Agglutination reaction device having geometrically modified chambers

A device for performing agglutination immunoassay reactions and the like is disclosed. The device includes a first hydrophilic layer, a second liquid-occlusive layer parallel to and overlying the first layer, and a third layer parallel to and overlying the second layer. The third layer has a transparent opening for observing particles. The second layer is interposed between and in adherent relationship to the first and third layers. The second layer has at least one slot defining a channel for directing liquid conducted by capillary action through the chamber defined in conjunction with the first and third layers. This chamber has a proximate zone and a distal zone. The geometry of the chamber is modified to provide preferably outwardly diverging walls and provide a flowpath having different rates of flow per unit area along the paths. This arrangement of different paths in the chamber allows agglutination reactions in the chamber to result in the formation of a non-random pattern of aggregated particles in the distal zone of the chamber. Such a non-random pattern is more readily observable than a random pattern of aggregated particles.

BACKGROUND OF THE INVENTION 
The present invention is directed to an improved 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 particular 
improvement involves varying the geometry, namely the width and/or depth, 
of a portion of the path along which liquid in the device flows by 
capillary action. 
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 involves placing 
liquid reagents on a glass slide and generally rocking or swirling 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. Pat. No. 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 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. In one aspect, the 
present invention is directed to a device in which the agglutination 
reaction can be rapidly performed and monitored with a minimum of sample 
material. In another aspect, the present invention is directed to 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 another aspect, the present invention is 
directed to devices for performing agglutination reactions having enhanced 
performance properties through utilization of a means for controlling the 
flow of liquid through the reaction chamber of the performance properties 
through utilization of a means for controlling the flow of liquid through 
the reaction chamber of the device, namely, through modification of the 
geometric configuration of the agglutination reaction chamber or the 
internal shape of the chamber so as to provide a non-random patterned 
array of aggregated agglutinates which non-random pattern is more easily 
observable than agglutinates aggregated in a random array. 
SUMMARY OF THE INVENTION 
The present invention provides a device for performing agglutination 
reactions comprising: in adherent relationship, a first wettable layer, a 
second liquid-occlusive layer parallel to and overlying the first layer, 
and a third layer parallel to and overlying the second layer and having a 
window for observing particles. The second layer is interposed between, 
and is in adherent relationship to, the first and third layers. The second 
layer has at least one general slot therein defining a channel for 
directing liquid conducted by capillary action through a chamber defined 
by the slot in conjunction with the first and third layers. Agglutination 
reactions can be performed in the chamber. The chamber has a proximate 
zone and a distal zone. The aforesaid slot in the second layer defines at 
least approximately parallel walls in the proximate zone thereby defining 
a first path of approximately constant width. The aforesaid slot in the 
second layer also defines walls in the distal zone which are spaced to 
define a second path of increased width compared to the first path. This 
arrangement of such different paths in the chamber at least in part 
enables agglutination reactions in the chamber to result in the formation 
of a surprisingly non-random pattern of aggregated particles in the distal 
zone of the chamber. Such a non-random pattern is more readily observable 
through the third layer than if a random pattern of aggregated particles 
occurred instead. 
The present invention also provides in particular for such a device for 
performing agglutination reactions in which the second layer has at least 
one general slot therein defining a channel for directing liquid conducted 
by capillary action through a chamber defined by the slot and by the first 
and third layers and within which chamber agglutination reactions can be 
performed. The chamber has a proximate zone and a distal zone. The slot in 
the second layer defines walls in the proximate zone which with the first 
and third layers define a first path of approximately constant depth. The 
slot in the second layer also defines walls in the distal zone which with 
the first and third layers define a second path of increasing depth 
compared to the first path whereby agglutination reactions in the chamber 
result in the formation of a non-random pattern of aggregated particles in 
the distal zone of the chamber which non-random pattern is more readily 
observable through the window of the third layer than if a random pattern 
of aggregated particles occurred instead. 
