Abstract:
Test cells have a first sorbent strip with a sample receiving location and defining a first migration path, a distinct second sorbent strip which receives buffer solution and at least partially defines a second migration path distinct from and elongated relative to the first migration path, conjugate supported by the second strip, a test site located at a junction of the first and second strips and having an immobilized ligand-binding mechanism, and a divider which directs a first amount of the buffer to the first strip to move the sample to the test site and a second amount to the second strip to move the conjugate to the test site. The first and second migration paths have first and second lengths chosen so that ligand in the sample reaches the test site and binds to the immobilized ligand-binding mechanism prior to the conjugate reaching the test site.

Description:
RELATED APPLICATIONS 
       [0001]    This application relates to PCT/US2006/008688 filed Mar. 10, 2006 published as WO 2006/099191 A2, U.S. Ser. No. 11/908,071 filed Sep. 7, 2007, and U.S. Ser. No. 61/338,303 filed Feb. 16, 2010 all of which are hereby incorporated by reference herein in their entireties. 
     
    
     BACKGROUND OF THE INVENTION 
       [0002]    1. Field of the Invention 
         [0003]    This invention relates broadly to immunoassay devices and the methods for their use. More particularly, this invention relates to chromatographic rapid test strips for detection of a ligand in a body fluid. 
         [0004]    2. State of the Art 
         [0005]    Many types of ligand-receptor assays have been used to detect the presence of various substances, often generally called ligands, in body fluids such as blood, urine, or saliva. These assays involve antigen antibody reactions, synthetic conjugates comprising radioactive, enzymatic, fluorescent, or visually observable polystyrene or metal sol tags, and specially designed reactor chambers. In all these assays, there is a receptor, e.g., an antibody, which is specific for the selected ligand or antigen, and a means for detecting the presence, and in some cases the amount, of the ligand-receptor reaction product. Some tests are designed to make a quantitative determination, but in many circumstances all that is required is a positive/negative qualitative indication. Examples of such qualitative assays include blood typing, most types of urinalysis, pregnancy tests, and AIDS tests. For these tests, a visually observable indicator such as the presence of agglutination or a color change is preferred. 
         [0006]    Even the qualitative assays must be very sensitive because of the often small concentration of the ligand of interest in the test fluid. False positives can also be troublesome, particularly with agglutination and other rapid detection methods such as dipstick and color change tests. Because of these problems, so-called “sandwich” assays and other sensitive detection mechanisms which use metal sols or other types of colored particles have been developed. 
         [0007]    In a “sandwich” assay, a target analyte such as an antigen is “sandwiched” between a labeled antibody and an antibody immobilized onto a solid support. The assay is read by observing the presence and/or amount of bound antigen-labeled antibody complex. In a “competition” immunoassay, antibody bound to a solid surface is contacted with a sample containing an unknown quantity of antigen analyte and with labeled antigen of the same type. The amount of labeled antigen bound on the solid surface is then determined to provide an indirect measure of the amount of antigen analyte in the sample. 
         [0008]    Because these and other assays can detect both antibodies and antigens, they are generally referred to as immunochemical ligand-receptor assays or simply immunoassays. 
         [0009]    Solid phase immunoassay devices, whether of the sandwich or competition type, provide sensitive detection of an analyte in a biological fluid sample such as blood, urine, or saliva. Solid phase immunoassay devices incorporate a solid support to which one member of a ligand-receptor pair, usually an antibody, antigen, or hapten, is bound. Common early forms of solid supports were plates, tubes, or beads of polystyrene which were well known from the fields of radioimmunoassay and enzyme immunoassay. In the last decade, a number of porous materials such as nylon, nitrocellulose, cellulose acetate, glass fibers, and other porous polymers have been employed as solid supports. 
         [0010]    A number of self-contained immunoassay kits using porous materials as solid phase carriers of immunochemical components such as antigens, haptens, or antibodies have been described. These kits are usually dipstick, flow-through, or migratory in design. 
         [0011]    In the more common forms of dipstick assays, as typified by home pregnancy and ovulation detection kits, immunochemical components such as antibodies are bound to a solid phase. The assay device is “dipped” for incubation into a sample suspected of containing unknown antigen analyte. Enzyme-labeled antibody is then added, either simultaneously or after an incubation period. The device is then washed and inserted into a second solution containing a substrate for the enzyme. The enzyme-label, if present, interacts with the substrate, causing the formation of colored products which either deposit as a precipitate onto the solid phase or produce a visible color change in the substrate solution. 
