ASSAY DEVICES FORMED FROM MATERIALS HAVING DIFFERENT POROSITIES

Abstract: An assay device which includes a first absorbent material having a surface upon which is placed a binder for an analyte, and a second absorbent material which is contiguous with and preferably surrounds the first absorbent material. In one aspect the second absorbent material is less porous than the first absorbent material and guides the flow of a test sample to the first absorbent material and the binder supported thereon. In another aspect, the first absorbent material is less porous than the second absorbent material, and the first absorbent material serves to guide the flow of washing liquids to the second absorbent material, whereby such washing liquids are absorbed by the second absorbent material.

Description: ASSAY DEVICES FORMED FROM MATERIALS HAVING DIFFERENT POROSITIES This invention relates to assay devices which, in particular, may be used in conducting so-called dynamic assays or "flow through" assays. More particularly, this invention relates to assay devices formed from materials having different porosities. Immunological assays may be conducted by using an apparatus which contains an absorbent material or an absorbent zone which induces a flow of liquid containing an analyte through a membrane, which supports an antigen, antibody, or other type of binder for the analyte. U.S. Patent No. 4,366,241, issued to Tom, et al. , discloses a flow-through assay device and assays employing such a device. The assay device has an immunosorbing zone to which is fixed a member of an immunological pair. Located adjacent to the immunosorbing zone is a liquid absorbing zone which draws a liquid sample through the immunosorbing zone. The absorbing zone can control the rate at which the liquid sample is drawn through the immunosorbing zone. U.S. Patent No. 4,632,901, issued to Valkirs, et al., discloses an apparatus and process for conducting immunoassays wherein an antibody such as a monoclonal antibody is bound to a membrane or filter. An absorbent material is located beϊow the membrane or filter which induces the flow of a fluid sample through the membrane or filter. Analyte in the fluid sample will bind with the antibody on the membrane or filter. Labeled antibody against the analyte may then be added. A washing step then removes the unbound labeled antibody. The presence of labeled antibody on the membrane or filter following the washing step is indicative of the presence of analyte in the sample being assayed. In accordance with an aspect of the present invention, there is provided an assay device which comprises a first absorbent material and a second absorbent material. The first absorbent material has a surface upon which is placed at least one binder for an analyte. The second absorbent material guides the flow of a sample to be assayed to the at least one binder for the analyte. The second absorbent material is contiguous with the first absorbent material and has a porosity which is less than that of the first absorbent material. In a preferred embodiment, the second absorbent material surrounds the first absorbent material. For example, the first absorbent material may be in the form of a cylinder, and the second absorbent material encircles or surrounds the cylinder, upon the surface of which is supported at least one binder for an analyte. When a sample is applied to the top of the assay device, the absorbent material having the lesser porosity guides or directs the flow of the sample to the first absorbent material or cylinder, whereby the sample flows through the at least one binder for the analyte, and is absorbed by the first absorbent material. The first absorbent material, in one embodiment, has a pore volume of from about 0.001 cc/g to about 0.400 cc/g, preferably from about 0.010 cc/g to about 0.280 cc/g. The first absorbent material also has a pore diameter of from about 0.005u to about 125u, preferably from about 0.03u to about 3u. In a preferred embodiment, the first absorbent material is a porous ceramic material. The surface of the first absorbent material may be pretreated with wetting agents or polymers to speed or slow the absorption time as desired. The surface of the first absorbent material may also be treated with surfactants and/or proteins to reduce non-specific binding, or the surface may be treated with particles such as latex particles or immunoprecipitates which act as reagent carriers. The second absorbent material, in one embodiment, is non-porous, and has a density of from about 1.8 g/cc to about 5.0 g/cc, preferably from about 2.2 g/cc to about 4.4 g/cc. Preferably, the second absorbent material is a non-porous ceramic material. As is the case with the first absorbent material, the surface of the second absorbent material may be pretreated with wetting agents or polymers to speed or slow the absorption time, and/or may be treated with surfactants and/or proteins. The first and second absorbent materials may be formed from the same type of material (eg., porous plastics, fibrous materials, ceramics, etc.), wherein such material is processed so as to form a porosity gradient from the higher porosity first absorbent material to the lower porosity, or non-porous second absorbent material. The porosity gradient may be formed, for example, by dry-pressing a ceramic powder in a mold. The lower die of the mold may have a movable center. ■ The center part may be raised during the dry powder filling step, thereby reducing the volume of dry powder above it. The center part may be retracted during the compression process, thereby providing a more porous center of the finished part. In one embodiment, the absorbent may be a non-fibrous porous plastic material which is "wettable" and capable of providing for capillary flow into the plastic. Examples of non-fibrous plastic absorbent materials which may be used include polyolefin, polyester, porous polyvinyl