Additionally, the present invention provides a device for performing 
simultaneously a plurality of agglutination reactions. The device 
comprises: in adherent relationship, a first wettable layer, a second 
liquid-occlusive layer parallel to and overlying the first layer, and a 
third layer parallel to and overlying the second layer and having windows 
for observing particles. The second layer is interposed between and is in 
adherent relationship to the first and third layers. The general slot of 
the second layer has a plurality of slotted arms (also slots) in radial 
spatial relationship to each other. These radiating slots respectively 
define channels for directing liquid conducted by capillary action through 
chambers respectively defined by the slots in conjunction with the first 
and third layers. Agglutination reactions can be performed simultaneously 
in these chambers. Each of the chambers has a proximate zone and a distal 
zone, and each of the slots defines at least approximately parallel walls 
in the corresponding proximate zone thereby defining a corresponding first 
path of approximately constant width and defines walls in the 
corresponding distal zone which are spaced to define a corresponding 
second path of increased width compared to the first path. Agglutination 
reactions performed in the corresponding chambers can result in the 
formation of a non-random pattern of aggregated particles in the distal 
zone of each chamber which non-random pattern is more readily observable 
through the window of the third layer than if a random pattern of 
aggregated particles occurred instead. 
An agglutination reaction device of the present invention additionally can 
include a sample receiving well contiguous with the ingress of the 
agglutination chamber. 
In an 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 U.S. patent application, Ser. No. 07/138,253, filed on Dec. 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.times.12.5 mm.times.1.5 mm 
(1.times.w.times.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 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. It has 
recently been found, and is the subject of a United States Patent 
Application entitled "Improved Agglutination Reaction Device Utilizing A 
Porous Absorbent Material", and filed event date herewith, that a porous, 
absorbent material such as an absorbent 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). 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. 
The present invention is directed to devices for performing agglutination 
reactions having improved properties including improved means for 
controlling the rate of liquid flow per unit area through the 
agglutination chamber so as to produce a non-random pattern of aggregated 
agglutinated particles. 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.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The present invention is directed to improved devices suitable for 
performing agglutination reactions. Surprisingly, it has been found that 
the devices of the present invention, for performing agglutination 
reactions, provide enhanced properties over prior art devices. The devices 
of the present invention utilize a means for controlling the rate of flow 
per unit area of liquid through the reaction chamber of the device. In 
particular these means consist of modifying the geometric configuration of 
the chamber or the internal shape of the chamber as illustrated in FIGS. 
1, 2, 3, 4, 5 and 6. 
FIG. 1 represents an embodiment of a device for performing agglutination 
reactions according to the invention. This embodiment has, 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 viewing 
area, for observing particles. The first layer (1) is made of a 
liquid-occlusive material having a water-wettable surface. In this 
embodiment 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 (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 general 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 
layer (1) and third layer (3) 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) 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). 
The embodiment illustrated by FIG. 1 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 (8) onto 
the surface of layer (1). 
In the embodiment illustrated by FIG. 1, the overall rate of liquid flow 
through the agglutination chamber 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 FIG. 1 are laminated respectively 
together. As used in the present specification, the absorbent 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. In a more preferred embodiment, layer (3) as 
shown in FIG. 1 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. 
For example, when a solution of cells is introduced into the receiving well 
of a device of the invention, which well is in liquid communication with 
the proximate end of the reaction chamber; and the chamber contains 
antibodies directed against antigens on the cells and which antigens are 
dried onto the floor of the chamber, the solution will migrate through the 
chamber by capillary action, mix with the antisera, and the cells will 
aggregate. This will all occur without any centrifugation or mixing steps. 
Control of the overall rate of flow of the liquid through the channel is 
necessary because the agglutination reaction occurs preferably during the 
period of liquid flow. Sufficient incubation time is built into the period 
of liquid flow to achieve optimum reaction of the reagents. 
In FIGS. 1, 2, and 3, the general slot (25) in layer (2) defines at least 
approximately parallel walls (9) in the proximate zone (6) of the chamber 
thereby defining a first path of approximately constant width. Looking in 
the direction toward the distal end of the chamber, the general slot (2) 
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. FIG. 4 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 FIG. 4 
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 FIG. 4, 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 
configurations 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 FIG. 5. Alternatively, although less preferred, the side 
walls of the second path can be formed to provide a second path with an 
approximately rectangular shape as illustrated in FIG. 6. 
