Abstract:
Disclosed are the methods for the detection and/or quantification of an analyte in liquid sample. The same principle can be applied in the case of detection and/or quantification of more than one analyte present in the sample. Devices of the invention include a sample application zone, a mobilization zone containing a mobile or mobilizable first binder that is specific for the analyte, a absorption zone containing immobilized analyte or analyte analog, a mobilization zone containing a mobile or mobilizable labeled second binder capable of binding to the first binder, and a capture zone or test zone containing an immobilized third binder capable of binding to the complex of the analyte, the first binder and the second binder. The color development in the capture zone or the test zone is indicative of presence or the concentration of the analyte in the test sample.

Description:
U.S. PATENT DOCUMENTS  
       [0001]     U.S. Pat. No. 4,168,146 September 1979 Grubb  
         [0002]     U.S. Pat. No. 4,235,601 November 1980 Deutsch  
         [0003]     U.S. Pat. No. 4,366,241 December 1982 Tom  
         [0004]     U.S. Pat. No. 4,442,204 April 1984 Greenquist  
         [0005]     U.S. Pat. No. 4,703,017 October 1987 Campbell  
         [0006]     U.S. Pat. No. 4,743,560 May 1988 Campbell  
         [0007]     U.S. Pat. No. 5,073,484 December 1991 Swanson  
         [0008]     U.S. Pat. No. 5,451,504 September 1995 Fitzpatrick  
         [0009]     U.S. Pat. No. 5,451,507 September 1995 Skold  
         [0010]     U.S. Pat. No. 5,798,273 August 1998 Shuler  
         [0011]     U.S. Pat. No. 6,485,982 B1 November 2002 Charlton  
         [0012]     The above cited references and all other references cited in this application are hereby incorporated herein by reference.  
       FIELD OF INVENTION  
       [0013]     The present invention relates to lateral-flow methods and/or devices for determining the presence and/or concentration of analytes in fluid samples. The analytes in sample can be a small molecule possessing only one epitope or a large molecule possessing multiple epitopes. The present invention provides a direct or positive detection result (i.e. increasing signal with increasing analyte concentration) regardless of the analyte being small or large.  
       BACKGROUND OF THE INVENTION  
       [0014]     The use of fast test devices in home to test for pregnancy, ovulation, and drugs of abuse is now a commonplace. The wide variety of the test devices is available commercially. Almost all of these commercially available home test devices are based upon lateral flow technology and ligand-binder (or analyte-binder) assay. Many types of ligand-binder assays have been used to detect the presence or quantification of various substances, or frequently called analytes, in liquid sample. These assays often involve antigen antibody reactions, receptor reactions, and enzymatic reactions. Many of these assays are based on the highly specific interactions between the binding pairs and with one or more of the members of a binding pair attached to a mobile or immobilized solid phase material such as latex, glass fibers, glass beads, cellulose strips, or nitrocellulose membranes (U.S. Pat. No. 4,703,017; 4,743,560; 5,073,484). In almost all these assays, there is a binder, e.g., an antibody or a receptor, which is specific for the selected ligand or analyte. A means is used for indication of the presence or the amount of the ligand-binder reaction product. Many such tests are designed to make a quantitative or semi-quantitative determination. But in certain circumstances, it is just required as a positive/negative indication. Examples of such qualitative assays include blood typing and most types of urinalysis. For these tests, visually observable indicia of a color change are preferred.  
         [0015]     The requirements for the sensitivity and specificity of fast assays with lateral flow technology could be very high. For example, the pregnancy home test kit has the sensitivity of human Chorionic Gonadotropin (hCG) at 50 mlU/mL or even 25 mlU/mL, that is about 5 ng/mL or 2.5 ng/mL. For the analyte of large molecule, the sandwich assays and other sensitive detection methods, which use metal sols or other types of colored particles, have been developed. Examples of sandwich immunoassays performed on test strips are described in U.S. Pat. Nos. 4,168,146, 4,366,241, and 6,485,982.  