         [0012]    Flow-through type immunoassay devices were designed to obviate the need for extensive incubation and cumbersome washing steps associated with dipstick assays. Valkirs et al., U.S. Pat. No. 4,632,901, disclose a device comprising antibody (specific to a target antigen analyte) bound to a porous membrane or filter to which is added a liquid sample. As the liquid flows through the membrane, target analyte binds to the antibody. The addition of sample is followed by addition of labeled antibody. The visual detection of labeled antibody provides an indication of the presence of target antigen analyte in the sample. 
         [0013]    Korom et al., EP-A 0 299 359, discloses a variation in the flow-through device in which the labeled antibody is incorporated into a membrane which acts as a reagent delivery system. 
         [0014]    The requirement of multiple addition and washing steps with dipstick and flow-through type immunoassay devices increases the likelihood that minimally trained personnel and home users will obtain erroneous assay results. 
         [0015]    In migration type assays, a membrane is impregnated with the reagents needed to perform the assay. An analyte detection zone is provided in which labeled analyte is bound and assay indicia is read. See, for example, Tom et al., U.S. Pat. No. 4,366,241, and Zuk, et al. U.S. Pat. No. 4,596,275. The sensitivity of migration type assays is frequently reduced, however, by the presence or formation in the sample of undesirable solid components which block the passage of labeled analyte to the detection zone. Assay sensitivity also declines when migration assay devices are flooded with too much liquid sample. 
         [0016]    Migration assay devices usually incorporate within them reagents which have been attached to colored labels (i.e., conjugates), thereby permitting visible detection of the assay results without addition of further substances. See, for example, Bernstein, U.S. Pat. No. 4,770,853. Among such labels are gold sol particles such as those described by Leuvering in U.S. Pat. No. 4,313,734, dye sol particles such as described in U.S. Pat. No. 4,373,932 by Gribnau et al., dyed latex such as described by May et al., WO 88/08534, and dyes encapsulated in liposomes by Campbell et al., U.S. Pat. No. 4,703,017. These colored labels are generally limited in terms of the immobilization methods which are suitable. Moreover, they require a relatively large amount of ligand molecule and can involve expensive reagents, thereby adding to the cost. 
         [0017]    The “Related Applications” set forth above overcome many deficiencies of the prior art by providing “dual path” immunoassays which are highly sensitive, extremely reliable, accurate and inexpensive rapid detection devices. Generally, the dual path immunoassays include a first sorbent material having a first location for receiving a buffer solution (in the case of a dry conjugate system) or a conjugate solution (in the case of a liquid conjugate system) with the first sorbent material defining a first horizontal flow path, a second sorbent material having a second location for receiving a sample with the second sorbent material defining a second horizontal flow path distinct from the first flow path, and a test line or test site with immobilized antigens or antibodies or other ligand binding molecules such as aptamers, nucleic acids, etc. located in a test zone at a junction of the first and second sorbent materials. 
         [0018]    Various types of samples are effectively tested using the duel path immunoassays, including but not limited to whole blood, blood serum, urine, sputum, saliva, and feces. For most samples it is common to utilize buffer solution to cause the sample to flow along the second sorbent material. Thus, one method of using a dual path immunoassays involves (1) depositing a sample at the (second) location for receiving the sample, (2) depositing buffer solution at the same location of the sample, (3) waiting a period of time sufficient to permit the sample to reach the test zone, (4) after waiting, depositing additional buffer solution at the (first) location for receiving the buffer solution such that the additional buffer solution causes the conjugate to flow to the test zone, and (5) inspecting the test zone to determine whether the test is positive or negative. 
       SUMMARY OF THE INVENTION 
       [0019]    According to one aspect of the invention, a rapid detection dual path immunoassay device is provided that requires fewer steps to use than the previous dual path immunoassay devices. 
         [0020]    According to another aspect of the invention, a dual path immunoassay device is provided that is simple to use and provides accurate results. 
