Abstract:
An assay device for the performance of immunochromatographic assays and other assays has two principal parts, a first opposable component and a second opposable component. The first opposable component can contain a sample preparation means and a second opposable component can contain a suitable chromatographic medium for detection of an analyte. Alternative embodiments of the invention can also exist. For example, the first opposable component can have a sample preparation means and a chromatographic medium that is not in communication with the sample preparation means and the second opposable component can contain a communicating means that, when the two components are brought into opposition, establishes a communication between the sample preparation means and the chromatographic medium. Assay devices according to the present invention can be used for both unidirectional and bidirectional assays.

Description:
FIELD OF THE INVENTION 
     This invention relates to chromatographic assay devices. In particular, this invention relates to chromatographic assay devices which are used to qualitatively or quantitatively test for the presence of clinically important biological molecules. 
     BACKGROUND OF THE INVENTION 
     Chromatographic assay systems are well-known and frequently used analytical systems. These assay systems have a wide range of utilities. Recently, they have taken on an ever larger role in providing physicians with information to guide the diagnosis and treatment of a variety of disorders. 
     Among the most important of such systems are the “thin layer” systems in which a solvent moves across a thin, flat, absorbent medium. 
     The use of immunoassays as a means of testing for the presence and amount of clinically important molecules has been known for some time. As long ago as 1956, J. M. Singer reported the use of an immune based latex agglutination test for detecting a factor associated with rheumatoid arthritis (Singer, J. M., Plotz, C. M.,  Am. J. Med.  Vol. 22, pp 888-82). These techniques are used in a particularly popular form of chromatographic assays, known as immunochromatography. In their simplest forms, these tests use a disclosing reagent or particle which has been linked to an antibody to the molecule of interest. This combination is then mixed with the specimen and, if the molecule of interest is present, the disclosing reagent-linked antigens agglutinate with the molecule of interest, thereby giving an indication that the molecule of interest is present. The disclosing reagent or particle may be identifiable on the basis of color, magnetic properties, radioactivity or any number of other physical or chemical properties. The specific reactions which are employed vary with the nature of the molecule of interest and the sample which is to be tested. 
     Immunochromatographic assays fall into two principal categories: “sandwich” and “competitive”. Generally, “sandwich-type” immunochromatographic procedures call for mixing a sample containing a molecule of interest with antibodies to that molecule, which causes an antigen-antibody complex to be formed. The antibodies which are used in this procedure are typically linked to a disclosing molecule or reagent, such as dyed latex, colloidal gold or a radioisotope. This mixture is then applied to a chromatographic medium which contains a band or zone to which antibodies to the molecule of interest have also applied. This medium often takes the form of a device or strips which resembles a “dipstick.” When the complex of the molecule of interest and the antibodies with disclosing reagents or particles reaches the zone of the chromatographic medium with the antibodies, binding occurs and the bound disclosing particles or reagents are localized at the zone or band on the chromatographic medium. This indicates the presence of the molecule of interest in the sample. Quantitative results can sometimes be obtained in this manner. 
     In addition to immunochromatographic assays, it is also known to use enzyme-based chromatographic assays. These techniques are roughly analogous to immune-reaction based systems, but use an enzymatically catalyzed reaction instead of an antigen-antibody reaction. Other analogous chromatographic assays are also known. 
     The chromatographic techniques which are available to the clinician are not without their drawbacks. Sometimes the specimen which is to be tested contains cells or particulate matter which can add colors to the chromatographic medium thereby making it difficult to read the test. In some cases, such as tests using fecal samples, particulate matter within the sample can clog the pores of the chromatographic medium making immunochromatography very difficult, if not completely impossible. It is also important (and sometimes quite difficult) to apply the sample to the chromatographic medium so that the sample front will be applied to and move through the chromatographic medium and reach the area where binding is to occur in a uniform, straight-line manner. 
     Other problems associated with chromatographic devices and techniques which are available to the physician are those of sample preparation and waste generation. It is rarely possible to apply a sample (such as feces) or a sampling device (such as throat swab) directly to the chromatographic media. Several extraction and pretreatment reactions are usually required before the sample can be applied to the chromatographic medium. Conventionally, these preparatory steps are carried out by the physician or a technician in several small vessels, each of which (along with a transfer device, such as a pipette) is thereby contaminated with biological, chemical or radiological wastes, all of which can come into contact with the physician, technician and many others. 