chloride, and polystyrene. The porous plastic may be impregnated with an appropriate material such as a cellulosic material which fills in the pores of the plastic so as to provide a porous material with desired pore sizes. In one embodiment, the porous plastic may have a pore size of from about 5μ to 500μ, preferably from about lOu to 40u. The cellulosic material does not adversely affect the absorbency and wettability of the plastic. Examples of a cellulosic material which may be employed are cotton linters and starch, such as arrowroot starch. The porous plastic may be a hydrophobic porous plastic which is rendered hydrophilic, or "wettable", by the addition of a wetting agent such as a surfactant. The surfactant may be applied to all or a portion of the porous plastic material (eg., after the porous plastic support is impregnated with a cellulosic material). The plastic, which is hydrophobic, is contacted with a surfactant so as to render a portion of the surface of the porous plastic hydrophilic, or wettable. The binder used in the assay is applied to the "wettable" portion of the porous material. If a limiting amount of surfactant is applied, the flow rates of specimen and of labeled antibody into the absorbent will be slowed, thus affecting the sensitivity of the assay. The surfactant may be applied to the entire surface of the support or to a_ portion of the surface, thus rendering either the entire surface or a portion of the surface hydrophilic. In another embodiment, the absorbent may be formed from pressed fiber material. The pressed fiber is also hydrophilic, or wettable, and capable of providing for capillary flow into the fiber disc. The fibers are pressed so as to provide a desired porosity gradient. The surface of the absorbent material which supports the binder is preferably a smooth surface in that it enhances the ability to read tracer on such surface. Examples of fibrous materials which may be employed include, but are not limited to, glass fibers, polyesters, polyamides, cellulose fibers and the like. The fibrous materials may be placed in a pressing die and then pressed by a punch having an inverted conical-shaped cavity, whereby there is formed a porosity gradient. Binder may be applied directly to the surface of the absorbent materials, or may be supported by solid particles such as latex particles, or by diatomaceous earth, porous glass, resin particles or the like. The surface of the absorbent material may be coated or treated with a material to prevent non-specific adsorption, e.g., BSA, or any other blocking substance known in the art. In a preferred embodiment, the absorbent may be made of a porous ceramic material. The ceramic absorbent may be defined as a non-fibrous, inorganic, porous matrix. The ceramic material is formed so as to provide a porosity gradient from the higher porosity first absorbent material to the lower porosity second absorbent material. The device may be employed in connection with a variety of various assays. Such assays include, but are not limited to, competitive assays, sandwich assays, enzyme-linked immunosorbent (ELISA) assays, agglutination assays, indirect assays, and other assays known to those skilled in the art. In a competitive assay an analyte and tracer compete with a binder specific for the analyte and tracer. The tracer is the analyte or an appropriate analogue thereof which is coupled to a detectable label or marker. The tracer and analyte compete for a limited number of binding sites on the binder and the amount of tracer which is bound to the binder is inversely proportional to the amount of analyte in the sample. The amount of tracer, and the amount of analyte as well can be determined by measuring the amount of label present. The label or marker which is part of the tracer may be a detectable marker such as a radioactive isotope of, for example, iodine, cobalt, or tritium, an enzyme, a fluorescent dye, an absorbing dye, a luminescent substance, a spin label, biotin, a colored particle or any other labeling substance known to one of ordinary skill in the art. A preferred label is comprised of colored particles such as colloidal gold. When a sandwich assay is employed, the binder which is specific for an analyte, is contacted with a sample containing or suspected of containing analyte. Analyte present in the sample will bind with the binder. After the sample has flowed past the binder into the absorbent material, the analyte-binder complex is then contacted with a tracer. The tracer is a ligand which is specific for the analyte to be assayed. For example, the tracer can be an antibody elicited in response to the analyte being assayed. The ligand is preferably labeled with a detectable marker as described above, and the amount of analyte present in the sample is determined by the amount of label present on the surface of the absorbent. In an indirect sandwich assay, analyte bound to the supported binder is contacted with a binder for the analyte which becomes bound to analyte bound to the supported binder. The tracer used in the assay is a labeled ligand which is bound by the binder bound to the analyte bound to the supported binder. In an ELISA assay, the tracer or ligand is labeled with an enzyme and the amount of analyte present in the sample to be assayed is determined by the amount of bound enzyme label present. An ELISA assay may be run as a sandwich assay or a competitive assay. In an agglutination assay, a binder consisting of particles sensitized with an antigen or antibody specific for an analyte is contacted with a sample suspected of containing the analyte. The presence of analyte is evidenced by agglutination of the solid particles. The binder which is used in an assay is dependent upon the analyte being assayed. For example, if the analyte is an antigen or hapten, the binder may be an antibody or naturally occurring substance which is specific for the analyte. If the