Abstract:
Lateral flow device and method are provided for performing semi-quantitative visual or instrument-based detection in a test sample of a large molecule analyte that contains two binding domains, α and β. The device contains two reagents, a contrast reagent and an indicator reagent, and an analyte test zone (ATZ) that contains a fixed number of immobilized α-domain-specific binding sites. The contrast and indicator reagents each contain signal generators such that the two reagents contrast each other in different regions of a visual, spectrophotometric, calorimetric or fluorometric spectrum. The contrast reagent is prebound to an α-region-fragment of the analyte of interest and, therefore, does not react with the analyte of interest in the test sample. The indicator reagent is attached to a binding ligand specific for the β-domain of the analyte of interest. When a test sample is applied to the device, the analyte, if present, reacts with the indicator reagent and competes with the contrast reagent for the ATZ&#39;s α-domain binding sites. The analyte concentration in the test sample is evaluated by comparing the ratio of contrast and indicator signals in the ATZ with signal ratios for known analyte concentrations.

Description:
This application is based on and claims the benefit of U.S. Provisional Application No. 60/048,902, filed Jun. 5, 1997, and the contents thereof are incorporated herein by reference. 
    
    
     BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present invention relates generally to a diagnostic device and more specifically to a lateral flow assay device (“LFD”) that provides quantitative or semi-quantitative results. 
     2. Background Information 
     Assays are needed and used for detecting the presence of analytes in liquid test samples in fields such as clinical and forensic medicine, environmental testing, food contaminant testing, and drug use testing. In particular demand are single step assays that are based on reactions between specifically reactive substances that can be conducted outside of the laboratory setting and in remote sites. 
     At present, there are a number of over-the-counter home testing and health care professional diagnostic devices that act to both collect human body fluids and perform a diagnostic assay. Some of these devices are used for midstream urine collection, while other devices involve dipstick collection of a fluid biological sample placed in a receptacle. Both types of diagnostic devices can employ a lateral flow technology with common features, including an absorbent wick, matrix and reaction zones. 
     Using such a device involves applying a biological or aqueous test sample to the matrix. The matrix usually is a porous carrier material. When the matrix does not have sufficient absorbent capacity, a wick is used to transfer the sample to the matrix. The test sample is applied to the wick or one end of the matrix strip and moves by capillary action in one direction along the matrix to a reaction zone. 
     The reaction zone contains a first reactant. The first reactant is usually diffusible conjugate formed from an antibody, or other ligand, and a marker substance. The first reactant is specifically selected to react with a component of interest in the test sample. The sample enters the reaction zone and the component of interest binds with the first reactant to form complexes. 
     The reactant complexes, any unreacted sample, and conjugate move by capillary action out of the reactant zone and into a single test capture zone. Within the test capture zone is a second reactant that is immobilized to the matrix. The reactant complexes that contain test capture zone binding sites are retained at the test capture site through the formation of a sandwich reaction product. Those reactant complexes that do not contain test capture binding sites move by capillary action out of the test capture zone, into a control capture zone where they are bound or continue to move along the matrix and out of the capture zones. The control capture zone usually contains an antibody that has a specificity for binding the conjugate. 
     LFD formats require a control indicator to insure that the test was performed properly. This indicator is a validity test, i.e., it shows whether enough sample has migrated past the test capture binding site and, therefore, that the test procedure is performing properly. 
     Two formats exist for test and control zones on LFDs. In the format used in the overwhelming majority of LFDs, each zone forms a line across the matrix strip. The first line (i.e., the line first touched by the sample&#39;s fluid front after moving through the reaction zone) is the test line. Parallel and adjacent to the test line is the control line. The second format, a +/− indicator system, is present in a few formats (see U.S. Pat. Nos. 4,916,056, 5,008,080 and 5,075,078). Several other indicator configurations, including dots, curved lines and triangles, are possible but have not received wide commercial use. 