         [0021]    In one embodiment, a dry conjugate dual path immunoassay device system is provided and includes a test cell with a first location for receiving a sample and a second location for receiving a buffer solution. A first sorbent material is provided for directing a horizontal flow path for the sample. Means are provided for dividing the buffer solution received at the second location such that some of the buffer solution is directed to the first sorbent material which directs the sample, and some of the buffer solution is directed to a second sorbent material. In one embodiment, the second sorbent material takes an elongated path (e.g., curved, angled, or tortuous path) to the test zone which is located at the junction of the elongated path of the second sorbent material with the first sorbent material. In another embodiment, the second sorbent material is provided with a delay element along its length. In yet another embodiment, second and third sorbent materials are provided with the third sorbent material initially receiving the buffer solution and having a slower flow characteristic than the first sorbent material, and the second sorbent material in contact with the third sorbent material and forwarding the buffer solution to the test zone which is located at the junction of the second sorbent material and the first sorbent material. Regardless, conjugate is provided at a location along the second sorbent material. Preferably, the flow path provided by the first sorbent material is a direct path to the test zone. The test zone preferably includes one or more test lines or test sites with immobilized antigens or antibodies or other ligand binding molecules such as aptamers, nucleic acids, etc. 
         [0022]    Where the test cell of the invention is provided in a housing, the housing is provided with a first opening adjacent the first location and a second opening adjacent the second location. A viewing window is provided in the housing above the test line. 
         [0023]    Various means for dividing the buffer solution received at the second location are provided. A first means for dividing the buffer solution is a wedge element (V-shaped or triangular in cross-section) located at the location for receiving the buffer solution (e.g., an opening in a housing), where the apex of the V or triangle is directed upward. As drops of buffer solution are dropped into the buffer receiving opening, the apex divides the drops into two streams. The first stream is directed to the first sorbent material. The second stream is directed to the second elongated path sorbent material. 
         [0024]    A second means for dividing the buffer solution received at the second location is a flow control material which is chosen to permit buffer to flow but which is resistant to receiving sample. The flow control material is coupled to both the first and second sorbent materials. 
         [0025]    A third means for dividing the buffer solution received at the second location is an arrangement where the second location is an opening in a housing sized to receive the nib of a buffer dispenser, and a vertical wall recessed in the housing opening is provided to divide the buffer solution. Preferably, the first sorbent material is provided on one side of the vertical wall, and the second sorbent material is provided on the other side of the vertical wall. 
         [0026]    According to one aspect of the invention, the means for dividing the buffer solution may be arranged so that approximately half of the buffer solution is directed toward the first sorbent material and the other half of the buffer solution is directed toward the second sorbent material. 
         [0027]    According to another aspect of the invention, the means for dividing the buffer solution may be arranged so that a desired first proportion of the buffer solution is directed toward the first sorbent material and a desired different second proportion of the buffer solution is directed toward the second sorbent material. 
         [0028]    In one embodiment, the locations for receiving the sample and the buffer solution are near or adjacent each other. 
         [0029]    In the preferred embodiment of the invention, the first sorbent material and second sorbent material are separate pieces which overlie one another at the test site junction, and the test line is printed on one or both of the sorbent materials at the junction. The systems of the invention preferably also include a control line or site which may be seen from the viewing window. 
         [0030]    According to one aspect of the invention, a test cell as summarized above may be used by (1) depositing a sample at the (first) location for receiving the sample, (2) depositing buffer solution at the (second) location for receiving the buffer solution, and (3) after a desired period of time, inspecting the test zone to determine whether the test is positive or negative. It is noted that sufficient buffer is deposited at the second location to cause the sample to move along the first sorbent material to the test zone, as well as to traverse the path of the second (and where provided, third) sorbent material and cause the conjugate to move to the test zone. Because of the arrangement of the first sorbent material as well as the second (and where provided, third) sorbent material, the sample is brought to the test zone in advance of the conjugate. 
         [0031]    In one embodiment of the invention, the materials, thicknesses and lengths of the first and second sorbent materials are chosen to adjust the timing regarding the sample and conjugate reaching the test site. 