FIG. 2 represents another embodiment of a device, in the form of a 
laminated card when the layers shown in FIG. 2 are adhered together, for 
performing agglutination reactions. This embodiment has, 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 embodiment represented 
by FIG. 1, this embodiment also utilizes a third layer (3) made from a 
clear, liquid-occlusive, preferably non-wettable film, such as a clear 
polycarbonate film or a non-wettable cellophone 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 
general 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 embodiment represented by FIG. 1, 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 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) 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 FIGS. 1 and 2 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 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). 
However, in the embodiment of FIG. 2 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. In this 
embodiment, the non-impregnated region (4) which is in liquid 
communication with the distal zone (7) of the chamber serves as 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 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 FIG. 2, 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. 3 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 card having in this particular illustration six agglutination 
chambers radiating from a common liquid receiving well. The device of FIG. 
3 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 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, 
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). 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), preferably filter paper, projecting from 
a generally annular ring (27) of such porous material, 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). 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, preferably positioned partially in the distal ends, 
when the structures of FIG. 3 are laminated respectively together. 
The third layer (3) of the device represented by FIG. 3 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 (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. 
If desired, the flow rate per unit area in the distal zone of the reaction 
chamber of an embodiment of the invention 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 by 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 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. 
All types of agglutination-based assays can be accommodated with a device 
according to the present invention. In some instances, a soluble reagent 
can be dried as spots or strips in the reaction chamber, for example, in 
blood typing. In other instances, a particulate reagent, such as a latex 
reagent, can be dried in the chamber. In yet another approach, 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 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. 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 advantageous 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. It is 
important to note that by utilizing means for controlling the rate of flow 
per unit area of liquid through the reaction chamber, particularly through 
the distal zone of the chamber, of a device according to the present 
invention, namely by modification of the geometric configuration of the 
distal zone 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 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 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. 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. 
It is required that a surface, preferably the bottom surface, of an 
agglutination chamber of the present invention be hydrophilic or wettable 
such that capillary flow is induced when a sample is placed in contact 
with the ingress of the proximate zone of the chamber. This can be 
accomplished by using a hydrophilic or water-wettable material for the 
surface. 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. 
Suitable materials for preparing a wettable layer for various embodiments 
of the invention include, for example, cellulose acetate butyrate, a 
wettable nylon material, or a layer coated with an acrylic latex emulsion 
to render the surface water-wettable. The "roof" of an agglutination 
chamber of the invention may be either wettable or non-wettable. 
The small size of the reaction devices of 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. 
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. 
EXAMPLE 1 
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 FIG. 1. To prepare the 
wettable base layer, 1 mil thick nylon film (Capran Emblem 2500, Allied 
Signal, Morristown, N.J.) was first laminated onto a paperboard backing 
(Westvaco Hi Yield PrintKote, 16 mil, New York, N.Y.) through the use of a 
two-sided adhesive layer (Fasson Fastape A, Fasson Specialty Division, 
Avery, Painesville, Ohio). Base subassemblies (3".times.6", i.e., 3 
inches.times.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 FIG. 1 from a second sheet of two-sided 
adhesive (3.1 mil, Specialty Tapes, Division of RSW Inc., Racine, Wis.) 
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 
(2.5.times.19 millimeter, 1CHR, Whatman, Clifton, N.J.) which have a layer 
of one-sided adhesive (ARCare 7597, Adhesive Research, Glen Rock, Pa.) 
laminated 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, Ind.) 
was die-cut as shown in item (3) of FIG. 1, and laminated onto the 
Base/core/paper subassembly using a mechanical laminator set at 50 psi and 
0.2 ft/sec. 
EXAMPLE 2 
Laminate disposable cards were prepared using a 3".times.6" piece of 
paperboard coated with a wettable acrylic latex emulsion coat (Part 
150HT(26-1), Daubert Coated Products, Dixon, Ill.) 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, Ill.) 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 0.1M (Molar) acetate buffer at a pH of 4.0. These 
cells were overcoated with 1% (weight/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 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 the laminate disposable cards. 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. 
EXAMPLE 4 
Laminate disposable cards were prepared as described in Example 2 with a 
flared channel design as shown in FIG. 1. 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 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 FIG. 4. In channels where there was not 
any HBsAg present, the Duracytes did not aggregate and no band of cells 
was visible.