     Using existing lateral flow devices, test results are based on the visual detection of a threshold using a single color for the test result indicator. The test results are determined in one of two methods. In one method, the test results are determined from the total number of discrete lines or bands containing the indicator color (see e.g., U.S. Pat. Nos. 4,425,438, 5,073,484, 5,229,073 and 5,451,504). In the alternative method, the test results are determined from the migration distance of a continuous color front (see e.g., U.S. Pat. No. 5,416,000). Discriminating the last line or band or where the color front ends is difficult in these methods and results in uncertainty. The uncertainty in determining the last line/band or the end of the color front is due to the lack of competition for unbound immobilized binding sites and lot-to-lot manufacturing variability in the number of immobilized binding sites present on the device. Thus, a need exits for a lateral flow device that provides accurate, reproducible, semi-quantitative results with a single line or region independent of the number of binding sites present. The present invention satisfies this need and provides related advantages as well. 
     SUMMARY OF THE INVENTION 
     The present invention provides a sandwich format LFD with a single test line that is compatible with semi-quantitative detection of large molecule analytes (molecular weight is greater than 3,000 daltons) at low concentrations. Marker competition for binding sites at the single test line within an analyte test zone (ATZ) will indicate zero, low or high concentrations of the analyte of interest. 
     In accordance with one embodiment of the present invention, a sandwich format LFD with multiple test lines within the ATZ allows for titration of the analyte concentration and semi-quantitative detection. 
     In another aspect of the present invention, an analyte capture zone referred to as an analyte modulating zone (AMZ) is included in a location reached by the sample prior to reaching the ATZ. The AMZ alters performance by removing a fraction of the analyte and, thereby, increases the detectable range of analyte concentration. 
     In another aspect of the present invention, an LFD is provided in which a differential amount of immobilized binding ligand in a specified gradient among the multiple test lines is used to modulate the range of concentrations of analyte competing for the limited number of binding sites on the test line. This modulation allows the LFD to accommodate a particular range of concentrations according to the specification of the test. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 is a schematic representation of a sandwich format LFD with a single test line that is compatible with semi-quantitative detection of large molecule analytes. 
     FIG. 2 is a schematic representation of a sandwich format LFD with multiple test lines that is compatible with semi-quantitative detection of large molecule analytes. This particular format includes a control line. 
     FIG. 3 is a schematic representation of an alternative embodiment of a sandwich format LFD that includes a reference line. 
     FIG. 4 is a graphical representation of a linear least squares fit of the average color intensity of the sum of lines 1-4 as measured on a Minolta CR-241 color analyzer of the multiple test line format (depicted in FIG. 2) for increasing concentrations of human chorionic gonadotropin(hCG). 
     FIG. 5 is a graphical representation of the average last line seen visually in the multiple line format (depicted in FIG. 2) for increasing concentrations of hCG. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The invention provides improved immunological and other specific binding assay methods and devices in a single or multi-analyte format, using contrast or control and indicator reagents in an LFD to instrumental or non-instrumental visual endpoints. 
     Sandwich LFD With Single Test Line 
     A sandwich format lateral flow device (“LFD”) with a single test line for the determination and semi-quantitative detection of a large molecule analyte in a biological or aqueous test sample is illustrated in FIG.  1 . The various dimensions are not necessarily drawn to scale in any of the Figures. 
     The LFD  10  includes a porous membrane  12  made, for example, of nitrocellulose, glass fiber, or nylon. Other appropriate membrane materials can be used depending on the characteristics of pore size, binding capacity, the particular biological or aqueous test sample and assay procedure. Usually, the porous membrane  12  can have a pore size between 2 and 10 microns. In one form, the porous membrane  12  will have a length between approximately 50-100 mm and a width between approximately 3.5-8.0 mm. The porous membrane  12  also will have a thickness, for example, between approximately 50-300 microns and a capillary rise characteristic, relative to an aqueous solution, between approximately 10-50 mm/min. However, the specific characteristics and dimensions of the porous membrane  12  are not critical and can be modified as necessary to achieve desired results of speed and a positive test result. 
     As shown in FIG. 1, a wicking pad  14  overlaps the porous membrane  12  at one end  16  of membrane  12  such that it is in liquid transfer contact with the porous membrane  12 . The wicking pad  14  can be made of glass fiber, fibrous cellulose or other suitable materials. The dimensions of the wicking pad  14 , although usually not critical, can be, for example, between 20-50 mm in length, between 3.5-8 mm in width and 0.5-2.0 mm in thickness. 
     The LFD  10  includes a conjugate zone  18 . The conjugate zone  18  can be a region of the wicking pad  14  or the porous membrane  12  or one or more separate reagent pads that are in liquid transfer contact with the wicking pad  14  or porous membrane  12 . The wicking pad  14 , porous membrane  12  and conjugate zone  18  can be held in place by a strip of tape (not shown). In other embodiments, the wicking pad  14 , porous membrane  12  and conjugate zone  18  can be held in place by an adhesive material or by the natural constriction of a container (not shown) housing the LFD components and preventing their contamination. 