         [0016]     However, these techniques have not solved all of the problems encountered in these rapid detection methods, especially for the analytes of small molecular weight. Due to the size limitation, the format of sandwich assay cannot be used since the small size of analyte does not allow simultaneous binding of more than one binder. One solution to this problem is classic competitive immunoassay. In competitive immunoassay, the label is typically a labeled analyte or analyte analog that competes with unlabeled analyte present in the sample for binding to an antibody. Or the binder (usually the antibody) is labeled, and the analyte in the test sample competes with the immobilized analyte or analyte analog. Examples of competitive immunoassay devices are those disclosed by U.S. Pat. Nos. 4,235,601 and 4,442,204. The widely used classic competitive assay format is usually not very sensitive. The other troublesome issue for this type of competitive assay is that the intensity of assay signal is inversely proportional to the analyte concentration. The higher the analyte concentration in the test sample, the weaker the assay signal. This is opposite of human instinct. It may not be a serious issue for professional users, but is could be awkward for ordinary consumers who are lack of experience and training. U.S. Pat. Nos. 5,451,504, 5,451,507, and 5,798,273 revealed several methods for detection small analytes using lateral flow tests with positive readout (i.e., increase the signal with increasing analyte concentration). U.S. Pat. No. 5,451,504 employs a method with three specific zones (mobilization, trap, and detection). The mobilization zone contains mobilizable binder (the labeled antibody) specific to the analyte in the sample. In the trap zone, the immobilized analyte analog traps or stops the labeled antibody that not bound with analyte. The detection zone indicates the presence of analyte by capturing the labeled analyte-antibody complex.  
         [0017]     U.S. Pat. No. 5,451,507 disclosed a two-zone method. The first zone has immobilized reagent that capable of binding the analyte or labeled analyte analog. The second zone binds to the labeled analytes or labeled analyte analog only when the analyte is present in test sample. The distance the labeled analyte or labeled analyte analog migrates into the second zone is directly related to the concentration of analyte.  
         [0018]     U.S. Pat. No. 5,798,273 discloses an approach that contains a capture zone with immobilized analyte or analyte analog and one or more read-out zones to bind labeled analyte-analog. A disadvantage of this method is the requirement to premix the test sample with antibody and labeled analyte or labeled analyte analog prior to application the mixture to the test device.  
       SUMMARY OF THE INVENTION  
       [0019]     It is an object of this invention to provide a method for detection analyte in liquid with positive readout format. In principle, this disclosed approach can be applied to a vast array of analytes no matter the analyte is small molecule or large molecule, as long as there is a correspondent specific binder such as antibody against the target analyte.  
         [0020]     It is a further object of this invention to improve the assay sensitivity for small molecules and the readability of such assays for semi-quantitative or quantitative purposes. The disclosed invention aims at achieving the above mentioned objects with minimum change of assay format or reagent arrangement regarding to the current lateral flow technology. 
     
    
     DETAILED DESCRIPTION OF EMBODIMENTS  
       [0021]     This invention discloses a method to determine a substance or substances in liquid sample. The substance being interested for identifying is defined as analyte. The analyte can be small molecule such as controlled drug, or large molecule such as protein. The pre-requisite for using this method is there must be a binder or binders. The definition of a binder is a substance that can specifically bind to the target molecule, or conjugate, or complex. For detecting analyte, the binder is required to specifically bind to its correspondent analyte or analyte analog. A binder for one analyte can itself be a binding target of another binder. A binder may have one binding site or several binding sites for its analyte or analyte analog. Two different binders may simultaneously bind to the same target analyte to form a sandwich without exclusion to each other. Antibody is a good example of such binder. The binder for one target can be the same substance or a mixture of substances, such as monoclonal antibody or polyclonal antibodies as long as the specificity is satisfied.  