analyte is an antibody, the binder may be an antibody, an antigen, or a naturally occurring substance which is specific for the analyte. The assays hereinabove described may be employed for determining a wide variety of analytes. Examples of analytes which may be assayed in accordance with the present invention include, but are not limited to, drugs, hormones, macromolecules, antibodies, microorganisms, toxins, polypeptides, proteins, polysaccharides, nucleic acids, etc. The selection of a suitable analyte is deemed to be within the scope of those skilled in the art. One can conduct such assays by placing a binder for an analyte (antigen, hapten, antibody) on at least a portion of the surface of the first absorbent material. The binder may be placed on the surface of the first absorbent material in the form of a spot or dispensed, sprayed, or printed onto the membrane surface to produce symbolic forms. The binder may be placed directly on the surface of the first absorbent material (either by passive or covalent methods of attachment) or may be supported on solid particles which are placed on the surface of the first absorbent material. The surface of the first and second absorbent materials is then contacted with a sample suspected of containing an analyte. The binder which is supported on the surface of the the first absorbent material is specific for the analyte. An analyte present will bind with the binder on the surface of the first absorbent material and form an analyte binder complex. Upon application of the sample to the surfaces of the first and second absorbent materials, the flow of the sample will be directed to the first absorbent material and to the binder placed thereon. Such flow of the sample is due to the lower porosity of the second absorbent material, which creates a "funneling" effect whereby the sample flows toward the more porous first absorbent material, thus providing for optimal binding of analyte in the sample of the binder. The surface of the first and second absorbent materials is then contacted with tracer simultaneously with or subsequent to contact with the sample. The flow of tracer also will be directed to the first absorbent material. The tracer is a labeled form of a ligand and the ligand employed is dependent on the assay format. In a competitive assay, the ligand of the tracer is one which is bound by the binder for the analyte. In a sandwich assay, the ligand of the tracer is bound by the analyte (direct assay) or bound by a binder for the analyte (indirect assay). When a sandwich assay is employed, the tracer is preferably a labeled form of a ligand which is specific for the analyte. In the assay, the analyte and tracer become bound to the supported binder (the tracer is directly bound to supported binder in a competitive assay and bound to the supported binder through the analyte in a sandwich assay) and therefore remain on the surface of the first absorbent material. The binding occurs while flowing the sample and tracer past the supported binder and any unbound portions of flow into the absorbent by means of capillary movement through the first absorbent material. The rate of flow of the sample through the assay device is dependent upon the porosity of the device and the treatment, if any, given to the surface of the device. In general, 0.25ml of sample will flow through about 1cm of absorbent material in a period of time of from about 2 seconds to about 60 minutes, preferably from about 6 seconds to about 45 minutes, and more preferably from about 10 seconds to about 20 seconds. After one or more of the steps, one may which to wash the surface which supports the binder, prior to a subsequent step. In the case of a sandwich assay, the surface may be washed subsequent to sample addition and prior to tracer addition and/or subsequent to tracer addition and prior to the development thereof. The wash may contain standard aqueous buffers; eg., phosphate and Tris buffers. The wash may also contain detergents and chaotropic agents to minimize nonspecific binding. The method of determination of analyte in the assayed sample depends upon the type of marker used in conjunction with the ligand or tracer. If a dye is used as label or marker, it will appear on the surface of the first absorbent material and remain on the surface following any washing steps. In the case of an enzyme label, a suitable, usually precipitating, substrate is used to provide color. The presence of radioactive, fluorescent, luminescent, enzyme, chromogen, spin label, biotin, gold particles or other types of markers which remain on the surface of the first absorbent material can be determined by any means known in the art. The assay may be qualitative or a quantitative assay, and the term "determining", as used herein, means qualitative and/or quantitative determining of analyte. In accordance with another aspect of the present invention, there is provided an assay device which comprises a first absorbent material and a second absorbent material. The first absorbent material has a surface which has at least one binder for an analyte. The second absorbent material is contiguous with the first absorbent material. The first absorbent material has a porosity which is less than that of the second absorbent material, whereby the flow of liquid applied to the surface of the device is guided from the first absorbent material to the second absorbent material. In a preferred embodiment, the second absorbent material surrounds the first material. For example, the first absorbent material may be in the form of a cylinder and the second absorbent material encircles or surrounds the cylinder, as hereinabove described. In this aspect of the present invention, a small amount of a sample suspected of containing analyte may be placed on top of the first absorbent material, the surface of which contains at least one binder for the analyte. The sample remains in contact with