglutinated particles in the semicircular end.
Figure 10 is a schematic diagram illustrating regions of different flow rate per unit area outward from the receiving well for an agglutination chamber having an approximately rectangular shape, and illustrating a band of agglutinated particles in the rectangular area of increased width.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention is directed toward improved devices suitable for performing agglutination reactions.
One embodiment of the invention is shown in Figure 1. The device (10) as shown in Fig. 1 generally comprises a first surface (12) and a parallel second surface (14) one of which has channels (16) formed therein such that when the two surfaces are placed together a chamber or microchamber (18) is formed through which liquid will flow by capillary action. In order to have capillary flow it i5 rsquired that one of the channel surfaces be water wettable (hydrophilic). Preferably, the surfaces are designed such that they create a plurality of chambers or microchambers (18) when assembled. Each of the channels (18) has a strip 5lJBSTlTUTE 5HEET
, W092/~973 PC~/VS9t/08S~3 ~ ~ . " ~
of porous, absorbent material, preferably a cellulosic material, more preferably paper, at the distal end of each channel for controlling the overall rate of flow of liquid through the channel. The aforesaid porous material, for example paper, is to be distinguished from water-soluble materials such as dried coatings of water-solùble polymers such as polyvinylpyrrolidone, polyvinylalcohol, gelatin, or bovine serum albumin. The porous absorbent material utilized in present invention is itself generally not water-soluble.
The surface having channels grooved therein also preferably has a sample receiving well (20) which is open to each of the channels (16). The receiving well (20) is positioned such that it is not covered by the second surface (14) as shown in Fig. 1. A liquid sample can be placed in the receiving well (20), such that it can be wicked into the chambers (18) by capillary action. Thus it is possible to form a device having multiple chambers and one large sample receiving well such that a single drop of sample can be placed in the well and wicked into the multiple chambers. Each chamber can then perform a different agglutination assay on a single sample, the porous absorbent strip in the distal end of each channel advantageously controlling the overall rate of liquid flow through the channel.
It is a further objective of the present invention to provide a device which is small enough to be utilized in automated instrumentation and monitored by automated means for the presence of agglutinated particles in each of the chambers. Thus in another embodiment of the agglutination device, the lower surface (12) will be opaque and the upper surface (14) will be transparent to transmitted or reflected light where an optical scanner can be positioned above the device (10). Thus, the opaque lower surface (12) gives a better background for detection SUBSTITUTE SH~ET
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2~3~3 ~
of agglutinated particles in this embodiment.
For example, when a solution of cells is introduced to one end of a chamber (18), containing antibodies directed against antigens on the cells and dried onto the interior of the channels (16), the solution will migrate ~hrough the channel, mix with the antisera, and the cells will aggregate. This will all occur without any centrifugation or mixing steps. Control of the flow of the liquid through the channel is necessary because the agglutination reaction occurs preferably during the period of liquid flow. Sufficient incubation time must be incorporated into the period of liquid flow to achieve optimum reaction of the reagents.
The rate of liquid flow through a capillary chamber in the present embodiment is controlled by means of a strip of porous, absorbent material (22) positioned in the distal end of each channel (16) of the agglutination reaction chamber. Preferably the porous material is a cellulosic material, more preferably paper such as Whatman filter paper. In this embodiment represented in Figure 1, one can consider the reaction zone of the agglutination chamber as ending just at the beginning of the strip of paper (22). It has been found that porous material such as paper utilized as the fluid flow control means provides advantages in both manufacturing and performance over the utiIization of coatings of water-soluble materials such as polyvinylpyrrolidone (PVP).
Figures 3 and 4 represent other embodiments of a device for performing agglutination reactions according to the invention. These embodiments have, in adherent relationship, a first wettable, but liquid-occlusive, layer (1), a second liquid-occlusive layer (2) parallel to and overlying ~he first layer (1), and a third liquid-occlusive, preferably non-wettable, layer (3) parallel to and overlying the second layer (2) and having a window, or r~ s~EEr .. . . . . .
... . ;. -.. - ~,. ~ . .... .. ... . . .
.. ~, . .... ~ .. .... - . 1 ~ ;.. ,.; .. .. . . .
W092/08973 ~CT/US91/08~73 ,..
9 2~3 viewing area, for observing particles. The first layer (l) is made of a liquid-occlusive material having a water-wettable surface. In this embodinlent the third layer (3) is made from a clear, liquid-occlusive, non-wettable, film, such as a clear polycarbonate film, and therefore also serves as a window, or viewing area, for observing particles in the agglutination chamber. The second layer (2) is interposed between, and is adhered to, the first layer (l) and third layer (3), for example by means of an adhesive on each side of layer (2) facing the topside of the first layer (l) and the underside of the third layer (3) respectively. The second layer (2) has a slot (25) cut through its thickness defining a channel for directing liquid for conduction by capillary action through the chamber defined by the slot (25) in conjunction with the first (l) and third (3) layers respectively.
In other words, when the first, second and third layers are laminated together, a portion of each of the first and third layers serve respectively as the floor and roof of the agglutination chamber with part of the walls of the slot (25) of the second layer (2) defining the walls (9) of the chamber. The agglutination reaction chamber has a proximate zone (6) and a distal zone (7), which proximate zone (6) for ex~mple in Figure 4 is represented by the génerally rectangular portion of the slot (25) of the second layer (2) with the distal zone (7) being represented by the deltoid or flared portion of the slot (25) of the second layer (2).
Each of the embodiments illustrated by Figures 3 and 4 has a well-defining slot (8) in the third layer (3) and a corresponding second well-defining slot (5) in the second layer (2) of the same size and configuration as the well~defining slot (8) in the third layer (3). The well-defining slot (5) in the second layer (2) is positioned directly below the well-defining slot (8) in the third ... .
SUBSTITWTE SHEET
WO92/Og973 PC~/US91/08573 ~: .
21 0036~
layer (3) such that when all three layers are laminated together, the second well-defining slot (5) in conjunction with the well-defining slot (8) along with the corresponding portion of the first layer define a well for receiving liquid, the well being in liquid communication with the proximate zone (6) of the chamber. The bottom of the well is formed from a corresponding circular portion of the first layer (1) which portion can be considered to be the projection of the outline of slots t5) and (8) onto the surface of layer ~1).
The overall rate of liquid flow through the agglutination chamber in these embodiments is controlled by means of a strip of porous absorbent material (4), pLeferably filter paper, in liquid communication with the chamber and positioned adjacent to the distal end of the chamber, and preferably extending partially into the distal end of the chamber, when the structures of Figures 3 and 4 are laminated respectively together. In a more preferred embodiment, layer (3) as shown in Figures 3 and 4 has a slot (28), of slightly larger dimensions as the strip of porous paper (4), such that when the respective layers are adhered together, the strip of porous absorbent material (4) lies partially within the slot (28), more particularly so that a front minor portion of the strip (4) lies within the distal zone (7) of the slot (25) with the remaining major portion of the strip lying within slot (28), so as to prevent disadvantageous formation of microcapillary channels at the sides of and along the length of the strip (4).
The resulting laminated structure, can be thought of as being in the form of a thin, disposable card with the paper strip (4) being in liquid communication with the distal zone (7) of the agglutination chamber.
Figures 5 and 6 represent other embodiments of a device, in the form of a laminated card when the layers SL)B5TITUTE 5~11EET
ll 21~ 3 shown in the Figures are adhered together, for performing agglutination reactions. These embodiments have, in adherent relationship, a first wettable, but liquid-occlusive, layer (1), a second layer (2) parallel to and overlying the first layer (1), and a third liquid-occlusive, preferably non-wettable, layer (3) parallel to and overlying the second layer (2) and having a window, or viewing area, for observing particles. The first layer (1) is made of a liquid-occlusive material having a water-wettable surface. As in the embodiments represented by Figures 3 and 4, these embodiments also utilize a third layer (3) made from a clear, liquid-occlusive, preferably non-wettable film, such as a clear polycarbonate film or a non-wettable cellophane tape, which therefore also serves as a window for observing particles in the agglutination chamber. The second layer (2) is interposed between, and is adhered to, the first layer (1) and third layer (3), for example by means of an adhesive on each side of layer (2) facing the topside of the first layer (1) and the underside of the third layer (3) respectively. The second layer (2) has a slot (25) cut through its thickness defining a channel for directing liquid for conduction by capillary action through the chamber defined by the slot (25) in conjunction with the first (1) and third (3) layers respectively. .
As in the embodiments represented by Figures 3 and 4, when the first, second and third layers axe laminated together a portion of each of the first and third layers serve respectively as the floor and roof of the agglutination~chamber with part of the walls of the slot (25) of the second layer (2) defining the walls (9) of the chamber, the other part of the walls of slot (25) defining the walls of the circular receiving well (5). The agglutination reaction chamber has a proximate zone (6~ and a distal zone (7), which proximate zone (6) for example in .