         [0022]      FIG. 1  is a schematic top view of test strip;  
         [0023]      FIG. 2  is the schematic top view of Format 1;  
         [0024]      FIG. 3  is the schematic top view of Format 1.1;  
         [0025]      FIG. 4  is the schematic top view of Format 1.2;  
         [0026]      FIG. 5  is the schematic top view of Format 1.3;  
         [0027]      FIG. 6  is the schematic top view of Format 1.4;  
         [0028]      FIG. 7  is the schematic top view of Format 2;  
         [0029]      FIG. 8  is the schematic top view of Format 2.1;  
         [0030]      FIG. 9  is the schematic top view of Format 2.2;  
         [0031]      FIG. 10  is the schematic top view of Format 2.3;  
         [0032]      FIG. 11  is the schematic top view of Format 2.4;  
         [0033]      FIG. 12  is the schematic top view of Format 3;  
         [0034]      FIG. 13  is the schematic top view of Format 3.1;  
         [0035]      FIG. 14  is the schematic top view of Format 3.2;  
         [0036]      FIG. 15  is the schematic top view of Format 3.3;  
         [0037]      FIG. 16  is the schematic top view of Format 3.4;  
         [0038]      FIG. 17  is the schematic top view of Format 3.5;  
         [0039]      FIG. 18  is the schematic top view of Format 4;  
         [0040]      FIG. 19  is the schematic top view of Format 4.1;  
         [0041]      FIG. 20  is the schematic top view of Format 4.2;  
         [0042]      FIG. 21  is the schematic top view of Format 4.3;  
         [0043]      FIG. 22  is the schematic top view of Format 4.4;  
         [0044]      FIG. 23  is the schematic top view of Format 4.5; 
     
    
       [0000]     Format 1 (Detection of Single Analyte)  
         [0045]     A test device is used in this invention to determine an analyte in liquid. The device comprises a strip  10 . The strip  10  contains two portions made of permeable materials, the first portion  11  and the second portion  12 . Those two portions are connected on the same plane. The capillary liquid flow can pass the junction  13  and move from the first portion  11  to the second portion  12 . The first portion  11  is in up stream position, and the second portion  12  is in down stream position (as shown in  FIG. 1 ). Before assay, the strip is in dry status.  
         [0046]     The first portion  11  contains a sample application site  14  for liquid sample application. Additional liquid containing no analyte may also be applied onto the sample application site  14  or up stream of sample application site  14  for the purpose of washing or signal development (as shown in  FIG. 2 ).  
         [0047]     On the first portion  11  down stream of sample application site  14 , there is a first binder site  15 . The first binder is pre-loaded onto this site. This binder can specifically bind to the analyte or the analyte analog. The first binder is in dry status before liquid application. This binder is mobilizable by liquid sample and carried by the liquid flow. The amount of liquid applied onto the first portion  11  should be sufficient to carry this binder down stream. The liquid should reach at least the area down stream of the test site  18  on the second portion  12 .  
         [0048]     The second portion  12  contains an absorption site  16  down stream of the first binder site  15 . The absorption site  16  contains immobilized analyte or analyte analog. If the first binder has vacant binding site or sites (that is, the binding site(s) not occupied by the analyte or analyte analog), the binder can bind to the immobilized analyte or analyte analog at this site, and stops to move along the liquid flow. Thus, if the first binder contains vacant binding site, it can be absorbed at absorption site  16 , and cannot be further carried away by the liquid flow. If there is analyte in the test sample, the first binder can form complex with analyte. If all of the binding sites of the first binder in the said complex has been occupied by the analyte in the test sample, the complex of first binder and analyte will not be absorbed at absorption site  16 . Such complex can be carried further by the liquid flow. The amount of immobilized analyte or analog is high enough that the first binder without forming a complex with its analyte will be absorbed at the absorption site  16 .  