         [0032]    In a fourth generation assay, two paths are provided for directing sample to two test zones, and two elongated paths are provided for carrying buffer and conjugate to the test zones. A first test zone is provided at the intersection of one of the elongated paths with one of the two sample paths, and a second test zone is provided at an intersection of the other elongated path and the second of the sample paths. In the fourth generation assay, one of the test zones may test for antigens in the sample while the other of the test zones may test for antibodies in the sample. 
         [0033]    Additional objects and advantages of the invention will become apparent to those skilled in the art upon reference to the detailed description taken in conjunction with the provided figures. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0034]      FIG. 1  is a schematic view of a first embodiment of the invention. 
           [0035]      FIG. 2  is a schematic view of a second embodiment of the invention. 
           [0036]      FIG. 3   a  is a schematic view of a third embodiment of the invention. 
           [0037]      FIG. 3   b  is a schematic view of an alternative third embodiment of the invention. 
           [0038]      FIG. 4  is a schematic view of a fourth embodiment of the invention. 
           [0039]      FIG. 5  is a schematic view of a fifth embodiment of the invention. 
           [0040]      FIG. 6  is a schematic view of a sixth embodiment of the invention. 
           [0041]      FIG. 7  is a side view of a first embodiment of a buffer divider. 
           [0042]      FIG. 8  is a top view of a second embodiment of a buffer divider. 
           [0043]      FIG. 9   a  is a top view of a third embodiment of a buffer divider without the dropper. 
           [0044]      FIG. 9   b  is a side view of the third embodiment of the buffer divider. 
           [0045]      FIG. 10  is a cross-sectional view of a buffer button. 
       
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
       [0046]    Before turning to the Figures, the previously incorporated applications are noted as providing examples of dual path immunoassay devices relevant to the present invention. In particular, many of the same elements described in the embodiments of the previously incorporated applications are utilized as the building blocks for the embodiments of the invention which are further described below. As a result, details of those elements are not further described, and attention may be paid to those applications for those details. 
         [0047]    Turning now to  FIG. 1 , a first embodiment of a dry conjugate dual path immunoassay device system  1000  is provided. System  1000  and includes a test cell  1010  with a first location  1026  for receiving a sample and a second location  1024  for receiving a buffer solution. A first sorbent or bibulous material  1032  is provided for directing a horizontal flow path  1061  for the sample. Means discussed below with reference to  FIGS. 7-9   b  are provided for dividing the buffer solution received at the second location  1024  such that some of the buffer solution is directed to the first sorbent material  1032  which directs the sample, and some of the buffer solution is directed to a second sorbent or bibulous material  1030 . In the embodiment of  FIG. 1 , the second sorbent material  1030  is seen to take an elongated path  1062  to the test zone  1033  which is located at the junction of the elongated path of the second sorbent material  1030  with the first sorbent material  1032 . The test zone  1033  includes one or more test lines or indicators (e.g.,  1050 A,  1050 B) having immobilized antigens or antibodies on either sorbent material  1030  or sorbent material  1032 . Preferably, a control line  1060  is provided downstream of the test line(s), and an optional reservoir or wicking zone  1037  may be provided as well. Conjugate  1039  having desired antigens or antibodies with attached colored markers is immobilized along the elongated path  1062  in or on the second sorbent material  1030  upstream of the test zone  1033 . As shown in  FIG. 1 , the conjugate  1039  is preferably located toward the test zone end of the elongated path. The elongated path  1062  of the second sorbent material  1030  of  FIG. 1  is shown to be tortuous and more than five times the length of the horizontal flow path  1061  for the sample. In this manner, if sample is first deposited into the first location  1026 , and buffer is then deposited in the second location  1024 , the buffer will cause the sample to reach the test zone  1033  via path  1061  and permit antigens or antibodies in the sample to bind to the antibodies or antigens in the test lines  1050 A,  1050 B in advance of the conjugate  1039  being carried by buffer reaching the test zone via the elongated path  1062 . 