. ... .... . . ....... . ... . .
W092/0~973 ~C~`/US91/08573 21~3~ ~
Figure 6 is represented by the generally rectangular portion of the slot (25) o~ the second layer (2) with the distal zone (7) being represented by the deltoid or flared portion of the slot (25) of the second layer (2).
Each of the embodiments illustrated by Figures 5 and 6 has a well-defining slot (8) in the thixd layer (3) and a corresponding second well-defining slot (5) in the second layer (2) of the same size and configuration as the well-defining slot (8) in the third layer (3). The well-defining slot (5) in the second layer (2) is positioned directly below the well-defining slot (8) in the third layer (3) such that when all three layers are laminated together, the second well-defining slot (5) in conjunction with the well-de~ining slot (8) along with the corresponding portion of the first layer define a well for receiving liquid, the well being in liquid communication with the proximate zone (6) of the chamber. The bottom of the well is formed from a corresponding circular portion of the first layer (1) which portion can be considered to be the projection of ~he outline of slots (S) and (6) onto the surface of layer (1).
However, in the embodiments of Figures 5 and 6, the second layer (2) is made of a liquid absorbent material, such as absorbent paper, selectively impregnated through its thickness with a substance, such as a water-repellent ink, to form an impregnated region (26) and a non-impregnated region (~). The non-impregnated region (4) is liquid absorbent and the impregnated region (26) is liquid-occlusive. In these embodiments, the non-impregnated region (4) which is in liquid communication with the distal zone (7) of the chamber serves as the means for controlling the overall rate of liqui.d flow through the agglutination chamber. The second la~er (2) also has a slot (25) in the impregnated region (26) defining a channel for directing liquid conducted by capillary action through SlJB51~1TlL)T~ SHE:~;T
- .. . `.` . ` .. .. ~. . ` . . , ~ . . . .. . .. .. .
W092/08973 PCT/U~l/0~573 ~,.'.
13 21~3~3 a chamber defined by the slot (25) in conjunction with the first layer (1) and third layer (3). This chamber also has a proximate zone (6) and a distal zone (7). It is within this chamber that agglutination reactions can be performed.
As can be seen from Figures 5 and 6, the non-impregnated region (4) is located adjacent to the distal end of the agglutination chamber and is in liquid communication with the chamber.
Also in the embodiments illustrated by Figures 5 and 6 there is a well-defining slot (8) in the third layer (3) and a corresponding second well-defining slot (5) in the second layer (2) of the same size and configuration as the well-defining slot (8) in the third layer (3). The well-defining slot (5) in the second layer (2) is positioned directly below the well-defining slot (8) in the third layer (3) such that when all three layers are laminated together, the second well-defining slot (5) in conjunction with the well-defining slot (8) along with the corresponding portion of the first layer (l) define a circular well for receiving liquid, the well being in liquid communication with the proximate zone (6) of the chamber. The bottom of the well is formed from a corresponding circular portion of the first layer (l).
Figure 7 illustrates an exploded, plan view of a preferred embodiment of the invention. This embodiment provides for performing a plurality of agglutination reactions utilizing a minimal amount of liquid sample. The -device in assembled form can be thought of a relatively thin, laminated, disposable structure having in this particular illustration six agglutination chambers radiating from a common liquid receiving well. The device of Figure 7 comprises, in adherent relationship, an approximately circular first wettable but liquid-occlusive layer (1), an approximately circular second liquid-occlusive layer (2) parallel to and overlying the first , SVBSTITUTE SHEET
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W092/08973 PCT/US91/~8573 2~0~363 layer (1), and a third liquid-occlusive layer (3) parallel to and overlying the second layer (2). These respective layers can be bonded together, for example, by means of an adhesive between the respective layers. In this embodiment the third layer (3) is made of a circular clear plastic film, such as a polycarbonate film, thereby providing windows, or viewing areas, ~or observing particles in the six radiating agglutination chambers. The second layer (2), interposed between and in adherent relationship to the first and third layers has a slot (25) in the form of a central, circular portion (5) having si~ radial, slotted arms extending outward therefrom. ~hese radial arms of the slot (25) define six channels for directing liquid conducted by capillary action through chambers respectively defined by the radial, slotted arms in conjunction with the first layer (1) and the third layer ~3). Within the resulting six chambers agglutination reactions can be performed simultaneously. Each of the six chambers has a generally rectangular proximate zone (6) and a generally flared or deltoid shaped distal zone (7). The overall rate of liquid flow through each agglutination chamber in this embodiment is controlled by means of a strip of porous absorbent material (4)r preferably filter paper, projecting from a generally annular ring (27) of such porous material having a hole (28), into the distal zone (7) of each of the channels defined by the radial, slotted arms. The annular ring (27) is selectively impregnated through its thickness with a substance to provide alternating non-impregnated liguid absorbent regions (4) and impregnated liquid-occlusive regions (26). These non-impregnated strips (4) of paper projecting from the annular ring (27) are in liquid communication with the chambers and are positioned adjacent to the distal ends of the chambers, pre~erably positioned partially in the distaI ends, when the structures of Figure 7 are laminated respectively together.
SUBSTITUTE SI~EE:T
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.. -.. .... , .. , -.. . ... , , .. , . , ., ..... . . ,-, ~, .. .. ,.. ..
W092/08973 PCI/US9l/0~S73 2~3~3 The third layer (3) of the device represented by Figure 7 has a circular well-defining slot (8), and the second layer has a corresponding circular second well-defining slot (5) of the same size and configuration as the well-defining slot (8) in the third layer (3). The well-defining slot (5) of the second layer (2) is positioned directly below the well-defining slot in the third layer (3) in the assembled configuration. Thus the second well-defining slot (5) in conjunction with the well-defining slot ~8) in the third layer (3) and the respective circular portion of the first layer (l) define a well for receiving liquid, the well being in liquid communication with the proximate zone (6) of each of the chambers.
The resulting, generally circular laminated structure, can be thought of as being in the form of a relatively thin, disposable card with the fluid-absorbent paper strip (4) being in liquid communication with the distal zone (7) of the agglutination chamber. Thus, in all of the embodiments of the present invention, the device of the invention has means for controlling overall rate of liquid flow through the reaction chamber which is a porous absorbent material in liquid communication with the chamber, which material is typically positioned adjacent to, and usually extending partially into, the distal end of the chamber. However, it has been found that the utility of devices of the present invention, for performing agglutination reactions, can be enhanced further by utilizing an additional means for controlling the flow of liquid through the reaction chamber of the device, by modifying the geometric configuration of the chamber or the internal shape of the chamber as illustrated in Figures 4, 6, 7 and 8.
For sxample, in Figures 4, 6, and 7, the general slot ~25) in layer (2) defines at least approximately parallel walls (9) in the proximate zone (6) of the chamber SUBSTITUTE SHEc:T
` - . , . .... , ., " . .. ... ,..... ; . . ...... ..
WO9~/08973 PCr/US91/08573 2~ 3~3 ~, thereby defining a first path of approximately constant width. Looking in the direction toward the distal end of the chamber, the general slot (25) defines walls in the distal zone (7) which are spaced to define a second path of increased width compared to the first path of the proximate zone (6). It has been found that agglutination reactions perfo~ed in such a chamber advantageously can result, surprisingly, in the formation of one or more patterned formations, such as, for example bands, of agglutinated particles in the distal zone (7) of the chamber which patterns are more easily observable through the window of the third layer (3) than non patterned aggregates of agglutinated particles which generally result in agglutination chambers of the prior art. Figure 8 shows a schematic representation of an approximately semicircular band (27) of agglutinated particles in the zone of increasing chamber width, namely in the flared (here approximately deltoid-shaped) "second path" of the chamber in the distal zone (7) of the chamber. As represented in schematic form in Figure 8 through the use of arrows of different length along the reaction path in the chamber, the walls in the distal zone (7) are spaced to provide a decreased liquid flow rate per unit area of liquid path along this second path. In Figure 8, the shorter arrows are, of course, intended to represent smaller flow rate per unit area of path, compared to that represented by the longer arrows.
While deltoid-shaped configurations of the second path of the distal zone in the chambers is preferred, it has been found that other geometric conigurations for this so-called "second path" provide advantageous patterned formations of agglutinated particles. For example, the side walls in the second path can be formed to be convex giving an approximately semicircular or bowl-shaped configuration to the second path as illustrated in SUBS~ E SH. -~ ~
-WO92/08973 PCT/US~1/08573 .
2~ ~3~3 Figure 9. Alternatively, although less pxeferred, the side walls of the second path can be formed to provide a second path with an approximately rectangular shape as illustrated in Figure 10.