     The conjugate zone  18  contains two reagents. The first reagent is a contrast reagent that is attached to a portion or fragment of the large molecule analyte of interest (referred to herein as the “α-region-fragment”) by covalent or ionic binding, adsorption or other means of attachment known in the art to form a contrast binding ligand (CL). The CL can be dried, reconstitutable, liquid-dispersible, diffusible, colored-latex beads. Usually, the latex beads are light in color; for example, yellow. Instead of colored-latex beads, the contrast reagent can be a colored dye molecule, an enzyme and dye combination, or a fluorescent, luminescent or radioactive molecule. 
     The second reagent contained in the conjugate zone  18  is a dried, reconstitutable, liquid dispersible, diffusible indicator reagent that is attached to a binding ligand specific for a second region (“β-region”) of the large molecule analyte of interest to form an indicator binding ligand (IL). The α-region-fragment and β-region of the large molecule analyte must be separate non-cross-reacting units. The indicator reagent can be colloidal gold particles, enzyme/dye combinations, colored latex particles, carbon particles, or fluorescent, luminescent or radioactive particles that can be visibly or otherwise distinguished from the CL. 
     One or both of the first and second reagents can be uniformly impregnated or dispersed within the conjugate zone  18  before they are contacted by the test sample. Alternatively, for example, the conjugate zone  18  can be coated with one or both reagents and the reagents dispersed throughout the conjugate zone when contacted by the sample. Or the two reagents can be longitudinally spaced apart within the conjugate zone  18  and dispersed throughout the conjugate zone when contacted by the sample. 
     As shown in FIG. 1, the LFD  10  includes an absorption pad  30  at the second end  32  of the porous membrane  12  opposite the wicking pad  14 . Between the absorption pad  30  and the conjugate zone  18  are one test line  22 , a space  20  between the conjugate zone  18  and the test line  22 , and a second space  24  between the test line  22  and absorption pad  30 . The test line  22  is located on the porous membrane  12  and contains an immobilized binding ligand specific for the α-region-fragment of the analyte of interest. 
     The absorption pad  30  can be a glass fiber or fibrous cellulose pad or other suitable material in liquid transfer contact with the porous membrane  12 . The absorption pad  30  collects unreacted reagent and sample and acts as a wick to remove any background material from the test line  22 . 
     A test sample containing an analyte moves along the wicking pad  14 , shown in FIG. 1, to the conjugate zone  18  by capillary action. When the sample comes into contact with the IL in the conjugate zone  18 , it reacts to form an analyte-IL complex. The CL attached to the α-region-fragments moves along the wicking pad with, but does not react with, the analyte or the analyte-IL complex. 
     When the test sample&#39;s fluid front reaches the test line  22 , a competition for the immobilized α-region-specific ligand&#39;s binding sites occurs among the CL, analyte-IL complexes, and uncomplexed analyte. For purposes of illustration only, for the following example the contrast reagent is yellow latex beads and the indicator reagent is colloidal gold particles. When no analyte is present in the test sample, only α-region-fragments-yellow latex beads (CL) will attach to the α-region-fragment-specific ligands immobilized on test line  22  and test line  22  will appear yellow. When there is a small concentration of analyte in the sample, the CL and IL will compete for the limited number of binding sites on the α-region-fragment-specific ligand immobilized on the test line  22 , with the yellow-colored beads predominating over the red of the colloidal gold complex forming a brown color. As the concentration of analyte increases, the concentrations of analyte-IL complexes increases and competes more effectively for the limited number of α-region-fragment-specific ligand binding sites, and the test line  22  will become more red in color. 
     The above-described transition in color in the test line  22  from yellow to brown to red forms the basis of a semi-quantitative single line test. Comparable transitions can be achieved by substituting other contrast and indicator reagents for the latex beads and colloidal gold. In each case, a visual or otherwise detectable test line  22 , or color or signal transition pattern for multiple regions, will develop whether the test is negative or positive and will serve as a procedural control for test validity. 
     After moving into the test line  22 , any unbound sample constituents and reagents continue to move up the porous membrane  12  into the absorption pad  30 , which acts as a wick to pull sample upward, thus washing out the test line  22  area of any background material. 
     Sandwich LFD With AMZ 