         [0048]    A second embodiment of a dry conjugate dual path immunoassay device system  1100  is seen in  FIG. 2 . System  1100  and includes a test cell  1110  with a first location  1126  for receiving a sample and a second location  1124  for receiving a buffer solution. A first sorbent or bibulous material  1132  is provided for directing a horizontal flow path  1161  for the sample. Means discussed below with reference to  FIGS. 7-9   b  are provided for dividing the buffer solution received at the second location  1124  such that some of the buffer solution is directed to the first sorbent material  1132  which directs the sample, and some of the buffer solution is directed to a second sorbent or bibulous material  1130 . In the embodiment of  FIG. 2 , the second sorbent material  1130  is seen to take an elongated path  1162  to the test zone  1133  which is located at the junction of the elongated path of the second sorbent material  1130  with the first sorbent material  1132 . The test zone  1133  includes one or more test lines or indicators (e.g.,  1150 A,  1150 B) having immobilized antigens or antibodies on either sorbent material  1130  or sorbent material  1132 . Preferably, a control line  1160  is provided downstream of the test line(s), and an optional reservoir or wicking zone  1137  may be provided as well. Conjugate  1139  having desired antigens or antibodies with attached colored markers is immobilized along the elongated path  1162  in or on the second sorbent material  1130  upstream of the test zone  1133 . As shown in  FIG. 1 , the conjugate  1139  is preferably located toward the test zone end of the elongated path. The elongated path  1162  of the second sorbent material  1130  of  FIG. 1  is shown to be curved and approximately four times the length of the horizontal flow path  1161  for the sample. In this manner, if sample is first deposited into the first location  1126 , and buffer is then deposited in the second location  1124 , the buffer will cause the sample to reach the test zone  1133  via path  1161  and permit antigens or antibodies in the sample to bind to the antibodies or antigens in the test lines  1150 A,  1150 B in advance of the conjugate  1139  being carried by buffer reaching the test zone via the elongated path  1162 . 
         [0049]    Alternate third embodiments of a dry conjugate dual path immunoassay device system  1200   a ,  1200   b  are seen in  FIGS. 3   a  and  3   b . The alternate third embodiments are similar to the second embodiment system  1100  except that the instead of providing elongated paths  1262   a ,  1262   b  for the buffer which are approximately four times the length of the sample paths  1261   a ,  1261   b , the curved paths  1262   a ,  1262   b  are about twice as long as the sample paths, but are provided with a delay element  1295   a ,  1295   b  along their lengths. In the embodiment of  FIG. 3   a , the delay element  1295   a  is a narrowing or bottleneck in the flow path which limits the flow capacity therethrough. In the embodiment of  FIG. 3   b , the delay element  1295   b  is a viscous material (e.g., sugar) or a blocking agent (e.g., BSA—bovine serum albumin). Delay element  1295   b  is utilized to delay the flow of buffer along the second sorbent material  1230   a ,  1230   b  such that buffer deposited into the second location  1216   a ,  1216   b  (and divided by means discussed below with reference to  FIGS. 7-9   b ) will cause the sample deposited into the first location  1226   a ,  1226   b  to reach the test zone  1233   a ,  1233   b  via path  1261   a ,  1261   b  and permit antigens or antibodies in the sample to bind to the antibodies or antigens in the test line  1250   a ,  1250   b  in advance of the conjugate  1239   a ,  1239   b  being carried by buffer reaching the test zone via the elongated path  1262   a ,  1262   b  which includes the delay element  1295   a ,  1295   b . In addition, if desired, multiple delay elements may be provided along the second sorbent material. The alternate third embodiments also preferably include control lines in zones  1250   a ,  1250   b  and wicking zones  1237   a ,  1237   b.    
         [0050]    A fourth embodiment of a dry conjugate dual path immunoassay device system  1300  is seen in  FIG. 4 . The fourth embodiment is similar to the alternate third embodiment systems  1200   a ,  1200   b  except that the instead of providing a delay element along the length second sorbent or bibulous material, a third sorbent or bibulous material  1330 A is provided between the second receiving location  1324  and the second sorbent material  1330 . The third sorbent material  1330 A is selected to have a slower flow characteristic than either the first sorbent material  1332  or the second sorbent material  1330 . The conjugate material  1339  is preferably located along the second sorbent material  1330 , although it could be located along the third sorbent material  1330 A. The test zone  1333  with one or more test lines  1350 A,  1350 B is located at the intersection of the first sorbent material  1332  and the second sorbent material  1330 , and a control zone  1360  with a control indicator as well as a wicking zone  1337  are preferably provided downstream of the test zone  1333 . With the provided arrangement, if sample is first deposited into the first location  1326 , and buffer is then deposited in the second location  1324  (and divided by means discussed below with reference to  FIGS. 7-9   b ), the buffer will cause the sample to reach the test zone  1333  via path  1361  and permit antigens or antibodies in the sample to bind to the antibodies or antigens in the test lines  1350 A,  1350 B in advance of the conjugate  1339  being carried by buffer reaching the test zone via the elongated path  1362  which includes the third sorbent material  1330 A and the second sorbent material  1330 . 