Moreover, if desired, the flow rate per unit area in the distal zone of the reaction chamber can be gradually decreased along the general direction of flow by gradually increasing the space between the floor and the roof of the chamber along the direction of liquid flow, for example by gradually bowing the roof of the chamber in the distal zone upward and/or gradually bowing the floor of the chamber in the distal zone downward. It has been found that such modification of the space between the floor and the roof of the chamber in the distal zone of the chamber can also contribute to the formation of regular patterns of agglutinated particles being formed in the distal zone of the chamber. For example, the space between the floor and the roof of the chamber can be gradually increased by stamping a spherical dome-shaped or cylindrical dome-shaped configuration in an area of the third layer (3) in such manner that when the third layer is adhered to the second layer (2) the dome in the third layer overlies the distal zone of the reaction chamber. Another example or a way to provide a gradually increasing space between the floor and the roof of the distal zone of the reaction chamber is to stamp a spherical bowl-shaped or cylindrical bowl-shaped depression in the base or first layer (l) in such manner that when the first layer (l) is adhered to the second layer (2) the bowl_shaped depression occurs in the floor of the distal zone of the reaction chamber.
All types of agglutination-based assays can be accommodated with a device according to the present invention. In some instances, a soluble reag~nt can be dried as spots or strips in the reaction chamber, for example, in blood typing. In other instances, a .
:
SUBSTIT~ITE SHEE~
W092/08973 P~/VS91/08573 2~363 ~ , particulate reagent, such as a latex reagent, can be dried in the chamber. In yet another approachr a reagent can be dispersed in a solution which is placed in the chamber.
One preferred reagent solution is microparticulates in a solution of dextran and sucrose. Preferably, the microparticulate reagent is mixed in a solution of about 2.5 to about 5.0 percent by weight dextran and from about to about 20 percent by weight sucrose. Another preferred solution for mixing reagents is FICOLL (a trademark by Sigma Chemical Co., St. Louis, MO for a nonionic synthetic polymer of sucrose) from about 20 to about 3~ percent by weight. Also, depending on the requirements of the assay, the flow of the liquid through the chamber can be controlled as described above to accommodate any necessary incubation times and assay sequences.
A particularly unique feature of the present invention is that it protTides for the ability to simultaneously perform multiple assays while utilizing a very small amount of sample material, for instance, a single drop. Also, the agglutination a~say is essentially self-performing once the drop has been added to the agglutination reaction device. Moreover, in those embodiments of the invention utilizing an additional means for controlling the flow of liquid through the reaction chamber of the device, namely by modification of the geometric configuration of the chamber or the internal shape of the chamber as discussed above, additional enhanced results can be obtained such as enhanced observability of aggregates of agglutinated particles in the distal zone of the reaction chamber.
A device of the invention is especially suitable for use in an automated fashion where the agglutination reaction can be monitored by an optical scanner. For example, the construction of the agglutination reaction 5VE~STITIIJTE SH ET
- . ~ . ~ . . . . ............................... .
.
W092/08973 PCT~US91/~8573 ~, .
2l~3~3 device enables one to use an image analysis system available from Olympus (CUE-2, LaXe Success, N.Y.) to determine the quantity and concentration of agglutinated material. The agglutination reaction device is illuminated, such ~hat transmitted or reflected light can be read by the reader. The image is then compùter analyzed to determine the quantity of agglutination which has occurred and to enhance the image for very accurate and sensitive determinations. By confining the sample to a chamber such as formed in the agglutination reaction device, there is no problem with curvatures of droplets or water which could interfere with the image seen by the reader. Thus, the uniformity of the reacted sample and reagents achieved by the agglutination reaction device provides an excellent imaging format for a reader or other imaging devices. Besides being able to read the transmission of light through the bottom of the agglutination reaction device, it is also possible to read reflected light because the sample and reacted reagents are confined to capillary chambers formed by the agglutination reaction device.
Again referring to Figure 1, one embodiment of the present invention is shown. Figure 1 shows a perspective view of device (10) constructed of two layers of material, a bottom layer (12) covered by a top layer (14). Layer (12) has a plurality of channels (16~ and a sample well (20) formed into the surface. The sample well (20) is contiguous with the ingress of each of the chambers (16). In the construction of the device (10), either the bottom layer (12) or the upper la~er (14) can be opaque.
Preferably the layer which is further from an optical scanning device is opaque to enhance the background. It is required that the bottom surface (12) be hydrophilic or wettable such that the capillary flow is induced when a sample is placed in contact with the ingress of a chamber SUBS I ITUTE S~cT
.
W~92/08973 PCT/~S91~08~73 '~L0~3~
(18). This can be accomplished by using a hydrophilic material for the surface (12); however, it is also possible to chemically treat or coat otherwise non-wettable (hydrophobic) materials such that they become wettable.
This preparation of a wettable surface can also be used to influence the flow rate in the capillary chamber (18) so formed.
Suitable materials for preparlngia wettable layer for various embodiments of the invention include, for example, cellulose acetate butyrate, or a wettable nylon material, or a layer coated with an acrylic latex emulsion to render the surface water-wettable. For example in one embodiment of the invention, an agglutination reaction device is prepared by molding a layer of cellulose acetate butyrate (CAB), commercially available from Eastman Chem.
Prod. Inc., Kingsport, TN, to have a plurality of channels (16) from about .010 to about 5 millimeters in depth and from about 0.1 to about 10 millimeters in width. ~ach of the channels (16) extends from a larger well (20) molded into one end of the layer (12~ of CAB. Next, a piece of transparent tape (14) sufficient to cover all the channels (16~ molded into the CAB is applied to the surface (12) to form the capillary chambers (18). A section of adhesive cellophane tape can be used to provide the upper cover or surface for the recessed surface (14) to form the capillary chambers (18). Other non-wettable (hydrophobic) materials can be used to form the upper surface (14) of the chambers (18). The preferred overall dimensions of this embodiment of the an agglutination reaction device of the in~ention is from about 10 to about 75 mm in length, from about 5 to about 20 mm in width and from about 0.5 to about 5.0 mm thick. The dimensions of the channels in the wettable surface are preferably from about 0.01 to about 5.0 mm in depth and from about 0.1 to about 10.0 mm in width.
The very small size of the reaction devices of SUBSTITUTE SHEE:T
.;;,~
21 21~363 the invention allows for the rapid and convenient handling of a plurality of devices and therefore samples. A device can then be loaded into an automated apparatus which indexes and scans the individual channels for the assay result and records this information for future access. The ~mall dimensions of the agglutination reaction device also provide for efficient use of sample and reagents.
The following examples are provided to further illustrate embodiments of the invention and should not be construed as a limitation on the scope of the invention.
Laminate disposable cards were prepared by assembling together a wettable base layer, a die cut adhesive core layer, paper strip assemblies, and a clear polycarbonate top assembly as shown in Figure 3. To prepare the wettable base layer, 1 mil thick nylon film (Capran Emblem 2500, Allied Signal, Morristown, New Jersey) was first laminated onto a paperboard backing (Westvaco Hi Yield PrintKote, 16 mil, New York, NY) through the use of a two-sided adhesive layer (Fasson Fastape A, Fasson Specialty Division, Avery, Painesville, OH). Base subassemblies (3"X6", i.e., 3 inches X 6 inches) were cut from this material, using care to keep the exposed nylon surface clean. Steel rule dies were prepared to cut the channel shapes as shown in Figure 3 from a second sheet of two-sided adhesive (3.1 mil, Specialty Tapes, Division of RSW Inc., Racine, WI) which has release liner on both adhesive surfaces. One piece of release liner was removed from the die-cut part and this adhesive layer was placed onto the nylon surface of the base subassembly. Pieces of filter paper (2.5Xl9 millimeter, 1 CHR, Whatman, Clifton, New Jersey) which have a layer of one-sided adhesive (ARCare 7S97, Adhesive Research, Glen RocX, PA) laminated ;SVBST~TUTE ~;HEET
W O 92/08973 P~r~US~1/08573 2iO~363 to one surface were positioned on the base/core subassemblies with the one-sided adhesive away from the card. Finally, a sheet of clear polycarbonate film (GE
Part 8040-112, Cadillac Plastics, Evansville, IN) was die~
cut as shown in it~m (3) of Figure 3, and laminated onto the Base/core/paper subassembly using a mechanical laminator set a' 50 psi and 0.2 ft/sec.
Laminate disposable cards were prepared using a 3--X6 piece of paperboard coated with a wettable acrylic latex emulsion coat (Part 150HT(26-1), Daubert Coated Products, Dixon, IL) in place of the nylon base subassemblies described in Example 1. Die-cut core layers were prepared using 3.1 mil two-sided adhesive (ARCare 7580, Adhesive Research, Glen Rock, PA). All other steps in card assembly were identical to those of Example 1.