     For analytes that occur in high concentrations, the range over which the analyte is determined can be increased by preparing an AMZ that intercepts a portion of the analyte prior to the sample reaching the test line  22  in FIG. 1. A capture antibody or other capture molecule can be attached to the wicking pad  14  or, for example, in space  20  on porous membrane  12  by known methods. The capture molecule lowers the concentration of analyte in a test sample by removing a fraction of the analyte and, thereby, extends the range of the color or other signal transition zone detected at test line  22 . The fraction of the analyte removed in the AMZ can be empirically established during manufacturing and quality control procedures for the LFD  10  and specific analyte. 
     The AMZ is described in greater detail in Example I for a sandwich LFD with a single test line, where an AMZ has extended the range of the test. The AMZ also can be used and applied to other sandwich LFD formats. 
     Sandwich LFD With Reference Line 
     The sandwich format LFD  10  of FIG. 1 also can be modified to accommodate a reference line  226  as shown in FIG.  3 . Functionally, the reference line  226  provides a check for samples containing very low amounts of analyte. 
     Structurally, the LFD  210  of FIG. 3 differs for the LFD  10  for FIG. 1 in the following ways. There are two separate species of contrast reagents used in the conjugate zone  218 . The first species is the CL of the analyte of interest described above for FIG.  1 . The second species is created by attaching the contrast reagent to a capturable label that has a specific capturing binding partner. One example of such a capturable label and capturing binding partner is biotin and streptavidin. Other suitable labels and binding partners are known in the art and can be incorporated by standard methods. 
     The capturing binding partner is immobilized on the porous membrane  212  to form a reference line  226 . The reference line  226  is located at the second end  232  of the porous membrane  212  between the conjugate zone  218  and the absorption pad  230 . The reference line  226  usually is distanced from the conjugate zone  218  by approximately 2-4 mm and absorption pad  230  by approximately 5-50 mm to create a first space  224  and second space  228  on the porous membrane  212 . 
     When the sample moves up the porous membrane  212  from the conjugate zone  218 , the contrast reagents will travel with it. The contrast reagent attached to the α-region-fragment (CL) will compete for immobilized α-region-fragment-specific ligand binding sites on ATZ  222  with analyte-IL complexes as described for FIG.  1 . The contrast reagent attached to the capturable label will bind to the capturing binding partner immobilized at the reference line  226 . 
     For purposes of illustration only, for the following that contrast reagent is yellow latex beads and the indicator reagent is colloidal gold particles. If there is no analyte in the sample, the color of the reference line  226  and test line  222  will be the same color. When there is analyte in the sample, the reference line  226  will be yellow while the test line  222  will be brown to red in color, depending on the concentration of analyte in the sample. The color of the reference line  226  thus provides a basis for color comparison with the color of the test line  222 . Comparable reference line and test line comparisons can be achieved with other contrast and indicator reagents. 
     Sandwich LFD With Multiple Test Regions 
     A sandwich LFD format with multiple parallel test lines (regions) for the determination and titration of an immunologically reactive large molecule analyte in a test sample is illustrated in FIG.  2 . This format enables the user to determine zero, from lower and higher concentrations of analyte, by the use of a scale to correlate the location of a color transition zone. The test lines that develop are detectable on the porous membrane due to binding of the analyte-IL complex to the limited number of immobilized binding sites on the ATZ. 
     Using multiple parallel test lines allows for a titration of the analyte concentration. Zero or subthreshold levels of analyte will not form lines within the matrix. Small amounts of IL will be completely captured by the first or second line. Larger amounts of analyte will be captured by subsequent lines as the analyte sequentially saturates the binding sites. The analyte, therefore, is quantitated by the color or signal transition pattern. 
     The LFD  110  includes a porous membrane  112  made of nitrocellulose, glass fiber, nylon or other suitable materials. The specific dimensions of the porous membrane  112  are not critical and maybe modified as necessary to achieve desired results of speed and positive test results. 
     As shown in FIG. 2, a wicking pad  114  overlaps the porous membrane  112  at one end  116  of membrane  112  such that it is in liquid transfer contact with the porous membrane  112 . The wicking pad  114  can be made of glass fiber, fibrous cellulose or other suitable materials. 
     The LFD  110  includes a conjugate zone  118 . In FIG. 2, as in FIG. 1, the conjugate zone  118  can be either a region of the wicking pad  114  or porous membrane  112  or one or more separate reagent pads in liquid transfer contact with the wicking pad  118  or porous membrane  112 . The wicking pad  114 , porous membrane  112  and conjugate zone  118  can be held in place by a variety of methods known in the art. 
     The conjugate zone  118  contains two reagents. The first reagent is a labeled control reagent. It, for example, can be dried, colored, liquid dispersible, diffusible latex beads. The label is one that has a specific binding partner, e.g., biotin and streptavidin. The beads are usually light in color; for example, yellow. As an alternative to colored-latex beads, the control reagent may be a light-colored dye molecule, an enzyme and dye combination, or a fluorescent, luminescent or radioactive molecule. 