         [0051]    A fifth embodiment of a dry conjugate dual path immunoassay device system  1400  is seen in  FIG. 5  and provides a fourth generation immunoassay device. The fifth embodiment is similar to the fourth embodiment except that two separate samples are established, two elongated paths are established for the buffer (and conjugate), and two test zones are established; typically, one for testing antigens and the other for testing antibodies. More particularly, system  1400  includes a includes a test cell  1410  with a first location  1426  for receiving a sample and a second location  1424  for receiving a buffer solution. A first sorbent or bibulous material  1432   a ,  1432   b  is provided for directing horizontal flow paths  1461   a ,  1461   b  for the sample. The first sorbent material may be made from a single piece of material or multiple pieces of material. Means discussed below with reference to  FIGS. 7-9   b  are provided for dividing the buffer solution received at the second location  1124  such that some of the buffer solution is directed to the first sorbent material  1432   a ,  1432   b  which directs the sample, and some of the buffer solution is directed to the third sorbent or bibulous material  1430 A (selected to have a slower flow characteristic than either the first sorbent material  1432   a ,  1432   b  or the second or fourth sorbent material  1430   a ,  1430   b ). In the embodiment of  FIG. 5 , the third sorbent material  1430 A is arranged as a “T”, although the material could be formed into other shapes (such as a “V”, an “M”, or a dash, by way of example only). Regardless, the third sorbent material  1430 A is in contact with second sorbent material  1430   a ,  1430   b  (shown as two separate pieces in FIG.  5 —although depending upon the arrangement of the third sorbent material, the second sorbent material could be a single piece such as in the shape of a bracket ([), an “E”, by way of example only). This provides two elongated path  1462   a ,  1462   b  to the respective test zones  1433   a ,  1433   b  which are respectively located at the junction of the second sorbent material  1430   a  with the first sorbent material  1432   a , and the junction of the second sorbent material  1430   b  with the first sorbent material  1432   b . The test zones  1433   a ,  1433   b  include one or more test lines or indicators (e.g.,  1450 A,  1450 B) having immobilized antigens or antibodies on either sorbent material  1430   a ,  1430   b  or sorbent material  1432   a ,  1432   b . Preferably, control lines  1460   a ,  1460   b  is provided downstream of the test lines, and an optional reservoir or wicking zone  1437   a ,  1437   b  may be provided as well. Conjugate  1439   a ,  1439   b  having desired antigens or antibodies with attached colored markers is immobilized along the elongated paths  1462   a ,  1462   b  preferably in or on the second sorbent material  1430   a ,  1430   b  upstream of the test zones  1433   a ,  1433   b . As shown in  FIG. 5 , the conjugate zones are preferably located toward the test zone ends of the elongated paths. With the provided arrangement, if sample is first deposited into the first location  1426 , and buffer is then deposited in the second location  1424 , the buffer will cause the sample to reach the test zones  1433   a ,  1433   b  via paths  1461   a ,  1461   b  and permit antigens and/or antibodies in the sample to bind to the antibodies and/or antigens in the test lines  1450 A,  1450 B in advance of the conjugate  1439   a ,  1439   b  being carried by buffer reaching the test zone via the elongated paths  1462   a ,  1462   b.    
         [0052]    In the fourth and fifth embodiments, the third sorbent or bibulous material which has a slower flow characteristic relative to the first sorbent material may be a small pore membrane (e.g., nitrocellulose or nylon membrane having a pore size of 3 to 30 microns), glass fibers, or cellulose, polyester, rayon or other known synthetic materials. As will be appreciated by those skilled in the art, other materials could be utilized to control the speed of the buffer flow through the third sorbent material and thereby control arrival timing of the buffer with the conjugate at the test site relative to the arrival of the sample. 