Fixed human erythrocytes (Duracytes TM, Abbott Laboratories, North Chicago, IL) were coated with affinity purified goat antibodies directed against Hepatitis s surface antigen (HBsAg) at a ~inal concentration of 100 ug/ml (micrograms/milliliter) in the presence of 0.05%
(weight/volume) chromic chloride in 0.1 M (Molar) acetate buffer at a pH of 4Ø These cells were overcoated with 1%
(weight to volume; w/v) human serum albumin (Sigma Chemical Co., St. Louis, MO) in 25 mM (millimolar) Tris-HCl (pH =
7.4) buffer and then resuspended with 0.1% bovine serum albumin (BSA)(Sigma Chemical Co., St. Louis, MO) in phosphate buffered saline (pH=7.4) containing 5%
(volume/volume) normal goat serum at a final cell concentration of 10% (volume/volume). Serum samples (20 SLJ~S7-lTllTE 511E~:ET
W ~ 92/08973 PC~r/US9~/08573 - 2 1 ~ 3 ul; i.e., 20 microliter) containing either 0, 6.25, or 25 ng/ml (nanograms/milliliter) of HBsAg were mixed with 10 ul (microliter) aliquots of these coated Duracytes and the solution was immediately added to the sample addition well of laminate disposable cards prepared as described in Example 1. The solutions flowed rapidly through the capillary channel (1-2 seconds; sec) and then slowly flowed into the paper strips. It took approximately 7 minutes for the liquid to completely saturate the paper strip. After the paper strips had completely wetted, agglutinated reaction products of the Duracyte cells could be observed within certain of the capillary channels of ~he laminate disposable cards. As seen in Table 1, Duracytes which had been mixed with samples containing HBsAg agsregated, whereas the duracytes which were mixed with sera which did not contain HBsAg, did not aggregate.
HBsAq Concentration Aqqreqation 6.25 ng/ml +/-25 ng/ml +
Laminate disposable cards were prepared as described in Example 2 with a flared channel design as shown in Figure 4. Duracytes coated with anti-HBsAg (Example 3) were mixed with sera containing various concentrations of HBsAg and were introduced into the laminate disposable cards having flared channels. After 5 SUBS~lTllTE S~tEtT
-,,, . ., . I . , .
--, W O 92/08973 P~r/VS91/08573 21003~3 minutes, aggregated particles appeared and formed into an easily visible band of agglutinates which stretched across the flared portion of the channel as shown in Figure 8. In channels where there was not any HBsAg present, the Duracytes did not aggregate and no band of cells was visible.
.
a porous absorbent material such as an absorbent paper in liquid communication with the chamber, typically at the distal end of the chamber.
In the agglutination reaction chamber of the present invention the reagent can be present in dried spots or strips. It is also possible to suspend the reagent in a water-soluble polymer.
A copending United States Patent Application, Serial Number 07/138,253, filed on December 23, 1987, entitled "Agglutination Reaction Device" (the disclosure of which is hereby specifically incorporated herein by reference), teaches an agglutination reaction chamber which is constructed to be very small in size to accommodate automated and efficient use of sample and reagents.
Typically, the length of such a chamber is from about 10 to about 75 millimeters (mm), the channels are from about 0.01 to about 5.0 mm in depth and from about 0.1 to about 10.0 mm in width. A typical overall size for such an agglutination reaction device having four chambers and a sample receiving well is about 37.5 mm x 12.5 mm x 1.5 mm (1 x w x h).
The aforesaid copending United States Patent Application also generally discloses a means for controlling the flow of fluid in an agglutination reaction chamber involving the configuration of the channel or geometric formations within the channel such as ridges, particularly ridges formed in the channel which extend across the entire width of the channel and for at least a portion of the len~th of the channel. The aforesaid copending United States Patent Application also discloses SU8~3TITUTE SHEET
. .. ... . - ... `. . , . . - . . ,.. .. - . .. . . ., ., . . . ,. . . . . .:: , ~
WO9~/08973 PCT/~JS91/~8573 2~ ~363 another means for controlling the flow of fluid in the chamber, namely utilization of a water-soluble material, such as a water-soluble polymer, (e.g., polyvinylpyrrolidone, polyvinylalcohol, gelatin, or bovine serum albumin) dried in portions of the channel.
However, it has been found that such expedients, while useful in helping to control the overall rate of liquid (fluid) flow in the channels, can be difficult to employ so as to obtain consistently uniform results. For example, where a water-soluble polymer such as polyvinlypyrolidone is utilized, it has been found that it can be difficult to obtain dried coatings of the polyvinylpyrrolidone so as to obtain consistent stability of overall flow of liquid in the channels. Also, there are advantages with respect to the ease of manufacture of devices utilizing a porous absorbent medium such as paper compared to utili~ation of coatings such ae dried polyvinylpyrrolidone.
The present invention is directed to devices for performing agglutination reactions having impro~ed properties including improved means for controlling the overall rate of liquid flow through the agglutination chamber. The present invention also is directed to such devices constructed in the form of convenient, disposable structures, such ~s disposable, laminated cards, optionally mounted in disposable rigid containers.
BRIEF DESCRIPTION OF THE DR~WINGS
Figure 1 is a top perspective view of one embodiment of the present agglutination reaction device showing a rec~angular sample receiving well and four reaction chambers.
Figure 2 is a top perspective view of one SUE~STITUTE 5HIEE:T
~.~
'2~00363 embodiment of a bottom surface of the present agglutination reaction device showing a rectangular sample receiving well and four channels ~for chambers) having strips of porous material at their distal ends.
Figure 3 is an exploded, top perspective view of another embodiment showing a three layer structure comprising a first or base layer, a second layer showing a cutout for a round receiving well and a generally straight agglutination chamber, a strip of porous absorbent material, and a third or top layer.
Figure 4 is an exploded, top perspective view of another embodiment showing a three layer structure comprising a first or base layer, a second layer showing a cutout for a round receiving well and an agglutination chamber having a flared portion at its distal end, a strip of porous absorbent material, and a third or top layer.
Figure 5 is an exploded, top perspective view of another embodiment showing a three layer structure comprising a first or base layer, a second layer showing a cutout for a round receiving well and a generally straight agglutination chamber with an integral porous absorbent strip in the second layer at the distal end, and a third or top layer.
Figure 6 is an exploded, top perspective view of another embodiment showing a three layer structure comprising a first or base layer, a second layer showing a cutout for a round receiving well and an agglutination chamber having a flared portion at its distal end with an integral porous absorbent strip in the second layer at the distal end of the chamber, and a third or top layer.
Figure 7 is a top plan view of another embodiment showing the parts of a laminated structure comprising a base layer, a second layer~having a cutout for a round receiving well and multiple radiating agglutination chambers having flared distal zones, an annular structure SUBSTITUTE SHEET
- .
... ~.. . . . .... . .. . . .
WO9~/08973 PCT/US~1/08573 2~003~3 6 ,' having alternating liquid absorbent regions (4) and liquid-occlusive regions (26), and another round layer which in cooperation with the annular structure forms the top layer.
Fi~ure 8 is a schematic diagram illustrating regions of different flow rate per unit area outward from the receiving well for an agglutination chamber having a flared distal end, and illustratiny a band of agglutinated particles in the flared distal end.
Figure 9 is a schematic diagram illustrating regions of different flow rate per unit area outward from the receiving well for an agglutination chamber having semicircular, or bowl-shaped, distal end, and illustrating a band of agglutinated particles in the semicircular end.
Figure 10 is a schematic diagram illustrating regions of different flow rate per unit area outward from the receiving well for an agglutination chamber having an approximately rectangular shape, and illustrating a band of agglutinated particles in the rectangular area of increased width.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention is directed toward improved devices suitable for performing agglutination reactions.
One embodiment of the invention is shown in Figure 1. The device (10) as shown in Fig. 1 generally comprises a first surface (12) and a parallel second surface (14) one of which has channels (16) formed therein such that when the two surfaces are placed together a chamber or microchamber (18) is formed through which liquid will flow by capillary action. In order to have capillary flow it i5 rsquired that one of the channel surfaces be water wettable (hydrophilic). Preferably, the surfaces are designed such that they create a plurality of chambers or microchambers (18) when assembled. Each of the channels (18) has a strip 5lJBSTlTUTE 5HEET
, W092/~973 PC~/VS9t/08S~3 ~ ~ . " ~
of porous, absorbent material, preferably a cellulosic material, more preferably paper, at the distal end of each channel for controlling the overall rate of flow of liquid through the channel. The aforesaid porous material, for example paper, is to be distinguished from water-soluble materials such as dried coatings of water-solùble polymers such as polyvinylpyrrolidone, polyvinylalcohol, gelatin, or bovine serum albumin. The porous absorbent material utilized in present invention is itself generally not water-soluble.