     Another limitation on the chromatographic devices which are available to the clinician is their inability to perform 2 directional or 2 dimensional chromatography. These chromatographic techniques have long been known to be powerful analytical tools but their complexity relative to simple unidirectional chromatography has made it difficult to apply them in the physician&#39;s office. 
     SUMMARY OF THE INVENTION 
     In a chromatographic device according to the present invention there are two principal parts, a first and a second opposable component. The first opposable component contains a sample preparation means and the second opposable component contains a suitable chromatographic medium. In operation, a sample is placed on the sample preparation means (along with suitable reagents and solvents) and the sample is thereby prepared for application to the chromatographic mechanism, but is not applied to the chromatographic medium until after the preparatory reactions have taken place. The two opposable components are then brought into opposition, thereby bringing the sample preparation means on the first component into contact with the chromatographic medium on the second component. This applies the treated sample to the chromatographic medium and the chromatographic process then begins. The result can be observed via an appropriate opening or transparent portion of the device. 
     In another embodiment of the invention, the first opposable component has a sample preparation means and a chromatographic medium which is not in communication with the sample preparation means. The second opposable component contains a communicating means which, when the two components are brought into opposition, establishes a communication between the sample preparation means and the chromatographic medium. In use, the sample is applied to the sample preparation means and the required preparatory reactions are conducted. Once this is accomplished, the two components are brought into opposition and the chromatography is begun. 
     By locking the two opposed components of the device together, one can permanently encase the sample as well as all materials which have been exposed to the sample or to the chemical, biological or radiological materials used in the assay. 
     The two opposable components can be constructed of any material which provides suitable mechanical support or the desired degree of protection from the materials which are used in the test. The sample preparation means and the chromatographic medium will necessarily be adapted to the requirements of the particular assay which is to be performed. In an advantageous embodiment, the sample preparation means is an absorbent pad which can retain a suitable quantity of fluid and which has a pore size which allows the absorbent pad to filter out particulate matter which should not be applied to the chromatographic medium. 
     This unique construction provides a simple, self-contained device which permits the reliable execution of chromatographic assays by persons having only basic laboratory skills and without the risk or expense associated with the generation or leakage of biological, chemical or radiological wastes. This device also facilitates the sequential execution of pretreatment and chromatographic steps in a self-contained device. 
     This invention also provides a simple device which makes it possible to routinely conduct two directional or two dimensional chromatography in the physician&#39;s office. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1A is a drawing of a chromatographic assay device according to the present invention with a first opposable component and a second opposable component in which the first component includes a sample preparation means and the second opposable component includes a chromatographic medium, shown in open position; 
     FIG. 1B is a drawing of the chromatographic assay device of FIG. 1A shown in closed position; 
     FIG. 2A is a drawing of an assay device for performing a bidirectional immunochromatographic assay for an antibody, shown in open position; 
     FIG. 2B is a drawing of the assay device of FIG. 2A in closed position; 
     FIG. 3A is a drawing of a chromatographic assay device for the detection of  Helicobacter pylori  antibodies; and 
     FIG. 3B is a drawing of the assay device of FIG. 3A in closed position showing the band visible through a window that indicates the presence of antibody. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     A chromatographic assay device  10  according to the present invention is shown in FIG.  1 A. It has a first opposable component  12  and a second opposable component  14 . 
     The first component  12  includes a sample preparation means  16 . Preferably, sample preparation means  16  is an absorbent pad which, besides serving as the locus of preparatory reactions which may be required, will serve to apply the treated sample to the chromatographic medium  18  on the second opposable component  14 . Sample preparation means  16  can be made of any suitable material, such as cellulose, paper, nylon or non-woven synthetic fabrics. If filtration of particulate matter is desired, as in the case of whole blood or fecal samples, this function will be governed by the pore size of the material, which can be selected accordingly. A sample or, optionally, a sampling device, can be placed by the operator upon the sample preparation means  16  and appropriate reagents can be added. If desired, some or all of the reagents for the treatment of the sample can be made part of the sample preparation means  16 , such as by impregnation in an absorbent pad. 