         [0049]     The second portion  12  contains a colored conjugate site  17  down stream of the absorption site  16 . This colored conjugate site  17  contains a colored conjugate made of colored particulate and the second binder. The second binder can specifically bind to the first binder regardless of the first binder already binds to analyte or not. Through the binding to the first binder, the second binder can form a complex with first binder, or form a complex with the complex of first binder and the analyte(s). The colored conjugate can be mobilized and carried by the liquid flow. The colored particulate can be colored latex, metal colloid, black carbon particle, fluorescent particle, phosphorescent particle, or liposome.  
         [0050]     The second portion  12  contains a test site  18  down stream of the colored conjugate site  17 . The test site  18  contains immobilized third binder. The third binder can specifically bind to the first binder regardless of the first binder is in free form, or bound with analyte, or formed complex with second binder. The second binder and the third binder can bind with first binder at same time, without exclusion to each other.  
         [0051]     The second portion  12  may also contain a control site  19  down stream of test site  18 . The control site  19  contains a forth binder immobilized at this site. The forth binder can bind to the second binder in the colored conjugate. In the assay, the control site  19  develops color when colored conjugate reached its place to indicate a valid assay condition.  
         [0052]     At down stream of the control site  19  of the second portion  12 , the device may contain an absorbent material  20  in close contact with the second portion  12 . That material  20  can absorb liquid to function as sink to facilitate the capillary liquid flow.  
         [0053]     If there is a detectable level of analyte in the applied test sample, some of the mobilized first binder will form complex with analyte in the way that its binding site is occupied by the analyte. Such non-vacant complex will pass the absorption site  16  and meet the mobilizable colored conjugate. The second binder of the colored conjugate will specifically bind to the first binder of the non-vacant complex, form a complex and flow along the liquid. When liquid flow carries the complex to the test site  18 , the immobilized third binder will catch it through binding to the first binder in the complex. Therefore, there will be color development at the test site  18 . The more the analyte in the test sample, the more the non-vacant complex of the first binder and analyte, the more the complex of colored conjugate and the non-vacant complex, and finally the more the color development at the test site  18 . Thus, the more the analyte in the test sample, the darker the color development at the test site  18 . If there is no detectable level of analyte in the sample, the first binder would be absorbed at the absorption site  16 . The second binder of the colored conjugate could not form a complex with the first binder. There would be no detectable color development at the test site  18 . Therefore, the color intensity at the test site  18  is indicative of the presence or absence of analyte, and its concentration.  
         [0000]     Format 1.1  
         [0054]     This is a modification to Format 1. This format is same as the Format 1 except that the first binder site  15  is on the second portion  12  (not on the first portion  11  as in Format 1), down stream of sample application site  14  and up stream of absorption site  16  (as shown in  FIG. 3 ).  
         [0000]     Format 1.2  
         [0055]     This is a modification to Format 1. The absorption site  16  is on the first portion  11  of strip  10  (not on the second portion  12  as in Format 1), and is down stream of first binder site  15 . The other conditions are the same as the Format 1 (as shown in  FIG. 4 ).  
         [0000]     Format 1.3  
         [0056]     This is a modification to Format 1. The colored conjugate site  17  and the absorption site  16  are on the first portion  11  of strip  10  (not on the second portion  12  as in Format 1). The colored conjugate site  17  is down stream of absorption site  16 , and the absorption site  16  is down stream of first binder site  15 . The other conditions are the same as the Format 1 (as shown in  FIG. 5 ).  
         [0000]     Format 1.4  
         [0057]     This is a modification to Format 1. There is same arrangement for the strip  10  as in Format 1 except there is no first binder site  15  on the strip  10 . The first binder is pre-mixed with the testing sample. Then, the mixture of first binder and testing sample is applied onto the sample application site  14  (as shown in  FIG. 6 ).  
         [0000]     Format 2 (detection of multiple analytes)  
         [0058]     The strip  21  contains two portions made of permeable materials. The first portion  22  and the second portion  23 . Both are connected on the same plane. The capillary liquid flow can move from the first portion  22  to the second portion  23  through passing the junction  24 . The first portion  22  is in up-stream position, and the second portion  23  is in down-stream position (as shown in  FIG. 7  for two analytes). Before assay, the strip is in dry status.  