The surface having channels grooved therein also preferably has a sample receiving well (20) which is open to each of the channels (16). The receiving well (20) is positioned such that it is not covered by the second surface (14) as shown in Fig. 1. A liquid sample can be placed in the receiving well (20), such that it can be wicked into the chambers (18) by capillary action. Thus it is possible to form a device having multiple chambers and one large sample receiving well such that a single drop of sample can be placed in the well and wicked into the multiple chambers. Each chamber can then perform a different agglutination assay on a single sample, the porous absorbent strip in the distal end of each channel advantageously controlling the overall rate of liquid flow through the channel.
It is a further objective of the present invention to provide a device which is small enough to be utilized in automated instrumentation and monitored by automated means for the presence of agglutinated particles in each of the chambers. Thus in another embodiment of the agglutination device, the lower surface (12) will be opaque and the upper surface (14) will be transparent to transmitted or reflected light where an optical scanner can be positioned above the device (10). Thus, the opaque lower surface (12) gives a better background for detection SUBSTITUTE SH~ET
: . ~ . . .. . . . ... . . . .... . . .
2~3~3 ~
of agglutinated particles in this embodiment.
For example, when a solution of cells is introduced to one end of a chamber (18), containing antibodies directed against antigens on the cells and dried onto the interior of the channels (16), the solution will migrate ~hrough the channel, mix with the antisera, and the cells will aggregate. This will all occur without any centrifugation or mixing steps. Control of the flow of the liquid through the channel is necessary because the agglutination reaction occurs preferably during the period of liquid flow. Sufficient incubation time must be incorporated into the period of liquid flow to achieve optimum reaction of the reagents.
The rate of liquid flow through a capillary chamber in the present embodiment is controlled by means of a strip of porous, absorbent material (22) positioned in the distal end of each channel (16) of the agglutination reaction chamber. Preferably the porous material is a cellulosic material, more preferably paper such as Whatman filter paper. In this embodiment represented in Figure 1, one can consider the reaction zone of the agglutination chamber as ending just at the beginning of the strip of paper (22). It has been found that porous material such as paper utilized as the fluid flow control means provides advantages in both manufacturing and performance over the utiIization of coatings of water-soluble materials such as polyvinylpyrrolidone (PVP).
Figures 3 and 4 represent other embodiments of a device for performing agglutination reactions according to the invention. These embodiments have, in adherent relationship, a first wettable, but liquid-occlusive, layer (1), a second liquid-occlusive layer (2) parallel to and overlying ~he first layer (1), and a third liquid-occlusive, preferably non-wettable, layer (3) parallel to and overlying the second layer (2) and having a window, or r~ s~EEr .. . . . . .
... . ;. -.. - ~,. ~ . .... .. ... . . .
.. ~, . .... ~ .. .... - . 1 ~ ;.. ,.; .. .. . . .
W092/08973 ~CT/US91/08~73 ,..
9 2~3 viewing area, for observing particles. The first layer (l) is made of a liquid-occlusive material having a water-wettable surface. In this embodinlent the third layer (3) is made from a clear, liquid-occlusive, non-wettable, film, such as a clear polycarbonate film, and therefore also serves as a window, or viewing area, for observing particles in the agglutination chamber. The second layer (2) is interposed between, and is adhered to, the first layer (l) and third layer (3), for example by means of an adhesive on each side of layer (2) facing the topside of the first layer (l) and the underside of the third layer (3) respectively. The second layer (2) has a slot (25) cut through its thickness defining a channel for directing liquid for conduction by capillary action through the chamber defined by the slot (25) in conjunction with the first (l) and third (3) layers respectively.
In other words, when the first, second and third layers are laminated together, a portion of each of the first and third layers serve respectively as the floor and roof of the agglutination chamber with part of the walls of the slot (25) of the second layer (2) defining the walls (9) of the chamber. The agglutination reaction chamber has a proximate zone (6) and a distal zone (7), which proximate zone (6) for ex~mple in Figure 4 is represented by the génerally rectangular portion of the slot (25) of the second layer (2) with the distal zone (7) being represented by the deltoid or flared portion of the slot (25) of the second layer (2).
Each of the embodiments illustrated by Figures 3 and 4 has a well-defining slot (8) in the third layer (3) and a corresponding second well-defining slot (5) in the second layer (2) of the same size and configuration as the well~defining slot (8) in the third layer (3). The well-defining slot (5) in the second layer (2) is positioned directly below the well-defining slot (8) in the third ... .
SUBSTITWTE SHEET
WO92/Og973 PC~/US91/08573 ~: .
21 0036~
layer (3) such that when all three layers are laminated together, the second well-defining slot (5) in conjunction with the well-defining slot (8) along with the corresponding portion of the first layer define a well for receiving liquid, the well being in liquid communication with the proximate zone (6) of the chamber. The bottom of the well is formed from a corresponding circular portion of the first layer (1) which portion can be considered to be the projection of the outline of slots t5) and (8) onto the surface of layer ~1).
The overall rate of liquid flow through the agglutination chamber in these embodiments is controlled by means of a strip of porous absorbent material (4), pLeferably filter paper, in liquid communication with the chamber and positioned adjacent to the distal end of the chamber, and preferably extending partially into the distal end of the chamber, when the structures of Figures 3 and 4 are laminated respectively together. In a more preferred embodiment, layer (3) as shown in Figures 3 and 4 has a slot (28), of slightly larger dimensions as the strip of porous paper (4), such that when the respective layers are adhered together, the strip of porous absorbent material (4) lies partially within the slot (28), more particularly so that a front minor portion of the strip (4) lies within the distal zone (7) of the slot (25) with the remaining major portion of the strip lying within slot (28), so as to prevent disadvantageous formation of microcapillary channels at the sides of and along the length of the strip (4).
The resulting laminated structure, can be thought of as being in the form of a thin, disposable card with the paper strip (4) being in liquid communication with the distal zone (7) of the agglutination chamber.
Figures 5 and 6 represent other embodiments of a device, in the form of a laminated card when the layers SL)B5TITUTE 5~11EET
ll 21~ 3 shown in the Figures are adhered together, for performing agglutination reactions. These embodiments have, in adherent relationship, a first wettable, but liquid-occlusive, layer (1), a second layer (2) parallel to and overlying the first layer (1), and a third liquid-occlusive, preferably non-wettable, layer (3) parallel to and overlying the second layer (2) and having a window, or viewing area, for observing particles. The first layer (1) is made of a liquid-occlusive material having a water-wettable surface. As in the embodiments represented by Figures 3 and 4, these embodiments also utilize a third layer (3) made from a clear, liquid-occlusive, preferably non-wettable film, such as a clear polycarbonate film or a non-wettable cellophane tape, which therefore also serves as a window for observing particles in the agglutination chamber. The second layer (2) is interposed between, and is adhered to, the first layer (1) and third layer (3), for example by means of an adhesive on each side of layer (2) facing the topside of the first layer (1) and the underside of the third layer (3) respectively. The second layer (2) has a slot (25) cut through its thickness defining a channel for directing liquid for conduction by capillary action through the chamber defined by the slot (25) in conjunction with the first (1) and third (3) layers respectively. .
As in the embodiments represented by Figures 3 and 4, when the first, second and third layers axe laminated together a portion of each of the first and third layers serve respectively as the floor and roof of the agglutination~chamber with part of the walls of the slot (25) of the second layer (2) defining the walls (9) of the chamber, the other part of the walls of slot (25) defining the walls of the circular receiving well (5). The agglutination reaction chamber has a proximate zone (6~ and a distal zone (7), which proximate zone (6) for example in .
. ... .... . . ....... . ... . .
W092/0~973 ~C~`/US91/08573 21~3~ ~
Figure 6 is represented by the generally rectangular portion of the slot (25) o~ the second layer (2) with the distal zone (7) being represented by the deltoid or flared portion of the slot (25) of the second layer (2).
Each of the embodiments illustrated by Figures 5 and 6 has a well-defining slot (8) in the thixd layer (3) and a corresponding second well-defining slot (5) in the second layer (2) of the same size and configuration as the well-defining slot (8) in the third layer (3). The well-defining slot (5) in the second layer (2) is positioned directly below the well-defining slot (8) in the third layer (3) such that when all three layers are laminated together, the second well-defining slot (5) in conjunction with the well-de~ining slot (8) along with the corresponding portion of the first layer define a well for receiving liquid, the well being in liquid communication with the proximate zone (6) of the chamber. The bottom of the well is formed from a corresponding circular portion of the first layer (1) which portion can be considered to be the projection of ~he outline of slots (S) and (6) onto the surface of layer (1).
