Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/5302—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

Definitions

• 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.
• 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.
• U.S. Patent 4,596,695 discloses an agglutination reaction chamber for reacting immunochemical 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 flow the immunochemical particle reagents can react.
• An object of the present invention is to provide
• SUBSTITUTESHEET 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 capable of performing multiple, highly sensitive, diagnostic tests simultaneously on a single sample in a single device.
• 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.
• 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.
• 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.
• the present invention is directed to a device having an agglutination reaction chamber for performing agglutination immunoassay reactions.
• 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 agglutination reaction chamber is formed for conducting fluid by capillary action.
• the agglutination reaction chamber can additionally include a sample receiving well contiguous with the ingress of the chamber formed by the first and
• 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 through the chamber comprises utilizing a porous absorbent material such as an absorbent paper in liquid communication with the chamber, typically at the distal end of the chamber.
• the reagent in the agglutination reaction chamber of the present invention 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.
• 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 length of the channel.
• the aforesaid copending United States Patent Application also discloses
• SUBSTITUTESHEET 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.
• 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.
• the present invention is directed to devices for performing agglutination reactions having improved 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 as disposable, laminated cards, optionally mounted in disposable rigid containers.
• Figure 1 is a top perspective view of one embodiment of the present agglutination reaction device showing a rectangular sample receiving well and four reaction chambers.
• Figure 2 is a top perspective view of one
• SUBSTITUTE SHEET 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
• BSTITUTE SHEET 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.
• Figure 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 illustrating 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.
• 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.
• one of the channel surfaces be water-wettable (hydrophilic) .
• 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
• 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-soluble 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.
• 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.
• 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).
• the opaque lower surface (12) gives a better background for detection
• 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.
• the porous material is a cellulosic material, more preferably paper such as Whatman filter paper.
• porous material such as paper utilized as the fluid flow control means provides advantages in both manufacturing and performance over the utilization 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 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
• the first layer (1) is made of a liquid-occlusive material having a water- wettable surface.
• 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 is made of 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) 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.
• 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 example in Figure 4 is represented by the generally 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
• SUBSTITUTESHEET 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 (5) 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), preferably 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.
• a strip of porous absorbent material (4) preferably filter paper
• 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
• 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
• SUBSTITUTE SHEET 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.
• 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.
• 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
• SUBSTITUTESHEET Figure 6 is represented by the generally 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 5 and 6 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 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 (5) and (6) onto the surface of layer (1).
• 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 (4) .
• the non-impregnated region (4) is liquid absorbent and the impregnated region (26) is liquid-occlusive.
• 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 liquid flow through the agglutination chamber.
• the second layer (2) also has a slot (25) in the impregnated region (26) defining a channel for directing liquid conducted by capillary action through
• SUBSTITUTE SHEET 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.
• the non-impregnated region (4) is located adjacent to the distal end of the agglutination chamber and is in liquid communication with the chamber.
• FIG. 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 (1) 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 (1).
• 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
• SUBSTITUTE SHEET 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.
• the third layer (3) is made of a circular clear plastic film, such as a polycarbonate film, thereby providing windows, or viewing areas, for 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 six radial, slotted arms extending outward therefrom.
• These 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) .
• 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), 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 liquid absorbent regions (4) and impregnated liquid- occlusive regions (26).
• 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, preferably positioned partially in the distal ends, when the structures of Figure 7 are laminated respectively together.
• 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.
• 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 (1) 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.
• 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.
• the general slot (25) in layer (2) defines at least approximately parallel walls (9) in the proximate zone (6) 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 performed 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.
• 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.
• 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.
• 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
• 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.
• 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.
• 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 of 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 (1) in such manner that when the first layer (1) is adhered to the second layer (2) the bowl-shaped depression occurs in the floor of the distal zone of the reaction chamber.
• agglutination-based assays can be accommodated with a device according to the present invention.
• a soluble reagent can be dried as spots or strips in the reaction chamber, for example, in blood typing.
• a soluble reagent can be dried as spots or strips in the reaction chamber, for example, in blood typing.
• a soluble reagent can be dried as spots or strips in the reaction chamber, for example, in blood typing.
• SUBSTITUTE SHEET particulate reagent such as a latex reagent
• 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.
• the microparticulate reagent is mixed in a solution of about 2.5 to about 5.0 percent by weight dextran and from about 15 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 30 percent by weight.
• FICOLL a trademark by Sigma Chemical Co., St. Louis, MO for a nonionic synthetic polymer of sucrose
• 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 provides 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 assay 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.
• the construction of the agglutination reaction can be monitored by an optical scanner.
• SUBSTITUTE SHEET device enables one to use an image analysis system available from Olympus (CUE-2, Lake Success, N.Y.) to determine the quantity and concentration of agglutinated material.
• the agglutination reaction device is illuminated, such that transmitted or reflected light can be read by the reader.
• the image is then computer analyzed to determine the quantity of agglutination which has occurred and to enhance the image for very accurate and sensitive determinations.
• 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.
• 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.
• Figure 1 shows a perspective view of device (10) constructed of two layers of material, a bottom layer (12) covered by a top layer
• Layer (12) has a plurality of channels (16) and a sample well (20) formed into the surface.
• either the bottom layer (12) or the upper layer (14) can be opaque.
• 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
• SUBSTITUTESHEET (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 preparing a 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.
• 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.
• CAB cellulose acetate butyrate
• Each of the channels (16) extends from a larger well (20) molded into one end of the layer (12) of CAB.
• 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 invention 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.
• SUBSTITUTE SHEET 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 small dimensions of the agglutination reaction device also provide for efficient use of sample and reagents.
• 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.
• 1 mil thick nylon film Capran Emblem 2500, Allied Signal, Morristown, New Jersey
• a paperboard backing Westvaco Hi Yield PrintKote, 16 mil, New York, NY
• 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 2.5X19 millimeter, 1 CHR, Whatman, Clifton, New Jersey
• ARCare 7597 Adhesive Research, Glen Rock, PA
• 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.
• EXAMPLE 3 Fixed human erythrocytes (Duracytes TM, Abbott Laboratories, North Chicago, IL) were coated with affinity purified goat antibodies directed against Hepatitis B surface antigen (HBsAg) at a final 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 4.0. These cells were overcoated with 1% (weight to volume; w/v) human serum albumin (Sigma Chemical Co., St.
• 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 the laminate disposable cards. As seen in Table 1, Duracytes which had been mixed with samples containing HBsAg aggregated, whereas the duracytes which were mixed with sera which did not contain HBsAg, did not aggregate.
• 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

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• 1991
  • 1991-11-15 EP EP19920901199 patent/EP0557447A4/en not_active Withdrawn
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