         [0059]     The first portion  22  contains a sample application site  25  for liquid sample application. Additional liquid containing no analyte may also be applied onto the sample application site  25  or the up-stream of sample application site  25  for the purpose of washing or signal development.  
         [0060]     On the first portion  22  down stream of sample application site  25 , there is a first binder site  26 . The first binders are loaded onto this site. For detection two or more than two analytes, this invention requires each first binder having its correspondent target analyte. Each analyte has a correspondent first binder that can specifically bind it. Each first binder can be the same substance or a mixture of substances, such as monoclonal antibody or polyclonal antibodies as long as the specificity is satisfied. Those first binders are deposited at the first binder site  26 , down stream of sample application site  25 . The first binders can be mobilized and carried away by the liquid flow. The first binders can be loaded onto this site either in the mixture of first binders or with each first binder separately. The site  26  may contain one zone or several zones correspondent to the number of analyte.  
         [0061]     On the second portion  23  of strip  21 , there is an absorption site  27  down stream of junction  24 . For each analyte in the test sample, there is such analyte or its analyte analog immobilized at the absorption site  27 . The amount of immobilized analyte(s) or their analog(s) is high enough that the first binder(s) without forming a complex with its analyte will be absorbed at the absorption site  27 . The analytes or analogs can be loaded onto this site either with the mixture or with each component separately in manufacturing process. The site  27  may contain one zone or several zones correspondent to the number of analyte.  
         [0062]     On the second portion  23 , down stream of absorption site  27 , there is colored conjugate site  28 . For each analyte, there is a colored conjugate made of colored particulate and the correspondent second binder. Each second binder can specifically bind to its correspondent first binder of correspondent analyte. This site contains colored conjugates that each one can specifically catch the correspondent first binder that passed the absorption site  27 . The colored conjugate(s) can be mobilized and carried by the liquid flow. The different colored conjugates can be pre-mixed or separately loading onto the colored conjugate site  28  in manufacturing process. The site  28  may contain one zone or several zones correspondent to the number of analyte  
         [0063]     On the second portion  23 , down stream of colored conjugate site  28 , there is a test site  29  for multiple analytes assay. In this site, there are number of zones correspondent to the number of analytes. Each zone is correspondent to each analyte. Each zone for a specific analyte contains an immobilized third binder that can specifically bind to the correspondent first binder for that analyte. The binding of the third binder is not competitive or mutually exclusive with the binding of colored conjugate to the corresponding first binder. In this way, in the case of test sample contains detectable level of one analyte or detectable level of analytes, there will be certain portion of the correspondent first binder or first binders catching the analyte or analytes that their binding site saturated by the analyte or analytes. The non-vacant complex can pass the absorption site  27 , caught by the correspondent colored conjugate, and finally caught by the correspondent third binder on the correspondent zone in the test site  29 . Similar to the single analyte assay situation, the more the analyte or analytes, the more the correspondent non-vacant complex(s) that can pass the absorption site  27 , the more the passed non-vacant complex(s) caught by the correspondent colored conjugate(s), and finally the stronger the color development on the correspondent zone(s) of test site  29 .  FIG. 7  illustrates a test strip for identifying two analytes.  
         [0064]     The second portion  23  may also contains a control site  30  down stream of test site  29 . The control site  30  contains forth binder or binders that is immobilized at that site. The forth binder(s) can bind to the second binder(s) in the colored conjugate(s). In the assay, the control site  30  develops color when colored conjugate(s) reached its place to indicate a valid assay condition. The control site  30  may contain one zone or several zones correspondent to the number of analyte.  
         [0065]     At down stream of the control site  30  of the second portion  23 , the device may contain an absorbent material  31  in close contact with the second portion  23 . That material  31  can absorb liquid to function as sink to facilitate the capillary liquid flow.  