However, in the embodiments of Figures 5 and 6, the second layer (2) is made of a liquid absorbent material, such as absorbent paper, selectively impregnated through its thickness with a substance, such as a water-repellent ink, to form an impregnated region (26) and a non-impregnated region (~). The non-impregnated region (4) is liquid absorbent and the impregnated region (26) is liquid-occlusive. In these embodiments, the non-impregnated region (4) which is in liquid communication with the distal zone (7) of the chamber serves as the means for controlling the overall rate of liqui.d flow through the agglutination chamber. The second la~er (2) also has a slot (25) in the impregnated region (26) defining a channel for directing liquid conducted by capillary action through SlJB51~1TlL)T~ SHE:~;T
- .. . `.` . ` .. .. ~. . ` . . , ~ . . . .. . .. .. .
W092/08973 PCT/U~l/0~573 ~,.'.
13 21~3~3 a chamber defined by the slot (25) in conjunction with the first layer (1) and third layer (3). This chamber also has a proximate zone (6) and a distal zone (7). It is within this chamber that agglutination reactions can be performed.
As can be seen from Figures 5 and 6, the non-impregnated region (4) is located adjacent to the distal end of the agglutination chamber and is in liquid communication with the chamber.
Also in the embodiments illustrated by Figures 5 and 6 there is a well-defining slot (8) in the third layer (3) and a corresponding second well-defining slot (5) in the second layer (2) of the same size and configuration as the well-defining slot (8) in the third layer (3). The well-defining slot (5) in the second layer (2) is positioned directly below the well-defining slot (8) in the third layer (3) such that when all three layers are laminated together, the second well-defining slot (5) in conjunction with the well-defining slot (8) along with the corresponding portion of the first layer (l) define a circular well for receiving liquid, the well being in liquid communication with the proximate zone (6) of the chamber. The bottom of the well is formed from a corresponding circular portion of the first layer (l).
Figure 7 illustrates an exploded, plan view of a preferred embodiment of the invention. This embodiment provides for performing a plurality of agglutination reactions utilizing a minimal amount of liquid sample. The -device in assembled form can be thought of a relatively thin, laminated, disposable structure having in this particular illustration six agglutination chambers radiating from a common liquid receiving well. The device of Figure 7 comprises, in adherent relationship, an approximately circular first wettable but liquid-occlusive layer (1), an approximately circular second liquid-occlusive layer (2) parallel to and overlying the first , SVBSTITUTE SHEET
:: ' .. . ,; . , . . . .. , , ,, ~ ~ ~
W092/08973 PCT/US91/~8573 2~0~363 layer (1), and a third liquid-occlusive layer (3) parallel to and overlying the second layer (2). These respective layers can be bonded together, for example, by means of an adhesive between the respective layers. In this embodiment the third layer (3) is made of a circular clear plastic film, such as a polycarbonate film, thereby providing windows, or viewing areas, ~or observing particles in the six radiating agglutination chambers. The second layer (2), interposed between and in adherent relationship to the first and third layers has a slot (25) in the form of a central, circular portion (5) having si~ radial, slotted arms extending outward therefrom. ~hese radial arms of the slot (25) define six channels for directing liquid conducted by capillary action through chambers respectively defined by the radial, slotted arms in conjunction with the first layer (1) and the third layer ~3). Within the resulting six chambers agglutination reactions can be performed simultaneously. Each of the six chambers has a generally rectangular proximate zone (6) and a generally flared or deltoid shaped distal zone (7). The overall rate of liquid flow through each agglutination chamber in this embodiment is controlled by means of a strip of porous absorbent material (4)r preferably filter paper, projecting from a generally annular ring (27) of such porous material having a hole (28), into the distal zone (7) of each of the channels defined by the radial, slotted arms. The annular ring (27) is selectively impregnated through its thickness with a substance to provide alternating non-impregnated liguid absorbent regions (4) and impregnated liquid-occlusive regions (26). These non-impregnated strips (4) of paper projecting from the annular ring (27) are in liquid communication with the chambers and are positioned adjacent to the distal ends of the chambers, pre~erably positioned partially in the distaI ends, when the structures of Figure 7 are laminated respectively together.
SUBSTITUTE SI~EE:T
.
.. -.. .... , .. , -.. . ... , , .. , . , ., ..... . . ,-, ~, .. .. ,.. ..
W092/08973 PCI/US9l/0~S73 2~3~3 The third layer (3) of the device represented by Figure 7 has a circular well-defining slot (8), and the second layer has a corresponding circular second well-defining slot (5) of the same size and configuration as the well-defining slot (8) in the third layer (3). The well-defining slot (5) of the second layer (2) is positioned directly below the well-defining slot in the third layer (3) in the assembled configuration. Thus the second well-defining slot (5) in conjunction with the well-defining slot ~8) in the third layer (3) and the respective circular portion of the first layer (l) define a well for receiving liquid, the well being in liquid communication with the proximate zone (6) of each of the chambers.
The resulting, generally circular laminated structure, can be thought of as being in the form of a relatively thin, disposable card with the fluid-absorbent paper strip (4) being in liquid communication with the distal zone (7) of the agglutination chamber. Thus, in all of the embodiments of the present invention, the device of the invention has means for controlling overall rate of liquid flow through the reaction chamber which is a porous absorbent material in liquid communication with the chamber, which material is typically positioned adjacent to, and usually extending partially into, the distal end of the chamber. However, it has been found that the utility of devices of the present invention, for performing agglutination reactions, can be enhanced further by utilizing an additional means for controlling the flow of liquid through the reaction chamber of the device, by modifying the geometric configuration of the chamber or the internal shape of the chamber as illustrated in Figures 4, 6, 7 and 8.
For sxample, in Figures 4, 6, and 7, the general slot ~25) in layer (2) defines at least approximately parallel walls (9) in the proximate zone (6) of the chamber SUBSTITUTE SHEc:T
` - . , . .... , ., " . .. ... ,..... ; . . ...... ..
WO9~/08973 PCr/US91/08573 2~ 3~3 ~, thereby defining a first path of approximately constant width. Looking in the direction toward the distal end of the chamber, the general slot (25) defines walls in the distal zone (7) which are spaced to define a second path of increased width compared to the first path of the proximate zone (6). It has been found that agglutination reactions perfo~ed in such a chamber advantageously can result, surprisingly, in the formation of one or more patterned formations, such as, for example bands, of agglutinated particles in the distal zone (7) of the chamber which patterns are more easily observable through the window of the third layer (3) than non patterned aggregates of agglutinated particles which generally result in agglutination chambers of the prior art. Figure 8 shows a schematic representation of an approximately semicircular band (27) of agglutinated particles in the zone of increasing chamber width, namely in the flared (here approximately deltoid-shaped) "second path" of the chamber in the distal zone (7) of the chamber. As represented in schematic form in Figure 8 through the use of arrows of different length along the reaction path in the chamber, the walls in the distal zone (7) are spaced to provide a decreased liquid flow rate per unit area of liquid path along this second path. In Figure 8, the shorter arrows are, of course, intended to represent smaller flow rate per unit area of path, compared to that represented by the longer arrows.
While deltoid-shaped configurations of the second path of the distal zone in the chambers is preferred, it has been found that other geometric conigurations for this so-called "second path" provide advantageous patterned formations of agglutinated particles. For example, the side walls in the second path can be formed to be convex giving an approximately semicircular or bowl-shaped configuration to the second path as illustrated in SUBS~ E SH. -~ ~
-WO92/08973 PCT/US~1/08573 .
2~ ~3~3 Figure 9. Alternatively, although less pxeferred, the side walls of the second path can be formed to provide a second path with an approximately rectangular shape as illustrated in Figure 10.
Moreover, if desired, the flow rate per unit area in the distal zone of the reaction chamber can be gradually decreased along the general direction of flow by gradually increasing the space between the floor and the roof of the chamber along the direction of liquid flow, for example by gradually bowing the roof of the chamber in the distal zone upward and/or gradually bowing the floor of the chamber in the distal zone downward. It has been found that such modification of the space between the floor and the roof of the chamber in the distal zone of the chamber can also contribute to the formation of regular patterns of agglutinated particles being formed in the distal zone of the chamber. For example, the space between the floor and the roof of the chamber can be gradually increased by stamping a spherical dome-shaped or cylindrical dome-shaped configuration in an area of the third layer (3) in such manner that when the third layer is adhered to the second layer (2) the dome in the third layer overlies the distal zone of the reaction chamber. Another example or a way to provide a gradually increasing space between the floor and the roof of the distal zone of the reaction chamber is to stamp a spherical bowl-shaped or cylindrical bowl-shaped depression in the base or first layer (l) in such manner that when the first layer (l) is adhered to the second layer (2) the bowl_shaped depression occurs in the floor of the distal zone of the reaction chamber.