     The second reagent contained in the conjugate zone  118 , is a dried, reconstitutable, liquid dispersible, diffusible indicator reagent that is attached to a binding ligand specific for a region (“β-region”) of the large molecule analyte of interest to form an indicator binding ligand (IL). The indicator reagent can be colloidal gold particles, enzyme/dye combinations, colored latex particles, carbon particles, or fluorescent, luminescent or radioactive molecules that can be visibly or otherwise distinguished from the control reagent. 
     One or both of the first and second reagents can be uniformly impregnated or dispersed within the conjugate zone  118  before they are contacted by the test sample. Alternatively, for example, the conjugate zone  118  can be coated with one or both reagents and the reagents dispersed throughout the conjugate zone when contacted by the sample. Or the two reagents can be longitudinally spaced apart within the conjugate zone  118  and dispersed throughout the conjugate zone when contacted by the sample. 
     As shown in FIG. 2, the LFD  110  includes between 2 and 20, usually about 7, multiple test lines  122 , a space  120  between the conjugate zone  118  and the test lines  122 , and a space  124  between the test lines  122  and an absorption pad  130  at the second end  132  of the porous membrane  112 . The test lines  122  contain a binding ligand specific for a second region (α-region) of the analyte of interest that is immobilized on the porous membrane  112 . The α- and β-regions of the large molecule analyte must be separate non-cross-reacting units. 
     A control line  148  is located on the porous membrane  112  near end  132 . The control line  148  contains an immobilized binding partner (e.g., streptavidin) for the label attached to the control reagent (e.g., biotin). 
     The absorption pad  130  can be glass fiber, fibrous cellulose or other suitable material in liquid transfer contact with the porous membrane  112 . The absorption pad  130  collects unreacted reagent and sample constituents and acts as a wick to remove any background material from the test lines  122 . 
     A test sample containing an analyte moves along the wicking pad  114  of FIG. 2 to the conjugate zone  118  by capillary action. When the sample comes into contact with the IL in the conjugate zone  118 , it reacts to form an analyte-IL complex. The labeled control reagent moves along the wicking pad  114  with, but does not react with, the analyte-IL complex or IL. When the sample&#39;s fluid front reaches the test lines  122 , only the analyte-IL complex and any uncomplexed analyte will bind to the α-region-specific ligand immobilized on test lines  122 . 
     For purposes of illustration only, for the following the control reagent is yellow latex beads and the indicator reagent is colloidal gold particles. When there is no analyte, no test lines have color or are otherwise visible. As the concentration of analyte increases in the test sample, the concentration of analyte-IL complexes increases and saturates the limited number of α-region-specific ligand binding sites at test lines  122 , creating a situation where more of the test lines  122  will become red in color. 
     The labeled yellow-colored latex beads will continue to migrate and then react with the label&#39;s binding partner immobilized on control line  148 . The control line  148  will be colored both in the presence and absence of analyte. The color of the control line  148  will be distinct from the test lines  122 . After moving through the test lines  122  and control lines  148 , the sample continues to move up the porous membrane  112  onto the absorption pad  130  which collects unreacted reagents and sample and acts as a wick to remove from the test line area  122  any background material. 
     Examples of the above described LFDs used to detect hCG in urine samples are found in Examples I, II, and III below. Other large molecule analytes of interest include drugs, hormones, synthetic chemicals, pollutants, trace compounds, toxins and microorganisms in biological fluids, food, water or air. Additional detection analytes that are either present in fluids or can be introduced into a liquid system and appropriate reagents will be known to those of skill in the art. 
     Multiple test lines in the ATZ (as shown in FIG. 2, area  122 ) could be used in the format of FIGS. 1 and 3 and the resulting color or signal transition pattern in the ATZ is then compared with that of patterns from known analyte concentrations. 
     Devices described herein can include more than one IL, wherein each IL has different and spectrally non-overlapping visual, spectrophotometric, colorimetric or fluorometric properties. 
     EXAMPLE I 
     SANDWICH LFD WITH SINGLE TEST LINE 
     Preparation of Test Strips 
     Pregnancy test strips (SA Scientific, San Antonio, Tex.) containing dried anti-β hCG monoclonal antibody conjugated to colloidal gold on a glass fiber conjugate pad were obtained. These strips contained immobilized anti-α hCG on a test line and immobilized anti-mouse IgG on a control line on a nitrocellulose membrane similar to what is depicted in FIG.  1 . The conjugate pad was carefully removed intact using a razor blade. To the region of nitrocellulose membrane corresponding to space  20  in FIG. 1, varying amounts of monoclonal anti-β hCG capture antibody (0, 250, 500, 1000, 2000 ng) were added, dried, washed and dried again. 