         [0053]    A sixth embodiment of a dry conjugate dual path immunoassay device system  1500  is seen in  FIG. 6  and also provides a fourth generation immunoassay device. The sixth embodiment is similar to the fifth embodiment except that instead of providing a third sorbent material having slower flow characteristics, the elongated paths for the buffer which eventually carries the conjugate are tortuous or serpentine, and are at least five times the length of the sample flow path. More particularly, system  1500  includes a includes a test cell  1510  with a first location  1526  for receiving a sample and a second location  1524  for receiving a buffer solution. A first sorbent or bibulous material with branches  1532   a ,  1532   b  is provided for directing horizontal flow paths  1561   a ,  1561   b  for the sample. The first sorbent material may be made from a single piece of material or multiple pieces of material. Means discussed below with reference to  FIGS. 7-9   b  are provided for dividing the buffer solution received at the second location  1524  such that some of the buffer solution is directed to the first sorbent material  1532   a ,  1532   b  which directs the sample, and some of the buffer solution is directed to the second sorbent or bibulous material  1530   a    1530   b  which provides two elongated path branches  1562   a ,  1562   b  to the respective test zones  1533   a ,  1533   b  which are respectively located at the junction a first branch of the second sorbent material  1530   a  with a first branch of the first sorbent material  1532   a , and the junction of a second branch of the second sorbent material  1530   b  with a second branch of the first sorbent material  1532   b . The test zones  1533   a ,  1533   b  include one or more test lines or indicators (e.g.,  1550 A,  1550 B) having immobilized antigens or antibodies on either sorbent material  1530   a ,  1530   b  or sorbent material  1532   a ,  1532   b . Preferably, control lines  1560   a ,  1560   b  is provided downstream of the test lines, and an optional reservoir or wicking zone  1537   a ,  1537   b  may be provided as well. Conjugate  1539   a ,  1539   b  having desired antigens or antibodies with attached colored markers is immobilized along the elongated paths  1562   a ,  1562   b  preferably in or on the second sorbent material  1530   a ,  1530   b  upstream of the test zones  1533   a ,  1533   b . As shown in  FIG. 6 , the conjugate zones are preferably located toward the test zone ends of the elongated paths. With the provided arrangement, if sample is first deposited into the first location  1526 , and buffer is then deposited in the second location  1524 , the buffer will cause the sample to reach the test zones  1533   a ,  1533   b  via paths  1561   a ,  1561   b  and permit antigens and/or antibodies in the sample to bind to the antibodies and/or antigens in the test lines  1550 A,  1550 B in advance of the conjugate  1539   a ,  1539   b  being carried by buffer reaching the test zone via the elongated paths  1562   a ,  1562   b.    
         [0054]    In all the previous embodiments, where the test cell of the invention is provided in a housing, the housing is provided with a first opening adjacent the first location and a second opening adjacent the second location. A viewing window is provided in the housing above the test line. Where a control line is provided, the viewing window may extend over the test line and control line, or a separate viewing window may be provided over the control line. 
         [0055]    In all of the previous embodiments, it is preferred that the locations for receiving the sample and the buffer solution are provided near or adjacent each other. 
         [0056]    Turning now to  FIGS. 7-9   b , various dividers which divide the buffer solution received at the second location are provided. A first divider for the buffer solution is seen in  FIG. 7  and comprises a wedge-shaped element  1601  (V-shaped or triangular in cross section) located at the location for receiving the buffer solution (e.g., at an opening in a housing), where the apex  1602  of the wedge is directed upward. As drops of buffer solution are dropped into the buffer receiving opening, the apex divides the drops into two streams. The first stream is directed to the first sorbent material. The second stream is directed to the second elongated path sorbent material. If the wedge apex is located in the middle of the buffer receiving location, half of the buffer solution is directed toward the sample receiving location and first sorbent material, and the other half is directed toward the second sorbent material. If the wedge apex is located to one side or the other, more of the buffer may be directed one way or to the other. 
         [0057]    A second divider for the buffer solution received at the second location is shown in  FIG. 8  and comprises a flow control material  1601   a . Flow control material  1601   a  is chosen to permit buffer to flow but is resistant to receiving sample. An example would be a very small pore size material such as cotton or cellulose paper (preferably with pores less than 3 microns). The flow control material  1601   a  is coupled to both the sample receiving location (e.g., pad) and the second (or third) sorbent material. 