         [0000]     Format 2.1  
         [0066]     This format is same as the Format 2 except that the first binder site  26  is on the second portion  23  (not on the first portion  22  as in Format 2), down stream of sample application site  25  and up stream of absorption site  27  ( FIG. 8  illustrates a example strip for two analytes).  
         [0000]     Format 2.2  
         [0067]     This is a modification to Format 2. The absorption site  27  is on the first portion  22  (not on the second portion  23  as in Format 2), and is down stream of first binder site  26 . The other conditions are the same as the Format 2 ( FIG. 9  illustrates a example strip for two analytes).  
         [0000]     Format 2.3  
         [0068]     This is a modification to Format 2. The colored conjugate site  28  and the absorption site  27  are on the first portion  22  (not on the second portion  23  as in Format 2). The colored conjugate site  28  is down stream of absorption site  27 , and the absorption site  27  is down stream of first binder site  26 . The other conditions are the same as the Format 2 ( FIG. 10  illustrates a example strip for two analytes).  
         [0000]     Format 2.4  
         [0069]     This is a modification to Format 2. There is same arrangement for the strip  21  as in Format 2 except there is no first binder site  26  on first portion  22 . The first binders are pre-mixed with the testing sample. Then, the mixture of first binders and testing sample is applied onto sample application site  25  ( FIG. 11  illustrates a example strip for two analytes).  
         [0000]     Format 3  
         [0070]     This Format is a modification to Format 1. The definitions of the first binder, second binder, third binder, and the forth binder are the same as in Format 1. The device contains strip  32  with first permeable portion  33  and second permeable portion  34 . The liquid flow can pass the junction  35  from first portion  33  to the second portion  34 . The first portion  33  contains sample application site  36 , first binder site  37 . The second portion  34  contains absorption site  38 , colored conjugate site  39 , third binder site  42 , test site  40 , and may also contain control site  41 . The difference between Format 1 and this Format is that the third binder at site  42  is not immobilized in Format 3. Instead, it can be mobilized and carried by the liquid flow. In order to enhance the assay sensitivity, there is a fifth binder that can specifically bind to the third binder. The fifth binder is immobilized at test site  40  in Format 3. The binding of the fifth binder to its target, the third binder, is not affected whether the third binder has bound to its specific target or not. The site  39  can be either in the upstream of site  42  or in the down stream of site  42 , as long as both the site  39  and site  42  are in the down stream of site  38  and up stream of site  40  ( FIG. 12  illustrates the Format 3 with site  39  in up stream of site  42 ). At down stream of the control site  41 , the device may contain an absorbent material  43  in close contact with the second portion  34 . That material  43  can absorb liquid to function as sink to facilitate the capillary liquid flow.  
         [0000]     Format 3.1  
         [0071]     This is a modification to Format 3. This format is same as the Format 3 except that the first binder site  37  is on the second portion  34  (not on the first portion  33  as in Format 3), down stream of sample application site  36  and up stream of absorption site  38  (as shown in  FIG. 13  with site  39  in up stream of site  42 ).  
         [0000]     Format 3.2  
         [0072]     This is a modification to Format 3. The absorption site  38  is on the first portion  33  of strip  32  (not on the second portion  34  as in Format 3), and is down stream of first binder site  37 . The other conditions are the same as the Format 3 (as shown in  FIG. 14  with site  39  in up stream of site  42 ).  
         [0000]     Format 3.3  
         [0073]     This is a modification to Format 3. In the case of site  39  is up stream of site  42 , the colored conjugate site  39  and the absorption site  38  are on the first portion  33  (not on the second portion  34  as in Format 3). The colored conjugate site  39  is down stream of absorption site  38 , and the absorption site  38  is down stream of first binder site  37 . In the case of site  42  is up stream of site  39 , the third binder site  42  and the absorption site  38  are on the first portion  33  (not on the second portion  34  as in Format 3). The third binder site  42  is down stream of absorption site  38 , and the absorption site  38  is down stream of first binder site  37 . The other conditions are the same as the Format 3 (as shown in  FIG. 15  with site  39  in up stream of site  42 ).  