All types of agglutination-based assays can be accommodated with a device according to the present invention. In some instances, a soluble reag~nt can be dried as spots or strips in the reaction chamber, for example, in blood typing. In other instances, a .
:
SUBSTIT~ITE SHEE~
W092/08973 P~/VS91/08573 2~363 ~ , particulate reagent, such as a latex reagent, can be dried in the chamber. In yet another approachr a reagent can be dispersed in a solution which is placed in the chamber.
One preferred reagent solution is microparticulates in a solution of dextran and sucrose. Preferably, the microparticulate reagent is mixed in a solution of about 2.5 to about 5.0 percent by weight dextran and from about to about 20 percent by weight sucrose. Another preferred solution for mixing reagents is FICOLL (a trademark by Sigma Chemical Co., St. Louis, MO for a nonionic synthetic polymer of sucrose) from about 20 to about 3~ percent by weight. Also, depending on the requirements of the assay, the flow of the liquid through the chamber can be controlled as described above to accommodate any necessary incubation times and assay sequences.
A particularly unique feature of the present invention is that it protTides for the ability to simultaneously perform multiple assays while utilizing a very small amount of sample material, for instance, a single drop. Also, the agglutination a~say is essentially self-performing once the drop has been added to the agglutination reaction device. Moreover, in those embodiments of the invention utilizing an additional means for controlling the flow of liquid through the reaction chamber of the device, namely by modification of the geometric configuration of the chamber or the internal shape of the chamber as discussed above, additional enhanced results can be obtained such as enhanced observability of aggregates of agglutinated particles in the distal zone of the reaction chamber.
A device of the invention is especially suitable for use in an automated fashion where the agglutination reaction can be monitored by an optical scanner. For example, the construction of the agglutination reaction 5VE~STITIIJTE SH ET
- . ~ . ~ . . . . ............................... .
.
W092/08973 PCT~US91/~8573 ~, .
2l~3~3 device enables one to use an image analysis system available from Olympus (CUE-2, LaXe Success, N.Y.) to determine the quantity and concentration of agglutinated material. The agglutination reaction device is illuminated, such ~hat transmitted or reflected light can be read by the reader. The image is then compùter analyzed to determine the quantity of agglutination which has occurred and to enhance the image for very accurate and sensitive determinations. By confining the sample to a chamber such as formed in the agglutination reaction device, there is no problem with curvatures of droplets or water which could interfere with the image seen by the reader. Thus, the uniformity of the reacted sample and reagents achieved by the agglutination reaction device provides an excellent imaging format for a reader or other imaging devices. Besides being able to read the transmission of light through the bottom of the agglutination reaction device, it is also possible to read reflected light because the sample and reacted reagents are confined to capillary chambers formed by the agglutination reaction device.
Again referring to Figure 1, one embodiment of the present invention is shown. Figure 1 shows a perspective view of device (10) constructed of two layers of material, a bottom layer (12) covered by a top layer (14). Layer (12) has a plurality of channels (16~ and a sample well (20) formed into the surface. The sample well (20) is contiguous with the ingress of each of the chambers (16). In the construction of the device (10), either the bottom layer (12) or the upper la~er (14) can be opaque.
Preferably the layer which is further from an optical scanning device is opaque to enhance the background. It is required that the bottom surface (12) be hydrophilic or wettable such that the capillary flow is induced when a sample is placed in contact with the ingress of a chamber SUBS I ITUTE S~cT
.
W~92/08973 PCT/~S91~08~73 '~L0~3~
(18). This can be accomplished by using a hydrophilic material for the surface (12); however, it is also possible to chemically treat or coat otherwise non-wettable (hydrophobic) materials such that they become wettable.
This preparation of a wettable surface can also be used to influence the flow rate in the capillary chamber (18) so formed.
Suitable materials for preparlngia wettable layer for various embodiments of the invention include, for example, cellulose acetate butyrate, or a wettable nylon material, or a layer coated with an acrylic latex emulsion to render the surface water-wettable. For example in one embodiment of the invention, an agglutination reaction device is prepared by molding a layer of cellulose acetate butyrate (CAB), commercially available from Eastman Chem.
Prod. Inc., Kingsport, TN, to have a plurality of channels (16) from about .010 to about 5 millimeters in depth and from about 0.1 to about 10 millimeters in width. ~ach of the channels (16) extends from a larger well (20) molded into one end of the layer (12~ of CAB. Next, a piece of transparent tape (14) sufficient to cover all the channels (16~ molded into the CAB is applied to the surface (12) to form the capillary chambers (18). A section of adhesive cellophane tape can be used to provide the upper cover or surface for the recessed surface (14) to form the capillary chambers (18). Other non-wettable (hydrophobic) materials can be used to form the upper surface (14) of the chambers (18). The preferred overall dimensions of this embodiment of the an agglutination reaction device of the in~ention is from about 10 to about 75 mm in length, from about 5 to about 20 mm in width and from about 0.5 to about 5.0 mm thick. The dimensions of the channels in the wettable surface are preferably from about 0.01 to about 5.0 mm in depth and from about 0.1 to about 10.0 mm in width.
The very small size of the reaction devices of SUBSTITUTE SHEE:T
.;;,~
21 21~363 the invention allows for the rapid and convenient handling of a plurality of devices and therefore samples. A device can then be loaded into an automated apparatus which indexes and scans the individual channels for the assay result and records this information for future access. The ~mall dimensions of the agglutination reaction device also provide for efficient use of sample and reagents.
The following examples are provided to further illustrate embodiments of the invention and should not be construed as a limitation on the scope of the invention.
Laminate disposable cards were prepared by assembling together a wettable base layer, a die cut adhesive core layer, paper strip assemblies, and a clear polycarbonate top assembly as shown in Figure 3. To prepare the wettable base layer, 1 mil thick nylon film (Capran Emblem 2500, Allied Signal, Morristown, New Jersey) was first laminated onto a paperboard backing (Westvaco Hi Yield PrintKote, 16 mil, New York, NY) through the use of a two-sided adhesive layer (Fasson Fastape A, Fasson Specialty Division, Avery, Painesville, OH). Base subassemblies (3"X6", i.e., 3 inches X 6 inches) were cut from this material, using care to keep the exposed nylon surface clean. Steel rule dies were prepared to cut the channel shapes as shown in Figure 3 from a second sheet of two-sided adhesive (3.1 mil, Specialty Tapes, Division of RSW Inc., Racine, WI) which has release liner on both adhesive surfaces. One piece of release liner was removed from the die-cut part and this adhesive layer was placed onto the nylon surface of the base subassembly. Pieces of filter paper (2.5Xl9 millimeter, 1 CHR, Whatman, Clifton, New Jersey) which have a layer of one-sided adhesive (ARCare 7S97, Adhesive Research, Glen RocX, PA) laminated ;SVBST~TUTE ~;HEET
W O 92/08973 P~r~US~1/08573 2iO~363 to one surface were positioned on the base/core subassemblies with the one-sided adhesive away from the card. Finally, a sheet of clear polycarbonate film (GE
Part 8040-112, Cadillac Plastics, Evansville, IN) was die~
cut as shown in it~m (3) of Figure 3, and laminated onto the Base/core/paper subassembly using a mechanical laminator set a' 50 psi and 0.2 ft/sec.
Laminate disposable cards were prepared using a 3--X6 piece of paperboard coated with a wettable acrylic latex emulsion coat (Part 150HT(26-1), Daubert Coated Products, Dixon, IL) in place of the nylon base subassemblies described in Example 1. Die-cut core layers were prepared using 3.1 mil two-sided adhesive (ARCare 7580, Adhesive Research, Glen Rock, PA). All other steps in card assembly were identical to those of Example 1.
Fixed human erythrocytes (Duracytes TM, Abbott Laboratories, North Chicago, IL) were coated with affinity purified goat antibodies directed against Hepatitis s surface antigen (HBsAg) at a ~inal concentration of 100 ug/ml (micrograms/milliliter) in the presence of 0.05%
(weight/volume) chromic chloride in 0.1 M (Molar) acetate buffer at a pH of 4Ø These cells were overcoated with 1%
(weight to volume; w/v) human serum albumin (Sigma Chemical Co., St. Louis, MO) in 25 mM (millimolar) Tris-HCl (pH =
7.4) buffer and then resuspended with 0.1% bovine serum albumin (BSA)(Sigma Chemical Co., St. Louis, MO) in phosphate buffered saline (pH=7.4) containing 5%
(volume/volume) normal goat serum at a final cell concentration of 10% (volume/volume). Serum samples (20 SLJ~S7-lTllTE 511E~:ET
W ~ 92/08973 PC~r/US9~/08573 - 2 1 ~ 3 ul; i.e., 20 microliter) containing either 0, 6.25, or 25 ng/ml (nanograms/milliliter) of HBsAg were mixed with 10 ul (microliter) aliquots of these coated Duracytes and the solution was immediately added to the sample addition well of laminate disposable cards prepared as described in Example 1. The solutions flowed rapidly through the capillary channel (1-2 seconds; sec) and then slowly flowed into the paper strips. It took approximately 7 minutes for the liquid to completely saturate the paper strip. After the paper strips had completely wetted, agglutinated reaction products of the Duracyte cells could be observed within certain of the capillary channels of ~he laminate disposable cards. As seen in Table 1, Duracytes which had been mixed with samples containing HBsAg agsregated, whereas the duracytes which were mixed with sera which did not contain HBsAg, did not aggregate.