         [0058]    A third divider  1601   b  for the buffer solution received at the second location is shown in  FIGS. 9 and 9   a  and comprises an opening  1603  in a housing  1604  sized to receive the nib  1605  of a buffer dispenser, and a vertical wall  1606  recessed in the housing opening which divides the buffer solution. Preferably, the sample receiving location or pad is provided on one side of the vertical wall, and the second (or third) sorbent material is provided on the other side of the vertical wall. It will be appreciated that the wall  1606  can take any desired shape as long as it is located appropriately to divide drops of buffer being delivered by the buffer dispenser nib. Thus wall may divide the buffer solution so that approximately half of the buffer solution is directed toward the first sorbent material and the other half of the buffer solution is directed toward the second sorbent material, or may divide the buffer solution so that a desired first proportion of the buffer solution is directed toward the first sorbent material and a desired different second proportion of the buffer solution is directed toward the second sorbent material. 
         [0059]    With respect to all of the above test cell embodiments and buffer divider embodiments, if desired, the buffer may be packaged as part of the test cell by providing a “buffer button”  1780  as seen in  FIG. 10  at the second location. For example, if a housing is provided, the buffer button may constitute a module located in a housing opening above the second location. The module could constitute a flexible plastic upper member  1781  and a rupturable (e.g., foil or plastic) lower member  1782 , with the buffer solution  1783  contained therebetween. As a result, when the flexible plastic upper member is pressed, the force is translated to the buffer solution which causes the lower member to rupture and release the buffer solution which is then divided by the buffer dividing means. Alternatively, contained within the module may be a dividing element (e.g., a vertical plastic element connected to the flexible plastic upper member) which causes the lower container to rupture and which will automatically divide the buffer solution contained within the module without need for a separate means for dividing the buffer solution. As another alternative, a wedge (such as wedge  1600 ) or a wall (such as wall  1606 ) contained within the housing below the buffer button can be used to rupture the lower member when the buffer button is pressed. 
         [0060]    According to one aspect of the invention, any of the test cells described above may be used by (1) depositing a sample at the (first) location for receiving the sample, (2) depositing a predetermined amount of buffer solution at the (second) location for receiving the buffer solution, and (3) after a desired period of time, inspecting the test zone(s) and where provided the control zone(s) to determine whether the test is positive or negative. It is noted that sufficient buffer is deposited at the second location to cause the sample to move along the first sorbent material to the test zone, as well as to traverse the path of the second (and where provided, third) sorbent material and cause the conjugate to move to the test zone. Because of the arrangement of the first sorbent material as well as the second (and where provided, third) sorbent material, the sample is brought to the test zone in advance of the conjugate. The step of depositing a sample may involve depositing blood, serum, spittum, feces, or other bodily fluid at the first location via a dropper, a swab, a loop or other depositing means known in the art. The step of depositing a predetermined amount of buffer solution may comprise utilizing a dropper, pressing a buffer button, or utilizing any other depositing means known in the art. 
         [0061]    It will be appreciated that the materials, thicknesses and lengths of the first and second sorbent materials are chosen to adjust the timing regarding the sample and conjugate reaching the test site(s). 
         [0062]    There have been described and illustrated herein several embodiments of immunoassays and methods of their use. While particular embodiments of the invention have been described, it is not intended that the invention be limited thereto, as it is intended that the invention be as broad in scope as the art will allow and that the specification be read likewise. For example, while the specification discusses ligand binding using antigen/antibody reactions, other ligand binding mechanisms such as aptamer binding, nucleic acid binding, enzymatic binding, etc. may also be used. Also while particular buffer dividers have been described which divide the buffer for the sample flow path and the conjugate flow path, it will be appreciated that other dividers could be utilized. Further, it should be appreciated that chemical agents such as sugar, BSA, detergent, etc., or biological agents (serum, antibody, antigen) may be added in one or both of the sorbent strips in order to delay or enhance flow rate for the buffer or for the buffer/sample solution. These modifications could be additionally utilized to enhance sensitivity or block non-specific binding for the assay. It will therefore be appreciated by those skilled in the art that yet other modifications could be made to the provided invention without deviating from its spirit and scope as claimed.