         [0000]     Format 3.4  
         [0074]     This is a modification to Format 3. There is same arrangement for the strip  32  as in Format 3 except there is no first binder site  37  on the strip  32 . The first binder is pre-mixed with the testing sample. Then, the mixture of first binder and testing sample is applied onto the sample application site  36  (as shown in  FIG. 16  with site  39  in up stream of site  42 ). The other conditions are the same as the  
         [0000]     Format 3.  
         [0000]     Format 3.5  
         [0075]     This is a modification to Format 3. The third binder site  42 , colored conjugate site  39 , and the absorption site  38  are on the first portion  33  (not on the second portion  34  as in Format 3). The third binder site  42  and the colored conjugate site  39  are down stream of absorption site  38 , and the absorption site  38  is down stream of first binder site  37 . The other conditions are the same as the Format 3 (as shown in  FIG. 17  with site  39  in up stream of site  42 ).  
         [0000]     Format 4  
         [0076]     This Format is a modification to Format 2 for detecting more than one analyte. The definitions of the first binders, second binder(s), colored conjugate(s), third binders, and the forth binder(s) are the same as in Format 2. The device contains strip  44  with first permeable portion  45  and second permeable portion  46 . The liquid flow can pass the junction  47  from first portion  45  to the second portion  46 . The first portion  45  contains sample application site  48 , first binder site  49 . The second portion  46  contains absorption site  50 , colored conjugate site  51 , third binder site  54 , test site  52 , and may also contain control site  53 . The difference between Format 2 and this Format is that the third binders at site  54  are not immobilized in Format 4. Instead, they can be mobilized and carried by the liquid flow. The site  51  can be either in the up stream of site  54  or in the down stream of site  54 , as long as both the site  51  and site  54  are in the down stream of site  50  and up stream of site  52 . In order to enhance the assay sensitivity, there are fifth binders that can specifically bind to the third binders. Each fifth binder can specifically bind to the correspondent third binder. Each third binder has correspondent fifth binder. The fifth binders are immobilized at test site  52  in Format 4. Each fifth binder has one zone at test site  52 . The number of zones of site  52  is the same as the number of analytes. Each analyte has correspondent zone in site  52 . The binding of each fifth binder to its target, the correspondent third binder, is not affected whether that third binder has bound to its specific target or not ( FIG. 18  illustrates the Format 4 for two analytes with site  51  in up stream of site  54 ). At down stream of the control site  53 , the device may contain an absorbent material  55  in close contact with the second portion  46 . That material  55  can absorb liquid to function as sink to facilitate the capillary liquid flow.  
         [0000]     Format 4.1  
         [0077]     This is a modification to Format 4. This format is same as the Format 4 except that the first binder site  49  is on the second portion  46  (not on the first portion  45  as in Format 4), down stream of sample application site  48  and up stream of absorption site  50  (as shown in  FIG. 19  for two analytes with site  51  in up stream of site  54 ).  
         [0000]     Format 4.2  
         [0078]     This is a modification to Format 4. The absorption site  50  is on the first portion  45  (not on the second portion  46  as in Format 4), and is down stream of first binder site  49 . The other conditions are the same as the Format 4 (as shown in  FIG. 20  for two analytes with site  51  in up stream of site  54 ).  
         [0000]     Format 4.3  
         [0079]     This is a modification to Format 4. In this case of site  51  is up stream of site  54 , the colored conjugate site  51  and the absorption site  50  are on the first portion  45  (not on the second portion  46  as in Format 4). The colored conjugate site  51  is down stream of absorption site  50 , and the absorption site  50  is down stream of first binder site  49 . In the case of site  54  is up stream of site  51 , the third binder site  54  and the absorption site  50  are on the first portion  45  (not on the second portion  46  as in Format 4). The third binder site  54  is down stream of absorption site  50 , and the absorption site  50  is down stream of first binder site  49 . The other conditions are the same as the Format 4 (as shown in  FIG. 21  for two analytes with site  51  in up stream of site  54 ).  