HBsAq Concentration Aqqreqation 6.25 ng/ml +/-25 ng/ml +
Laminate disposable cards were prepared as described in Example 2 with a flared channel design as shown in Figure 4. Duracytes coated with anti-HBsAg (Example 3) were mixed with sera containing various concentrations of HBsAg and were introduced into the laminate disposable cards having flared channels. After 5 SUBS~lTllTE S~tEtT
-,,, . ., . I . , .
--, W O 92/08973 P~r/VS91/08573 21003~3 minutes, aggregated particles appeared and formed into an easily visible band of agglutinates which stretched across the flared portion of the channel as shown in Figure 8. In channels where there was not any HBsAg present, the Duracytes did not aggregate and no band of cells was visible.
.
Claims (22)
- Wo 92/08973 PCT/US91/08573 WHAT IS CLAIMED IS:
l. A device for performing agglutination reactions comprising: in adherent relationship, a first wettable layer, a second liquid-occlusive layer parallel to and overlying said first layer, and a third layer parallel to and overlying said second layer and having a window for observing particles, said second layer interposed between and in adherent relationship to said first and third layers, said second layer having at least one slot therein defining a channel for directing liquid conducted by capillary action through a chamber defined by said slot in conjunction with said first and third layers and within which chamber agglutination reactions can be performed, said chamber having a proximate zone and a distal zone and means for controlling overall rate of liquid flow through said chamber, said means comprising a porous absorbent material in liquid communication with said chamber and positioned adjacent to the distal end of said chamber. - 2. The device of claim l wherein said means for controlling overall rate of liquid flow is positioned partially within said distal zone of said chamber.
- 3. The device of claim 2 wherein said porous absorbent material comprises a cellulosic material.
- 4. The device of claim 3 wherein said cellulosic material comprises paper.
- 5. The device of claim 4 wherein said paper comprises filter paper.
- 6. The device of claim 1 wherein said third layer is non-wettable.
- 7. The device of claim 1 wherein said slot defines at least approximately parallel walls in said proximate zone thereby defining a first path of approximately constant width and defining walls in said distal zone which are spaced to define a second path of increased width compared to said first path whereby agglutination reactions in said chamber result in the formation of one or more bands of agglutinated particles in said distal zone of said chamber which bands are visibly observable through said window of said third layer.
- 8. The device of claim 7 wherein said slot defines walls in said distal zone which are spaced to provide said second path such that agglutination reactions in said chamber result in said bands of agglutinated particles being approximately semicircular in configuration.
- 9. The device of claim 7 wherein said slot defines walls in said distal zone which are spaced to provide a decreased flow rate per unit area of liquid along said second path.
- 10. The device of claim 7 wherein said slot defines walls in said distal zone which are spaced to provide said second path with a flared configuration relative to said first path.
- 11. The device of claim 10 wherein said flared configuration is approximately deltoid in shape.
- 12. The device of claim 10 wherein said flared configuration is approximately semicircular in shape.
- 13. The device of claim 7 wherein said slot defines walls in said distal zone which are spaced to provide said second path with an approximately rectangular shape.
- 14. The device of claim 1 in which said third layer contains a well-defining slot and said second layer contains a corresponding second well-defining slot of the same size and configuration as said well-defining slot in said third layer and positioned directly below said well-defining slot in said third layer, wherein said second well-defining slot in conjunction with said well-defining slot in said third layer and said first layer define a well for receiving liquid, said well being in liquid communication with said proximate zone of said chamber.
- 15. A device for performing simultaneously a plurality of agglutination reactions comprising: in adherent relationship, a first wettable layer, a second liquid-occlusive layer parallel to and overlying said first layer, and a third layer parallel to and overlying said second layer and having windows for observing particles, said second layer interposed between and in adherent relationship to said first and third layers, said second layer having a plurality of slots in radial spatial relationship to each other, said slots respectively defining channels for directing liquid conducted by capillary action through chambers respectively defined by said slots in conjunction with said first and third layers and within which chambers agglutination reactions can be performed simultaneously, each of said chambers having a proximate zone and a distal zone and means for controlling overall rate of liquid flow through the chamber, said means comprising a porous absorbent material in liquid communication with said chamber and positioned adjacent to the distal end of said chamber.
- 16. The device of claim 15 in which said third layer has a well-defining slot and said second layer has a corresponding second well-defining slot of the same size and configuration as said well-defining slot in said third layer and positioned directly below said well-defining slot in said third layer, wherein said second well-defining slot in conjunction with said well-defining slot in said third layer and said first layer define a well for receiving liquid, said well being in liquid communication with said proximate zone of each of said chambers.
- 17. A device for performing agglutination reactions comprising: a first wettable layer, a second layer parallel to and overlying said first layer and having a window for observing particles, and chamber forming means such that when said first and second layers are brought into contact with said chamber forming means a chamber for performing agglutination reactions is formed therebetween, said chamber having a proximate zone and a distal zone and means for controlling overall rate of liquid flow through said chamber, said means comprising a porous absorbent material in liquid communication with said chamber and positioned adjacent to the distal end of said chamber.
- 18. The device of claim 17 wherein said means for controlling overall rate of liquid flow is positioned partially within said distal zone of said chamber.
- 19. The device of claim 17 wherein said porous absorbent material comprises a cellulosic material.
- 20. The device of claim 19 wherein said cellulosic material comprises paper.
- 21. The device of claim 20 wherein said paper comprises filter paper.
- 22. The device of claim 17 wherein said second layer is non-wettable.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US61489590A | 1990-11-16 | 1990-11-16 | |
| US614,895 | 1990-11-16 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2100363A1 true CA2100363A1 (en) | 1992-05-17 |
Family
ID=24463154
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2100363 Abandoned CA2100363A1 (en) | 1990-11-16 | 1991-11-15 | Agglutination reaction device utilizing porous absorbent material |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP0557447A4 (en) |
| AU (1) | AU658593B2 (en) |
| CA (1) | CA2100363A1 (en) |
| TW (1) | TW201824B (en) |
| WO (1) | WO1992008973A1 (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5518887A (en) * | 1992-03-30 | 1996-05-21 | Abbott Laboratories | Immunoassays empolying generic anti-hapten antibodies and materials for use therein |
| WO2006047831A1 (en) * | 2004-11-03 | 2006-05-11 | Agen Biomedical Limited | Detection device and method |
| WO2017120464A1 (en) * | 2016-01-08 | 2017-07-13 | Siemens Healthcare Diagnostics Inc. | Heating element for sensor array |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA1292176C (en) * | 1985-09-18 | 1991-11-19 | Joel M. Blatt | Volume metering capillary gap device for applying a liquid sample onto a reactive surface |
| DE3684014D1 (en) * | 1985-10-15 | 1992-04-02 | Pan American Trading Co Ltd | INJECTION DEVICE FOR LIQUID CEMENT. |
| US4906439A (en) * | 1986-03-25 | 1990-03-06 | Pb Diagnostic Systems, Inc. | Biological diagnostic device and method of use |
| DE3889885T2 (en) * | 1987-12-23 | 1994-12-15 | Abbott Lab | Agglutination reaction device. |
-
1991
- 1991-11-15 AU AU90821/91A patent/AU658593B2/en not_active Ceased
- 1991-11-15 WO PCT/US1991/008573 patent/WO1992008973A1/en not_active Application Discontinuation
- 1991-11-15 EP EP19920901199 patent/EP0557447A4/en not_active Withdrawn
- 1991-11-15 CA CA 2100363 patent/CA2100363A1/en not_active Abandoned
-
1992
- 1992-01-29 TW TW81100682A patent/TW201824B/zh active
Also Published As
| Publication number | Publication date |
|---|---|
| EP0557447A1 (en) | 1993-09-01 |
| WO1992008973A1 (en) | 1992-05-29 |
| EP0557447A4 (en) | 1994-06-01 |
| AU9082191A (en) | 1992-06-11 |
| TW201824B (en) | 1993-03-11 |
| AU658593B2 (en) | 1995-04-27 |
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