     The chromatographic medium  18  on the second opposable component  14  is comprised of suitable material such as nitrocellulose, nylon or silica. The chromatographic medium  18  can be pre-treated or modified to suit the needs of the assay which is to be performed. For example, when the device is to be used to perform a “sandwich” immunochromatography, zone  20  of the chromatographic medium  18  can be impregnated with antibodies, usually to the molecule of interest, which will serve to facilitate binding with those molecules and which may be bound to antibodies which are in turn attached to disclosing particles or reagents. 
     Optionally, the chromatographic medium  18  may include other zones which will serve as controls for the assay procedure. An example of such a control zone is zone  22  of FIG. 1A, to which a small amount of the molecule of interest (or a cross-reactant) has been applied. Labeled antibodies to the molecule of interest will agglutinate zone  22 , thereby demonstrating that an absence of agglutination in zone  20  is not attributable to a lack of labeled antibodies in the sample which is applied to the device  10  in an immunochromatographic assay. 
     The bodies of opposable components  12  and  14  are preferably made of plastic which is impervious to moisture. In the embodiment which is shown in FIG. 1A, components  12  and  14  are joined by a hinge  24  and have locking means  26  and  28  which are engaged when the two components are brought into opposition. A sealing ridge or gasket  30  may be provided around the perimeter of the opposable components  12  and  14  to guard against leakage of samples or reagents. 
     FIG. 1B shows device  10  after the opposable components  12  and  14  have been brought into opposition. The chromatographic medium  18  (including zones  20  and  22 ) is visible through window  32 . 
     The present invention will now be described by reference to the following non-limiting examples. 
     EXAMPLE 1 
     A Strep B assay was performed in the following manner. 
     A device such as is shown in FIGS. 1A and 1B was provided with a sample preparation means which was adapted to receive a throat swab. A throat swab from a patient was placed on the sample preparation means and wetted with a suitable extraction reagent. After a suitable interval, an absorbent pad impregnated with anti-Strep B antibodies labeled with pink colloidal gold was added to the sample preparation means. The components were then brought into opposition so that the absorbent pad in the sample preparation means was brought into contact with the chromatographic medium on the second opposable component, thereby allowing the sample (including the labeled antigen/antibody complex) to migrate up the chromatographic medium. A band of the chromatographic medium was provided with anti-Strep B antigens and, when the sample reached this band, binding of the labeled complex occurred in that region, indicating the presence of Strep B antigen. 
     In a variation of this procedure, the impregnated absorbent pad can be placed in the second opposable component and brought into contact with the swab when the two components are brought into opposition. In another variation, the absorbent pad can be eliminated altogether and the labeled antibodies can be added to the swab as a liquid suspension and the swab can be used to apply the sample to the chromatographic medium when the two components are brought into opposition. 
     EXAMPLE 2 
     A bi-directional immunochromatographic assay for antibodies to  H. pylori  was performed in the following manner. 
     An assay device  50 , as shown in FIG. 2A, having a first opposable component  52  and a second opposable component  54 , was used. The first opposable component  52  included a first sample preparation means  56  which was in contact with one end of the chromatographic medium  58 . The chromatographic medium  58  was made of nitrocellulose and included a zone  60  at which were immobilized specific antigen of  H. pylori . The second opposable component  54  included an absorbent pad  62  and a second sample preparation means  64 . 
     The serum specimen to be tested for the presence of antibodies to  H. pylori  was placed on the first sample preparation means  56  and thereby applied to the chromatographic medium  58 . As the serum sample migrated up the chromatographic medium  58 , antibodies specific to  H. pylori  antigen reacted with the antigen immobilized in zone  60  and were retained in that zone. A suspension of anti-human IgG antibody labelled with pink colloidal gold was then placed on the second sample preparation means  64 , and the two opposable components were brought into opposition by closing hinge  66  which joined the two components. The device then appeared as in FIG.  2 B. 