     The conjugate pad was carefully and uniformly coated with a total of 3 ul (in 0.2 ul drops) of a 10 mg/ml solution of yellow latex beads (200 nm particles, carboxylated) covalently conjugated with affinity purified α-region of hCG (20 ng/mg latex). The pad was then dried at 50° C. The conjugate pad was then placed back in its original position on the modified test strip and secured with cellophane tape. 
     Test Procedure 
     Negative urine samples were spiked with hCG to achieve concentrations of 0, 25, 225, 450, 1100, 2500, 10,000 and 100,000 mIU/ml. A 0.3 ml aliquot from each of the 8 spiked urine samples was added to separate microfuge tubes. The wicking pad of each test strip was placed in contact with the urine. Each strip contained a set amount of immobilized capture antibody and a conjugate pad as described above. 
     After five minutes, the ATZ on each test strip was then visually scored for color type as either yellow (Y), brown (B), red-brown (RB), or red (R). The test results are summarized in Table I. 
     The results in Table I show a semi-quantitative capability arising from the use of a yellow-colored latex and red colloidal gold combination. The results also show that there is an improvement in the range of concentrations indicated by the brown-colored line achieved by removing excess hCG at the higher immobilized anti-β monoclonal antibody levels in the AMZ. The additional antibody did not compromise sensitivity, and there were no false positives due to non-specific binding of the colloidal gold to the test line. At 1000 and 2000 ng of anti-β hCG capture antibody, the brown color extended to 1000 mIU/ml, which is 3-5 times higher than the range seen in the control, 0 ng, antibody strips. 
     EXAMPLE II 
     SANDWICH LFD WITH REFERENCE LINE 
     Preparation of Reference Line Test Strips 
     The preparation pregnancy strips (SA Scientific, San Antonio, Tex.) with reference lines containing immobilized streptavidin required temporary removal of the conjugated pads from the pregnancy strips to avoid damaging the pads during preparation of test strips corresponding to FIG.  3 . While each conjugate pad was removed, a coating of 3 ul biotin-labeled yellow latex beads (10 mg/ml) and yellow latex beads covalently conjugated with α-region-fragments of hCG (10 mg/ml) were added to the reagent pad as described above in Example I. 
     The reference line  226  can be prepared by immobilizing streptavidin directly on the porous membrane using passive adsorption or other methods known in the art. 
     Test Procedure 
     Test procedures were identical to those in Example I, except that only 0, 25, and 10,000 mIU/ml samples were tested. 
     Test Results 
     In the negative sample, the yellow color in the test line  222  was indistinguishable from the reference line  226 . The 25 mIU/ml sample displayed a weak brown color relative to the yellow reference line. The 10,000 mIU/ml sample yielded a solid red test line and a yellow reference line. 
     EXAMPLE III 
     SANDWICH LFD WITH MULTIPLE TEST LINE ATZ 
     Preparation of Test Strips 
     Custom test strips corresponding to FIG. 2 were prepared that contained 7 anti-α hCG antibody test lines spaced 1 mm apart on the nitrocellulose membrane. A line of streptavidin was immobilized on the control line  148  as described for the reference line  226  in Example II. 
     Yellow latex-BSA conjugated with biotin (3 ml at 10 mg/ml) was added to the anti-β hCG monoclonal antibody-colloidal gold treated conjugate pad and air dried. The pad was secured to the test strips with cellophane tape to the backing. 
     Seven test lines  122  were prepared as described for test lines  22  in Example I. 
     Test Procedure 
     The following procedure was performed in triplicate using the test strips prepared above. The strips were placed in 0.5 ml of hCG negative urine that had been spiked with varying amounts of hCG (0-51,200 mIU/ml). The strips were allowed to develop for 10 minutes. The strips were then dried for 2 hours, at 50° C. and color intensity determined on a colorimeter using the Yxy color field (Minolta CR-241, 0.3 discrimination aperture). The signal intensities were then calculated using the sum of the first four lines (net 1-4) for the Y color space. The data is presented in Table II. A plot was then made correlating the Net 1-4 with the log hCG concentration, shown in FIG. 4. A linear least squares fit was derived and values were calculated from that fit for each hCG concentration tested, and compared to the known values (Table III). A visual discrimination of the line number containing the last visible red line was also performed on the test strips developed above. The results were summarized in Table IV, and the averages were plotted in FIG.  5 . 
     Test Results 
     The multi-test line format allows for quantitating the level of hCG in urine. Calculated values differed from known values on average by 13%, with a range of from 0.6-26.3%. A visual semi-quantitative method allows the user to place the hCG concentration into 5-7 ranges of hCG within a range of 50 mIU/ml to greater than 50,000 mIU/ml. 
     Although the invention has been described with reference to the examples provided above, it should be understood that various modifications can be made without departing from the spirit of the invention. 
     