         [0000]     Format 4.4  
         [0080]     This is a modification to Format 4. There is same arrangement for the strip  44  as in Format 4 except there is no first binder site  49  on the strip  44 . The first binders are pre-mixed with the testing sample. Then, the mixture of first binders and testing sample is applied onto the sample application site  48  (as shown in  FIG. 22  for two analytes with site  51  in up stream of site  54 ). The other conditions are the same as the Format 4.  
         [0000]     Format 4.5  
         [0081]     This is a modification to Format 4. The third binder site  54 , colored conjugate site  51 , and the absorption site  50  are on the first portion  45  (not on the second portion  46  as in Format 4). The third binder site  54  and the colored conjugate site  51  are down stream of absorption site  50 , and the absorption site  50  is down stream of first binder site  49 . The other conditions are the same as the Format 4 (as shown in  FIG. 23  for two analytes with site  51  in up stream of site  54 ).  
       EXAMPLE  
       [0082]     Colored Conjugate Preparation. Mono-dispersed gold colloidal particles were prepared in accordance with the method of Frens, Controlled Nucleation for the Regulation of the Particle Size in Mono Dispersed Gold Solutions (1973). Briefly, the colloidal gold was prepared by reducing a 0.01% solution of gold chloride with 2% sodium citrate to produce gold particles of approximately 45 nm in diameter. The gold sol was adjusted to pH9.04 with Potassium carbonate. The Donkey anti-Goat IgG (obtained from Lampire Biological Laboratories, Inc.) was added at 7.5 mg of antibody per 1000 mL of gold sol solution. The conjugate was separated from the free antibody through centrifugation. The gold conjugate was in solution containing 1.14 mg/mL of BSA, 2.27 mM of Tris, pH8.0, 0.02% of sodium azide, and 0.01% of Triton X-100.  
         [0083]     The reagent of human chorionic gonadotropin (hCG) was obtained from Sigma-Aldrich Chemical Company. The hCG was diluted to 200 lU/mL in 50 mM potassium phosphate buffer, pH7 with 0.05 mg/mL of BSA, 0.05% of sodium azide, and 0.2% of mannitoal.  
         [0084]     The permeable material of nitrocellulose membrane HiFlow Plus HF09004 was obtained from Millipore Corporation. The reagents were striped on the nitrocellulose membrane according to Format 1. Donkey anti-Goat IgG solution of 0.2 mg/mL was striped onto test site of the nitrocellulose membrane strip. The second binder site was pre-blocked with 10 mg/mL of BSA and 2.5% of Dextran 150K. Sucrose was added into gold conjugate at 10% concentration. The gold conjugagte in 10% sucrose solutioin was striped onto second binder site pre-blocked with BSA and Dextran. The hCG solution of 200lU/mL was striped onto absorption site and immobilized by drying at air.  
         [0085]     The Goat anti-beta-hCG was obtained from Lampire Biological Laboratories. Prepare the Goat anti-hCG at 0.05 mg/mL in 10 mM Tris buffer, pH8.0 with 5 mg/mL BSA, 0.1% sodium azide, and 0.05% Triton X-100. Mix the Goat anti-hCG with 100 mlU/mL of hCG or negative urine.  
         [0086]     Assay procedure. Add 4-5 microliter of the mixture onto one end of strip, followed by washing with 10 mM Tris buffer, pH8.0 with 5 mg/mL BSA, 0.1% sodium azide, and 0.05% Triton X-100. Mix the Goat anti-hCG with 200mlU/mL of hCG or negative urine.  
         [0000]     Result  
         [0087]     The test site showed purple color with the mixture contains 200mlU/mL of hCG. The test site showed no purple color with the mixture contains no hCG.