     When the two components were brought into opposition, the absorbent pad  62  was brought into contact with the first sample preparation  56  and drew the serum sample out of the first sample preparation means  56  and the chromatographic medium  58 . Simultaneously, the second sample preparation means  64  was brought into contact with the opposite end of the chromatographic medium  58 , thereby applying the solution of labeled anti-human IgG to the chromatographic medium  58 . This caused a reversal of flow along the chromatographic medium  58  with the serum sample flowing back toward the first sample preparation means  56 , followed by the labelled anti-human IgG antibodies. As the labelled anti-human IgG antibodies reached zone  60 , they were bound to that zone, which resulted in a colored strip which was visible through window  68  and indicated the presence of  H. pylori  antibody. 
     EXAMPLE 3 
     An assay for fecal occult blood was conducted as follows. 
     A fecal sample was collected in a conventional manner by having a patient smear a specimen card with feces. 
     In a conventional assay procedure, the card would be placed in a solution which extracts hemoglobin, the extraction solution would then be filtered and then the filtered solution would be tested for the presence of hemoglobin. Besides its obvious aesthetic drawbacks, this procedure requires the use of a filter, an extraction vessel and a transfer pipette, all of which present disposal and/or sterilization problems. This also presents an unavoidable risk of spillage with resulting contamination. 
     By using an assay device according to the present invention, the use of ancillary vessels and the attendant contamination disposal problems were virtually eliminated. 
     The sample card was placed on the sample preparation means of a device such as is shown in FIGS. 1A &amp; 1B. The sample preparation means included an absorbent pad made of cellulose. A quantity of extraction solution containing dye-labeled anti-human hemoglobin antibodies was also added to the sample preparation means and was allowed to extract hemoglobin from the sample for about one minute. The device was then closed and the extraction solution, along with the extracted hemoglobin which was bound to the dye labeled antibodies, migrated up the nitrocellulose chromatographic mechanism. The particulate matter in the feces was filtered out by the cellulose and was not applied to the chromatographic medium. As the hemoglobin migrated up the nitrocellulose, it reached a zone to which anti-human hemoglobin antibodies were immobilized and the labelled hemoglobin was localized in that zone. The resulting band of color indicated the presence of fecal occult blood. 
     This assay was accomplished without any extraneous vessels and the test device completely encased all specimens and reagents, greatly reducing the possibility of exposure to these materials and any infectious agents which they may contain. 
     EXAMPLE 4 
     An assay for the presence of  H. pylori  antibodies was conducted using a device  100  as shown in FIGS. 3A and 3B. The device has first and second opposable components  102  and  104 . The first opposable component  102  contains a first application pad  106  which has been impregnated with an inert dye. Adjacent to and in communication with first application pad  106  is a nitrocellulose chromatographic medium  108 . At the end of the chromatographic medium which is opposite to the first application pad  106  is a reagent-impregnated reagent pad  110 . The reagents which are impregnated on the reagent pad  110  include anti-human IgG antibodies which have been labeled with pink colloidal gold. The second opposable component  104  contains an absorbent pad  112  and a second application pad  114 . 
     In operation, a buffer solution was added to the second application pad  114  and the serum which was to be tested was added to the first application pad  106 . The serum sample migrated across the chromatographic medium  108  and, as was indicated by the progress of the inert dye, passed zone  116  of the chromatographic medium  108 . ( H. pylori  antigen had previously been immobilized in zone  116 .) After this occurred, the two components were brought into opposition and the absorbent pad  112  was brought into contact with the first application pad  106 , thereby causing the flow of the serum along the chromatographic medium  108  to be reversed. This also brought the second application pad  114  into contact with the reagent pad  110  and caused the buffer solution to be applied to the chromatographic medium  108  along with the labeled antibody reagent. When the buffer solution reached zone  116 , the labeled antibody bound to the anti- H. pylori  antibody that was itself bound to the  H. pylori  antigen that was located at the Zone  116 , causing a pink band to develop, indicating the presence of anti- H. pylori  antibody. The pink band in zone  116  was visible through window  118  as shown in FIG.  3 B. 
     While the foregoing examples have described a variety of sample preparation procedures which can be performed in a device according to this invention, these examples are not an exhaustive listing of such procedures. In addition to sample dilution or the addition of extraction reagents or labels, it is possible to macerate tissue in a properly configured sample preparation means, such as by mechanical action before or after the components of the device are brought into opposition. 
     The assay device of the present invention is especially well suited to running several chromatograms simultaneously, a significant advantage when a quantitative or semi-quantitative assay is required.