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE I 
               
             
             
               
                   
               
               
                 anti-β hCG capture mAb immobilized 
               
             
          
           
               
                 mIU/ml hCG 
                 0 ng 
                 250 ng 
                 500 ng 
                 1000 ng 
                 2000 ng  
               
               
                   
               
             
          
           
               
                 0 
                 Y 
                 Y 
                 Y 
                 Y 
                 Y 
               
               
                 25 
                 B 
                 B 
                 B 
                 B 
                 B 
               
               
                 225 
                 B 
                 B 
                 B 
                 B 
                 B 
               
               
                 450 
                 R 
                 RB 
                 RB 
                 B 
                 B 
               
               
                 1100 
                 R 
                 RB 
                 R 
                 RB 
                 RB 
               
               
                 2500 
                 R 
                 R 
                 R 
                 R 
                 R 
               
               
                 10,000 
                 R 
                 R 
                 R 
                 R 
                 R 
               
               
                 100,000 
                 R 
                 R 
                 R 
                 R 
                 R  
               
               
                   
               
             
          
         
       
     
     
       
         
               
               
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE II 
               
               
                   
               
               
                 strip 
                 1 
                 2 
                 3 
                 4 
                 5 
                 6 
                 7 
                 8 
                 9 
                 10 
                 11 
                 12 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 line 
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                   
                 1 
                 90.19 
                 84.83 
                 75.14 
                 66.59 
                 40.86 
                 29.92 
                 34.01 
                 27.76 
                 16.19 
                 15.78 
                 23.76 
                 19.15 
               
               
                   
                 1 
                   
                 83.98 
                 75.23 
                 71.24 
                 55.68 
                 43.1 
                 41.3 
                 23.74 
                 22.68 
                 20.92 
                 21.56 
                 15.85 
               
               
                   
                 1 
                   
                 84.43 
                 75.06 
                 55.39 
                 57.77 
                 47.08 
                 35.69 
                 31.55 
                 26.71 
                 18.24 
                 22.12 
                 19.92 
               
               
                 1 ave 
                   
                 90.19 
                 84.41 
                 75.143 
                 64.41 
                 51.44 
                 40.03 
                 37 
                 27.68 
                 21.86 
                 18.313 
                 22.48 
                 18.31 
               
               
                   
                 2 
                 89.7 
                 91.99 
                 87.34 
                 86.9 
                 76.62 
                 71.4 
                 69.63 
                 56.71 
                 42.97 
                 50.77 
                 41.73 
                 19.14 
               
               
                   
                 2 
                   
                 89.65 
                 86.71 
                 85.54 
                 83.53 
                 75.26 
                 75.41 
                 58.43 
                 55.34 
                 44.94 
                 34.6 
                 24.47 
               
               
                   
                 2 
                   
                 87.68 
                 88.23 
                 88.69 
                 81.89 
                 76.73 
                 72.33 
                 63.66 
                 58.68 
                 52.19 
                 35.24 
                 22.13 
               
               
                 2 ave 
                   
                 89.7 
                 89.77 
                 87.427 
                 87.04 
                 80.68 
                 74.46 
                 72.46 
                 59.6 
                 52.33 
                 49.3 
                 38/10 
                 21.91 
               
               
                   
                 3 
                 89.25 
                 88.32 
                 87.9 
                 87.56 
                 84.09 
                 81.26 
                 80.88 
                 73.47 
                 72.46 
                 71.29 
                 60.04 
                 37.03 
               
               
                   