the at least one binder for a time sufficient to form an analyte-binder complex. In this assay procedure, when one wishes to wash the assay device through one or more washing steps, one may employ large volumes of washing agents. Because the first absorbent material has a lower porosity than the second absorbent material, the first absorbent material directs the flow of the washing agents to the second absorbent material, which absorbs the larger volumes of washing agents. The first absorbent material, in one embodiment, is a non-porous material which has a density of from about 1.8 g/cc to about 5.0 g/cc, preferably from about 2.2 g/cc to about 4.4 g/cc. In one embodiment, the first absorbent material is a non-porous ceramic. The second absorbent material, in one embodiment, has a pore volume, of from about O.OOlcc/g to about 0.400 cc/g preferably from about 0.010 cc/g to about 0.280 cc/g and has a pore diameter of from about 0.005u to about 125u, preferably from about 0.030u to about 3u. In one embodiment, the second absorbent material is a porous ceramic material. The first and second absorbent materials may be formed from materials such as those hereinabove described; however, the porosity gradient which is formed is from the higher porosity second absorbent material to the lower porosity first absorbent material, which has a surface having at least one binder for the analyte. The surfaces of the first and second absorbent materials may be pretreated with wetting agents or polymers to speed or slow the absorption time. The surfaces of the first and second absorbent materials may also be treated with surfactants and/or proteins to reduce non-specific binding. Assays which may be employed using this assay device include those hereinabove described. In conducting assays with the device, a binder for analyte may be placed on at least a portion of the surface of the first absorbent material by methods such as those hereinabove described. A small amount of a sample suspected of containing analyte is placed on the surface of the first absorbent material, and remains upon the surface of the first absorbent material for a time sufficient to form an analyte-binder complex. After the application of sample, and prior to the addition of tracer and/or developing reagents, the surface of the first absorbent material may be washed with larger volumes of washing agents. As the washing agents are applied to the device, the washing agents, upon washing the sample from the first absorbent material, are directed from the less porous first absorbent material to the more porous second absorbent material, whereby the sample and washing agents are absorbed into the second absorbent material. Such is also true for any additional washing steps which may need to be employed in the assay process. It is to be understood, however, that the scope of the present invention is not to be limited to the specific embodiments described above. The invention may be practiced other than as particularly described and still be within the scope of the accompanying claims.

Claims: WHAT IS CLAIMED IS: 1. An assay device comprising: a sample receiving surface, said sample receiving surface including a first surface portion having at least one binder for an analyte, said first surface portion being formed from a first absorbent material; and a second surface portion for guiding the flow of a liquid sample to be assayed to said at least one binder for an analyte, said second surface portion being contiguous with said first surface portion, said second surface portion being formed from a second absorbent material, and said second absorbent material having a porosity which is less than that of said first absorbent material. 2. The device of Claim 1 wherein said first absorbent material has a pore volume from about O.OOlcc/g to about 0.400 cc/g. 3. The device of Claim 2 wherein said first absorbent material has a pore volume of from about 0.010 cc/g to about 0.280 cc/g. 4. The device of Claim 1 wherein said second absorbent material has a density of from about 1.8g/cc to about 5.0 g/cc. 5. The device of Claim 4 wherein said second absorbent material has a density of from about 2.2 g/cc to about 4.4 g/cc. 6. - The device of Claim 1 wherein said first absorbent material is a porous ceramic material. 7. The device of Claim 1 wherein said second absorbent material is a non-porous ceramic material. 8. An assay device, comprising: a sample receiving surface, said sample receiving surface including a first surface portion having at least one binder for an analyte, said first surface portion being formed from a first absorbent material; and a second surface portion, said second surface portion being contiguous with said first surface portion, said second surface portion being formed from a second absorbent material, wherein said first absorbent material has a porosity which is less than that of said second absorbent material, and wherein said first surface portion is capable of directing the flow of liquid applied to said device to said second surface portion. 9. The device of Claim 8 wherein said first absorbent material has a density of from about 1.8 g/cc to about 5.0 g/cc. 10. The device of Claim 9 wherein said first absorbent material has a density of from about 2.2 g/cc to about 4.4 g/cc. 11. The device of Claim 8 wherein said second absorbent material has a pore volume of from about 0.001 cc/g to about 0.400 cc/g. 12. The device of Claim 11 wherein said second absorbent material has a pore volume of from about 0.010 cc/g to about 0.280 g/cc. 13. The device of Claim 8 wherein said first absorbent material is a non-porous ceramic material. 14. The device of Claim 8 wherein said second absorbent material is a porous ceramic material.

Inventors: SCHRENK W JUERGEN, SCHRENK, W., JUERGEN
Applicants: CARTER WALLACE, CARTER-WALLACE, INC.
Application year: 1993
Main classification: G01N33
IPC codes: B01L3, G01N33