                 3 
                   
                 89.64 
                 87.25 
                 87.78 
                 85.7 
                 87.09 
                 83.17 
                 82.61 
                 74.98 
                 66.3 
                 53.23 
                 40.99 
               
               
                   
                 3 
                   
                 87.54 
                 87.65 
                 89.65 
                 86.06 
                 83.98 
                 81.52 
                 77.9 
                 73.01 
                 70.8 
                 55.12 
                 36.21 
               
               
                 3 ave 
                   
                 89.25 
                 88.5 
                 87.6 
                 88.33 
                 85.28 
                 84.11 
                 81.86 
                 77.99 
                 73.48 
                 69.463 
                 56.13 
                 38.08 
               
               
                   
                 4 
                 88.46 
                 88.9 
                 87.6 
                 88.53 
                 86.25 
                 850.01 
                 86.31 
                 81.64 
                 84.78 
                 81.14 
                 73.14 
                 53.43 
               
               
                   
                 4 
                   
                 86.64 
                 86.52 
                 87.27 
                 84.89 
                 87.3 
                 84.22 
                 85.48 
                 80.75 
                 79.39 
                 73.43 
                 59.19 
               
               
                   
                 4 
                   
                   
                 87.25 
                 89.26 
                 87.17 
                 87.45 
                 85.29 
                 83.88 
                 80.74 
                 81.08 
                 72.94 
                 61.02 
               
               
                 4 ave 
                   
                 88.46 
                 87.77 
                 87.123 
                 88.35 
                 86.1 
                 86.59 
                 85.27 
                 83.67 
                 82.09 
                 80.537 
                 73.17 
                 57.88 
               
               
                 Total 
                   
                 357.6 
                 350.5 
                 337.29 
                 328.1 
                 303.5 
                 285.2 
                 276.6 
                 248.9 
                 229.8 
                 217.61 
                 188.97 
                 136.2 
               
               
                 Net 1-4 
                   
                 0 
                 7.143 
                 20.307 
                 29.47 
                 54.1 
                 72.41 
                 81.01 
                 108.7 
                 127.8 
                 139.99 
                 168.63 
                 221.4 
               
               
                 mIU/ml 
                   
                 1 
                 50 
                 100 
                 200 
                 400 
                 800 
                 1600 
                 3200 
                 6400 
                 12800 
                 25600 
                 51200 
               
               
                 log mIU/ml 
                   
                 0 
                 1.699 
                 2 
                 2.301 
                 2.602 
                 2.903 
                 3.204 
                 3.505 
                 3,806 
                 4.1072 
                 4.4082 
                 4.709 
               
               
                   
               
             
          
         
       
     
     
       
         
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE III 
               
               
                   
               
               
                 mIU/ml hCG 
                 Ave Line 1-4 net Y 
                 Calculated mIU/ml 
                 % difference  
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 50 
                 7.1 
                 61.5 
                 23 
               
               
                 100 
                 20.3 
                 104 
                 4.2 
               
               
                 200 
                 29.2 
                 150 
                 24.7 
               
               
                 400 
                 54.1 
                 402 
                 0.6 
               
               
                 800 
                 72.4 
                 836 
                 4.5 
               
               
                 1600 
                 81 
                 1179 
                 26.3 
               
               
                 3200 
                 108.7 
                 3569 
                 11.5 
               
               
                 6400 
                 127.8 
                 7659 
                 19.7 
               
               
                 12800 
                 140 
                 12474 
                 2.5  
               
               
                   
               
             
          
         
       
     
     
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
                 TABLE IV 
               
               
                   
                   
               
               
                   
                 Last line #1 
                 Last Line #2 
                 Last line #3 
                 Average  
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                 50 
                 1 
                 1 
                 1 
                 1 
               
               
                 100 
                 1 
                 1 
                 1 
                 1 
               
               
                 200 
                 1 
                 2 
                 1 
                 1.3 
               
               
                 400 
                 2 
                 2 
                 2 
                 2 
               
               
                 800 
                 3 
                 2 
                 2 
                 2.3 
               
               
                 1600 
                 3 
                 2 
                 3 
                 2.6 
               
               
                 3200 
                 4 
                 3 
                 3 
                 3.6 
               
               
                 6400 
                 4 
                 4 
                 5 
                 4.3 
               
               
                 12800 
                 6 
                 5 
                 5 
                 5.3 
               
               
                 25600 
                 6 
                 6 
                 7 
                 6.3 
               
               
                 51200 
                 7 
                 7 
                 7 
                 7